STRUCTURAL PROPERTIES OF ZEIN-XANTHAN

GUM BIOFILMS

Crislene B. Almeida1*

, José F. Lopes Filho1

1*

Universidade Estadual Paulista - UNESP, Campus São José do Rio Preto - crislene2002@yahoo.com.br

Structural and optical characteristics of zein based biofilms produced with four xanthan gum concentrations, 0 to

0.04%, were analyzed in this study. Scanning electronic microscopy (SEM) and optical microscopy were performed to

identify if the incorporation of the material into the matrix film, formed a homogeneous structure, as well as to

characterize its constituents as the color and shape. SEM showed a homogeneous matrix for the control (0% xanthan)

with better lipids distribution. However, when the samples were investigated by Optical Microscopy, lipids globules in

the control biofilm appeared larger and more dispersed in the matrix than the others samples. Transparency/opacity test

measurements by spectrophotometry indicated that xanthan addition to the matrix of the film lowered significantly their

transparencies properties. Overall, the addition of xanthan gum favored lipids dispersion in the matrix making the

biofilms more homogeneous, although whit less transparency.

Keywords: zein, xanthan gum, biofilms.

Introduction

Zein, the main corn protein and alcohol soluble, is commercially produced from corn gluten,

a subproduct of corn wet milling process. This protein has low biologic value due to the aminoacids

unbalance: high contents of leucin and glutamine and low contents of lysine and triptophan (1-2-3).

A great differential of the zein, as compared to the other corn protein, is the polymerization

characteristics. It has two times more potential than the necessary to produce linear

polyamide/polyester polymers.

According to Morris (4) the xanthan or xanthan gum is an important food additive due to its

functional properties as well as the improvement in several food characteristics. Xanthan is a

natural polymer, hydrophilic, produced by microorganisms of the gender Xanthomonas campestris.

The product is obtained through sugar fermentation by specials types of the bacteria (5). Because

of the excellent rehological properties it has been used as thickener, stabilizer, emulsifier, and

suspension agent in several products and process by chemical, cosmetics and food industries,

among others (6).

According to Lai and Padua (7), zein biofilms present good transparency and with

plasticizers addition to the matrix, the material becomes more flexible, although some properties

can be modified as, for example, increasing opacity (8-9). In order to improve the material

characteristics, co-polymerizations and different polymeric blends have been produced and

characterized (10).

Anais do 10o Congresso Brasileiro de Polímeros – Foz do Iguaçu, PR – Outubro/2009

During polymeric materials development, a physical mixture of two or more polymers

forming a polymeric blend has, most of the time, attracted more attention than the polymers

synthesis. This happened mainly because the combination of polymers properties resulting in

materials with different properties that, several times, are better than the individual polymer

properties. This proceeding is easier and less expensive than investigation of new synthetics route.

The final polymeric blends properties depend on miscibility of the constituents or morphological

structure of each phase in the case of heterogeneous blend (11).

For understanding polymer-polymer miscibility, Scanning Electronic Microscopy (SEM),

among other techniques, has been used (12). Material incorporation in the matrix can form a

homogeneous or heterogeneous structure depending on their interactions. Optical Microscopy and

SEM allow the identification of material incorporation in the matrix and permit its characterization

through the color and shape.

The transparency/opacity of the material shows its capacity to block the light. A low

transparency or a high opacity indicates that the material is a good light blocker. Biofilms to use as

packing or food covering should have a high transparency when original characteristics of the

packed product have to be visible (13). However the material can become more susceptible to the

heat (14).

The aim of this research were to produce composites biofilms with zein-xanthan gum at

different xanthan concentrations, and to determine its optical and structural characteristics. SEM,

Optical Microscopy, and transparency/opacity determinations were done to characterize the

biofilms.

