Unearthing novel and multifunctional peptides in peptidome of fermented chhurpi cheese of Indian Himalayan region.

Fermented foods of the Indian Himalaya are unexplored functional resources with high nutritional potential. Chhurpi cheese, fermented by defined native proteolytic lactic acid bacteria of Sikkim was assessed for ACE inhibitory, HOCl reducing, and MPO inhibitory, activity across varying stages of gastrointestinal (GI) digestion. The enhanced bioactivity of Lactobacillus delbrueckii WS4 chhurpi was associated with the generation of bioactive and multifunctional peptides during fermentation and GI digestion. Qualitative and quantitative in silico tools were employed for prediction of ACE inhibitory activity of novel chhurpi peptides. Selected peptides, with highest predictive ACE inhibitory potential were synthesized and in vitro validation revealed the ACE inhibitory potential of peptides HPHPHLSFM and LKPTPEGDL. LKPTPEGDL showed the most potent ACE inhibitory activity (IC₅₀ of 25.82 ± 0.26 µmol) which slightly decreased upon GI digestion. The peptides demonstrated a non-competitive type mixed ACE inhibition modality. Furthermore, the two peptides exerted observable HOCl reducing and MPO inhibitory activity, demonstrating their antioxidative potential. HPHPHLSFM exhibited superior HOCl reduction (EC₅₀ of 0.29 ± 0.01 mmol), while LKPTPEGDL demonstrated higher MPO (IC₅₀ of 0.29 ± 0.01 mmol) inhibition. Molecular docking of the two peptides with MPO revealed proline and aspartate near peptidyl C-terminus to bind with enzyme catalytic residues. This study presents the first peptidome analysis of chhurpi produced through controlled fermentation and identifies novel peptides with MPO and ACE inhibitory activity. Furthermore, it marks the first synthesis and in vitro bioactivity validation of bioactive peptides from chhurpi cheese, highlighting its multifunctional potential.