Research On Hydrolysis Conditions Of Casein By Immobilized Trypsin To Generate Casein Phosphopeptides

Casein, with essential amino acid for growth, is the most abundant protein in milk. Recently, many researches demonstrated that casein not only had nutrition function but also had very important physiological functions, inaddition, casein was an important source of bioactive peptides, as the "carrier of minerals". CPPs can prevent the divalent metal ion precipitating in the intestinal environment of animals, and increase the concentration of intestinal soluble minerals, which will promote the absorption and utilization of calcium, iron, zinc, selenium by the intestinal mucosa, especially for calcium. Presently, CPPs is the only active peptide which can promote calcium absorption, furthermore, it also plays an important role on improving the body immunity and the reproductive performance, and so on.In this paper, the immobilized conditions of trypsin were studied, and cross-linking-adsorption was used to immobilized trypsin. The carrier was chitosan, and the crosslinking agent was glutaraldehyde. These factors which were enzyme dosage, glutaraldehyde concentration, pH, temperature, cross linking time and adsorption time influencing on immobilized enzyme. Activity and dynamic recovery were researched. The significant factors were chose for L₉(3⁴) orthogonal experiments, enzyme activity was chose as experimental indicator, and then the immobilized conditions of trypsin were optimized, so as to obtain the best reaction conditions for immobilized.When chitosan was used to immobilized trypsin, the best immobilized conditions were as follows: enzyme dosage 14mg, glutaraldehyde concentration 0.2%, pH7.5, temperature 35, crosslinking time 2h and adsorption time 5h. On the best immobilized conditions, the immobilized enzyme activity and the dynamic recovery were 397.29U and 76.57%, respectively.Immobilized enzyme which had been prepared was used to fill column, and then continuous hydrolysis of casein to generate CCPs.â–³pH and N/P of hydrolysis solution were chose as experimental indicators. These factors which were pH, substrate concentration, temperature and flow velocity of substrate were discussed in the hydrolysis process. The best conditions for hydrolysis were as follows: temperature 60℃, substrate concentration 4%, pH 9.0 and speed of constant-current pump 20r/min.The molecular weight distribution of CCPs was measured by using Sephadex gel (Sephadex G-25) column and 0.01MHC1 was used as the mobile phase. According to principle of protein separation by Sephadex G-25, we could acquire the standard curve equation: lgM=4.7856-41.984Kav, so that we could gain the relationship between molecular weight and elution volume, and then on the basis of the elution volume of product, we could calculate the molecular weight. The flow rate of mobile phase was 0.4mL/min, detection wavelength was 280nm and column pressure was 0.3MPa, we could gain an ideal chromatogram of standard product. Under these conditions, elution volume of the main peaks were measured, which were 11.49mL and 12.01mL respectively, and the molecular weights were also measured, which were 25000 and 4500 respectively.