Research On Casein Enzymatic Hydrolysis Condition To Prepare Casomorphin

Casein, with essential amino acid for indidual growth ,is the most abundant protein in milk.Recently,many researches demonstrated that casein was not only source of nutrients,but may also be ones of biologically active peptides, among which casomorphins has received the most attention for its special physiological action,such as inhibiting intestinal movement,accelerating immunity and accelarating insulin secretion.Many research works have shown that a large number of bioactive peptides could be found when some kinds of rich proteins could be hydrolyzed by proper proteinase.The enzymatic hydrolysis of casein by trypsin, papain, subtilisin and pepsin,under their condition were systematicaly studied.The effects of operating parameters — enzyme concentration,substrate concentration and time—on the degree and the speed of hydrolysis of hydrolysed protein was measured by colonmetic method.Taking DH as index,orthogonal design of four factors three levels was taken to optimize the hydrolysis condition of casein with proteases.The optimal hydrolisis condition was received.The optimal hydrolysis condition of casein by trypsin were: enzyme concentration 1.5%, temprature 55℃, pH 7.5, substrate concentration 6% and the DH is 16.67%;the optimal hydrolysis condition of casein by papain were: enzyme concentration 0.8%, temprature 70℃,pH 6.0,substrate concentration 2% and the DH is 12.14%;the optimal hydrolysis conditionof casein by subtilisin were:enzyme concentration 0.6%? temprature 70"C,pH 8.0,substrate concentration 2% and the DH is 10.05%.The relations between Degree of Hydrolysis and distribution of Molecular Weight were obtained according to the equation: lgA/ =4.82216-2.81531Kav.The Sephadex G-15 column (1.7cmx57.5cm)were used to isolate the bioactive peptides obtained from the casein hydrolyzed by trypsin-pepsin, subtilisin -pepsin, papain-pepsin.Detecting B-casomorphin-7 through RP—HPLC. a ODS columnC 250mmx4.6mm, 4.7/rni) was used, the mobile phase was acetonitrile-H2O(20:80).The flow rate was lml/min and detective wavelength was 215nm,the column temprature was 37 ℃, column temprature pressurewas 9.6MP. retention time was 29.912min.RP-HPLC was used to detect collecting component detached by Sephadex G-lS.We attained chromatogram chart with the same retention time as 3 -CM-7 standard sample.According to the chromatograms,there were 4 samples have the same reservation time as P -CM-7 in trypsin-pepsin and only 1 sample have the same reservation time in subtilisin -pepsin.