CHARACTERISATION AND ANTIMICROBIAL ACTIVITY OF …vuir.vu.edu.au/32304/1/TAWAKKAL Intan Syafinaz -...

CHARACTERISATION AND ANTIMICROBIAL ACTIVITY OF

POLY(LACTIC ACID)/KENAF BIO-COMPOSITES CONTAINING A

NATURAL AGENT

A thesis submitted for the degree of Doctor of Philosophy

by

Intan Syafinaz Mohamed Amin Tawakkal

B. Eng. (Process and Food), M. Sc.

College of Engineering and Science

Victoria University

Melbourne, Australia

October 2016

i

Abstract

The use of materials based on poly(lactic acid) (PLA) as alternatives to petroleum-based

polymers for a range of applications has increased in recent years. In the case of food packaging

in particular, PLA has experienced growth in combination with the use of a wide range of other

materials and/or additives derived from natural and renewable resources. However, the initial

costs associated with new technologies to produce PLA and/or other bio-based polymers are

typically high, so new techniques are required to reduce costs without compromising material

properties and biodegradability. Naturally sourced lignocellulose fibres such as kenaf

(Hibiscus cannabinus L.) are often used as cost-reducing fillers and/or reinforcing agent for

biopolymers such as PLA.

This study explores the characteristics and antimicrobial (AM) activity of PLA and PLA/kenaf

composites incorporated with thymol, a natural bio-active AM substance/agent. The production

of PLA/kenaf composites containing thymol is intended for potential use in AM packaging

applications such as rigid and/or flexible packaging and coatings. Composites of PLA

incorporated with 5 to 40% w/w kenaf fibre loadings and thymol concentrations ranging from

5 to 30% w/w were prepared and compared with control systems containing either no kenaf or

no thymol. Kenaf fibres can be treated by alkalisation to improve compatibility with polymer

matrices. It was found that the PLA/kenaf composites containing treated kenaf possessed

significantly higher tensile strength and stiffness than composites prepared with untreated

kenaf. Micrograph images of the fracture surface revealed better adhesion between the treated

kenaf fibres and the matrix, thereby resulting in improved reinforcement of the composite.

Moreover, PLA/kenaf composites containing thymol exhibited lower tensile strength than

those without thymol, suggesting a possible plasticizing effect in the presence of the additive.

ii

The PLA systems containing treated kenaf were selected as the main formulation with which

to systematically investigate the effect of different processing conditions and formulations on

mechanical and thermal properties, the interaction and quantification of AM agent, the

migration rate of AM agent from the materials as well as the potential AM properties of these

systems. A study of PLA/kenaf composites containing 5% w/w thymol demonstrated that the

addition of fibre (10 to 40% w/w) to the composites affected the tensile properties of the system

more than the incorporation of thymol alone. Thermogravimetric analyses of PLA and

PLA/kenaf composites incorporated with 5% and 10% w/w thymol revealed no significant

changes in the decomposition temperature. Analysis by differential scanning calorimetry,

however, showed a decrease in all of the key thermal transitions with the addition of thymol.

Moreover, the quantification of thymol in PLA and PLA/kenaf composites after thermal

processing, as well as the interactions amongst the PLA matrix, kenaf fibres and the AM agent,

were investigated. The PLA incorporating a fibre content in the range of 10 to 40% w/w

retained less thymol upon processing than PLA alone and the PLA/kenaf composites

containing the highest fibre loadings exhibited the lowest thymol retention. The observed losses

were attributed to the higher mechanical shear required during the mixing process which may

lead to thermal degradation, as well as the creation of voids in the composites that can facilitate

the release of thymol from the system.

In order to investigate the migration of thymol from the materials, the PLA and PLA/kenaf

films containing 10% w/w thymol were prepared and placed in contact with food simulants

comprised of 15% and 95% v/v ethanol/water mixtures at different temperatures. First-order

kinetics, diffusion modelling and Fick’s law modelling were used to describe the release of the

AM agent. The release rate of thymol into 95% v/v ethanol/water at different temperatures

displayed Fickian behaviour, with diffusion coefficient values between 1 and 100 10-11 m2 s-

1 and with close to 100% of thymol being released. The release rate of thymol was temperature

iii

dependent and was affected by the percentage of ethanol in the simulant. In the case of PLA

and PLA/kenaf films, a faster release occurred in 95% v/v ethanol/water than in 15% v/v

ethanol/water with the composite film exhibiting a higher diffusion coefficient in each case.

The AM activity and stability under different storage conditions for the PLA-based materials

containing kenaf fibres and thymol were also investigated. Microbial reductions of 6.8 log

CFU mL-1 in tryptone soy broth after two days at 37°C and 3.1 log CFU cm-2 on processed

meat samples after 30 days at 10°C were observed in PLA film containing 30% w/w kenaf and

10% w/w thymol. The PLA/kenaf films containing 20 to 30% w/w thymol showed significant

inhibition of microbial growth in solid and liquid media compared to active PLA. The

composite films also inactivated Escherichia coli (E. coli) that was inoculated on the surface

of meat samples. The PLA/kenaf films containing thymol that were wrapped with aluminium

foil were able to retain the additive in the films after 3 months of storage at ambient

temperature, however films that were not wrapped in foil lost some thymol to the atmosphere.

Therefore, the development of active PLA composite films containing the natural fibre as a

filler demonstrates a strong potential for the development of active packaging films that can

extend the shelf life of certain foods.

iv

Declaration

“I, Intan Syafinaz Mohamed Amin Tawakkal, declare that the PhD thesis by Publication

entitled Characterisation and Antimicrobial Activity of Poly(lactic acid)/Kenaf Bio-

composites Containing a Natural Agent is no more than 100,000 words in length including

quotes and exclusive of tables, figures, appendices, bibliography, references and footnotes.

This thesis contains no material that has been submitted previously, in whole or in part, for the

award of any other academic degree or diploma. Except where otherwise indicated, this thesis

is my own work”.

Intan Syafinaz Mohamed Amin Tawakkal Date: 26 September 2016

v

Acknowledgments

All praises be to the Mighty Allah, the Merciful and the Beneficent for the strength, patience,

determination and blessing in the completion of this study. It is impossible for me to complete

this thesis all by myself without the guidance and help from many important people.

Thank you to my principal supervisor, Professor Stephen W. Bigger who guides me

continuously in all aspect of scientific research. I thank him for assisting me to maintain focus

and for his attention to details. I am grateful for his encouragement, supports, constructive

criticisms and contribution to the progress of this thesis.

Thank you to my associate supervisor, Dr Marlene J. Cran, for sharing outstanding knowledge

and for providing helpful advices and suggestions. I thank her for helping me with the sample

analyses and for her contribution to journal submissions. Without her guidance, editorial and

artworks assistance, this thesis by publication would not have been possible.

Thank you to all the administrative and laboratory technicians in Victoria University (Werribee

Campus) especially to Joseph Pelle, Stacy Lloyd, Sarah Fraser and Min Thi Nguyen. Thank

you to Mike Allen, senior research assistance of RMIT, for assisting me throughout the

preparation of my samples and for sharing his knowledge and experiences.

Thank you to my colleagues from College of Engineering and Science and College of Health

and Biomedicine who have made my experience at Werribee Campus enjoyable and

worthwhile especially to Kuorwel Kuai Kuorwel, Aprianita Aprianita, Qomarudin Qomarudin,

Anusuya Joshi, Rabia Ashraf, Zeinab Ahmed, Nuwan, Mokhamad Nur, Narges Dargahi,

Rahmi Nurdiani, Chathuri Piyadasa, Baidya Nath Prasad Sah, Md Toheder Rahaman, Ghofran

and Zuhair Nor. Thank you for being supportive all the way.

vi

I gratefully acknowledge the Ministry of Education Malaysia and Universiti Putra Malaysia

(UPM) for providing the PhD scholarship as well as Victoria University for the publication

incentives.

I would like to thank my husband, Zuhair Nor for his endless love, patience, support and

motivation in the past, now and future.

A special thanks to my family especially my beloved mother Rahimah Mahmod. I am

particularly grateful for her valuable assistance in several trips to Australia for taking care of

my daughter Qaseh Zuhair during my final stage of thesis writing.

vii

Publications from this Work

Papers in Refereed Journals

Tawakkal, I. S. M. A., Cran, M. J., Miltz, J. and Bigger, S. W. (2014). A Review of Poly(Lactic

Acid)-Based Materials for Antimicrobial Packaging. Journal of Food Science, 79(8), 1477-

1490.

Tawakkal, I. S. M. A., Cran, M. J. and Bigger, S. W. (2014). Effect of Kenaf Fibre Loading

and Thymol Concentration on the Mechanical and Thermal Properties of PLA/Kenaf/Thymol

Composites. Industrial Crops and Products, 61, 74-83.

Bigger, S. W., Cran, M. J. and Tawakkal, I. S. M. A. (2015). Two Novel Algorithms for the

Thermogravimetric Assessment of Polymer Degradation under Non-Isothermal Conditions.

Polymer Testing, 43, 139-146.

Tawakkal, I. S. M. A., Cran, M. J. and Bigger, S. W. (2016). Interaction and Quantification of

Thymol in Active PLA-Based Materials Containing Natural Fibers. Journal of Applied

Polymer Science, 132, 42160 (1 of 11).

Tawakkal, I. S. M. A., Cran, M. J. and Bigger, S. W. (2016). Release of Thymol from PLA-

Based Antimicrobial Films Containing Kenaf Fibres as Natural Filler. LWT-Food Science and

Technology, 66, 629-637.

Tawakkal, I. S. M. A., Cran, M. J. and Bigger, S. W. (2016). The Influence of Chemically

Treated Natural Fibers Containing Thymol in Poly(Lactic Acid)-Based Antimicrobial

Composites for Packaging. Polymer Composites (early view article).

Tawakkal, I. S. M. A., Cran, M. J. and Bigger, S. W. (2016). Effect of Poly(Lactic Acid)/Kenaf

Composites Incorporated with Thymol on the Antimicrobial Activity of Processed Meat.

Journal of Food Processing and Preservation (accepted for publication).

viii

Conference/Symposium/Colloquium Presentations

Intan S. M. A. Tawakkal, Marlene J. Cran and Stephen W. Bigger, Development of Active

Poly(Lactic Acid) Composites Reinforced with Natural Fibres, paper presented at the

Malaysian Postgraduate Colloquium, Melbourne, Australia, 19-20 December 2015.

Intan S. M. A. Tawakkal, Marlene J. Cran and Stephen W. Bigger, Development of Active

Poly(Lactic Acid) Composites Reinforced with Natural Fibres, poster presented at the 27th

IAPRI Symposium on Packaging, Valencia, Spain, 8-11 June 2015.

Intan S. M. A. Tawakkal, Marlene J. Cran, and Stephen W. Bigger, Antimicrobial Activity of

Poly(Lactic Acid)/Kenaf Composites Containing a Natural Agent, poster presented at the 5th

AIFST Food Science Summer School, Melbourne, Australia, 28-30 January 2015.

Intan S. M. A. Tawakkal, Marlene J. Cran, and Stephen W. Bigger, Characteristics of Active

Natural Fibre Reinforced Poly(Lactic Acid) Composites, paper presented at the 19th IAPRI

World Conference on Packaging, Melbourne, Australia, 15-19 June 2014.

Stephen W. Bigger, Marlene J. Cran and Intan S. M. A. Tawakkal, Developments in

Antimicrobial Food Packaging Research, paper presented at the 19th IAPRI World Conference

on Packaging, Melbourne, Australia, 15-19 June 2014.

Stephen W. Bigger, Marlene J. Cran and Intan S. M. A. Tawakkal, Thermal Measurements in

Assessing the Performance of Poly(lactic acid)/Natural Fibre Composites for Food Packaging

Applications, paper presented at the 248th ACS National Meeting & Exposition, California,

USA, 10-14 August 2014.

ix

Awards and Achievements

Won the Best Presenter Award in the Malaysian Postgraduate Colloquium, Melbourne,

Australia, 19-20 December 2015 with presentation entitled Development of Active Poly(Lactic

Acid) Composite Reinforced Natural Fibres, Melbourne, Australia, 19-20 December 2015.

Winner of the 2nd runners up place in the Victoria University Three Minute Thesis Competition

with presentation entitled Love Food and Nature…Hate waste!, Melbourne, Australia, 27

September 2013.

x

Details of Included Papers: Thesis by Publication

Included papers update:

Chapter 3 is now published in Polymer Composites.

Chapter 8 is accepted for publication in Journal of Food Processing and Preservation.

xi

Table of Contents

Abstract ......................................................................................................................... i

Declaration .................................................................................................................. iv

Acknowledgments......................................................................................................... v

Publications from this Work ...................................................................................... vii

Details of Included Papers: Thesis by Publication ....................................................... x

List of Unpublished Figures...................................................................................... xiii

List of Unpublished Tables ....................................................................................... xiv

List of Abbreviation and Nomenclature ................................................................... xiv

Chapter 1 – Introduction ............................................................................................... 1

1.1 Overview .......................................................................................................................... 1 1.1.1 Antimicrobial Packaging Systems ............................................................................ 1

1.1.2 Ternary AM packaging systems ............................................................................... 4

1.2 Aims of this Work ............................................................................................................ 7 1.3 Thesis Outline .................................................................................................................. 8

Chapter 2 – Literature Review .................................................................................... 11

Chapter 3 – The Influence of Fiber Chemical Treatment ........................................... 28

3.1 Abstract ..................................................................................................................... 32 3.2 Introduction ............................................................................................................... 33 3.3 Materials and Methods .............................................................................................. 35

3.3.1 Materials ............................................................................................................ 35

3.3.2 Preparation of Composites ................................................................................. 36

3.3.3 Imaging of Fibers and Composites .................................................................... 37

3.3.4 Infrared Analyses ............................................................................................... 37

3.3.5 Tensile Testing ................................................................................................... 38

3.3.6 Thermal Property Testing .................................................................................. 38

3.3.7 Disintegration Studies ........................................................................................ 39

3.4 Results and Discussion .............................................................................................. 40 3.4.1 Structural Analysis ............................................................................................. 40

3.4.2 Processing of PLA and Composites ................................................................... 41

3.4.3 Composite Morphology ..................................................................................... 43

3.4.4 Mechanical Properties ........................................................................................ 44

xii

3.4.5 Thermal Properties ............................................................................................. 49

3.4.6 Thermogravimetric Analysis ............................................................................. 52

3.4.7 Decomposition in Compost................................................................................ 55

3.5 Conclusions ............................................................................................................... 57 3.6 References ................................................................................................................. 59

Chapter 4 – Effect of Kenaf Fibre Loadings ............................................................... 78

Chapter 5 – Novel Algorithms for TG Analysis ......................................................... 91

Chapter 6 – Thymol Interactions in PLA Composites .............................................. 102

Chapter 7 – Release of Thymol from PLA Films ..................................................... 116

Chapter 8 – Antimicrobial Activity and Storage Stability of Composite Films ....... 128

8.1 Abstract ................................................................................................................... 132 8.2 Introduction ............................................................................................................. 133 8.3 Materials and Methods ............................................................................................ 135

8.3.1 Materials .......................................................................................................... 135

8.3.2 Production of PLA/Kenaf/Thymol Films ........................................................ 136

8.3.3 Antimicrobial Activity of Films on Solid Media ............................................. 137

8.3.4 Antimicrobial Activity of Films in Liquid Media............................................ 137

8.3.5 Antimicrobial Activity of Films on Processed Food ....................................... 138

8.3.6 Storage Stability of Thymol in AM Films ....................................................... 140

8.4 Results and Discussion ............................................................................................ 141 8.4.1 Antimicrobial Activity of Films In-vitro ......................................................... 141

8.4.2 Antimicrobial Activity on Real Foods ............................................................. 146

8.4.3 Effect of Different Storage Conditions on Thymol Retention ......................... 151

8.5 Conclusion ............................................................................................................... 154 8.6 References ............................................................................................................... 155

Chapter 9 – Conclusions ........................................................................................... 159

9.1 General Conclusions ................................................................................................... 159 9.1.1 Effect of Kenaf Fibre Surface Treatment ............................................................. 159

9.1.2 Effect of Kenaf Loadings and Thymol Concentrations ....................................... 160

9.1.3 Interaction and Retention of Thymol ................................................................... 160

xiii

9.1.4 Migration of Thymol............................................................................................ 161

9.1.5 Antimicrobial Activity and Storage Stability ...................................................... 162

9.2 Significance of the Findings ....................................................................................... 162 9.3 Recommendation for Further Research ...................................................................... 163

Appendix A: Properties of Polymer and AM Agent ................................................. 165

Appendix B: Supplementary Figures Pertaining to the Properties of the PLA-based

Materials ................................................................................................................... 167

Appendix C: Supplementary Figures Pertaining to the Migration of Thymol into Food

Simulants................................................................................................................... 168

Appendix D: Supplementary Figures Pertaining to Antimicrobial Activity of PLA-

based Materials containing Thymol on Solid and Liquid Media .............................. 170

References ................................................................................................................. 173

List of Unpublished Figures

Figure 3.1 Infrared spectra of untreated and treated kenaf fibers ............................................ 40

Figure 3.2 Intensity of the IR band mapped over PLA sample ............................................... 41

Figure 3.3 Normalized torque as a function of time for PLA melts ........................................ 42

Figure 3.4 Scanning electron micrographs of PLA composites .............................................. 44

Figure 3.5 Normalized tensile properties of PLA composites ................................................. 45

Figure 3.6 DSC thermograms of PLA and PLA composites ................................................... 51

Figure 3.7 TGA profiles of PLA and PLA composites ........................................................... 54

Figure 3.8 Images of PLA and PLA containing TK fiber and/or thymol ................................ 56

Figure 8.1 Antimicrobial activity of films against E. coli after 24 h of incubation ............... 142

xiv

Figure 8.2 Influence of thymol and kenaf loadings on the zone of inhibition ....................... 142

Figure 8.3 Liquid media antimicrobial activity of PLA formulations ................................... 145

Figure 8.4 Death rate of E. coli inoculated on deli chicken slice .......................................... 148

Figure 8.5 Fungal coverage on the surface of deli chicken slice samples ............................. 149

Figure 8.6 Percentage of fungal coverage per unit area (ƒ) of deli chicken slice samples .... 150

Figure 8.7 Normalized thymol peak area in films up to 3 months of storage ........................ 152

List of Unpublished Tables

Table 3.1 Thermal analysis parameters obtained from DSC thermograms ............................. 50

Table 8.1 Percentage of thymol in PLA films after thermal processing ................................ 136

Table 8.2 Antimicrobial activity of PLA and PLA/kenaf films against E. coli on chicken

slice ........................................................................................................................................147

List of Abbreviation and Nomenclature

AIT Allyl isothiocyanate AM Antimicrobial ANOVA Analysis of variance AOX Antioxidant AP Active packaging ASTM American society for testing

and materials ATCC American type culture

collection ATR Attenuated total reflectance BHI Brain-heart infusion BHT Butylated hydroxytoluene CFU Colony forming units DMA Dynamic mechanical

analysis

DSC Differential scanning calorimetry

DTA Differential thermal analysis

E. coli Escherichia coli EDTA Ethylenediaminetetraacetic

acid EOs Essential oils EVA Ethylene vinyl acetate EVOH Ethylene vinyl alcohol FDA Food and drug

administration FID Flame ionization detector FTIR Fourier transform infrared GC Gas chromatography

xv

GRAS Generally recognized as safe

GTA Glycerol triacetate JPEG Joint photographic experts

group LAE Lauric arginate LDPE Low-density polyethylene LLDPE Liner low-density

polyethylene L/P Length to diameter aspect

ratio MC Methylcellulose MCC Microcrystalline cellulose MFC Microfibrillated cellulose MIC Minimum inhibitory

concentration OLE Olive leaf extract PBS Poly(butylene succinate) PCL Poly(ε-caprolactone) PE Polyethylene PEG Polyethylene glycol PET Poly(ethylene terephthalate) PHB Polyhydroxybutyrate PLA Poly(lactic acid) PP Polypropylene PS Polystyrene PVC Polyvinyl chloride PVOH Polyvinyl alcohol RH Relative humidity SAS Statistical analysis software SB Sodium benzoate SEM Scanning electron

microscopy SPI Soy protein isolate SSE Sum of squared errors TBHQ Tertbutylhydroquinone TG Thermogravimetric

TK Treated kenaf TS Tensile strength TSB Tryptone Soy Broth TSBP Thermoplastic sugar beet

pulp UK Untreated kenaf D Diffusion coefficient E Young’s modulus Ea Activation energy of

diffusion ε Elongation at break f Percentage of fungal

coverage ∆Hcc Enthalpy of cold

crystallisation ∆Hm Enthalpy of melting k1 Diffusion rate constant k2 Kinetic rate constant l Film thickness mt Amount of AM agent

released from the film m Equilibrium amount of AM

agent released from the film µ Specific death rate N Population surviving N0 Initial population R Ideal gas constant r2 Correlation coefficient T Temperature t Time Tcc Cold crystallisation

temperature Tg Glass transition temperature Tm Melting temperature v0 Initial rate of release of the

AM agent %Xc Percentage of crystallinity

1

Chapter 1 – Introduction

This chapter presents an overview of antimicrobial packaging with a focus on materials based

on poly(lactic acid) (PLA) in combination with natural bio-active agents and plant-based fillers.

The overall aims and the thesis outline are also presented in this chapter.

1.1 Overview

1.1.1 Antimicrobial Packaging Systems

Traditional food packaging materials and technologies are designed to provide a physical

barrier to passively protect food products from physical, chemical and biological contamination

that can lead to the deterioration of the flavour, odour, colour, sensory and textural properties

of foods (Risch, 2009). In addition to these functions, more recently developed active

packaging (AP) systems are designed to specifically interact with the biochemical or chemical

processes in the headspaces or on the surface of food products in order to maintain the safety

and quality of the produce, and ultimately, reduce food wastage (Muriel-Galet et al., 2013; Qin

et al., 2015a; Ramos et al., 2013). Antimicrobial (AM) packaging, a form of AP, has recently

gained attention in the research and industrial sectors with the incorporation of bio-based

materials and natural bio-active agents/substances to impart AM activity. Antimicrobial

packaging offers benefits over traditional food preservatives including the provision of a

continuous AM effect on foods for extended times and the minimization of interactions with

and possible inactivation of AM agents by food components (Muriel-Galet et al., 2012).

In AM packaging systems the packaging material, in the form of films or sheets, is used as a

vector for the retention of preservatives. Moreover, bio-based polymers are preferable to

petroleum-based polymers such as polyethylene terephthalate (PET) due to environmental and

food safety reasons (e.g. migration of hazardous additives into foods), and natural, non-toxic

2

bio-active agents are also preferable due to health concerns and ecological issues (Kim and

Lee, 2012; Kuorwel et al., 2013; Qin et al., 2015b). These two main components of AM

packaging system which can be generated or produced from renewable resources and are free

from toxicity risks associated with the components themselves or migration of undesirable

substances into the food, are thus highly in demand (Jamshidian et al., 2010). Natural bio-

active agents can be integrated into polymeric materials using several techniques including

surface coating of formed articles, immobilizing the agents by chemical grafting, direct

addition during processing and using polymers that possess intrinsic AM activity such as

chitosan (Fernández-Pan et al., 2015; Peretto et al., 2014).

There are a number of natural AM bio-active agents such as organic acids, bacteriocins (e.g.

nisin and lacticin), essential oils (e.g. thymol, carvacrol and linalool), plant extracts (e.g. olive

leaf extract), enzymes (lysozyme and peroxidase), chelating agents (e.g. amino acids), metals

(e.g. silver) that have been incorporated into AM packaging materials to provide AM activity.

A wide range of essential oil (EO) extracts including thymol have been incorporated directly

into packaging materials. Thymol (2-isopropyl-5-methylphenol) is a white crystalline

substance with a pleasant aromatic odour that is found in the EO of thyme (Thymus vulgaris)

and it possesses AM and antioxidant properties for a wide spectrum of microorganisms such

as bacteria, mould and yeast (Burt, 2004; Nostro et al., 2007). Thymol has the potential to

inhibit Gram-positive bacteria and to a lesser extent, Gram-negative bacteria, including the

pathogenic strains of Escherichia coli, Staphylococcus aureus and Bacillus cereus (Burt, 2004;

Falcone et al., 2005; Petchwattana and Naknaen, 2015). The AM activity of thymol has been

attributed to its hydrophobic nature whereby it is most likely to partition in the lipid layer of

bacteria cell membranes and mitochondria resulting in the leakage of cell contents (Burt, 2004;

Guarda et al., 2011).

3

For food applications in particular, thymol is highly suitable as a food preservative and it is

classified by the US Food and Drug Administration as a Generally Recognized as Safe (GRAS)

food additive. Recently, the AM activity of thymol against spoilage bacteria isolated from fresh

produce has been reported by Zheng et al. (2013). According to Burt (2004), the concentration

of thymol required to achieve a significant AM effect is ca. 0.5-20 μL g-1 in food products such

as fresh meat, meat products, fish, dairy products, vegetables and fruit. Thymol residues in

food must be less than 50 mg kg-1 in order to be considered safe for consumption as reported

by the World Health Organization (Tao et al., 2014). Nevertheless, there are a number of

limitations associated with the use of AM substances extracted from essential oils such as

limited water solubility, ease of degradation or chemical reactivity, high volatility, and the

potential to alter organoleptic properties of foods when used in high concentrations (Guarda et

al., 2011; Rodríguez-Martínez et al., 2016; Tao et al., 2014).

Several polymers including petroleum-based and biodegradable polymers have been

investigated for thymol incorporation including starch-based polymers (Kuorwel et al., 2013),

zein (Li et al., 2012; Mastromatteo et al., 2009), soy protein isolate (Emiroglu et al., 2010; Hu

et al., 2012), pullulan (Gniewosz and Synowiec, 2011), polycaprolactone (Del Nobile et al.,

2009; Sanchez-Garcia et al., 2008), PLA (Ramos et al., 2014; Tawakkal et al., 2016; Wu et al.,

2014), polybutylene succinate (Petchwattana and Naknaen, 2015), polypropylene (Ramos et

al., 2012), low density polyethylene (Cran et al., 2010), and linear low density polyethylene

(Torres et al., 2014). Most of the aforementioned studies have concentrated on the mechanical,

thermal and water vapour barrier properties, the quantification and the migration rate of AM

agent from the polymers as well as the potential AM activity of these systems against targeted

microorganisms. Some studies have also focused on ternary systems where the inclusion of

various additives, fillers and other polymers have been combined, with the main aim being to

4

improve material performance, enhance AM and/or antioxidant properties, reduce costs, and

expand into new applications.

1.1.2 Ternary AM packaging systems

The utilisation of bio-based materials, particularly in food packaging applications such as rigid

and flexible sheets and/or films, edible films and coatings, has become popular due to the

environmental benefits associated with these being renewable resources and the growing

economic pressure to reduce the dependence on fossil resources (Johansson et al., 2012).

Moreover, consumer preferences for natural food products with few or no preservatives and

minimal microbial contamination while using disposable, potentially biodegradable and

recyclable packages, has generated a growing interest in the use of bio-based materials in AM

packaging. Formulations of PLA containing various types of AM and antioxidant agents have

been investigated by a number of researchers (Byun et al., 2010; Hwang et al., 2013; Jin and

Zhang, 2008; Liu et al., 2010; Rodríguez-Martínez et al., 2016). Of the reported studies, many

have investigated the incorporation of non-volatile antioxidants in particular (e.g. resveratrol,

α-tocopherol and butylated hydroxytoluene (BHT)) and immobilized AM agents (e.g. nisin and

lysozyme) with little attention having been devoted to investigating a ternary system of an

antimicrobial PLA containing a bio-filler. One of the reasons may be attributed to there being

less focus on the development of bio-composite materials containing natural fibre fillers in

packaging applications. Moreover, the initial costs associated with new technologies to produce

biopolymers such as PLA are typically high so new techniques must be sought to reduce costs

without compromising material properties (Mensitieri et al., 2011). For example, naturally-

sourced fibres are commonly used as cost-reducing fillers for PLA (Gurunathan et al., 2015;

Saba et al., 2015).

5

In a recent study, PLA was incorporated with nanocellulosic-fillers derived from plants such

as microfibrillated cellulose (MFC), cellulose whiskers or cellulose crystals to produce

nanocomposite materials that have advanced, high-performance characteristics (Arjmandi et

al., 2016; Herrera et al., 2015). The incorporation of nanocellulosic fillers such as

microfibrillated cellulose improves polymer mechanical properties such as tensile strength and

modulus as well as water vapour barrier properties in a more efficient manner even at low filler

loading than is achieved in conventional micro- and macro-filler composite materials

(Arjmandi et al., 2016; Halász and Csóka, 2012; Herrera et al., 2015; Siró and Plackett, 2010).

There have been few reports that have investigated PLA-based nanocomposites intended for

AM food packaging applications (Abdulkhani et al., 2015; Almasi et al., 2014; Liu et al., 2014;

Salmieri et al., 2014). Almasi et al. (2014) prepared solvent cast PLA-based nanocomposite

films incorporated with modified cellulose nanofibre (MCNF) and tert-butylhydroquinone

(TBHQ) antioxidant with the aim of determining the efficacy of the antioxidant films to

prolong the shelf-life of fatty foods. They reported that the release rate of TBHQ in soybean

oil was significantly decreased by the presence of MCNF, which was enough to delay the

induction of the oxidation of soybean oil stored for 6 months. Nevertheless, the mechanical

separation of plant fibres into smaller elementary constituents typically requires high energy

input and the lack of compatibility with hydrophobic polymers can result in significant

aggregation of these nanosized fillers (Dufresne, 2013; Herrera et al., 2015; Oksman et al.,

2015). Moreover, possible migration of nanosubstances can potentially have an adverse impact

on human health (Abdul Khalil et al., 2012; Bumbudsanpharoke and Ko, 2015).

Natural lignocellulose fibres derived from a variety of plants such as wood, kenaf, jute, flax,

ramie, hemp, pineapple leaf and bamboo could be used as reinforcements in biopolymers to

generate composite materials with opportunities in biodegradable packing applications. Natural

fibres possess advantages such as low density, relatively high specific strength and stiffness

6

and impart less equipment abrasion (Awal et al., 2015; Wambua et al., 2003). These advantages

have also initiated much attention in replacing inorganic reinforcement materials (e.g. glass

fibre) with abundantly available and less expensive natural fibres. It is important to note,

however, that there are sometimes high costs associated with the production of nanofibre fillers

from natural materials that can be comparable to the production of some synthetic nanofibres

(Johansson et al., 2012). Although polymers that are reinforced/filled with natural fibres are

generally not as strong as those reinforced with fillers such as cellulose nanofibres, the

moderate strength and light weight characteristics of the former still make these suitable to be

implemented in food packaging applications, particularly for ready-to-eat food products (Pilla,

2011).

Bast fibres obtained from the outer stem layer of the kenaf plant (Hibiscus cannabinus L.) have

good mechanical properties that enable this material to be used as a filler and reinforcing agent

in biopolymer composites (Abdul Khalil et al., 2010; Saba et al., 2015). Moreover, the

incorporation of natural fibres into PLA matrices in order to reduce material costs and improve

mechanical properties has recently gained attention. Furthermore, such composites can be

produced via commercial plastic processing methods and the incorporation of the natural fibres

is believed to enhance compostability and biodegradability (Halász and Csóka, 2012; Kwon et

al., 2014). In the current study, a novel ternary system comprising PLA, kenaf fibres and

thymol, was investigated with a view towards developing a biodegradable and active packaging

material.

7

1.2 Aims of this Work

The overall aim of this study is to develop an AM packaging system from PLA-filled kenaf

fibre bio-composites that incorporates a natural AM agent such as thymol. The specific aims

are as follows:

To prepare AM PLA and PLA/kenaf composites via melt blending and heat pressing and

to determine the mechanical, thermal and morphological properties of these materials at

various kenaf loadings and thymol concentrations;

To evaluate the degradability of AM PLA and PLA/kenaf composites under controlled

composting conditions;

To investigate the interaction of thymol between the PLA matrices and kenaf fibres filler

and to determine the retention of thymol in the PLA-based materials following thermal

processing;

To investigate the migration of AM agent from a ternary system comprised of PLA, kenaf

fibres and thymol by diffusion, kinetics analyses and Fick’s law modelling;

To examine the temperature dependency of the release rate of thymol from the PLA and

PLA/kenaf composite films into fatty and aqueous food simulants;

To evaluate the effectiveness of AM PLA and PLA/kenaf composites incorporated with

thymol against E. coli or inoculated on the surface of processed meat samples;

To investigate the loss of thymol to the atmosphere from these packaging materials and of

their thymol retention during storage.

8

1.3 Thesis Outline

This thesis is comprised of seven papers that are presented in separate chapters. Declarations

of co-authorship and co-contribution of these papers are included at the start of the respective

chapters. Five of these papers have been published in peer-reviewed journals with a further two

manuscripts submitted.

Chapter 2 presents the literature review with an emphasis on PLA-based materials intended for

use in AM packaging applications. This chapter reports the recent findings of the engineering

characteristics and the AM activity of PLA-based materials incorporated and/or coated with

AM agents. The preparation of PLA-based materials incorporated with different AM agents

and by using various techniques is also reviewed and critically discussed in this chapter.

In Chapter 3, the effects of untreated and alkali treated kenaf fibres incorporated into the PLA

containing thymol in a range of 5 to 10% w/w are examined. The compatibility between the

treated kenaf and the PLA matrix as depicted by micrograph images is reported. In this chapter,

the PLA/kenaf composites containing untreated and/or treated kenaf has been fixed at 30%

w/w kenaf loading. The findings of the mechanical, thermal and morphological properties of

the PLA and PLA/kenaf composites containing either no kenaf or no thymol are also discussed.

The degradation properties of the studied materials under controlled composting conditions are

also presented in this chapter.

It is important to note that the PLA systems containing treated kenaf were selected as the main

composite formulation in which to systematically investigate the mechanical and thermal

properties, the interaction and retention of AM agent, the migration rate of AM agent from the

material and the AM efficacy in laboratory media and on a real foodstuff. Such systems are

reported in the remaining chapters (4 to 7).

9

Chapter 4 provides a detailed examination on the effect of different kenaf fibre loadings ranging

from 10 to 40% w/w and thymol concentrations in a range of 5 to 10% w/w on the mechanical,

thermal, rheological and morphological properties of the AM materials. The possible

packaging applications such as rigid and/or flexible packaging as well as coatings of this

particular material can be postulated from these studies.

A standalone Chapter 5 provides an insight into polymer degradation under non-isothermal

conditions presented by two novel algorithms. The algorithms are validated using model data

and applied to thermogravimetric (TG) data obtained during the degradation of PLA and

PLA/kenaf composites containing thymol under non-isothermal conditions. The novel kinetic

approach developed in this chapter was used in subsequent chapters to investigate the nature

of the interactions between the various components of the composite systems.

Chapter 6 examines the retention of thymol in PLA and PLA/kenaf films following thermal

processing by using thermogravimetric and solvent extraction techniques. A detailed

discussion of the interactions amongst the PLA matrix, the kenaf fibres and the AM agent in

these ternary systems is presented in this chapter. The potential applications of these systems

as AM food-packaging materials are also considered.

The diffusivity characteristics of thymol incorporated into the packaging materials with the aim

to prolong the shelf life of food products are discussed in Chapter 7. The migration of thymol

from the AM PLA and PLA/kenaf films into food simulants is examined. The food simulants

used in this study were 15% v/v ethanol/water mixture and 95% v/v ethanol/water mixtures

with these simulants representing aqueous and fatty foods respectively. The release rates of

thymol into the simulants at different temperatures were determined by using first-order

kinetics analysis, diffusion modelling and Fick’s law modelling and these are compared and

discussed in this chapter.

10

Chapter 8 examines the effectiveness of AM PLA and PLA/kenaf composite films incorporated

with thymol against E. coli or inoculated on the surface of processed meat samples. The

influences of thymol concentration and kenaf fibres loading on the AM activity of the films are

also reported. The capability of PLA and PLA/kenaf films containing thymol as an AM food

packaging system is explored in this chapter. Moreover, the loss of thymol to the atmosphere

from these packaging materials and their thymol retention during storage under different

storage conditions is examined in this chapter.

Finally, the overall conclusions of this work, the significance of the findings as well as the

recommendations for future research are stated in Chapter 9.

11

Chapter 2 – Literature Review

A review of Poly(Lactic Acid)-Based Materials for Antimicrobial Packaging

Overview

Chapter 2 presents the literature review of PLA-based materials intended for use in AM food

packaging applications. The engineering characteristics and the AM activity of PLA-based

materials incorporated and/or coated with AM agents are reported in this chapter. The current

trends in the production of PLA-based materials containing AM and/or antioxidant agents are

also presented and discussed.

The paper entitled “A Review of Poly(Lactic Acid)-Based Materials for Antimicrobial

Packaging” by Tawakkal I. S. M. A., Cran M. J., Miltz J. and Bigger S. W. was published in

the Journal of Food Science, 79(8), 1477-1490, 2014.

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ceA Review of Poly(Lactic Acid)-Based Materials forAntimicrobial PackagingIntan S. M. A. Tawakkal, Marlene J. Cran, Joseph Miltz, and Stephen W. Bigger

Abstract: Poly(lactic acid) (PLA) can be synthesized from renewable bio-derived monomers and, as such, it is analternative to conventional petroleum-based polymers. Since PLA is a relatively new polymer, much effort has beendirected toward its development in order to make it an acceptable and effective option to the more traditional petroleum-based polymers. Commercially, PLA has received considerable attention in food packaging applications with a focuson films and coatings that are suitable for short shelf life and ready-to-eat food products. The potential for PLA to beused in active packaging has also been recognized by a number of researchers. This review focuses on the use of PLAin antimicrobial systems for food packaging applications and explores the engineering characteristics and antimicrobialactivity of PLA films incorporated and/or coated with antimicrobial agents.

Keywords: active packaging antimicrobial, composites, poly(lactic acid)

IntroductionTraditional synthetic polymers made from petrochemicals that

currently dominate the food packaging sector possess most of thespecific properties required of useful packaging materials such aseffective gas barrier properties (oxygen, water vapor, aroma, andlight), transparency, ability to be sealed, chemical resistance, me-chanical strength, as well as ease of processing. However, the long-term environmental sustainability of these polymers is now beingquestioned and interest is shifting to the search for biodegradable,biocompostable polymers, and composites as alternatives to con-ventional polymers (Mohanty and others 2005; Avella and others2009; Ghosh and others 2011; Kuorwel and others 2011a; Pilla2011). Commercial and academic interest in utilizing biodegrad-able polymers for food packaging applications has increased in re-cent years (Siracusa and others 2008; Ahmed and Varshney 2011;La Mantia and Morreale 2011). These materials also have the po-tential to reduce environmental impacts associated with the man-agement of petroleum-based packaging wastes. There are 3 maincategories of biodegradable polymers: (i) polysaccharides (starchand cellulose), proteins (gelatin and casein), and lipids that are de-rived directly from natural raw materials and renewable resources;(ii) poly(lactic acid) (PLA) that are chemically synthesized frombio-derived monomers; and (iii) poly(hydroxybutyrate) (PHB),poly(hydroxyvalerate), and poly(hydroxyalkanoates) that are madefrom microbiologically produced materials or genetically modifiedbacteria (Chandra and Rustgi 1998; Averous 2008).

The shelf life of most food products depends upon the bi-ological, chemical, and physical interactions between the food,package, and the ambient environment (Robertson 2010). Highlevels of bacteria and microorganism in food products can po-tentially generate undesirable deteriorations in flavor, odor, color,sensory, and textural properties and may even become harmful to

MS 20131583 Submitted 10/31/2013, Accepted 5/27/2014. Authors Tawakkaland Bigger are with College of Engineering and Science, Victoria Univ., PO Box14428, Melbourne, 8001, Australia. Author Cran is with Inst. for Sustainabilityand Innovation, Victoria Univ., PO Box 14428, Melbourne, 8001, Australia. Au-thor Miltz is with Dept. of Biotechnology and Food Engineering, Technion-IsraelInst. of Technology, Haifa, 32000, Israel. Direct inquiries to author Cran (E-mail:[email protected]).

human health (Raouche and others 2011). The effort toward ef-ficient food preservation continues to increase to fulfill consumersdemand for natural, fresh, safer, and healthier products (Pilizota2012). Over the last few decades, the active packaging (AP) con-cept has been established and developed in order to improve thequality and extended the shelf life of food products (Miltz and oth-ers 1995; Rooney 1995; Appendini and Hotchkiss 2002). Some ofthe common AP concepts include gas scavengers, carbon dioxideemitters, moisture absorbing systems, and antioxidant and/or an-timicrobial releasing and/or containing systems (Brody and others2001; Suppakul and others 2003; Lopez-Rubio and others 2006;Perez-Perez and others 2006).

To date, one of the extremely challenging AP technologiesis the use of antimicrobial additives that can minimize the riskof food spoilage and contamination by suppressing the activi-ties of targeted microorganisms (Labuza and Breene 1989; Limand Mustapha 2003; Mauriello and others 2005; Joerger 2007;Lopez-Mendoza and others 2007; Sung and others 2013; Guoand others 2014). A wide range of additives have been successfullyincorporated directly into food products as well as into packagingmaterials, including organic acids and their salts, bacteriocins, en-zymes, chelators such as ethylenediaminetetraacetic acid (EDTA),lactoferrin, and a range of plant extracts (Cooksey 2005; Sanchez-Garcia and others 2008; Jin and Niemira 2011; Barbiroli and others2012; Jokar and others 2012; Torres and others 2014). This tech-nology also combines antimicrobial additives with the packagingmaterial that slowly and continuously release the additives over therequired period of time and maintain a high concentration of theantimicrobial in the product (Quintavalla and Vicini 2002).

Different materials and methods can be used in order to developantimicrobial packaging systems. For instance, petroleum-basedand biodegradable polymers have been studied as potential candi-dates for the incorporation of antimicrobial substances in foodpackaging applications. These include polyolefins (polypropy-lene, low- and high-density polyethylene) and polyesters such aspoly(ethylene terephthalate) (PET) and poly(caprolactone) (PCL)(Limjaroen and others 2003; Del Nobile and others 2009; Ramosand others 2012; Bastarrachea and others 2013). An antimicrobialactivity between an antimicrobial package and food product canbe achieved by either direct contact using a nonmigratory sys-tem or by indirect contact using a volatile antimicrobial releasing

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Antimicrobial PLA packaging materials . . .

system (Guarda and others 2011). In addition, Sung and others(2013) have noted that even though there has been much researchactivity on the development of antimicrobial packaging systemsthat utilize various plastic materials and antimicrobial agents, onlya few commercial antimicrobial packaging products are found inthe market. They speculated that this might be due to strict safetyand hygiene regulations, as well as limited consumer acceptanceof high cost.

PLA polymer

Synthesis and properties of PLAThe PLA polymers belong to the class of aliphatic polyesters

that can be produced either by carbohydrate fermentation or bychemical synthesis of the lactic acid (LA) monomer (Averous 2008;Jamshidian and others 2010). The LA monomer can be obtainedfrom renewable resources including starch-rich products such ascorn, sugar beet, tapioca, and wheat (Auras and others 2004). Thechemical structure of LA is such that it contains an asymmetric car-bon atom creating 2 optically active configurations known as theL- and D-isomers. The ratio of L- to D-monomer units affectssome of the key macromolecular properties such as the degreeof crystallinity, melting temperature, and the ease of processing(Gupta and others 2007; Lim and others 2011). The synthesisof PLA is a relatively complex multistep process that starts fromthe production of LA, followed by the formation of the lactidemonomer and finally, the polymerization process itself. Commer-cial PLA resins are usually produced from L-lactide with the result-ing polymer, poly(L-lactic acid), being a semicrystalline materialthat has relatively high melting and glass transition temperatures(Albertsson and others 2011).

The beneficial properties of PLA include high mechanicalstrength, excellent thermoforming ability, biocompatibility, abilityto be easily composted, and monomer renewability. In addition,the production of PLA has been reported to result in 15% to60% lower carbon emissions and 25% to 55% lower energy con-sumption than petroleum-based polymers (Vink and others 2003;Dorgan and others 2006). This bio-based polymer can be pro-cessed by conventional plastic processing methods (which is ofa commercial importance) to produce a range of products thatare both recyclable and/or compostable. However, its inherentbrittleness, low thermal resistance, and poor water-vapor barrierproperties are disadvantages that currently limit its widespread usein packaging (Bhardwaj and Mohanty 2007).

A recent life cycle assessment (LCA) of PLA and PET for drink-ing water bottles comparing their impact on the environment wasreported by Gironi and Piemonte (2011). In this study it was foundthat PLA has lower environmental impacts than PET in terms offossil fuel resources and recycling capability. However, it was alsonoted that a reliance on PLA production can have adverse effectssuch as the clearing of forests for growing starch crops and the as-sociated excessive water use. Such production may also indirectlyaffect human health and the environment because of the use ofpesticides. Thus, it is important to note that LCA studies requirea sufficient amount of data and information in order to avoid biasin the final findings.

Trends in PLA-based materialsA range of other polymers, additives, and fillers have been suc-

cessfully combined with PLA in order to enhance material per-formance, reduce costs, and expand PLA into new applications(Martin and Averous 2001). To date, the combination of

biodegradable polymers and biofillers with PLA is receiving con-siderable academic and commercial attention mainly for the pur-pose of creating materials that potentially have lower environ-mental impacts than conventional synthetic polymers. The use ofnatural fibers as fillers or reinforcing agents (for example, wood,kenaf, flax, hemp, and ramie) and other biodegradable polymers(for example, starch, protein, PCL, and PHB) in PLA blends orcomposites is expected to considerably lower the price of the re-sulting products (due to the renewability and abundance of the rawmaterials) as well as to improve the properties that these materialsoffer without compromising the biodegradability (Akil and others2011).

In addition, the use of nanoadditives, nanofibers, and nanocom-posites, in particular, are some of the current approaches used infood packaging applications. Various types of nanofillers such asclay, silica, talc, montmorillonite, and nanofiber polymers havebeen utilized in order to improve the properties of PLA-based ma-terial composites (Sinha Ray and Okamoto 2003; Iwatake and oth-ers 2008; Torres-Giner and others 2008; Rhim and others 2009;Ray 2011; Abdul Khalil and others 2012; Tenn and others 2012).Packages containing nanomaterials are apparently more acceptableto the average consumer than nanotechnology-engineered foodproducts (Siegrist and others 2007). However, some of the maindrawbacks of PLA-based nanomaterials include their high cost,due to energy consumption in their production, as well as thepossible migration of nanoparticles that might have a potentialimpact on human health (Abdul Khalil and others 2012; Rhimand others 2013).

PLA as a packaging materialWith its classification being generally recognized as safe

(GRAS), PLA has been approved for use in food packaging, in-cluding direct contact applications (Conn and others 1995). Inaddition, PLA is a good candidate for a variety of packaging ap-plications due to its close similarity to commercial thermoplasticssuch as PET (Auras and others 2005). In the last decade, PLAhas been developed for a wide range of primary packaging ap-plications including oriented and flexible films, extruded and/orthermoformed packages suitable for common applications suchas food and beverage containers, cups, overwrap, blister packages,as well as coated paper and board (Tullo 2000; Groot and others2011). Recently, a Danish dairy company has used PLA, that wasclaimed to be biodegradable, for yoghurt cups that were tradi-tionally made from high-impact polystyrene (Jessen 2007). Othercommercial examples include the use of PLA for the production oflunch boxes and fresh food packaging (Mutsuga and others 2008),and containers for packaging of bottled water, bottled juices andyogurts (Ahmed and others 2009). Blends of PLA with starches,proteins, and other biopolymers have also been studied in or-der to develop fully renewable and degradable packaging materials(Raghavan and Emekalam 2001; Ke and Sun 2003; Suyatma 2004;Yew and others 2005; Bhatia and others 2007).

PLA antimicrobial packagingThe potential of PLA for use in antimicrobial packaging ap-

plications has been investigated in recent years by a number ofresearchers (Mustapha and others 2002; Rhim and others 2009;Chen and others 2012; Li and others 2012a; Jamshidian andothers 2013; Fei and others 2014). There are also a number ofpatents worldwide on PLA-based materials containing antimicro-bial agents (Auras and others 2010; Buonocore and others 2012;Chen and others 2012; Liu and others 2012). Several substances

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ceAntimicrobial PLA packaging materials . . .

such as organic acids, bacteriocins (for example, nisin), plant ex-tracts (for example, lemon extract), essential oils and extracts (forexample, thymol), enzymes (for example, lysozyme), chelatingagents (for example, EDTA), metals (for example, silver) havebeen incorporated into PLA to provide antimicrobial activity. Inparticular, PLA with the addition of natural antimicrobial agentssuch as nisin, lysozyme, and silver zeolite has shown inhibitoryeffects against selected microorganism such as Listeria monocyto-genes, Escherichia coli, Staphylococcus aureus, and Micrococcus lysodeikti-cus. Natural antimicrobial agents have also been incorporated intocoatings on the surface of PLA and these were shown to be effec-tive against spoilage and pathogenic microorganisms (Del Nobileand others 2009; Jin and others 2009; Liu and others 2009a; Rhimand others 2009). According to Jamshidian and others (2010), onlya few studies have investigated the potential of PLA in general APapplications although there are a number of examples that use PLAin antimicrobial food packaging applications.

