RU2803350C1 - Bifidobacterium longum 174 strain for the preparation of region-specific probiotic preparations for the prevention and personalized treatment of diseases of the gastrointestinal tract in residents of the karachay-cherkess republic and/or for enrichment of the traditional fermented milk drink gypy airan based on indigenous kefir grains - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method consists in isolating the bacterial strain Bifidobacterium longum 174 from the intestines of residents of a particular ethno-geographical community (Karachay-Cherkess Republic), which can be used to prepare region-specific probiotics for the treatment and prevention of diseases of the gastrointestinal tract in residents of Karachay-Cherkess Republic, as well as to enrich the traditional fermented milk drink Gypy Airan based on indigenous kefir grains.

EFFECT: treatment and prevention of gastrointestinal diseases.

3 cl, 1 tbl

Description

Field of technology to which the invention relates

The present invention relates to the field of microbiology and biotechnology.

The claimed strain was isolated from a resident of a specific geographic community; can be used to prepare region-specific probiotics for the treatment and prevention of gastrointestinal diseases in residents of the Karachay-Cherkess Republic.

State of the art

Modern ideas about the composition of the intestinal microbiota and its impact on human health are based mainly on studies of part of the population of Europe and North America. However, these data cannot be directly extrapolated to individuals from other populations. Even among well-studied human cohorts, individual characteristics such as location and ethnicity need to be taken into account along with microbiome data to more accurately diagnose and treat various diseases, in particular diseases of the gastrointestinal tract. Thus, large-scale studies have shown extensive personalization of the taxonomic composition of the intestinal microbiome [Gaulke, C.A., 2018]. It is also known that the composition of the intestinal microbiota in individuals of different ethnic origins - Dutch, Ghanaian, Moroccan, Turkish, African and South Asian Surinamese living in the same city in Holland, has characteristic ethnic profiles, despite prolonged exposure to the same environmental conditions, in particular elevated levels of bifidobacteria have been identified in certain ethnic groups. Ethnicity explains much more differences in the composition of the microbiome between individuals than any other characteristic taken into account in this study [Deschasaux M., 2018; Deschasaux M., 2019].

In addition, significant differences are known in the taxonomic composition of the microbiota of residents of the United States of America of different ethnic origins - Asians, African Americans, Latin Americans, Europeans, which also demonstrates the need to take into account ethnic diversity both in microbiota research and in the development of methods of probiotic therapy for gastrointestinal diseases [Brooks, A. W., 2018].

Significant differences in microbiomes were also revealed, apparently related to geography in the study of the fecal microbiota of Indians from the Amazon, Venezuela and residents of rural communities in Malawi, who historically lived compactly in certain geographical conditions and used a relatively monotonous diet [Yatsunenko T. et al., 2012]. It is known that ethnic-dependent diversity of the intestinal microbiota can be traced in infants even before the introduction of complementary foods [Jia Xu et.al., 2020]. Marked differences in the composition of gut microbial communities were found between Danes and Indians [Camila Alvares-Silva et.al., 2021]. It has been found that in countries with high ethnic diversity, such as Malaysia, differences in the gut microbiota between ethnic groups also persist, despite geographical separation over time, and the composition of the microbiota of migrants shares similarities with the population in the country of origin [Jacky Dwiyanto et al., 2021].

Thus, the taxonomic composition of the gut microbiome is associated with both the ethnicity and geographic location of the individual. This connection must be taken into account in a certain way for the development of drugs based on normal representatives of the human microbiome for use as group-personalized probiotic therapy.

