RU2280465C2 - Method for preparing probiotic - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method involves addition of strains of lactobacillus microorganisms L. fermentum 39 and L. plantarum 8PA3 to the nutrient medium, their combined culturing in the nutrient medium containing the defatted milk hydrolyzate diluted with the purified water and agar-agar. The culturing process is carried out for two steps. At the first step strains diluted in the ratio = 1:1 are cultured at temperature 37°C for 24 h. At the second step the prepared biomass of the first generation is mixed with the fresh nutrient medium in the ratio = 1:1000, and the culturing process is continued at the same temperature for 24 h. Invention provides enhancing the viability of microorganisms, their antagonistic activity and the yield of the biomass. Invention can be used in preparing biologically active supplements and products for the rational nutrition base on living microorganisms.

EFFECT: improved preparing method of probiotic.

2 tbl, 2 dwg

Description

The invention relates to medical and food biotechnology and can be used for the preparation of biologically active additives and functional food products based on live bacteria.

It is widely known drugs aimed at restoring normal intestinal biocenosis, in particular eubiotics - bacterial preparations from living microorganisms - representatives of obligate intestinal microflora, these are colibacterin, lactobacterin, bifidumbacterin, bificol, etc. dry preparations (see the book under the editorship of V.A. Knyazhev. Dysbacterioses. Theory and practice. Nizhny Novgorod, 1999, pp. 70-73).

With all the positive qualities of these drugs: long shelf life, ease of transportation and use, and so on, there are a number of drawbacks - firstly, due to the freeze-drying process, the cells of microorganisms are in deep suspended animation, it takes 8-10 hours to go into their active physiological state (the digestion process in children is 4-6 hours), in addition, after the lyophilization process, the cells lose specific receptors (adhesins), with the help of which they are fixed on the mucous membranes, as a result, their stay in the intestine thus, the therapeutic effect is reduced. Also, during the drying process, most of the metabolites (in particular, fatty acids), which contain additional therapeutic factors, are destroyed.

It is known that to improve the quality of drugs, in particular digestibility and therapeutic effect, yeast is prepared from bifidobacteria and lactobacilli, which are introduced into milk, see, for example, a method for preparing a probiotic based on live bifidobacteria and lactobacilli according to RF patent No. 2060673, A 23 C 9/12, C 12 N 1/20, 1996

The disadvantage of this method is that the basis of the probiotic is whole milk, into which the yeast is introduced. The pH of the final product is 4.4-4.8, and the concentration of live bacteria is only 10 ⁸ .

As a prototype, a method of preparing starter culture for producing fermented milk lactobacterin was adopted (see methodological recommendations “Use of milk lactobacterin in the treatment of diseases that occur against the background of intestinal dysbiosis.” Gorky, 1980, p.7).

The method includes preparing a milky-yeast medium containing a skim milk hydrolyzate (skim milk hydrolyzate) + 5% yeast autolysate and + 0.2% glucose (carbohydrate). Lactobacillus strains Lb fermenti 90-TC-4 or Lb plantamm 8-PA-3 are used, which are seeded in milk-yeast medium at a rate of 200-300 billion per 10 l of medium and incubated for 20-22 hours.

Sourdough has the appearance of a muddy liquid of a dirty yellow color with an acidic taste. 1 ml of starter culture contains at least 10 ⁶ -10 ⁸ live bacteria, which means there are fewer in the final product.

The product has a fairly high antagonistic activity to pathogenic and conditionally pathogenic microbes.

The disadvantage of this method is the rather short shelf life of the starter culture (1-2 days). Ferment is used only as part of a fermented milk product.

These shortcomings are eliminated by the proposed solution.

The problem to be solved is the improvement of the method for preparing a probiotic (therapeutic product) based on live strains of microorganisms with an increased shelf life (up to 60 days).

The technical result is an increase in the viability of bacteria, their antagonistic activity and yield of biomass.

