(255x) Analyzing the Chemical and the Physical Characteristics of Crosslinked Zein Gel Films Cast from Acetic Acid Solutions

Zein is the most abundant protein found in corn and large amounts are obtained as a byproduct of ethanol production ^[1]. Zein is amphiphilic and insoluble in pure water and is highly soluble in various ethanol/water solutions and acetic acid/water solutions. Zein has a wide range of utilization area. Its film forming capabilities makes zein one of the most commonly chosen plant-based materials for creating various analytical platforms. In this study biodegradable zein gel films were produced by solution casting and the mechanical, chemical and surface properties of the films were analyzed with different techniques. 70% acetic acid aqueous solution was used as the solvent due to its good dissolution properties for zein and due to its ability to produce very smooth surfaces with excellent surface topography ^{[3], [4]}. The concentration of zein was kept constant at 1:5 (g/ml) ratio. Oleic acid was used as a plasticizer at a 1:1 (g/g) zein to oleic acid ratio to increase flexibility and ductility of the film. For a better blending of the plasticizer into the zein matrix, monoglyceride at a 1:0.15 (g/g) oleic acid to monoglyceride ratio was used as the emulsifier. This study particularly focuses on the effect of crosslinking on the surface, mechanical and chemical properties of zein gel films. Glutaraldehyde was used as the crosslinker and was added to the zein solutions in different ratios and the solutions were allowed to crosslink for 20 hours after sonication. The solutions were cast on cured PDMS films and dried under the fume hood until complete evaporation of unbound solvent was obtained.

Chemical changes occurring due to crosslinking were studied with FTIR and FT-Raman spectroscopies. The surface properties of the contact side of the films with PDMS were analyzed with water contact angle measurements and Atomic Force Microscopy. The tensile mechanical properties were analyzed with a texture analyzer. FTIR spectra of zein gel films show typical characteristics of proteins at Amide I, II and III bands. In addition, some of the peaks were also seen as an indication of amino acid content of zein such as tyrosine or phenylalanine. The addition of glutaraldehyde at ratios of 4%, 8%, 10% and 12% with respect to the amount of zein were found to have a significant effect on the formation of peaks in the range 900-1100 cm^-1. There is a significant increase in the total intensity of this range. This increasing intensity, along with the increasing intensity in the 1230-1240 cm^-1 range, suggests an increasing number of crosslinking bond formation between zein and glutaraldehyde. These changes suggest that the interaction between glutaraldehyde and zein happens through the OH groups of glutamine at the end of which C-O-C bond formation occurs ^[5]. Amide I bands of the FTIR spectra were further analyzed for secondary structure changes and the results showed that the addition of increasing amounts of glutaraldehyde resulted in conversion of some of the α-helix into β-turns consistent with the expectation that crosslinks would limit the formation of α-helix and in crosslink points these constraints would results in the rearrangement of zein in the form β-turns. Characteristics of zein protein were also studied with FT-Raman experiments. The formation of Amide I band at 1600-1700 cm^-1 and Amide III band at 1200-1400 cm^-1 are mainly due to the C=O stretching and N-H bending respectively ^[6]. The peak with the highest intensity was observed in 2800-3000 cm^-1 range which represents C-H stretching ^[6]. The increasing glutaraldehyde concentration caused an intensity increase in this range which is due to the increasing amount of aliphatic residues that include more C-H bonds. Increase in the intensity of the peaks at 2800-3000 cm^-1 range of FT-Raman were also sensitive to increasing thickness of the zein gel films. Similar results were shown in a previous study in which a method of determining the thickness of the films through FT-Raman spectra was suggested for the first time ^[7].

AFM images showed that the surface of the crosslinked films were covered with needle-like structures while control films had fewer structures on their surfaces that were shorter and thicker. Control films had the smallest surface roughness value of 1.633 nm in a 0.5 μm x 0.5 μm scanning area while the roughness increased as the ratio of glutaraldehyde increased. Films with 12% glutaraldehyde ratio had the highest roughness value of 4.902 nm over a 0.5 μm x 0.5 μm scanning area. Water contact angle measurements did not show much of a trend between the degree of crosslinks and uncrosslinked films. Control films had an the highest average WCA value of 87.850 degrees while the lowest average WCA value was measured with 8% crosslinked films. Tensile strength measurements also showed that as the glutaraldehyde ratio increased the youngâ??s moduli of the films increased indicating increasing stiffness.

In summary, this study focuses on the effects of crosslinking on the mechanical, chemical and surface properties of zein gel films cast from acetic acid solutions. The formation of new bonds due to the interaction between glutaraldehyde and zein molecules were clearly observed in FTIR results. FT-Raman results were consistent with FTIR results suggesting chemical changes due to increasing amount of glutaraldehyde. Increasing crosslinker amount increased the stiffness of the films as well as the surface roughness of the films. Lastly, surface wettability of the films were not consistently affected from the addition of crosslinker in different ratios.

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[6] E. C. Y. Li-Chan, â??The applications of Raman spectroscopy in food science,â? Trends Food Sci. Technol., vol. 7, no. 11, pp. 361â??370, Nov. 1996.

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