Purification and identification of potentially bioactive peptides from enzyme-modified cheese

Abstract

Antihypertensive peptides inhibiting angiotensin I-converting enzyme have been isolated from enzymatic hydrolysates of various food materials, but no information is available on the isolation of antihypertensive peptides from enzyme-modified cheese. In this study, several bioactive peptides, mainly potential antihypertensive peptides from enzyme-modified cheese prepared by commercial and Lactobacillus casez enzymes, were purified and identified. Enzyme-modified cheese samples were prepared by combination of Neutrase® (1883.0 U/ml), L. casei enzymes (amino peptidase activity 86.4 leucine aminopeptidase U/g), and Debitrase™ (22.0 leucine aminopeptidase U/g). The water-soluble fractions of the enzyme-modified cheeses that were prepared by different enzymes were subjected to reverse-phase HPLC on a Delta Pak C18 column. Each peak was purified on the same column using a binary gradient. One peak from the Neutrase® digest, five peaks from the Neutrase®-Debitrase™ digest, and two peaks from the Neutrase®-Lactobacillus enzyme digest were purified and identified by API mass spectrometry. On the basis of their molecular masses, amino acid sequences of purified peptides were identified. β-Casomorphin with a sequence like that of β-casein (YPFPGPI f 60-66) was found after the Neutrase® digest. All of the peptides purified from the digests with combination of Neutrase® and Debitrase™ or Neutrase® and L. casei enzymes contained active sites in their sequences. The presence of sites containing potential antihypertensive peptides suggests that the purified peptides may have antihypertensive properties. Thus, the enzyme-modified cheese process, mainly designed to produce flavor ingredients, may simultaneously produce bioactive peptides, which are considered to be of physiological importance.