Current technologies enable effective antimicrobial packages tobe prepared from PLA that has been blended with different com-patible materials and plasticizers. The consumer preference fornatural food products with few or no preservatives, with mini-mal microbial contamination while using sustainable packages hasgenerated a growing interest in the use of PLA in antimicrobialpackaging. An example of a commercial antimicrobial PLA pack-aging product is AntipackTM produced by Handary in Belgium,which is a film manufactured from a PLA-/starch-based materialincorporated with an antifungal agent. This product is claimed toprevent the growth of yeast and mold during the shelf life pe-riod by gradually releasing chitosan-containing natamycin ontothe surface of solid foods such as cheese, fruits, vegetables, meat,and poultry (Szafranska 2012).

Furthermore, it is believed that PLA can perform as a suit-able carrier of antimicrobial agents without showing any indi-vertible impact on the compositing and potential biodegradationprocess. This is possible if the rate of dissipation of the antimi-crobial agent or the controlled release during the shelf life of thesystem as a packaging material is fully understood, systematicallyperformed, and accurately controlled (Balasubramanian and oth-ers 2009). Therefore, the study of antimicrobial agent migrationin the system will be very important in the future to ensure theagent is dissipated before the packaging materials is disposed of inlandfill. Ramos and others (2014) observed some improvement inthe biodegradation of extruded PLA films containing thymol andsilver nanoparticles as the antioxidant and antimicrobial agents,respectively, using a disintegration test in composting conditions.

Preparation of antimicrobial PLA-based materialsThe choice of processing technique may significantly affect the

properties of the resultant antimicrobial films, especially thosemade from biodegradable polymers (Rhim and others 2006). Ac-cording to Han (2005), different factors affect the selection of theprocessing technique when preparing an antimicrobial packagingfilm. These include the type and properties of the polymer, thecharacteristics of the antimicrobial agent (such as polarity, compat-ibility, and thermal stability), storage temperature, type of packedfoods and targeted microorganisms, and the residual antimicrobialactivity after manufacturing.

It is also important to note that the selection of the processingmethod for the preparation of antimicrobial PLA-based materi-als depends on the type of system being developed. Basically, theantimicrobial system can be either a direct contact one using anonmigratory antimicrobial agent or an indirect contact one us-

ing a volatile antimicrobial releasing system (Guarda and others2011; Jin and Niemira 2011). In particular, the coating methodis suitable for nonmigratory antimicrobial systems where a highconcentration of antimicrobial agent is required on the surface ofthe film (Appendini and Hotchkiss 2002; Jin and others 2009).Solvent casting and extrusion are more suitable for migratory an-timicrobial systems where the release of a volatile antimicrobialagent from the packaging material to the packaging headspace orthe surface of the food product is required (Jin and Niemira 2011;Guo and others 2014).

In these systems, mathematical diffusion modeling can be usedto predict the release profile of the antimicrobial agents. The initialand boundary conditions can be used to determine the distributioncoefficient (Crank 1979). Moreover, a number of factors need tobe considered in order to use the most appropriate diffusion modelincluding film thickness, the ways in which the antimicrobial agentdiffuses, and the volume of food simulant into which the migrationoccurs (Perez-Perez and others 2006). The various mass transfermodels that can be used to analyze the migration of antimicrobialagents can also account for the influence of the molecular weight,ionic charge, and solubility of the agents (Han 2003).

The effectiveness of antimicrobial PLA-based material dependsalso on the selection of antimicrobial agents. Some volatile ad-ditives have low resistance to the conditions experienced duringprocessing and fabrication that include high temperatures, shear,and pressure at the extrusion stage (Han 2003). The polarity andmolecular weight of the antimicrobial agent are crucial materialparameters that dictate the solubility or compatibility of the agentin the PLA matrix. For example, olive leaf extract (OLE) preparedusing water as the solvent was found to be incompatible with PLAwhereas OLE dissolved in chloroform and incorporated into aPLA film containing methylcellulose (MC) demonstrated a signif-icant inhibition of S. aureus (Ayana and Turhan 2009).

Antimicrobial substances can be incorporated into PLA viawet or dry processing techniques, similar to those used for otherbiodegradable and synthetic polymers (Brody and others 2001).The wet processing technique consists of solvent casting/solventevaporation using ambient or low temperature during mixing.According to Appendini and Hotchkiss (2002), biopolymers aregood candidates for this type of film production when compared topolyolefins and other hydrophobic polymers. For the productionof solvent cast films, PLA can be dissolved in different solvents suchas chloroform (Rhim and others 2009), ethyl acetate (Mascheroniand others 2010), as well as methylene chloride (Jin and others2010) and in many cases, both the PLA and the associated antimi-crobial agents can be dissolved in the same solvent.

Solvent casting of additives onto a polymeric substrate involvessolubilization, casting, and drying. This process is one of the morecommonly used methods for laboratory-scale preparation of an-timicrobial films from biopolymers (Kuorwel and others 2011a).The solvent casting method is also suitable for heat-sensitive an-timicrobial agents such as bacteriocins (for example, nisin) andrather volatile compounds such as essential oils and their extracts(for example, thymol and carvacrol). Although some bacteriocinsand peptides are relatively heat resistant, it is believed that theirantimicrobial activity may be higher when minimal heating is ap-plied during processing (Appendini and Hotchkiss 2002; Liu andothers 2009a).

Dry or thermal processing techniques include the standard con-ventional processing methods used in the plastics industry suchas extrusion, compression molding, blow molding, and injec-tion molding. Extrusion is the most common method used for

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commercially processing polymeric packaging films (Raouche andothers 2011). Compression molding of PLA can also be used toproduce films that are relatively strong, thermally stable but brittlewith a low thermal resistance compared to solvent-cast PLA filmsthat are more ductile (Rhim and others 2006).

PLA is processed during extrusion at a temperature higher than160 °C, which is crucial in order to ensure optimal melt vis-cosity as well as to complete the melting of the crystalline phasein the PLA matrix. This temperature is generally lower than theprocessing temperature of common thermoformed food pack-ages made of PS, and PET. In addition, PLA can be processedusing standard equipment with minimal equipment modifications(Jamshidian and others 2010; Lim and others 2011). It has been re-ported that PLA can be successfully extruded and/or compressionmolded with organic and inorganic antimicrobial agents and theircombinations (Del Nobile and others 2009; Jin and others 2009;Busolo and others 2010; Prapruddivongs and Sombatsompop2012). These processing techniques enable the antimicrobial agentto be evenly distributed in the amorphous regions of the polymericmaterial and can regulate its slow release from the film and main-tain adequate concentrations against microorganisms (Suppakul2004; Liu and others 2009a).

For antimicrobial PLA films, high temperatures and shear duringextrusion processing can lead to a partial loss of the antimicrobialagent or its activity, especially if the antimicrobial agent has lowthermal stability or high volatility (Han 2003; Del Nobile and oth-ers 2009). Therefore, the polymer and/or the antimicrobial agentmay require modification prior to film processing in order to in-crease the compatibility between the 2 components (Kuorwel andothers 2011a). Liu and others (2009a) have modified the steps inthe processing method of co-extruded PLA membranes containing5% (w/w) Nisaplin as the antimicrobial agent and lactide and/orglycerol triacetate (GTA) as the plasticizers. They reported that themaximum temperature at which Nisaplin retains its bioactivity is120 °C but the temperature required to melt PLA is 160 °C. Inorder to address this problem, the processing temperature was re-duced from 160 to 120 °C, by the addition of plasticizers, prior toblending with the antimicrobial agent. The incorporation of plas-ticizers lowered the temperature profile during manufacturing ofthe antimicrobial films. The resulting PLA/Nisaplin films showedno antimicrobial activity whereas, PLA/lactide and PLA/GTAblended membranes containing Nisaplin prevented the growth ofL. monocytogenes in brain–heart infusion (BHI) broth.

It is clear that more research needs to be conducted to investigatethe retention of natural antimicrobial agents when high temper-atures are applied during processing. Microencapsulation is oneimportant technique that can reduce the loss or the inactivation ofantimicrobial agents by protecting volatile and heat-sensitive agentsduring thermal processing (Martins and others 2009; Guarda andothers 2011; Joo and others 2012). The stability of microencap-sulated antimicrobial agent during processing and storage is cer-tainly a challenge and there are various possible negatives in suchprocesses, including high cost as well as the complexity of theproduction process (Zuidam and Shimoni 2010).

Other studies have focused on coating methods where the an-timicrobial substance is coated onto the surface of a primary poly-meric film or substrate (Jin and Gurtler 2011; Jin and Niemira2011; Li and others 2012a). The 2 common types of coatingmethods are spray gun coating and diffusion coating with eachof these techniques involving the prior dissolution of the antimi-crobial agent. Other coating techniques such as corona discharge,electro spinning, as well as sonification have been utilized to pro-

duce antimicrobial-coated PLA films and membranes (Li and oth-ers 2009; Rhim and others 2009; Theinsathid and others 2012).It was claimed that these methods do not significantly influencethe loss of antimicrobial agent or result in an overall reduction inthe mechanical properties of the films.

PLA-based materials containing antimicrobial agents can alsobe prepared in the form of active PLA nanofiber-based systemsby using nanotechnology (Xu and others 2006; Torres-Giner andothers 2008; Vega-Lugo and Lim 2009; Vargas-Villagran and oth-ers 2012). This recent and efficient technology features the useof electrical forces to produce ultrathin fibers known as polymernanofibres. More than 200 types of both biodegradable and non-biodegradable polymers have been designed and prepared by theelectrospinning technique for specific applications and their prop-erties have been characterized (Torres-Giner 2011). For instance,electrospun chitosan nanofiber mats made from a chitosan/PLAblend have been successfully prepared by Torres-Giner and others(2008). The main advantages of including the antimicrobial agentin the nanofiber in this way are that it can improve the materialproperties (for example, gas barrier and mechanical properties)and the release rate of the antimicrobial agent due to the highsurface area and small pore size (micrometer to nanometer) (Kay-aci and others 2013; Tanadi 2014). However, the disadvantagesare that these materials are currently expensive to produce. Also,in some cases nanofiber mats might potentially disturb the releaseof the antimicrobial agent. Furthermore, these materials may notmeet the requirements for FDA approval of biocides (Torres-Giner2011).

Characteristics of antimicrobial PLA-based materialsChanges may occur in the mechanical and physical properties

of many packaging materials after the incorporation of antimicro-bial agents. When the antimicrobial agent is compatible with thepackaging material, it can be incorporated with minimal physico–mechanical property deterioration. Conversely, an excess amountof an incompatible antimicrobial agent may reduce the physico–mechanical properties of the resulting composite material (Han2003; Cooksey 2005). It is important to note that in the caseof antimicrobial agents derived from natural polymers such aspolysaccharides (for example, chitosan), the melt blending of 2compatible polymers containing different glass transition temper-atures may significantly alter the properties of the material. Theengineering characteristics of various polymeric antimicrobial ma-terials for food packaging have been reviewed by Bastarrachea andothers (2011). They reported that a few changes occurred in themechanical, thermal, and gas barrier properties of the polymer aswell as in the surface morphology as a result of the addition of theantimicrobial agent. According to their literature review, only afew studies have measured the changes in PLA films loaded withantimicrobial agents.

In a recent study, Liu and others (2009a) extruded a thin mem-brane of PLA/pectin–Nisaplin microparticles with 1% (w/w) and9% (w/w) concentrations of Nisaplin/pectin. They reported thatthe extruded PLA films impregnated with solvent-cast pectin–Nisaplin significantly reduced the tensile strength (by 49%), ten-sile modulus (by 41%), and toughness (by 51%) but no significantchange in the flexibility occurred when compared to PLA/pectinmembranes. They speculated that this phenomenon occurreddue to the incompatibility between the inactive components inthe Nisaplin (for example, 2.5% nisin, milk solids and salt) withthe PLA. Nonetheless, the results seem contradictory because onewould expect the flexibility to change along with the observed


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reduction in the tensile modulus and toughness. No explanationof this behavior was given.

In another study, Liu and others (2010) incorporated both 5%(w/w) loadings of Nisaplin and EDTA into plasticized PLA/GTAfilms and found a significant reduction in the elongation at break(from 108.5% to 62.5%) and in the impact strength (from 5.4 to3.4 J/cm3) when compared to the plasticized only PLA. Althoughthe PLA flexibility increased by adding GTA, the reduction inthe mechanical properties of plasticized PLA incorporated withantimicrobial agents might be attributed to the “filler effect” (Liuand others 2010). The reported findings however, were unableto resolve the contradiction of the effect of the antimicrobialagent on the engineering properties. Generally, modification ofthe properties of neat PLA, or other biopolymers, by the addi-tion of plasticizers is performed in order to increase the flexibilityand to reduce the glass transition temperature (Tg) (Ljungberg andWesslen 2002; Ozkoc and Kemaloglu 2009).

Recently, Prapruddivongs and Sombatsompop (2012) studiedthe effects of incorporating natural fibers such as wood flour onthe mechanical properties of antimicrobial films. They reportedthat the addition of 1.5% (w/w) triclosan produced a slight effecton the mechanical properties of neat PLA. However, the inclusionof 10% (w/w) wood flour in the polymeric system containing 1.5%(w/w) triclosan was more pronounced than that of the inclusionof triclosan. The increase of wood flour loadings from zero to10% (w/w) increased the stiffness of the active composites and, atthe same time, reduced the flexibility and toughness. The latterobservations are in agreement with those reported by Huda andothers (2006) who also investigated the mechanical properties ofPLA/wood composites and reported similar property changes.The incorporation of triclosan into the composite system did notcause any significant effect on the material properties and thismight be due to the low percentage of antimicrobial agent in theformulation.

The type of fibers used and the uniformity of their dispersionwithin the matrix significantly affects the mechanical propertiesof polymer fiber composites. Key governing factors affecting themechanical properties include the cellulose content of the fibers,their orientation, length, and diameter. According to Mukherjeeand Kao (2011), the diameter of the fibers affects the length todiameter aspect ratio (L/D) and this may affect the mechanical per-formance of the composite. If the L/D ratio is too low, there willbe insufficient stress transfer and the resulting reinforcement effectwill be less significant. Conversely, if the L/D ratio is too high,the fibers may become entangled during mixing, and thus a lowerstress transfer will be obtained (Nando and Gupta 1996). Bonillaand others (2013) investigated the effect of different particle sizesand loadings of chitosan powder (5% to 10%, w/w) incorporatedinto PLA on the physico–mechanical properties and antimicrobialactivity of the resulting system. They observed that these filmssuccessfully inhibited the growth of total aerobic mesophilic andcoliform microorganisms in minced pork meat as compared to thecontrol, especially at a lower particle size of the chitosan powder.However, the incorporation of chitosan powder resulted in a lessrigid and less stretchable film.

Generally, the mechanical properties of antimicrobial coatedpolymeric materials are not expected to be affected considerablyby the antimicrobial coating. In these cases, the polymeric materialis a supporting layer for the coating. However, some interactionbetween the polymer and coating system may occur at the in-terface between them, particularly when the antimicrobial agentconcentration is high. Jin and others (2009) have developed an

antimicrobial film consisting of PLA and pectin with nisin loadedinto the film at 1% (w/w) concentration by a diffusion coatingmethod. Although they reported that the coating of nisin did notaffect the overall properties of the PLA/pectin film, significantchanges in the mechanical properties were reported with the ad-dition of approximately 20% (w/w) pectin and 6.7% (w/w) waterinto the PLA films.

Theinsathid and others (2012) reported some increase in theflexibility of PLA films (up to 14%) by incorporating 0.28% (w/w)of lauric arginate (LAE) via a coating method. However, it wasreported that the change in elongation at break was not statisticallysignificant. A corona discharge was used to modify the surfaces ofthe PLA films in the study to produce a more hydrophilic PLA aswell as to increase the surface roughness. The surfaces of the mod-ified films were expected to enhance the compatibility betweenthe PLA and LAE. The authors reported that the slight increasein flexibility might be due to the weak secondary attraction forcesbetween anionic charge on the surface of PLA and the cationicsurfactant LAE. Similar findings were reported by Li and others(2012a) comparing uncoated films consisting of PLA and ther-moplastic sugar beet pulp (TSBP) with PLA/TSBP films coatedwith Nisaplin in PLA/dichloromethane (DCM). They reported aslight increase in the percent of elongation at break (by 9%) forthe coated films and a significant reduction in tensile modulus (by64%) and tensile strength (by 36%). The reduction in the tensileproperties was suggested to be due to the dissolution of PLA inDCM resulting in a decrease of the PLA’s crystallinity as confirmedby dynamic mechanical analysis (DMA) and microscopy images.

There are only a few studies that have investigated the thermalproperties of antimicrobial PLA packaging films. This might bedue to the small or insignificant changes in the thermal profile byusing thermogravimetric analysis (TGA) of the PLA films incor-porated with antimicrobial agents as a result of the small samplesize. The inclusion of low concentrations of antimicrobial agentsat levels less than 10% (w/w) for example, will usually have only avery slight effect on the Tg, crystallinity temperature (Tc), meltingtemperature (Tm), or the percentage of crystallinity (Xc). In oneexample, Liu and others (2010) studied the thermal properties ofPLA incorporated with 5% (w/w) nisin and EDTA that was plas-ticized by using 30% (w/w) GTA. A comparison between PLAfilms and the antimicrobial PLA films containing Nisaplin andEDTA showed that the antimicrobial agents had a smaller effecton the Tg than the plasticizer in the PLA film with 30% (w/w)GTA. From this study, it was suggested that the plasticizer contentis more important than that of the antimicrobial agent in termsof the thermal profile of the materials. Furthermore, it was foundthat only a large amount of plasticizer (that is, 30%, w/w, GTA)incorporated into PLA lead to a significant decrease in Tc and Tm.

Prapruddivongs and Sombatsompop (2012) have reported thatPLA/wood composites and antimicrobial PLA/wood composites(incorporated with triclosan) have lower Tg values than neat PLA.This is due to the reduction in the PLA content and the hydrophiliccharacteristic of wood as well as that of triclosan. In this study, thepercent crystallinity of PLA/wood composites was reduced from34.4% to 33.1% with the addition of 1.5% (w/w) of triclosan.A similar trend was observed for the percent crystallinity of an-timicrobial PLA and neat PLA film with a reduction of 7.9% and6.5%, respectively. However, no explanation for these phenom-ena was given. These findings are contradicted by the findingsby Lui and others (2010) who reported that a PLA/Nisaplin–EDTA system exhibited a higher degree of crystallinity (61.1%)compared to neat PLA (57.9%). When an antimicrobial PLA film




demonstrates a lower Tg value compared to a neat PLA film, it canbe speculated that the antimicrobial agent is acting like a plastisizerand at the same time it will alter the thermogravimetric profile ofthe material. Lowering the Tg value of antimicrobial films due tothe inclusion of antimicrobial agents may also reduce the requiredtemperature profile during processing.

Most of the aforementioned studies have concentrated on themechanical and thermal properties of antimicrobial PLA films,which are critical to developing effective film products. However,the retention of antimicrobial agents after thermal processing andthe controlled release and migration of the antimicrobial agentsfrom PLA films afterwards, have not been adequately and system-atically addressed in the literature. These properties are of utmostimportance in antimicrobial films in initiating and maintainingeffective antimicrobial activity. Kumar and Munstedt (2005) in-vestigated the release of silver ions from polyamides with a lowpercentage of crystallinity and found that these films inhibited thegrowth of microorganisms such as E. coli and S. aureus. They re-ported that the high crystallinity of polymeric materials results ina lower and slower release of antimicrobial agents to the surface ofthe film and hence affects its antimicrobial efficacy.

It is known that the hydrophilic nature of biopolymers such aspolysaccharides lead to their low mechanical and water resistanceproperties. Thus like many other biopolymers, PLA may requiresome additional processing or modification in order to developuseful antimicrobial materials. This is mainly due to the intrinsicproperties of unmodified PLA that include its high brittleness,poor water-vapor barrier, low crystallinity, slow biodegradationrate, hydrophobicity, and lack of reactive side-chain groups. Severaltypes of modification have been developed to address these inade-quacies and include chemical modification (for example, grafting,polymerization), addition of plasticizers (to reduce brittleness),blending with other biopolymers or biofibers, and the addition ofcompatibilizers to enhance its miscibility with otherwise incom-patible polymers (Wu 2005; Yu and others 2006; Xu and others2008; Signori and others 2009; Suryanegara and others 2009; Taiband others 2009; Van Den Oever and others 2010; Tudorachi andLipsa 2011; Tawakkal and others 2012; Faludi and others 2013)

An important area that requires much further attention is thestability during the development of the antimicrobial packagingduring production, distribution, and storage. In particular, thestorage stability of the antimicrobial agents in the PLA materials iscrucial and needs to be predicted from an end use standpoint. Thisis because antimicrobial packaging materials are often transportedto and stored in warehouses under different temperature and hu-midity conditions that may lower the concentration and decreasethe effectiveness of the antimicrobial agent (Suppakul and others2011; Li and others 2012b). For instance, high storage temper-atures might result in increasing the migration rate of the activeagents and thus, reduce the antimicrobial activity of the activepackage system. To date, there is limited published scientific lit-erature available on the loss and retention of antimicrobial agentsin PLA-based materials during storage, especially at longer storagedurations.

Antimicrobial activity of PLA films incorporated withantimicrobial agents

This section focuses on the capability of a PLA matrix to haveincorporated within it various types of natural and synthetic an-timicrobial agents via different methods of preparation. The pur-pose is to produce packaging systems that inhibit the growth ofmicroorganisms. The antimicrobial activity of various types of

natural and synthetic antimicrobial agents impregnated in PLAvia melt blending, solvent casting, as well as their combinationsis presented in Table 1. These antimicrobial PLA-based materi-als are purported to show inhibitory activity against the growthof different microorganisms such as E. coli O157:H7, S. aureus,Samonella, L. monocytogenes, S. typhimurium, Fusarium proliferatum,Botrytis cinerea, F. moniliforme, Aspergillus ochraceus, yeasts as well asmolds. From Table 1, it is clear that most of the current studiesare concerned with impregnated nonvolatile antimicrobial agentssuch as bacteriocin, enzymes, metals, as well as chelating agents.

Del Nobile and others (2009) have studied the effect of lemonextract, thymol, and lysozyme incorporated directly into PLA viaa melt extrusion process. They reported that among the antimicro-bial agents under investigation, lysozyme demonstrated the highestmicrobial inhibition against M. lysodeikticus when compared to thevolatile antimicrobial agents (lemon extract and thymol). This wasattributed to the good thermal resistance of lysozyme. Jin andZhang (2008) have prepared PLA/nisin films and reported thatthese successfully inhibited the growth of L. monocytogenes in BHIbroth and liquid egg white up to approximately 50% reductionwhen compared to the control. They reported that the PLA/nisinfilm is less effective against E. coli O157:H7 in culture mediumagar than it is against L. monocytogenes.

Table 1 also shows that some researchers have investigated theincorporation of antimicrobial agents derived from metals, includ-ing silver ions, into PLA (Li and others 2009; Busolo and others2010; Fernandez and others 2010). Fernandez and others (2010)studied the effect of different processing techniques that were usedto impregnate silver ions into PLA/zeolite films on the antimi-crobial activity of the resulting films against S. aureus and E. coli.In their findings, the solvent-cast PLA/silver zeolite reduced thecolonies of S. aureus and E. coli up to 0.8 and 1.02 log CFU/mLrespectively, which were greater than the reductions observed inthe melt blended and compression molded samples.

Although in most studies PLA was incorporated with non-volatile and volatile antimicrobial agents, some have used com-pounds that would typically be added directly into foods. Jin andothers (2010) explored the effect of food additives such as potas-sium sorbate and sodium benzoate (SB) incorporated into PLA.These antimicrobial PLA films were prepared via the solvent cast-ing method in order to investigate the inhibitory effect againstE. coli O157:H7 and microflora in strawberry puree. The filmsshowed greater inhibitory effects against E. coli when comparedwith the direct addition of the agents into the real food. It wassuggested that the direct addition of antimicrobial agents into atargeted food might lead to a loss of activity due to the instantreaction with other components in the food such as lipids or pro-teins. Conversely, a more continuous antimicrobial effect couldoccur in the PLA films due to the slow release of the antimicrobialagents from the film and thus enable high enough concentrationsagainst microorganisms to be sustained with time.

Encapsulation of antimicrobial agents is an emerging techniqueto introduce antimicrobial activity into polymeric substrates. Thisis particularly useful when volatile agents are used and thermalprocesses are required. Joo and others (2012) encapsulated trans-2-hexenal (which is a volatile antimicrobial agent) with beta-cyclodextrin (β-CD) and impregnated the complex into PLA viaextrusion processing. They reported that during 7 d of storage at23 °C the PLA/β-CD/trans-2-hexenal pellets completely inhib-ited the growth of A. solani in potato dextrose agar media whencompared to the PLA/β-CD-trans-2-hexenal sheets. Encapsu-lation of volatile antimicrobial agents into β-CD has also been


R:Co

ncise

Revie

wsin

Food

Scien


Tab

le1–

Applica

tions

of

PLA

asa

pri

mar

yan

d/o

rse

condar

ym

ater

ialin

anti

mic

robia

lfilm

and

coat

ing

syst

ems

des

igned

for

food

pac

kag

ing.

Poly

mer

Antim

icro

bia

lfo

rmula

tion

agen

tsLoad

ings

aM

icro

org

anis

ms

Subst

rate

sA

pplica

tionsb

Com

men

tsR

efer

ence

s

PLA

/Mic

rocr

ysta

lline

cellu

lose

(MC

C)

Silv

erna

nopa

rtic

les

5%(w

/w)

E.

coli

Aga

rm

edia

SCPL

A/M

CC

/silv

erna

noco

mpo

sites

inhi

bite

dth

egr

owth

ofE

.co

liat

7.36

mm

ofzo

neof

inhi

bitio

n

Ali

and

Noo

ri(2

014)

PLA

Chi

tosa

n5%

and

10%

(w/w

)A

erob

icm

esop

hilic

and

colif

orm

mic

roor

gani

sms

Min

cepo

rkm

eat

EPL

A/c

hito

san

show

edan

antim

icro

bial

activ

ityag

ains

tm

icro

orga

nism

sas

com

pare

dto

nonc

oate

dsa

mpl

esat

2lo

gC

FU/m

L

Bon

illa

and

othe

rs(2

013)

PLA

Silv

er-b

ased

nano

clay

1%to

10%

(w/w

)Sa

mon

ella

spp.

Aga

rm

edia

Liqu

idm

edia

SCPL

Aco

ntai

ning

10%

(w/w

)sil

ver-

base

dna

nocl

aysig

nific

antly

inhi

bits

the

grow

thof

Sam

onel

lasp

p.at

99.9

9%C

FUre

duct

ion

Bus

olo

and

othe

rs(2

010)

PLA

Lem

onex

trac

tT

hym

olLy

sozy

me

3%to

7%(w

/w)

7%to

15%

(w/w

)3%

to10

%(w

/w)

M.

lyso

deik

ticus

and

Pseu

dom

onas

spp.

Aga

rm

edia

Liqu

idm

edia

EPL

Are

tain

edsli

ght

antim

icro

bial

activ

ityof

10%

lyso

zym

eag

ains

tM

.ly

sode

iktic

usLy

sozy

me

dem

onst

rate

dhi

gher

ther

mal

resis

tanc

e

Del

Nob

ilean

dot

hers

(200

9)

PLA

/MC

CSi

lver

nano

part

icle

s1%

(w/w

)S.

aure

usan

dE

.co

liLi

quid

med

iaA

gar

med

iaE

MC

Cre

info

rced

the

stre

ngth

prop

ertie

sof

PLA

.PL

A/M

CC

/silv

erna

noco

mpo

sites

dem

onst

rate

dsig

nific

ant

inhi

bitio

nof

E.

coli

than

S.au

reus

Fort

unat

iand

othe

rs(2

012)

PLA

/Zeo

lites

Silv

er(A

g+)

5%(w

/w)

S.au

reus

and

E.

coli

Aga

rm

edia

Liqu

idm

edia

SC/M

MSo

lven

tca

sted

PLA

/silv

erze

olite

redu

ceth

eco

loni

esup

to0.

8an

d1.

02lo

gC

FU/m

Lfo

rS.

aure

usan

dE

.co

li,re

spec

tivel

y,as

com

pare

dto

neat

PLA

film

s

Fern

ande

zan

dot

hers

(201

0)

PLA

/Iso

late

dso

ypr

otei

n(I

SP)

Thy

mol

Nat

amyc

in2.

5%to

25%

(w/w

)0.

33%

to0.

52%

(w/w

)A

sper

gillu

ssp

.,S.

cere

visia

eE

.co

li,an

dS.

aure

usA

gar

med

iaTo

mat

osli

ceC

hees

esli

ceSC

PLA

/ISP

film

cont

aini

ngth

ymol

inhi

bite

dth

egr

owth

ofE

.co

lian

dS.

aure

usbu

tno

zone

ofin

hibi

tion

for

mol

dor

yeas

tw

asob

serv

ed

Gon

zale

zan

dIg

arza

bal

(201

3)

PLA

Lact

icac

id(L

A)

Sodi

umbe

nzoa

te(S

B)

ED

TA

2.5

mg/

gfo

ral

lE

.co

liO

157:

H7

and

S.sta

nley

Aga

rm

edia

SCA

pple

coat

edw

ithPL

A/S

B+L

Aan

dPL

A/S

B+L

A+E

DT

Ain

hibi

tE

.col

iO15

7:H

7an

dS.

stanl

eyat

4.7

log

CFU

/cm

2af

ter

14d

ofst

orag

e

Jinan

dN

iem

ira

(201

1)

(Con

tinue

d)




Tab

le1–

Conti

nued

.

Poly

mer

Antim

icro

bia

lfo

rmula

tion

agen

tsLoad

ings

aM

icro

org

anis

ms

Subst

rate

sA

pplica

tionsb

Com

men

tsR

efer

ence

s

PLA

Ally

liso

thio

cyan

ate

(AIT

)N

isin

Zin

cox

ide

100

to50

0μ

L25

0m

g/g

250

mg/

gSa

lmon

ella

Liqu

ideg

gal

bum

enSC

Gla

ssja

rco

ated

with

PLA

/AIT

/ni

sinre

duce

dSa

lmon

ella

grow

that

<10

log

CFU

/mL

afte

r21

dof

stor

age

Jinan

dG

urtle

r(2

011)

PLA

Nisi

nE

DT

ASo

dium

benz

oate

(SB

)Po

tass

ium

sorb

ate

(PSo

rb)

250

mg/

g25

0m

g/g

47m

g/g

45m

g/g

E.

coli

O15

7:H

7m

olds

and

yeas

tsSt

raw

berr

ypu

ree

SCPL

A/S

B+P

Sorb

film

sin

hibi

tgr

eate

rE

.co

liO

157:

H7

and

mic

roflo

raco

mpa

red

todi

rect

addi

tion

ofSB

+PSo

rbto

food

Jinan

dot

hers

(201

0)

PLA

Nisi

n80

to50

0m

g/g

L.

mon

ocyt

ogen

esSk

imm

ilkLi

quid

egg

whi

teSC

Gla

ssja

rco

ated

with

PLA

/250

mg

nisin

inac

tivat

edce

llof

L.

mon

ocyt

ogen

esin

liqui

deg

gw

hite

and

skim

milk

atst

orag

ete

mpe

ratu

reof

4an

d10°C

Jin(2

010)

PLA

/Pec

tinN

isin

1%(w

/v)

L.

mon

ocyt

ogen

esLi

quid

egg

Whi

teO

rang

eju

ice

BH

Iliq

uid

med

iaE+C

PLA

/pec

tinco

ated

with

nisin

redu

ces

mor

ece

llsof

L.

mon

ocyt

ogen

esup

to4.

5lo

gC

FU/m

Lin

liqui

deg

gw

hite

afte

r48

hat

24°C

.Pe

ctin

faci

litat

edth

eac

cess

and

adso

rptio

nof

nisin

Jinan

dot

hers

(200

9)

PLA

Nisi

n0.

25g/

gL

.m

onoc

ytog

enes

,E

.co

liO

157:

H7,

and

S.en

terit

idis

Liqu

ideg

gw

hite

Ora

nge

juic

eLi

quid

med

iaA

gar

med

ia

SCPL

A/n

isin

film

sin

hibi

tL

.m

onoc

ytog

enes

inB

HI

and

liqui

deg

gw

hite

upto

appr

oxim

atel

y50

%re

duct

ion

com

pare

dto

cont

rol(

4.5

log

CFU

/mL)

;PL

A/n

isin

isle

ssin

effe

ctiv

eag

ains

tE

.co

liO

157:

H7

incu

lture

med

ium

agar

Jinan

dZ

hang

(200

8)

(Con

tinue

d)


R:Co

ncise

Revie

wsin

Food

Scien


Tab

le1–

Conti

nued

.

Poly

mer

Antim

icro

bia

lfo

rmula

tion

agen

tsLoad

ings

aM

icro

org

anis

ms

Subst

rate

sA

pplica

tionsb

Com

men

tsR

efer

ence

s

PLLA

Tra

ns-2

-hex

enal

trap

ped

inβ

-cyc

lode

xtri

ns(7

0:30

)β

-CD

s:Tra

ns-2

-hex

enal

A.

sola

ni,A

.ni

ger,

B.

ciner

ea,a

ndC

.ac

utat

umPe

nicil

lium

sp.

Aga

rm

edia

Liqu

idm

edia

EPL

A/β

-CD

-tra

ns-2

-hex

enal

pelle

tsco

mpl

etel

yin

hibi

tth

egr

owth

ofA

.so

lani

whe

nco

mpa

red

toth

ePL

A/β

-CD

-tra

ns-2

-he

xena

lshe

et

Joo

and

othe

rs(2

012)

PLA

Tri

clos

an/c

yclo

dext

rin

incl

usio

nco

mpl

ex(T

R/C

D-I

C)

5%(w

/w)

E.

coli

and

S.au

reus

Aga

rm

edia

ES

PLA

cont

aini

ngT

R/C

D-I

Cna

nofib

ers

show

edsli

ght

impr

ovem

ent

antib

acte

rial

activ

ityag

ains

tS.a

ureu

sand

E.

coli

com

pare

dto

PLA

nano

fiber

sco

ntai

ning

only

tric

losa

nw

ith2.

8to

3.0

cmzo

neof

inhi

bitio

n,re

spec

tivel

y

Kay

acia

ndot

hers

(201

3)

PLLA

Silv

erna

nopa

rtic

les

5%(w

/w)

E.

coli

and

S.au

reus

Aga

rdi

ffusio

nLi

quid

med

iaSC

PLLA

/nan

osilv

erex

hibi

tst

rong

antim

icro

bial

prop

ertie

sat

5m

mzo

neof

inhi

bitio

n

Lian

dot

hers

(200

9)

PLA

/Sug

arbe

etpu

lp(S

BP)

Nisa

plin

AIT

0.51

mg/

cm2

20.4

μL/

cm2

L.

mon

ocyt

ogen

esan

dSa

mon

ella

Liqu

idm

edia

E+C

PLA

/SB

Pco

ated

with

Nisa

plin

and

AIT

inhi

bit

cells

ofL

.m

onoc

ytog

enes

upto

3.91

and

4.77

log

CFU

/mL,

resp

ectiv

ely,

inB

HI

brot

haf

ter

48h

at24°C

Lian

dot

hers

(201

2a)

PLA

/Pla

stic

izer

Gly

cero

ltria

ceta

te(G

TA

)

Nisa

plin

ED

TA

5%(w

/w)

5%(w

/w)

E.

coli

O15

7:H

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stic

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acid

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ical

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ityof

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and

othe

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(Con

tinue

d)




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le1–

Conti

nued

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mer

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icro

bia

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rmula

tion

agen

tsLoad

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org

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ms

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rate

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men

tsR

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ence

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pret

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men

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timic

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coli

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9)

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sites

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elia

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tinue

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R:Co

ncise

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wsin

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Scien


Tab

le1–

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nued

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mer

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icro

bia

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rmula

tion

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tsLoad

ings

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icro

org

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ms

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rate

sA

pplica

tionsb

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men

tsR

efer

ence

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ium

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ease

dby

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4.80

and

5.13

CFU

/mL

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thid

and

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012)

PLA

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icac

id(L

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ctat

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L)5%

to15

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ease

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dity

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lene

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the

film

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thid

and

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011)

PLA

/Saw

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part

icle

(SP)

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ocin

0.2%

(w/v

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onoc

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enes

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rm

edia

Raw

slice

mea

tC

PLA

/SP

cont

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inpr

econ

ditio

nby

dry

heat

trea

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tin

hibi

ted

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mon

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raw

slice

mea

tat

2lo

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cle

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in14

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age

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013)

a Nom

inal

load

ings

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port

edin

liter

atur

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vent

cast

ing;

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sion;

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g;M

M,m

elt

mix

ing;

ES,

elec

tros

pinn

ing.

investigated by others using different types of polymeric materials(Raouche and others 2011; Ramos and others 2012).

Other materials such as fillers and plasticizers can be used tofacilitate and increase the activity of antimicrobial agents in PLA.Some fillers such as natural fibers (for example, wood flour) andother polysaccharides (for example, pectin) can be used for thispurpose. Prapruddivongs and Sombatsompop (2012) studied theincorporation of wood flour particles as an antibacterial promoterfor triclosan-based wood flour/PLA composites. In these systemsa higher loading of wood flour particles resulted in the migra-tion of more triclosan onto the composite surface and a subse-quent inhibition of the growth of nonpathogenic E. coli ATCC25922 in a growing liquid media. They reported that by usingthe plate agar count technique, 1.5% (w/w) of triclosan in 10%(w/w) wood flour/PLA composite reduced the viable cell count ofE. coli by 40% after 1 h of contact time in nutrient broth, whichwas greater than a PLA/triclosan film without wood flour. Theyspeculated that since the wood flour is hydrophilic, the PLA alsobecomes more hydrophilic and thus, more water molecules canbe adsorbed on the composite surface thereby facilitating the mi-gration of the antimicrobial agent to the surface of the film. Liuand others (2009a) reported that a neat PLA film did not exhibitany antimicrobial activity with a small concentration of Nisiplin.Nevertheless, they found that co-extrusion of PLA and pectin–Nisaplin composites demonstrated a higher antimicrobial activitycompared to neat PLA and suggested that the pectin protects thebiological activity of the composite films and thus, significantlyinhibits the growth of the test species L. monocytogenes. Othersystems effective against L. monocytogenes include PLA/nanoclaycomposites using Cloisite 30B (Rhim and others 2009), althoughthe use of nanoclays is yet to be fully established. Liu and others(2012) have patented their active materials combination containingPLA with bacteriocin (for example, nisin, generally in the formof Nisaplin R©) and plasticizer (for example, LA, lactide, triacetin,GTA), and as an option at least one pore forming agent. Recently,Kayaci and others (2013) have prepared antimicrobial nanofibres ornanowebs using an electrospinning method that comprises a PLAand triclosan/cyclodextrin inclusion complex (TR/CD-IC). Thetriclosan/cyclodextrin complex consisted of both beta (β) andgamma (γ ) complex. They reported that the zones of inhibi-tion of E. coli and S. aureus were wider for the PLA containingTR/CD-IC nanowebs, suggesting that both PLA/TR/β-CD-ICand PLA/TR/γ -CD-IC nanowebs have better antibacterial prop-erties than the PLA containing triclosan.

It is therefore clear that PLA has the potential to be used as a pri-mary polymeric material in antimicrobial packaging applicationssince both volatile and nonvolatile additives demonstrate reason-ably good compatibility with this polymer. Nevertheless, futureand systematic work needs to be performed in order to confirmthe potential of such antimicrobial systems through studies withfood stimulants as well as with real food products.

Antimicrobial activity of PLA films coated withantimicrobial agents

Due to the heat sensitivity of antimicrobial agents, they are of-ten either coated onto the packaging material after it is formedor added to cast films. For example, natural polyphenolics donot tolerate the high temperatures that are typically encoun-tered during thermal processing of polymeric materials (Appendiniand Hotchkiss 2002). Furthermore, antimicrobial agents are oftencoated onto the surface of packaging materials in order to pro-vide a high concentration of the agent on the surface of the food




product where microbial contamination is more significant (Jin2010; Kuorwel and others 2011b). Only a few reports were foundin the literature on the activity of antimicrobial agents coated ontoPLA films or PLA incorporated with antimicrobial agents thathave been then solution coated onto other substrates. These sub-strates can be glass jars, plastic films, or coated directly onto foodproducts.

Table 1 depicts the antimicrobial activity of different types of an-timicrobial agents incorporated in PLA via coating, solvent castingas well as their combinations with primary and/or secondary mate-rials. Jin and others (2009) studied the effectiveness of PLA/pectinfilms coated with nisin against L. monocytogenes. They reported thatPLA/pectin and PLA films coated with nisin (1%, w/v) were sig-nificantly different in terms of their antimicrobial activity based onthe agar diffusion test. In this system, nisin can be easily coated onPLA/pectin due to the rough surface of the composite as comparedto the relatively smooth surface of neat PLA films. The presenceof pectin also facilitates the access and adsorption of nisin. Thenisin-coated PLA/pectin system was found to reduce the count ofL. monocytogenes up to 3.7 and 4.5 log CFU/mL in orange juiceand liquid egg white, respectively.

In another study, Theinsathid and others (2012) prepared PLAfilms coated with LAE at various coating loadings from zero to2.6% (w/w). An average 4 mm zone of inhibition was observed forall antimicrobial films starting from 0.07% to 0.28% (w/w) of LAEloading. The populations of L. monocytogenes and S. typhimuriumwere decreased with a higher loading of LAE (2.6%, w/w) by log 2to 3 CFU/mL compared to the uncoated PLA films, as expected.

Table 1 also lists systems in which direct contact of the an-timicrobial agent with the food product as well as the packagingmaterial occurs. For example, Jin and Niemira (2011) preparedPLA coating systems using SB, LA, and EDTA. They coated ap-ples with solvent-cast PLA/(SB+LA) and PLA/(SB+LA+EDTA)consisting of 2.5 mg of each antimicrobial substance per gram ofPLA. They reported that PLA/(SB+LA) films inhibited E. coliO157:H7 and Salmonella stanley at 4.7 log CFU/mL after 14 d ofstorage. Jin (2010) coated glass jars with solvent-cast PLA and 250mg of nisin and reported that in liquid egg white and in skim milkL. monocytogenes were inactivated by this system at storage temper-atures of 4 and 10 °C. It is important to note that an antimicrobialfilm is more effective against microbial growth when the food andthe packaging material are in direct contact but this is not alwayspractical (Brody and others 2001; Appendini and Hotchkiss 2002).

Generally, if the concentration of an antimicrobial agent is at orabove the minimum inhibitory concentration (MIC) on the foodsurface, the system will maintain effective antimicrobial activity(Suppakul 2004). According to Rardniyom (2009), a good selec-tion of an antimicrobial agent together with the combination ofdifferent types of tested foods or food simulants is an importantfactor to be considered when designing an antimicrobial package.Since PLA is a relatively new polymer, it requires further investiga-tion, particularly with regard to the antimicrobial activity of PLAsystems that are intended for targeted food packaging materials.

ConclusionsThe development of antimicrobial packaging materials based on

PLA polymers is expected to grow in the future with major focuseson enhancing food safety and quality and concurrently exploringalternatives to synthetic polymers made from petrochemicals thatare less environment friendly. To date, there has been little exten-sive research reported on antimicrobial PLA films and coatings butnonetheless there are already some commercial products available

in the marketplace. Of the reported studies, some have investi-gated the activity of the antimicrobial films by determining thediffusion of antimicrobial agents in solid and liquid media as wellas with real food products. Others have investigated the physico–mechanical, thermal, and other properties of antimicrobial PLAsystems. In general, PLA films offer an effective matrix to an-timicrobial agents and have the potential for use in commercialpackaging applications.

The development of antimicrobial PLA films with improvedphysical and mechanical properties and antimicrobial activity isstill a challenge due to the inherent brittleness and hydropho-bicity of this polymer. Some of these material limitations can beovercome with the implementation of current and advanced tech-nologies such as nanotechnology. In the near future, antimicrobialPLA materials intended for food contact packaging applicationsmay rival conventional petroleum-based polymeric materials, theformer having significant environmental benefits.

AcknowledgmentsThe authors gratefully acknowledge the Ministry of Education

Malaysia and Univ. Putra Malaysia (UPM) for providing the PhDscholarship for Intan Tawakkal.

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Chapter 3 – The Influence of Fibre Chemical Treatment

The Influence of Chemically Treated Natural Fibers Containing Thymol in Poly(Lactic

Acid)-Based Antimicrobial Composites for Packaging

Overview

Chapter 3 presents a study of the effect of untreated and alkali treated kenaf fibres in PLA/kenaf

composites with and without thymol. In this chapter, the preparation of PLA/kenaf composite

formulations was fixed at 30% w/w kenaf loading. The findings of the mechanical, thermal and

morphological properties of the PLA and PLA/kenaf composites are discussed. The

degradation properties of the studied materials under controlled composting conditions are also

reported and discussed.

The manuscript entitled “The Influence of Chemically Treated Natural Fibers Containing

Thymol in Poly(Lactic Acid)-Based Antimicrobial Composites for Packaging” by Tawakkal I.

S. M. A., Cran M. J. and Bigger S. W. was submitted for peer review in the journal, Polymer

Composites, 2016.

Note:

At the time of submission, this work was under review but has since been published in Polymer

Composites, Tawakkal, I. S. M. A., Cran, M. J., & Bigger, S. W. (2016). The Influence of

Chemically Treated Natural Fibers in Poly(Lactic Acid) Composites Containing Thymol (early

view article) and is attached following this chapter. Moreover, an article for the Society of

Plastics Engineers (SPE) online magazine based on this paper/chapter which further

demonstrates its impact on the field is included following the full publication.

29

30

31

The Influence of Chemically Treated Natural Fibers in Poly(Lactic Acid) Composites

Containing Thymol

Intan S. M. A. Tawakkal1, Marlene J. Cran2* and Stephen W. Bigger1

1 College of Engineering and Science, 2 Institute for Sustainability and Innovation,

Victoria University, Melbourne, Australia

*Corresponding author Email: [email protected]

Institute for Sustainability and Innovation, Victoria University

PO Box 14428, Melbourne, Victoria 8001, Australia

Ph. +61 3 9919 7642

mailto:[email protected]

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3.1 Abstract

The mechanical, thermal and morphological properties of poly(lactic acid) (PLA) composites

incorporated with 30% w/w untreated kenaf (UK) or alkali treated kenaf (TK) fibers was

explored together with the inclusion of 5-10% w/w thymol for potential antimicrobial

packaging material applications. The TK fiber composites had significantly higher tensile

strength than those containing UK fibers. Scanning electron micrograph images suggested a

better adhesion between the TK fibers and the matrix was achieved resulting in improved

reinforcement of the PLA/TK fiber composite. The neat PLA and composites containing 10%

w/w thymol exhibited lower processing torque, tensile strength and glass transition

temperatures than those without thymol suggesting a possible lubricating and/or plasticizing

effect. However, the incorporation of thymol into PLA at this level as well as into the

composites did not influence the flexibility of the materials as a whole. Decomposition in

compost at 58°C progressed rapidly for neat PLA and PLA/thymol resulting in complete

disintegration within 35 days. The presence of kenaf slightly inhibited the degradation

although complete disintegration of the composite was achieved within 48 days. The results

suggest that PLA composites containing kenaf have the potential to be developed as rigid and

disposable food packaging materials from biodegradable and renewable resources.

Keywords

Poly(lactic acid); Natural fibers; Polymer composites; Mechanical properties; Thermal

properties

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3.2 Introduction

To date, many researchers have studied bio-composite materials that are either derived from

natural resources or renewable resources for various applications where the focus has been on

creating materials that have a perceived low environmental impact. Poly(lactic acid) (PLA) is

a relatively new and promising bio-based thermoplastic polyester that can be derived from

renewable, bio-derived monomers obtained from a range of plants containing polysaccharides

(Jamshidian et al., 2010). For packaging applications, in particular, this polymer provides good

strength and optical properties, low toxicity, is readily compostable and is easily formed using

equipment that processes conventional, commercial polyolefin plastics (Jamshidian et al.,

2010; Obuchi and Ogawa, 2011). Although the potential of PLA for use in packaging

applications has been established (Tawakkal et al., 2014b), there are some inherent limitations

of this material such as its high production costs, poor water vapour and gas barrier properties,

low heat resistance and brittleness, that limit its widespread application in this industry (Auras

et al., 2005; Suryanegara et al., 2009).

