Preparation Of Casein Phosphopeptides Derived From Enzymatic Hydrolysis Of Buffalo Casein

Casein phosphopeptides (CPP), containing phosphoseryl cluster, were derived from casein by hydrolying with a single enzyme or a combination of enzymes. In this study, buffalo casein was used to prepare CPP, which can promote to deepen processing, enrich the production and enhance the added value of buffalo milk. Thus, it is worthy to do research on buffalo casein phosphorpeptides.Effects of Alcalase enzyme and Neutrase enzyme on production of buffalo CPP were investigated with single factor. The results showed that Alcalase was better than Neutrase. The orthogonal test has been carried out to obtain the optimum conditions for hydrolysis of buffalo casein by Alcalase in which the ratio of enzyme to substrate (E/S) was 3.0% (W/W), the substrate concentration was 5.0% (W/V), the hydrolysis reaction occurred at 70℃and pH 7.0. The degree of hydrolysis was determined to be 20.05% under the conditions.The technology parameter of preparing crude CPP were obtained through the investigation of enzymolysis experiment with Alcalase. The N/P (mol/mol) of the crude buffalo CPP, moisture and ash content in crude buffalo CPP were determined to be 20, 6.5% and 8.0%, respectively.The crude buffalo CPP were purified using macroporous alkali anion-exchange chromatography and alkalescence anion-exchange chromatography. The N/P of buffalo CPPⅠobtained from the macroporous alkali anion-exchange chromatography, and that of buffalo CPPⅡobtained from alkalescence anion-exchange chromato- graphy were determined to be 4 and 6 respectively. These results demomstrated that using macroporous alkali anion-exchange chromatography was more effective in purification of buffalo CPP compared with using alkalescence anion-exchange chromatography. The molecular weight of crude buffalo CPP, CPPⅠand CPPⅡwas found to be 1,260 Da, 970 Da and 1,090 Da respectively using HPLC.The function test demonstrated that buffalo CPP can prevent the formation of calcium phosphate and ferrous phosphate. The inhibiting effect was increased with the diminishing of the N/P and the increasing of buffalo CPP dose. The pH of 8 was efficient for buffalo CPP to prevent the formation of calcium phosphate. However, temperature variation had no great effect on the buffalo CPP's function when the temperature was below 100℃. Buffalo CPP which worked along with citric acid or xylitol, exhibited synergistic effect on the preventing of formation of calcium phosphate.