RU2757600C1 - Method for obtaining biological products of living microorganisms with an extended period of preservation of a high titer of viable cells by immobilization in gels based on silanol derivatives of humic substances - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and can be used to stabilize biological products containing live microorganisms. The method provides for the preparation of a gel from alkaline solutions of humic substances (HS) and 3-aminopropyltrialkoxysilane (APTS) by titration with an acetic acid solution to a pH value close to neutral, followed by the addition of APTS, immobilization of microorganism cells in gels by mixing a suspension of bacteria and gel in a given ratio, the stage of keeping for several hours until the gel thickens.

EFFECT: invention allows microorganisms being kept in an active state for a long time.

12 cl, 6 dwg, 1 tbl, 6 ex

Description

The invention relates to the field of biotechnology, which uses the culture of living microorganisms, for example, biotechnology for cleaning natural and man-made objects from pollution, as well as food biotechnology. More specifically, the invention can be applied to stabilize biological products containing live microorganisms in order to prolong the maintenance of a high titer of living microorganisms and high technological properties of the corresponding biological products.

For the effective use of biological products containing living microorganisms, it is important that the concentration of viable cells is at a level sufficient for the manifestation of the target technological properties. So, to clean the environment from oil products, biological products must have a concentration of hydrocarbon-oxidizing bacteria (HCB), not less than 10 ⁸ cells / g (RU2618699C1; Guide ..., 2018), and for the product to implement probiotic properties, it must contain cells of probiotic bacteria not less than 10 ^6-8 CFU / ml, depending on the type of microorganisms and the purpose of their use (Minelli, Benini, 2008).

Since individual organisms cannot exist forever, when using living microorganisms, it is important to slow down their dying off. For this, various methods are used, for example, drying preparations, storage at low temperatures (Sidyakina, 1985), which is most effective, but increases the cost and complicates the technological process of obtaining biological products. A common way of preserving microorganisms is immobilization - the fixation of cells on or in a carrier using physical or chemical forces. The main types of immobilization are the inclusion of cells in a substance, as a rule, in a gel, or attachment to a solid surface (Efremenko, 2018). Inclusion in dense gels is used when the target product is any cell metabolite (amino acids, glucose-fructose syrups, etc.), and it is necessary to use high volumetric cell concentrations. For technologies that require the introduction of microorganisms that must begin to multiply and spread in the environment of use, it is important that the cells can leave the carrier - by desorption from the surface or destruction of the carrier. For example, it is necessary that bacteria easily leave the carrier and be evenly distributed throughout the volume during bioremediation of soil and water, when used as ferments for silage, in food products.

From the state of the art, a method for storing microorganisms is known, which consists in growing microorganisms with their subsequent immobilization on synthetic fibers made of nylon, polypropylene, lavsan or others (RF Patent No. 2008348). The disadvantage of this method is the difficulty of using suspensions of such preparations for the treatment of large objects.

There are many ways to immobilize probiotic cultures of microorganisms in gels (Mitropoulouet G. et all., 2013). The most common gel base material is alginate, which forms gels with sufficient density and strength. In biotechnologies for obtaining various products of microbial metabolism, gels from alginate and polyvinyl alcohol are most often used (Efremenko, 2018). However, if it is necessary to release cells before using a biological product, these polymers are difficult to use due to their strength. Therefore, we paid attention to gels that are easily destroyed before or during use.

A known method of producing a gel based on water-soluble silanol derivatives of humic substances (SPGV) (patent RU 2530024), which are modified in such a way that they are able to be strongly sorbed on mineral or organic hydroxyl-containing substances. According to the method, an alkaline solution of humates is titrated with an inorganic acid to pH 3-7, after which aminoorganosilane is added, kept at a certain temperature, water is removed and used to obtain films. This method was chosen by us as a prototype due to the water solubility of the obtained composite polymers from SPGV, the known properties of humates to be soil ameliorants (Dagurov et al., 2005) and biologically active additives (RU2259147C2), as well as the ability of humates to increase the number of viable bacteria during long-term storage ( Nikolaev et al., 2020). As a result of the modification of the prototype method, the following changes were made: an organic acid (acetic acid) was used to acidify the medium, which introduced a food source for microorganisms into the medium; as a surface for the attachment of the gel, the surface of living cells containing OH groups was used, which is necessary for the attachment of the gel and, at the same time, for the immobilization of microorganisms in it; the resulting gel was used directly in the production medium (without removing water) as a matrix containing live bacteria of different species.

The technical result of the proposed solution is to obtain a new carrier of living microorganisms capable of dissolving in aqueous media, ensuring the maintenance of a high titer of viable cells, which leads to an increase in the shelf life of biological products intended for storage without additional cooling, the relative ease of obtaining, as well as the presence in the biological product of substances with additional useful technological and consumer properties - humates.