This result is achieved by the fact that in the method of preparing a probiotic based on live strains of lactobacilli using milk-yeast nutrient medium (MDS), including a skim milk hydrolyzate, yeast autolysate, carbohydrate as an energy source, by culturing producer strains in it at a temperature of 37 ^± 1 ° C, packaging of the preparation, culture medium further contains agar-agar diluted with purified water skim milk hydrolyzate with amino nitrogen content of 130-200 mg% as carbohydrate and polzujut raffinose, and co-cultivation of lactobacilli strains L. plantarum 8 L.fermentum PA 3 and 39 are in two phases: the first phase diluted in the medium the strains are combined in a ratio of 1: 1 and cultured for 24 ^± 1 hours, in a second step the obtained biomass first generations are mixed with fresh nutrient medium in a ratio of 1: 1000 and cultivation is continued for 24 ^± 1 h at the same temperature.

Thus, an optimal complex has been proposed that allows one to obtain a preparation from living bacteria with a longer shelf life.

Joint cultivation of synergist strains L.plantarum 8 PA 3, L.fermentum 39 allowed to accelerate the process of biomass growth and increase its yield.

Also, with the joint cultivation of these synergist strains, their level of anatagonistic activity increased in relation to the widespread species of fungi of the genus Candida C. albicans. This species is etiologically significant in 60-80% of cases of fungal etiology. It is known that the antagonistic properties of lactobacilli are due to their ability to produce bacteriocins or bacteriocin-like substances. The strains L.plantarum 8 PA 3, L.fermentum 39, L.fermentum 90-TC-4 are producers of bacteriocins. L.plantarum 8 PA 3 is resistant to bacteriocins, both to its own and to bacteriocins produced by L.fermentum 39, L.fermentum 90-TC-4. L. fennentum 39 is resistant to the action of its own bacteriocins, while L.fermentum 90-TC-4 is sensitive to the action of its own bacteriocins. And strains of lactobacilli resistant to their own bacteriocins create selective advantages, both for their own growth and for strains resistant to the action of their bacteriocides. Thus, strains of L. plantarum 8 PA 3 and L.fermentum 39 can be cultivated together with a high level of biomass yield, in contrast to L.fermentum 90-TC-4.

Another reason for choosing the strain L.fementum 39 is its high adhesive activity (bacteriosorption) to mucin and fibronectin, which are receptors for lactobacilli adhesins on eukaryotic cells, which is important in ensuring the colonization of lactobacilli of the gastrointestinal tract.

The use of skim milk hydrolyzate diluted with purified hydrolyzed water as the basis of MDS increases the supply of hydrogen and oxygen to lactobacilli (which are microaerophiles), sodium hydroxide satisfies the cell’s need for inorganic elements, agar-agar allows to increase the viscosity of the medium, which allows lactobacilli to grow uniformly throughout the entire thickness environment (on liquid media, near-bottom growth), that is, evenly consume growth factors and nutrients throughout the volume, and the replacement of carbohydrate and glucose to raffinose (fructo-oligosaccharide), in addition to meet the needs of the cells in carbohydrate, promotes the selective stimulation of the growth and revitalization of mineral metabolism representatives of the intestinal microflora (in particular, lactobacilli).

The method is as follows. The MDS medium is prepared according to the following scheme: skim milk is boiled for 2-3 minutes, cooled to 45 ° C, adjusted to pH 20% with sodium hydroxide solution, add pancreatin, mix, add chloroform, put in a thermostat, stir for the first hour, then filter amine nitrogen is installed through a paper filter, 2: 1 purified water is diluted, agar-agar solution is prepared from part, yeast autolysate from pressed yeast diluted in water, infused in a thermostat, then autoclaved and profiled is added to the remaining part. iltrated, and raffinose, all mixed and sterilized.

The cultivation of producer strains of L. plantarum 8 PA 3, L.fermentum 39 is carried out at a temperature of 37 ^± 1 ° C in two stages together, at the first stage, the strains diluted in MDS are combined in a 1: 1 ratio and cultivated for 24 ^± 1 hours, at the second stage the obtained biomass of the first generation is mixed with fresh MDS in a ratio of 1: 1000 and continue to cultivate another 24 ^± 1 hour at the same temperature. The second generation of strains goes to the final preparation, then at the end of cultivation the biomass is packaged in 5 ml taking into account the daily dose of the probiotic. Experiments show that this mode is optimal for achieving a result.