Bifidobacteria are one of the main representatives of the normal microbiota of the human intestine [Turroni, 2009], and along with lactobacilli, they are most often used as probiotic microorganisms. According to the generally accepted definition, probiotics are live microorganisms that, when administered in adequate quantities, have a beneficial effect on the host [Hill C. et al., 2016]. The most important characteristics of an effective probiotic microorganism are considered, in particular, its ability to adhere to the cells of the host organism, which is the first and most important stage of colonization, as well as the ability to inhibit opportunistic and pathogenic microorganisms [Shokryazdan P., 2017]. However, due to the fact that existing probiotics using exogenous bifidobacteria (RF patent WO2009051510A1, IPC C12N11/14, publ. 2009-94-23), unlike their own strains (autostrains), are usually incapable of long-term colonization intestines of a particular person, successful probiotic therapy requires long-term intake of probiotics in high doses. At the same time, strains of bifidobacteria characteristic of a specific ethno-geographical group, which has its own nutritional characteristics, environmental conditions, and historically formed genetic characteristics, will be more consistent with the human body living in a specific region and belonging to a specific ethnic group, and , accordingly, have a higher probiotic potential.

In the USSR, a method was proposed for correcting intestinal microflora using autostrains of lactobacilli [Korshunov V.M., Sinitsyna N.A., Pinegin B.V. Method for treating dysbiosis // RF Patent No. SU1286212]. Also, later, a method was developed for producing an autoprobiotic based on an anaerobic consortium of bacteria for the personal correction of dysbiotic disorders [Suvorov A.N., Ermolenko E.I., Kotyleva M.P., Tsapieva A.N. Method for producing an autoprobiotic based on an anaerobic consortium of bacteria //RF Patent RU2734896C2]; method for obtaining an autoprobiotic with increased physiological activity [Denisov D.E., Kuznetsova T.N. A method for producing an autoprobiotic containing live bifidobacteria and lactobacilli //RF Patent RU 2580002 C1], a method for obtaining a personalized autoprobiotic product [Simanenkov V.I., et al. A method for obtaining a personalized autoprobiotic product in the form of a lactic acid starter based on autostrains of lactobacilli and a method for treating the syndrome irritable bowel, accompanied by intestinal dysbiosis, obtained with a personalized autoprobiotic product // RF Patent RU2013120765A]. A composition with various variants of lactobacilli is also known, providing personalization for the correction of intestinal dysbiosis [Aleshkin V.A., et al. Composition for the correction of intestinal dysbiosis //RF Patent RU2431492C1] and a probiotic composition for groups of people combined by blood groups [Probiotics compound for crowds with blood fluids of A,B, and method and application of probiotics compound //Patent CN105105115A]. A known method and system for personalizing probiotic preparations based on the assessment of family, genetic, geographic/ethnic information about the patient [Michael F. Roizen, Mehmet C. Oz. Methods and Systems for improving human health using targeted probiotics//Patent US20090287506A1].

The Karachay-Cherkess Republic is a region of the North Caucasus in the south of the European part of Russia. The majority of the population is made up of indigenous Karachais and Circassians, who largely continue to maintain traditional rural lifestyles and dietary habits. In particular, the Karachays continue to consume abundantly of traditional fermented milk drinks - ayran and kefir (“Gypy ayran”). Kefir is prepared on the basis of autochthonous kefir grains, consisting of yeast and lactic acid bacteria of the genus Lactobacillus [Khadzhieva, 2008]. Increasing consumer demand for more natural and healthy food, interest in prebiotics and probiotics are actively popularizing national fermented milk drinks. Innovation in this segment has favorable prospects [Batdyeva, 2019].

Studies of the intestinal microflora of residents of the Karachay-Cherkess Republic found significant differences in the composition of the species level of microflora compared to residents of European Russia. In particular, elevated levels of Butyricimonas were observed, as well as the presence of some rare species of Lactobacillus and Prevotella. Strains of Lactobacillus ruminis were discovered that were not previously considered characteristic of the human intestinal microflora. Seven bacterial strains represented new, previously unknown species of normal anaerobic intestinal bacteria, the genera Dialister, Butyricimonas, Collinsella, Prevotella and Ruminococcus [Katchieva et al., 2018].

