Identification, Viability, and Membrane Potential during the Cryopreservation of Autochthonous Lactic-Acid Bacteria Isolated from Artisanal Adobera Cheese from Los Altos de Jalisco

Abstract

Lactic acid bacteria (LAB) comprise a group of microorganisms responsible for developing the sensory and chemical characteristics of several foods and fermented products, particularly cheese. For this reason, after isolation and identification of LAB, validated protocols and procedures for their long-term preservation without compromising its integrity and technological properties, as well as methodologies aiming to assess their viability and integrity are paramount. This study aimed to isolate and identify autochthonous LAB from artisanal Adobera cheese and determine the effect of LAB cryopreservation with thioglycolate broth and glycerol on their viability, membrane integrity, and kinetics. Sixteen LAB were isolated and genetically identified from artisanal cheese samples; eleven of those strains were selected (genus Lactobacillus, Leuconostoc, Streptococcus, and Lactococcus) and included in the cryo-preservation assay. The initial average concentration of the bacterial suspensions was 6.89 log10 CFU mL−1; increasing to 8.9 log10 CFU mL−1 21 days later and slightly reduced at day 42 post-preservation (losses below one logarithm). About 77% of the cells maintained their membrane potential 180 days after their preservation and showed normal Kinetic parameters, maintaining normal adaptation times (Lag phase) and Log phases (9 h average), before reaching the stationary phase. The proposed protocol constitutes a viable alternative to the long-term preservation of different LAB genera because it keeps their viability and integrity. Using flow cytometry allowed the enumeration of viable LAB and provide evidence of the integrity of their membrane.