Growth-independent CLA production by Bifidobacterium breve JKL2022 and the potential transcriptional regulatory role of TetR in linoleic acid isomerase expression.

BACKGROUND: Microbial production of conjugated linoleic acid (CLA) has garnered wide attention for the possibility to increase the CLA content in food products, therefore achieving higher concentrations of beneficial compounds for consumers. However, this approach has only been done using metabolically active cells, particularly in Bifidobacterium spp., thus being a major limitation given the anaerobic and fastidious nature of bifidobacteria. In this study, we aimed to investigate the capacity of Bifidobacterium breve JKL2022 (KACC81214BP) to convert free linoleic acid (LA) into CLA using growing cells and postbiotic preparations (washed cells and crude protein extracts) as catalysts. RESULTS: Bifidobacterium breve JKL2022 demonstrated high CLA production as early as 6 h and continued to increase until 12-15 h of incubation. Moreover, CLA production was observed in JKL2022 washed cells (97.42 ± 3.64%) and crude protein fractions (33.87 ± 4.05%- 103.65 ± 2.70%) obtained after cell lysis, highlighting its superior CLA-converting activity compared to the B. breve JCM strains. In vitro CLA reaction conditions were optimal at pH 7.0, following the first-order kinetics within the first 5 min of reaction, and the extraction efficiency of the isopropanol-hexane protocol increased after adjusting the pH to 5.0-5.5. Finally, RT-qPCR and in silico analysis revealed a strong correlation between the expression levels of lai (JKL2022_00014) and tetR (JKL2022_00217) genes, suggesting the potential role of TetR in upregulating the lai gene expression in JKL2022 that could explain the LA conversion in washed JKL2022 cells. CONCLUSIONS: The ability of B. breve JKL2022 strain to convert free LA to CLA during growth, as well as using washed cells and crude protein extracts, suggests strain specificity and superior enzymatic activity. In addition to its potential application as a probiotic strain with CLA-enhancing properties, washed JKL2022 cells or crude protein extracts can be developed as postbiotic preparations for the same purpose.