Elaboration and characterization of antifungal properties of biodegradable film added with cinnamon oil

P. Hernández Carranza1, S. Mendoza Vázquez2, J. A. Guevara García2, S. Cid Pérez1, R. Ávila Sosa1 and C. E. Ochoa Velasco*1 1 Biochemistry-Food Department, Chemical Sciences Faculty, Benemérita Universidad Autónoma de Puebla, Ciudad

Universitaria, 72573 Puebla, Puebla, Mexico 2 Bioinorganic and Bioremediation Laboratory, Universidad Autónoma de Tlaxcala, San Luis Apizaquito, 90300 Apizaco

Tlaxcala, Mexico.

The aim of this research was to elaborate and characterize a film based on hydroxypropylmethylcellulose (HPMC), casein, glycerol and cinnamon essential oil (CEO). To evaluate the minimal inhibition concentration (MIC) of CEO, different concentration were used (0, 1, 1.5, 2, or 4 %) against Aspergillus niger and Penicillium spp. Edible films were characterized with Fourier-transform infrared spectroscopy (FT-IR). Results showed MIC values of 2% of CEO for both microorganisms. FT-IR shows that increase CEO concentration improves the interrelation between the components and the crystallinity, which permits to estimate the mechanic resistance of the film. The use of CEO based on HPMC should be used against spoilage molds and could be used as a biodegradable package for foods.

Keywords: cinnamon oil; biodegradable film; antifungal properties

1. Introduction

Essential oils are secondary metabolites present in plants containing low molecular weight aliphatic compounds, monoterpenes, sesquiterpenes and phenylpropanes; substances with antibacterial and/or antifungal activity [1], cinnamon essential oil (CEO) is rich in cinnamaldehyde, although it also contains linalool, eugenol and other phenolic compounds [2]. CEO is considered an antimicrobial, which means that it can inactivate microorganisms or control their growth [3], because it inhibits the production of intracellular enzymes, such as amylases and proteases, causing wall deterioration and a high degree of cell lysis [4]. In the last decades, the use of natural antimicrobials in packaging materials has become an alternative for food industry, whose main objective is to offer fresh foods with an extended shelf life [5]. Studies on the use of essential oils with antimicrobial properties have been reported by Pranoto [6] who described the benefits of adding garlic essential oil to edible films. On the other hand, Seydim and Sarikus [7] showed antimicrobial activity of garlic, oregano and rosemary essential oils in edible films. Kechichian [8] investigated the application of cinnamon and clove essential oils in starch films; therefore, the use of essential oils with antimicrobial properties generates a protective effect, which offers benefits in food conservation, in addition with this application it is possible to reduce the production of traditional packaging materials. Recently one food research challenge is not only to develop edible films added with natural substances like antimicrobials, but also to replace the use of synthetic polymers as edible coatings and packaging, in this regard new materials are being used such as hydroxypropylmethylcellulose (HPMC), which has high films forming capacity [9], is partially esterified by methyl groups and contains a small proportion of hydroxypropyl groups substitutions, allowing its use as a biodegradable material [10,11]. HPMC has been used as a coating matrix due to its selective permeability to gases, which allows to reduce the respiratory rate in fruits, delaying their deterioration [12], it also has some ability to form films with neutral sensory properties; however, its high permeability makes it necessary to incorporate lipids in their formulations [13], the addition of essential oils obtained from woody barks such as cinnamon, could help to improve HPMC edible films properties, generating biodegradable coatings [14], obtaining a product with two functionalities, a coating material and also biodegradable, therefore, the aim of this study was to elaborate and characterize a HPMC and casein edible film added with CEO to inhibit the growth of spoilage molds.

2. Materials and methods

2.1 Microorganisms

Aspergillus niger and Penicillium spp. strains were obtained from the laboratory of Facultad de Ciencias Básicas, Ingeniería y Tecnología, of Universidad Autónoma de Tlaxcala, Mexico. Cultures were maintained in acidified potato-dextrose agar (PDA) for 5 day at 28 ° C, after this time a spore suspension was made by removing the culture surface with 10 mL of peptone water. The number of spores present in the suspension was determined using a hemocytometer and an optical microscope (Zeiss Primo Star, Göttingen, Germany), and expressed as number of spores per milliliter (spores mL−1).

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2.2 Reagents

HPMC, glycerol, casein, citric acid, dibasic sodium phosphate and potassium bromide were purchased from J.T. Baker, CEO from Sigma-Aldrich, and PDA and peptone from BD Bioxon.