Naturally sourced fibers or lignocellulosic materials such as wood, kenaf, jute, ramie and flax

are commonly used as cost-reducing biofillers and/or reinforcements for PLA and can render

the PLA more environmentally friendly in the sense that these can enhance the bio/hydro-

degradability of the polymer (Gurunathan et al., 2015; Valdés et al., 2014). Furthermore, PLA

generally exhibits a better compatibility and interaction with the natural fibers than petroleum-

based polymers during processing (Shanks et al., 2004). Thus, the incorporation of natural

fibers into PLA matrices has recently gained attention in order to reduce costs, improve

mechanical properties as well as enhance compostability and biodegradability (Halász and

Csóka, 2012; Kwon et al., 2014). Bast fibers obtained from the outer stem layer of the kenaf

plant (Hibiscus cannabinus L.) have good mechanical properties that enable this material to be

used as a reinforcement in bio-polymer composites as an alternative to glass fibers (Abdul

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Khalil et al., 2010). However, the inherent hydrophilicity of this fiber requires that some

chemical modifications need to be made in order to impart good surface adhesion between the

polymer and the filler. Moreover, for potential applications related to disposable packaging

materials such as food service products, alkali fiber treatment is an important fiber processing

step. Such treatments are required to: (i) remove impurities thereby producing a safer

packaging materials for food contact and (ii) improve the interaction between the polymer

matrix and the fibers (Johar et al., 2012). There are several well-established and recommended

pre-treatments for kenaf fibers including graft copolymerization, the use of coupling agents, as

well as alkali chemical treatments (Johar et al., 2012). Such modifications can improve the

wettability of the fibers with polymer matrices, reduce moisture absorption and ease of

processing. Nevertheless, the ability of fibers to reinforce PLA is also influenced by their size,

quantity and dispersion as well as processing conditions (Gurunathan et al., 2015; Xia et al.,

2015)

In addition to the use of biofillers, the use of some naturally derived additives can also provide

additional functional properties of PLA packaging materials (Tawakkal et al., 2016). For

example, the incorporation of essential oil extracts such as thymol (2-isopropyl-5-

methylphenol) into packaging materials may extend the shelf life of packaged food products

by minimizing the growth of microorganisms due to the inherent antimicrobial (AM) properties

of this extract. Thymol is a well-established natural AM agent that can reportedly exhibit

antioxidant and antimicrobial activity against wide spectrum of microorganisms such as

bacteria, fungi, mould and yeast (Del Nobile et al., 2009; Ramos et al., 2014b; Tao et al., 2014).

For a continuous AM activity, a migratory release system can be used where volatile additives

such as thymol, that often seem a safer alternative to synthetically derived chemical additives,

are released from the package into the headspace surrounding the food product (Han, 2003).

Moreover, the antimicrobial activity of PLA-based materials can be increased with the

35

inclusion of hydrophilic fibers that promote and facilitate the release of the AM agent

(Prapruddivongs and Sombatsompop, 2012; Tawakkal et al., 2016). The combination of the

main elements in a composite system comprised of a PLA matrix, kenaf and natural AM

substances could potentially improve the key properties and contribute to the sustainability of

the system as a whole. However, few studies have reported the development of bio-composites

containing both natural fibers and AM additives for use as food packaging materials (Tawakkal

et al., 2014a).

The aim of this study was to explore the mechanical, thermal and morphological properties of

PLA composites incorporated with untreated or alkali treated kenaf fibers and thymol.

Moreover, a visual qualitative analysis of the degradation under thermophilic aerobic

conditions was also performed as a preliminary assessment of the compostability of these PLA-

based composites.

3.3 Materials and Methods

3.3.1 Materials

Film and bottle grade poly(lactic acid) 7001D IngeoTM; specific gravity 1.24; weight average

molecular weight, Mw = 1.10 105 g mol-1 (Othman et al., 2012); melt flow index (MFI) 6

g/10 min at 210°C and 2.16 kg; melting temperature range 145-160°C was purchased from

NatureWorks LLC, USA. Short kenaf fiber bundles (bast) were purchased from Ecofiber

Industries, Australia. The aspect ratio (L/D) of the kenaf fibers bundle was approximately 9

with an average length of 920 ± 0.40 μm and an average diameter of 100 ± 0.03 μm. The

diameter and length of the fiber bundles were determined by using scanning electron

microscopy (SEM) with approximately 90 fiber images that were measured and recorded.

Generally, raw kenaf bast fibers consist of ca. 63.5% cellulose, 17.6% hemicellulose, 12.7%

lignin and 4% extractive (Jonoobi et al., 2009). Thymol (T0501, MW = 150.22 g mol-1), a

36

white crystalline substance with purity of 99.5% was purchased from Sigma-Aldrich Pty. Ltd.,

Australia. Sodium hydroxide (NaOH) (MW = 40 g mol-1) and acetic acid (CH3COOH) (MW

= 60.05 g mol-1) with purity of 100% were purchased from Merck Chemicals, Australia.

3.3.2 Preparation of Composites

The surface treatment of the kenaf was performed by immersing it in 5% (w/v) NaOH for 2 h

at room temperature. Acetic acid was used to adjust the pH during the washing and rinsing of

the fibers with distilled water. The treated fibers were removed from the solution and dried

overnight in an air circulating oven at 105°C. Prior to mixing with the PLA resin, both the

treated and untreated fibers as well as the PLA resin were dried in an oven at 60°C overnight

before blending with thymol (Ibrahim et al., 2011). Drying of the fibers before mixing is

necessary in order to avoid fibers debonding at the fiber-matrix interface due to the presence

of moisture on the fibers surface (Gurunathan et al., 2015) and drying also avoids hydrolysis

of the polymer. Formulations containing a 30% w/w kenaf loading were prepared with thymol

concentrations at zero, 5 or 10% w/w.

The neat PLA and PLA composites containing thymol were compounded using an internal

mixer with torque recording capabilities (Haake PolyLab OS, Germany) at 50 rpm and 155°C

for 8 min. The PLA resin was introduced in the chamber and mixed for ca. 2 min to obtain a

constant torque. The kenaf fibers and/or thymol were then added step by step in small amounts

during the next 7 to 8 min whilst the mixture was continuously mixed. Films and slabs of 0.3

and 3 mm thickness respectively were prepared using a laboratory press (L0003, IDM

Instrument Pty. Ltd., Australia). The samples were preheated at 150°C for 2 min and pressed

at the same temperature for 3 min under a force of 50 kN before cooling to 30°C. A hand-held

micrometer (Hahn & Kolb, Stuttugart, Germany) was used for measuring film thickness.

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3.3.3 Imaging of Fibers and Composites

Scanning electron microscopy (SEM) was performed to observe the morphology of the tensile

fracture surface of the composites using a JOEL NeoScope (JCM-5000) scanning electron

microscope (SEM) instrument under high vacuum and using an accelerating voltage of 10 kV.

Prior to imaging, all samples were coated with a thin layer of gold (up to 6 nm) using a

NeoCoater device (MP19020NCTR).

3.3.4 Infrared Analyses

A Shimadzu IR Prestige Fourier transform infrared (FTIR) spectrophotometer with attenuated

total reflectance (ATR) technique was used to undertake the infrared spectral analyses. All

spectra were recorded in absorbance mode in the range of 550-4000 cm-1 with a resolution of

4 cm-1 and with 32 scans recorded at every point using Happ-Genzel apodization. A small

portion of thymol, and untreated and/or treated fibers were mixed in an agate mortar and pestle

with a small amount of paraffin oil. The sample was then applied to a KBr disc and its FTIR

spectrum recorded. This technique was required in order to avoid agglomeration of the kenaf

fibers and to dilute the high concentration of thymol so the respective peaks could be identified.

Ten scans were performed for each acquisition.

For FTIR mapping analysis, the spectra were acquired using a Bruker Hyperion 2000 IR

microscope (Bruker Optik Gmbh, Ettlingen, Germany) equipped with a liquid nitrogen cooled

detector. The apparatus was connected to the infrared microspectroscopy beamline at the

Australian Synchrotron (Clayton, Australia). The microscope was coupled to a Bruker Vertex

70 spectrometer (Bruker Optik Gmbh, Ettlingen, Germany). The line scan and spectral

mapping were performed on the surface of PLA film containing 10% w/w thymol and were

collected in transmission mode by scanning the film using a computer-controlled microscope

stage at an aperture of 10 µm 10 µm with a 25 IR objective. The spectral data were collected

38

in the IR range 4000-800 cm-1 at a resolution of 4 cm-1 and with 64 scans. All spectra were

processed using Opus 6.0 software (Bruker Optics, Gmbh, Ettlingen, Germany). A two

dimensional (2D) IR map was produced using the software, where integrated areas were

calculated for bands attributable to the PLA carbonyl group and thymol ring aromatic

functional group.

3.3.5 Tensile Testing

Tensile tests were performed using a Model 4301 Instron Universal Testing Machine with a

load cell of 1 kN. Eight tensile specimens were prepared from each sample set via heat press

moulding using a dog-bone die. The tests were conducted at a cross-head speed of 5 mm min-1

on dumbbell-shaped specimens in accordance with the ASTM D638 Type V method until

tensile failure was detected and at least six replicate specimens were taken as an average in

accordance with the standard method. The average tensile strength, tensile modulus and

percentage extension at break were measured and calculated using Instron BlueHill Series IX

software. The tensile properties were then normalized against PLA control without filler as

well as PLA films containing 5% and 10% w/w thymol to enable a comparison between the

studied systems.

3.3.6 Thermal Property Testing

A differential scanning calorimeter (Mettler Toledo DSC1) was used to obtain the thermal

properties of the PLA and composites under an inert gas (nitrogen) atmosphere. The samples

of ca. 8-9 mg each were weighed and sealed in aluminium crucibles. The heating was

performed over the range 30 to 300°C at a rate of 10°C min-1 and with a nitrogen flow rate of

20 mL min-1. The glass transition temperature (Tg), cold crystallization temperature (Tcc),

melting temperature (Tm), cold crystallization enthalpy (ΔHcc) and melting enthalpy (ΔHm)

were obtained from the DSC thermograms using the first heating scan. The maximum

39

percentage crystallinity (%Xc) of each sample during the DSC heating was determined by

calculating the ratio between the melting enthalpy of the sample and that of 100% crystalline

PLA (Du et al., 2014) as shown in Equation (1):

%Xc = ΔHm/ΔHm0 x 100/w (1)

where w is the weight fraction of PLA in the formulations and ΔHm0 is the enthalpy of melting

for 100% crystalline PLA (93.7 J g-1) (Lee et al., 2009; Liao et al., 2007). The samples were

also characterized using a thermogravimetric analyser (TGA) (Mettler Toledo TGA/DSC1) in

order to determine their thermal stability. The mass-loss curves of the active composites were

recorded when heated from 30 to 500°C at a heating rate of 5ºC min-1 and at a flow rate of

nitrogen of 0.2 L min-1. The data were obtained via the first derivative of the original curve in

order to record the onset of thermal degradation.

3.3.7 Disintegration Studies

A study of the decomposition under composting conditions was performed on composite

samples cut into pieces (20 20 3mm). Samples were buried in a commercial compost at 5

cm depth in perforated boxes made of PE and incubated in the oven at 58°C. Aerobic conditions

were maintained by mixing the compost periodically and by the addition of water to maintain

a moisture content as well as by monitoring the relative humidity of headspace above the

compost at ca. 60% according to ASTM D5338. Samples were removed from the compost

after 7, 14, 21, 28, 35 and 48 days, were immediately washed with distilled water to remove

traces of compost and then photographed.

40

3.4 Results and Discussion

3.4.1 Structural Analysis

To confirm the effect of alkali treatment on the kenaf fibers, surface FTIR spectra of UK and

TK fibers were obtained and are shown in Figure 3.1. In the spectrum of the UK fibers, a sharp

absorption peak at approximately 1740 cm-1 is observed which corresponds to the carbonyl

group (>C=O) stretch. The existence of this peak in the UK fibers has been ascribed to the

acetyl and ester group in hemicellulose or carboxylic acid group in the ferulic and p-coumeric

components of lignin (Johar et al., 2012). This particular peak is not apparent in the case of the

TK fibers which indicates the elimination of non-lignocellulose components due to the alkali

treatment (Cao et al., 2007; Johar et al., 2012). A similar finding was also reported by Cao et

al. (2007) who treated kenaf fibers with 5 to 15% (v/w) NaOH solution at 25°C for 2 h.

Figure 3.1 Infrared spectra of untreated and treated kenaf fibers in the region 1900-1500 cm-1.

The FTIR structural analysis of thymol and PLA containing 10% w/w thymol in this study have

been discussed in detail previously (Tawakkal et al., 2016). However, in that study the presence

and distribution of thymol at the surface of the PLA containing 10% w/w thymol film was not

41

investigated and was therefore further analysed using an IR mapping technique in the present

study. Figure 3.2 shows the intensity of the IR band mapped over PLA containing 10% w/w

thymol film at 1618 and 1700 cm-1 that represent the ring aromatic group of thymol and the

carbonyl group of PLA respectively (Tawakkal et al., 2016). Figure 3.2(a) shows that a small

amount of thymol is present on the film surface with a higher concentration of thymol detected

at the left bottom edge of the area tested. This finding is also consistent with the intensity

distribution of the PLA carbonyl group (see Figure 3.2(b)) and suggests that this technique can

be used to observe the distribution of thymol on the PLA matrix surface and within a relatively

small film area. Moreover, the localization of thymol molecules such as within the matrix or

the fibers and at the fiber-matrix interface is an important parameter in further understanding

the behaviour of thymol based bio-composites but is beyond the scope of the present study.

Figure 3.2 Intensity of the IR band mapped over PLA sample containing 10% w/w thymol at: (a) 1622 cm-1 (aromatic ring of thymol) and (b)

1700 cm-1 (carbonyl group of PLA).

3.4.2 Processing of PLA and Composites

The processing torque profiles of PLA and PLA filled with 30% w/w UK fibers in the presence

of thymol are shown in Figure 3.3. Generally, the addition of 5-10% w/w thymol to PLA and

PLA/kenaf composites imparts a lubricating effect during processing resulting in a significant

reduction in the normalized torque value. This lubricating effect could also be due to the

42

diffusion of thymol molecules to the walls of the internal mixer. Similar trends were observed

for active PLA filled with TK fibers containing thymol with no significant changes observed

in comparison with the PLA/UK fiber composites (Tawakkal et al., 2014a).

Figure 3.3 Normalized torque as a function of time for PLA melts of: (i) PLA/UK fiber composite, (ii) PLA/UK fiber composite with 10% w/w

thymol, (iii) neat PLA and (iv) PLA with 10% w/w thymol.

The peak in the melt torque observed at ca. 2 min for PLA composite containing UK fibers

(see Figure 3.3) corresponds to the time when the fibers are introduced into the mixer but this

peak is absent when thymol is incorporated into the PLA formulation. For the PLA containing

thymol, the torque decreased momentarily and achieved an equilibrium torque value almost

immediately where it was maintained throughout the mixing process. These results clearly

suggest that the thymol acts as a lubricant for the system. Furthermore, this finding is also

supported by the significantly lower equilibrium torque observed after ca. 8 min in the

formulation containing thymol compared with the formulation without thymol. Similar

findings have been reported by Sungsanit (2011) who studied the rheology of plasticized PLA

blends that contain the oligomer polyethylene glycol (PEG) at 5-20% w/w content. It was found

that higher loadings of PEG in the PLA blends produced a lower melt viscosity compared to

neat PLA.

43

It is interesting that the presence of thymol, which is a relatively small molecule compared to

typical PEG oligomers, has a noticeable effect on the viscosity of the melt. The lower PLA

melt viscosity observed in the presence of 10% w/w thymol may be due to the thymol

weakening the intermolecular forces between adjacent polymer chains, spacing them further

apart and creating a greater free volume (Sungsanit, 2011). The hydroxyl group in the thymol

is also expected to develop a hydrogen bond with the polymer and thus interfere with the

polymer-polymer interactions thereby acting in a similar fashion to that of an oligomeric

plasticizer (Cao et al., 2009).

3.4.3 Composite Morphology

The aim of chemical treatment of the fibers is to remove impurities, pectin, waxy substances,

lignin and hemicelluloses in order to create a rough fiber surface and increase hydroxyl group

accessibility for a better interaction with the matrix (Mwaikambo and Ansell, 1999). Hydrogen

bonding is also likely to occur in the composites between the hydroxyl groups in the UK and

TK fibers, the terminal hydroxyl groups of PLA and the carbonyl groups of the ester linkages

of PLA (Bax and Müssig, 2008). Figure 3.4(a) and (b) show SEM micrographs of the tensile

fracture surfaces of PLA composites containing TK fibers with and without thymol. For both

of these composites, it appears that the fibers are well dispersed throughout the polymer matrix.

For the PLA composite containing no thymol (Figure 3.4(a)), the TK fibers appear to be more

tightly bound to the matrix compared to the composite in which thymol is present (Figure

3.4(b)). This observation is in agreement with the work of Yousif et al. (2012) who treated

kenaf fibers with 6% (w/v) NaOH and observed a slight improvement in the interfacial

adhesion and the porosity of the epoxy/kenaf composites which prevented the debonding,

detachments or pull-out of fibers. The presence of thymol in the matrix appears to facilitate the

slippage of the fibers out of the matrix during fracture and this is seen in Figure 3.4(b) with the

44

presence of more protruding fibers at the matrix surface. Moreover, the thymol that is present

on the surface of the kenaf fibers will lower the coefficient of friction and further facilitate the

slippage of the fibers within the matrix. The smooth surfaces of some of the fibers, as well as

the voids, suggests that there is weaker adhesion between the fibers and the PLA matrix when

thymol is present in the formulation compared to systems where thymol is absent.

Figure 3.4 Scanning electron micrographs of PLA composites of: (a) PLA/TK fiber composite and (b) PLA/TK fiber composite with 10% w/w

thymol at 200 magnification.

3.4.4 Mechanical Properties

Figure 3.5 shows the effect of the presence of UK or TK fibers on the normalized tensile

properties of PLA composites containing zero, 5 or 10% w/w thymol. The tensile strength of

PLA filled with TK fibers and containing no thymol was slightly higher than that of the UK

composites as well as that of the neat PLA (see Figure 3.5(a)). The TK fibers act as

reinforcement and impart an approximately 8% increment in tensile strength, whereas no

significant changes in the tensile strength of the neat PLA were imparted in the case of the

composite containing the UK fibers. The slight improvement in the tensile strength is possibly

due to better interfacial adhesion between the matrix and the TK fibers as well as better mixing

or compatibility within the composite system (Ibrahim et al., 2011; Xia et al., 2015).

45

Figure 3.5 Normalized tensile properties of PLA composites with UK or TK fibers containing zero, 5 or 10% w/w thymol: (a) tensile

strength; (b) tensile modulus; and (c) percentage elongation at break.

46

The addition of 10% w/w thymol into the neat PLA and the PLA/kenaf composites containing

UK and TK caused a significant reduction in the tensile strength compared to those composites

without thymol. The percentage reduction in the tensile strength was found to be 11%, 24%

and 21% for neat PLA, and PLA composites containing UK and TK fibers, respectively. The

localized plasticizing effect between the PLA and the thymol whereby the thymol molecules

diffuse into the bulk of the matrix between the PLA chains is in some respects akin to the case

of PLA containing low molecular weight PEG plasticizer (Chieng et al., 2013). This suggests

that the thymol additive interferes with the interaction between the polymer matrix and the

fiber in the presence of the applied stress due to the aforementioned slippage effect (Ramos et

al., 2012). This finding is consistent with the observations made in assessing the SEM

micrograph images in Figure 3.4(b). In addition, the latter composites demonstrated lower

tensile strength than the neat PLA containing only 10% w/w thymol. Thus, thymol may

increase end slipping of fibers from the polymer matrix and subsequently reduce the tensile

strength. Similarly, Taib et al. (2009) reported a reduction up to 15% in the tensile strength of

PLA/30% w/w kenaf composites containing 10% w/w PEG. Interestingly, the PLA/kenaf

composite containing TK fibers and 10% w/w thymol exhibited a higher tensile strength (51

MPa) than the composite containing UK fibers and 10% w/w thymol (44 MPa). This suggests

that the reinforcement offered by the TK fibers may prevail over the fiber slippage effect of

thymol in PLA at this level of additive in the system. In addition, changes in the tensile strength

that may arise due to the effect of the thymol may not be significant at a low level of thymol

(5% w/w).

The effect of UK and TK fibers on the normalized tensile modulus and elongation at break of

the composites containing zero, 5 or 10% w/w thymol is shown in Figure 3.5(b) and (c)

respectively. As expected, the tensile modulus (or "stiffness") of the composites containing UK

or TK fibers is significantly greater than that of the neat PLA by ca. 80% (see Figure 3.5(b)).

47

This may be due to the inherently high stiffness of the fiber (Suryanegara et al., 2009), and this

finding is also in agreement with the work of Sujaritjun et al. (2013) who incorporated PLA

with 30% w/w bamboo fibers and found an increase in the tensile modulus of ca. 17%. In this

case, the tensile modulus of PLA filled with TK fibers was slightly lower than the composite

containing UK fibers. A similar finding was observed by Xia et al. (2015) who investigated

the tensile modulus of PLA composites containing untreated and alkaline treated jute fibers

reinforcements. Cao et al. (2007) reported that kenaf fibers treated with 5% NaOH solution are

of smaller fiber diameter, increasing the tensile strength of the fiber with no significant changes

in the tensile modulus when compared with untreated kenaf fiber. Nevertheless, it is important

to note that the removal of surface components from the fibers during the alkaline treatment

cannot be solely responsible for the observed decrease in the stiffness of the composite systems.

This is because the stiffness of composites containing natural fibers is mainly related to the

cellulose microfibrils which are the major structural components within the microstructure.

The reduction in tensile modulus also occurs upon the addition of 10% w/w thymol to the

systems containing kenaf, with no changes in the tensile modulus observed upon the addition

of 10% w/w thymol to neat PLA. This finding is in sharp contrast to the findings of Ramos et

al. (2014b), who reported a 15% reduction in the tensile modulus of PLA films impregnated

with 8% w/w thymol, although the reasons for the difference in findings are unclear.

Nonetheless, the decrease in the stiffness of the composites containing UK and TK fibers are

more pronounced due to the effect of the thymol as evidenced in Figure 3.4(d).

Figure 3.5(c) shows the effect of UK and TK fibers on the normalized elongation at break of

composites containing zero, 5 or 10% w/w thymol. In general, the elongation at break of these

composites decreases with the addition of the fibrous filler. This suggests that the kenaf does

not contribute to the elasticity or the final composite flexibility and this observation is likely to

be related to the high stiffness of the composites as indicated by the results in Figure 3.5(b). In

48

contrast, the elongations at break of PLA/flax composites were found to be 100% higher than

neat PLA, with no significant changes having been found between the untreated and treated

PLA/flax fibers composites (Xia et al., 2015). No significant changes in the elongation at break

of any of the composites containing UK and TK fiber were observed upon the addition of 5-

10% w/w thymol. Liu et al. (2010) incorporated 5% w/w loadings of both Nisaplin and EDTA

into plasticized PLA/glycerol triacetate films and observed a significant reduction of the

elongation at break from 108.5 to 62.5% compared to the plasticized PLA. However, a slight

increase in the elongation at break was found in extruded PP containing 8% w/w thymol

resulting in an increase in ductile properties (Ramos et al., 2012). The reason for this difference

in behaviour of the two systems is unclear, nonetheless it is possible that a higher loading of

10% w/w thymol in PLA may produce an enhancement of the elasticity properties of the

system. This has not been verified in the present study.

In the case of PLA/kenaf composites containing thymol, it appears that the presence of thymol

does not contribute to the flexibility of the composites as a whole. This finding is supported

by the work of Taib et al. (2009) who prepared PLA plasticized with PEG and found a higher

strain at break (42%) compared to neat PLA (4%), whereas the addition of 30% w/w kenaf in

the plasticised PLA reduced the stain at break significantly to 1%. The addition of UK and TK

significantly reduces the flexibility and has been attributed to the stiffening effect of kenaf.

The addition of kenaf to the system restricts the mobility of PLA chains and increases the

number of the stress-concentrated areas at the fiber ends that can ultimately contribute to the

mechanical failure of the composite (Taib et al., 2009). The further inclusion of a small

molecular species such as thymol essential oil to the PLA/kenaf composite system undoubtedly

increases the complexity of the system and further complicates any explanation of the

experimental observations. Nonetheless, it can be suggested that the type of active PLA/kenaf

composite under investigation in the present study is more suitable for use as a rigid packaging

49

material than a flexible packaging material due to the high stiffness of this material (Faludi et

al., 2013).

It is important to note that the mechanical properties of these composite systems also depend

on the fiber size distribution and/or aspect ratio of the fibers. High shear stresses developed

during the compounding with an extruder or mixer may lead to fiber damage or breakage

resulting in a smaller fiber aspect ratio (Kwon et al., 2014). Moreover, alkaline treatments

reduce the fiber diameter and thereby increase the fiber aspect ratio (Cao et al., 2012). The

effect of fiber size distribution of the alkali treated fibers containing AM additive composites

on the mechanical properties should be considered in the future in order to fully understand the

complexities of stress transfer in these ternary composite systems.

3.4.5 Thermal Properties

Table 3.1 summarises the key data obtained from the analysis of the DSC thermograms of PLA

and PLA composites containing UK or TK fibers with zero, 5 or 10% w/w thymol. Neat PLA

exhibited an onset Tg at 59.5°C, a Tcc at 93.3°C and Tm values at 142.3 (shoulder) and 149.6°C

(peak), the latter of which is higher than the value of 141.7°C reported by Byun et al. (2010).

The onset of Tg, Tcc, Tm of the PLA composites containing UK or TK fibers demonstrated no

significant differences. However, some of these values, such as Tg, Tcc, Tm, ∆Hcc and ∆Hm,

for both of the composites were slightly lower than that of neat PLA. The maximum values of

%Xc calculated using equation (1) for the composites containing UK or TK fibers was slightly

higher than that of neat PLA and this finding is in agreement with the work of Du et al. (2014)

who investigated PLA containing 30% w/w pulp fibers from hardwood, softwood and bleached

Kraft wood.

50

Table 3.1 Thermal analysis parameters obtained from DSC thermograms of neat PLA, PLA/kenaf and PLA/kenaf/thymol composites.

Formulation

%w/w thymol Tg /°C Tcc /°C Tm1/°C Tm2/°C ∆Hcc/J g-1 ∆Hm/J g-1 Xc /%

Neat PLA

0 59.5 93.3 142.3a 149.6 18.8 26.1 28.0

5 47.9 86.9 131.9 143.0 20.0 23.4 26.7

10 41.6 85.6 124.5 137.4 22.1 22.7 27.1

PLA/UK

fibers

0 59.6 93.5 138.1 146.2 19.7 19.4 29.8

5 46.6 82.9 123.3 134.6 16.0 15.9 26.3

10 30.1 75.7 115.5 129.3 15.9 15.7 28.1

PLA/TK

fibers

0 58.8 94.9 137.8 145.5 19.0 19.1 29.2

5 46.9 85.9 125.5 136.18 16.3 16.6 27.4

10 31.3 78.5 116.7 129.8 15.8 15.8 28.3 a Shoulder on the main PLA peak

Figure 3.6 presents the DCS thermograms of the PLA and PLA composite systems and, as

shown in Figure 3.6(a), the neat PLA exhibits a double melting peak with a dominant peak at

higher temperature (Tm2). With the addition of kenaf fibers, the double melting peaks become

more distinct. The appearance of these dual melting peaks is in accordance with other reports

where the addition of fibers resulted in the development of a small melting peak that shifted to

a lower temperature, Tm1, upon further addition of fibers (Du et al., 2014; Prapruddivongs and

Sombatsompop, 2012; Tawakkal et al., 2014a). The minor peak may be due to either a different

PLA crystal type formed only in presence of the fibers and/or melting of the transcrystalline

zone as reported by Yussuf et al. (2010). For the latter, this may also be due to recrystallization

of PLA crystals of different lamella thicknesses that can be verified by using DSC at different

51

heating ramp rates. Moreover, there are several other factors that can lead to multiple melting

peaks and these may have arisen from the melting, recrystallization, and remelting processes

during heating, the presence of more than one crystal type, different polymer morphologies,

different molecular weight species, the effects of orientation as well as physical aging.

Figure 3.6 DSC thermograms of PLA and PLA composites (a) neat PLA and PLA composites without thymol: (i) neat PLA, (ii) PLA/UK fiber, (iii) PLA/TK fiber and (b) neat PLA and PLA composites with 10% w/w

thymol: (i) neat PLA, (ii) PLA/UK fiber, (iii) PLA/TK fiber.

For the PLA and PLA composites containing 5-10% w/w thymol, the addition of the additive

resulted in a decrease in Tg (see Table 3.1 and Figure 3.6(b)). This might be attributed to the

resultant increase in the free volume of polymer matrix as well as segmental mobility of the

PLA chains that change the thermal properties in a way that is akin to a plasticizing effect

(Jamshidian et al., 2012; Tawakkal et al., 2014a). The incorporation of thymol in the PLA

decreases the Tg value which may be a similar effect as that previously observed in the case of

PLA containing low and high molecular weight PEG at a 10% w/w loading (Chieng et al.,

2013; Taib et al., 2009). Furthermore, Halász and Csóka (2012) reported a similar finding

52

where the addition of 10% w/w PEG decreased the Tg of neat PLA and PLA composite

containing 5% w/w microcrystalline cellulose (MCC). The observed plasticizing effect

reflected in the Tg values indicates that the thymol is solubilised with the PLA and this is

consistent with the observations made from the morphology images, tensile strength and tensile

modulus of neat PLA and PLA kenaf composites (UK and TK) containing thymol (see Figure

3.4(b) and Figure 3.5). It can be observed that, as the content of thymol was increased to 10%

w/w in the PLA film, the Tcc peak shifted to slightly lower temperature, was slightly sharpened,

and the ∆Hcc value was increased (see Figure 3.6 and Table 3.1). This may be attributed to a

nucleating effect where the presence of the additive enhances the ability of PLA to undergo

cold crystallization as reported for more complex composite PLA films (Silverajah et al.,

2012).

Two melting peaks were also observed for PLA containing thymol. The additional melting

peak exhibited by the active PLA formulation is more pronounced than that of the neat PLA

(see Figure 3.6) and this may also be related to the reorganization of the crystal structure. These

observations contrast with other findings in the literature where no additional melting peak

arose upon the addition of plasticizer such as low molecular weight PEG200 in PLA blends

(Chieng et al., 2013). In general, slight decreases were observed for the Tg, Tcc, and Tm of

PLA/kenaf composites containing 10% w/w thymol irrespective of the fiber pre-treatment.

3.4.6 Thermogravimetric Analysis

It is important to investigate the decomposition and degradation of composites at higher

temperature, especially if these materials are intended for use in food packaging applications.

Low levels of degradation products produced during the thermal processing of such polymers

may taint and/or contaminate foodstuffs upon contact thereby causing concerns regarding

53

organoleptic properties or indeed safety. Furthermore, the thermal stability of these materials

is also of relevance to the ultimate disposal in composting or land fill where degradation usually

takes place at elevated temperatures.

The thermogravimetric (TG) profiles of neat PLA and PLA composites containing UK and TK

fibers with zero or 10% w/w thymol are shown in Figure 3.7 in the form of the normalized

weight loss as a function of temperature. The corresponding derivative weight loss curves are

also shown in Figure 3.7 to enable a more detailed analysis of the TG data to be made. The

thermal degradation of neat PLA takes place in a single step with a maximum rate of weight

loss in the range of 280 to 355°C (Ramos et al., 2014b). In general, the maximum degradation

temperature of the neat PLA was higher than any of the PLA composites (see Figure 3.7(a))

and this is more clearly reflected in the derivative TG analysis curves. As expected, the

presence of fibers in the PLA destabilised the PLA matrix in the composite as has been

previously reported by Yussuf et al. (2010) whereby some portion of the polymer is replaced

with less thermally stable fibers in the composite materials. The temperature at which the

maximum rate of degradation occurs for the composite containing TK fibers was slightly higher

than that of the composite containing UK fibers with degradation temperatures of 331 and 328

°C respectively. This may be due to the better interfacial adhesion between the TK fibers and

the PLA matrix (Yu et al., 2010).

The TG profile of PLA containing a nominal 10% w/w thymol in the formulation exhibits two

main steps during the analysis (see Figure 3.7(b)). The first step occurs gradually over the

temperature range of ca. 30 to 300°C and is attributable to the release of thymol from the

polymer matrix. This step corresponds to a ca. 7 % w/w loss in total mass and suggests that the

neat PLA and PLA composites that were formulated with 10% w/w thymol retain ca. 7% w/w

of the thymol that was originally added to the formulation. Thymol release during melt

54

processing due to the high friction between the barrel and the screw, is primarily responsible

for the loss of thymol from the formulation (Graciano-Verdugo et al., 2010). This observation

is in agreement with the work by Ramos et al. (2014b) who investigated by TG analysis the

retention of thymol in a PLA matrix after processing. The temperature corresponding to the

maximum rate of release of thymol was slightly higher for the composites containing TK fibers

(148°C) compared to those containing UK fibers (141°C) suggesting that the thymol interacts

more strongly with the TK fibers. The second step observed in the TG profiles of the PLA and

PLA composites containing thymol occurs over the temperature range of ca. 310 to 350°C and

is attributable to the complete degradation of the polymer matrix.

Figure 3.7 TGA profiles of PLA and PLA composites (a) neat PLA and PLA composites without thymol: (i) neat PLA, (ii) PLA/UK fiber, (iii) PLA/TK fiber and (b) neat PLA and PLA composites with 10% w/w

thymol: (i) neat PLA, (ii) PLA/UK fiber, (iii) PLA/TK fiber.

55

The temperature corresponding to the maximum rate of degradation for the composite

containing TK fibers after the release of 10% w/w thymol was slightly higher than that of the

composite containing UK fibers. This may be due to the increased thermal stability of the

almost complete cellulosic material that comprises the TK fibers and the absence of other

components such as waxy substances, hemicellulose and lignin that may otherwise destabilize

the system (Ibrahim et al., 2011; Shukor et al., 2014). Overall, the addition of thymol to the

formulation seems to have little effect on the thermal stability of the PLA whereas the addition

of fiber decreases the thermal stability.

3.4.7 Decomposition in Compost

Although many studies have focused on the biodegradation and/or composting of PLA and

PLA incorporated bio-filler composites, few have investigated the composting of PLA-based

materials containing AM agents. Moreover, there is still a question of whether the AM property

of the material would compromise or affect the ultimate degradation by the microorganisms in

soil (Wang et al., 2015). In the present study, a preliminary and qualitative analysis of the

disintegration under controlled compost-like conditions of PLA and PLA/kenaf films

containing the AM agent thymol was conducted as a prelude to future controlled composting

studies of these materials. Figure 3.8 shows images of PLA and PLA composites containing

30% w/w TK fibers with and without 10% w/w thymol that were removed from controlled

composting conditions at different times. Overall, a considerable modification in colour of all

samples was observed with a change from clear to opaque for the neat PLA and PLA containing

10% w/w thymol and changes were also observed for the composites. A similar observation

was reported by Ramos et al. (2014a) who investigated the disintegration of PLA and PLA

nano-composites containing thymol and silver nanoparticles under controlled composting

conditions. These colour changes are primarily due to the hydrolytic degradation and

crystallization of the PLA matrix (Pantani and Sorrentino, 2013). Moisture absorption may lead

to hydrolysis that breaks down the macro-molecular chains of the polymer with a consequent

56

erosion of the sample surface as demonstrated by the whitening of the surfaces followed by

microbial assimilation (Martucci and Ruseckaite, 2015). In addition, the hydrolysis of PLA

may also be attributed to the presence of enzymes or catalysts (e.g. carboxylic acid (RCOOH))

as well as the higher temperature conditions in order to promote the reaction and degradation

processes. It is important to note that the degradation of PLA can be very slow in soil and this

may be due to the limited presence of naturally occurring enzymes required to degrade PLA in

the natural environment and at lower temperatures.

Figure 3.8 Images of PLA and PLA containing TK fiber and/or thymol at different stages of decomposition in compost.

57

One may expect that the incorporation of AM agent into the PLA-based films will delay the

degradation process. However, the neat PLA and PLA containing 10% w/w thymol

commenced degrading within 14 days as evidenced by sample fragmentation although no

significant differences were observed between the PLA formulations with and without thymol.

For composite samples containing kenaf, degradation was not apparent until 28 days under the

test conditions and in these cases, the PLA surrounding the fiber was observed to have

subsequently degraded, dislodging the fibers and roughening the surface. The differences in

the degradation rate of these two systems may be attributed to the diffusion through the

composites with the PLA readily adsorbing water and/or the resistance in water uptake with

the presence of filler, whereby a constant molecular mobility of water in the permeable

(amorphous) phase, forcing diffusion to follow a more tortuous path was created (Azwa et al.,

2013; Mathew et al., 2005). The relatively high degradation rate of the PLA under the aerobic

composting conditions is not unexpected under the high humidity and temperature (58°C)

conditions applied (Mathew et al., 2005). Compost monitoring such as pH, evolved CO2, and

the molar mass of PLA as well as microstructural analysis of the composites by SEM imaging

and DSC during biodegradation would be required for further investigation in order to depict

the effect of thymol on the biodegradation processes. Moreover, the changes in the properties

of the materials could be employed since the overall degradation of PLA is via a surface or

bulk erosion process.

3.5 Conclusions

Bio-based PLA composites containing TK or UK kenaf fibers and thymol were prepared and

characterized. The incorporation of thymol into the PLA/kenaf composites imparted a

lubricating effect whereby the equilibrium torque value was decreased during processing. A

relatively weak adhesion between the PLA matrix and the kenaf fibers was confirmed

microscopically in composites containing thymol. The tensile strength of PLA composites

58

containing TK fibers was slightly higher than that of composites containing UK fibers,

suggesting the alkaline treatment imparts a reinforcing effect within the polymer matrix. The

incorporation of the higher level of 10% w/w thymol into the PLA/kenaf composites decreased

the tensile strength and stiffness irrespective of the fiber pre-treatment with no significant

changes on the elasticity. Thermal analysis by DSC showed a general decrease in Tg, Tcc and

Tm in PLA and PLA composites containing 10% w/w thymol compared to the formulations

without thymol suggesting changes in the phase structure of the polymer. The TG

decomposition temperature of the PLA composite containing TK fibers and 10% w/w thymol

was slightly increased indicating an increase in the thermal stability of the PLA matrix. Under

controlled composting conditions, the degradation of the PLA and PLA containing TK fibers

progressed rapidly resulting in a complete loss within 48 days. In general, the presence of TK

fibers in combination with thymol in the PLA composite resulted in significant improvement

in the overall properties of the material suggesting that this composite has the potential to be

used as an active bio-based packaging material.

Acknowledgements

The authors gratefully acknowledge the Ministry of Education Malaysia and Universiti

Putra Malaysia (UPM) for providing the PhD scholarship for Intan S. M. A. Tawakkal and

would like to acknowledge the technical staff from RMIT University especially Mr. Mike Allan

for the preparation of the composite samples. The infrared mapping was undertaken on the

infrared microspectroscopy beamline at the Australian Synchrotron, Victoria, Australia. This

research received no specific grant from any funding agency in the public, commercial, or not-

for-profit sectors.

59

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The Influence of Chemically Treated Natural Fibers inPoly(lactic acid) Composites Containing Thymol

Intan S. M. A. Tawakkal,1 Marlene J. Cran,2 Stephen W. Bigger1

1College of Engineering and Science, Victoria University, Melbourne, Australia

2Institute for Sustainability and Innovation, Victoria University, Melbourne, Australia

The mechanical, thermal, and morphological propertiesof poly(lactic acid) (PLA) composites incorporated with30% w/w untreated kenaf (UK) or treated kenaf (TK)fibers was explored together with the inclusion of 5%–10% w/w thymol for potential antimicrobial packagingmaterial applications. The TK fiber composites hadsignificantly higher tensile strength than those contain-ing UK fibers. Scanning electron micrograph imagesshowed a better adhesion between the TK fibers andthe matrix was achieved resulting in improved rein-forcement of the PLA/TK fiber composite. The neatPLA and composites containing 10% w/w thymolexhibited lower processing torque, tensile strength,and glass transition temperatures than those withoutthymol suggesting a possible lubricating and/or plasti-cizing effect. However, the incorporation of thymol intoPLA at this level as well as into the composites did notinfluence the flexibility of the materials as a whole.Decomposition in compost progressed rapidly for neatPLA and PLA/thymol resulting in complete disintegra-tion within 35 days. The presence of kenaf slightlyinhibited the decomposition although complete disinte-gration of the composite was achieved within 48 days.The results suggest that PLA composites containingkenaf have the potential to be developed as rigid, com-postable food packaging items such as trays from bio-degradable and renewable resources. POLYM.COMPOS., 00:000–000, 2016. VC 2016 Society of PlasticsEngineers

INTRODUCTION

To date, many researchers have studied biocomposite

materials that are either derived from natural resources or

renewable resources for various applications where the

focus has been on creating materials that have a perceived

low environmental impact. Poly(lactic acid) (PLA) is a

relatively new and promising biobased thermoplastic

polyester that can be derived from renewable, bioderived

monomers obtained from a range of plants containing

polysaccharides [1]. For packaging applications, in partic-

ular, this polymer provides good strength and optical

properties, low toxicity, is readily compostable, and is

easily formed using equipment that processes conven-

tional, commercial polyolefin plastics [1, 2]. Although the

potential of PLA for use in packaging applications has

been established [3], there are some inherent limitations

of this material such as its high production costs, poor

water vapor and gas barrier properties, low heat resist-

ance, and brittleness that limit its widespread application

in this industry [4, 5].

Naturally sourced fibers or lignocellulosic materials

such as wood, kenaf, jute, ramie, and flax are commonly

used as cost-reducing biofillers and/or reinforcements for

PLA. These can render the PLA more environmentally

friendly in the sense that they can enhance the bio/hydro-

degradability of the polymer [6, 7]. Furthermore, during

processing PLA generally exhibits a better compatibility

and interaction with natural fibers than petroleum-based

polymers [8]. Thus, the incorporation of natural fibers

into PLA matrices has recently gained attention in order

to reduce costs and improve mechanical properties as

well as enhance compostability and biodegradability [9,

10]. Bast fibers obtained from the outer stem layer of the

kenaf plant (Hibiscus cannabinus L.) have good mechani-

cal properties that enable this material to be used as a

reinforcement in biopolymer composites as an alternative

to glass fibers [11]. However, the inherent hydrophilicity

of this fiber requires that some chemical modifications

need to be made in order to impart good surface adhesion

between the polymer and the filler. Moreover, for poten-

tial applications related to disposable packaging materials

such as food service products, alkali fiber treatment is an

important processing step. Such treatments are required

to: (i) remove impurities thereby producing safer packag-

ing materials for food contact, and (ii) improve the inter-

action between the polymer matrix and the fibers [12].

There are several well-established and recommended pre-

treatments for kenaf fibers including graft copolymeriza-

tion and the use of coupling agents as well as alkali

Correspondence to: M. J. Cran; e-mail: [email protected]

DOI 10.1002/pc.24062

Published online in Wiley Online Library (wileyonlinelibrary.com).

VC 2016 Society of Plastics Engineers

POLYMER COMPOSITES—2016

64

chemical treatments [12]. Such modifications can improve

the wettability of the fibers with polymer matrices, reduce

moisture absorption, and facilitate ease of processing.

Nevertheless, the ability of fibers to reinforce PLA is also

influenced by its size, quantity, and dispersion as well as

processing conditions [7, 13].

In addition to the use of biofillers, the use of some nat-

urally derived additives can also provide additional func-

tional properties of PLA packaging materials [14]. For

example, the incorporation of essential oil extracts such

as thymol (2-isopropyl-5-methylphenol) into packaging

materials may extend the shelf life of packaged food

products by minimizing the growth of microorganisms

due to the inherent antimicrobial (AM) properties of this

extract. Thymol is a well-established natural AM agent

that can reportedly exhibit antioxidant and AM activity

against wide spectrum of microorganisms such as bacte-

ria, fungi, mould and yeast [15–17]. For a continuous AM

activity, a migratory release system is needed where vola-

tile additives such as thymol, that often seem a safer

alternative to synthetically derived chemical additives, are

released from the package into the headspace surrounding

the food product [18]. Moreover, the AM activity of

PLA-based materials can be increased with the inclusion

of hydrophilic fibers that promote and facilitate the

release of the AM agent [14, 19]. The combination of the

main elements in a composite system comprised of a

PLA matrix, kenaf, and natural AM substances could

potentially improve the key properties and contribute to

the sustainability of the system as a whole. However, few

studies have reported the development of biocomposites

containing both natural fibers and AM additives for use

as food packaging materials [20].

The aim of this study was to explore the mechanical,

thermal and morphological properties of PLA composites

incorporated with untreated or alkali-treated kenaf fibers

and thymol. Moreover, a visual qualitative analysis of the

degradation under thermophilic aerobic conditions was

also performed as a preliminary assessment of the com-

postability of these PLA-based composites.

MATERIALS AND METHODS

Materials

Film and bottle-grade poly(lactic acid) 7001D

IngeoTM; specific gravity 1.24; weight-average molecular

weight Mw 5 1.1 3 105 g/mol [21]; melt flow index

(MFI) 6 g/10 min at 2108C and 2.16 kg; melting tempera-

ture range 1458C–1608C; was purchased from Nature-

Works LLC, The United States. Short kenaf fiber bundle

(bast) was purchased from Ecofiber Industries, Australia.

The aspect ratio (L/D) of the kenaf fibers was approxi-

mately 9 with an average length of 920 6 0.40 lm and an

average diameter of 100 6 0.03 lm. The diameter and

length of the fibers were determined by using scanning

electron microscopy (SEM) with more than 90 fibers

measured. Generally, raw kenaf bast fibers consist of

about 63.5% cellulose, 17.6% hemicellulose, 12.7% lig-

nin, and 4% extractive [22]. Thymol (T0501,

Mw 5 150.22 g/mol) a white crystalline substance with

purity of 99.5% was purchased from Sigma-Aldrich Pty.

Ltd., Australia. Sodium hydroxide (NaOH) and acetic

acid were purchased from Merck Chemicals, Australia.

Preparation of Composites

The surface treatment of the kenaf was performed by

immersing it in 5% w/v NaOH for 2 h at room tempera-

ture. Acetic acid was used to adjust the pH during the

washing and rinsing of the fibers with distilled water. The

treated fibers were removed from the solution and dried

overnight in an air circulating oven at 1058C. Prior to

mixing with the PLA resin, both the treated and untreated

fibers, as well as the PLA resin, were dried in an oven at

608C overnight before blending with thymol. Drying of

the fibers before mixing is necessary in order to avoid

fibers debonding at the fiber–matrix interface due to the

presence of moisture on their surface [7] and drying also

avoids hydrolysis of the polymer. Formulations containing

a 30% w/w kenaf loading were prepared with thymol

concentrations at zero, 5% or 10% w/w.

The neat PLA and PLA composites containing thymol

were compounded using an internal mixer with torque

recording capabilities (Haake PolyLab OS, Germany) at

50 rpm and 1558C for 8 min. The PLA resin was intro-

duced in the chamber and mixed for about 2 min to

obtain a constant torque. The kenaf fibers and/or thymol

were then added step-by-step in small amounts during the

next 7–8 min while the mixture was continuously mixed.

Films and slabs of 0.3 and 3 mm thickness, respectively,

were prepared using a laboratory press (L0003, IDM

Instrument Pty. Ltd., Australia). The samples were pre-

heated at 1508C for 2 min and pressed at the same tem-

perature for 3 min under a force of 50 kN before cooling

to 308C. A hand-held micrometer (Hahn & Kolb, Stuttu-

gart, Germany) was used for measuring film thickness.

Imaging of Fibers and Composites

Scanning electron microscopy (SEM) was performed

to observe the morphology of the tensile fracture surface

of the composites using a JOEL NeoScope (JCM-5000)

scanning electron microscope (SEM) instrument under

high vacuum and using an accelerating voltage of 10 kV.

Prior to imaging, all samples were coated with a thin

layer of gold (up to 6 nm) using a NeoCoater device

(MP19020NCTR).

Infrared Analyses

A Shimadzu IR Prestige Fourier transform infrared

(FTIR) spectrophotometer with an attenuated total reflec-

tance (ATR) attachment was used to undertake the

2 POLYMER COMPOSITES—2016 DOI 10.1002/pc

65

infrared spectral analyses. All spectra were recorded in

the range of 550–4000 cm21 with a resolution of 4 cm21

and with 32 scans recorded at every point using Happ-

Genzel apodization. A small portion of thymol, untreated

and treated fibers was mixed in an agate mortar and pes-

tle with a few drops of paraffin oil. The sample was then

applied to a KBr disc and its FTIR spectrum recorded.

Ten scans were performed for each acquisition.

For FTIR mapping analysis, the spectra were acquired

using a Bruker Hyperion 2000 IR microscope (Bruker

Optik Gmbh, Ettlingen, Germany) equipped with a liquid

nitrogen cooled detector. The apparatus was connected to

the infrared microspectroscopy beamline at the Australian

Synchrotron (Clayton, Australia). The microscope was

coupled to a Bruker Vertex 70 spectrometer (Bruker

Optik Gmbh, Ettlingen, Germany). The line scan and

spectral mapping were performed on the surface of PLA

film containing 10% w/w thymol and were collected in

transmission mode by scanning the film using a

computer-controlled microscope stage at an aperture of

10 mm 3 10 mm with a 253 IR objective. The spectral

data were collected in the IR range 4,000–800 cm21 at a

resolution of 4 cm21 and with 64 scans. All spectra were

processed using Opus 6.0 software (Bruker Optics, Gmbh,

Ettlingen, Germany). A two-dimensional (2D) IR map

was produced using the software where integrated areas

were calculated for bands attributable to the PLA car-

bonyl group and thymol ring aromatic functional group.