An example implementation of the method.

At the first stage, a whole skim milk hydrolyzate and a yeast autolysate are prepared for the future.

The whole skim milk hydrolyzate is prepared as follows: skim milk is boiled (category I-II) for 2-3 minutes, cooled to 45 ^± 1 ° C, pancreatin is added at a rate of 1 g / l, previously diluted in a small amount heated to 44 ^± 1 ° C of water, stir, put in a thermostat and incubated at a temperature of 40 ^± 1 ° C, constantly mix for 4 hours with correction of active acidity, then add 1-2% chloroform, close with a rubber stopper and put in a thermostat. During the first hours, skim milk is mixed several times, (the cork is punctured after shaking to remove chloroform vapor). After 4 hours, the pH was adjusted to 4.3 ^± 0.1 UNITS with a 30% solution of acetic acid, boiled, stirring, for 15 minutes, filtered through a paper filter. The finished hydrolyzate should contain 200-300 mg /% amine nitrogen. The hydrolyzate is stored for future use under chloroform (1% by volume) at a temperature of 4 ^± 1 ° C.

The yeast autolysate is prepared as follows: 1 kg of pressed yeast is diluted in 1 liter of water and placed in a thermostat at a temperature of 56 ^± 1 ° C for 72 ^± 2 hours. After that, the resulting suspension is autoclaved at a temperature of 115 ^± 1 ° C for 15 ^± 1 minutes. The autolysate is filtered through a cotton-gauze filter, washing the precipitate with such an amount of water that the total amount of the filtrate is 4.0 ^± 0.1 L (stored for the future in a cold chamber at a temperature of 4 ^± 1 ° C).

The following formulation of the nutrient medium (MDS) is prepared from the obtained main raw materials:

Skim milk hydrolyzate 0.633 L 658.32 g 63.27% Purified water 0.32 L 320 g 30.75% Yeast autolysate 0.05 L 51.5 g 4.95% Agar agar 0.75 g 0.75 g 0.07% Raffinose 10.0 g 10.0 g 0.96% Total: 1040.57 g one hundred%

NaOH 20% solution from 0.00005 to 0.001 L to establish a pH of 7.1-7.2 UNITS. Nutrient sterilization mode: 20 minutes at 0.5 atm.

The order of preparation.

To prepare MDS, the whole hydrolyzate (amine nitrogen 200-300 mg%) is diluted with purified water based on 0.633 l of the whole hydrolyzate 0.32 l of purified water, i.e. 2: 1 (amine nitrogen 130-200 mg%).

Next, 100 ml of the diluted hydrolyzate is taken, 0.75 g of agar-agar is added and heated in a water bath until the agar is completely dissolved.

To the remaining part of the diluted hydrolyzate of 0.853 L, add 0.05 L of yeast and 10.0 g of raffinose, mix, add the diluted agar, mix, adjust the pH to 7.1 IU with a 20% sodium hydroxide solution, mix, pour into the desired volumes and sterilize 20 minutes at 0.5 atm.

As producer strains, L.plantarum 8 PA 3 and L.fermentum 39, having high antagonistic activity against C. albicans, are used.

The cultivation of producer strains is carried out according to the following method.

Generation I - a dry strain of L. plantarum 8 RA 3 is diluted with 0.007 L of MDS, dissolved for 10 minutes, shaking at room temperature, the strain L.fermentum 39 is also prepared. Then they are poured together in a 1: 1 ratio of 0.3 l MDS and cultured for 24 hours at a temperature of 37 ^± 1 ° C.

II generation. 0.001 L of the 1st generation are added to 1 liter of fresh MDS medium (i.e., from 0.314 L of the 1st generation 314 liters of the 2nd generation are obtained) and cultivated for 24 hours at a temperature of 37 ^± 1 ° С.

At the end of the biomass cultivation, the ready-to-use preparation is packaged in 5 ml vials, hermetically closed with a rubber stopper and an aluminum cap. The drug is stored at a temperature of +6 ^± 2 ° C for up to 60 days with the preservation of live microbial cells in 1 ml of a liquid preparation of 10 ⁹ -10 ¹¹ CFU / ml.

