Identification and Quantification of Bioactive Peptides in Milk and Kefir

Milk provides a wide range of bioactive substances, such as antimicrobial peptides and proteins. Our study aimed to identify novel antimicrobial peptides naturally present in milk. The components of an endogenaus bovine milk peptide database were virtually screened for charge, amphipathy, and predicted secondary structure. Thus, 23 of 248 screened peptides were identified as candidates for antimicrobial effects. After commercial synthesis, their antimicrobial activities were determined against E.coli NEB5a, E. coli ATCC25922, and Bacillus subtilis ATCC6051. In the tested concentration range (<2mM), bacteriostatic activity of 14 peptides was detected including nine peptides inhibiting both Gram-positive and Gram-negative bacteria. The most effective fragment was TKLTEEEKNRLNFLKKISQRYQKFALPQYLK corresponding to as2-casein151 _181, with minimum inhibitory concentration (MIC) of 4.0 µM against B. subtilis ATCC6051, and MIC of 16.2 µM against both E. coli strains. Circular dichroism spectroscopy revealed conformational changes of most active peptides in a membrane-mimic environment, transitioning from an unordered to a-helical structure.

To evaluate if the recently identified antimicrobial milk peptides as2-casein182-207 and as2-casein151-181 can be of physiological or technological relevance, their presence in commercial milk was investigated. Thus, a UHPLC-ESI-MS/MS scheduled multiple reaction monitaring method was developed for quantification using stable isotope-labeled peptides. The developed protocol gave very good linear response (R2 >0.99) for both peptides, recovery>95% and coefficients of variations of 5.51% and 4.79%. The Iimits of detection (0 .3 nM as2- casein182-207 and 0.5 nM as2-casein151-181) were low enough to detect both peptides without further enrichment. The highest peptide concentration was recorded in pasteurized milk (1.36 µM as2-casein182-207 and 0.20 µM as2-casein151-181). The concentration of as2-casein182-207 was in a range that was previously reported to act bacteriostatic against Bacillus subtilis. Industrial milk processing led to a significant loss of both peptides resulting in a mean concentration of 0.47 µM as2-casein182-207 and 0.098 µM as2-casein151-181 in ultrahigh-temperature milk.

Health-promoting effects of kefir may be partially caused by bioactive peptides. To evaluate their formation or degradation during gastrointestinal digestion, we monitared changes of the peptide profile in a model of (1) oral, (2) gastric, and (3)
small intestinal digestion of kefir. Matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses revealed clearly different profiles between digests 2/3 and kefir/digest 1. Subsequent UPLC-ESI-MS/MS identified 92 peptides in total (25 , 25, 43 , and 30, partly overlapping in kefir and digests 1, 2, and 3, respectively), including 16 peptides with ascribed bioactivity. Relative quantification in scheduled multiple reaction monitoring mode showed that many bioactive peptides were released by simulated digestion. Most prominently, the concentration of angiotensin-converting enzyme inhibitor ß-casein203-209 increased approximately 10 000-fold after combined oral, gastric, and intestinal digestion. Thus, physiological digestive processes may promote bioactive peptide formation from proteins and oligopeptides in kefir.