A Comparative study on Physio-Chemical Properties of Recovered Gelatin from Beluga (Huso huso) fish skin by Enzymatic and Chemical Methods

Gelatin is a water-soluble protein mixture that is obtained by partial hydrolysis of collagen, which forms the major protein in bones, cartilage, and skin. Gelatin is made from collagen fibers, which is low in protein, cholesterol, fat and carbohydrates, with a special positive effect on human health. Gelatin is one of the most notable natural biopolymers, the most important source of this hydrocolloid is pig. Trying to find suitable gelatin supplements for food products is increasing. Fish gelatin is one of the most suitable mammalian gelatinous substitutes and is accepted as a Halal (Kosher) food item. The purpose of this study was to extract gelatin from sturgeon Beluga skin using pepsin enzyme and then investigate the effect of the enzyme on the improvement of physiochemical and its functional properties in comparison to the gelatin extracted by chemical method.

Pre-treatment and extraction of gelatin and application of factorsSkin Preparation was performed according to Feng et al. (2013) with slight changes in pre-treatment steps. A solution of 3.5% NaCl was used to remove non-collagenic proteins and 0.5% sodium carbonate solution (Na2CO3) to remove lipid from the skin. The initial pretreatment was carried out with a solution of 3.5% sodium chloride at a rate of 1:10 w / v at a speed of 180 rpm for 6 hours which was replaced every 3 hours with the water. Extraction by was carried out following Tong et al. (2013) method. Gelatin was obtained from pre-treated skin in distilled water at temperature of 30, 40 and 50 degrees Celsius and a percentage of enzymes (0.01, 0.055 and 0.1) at different pH (2, 3 and 4) for 6 hours and 45 minutes in hot water bath. Then, the mixture was kept in the boiling water bath, for 5 minutes to inactivate the enzyme. The solution was passed through a cleaning cloth and then centrifuged at 3500 rpm for 20 min and finally was lyophilized in a freeze-drier. In this research, the Response surface methodology response (RSM) method was used to optimize the experimental treatments. The central composite rotatable design was used to optimize the gelatin enzyme extraction process.

The α chains were clearly visible in the sample extracted by the enzyme, with molecular weight of 130 kDa (treatment 5), while the α2 chain is much weaker in the extracted gelatin by chemical method. By decreasing enzyme ratios, chains with molecular weights of less than 130 kDa disappeared gradually in chemical samples as well as in an enzyme-extracted sample (treatment 19). The gelatin extracted by the enzymatic method contains α1 and α2 chains. In the average amount of enzyme (treatment 17), the α2 chains were relatively weaker than the maximum value of the enzyme (treatment 5), and these chains almost disappeared in the minimum amount of enzyme (treatment 19). These chains are weaker in the chemical extracted gelatin than that of treatment 5. Moreover, it can be seen that in the gelatin extracted by the enzymatic method, the ratio of the α2 / α1 chain is about 2 (the intrinsic ratio in collagen I), which shows that the inherent structure of gelatin is preserved.The highest and lowest amidic wavelength A was obtained for treatment 17 and treatment 19, the highest and lowest wavelength amide I for treatment 5 and treatment 19, the highest and lowest amid II wavelengths for chemical extracted treatments and treatment 17, The highest and lowest ratio of amide III to amide 1454 is related to treatments 5 and 19, and the highest and lowest amount of amide B is related to treatment 19 and 5.The behavior of the viscoelastic modulus of the treatments extracted with the pepsin enzyme (treatment 5, 17 and 19) and the chemically prepared sample showed that at temperatures lower than 20 ° C the storage or elastic modulus (G ') and the loss or viscous modulus (G') decreased with increasing temperature and the elastic modulus was larger than the loss modulus (G' >G'' ) indicating that the samples are still jelly-like (with the exception of treatment 19 that had the weakest gel strength).In all gelatin samples extracted using pepsin and chemical method, first, a relaxation stress index (viscosity increase at the beginning of the graph), and then a thinning non-Newtonian behavior (pseudo-plastic) was obvious during the shear rate. In fact, in the behavior of pseudo-plastic, the viscosity of the fluid is related to the shear rate and has decreased with increasing shear rate. Non-Newtonian viscosity of treatments prepared by the enzymatic method (except treatment 19) was higher than that of the chemically prepared sample at different applied shear rate.