Improving the viability of powdered Lactobacillus fermentum Lf01 with complex lyoprotectants by maintaining cell membrane integrity and regulating related genes

In this study, Lactobacillus fermentum Lf01, which was screened out in the early stage of the experiment, had better fermentation performance as the research objectives, and was prepared into powder by vacuum freeze-drying technology. We used response surface methodology to optimize the composition of the mixture used to protect powdered L. fermentum. Our data demonstrated that 10% skim milk, 12% sucrose, 0.767% tyrosine, and 2.033% sorbitol ensured the highest survival rate (92.7%) of L. fermentum. We have initially explored the potential mechanism of the complex protectants through the protection effect under the electron microscope, and the analysis methods of Fourier transform infrared spectroscopy and transcriptomics. The complex protectants could effectively maintain the permeability barrier and structural integrity of cell membrane and avoid the leakage of cell contents. Transcriptomic data have also indicated that the protective effect of the complex protectants on bacteria during freeze-drying was most likely achieved through the regulation of related genes. We identified 240 differential genes in the treatment group, including 231 up-regulated genes and 9 down-regulated genes. Gene ontology (GO) and Kyoto encyclopaedia of genes and genomes (KEGG) analyses of differential expression genes (DEGs) indicated that genes involved in amino acid metabolism, carbohydrate metabolism, membrane transport, fatty acid biosynthesis and cell growth were significantly up-regulated. These new results provided novel insights into the potential mechanism of lyoprotectants at the cellular level, morphological level, and gene level of the bacteria. PRACTICAL APPLICATIONS: In our study, a strain of Lactobacillus fermentum Lf01 with good fermentation performance was selected to be prepared into powder by freeze-drying technique. Bacterial cells were unavoidably damaged during the freeze-drying process. As a result, we investigated the protective effects on L. fermentum of ten distinct freeze-dried protectants and their mixtures. We were also attempting to explain the mechanism of action of the complex protectants at the cellular level, morphological level, and gene level of the bacteria. This presents very important theoretical and practical significance for the preservation of strains and the production of commercial direct-investment starter.