Long-term survival of Enterococcus faecium under different conditions of cell stabilization and immobilization

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Abstract

Lactic acid bacteria (LAB) play an important role in biotechnology and biomedicine. Their most important disadvantage is the rapid death of crops and preparations during storage. Studying ways to increase the survival time of lactic acid bacteria under various conditions is an urgent scientific and applied task and was the goal of this work. The object was the lactic acid bacterium Enterococcus faecium. It has been shown that in aging planktonic cultures, bacteria quickly lose viability (the number of viable cells decreases by 2–4 orders of magnitude in 1 month). The development cycle of the E. faecium population under these conditions ends with the formation of cyst-like resting cells of two types: L-forms and hypometabolic cells. The use of chemical stabilizers, humic substances (typical soil components), and increases the number of surviving cells by 2–3 times. With surface immobilization (adsorption) on organosilanol or inorganic carriers (organosilane, silica), the number of cells surviving under starvation conditions increases by 1.25–3 times. The most effective approach was the immobilization of cells in silanol-humate gels (increasing the number of surviving cells up to 35 times relative to the control). The data obtained reveal the mechanisms and forms of survival of LAB in natural conditions (state of hypometabolism, the presence of specialized forms of dormancy), and can also be used to develop methods for long-term storage of LAB in their biological products.

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About the authors

O. A. Galuza

Federal Research Center of Biotechnology RAS; Bavar+ JSC

Author for correspondence.
Email: olesya_galuza@mail.ru

Institute of Microbiology named after. S.N. Vinogradsky

Russian Federation, 119071, Moscow; 127206, Moscow

G. I. El-Registan

Federal Research Center of Biotechnology RAS

Email: olesya_galuza@mail.ru

Institute of Microbiology named after. S.N. Vinogradsky

Russian Federation, 119071, Moscow

T. A. Kanapatski

Federal Research Center of Biotechnology RAS

Email: olesya_galuza@mail.ru

Institute of Microbiology named after. S.N. Vinogradsky

Russian Federation, 119071, Moscow

Yu. A. Nikolaev

Federal Research Center of Biotechnology RAS

Email: olesya_galuza@mail.ru

Institute of Microbiology named after. S.N. Vinogradsky

Russian Federation, 119071, Moscow

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2. Fig. 1. Dynamics of changes in the number of viable E. faecium cells in a population grown in skim milk, when stored in static conditions with access to atmospheric oxygen for 1 month. The numbers indicate the phases of death: 1 – stationary, 2 – rapid death, 3 – plateau.

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3. Fig. 2. The proportion (% of control) of surviving E. faecium cells stored for 1 month in the control (without adding HS) and experimental variants with the addition of HS at a concentration of 0.5 g/l (column marked with number 1) and 0.15 g/l (column marked with number 2).

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4. Fig. 3. The proportion (% of control) of surviving E. faecium cells stored for 1 month in the control (without adding HS) and experimental variants with the addition of HS at a concentration of 0.15 g/l and during storage with limited access (column marked with number 1) and without limiting access to atmospheric oxygen (column marked with number 2).

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5. Fig. 4. E. faecium cells adsorbed on the surfaces of gel particles or in the gel: a – Enterosgel; b – Polysorb; c – SGG based on Paohumus humate. 1 – Particles of polymethylsiloxane polyhydrate; 2 – particles of finely dispersed silica (Polysorb); 3 – adsorbed E. faecium cells. Fluorescence microscopy.

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6. Fig. 5. The proportion (% of control) of surviving cells of the E. faecium population grown in milk during storage for a month in the control (without the addition of stabilizers) and experimental variants with the addition of stabilizers.

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7. Fig. 6. The proportion (% of control) of surviving E. faecium cells grown in milk during storage for 1 month in the control (planktonic culture) and experimental variants in the SHG.

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8. Fig. 7. The proportion (% of control) of surviving cells of the E. faecium population grown during heterophase cultivation with the addition of sorbents and subsequent storage for 1 month under different conditions of oxygen access: with limited oxygen access (solid filled columns) and with oxygen access (hatched columns).

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9. Fig. 8. Electron micrographs of thin sections of E. faecium cells grown in milk to the stationary growth phase (a, b) and stored for 6 months in a static mode (c-g). Designations: VC – ultrastructure of vegetative cells (a, b); type I CPC with a thickened cell wall (CL); type II CPC with a thick multilayer CL and dense cytoplasm, compacted nucleoid (CN); L-ф – L-form cells without CL (c); CPM – cytoplasmic membrane (b, d, g).

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10. Fig. 9. Colonies of E. faecium dissociants in a population obtained from cells grown in skim milk for 24 h (a) and stored for 6 months (b). 1 – Colonies of the dominant S-morphotype up to 2 mm in size; 2 – colonies of the Sb-morphotype 2–3 mm in size; 3 – colonies of the Sm-morphotype less than 1 mm in size, translucent. Light microscopy.

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