Obtaining paraprobiotics from Lactobacilus acidophilus, Lacticaseibacillus casei and Bifidobacterium animalis using six inactivation methods: Impacts on the cultivability, integrity, physiology, and morphology

This study aimed to determine the suitable inactivation conditions of ultrasound and other five methods [heat, low pH, high pH, gamma irradiation and supercritical carbon dioxide (CO₂)] to obtain paraprobiotics from Lactobacilus acidophilus, Lacticaseibacillus casei and Bifidobacterium animalis. The impact of each method at distinct conditions of inactivation was assessed by plate culturing and, the physiological and morphological aspects of the inactivated cells were assessed by a combined approach of flow cytometry and field-emission scanning electron microscopy analysis. The ability to grow on culture media after exposure to distinct processes or different conditions of the same process largely varied among the tested strains. The exposure to the best conditions defined for the distinct processes for inactivation of each strain resulted in distinct (p < 0.05) percentages of cells with active enzymatic metabolism and integrity of membranes. After the ultrasound treatment, L. casei presented the highest and lowest percentages of enzymatic activity (86.2%) and damage to membrane integrity (4.6%) among the tested strains, respectively. Morphological characteristics of L. casei were similar after exposure to the distinct processes tested. Otherwise, ultrasound deeply affected the morphological aspects of L. acidophilus and B. animalis, and compromised the enzymatic activity in a great percentage of cells of these bacteria (>80%). The extension of damage to the cell surface, changes in cell roughness, lysed cells and cell debris varied with the tested strain and the inactivation method. Results indicate that the inactivation method defines the physiological and morphological conditions in each derived paraprobiotic that must be optimized for each strain. Findings suggest ultrasound as a feasible method to obtain L. casei paraprobiotics with an active metabolism.