Tensile Testing

Tensile tests were performed using a Model 4301 Ins-

tron Universal Testing Machine with a load cell of 1 kN.

Six tensile specimens were prepared from each sample

set via heat press molding using a dog-bone die. The tests

were conducted at a cross-head speed of 5 mm/min on

tensile specimens with dimensions of 63 mm 3 10 mm

3 2 mm in accordance with the ASTM D638 [23] Type

V method until tensile failure was detected. The average

tensile strength, tensile modulus and percentage extension

at break were measured and calculated using Instron

BlueHill Series IX software.

Thermal Property Testing

A differential scanning calorimetry (DSC) instrument

(Mettler Toledo) was used to study the thermal properties

of the PLA and composites under an inert gas (nitrogen)

atmosphere. Samples of about 8–9 mg each were weighed

and sealed in aluminum crucibles. The heating was per-

formed over the range 308C–3008C at a rate of 108C/min

and with a nitrogen flow rate of 20 mL/min. The glass

transition temperature (Tg), cold crystallization tempera-

ture (Tcc), melting temperature (Tm), cold crystallization

enthalpy (DHcc), and melting enthalpy (DHm) were

obtained from the DSC thermograms using the first heat-

ing scan. The maximum percentage crystallinity (%Xc) of

each sample during the DSC heating was determined by

calculating the ratio between the melting enthalpy of the

sample and that of 100% crystalline PLA [24] as shown

in Eq. 1:

%Xc5DHm=DHm03 100=w (1)

where w is the weight fraction of PLA in the formulations

and DHm0 is the enthalpy of melting for 100% crystalline

PLA (93.7 J/g) [25, 26]. The samples were also character-

ized using a thermogravimetric analyzer (TGA) (Mettler

Toledo TGA/DSC1) in order to determine their thermal sta-

bility. The mass-loss curves of the active composites were

recorded when heated from 308C to 5008C at a heating rate

of 58C/min and at a nitrogen flow rate of 0.2 L/min.

Disintegration Studies

A qualitative/visual study of the decomposition under

composting conditions was performed on composite sam-

ples cut into pieces (20 3 20 3 3 mm). Samples were

buried in a commercial compost at 5-cm depth in perfo-

rated boxes and incubated at 588C. Aerobic conditions

were maintained by mixing the compost periodically and

by the addition of water to maintain a moisture content

equivalent to 60% relative humidity. Samples were

removed from the compost after 7, 14, 21, 28, 35, and 48

days, were immediately washed with distilled water to

remove traces of compost, and then photographed.

RESULTS AND DISCUSSION

Structural Analysis

To confirm the effect of alkali treatment on the kenaf

fibers, surface FTIR spectra of UK and TK fibers were

obtained and are shown in Fig. 1. In the spectrum of the

UK fibers, a sharp absorption peak at approximately

1,740 cm21 is observed which corresponds to the carbonyl

group (>C@O) stretching. The existence of this peak in

the UK fibers has been ascribed to the acetyl and ester

group in hemicellulose or carboxylic acid group in the

ferulic and p-coumeric components of lignin [12]. This

particular peak is not apparent in the case of the TK fibers

which indicate the elimination of non-lignocellulose com-

ponents due to the alkali treatment [12, 27]. A similar find-

ing was also reported by Cao et al. [27] who treated kenaf

fibers with 5%–15% v/w NaOH solution at 258C for 2 h.

The FTIR structural analysis of thymol and PLA con-

taining 10% w/w thymol in this study have been dis-

cussed in detail previously [14]. However, in that study

the presence and distribution of thymol at the surface of

the PLA containing 10% w/w thymol film was not inves-

tigated and was therefore further analyzed using an IR

mapping technique in the present study. Figure 2 shows

the intensity of the IR band mapped over PLA film con-

taining 10% w/w thymol at 1,618 and 1,700 cm21 that

DOI 10.1002/pc POLYMER COMPOSITES—2016 3

66

represent the ring aromatic group of thymol and the car-

bonyl group of PLA, respectively [14]. Figure 2a shows

that a small amount of thymol is present on the film sur-

face with a higher concentration of thymol detected at the

left bottom edge of the area tested. This finding is also

consistent with the intensity distribution of the PLA car-

bonyl group (see Fig. 2b) and suggests that this technique

can be used to observe the distribution of thymol on the

PLA matrix surface and within a relatively small film

area. Moreover, the localization of thymol molecules

within the matrix or the fibers and at the fiber–matrix

interface is an important parameter in further understand-

ing the behavior of thymol-based biocomposites but is

beyond the scope of the present study.

Processing of PLA and Composites

The processing torque profiles of PLA and PLA filled

with 30% w/w UK fibers in the presence of thymol are

shown in Fig. 3. Generally, the addition of 5%–10% w/w

thymol to PLA and PLA/kenaf composites imparts a

lubricating effect during processing resulting in a signifi-

cant reduction in the normalized torque value. This lubri-

cating effect could also be due to the diffusion of thymol

molecules to the walls of the internal mixer. Similar

trends were observed for active PLA filled with TK fibers

containing thymol with no significant differences

observed in comparison with the PLA/UK fiber compo-

sites [20].

The peak in the melt torque observed at about 2 min

for PLA composite containing UK fibers (see Fig. 3) cor-

responds to the time when the fibers are introduced into

the mixer but this peak is absent when thymol is incorpo-

rated into the PLA formulation. For the PLA containing

thymol, the torque decreased momentarily and achieved

an equilibrium torque value almost immediately where it

was maintained throughout the mixing process. These

results clearly suggest that the thymol acts as a lubricant

for the system. Furthermore, this finding is also supported

FIG. 2. Intensity of the IR band mapped over PLA sample containing 10% w/w thymol at: (a) 1,622 cm21

(aromatic ring of thymol) and (b) 1,700 cm21 (carbonyl group of PLA). [Color figure can be viewed in the

online issue, which is available at wileyonlinelibrary.com.]

FIG. 3. Normalized torque as a function of time for melts of: (i) PLA/

UK fiber composite, (ii) PLA/UK fiber composite with 10% w/w thy-

mol, (iii) neat PLA, and (iv) PLA with 10% w/w thymol.FIG. 1. Infrared spectra of untreated and treated kenaf fibers in the

region 1,900–1,500 cm21.


67

by the significantly lower equilibrium torque observed

after about 8 min in the formulation containing thymol

compared with the formulation without thymol. Similar

findings have been reported by Sungsanit [28] who stud-

ied the rheology of plasticized PLA blends that contain

oligomer polyethylene glycol (PEG) at 5%–20% w/w

content. It was found that higher loadings of PEG in the

PLA blends produced a lower melt viscosity compared

with neat PLA.

It is interesting that the presence of thymol, which is a

relatively small molecule compared with typical PEG

oligomers, has a noticeable effect on the viscosity of the

melt. The lower PLA melt viscosity observed in the pres-

ence of 10% w/w thymol may be due to the thymol

weakening the intermolecular forces between adjacent

polymer chains, spacing them further apart and creating a

possible free volume [28]. The hydroxyl group in the thy-

mol is also expected to develop a hydrogen bond with the

polymer and, thus interfere with the polymer–polymer

interactions thereby acting in a similar fashion to that of

an oligomeric plasticizer [29].

Composite Morphology

The aim of chemical treatment of the fibers is to

remove impurities, pectin, waxy substances, lignin, and

hemicellulose in order to create a rough fiber surface and

increase hydroxyl group accessibility for a better interac-

tion with the matrix [30]. Hydrogen bonding is also likely

to occur in the composites between the hydroxyl groups

in the UK and TK fibers, the terminal hydroxyl groups of

PLA, and the carbonyl groups of the ester linkages of

PLA [31]. Figure 4a and b show SEM micrographs of the

tensile fracture surfaces of PLA composites containing

TK fibers with and without thymol. For both of these

composites, it appears that the fibers are well dispersed

throughout the polymer matrix. For the PLA composite

containing no thymol (Fig. 4a), the TK fibers appear to

be more tightly bound to the matrix compared with the

composite in which thymol is present (Fig. 4b). This

observation is in agreement with the work of Yousif et al.

[32] who treated kenaf fibers with 6% w/v NaOH. They

observed a slight improvement in the interfacial adhesion

and the porosity of the epoxy/kenaf composites that pre-

vented the de-bonding, detachment or “pull-out” of fibers.

The presence of thymol in the matrix appears to facilitate

the slippage of the fibers out of the matrix during fracture

and this is seen in Fig. 4b with the presence of more pro-

truding fibers at the matrix surface. Moreover, the thymol

that is present on the surface of the kenaf fibers will

lower the coefficient of friction and further facilitate the

slippage of the fibers within the matrix. The smooth

surfaces of some of the fibers, as well as the voids, sug-

gests that there is weaker adhesion between the fibers and

the PLA matrix when thymol is present in the formulation

compared with systems where thymol is absent.

Mechanical Properties

Figure 5 shows a photograph of the prepared tensile

specimens containing 30% w/w TK fibers and typical

stress-strain curves of the PLA and PLA composites con-

taining 5% w/w thymol. In all cases, the curves are con-

sistent with a brittle failure mode that is common for

fiber-reinforced PLA materials [33]. Figure 6 shows the

effect of the presence of UK or TK fibers on the tensile

properties of PLA composites containing zero, 5% or

10% w/w thymol. The tensile strength of PLA filled with

TK fibers and containing no thymol was slightly higher

than that of the UK composites as well as that of the neat

PLA (see Fig. 6a). The TK fibers act as reinforcement

and impart an approximate 8% increase in tensile strength

whereas no significant changes in the tensile strength of

the neat PLA were imparted in the case of the composite

containing the UK fibers. The slight improvement in the

tensile strength is possibly due to better interfacial adhe-

sion between the matrix and the TK fibers as well as

FIG. 4. Scanning electron micrographs of: (a) PLA/TK fiber composite and (b) PLA/TK fiber composite

with 10% w/w thymol at 2003 magnification.


68

better mixing or compatibility within the composite sys-

tem [13, 34].

The addition of 10% w/w thymol into the neat PLA

and the PLA/kenaf composites containing UK and TK

caused a significant reduction in the tensile strength com-

pared with those composites without thymol. The percent-

age reduction in the tensile strength was found to be

11%, 24%, and 21% for neat PLA, PLA composites con-

taining UK, and those containing TK fibers, respectively.

The localized plasticizing effect between the PLA and the

thymol whereby the thymol molecules diffuse into the

bulk of the matrix between the PLA chains is, in some

respects, akin to the case of PLA containing low molecu-

lar weight PEG plasticizer [35]. This suggests that the

thymol additive interferes with the interaction between

the polymer matrix and the fiber in the presence of the

applied stress due to the aforementioned slippage effect

[36]. This finding is consistent with the observations

made in assessing the SEM micrograph images in Fig. 4.

In addition, the latter composites demonstrated lower ten-

sile strength than the neat PLA containing only 10% w/w

thymol. Thus, thymol may increase end slipping of fibers

from the polymer matrix and subsequently reduce the ten-

sile strength. Similarly, Taib et al. [37] reported a reduc-

tion of up to 15% in the tensile strength of PLA/30% w/

w kenaf composites containing 10% w/w PEG. Interest-

ingly, the PLA/kenaf composite containing TK fibers and

10% w/w thymol exhibited a higher tensile strength (51

MPa) than the composite containing UK fibers and 10%

w/w thymol (44 MPa). This suggests that the reinforce-

ment offered by the TK fibers may prevail over the fiber

slippage effect of thymol in PLA at this level of additive

in the system. In addition, changes in the tensile strength

that may arise due to the effect of the thymol may not be

significant at a low level of thymol (5% w/w).

The effect of UK and TK fibers on the tensile modulus

and elongation at break of the composites containing

zero, 5% or 10% w/w thymol is shown in Fig. 6b and c,

respectively. As expected, the tensile modulus (or

“stiffness”) of the composites containing UK or TK fibers

is significantly greater than that of the neat PLA by about

80% (see Fig. 3b). This may be due to the inherently

high stiffness of the fiber [4]. This finding is also in

FIG. 5. Photograph of tensile specimens (a), and typical stress–strain

curves (b) of composites containing 5% w/w thymol: (i) PLA, (ii) PLA

with 30% w/w TK fibers, and (iii) PLA with 30% w/w UK fibers.

[Color figure can be viewed in the online issue, which is available at

wileyonlinelibrary.com.]

FIG. 6. Tensile properties of PLA composite with UK or TK fibers

containing zero, 5% or 10% w/w thymol: (a) tensile strength, (b) tensile

modulus, and (c) percentage elongation at break.


69

agreement with the work of Sujaritjun et al. [38] who

incorporated PLA with 30% w/w bamboo fibers and

found an increase in the tensile modulus of about 17%. In

this case, the tensile modulus of PLA filled with TK

fibers was slightly lower than the composite containing

UK fibers. A similar finding was observed by Xia et al.

[13] who investigated the tensile modulus of PLA compo-

sites containing untreated and alkaline-treated jute fiber

reinforcements. Cao et al. [27] reported that treating kenaf

fibers with 5% NaOH solution reduces the size of fiber

diameter and increases the tensile strength of the fiber

with no significant changes in the tensile modulus when

compared with untreated kenaf fiber. Nevertheless, it is

important to note that the removal of surface components

from the fibers during the alkaline treatment cannot be

solely responsible for the observed decrease in the stiff-

ness of the composite systems. This is because the stiff-

ness of composites containing natural fibers is mainly

related to the cellulose microfibrils that are the major

structural components within the microstructure. The

reduction in tensile modulus also occurs upon the addition

of 10% w/w thymol to the systems containing kenaf with

no changes in the tensile modulus observed upon the

addition of 10% w/w thymol to neat PLA. This finding is

in sharp contrast with the findings of Ramos et al. [16]

who reported a 15% reduction in the tensile modulus of

PLA films impregnated with 8% w/w thymol but the rea-

sons for the difference in findings are unclear. Nonethe-

less, the decrease in the stiffness of the composites

containing UK and TK fibers are more pronounced due to

the effect of the thymol as evidenced in Fig. 4b.

Figure 6c shows the effect of UK and TK fibers on the

elongation at break of composites containing zero, 5% or

10% w/w thymol. In general, the elongation at break of

these composites decreases with the addition of the

fibrous filler. This suggests that the kenaf does not con-

tribute to the elasticity or the final composite flexibility

and this observation is likely to be related to the high

stiffness of the composites as indicated by the results in

Fig. 6b. In contrast, the elongations at break of PLA/flax

composites were found to be 100% higher than neat PLA

with no significant changes having been found between

the untreated and treated PLA/flax fibers composites [13].

No significant changes in the elongation at break of any

of the composites containing UK and TK fiber were

observed upon the addition of 5%–10% w/w thymol. Liu

et al. [39] incorporated 5% w/w loadings of both Nisaplin

and EDTA into plasticized PLA/glycerol triacetate films

and observed a significant reduction of the elongation at

break from 108.5 to 62.5% compared with the plasticized

PLA. However, a slight increase in the elongation at

break was found in extruded PP containing 8% w/w thy-

mol resulting in an increase in ductile properties [36].

The reason for this difference in behavior of the two sys-

tems is unclear, nonetheless it is possible that a higher

loading of 10% w/w thymol in PLA may produce an

enhancement of the elastic properties of the system but

this has not been verified in the present study.

In the case of PLA/kenaf composites containing thy-

mol, it appears that the presence of thymol does not con-

tribute to or enhance the flexibility of the composites as a

whole. This finding is supported by the work of Taib

et al. [37] who prepared PLA plasticized with PEG and

found a higher strain at break (42%) compared with neat

PLA (4%) whereas the addition of 30% w/w kenaf in the

plasticized PLA reduced the stain at break significantly to

1%. The addition of UK and TK significantly reduces the

flexibility and has been attributed to the stiffening effect

of kenaf. The addition of kenaf to the system restricts the

mobility of PLA chains and increases the number of the

stress-concentrated areas at the fiber ends that can ulti-

mately contribute to the mechanical failure of the com-

posite [37]. The further inclusion of a small molecular

species such as thymol to the PLA/kenaf composite sys-

tem undoubtedly increases the complexity of the system

and further complicates any explanation of the experi-

mental observations. Nonetheless, it can be suggested that

the type of active PLA/kenaf composites under investiga-

tion in the present study is more suitable for use as a

rigid packaging material than a flexible packaging mate-

rial due to the high stiffness of this composite [40].

Whence, these materials are potentially suitable for ther-

mal processing into food trays or containers with moder-

ate strength requirements for the packaging of ready-to-

eat food products such as fruits and processed deli meats.

It is important to note that the mechanical properties

of these composite systems also depend on the fiber size

distribution and/or aspect ratio of the fibers. High shear

stresses developed during the compounding with an

extruder or mixer may lead to fiber damage or breakage

resulting in a smaller fiber aspect ratio [9]. Moreover,

alkaline treatment reduces the fiber diameter and thereby

increases the fiber aspect ratio [41]. The effect of fiber

size distribution of the alkali-treated fibers on the

mechanical properties of composite containing AM addi-

tives should be considered in the future in order to fully

understand the complexities of stress transfer in these ter-

nary composite systems.

Thermal Properties

Table 1 shows a summary of the key data obtained

from the analysis of the DSC thermograms of PLA and

PLA composites containing UK or TK fibers with zero,

5% or 10% w/w thymol. Neat PLA exhibited an onset Tg

at 59.58C, a Tcc at 92.28C, and Tm values at 142.3

(shoulder), and 149.68C (peak), the latter of which is

higher than the value of 141.78C reported by Byun et al.

[42]. The onset of Tg, Tcc, and Tm of the PLA composites

containing UK or TK fibers demonstrated no significant

differences however, some of these values such as Tg,

Tcc, Tm, DHcc, and DHm for both of the composites were

slightly lower than that of neat PLA. The maximum


70

values of %Xc calculated using Eq. 1 for the composites

containing UK or TK fibers was slightly higher than that

of neat PLA and this finding is in agreement with the

work of Du et al. [24] who investigated PLA containing

30% w/w pulp fibers from hardwood, softwood and

bleached Kraft wood.

Figure 7 presents the DCS thermograms of the PLA

and PLA composite systems and, as shown in Fig. 7a, the

neat PLA exhibits a double melting peak with a dominant

peak at higher temperature (Tm2). With the addition of

kenaf fibers, the double melting peaks become more dis-

tinct. The appearance of these dual melting peaks is in

accordance with other reports where the addition of fibers

resulted in the development of a small melting peak that

shifted to a lower temperature, Tm1, upon further addition

of fibers [19, 20, 24]. The minor peak may be due to

either a different PLA crystal type formed only in pres-

ence of the fibers and/or the melting of the transcrystal-

line zone as reported by Yussuf et al. [43].

For the PLA and PLA composites containing 5%–10%

w/w thymol, the addition of the additive resulted in a

decrease in Tg (see Table 1 and Fig. 7b) and this might

be attributed to the resultant increase in the free volume

of polymer matrix as well as segmental mobility of the

PLA chains that change the thermal properties in a way

that is akin to a plasticizing effect [20, 44]. In particular,

the incorporation of thymol in the PLA decreases the Tg

value which may be a similar effect as that previously

observed in the case of PLA containing low and high

molecular weight PEG at a 10% w/w loading [35, 37].

Furthermore, Hal�asz and Cs�oka [10] reported a similar

finding where the addition of 10% w/w PEG decreased

the Tg of neat PLA and PLA composite containing 5% w/

w microcrystalline cellulose (MCC). The observed plasti-

cizing effect reflected in the Tg values indicates that the

thymol is miscible with the PLA and this is consistent

with the observations made from the morphology images,

tensile strength, and tensile modulus of neat PLA and

PLA kenaf composites (UK and TK) containing thymol

(see Figs. 4b and 5). It can be observed that, as the con-

tent of thymol is increased to 10% w/w, the Tcc peak

becomes broader and shifts to lower temperatures, which

suggests that the additive enhances the ability of PLA to

undergo cold crystallization [45].

Two melting peaks were also observed for PLA con-

taining thymol. The additional melting peak exhibited by

the active PLA formulation is more pronounced than that

of the neat PLA (see Fig. 4) and this may also be related

to the reorganization of the crystal structure. These obser-

vations contrast with other findings in the literature where

no additional melting peak arose upon the addition of

plasticizer such as low molecular weight PEG200 in PLA

blends [35]. In general, slight decreases were observed

for the Tg, Tcc, and Tm of PLA/kenaf composites contain-

ing 10% w/w thymol irrespective of the fiber pre-

treatment.

Thermogravimetric Analysis

It is important to investigate the decomposition and

degradation of composites at higher temperature espe-

cially if these materials are intended for use in food pack-

aging applications. Low levels of degradation products

TABLE 1. Thermal analysis parameters obtained from DSC thermograms of neat PLA, PLA/kenaf, and PLA/kenaf/thymol composites

Formulation % w/w thymol Tg (8C) Tcc (8C) Tm1 (8C) Tm2 (8C) DHcc (J/g) DHm (J/g) Xc (%)

Neat PLA

0 59.5 93.3 142.3a 149.6 18.8 26.1 28.0

5 47.9 86.9 131.9 143.0 20.0 23.4 26.7

10 41.6 85.6 124.5 137.4 22.1 22.7 27.1

PLA/UK fibers

0 59.6 93.5 138.1 146.2 19.7 19.4 29.8

5 46.6 82.9 123.3 134.6 16.0 15.9 26.3

10 30.1 75.7 115.5 129.3 15.9 15.7 28.1

PLA/TK fibers

0 58.8 94.9 137.8 145.5 19.0 19.1 29.2

5 46.9 85.9 125.5 136.18 16.3 16.6 27.4

10 31.3 78.5 116.7 129.8 15.8 15.8 28.3

aShoulder on the main PLA peak.

FIG. 7. DSC thermograms of (a) neat PLA and PLA composites with-

out thymol: (i) neat PLA, (ii) PLA/UK fiber, (iii) PLA/TK fiber and (b)

neat PLA and PLA composites with 10% w/w thymol: (i) neat PLA, (ii)

PLA/UK fiber, (iii) PLA/TK fiber.


71

produced during the thermal processing of such polymers

may taint and/or contaminate foodstuffs upon contact

thereby causing concerns regarding organoleptic proper-

ties or indeed safety. Furthermore, the thermal stability of

these materials is also of relevance to the ultimate dis-

posal in composting or land fill where degradation usually

takes place at elevated temperatures.

The thermogravimetric (TG) profiles of neat PLA and

PLA composites containing UK and TK fibers with zero

or 10% w/w thymol are shown in Fig. 8 in the form of the

normalized weight loss as a function of temperature. The

corresponding derivative weight loss curves are also shown

in Fig. 8 to enable a more detailed analysis of the TG data

to be made. The thermal degradation of neat PLA takes

place in a single step with a maximum rate of weight loss

in the range of 2808C–3558C [16]. In general, the maxi-

mum degradation temperature of the neat PLA was higher

than any of the PLA composites (see Fig. 8a) and this is

more clearly reflected in the derivative TG analysis curves.

As expected, the presence of fibers in the PLA destabilized

the PLA matrix in the composite as has been previously

reported by Yussuf et al. [43]. The temperature at which

the maximum rate of degradation occurs for the composite

containing TK fibers was slightly higher than that of the

composite containing UK fibers with degradations temper-

atures of 3318C and 3288C, respectively. This may be due

to the better interfacial adhesion between the TK fibers

and the PLA matrix [46].

The TG profile of PLA containing a nominal 10% w/

w thymol in the formulation exhibits two main steps dur-

ing the analysis (see Fig. 8b). The first step occurs gradu-

ally over the temperature range of about 308C–3008C and

is attributable to the release of thymol from the polymer

matrix. This step corresponds to about 7% loss in total

mass and suggests that the neat PLA and PLA composites

that were formulated with 10% w/w thymol retain about

7% w/w of the thymol that was originally added to the

formulation. Thymol release during melt processing due

to the high friction between the barrel and the screw, is

primarily responsible for the loss of thymol from the for-

mulation [47]. This observation is in agreement with the

work by Ramos et al. [16] who investigated by TG analy-

sis the retention of thymol in a PLA matrix after process-

ing. The temperature corresponding to the maximum rate

of release of thymol was slightly higher for the compo-

sites containing TK fibers (1488C) compared with those

containing UK fibers (1418C) suggesting that the thymol

interacts more strongly with the TK fibers. The second

step observed in the TG profiles of the PLA and PLA

composites containing thymol occurs over the temperature

range of about 3108C–3508C and is attributable to the

complete degradation of the polymer matrix.

FIG. 8. TGA profiles of (a) neat PLA and PLA composites without thymol: (i) neat PLA, (ii) PLA/UK

fiber, (iii) PLA/TK fiber and (b) neat PLA and PLA composites with 10% w/w thymol: (i) neat PLA, (ii)

PLA/UK fiber, (iii) PLA/TK fiber.


72

The temperature corresponding to the maximum rate

of degradation for the composite containing TK fibers

after the release of 10% w/w thymol was slightly higher

than that of the composite containing UK fibers. This

may be due to the increased thermal stability of the

almost complete cellulosic material that comprises the TK

fibers and the absence of other components such as waxy

substances, hemicellulose, and lignin that may otherwise

destabilize the system [34, 48]. Overall, the addition of

thymol to the formulation seems to have little effect on

the thermal stability of the PLA whereas the addition of

fiber decreases the thermal stability.

Decomposition in Compost

Although many studies have focused on the biodegrada-

tion and/or composting of PLA and PLA incorporated bio-

filler composites, few have investigated the composting of

PLA-based materials containing AM agents. Moreover,

there is still a question of whether the AM property of the

material would compromise or affect the ultimate degrada-

tion by the microorganisms in soil [49]. In the present

study, a preliminary and qualitative analysis of the disinte-

gration under controlled compost-like conditions of PLA

and PLA/kenaf films containing the AM agent thymol was

conducted as a prelude to future controlled composting

studies of these materials. Figure 9 shows images of PLA

and PLA composites containing 30% w/w TK fibers with

and without 10% w/w thymol that were removed from

controlled composting conditions at different times. Over-

all, a considerable change in color of all samples was

observed with a change from clear to opaque for the neat

PLA and PLA containing 10% w/w thymol and changes

also observed for the composites. A similar observation

was reported by Ramos et al. [50] who investigated the

disintegration of PLA and PLA nano-composites contain-

ing thymol and silver nanoparticles under controlled com-

posting conditions. These color changes are primarily due

to the hydrolytic degradation and crystallization of the

PLA matrix [51]. Moisture absorption may lead to hydro-

lysis that breaks down the macromolecular chains of the

polymer with a consequent erosion of the sample surface

as demonstrated by the whitening of the surfaces followed

by microbial assimilation [52].

One may expect that the incorporation of an AM agent

into the PLA-based films will delay the degradation pro-

cess. However, the neat PLA and PLA containing 10%

w/w thymol commenced degradation within 14 days as

evidenced by sample fragmentation although no signifi-

cant differences were observed between the PLA formula-

tions with and without thymol. This may be due to the

inherent volatility of thymol, an essential oil extract, that

one expects will be released into the atmosphere under

the thermal conditions in the compost. For composite

samples containing kenaf, degradation was not apparent

until 28 days under the test conditions and in these cases,

the PLA surrounding the fiber was observed to have sub-

sequently degraded, dislodging the fibers and thereby

roughening the surface. The differences in the degradation

rate of these two systems may be attributed to the resist-

ance in water uptake and the diffusion through the com-

posites with the PLA readily adsorbing water [53, 54].

The relatively high degradation rate of the PLA under the

aerobic composting conditions is to be expected under the

high humidity and temperature (588C) conditions applied

[54]. A more comprehensive biodegradation test such as

that outlined in ASTM D5538 [55] is recommended to

fully analyze the compostability of the materials devel-

oped in this study. Compost monitoring such as pH,

evolved CO2, and the molar mass of PLA as well as

microstructural analysis of the composites by SEM imag-

ing and DSC during biodegradation is required for further

investigation in order to illustrate the effect of thymol on

the biodegradation processes.

CONCLUSIONS

Biobased PLA composites containing TK or UK kenaf

fibers and thymol were prepared and characterized. The

FIG. 9. Images of PLA and PLA containing TK fiber and/or thymol at

different stages of decomposition in compost. Sample sizes at day 0 are

about 20 3 20 mm. [Color figure can be viewed in the online issue,

which is available at wileyonlinelibrary.com.]


73

incorporation of thymol into the PLA/kenaf composites

imparted a lubricating effect whereby the equilibrium tor-

que value was decreased during processing. A relatively

weak adhesion between the PLA matrix and the kenaf

fibers was confirmed microscopically in composites con-

taining thymol. The tensile strength of PLA composites

containing TK fibers was slightly higher than that of

composites containing UK fibers suggesting the alkaline

treatment imparts a reinforcing effect within the polymer

matrix. The incorporation of the higher level of 10% w/w

thymol into the PLA/kenaf composites decreased the ten-

sile strength and stiffness irrespective of the fiber pre-

treatment with no significant changes in the elasticity.

Thermal analysis by DSC showed a general decrease in

Tg, Tcc, and Tm in PLA and PLA composites containing

10% w/w thymol compared with the formulations without

thymol suggesting changes in the phase structure of the

polymer. The TG decomposition temperature of the PLA

composite containing TK fibers and 10% w/w thymol

was slightly increased indicating an increase in the ther-

mal stability of the PLA matrix. Under qualitative con-

trolled composting conditions, the disintegration of the

PLA and PLA containing TK fibers progressed rapidly

resulting in a complete loss within 48 days. In general,

the presence of TK fibers in combination with thymol in

the PLA composite resulted in significant improvement in

the overall properties of the material suggesting that this

composite has the potential to be used as a rigid, active

biobased packaging material.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the Ministry of Edu-

cation Malaysia and Universiti Putra Malaysia (UPM) for

providing the PhD scholarship for Intan S. M. A. Tawakkal

and would like to acknowledge the technical staff from

RMIT University especially Mr. Mike Allan for the prepara-

tion of the composite samples. The infrared mapping was

undertaken on the infrared microspectroscopy beamline at

the Australian Synchrotron, Victoria, Australia. This

research received no specific grant from any funding agency

in the public, commercial, or not-for-profit sectors.

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75

Novel food packaging from biodegradable,biocomposites with antimicrobial properties

A new biodegradable composite system comprised of a biopolymer,

natural fibers, and an essential oil extract shows promise for

antimicrobial food packaging applications.

Authors: Intan S.M.A. Tawakkal, Marlene J. Cran, and Stephen W. Bigger

Single-use food contact packaging materials are

typically made from common plastics including

polyethylene and poly(ethylene terephthalate).

Although these materials are recyclable, vast

quantities end up in landfill where they resist

degradation for long periods of time. Bio-derived

polymers are gaining popularity as replacements

for these traditional packaging materials with

poly(lactic acid) (PLA) among the more promising

biopolymers in this space. However, PLA cannot

rival these materials in terms of costs so creative

solutions are needed to make PLA a more

financially viable alternative for packaging.

Fortunately, PLA is highly compatible with many

natural fibers and their composites can

substantially reduce the costs often without

detriment to the mechanical integrity of the

resulting products1. For example, filling PLA with

abundant, natural plant fibers such as kenaf

(Hibiscus cannabinus), is a common practice that

can both lower the production costs and impart

reinforcement to this naturally brittle polymer2.

In order to use kenaf fibers in PLA composites,

the use of an alkaline chemical treatment is

required to remove surface impurities and to

increase the compatibility of the fibers with the

PLA matrix.

In recent years, PLA has also become an

important polymer in the field of active food

packaging materials where systems are designed

specifically to interact with the food product and

the environment inside and outside the package.

Antimicrobial (AM) packaging is one such aspect

of active packaging where PLA is beginning to

excel. As the name suggests, AM packaging

utilizes agents such as thymol, an essential oil

extract (EEO) obtained from the thyme plant, to

impart an inhibitory effect against microbial

growth that can eventually result in food

spoilage. One relatively new and promising field

of PLA packaging is the use of both fillers and AM

agents to form ternary composites3, 4.

In ternary systems comprised of PLA, alkali-

treated kenaf (TK), and thymol, typical levels of

the fibrous filler of 30-40% w/w are commonly

used with up to 10% w/w thymol required to

impart adequate AM activity. As shown in Figure

1, the processability of PLA composites

containing 30% kenaf is improved following the

addition of thymol with and the treated fibers are

slightly easier to process than the untreated

kenaf (UTK) fibers. In these cases, the addition of

thymol imparts a welcome lubricating effect,

thus reducing the processing energy required to

form the composite4.

Figure 1. Effect of addition of thymol on

rheological properties of PLA/kenaf composites.

76

The resulting mechanical properties of the

ternary PLA composites are found to be

appropriate for their intended use as rigid,

compostable food containers or trays. Figure 2

shows that the tensile strength in particular is

improved with both increasing kenaf content and

when the fibers are alkali treated. A slight

decrease is observed when thymol is added and

this may be due to the plasticizing effect

suggested previously and a potential slippage of

the fibers within the PLA matrix3.

Figure 2. Effect of increasing kenaf content, fiber

treatment and the presence of thymol on

composite tensile strength.

Unlike conventional rigid food trays which can be

recycled or disposed to landfill, PLA/kenaf/

thymol composites are suitable for composting.

As shown in Figure 3, under typical compost

conditions, the ternary composite is observed to

completely disintegrate into the compost

medium within 48 days4. The volatile nature of

EEOs such as thymol can assist in the liberation of

these agents, particularly under the relatively

high temperatures encountered in compost

environments. When the agents are released,

the composting microorganisms can flourish,

decompose, and assimilate the composite.

Through our recent work we have identified the

potential for PLA composites to compete with

conventional rigid packaging and there is still

considerable scope for the further development

of such biocomposites for food packaging. This is

particularly the case in the areas of active and AM

packaging where extending the shelf-life of food

products is becoming a vital issue but where

environmental sustainability and resource

management is of equal importance.

Figure 3. Amount of PLA or PLA/kenaf/thymol

composite in compost environment as a function of

time.

References:

1 T. Mukherjee and N. Kao, PLA basedbiopolymer reinforced with natural fibre: Areview, J. Polym. Environ. 19 (3), pp. 714-725,2011. doi:10.1007/s10924-011-0320-6

2 M. U. Wahit, N. I. Akos and W. A. Laftah,Influence of natural fibers on the mechanicalproperties and biodegradation of poly(lactic acid)and poly(ε-caprolactone) composites: A review, Polym. Compos. 33 (7), pp. 1045-1053, 2012.doi:10.1002/pc.22249

3 I. S. M. A. Tawakkal, M. J. Cran and S. W.Bigger, Effect of kenaf fibre loading and thymolconcentration on the mechanical and thermalproperties of PLA/kenaf/thymol composites, Ind.Crops Prod. 61 pp. 74-83, 2014.doi:10.1016/j.indcrop.2014.06.032

4 I. S. M. A. Tawakkal, M. J. Cran and S. W.Bigger, The influence of chemically treatednatural fibers in poly(lactic acid) compositescontaining thymol, Polym. Compos. (early view),pp. 2016. doi:10.1002/pc.24062

77

78

Chapter 4 – Effect of Kenaf Fibre Loadings

Effect of Kenaf Fibre Loading and Thymol Concentration on the Mechanical and

Thermal Properties of PLA/Kenaf/Thymol Composites

Overview

In this chapter, the PLA systems containing treated kenaf were selected as the main formulation

with which to further investigate the effect of kenaf fibre loadings and thymol concentrations

on the mechanical, thermal and rheological properties of the AM materials. The possible

packaging applications such as rigid and/or flexible packaging as well as coatings of this

particular material can be suggested from these studies.

The paper entitled “Effect of Kenaf Fibre Loading and Thymol Concentration on the

Mechanical and Thermal Properties of PLA/Kenaf/Thymol Composites” by Tawakkal I. S. M.

A., Cran M. J. and Bigger S. W. was published in Industrial Crops and Products, 61, 74-83.

79

80

Industrial Crops and Products 61 (2014) 74–83

Contents lists available at ScienceDirect

Industrial Crops and Products

jo u r n al homep age: www.elsev ier .com/ locate / indcrop

Effect of kenaf fibre loading and thymol concentration on themechanical and thermal properties of PLA/kenaf/thymol composites

Intan S.M.A. Tawakkala, Marlene J. Cranb,∗, Stephen W. Biggera

a College of Engineering and Science, Victoria University, PO Box 14428, Melbourne 8001, Australiab Institute for Sustainability and Innovation, Victoria University, PO Box 14428, Melbourne 8001, Australia

a r t i c l e i n f o

Article history:Received 2 April 2014Received in revised form 4 June 2014Accepted 19 June 2014Available online 12 July 2014

Keywords:Polymer-matrix composites (PMCs)Mechanical propertiesRheological propertiesThermal propertiesPoly(lactic acid)

a b s t r a c t

Composites of poly(lactic acid) (PLA) containing up to 40% (w/w) of kenaf fibre and up to 10% (w/w) of thy-mol were studied to evaluate the mechanical and thermal properties. These composites were comparedwith control systems containing either no fibre or no thymol and were prepared using melt blendingand compression moulding techniques. The composites with 10% (w/w) thymol had the lowest tensilestrength with slightly higher flexibility compared with those systems containing lower concentrationsof this additive. The tensile properties of composites containing 5% (w/w) thymol demonstrated that theaddition of fibre to the PLA/kenaf composites was affected more than the incorporation of the thymolalone. Thermogravimetric analysis of neat PLA and PLA/kenaf composites incorporated with 5% and 10%(w/w) thymol revealed no significant changes in the decomposition temperature. Analysis by differentialscanning calorimetry, however, showed a decrease in all of the key thermal transitions with the additionof 5% and 10% (w/w) thymol into the neat PLA and PLA/kenaf composites. The results of the mechanicaland thermal properties tests suggest that thymol acts as a plasticizing agent in this composite.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

The development of active packaging (AP) materials based onbiopolymers is expected to grow in the next decade with majorfocuses on extending the shelf life of foods, enhancing food safetyand quality, providing viable alternatives to petroleum based poly-mers and reducing environmental impacts (Bonilla et al., 2013; Jinand Zhang, 2008). To date, many researchers have studied packag-ing materials that are either derived from natural resources or aremade from renewable resources in order to fulfil the requirementsset by some regulatory agencies and/or those of consumers whoprefer more environmentally friendly packaging that minimizespollution (Kuorwel et al., 2011a).

Biopolymers such as poly(lactic acid) (PLA) can be synthesizedfrom renewable, bioderived monomers obtained from a range ofmaterials including starch from corn (Auras et al., 2005). SincePLA is a relatively new biopolymer, considerable effort has beenplaced into making it as acceptable and effective as its commercialpetroleum-based counterparts (Auras et al., 2005). The potential ofPLA for use in AP packaging applications has been investigated by anumber of researchers (Ali and Noori, 2014; González and Igarzabal,

∗ Corresponding author. Tel.: +61 399197642.E-mail address: [email protected] (M.J. Cran).

2013; Jin, 2010; Mustapha et al., 2002; Rhim et al., 2009). Cur-rent AP technologies enable effective antimicrobial (AM) packagingmaterials to be prepared from PLA that have been incorporatedor blended with different compatible materials, additives and/orplasticizers.

Several classes of AM additives are used in packaging withthose derived from natural sources such as herbs and plant speciesof particular importance. Thyme, for example, contains volatileessential oils and extracts that are rich in terpenoids, particularlymonoterpenoid phenols such as thymol and carvacrol. Thymol(2-isopropyl-5-methylphenol) is known to exhibit antimicrobialactivity against several microorganisms such as bacteria, fungi,mould and yeast (Gutierrez et al., 2009) and it is classified in the“Generally Recognized as Safe (GRAS)” category by the US Food andDrug Administration (Persico et al., 2009). Thymol is ideally suitedfor food packaging applications requiring a migratory release thatcan provide continuous AM activity from the package to the food(González and Igarzabal, 2013; Kuorwel et al., 2011b; Ramos et al.,2012). Although there are few reports of PLA films and coatingscontaining AM agents, there are some commercial products avail-able in the marketplace such as the controlled release antifungalPLA/starch film ANTIPACKTM AF (Szafranska, 2013). Of the reportedstudies, some have investigated the microbial activity of such activefilms by the diffusion of AM agents in solid and liquid media as wellas in related real food products with little attention having been

http://dx.doi.org/10.1016/j.indcrop.2014.06.0320926-6690/© 2014 Elsevier B.V. All rights reserved.

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I.S.M.A. Tawakkal et al. / Industrial Crops and Products 61 (2014) 74–83 75

devoted to studying the physico-mechanical, thermal and otherproperties of the active material systems (Bastarrachea et al., 2011).

The combination of natural fibres into PLA is receiving attentionmainly for its potential to create materials that are more envi-ronmentally sustainable. The use of these natural fibres as fillersor reinforcements in PLA composites is expected to considerablylower the overall cost of the material due to the renewability andabundance of these raw materials (Abdul Khalil et al., 2012a; Akilet al., 2011; Faruk et al., 2012). Furthermore, the production ofbiopolymers incorporated with natural fibres offers several otheradvantages including improved compatibility and better mechan-ical properties, improved abrasion resistance as well as enhancedbiodegradability (Anuar et al., 2012; Du et al., 2014; Tawakkal et al.,2012). Several studies have reported the treatment of fibres as ameans to create better compatibility between the polymer matrixand the fibres although this has the potential to produce wastechemicals. Treatments include the use of alkaline agents (Hudaet al., 2008; Ibrahim et al., 2011) and silane agents (Le Moigne et al.,2014; Tran et al., 2014) which would be expected to alter the sur-face chemistry of the fibres and promote fibre/PLA compatibility.However, the environmental impact of the chemical treatment ofkenaf fibres has not been addressed but it could potentially be over-come by treating the chemical waste before it is released to theenvironment.

Examples of relatively inexpensive and widely available plantfibres that can be used as fillers include wood flour, jute, hemp,flax, kenaf, bamboo, oil palm empty fruit bunch, rice husk and ramie(Akil et al., 2011; Harmaen et al., 2013; Van Den Oever et al., 2010).Non-wood lignocellulosic fibre such as kenaf (Hibiscus cannabinusL.) with approximately 53.4% (w/w) cellulose content (Abdul Khalilet al., 2010) has created considerable interest as a reinforcementfiller in polymer composites (Ali et al., 2014; Deka et al., 2013; Holtet al., 2014). Many studies have been conducted in order to inves-tigate the effect of kenaf fibres when these are used as the filler forPLA and the results generally show that a positive reinforcing effectis achieved (Anuar et al., 2012; Bax and Müssig, 2008; Bledzki et al.,2009; Huda et al., 2008; Oksman et al., 2003; Plackett et al., 2003).However, few studies have reported the development of PLA com-posites with natural fibres for AP applications such as AM packagingmaterials for food products. In one recent study, Prapruddivongsand Sombatsompop (2012) found that the incorporation of naturalfibres such as wood flour into PLA affects the mechanical propertiesof the resulting films. They reported that the addition of 1.5% (w/w)triclosan AM agent produced only a slight effect on the mechanicalproperties of neat PLA. However, the inclusion of 10% (w/w) woodflour into the polymeric system was more pronounced than that ofthe triclosan inclusion. An increase in wood flour content from 0to 10% (w/w) enhanced the stiffness of the composites and at thesame time, reduced the flexibility, tensile strength and toughness.

The incorporation of natural fibres into bio-based plasticstogether with the addition of naturally-derived AM agents thathave minimal impact on the environment is likely to lead to the nextgeneration of packaging materials. The aim of the present study isto investigate the effect of kenaf fibre content and thymol concen-tration on the mechanical and thermal characteristics of PLA andPLA/kenaf composites.

2. Experimental

2.1. Materials

Poly(lactic acid) (7001D IngeoTM; specific gravity 1.24; meltflow index (MFI) 6 g/10 min at 210 ◦C and 2.16 kg; melting tem-perature range 145–160 ◦C) was purchased from NatureWorks LLC,USA. Kenaf fibre (bast) was purchased from Ecofibre Industries,

Australia. The aspect ratio (L/D) of the kenaf fibres was approxi-mately 9 with an average length of 920 �m and an average diameterof 104 �m. These dimensions were obtained from scanning elec-tron micrograph images. Thymol (T0501) with purity of 99.5%was purchased from Sigma–Aldrich Pty. Ltd., Australia. Sodiumhydroxide and acetic acid were purchased from Merck Chemicals,Australia.

2.2. Preparation of neat PLA and PLA composites

Kenaf fibres were soaked in a 5% (w/v) sodium hydroxide solu-tion for 2 h. The fibres were filtered and washed with distilled waterbefore being neutralized by acid treatment upon the addition of afew drops of acetic acid (Amel et al., 2013). The fibres were thenfiltered, washed and rinsed with distilled water to remove excessacid and neutrality was confirmed using a pH meter. Finally, thefibres were dried overnight in an air circulating oven at 105 ◦C. Priorto mixing, the PLA resin and fibres were dried in an oven at 60 ◦Covernight before blending with 5% and 10% (w/w) thymol.

The composites were prepared by melt-blended using an inter-nal mixer (Haake PolyLab OS, Germany) at a fixed screw speed of50 rpm. The processing temperature and time were set at 155 ◦Cand 8 min respectively and a total mass of 50 g was required to fillthe mixing chamber. The mixing torque curves together with thestock temperature (actual temperature of the blend) were simulta-neously monitored and recorded using Polysoft OS software duringthe mixing process. A laboratory press (L0003, IDM Instrument Pty.Ltd., Australia) was used to prepare films (thickness ca. 0.3 mm) andslabs (thickness ca. 3 mm) for mechanical testing. The materialswere preheated at 150 ◦C for 3 min and pressed at the same tem-perature for 5 min under a pressure of 50 kN before quench coolingto 30 ◦C under pressure.

2.3. Composite characterization

2.3.1. Composite morphologyThe morphology of the materials was investigated by scan-

ning electron microscopy (SEM). Images of the treated kenaf fibresand tensile fracture surface of neat PLA and PLA/kenaf compositesincorporated with thymol were obtained using a JOEL NeoScope(JCM-5000) scanning electron microscope. Samples were mountedon an aluminium sample holder and coated with up to 6 nm of goldusing a NeoCoater (MP19020NCTR) under high vacuum and usingan accelerating voltage of 10 kV.

2.3.2. Mechanical propertiesFor each set of composite samples, six tensile specimens were

prepared and tested using a Model 4301 Instron Universal TestingMachine with a load cell of 1 kN. Each tensile test was conductedusing a cross-head speed of 5 mm min−1 on dumbbell-shaped spec-imens with dimension of 10 × 63 × 3 mm in accordance with theASTM D638 Type V standard method until tensile failure wasdetected. The average tensile strength, Young’s modulus and per-centage extension at break were calculated from the stress–straincurves.

2.3.3. Thermal analysisThermal properties of the samples were analyzed using a Met-

tler Toledo differential scanning calorimetry (DSC) instrumentequipped with an intracooler system and under an inert nitro-gen gas atmosphere. The thermal properties of the materials weredetermined and analyzed in accordance with ASTM E1131 standardmethod using nitrogen gas. Samples of ca. 10–12 mg each wereweighed and sealed in aluminium crucibles and were then heatedfrom 30 to 300 ◦C at a rate of 10 ◦C min−1 with a nitrogen flow rate

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76 I.S.M.A. Tawakkal et al. / Industrial Crops and Products 61 (2014) 74–83

Fig. 1. Normalized torque as a function of time for melts of neat PLA and PLA con-taining 10% and 40% (w/w) kenaf.

of 20 mL min−1. The glass transition temperature (Tg), cold crystal-lization temperature (Tcc), melting temperature (Tm) and meltingenthalpy (�Hm) were obtained from the DSC thermograms andthe percentage of crystallinity (%Xc) of each sample was calculatedusing equation (1):

%Xc = �Hm

�H◦m× 100 (1)

where �H◦m is the melting enthalpy of 100% crystalline PLA witha value of 93.1 J g−1 (Henton et al., 2005). The percentage of crys-tallinity of neat PLA and PLA/kenaf composites was calculatedproportional to the PLA weight percentage in the composites.

A Perkin Elmer TGA 7 was used to obtain the decompositiontemperatures of the materials and to determine the approximateweight percentage of thymol that was retained in the samples afterprocessing. The PLA, fibres and composites were heated from 30 to500 ◦C at a heating rate of 5 ◦C min−1 and under a nitrogen flow rateof 0.2 L min−1.

3. Results and discussion

3.1. Characterization of the mixing process

The torque value of the formulations during mixing was moni-tored in order to ascertain that homogeneous mixing had occurredafter a sufficient processing time. Upon achieving a consistenttorque in the stabilization zone it was deemed that the filler hadbeen well mixed and evenly distributed throughout the matrix(Balakrishna et al., 2013; Premalal et al., 2002). Fig. 1 shows thenormalized mixing torque curves of neat PLA and PLA/kenaf com-posites at zero, 10% and 40% (w/w) kenaf loadings. For neat PLA(Fig. 1 (a)), the initial torque peak or maximum torque (�1) wasdetected at the beginning of processing after which a rapid decreasein the torque occurred before a constant value was attained in thestabilization zone. The �1 peak corresponds to the highest mechan-ical shear forces needed to cause the cold PLA resin to flow. As thePLA becomes completely molten, the torque rapidly decreases. Foreach of the PLA/kenaf composite formulations, the �1 peak was alsoobserved during processing and the intensity of this peak decreasedas the fibre content increased due to the concomitant decrease inthe amount of PLA in the formulation. After the initial mixing, asecondary torque peak (�2) was recorded in each melt after approx-imately 2 min of mixing and this peak is due to the presence of thefibres in the melt.

