Biosynthesis of the acid protease produced by Lacticaseibacillus casei LBC 237 and Limosilactobacillus fermentum LBF 433 and their potential application in the bovine milk clotting

This study aimed to produce and evaluate proteases from Lacticaseibacillus casei (LBC 237) and Limosilactobacillus fermentum (LBF 433) for milk coagulation. Proteases were assessed for acid protease activity (PA) and stability within temperature range of 30–75 °C and pH levels of 5.0, 5.5, and 6.8. Substrate concentration (0.025 %–2.5 %) and storage conditions (up to 120 days) were also investigated. Milk-clotting activity (MCA) and the effect of NaCl (1–4 %) on coagulation were evaluated, with subsequent characterization of resulting clots in terms of molecular size, structure, and flavor profile. Chemical changes in fermentation, proteases, and clots were determined. The proteases showed better PA at 60 °C and a pH of 5.5, with no change over 120 days. LBC-P demonstrated a higher affinity for the substrate. The proteases generated bands at ∼35 kDa, similar to bovine chymosin bands, and LBC-P exhibited MCA (480 SU/mL). Clots formed using LBC-P and bovine chymosin displayed similar bands (50 kDa, 35 kDa, and 15 kDa), while the LBC-P clot exhibited a more porous structure. LBC-P was inactivated by NaCl concentrations (≥1 %), and the clots showed changes in amides I. The bitterness intensity was lower for the clot obtained with LBC-P. While both LBC-P and LBF-P showed potential for protein hydrolysis, only LBC-P demonstrated potential for milk coagulation for cheese production. This study suggests acid proteases with high hydrolytic activity. In conclusion, L. casei (LBC 237) derived proteases show potential for milk coagulation, offering a promising alternative to bovine chymosin in yellow cheese production.