Materials and Methods

The biofilms were prepared mixing 20% of Corn zein, regular grade (F4000, Freeman

Industries, Inc., Tuckahoe, NY), 0.06% of glycerol (Merck, Brazil), 14% of oleic acid 90% (Synth,

Brazil), and 0.01% of sorbitan/emulsifier (Duas Rodas Industrial Ltda, Brazil). All components

were solubilized in aqueous ethanol solution 75% (w/v). For each treatment 10mL total solution of

zein-biomaterials were prepared by dissolving granular zein in a 75% aqueous ethanol solution, to a

concentration of 16% (w/v) at room temperature. Oleic acid was added at a ratio of 70g/100g zein

(w/w), while stirring the solution on a water bath at 65°C at 3500rpm using a vortex type stirrer

(Phoenix AP-56, Brazil). Following, glycerol and sorbitan were added at a ratio according

concentration previously stated. After 10min the filmogenic solution was submitted to a 20mHz

ultra sonic frequency (Fisher Scientific®

) by others 10min. The following xanthan concentrations

(w/v), used as the sorbitan substitutes, represent the treatments performed: 0.01%, 0.02%, 0.03%,

Anais do 10o Congresso Brasileiro de Polímeros – Foz do Iguaçu, PR – Outubro/2009

and 0.04%. The filmogenic solutions were casting on rectangular acrylic plates and maintained at

room temperature (25°C) for 48h to dry. After drying the films formed were peeled out and stored

inside of dissecators at 58% relative humidity until analyses begin.

The thickness of the films were obtained by the arithmetic mean of six values measured in

six randomized points of each sample using a digital micrometer with 0.001mm resolution

(Digimess model).

Scanning Electronic Microscopy (SEM) is a common technique to analyze the

microstructure of biodegradable films. This technique has been used at decades to study the global

structure of proteins, mainly the quarternary conformations (15). For this analysis, films samples of

12mm diameter in duplicate, were fixed on stubs with conductive double face cupper ribbon and

covered with 35nm gold (EMITECH K550). Samples were observed on an electronic microscope

(LEO 435 VP) at 15 kV and 100µm magnification in a climatized room.

Optical microscopy was used to identify the formed compounds of the films through Xilidine

Ponceau pH 3.5 coloration techniques, which permits the detection of total cationic protein radicals

(16). The coloration technique of Periodic Acid & Schiff was used to identify neutral

polysaccharides and glycoproteins (17). The samples, in duplicate, were treated directly with

colorant techniques without previous fixation and dehydration because of the zein solubility in

alcohol solutions, which are used to fix the material. Instead of the fixation by the ethanol based

solution, the laminas were dried in oven (Odontobras ECB 1.2 Digital) at 37°C for 24h and

mounted with Canadian Balsam. After 24h the samples were analyzed at room temperature in an

optical microscope (Olympus BX 60) with an image capture system (Olympus DP 71). Different

points in the sample were observed with 500µm of magnification (10X).

Films apparent transparency was determined through a spectrophotometer UV-Vis (Quimis-

Brazil) as proposed by Gounga et al. (18). Samples of rectangular shape were applied in internal

wall of the cuvette and three replications of readings were done for each film at 600nm. Films

transparency was calculated dividing absorbance at 600nm by the film thickness.

ANOVA (Analyses of Variance) was performed considering a randomized design

experimental and Tukey tests applied to compare data means at 5% probability using a

computational program ESTAT, version 2.0, according to Banzatto and Kronka (19).

Results and Discussion

Thickness average of the films was 0.12 ± 0.03mm. The homogeneity of the samples can be

observed by electromicrographs shown in Figure 1. From this Figure it is observed that an increase

on xanthan concentration promoted changes in the surface of the material, interfering in the

compounds distribution. The control (Figure 1a) presented the best fat globules distribution, which

Anais do 10o Congresso Brasileiro de Polímeros – Foz do Iguaçu, PR – Outubro/2009

is identified by the black points in the picture. Apparently, there was no formation of a continuous

layer of the matrix in the others films with xanthan concentrations higher than 0%. Similar

observations characterized by several points in the material surface, were found for zein biofilms in

the work done by Corradini and Ghanbarzadeh et al. (20- 21). The first insight suggests that these

points are micro bulbs entrapped inside the matrix or spaces occupied by glycerol before the drying

process (22). However, there is a possibility of phase separation between zein and glycerol due to a

low interaction between these two compounds (20).

a b

c d Figure 1. Scanning Electronic Micrographs of zein- xanthan biofilms: a) 0% xanthan (control); b)

0.01% xanthan; c) 0.02% xanthan; d) 0.04% xanthan.

