PubMed · 13349834
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W PILNIAK. 1956-06-25. [Lyophilization].. https://pubmed.ncbi.nlm.nih.gov/13349834/
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Maintaining the viability of lactic acid bacteria during dehydration and subsequent storage remains a significant challenge. Here, we employed single-cell RNA sequencing to reveal the heterogeneous stress responses of Lactobacillus delbrueckii subsp. bulgaricus, identifying seven distinct transcriptional clusters across the liquid culture, freeze-drying, and storage phases. The dominant clusters in the freeze-drying and storage were not completely consistent, showing significant functional differentiation. Genomic stability may be important for survival during freeze-drying and storage, while intracellular energy homeostasis appears important for viability during storage. The magnesium transporter mgtB was highly expressed in clusters tolerant to freeze-drying and storage, suggesting a critical role for Mg2+ homeostasis. Further experimental validation confirmed that Mg2+ treatment significantly bolstered stress resistance, increasing immediate post-freeze-drying survival by over 2-fold (up to 92.90%) and post-storage survival by over 5-fold (up to 5.98%). Proteomic data indicated that Mg2+ supplementation correlated with the maintenance of several biological functions potentially relevant to bacterial survival during freeze-drying and storage, including DNA repair, translation, and central carbon metabolism. These findings provide a map of microbial stress resistance through population heterogeneity and offer a potential strategy that may be adapted for enhancing the stability of other industrial lactic acid bacteria products.
The survival of a variety of species of microorganism following storage for up to 20 years has been analyzed. The organisms were freeze-dried, sealed in ampoules under vacuum (<1 Pa) and stored in the dark at 5 degrees C. The yeast that was tested, Saccharomyces cerevisiae, showed only 8% survival when recovered shortly after freeze-drying, but subsequent loss during storage was the least among all the tested microorganisms. The decrease in the logarithm of survival per year (log survival) was -0.010, which corresponds to a survival rate of 97.7% per year. The Gram-negative bacteria tested, Escherichia coli, Pseudomonas putida, and Enterobacter cloacae, showed 42.6, 33.5, and 50.8% survival shortly after freeze-drying, which was higher than the corresponding survival of S. cerevisiae, but the subsequent loss during storage was greater than S. cerevisiae, the log survival figures being -0.041, -0.058, and -0.073 per year. These values correspond to survival rates of 91.0, 87.5, and 84.5% each year. The Gram-positive bacteria tested, Lactobacillus acidophilus and Enteroccoccus faecium, showed 62.5 and 85.2% survival shortly after freeze-drying, which was even higher than that of the Gram-negative species, and these organisms also showed better survival during storage than Gram-negative bacteria; their log survival rates were -0.018 and -0.016 per year, which corresponded to survival rates of almost 96% per year. Comparison of these results with other published data for different drying conditions suggests that survival during storage is strongly influenced by the degree of vacuum under which the ampoules were sealed. The excellent survival after freeze-drying of each species might be attributable to the high level of desiccation and to sealing under vacuum.
Sample preparation is considered one of the limiting steps in microbial metabolome analysis. Eukaryotes and prokaryotes behave very differently during the several steps of classical sample preparation methods for analysis of metabolites. Even within the eukaryote kingdom there is a vast diversity of cell structures that make it imprudent to blindly adopt protocols that were designed for a specific group of microorganisms. We have therefore reviewed and evaluated the whole sample preparation procedures for analysis of yeast metabolites. Our focus has been on the current needs in metabolome analysis, which is the analysis of a large number of metabolites with very diverse chemical and physical properties. This work reports the leakage of intracellular metabolites observed during quenching yeast cells with cold methanol solution, the efficacy of six different methods for the extraction of intracellular metabolites, and the losses noticed during sample concentration by lyophilization and solvent evaporation. A more reliable procedure is suggested for quenching yeast cells with cold methanol solution, followed by extraction of intracellular metabolites by pure methanol. The method can be combined with reduced pressure solvent evaporation and therefore represents an attractive sample preparation procedure for high-throughput metabolome analysis of yeasts.