The proposed method for obtaining immobilized preparations of living microorganisms includes the stage of growing microorganisms, the stage of preparing a gel from alkaline solutions of humic substances (HS) and 3-aminopropyltrialkoxysilane (APTS) by titration with an acetic acid solution to a pH value close to neutral, followed by the addition of APTS, the stage of mixing suspension of bacteria and gel, the stage of incubation for several hours until the gel thickens. The resulting gel can be stored for several months, while maintaining a high titer of viable bacteria. Before use, the gel is dissolved in an aqueous medium and used as a source of bacteria.

In accordance with the proposed method to obtain a gel based on water-soluble silanol derivatives of humic substances (SPGV), various preparations of humic substances can be used.

Aminoorganosilanes, preferably, but not exclusively, 3-aminopropyltrialkoxysilane, can be used for the production of SPGL in accordance with the proposed method.

In accordance with the proposed method, organic acids, preferably, but not exclusively, acetic acid can be used to produce SPGV.

In accordance with the proposed method, gram-positive and gram-negative bacteria of different physiological groups can be used - for example, aerobic chemoorganotrophic hydrocarbon-oxidizing bacteria and aerotolerant lactic acid bacteria grown on optimal media for them until the stationary growth phase.

In accordance with the proposed method, a 10-15% HS solution is brought with an acid to pH 6-7, depending on the properties of bacteria, 2.5-5% APTS is added to it, or the pH is adjusted in a mixture of HS and APTS, after which a suspension of bacteria is added to the neutralized mixture in the amount of 0.4-0.5 of the volume of the obtained SPGV solution and left for several hours until the gel thickens.

The obtained preparations can be stored under ambient conditions for several months and before use they can be dissolved in aqueous media in the required proportion.

The above set of features included in three independent claims is not known from the prior art. Within the scope of the above set of features, the claimed technical result is achieved, which is confirmed below. Neither in the scientific, technical, nor in the patent literature was found information about the possible influence of the distinctive features of independent claims (obtaining gels based on silanol derivatives of humic substances using organic acids, introducing living microorganisms into such gels as a solid surface for attaching a gel, using these gels to extend the viability of microorganisms) on the technical result achieved when implementing the claimed method.

FIG. 1 shows the type of gel based on SPGV (from Powhumus humate) after hardening.

FIG. 2 shows the content of viable cells (CFU / ml) of A. seifertii immobilized in a gel based on Powhumus humate during 7 months of incubation, where K is the control culture, Op is the culture in the gel (experiment).

FIG. 3 shows the content of viable cells (CFU / ml) of P. aeruginosa immobilized in a gel based on Baikal humate during 3.5 months of incubation (experiment) in comparison with the control.

FIG. 4 shows the content of viable cells (CFU / ml) of E. faecium immobilized in a gel based on Baikal humate during 28 days of incubation (experiment) in comparison with the control.

FIG. 5 shows the morphology of P. aeruginosa cells aged 24 h (A), 2 months (B), and 2 months in a gel based on Baikal humate (C). A and B - phase contrast, C - DAPI color, inversion (negative).

FIG. 6 shows respiratory rate (accumulation of CO ₂₎ in cultures of P.aeruginosa (A) and A.seifertii (B) of different ages and during storage in a gel based humate Baikal.

When we created the proposed innovative method for stabilizing biological products (i.e. long-term maintenance of a high titer of viable cells of microorganisms), we proceeded from several preconditions: microorganisms immobilized in gels have increased stress resistance and survival due to being in a state close to or identical to the biofilm phenotype ( Efremenko, 2018; Z'ur et al., 2016), and for this reason they settled on gel biocomposites. The resulting gel should be readily soluble in aqueous media before use. The technology for producing the gel should be relatively simple, preferably in a single reactor. The components of the gel should be readily available, inexpensive, and desirably have additional beneficial properties.

The essence of the method and its technical feasibility are illustrated by the following examples.

Example 1. Obtaining SPGV gel using potassium and sodium humate obtained from leonardite, Powhumus (Humintech, Germany)

The organosilane used was 3-aminopropyltriethoxysilane (APTS) (AGM-9, OOO Penta 91, Russia). 1-1.5 g of humate was ground in a mortar and dissolved in 10 ml of distilled water with vigorous stirring on a magnetic stirrer, the insoluble fraction by centrifugation (5000 g, 5 minutes), the supernatant was poured into a beaker and 0.3-0.5 ml of APTES was added with vigorous stirring on a magnetic stirrer, after which it was titrated with a solution of glacial acetic acid to pH 6-7. The resulting mixture was left at room temperature. Thickening (gelation) of the reaction mixture took place within 2-12 hours. The resulting gel was homogeneous, had moderate strength properties - it did not flow out of the test tubes when they were turned over (Fig. 1), retained its properties for several months, and dissolved with the addition of water and vigorous stirring.

