Induction Of Viable But Nonculturable State In Lacticaseibacillus Casei Zhang And Transcriptome Study

Lactic acid bacteria（LAB）VBNC（Viable but non-culturable）state refers to a dormant state of LAB under unsuitable environmental conditions,although the bacteria still have vitality,they cannot grow and reproduce on traditional media.In recent years,with the development of biotechnology and modern analytical technology,research on the VBNC state of LAB has gradually deepened.During the production and application of LAB,various unsuitable environmental pressures can lead to the formation of VBNC states.The presence of VBNC state of LAB has a significant impact on the shelf life and quality of fermented foods.VBNC state LAB may affect the taste,acidity,and production of harmful metabolites of products and it will also affect the probiotic effects of lactic acid bacteria and the quality evaluation of lactic acid bacteria.In this study,Lacticaseibacillus casei Zhang（L.casei Zhang）was selected as the research object to screen the induction conditions for entering the VBNC state,analyze the morphological changes of VBNC cells and detect the ability of VBNC state cell biofilm formation,analyze the differential expression genes of VBNC state cells through transcriptome methods,and preliminarily explore its formation mechanism.The main research findings are as follows:（1）A variety of compound conditions induced,only at 4℃,4%Na Cl liquid MRS conditions can successfully induce L.casei Zhang into the VBNC state,the number of cultured cells decreased to 0 at 235 d;the number of VBNC cells reached 3.46×10⁵pieces/m L.by morphological observation VBNC cell length shortened from 1.55±0.19μm.（2）VBNC cells produced more biofilm than normal cells and stabilized after 18 h,with an OD₅₉₅nm of 1.924.The ATP concentration of VBNC cells reached 2.85μmol/m L,and the c AMP concentration of VBNC cells was significantly higher than that of normal cells,reaching 39.82 nmol/m L.（3）Transcriptomic sequencing and analysis of L.casei Zhang VBNC cells and normal cells induced under 4℃,4%Na Cl liquid MRS conditions were performed by transcriptomic methods.The differentially expressed genes were classified and functionally analyzed by GO annotation,and the results showed that the genes were mainly enriched in functional pathways such as protein transport,glycolytic process,and pyridoxal phosphate.The metabolic pathways of the expressed genes were investigated by KEGG,and their main enriched metabolic pathway was the propionate metabolic pathway.At the transcriptional level,it was shown that amino acid and protein synthesis in L.casei Zhang VBNC state cells induced under 4℃,4%Na Cl liquid MRS conditions did not stop,and there was still some carbohydrate-related energy metabolism,while a certain amount of protein and amino acid was synthesized to maintain the basal metabolic process in VBNC state cells.To conclude,the investigation of the VBNC state among LAB delivers fresh perspectives and concepts for their application in the food industry,as well as discovering non-culturable LAB in ecologic fields.Given the ongoing research on the molecular mechanism of VBNC state among LAB,we are hopeful for further advances and improvements in the future application research of these bacterial strains.