Optimising Conditions for the Preservation of Extremely Oxygen Sensitive Obligate Anaerobes of the Gut Microbiota as Candidates for Probiotic Strains

Obligate anaerobic difficult culture bacteria, which make up the bulk of the microbiota of the large intestine, are potential candidates for the development of highly effective new generation probiotics, since they are capable of synthesizing a variety of metabolites, including short-chain fatty acids, which have a stimulating effect on both commensal bacteria and host cells. However, for their long-term preservation, it is necessary to carefully select the preservation method and protective components. To evaluate the efficacy of using different cryoprotectants to increase the viability of obligate anaerobic difficult culture bacteria when preserved by lyophilisation and cryopreservation methods. The studies showed that freeze-drying contributed most to the preservation of the viability of Faecalibacterium prausnitzii, Anaerostipes hadrus, Eubacterium hallii, provided that they were used as a stabilizing medium, which included inulin, cysteine and riboflavin, ensuring the preservation of the initial bacterial titer for 30 days. Among the studied bacteria, the effectiveness of cryopreservation was shown for A. hadrus, since it ensured the survival of bacteria at the initial level for 14 days of storage, regardless of the cryopreservative used, but by the 30th day their viability decreased significantly (by 100 times when using liquid as a cryopreservant). nutrient medium 110 with minced meat and carbohydrates with the addition of glycerin, 10 000 with the commercial cryopreservative CRYOINSTANT). The results obtained allow us to recommend lyophilization as the most optimal method for long-term storage of probiotic strains of bacteria using highly effective stabilizers.

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