Example 2. Obtaining SPGV gel using potassium and sodium humate obtained from brown coal, Baikal ("AgroTechHumat", RF)

The organosilane used was APTS of the same manufacturer as in Example 1. 10-15 g of humate was dissolved in 100 ml of distilled water with vigorous stirring on a magnetic stirrer, the pH was adjusted to 6-7 with glacial acetic acid, 5-10 ml of an aqueous solution was added APTES (50%) with pH 6-7, adjusted with glacial acetic acid. The resulting mixture was left at room temperature, within 2-12 hours thickening (gelation) of the reaction mixture occurred. The resulting gel was homogeneous, possessed moderate strength properties - it did not flow out of 20 ml tubes when they were turned over, retained its properties for several months, and dissolved with the addition of water and vigorous stirring.

The above examples 1 and 2 prove the technical feasibility of the proposed method for producing gels based on SPGV using various preparations of humates, acetic acid and methods of mixing with APTS and titration methods.

Example 3. Storage of cultures of hydrocarbon-oxidizing bacteria and yeast in SPGV gels based on Powhumus humate

Cultures of hydrocarbon-oxidizing bacteria (Gram-positive Rhodococcus erythropolis, Gram-negative Acinetobacter seifertii and Pseudomonas aeruginosa) and one yeast organism (Yarrovia lypolytica) were grown in LB medium until the stationary growth phase, after which they were mixed with a freshly obtained gel based on humate 3 ml Powhum gel, poured into Eppendorf tubes (1 ml each) or Falcon tubes with a volume of 15 ml (5 ml gel) and stored for several months, after which they were resuspended in sterile saline and the titer of viable cells (CFU) was determined.

FIG. 2 shows the curves of the content of viable A.seifertii cells immobilized in the SPGV gel based on Powhumus humate during 7 months of incubation. Control culture of bacteria rapidly dying out and after 1 month the number of viable cells was below the value of 10 ⁸ cfu / ml. The cells included in the silanol-humic gel continued to multiply during the first month, their titer increased several times, after which they began to die off at a rate 5 times lower than in the control. After 4-7 months of storage, the CFU titer in the experimental variant was 150-40 times higher than in the control one.

For other cultures, similar curves of the CFU content versus time were obtained in the experimental and control variants. In all cases, after several months of storage, the CFU titer in the experimental variants was 1-2 orders of magnitude higher than in the control (Table 1).

Table 1. The excess of the titer of viable cells (CFU / ml) of cultures of R. erythropolis, A. seifertii, P. aeruginosa and Y. lypolytica in the experimental variant over the control after 4 months of storage.

Microorganism CFU ratios
Experience / control R. erythropolis eleven A. seifertii 150 P. aeruginosa eleven Y. lypolytica eight

Example 4. Storage of cultures of hydrocarbon-oxidizing bacteria in SPGV gels based on Baikal humate

For one of the hydrocarbon-oxidizing bacteria, P. aeruginosa, storage for 3.5 months was studied during immobilization in a gel based on another humate, Baikal. FIG. 3 shows the curves of the CFU titer in the control variant and when cells are included in the gel based on 10% humate content. For a gel based on a 15% humate content, the data are similar and not presented. Figure 3 clearly shows that inclusion in the gel prevents cell death within 2 months, leads to an increase in the number of living cells by 1.5 times, while during the same time in the control, the CFU titer decreased by 40 times.

Example 5. Storage of cultures of lactic acid bacteria in SPGV gels

Lactic acid bacteria are a group of bacteria important for medicine, food industry and biotechnology, which is characterized by high rates of death, therefore storage in SPGV gels was also tested on this group of bacteria. Lactic acid bacterium Enterococcus faecium were grown in milk at ³⁰ ° C and stored for 4 weeks at room temperature in a flask under stationary conditions (control) or placed in a gel based on humates Powhumus or Baikal.

FIG. 4 it can be seen that during 4 weeks of storage, the titer of viable cells decreased in the control by almost 4 orders of magnitude. The inclusion of Powhumus in the gel based on humate slowed down the death, which caused the titer of viable cells to exceed the control variants by 30 times after 4 weeks of storage. Immobilization of cells in a gel based on Baikal humate was less effective - the excess of the CFU titer in the experiment over the control was 6 times, which is associated with the different quality of humate preparations, and that it is necessary to take into account when developing methods for stabilizing biological products.