Fig. 2 shows the normalized �2 and equilibrium torque (�eq)of PLA/kenaf formulations at various kenaf contents ranging from

Fig. 2. Normalized secondary torque, �2 (�) and equilibrium torque, �eq (�) formelts of neat PLA and PLA/kenaf composites.

zero to 40% (w/w). The results show that as the kenaf content isincreased, the �2 and �eq values increase almost linearly wherethe PLA/kenaf composite containing 40% (w/w) kenaf exhibits thehighest secondary and equilibrium torque values of any of the sys-tems that were tested. The increase in �2 with increasing kenafcontent is attributed to the increase in mechanical shear that isrequired to mix larger amounts of kenaf into smaller amounts ofmolten polymer. This occurs towards the end of the processingperiod where the equilibrium torque is observed, the latter of whichis also dependent on the kenaf content.

Fig. 3 shows the normalized torque curve for neat PLA andthose curves for neat PLA to which 5% or 10% (w/w) thymol wasadded after approximately 2 min in the mixer. The addition of thy-mol causes the torque to decrease momentarily but eventually theequilibrium torque is achieved as soon as the thymol is mixedthroughout the melt. The observed decrease in the torque uponaddition of thymol suggests that the additive acts as a lubricant orplasticizer. The behaviour of thymol as a plasticizer in the systemis confirmed by the final normalized torque readings in the stabi-lization zone (see inset in Fig. 3). These normalized �eq values werefound to be 0.16 and 0.10 for the systems containing 5% and 10%(w/w) thymol respectively compared to the neat PLA that produceda normalized �eq of 0.18.

Fig. 4 depicts the normalized torque curves for PLA compositeswith 40% (w/w) kenaf containing zero, 5% or 10% (w/w) thymol.

Fig. 3. Normalized torque as a function of time for melts of PLA containing zero,5% and 10% (w/w) thymol. Inset: corresponding normalized equilibrium torque foreach of these systems.

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Fig. 4. Normalized torque as a function of time for melts of PLA/kenaf composite(40%, w/w, kenaf) containing zero, 5% and 10% (w/w) thymol. Inset: correspondingnormalized equilibrium torque for each of these systems.

The addition of 10% (w/w) thymol into the melt after 2 min hasa significant effect on the rheology as the �2 and �eq values forthis formulation were found to be significantly lower than theircounterparts in the other two formulations. It can therefore be sug-gested that by increasing the thymol concentration in the melt, onecan reduce the mechanical shear forces due to the plasticizing andmixing effect of this AM agent. A similar finding was observed byXue et al. (2008) who studied the effect of water content on the rhe-ological behaviour of starch. In the case of the latter study it wasfound that the torque decreased with increasing water content.

3.2. Fracture surface imaging

The SEM image of neat PLA shown in Fig. 5(a) reveals a relativelysmooth surface in which there is evidence of boundaries presum-ably created upon the stress fracture due to the brittleness of the

PLA. The inherently brittle nature of PLA (Mukherjee and Kao, 2011)is confirmed by the large cracks that appear on the surface. Theaddition of 10% (w/w) thymol to PLA results in reduced surfacecracking (see Fig. 5(b)) and this is also consistent with the notionthat thymol behaves as a plasticizing agent in the system (see alsoFig. 3).

Fig. 5(c) and (d) shows SEM images of a tensile fracture surfaceof PLA formulations that contain 40% (w/w) kenaf. It can be seenthat in each of these composites the kenaf fibres are well dispersedthroughout the polymer matrix. In the case of the composite con-taining no thymol (Fig. 5(c)) the kenaf fibres appear to be moretightly bound to the matrix compared to the composite in whichthymol is present (Fig. 5(d)). In the latter case the presence of pro-truding fibres at the surface suggests that the thymol may be moreconcentrated at the interface between the fibres and the polymermatrix thereby acting as a lubricant that facilitates the slippageof the fibres out of the matrix during fracture. This behaviour isalso consistent with the lower equilibrium torque observed in thecomposite melts containing thymol compared to those where nothymol was present (see Fig. 4). The smooth surfaces of some ofthe fibres together with the voids that can be seen in Fig. 5(d) alsosuggests that there is weak adhesion between the fibres and thePLA matrix when thymol is present in the formulation.

3.3. Tensile properties

The effect of incorporating different amounts of kenaf on thetensile strength of the PLA formulations is shown in Fig. 6 where thenormalized tensile strength (TS), Young’s modulus (E) and percentelongation at break (ε) are given for systems containing variouskenaf loadings and zero, 5% and 10% (w/w) thymol.

3.3.1. Tensile strengthIn Fig. 6(a) it is evident that at kenaf loadings from 10–30%

(w/w), the tensile strength of the PLA/kenaf formulations contain-ing zero and 5% (w/w) thymol is slightly increased by up to ca. 11%and 14% respectively compared with the neat PLA. This suggests

Fig. 5. Scanning electron micrograph images at 500× magnification of: (a) neat PLA and (b) PLA containing 10% (w/w) thymol; and images at 50× magnification of: (c) PLAcontaining 40% (w/w) kenaf and (d) PLA containing 40% (w/w) kenaf and 10% (w/w) thymol.

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Fig. 6. Tensile properties of PLA composites containing: (�) zero, (�) 5% (w/w) and(�) 10% (w/w) thymol: (a) tensile strength (TS); (b) Young’s modulus (E); and (c)percent elongation at break (ε).

that the kenaf fibres act not only to fill the PLA but also providereinforcement for the composite. This reinforcement may occurthrough a mechanism in which the applied stress is transferredfrom one fibre to the next thereby enabling an even distribution ofthe stress throughout the material (Avella et al., 2009). The roughsurface topography of the kenaf fibre as apparent in the SEM image(see Fig. 7) may increase the adhesive characteristics and thus facil-itate the stress transfer during an applied load due to the goodmechanical interlocking between the kenaf fibres and the matrix.The rough surface of kenaf fibre results from the removal of somelignin, pectin, hemicellulose as well as unwanted impurities fromthe surface during the alkali treatment of the fibres. Similar find-ings have been reported by Ibrahim et al. (2010) who incorporated10–30% (w/w) kenaf fibre into a PLA matrix. They reported thatthe tensile strength of the composite increased with an increasein the amount of the fibre in the composite. However, the tensile

Fig. 7. Scanning electron micrograph image of kenaf fibre at 20× magnification.

strength of their composite materials dropped at above 30% (w/w)kenaf content. They speculated that the amount of matrix is prob-ably insufficient to wet out the fibre and fully transfer the stresseffectively at such high fibre loadings. Furthermore, this may alsobe due to high fibre–fibre interaction and agglomeration of fibresin the PLA matrix. Nevertheless, the latter result is in agreementwith the current findings whereby the tensile strength exhibiteda plateau from 30 to 40% (w/w) of kenaf loading. This suggeststhat the fibres were well distributed and had a sufficient extentof interaction with the PLA matrix.

No significant difference between the tensile strength of thePLA/kenaf formulations without thymol or those containing 5%(w/w) thymol was observed across the range of kenaf loadings thatwere tested. This may be due the level of thymol being too smallto affect any change in the tensile strength that could be reliablydetected given the inherently low sensitivity of tensile testing asan experimental technique in such cases. Thus any changes in thetensile strength that may arise due to the plasticizing effect of thethymol in the formulation may not be seen at such low levels of thy-mol. Furthermore, a small amount of the thymol might have beenlost from the formulation as a result of the thermal processing, theeffect of which may be more critical at low levels of thymol.

Similarly, no significant change in the tensile strength ofthe PLA/kenaf formulations containing 10% (w/w) thymol wasobserved with increased kenaf content over the range of formu-lations prepared in the study (see Fig. 6(a)). For these formulationsthe average tensile strength was found to be ca. 50 MPa with 60 MPafor neat PLA. The inclusion of thymol at the higher level of 10%(w/w) compared to the other series of formulations has a mea-surable effect on the tensile strength and it appears to negate theincrease in tensile strength that would normally be imparted by thekenaf fibres in the absence of thymol. This may again be attributedto the plasticizing effect of thymol within the PLA matrix and is con-sistent with the observations made from the micrograph images ofthe tensile fracture surface (Fig. 5). In particular, the thymol mayinterfere with the interaction and/or stress transfer between thePLA matrix and the kenaf fibres during the applied stress. As such,this disturbance of stress transfer is likely to facilitate the end slipof fibres from the polymer matrix, particularly in the presence ofthe plasticizing agent (see also Fig. 5(d)).

Kramer (2009) reported a decrease in the tensile strength ofthermoplastic starch films upon coating with AM additives and ithas been suggested that the interactions between the AM addi-tives and the polymeric matrix affects the intermolecular forceswithin the matrix in a similar manner to water acting as a plasti-cizer (Chabrat et al., 2012). Taib et al. (2009) studied the effect of

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the plasticizer poly(ethylene glycol) (PEG) on the tensile strength ofPLA/kenaf composites and found that a plasticized PLA/kenaf com-posite had a lower tensile strength than that of an un-plasticizedcomposite at 40% (w/w) kenaf content. Moreover, the effect of thy-mol addition observed in the present study is in agreement with thefindings of Prapruddivongs and Sombatsompop (2012) who foundthat an addition of 0.5–1.5% (w/w) triclosan into PLA compositescontaining 10% (w/w) wood flour resulted in a reduction in thetensile strength of the composites from 45 to 40 MPa.

3.3.2. Young’s modulusFig. 6(b) shows the effect of various kenaf loadings on the

Young’s modulus of PLA composites containing zero, 5% and 10%(w/w) thymol. Overall, these results suggest an almost linearincrease in the Young’s modulus with the addition of kenaf fibreat all concentrations of thymol for which the composite contain-ing 40% (w/w) kenaf showed the highest stiffness at approximately1500 MPa. This may be due to the kenaf fibres imparting an over-all increase in stiffness to the composite as the kenaf content isincreased. This is in agreement with the findings of Avella et al.(2009) who suggested that the modulus of the fibre component isthe dominant contributor to the tensile modulus of the compos-ite. A general upward trend in the Young’s modulus with increasedfiller content in the composite was also observed in the case ofpolypropylene (PP)/wood fibre composites where the stiffness ofthe composite was found to be dependent on the filler contentand homogeneity of the filler dispersion (Karmarkar et al., 2007).These findings are also in agreement with a study of PLA/woodflour/triclosan composites where an increase in wood flour from0 to 10% (w/w) enhanced the stiffness and concurrently reducedthe flexibility and toughness (2012). In the present study, the plas-ticizing effect of the thymol that was observed in the results of thetensile tests in Fig. 6(a) is also reflected in the Young’s modulus datain Fig. 6(b) particularly at the higher kenaf loadings. For example, atkenaf loadings greater than 20% (w/w), a concomitant decrease inthe Young’s modulus that occurs upon an increase in thymol con-centration can be clearly seen. Indeed, the addition of 10% (w/w)thymol to the composite formulation in general results in a markedreduction in stiffness compared to the composites without thy-mol and is consistent with the notion that the thymol may beconcentrated at the interface between the kenaf fibres and the poly-mer matrix as discussed in relation to Fig. 5(d). A decrease in theYoung’s modulus of PP films impregnated with 8% (w/w) thymolsupports the present findings (Ramos et al., 2012). It can thereforebe suggested that the addition of thymol into PLA/kenaf compos-ites decreases the stiffness of the materials due to the plasticizingeffect of the AM agent.

3.3.3. Elongation at breakThe effect of various kenaf loadings on the elongation at break

of the PLA composites containing zero, 5% and 10% (w/w) thy-mol is shown in Fig. 6(c). As expected, the elongation at break ofthese composites generally decreases with increasing amounts ofkenaf. This suggests that the presence of the kenaf fibres does notcontribute to the elasticity or the final composite flexibility. Theobserved decrease in flexibility of the composites is likely to berelated to the high stiffness of the composites as is apparent inFig. 6(b). The effect of thymol addition on the elongation at break ofthe composites can also be seen in Fig. 6(c). The PLA/kenaf compos-ites containing 10% (w/w) thymol exhibited slightly higher valuesof the elongation at break compared with the other formulationsin the series. This increase may be due to the AM agent causinga decrease in the compatibility between the PLA matrix and thefiller in the composite system or the possible slippage effect asevidenced in the SEM images above (see Fig. 5(d)). According toAbdul Khalil et al. (2012b), poor fibre-matrix bonding decreases the

Fig. 8. DSC thermograms of (a) neat PLA and PLA containing: (b) 40% (w/w) kenaf,(c) 10% (w/w) thymol and (d) 40% (w/w) kenaf and 10% (w/w) thymol.

strength, stiffness, interfacial adhesion and increases the flexibilityof a composite due to the capability of the filler particles to split andfall apart. In the present study, the kenaf fibres may have becomeless stiff in the presence of thymol, which is a volatile essential oilextract. In the case of non-volatile agents such as nisaplin and EDTAincorporated into plasticized PLA/glycerol triacetate films, a signif-icant reduction of the elongation at break from 108.5 to 62.5% andthe impact strength from 5.4 to 3.4 J mL−1 in comparison with theplasticized PLA has been reported (Liu et al., 2010).

3.4. Thermal properties

3.4.1. Differential scanning calorimetric analysisThe thermal properties of composite samples were studied by

DSC analysis. The DSC thermograms of neat PLA and PLA compositesare shown in Fig. 8. Neat PLA exhibited a Tg peak at 61.7 ◦C andTcc peak at 107.8 ◦C (see Fig. 8(a)). The peak melting temperature,Tm, was observed to be 152.5 ◦C which is in the typical range for asemi-crystalline polymer (Byun et al., 2010).

The values of Tg, Tcc, Tm, �Hm and %Xc, of the various formula-tions were determined from the complete DSC analysis and theseare summarized in Table 1. It was found that the incorporation ofkenaf at 10% or 40% (w/w) (Fig. 8(b)) into the PLA matrix resultedin no significant change to the Tg, whereas a slight increase in theTg seemed to occur at a 10% (w/w) loading of kenaf (see Table 1).These results seem contrary to the findings of Rahman et al. (2012)who reported that the Tg of PLA filled with natural fibres demon-strated a decreasing trend in Tg with increasing fibre content. Itremains unclear as to why such a trend was not observed in thecurrent study. Nonetheless, the Tcc peak of PLA that has a high con-tent of fibre (40% (w/w) kenaf) is observed at a lower temperature(101.7 ◦C) compared to neat PLA (107.8 ◦C). This effect has beenpreviously reported for composites of PLA/kenaf and PP/kenaf andwas attributed to the kenaf fibres acting as a nucleating agent (Hanet al., 2012).

The incorporation of 10% (w/w) thymol in neat PLA and in thePLA/kenaf composites resulted in a decrease in Tg and Tcc (seeFig. 8(c) and (d) as well as Table 1). This may be due to the plas-ticizing effect of thymol that lowers the Tg in particular and this

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Table 1Thermal analysis values obtained from DSC thermograms of neat PLA, PLA/kenaf and PLA/kenaf/thymol composites.

Kenaf content/% (w/w) Thymol content/% (w/w) Tg/◦C Tcc/◦C Tm1/◦C Tm2/◦C �Hm/J g−1 Xc/%

0 0 61.7 107.8 147.5a 152.5 26.1 28.05 53.0 101.3 139.6 147.2 24.6 27.8

10 44.5 94.5 132.6 142.7 22.1 26.4

10 0 62.2 107.7 146.1 152.4 23.3 27.85 52.5 98.7 137.6 147.2 19.9 27.6

10 39.1 90.6 128.7 140.4 16.6 26.2

40 0 61.7 101.7 141.0 148.8 15.4 27.65 50.4 91.9 132.7 143.1 13.5 26.3

10 37.4 84.5 113.8 135.5 12.2 26.1

a Shoulder on the main PLA peak.

observation is consistent with the rheology and tensile propertiesresults that were obtained for neat PLA and the PLA/kenaf com-posites containing thymol (see Figs. 2, 3 and 6). Similar findingshave been reported by Prapruddivongs and Sombatsompop (2012)whereby the addition of 1.5% (w/w) triclosan decreased the Tg ofneat PLA and PLA containing 10% (w/w) wood flour.

In the present study, however, the neat PLA containing 10%(w/w) thymol demonstrated a narrower Tg peak than the PLA/kenafcomposite containing 10% (w/w) thymol (see Fig. 8(c) and (d)) andthis may be due to the lower amount of PLA present in the compos-ite material. Fig. 8(c) also shows that the Tcc peak narrowed withthe addition of 10% (w/w) thymol in PLA and shifted to a lower tem-perature. A similar finding has been reported by Byun et al. (2010).Nevertheless, the addition of thymol to the PLA/kenaf compositescontaining 40% (w/w) kenaf resulted in a lower Tcc with a slightlybroader peak (Fig. 8(d)).

The neat PLA exhibited what can be considered to be a singlemelting peak at 152.2 ◦C with a possible shoulder at ca. 147.5 ◦Cwhereas two distinct melting peaks were observed for all ofthe PLA/kenaf composites. Both melting peaks of the compositesshifted to lower temperatures as the fibre loading was increased.This behaviour is consistent with other studies where it has beenreported that an increase in the fibre content results in the appear-ance of a small, secondary melting peak which shifts to lowertemperatures (Prapruddivongs and Sombatsompop, 2011). Theminor peak may be due to either a different PLA crystalline phasethat forms only in presence of the fibres and/or melting of thetranscrystalline zone (Prapruddivongs and Sombatsompop, 2012).

The addition of higher loadings of kenaf also decreased the peakTm temperatures and the �Hm of PLA across the range of systemsthat were studied. For example, at zero thymol concentration theTm decreases from 152.5 ◦C (pure PLA) to 141.0 ◦C (Tm1 at 40%(w/w) loading kenaf). The �Hm for these corresponding samplesdecreased from 26.1 to 15.4 J g−1 respectively with no significantchanges on the percentage of crystallinity, Xc. Byun et al. (2010)also observed a decrease in the Tm of PLA films with addition of PEG.

It may be expected that the addition of fillers or additives such askenaf fibre or thymol could result in the appearance of a second Tmpeak in the DSC thermogram where the presence of the additivecauses a secondary crystalline phase at the boundary of the bulkpolymer and the additive. For example, in the present study, twomelting peaks are observed at 132.6 and 142.7 ◦C for PLA contain-ing 10% (w/w) thymol with zero kenaf loading. Both of these Tmpeaks appear at correspondingly lower temperatures than those ofthe PLA/kenaf composite containing 40% (w/w) kenaf loading butwithout thymol which appear at 141.0 and 148.8 ◦C (see Table 1).This observation is in agreement with the other research findingswhere a minor secondary melting peak appeared in a PLA film for-mulation containing a plasticizer such as PEG (Byun et al., 2010).Furthermore, a weaker secondary Tm peak is observed in the PLAcomposite containing both kenaf (40%, w/w) and thymol (10%, w/w)and both of these melting peaks appear at lower temperatures than

the corresponding peaks in the formulations that have only one ofthe two additives (see Fig. 8(d)). This may also be due to the loweramount of PLA in the PLA/kenaf composite system. Interestingly,the Xc values for the systems containing thymol show no significantdifference (see Table 1).

3.4.2. Thermogravimetric analysisFig. 9 shows the thermogravimetric analysis (TGA) profiles of

neat PLA and PLA/kenaf composites containing 10 and 40% (w/w)kenaf in the form of the normalized weight percentage as a func-tion of temperature. There is a small but noticeable step in the firststages of the profiles of the composites that is due to the presence ofwater in the kenaf fibres and this step is more evident in the profileof the composite containing 40% (w/w) kenaf. A single degrada-tion step was observed for neat PLA with no char residue apparentat temperatures above ca. 380 ◦C. The rate of weight loss of neatPLA passes through a maximum in the degradation temperaturerange between 280 ◦C and 355 ◦C with the maximum occurring at333 ◦C (see Fig. 9(b)). As expected, the addition of kenaf fibres to the

Fig. 9. Thermogravimetric profiles of (a) PLA containing zero, 10% and 40% (w/w)kenaf; and (b) first derivatives of these systems.

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composite decreases the temperature at which the onset of PLAdegradation occurs. This is also reflected in the derivative curveswhere the maximum rate of degradation for the neat PLA and PLAcontaining 10% (w/w) and 40% (w/w) kenaf occurs at the temper-atures of 333, 332 and 321 ◦C respectively. The third degradationtemperature of the composite containing 40% kenaf content wasobserved at approximately 370 ◦C with a small but noticeable peak.This may be due to the degradation of a non-cellulosic substancesuch as lignin, waxy material or sugar mallards (Bourmaud et al.,2010). Generally, a lower degradation temperature is observed forplant fibres alone under nitrogen gas as compared to air (Van deVelde and Baetens, 2001). However, the fibre degradation peakwould be expected to be more pronounced under an air atmosphere(Bourmaud et al., 2010).

The degradation temperature of PLA/kenaf composites there-fore demonstrates a decreasing trend with increasing kenafcontent. This occurs due to some portions of the PLA matrix hav-ing been replaced with the less thermally stable kenaf fibres thatreduce the thermal stability of the polymer matrix as a whole(Rahman et al., 2012; Yussuf et al., 2010). This result is in agree-ment with the findings of Yussuf et al. (2010) who investigatedthe thermal stability of neat PLA and PLA/kenaf composite samplescontaining 20% (w/w) kenaf using TGA. They observed that the com-posite degraded at 321 ◦C compared with neat PLA which degradedat 323 ◦C at a weight loss of 10%. In addition, the latter compositesalso showed obvious char residues at the end of the degradationprocess and this is consistent with the data shown in Fig. 8. In thepresent study the char residues of composites containing 40% and10% (w/w) kenaf were 10% and 4% respectively at a degradationtemperature of 380 ◦C. It is interesting to note that the presenceof cellulose in the kenaf fibres, which is a lignocellulosic material,results in the production of char residue which, according to Mitraet al. (1998), may improve the fire retardancy of the material.

The TGA profiles of neat PLA and PLA containing 5% and 10%(w/w) of thymol are shown in Fig. 10(a). The profile of the neatPLA exhibits a single step across the temperature range of ca.30–400 ◦C whereas the profile of the formulations containing thy-mol exhibits two main steps during the analysis. In the case of thelatter, the first of these steps occurs gradually over the tempera-ture range of ca. 30–300 ◦C and is attributable to the evaporationand/or loss of thymol from the polymer matrix. This observationis in agreement with the work of Ramos et al. (2012) who inves-tigated by TGA the retention of AM agents such as thymol and/orcarvacrol in a PP matrix after processing. In that study it was sug-gested that the evaporation and/or volatilization of thymol fromthe polymer matrix commenced at a specific temperature withinthe range of 100–200 ◦C and not immediately after the tempera-ture was increased during the TG analysis. This behaviour can alsobe seen in Fig. 10. The second step observed in the profile of thethymol-containing formulations (see Fig. 10) occurs over the tem-perature range of ca. 310–330 ◦C and is attributable to the almostcomplete degradation of the polymer matrix as evidenced by theabsence of a persistent char at high temperatures.

Fig. 10(b) shows the TGA profiles of PLA composites containing40% (w/w) kenaf and containing thymol at zero, 5% and 10% (w/w).These profiles relate directly to the profiles of the PLA formulationscontaining kenaf alone (see Fig. 9) as well as those containing thy-mol alone (see Fig. 10) in that a single degradation step is observedover the temperature range from ca. 30–200 ◦C. This is followedby a second step due to the thermal degradation of the polymermatrix as well as there being evidence of a char residue at highertemperatures. The initial weight loss observed in the temperaturerange of ca. 30–100 ◦C for the PLA/kenaf composite without thymolin Fig. 10 is due to the evaporation of water that is present in thekenaf fibres (see also Fig. 9) whereas the initial weight loss of thosecomposites containing thymol is due to the evaporation of water

Fig. 10. Thermogravimetric profiles of (a) PLA containing zero, 5% and 10% (w/w)thymol; and (b) PLA/kenaf composite (40%, w/w, kenaf) containing zero, 5% and 10%(w/w) thymol.

as well as the degradation and/or evaporation of thymol. The latterpredominantly occurs in the temperature range of 100–200 ◦C.

It is also clear that the higher the kenaf content in the composite,the higher will be the moisture content. The affinity of the kenaffibres for water may be due to the alkali surface treatment thatleads to an increase in the extent of surface roughness as well asthe hydroxyl group accessibility (Karmakar, 1999; Vilaseca et al.,2007). From the data presented in Fig. 10(b), it can be determinedthat the PLA/kenaf composites that were formulated with 5% and10% (w/w) thymol retain about 3% and 7% (w/w) of the thymol thatwas originally added to the formulation respectively. Thus there hasbeen some loss of the volatile AM agent during processing (Ramoset al., 2012). The moisture that is present within the kenaf fibresmay also facilitate the loss of the AM agent.

It can be seen from the weight loss curves in Fig. 10(a) and(b) that the loss of thymol from the composites containing kenafstarted right at the beginning of the heating process as comparedto the loss of thymol from the neat PLA where a delay is observed.This immediate loss of thymol from the composite containing kenafmay be due partially to the ends of the kenaf fibres facilitating theescape of the thymol. This is consistent with the micrograph imagesin Fig. 5 where PLA containing 40% (w/w) kenaf and 10% (w/w) thy-mol that show the presence of voids in the facture surface that werepossibly created by the fibres in the composite.

4. Conclusions

The characterization of PLA and PLA composites was suc-cessfully performed using mechanical and thermal analyticaltechniques in order to investigate the effects of the natural fillerkenaf as well as the AM additive thymol on the stability and per-formance of the material during processing.

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The results suggest that the addition of thymol to PLA andPLA/kenaf composites imparts a plasticizing effect that modifies therheology and subsequent mechanical properties of the processedmaterials. In particular, the equilibrium torque value of each ofthe materials was decreased during processing with the additionof thymol thereby offsetting, to some extent, the effects of theincompatibility between the PLA matrix and the kenaf fibres. Thisobservation was supported by evidence obtained from high magni-fication images of the fracture surface of the PLA/kenaf composites.

The incorporation of thymol into PLA/kenaf composites alsodecreased the tensile strength with no significant change observedupon increasing the kenaf fibre loading. The tensile strength andstiffness of PLA/kenaf composites however increased with higherfibre loadings thereby imparting a reinforcement effect within thePLA composite. Moreover, it was found that an acceptable tensilestrength was attained by the PLA/kenaf composites containingthymol compared to the commercial neat PLA thereby renderingthese materials as potential candidates for commercial packagingsystems. Nonetheless, the effect of AM agent addition on theimpact strength of the material might also be an interestingproperty to investigate prior to developing these systems forcommercial applications.

The DSC analyses showed a general decrease of Tg, Tcc, Tm and�Hm, with no significant changes in %Xc, for PLA and PLA/kenafcomposites containing 5% and 10% (w/w) thymol. It was notedthat an increased loading of kenaf fibres in the PLA resulted ina decrease in the TGA decomposition temperature indicating areduced thermal stability of the PLA. The TGA results also sug-gested that thymol can be released from the PLA/kenaf matrix atrelatively low temperatures and as such has the potential to impartAM activity. Further work in our laboratory is currently underwayto evaluate the kinetics of thymol release from these systems andtheir subsequent AM activity in order to develop effective activefood packaging materials.

Acknowledgments

The authors gratefully acknowledge the Ministry of EducationMalaysia and Universiti Putra Malaysia (UPM) for providing the PhDscholarship for Intan Tawakkal and would like to acknowledge Mr.Mike Allan and the technical staff from the Royal Melbourne Insti-tute Technology, Melbourne for their invaluable assistance with thepreparation of the composites samples.

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91

Chapter 5 – Novel Algorithms for TG Analysis

Two Novel Algorithms for the Thermogravimetric Assessment of Polymer Degradation

under Non-Isothermal Conditions

Overview

This standalone chapter provides an insight into polymer degradation under non-isothermal

conditions as presented by two novel algorithms. The algorithms were validated using model

data and applied to thermogravimetric (TG) data obtained during the degradation of PLA and

PLA/kenaf composites containing thymol under non-isothermal conditions.

The paper entitled “Two Novel Algorithms for the Thermogravimetric Assessment of Polymer

Degradation under Non-Isothermal Conditions” by Bigger S. W., Cran M. J. and Tawakkal I.

S. M. A. was published in Polymer Testing, 43, 139-146.

92

93

ilable at ScienceDirect

Polymer Testing 43 (2015) 139e146

Contents lists ava

Polymer Testing

journal homepage: www.elsevier .com/locate/polytest

Property modelling

Two novel algorithms for the thermogravimetric assessmentof polymer degradation under non-isothermal conditions

Stephen W. Bigger a, *, Marlene J. Cran a, Intan S.M.A. Tawakkal b

a Institute for Sustainability and Innovation, College of Engineering and Science, Victoria University, PO Box 14428, Melbourne, 8001,Australiab College of Engineering and Science, Victoria University, PO Box 14428, Melbourne, 8001, Australia


Article history:Received 23 January 2015Accepted 2 March 2015Available online 13 March 2015

Keywords:Arithmetic algorithmThermogravimetryNon-isothermal degradation kineticsApparent activation energyArrhenius A-factor

* Corresponding author. Tel.: þ61 3 9919 2959.E-mail address: [email protected] (S.W.

http://dx.doi.org/10.1016/j.polymertesting.2015.03.00142-9418/Crown Copyright © 2015 Published by E

a b s t r a c t

Two novel algorithms are presented for processing thermogravimetric (TG) data obtainedduring the degradation of a polymer in a single step mechanism under non-isothermalconditions. The first algorithm assesses three characteristics computed from the TG pro-file against a theoretical data set, and identifies likely kinetic models to fit the experi-mental data. The second algorithm provides an iterative arithmetic method to extract theapparent activation energy, Ea, and Arrhenius A-factor, A, from TG data without simplifyingassumptions. The algorithms are validated using model data and applied to data for thenon-isothermal degradation of poly(ethylene adipate), poly(lactic acid) (PLA) and a foodpackaging PLA composite formulation containing kenaf, a natural fibre. The analysis ofpoly(ethylene adipate) produced Ea ¼ 137 kJ mol�1 and log10A ¼ 8.71 (first-order kineticmodel). The kenaf fibre destabilizes PLA, lowering its Ea from 190 kJ mol�1 to 150 kJ mol�1

(contracting volume model).Crown Copyright © 2015 Published by Elsevier Ltd. All rights reserved.

1. Introduction

The scientific literature on the application of thermog-ravimetric analysis (TGA) and differential thermal analysis(DTA) techniques experienced a huge expansion in thedecade following the late 1960's [1]. These techniques havesince been used as valuable tools to investigate syntheticpolymer degradation as well as a wide range of other ma-terials, including cellulose [2,3] and various solids such ascarbonates [4e6] and active solids [7]. Early reviews in thearea include the work of Mitchell and Chiu [8e11] andMurphy [12,13]. More recently, Vyazovkin et al. [14] pub-lished a comprehensive account of methods to evaluatekinetic parameters such as the activation energy, ArrheniusA-factor and the reaction model, from data obtained viaTGA, DTA and differential scanning calorimetry (DSC) ex-periments. The work covers the most common kinetic

Bigger).

02lsevier Ltd. All rights reserve

94

methods, including the model-free (iso-conversional) onesas well as model fitting.

The application of TGA to the study of polymer degra-dation has been primarily focused on deriving kinetic pa-rameters, such as the apparent activation energy andArrhenius A-factor, that in some cases may be used as in-dicators of thermal stability. To obtain these parameters,TGA experiments can be performed either isothermally at aseries of constant temperatures or non-isothermally undera known, usually linear, temperature ramp at a number ofdifferent heating rates. Clearly, the major advantage of thelatter over the former is that less time is usually required toexperimentally obtain the data for analysis. Nonetheless,criticisms that are often leveled at non-isothermal methodsinclude: (i) the fact that the mechanism, kinetic model, andhence the kinetic parameters, may change with tempera-ture over the course of the experiment, (ii) over extremetemperature ranges, and particularly under oxidizing con-ditions, the material at the end of the experiment bearslittle chemical resemblance to the startingmaterial and (iii)

d.


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S.W. Bigger et al. / Polymer Testing 43 (2015) 139e146140

at high heating rates there may be thermal lag effects[1,14,15].

In order to extract kinetic parameters from experi-mentally acquired TGA data, numerous kinetic models andmethods of mathematically processing the data in accor-dance with the proposed model have been suggested. Earlypapers by Dollimore et al. [16,17] present numerical criteriaderived from theoretical modeling that can be used to helpidentify suitable kinetic models that may be applied infitting a given set of data. The mathematical methods[18,19] for processing thermogravimetric (TG) data inaccordance with a given model fall into two main cate-gories, namely the integral methods [14,20] and differentialmethods. The respective merits of these are extensivelydiscussed in the literature [14,15,21].

Many of the earlier studies in this field describe ways inwhich experimental data can be analyzed in accordancewith an appropriate kinetic model, given that the requiredmathematical manipulations are often very difficult.Indeed, some of the early methods for processing experi-mental TGA results can only be described as tedious andcumbersome at best, and alternative methods were widelyexplored in an attempt to simplify the procedures. How-ever, simplifying assumptions and approximations mayhave a serious effect on the outcome.

Notwithstanding the extreme caution one must use inapplying the results derived fromTGA studies, particularly tothe prediction of polymer service lifetimes, the techniquecan provide in some cases a useful basis on which tocompare the apparent stability of polymeric materials. Suchcomparative data can be used in conjunction with otherstability information in order to provide a more completeunderstanding of the overall stability of thematerial. In viewof the potentially useful information that can be derivedfromTGA experiments, and the advent ofmodern computer-based iterative numerical methods that can overcome manyof the previously encountered difficulties, or indeed im-possibilities, associated with solving complex systems ofequations that do not conform to closure, a computer-basedalgorithmic procedure was developed for: (i) the analysis ofnon-isothermal TGA data and (ii) the verification that thechosen analysis is appropriate. Indeed, an integral numericalapproach to solving isoconversional systems has been her-alded by Vyazovkin and Dollimore [22] and Vyazovkin[23,24] as a means by which kinetic parameters can bemoreaccurately determined from experimental TG data.

2. Theory

The recent review by Vyazovkin et al. [14] provides thebasic theory and recommendations for performing com-putations on data acquired from thermal analyticalmethods. The following is intended as a brief overview ofthe basic theoretical principles that apply directly to theapproach that is described in the current work.

2.1. Kinetic equation for non-isothermal TGA

For the case of non-isothermal TGA, the followingequation applies and is derived in the SupplementaryMaterial:

95

gðaÞ ¼ ðAEa=RbÞZ∞�

expð�xÞ�x2�dx (1)
x
where g(a) ¼ 1/dG(a)/da, a is the extent of the reaction(conversion), A is the Arrhenius A-factor, Ea is the apparentactivation energy, R is the ideal gas constant, b is theheating rate and x ¼ Ea/RT. The function G(a) describes thekinetics of the system. Eq. (1) can be written more simplyas:

gðaÞ ¼ ðAEa=RbÞ � pðxÞ (2)

where the function p(x) represents the integralpðxÞ ¼ R∞

x ½expð�xÞ=x2�dx

2.2. Differential and isoconversional data analysis equations

For a given process where (T, a) data exist and the ki-netic form of G(a) along with the heating rate, b, are known,the following equation applies:

ln½ðda=dTÞ=f ðaÞ� ¼ InðA=bÞ � Ea=RT (3)

The application of this equation is representative of oneof the so-called “differential” methods for analyzing TGAdata, and the derivation of eq. (3) is given in theSupplementary Material. A plot of ln[(da/dT)/f(a)] versus1/T will enable the value of the apparent activation energy,Ea, to be calculated from the gradient of the plot. TheArrhenius A-Factor can be calculated from the intercept.

A differential isoconversional method devised byFreidman [25] has led to the commonly used equation:

lnhbðda=dtÞa;i

i¼ In½f ðaÞ � A� � Ea

�RTa;i (4)

where (da/dT)a,i is the derivative of the a versus T curve at afixed degree of conversion, ai, and Ta,i is the correspondingtemperature at this point. At a fixed degree of conversion,f(a) will be a constant for a given set of experimental TGcurves that are run under different heating rates and, assuch, the ln[f(a)�A] term in eq. (4) will be a constant for theset of runs. Consequently, a plot of ln[b(da/dt)a,i] versus1/Ta,i will yield a straight line from which a value of theapparent activation energy can be obtained from thegradient.

2.3. Non-isothermal TGA data analysis algorithms

Algorithms were devised to facilitate a convenientmethod of analysis for a wide range of non-isothermal TGdata. These computer-based algorithms are: (i) an algo-rithm for identifying suitable kinetic models for fitting theexperimental data based on theoretical reference charac-teristics calculated by Dollimore et al. [16] for a given set of(T, a) input data and (ii) an iterative arithmetic algorithmthat solves eq. (2) without invoking assumptions or ap-proximations in order to extract the two Arrhenius pa-rameters. The overall approach delivers the so-called“kinetic triplet” information and also enables one to assesswhether the analysis has been appropriate in so far as the

Table 1Values of the model fitting parameter, r, and the corresponding linear

S.W. Bigger et al. / Polymer Testing 43 (2015) 139e146 141

degradation occurred by a single mechanism over thetemperature range.

2.3.1. Algorithm to identify kinetic modelThis algorithm systematically calculates from the

experimental (T, a) data the values of three characteristicparameters (da/dT)max, amax and DT, where (da/dT)max isthe maximum value of the derivative of a with respect totemperature, amax is the value of a at which the maximumderivative value occurs, and DT is the half-height width ofthe da/dT versus T plot. The parameters are then comparedwith the corresponding ranges for these given in thereference data [16] for a number of different kinetic models,and a fit parameter, r, is calculated for each model. The fitparameter is such that 0� r� 1 and r¼ 1 is deemed to be a“perfect” fit. Details of the calculation of the fit parameterand the four possible cases under which it is calculated aregiven in the Supplementary Material.

2.3.2. Iterative numerical TG analysis algorithmTaking the natural logarithm of both sides of eq. (2) and

allowing for separate experimental measurements atdifferent values of ai as well as allowing for different valuesof the activation energy yields:

di�ai; Ea;j

� ¼ ln½gðaiÞ� � ln½pðxiÞ� ¼ AEa;j�Rb (5)

where di(ai, Ea,j) represents a single value of a differencefunction that exists for a given value of ai and its corre-sponding xi value. The value of xi at a given value of Ea,j iscalculated from xi ¼ Ea,j/RTi where Ti is the temperaturecorresponding to the particular ai value. Details of theapplication of eq. (5) in an iterative process to identify theoptimum value of Ea and the corresponding value of theArrhenius A-factor associated with the experimental dataare provided in the Supplementary Material.

regression coefficient, R2, pertaining to the g(a) versus p(x) plot obtainedfrom the analysis of the contracting volume test data set under the variouskinetic models.

Kinetic Model g(a) r R2

AcceleratoryPn Power law a1/n 0.69 note[1]

E1 Exponential law ln(a) 0.55 0.6275[2]

SigmoidalA2 Avrami-Erofeev [eln(1ea)]1/2 0.95 0.9851A3 Avrami-Erofeev [eln(1ea)]1/3 0.95 0.9914A4 Avrami-Erofeev [eln(1ea)]1/4 0.69 0.9929B1 Prout-Tompkins [eln(a/(1ea))] þ C 0.64 note[3]

DeceleratoryGeometricalR2 Contracting area 1e(1ea)1/2 0.88 0.9950R3 Contracting volume 1e(1ea)1/3 0.91 0.9996DiffusionD1 One dimensional a2 0.79 0.8955D2 Two dimensional (1ea)ln(1ea) þ a 0.79 0.9531D3 Three dimensional [1e(1ea)1/3]2 0.66 0.9979D4 Ginstling-Brounshtein 1e(2/3)ae(1ea)2/3 0.73 0.9777Reaction OrderF1 First order eln(1ea) 0.87 0.9582F2 Second order 1/(1ea) 0.58 0.5440F3 Third order 1/(1ea)2 0.16 0.0735

Notes.1. Not fitted.2. No convergence using fitting algorithm. Fitted using derivative method.3. No convergence using fitting algorithm.

3. Experimental section

Poly(lactic acid) (7001D Ingeo™; specific gravity 1.24;melt-flow index 6 g/10 min at 210 �C and 2.16 kg; meltingtemperature range 145e160 �C) was obtained fromNatureWorks LLC, USA. Kenaf fibre (bast) was purchasedfrom Ecofibre Industries, Australia.

To improve compatibility with the PLA matrix andremove any impurities, a preliminary surface treatment ofthe kenaf was performed by immersing it in 5% w/v NaOH(Merck Chemicals, Australia) for 2 h at room temperature.The fibres were thenwashed with and rinsed with distilledwater. Acetic acid (Merck Chemicals, Australia) was used toadjust the pH to neutrality prior to a final rinse withdistilled water. The fibres were filtered and dried overnightin an air circulating oven at 105 �C.

Prior to mixing, the PLA resin was dried in an oven at60 �C. The PLA and treated kenaf fibres (40% w/w) werecompounded using an internal mixer (Haake PolyLab OS,Germany) at 50 rpm and 155 �C for 8 min. The material wasthen collected from the mixer, cooled and preheated in alaboratory press (L0003, IDM Instruments Pty. Ltd.,Australia) at 150 �C for 2 min. It was pressed at the sametemperature for 3 min under a pressure of 50 kN before

96

quench cooling to 30 �C under pressure to produce eitherfilms (0.3 mm thickness) or slabs (3 mm thickness) ofmaterial for analysis.

A Mettler Toledo TGA/DSC1 STARe system thermogra-vimetric analyzer was used to undertake the TG analyses.The PLA and composite samples were heated from 30 to500 �C at a rate of 5 K min�1 and under a nitrogen atmo-sphere at a flow rate of 0.2 L min�1.


4.1. Validation of the model identification and data fittingprotocols

In order to validate the previously described modelidentification and data fitting algorithms, a TG profilewhose characteristics are known [18] was deconstructedand its (T, a) data analyzed as test data. The characteristicsof these data are: contracting volume kinetic model,Ea ¼ 100 kJ mol�1, A ¼ 1.883 � 1015 min�1 and heating rateb ¼ 1.000 K min�1.

Table 1 lists the values of r that were produced by themodel identification algorithm along with the corre-sponding values of the linear regression coefficient, R2,obtained from the analysis of plots of g(a) versus p(x) thatwere also generated over the range of common kineticmodels. Inspection of the values of r confirms that the testdata conform to a contracting volume model (r ¼ 0.913), asexpected. However, it is also apparent that other modelssuch as the Avrami-Erofeev A2 and A3 models (r ¼ 0.953 ineach case) may also be appropriate choices for fitting thedata.

Fig. 2. Plot of g(a) versus p(x) for the test data [18] where Ea ¼ 100 kJ mol�1

and g(a) ¼ 1e(1ea)1/3 (i.e. contracting volume model). The value of theapparent activation energy was that determined by the iterative numericalalgorithm for solving eq. (2).


Fig. 1 shows a plot of the linear regression coefficient, R2,versus r that was constructed from the datawhose analysesare given in Table 1. Although some clustering around thehigher values of the fit parameters is observed, the plotshows a reasonably good overall correlation between thetwo variables, suggesting that the proposed model identi-fication algorithm can be used to distinguish suitable ki-netic models for fitting the TG data.

The clustering of the data associated with the variousmodels (Fig.1) is a possiblemanifestation of the establishedfact that the same kinetic process can be described bydifferent reaction models as well as by combinations ofdifferent apparent activation energy and Arrhenius A-fac-tor values [14]. It has been suggested that almost anymodelcan satisfactorily fit data if wide variations in the apparentactivation energy and Arrhenius A-Factor are allowed [14].Indeed, in the case of solid-state kinetics, this becameapparent through the early work of Huttig et al. [26], andBruzs [27], and had led to the iso-conversional method ofevaluation proposed by Kujirali and Akahira [28].

The iterative numerical application of eq. (5) to the testdata produces a convergence for the contracting volumekinetic model at an apparent activation energy value ofEa ¼ 100 kJ mol�1 precisely (see also SupplementaryMaterial), as expected from the prior knowledge of theinput test data. The corresponding value of the A-factorproduced by the algorithm is A¼ 1.906 � 1015 min�1 whichis acceptably close to the expected value ofA ¼ 1.883 � 1015 min�1. The appropriate fit of the test datato the expected contracting volume kinetic model is pre-empted by the value of r being close to unity, as indicated inTable 1. The fit of the model can be more authoritativelyconfirmed by plotting g(a) versus p(x), as shown in Fig. 2.

The linearity of the plot of g(a) versus p(x) in accordancewith eq. (2) confirms that the model is appropriate andprovides a high level of confidence in the value of theapparent activation energy that is obtained under the givenmodel for the process (see Fig. 2). It also validates theassumption that the apparent activation energy remainsconstant over the temperature range of the experiment,suggesting that the mechanism does not change over thecourse of the applied temperature ramp. Furthermore, the

Fig. 1. Plot of r values produced by the model identification algorithm forthe test data [18] versus corresponding values of the linear regression co-efficient, R2, obtained from the regression analysis of g(a) versus p(x) plotsfor the selection of common kinetic models.

97

value of the apparent activation energy obtained from thegradient of the plot (see eq. (2)) is 97.9 kJ mol�1 and isacceptably close to that obtained by the iterative arithmeticalgorithm. A value for the apparent activation energy canalso be determined by the differential method from anappropriate plot in accordance with eq. (3) whereG(a)¼ 3(1ea)2/3 in the case of a contracting volume model.Such a plot is shown in Fig. 3.

The gradient and intercept of the plot shown in Fig. 3can be used to calculate values of the kinetic parameters:Ea ¼ 93.7 kJ mol�1 and A ¼ 1.606 � 1014 min�1. Clearly,these values obtained via a differential method are signif-icantly lower than the expected values and reflect theproblem identified by Vyasovkin et al. [14] in that differ-entiation of “integral” type data, such as those data ob-tained from TGA experiments, tends to magnify the noise(as is clearly apparent at low levels of conversion). There-fore, it has been suggested that integral and differentialmethods are best suited for, respectively, analyzing integral(e.g. TGA) and differential (e.g. DSC, DTA) data [14].

4.2. Thermal degradation of poly(ethylene adipate)

The application of TGA to studying the thermal degra-dation of poly(ethylene adipate) is reported [21] in a paper

Fig. 3. Differential method plot of ln[(da/dT)/G(a)] versus 1/T in accordancewith eq. (3) for the test data [18]. In this case G(a) ¼ 3(1ea)2/3 for a con-tracting volume model.


that compares a range of kinetic models along with an iso-conversional method. The relevant data in this referencepaper pertaining to four different experimental heatingrates were deconstructed and processed (up to a¼ 0.8 in allcases) as above in order to further assess the proposedalgorithms.

Table 2 lists the values of the model fitting parameter, r,and the average of this parameter, rave, across the differentexperimental heating rates obtained from the analysis ofthe reference data [21]. The processed data indicate thatthe best kinetic fit may lie amongst the R3, D2, D3, D4 or F1models, each of which has a rave value of ca. 0.9 or above.The numerical algorithm was used to fit the data at thedifferent heating rates to each of the models listed in Table2 and these results appear in Table 3.

Table 3 lists the values of the apparent activation energy,Ea,calc, and the decadic logarithm of the A-factor, log10Acalcthat were obtained using the iterative arithmetic algorithmto analyze the reference data [21]. Also listed in this tablefor comparison are Ea,ref and log10Aref that are the overallvalues of the kinetic parameters reported in the literature[21].

The data in Table 3 exhibit wide variation in the kineticparameters across the range of kinetic models, againillustrating that a given set of kinetic data may be fitted by anumber of different kinetic triplets [14]. With the exceptionof the data obtained at a heating rate of 10 K min�1, there isa consistent downward trend in the apparent activationenergies as the heating rate is increased. This is consistentwith the notion that for a given reaction model the kineticparameters are expected to vary with heating rate [14], andhighlights a possible limitation of non-isothermal gravi-metric methods for unambiguously determining rate pa-rameters within a given kinetic model.

Table 2Values of the model fitting parameter, r, and the average of thisparameter, rave, across the different experimental heating rates obtainedfrom the analysis of reference data [21] for the thermal decomposition ofpoly(ethylene adipate).

Kinetic Model Heating rate, b/K min�1 rave

5 10 15 20

AcceleratoryPn Power law 0.01 0.07 0 0 0.02 ± 0.02E1 Exponential law 0.52 0.52 0.52 0.52 0.52 ± 0.01

SigmoidalA2 Avrami-Erofeev 0.78 0.80 0.74 0.78 0.77 ± 0.02A3 Avrami-Erofeev 0.74 0.77 0.70 0.74 0.74 ± 0.02A4 Avrami-Erofeev 0 0 0 0 0B1 Prout-Tompkins 0 0.03 0 0 0.01 ± 0.01

DeceleratoryGeometricalR2 Contracting area 0.92 0.94 0.76 0.85 0.87 ± 0.06R3 Contracting volume 0.91 0.92 0.86 0.91 0.90 ± 0.02DiffusionD1 One dimensional 0.68 0.72 0.54 0.61 0.64 ± 0.06D2 Two dimensional 0.97 0.97 0.91 0.94 0.95 ± 0.02D3 Three dimensional 0.97 0.98 0.96 0.99 0.98 ± 0.01D4 Ginstling-Brounshtein 0.97 0.95 0.93 0.96 0.95 ± 0.01Reaction OrderF1 First order 0.94 0.94 0.98 0.98 0.96 ± 0.02F2 Second order 0.82 0.83 0.92 0.87 0.86 ± 0.04F3 Third order 0.57 0.56 0.71 0.64 0.62 ± 0.06

98

The TGA data for the decomposition of poly(ethyleneadipate) were analyzed using a model-free approach usingthe isoconversional method proposed by Friedman [25] inaccordance with eq. (4). The relevant plot of the processeddata appears in Fig. 4.

