Isolation and Characterization of Caseinophosphopeptides from Buffalo Milk

摘要

Milk is a complex mixture of molecular species, containing bioactivity, which confer special properties for the infant development and growth beyond basic nutrient. Many of the bioactivities of the milk are attributed to the proteins and peptides secreted into the milk by mammary gland. The bioactivity of several milk proteins are latent; and are released during proteolytic digestion of the protein (in vivo)or during food processing (in vitro). Caseins, the ma/or milk proteins are the source of manybioactive peptides. Tryptic hydrolysis of casein yield phosphorylated peptides called Caseinophosphopeptides(CPP), which possess bioactive properties such as mineral binding, antioxidative and immunological activities. The present investigations have been done with an aim to characterize CPP of buffalo milk origin. Casein was prepared by Isoelectric precipitation method from buffalo milk. Isoelectrically prepared casein was further fractioned into alpha-, beta- and kappa-caseins on the basis of their differential solubility in urea. The caseins and their fractions were hydrolyzed with tyrpsin for different time interval to determine the degree of hydrolysis. The Caseinophosphopeptides (CPPs) were isolated from hydrolyzed solution of casein based on theprinciple that CPPs were soluble at pH 4.6 and aggregated with divalent cation such as calcium at neutral pH and precipitated out using ethanol. The CPP whole and obtained from different fractions were resolved on Tricine SDS-PAGE. The electrophoretic pattern revealed that CPP has a low molecular weight of 3-4kDa. The CPPs were further resolved on RP-HPLC. The elution profile of whole CPP showed three major peaks with retention times 13.216 min, 14.304 min and 17.711 min.