Purification and identification of novel peptides with inhibitory effect against angiotensin I-converting enzyme and optimization of process conditions in milk fermented with the yeast Kluyveromyces marxianus

• Two novel ACE-inhibitory peptides (VLSRYP and LRFF) were isolated and identified from the yeast fermented milk.
• VLSRYP and LRFF behaved as competitive ACE inhibitors with IC50 values of 36.7 and 116.9 µM, respectively.
• The optimum process conditions are determined for the maximum production of ACE-inhibitory peptides.
• Response surface methodology was used to achieve the optimal process conditions.
• Rotation speed is found to be the most significant factor influencing the ACE inhibition.

Angiotensin-converting enzyme (ACE) inhibitory peptides from milk fermented with the yeast Kluyveromyces marxianus Z17 were identified. The optimum process conditions for the production of bioactive peptides were also established. Two novel peptides exhibiting ACE-inhibitory activity were identified from the fermented milk using Sephadex G-15 gel filtration, reversed phase-high performance liquid chromatography and MALDI/TOF-TOF MS/MS. The sequences of the two novel peptides were VLSRYP and LRFF with IC50 values of 36.7 and 116.9 µM, respectively. Lineweaver–Burk plots revealed that both peptides behaved as competitive ACE inhibitors. Response surface methodology was used to determine the optimum process conditions for the production of ACE-inhibitory peptides. The results showed that the most significant factors affecting ACE inhibition were fermentation temperature, inoculum level and rotation speed. The maximum ACE-inhibitory activity (81.23%) was observed at temperature 32 °C, initial pH 6.5, inoculum level 6% and rotation speed of 189 rpm. The peptide content and peptidase activity (carboxypeptidase and aminopeptidase) had a significant positive effect on ACE inhibition, while endoproteinase activity showed an insignificant effect. The results of this study may contribute to the development of a functional beverage with antihypertensive effects.