1 STRUCTURAL PROPERTIES OF ZEIN-XANTHAN GUM BIOFILMS Crislene B. Almeida 1*, José F. Lopes Filho 1 1* Universidade Estadual Paulista - UNESP, Campus São José do Rio Preto - 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).
2 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%,
3 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
4 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
5 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.
6 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, ± 0,29 b Zein + Xanthan 0.01% ± 0, ± 0,95 a Zein + Xanthan 0.02% ± 0, ± 0,26 b Zein + Xanthan 0.03% ± 0, ± 1,13 b Zein + Xanthan 0.04% ± 0, ± 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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