The Friedman method of analysis of the data producesapparent activation energy values of Ea ¼ 156 kJ mol�1

(a¼ 0.2) and Ea¼ 146 kJ mol�1 (a¼ 0.8). Themagnitudes ofthese values are consistent with those reported in theoriginal source but calculated using a different method[21]. The decrease in the apparent activation energy withincreased extent of conversion suggests the assumptionthat the activation energy remains constant throughout theprocess, which is inherent in the data processing algorithm,is not strictly valid. Nonetheless, the variation of ca.10 kJ mol�1 is considered to be small and inconsequential.For systems where this assumption is not valid, a correctionprocedure similar to the one described by Vyazovkin [24],for handling integral isoconversional datawould need to beinvoked.

The apparent activation energy range determined in theFriedman analysis is closest to those values of this param-eter that are produced by the iterative arithmetic algorithmfor the R2, R3 and F1 models (see Table 3). The R3 and F1models were identified by the model identification algo-rithm as being appropriate candidates for data fitting withrespective average fit parameters of rave ¼ 0.90 ± 0.02 andrave ¼ 0.96 ± 0.02 (see Table 2). The quality of this fit is alsoconfirmed by the respective linear regression coefficientsassociated with the g(a) versus p(x) plots for these systemsdetermined at a heating rate of 5 K min�1, which areR2 ¼ 0.9913 and R2 ¼ 0.9753, respectively. Given the highlynon-linear nature of the functions g(a) and p(x), it isanticipated that the extent to which a plot of g(a) versusp(x) conforms to linearity will be a sensitive indicator of thegoodness of fit of the model and, consequently, the validityof the assumption that the apparent activation energy re-mains constant over the temperature range of theexperiment.

Considering only those models where the fit parameterwas 0.9 or above (i.e. the R3, D2, D3, D4 or F1), the con-gruency between the apparent activation energies deliv-ered by the iterative arithmetic algorithm, Ea,calc and thecorresponding reference values [21], Ea,ref can be tested byplotting these quantities against each other (Fig. 5).

Plots similar to that shown in Fig. 5 can be constructedfor the data obtained at the other heating rates and all showa similar trend. The regression line of best fit through thedata and that passes through the origin shows a reasonablyhigh level of correspondence between the variables in asfar as its gradient is close to unity and the linear regressioncoefficient is also reasonably high. Thus, the values of theapparent activation energy delivered by the iterativearithmetic algorithm are reasonably consistent with thosevalues reported in the literature obtained at a heating rateof 5 K min�1. The differences between the Ea,calc values forthe R3 and F1 models and the corresponding valuesdelivered by the model-free analysis [25] may be due inpart to the assumption inherent in the arithmetic algorithmthat the apparent activation energy remains constant overthe temperature range of the experiment.

Table 3Values of the apparent activation energy, Ea,calc, and the decadic logarithm of the A-factor, log10Acalc that were obtained from reference data [21] for thethermal decomposition of poly(ethylene adipate) at different heating rates. The overall values reported for the kinetic parameters (Ea,ref, log10Aref) [21] arelisted for comparison.

Kinetic Model Ea,calc/kJ mol�1 Ea,ref/kJ mol�1 log10Acalc[1] log10Aref

[1]

Heating rate b/K min�1 Heating rate b/K min�1

5 10 15 20 5 10 15 20

SigmoidalA2 Avrami-Erofeev 64 51 60 57 123.0 2.47 1.50 2.38 2.22 7.68A3 Avrami-Erofeev 40 31 37 35 114.7 0.34 �0.27 0.40 0.33 7.00B1 Prout-Tompkins nc[2] nc nc nc 118.1 nc nc nc nc 8.35

DeceleratoryGeometricalR2 Contracting area 131 100 125 115 153.1 7.83 5.25 7.41 6.62 9.74R3 Contracting volume 133 99 126 117 159.6 7.85 5.01 7.34 6.65 10.13DiffusionD1 One dimensional 261 209 250 228 220.6 18.79 14.29 17.59 15.73 15.23D2 Two dimensional 269 211 257 237 246.1 19.26 14.23 17.95 16.28 17.14D3 Three dimensional 275 208 263 243 280.9 19.19 13.41 17.89 16.23 19.46D4 Ginstling-Brounshtein 270 203 259 236 257.5 18.72 12.94 17.51 15.58 17.46Reaction OrderF1 First order 137 110 128 123 174.7 8.71 6.46 8.00 7.66 11.93F2 Second order 8 5 5 4 237.1 �2.51 �2.61 �2.44 �2.46 17.30

Notes.1. Units of A are s�1.2. Data were not convergent (nc) using the analysis algorithm.


4.3. Thermal degradation of poly(lactic acid)/kenafcomposites

To further test the applicability of the proposed algo-rithms, samples of poly(lactic acid) (PLA) that were filledwith 40% w/w of the natural fibre kenaf were degraded innitrogen over the temperature range of 200 to 500 �C.These materials are of current interest as a naturallyderived substrate for formulations of novel packaging ma-terials that contain natural antimicrobial agents [29]. Fig. 6shows plots of a versus temperature for both the neat PLAand the filled sample. The plot for the composite clearlyshows two steps: the first being due to the thermaldegradation of PLA and the second due to the decomposi-tion of the kenaf that ultimately leads to a char residue.

The model identification algorithm identified the con-tracting volume model for degradation (R3) as the most

Fig. 4. Differential isoconversional method plot of ln[b(da/dT)a,i] versus1/Ta,i for the thermal decomposition of poly(ethylene adipate) in accordancewith the method devised by Freidman [25].

99

appropriate basis on which to analyze the TG data per-taining to the degradation of the PLA. The kinetic analysesof these data in accordance with the R3 model suggest thatthe apparent activation energy for the degradation of PLA isca. 190 kJ mol�1, whereas in the presence of the kenaf fillerthis value is significantly lowered to ca. 150 kJ mol�1. Thissuggests the addition of the kenaf to the PLA matrix de-stabilizes the polymer.

Furthermore, the data in Fig. 6 corresponding to thedegradation of the TK fibres (i.e. the data from ca. 350 to460 �C) were analyzed separately and found to conformclosely to a first-order kinetic process (i.e. g(a)¼ eln(1ea))and were fitted accordingly. The analysis produced a valueof Ea ¼ 314 kJ mol�1 for this process which is very highcompared to the value of ca. 169 to 170 kJ mol�1 obtainedby Yao et al. [30]. This difference may be due to the alkali

Fig. 5. Plot of Ea,calc, the apparent activation energy obtained using theiterative arithmetic algorithm for the R3, D2, D3, D4 and F1 models versusEa,ref, the corresponding overall apparent activation energy reference valuereported for the thermal decomposition of poly(ethylene adipate) [21] at aheating rate of 5 K min�1.

Fig. 6. Plot of a versus T for: (i) neat PLA and (ii) PLA filled with 40% w/wkenaf. The samples were degraded in nitrogen over the temperature range of200 to 500 �C at a heating rate of 5 K min�1.


pre-treatment of the fibres used in the present study whichchemically alters much of the lignin, hemicellulose andother material on their surface that may, in turn, destabilizeand/or catalyze the decomposition of the kenaf [31,32].Discrepancy may also arise as a result of the data selectedfor analysis being convoluted in the early part of the tracewith those data associated with the final part of the PLAdecomposition. It is anticipated, however, that failure to de-convolute these data prior to analysis would not be solelyresponsible for the large difference between the twovalues.

5. Conclusions

Iterative arithmetic techniques can, where appropriate,provide a convenient means by which integral type ther-mochemical data can be processed for the routine assess-ment of polymer degradation kinetics. The modelidentification algorithm exhibits significant promise as amethod by which applicable kinetic models can be identi-fied. The iterative arithmetic algorithm for the calculationof the kinetic parameters provides a way of solving theArrhenius integral and associated equations without thenecessity of making mathematical approximations andassumptions in the processing of the TG experimental data.Nonetheless, it is expected that such analyses will onlyhave validity in the case of simple systems where a singledegradation mechanism prevails across the range of tem-peratures and heating rates used in the experiment. Thelinearity of the g(a) versus p(x) plot serves as a check on thevalidity of any assumptions that may have been made.

The decomposition of poly(ethylene adipate) may bedescribed by a kinetic triplet involving a first-order (F1)mechanism with an apparent activation energy of137 kJ mol�1 and log10A ¼ 8.71. Neat PLA and PLA com-posites each decompose in accordancewith the contractingvolume (R3) model where the addition of kenaf filler to thePLA destabilizes the latter, lowering its apparent activationenergy from ca. 190 kJ mol�1 to ca. 150 kJ mol�1.

Acknowledgements

The authors gratefully acknowledge the Ministry ofEducation, Malaysia and the Universiti Putra Malaysia

100

(UPM) for providing the PhD scholarship for Ms IntanTawakkal and would like to acknowledge Mr Mike Allanfrom RMIT University for assistance in the preparation ofthe composite material.

Appendix A. Supplementary data

Supplementary data related to this article can be foundat http://dx.doi.org/10.1016/j.polymertesting.2015.03.002.

References

[1] J.H. Flynn, Thermogravimetric analysis and differential thermalanalysis, in: H.H.G. Jellinek (Ed.), Aspects of Degradation and Sta-bilization of Polymers, Elsevier, Amsterdam, 1978, pp. 573e615.

[2] M.J. Antal, G. Varhegyi, E. Jakab, Cellulose pyrolysis kinetics: Revis-ited, Ind. Eng. Chem. Res. 37 (4) (1988) 1267e1275.

[3] V. Mamleev, S. Bourbigot, J. Yvon, Kinetic analysis of the thermaldecomposition of cellulose: the main step of mass loss, J. Anal. Appl.Pyrol. 80 (1) (2007) 151e165.

[4] D. Dollimore, The application of thermal analysis in studying thethermal decomposition of solids, Thermochim. Acta. 203 (1992)7e23.

[5] S. Maitra, N. Bandyopadhyay, J. Pal, Application of non-Arrheniusmethod for analyzing the decomposition kinetics of SrCO3 andBaCO3, J. Am. Ceram. Soc. 91 (1) (2008) 337e341.

[6] D. Dollimore, P. Tong, K.S. Alexander, The kinetic interpretation ofthe decomposition of calcium carbonate by use of relationshipsother than the Arrhenius equation, Thermochim. Acta 282e283(1996) 13e27.

[7] D. Dollimore, Thermodynamic, kinetic and surface texture factors inthe production of active solids by thermal decomposition, J. Therm.Anal. 38 (1992) 111e130.

[8] J. Mitchell Jr., J. Chiu, Analysis of high polymers, Anal. Chem. 4(1969) 248Re298R.

[9] J. Mitchell Jr., J. Chiu, Analysis of high polymers, Anal. Chem. 43 (5)(1971) 267Re334R.



[12] C.B. Murphy, Thermal analysis, Anal. Chem. 44 (5) (1972)513Re524R.

[13] C.B. Murphy, Thermal analysis, Anal. Chem. 46 (5) (1974)451Re459R.

[14] S. Vyazovkin, A.K. Burnham, J.M. Criado, L.A. P�erez-Maqueda,C. Popescu, N. Sbirrazzuoli, ICTAC Kinetics Committee recommen-dations for performing kinetic computations on thermal analysisdata, Thermochim. Acta 520 (1) (2011) 1e19.

[15] S. Vyazovkin, C.A. Wight, Isothermal and non-isothermal kinetics ofthermally stimulated reactions of solids, Int. Rev. Phys. Chem. 17 (3)(1998) 407e433.

[16] D. Dollimore, T.A. Evans, Y.F. Lee, G.P. Pee, F.W. Wilburn, The sig-nificance of the onset and final temperatures in the kinetic analysisof TG curves, Thermochim. Acta 196 (2) (1992) 255e265.

[17] D. Dollimore, T.A. Evans, Y.F. Lee, F.W. Wilburn, Correlation betweenthe shape of a TG/DTG curve and the form of the kinetic mechanismwhich is applying, Thermochim. Acta 198 (2) (1992) 249e257.

[18] M.E. Brown, C.A.R. Phillpotts, Non-isothermal kinetics, J. Chem.Educ. 55 (9) (1978) 556e560.

[19] A. Khawam, D.R. Flanagan, Solid-state kinetic models: basics andmathematical fundamentals, J. Phys. Chem. B 110 (35) (2006)17315e17328.

[20] R.E. Lyon, An Integral Method of Non-isothermal Kinetic Analysis,Office of Aviation Research, Washington DC, July 1996, p. 20. ReportDOT/FAA/AR-96/68.

[21] K. Chrissafis, Kinetics of thermal degradation of polymers, J. Therm.Anal. Calorim. 95 (1) (2009) 273e283.

[22] S. Vyazovkin, D. Dollimore, Linear and nonlinear procedures inisoconversional computations of the activation energy of non-isothermal reactions in solids, J. Chem. Inf. Comput. Sci. 36 (1)(1996) 42e45.

[23] S. Vyazovkin, Evaluation of activation energy of thermally stimu-lated solid-state reactions under arbitrary variation of temperature,J. Comput. Chem. 18 (3) (1997) 393e402.


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[24] S. Vyazovkin, Modification of the integral isoconversional method toaccount for variation in the activation energy, J. Comput. Chem. 22(2) (2001) 178e183.

[25] H.L. Friedman, Kinetics of thermal degradation of char-formingplastics from thermogravimetry. Application to a phenolic plastic,J. Polym. Sci. C Polym. Symp. 6 (1) (1964) 183e195.

[26] G.F. Huttig, A. Meller, E. Lehmann, Active oxides. LIV. Rate ofdecomposition of zinc carbonate into zinc oxide and carbon dioxide,Z. Physik. Chem. B19 (1932) 1e21.

[27] B. Bruzs, Velocity of thermal decomposition of carbonates, J. Phys.Chem. 30 (5) (1926) 680e693.

[28] T. Kujirai, T. Akahira, Effect of temperature on the deterioration offibrous insulating materials, Sci. Pap. Inst. Phys. Chem. Res. (Jpn) 2(21) (1925) 223e252.

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[29] I.S.M.A. Tawakkal, M.J. Cran, S.W. Bigger, Effect of kenaf fibre loadingand thymol concentration on the mechanical and thermal proper-ties of PLA/kenaf/thymol composites, Ind. Crops Prod. 61 (2014)74e83.

[30] F. Yao, Q. Wu, Y. Lei, W. Guo, Y. Xu, Thermal decomposition kineticsof natural fibers: activation energy with dynamic thermogravi-metric analysis, Polym. Degrad. Stab. 93 (1) (2008) 90e98.

[31] S. Ouajai, R.A. Shanks, Composition, structure and thermal degra-dation of hemp cellulose after chemical treatments, Polym. Degrad.Stab. 89 (2) (2005) 327e335.

[32] A.R. Bertoti, S. Luporini, M.C.A. Esperidi~ao, Effects of acetylation invapor phase and mercerization on the properties of sugarcane fi-bers, Carb. Polym. 77 (1) (2009) 20e24.






































102

Chapter 6 – Thymol Interactions in PLA Composites

Interaction and Quantification of Thymol in Active PLA-Based Materials Containing

Natural Fibers

Overview

Chapter 6 examines the quantification/retention of thymol in PLA and PLA/kenaf films after

thermal processing with a discussion on the interactions of thymol between the PLA matrix

and the kenaf fibre filler. The algorithms of the thermogravimetric (TG) assessment of the PLA

and PLA/kenaf composites containing thymol degradation under non-isothermal was used as a

tool to investigate the interactions between the thymol, the kenaf fibres and the PLA matrix as

reported in this chapter. The potential applications of these systems as AM food-packaging

materials are also considered.

The paper entitled “Interaction and Quantification of Thymol in Active PLA-Based Materials

Containing Natural Fibers” by Tawakkal I. S. M. A., Cran M. J. and Bigger S. W. was published

in the Journal of Applied Polymer Science, 132, 42160 (1-11), 2016.

A part of the research work was selected as the Cover Image in Journal of Applied Polymer

Science, Special Issues: Bio-Based Packaging, Volume 133, Issues1-2, 2016.

103

104

Interaction and quantification of thymol in active PLA-basedmaterials containing natural fibers

Intan S. M. A. Tawakkal,1 Marlene J. Cran,2 Stephen W. Bigger1

1College of Engineering and Science, Victoria University, Melbourne 8001, Australia2Institute for Sustainability and Innovation, Victoria University, Melbourne 8001, AustraliaCorrespondence to: M. J. Cran (E - mail: [email protected])

ABSTRACT: The quantification of thymol, a commercial essential oil extract that is an antimicrobial (AM) agent, in poly(lactic acid)

(PLA) and PLA/kenaf composites was investigated to explore the potential of these systems as AM food-packaging materials. Neat

PLA and PLA/kenaf composites containing thymol (5–10 wt %) were prepared via melt blending and compression molding. The

quantification of the thymol content in PLA and PLA/kenaf composites after processing as well as the interactions between the PLA

matrix, kenaf fibers and the AM agent were investigated. The PLA/kenaf composites in the range of 10 to 40 wt % fiber content

retained less thymol upon processing than PLA alone and the composites containing the highest fiber loadings demonstrated the low-

est thymol retention. The observed losses were attributed to the higher mechanical shear that exists during the mixing process as well

as the creation of voids in the composites that facilitate the release of thymol from the system. VC 2015 Wiley Periodicals, Inc. J. Appl.

Polym. Sci. 2015, 132, 42160.

KEYWORDS: biopolymers and renewable polymers; composites; fibers; packaging; thermal properties

Received 16 December 2014; accepted 5 March 2015DOI: 10.1002/app.42160

INTRODUCTION

The utilization of biocomposites for active food packaging is

currently under investigation with a major purpose being to

reduce environmental pollution as well as recover biodegradable

polymers.1–3 Nowadays, renewable polymers such as PLA that

are derived and synthesized from plant materials are widely

used for films and coatings as well as matrices for incorporating

naturally sourced additives such as antimicrobial (AM) agents

that prolong the shelf life of packaged food products.4–7 Several

additives have been incorporated directly into polymers includ-

ing organic acids, enzymes, bacteriocins, chelators and a range

of plant extracts.8–11

For food-packaging applications, the concept of AM agent

migration is used to provide continuous AM activity to food

products. This can be achieved by using volatile additives

derived from plant extracts (e.g., essential oils) whereby these

natural agents are considered to be much safer than syntheti-

cally derived chemical agents.12 Thymol and carvacrol which are

the major constituents of thyme essential oil can act as antioxi-

dants and AM agents. These compounds are amongst the most

currently studied natural additives that can be incorporated into

packaging materials.13–17 However, essential oils have low ther-

mal stability and high volatility and so their exposure to high

temperature, shearing and pressure during processing (e.g.,

extrusion, injection and blown molding) often results in their

loss from the matrix and consequently a reduction in the AM

activity of the system.15 For instance, extruded PLA demon-

strated a loss of thymol with lower inhibition of Listeria mono-

cytogenous.11 Furthermore, PLA has a relatively lower melting

temperature than many commercial food-packaging materials

such as poly(ethylene terephthalate). Nevertheless, the process-

ing temperature of PLA using an extruder is normally greater

than 150�C and this is crucial to ensure optimal melt viscosity

as well as the complete melting of the crystalline phase in the

matrix during extrusion.18 Such conditions enable the AM agent

to be evenly distributed in the amorphous regions of the poly-

meric material and thus regulate a slow release of the agent

from the film.19

Plasticizers and fillers such as polyethylene glycol (PEG), natural

fibers and nanofillers are used to facilitate the controlled release

and to increase the activity of AM agents. Recent studies by

Ramos et al.20 reported an increment in the thermal stability of

the essential oil thymol upon processing due to the incorpora-

tion of nanoclay (e.g., montmorilonite) into active PLA films

where the nanoclay was incorporated to control the thymol

release in the active films. Liu et al.21 reported that the incorpo-

ration of plasticizers during the extrusion process lowered the

temperature profile during the manufacturing of AM films.

VC 2015 Wiley Periodicals, Inc.

WWW.MATERIALSVIEWS.COM J. APPL. POLYM. SCI. 2015, DOI: 10.1002/APP.4216042160 (1 of 11)

105

http://www.materialsviews.com/

In that study, PLA/Nisaplin films showed no AM activity

whereas PLA blended with the lactide dimer of PLA and PLA

plasticized with glycerol triacetate (GTA) that were used to cre-

ate membranes containing Nisaplin each prevented the growth

of L. monocytogenes in (BHI) broth. Prapruddivongs and Som-

batsompop22 found that a higher loading of wood flour (10 wt

%) resulted in facilitating the release of more triclosan onto a

PLA composite surface due to the hydrophilic nature of the

wood flour causing water molecules to be absorbed by the sur-

face of the composite. Similar findings were reported by Worap-

rayote et al.23 where sawdust particles helped to embed pediocin

into coated PLA composites and significantly inhibited the

growth of L. monocytogenes in agar media and sliced pork

mince. Nevertheless, in these studies, there is little information

on the quantification of AM agents after the processing of the

active PLA-based materials containing natural fibers.

It is clear that more research needs to be conducted to investi-

gate the quantification of AM agents when high temperature,

shear and pressure are applied during processing. The release

profile and AM inhibition activity in order to produce efficient

active films or materials also requires further investigation along

with the possible interaction of the AM agent with other addi-

tives in the matrix. To date, it appears that no study in the liter-

ature has systematically addressed the quantification of thymol

or other essential oil AM agents after plastic thermal processing

for PLA-based materials containing natural fibers. In the current

study, a natural fibrous substance, namely kenaf fiber (Hibiscus

cannabinus L.), is of interest as a reinforcing filler for PLA-

based composites. The composite material is expected to be

advantageous when compared to many synthetic composite

materials due to the renewability of the raw materials from

which it is comprised and its propensity to be environmentally

friendly.2,23,24 Moreover, the incorporation of the natural fibers

as a filler in the biopolymer can also improve its mechanical

properties, reduce abrasion resistance during processing, pro-

mote good compatibility and enhance biodegradability.25,26

Tawakkal et al.7 reported that the PLA/kenaf composites dem-

onstrated improved mechanical properties such as tensile

strength and stiffness compared with commercial PLA due to

the reinforcement of the kenaf fibers in the PLA matrix. How-

ever, the increased stiffness and hence less flexibility means that

these materials are perhaps more suitable to be developed as

rigid food-packaging materials.

The objective of this study is to quantitatively investigate the

retention of thymol incorporated into PLA and PLA/kenaf com-

posite films during processing as well as the thermal release

kinetics of thymol from these materials. These parameters are of

importance in order to understand the interactions amongst the

matrix, natural fibers and AM agents in the films and the ability

of the films to initiate and maintain effective AM activity.

EXPERIMENTAL

Materials

Poly(lactic acid) (7001D IngeoTM; specific gravity 1.24; melt

flow index (MFI) 6 g/10 min at 210�C and 2.16 kg; melting

temperature range 145–160�C) was obtained from NatureWorks

LLC. Mechanically separated kenaf fiber (bast) was purchased

from Ecofibre Industries, Australia. The thymol (T0501, purity

of 99.5%) was purchased from Sigma Aldrich Pty. Ltd., Aus-

tralia. Sodium hydroxide and acetic acid were purchased from

Merck Chemicals, Australia. Un-denatured ethanol was pur-

chased from Chem-Supply Pty Ltd., Australia. Isooctane (2,2,4-

trimethylpentane, 36006) was purchased from Sigma Aldrich,

Australia.

Preparation of Active PLA/Kenaf/Thymol Composites

The kenaf fiber surface treatment was performed by immersing

fibers in 5% w/v sodium hydroxide (NaOH) for 2 h at room

temperature. Acetic acid was used to adjust the pH (until neu-

tralized) during the process of washing and rinsing the fibers

with distilled water. Treated kenaf (TK) fibers were filtered from

the solution, washed, and later dried overnight in an air oven at

105�C. The dried fibers were then ground and sieved using a

300–500 mm aperture sieve. The aspect ratio (L/D) of the kenaf

fibers was approximately 9 with an average length of 920 mm

and an average diameter of 104 mm. The preparation of

untreated kenaf (UK) and TK doped with thymol was per-

formed by immersing 20 g of fibers in 800 mL of 10–25% v/w

thymol/ethanol solution and stirring for 1–2 h. The doped

fibers were then filtered from the solution and dried overnight

in a laminar flow cabinet in order to evaporate the remaining

ethanol. The micrograph images of TK and TK doped with 25

wt % thymol can be seen in Figure 1. These show that the dop-

ing of the TK was successful as a smooth fiber surface is

observed on the doped TK sample. Prior to mixing, the PLA

resin was dried in an oven at 60�C overnight before blending

with kenaf (undoped and doped with thymol) at various con-

centrations in the range zero to 40 wt %.

To produce active PLA as well as PLA/kenaf films, the PLA, UK

or TK fibers (zero to 40 wt %) or thymol were compounded

using an internal mixer (Haake PolyLab OS, Germany) at

50 rpm and at a processing temperature of 155�C for 8–10 min.

The samples were prepared by using a laboratory press (L0003,

IDM Instrument Pty. Ltd., Australia). The PLA and composites

were preheated at 150�C for 2 min and pressed at the same

temperature for 3 min under a pressure of 50 kN before quench

cooling to 30�C under pressure. The average thickness of the

heat pressed PLA and PLA/kenaf films incorporated with thy-

mol were 0.19 6 0.03 and 0.25 6 0.05 respectively. A hand-held

micrometer (Hahn & Kolb, Stuttugart, Germany) was used for

measuring the film thickness.

Infrared Analyses

The infrared spectral analysis of PLA and PLA/kenaf composite

film samples was performed using a Shimadzu IR Prestige Fou-

rier transform infrared (FTIR) spectrophotometer and utilizing

the attenuated total reflectance (ATR) technique. For thymol

and kenaf fiber samples, a small portion of thymol or kenaf

fiber powder was mixed in an agate mortar and pestle with a

few drops of paraffin oil. The sample was then applied to a KBr

disc and its FTIR spectrum recorded. All spectra were recorded

in absorbance mode in the range of 550–4000 cm21 with a

resolution of 4 cm21 and with 32 scans recorded at every point

using Happ-Genzel apodization. Ten scans were performed for

each acquisition.

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Thermogravimetric Analyses

A Mettler Toledo (TGA/DSC 1 Star System) was used to under-

take the thermogravimetric (TG) analyses. The weight percent-

age of thymol that was retained in the samples after processing

was measured from the normalized weight loss curve and the

derivative of the weight loss curve, the latter being used to iden-

tify the start and end points of the process. The PLA and PLA/

kenaf composite samples containing thymol were heated from

30 to 500�C at a heating rate of 5�C min21 and under a nitro-

gen atmosphere flow rate of 0.2 L min21.

Thymol Quantification in PLA and PLA/Kenaf Composites

Reflux extraction followed by gas chromatography (GC) was

used to analytically determine the weight percentage of thymol

that was retained in the samples after processing. One gram of

compressed sample was extracted at 100�C for 2–5 h using

100 mL of isooctane or 95% ethanol. An aliquot of the solution

was analyzed using GC. The conditions applied in the GC

instrument were as follows: injected volume, 1.0 mL; initial col-

umn temperature, 80�C; heating rate, 5�C min21 up to 120�C,

held at this temperature for an additional 5 min; injector tem-

perature, 250�C; FID detector temperature, 300�C; flow rate,

2 mL min21; splitting; carrier gas, nitrogen. A standard curve

for thymol was also prepared and the thymol content of the

samples was calculated using this curve.

Thymol Release Using TG Kinetic Analyses

The application of non-isothermal techniques for the determi-

nation of kinetic parameters of reaction measured by loss in

weight has been long established.27 In the current study, non-

isothermal TG kinetic analyses of the release of thymol from

PLA and PLA/kenaf composite samples were performed by a

computer-based iterative numerical method using original soft-

ware. The software was developed to execute an integral solu-

tion of the general kinetic equation pertaining to TG analysis:

gðaÞ 5 ðAEa=RbÞ3 p xð Þ (1)

where a is the degree of conversion at time t in the process, A

is the Arrhenius A-factor, Ea is the apparent activation energy

for the process, R is the ideal gas constant and b is the heating

rate. The function p(x) represents the integral:

p xð Þ 5

ð1x

exp –xð Þ=x2� �

dx (2)

where x 5 Ea/RT and T is the absolute temperature The data

pertaining to the release of thymol were analyzed according to a

3D diffusional model28 and an algorithm developed from the

work of Dollimore et al.29 was used to confirm that this model

was the most appropriate one needed to fit the data. For a 3D

diffusion model:

gðaÞ 5 ½1– ð1–aÞ1=3�2 (3)

All TGA profiles were analyzed up to 85% conversion with

respect to the corresponding first step in the TG analysis profile

in order to extract the apparent activation energy and Arrhenius

A-Factor data.

Morphology of Fibers and Composites

Scanning electron microscopy (SEM) was conducted to observe

the morphology of TK, TK doped with thymol as well as that

of the composites. The composite films were immersed in liquid

nitrogen and then fractured in order to create a fracture surface

of the films for observation. All micrographs were obtained

using a JOEL NeoScope (JCM-5000) scanning electron micro-

scope. Samples were coated with a thin layer of gold (6 nm)

using a NeoCoater (MP19020NCTR) device under high vacuum

and using an optimal accelerating voltage of 10 kV to avoid

charging effects.

RESULTS AND DISCUSSION

Structural Properties

Figure 2 shows the FTIR spectra of the neat PLA, thymol and

TK fibers. The absence of the OAH stretching band in the spec-

trum of the neat PLA confirms that this particular grade of

PLA (7001D) is hydrophobic. The carbonyl (>C@O) stretching

peak at 1746 cm21 is due to the carbonyl group in the lactic

acid ester moiety of PLA.30 The deformational vibrations of

CAH in the methyl group of PLA are also observed in the range

Figure 1. Scanning electron micrographs of: (a) TK fiber at 5003 magnification and (b) TK fiber doped with 25 wt % thymol at 5003 magnification.



107



of 1300 to 1500 cm21.30 The peak at 1180 cm21 is due to the

stretching vibration of CAOAC and another asymmetric

stretching vibration of CAOAC is observed in the range of

1150–1060 cm21.31

The FTIR spectrum of thymol exhibits a number of major peaks

as seen in Figure 2 and these are also tabulated in Table I along

with the major peaks observed in the spectra of PLA and PLA/

TK composites containing thymol. The following spectral fea-

tures are apparent in Figure 2: a broad band due to the OAH

hydroxyl group stretching vibration appears in the range of

3400–3500 cm21; the CAH methyl group stretching at

2945 cm21; a strong absorption due to the phenolic CAO

stretching in the region at 1215 cm21; CAC stretching at

1419 cm21; and strong peaks due to isopropyl stretching and

ring aromatic CAH bending at 1288 cm21 and 810 cm21

respectively.32 Moreover, the FTIR spectrum of the TK fibers

exhibits a broad OAH band at 3500-3400 cm21 with the

expected absence of a sharp, carbonyl group absorption at

approximately 1700 cm21. This is due to the removal of ester

groups in the hemicellulose during the alkali treatment of the

surface of the kenaf fibers.33 A similar observation was reported

by Himmelsbach et al.34 who found that the ester groups of

hemicellulose or the ester linkage of the carboxylic group of

ferulic and p-coumaric acids of lignin and/or hemicellulose dis-

appeared in the spectrum of cellulose fibers.

From Table I, it can be noted that the spectra of the PLA and

PLA/TK composites containing thymol are similar. This could

be due to the high content of PLA present in the surface of the

pressed films. Nevertheless, each of the PLA and PLA/TK com-

posites containing 20 wt % thymol demonstrated a significant

shift of O2H group absorptions that appear in the regions of

approximately 3510 cm21 respectively as compared with the

thymol spectrum. This suggests that the thymol interacts with

the PLA and/or the TK fibers. Intermolecular hydrogen bonding

is presumed to exist between thymol and TK as well as thymol

and PLA. Furthermore, hydrogen bonding is likely to be present

in the composite between the hydroxyl groups in the TK fibers

and the terminal hydroxyl groups of PLA,35 the carbonyl groups

of the ester linkages of PLA36 as well as the thymol terminal

hydroxyl group. The FTIR spectrum of the PLA/TK composite

containing 20 wt % TK and 20 wt % thymol was similar to that

of the PLA containing 20 wt % thymol (see Table I). A small

but noticeable peak broadening of the hydroxyl group absorp-

tion was observed in the active PLA/TK composite compared to

PLA containing 20 wt % thymol. This is attributable to the

presence of the hydrophilic TK fibers in the composite [see Fig-

ure 3(a)].

Figure 3(b) shows the normalized carbonyl absorptions of neat

PLA and PLA/TK composites containing 20 wt % thymol, where

a slight hypsochromic shift in the carbonyl peak (at 1755 cm21)

of the PLA is observed upon the addition of thymol to the PLA.

A small shoulder on the peak is observed in the carbonyl group

absorption at 1750 cm21 for PLA containing 20 wt % thymol

and this shoulder becomes more pronounced with the presence

of TK fibers in the film. The peak shift and presence of the peak

shoulder support the notion of an intermolecular interaction

existing between the PLA and thymol. A similar observation was

made by Prapruddivongs and Sombatsompop22 who investigated

the interaction between PLA, 10 wt % wood flour and 1.5 wt %

triclosan by using FTIR. They reported that the incorporation of

triclosan and wood flour into PLA broadened the carbonyl

absorbance peak and caused carbonyl peak splitting at wavenum-

bers of 1753 and 1746 cm21. The data listed in Table I suggest

that overall the PLA/TK composite demonstrates similar spectral

features to those of neat PLA. The hydroxyl group absorption at

a wavenumber of ca. 3515 cm21 is possibly due to the low

amount of fibers on the surface of the pressed film. These fibers

are expected to create a surface roughness and may inhibit the

resolution of the ATR technique compared to the case of PLA

alone.

Thermal Properties

Figure 4 shows the normalized weight loss as a function of tem-

perature for PLA composites containing TK and 10 wt % thy-

mol. The profiles typically show an initial step that occurs over

the temperature range of ca. 90–300�C and corresponds to the

degradation/release of thymol from the matrix.13 The second,

Figure 2. FTIR spectra of PLA, thymol and TK fiber.



108



more pronounced step at 300–370�C corresponds to the degra-

dation of the PLA that presumably occurs by thermal depoly-

merization and decomposition.37 As the TK loading in the

formulation is increased the level of char remaining in the sys-

tem at elevated temperatures (ca. 390�C and above) is observed

to increase accordingly.

The quantification of thymol in the PLA and PLA/TK compo-

sites can be calculated from the TG profile as in the work of

Ramos et al.20 who investigated the retention of thymol in a

PLA matrix and found that there was some loss of this volatile

AM agent during processing. An example of a detailed analysis

of the TG profiles obtained in the current study is shown in

Figure 5 that shows the first derivative with respect to tempera-

ture, dw/dT, of the TG weight loss profiles. The value of the ini-

tial temperature at which thymol is released, Trel, decreases by

ca. 3.8�C upon the addition of 40 wt % TK to the formulation

(see Figure 5) suggesting that the addition of TK to the polymer

facilitates the loss of thymol from the PLA matrix. Similarly, a

smaller decrease of ca. 1.7�C in Trel is also observed in the case

where 10 wt % of TK is present in the formulation although

this decrease may not be significant. These findings are also

supported by the observation that the temperature at which the

maximum rate of degradation of PLA occurs, Tdeg, is lower in

the case of the sample with the highest loading of fiber than in

the case of PLA alone.

The values of Trel, Tdeg and the percentage of char residue of

the various formulations were determined from the complete

TG analyses and these are summarized in Table II. Overall, it

was found that the Trel value of PLA/TK composites decreased

with increasing TK fiber loading from 10% to 40 wt % over the

temperature range of 149.9 to 144.0�C. Thus, it is clear from

the results in Table II that the addition of TK to the formula-

tion decreases the temperature at which thymol is released from

the matrix at maximum rate and also decreases the temperature

at which the maximum rate of degradation of the PLA occurs

under the temperature ramp. The latter suggests that the addi-

tion of fiber destabilizes the polymer to some extent.38 More-

over, the addition of 5–10 wt % thymol has no significant effect

on the value of the Tdeg of PLA and this finding is in agree-

ment with the work of Ramos et al.20 who investigated the TG

properties of PLA containing 8 wt % thymol. Thus, the addition

of thymol to the formulation has little effect on the thermal sta-

bility of the material as a whole whereas the addition of TK

affects the thermal stability.

Quantification of Thymol

The effect of adding TK to the formulation on the quantifica-

tion of thymol released from the matrix can be further explored

by plotting the normalized Trel values of PLA/kenaf composites

containing 5 and 10 wt % thymol at various TK loadings in the

range of zero to 40 wt % (see Figure 6). An almost linear

Table I. Major Peaks of Thymol, PLA and PLA Containing Thymol and TK Fibers

Functional group Thymol PLA PLA/thymola PLA/TKb PLA/thymol/TKc Notes

OH 3483.6 – 3510.9 3505.8 3514.5 Significant shift of thymolOAH band incorporated incomposite; peak broadeningwith presence of TK

CH 2978.2 2951.2 2945.4 2951.2 2947.4 Significant shift of thymolCAH stretch when incorpo-rated in PLA and PLA/TK

C@O – 1747.6 1750.5 1747.6 1755.4 Small shift in C@O absorptionacross the active composites;peak broadening in activecomposites cf. neat PLA

Ring CH 945.2 – 947.1 – 947.1 Small shift of thymol ring CAHstretch when incorporated inPLA and PLA/TK

CH 2881.8 – 2875.9 – 2875.9 No significant shift

Ring 1622.2 – 1620.3 – 1618.4 No significant shift; weak peak

Ring 1581.7 – 1585.6 – 1585.6 No significant shift; weak peak

CC 1419.7 – 1423.5 – 1419.7 No significant shift; weak peak

CH or CC isopropyl 1288.5 – 1292.4 – 1292.4 No significant shift; weak peak

COC – 1180.5 1180.5 1180.5 1182.5 No significant shift; peakbroadening in composites cf.neat PLA

COC – 1078.3 1082.1 1078.3 1084.1 No significant shift; peakbroadening in composites cf.neat PLA

Ring CH 808.2 – 810.1 – 810.1 No significant shift

a thymol at 20 wt %; b TK fiber at 20 wt %; c PLA containing 20 wt % TK fiber and 20 wt % thymol



109



reduction in the Trel with the addition of TK fiber was observed

at each of the concentrations of thymol for which the composite

containing 40 wt % kenaf showed the lowest Trel (see Figure 6

and Table II). This suggests that the presence of the less ther-

mally stable fibers may also destabilize the PLA-thymol matrix.

Moreover, no significant change was observed for the Trel of

PLA and PLA/TK composite formulations containing 5 and 10

wt % thymol.

The effect of fillers such as TK on the release of thymol from the

polymer matrix has important implications particularly with

regard to the loss of the active agent during processing. The TG

analysis technique can be used to provide indirect confirmation

of the presence of AM agents in the polymer matrix after thermal

processing39 and so to this end it was decided to utilize the tech-

nique to investigate the effect of the TK filler on the quantifica-

tion of thymol in the PLA system following thermal processing.

The results from the TG experiments were corrected for the

inherent water content of the TK fibers and the analytical results

were also verified by solid–liquid extraction and GC analysis.19

Figure 7 shows the weight percentage of thymol that remains in

the PLA formulation following thermal processing for neat PLA

and PLA/TK composites containing different TK loadings where

the analyses were conducted using the TG technique and inde-

pendently confirmed by extraction followed by GC analysis.

There is an acceptable level of consistency between the results

obtained using the two techniques. The results suggest that the

unfilled PLA formulation exhibits the highest level of thymol

(ca. 8 wt %) following thermal processing and that the ability

of the PLA to retain thymol as such decreases upon increased

loadings of the TK filler. Generally, during the thermal mixing

process, friction occurs between the barrel and screw that mayFigure 3. FTIR spectra showing: (a) hydroxyl group and (b) carbonyl

group absorptions for: (i) neat PLA, (ii) PLA containing 20 wt % thymol,

and (iii) PLA/TK fiber composite containing 20 wt % thymol.

Figure 4. Normalized weight loss as a function of temperature for: (i)

neat PLA and PLA containing: (ii) 10 wt % thymol, (iii) 10 wt % TK and

10 wt % thymol and (iv) 40 wt % TK and 10 wt % thymol. The thermo-

grams were obtained using a heating rate of 5�C min21.

Figure 5. Plots of the first derivative with respect to temperature, dw/dT,

of the respective TG weight loss profiles shown in Figure 4 for PLA con-

taining: (i) 10 wt % thymol, (ii) 10 wt % TK and 10 wt % thymol and

(iii) 40 wt % TK and 10 wt % thymol.



110



lead to the degradation of the AM agents.40,41 In the current

study, it was found that the degradation/release temperature of

thymol Trel in active PLA film (see Table II) was lower (149�C)

than the processing temperature (155�C) at which the melt was

mixed for 8–10 min. The latter resulted in a considerable loss of

thymol from the PLA formulation containing 10 wt % thymol.

A similar finding was observed by Ramos et al.13,20 where ca.

75% of the initial thymol remained after processing. Active

PLA-based formulations containing butylated hydroxytoluene

(BHT) underwent similar losses due to factors such as poor

mixing in the extruder, evaporation and thermal degradation of

BHT.42

The effect of TK loading on the retention of thymol in the for-

mulation can be measured by comparing the residual thymol

concentration following processing with the nominal thymol

concentration in the formulation. For example, Figure 8 shows

plots of wT, the weight percentage of thymol in the PLA formu-

lation following thermal processing as determined using TG

analysis versus wF, the nominal weight percentage of thymol in

the formulation for systems containing two different TK load-

ings as well as a plot for a control sample (zero TK loading).

The PLA composite containing 10 wt % thymol and the higher

TK loading (40 wt %) exhibited lower thymol retention com-

pared to the PLA composite containing 10 wt % thymol and 10

wt % TK loading. However, this effect is not as pronounced at

the lower thymol concentration of 5 wt %. Overall, it was found

that the final weight percentage of thymol in the PLA/TK com-

posites containing 40 wt % TK could be lower than the nomi-

nal weight percentage by up to ca. 30%. Furthermore, the

Table II. Thermal Parameters Obtained from the Analyses of the TG Pro-

files of PLA and PLA/ TK Composites Containing Zero, 5% and 10 wt %

Thymol.

Thymolcontent(wt %)

TK content(wt %) Trel (�Ca) Tdeg (�C)

%Charresidueat 400�C

0 0 – 352.9 0.5

10 – 346.4 4.2

40 – 332.5 11.2

5 0 149.9 354.8 1.4

10 148.2 341.8 5.0

40 146.8 328.7 12.1

10 0 147.8 352.3 1.7

10 146.1 348.3 5.2

40 144.0 328.1 13.2

a Trel determinations were performed in triplicate.

Figure 6. Normalized maximum release rate temperature, Trel, of thymol

from PLA and PLA/TK composites containing: 5 wt % (w) and 10 wt %

( ) thymol. The determinations were performed in triplicate.

Figure 7. Weight percentage of thymol in the PLA formulation following

thermal processing as determined using TGA, wT, for neat PLA and PLA/

TK composites containing different kenaf loadings. Analyses were con-

ducted using: TG analysis (w) and extraction/GC ( ). The determina-

tions were performed in triplicate.

Figure 8. Plots of the weight percentage of thymol in the PLA formula-

tion following thermal processing as determined using TGA, wT, versus

the nominal weight percentage of thymol in the formulation, wF, for sys-

tems containing: zero (�), 10 wt % (�) and 40 wt % ( ) TK loading.



111



results confirm that as the TK loading is increased, the retention

of thymol during processing is decreased.

Analyses similar to those depicted in Figure 8 were conducted

at all loadings of TK used in this study and the gradients of the

plots, dwT/dwF, along with the corresponding linear coefficient

of determination (r2) are presented in Table III. In all cases the

gradient of the neat PLA (control) is greater than the gradient

obtained for the composites and there is a concomitant decrease

in the gradient with an increase in the TK loading. This con-

firms that the presence of TK in these latter systems decreases

the retention of thymol during processing with retentions rang-

ing from ca. 85% (neat PLA) down to ca. 69% (40 wt % TK

loading) for a thymol concentration of up to 10 wt %.

In the case of PLA/TK composites, the increased loss of thymol

at higher TK loadings during processing may be attributable to

the higher mechanical shear that exists during the mixing of the

composites and that most likely contributes to the loss of thy-

mol from the system through evaporation. The presence of a

high content of fiber in the PLA-thymol matrix may also lead

to the creation of voids that facilitate the release of thymol

from the composite. This suggestion is consistent with the SEM

images as seen in Figure 9 where voids and loose fibers are

observed on the facture surface of PLA/kenaf composites con-

taining 10 and 30 wt % thymol. Furthermore, the heat evolved

during the mixing process might liberate moisture in the TK

fiber to produce steam and subsequently facilitate the evapora-

tion of thymol. Indeed, the loss of thymol through a process

akin to steam distillation is possible as the boiling point of thy-

mol is lowered in the presence of steam. Mulvaney43 reported

that the boiling point of thymol is depressed in the presence of

steam, allowing it to evaporate at a temperature below that at

which the deterioration of the material becomes appreciable.

TG Kinetics Analysis

The apparent activation energy (Ea) for the release of thymol

from both active PLA and PLA/TK composites containing 10,

20 or 30 wt % thymol was calculated by applying a 3D-

diffusion model [see eqs. (1) and (3)] based on the TG analysis

results (release of thymol, Trel curve). The results are given in

Table IV along with: (i) the corresponding Arrhenius A-factors,

(ii) the goodness of fit to the 3D diffusion analysis model as

determined on a scale of zero to unity from the computer algo-

rithm, and (iii) the linear regression analyses of the plots of

g(a) versus p(x), including the corresponding coefficient of

determination, r2 of the latter. The linearity reflected in the

regression analyses demonstrates that the TG fitting protocol is

appropriate and provides some degree of confidence in the

derived value of the apparent activation energy. Figure 10 shows

a typical plot of g(a) versus p(x). In this case the plot pertains

to the analysis of the release of thymol from PLA containing 20

wt % TK and 30 wt % thymol. From the gradient of this plot

and the Arrhenius A-Factor, the value of Ea is calculated to be

68 kJ mol21 [see eqs. (1) and (2)] and this value corresponds

closely to the value of 65 kJ mol21 that was delivered by the

iterative computer analysis program.

The data listed in Table IV suggest that the apparent activation

energy for the release of thymol from PLA is ca. 46 kJ mol21.

As expected, this value does not appear to depend on the level

of thymol and repeated measurements of this parameter for six

replicates at three different thymol concentrations in the range

Table III. Linear Regression Analyses of PLA and PLA/TK Formulation

Containing Zero, 5% and 10 wt % Thymol

Composition dwT/dwF r2

Neat PLA 0.851 0.993

PLA110 wt % TK 0.746 0.996

PLA120 wt % TK 0.724 0.998

PLA130 wt % TK 0.692 0.990

PLA140 wt % TK 0.690 0.999

Figure 9. Scanning electron micrographs of: (a) PLA/TK composite containing 20 wt % TK and 10 wt % thymol at 1003 magnification and (b) PLA/

TK composites containing 20 wt % TK and 30 wt % thymol at 2003 magnification.



112



of 10–30 wt % thymol yielded an average result of 46 6 9 kJ

mol21. Similarly, Soto-Valdez et al.44 reported that the apparent

activation energy of the diffusion of resveratrol (antioxidant)

from PLA films immersed in different food simulants and at

different resveratrol concentrations remained almost constant

between 175 and 177 kJ mol21. The addition of TK to the for-

mulation significantly increases the apparent Ea for the release

of thymol from the PLA composite matrix, presumably due to

the interaction between TK and thymol that was established

from the FTIR analysis (see Figure 3 and Table I). Interestingly,

it appears that the level of TK does not significantly affect the

value of Ea for thymol release, as there seems only to be a small

increase in the latter value when the TK loading is increased

from 20 to 40 wt %. Assuming that the Ea for the release of

thymol from these systems is independent of the level of thymol

and loading of TK over the respective ranges that were tested in

the current study, the Ea data for the PLA/TK composites con-

taining thymol can be averaged to produce an overall result of

65 6 4 kJ mol21. Clearly, by comparing this result with the

average Ea value for the release of thymol from PLA alone dem-

onstrates that the addition of TK to the formulation signifi-

cantly increases the apparent activation energy for the release of

thymol. This may be due to the interaction between the thymol

and the kenaf as well as the presence of the filler producing a

reduction in amorphous regions through which the additive

molecules can be released.44,45

The interaction between the TK and thymol is also confirmed

in the apparent activation energy for the release of thymol from

TK fibers doped with 25 wt % thymol. The results in Table IV

suggest that the latter Ea value (106 kJ mol21) is significantly

greater than that which is associated with the release of thymol

from either the PLA alone or the PLA/thymol/kenaf composites.

