In vitro biosafety and bioactivity assessment of the goat milk protein derived hydrolysates peptides

Abstract

Goat milk and protein hydrolysate peptides have generated significant interest due to their diverse range of biological functions. To produce these peptides, casein (CP), and whey proteins (WP) were partially purified from goat milk and subsequently hydrolyzed with gastrointestinal endopeptidases such as pepsin, trypsin, and α-chymotrypsin based on in silico selection to achieve gastrointestinal stable peptides. The hydrolysates were characterized using various techniques, including zeta sizer, polydispersity index (PDI), zeta potential, and reverse-phase high-performance liquid chromatography (RP-HPLC). The resulting peptides were then purified using RP-HPLC. To assess cytotoxicity, an MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) assay was performed with fluorescence microscopy. Finally, cytokine levels were determined using enzyme-linked immunosorbent assay (ELISA). In comparison to WP and CP, the casein protein hydrolysates (CPH) and whey protein hydrolysates (WPH) peptides average particle size and PDI were decreased. The zeta potential of casein and whey peptides hydrolysates there were no differences. The RP-HPLC analysis revealed the production of peptides through the hydrolysis of CP and WP. These hydrolysates peptides were found to possess diverse biological activities, as evidenced by their significant inhibition of α-amylase, pancreatic lipase (PL), and angiotensin-converting enzyme (ACE), as well as their antibacterial properties against selected pathogens. Further investigations were carried out to understand the mode of action of these peptides using fluorescence and scanning electron microscope (SEM) microscopy. This study provides the first evidence of gastrointestinal stable peptides derived from goat milk with inhibitory activity against α-amylase, PL, and ACE, as well as antibacterial properties against specific pathogens. Additionally, the peptides demonstrated significant cytokine inhibition and low toxicity toward the HT-29 cell line. This study strongly suggests that the biologically active peptides responsible for the health-related bioactive properties described here, which are considered functional and nutraceutical ingredients, should be identified and validated in subsequent research, particularly the most effective hydrolysate peptides derived from goat milk protein. Goat milk is widely recognized as an excellent source of milk protein. In this study, bioactive peptides were produced using casein and whey proteins from goat milk, which exhibited inhibitory activity against angiotensin converting enzyme, α-amylase, and pancreatic lipase, as well as antibacterial properties. Goat milk protein is a promising source for developing high-quality protein products with excellent safety standards that have potential applications in the pharmaceutical and food industries.