Also, differences were found in the species composition of bifidobacteria between residents of the Karachay-Cherkess Republic and residents of Moscow. Although in both cases the most common species was Bifidobacterium longum, B. adolescentis strains were significantly more often isolated from residents of the Karachay-Cherkess Republic. Strains of B. catenulatum and B. angulatum were isolated only from residents of the Karachay-Cherkess Republic, while B. dentium, B. pseudocatenulatum, B. animalis and B. bifidum were isolated only from residents of Moscow. In addition, only in residents of the Karachay-Cherkess Republic, strains of bifidobacteria were identified, most likely belonging to “rare” species not identified using the MALDI-TOF method [Aibazova et al., 2019].

Disclosure of the invention

The objective of the present invention is to obtain a strain of microorganisms of the species Bifidobacterium longum 174 with high adhesive ability to the cells of residents of the Karachay-Cherkess Republic, expressed antagonistic activity against opportunistic bacteria, rapidly multiplying on artificial nutrient media.

Strain Bifidobacterium longum 174 was isolated from the intestinal contents of a healthy human resident of the Karachay-Cherkess Republic and deposited in the collection of microorganisms of the State Scientific Center for Applied Microbiology and Biotechnology "GKPM - OBOLENSK" under No. B-9674.

The inventive strain can be used in the production of dietary supplements, bacterial preparations, fermented and non-fermented food products, starter cultures, fermented milk products, etc. products containing bifidobacteria. In addition, the Bifidobacterium longum 174 strain can be used to enrich a traditional kefir drink (“Gypy ayran”) based on indigenous kefir grains. Strain Bifidobacterium longum 174 is antagonistically active against opportunistic microorganisms and has a high adhesive ability to the cells of residents of the Karachay-Cherkess Republic. This allows it to be used as a biologically active component in probiotic products for group-personalized prevention and treatment of gastrointestinal diseases in a specific ethno-geographical community - residents of the Karachay-Cherkess Republic.

For the initial selection, 18 strains of intestinal bifidobacteria isolated from residents of the Karachay-Cherkess Republic and identified as Bifidobacterium longum were used. This type is the most common among residents of the Karachay-Cherkess Republic. Isolation of bacteria: fresh feces of a clinically healthy person, after collecting anamnesis and carrying out additional examinations, were subjected to serial tenfold dilution in saline solution. Inoculation was carried out on Petri dishes with dense Bifidum-agar medium (Obolensk). The dishes were placed anaerobically in an anaerostat for 48 hours using AE-01 anaerostats and Anaerogas gas packages (NIKI MLT, Penza). After incubation, the grown colonies were counted, the morphology of the colonies grown on agar was studied, a bacterioscopic examination of typical colonies was carried out, and they were subcultured to isolate pure cultures. To identify the bacterium, we used matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (Matrix-Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry, MALDI-TOF MS) [Chaplin et al., 2015]. The strains were pre-prepared by mixing biomaterial on a mass spectrometer substrate from a bacterial colony and a special matrix (2',5' dihydroxybenzoic acid). Each sample was placed on a specific section of the plate. The sample was then placed into the instrument and exposed to nanosecond laser pulses. Based on the instrument software database (MALDI Biotyper 3.0 (Bruker Daltonics, Germany)), strain Bifidobacterium longum 174 was identified with 99.9% accuracy.

To identify strains of Bifidobacterium longum that are most characteristic of residents of the Karachay-Cherkess Republic, a genetic analysis was carried out using the VNTR method [Matamoros et al., 2011], which made it possible to identify several groups of genetically similar strains, i.e. having similar VNTR profiles. The most common profile was found in 8 strains.

As the most promising regional probiotic from the indicated 8 strains, the Bifidobacterium longum 174 strain was subsequently selected based on the highest ability to adhere to the erythrocytes of donors from the Karachay-Cherkess Republic, as well as a wide spectrum of antagonistic activity against a set of indicator opportunistic bacteria using the method of delayed antagonism.