Whence it can be proposed that the observed increased loss of

thymol that occurs on processing TK containing PLA compo-

sites (see Figures 6–9) may be due to the presence of the TK

which increases friction during processing as well as creates a

more open amorphous structure in the resulting material

thereby facilitating the release of the thymol. The data in Table

IV are also consistent with the notion that the alkaline chemical

pre-treatment of kenaf fibers enhances its compatibility and

attractive interactions with substrates such as PLA.46,47 In the

case of its interaction with thymol it can be seen that the appa-

rent Ea for thymol release from TK (i.e. 106 kJ mol21) is

greater than that for its release from UK where the value of the

latter is 96 kJ mol21. This difference in thymol release from the

doped fibers containing 25 wt % thymol is also reflected in the

release of thymol from the corresponding PLA composites. The

data in Table IV are consistent in this regard and show that the

addition of UK doped with thymol to the PLA composite sys-

tem lowers as expected the apparent Ea for the release of thy-

mol from the system.

Table IV. Kinetics Analyses of TG Data for PLA and PLA/Kenaf Composites Containing Thymol

g(a) vs p(x)

Linear regression analysis

Formulation Ea (kJ mol21)Ea (ave)a

(kJ mol21) A (min21)3D DiffModel Fit Regression equation r2

PLA120Th 46 46 6 9 6.33E103 0.922 y 5 8.948E106x 2 2.136E-02 0.981

PLA130Th 44 1.50E103 0.850 y 5 1.647E106x 2 3.666E-03 0.995

PLA110Th130UTKb 52 53 6 6 3.44E104 0.953 y 5 4.551E107x 2 4.587E-03 0.988

PLA110Th120TK 63 6.26E105 0.919 y 5 6.202E108x 1 1.991E-02 0.961

PLA120Th120TK 58 65 6 4 2.72E105 0.940 y 5 3.864E108x 2 6.240E-03 0.998

PLA130Th120TK 65 1.64E106 0.905 y 5 2.681E109x 2 4.579E-03 0.998

PLA110Th140TK 69 1.04E107 0.928 y 5 1.487E110x 1 5.603E-03 0.997

UK125Thb 96 98 6 4 1.80E111 0.960 y 5 4.577E114x 1 1.584E-03 0.977

TK125Thb 106 105 6 1 4.32E112 0.906 y 5 1.583E116x 2 6.636E-03 0.991

a Ea averaged over: six different systems for PLA1thymol; five replicates for PLA1thymol1UK; four different systems for PLA1thymol1TK; three repli-cates for UK1thymol; two replicates for TK1thymol.b Fibers doped with thymol.

Figure 10. A typical plot of g(a) versus p(x) for PLA/TK composite con-

taining 20 wt % TK and 30 wt % thymol.



113



CONCLUSIONS

The FTIR analysis of the active PLA and PLA/TK composites

containing thymol showed that the thymol interacts with PLA

and kenaf as revealed by the observed significant shifts in the

various FTIR absorption bands. Active PLA/KF composites

retain less thymol upon processing than PLA alone and the

PLA/KF composites containing the highest fiber loadings dem-

onstrated the lowest retained thymol content. This is despite the

fact that the apparent activation energy for thymol release from

the PLA/TK composites containing thymol being greater than

that found for the release of thymol from PLA alone. It would

therefore appear that the disruption to the crystalline regions

caused by the addition of kenaf, along with the concomitant

creation of voids and the resulting decrease in tortuosity, facili-

tate the release of the active agent thymol from the composite.

These effects seem to overshadow the intermolecular attractions

that occur as a result of hydrogen bonding between the compo-

nents in the composite. Clearly, the interactions between PLA,

thymol and kenaf when together in a polymer composite are

complex and it is difficult to make any further generalizations

based on the data obtained so far. Nonetheless, the exploration

of the interactions that exist between the pairs of separate com-

ponents in these systems can give valuable insight into the

mechanism of AM loss during processing and assist in identify-

ing measures that will minimize such losses in future commer-

cial applications.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the Ministry of Education,

Malaysia and the Universiti Putra Malaysia (UPM) for providing

the PhD scholarship for Intan Tawakkal and acknowledge the assis-

tance of technical staff from RMIT University especially Mr. Mike

Allan for the preparation of the composite samples.

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115



116

Chapter 7 – Release of Thymol from PLA Films

Release of Thymol from PLA-Based Antimicrobial Films Containing Kenaf Fibres as

Natural Filler

Overview

In this chapter, the migration of thymol from the PLA and PLA/kenaf films into food simulants

is examined. The release rates of thymol into simulants at different temperatures were

determined by using first-order kinetics, diffusion modelling and Fick’s law modelling with a

comparison of the various models. The final material appearance after the release of thymol

into simulants is also reported and discussed.

The paper entitled “Release of Thymol from PLA-Based Antimicrobial Films Containing

Kenaf Fibres as Natural Filler” by Tawakkal I. S. M. A., Cran M. J. and Bigger S. W. was

published in LWT-Food Science and Technology, 66, 629-637, 2016.

117

118

Release of thymol from poly(lactic acid)-based antimicrobial filmscontaining kenaf fibres as natural filler

Intan S.M.A. Tawakkal a, Marlene J. Cran b, *, Stephen W. Bigger b

a College of Engineering and Science, Victoria University, PO Box 14428, Melbourne, 8001, Australiab Institute for Sustainability and Innovation, Victoria University, PO Box 14428, Melbourne, 8001, Australia


Article history:Received 7 July 2015Received in revised form6 November 2015Accepted 7 November 2015Available online 11 November 2015

Keywords:Active packagingAntimicrobialThymolMigrationPoly(lactic acid)

a b s t r a c t

The migration of thymol, a natural antimicrobial (AM) substance, from poly(lactic acid) (PLA) filmscontaining 300 g/kg kenaf fibres into food simulants is reported. Neat PLA and PLA/kenaf films containing100 g/kg thymol were prepared via melt blending and heat pressing and were placed in contact with150 mL/L and 950 mL/L ethanol/water mixtures at different temperatures. First-order kinetics, diffusionmodelling and Fick's law modelling were used to describe the release. The release rate of thymol into950 mL/L ethanol/water at different temperatures displays Fickian behavior with diffusion coefficientvalues between 1 and 100 � 10�11 m2 s�1 with close to 100% of thymol being released. The release rate ofthymol is temperature dependent and is affected by the percentage of ethanol in the simulant. In the caseof neat PLA and PLA/kenaf films, a faster release occurred in 950 mL/L ethanol/water than in 150 mL/Lethanol/water with the composite film exhibiting a higher diffusion coefficient in each case.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Controlled release systems in food packaging have experiencedconsiderable growth recently due to developments in active pack-aging concepts such as the integration of antioxidant (AOX) and/orantimicrobial (AM) substances into packages in order to improvethe quality and safety of food products. In these systems, low mo-lecular mass compounds and/or substances are released from thepackage in a slow and controlled manner to maintain an adequateconcentration of the substance in the packed food for a certainperiod of time. The release of substances that involve migration isthe result of diffusion, dissolution and equilibrium processes(Crank,1979). There are various factors that influence themigrationof a substance from the packaging material including the filmfabrication method, the volatility and polarity of the substance, thechemical interaction between the substance and polymer chains,hydrophobicity and hydrophilicity of the polymer as well as foodproperties and composition (Suppakul, Miltz, Sonneveld, & Bigger,2003).

Poly(lactic acid) (PLA) is a polyester synthesized from therenewable, bio-derived, monomer lactic acid and can be used as AM

films and/or membranes for a different range of applications(Auras, Harte,& Selke, 2004; Karami, Rezaeian, Zahedi,& Abdollahi,2013). This GRAS (Generally Recognized As Safe) grade polymer canbe used in contact with food and fabricated via conventional pro-cessing procedures (Jamshidian, Tehrany, Imran, Jacquot, &Desobry, 2010). The use of fillers in combination with PLA hasbeen widely studied with aims to improve physicomechanicalproperties, reduce production costs and enhance biodegradability.Naturally derived additives or fillers such as starch and cellulosecan be combined with PLA and other active agents such as AM andAOX compounds. For example, Hwang et al. (2013) studied themigration of a-tocopherol and resveratrol from poly(L-lactic acid)(PLLA)/starch blend films into ethanol and found that the neat PLLAcontaining a-tocopherol had a lower release rate than the PLLA/starch blend films at 43 �C in 100% ethanol simulant with diffusivitycoefficients of 89 � 10�11 and 282 � 10�11 cm2 s�1 respectively. Asimilar trend was observed for resveratrol, a non-volatile AOXcompound with diffusion rate of 25 � 10�11 cm2 s�1 in neat PLLAand 40 � 10�11 cm2 s�1 in the PLA/starch films. Fortunati et al.(2012) prepared PLA AM films with 50 g/kg microcrystalline cel-lulose (MCC) and 10 g/kg silver nanoparticles by extrusion andinjection molding techniques. The PLA films with silver nano-particles and MCC had greater AM activity against Escherichia colidue to the presence of MCC; however, it had less AM activity thanPLA films with silver nanoparticles. Although there are examples of* Corresponding author.

E-mail address: [email protected] (M.J. Cran).

Contents lists available at ScienceDirect

LWT - Food Science and Technology

journal homepage: www.elsevier .com/locate/ lwt

http://dx.doi.org/10.1016/j.lwt.2015.11.0110023-6438/© 2015 Elsevier Ltd. All rights reserved.

LWT - Food Science and Technology 66 (2016) 629e637

119

the controlled release of AM and AOX substances from PLA mate-rials (Busolo & Lagaron, 2013; Fernandez, Soriano, Hernandez-Munoz, & Gavara, 2010; Hwang et al., 2013; Iniguez-Franco et al.,2012; Llana-Ruiz-Cabello et al., 2015), few reports have combinedPLA with natural fibres in order to control the release of activesubstances from composite films.

Antimicrobial packaging systems can be categorised as eithermigratory or non-migratory systems. In the former, AM substancesmigrate from the packaging material into the headspace of thepackage and onto the food surface, whereas in the latter, AM sub-stances are immobilised onto the packaging material which isplaced in direct contact with the foodstuff to facilitate its activity(Han, 2003). The integration of PLA with AM substances has beeninvestigated by a number of researchers (Del Nobile et al., 2009; Jin,2010; Qin et al., 2015; Rhim, Hong, & Ha, 2009). Of the reportedstudies, many have investigated the inhibition of targeted micro-organismswith little attention having been devoted to studying therelease rate from the active systems. This might be due to thepreponderance of non-volatile and/or immobilised AM substances(e.g. nisin, chitosan, lysozyme and peptide) incorporated into PLAfilms rather than volatile AM substances (Green, Fulghum, &Nordhaus, 2011; Rhim, 2013; Tawakkal, Cran, Miltz, & Bigger,2014b). Several AM substances have been incorporated directlyinto polymers including a range of volatile plant extracts such asbasil, thymol, linalool, methyl cinnamate and cavacrol (Cran,Rupika, Sonneveld, Miltz, & Bigger, 2010; Del Nobile, Conte,Incoronato, & Panza, 2008; Fern�andez-Pan, Mat�e, Gardrat, &Coma, 2015; Rubilar et al., 2013; Suppakul, 2004; Suppakul,Sonneveld, Bigger, & Miltz, 2011; Tawakkal, Cran, & Bigger, 2015).

Thymol, an essential oil extract that has GRAS status, can beused in contact with food products and like other volatile AMsubstances, the migration of this substance into real food productsis complex. In some studies, active AM films containing thymolwere evaluated in vitro and in vivo against a wide spectrum ofmicroorganisms such as bacteria, mould and yeast (Kuorwel, Cran,Sonneveld, Miltz, & Bigger, 2011; Wu et al., 2014). In a recent studyby Petchwattana and Naknaen (2015), extruded films of poly(-butylene succinate) (PBS) containing thymol demonstrated AMactivity against E. coli and Staphylococcus. Moreover, the release rateof thymol from the PBS films into 950 mL/L ethanol/water wasfound to be 5.9� 10�14 m2 s�1. Ramos, Beltr�an, Peltzer, Valente, andGarrig�os (2014a) reported that the migration of thymol frompolypropylene (PP) films into 950 mL/L ethanol at 40 �C conformedto Fick's law with a diffusion coefficient of 1.0 � 10�14 m2 s�1. Ingeneral, an AM substance can be released into a food simulant byswelling-controlled release with three main steps involved: (i) theabsorption of fluid (penetrant) from the food simulant which leadsto the swelling effect of the polymer, (ii) the active substancemolecule being dissolved or dispersed in the polymeric matrix and(iii) the active substance migrating to the food simulant (Del Nobile& Conte, 2013).

The quantification of volatile AM substances in active films is animportant consideration given that high pressure, shear forces andtemperatures are required for film processing. According toRaouche, Mauricio-Iglesias, Peyron, Guillard, and Gontard (2011), ahigher temperature of ca. 160e190 �C was needed to process PLAusing extrusion which may easily degrade and evaporate the vol-atile AM substance during thermal processing. The retention ofvolatile additives such as thymol in PLA film was found to beapproximately 70e80% after thermal fabrication (Tawakkal et al.,2015). Such retention percentages were significantly higher thanthose found in polyolefin film systems containing similar volatileadditives. For example, Ramos, Jim�enez, Peltzer, and Garrig�os(2012) reported much lower retention of thymol and carvacrol(ca. 25e45% respectively) in PP formulations. The marked

reduction in the retention of these additives in polyolefin filmsupon thermal processing may result from the reduced compati-bility between the polymer matrix and the natural additives as wellas the processing parameters such as temperature, time and screwrotation (Del Nobile et al., 2009).

There are clearly many examples of the release of a wide rangeof AM substances from homopolymers such as PLA. However, littleattention has been devoted to evaluating the release of AM agentsfrom ternary composite systems, particularly taking into accountthe possible swelling of polymeric materials immersed in foodsimulants as well as the effects of natural fillers on the release rate.Moreover, the release of volatile AM substances from polymericmaterials also has been restricted to mainly hydrophobic andmoderate hydrophilic polymeric matrices (Buonocore, Del Nobile,Panizza, Corbo, & Nicolais, 2003; Herath, 2009). The aim of thecurrent work was therefore to investigate the release of a naturallyderived AM agent from a novel ternary system comprised of PLA,kenaf fibers and thymol. In particular, the release of thymol fromPLA and PLA/kenaf composite and its diffusion kinetics werestudied.

2. Experimental section

2.1. Materials

Poly(lactic acid) (7001D Ingeo™; specific gravity 1.24; meltingtemperature 152.5 �C (Tawakkal, Cran, & Bigger, 2014a)) was ob-tained from NatureWorks LLC, USA. Mechanically separated kenaffibre (bast) was purchased from Ecofibre Industries, Australia.Thymol (T0501, purity of 99.5%) was purchased from Sigma AldrichPty. Ltd., Australia. Sodium hydroxide and acetic acid were pur-chased from Merck Chemicals, Australia. Un-denatured ethanolwas purchased from Chem-Supply Pty Ltd., Australia. Isooctane(2,2,4-trimethylpentane, 36006) was purchased from SigmaAldrich, Australia.

2.2. Production of PLA/kenaf/thymol films

Kenaf fibres were soaked in 0.05 g/mL sodium hydroxide for 2 hat room temperature. The fibers were then filtered and washedwith distilled water prior to acid treatment to affect neutralizationby adding a few drops of acetic acid. The fibres were then filtered,washed and rinsed with distilled water to remove the acetic acid,the latter being confirmed using a pH meter (inoLab® pH7110,WTW GmbH, Germany). Finally, the fibres were dried overnight inan oven at 105 �C. Prior to mixing, PLA resin and kenaf fibres werefurther dried in an oven at 60 �C overnight before mixing withthymol at 100 g/kg concentration.

The film samples were prepared firstly by melt-blending thecomponents in an internal mixer (Haake PolyLab OS, Germany) at155 �C for 8 min and 50 rpm followed by heat pressing, according toa method previously reported by Tawakkal, Cran, and Bigger(2014a). The PLA was added to the mixer first and the kenaf andthymol were introduced once the polymer was molten in order toavoid unnecessary loss of thymol. In the current study, a 300 g/kgloading of kenaf fibres was used to produce the PLA/kenaf com-posite that has moderate flexibility as well as high strength andstiffness compared with unfilled PLAwhich is more suitable for theproduction of rigid packaging applications (Tawakkal, Cran, &Bigger, 2014a). In a second step, a laboratory press (L0003, IDMInstrument Pty. Ltd., Australia) was used to prepare films. Thesamples were preheated at 150 �C for 3 min without applyingpressure until the material melted, and then pressed at the sametemperature for 2 min under a force of 20 kN before quench coolingto 30 �C under pressure. The average thicknesses of the pressed

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120

neat PLA and PLA/kenaf films incorporated with thymol were0.19 ± 0.03 and 0.25 ± 0.05mm respectively. The film thickness wasmeasured using a hand-held micrometer (Hahn & Kolb, Stuttugart,Germany).

2.3. Quantification of thymol in PLA and PLA/Kenaf films

One gram of film sample was cut into pieces (0.25 cm2) andimmersed in a round bottom flask containing 150 mL of isooctanefor solideliquid extraction. Isooctane was used as it promotes theswelling of the polymer and a typical reflux extraction was per-formed at 100 �C for 4e5 h to extract thymol from the films. Thequantification was achieved with a gas chromatography (GC) in-strument (Varian 8200Cx) equipped with a fused silica capillarycolumn (DB5; 30 m � 0.25 mm i.d.; thickness 0.25 mm; J & WScientific, USA). The conditions applied in the GC instrument wereas follows: injected volume: 1.0 mL, initial column temperature:80 �C; heating rate: 5 �C min�1 up to 120 �C, held at this temper-ature for an additional 5 min; injector temperature: 250 �C; FIDdetector temperature: 300 �C; flow rate: 2 mL min�1; splitting;carrier gas: nitrogen. Standard solutions of thymol in isooctane atconcentrations from 0.05 to 1.0 mg mL�1 were prepared and usedto produce a calibration curve. The experiments were performed intriplicate. The retention of thymol in the neat PLA and PLA/kenaffilms following thermal processing was 73% and 62% respectively asreported by Tawakkal et al. (2015). The loss of thymol observed inthe present study for composite films seems to be at an acceptablelevel compared to petroleum-based (polyolefin) films containingvolatile additives.

2.4. Migration of thymol into food simulants

The release of thymol from neat PLA and PLA/kenaf films intoaqueous food simulants was studied at different temperatures. Therelease was examined via a total immersion migration test (EC.,1997) using 950 and 150 mL/L ethanol/water. Ethanol iscommonly used as food simulant to investigate themigration of AMsubstances from the PLA matrix. The 950 and 150 mL/L ethanol/water simulants are fatty and aqueous food simulants respectively.The sorption of ethanol by the PLA matrix may lead to the creationof voids and/or swelling of the matrix where it can penetrate thePLA chains and promote the migration of the active substance(Mascheroni, Guillard, Nalin, Mora, & Piergiovanni, 2010). In thecase of the 950 mL/L ethanol/water simulant the experiments wereperformed at 30, 40, 50 and 60 �C and for the 150 mL/L ethanol/water simulant experiments were performed at 60, 65, 75 and83 �C. In the latter experiments, the test temperatures were higherthan recommended in the standard methods (EC., 1997) in order toaccelerate the migration of thymol. Studying the release rates ofthymol from the neat PLA and PLA/kenaf films by using fatty andaqueous food simulants is important in order to assess the in vitroand in vivo AM activity of these systems against targetedmicroorganisms.

Samples of film weighing ca. 0.5e0.6 g were immersed in100 mL of simulant in a three-neck round bottom flask with theratio of simulant volume per area film being ca. 2.7e4.7 mL cm�2.The flask was immersed in an oil bath that was placed on a mag-netic stirrer heating plate and the simulant was gently agitatedusing magnetic stirring at 60 rpm. The flask was connected to acondenser and the stirring speed and temperature were fixed andmonitored throughout the experiment. The amount of AM agentreleased from the films was monitored until equilibrium wasattained. A 0.2 mL sample of the simulant solution was collectedperiodically during the experiment and 1 mL aliquots were injectedinto the GC. The quantification of thymol in all simulants was

performed using the same method and calibration curve describedin the previous section. The thymol quantification was performedin triplicate.

2.5. Data analysis

The migration of thymol from the PLA and PLA/kenaf compositefilms was analysed using three data analysis treatments: (i) overallkinetics, (ii) diffusion models in accordance with Cran et al. (2010)and Kuorwel, Cran, Sonneveld, Miltz, and Bigger (2013) and (iii)Fick's diffusion law model.

2.5.1. Overall kinetics analysisBy considering the overall diffusion process to be a single pro-

cess that obeys first-order kinetics, equations describing themigration of an additive from a polymeric film into simulant withtime have been described by Miltz (1987) and Crank (1979). Therelease of the AM agent into the simulant was initially analysed forits fit to a first order kinetics model. In the case of a first-ordersystem, Equation (1) applies:

ln�1� mt

m∞

�¼ �k1t (1)

wheremt is themass of additive released from the film at time t,m∞is the amount of additive released from the film at equilibrium(t ¼ ∞) and k1 is the first-order rate constant. From Equation (1), aplot of ln(1 e mt/m∞) versus time should be a straight line with aslope of -k1. The apparent first-order rate constants were calculatedusing Equation (1) and the initial release rates, v0, of the AM agentwere calculated using Equation (2) (Kuorwel et al., 2013):

v0 ¼ m∞k1 (2)

2.5.2. Diffusion modelIn the diffusion model, the release of the AM agent from the film

into the simulant is considered in two stages, namely the short-term and the long-term (Crank, 1979; Miltz, 1987). This diffusionmodel is based on a geometry whereby the release is considered tooccur from both sides of the film. The diffusion data were analysedusing Equation (3) for short-term migration and the correspondingrate constants were calculated using Equation (4) for long-termmigration.

Short-term migration is defined as the time for which mt/m∞ <0.6:

mt

m∞¼ 4

�Dtpl2

�12

(3)

where D is the diffusion coefficient and l is the thickness of the film.A plot ofmt/m∞ versus t½ should yield a straight line fromwhich thediffusion coefficient can be obtained.

For long-term migration mt/m∞ > 0.6 and Equation (4) applies:

mt

m∞¼ 1�

�8p2

�exp

�� p2Dt

l2

�(4)

2.5.3. Fick's law modelThe diffusion coefficient of the AM agent can be determined

from its release versus time data, by fitting these data to Fick'ssecond law. Equation (5) can be derived from Fick's second law inthe case of one dimensional diffusion from a limited volume of filmthat is in contact with an infinite volume of solution (Crank, 1979).

I.S.M.A. Tawakkal et al. / LWT - Food Science and Technology 66 (2016) 629e637 631

121

mt

m∞¼ 1� 8

p2

X∞m¼0

1

ð2mþ 1Þ2exp

"�Dð2mþ 1Þ2p2t

l2

#(5)

2.5.4. Diffusion activation energyThe effect of temperature on the release rate of the AM agent

was modeled in accordance with the Arrhenius equation (Suppakul2004):

D ¼ D0exp�EaRT

�(6)

whereD is the diffusion coefficient,Do is the pre-exponential factor,Ea is the activation energy for the diffusion process, R is the ideal gasconstant, and T is the absolute temperature.


3.1. Release of thymol into simulants

Fig. 1(a) and (b) show plots of the mass fraction mt/m∞ versustime, t and the overall kinetic analysis for the release of thymolfrom neat PLA and PLA/kenaf films into 950 mL/L ethanol/water atthe four temperatures studied. The thymol release reached equi-libriumwithin ca. 9 h at the lowest temperature of 30 �C (Fig. 1(a)).A similar observation was found for PLA containing resveratrolwhere the system achieved equilibrium after ca. 14 h using thesame food simulant at 33 �C (Soto-Valdez, Auras,& Peralta, 2011). Incomparison, the times required to achieve the equilibrium con-centration of thymol in different polymeric systems with the samefood simulant at 40 �C were found to be 50 h for PBS/thymol filmsand 150 h for PP/thymol films (Ploypetchara, Suppakul, Atong, &Pechyen, 2014; Ramos, Beltr�an, Peltzer, Valente, et al., 2014a) and2 h for starch/thymol films in isooctane at 35 �C (Kuorwel et al.,

2013). In the current work, it was found that increasing the tem-perature to say 60 �C, increases the release rate of thymol andequilibrium was attained within 1.4 h. At the end of these experi-ments, ca. 87e100% and 82e95% of the thymol was released fromthe neat PLA and PLA/kenaf films respectively into 950 mL/Lethanol/water over the temperature range of 30e60 �C.

The initial release rates and the overall rate constants for therelease of thymol that were calculated from the data in Fig. 1(b) arepresented in Table 1. For both neat PLA and PLA/kenaf films, theinitial release rates and the overall rate constants for thymol releaseinto 950 mL/L ethanol/water consistently increased with an in-crease in temperature from 30 to 60 �C. This is consistent with thefindings of Kuorwel et al. (2013) where an increase in temperaturewas found to have a significant effect on the migration of the ad-ditive from the film. From the results in Table 1 a similar observa-tion can also be made in the case of the 150 mL/L ethanol/watersimulant over the range of the four temperatures studied. However,in the latter case the rates are significantly lower compared withthe 950 mL/L ethanol/water case. The experimental results werefurther analyzed by using the diffusion model in which the short-term and the long-term migration periods were considered.

Fig. 2(a) and (b) show the respective plots of mt/m∞ versus t1/2

for the short-term release of thymol and ln(1 emt/m∞) versus t forthe long-term release of thymol from the neat PLA and PLA/kenaffilms into 950 mL/L ethanol/water at 30 �C. A similar behavior wasobserved at each of the temperatures that were studied and thelinearity of the plots suggests that the data conform well to thediffusion model given in Equation (3) for short-term release. Valuesof the apparent diffusion coefficient at each of the various tem-peratures were determined from the gradients of these plots andare also presented in Table 1. For the long-term release, the linearityof the ln(1 emt/m∞) versus t plots also confirms the data reliably fitthe model presented in Equation (4). Indeed, all results exhibitedgood linear correlation with correlation coefficients (r2 values)

Fig. 1. Plots of: (a) the mass fractionmt/m∞ versus t, and (b) ln(1 �mt/m∞) versus t for the release of thymol from: (i) PLA and (ii) PLA/kenaf films into 950 mL/L ethanol/water at: -30 �C, , 40 �C,C 50 �C and B 60 �C where mt is the mass of thymol released from the film at time t and m∞ is the amount of thymol released from the film at equilibrium (t ¼∞).


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greater than 0.97.The results in Table 1 confirm that the diffusion coefficients of

thymol from the neat PLA and PLA/kenaf films into the 950 mL/Lethanol/water increase with increasing temperature. As expected,the diffusion rates of the composite films are higher than thoseobtained for the neat PLA films. This may be attributed in part to thepresence of the kenaf fibre filler in the polymer matrix that createsvoids thereby enabling the release of thymol from the film. Thesefindings are consistent with the result obtained for the retention ofthymol in a previous study whereby following processing the

composite filmswere found to retain less thymol than neat PLA film(Tawakkal et al., 2015). It is important to note that the values of theD parameter found for the neat PLA and PLA/kenaf films containingthymol are one order of magnitude higher than those observed foractive low-density polyethylene (LDPE), PBS and PP containing asimilar AM agent and using 950 mL/L ethanol/water simulant (Cranet al., 2010; Ploypetchara et al., 2014; Ramos, Beltr�an, Peltzer,Valente, et al., 2014a). Moreover, and as expected, active PLAfilms containing a non-volatile and thermally stable agent (i.e.resveratrol) prepared by Soto-Valdez et al. (2011) exhibit diffusion

Table 1Kinetic and the diffusion analyses for the release of thymol from PLA and PLA/kenaf composite films.

Temperature/�C First order kinetic analysis Diffusion analysis Fickian diffusion

SSEa Short term SSE Long term SSE SSE

no � 105/g s�1 k1 � 105/s�1 D � 1012/m2 s�1 k2 � 105/s�1 D � 1012/m2 s�1

950 mL/L ethanol/water

PLA 30 5.2 14.0 0.220 41 0.003 13.7 0.197 0.29 0.01040 7.3 20.1 0.111 93 0.002 20.0 0.057 0.60 0.06050 30.2 66.6 0.147 236 0.004 69.2 0.082 1.63 0.09960 49.9 119.6 0.569 1124 0.002 117.8 0.451 5.75 0.373

PLA/kenaf 30 5.4 14.4 0.254 90 0.001 13.8 0.194 0.56 0.00140 7.9 23.5 0.100 162 0.003 24.1 0.060 1.14 0.07950 38.8 73.2 0.036 443 0.002 73.2 0.009 3.10 0.06560 46.1 141.0 3.686 1340 0.000 124.4 2.720 10.05 0.498

150 mL/L ethanol/water

PLA 60 0.8 4.8 0.086 28 0.001 5.2 0.053 0.17 0.00665 2.7 9.3 0.089 43 0.010 8.9 0.066 0.24 0.02675 12.3 46.0 0.325 132 0.003 45.9 0.248 0.66 0.07283 15.4 58.8 0.486 255 0.004 61.1 0.048 2.62 0.278

PLA/kenaf 60 1.8 7.0 0.395 75 0.002 7.2 0.327 0.40 0.01065 2.6 9.6 0.392 102 0.001 9.6 0.329 0.52 0.02275 10.0 36.7 0.460 195 0.001 35.9 0.373 0.90 0.09883 14.1 57.3 0.101 454 0.004 67.1 0.004 3.39 0.189

a SSE: Sum of squared errors.

Fig. 2. Plots of: (a) mass fractionmt/m∞ versus t1/2 and (b) ln(1 �mt/m∞) versus t for the release of thymol from: (i) PLA and (ii) PLA/kenaf films into 950 mL/L ethanol/water at 30 �Cwhere mt is the mass of thymol released from the film at time t and m∞ is the amount of thymol released from the film at equilibrium (t ¼ ∞).


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coefficients that are one order of magnitude lower than those ofPLA films containing a volatile AM agent such as thymol. Similartrends in the diffusion coefficients of thymol were observed for theneat PLA and PLA/kenaf films when immersed in the 150 mL/Lethanol/water simulant. However, the diffusion coefficients for thefilms immersed in the 150 mL/L ethanol/water simulant wereconsistently lower than those pertaining to the 950 mL/L ethanol/water simulant. The observed decrease in the diffusivity may beexplained by the lack of affinity between thymol and water thatleads to the limited solubility of thymol inwater. Moreover, PLA is ahydrophobic polymer but ethanol is sufficiently non-polar tofacilitate the swelling of the PLA matrix (Sato, Gondo, Wada,Kanehashi, & Nagai, 2013), dissolve the thymol and release it intothe bulk of the simulant. According to Manzanarez-L�opez, Soto-Valdez, Auras, and Peralta (2011), ethanol is also an aggressivesolvent for PLAwhich can penetrate into PLA chains and release theactive substance.

The lower concentration of ethanol in the 150 mL/L ethanol/water simulant may lead to a slight extent of swelling as well ashydrolysis of the PLA (Manzanarez-L�opez et al., 2011). Interestinglythis slight swelling of the PLAmatrix appears to be more significantthan the swelling effect of the kenaf fibres. The PLA/kenaf com-posites are semi-hydrophilic materials due to the presence of kenaffibres that act as a hydrophilic filler. One may therefore expect therate of diffusion of thymol from these composite systems (PLA/kenaf films) to increase as the water content of the simulant in-creases. Taib, Ramarad, Mohd Ishak, and Todo (2009) prepared PLA/kenaf composites containing polyethylene glycol (PEG) and re-ported that when the composite was immersed into the water, thefibres absorbwater leading to expansion of the fibres. Such an effectmay create internal stress in the adjacent matrix and resulting inthe formation of microcracks. However, the rate of diffusion ofthymol from the PLA/kenaf films was in fact found to be lower inthe 150 mL/L ethanol/water simulant than in the 950 mL/L ethanol/water simulant. It can therefore be suggested that a complex andstrong interaction exists between the kenaf, PLA and thymol in thecomposite systems. The PLA matrix may act as a coating agent tothe kenaf fibre filler, preventing the water from swelling the kenaffibres and releasing thymol that is associated with the fibres. Suchan interaction between the PLA, kenaf fibre and thymol was re-ported previously where these composite systems were studiedusing Fourier transform infrared (FTIR) spectroscopic and ther-mogravimetric (TG) analyses (Tawakkal et al., 2015). In that study,the activation energy for the thermal release of thymol, using a 3D

diffusion kinetic model, was found to be 46 kJ mol�1 in the case ofneat PLA containing thymol and 65 kJ mol�1 for the PLA/kenafcomposite system.

Fick's second law model was also used to calculate the diffusioncoefficient byminimizing the sum of the squared errors (SSE) of themeasured and calculated value (see Table 1). To determine the fit ofthe experimental data, Equation (5) was used and a non-linearregression function was applied to the data. Fig. 3 shows plots ofmt/m∞ versus t for the diffusion of thymol from PLA/kenaf film into950 mL/L ethanol/water at 30 �C where the data have been fittedusing the Fick's second law model. The satisfactory fit of theexperimental data suggests that the diffusion kinetics of thymolinto 950 mL/L ethanol/water from the composite can also beadequately described by Fick's model. The SSE values for all therelease models studied are also presented in Table 1. In general, theshort-term diffusion model presents the best fit for the releasekinetics with the lowest SSE values for all systems studied. This isfollowed by the Fickian diffusion model, with the first order modeland long-term diffusion models showing some higher SSE valuesfor some systems.

Using the diffusion coefficients obtained from the diffusionmodel, the effect of temperature on the diffusion coefficient for therelease of thymol into 950 and 150 mL/L ethanol/water wasdetermined and Arrhenius plots of the data are shown in Fig. 4. Theactivation energy for the diffusion process, Ea, was calculated fromthe slope of the ln(D) versus 1/T plot in each case (see Fig. 4) inaccordance with the Arrhenius equation (see Equation (6)). Theactivation energies for the release of thymol from the neat PLA andPLA/kenaf filmswere found to be: 90.6 and 76.2 kJ mol�1 in 950mL/L ethanol/water and 98.7 and 84.8 kJ mol�1 in 150 mL/L ethanol/water respectively. Significant differences were therefore found toexist amongst the Ea values for these active neat PLA and PLA/kenaffilms immersed in the 950 and 150 mL/L ethanol/water simulants.

The Ea values for the neat PLA films are significantly higher thanthose found for PLA/kenaf films and this is attributed mainly to thestrong interaction between the PLA and thymol that presumably isnot as strong in the presence of the kenaf filler. Furthermore, the Eavalues increase with an increase in water content of the simulantand this is consistent with the observations made in relation to thediffusion coefficients discussed above (see Table 1). The Ea valuesobtained in the present study are all considerably lower than thosereported elsewhere for the diffusion of butylated hydroxytoluene(BHT) from PLA/BHT films into 950 mL/L ethanol/water which was164.7 kJ mol�1 (Ortiz-Vazquez, Shin, Soto-Valdez, & Auras, 2011).This significant difference may be explained by the difference inmolecular interaction and hydrogen bonding that exists betweenthe polymeric matrix and the AM additive in these systems(Kuorwel et al., 2013). As expected, the trend in Ea values for thevarious systems when calculated using the Fick's law model areconsistent with those calculated by the diffusion model given thatthe latter model has been derived from the former model. Theactivation energies using the Fick's law model for the release ofthymol from the neat PLA and PLA/kenaf films were found to be:83.3 and 80.9 kJ mol�1 in 950 mL/L ethanol/water and 118.5 and96.3 kJ mol�1 in 150 mL/L ethanol/water respectively. These slightdifferences in the Ea values that were obtained using the differentmodels might be due to the use of the diffusion coefficients derivedfrom the short-term experimental data in the construction of theArrhenius plot.

3.2. Film appearance

Fig. 5 shows the images of neat PLA and PLA/kenaf films after therelease of thymol into 150 and 950 mL/L ethanol/water at thedifferent temperatures studied. A considerable change in color for

Fig. 3. Plot of the mass fraction mt/m∞ versus t for the release of thymol from the PLAfilm into 950 mL/L ethanol/water at 30 �C fitted using the Fick's law model where mt isthe mass of thymol released from the film at time t, and m∞ is the amount of thymolreleased from the film at equilibrium (t ¼ ∞).


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the neat PLA and PLA/kenaf films was observed after the releaseexperiments. These color changes are due to the effects of tem-perature as well as the presence of water in the simulants. The coloris observed to change from clear to opaque for the neat PLAimmersed in 150 mL/L ethanol/water with an increase in opacitywith increasing temperature. A similar observationwasmade in thecase of the 950 mL/L ethanol/water simulant. The opacity of ma-terials may be attributed to a polymer hydrolytic degradation

process which can be related to crystallization of the PLA matrix aswell as moisture absorption. The water molecules diffuse throughthe films, promoting hydrolysis leading to the formation of lowmolecular weight degradation by-products (Ramos, Beltr�an,Peltzer, Dominici, et al., 2014b). In addition, color changes frombrown to light brown are also observed for the compositesimmersed in 150 and 950 mL/L ethanol/water. For the PLA/kenafcomposites immersed in 150 mL/L ethanol/water, the PLA

Fig. 4. Arrhenius plots of ln(D) versus 1/T for the release of thymol into: (a) 950 mL/L ethanol/water and (b) 150 mL/L ethanol/water from:C PLA film andB PLA/kenaf filmwhere Dis the diffusion coefficient and T is the absolute temperature.

Fig. 5. Images of PLA and PLA/kenaf films after the release of thymol into 150 and 950 mL/L ethanol/water simulants at different temperatures.


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surrounding the fibres dissolved or delaminated from the filmsurface revealing the kenaf fibers and this effect was less apparentin the 950 mL/L ethanol/water simulant.

4. Conclusions

The results of this study suggest that thymol is readily releasedfrom neat PLA and PLA/kenaf films into 150 and 950 mL/L ethanol/water simulants and the process can be described by an overallfirst-order kinetics model that can be used to determine the initialrelease rate. The short- and long-term diffusion models alsoadequately describe the release of thymol from these systems andthe results are consistent with those obtained using a Fick's lawanalysis approach. The diffusion coefficient data suggest that theaddition of the kenaf filler to the PLAmatrix facilitates the release ofthymol from the matrix and that the diffusion follows an Arrheniusrelationship with temperature. Furthermore, an increase in polarityof the simulant that results from an increase in its water contentdecreases the propensity of thymol to be released from the com-posite system. Nonetheless, it is apparent that active PLA/kenafcomposites containing natural AM agents such as thymol are po-tential candidates to be explored further for use as active packagingsystems. Such systems have the advantage of being derived fromnatural sources, contain a naturally-derived AM agent and, as such,are expected to be more susceptible to biodegradation than theirsynthetically-based counterparts.

Acknowledgments

The authors gratefully acknowledge the Ministry of EducationMalaysia and Universiti Putra Malaysia (UPM) for providing thePhD scholarship for Intan S. M. A. Tawakkal. We would like toacknowledge Mr. Mike Allan and the technical staff from the RoyalMelbourne Institute Technology, Melbourne for their invaluableassistance with the preparation of the composite samples.

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Chapter 8 – Antimicrobial Activity and Storage Stability of Composite Films

Antimicrobial Activity and Storage Stability of Active PLA/Kenaf/Thymol Composites

Overview

The in vitro and in vivo analyses of AM activity of PLA and PLA/kenaf composite films against

E. coli or inoculated on the surface of processed meat samples are discussed. The influence of

thymol concentration and kenaf fibre loadings on the AM activity of the films are reported.

Moreover, the retention of thymol during storage under different storage conditions is

examined in this chapter. The capabilities of PLA and PLA/kenaf films containing thymol as

an AM packaging systems for real foodstuff can be suggested from the results.

The manuscript entitled “Effect of Poly(Lactic Acid)/Kenaf Composites Incorporated with

Thymol on the Antimicrobial Activity of Processed Meat.” by Tawakkal I. S. M. A., Cran M.

J. and Bigger S. W. has been accepted for publication in the Journal of Food Processing and

Preservation.

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130

131

Antimicrobial Activity and Storage Stability of Poly(Lactic Acid)/Kenaf/Thymol

Composites

Intan S. M. A. Tawakkal1, Marlene J. Cran2* and Stephen W. Bigger1

1. College of Engineering and Science, Victoria University, PO Box 14428, Melbourne,

8001, Australia.

2. Institute for Sustainability and Innovation, Victoria University, PO Box 14428,

Melbourne, 8001, Australia

*Institute for Sustainability and Innovation, Victoria University, PO Box 14428, Melbourne,

8001, Australia. Telephone: +61 3 9919 7642; E-mail: [email protected]


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8.1 Abstract

Bio-based composites comprised of poly(lactic acid) (PLA), kenaf fibres and thymol were

developed and their antimicrobial (AM) properties and stability under different storage

conditions investigated. The composite films containing 20-30% w/w thymol reduced E. coli

in tryptone soy broth after two days at 37°C and imparted a significant zone of inhibition in

contact with E. coli inoculated plates. The composite films also reduced E. coli inoculated on

the surface of the processed sliced chicken samples after 30 days at 10°C both in direct contact

and in the vapour phase. The thymol additive was retained in the PLA/kenaf films that were

wrapped with aluminium foil after 3 months of storage at ambient temperatures, however

unwrapped films lost some thymol to the atmosphere. The development of PLA/kenaf/thymol

composite films shows a strong potential for the development of active packaging systems in

order to extend the shelf life of some processed food products.

Keywords: antimicrobial activity, biopolymer, essential oil, natural fibres, food packaging

Practical Application: Packaging materials utilizing bio-based materials and a natural bio-

active agent, thymol, to impart antimicrobial activity were developed. The presence of a natural

bio-filler derived from kenaf fibres into a poly(lactic acid) matrix enhanced the release of

thymol and consequently inhibited the growth of microorganisms. This can potentially extend

the shelf life of some food products and can be further developed as a rigid packaging material

and/or coating.

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8.2 Introduction

Modern food packaging is designed to fulfil a range of functions in addition to providing a

primary container for food products (Risch, 2009). One such function is to minimize the usage

of preservatives added directly to food products in order to hinder foodborne illness outbreaks

that may result from microbial spoilage (Han, 2005). Various forms of antimicrobial (AM)

packaging have been implemented as alternatives to conventional preservatives to inhibit

microbial growth in food, maintain the quality of the produce and, ultimately, to reduce food

wastage (Qin et al., 2015; Ramos et al., 2013). There are several approaches used to integrate

AM agents into polymeric materials including direct addition during processing, surface

coating of formed articles, immobilizing AM agents by chemical grafting, and using polymers

that possess intrinsic AM activity (Fernández-Pan et al., 2015; Muriel-Galet et al., 2013;

Peretto et al., 2014; Wang et al., 2011). These techniques offer advantages over traditional

preservatives, including the provision of continuous AM effect on foods for extended times

and minimizing interactions and possible inactivation of AM agents with food components

(Muriel-Galet et al., 2012; Otoni et al., 2014).

In addition to active food packaging, the utilisation of bio-based materials in food packaging

applications such as edible films, flexible and rigid films and coatings has become increasingly

popular in recent years (Otoni et al., 2014; Peretto et al., 2014). In this case, the perceived

environmental benefits and the emerging economic pressures to reduce the dependence on

fossil resources are the primary driving forces behind the growing interest in bio-materials

(Kuorwel et al., 2011; Peretto et al., 2014; Petchwattana and Naknaen, 2015; Qin et al., 2015).

There is also an increasing consumer demand for natural, disposable, biodegradable and

recyclable food packaging materials as well as demand for high-quality fresh food products

(Rhim and Ng, 2007). Bio-based polymers such as poly(lactic acid) (PLA) have received much

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attention in the packaging research and development area over the past few decades. Most

importantly, materials such as PLA can be synthesized from renewable and bio-derived

monomers and are comparable to conventional petroleum based polymers in terms of their

mechanical properties and processability (Jamshidian et al., 2010). Poly(lactic acid) is suitable

for applications that involve delivery and release of additives and, under specific composting

conditions, PLA is biodegradable (Auras et al., 2004). More recently, PLA has been widely

used in advanced packaging applications such as antioxidant and AM films (Iniguez-Franco et

al., 2012; Liu et al., 2009; Llana‐Ruíz‐Cabello et al., 2015; Qin et al., 2015; Wu et al., 2014b).

With regard to the latter, the main focus has been directed towards the inhibition of targeted

microorganisms by PLA-based materials containing a range of AM agents.

Although there are considerable benefits in using biopolymers for packaging applications, a

major consideration is the current high cost of producing these materials (Jamshidian et al.,

2010). The incorporation of natural fillers into biopolymers such as PLA is receiving attention,

especially for the potential development of materials that have reduced environmental impacts

with subsequently lower production costs. In particular, plant fibres such as wood, flax, jute,

kenaf, hemp and sisal can be used as reinforcements and fillers in biopolymer materials. In

addition to enhanced biodegradability, the resulting bio-composites can have improved

mechanical strength and stiffness as well as reduced densities (Gurunathan et al., 2015; Liu et

al., 2005; Tawakkal et al., 2012). Although there are many studies that suggest PLA is a

suitable matrix for the incorporation of natural fibres in bio-composites (Awal et al., 2015;

Rajesh et al., 2015; Tawakkal et al., 2012; Xia et al., 2015), there are limited reports on the

combination of bio-based polymers with natural fibres and AM agents in packaging

applications. In one example, the inhibition of E. coli in liquid media for PLA/triclosan film

was enhanced when the film was incorporated with 10% w/w wood fibres (Prapruddivongs and

Sombatsompop, 2012). Another study of a commercial lignin-based material Arbofill kokos®

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containing short coconut fibres and 2% w/w thymol, a common AM agent, showed significant

inhibition of Gram-positive S. aureus after 20 h incubation, although no AM activity against

Gram-negative bacteria, E. coli, was observed (España et al., 2012).

In a recent study of the interaction and quantification of thymol in PLA/kenaf films, it was

suggested that the polymer may protect the AM agent during storage and the kenaf fibres may

subsequently trigger the AM activity once exposed to a humid environment (Tawakkal et al.,

2016a). Clearly, the retention of volatile AM agents such as thymol as well as the activity of

AM films can be influenced by different storage conditions and temperatures. Nevertheless,

few studies have reported the loss of AM agents during film storage particularly for AM PLA-

based materials containing natural fibres. This is an important consideration to ensure that

adequate AM activity is maintained against target microorganisms following the production,

transportation and storage of the packaging material. In this work, PLA biocomposites

incorporated with kenaf fibres and the AM agent thymol were developed and the AM activities

against E. coli were studied in vitro and on commercial deli chicken slice. The anti-fungal

activity of the AM film on the surface of deli chicken slice and the retention of AM agent in

the film during prolonged storage are also reported.

8.3 Materials and Methods

8.3.1 Materials

Polymer and kenaf fibre: Poly(lactic acid) (7001D IngeoTM; specific gravity 1.24; melting

temperature 154°C) (Tawakkal et al., 2014) was obtained from NatureWorks LLC, USA.

Mechanically separated kenaf fibre (bast) was purchased from Ecofibre Industries, Australia.

Antimicrobial agent: The thymol (T0501, purity of 99.5%) was a food-grade, Halal and Kosher

certified product purchased from Sigma Aldrich Pty. Ltd., Australia. Chemicals: Sodium

hydroxide and acetic acid were purchased from Merck Chemicals, Australia. Media: The media

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included tryptone soy broth (TSB) and nutrient agar purchased from Oxoid, USA,

bacteriological broth obtained from Amyl, Australia, and 3M Petrifilm™ for E. coli

enumeration purchased from 3M, USA. Microorganism: Escherichia coli (ATCC 8739) was

obtained from the culture collection of Food Science Australia, Werribee, Victoria, Australia.

8.3.2 Production of PLA/Kenaf/Thymol Films

The composite films were prepared as outlined in detail in our previous work (Tawakkal et al.,

2014, 2016a). Briefly, the kenaf fibres were subjected to an alkali treatment, washed,

neutralized and dried. The PLA polymer was also dried overnight prior to blending with thymol

and kenaf fibers in an internal melt mixer. Following mixing, the composites were pressed into

films for subsequent testing. The film formulations and the final retention of thymol in the

films following thermal processing are shown in Table 8.1. The retention of thymol was

determined using thermogravimetric analysis as reported in our previous work (Tawakkal et

al., 2016a).

Table 8.1 Percentage of thymol in PLA films after thermal processing as determined by thermogravimetric analysis.

Kenaf#/% w/w Thymol#/% w/w Thymol retained in films / % w/w

0 5 3.7 ± 0.2 30 5 3.1 ± 0.1 0 10 8.3 ± 0.2 10 10 7.6 ± 0.1 20 10 7.1 ±0.1 30 10 6.7 ± 0.1 40 10 6.8 ± 0.2 0 20 16.8 ± 0.3 20 20 13.8 ± 0.9 0 30 23.2 ± 1.5 20 30 19.7 ± 1.3

# Balance is PLA

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8.3.3 Antimicrobial Activity of Films on Solid Media

The microorganism E. coli was selected to demonstrate AM activity via an agar disc diffusion

assay (Kun and Marossy, 2013). The bacteria culture was stored in TSB broth containing 30%

v/v glycerol at -80°C and was sub-cultured in broth twice before being used. Samples of 0.1

mL of E. coli suspensions containing approximately 106 - 107 CFU mL-1 were spread over a

prepared agar surface in a Petri dish. Film discs of 6 mm diameter were placed in triplicate on

the Petri dish containing the agar. Plates were incubated at 37°C for 24 h and the diameter of

the resulting inhibition zone was measured directly after the incubation period using a digital

Vernier calliper. The presence of a clear zone of inhibition around the test films was taken as

indication of AM activity in the films and the test was performed in triplicate.