The given examples No. 3-5 prove the technical feasibility of the proposed method of storing all tested bacteria in silanol-humic gels and the high efficiency of this method in maintaining the viability of cells of various microorganisms for a long time.

Example 6. Determination of the state of bacterial cultures stabilized using SPGV gels based on Baikal humate, after 2 months of storage

The results obtained in examples 3 and 4 demonstrated that bacterial cells included in SPGV gels can multiply for some time, which may be due to the fact that the gels contain organic substances that can be consumed by microorganisms and serve as sources of energy and carbon - acetic acid added as a titrant (up to 1.5 g / l) and ethanol formed in the esterification reaction (up to 0.5 g / l).

The physiological state of cells was judged by two indicators - respiratory activity and the appearance of cultures, determined microscopically.

P.aeruginosa culture A.seifertii and after 2 months of storage in gel based humate Baikal (10%) was transferred to a medium without carbon source Raimond (5% by volume) and incubated on shaker at 30 ^C for 24 hours. The cells Breath initiated by heating and oxygen supply. The metabolic activity of cells was assessed by increasing the concentration of carbon dioxide in the culture medium, which was determined by transferring 1 ml of the medium into hermetically sealed vials (V = 18 ml) with 0.2 ml of phosphoric acid using a Kristall 5000 gas chromatograph (RF).

Microscopic studies were performed using phase contrast and fluorescence microscopy using an Axio Imager M2 microscope, Carl Zeiss Microscopy GmbH, Germany. Used fluorescent dye DAPI (DAPI).

After two months of staying in the Baikal humate-based gel, the P. aeruginosa culture looked like a growing active - it was dominated by elongated cells, there were no lysed and destroyed cells (Fig. 5). The length of the cells in the gel (Fig. 5B) exceeded the average cell length of the active young culture (Fig. 5A). In this case, in the control 2-month culture, the cells are characterized by shortened sizes, less refractoriness and fewer numbers (Fig. 5B). Low refractoriness is a sign of lysed or destroyed cells. For A.seifertii, the data are similar and not presented.

Both 2-month-old cultures stored in the gel showed higher respiration rates without added carbon source than both 2-day-old and 2-month-old controls (FIG. 6). Note that culture respiration could occur only due to the presence of endogenous carbon sources - the remains of the nutrient medium for a young culture, autolysates of cells for old cultures, and in the presence of a gel - also acetate and ethanol. The respiration rate of P. aeruginosa culture with gel exceeded control by 2-3 times, and A. seifertii - 1.2-1.7 times, which reflects the microscopically and by CFU titer greater safety of the cultures in the gel based on Baikal humate in comparison with the control culture.

This example illustrates the mechanism of effective maintenance of the number of viable cells in bacterial cultures in SPGV gels during long-term storage - by maintaining them in an active metabolic state, which allows this storage method to be used for a wide range of microorganisms.

Cited sources

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Claims (12)

1. A method of obtaining preparations of live microorganisms by immobilizing cells of microorganisms in gels based on silanol derivatives of humic substances (SPGV), including the stage of growing microorganisms, the stage of preparing a gel from alkaline solutions of humic substances (HS) and 3-aminopropyltrialkoxysilane (APXS) by titration with a solution of urea acid to a pH value close to neutral, followed by the addition of APTS, the stage of mixing the suspension of bacteria and the gel, the stage of holding for several hours until the gel thickens. 2. A method according to claim 1, characterized in that the preparations of living microorganisms are mixed with freshly prepared gels based on SPGV in a ratio of 0.4-0.5: 1 by volume. 3. The method according to claim 1, characterized in that gels based on SPGV are obtained by mixing solutions of humic substances and aminoorganosilane APTS, bringing the pH to neutral or slightly acidic values, holding for several hours. 4. The method according to claim 3, characterized in that the humic substances are used in the form of aqueous solutions at a concentration of 10-15%. 5. The method according to claim 3, characterized in that the aminoorganosilane APTS is used in pure form or in the form of a 50% aqueous solution. 6. A method according to claim 3, characterized in that humic substances and aminoorganosilane are mixed in a mass ratio of 7.5: 1-2: 1. 7. A method according to claim 3, characterized in that acetic acid is used to adjust the pH. 8. The method according to claim 3, characterized in that bringing the pH to neutral values is carried out both separately for solutions of humic substances and aminoorganosilane APTS, and for their mixture. 9. The method according to claim 1, characterized in that the microorganisms are grown to the stationary growth phase on nutrient media that are optimal for them. 10. The method according to claim 9, characterized in that the microorganisms can be gram-positive bacteria. 11. The method according to claim 9, characterized in that the microorganisms can be gram-negative bacteria. 12. The method according to claim 9, wherein the microorganisms can be yeast.

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