Comparative thermodynamic insights of proteolytic enzymes from two indigenous potent probiotics with hypocholesterolemic activity

The aim of this study was to isolate and characterize lactic acid bacteria (LAB) strains capable of producing proteases, evaluate their ability to release extracellular amino acids, and investigate their thermodynamic properties alongside their in vitro hypocholesterolemic potential when applied in functional yogurt formulations. Two LAB strains were identified: Lactobacillus gasseri ON877236, isolated from the breast milk of a nursing mother, and Lacticaseibacillus rhamnosus ON877389, obtained from the feces of an infant exclusively on a breast milk diet. Importantly, neither strain produced the biogenic amines histamine or tyramine. Both strains exhibited intrinsic resistance to vancomycin. Lcb. rhamnosus demonstrated intermediate resistance to azithromycin and amoxicillin-clavulanic acid. In contrast, L. gasseri remained susceptible to five other tested antibiotics: amoxicillin-clavulanic acid, oxytetracycline, ampicillin, azithromycin, and sulfamethoxazole-trimethoprim. The proteases produced by both strains displayed acidic properties, with maximal activity observed at pH 4. Thermodynamic analysis revealed that L. gasseri protease had a half-life of 693.14 min and a D-value of 2302.58 min at 30 °C, while those of Lcb. rhamnosus were 182.40 and 605.94 min, respectively. Compared to Lcb. rhamnosus, the L. gasseri protease demonstrated greater thermostability, reflected in its lower activation energy (Ea), longer half-life, higher D-value, and increased enthalpy (ΔH°) and Gibbs free energy (ΔG°). Furthermore, both strains exhibited notable in vitro cholesterol-lowering activity, degrading over 50% of cholesterol within 48 h and more than 90% after 96 h. These promising hypocholesterolemic effects support the potential of these LAB strains as functional probiotics with possible applications in nutraceuticals that may contribute in the management of cardiovascular disease, peripheral arterial disease, and stroke.