Research On Dairy Yeast Fermented Milk For The Production Of Angiotensin-converting Enzyme Inhibitory Peptides

In this study, dairy yeasts were isolated from Tibet kefir grains. The isolates were identified using physiological, biochemical characteristics and molecular method. The proteolytic activity, potential to generate bioactive peptides with milk fermentation and potential to be used as probiotics were investigated among the representative stains of different species. The process conditions for the production of angiotensin-converting enzyme (ACE) inhibitory peptides were optimized with the yeast Kluyveromyces marxianus Z17. Furthermore, the peptides responsible for the inhibitory activity were also purified and identified.Based on physiological, biochemical characteristics and molecular identification results, eight species of yeast were isolated and identified from Tibet kefir, including Saccharomyces cerevisiae, Pichia fermentans, Debaryomyces hansenii, Rhodotorula mucilaginosa, Candida zeylanoide, Candida parapsilosis, Kluyveromyces marxianus and Kazachstania unispora. Among the test samples, K. marxianus, Ka. unispora and P. fermentans were the highest three species in frequency of occurrence of yeast isolates. C. zeylanoides, C. parapsilosis and R. mucilaginosa were first found the occurrence in Tibet kefir. The results provided new information of yeast composition and biodiversity of Tibet kefir.Among the seven tested strains, K11, K14, H2 and Z17 tolerated low pH in simulated gastric juice and remained high survival rate in simulated intestinal juice. The strain Z17 can grow in 2.0% concentration of bile salt, followed by the Kll and K14 in 1.8%, and H2 in 1.5% bile salt. The four strains were able to degrade cholesterol from the growth medium and with resting cells. The S. cerevisiae K11 exhibited higher activity and achieved 65% degradation rate in grow medium and 57% with resting cells. The four strains showed ability to scavenge DPPH and hydroxyl radical. The result indicated that the selected yeast strains from Tibet kefir may be used as probiotics.The tested strains presented different proteolytic activity while growing in the milk. Among the test strains, R. mucilaginosa D2 and K. marxianus Z17 exhibited stronger proteolytic activity on milk protein, S. cerevisiae K11, P. fermentans K14, D. hansenii H2 and C. zeylanoides D3 were in the middle, C. parapsilosis D4 and Ka. unispora K21 were the weakest strains in proteolytic activity. The peptide fractions from milk fermented with K14, D2, D3, D4 and Z17 showed high ACE-inhibitory activity. The strains of D2, K14 and Z17 possessed good potential to produce ACE-inhibitory activity via milk fermentation, the IC50 values of peptides ranged from 53.9-227.6μg/mL. The strains K14 and Z17 showed higher DPPH radical scavenging activity (74.6% and 66.7% respectively) in fractions of molecule weight below 3kDa. The hydroxyl radical scavenging activity profile of peptide fractions showed characteristic differences among species and molecule sizes, ranging from 7.3% to 53.5%.Response surface methodology was used to determine the optimum process conditions for the production of ACE-inhibitory peptides. The results have shown that the most significant factors affecting ACE inhibition are fermentation temperature, inoculum level and rotation speed. The maximum ACE-inhibitory activity (81.23%) was observed at temperature 32℃, initial pH 6.5, inoculum level 6% and rotation speed of 189 r/min. The peptide content and peptidase activity (carboxypeptidase and aminopeptidase) had a significant positive effect on ACE inhibition, while endoproteinase activity showed insignificant effect. This result indicates that some of the ACE-inhibitory peptides were released intracellularly by the action of peptidases and then transported to medium across the cell membrane.The two novel peptides exhibiting ACE-inhibitory activity were identified from the fermented milk using Sephadex G-15 gel filtration, reverse phase-high performance liquid chromatography and MALDI/TOF-TOF MS/MS. The sequences of the two novel peptides are VLSRYP and LRFF with IC50 values of 36.7 and 116.9 μmol/L, which corresponds to the fragment of κ-casein f(31-36) and αsl-casein f(21-24), respectively. By retrieving relevant database of bioactive peptides and reference, these peptides identified in the present study have not been reported in milk fermented with LAB or casein hydrolysate. Lineweaver-Burk plots revealed that both peptides behaved as competitive ACE inhibitors.