Comparative analysis of thermal and ultrasound inactivation of Lactobacillus acidophilus LA-5: Viability by flow cytometry and plate count and postbiotic functional properties

Considering the potential of postbiotics to exert health effects similar to probiotics and the limited data available in the literature, this study aimed to evaluate the viability and functional properties of Lactobacillus acidophilus LA-5 under different heat and ultrasound treatment conditions. The probiotic microorganism L. acidophilus LA-5 was subjected to 16 different heat treatments (65 degrees C-95 degrees C, 5-90 min) and 16 different ultrasound treatments (98 mu m-320 mu m, 15-60 min). Cell damage levels (colony count and flow cytometry) and functional properties of the inactivated probiotics (postbiotics), including antimicrobial activity, probiotic-enhancing activity, and short-chain fatty acid (SCFA) production in Lacticaseibacillus casei 431 and Bifidobacterium culture media, were assessed. All heat treatments achieved complete inactivation, while only 9 ultrasound conditions were fully effective. In ultrasound-treated samples, flow cytometry and colony count results were inconsistent, suggesting possible formation of viable but non-culturable cells, complicating viability assessments. Heat-treated postbiotics significantly enhanced the growth of L. casei 431, achieving up to a 10.8-fold increase compared to the control (p < 0.05), while ultrasound-treated postbiotics showed minimal effect. The strongest antimicrobial activity was observed in heat-treated postbiotics, reducing Escherichia coli and Enterococcus faecalis populations by 14.6 % and 8.4 %, respectively, compared to 10.0 % and 4.0 % with ultrasound-treated samples. Both methods increased SCFA levels, with heat-treated postbiotics at 65 degrees C yielding the highest acetic and significantly elevated butyric acid concentrations (p < 0.05). These findings underscore the importance of selecting inactivation strategies tailored to desired postbiotic functions and highlight the need for complementary analyses and standardized criteria to ensure reproducibility and safety in postbiotic applications.