2.3 Edible film formation

Edible films were formed according to Herrera-Vázquez [11] methodology with modifications, 48.6 mL of 4% casein previously solubilized with dibasic sodium phosphate (0.2 N) and citric acid (0.1 N), 2.8 mL of glycerol and 0, 1, 1.5, 2 or 4 % v/v CEO, finally 48.6 mL of HPMC (9%) was added. The solution was kept under stirring for 15 min at 200 rpm, then solution was laminated onto a surface and dry for a period of 24-48 h, once the film was dry it was stored in sterile bags until use.

2.4 Antifungal activity of edible films

Spore suspension was inoculated in Petri dishes with acidified PDA, a 2x2 cm film square with different CEO concentrations (0, 1, 1.5, 2 or 4 %) were placed in plate center, plates were incubated at 28°C for 30 days, minimum inhibitory concentration (MIC) was determined at the concentration where no growth is presented. Each experiment was done in duplicate.

2.5 Chemical characterization

2.5.1 Fourier transform infrared spectroscopy (FT-IR)

Spectroscopy was performed for film components (HPMC and casein), a KBr pellet (mg pulverized feedstock with 100 mg KBr) was prepared. IR spectra were determined by FT-IR spectroscopy (Tensor II, Bruker, USA) in infrared region (4000 to 500 cm-1) with a resolution of 0.5 cm-1, data were processed and plotted with OriginPro 8 SR0 v8.0724 (B724) software. Spectra were determined as frequency (cm-1) vs transmittance. For HPMC and CEO edible films at different concentrations spectra were determined as absorbance vs wavelength.

2.5.2 Raman spectroscopy

Raman spectra were determined on HPMC and CEO edible films with a micro-Raman spectrometer (T64000, Horiba, USA), with a triple monochromator and a solid wavelength laser of 532.1 nm (Ventus, Laser Quantum, UK) with a power of 40 mW and a spectral resolution of 0.1 cm-1. The beam was focused on the sample (capillary 1 mm in diameter) to seal and irradiate, the scattered light was collected at 90° through a collimator and focused on the spectrometer input slit. Castor oil added to HPMC and casein edible film was analyzed as control.

3. Results and discussion

3.1 Antifungal activity of edible films

It was possible to incorporate CEO to edible films and all its components, Krochta [15] mentioned that cellulose derivatives such as carboxymethylcellulose, methylcellulose and HPMC are excellent compounds for edible films formation, since they are odorless, tasteless and biodegradable, in addition to their low application cost. To determine the antimicrobial effect of a substance it is necessary to consider some factors, including microorganism’s characteristics, such as inoculum size, physiological state and storage conditions [16]; in this study we start with a population of 1.6 x 104 spores mL-1 of Aspergillus niger and 2.3 x 104 spores mL-1 of Penicillium spp., being a suitable population to determine the antimicrobial effect of edible films added with CEO. Radial growth of both molds (Fig. 1) presents a delay for the adaptation phase until day 10. Aspergillus niger has a higher growth along incubation time, antifungal activity is more evident with edible films added with 1.5 % of CEO, at concentrations above 2 % of CEO microorganisms were inhibited. Pawar [17] reported that CEO has an inhibitory effect against hyphae growth and spore formation of A. niger. Matan [18] showed than CEO mixed with cloves at 2% concentration inhibits Aspergillus and Penicillium growth.

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Fig. 1 Effect of HPMC edible films added with CEO on Aspergillus niger (a) and Penicilluim spp. (b) growth.

3.2 Chemical characterization

Casein and HPMC FT-IR spectra, materials used in film formation are observed in Fig. 2, whereas functional groups bands assignments are consigned in Table 1. HPMC bands were interpreted according to Silverstein [19]. Results show a wide and strong absorption band with absorbances between 3500-3400 cm-1 corresponding to the vibration and stretching of the -OH (ν O-H) groups. Around 2900 cm-1 indicates a symmetrical elongation of C-H bonds (ν C-H), symmetrical vibration of C-H bonds (δ s Me) was determined between 1400-1350 cm-1, while asymmetric vibration out of phase of C-H (ν as Me) bonds occurs between 1500-1450 cm-1, signals can be attributed to methyl and hydroxypropyl groups characteristic of HPMC. These data agree with Alekseeva [20] who determined a wide band on 1358 cm-1 of C-H2 and C-H groups (3000-2800 cm-1) on hydroxyethylcellulose IR spectra, setting planar deformation of C-H and O-H links. On the other hand, in the region between 1650 and 1600 cm-1 is the vibration of elongation C-O (ν C-O) of cellulose rings, which is related to asymmetric vibration (ν as) of pyranoses in 1000-950 cm-1 region. Similarly, 1300-1250 cm-1 bands are indicative of cyclic epoxide (ν C-O-C) vibration. Shan [21] reported the characteristic bands of cellulose on 1200-1000 cm-1, they assigned a symmetrical stretching of ethylcellulose (C-O-C group at 1124 cm-1). Ethers characteristic vibrations are found between 1100-1000 cm-1 (ν C-O). Peak at 944 cm-1 near a weak band at 1053 cm-1 represents ether linkage vibration and vibrational rocking mode of -CH2 groups is in the range 850-800 cm-1. It was also determined that between 1091-1130 cm-1 an intense band is located and on this one shoulder (averaged at 1112 cm-1) indicative of C-O bonds of primary and secondary alcohols, glucose ethers as well as pyran ether-type bonds substituents are related to 1053 and 944 cm-1 bands respectively.