During biofilms formation it was observed difficulties to homogenize the solution as

xanthan concentrations increased, manly with 0.03% and 0.04%, when some lumps appeared as

spots in the film. In order to better characterize these lumps as well as the points found in the

surface, optical microscopy was performed to observe the homogeneity through protein and fat

globules distribution besides the presence of the small cracks.

Figures 2 and 3 show the images magnified 500µm obtained for each xanthan concentration

using the two kinds of sample preparation: Xilidine Ponceau pH 2.5 and Periodic Acid & Schiff

colorations, respectively. The red color in Figure 2 represents protein fraction and the white points

the fat globules. As xanthan concentration increases, fat globules sizes decreases, enhancing a better

homogenization of the material into the film matrix. From Figure 3 it is observed that increasing

Anais do 10o Congresso Brasileiro de Polímeros – Foz do Iguaçu, PR – Outubro/2009

xanthan concentration resulted in bluer colored sample. Figures 3d (0.04% xanthan) presents less

fat dispersed confirming the observations shown in Figure 2 (Xilidine Ponceau coloration).

The control sample also presents a slight purple color which indicates that the Periodic Acid

& Schiff reagent coloured the hydroxil radicals of the matrix structure (23).

a b

c d Figura 2. Optical microscopy with Xilidine Ponceau pH 2.5 coloration for zein- xanthan biofilms:

a) 0% xanthan (control); b) 0.01% xanthan; c) 0.03% xanthan; d) 0.04% xanthan.

a b

c d Figure 3. Optical microscopy with Periodic Acid & Schiff coloration for zein- xanthan biofilms: a)

0% xanthan (control); b) 0.01% xanthan; c) 0.03% xanthan; d) 0.04% xanthan.

Anais do 10o Congresso Brasileiro de Polímeros – Foz do Iguaçu, PR – Outubro/2009

After optical microscopy analyses, it can be concluded that, the observations previously

considered as porous on SEM images, are indeed fat globules dispersed into the film matrix. Thus it

is confirmed that xanthan addition improves homogenization of the compounds mainly with respect

to the size and disposition of the fat globules.

Transparency tests done by UV-vis spectrometer confirmed the observations of PAS analyses

(Table 1).

Table 1. Transparency of the zein-xanthan biofilms obtained by UV-vis spectrometry.

Material Absorbance at 600nm Transparency

Zein + Sorbitan 0.963 ± 0,04 7.570 ± 0,29 b

Zein + Xanthan 0.01% 0.822 ± 0,08 9.605 ± 0,95 a

Zein + Xanthan 0.02% 0.811 ± 0,03 7.277 ± 0,26 b

Zein + Xanthan 0.03% 0.623 ± 0,09 7.624 ± 1,13 b

Zein + Xanthan 0.04% 0.869 ± 0,04 5.565 ± 0,30 c

a,b,c – Means followed by the same letters in each column are not different by Tukey´s test (p<0.05)

Films with 0.01% xanthan demonstrated better transparency or smaller opacity as shown by

the peaks with less intensity in the PAS results. Thus, the addition of xanthan gum in the polymer

matrix decreases the transparency of the material.

Conclusions

It was possible to produce the biofilms composed by zein-xanthan gum. The film prepared

with 0.04% of xanthan presented, visually, less homogeneity due to the presence of lumps across

surface. However, after optical microscopy analyses, it can be concluded that the observations

previously considered as porous on SEM images, are indeed fat globules dispersed into the film

matrix. Thus, it is confirmed that xanthan addition improves homogenization of the compounds,

mainly with respect to the size and disposition of the fat globules. The addition of xanthan gum

favored lipids dispersion in the matrix making the biofilms more homogeneous, although whith less

transparency.

Acknowledgments

FAPESP, CAPES, Department of Animal Biology (UNESP-S.J.R.Preto, Brazil).

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