Mechanisms of survival of lactic acid bacteria in silanol-humate gels with organic acids

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Abstract

Bacterial survival under unfavorable growth conditions is one of the fundamental problems of microbiology. The applied aspect of this problem – long-term preservation of bacterial cell viability – is of particular importance for storage of lactic acid bacteria due to the biotechnological significance of this group of microorganisms and their high rates of death during long-term storage. The aim of this study was to investigate the long-term survival of lactic acid bacteria of different physiological groups (heterofermentative Enterococcus faecium M3185 and homofermentative Lactobacillus paracasei AK 508) in silanol-humate gels (SHG) containing various organic acids used as titrants in obtaining SHG (malic, lactic, acetic, ascorbic, citric). Placing cells in CHG with organic acids resulted in a significant increase in the titer of viable cells relative to the control during long-term storage (up to 200 times) and depended on the bacterial culture, the acid used and the storage period (up to 5 months). The experimentally proven reasons for the long-term survival of bacteria in CHG are: 1) most of the cells are in a state of hypometabolism and consume organic acids, which is evidenced by a decrease in their concentration during storage, as well as by the release of CO2 in the case of E. faecium (in this case, the metabolic rate is 1000 times lower than that of growing cells); 2) the absence of mass autolysis of cells, which is presumably due to the “disunity” of the cells in the gel and the impossibility of creating sufficient concentrations of autoregulators and autolysis enzymes; 3) some of the cells are in a state of rest, in the form of stress-resistant cyst-like cells. There is also reason to believe that when transferred to a gel, an alternative (biofilm) phenotype is formed, which has increased stress resistance. The results obtained indicate the feasibility of immobilizing lactic acid bacteria cells in SGG with organic acids for long-term storage.

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O. A. Galuza

S. N. Winogradsky Institute of Microbiology, Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences; OOO “Bavar+”

Author for correspondence.
Email: olesya_galuza@mail.ru
Russian Federation, Moscow, 119071; Moscow, 127206

G. I. El-Registan

S. N. Winogradsky Institute of Microbiology, Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences

Email: olesya_galuza@mail.ru
Russian Federation, Moscow, 119071

A. V. Vishnyakova

S. N. Winogradsky Institute of Microbiology, Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences

Email: olesya_galuza@mail.ru
Russian Federation, Moscow, 119071

Yu. A. Nikolaev

S. N. Winogradsky Institute of Microbiology, Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences

Email: olesya_galuza@mail.ru
Russian Federation, Moscow, 119071

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2. Fig. 1. Dynamics of changes in the number of viable cells of E. faecium (a) and L. paracasei (b) immobilized in SGH with different acids. The numbers indicate: 1 – control, non-stabilized culture stored in liquid LB medium; 2 – culture immobilized in SGH with citric acid; 3 – culture immobilized in SGH with malic acid; 4 – culture immobilized in SGH with lactic acid; 5 – culture immobilized in SGH with acetic acid; 6 – culture immobilized in SGH with ascorbic acid; 7 – culture immobilized in SGH with hydrochloric acid.

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3. Fig. 2. Dynamics of CO2 accumulation by E. faecium cells with the addition of: a ‒ lactic acid; b ‒ ascorbic acid; c ‒ acetic acid. The numbers indicate: 1 – control, non-stabilized culture stored in liquid LB medium; 2 – culture immobilized in SHG with hydrochloric acid; 3 – non-stabilized culture stored in liquid LB medium with the addition of lactic acid; 4 – culture immobilized in SHG with lactic acid; 5 – non-stabilized culture stored in liquid LB medium with the addition of ascorbic acid; 6 – culture immobilized in SHG with ascorbic acid; 7 – non-stabilized culture stored in liquid LB medium with the addition of acetic acid; 8 – culture immobilized in SHG with acetic acid.

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4. Fig. 3. Schematic diagram of the life cycle of E. faecium under different conditions in the presence of different food sources.

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5. Fig. 4. Dynamics of pH changes during storage of E. faecium (a) and L. paracasei (b) cultures immobilized in SGS with different acids. The numbers indicate: 1 – control, non-stabilized culture stored in liquid LB medium; 2 – culture immobilized in SGS with lactic acid; 3 – culture immobilized in SGS with malic acid; 4 – culture immobilized in SGS with citric acid; 5 – culture immobilized in SGS with acetic acid; 6 – culture immobilized in SGS with ascorbic acid.

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6. Fig. 5. The proportion (% of control) of viable cells of the E. faecium (a) and L. paracasei (b) populations in the control (without added acids) and experimental variants with the addition of organic acids – in the SHG (hatched columns) and the LB bacterial growth medium (solid filled columns), stored for 30 days. The numbers indicate: 1 – control; 2 – samples with the addition of ascorbic acid; 3 – samples with the addition of malic acid; 4 – samples with the addition of lactic acid; 5 – samples with the addition of citric acid; 6 – samples with the addition of acetic acid.

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7. Fig. 6. Micrographs (transmission electron microscopy) of E. faecium cells: a – cells in the stationary growth phase in liquid LB medium; b – cells grown in LB medium after 30 days of storage in the same medium, general view; c – same as b, enlarged. Designations: VC – vegetative cells; LC – lysed cells; CLC – cyst-like resting cell; LF – L-form.

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8. Fig. 7. Micrographs (transmission electron microscopy) of E. faecium cells stored for 1 month in SHG: a – with ascorbic acid, enlarged; b – with lactic acid; c – with acetic acid. Designations: VC – vegetative cell; CLC – cyst-like resting cell; K – capsule.

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9. Fig. 8. Micrographs (transmission electron microscopy) of L. paracasei cells: a – cells in the stationary growth phase in liquid LB medium; b – cells grown in LB medium after 30 days of storage in the same medium, general view; c – the same as b, enlarged. Designations: SC – stationary cells; LC – lysed cells; CLC – cyst-like resting cell; LF – L-form.

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10. Fig. 9. Micrographs (transmission electron microscopy) of L. paracasei cells stored for 1 month in SGG: a – with malic acid; b – with acetic acid. Designations: SC – stationary cell; CLC – cyst-like resting cell; K – capsule.

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