8.3.4 Antimicrobial Activity of Films in Liquid Media

The AM activity of films was tested in TSB liquid media. Cell cultures of E. coli in the

stationary phase (optical density of ca. 1 at 600 nm) were diluted in fresh TSB to an optical

density of 0.1 and grown for 1.5 h at 37°C until the growth reached the exponential phase

(optical density of ca. 0.6 at 600 nm). The cells were then diluted into TSB (1:100) to obtain

stock solution with a working concentration of ca. 105 CFU mL-1 (Shemesh et al., 2015). At

this stage, 0.1 mL of TSB containing the stock solution and 0.25 g of film were placed into

separate sterile tubes with an additional 10 mL of TSB and were incubated at 37°C for 24 h.

Serial dilutions were performed using 0.1% w/v peptone water and 1 mL of each final diluted

mixture was placed on a 3M Petrifilm™ count plate. Colonies were counted after incubation

at 37°C for 24 h and expressed in units of log CFU mL-1. Controls comprising inoculated TSB

in the absence of films were also tested and the bacteria enumeration was performed in

triplicate.

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8.3.5 Antimicrobial Activity of Films on Processed Food

Direct Food Contact Analysis

The AM activity of films on a real food product was tested. Processed sliced chicken with a

thickness of ca. 0.2 cm was purchased from a local supermarket (Fettayleh Smallgoods). The

samples were then placed on a sterile tray and were aseptically cut into 3 × 3 cm squares. The

samples were inoculated on the upper surface with 0.1 mL of E. coli suspension prepared in

accordance with Section 8.3.3. The inoculum was spread evenly over the surface of the chicken

slice using sterile spreaders to obtain an initial coverage of ca. 5.0 log CFU cm-2. After

inoculation, the samples were placed inside a laminar flow cabinet for 30 min to facilitate

bacterial attachment. Films of 5 × 5 cm were placed on the top of each inoculated sample which

were then packed into sterile stomacher bags and sealed using a plastic bag sealer device.

Inoculated samples in the absence of films were prepared as controls and all samples were

stored at 10°C to simulate mild temperature abuse (Guo et al., 2015). On each sampling day,

the samples were hand-massaged in 20 mL of 0.1% w/v peptone water for 1 min and placed in

a laboratory paddle blender masticator for a further 1 min. Serial dilutions ranging from 101 to

107 were made in 0.1% w/v peptone water. Following this, 1 mL of each final diluted mixture

was placed on 3M Petrifilm™ count plates and incubated for 24 h at 37ºC prior to enumeration.

The final bacterial cell density was expressed in units of log CFU cm-2 (Guo et al., 2015).

Microbial Death Rate

The death rate of E. coli inoculated onto the deli chicken slice samples was measured in

accordance with the procedure outlined by Bachrouri et al. (2002). In this procedure, equation

(1) can be used to determine specific death rate, µ:

ln(𝑁) = ln(𝑁0) − 𝜇𝑡 (1)

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where, N is the population surviving at any time t, and N0 is the initial population. The specific

death rate, µ, is obtained from the gradient of a plot of the equation (1) which was used to

compare the AM activities of the films.

Data Analysis

The bacteria enumeration in CFU mL-1 and CFU cm-2 were transformed to log10 values and

analysed using analysis of variance with SAS version 9.1 software (SAS Institute, NC, USA).

A Student t-test analysis was used to determine significant differences between treatment

means. A significant value was defined as one where p < 0.05.

Indirect Food Contact Analysis

Antimicrobial films with a constant diameter of ca. 25 mm were prepared in order to maintain

the same equilibrium surface area of the films which were then attached to the lids of petri

dishes containing untreated chicken slice to avoid direct contact with the samples and facilitate

headspace release of the AM agent. The petri dish and film assembly were sealed with

Parafilm™ and incubated at two temperatures, 10°C and 25°C, in duplicate. Samples without

test films and samples that were in contact with films containing no thymol were also tested as

controls. Images of the samples were taken every day for up to 18 days and were subjected to

image analysis to determine the area of mould growth.

Image Analysis

The percentage of fungal coverage on the surface of the chicken samples was measured in order

to investigate the antifungal activity of the AM films. Images of the samples in the form of

black and white photographic JPEG files were analysed using original imaging algorithm

software designed to systematically count the separate black and white pixel areas comprising

the image and compile a normalized area count of the black pixels (i.e. fungal coverage). The

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JPEG photographic standard stores images as a two-dimensional array of pixel information,

each pixel having a red, blue and green (R, B, G) component along with other information

associated with the pixel. The image processing algorithm firstly removes grey-scale shading

from the image file by systematically examining the R, G, B information associated with each

pixel and setting the pixel to either "white" (R = 255, G = 255, B = 255) or "black" (R = 0, G

= 0, B = 0) in accordance with a threshold set by the user. The algorithm then counts the

number of sequential black pixels in each line of the image array and compiles a count of these

which can be expressed as a percentage of the total area of the image (i.e. the total number of

pixels comprising the image).

8.3.6 Storage Stability of Thymol in AM Films

The release of thymol from the PLA and PLA/kenaf films was investigated under two different

storage conditions: unwrapped and wrapped with aluminium foil. The unwrapped films were

exposed to ambient conditions at ca. 24°C and 51% relative humidity (RH) whereas the

wrapped films were stored under: (i) ambient conditions and (ii) refrigeration at 4°C and ca.

95% RH. The amount of thymol retained in the films was measured using a Perkin-Elmer

Spectrum 100 Optica Fourier-transform infrared (FTIR) spectrophotometer (PerkinElmer,

Boston, USA) equipped with a diamond crystal attenuated total reflectance (ATR) accessory.

A total of 16 scans per run were conducted in the range of 4000-600 cm-1 at 4 cm-1 resolution.

Samples were placed on the surface of the ATR crystal and clamped in place to maintain a

consistent pressure and subsequent contact with the crystal. The peak area at wavenumber ca.

807 cm-1 which corresponds to the ring vibration of thymol was used to measure the amount

of thymol retained in the films starting from day zero to day 90.

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8.4 Results and Discussion

8.4.1 Antimicrobial Activity of Films In-vitro

Solid Media AM Activity

The PLA/thymol and PLA/kenaf/thymol films were tested in vitro in order to provide

preliminary information about the potential AM activity against E. coli, which is a common

microorganism found in perishable food products such as processed meats (Tao et al., 2014).

Figure 8.1 shows a typical image of the AM effect of these films against E. coli after 24 h of

incubation at 37°C. The results demonstrate that the PLA/thymol and PLA/kenaf/thymol films

containing 30% w/w thymol imparted a strong AM effect against E. coli with a clear zone of

inhibition observed directly below and around the test films. The clear zone indicates the

inhibition of E. coli colonies caused by the release of thymol from the film to the agar medium.

As expected, no inhibition of E. coli was evident for the control films containing no thymol.

The size of the inhibition zones was measured in order to investigate the effects of thymol

concentration and kenaf fibre loadings of the AM films against E. coli. Figure 8.2 shows the

average diameter of the inhibition zone following the incubation period for the PLA/thymol

and PLA/kenaf/thymol film formulations. No zone of inhibition was observed for the films

containing 5% w/w thymol, which is the lowest thymol concentration (data not shown). A

similar investigation of the AM activity of solvent cast soy protein edible films enriched with

oregano and thyme essential oils reported that the films containing 5% w/w thyme essential oil

showed the more intense AM activity against E. coli compared with films containing oregano,

with inhibition zone diameters of 49 and 45 mm respectively (Emiroglu et al., 2010). The

solvent casted soy protein edible films containing thyme essential oil in this study were found

to be effective in inhibiting the E. coli which is in contrast to the current studied films. The

observed differences may be attributed due to the different type of polymeric materials and

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agents used, the method of film preparation as well as implementation of migratory and/or non-

migratory system which influences the release and inhibition of the microorganisms. Generally,

the thyme plant contains essential oils that are highly enriched with terpenoids, particularly

monoterpenoid phenols such as thymol and carvacrol. Thymol (2-isopropyl-5-methylphenol),

the agent used in the present study, is a naturally derived additive which is commercially

available in a crystalline and colourless form.

Figure 8.1 Antimicrobial activity of films against E. coli after 24 h of incubation at 37°C: (a) PLA control, (b) PLA with 20% w/w kenaf fibres, (c) PLA with

30% w/w thymol and (d) PLA with 20% w/w kenaf fibres and 30% w/w thymol.

Figure 8.2 Influence of thymol and kenaf loadings on the zone of inhibition

of PLA/thymol and PLA/kenaf/thymol films against E. coli. Solid lines represent error bars and the numbers represent the inhibition zone diameters in mm.

(a) (a)

(c) (d)

(b)

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These current formulations may have a limited effect against E. coli at lower thymol content

since the AM agent must diffuse from the polymer composite through to the agar medium

(Suppakul et al., 2011). A similar MIC was reported for extruded poly(butylene succinate)

films containing 10% w/w thymol tested via the agar diffusion method (Petchwattana and

Naknaen, 2015). In another study, the zone of inhibition in extruded polypropylene films

containing 8% w/w thymol showed negligible zone of inhibition against E. coli although no

growth was observed directly under the film (Ramos et al., 2012). This finding is in accordance

with the MIC of 10% w/w thymol suggested in the present study.

As shown in Figure 8.2, it is clear that increasing the concentration of thymol from 10 to 30%

w/w increased the area of the inhibition zone and this effect is further enhanced with the

addition of more kenaf fibers. For the films containing 10% w/w thymol, a small increase in

the inhibition zone was observed with increasing kenaf loading. The PLA/kenaf/thymol film

containing the highest kenaf fibre content (40% w/w) exhibiting a ca. 10% increase in the size

of the zone of inhibition compared with the film containing the next highest kenaf loading

(30% w/w). The higher loading of kenaf fibres in the film may have increased the

hydrophilicity of the film resulting in water molecules being absorbed at the surface of the film

which may contribute to a potential burst release of the AM agent which may also have an

effect over the long term release profile. It is also clear from the results shown in Figure 8.2

that, for a given kenaf loading, increasing the thymol loading has a marked effect on the AM

activity of the film. For example, at a constant kenaf loading of 20% w/w, increasing the

concentration of thymol from 10 to 30% w/w produces a concomitant ca. three-fold increase

in the diameter of the zone of inhibition.

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Liquid Media AM Activity

The AM activity of PLA/thymol and PLA/kenaf/thymol films containing 10% w/w thymol and

30% w/w kenaf fibers against E. coli in a liquid media is represented in Figure 8.3(a). The

results show that the concentration of E. coli in the presence of PLA films containing 10% w/w

thymol was similar to those of the control (i.e. inoculated TSB that was not in contact with a

test film) after 24 h, indicating that the PLA/thymol film showed little inhibitory effect on the

growth of E. coli. After 48 h of incubation, the PLA/thymol film slightly reduced the cell count

compared with the control sample, suggesting that thymol could diffuse through the polymer

matrix of the film after an extended period of time (Nostro et al., 2007). After 24 h of

incubation the composite film containing 30% w/w kenaf fibres and 10% w/w thymol showed

a reduction in the E. coli population of ca. 1 log CFU mL-1 compared to both the control and

PLA/thymol film. In addition, no recovery of injured bacteria cells was observed after 48 h

incubation for both films containing thymol. It is clear that the PLA/kenaf/thymol film

imparted a significant inhibitory effect compared to the PLA/thymol film, which may be

attributed to the higher release rate of thymol from the composite film in the liquid media.

The presence of kenaf fibres in the polymer matrix may create voids thereby leading to the

release of thymol (Tawakkal et al., 2016b). This finding is consistent with the observation

made in relation to previous scanning electron microscope images where visible voids in the

composite materials are shown as well as the diffusion rate of thymol from the PLA/kenaf films

into fatty and aqueous food stimulants (Tawakkal et al., 2014; Tawakkal et al., 2016b). Similar

results have also been observed in other systems whereby it was suggested that the presence of

larger micro-voids in solvent-cast films of PLA/poly(ε-caprolactone) (PCL) containing thymol

facilitates the release of the AM agent to the surface of the film and improves its AM activity

against E. coli after 24 h in liquid media (Wu et al., 2014a). Moreover, hydrophilic fibres can

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swell when immersed in liquid media resulting in internal stresses generated in the adjacent

polymer matrix that may produce microcracks, further facilitating the release of thymol

(Tawakkal et al., 2016b).

Figure 8.3 Liquid media antimicrobial activity of PLA formulations (a) control; PLA containing 10% w/w thymol; PLA containing 10% w/w thymol and 30% w/w kenaf fibres; (b) control; PLA containing 30% w/w thymol; PLA containing 20% w/w kenaf fibres and 30% w/w thymol: () PLA control, ()

PLA/thymol and () PLA/kenaf/thymol films.

The results in Figure 8.3(b) show the inhibitory activity of films at higher thymol concentration

(30% w/w) against E. coli in liquid media where the kenaf loading was 20% w/w in the

PLA/kenaf composites. In this case, the higher concentration of AM agent in the films reduces

the population of E. coli in the liquid media almost immediately upon contact which may be

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due to the thymol that is present on the surface of the film. At this level of thymol, only a

moderate extent of AM activity was observed in the active PLA/thymol film up to 48 h

incubation period suggesting that a significant interaction persists between the PLA matrix and

thymol, thus preventing the liquid media from effectively swelling the film and releasing the

thymol (Qin et al., 2015). This may be attributed to the selective adsorption as well as possible

encapsulation of thymol in the polymer matrix. Figure 8.3(b) further suggests that when kenaf

fibres are introduced, the composite film exhibits significant AM activity by suppressing the

growth of E. coli at 24 h. This film reduces E. coli by ca. 2 log CFU mL-1 after 24 h with no

recovery of injured bacteria cells at 48 h. This finding is consistent with the results of the agar

disc diffusion tests (see Figure 8.1). In a similar study, it was found that a higher loading of

wood flour particles resulted in the release of more triclosan onto the composite surface and a

subsequent inhibition of the growth of E. coli in a liquid medium (Prapruddivongs and

Sombatsompop, 2012). Wu et al. (2014a) found that the AM activity of PLA/PCL films

containing up to 12% w/w thymol were higher against Gram-positive bacteria (L.

monocytogenes) than Gram-negative bacteria (E. coli) due to differences in the structures of

the cell walls (Wu et al., 2014a). Moreover, Del Nobile et al. (2008) prepared zein films

incorporated with thymol from 10 to 35% w/w loading and found that all the films effectively

inhibited the growth of Pseudomonas spp. (Del Nobile et al., 2008).

8.4.2 Antimicrobial Activity on Real Foods

Direct Contact AM Activity

To further evaluate the efficacy of the active PLA composites, samples were tested in contact

with a real food sample, namely deli chicken slice. Table 8.2 shows the population counts of

E. coli in log CFU cm-2 on the surface of the inoculated chicken samples as a function of storage

time at 10°C for each of the AM films. The results in Table 8.2 confirm that the population of

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E. coli decreased upon increasing the thymol concentration from 10 to 30% w/w. No significant

differences were observed between the bacteria counts for PLA films containing 10% w/w

thymol and the control (i.e. inoculated chicken sample that was not in contact with a test film)

in the first 13 days. As expected, the AM activities of the PLA/kenaf/thymol films are higher

than those obtained for the PLA/thymol films, with an inhibitory activity against E. coli that is

sustained for ca. 4 weeks. These findings are consistent with the results obtained in the previous

in vitro studies (see Figure 8.2 and Figure 8.3), with the AM agent in the PLA/kenaf/thymol

films found to have been activated or released almost instantaneously once the film is in direct

contact with the substrate. Guo et al. (2015) prepared edible films based on chitosan loaded

with different AM agents and found that the films reduced Listeria in a deli chicken slice to

3.7 log CFU cm-2 after 5 weeks at 10°C, with control samples reaching over 6.6 log CFU cm-2.

Emiroglu et al. (2010) found that isolated soy protein films containing thyme essential oil

reduced coliform counts to 5 log CFU g-1 in ground beef patties at 4°C during 12 days of

storage.

Table 8.2 Antimicrobial activity of PLA and PLA/kenaf films against E. coli on chicken slice stored at 10°C for 30 days.

E. coli counts/ log CFU cm-2 Kenaf#/% w/w

Thymol#/% w/w Day 2 Day 6 Day 13 Day 19 Day 30

†0 0 4.42bc 4.30b 4.15b 4.13a 3.64a 0 10 4.49bc 4.75a 4.32a 3.87b 3.55ab 0 20 4.55ab 3.83c 3.59d 3.33c 3.09b 0 30 4.44bc 3.73c 3.10f 2.64d 2.09c 30 10 4.72a 4.24a 3.80c 3.74b 3.10b 20 20 4.27c 3.77c 3.32e 2.71d 2.39c 20 30 3.19d 2.45d 1.22g ND ND

# Balance is PLA; abc values are expressed as mean ± standard deviation, different letters in the same column indicate significant difference (p < 0.05); ND = not detected; †control sample without films

The death rates of E. coli in the presence of the PLA/thymol and PLA/kenaf/thymol

formulations were determined for all systems and are plotted in Figure 8.4 as a function of the

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concentration of thymol in the formulation. The death rates were determined from the gradients

of plots of the logarithm of the surviving population versus time (see equation (1)), the data

having been derived from Table 8.2. Figure 8.4 shows that the death rate of E. coli in the

PLA/thymol and PLA/kenaf/thymol films increased with increasing thymol concentration. The

latter films showed significantly higher death rates than the PLA/thymol films, consistent with

the previous observations, with the PLA/kenaf/thymol film containing 30% w/w thymol

demonstrating the strongest inhibitory effect on the growth of E. coli on the chicken samples

with a death rate of ca. 0.19 day-1. The results also suggest that the incorporation of thymol

into PLA and PLA/kenaf films prevents the recovery of injured bacterial cells for up to 30 days

and this may be attributed to the strong AM properties of thymol.

Figure 8.4 Death rate of E. coli inoculated on deli chicken slice and stored at 10°C in contact with: (●) PLA/thymol and (■) PLA/kenaf/thymol

composite films.

Indirect Contact AM Activity

The coverage of fungi on the surface of chicken samples was measured via visual observation

together with semi-quantitative image analysis and the antifungal activity of all films was tested

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using a vapour phase method. Figure 8.5 shows the appearance of chicken samples placed in

petri dishes containing AM PLA and PLA/kenaf films at 25°C for up to 6 days. The images

show that fungal growth commences with the appearance of white cottony mycelium at day 4

which becomes more apparent on day 6, with green sporulating mould clearly observed. Fungi

such as moulds that are commonly found on meat and poultry include Aspergillus, Penicillium,

Rhizopus, Mucor, Cladosporium, Geotrichum, Thamnidium among others (Pitt and Hocking,

2009). Moulds also can grow on refrigerated meat, where it can be detected by rot spots of

various sizes and colours, unsightly scabs, slime, white cottony mycelium or highly coloured

sporulating mould (Pitt and Hocking, 2009).

Figure 8.5 Fungal coverage on the surface of deli chicken slice samples stored at 25°C in the presence of selected AM film formulations.

Figure 8.6(a) and (b), show that the percentage of fungal coverage per unit area (ƒ) gradually

increased during the storage period at storage temperatures of 10 and 25°C. In the case of the

control sample and samples that were in contact with films without thymol, a rapid growth of

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fungus was observed starting at day 4 whereas for samples in contact with the active films, no

fungal growth was observed until day 6 at 25°C. The presence of fungus was first observed on

the chicken samples stored at 10°C after 12 days of storage. Under both storage conditions, it

was observed that PLA/thymol and PLA/kenaf/thymol films containing high thymol

concentrations of 20 and 30% w/w completely inhibited fungal growth during the storage

period (results not shown). Although the headspace was not measured in the current study, this

result may be attributed to the release of a large amount of thymol from the active films into

the headspace (Del Nobile et al., 2008).

Figure 8.6 Percentage of fungal coverage per unit area (ƒ) of deli chicken slice samples stored at: (a) 25°C and (b) 10°C. Key: control, neat PLA film, ● PLA/kenaf film containing 30% w/w kenaf fibres, □ PLA/thymol film containing 10% w/w thymol, ○ PLA/kenaf/thymol film containing 10% w/w kenaf fibres and 10% w/w thymol and ∆

PLA/kenaf/thymol film containing 40% w/w kenaf fibres and 10% w/w thymol.

In general, the results show that the PLA/thymol and PLA/kenaf/thymol films containing 10%

w/w thymol and lower kenaf fibres loading (10% w/w) are slightly better at retarding fungal

growth than the control samples (see Figure 8.6(a) and (b)). However, active PLA films filled

with a high loading of kenaf fibres (such as 40% w/w) and containing 10% w/w thymol

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demonstrated significant antifungal activity at both storage temperatures. The latter finding is

consistent with the observed AM activity of PLA/kenaf/thymol films in contact with the solid

agar media (see Figure 8.2). Muriel-Galet et al. (2015) tested the AM activity of ethylene vinyl

alcohol (EVOH) films containing oregano essential oil and green tea extract on the fungal

species Penicillium expansum via the agar diffusion method in the vapour phase. It was

revealed that the EVOH films presented a strong effect against fungal growth after 12 days of

storage at 30°C. The results obtained from the current study also provide evidence of the

effectiveness of volatile AM agents when incorporated in PLA containing natural fibres at

improving the shelf-life of perishable food. Further evaluation of the effectiveness of the active

films against other microorganisms, on various real-food products, and at different storage

temperatures could also be considered for future studies.

8.4.3 Effect of Different Storage Conditions on Thymol Retention

Although many studies have focused on the release of thymol from the film into food simulants,

few have investigated the release of thymol into the atmosphere or surroundings as a function

of storage time and temperature. In one such example, Suppakul et al. (2011) reported that

there was no difference in AM activity of the AM LLDPE and films against E. coli at the time

when the film was originally produced and after 1 year of storage, although a reduction in

additive concentration was detected. In the present study, the release of thymol from the films

was determined by measuring the decrease of the FTIR band at 807 cm-1 over time (Sanchez-

Garcia et al., 2008). According to Tawakkal et al. (2016a), the most intense peaks at 738 and

807 cm-1 are assigned to ring vibrations of thymol. Figure 8.7 shows the normalized peak area

of thymol in the PLA/thymol and PLA/kenaf/thymol films containing 10% w/w thymol under

different storage conditions for up to 3 months. The peak area of thymol at ca. 807 cm-1 on day

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one to day 90 was referenced to the peak area of thymol on day zero in order to normalize the

peak area.

Figure 8.7 Normalized thymol peak area in films up to 3 months of storage under different storage conditions: (a) films unwrapped in aluminium foil and stored at

ambient temperature, (b) films wrapped in aluminium foil and stored at ambient temperature and (c) films wrapped with aluminium foil and stored at 4°C. Key: □

PLA/thymol film containing 10% w/w thymol; ○ PLA/kenaf/thymol film containing 10% w/w kenaf and 10% w/w thymol and ● PLA/kenaf/thymol film containing 30% w/w kenaf

and 10% w/w thymol.

As shown in Figure 8.7(a), the unwrapped PLA/kenaf films containing 30% w/w kenaf loading

showed a higher loss of thymol when stored under ambient conditions compared with those

formulations with a lower kenaf loading. The results also suggest that most of the loss of thymol

from the PLA/kenaf films containing the higher kenaf loading occurs during the first 8 days of

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storage and the films achieved an equilibrium thymol concentration after ca. 16 days of storage.

In a few cases, the concentration of thymol was detected to be more than the normalized value

and this may be attributed to the distribution/dispersion of thymol in the surface of pressed

films and subsequent migration of thymol to the surface. It is important to note that the

measurement of thymol concentration by using FTIR detects thymol in relation to the surface

of the pressed films with the infrared beam penetrating only a few microns below the surface.

Moreover, the release of thymol from the PLA/kenaf films exposed to the atmosphere may be

attributed to its high volatility as well as the presence of voids that facilitate the release of the

agent from the films. This is consistent with the micrograph images presented in a previous

study where voids and loose fibers are observed on the fracture surface of PLA/kenaf

composites containing 30% w/w kenaf loading (Tawakkal et al., 2014).

For the foil wrapped samples stored at ambient and refrigeration temperatures (see Figure

8.7(b) and (c) respectively), the thymol concentration was found to be more stable in the PLA

and PLA containing 10% w/w kenaf loading films, with ca. 85% thymol retained in these films

compared with the films stored in the open environment. As expected, the PLA and PLA/kenaf

films wrapped in the aluminium foil retained the thymol and a similar observation was found

by Kuorwel (2011) who prepared starch films incorporated with thymol. Interestingly, for the

PLA/kenaf/thymol films containing the higher fibre loadings, a slight loss of thymol was

observed for these films when wrapped in the aluminium foil and stored at 4°C. This may be

attributed to the presence of surface moisture on the film created by condensation that may

remove some of the AM agent on the surface when the film sample is prepared prior to the

FTIR measurement. In addition, the hydrophilicity of the kenaf fibres may enable the

condensate to swell the film and facilitate the thymol release to the environment. This

observation suggests that in any commercial application of these formulations, a protective

barrier layer and/or hermetic seal of the films should be implemented to avoid condensation

154

and subsequent loss of AM agent to the atmosphere during storage in order to preserve the

initial AM activity at the time of manufacture.

8.5 Conclusion

The AM efficacy of PLA films incorporated with kenaf fibers and thymol were tested against

E. coli and naturally occurring fungi. The PLA/thymol and PLA/kenaf/thymol films containing

high thymol concentrations showed a strong AM activity against E. coli with a significant

inhibition of the growth in both solid and liquid media. The composite films also significantly

reduced the population of E. coli on inoculated chicken slice samples when placed in direct

contact with the food. It is suggested the films retarded natural fungal growth in the chicken

slice samples via the release of thymol into the headspace surrounding the samples. The

PLA/thymol and PLA/kenaf/thymol films containing higher thymol content as well as higher

kenaf loading were more effective in controlling or limiting the fungal growth than those

containing lower loadings of these additives. After storage for 3 months at ambient

temperatures, a slight decrease in the retention of the AM additive was observed in

PLA/kenaf/thymol films containing 30% w/w kenaf and 10% w/w thymol that were not

wrapped with aluminium foil.

Acknowledgments

The authors gratefully acknowledge the Ministry of Education Malaysia and Universiti Putra

Malaysia (UPM) for providing the PhD scholarship for Intan S. M. A. Tawakkal and would

like to acknowledge Mr. Mike Allan and the technical staff from the Royal Melbourne Institute

Technology, Melbourne for their invaluable assistance with the preparation of the composite

samples.

155

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Bachrouri, M., Quinto, E., Mora, M., 2002. Survival of Escherichia coli O157: H7 during storage of yogurt at different temperatures. J. Food Sci. 67, 1899-1903.

Del Nobile, M.A., Conte, A., Incoronato, A.L., Panza, O., 2008. Antimicrobial efficacy and release kinetics of thymol from zein films. J. Food Eng. 89, 57-63.

Emiroglu, Z.K., Yemis, G.P., Coskun, B.K., Candogan, K., 2010. Antimicrobial activity of soy edible films incorporated with thyme and oregano essential oils on fresh ground beef patties. Meat Sci. 86, 283-288.

España, J.M., Fages, E., Moriana, R., Boronat, T., Balart, R., 2012. Antioxidant and antibacterial effects of natural phenolic compounds on green composite materials. Polym. Compos. 33, 1288-1294.

Fernández-Pan, I., Maté, J.I., Gardrat, C., Coma, V., 2015. Effect of chitosan molecular weight on the antimicrobial activity and release rate of carvacrol-enriched films. Food Hydrocoll. 51, 60-68.

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Iniguez-Franco, F., Soto-Valdez, H., Peralta, E., Ayala-Zavala, J.F., Auras, R., Gamez-Meza, N., 2012. Antioxidant activity and diffusion of catechin and epicatechin from antioxidant active films made of poly(l-lactic acid). J. Agric. Food Chem. 60, 6515-6523.


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Kun, E., Marossy, K., 2013. Evaluation methods of antimicrobial activity of plastics. Mater. Sci. Forum 729, 430-435.

Kuorwel, K.K., 2011. Incorporation of natural antimicrobial agents into starch-based material for food packaging. Victoria University, Melbourne, p. 286.

Kuorwel, K.K., Cran, M.J., Sonneveld, K., Miltz, J., Bigger, S.W., 2011. Antimicrobial activity of natural agents coated on starch-based films against Staphylococcus aureus. J. Food Sci. 76, 531-537.

Liu, L., Fishman, M.L., Hicks, K.B., Liu, C.K., 2005. Biodegradable composites from sugar beet pulp and poly(lactic acid). J. Agric. Food Chem. 53, 9017-9022.

Liu, L., Jin, T.Z., Coffin, D.R., Hicks, K.B., 2009. Preparation of antimicrobial membranes: coextrusion of poly(lactic acid) and Nisaplin in the presence of plasticizers. J. Agric. Food Chem. 57, 8392-8398.

Llana‐Ruíz‐Cabello, M., Pichardo, S., Baños, A., Núñez, C., Bermúdez, J.M., Guillamón, E., Aucejo, S., Cameán, A.M., 2015. Characterisation and evaluation of PLA films containing an extract of Allium spp. to be used in the packaging of ready-to-eat salads under controlled atmospheres. LWT-Food Sci. Technol. 64, 1354-1361.

Muriel-Galet, V., Cran, M.J., Bigger, S.W., Hernandez-Munoz, P., Gavara, R., 2015. Antioxidant and antimicrobial properties of ethylene vinyl alcohol copolymer films based on the release of oregano essential oil and green tea extract components. J. Food Eng. 149, 9-16.

Muriel-Galet, V., López-Carballo, G., Gavara, R., Hernández-Muñoz, P., 2012. Antimicrobial food packaging film based on the release of LAE from EVOH. Int. J. Food Microbiol. 157, 239-244.

Muriel-Galet, V., Talbert, J.N., Hernandez-Munoz, P., Gavara, R., Goddard, J., 2013. Covalent immobilization of lysozyme on ethylene vinyl alcohol films for nonmigrating antimicrobial packaging applications. J. Agric. Food Chem. 61, 6720-6727.

Nostro, A., Roccaro, A.S., Bisignano, G., Marino, A., Cannatelli, M.A., Pizzimenti, F.C., Cioni, P.L., Procopio, F., Blanco, A.R., 2007. Effects of oregano, carvacrol and thymol on Staphylococcus aureus and Staphylococcus epidermidis biofilms. J. Med. Microbiol. 56, 519-523.

Otoni, C.G., Pontes, S.F.O., Medeiros, E.A.A., Soares, N.d.F.F., 2014. Edible films from methylcellulose and nanoemulsions of clove bud (Syzygium aromaticum) and oregano (Origanum vulgare) essential oils as shelf life extenders for sliced bread. J. Agric. Food Chem. 62, 5214-5219.

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Peretto, G., Du, W.-X., Avena-Bustillos, R.J., Berrios, J.D.J., Sambo, P., McHugh, T.H., 2014. Optimization of antimicrobial and physical properties of alginate coatings containing carvacrol and methyl cinnamate for strawberry application. J. Agric. Food Chem. 62, 984-990.

Petchwattana, N., Naknaen, P., 2015. Utilization of thymol as an antimicrobial agent for biodegradable poly(butylene succinate). Mater. Chem. Phys. 163, 369-375.

Pitt, J.I., Hocking, A.D., 2009. Fungi and Food Spoilage. Springer-Verlag, New York.

Prapruddivongs, C., Sombatsompop, N., 2012. Roles and evidence of wood flour as an antibacterial promoter for triclosan-filled poly(lactic acid). Compos. Part B 43, 2730-2737.

Qin, Y., Liu, D., Wu, Y., Yuan, M., Li, L., Yang, J., 2015. Effect of PLA/PCL/cinnamaldehyde antimicrobial packaging on physicochemical and microbial quality of button mushroom (Agaricus bisporus). Postharvest Biol. Technol. 99, 73-79.

Rajesh, G., Prasad, A.R., Gupta, A., 2015. Mechanical and degradation properties of successive alkali treated completely biodegradable sisal fiber reinforced poly lactic acid composites. J. Reinf. Plast. Compos. 34, 951-961.

Ramos, M., Beltran, A., Valdes, A., Peltzer, M.A., Jimenez, A., Garrigós, M.C., Zaikov, G.E., 2013. Carvacrol and thymol for fresh food packaging. J. Bioequiv. Availab. 5, 154-160.


Rhim, J.W., Ng, P.K., 2007. Natural biopolymer-based nanocomposite films for packaging applications. Crit. Rev. Food Sci. Nutr. 47, 411-433.

Risch, S.J., 2009. Food packaging history and innovations. J. Agric. Food Chem. 57, 8089-8092.

Sanchez-Garcia, M.D., Ocio, M.J., Gimenez, E., Lagaron, J.M., 2008. Novel polycaprolactone nanocomposites containing thymol of interest in antimicrobial film and coating applications. J. Plast. Flim Sheet. 24, 239-251.

Shemesh, R., Goldman, D., Krepker, M., Danin-Poleg, Y., Kashi, Y., Vaxman, A., Segal, E., 2015. LDPE/clay/carvacrol nanocomposites with prolonged antimicrobial activity. J. Appl. Polym. Sci. 132, 41261 (41261 of 41268).

Suppakul, P., Sonneveld, K., Bigger, S.W., Miltz, J., 2011. Loss of AM additives from antimicrobial films during storage. J. Food Eng. 105, 270-276.

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Tawakkal, I.S.M.A., Cran, M.J., Bigger, S.W., 2014. Effect of kenaf fibre loading and thymol concentration on the mechanical and thermal properties of PLA/kenaf/thymol composites. Ind. Crops Prod. 61, 74-83.

Tawakkal, I.S.M.A., Cran, M.J., Bigger, S.W., 2016a. Interaction and quantification of thymol in active PLA-based materials containing natural fibers. J. Appl. Polym. Sci. 133, 42160 (42161 of 42111).

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Xia, X., Liu, W., Zhou, L., Liu, H., He, S., Zhu, C., 2015. Study on flax fiber toughened poly(lactic acid) composites. J. Appl. Polym. Sci. 132, 42573 (42571 of 42510).

159

Chapter 9 – Conclusions

9.1 General Conclusions

Thymol can be successfully incorporated into the commercially available biopolymer PLA as

well as PLA composites containing kenaf fibres to produce active packaging materials that

exhibit activity against E. coli both in vitro and on the surface of a real food product. The

mechanical, thermal, morphological, and biodegradation properties of PLA and PLA/kenaf

composites containing thymol were successfully characterized. In addition, the optimization of

material formulations and properties such as the effects of different kenaf fibre loadings and

thymol concentrations was achieved. In this chapter, conclusions are drawn based on the

discussion of the results presented in Chapters 3 to 8.

9.1.1 Effect of Kenaf Fibre Surface Treatment

In general, the presence of thymol in PLA and PLA/kenaf composites containing 30% w/w UK

and TK respectively imparted a plasticizing and/or lubricating effect. A relatively weak

adhesion between the PLA matrix and the fibres was confirmed microscopically in composites

containing thymol. The tensile strength of PLA composites containing TK fibres was slightly

higher than that of composites containing UK fibres suggesting the alkaline treatment imparts

a reinforcing effect within the polymer matrix. The incorporation of 10% w/w thymol into the

composites decreased the tensile strength and stiffness irrespective of the fibre pre-treatment

with no significant changes to the elasticity. Thermal analysis by DSC showed a general

decrease in Tg, Tcc, and Tm in PLA and PLA composites containing 10% w/w thymol

compared with the formulations without thymol, suggesting changes in the phase structure of

the polymer. The TG decomposition temperature of the PLA composite containing TK fibres

and 10% w/w thymol was slightly increased indicating an increase in the thermal stability

compared with the PLA composite containing the UK fibres. Under controlled composting

160

conditions, the degradation of the PLA and PLA containing TK fibres progressed rapidly

resulting in a complete loss within 48 days.

9.1.2 Effect of Kenaf Loadings and Thymol Concentrations

Further characterisation of PLA and PLA/kenaf composites containing TK was systematically

performed in order to investigate the effects of the natural filler kenaf as well as the AM

additive thymol on the stability and performance of the material during processing. The

incorporation of thymol into PLA/kenaf composites decreased the tensile strength with no

significant further changes observed upon increasing the kenaf fibre loading. The tensile

strength and stiffness of composites however increased with higher fibre loadings, thereby

imparting a reinforcement effect within the PLA composite. It was noted that an increased

loading of kenaf fibres in the PLA resulted in a decrease in the TGA decomposition temperature

indicating a reduced thermal stability of the PLA whereby some portion of the polymer is

replaced with less thermally stable fibres in the composite materials. These results are in

agreement with the results of kinetics analysis using two algorithms described in Chapter 5

where the PLA and PLA composite each decomposes in accordance with the contracting

volume (R3) model and where the addition of the kenaf filler to the PLA destabilizes the

polymer and lowers the apparent activation energy from 190 kJ mol-1 to ca. 150 kJ mol-1. The

model identification algorithm exhibits significant promise as a method by which applicable

kinetic models can be identified. Moreover, the developed kinetic approach was used as a tool

to investigate the interactions between the thymol, kenaf fibres and PLA of the composite

systems.

9.1.3 Interaction and Retention of Thymol

The FTIR analysis of the PLA and PLA/kenaf composites containing thymol showed that the

thymol interacts with PLA and kenaf as revealed by significant shifts in the various FTIR

161

absorption bands. Active PLA/kenaf composites retain less thymol upon processing than PLA

alone and the composites containing the highest fibre loadings demonstrated the lowest

retained thymol content. Nevertheless, the apparent activation energy calculated from the

kinetic model for thymol release from the PLA/TK composites was found to be greater than

that for the release of thymol from PLA alone, which is consistent with the intermolecular

attractions that occur as a result of hydrogen bonding between the components in the

composite. It would therefore appear that the disruption to the crystalline regions caused by the

addition of kenaf, along with the concomitant creation of voids and the resulting decrease in

tortuosity, facilitate the release of thymol from the composite. In addition, the increased

hydrophilicity due to water absorption into the created voids may increase the migration of

water into the polymer matrix and potentially increase the creation of further voids due to an

internal autocatalytic effect.

9.1.4 Migration of Thymol

It is suggested that thymol is readily released from PLA and PLA/kenaf films into 15% and

95% v/v ethanol/water simulants and the process can be described by an overall first-order

kinetics model that can be used to determine the initial release rate. The short- and long-term

diffusion models also adequately describe the release of thymol from these systems and the

results are consistent with those obtained using a Fick’s law analysis approach. The diffusion

coefficient data suggest that the addition of the kenaf filler to the PLA matrix facilitates the

release of thymol from the composite and that the temperature dependency of diffusion follows

an Arrhenius relationship. As expected, the release rate of thymol from the PLA film

formulations into fatty and aqueous food simulants was found to be temperature-dependent.

Furthermore, an increase in polarity of the simulant that results from an increase in its water

content decreases the propensity of thymol to be released from the composite system.

162

9.1.5 Antimicrobial Activity and Storage Stability

The PLA and PLA/kenaf films containing high thymol concentrations showed a strong AM

activity against E. coli with a significant inhibition of the growth in both solid and liquid media.

The PLA/kenaf/thymol composite films significantly reduced the population of E. coli on

inoculated chicken slice samples when placed in direct contact with the food compared with

the PLA/ thymol films. It is suggested that the films containing higher thymol content as well

as higher kenaf loading were more effective in controlling or limiting the fungal growth in the

chicken slice samples via vapour phase transmission of the AM agent than those containing

lower loadings of these additives. Moreover, compared to samples that were wrapped in

aluminium foil prior to storage, a slight decrease in the retention of the AM additive was

observed in PLA/kenaf/thymol films containing 30% w/w kenaf and 10% w/w thymol that

were not wrapped in the foil after storage for 3 months at ambient temperatures.

9.2 Significance of the Findings

Nowadays, there is increasing consumer demands for fresh, high-quality and natural foods with

minimum amounts of preservatives that are packaged in environmentally friendly materials

that prolong the shelf life of the foodstuff. The development of bio-based materials that are

renewable and biodegradable is of increasing priority. The use of bio-fillers incorporated into

PLA is receiving attention mainly for the potential these to create materials that are more

environmentally sustainable than currently used materials. The results of the current study

suggest the use of kenaf fibres as a natural filler or reinforcement in PLA composites will

enable the production of a low-cost AM packaging material that is environmentally sustainable

due to the renewability and abundance of the associated raw materials. The incorporation of

natural fibres into bio-based polymers together with the addition of naturally-derived AM

163

agents that have minimal impact on the environment is likely to lead to the next generation of

packaging materials.

The present study has demonstrated that the active PLA composites containing natural filler

have acceptable packaging properties as compared with commercial PLA. The good

mechanical strength of the material offers the potential application as a rigid packaging material

and/or coating. The high stiffness of active PLA/kenaf composites offers potential applications

in thin walled food containers, crates and food trays. Since the PLA/kenaf composites release

thymol faster than neat PLA, as reflected in the AM activity results (in vitro and in vivo), the

use of active PLA with the presence of kenaf filler for disposable food trays or containers is

suggested. Moreover, based on the observations made during the investigation of the

interaction and quantification of thymol in the active PLA and PLA/kenaf films as well as the

AM activity of the materials, it is suggested that the polymer may protect the AM agent during

storage while the presence of kenaf fibres may trigger the AM activity once the material

exposed to a humid environment. In addition, only a slight decrease in the retained AM additive

concentration was observed after storage suggesting that a sufficient amount of thymol

remained in the polymeric matrix to enable these materials to remain active.

9.3 Recommendation for Further Research

It was found that the active PLA-based materials containing the natural additive thymol and a

natural fibre filler are best suited for rigid packaging materials and ready-to-eat food products.

Whilst achieving its original aims, the current study has opened up new avenues of enquiry that

can be pursued in the future to possibly enable the commercial implementation of this or similar

AM systems:

The method of incorporating natural fibres and thymol into PLA during melt blending

and/or extrusion processing could be varied in order to further study the interaction and

164

quantification of thymol in the system. One suggestion is to dope the fibres with thymol

solutions prior to processing via melt mixing with PLA.

The effect of nano-fillers derived from kenaf fibres on the mechanical, thermal and AM

activity of PLA as well as the potential of these particles to migrate into food products

could be investigated to see if there are further advantages to be had by nano-sizing the

filler in these systems.

The effect of microencapsulation on the volatility of the AM agent during processing

could be investigated as a possible way of optimizing the retention of the AM agent in

the system prior to use as an AM packaging material.

The potential for incorporating other AM agents such as immobilized AM substances

onto PLA-based materials containing natural fibres as well as the inhibitory effect of

thymol against other spoilage or pathogenic microorganisms on various foodstuffs

could be investigated in other major studies.

The lifetime of the active PLA composites could be further investigated with studies

such as the effect of aging on the films and composites, the effect of molecular weight

changes over time, the effect of food contact on the properties of materials, the effect

of soil-based or model landfill degradation, as well as a full life cycle assessment of the

materials including the impact of increasing food preservation due to the addition of the

AM additives.

165

Appendix A: Properties of Polymer and AM Agent

Table A. 1 Properties of poly(lactic acid)

Poly(lactic acid)

Product Code 7001D IngeoTM Injection Stretch Blow Moulding Bottle Grade

Company NatureWorks LLC, USA

Structure

Molecular Formula (C3H4O2)n

Specific Gravity 1.24

Melt Flow Rate 6 g/10 min (210°C, 2.16kg)

Relative Viscosity 3.9-4.1

Crystalline Melt Temperature 145-155°C

Glass Transition Temperature 52-58°C

Crystallization Temperature 100-120°C

Transmission Rates Oxygen 550 cc-mil/m2/24 hr-atm

Carbon Dioxide 3000 cc-mil/m2/24 hr-atm

Water Vapour 325 g-mil/m2/24 hr-atm

Clarity Transparent

Tensile Yield Strength 60 MPa

Tensile Strength at Break 53 MPa

Tensile Modulus 3.6 GPa

Tensile Elongation 6%

Notched Impact Strength 16 J/m

Flexural Strength 83 MPa

Flexural Modulus 3.8 GPa

Heat Distortion Temperature 55°C

O

CH3

O

n

166

Table A. 2 Properties of thymol

Thymol

Product Code T0501

Assay ≥99.5%

Company Sigma-Aldrich Pty, Ltd.

Synonyms 2-Isopropyl-5-methlphenol;

5-Methyl-2-isopropylphenol;

5-Methyl-2-(1-methylethyl) phenol;

Structure

Molecular Formula C10H14O

Molecular Weight 150.22 g/mol

CAS Number 89-83-8

Appearance/Physical State Crystalline and colourless

pH 7 at 1 g/l

Density 0.965 g/cm3 at 25°C

Melting Point 48-51°C

Boiling Point 232°C

Flash Point 110°C

Solubility Slightly soluble in water and glycerol, very soluble in

alcohol and in ether, freely soluble in essential oils and in

fatty oils

OH

CH3

CH3 H3C

167

Appendix B: Supplementary Figures Pertaining to the Properties of the PLA-based

Materials

Figure B-1. Tensile properties of PLA/untreated kenaf composites containing: (□) zero, ()

5% (w/w) and () 10% (w/w) thymol: (a) tensile strength; (b) Young’s modulus and (c)

percent elongation at break

0

20

40

60

80

0 10 20 30 40

ten

sile

str

en

gth

/MP

a

0

500

1000

1500

2000

0 10 20 30 40

Yo

un

g's

mo

du

lus/M

Pa

0

5

10

15

0 10 20 30 40

elo

nga

tio

n a

t b

ea

k/%

UK Content/%

(a)

(b)

(c)

168

Appendix C: Supplementary Figures Pertaining to the Migration of Thymol into Food

Simulants

Figure C-1. Plot of (a) the mass fraction mt/m∞ versus t and (b) ln(1 - mt/m∞) versus t for the

release of thymol from (i) PLA and (ii) PLA/kenaf films into 150 mL/L ethanol/water at: ■

83°C, □ 75°C,●65°C and ○ 60°C where mt is the mass of thymol released from the film at

time t and m is the amount of thymol released from the film at equilibrium (t = ).

-5

-4

-3

-2

-1

0

0.0

0.2

0.4

0.6

0.8

1.0

0 10 20 30 40 50

t / s x 10-3

-6

-5

-4

-3

-2

-1

0

0 10 20 30 40 50

t / s x 10-3

(a) (b)

0.0

0.2

0.4

0.6

0.8

1.0 (i) (i)

(ii) (ii)

169

Figure C-2. Plot of (a) mass fraction mt/m∞ versus t1/2and (b) ln(1 - mt/m∞) versus t for the

release of thymol from (i) PLA and (ii) PLA/kenaf films into 150 mL/L ethanol/water at 60°C

where mt is the mass of thymol released from the film at time t and m is the amount of thymol

released from the film at equilibrium (t = ).

-3.5

-2.5

-1.5

-0.5

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0 20 40 60 80 100 120 140

time½/s½

-4.0

-3.0

-2.0

-1.0

0 10 20 30 40 50

time/s × 10-3

(i) (i)

(ii) (ii)

(a) (b)

170

Appendix D: Supplementary Figures Pertaining to Antimicrobial Activity of PLA-based

Materials containing Thymol on Solid and Liquid Media

Figure D-1. Plot of bacteria count of log CFU cm-2 versus day of the AM activity on meat

samples against E. coli for: (a) control without film, (b) PLA films containing 10% w/w thymol,

(c) PLA films containing 20% w/w thymol and (d) PLA films containing 30% w/w thymol.

y = -0.0258x + 4.4901R² = 0.9283

0.0

1.0

2.0

3.0

4.0

5.0

Control

y = -0.0465x + 4.3278R² = 0.8387

y = -0.067x + 4.2304R² = 0.9457

0.0

1.0

2.0

3.0

4.0

5.0

0 10 20 30 40

t/day

PLA20T 20KF20T

y = -0.0412x + 4.7719R² = 0.881

y = -0.0536x + 4.6671R² = 0.9501

0.0

1.0

2.0

3.0

4.0

5.0

6.0

PLA10T 30KF10T

y = -0.0809x + 4.3338R² = 0.9477

y = -0.1893x + 3.5648R² = 0.9992

0.0

1.0

2.0

3.0

4.0

5.0

0 10 20 30 40

t/day

PLA30T 20KF30T

(a) (b)

(c) (d)log

CF

U c

m-2 lo

g C

FU

cm

-2

171

Figure D-2. Fungal coverage on the surface of meat samples store at 10°C for the control,

neat PLA, PLA film containing 30% w/w kenaf, PLA film containing 10% w/w thymol and

PLA/kenaf film containing 10% w/w thymol and 40% w/w kenaf fibres.

172

Figure D-3. Antimicrobial effect of films against E. coli after 24h of incubation at 37°C of

(a) PLA film containing 20% w/w thymol and (d) PLA/kenaf film containing 20% w/w thymol

and 20% w/w kenaf fibres.

173

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