The drug can be obtained by a reactor method.

The test results obtained according to the described example of the drug are shown in tables 1.2 and in the graphs of figure 1.2.

Table 1 shows the viability of the bacteria in the preparation obtained by a known method (prototype) and the proposed. It can be seen that the number of living microbial cells persists with increasing storage time.

Table 2 shows the antagonistic activity of strains of lactobacilli, their complex and prototype in relation to fungi C. albicans.

The graphs of Fig. 1,2 respectively show the growth dynamics of lactobacilli during separate and co-cultivation (it is more active in co-cultivation - in the number of CFU / ml) and in the change in the optical density of the medium - it is higher during co-cultivation of L. plantarum 8 PA 3 strains and L.fermentum 39).

The drug can be used orally in 1 or 2 doses, 1 bottle per day before meals with water, milk, kefir, jelly, compote, cottage cheese, yogurt, etc. with a temperature not exceeding 30 ° C.

Indications for use: all diseases with an etiological factor, which is C. albicans (bacteriologically confirmed), all gastroenterological diseases, the use of antibiotics for any pathology, allergic diseases, gynecological diseases (thrush) and pregnant women in preparation for childbirth.

Perhaps the local use of the drug as vaginal swabs (2.5 ml per swab 2 times a day for 2 hours) or for the treatment of the oral cavity and gums (3-4 times a day between meals 1-2.5 ml per swab) .

Clinical testing of a probiotic prepared according to the method presented in the application was carried out in the following medical institutions of the city of Nizhny Novgorod: City Children's Clinical Hospital No. 27, Children's Clinic No. 49 of the Prioksky District Health Department, Sormovsky Interdistrict Skin and Venereologic Dispensary.

The indicated results were fully confirmed.

Obtained by the proposed method, the drug is conditionally called LL-complex.

Table 1 The number of live lactobacilli CFU / ml (LL complex) on various modifications of the MDS medium. Name of medium Storage time in days MDS (classic) According to the prototype MDS according to the proposed method The number of live microbial cells of lactobacilli in 1 ml of the drug in CFU / ml one 10 ¹² 10 ¹² 10 10 ¹⁰ 10 ¹² twenty 10 ⁹ 10 ¹² thirty 10 ⁹ 10 ¹² 40 10 ⁹ 10 ¹¹ fifty 10 ⁸ 10 ¹¹ 60 10 ⁷ 10 ¹¹ 70 10 ⁷ 10 ¹⁰

We studied the antagonistic activity of L. plantarum 8 PA 3, L.fermentum 39 LL-complex and starter culture for the production of sour milk lactobacterin (according to the prototype) in relation to C. albicans strains (41 strains) isolated from various biotopes of the human body (pharynx, sputum, intestines, reproductive tract, etc.).

table 2 Antagonistic activity of strains of lactobacilli, their complex in relation to fungi C.albicans Test strain Lack of antagonistic activity (%) Low degree of antagonistic activity (%) High degree of antagonistic activity (%) L.plantarum 8 RA 3 8.8 47.2 44 L.fermentum 39 5.8 fifty 44,2 LL-complex of the proposed method. 0 29.4 70.6 Ferment for the production of fermented milk lactobacterin (prototype) 8.8 47.2 44

Claims (1)

A method of preparing a probiotic based on live strains of lactobacilli using a milk-yeast nutrient medium, including a skim milk hydrolyzate, yeast autolysate and carbohydrate, by cultivating lactobacilli at a temperature of (37 ± 1) ° С, packaging the preparation, characterized in that the nutrient medium additionally contains agar-agar, diluted with purified water, a skim milk hydrolyzate with an amine nitrogen content of 130-200 mg%, raffinose is used as a carbohydrate, and the strain is co-cultured of lactobacilli L. fermentum 39 and L.plantarum 8 PA 3 are carried out in two stages: at the first stage, the strains diluted in the medium are combined in a 1: 1 ratio and cultured (24 ± 1) h; at the second stage, the obtained biomass of the first generation is mixed with fresh nutrient medium in a ratio of 1: 1000 and continue cultivation for (24 ± 1) h at the same temperature.

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