Morphological properties. When cultivated on a dense medium, Bifidum agar under anaerobic conditions forms round colonies, opaque, shiny, with smooth edges, without pigment, the diameter is 2-3 mm; colonies have a slightly convex profile and can be easily removed from a dense medium with a loop. In a liquid nutrient medium under anaerobic conditions they grow along the entire height of the volume, forming a loose sediment. In smears, the cells appear randomly arranged, gram-positive, polymorphic, with Y-shaped forked ends (rarely), and club-shaped swellings. It does not form spores or capsules and is immobile. Catalase negative.

Cultural characteristics. It is an obligate anaerobe; grows on Petri dishes with a dense medium, in a high column of agar in a test tube, and in test tubes with a liquid medium. The optimal growing temperature is 37-38°C, under anaerobic conditions. The growth of the Bifidobacterium longum 174 strain when cultivated in liquid nutrient media MRS and Bifidum medium after incubation at 37°C for 18 hours anaerobically reached OD600 values of 5.17±1.02 and 3.57±0.38, respectively.

Physiological and biochemical properties of the strain. To study the enzymatic profile, test kits were used to study enzymatic activity (API 50 CHL system (bioMerieux)) assessed by changes in the color of the medium: when fermenting sugars, yellow coloring is positive, violet coloring is negative. A positive result was detected for lactose, xylose, glucose, sucrose, maltose, mannose, arabinose, raffinose, ribose, inulin, sorbitol, mannitol, and a negative result for rhamnose.

Study of antagonistic activity. To quantify the antagonistic activity of the strain, a modification of the delayed antagonism method was used [Muriana et al., 1987]. To study the antagonistic activity of Bifidobacterium longum 174 strain, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, and Klebsiella pneumonia strains were used as indicator strains. As a nutrient medium for bifidobacteria, we used agarized medium Bifidum agar (FBUN “SSC PMB”, Obolensk); For the cultivation of indicator culture strains, the GRM medium was used (FBUN “SSC PMB”, Obolensk). Dishes with grown colonies of bifidobacteria were overloaded with cover agar containing 0.05 ml of an overnight culture of the indicator strain, incubated at 37°C for 18 hours, and the diameters of zones of growth inhibition of indicator strains around the colonies of bifidobacteria were measured. When studying the antagonistic activity of the Bifidobacterium longum 174 strain, it was revealed that the strain actively suppresses the growth of a number of opportunistic bacteria in vitro (Table 1).

Table 1 Indicator culture Radius of growth inhibition zones around colonies of a bifidobacteria strain, (Average±SD ^* , mm) Staphylococcus aureus 19.9±2 Escherichia coli 26.5±1.5 Pseudomonas aeruginosa 20.5±2 Enterococcus faecalis 29.1±1.8 Klebsiella pneumonia 25.2±1 Shigella flexneri 29.5±1 * SD - standard deviation

Thus, the Bifidobacterium longum 174 strain actively suppresses the growth of a number of opportunistic bacteria in vitro.

Adhesive abilities of Bifidobacterium longum 174 on red blood cells of donors - residents of the Karachay-Cherkess Republic. To study adhesive activity, a test-tube / expanded method was used to determine the adhesion of microbes to erythrocytes - the Brilis method [Brilis, 1986]. To study the adhesive properties, daily cultures of strains grown on a slanted nutrient medium were used. A daily culture of strains was washed off with buffered physiological phosphate solution, then washed twice by centrifugation at 1500 rpm. within 10 minutes. in a similar 0.1% phosphate-buffered saline with pH 7.2. A suspension was prepared from a suspension of bacteria with a concentration of 1 billion tons/ml. Heparinized (0.03 ml of heparin (5000 units/1 ml) per 5 ml of blood) human erythrocytes were washed twice with 0.1% phosphate-buffered saline at 300 rpm. Blood was taken from the ulnar vein from donors - residents of the Karachay-Cherkess Republic who did not have a history of chronic inflammatory diseases and acute intestinal diseases, diseases of the cardiovascular and circulatory systems. Then a 1% suspension of red blood cells (concentration of about 100 ml cells/ml) was prepared. A suspension of erythrocyte bacteria was mixed in different volumes (0.5 ml each) in a test tube. The mixture was incubated at 37 degrees Celsius for 30 minutes with shaking. A smear was prepared on defatted glass, dried in air, fixed in alcohol for 10 minutes and stained with Gram. The number of microbial cells adhering to 50 erythrocytes was microscopically counted. Subsequently, the microbial adhesion index was calculated as the average number of microbial cells on one erythrocyte participating in the adhesive process:

