Research On Immobilized Rennet Production Of Casein Glycomacropeptide

Casein Glycomacropeptide (CGMP) is a glycopeptide contains 64 amino acid produced by chymosin (or pepsin) cleavaging of κ-casein between Phe105-Met106.CGMP has a variety of biological activity. But amount of lactose and whey protein prevent the development of production CGMP on large scale. This paper studies on the craft of the production CGMP with enzymatic casein and immobilization of rennet process to reduce industrial costs, separate and purify CGMP in a variety of methods and identified the ingredients of CGMP sample in different purity.According to the experimental results of single factor, the immobilized enzyme main factors affecting were the total amount of enzyme, immobilizating time and concentration of glutaraldehyde by Minitab software. After the Design Expert software forecasting optimization analysis, glutaraldehyde concentration of 13%, the total amount of enzyme 200 mg/gram of carrier, immobilization time of 3h, immobilization time of 50 min.Enzymatic properties and stability of the immobilized rennet are measured, the optimum temperature is 55℃, the optimum pH is 5.5, and immobilized enzyme show good stability. The recovery rate of activity of immobilized enzyme reaches 26.59%. Comparison of the immobilized enzyme with the free enzyme in production of CGMP, they show the similar cotent of CGMP in the supernatant. The immobilized rennet can be reused for six times, which shows its good operating stability. The enzyme activity is still more than 80% after 2 months’ storing. In practical applications it can save costs and has good potentials for industrial applications.In the part of separation and purification CGMP, the impurity effect of the optimum conditions of ultrafiltration and ion exchange chromatography are studied. The results show the ultrafiltration pressure of 0.03Mpa and the pH 7 are determined in the condition of interception of 5000Da of ultrafiltration membrane. A sort of anion exchange resin D280 was selected and the condition of sample volume of 1/10BV, pH 6.5, elution rate of 1BV/h elution gradient of NaCl of 0.4,0.6,1.0mol/L. and each gradient elution volume of 1.5BV. Moreover, the casein glycomacropeptide the recovery rate reaches 75.68%.Three kinds of CGMP samples in different purity are made after spray-drying, then they are compared with Danish sample, the related physical and chemical indicators are detested according to the national standards. The results show that the glycosylation and purity of the sample 2 and sample 3 are in a higher degree and comply with health standards of production.