Hypoglycemic effect of alphaS1-casein hydrolysates extracted from milk of Gir cows

Introduction: Recent research in food and nutrition sciences has explored milk peptides for their nutraceutical and therapeutic benefits. In this study, an attempt was made to assess the hypoglycemic effect of αS1-casein (αS1-CSN) isolated from the milk of Gir through dipeptidyl peptidase – IV (DPP-IV) inhibition.

Methods: The enzymatic hydrolysis of extracted αS1-CSN was carried out using Flavourzyme, pepsin and trypsin individually at 1%, 2% and 4% enzyme–substrate (E-S) ratios for a period of 2 - 12 h. This hydrolysate was ultra-filtered through 10 and 3 kDa cutoff membranes in order to obtain low-molecular-weight peptides. Permeate of 3 kDa was subjected to preparatory RP-HPLC in order to collect time-bound fractions. Later, peptide sequencing was performed through LC-MS/MS. Furthermore, the α-CSN hydrolysate was assessed with a peptide transportation assay using the caco2 cell line model.

Results: The hydrolysate obtained from pepsin treatment after 4 h at a 4% E-S ratio showed maximum DPP-IV inhibition (92.08±1.77%) with an IC₅₀ value of 140.3 µg/mL, and the corresponding degree of hydrolysis was 11.22±1.06%. A total of 45 time-based (collected every 30 s) peaks were recorded and evaluated for % DPP-IV inhibition; among them, fraction 27 showed the highest DPP-IV inhibition (65.31±2.41%), and this fraction was subjected to UHPLC and LC-MS/MS analysis, through which 40 unique peptides were identified. Most of the peptides identified (~85%) belonged to the region of f193-213 and f137 to 154 in αS1-CSN. Among them, two low-molecular-weight peptides were synthesized, viz., IPNPIGSENSE and IQKEDVPSE, and assessed for DPP-IV inhibition; later, one showed the lowest IC50 value of 1.67mM. Cell viability was not affected with up to 100 µg of product per well, and about 10% of the peptides were transported from the apical to the basal chamber in a time of 2 h.

Conclusion: The developed hydrolysate of αS1-CSN could be a potential hypoglycemic product.