IAM = (SPA x 100%) / K.

where: SPA - average adhesion index - average number of microorganisms attached to one red blood cell

K - % of red blood cells with adherent microorganisms on their surface

Microorganisms were considered:

- non-adhesive with IAM from 1.00 to 1.75;

- low-adhesive - from 1.76 to 2.49;

- medium adhesive - from 2.50 to 3.99;

- highly adhesive > 4.00.

The strain is highly adhesive - adhesion index 7.3±0.25. For control, we used the intestinal strain Bifidobacterium longum 44 [Chaplin, 2015], previously isolated from a resident of Moscow. The adhesion index was 3.49±0.14 for the Bifidobacterium longum 44 strain. Thus, the Bifidobacterium longum 174 strain has a higher adhesive ability to the cells of donors - residents of the Karachay-Cherkess Republic than the strain isolated from a resident of Moscow and used for control.

When studying the spectrum of sensitivity to antibiotics, it was found that the Bifidobacterium longum 174 strain is sensitive to benzylpenicillin, amoxicillin, oxacillin, cefazolin, ceftriaxone, erythromycin, clarithromycin, tetracycline, and resistant to gentamicin, ciprofloxacin.

Carrying out the invention

The use of the strain is illustrated by the following examples.

Example 1. Use of Bifidobacterium longum 174 strain to obtain bacterial preparations.

The culture of the Bifidobacterium longum 174 strain is added to the Bifidum medium nutrient medium and cultivated at a temperature of 37-38°C until a bacterial biomass is obtained with a content of living cells of at least 10 ⁹ per 1 ml (18-20 hours anaerobically). Protective drying media (sucrose-gelatin media) are added to the resulting biomass. This suspension is poured into penicillin vials, frozen at a temperature of at least -20°C and lyophilized. The resulting bacterial preparation contains, after repeated dilution with water to the previous volume, at least 10 ⁸ living cells of bifidobacteria.

Example 2. Enrichment of the traditional fermented milk drink “Gypy-ayran” based on indigenous kefir grains with the Bifidobacterium longum 174 strain.

Indigenous kefir grains are inoculated into pasteurized cow's milk at a temperature of 37°C at a rate of 5-10g. grains: 300 ml milk. Maintain at a temperature of 18-21°C for 18-24 hours, in a glass or wooden container, without a lid, stirring every three hours.

Cultures of Bifidobacterium longum 174 are grown and lyophilized as described above, in Lyophilized cultures are dissolved with sterile water to the original volume and added to a freshly prepared one-day kefir drink (“Gypy ayran”) until a concentration of live bacteria is reached in the amount of 10 ⁷ CFU/ml.

References

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Claims (2)

1. The bacterial strain Bifidobacterium longum 174, deposited in the collection of microorganisms of the State Scientific Center for Applied Microbiology and Biotechnology "GKPM - OBOLENSK" under N B-9674, which has pronounced adhesive and antagonistic properties, as a region-specific probiotic microorganism for residents of the Karachay-Cherkess Republic . 2. Use of the strain according to claim 1 for the enrichment of the traditional kefir drink “Gypy Ayran” based on indigenous kefir grains.