The Research On Using Immobilized Alkaline Protease To Prepare Soybean Peptide And Its Antioxidant Activity

In order to prevent food spoilage, a certain amount of antioxidants are often added. the synthetic antioxidants are remains the most extensive additive in food industry,because these synthetic antioxidants existthe potential health hazards , making them the application in the food subject to certain restrictions. Development of the natural antioxidants with low toxicity and high efficiency has become the research hotspot in recent years.Recent studies have found that the peptides obtained from the hydrolysis of protein have good antioxidant activity, and the benefits of hydrolysis of soy protein isolates with the alkaline proteases have been widely recognized. However, hydrolysis with the free proteases has many disadvantages such as instability, the higher cost and so on, and thus its industrial application has been limited. The immobilized proteases not only retain the inherent advantages of the proteases, bot also have many advantages such as easier separation and recovery and greater stability.In the study, Alcalase 2.4L was immobilized using porous chitosan microsphere adsorption and glutaraldehyde cross-linking method. Based on the results of single-factor tests on the effect of pH, E/S ratio, temperature, glutaraldehyde concentration and time, the optimum conditions of immobilization of Alcalase 2.4L were determined using response surfae methodology. The optimal immobilizing condition was pH of 9.20, E/S ratio of 7512U/g, temperature of 44.58℃, glutaraldehyde concentration of 0.20 % and the incubation time of 8.11 hours. Under this condition, the recovery rate of enzyme activity reached 56.73 %.The km value, the optimum temperature, optimum pH, storage stability, thermal stability of immobilized alkaline protease and alkaline protease t were studyed in this present paper ,the result showed :Compared with the free Alcalase, the immobilized Alcalse showed higher thermal stability, pH stability and storage stability.The soybean protein isolates were hydrolysed using the immobilized Alcalase. The single-factor tests were conducted using the degree of hydrolysis as the target index. Based on the results of the single-factor experiment, the conditions for preparing soybean antioxidant peptides was optimized using orthogonal experimental methodology. The results indicated that the optimal condition was as the followings : temperature of 60℃, pH of 9.2 and enzyme loading of 0.20 g/g Substrate. Under this condition, the product had the DH of 24.34 % and the reducing power equivalent to 0.14 mg/ml Vc.Ultrafiltration of the soybean antioxidant peptides was performed and the antioxidant activity of the soybean peptides and the resultant fractions were assayed. The results showed that the content of the small peptides (molecular weight<1000) reached 65.7 %, the reducing power of the small peptides was equivalent to 0.345 mg/ml Vc and their inhibition rate of superoxide anion and hydroxyl groups were 82.5 % and 91.1 %,respectively.