Fig 2 FT-IR spectra characteristics bands of casein and HPMC.

ab

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Table 1. Band assignation for HPMC and casein FT-IR spectra.

HPMC CASEIN BAND (cm-1)

INT ASSIGNATION BAND (cm-1)

INT ASSIGNATION

3482 S ν (O-H) alcohol 3439 S ν (O-H) alcohol 2982 2935

M Methyl and hydroxypropyl groups

2929 W ν (CH2) as

1652 M ν C-O, benzene 2858 W ν (CH2) sim 1470 M δ asMe, δ asOCH,

δ asCCH+vib. Hexose (cellulose)

1382 M δ sMe + ν C-O-C cyclic anhydrides

1335 W ν C-O-C cyclic epoxid

1633 M I (C=O) amide

1052 S ν C-O-C de ether 1527 W II (NH) amide 941 S ν as de la pyranose 847 S CH2 groups 1039 W δ (C-O) alcohol

INT – Intensity; S – Strong; M – Medium; W – Weak FT-IR spectra of HPMC added with castor oil or CEO is showed in Fig. 3. In HPMC (gray solid line) hexose methylenes signal appears at 1500-1250 cm-1 range, which are completely modified suggesting a strong interaction between ring substituents and other film components, these interactions may be due to the presence of hydrogen bonds observed in the band a 3500 cm-1. A band growth and widening signal near 3415 cm-1 is also observed, and a second thin near 1747 cm-1, which is indicative of an interaction and/or the formation of new bonds between HPMC and casein, which is corroborated by amide signals I and II that move to higher frequencies (1647 and 1572 cm-1). In the film added with 4 % of CEO (black solid line), at shift at lower frequencies (3273 cm-1) compared to HPMC film (3415 cm-1) is observed, which is indicative of a reinforcement of hydrogen bonds to HPMC -OH groups. HPMC-casein interaction is reduced in this film which is related to 1747 cm-1 signal that moves at 1736 cm-1 (minimum peak) and a decrease of amide I and II vibrations with respect to HPMC, which in the spectrum are shown at 1539 and 1642 cm-1.

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Fig. 3 FT-IR spectra of HPMC (grey line); HPMC added with 1% of castor oil (black dashed line) and HPMC added with 4 % of CEO (black line) edible films.

On the other hand, HPMC and CEO are showed in Fig. 4, spectra showed the displacement of the alcohol band and ν (O-H) which indicates that hexose ring moves at a higher frequency. A growth and spread signal, whose maximum absorption is displaced to 3415 cm-1, is indicative of a reinforcement in hydrogen bonding network, involving –OH groups of HPMC when CEO concentration increases.

Fig 4 FT-IR spectra of HPMC edible films added with different concentrations of CEO.

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Figure 5 showed Raman spectra of films with different concentrations of CEO. Narrow bands correspond to the highest concentrations of CEO are observed, which implies that crystallinity is directly proportional to CEO concentration. Therefore, an increased interaction of hydrogen bonds in film formation and the inclusion of CEO allows films with higher crystallinity degrees, and could provide certain mechanical and stability characteristics in films. Ziani [22] determined that crystallinity is directly related to the formation of hydrogen bonds bridges and Van der Waals interactions between film biomolecules, which promotes the formation of ordered and compact structures.

Fig 5 Raman spectra (normalized at 1120 cm-1) of HPMC edible films added with different concentrations of CEO (1% red line, 1.5% crimson line, 2% blue line, 4% black line).

4. Conclusions

HPMC edible films added with CEO could be used as antifungal inhibiting spoilage molds and to be as a natural antifungal agent for preservation of foods. The film can be used as active packaging and provides new ways for enhancing safety and long lasting in food systems. On the other hand, the use of spectroscopic tools allowed to know films components, as well as properties such as crystallinity.

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