Development of Thermotolerant Lactobacilli Cultures with Improved Probiotic Properties Using Adaptive Laboratory Evolution Method

Probiotics play a significant role in functional foods. Heat stress and dehydration are the two principal mechanisms leading to inactivation and loss of probiotics viability in its production. There is a need to develop an industrial organism to withstand higher temperatures during its processing and storage. This current study aims to develop thermotolerant strains of Lacticaseibacillus casei N (N) and Lactobacillus helveticus NRRL B-4526 (H) by acclimatizing the wild-type strains to the higher temperature of 45 degrees C by adaptive laboratory evolution. A two-fold increase in biomass was observed in both acclimatized strains up to the 200th generation, which subsequently remained stable after 500 generations. The morphological change of these acclimatized strains was observed under scanning electron microscopy. Also, there was an increase in probiotic attributes of these acclimatized strains compared to their wild-types. Among two acclimatized strains, L. casei N-45 had shown higher tolerance in the acidic pH 3.0 (89.31%), the bile of 0.3% (84.45%), simulated gastric juice (79.12%), and simulated intestinal juice (73.86%). There was also an increase in salt tolerance (NaCl), radical scavenging activity, autoaggregation, coaggregation, and hydrophobicity of these adapted strains. The total protein profiling using 2D gel electrophoresis reveals the differences in protein expressions between wild-type and acclimatized strains. Specific protein spots from acclimatized strains of H-45 and N-45 were further subjected to MALDI-TOF MS/MS. Some of the identified proteins were recognized to play a role in RNA chaperones and protein synthesis during stress conditions.