Method for increasing the antagonistic activity and survival rate of autoprobiotic strains

Abstract

FIELD: biotechnology.SUBSTANCE: method is proposed for increasing the antagonistic activity and survival rate of autoprobiotic strains, characterized by the isolation of bifido- and lactobacilli from the host's feces, their cultivation on nutrient media and use in the form of autostrains for the treatment of dysbacteriosis, while sowing the biomass for isolating the autostrains, reseeding and washing the autostrains for preparing the autoprobiotic are carried out in the period from 10 to 12 a.m. local time.EFFECT: reduction in the manifestation of dysbacteriosis symptoms.1 cl, 2 ex

Description

[0001] The invention relates to microbiology, biotechnology and medicine and can be used in research and production bacteriological laboratories for the purpose of obtaining an autoprobiotic containing live bifidobacteria and lactobacilli, with increased antagonistic activity and survival rate as a raw material for the production of internal and local (topical, external) therapeutic and prophylactic agents.

[0002] STATE OF THE ART

[0003] The antimicrobial (antagonistic) activity of bifidobacteria against various types of opportunistic and pathogenic microorganisms is known in the scientific literature. The antimicrobial activity of bifidobacteria and the products of their secondary metabolism is an important competitive factor preventing the invasion and persistence of pathogenic and opportunistic bacteria. A variety of nonspecific mechanisms of antagonistic action of bifidobacteria against gram-positive and gram-negative microflora have been described, such as the inhibitory effect of organic acid molecules causing a local decrease in pH in the intestinal biotope, competition for nutrients and adhesion sites; the production of specific antimicrobial substances (bacteriocin-like inhibitory substances, BLIS); stimulation of the host's innate and adaptive immunity.However, a significant problem is the decreased antagonistic activity of bifidobacteria strains that occurs with the use of bifidobacteria-containing probiotic preparations. Therefore, research is needed to identify not only new bifidobacteria strains but also ways to enhance the antimicrobial (antagonistic) activity of probiotic strains and their metabolites.

[0004] Currently, various methods are used to enhance the antimicrobial activity of probiotic strains. A known method for increasing the antimicrobial activity of probiotic microorganisms is based on the cultivation of antagonistically active strains of bifido- and lactobacilli using dietary fiber (grape pomace) as growth stimulants. The basis of this method is the introduction of an additional component into the nutrient medium - grape pomace, which enhances the antimicrobial properties of probiotic bacteria (Makarova N.V., Ignatova D.F. Eremeeva N.B. Use of grape processing waste as a source of a complex of biologically active substances / / VSUET Bulletin. 2020. Vol. 82. No. 4. pp. 207-212. doi: 10.20914 / 2310-1202- 2020-4-207-212). However, the introduction of the proposed component into the environment requires the preliminary production of grape pomace, which requires additional time and resource costs for the implementation of the method.Another disadvantage of this method is that grape pomace may have its own antimicrobial properties, including against strains of bifido- and lactobacilli.

[0005] A method for increasing the antagonistic activity of some antagonist strains (Enterococcus faecalis, Escherichia coli M-17, Bacillus subtilis) is known (Semenov A.V. Method for increasing the antagonistic activity of bacteria, OSU Bulletin No. 6, pp. 100-103). This method increases the antagonistic activity of antagonist bacteria by activating the antagonist strain with components of the studied S. aureus culture, towards which increased antagonism is achieved. Moreover, this method uses cell wall fragments and exometabolites of the indicator staphylococcus strain. The main disadvantage of this method is that it does not increase the antagonistic activity of probiotic strains of bifidobacteria. The method is characterized by high labor intensity, multi-stage nature and a long implementation time.

[0006] A method for producing and a composition for increasing the effectiveness of probiotic microorganisms is known (Patent RU 2740140 C1), which consists of cultivating bifidobacteria in a medium with a reduced concentration of a limiting energy carbohydrate substrate with its fractional supply, at a temperature of 36 to 38 ° C, with a pH of the culture liquid in the range from 5.0 to 7.0, and terminating the cultivation process when the suspension contains at least 2⋅10 9CFU of bifidobacteria per ml, with at least 70% of the cells agglomerating into microcolonies, is achieved by removing the culture medium and replacing it with buffered saline, mixing and adsorbing it onto activated carbon particles no larger than 100 µm in amounts ranging from 10 to 40%, adding a sucrose-gelatin protective medium to 100%, and lyophilizing. The main disadvantage of this method is that it is aimed at protecting probiotic microorganisms from stress and death under the extreme conditions of the stomach and upper intestine, or during long-term storage, and does not enhance the antimicrobial (antagonistic) activity of bifidobacteria strains. This method is also characterized by high labor intensity, multi-stage nature, and a long time.

[0007] An autoprobiotic is a drug created on an individual basis by cultivating strains, samples of which are taken directly from the patient.

[0008] Various approaches to cultivating autoprobiotic organisms are known from the prior art.

[0009] A method for preparing an autoprobiotic based on an anaerobic bacterial consortium (Patent RU 2734896 C2), which involves preparing a fecal extract by resuspending it in a phosphate buffer, separating the supernatant by centrifugation, followed by mixing the supernatant with a saline solution and filtration, adding the resulting filtrate to a nutrient medium containing: yeast extract in an amount of 4.5-5.5 g, sodium chloride - 2.0-3.0 g, glucose monohydrate - 2.5-7.5 g, thioglycolic acid - 0.2-0.3 g, pancreatic casein hydrolysate - 10.0-18.0 g, L-cysteine ​​- 0.4-0.5 g, granulated agar - 0.6-0.8 g, gelatin - 0.3-0.7 g, distilled water - 1 l, into which a suspension of feces in a physiological solution containing vitamin K and glucose is then added, followed by cultivation under anaerobic conditions at a temperature of 37°C for 3-6 days, centrifugation, resuspension of the sediment in a stabilizing solution,containing 10% sucrose and 1% gelatin, yielding the target product. However, this method does not take into account the circadian rhythms of the biological activity of autologous strains.

[0010] A known method for producing an autoprobiotic containing live bifidobacteria and lactobacilli for restoring the intestinal microecology of humans and animals (Patent RU 2505304 C2) involves isolating live bifidobacteria and lactobacilli from the host intestine and using them as automicroorganisms. The method is characterized in that the bifidobacteria and lactobacilli are simultaneously isolated as a natural complex of indigenous microorganisms by diluting the host's colon contents with a liquid medium supplemented with selective components that do not inhibit bifidobacteria and lactobacilli, adding a solution of diluted saliva from the same host, culturing the mixture, and then subculturing it onto a nutrient medium for culturing bifidobacteria and lactobacilli without the addition of selective agents. However, this method does not take into account the circadian rhythms of the biological activity of the autostrains.

[0011] A method for producing an autoprobiotic containing live bifidobacteria and lactobacilli is known (Patent RU 2139070 C1). The proposed method allows the obtained biomass of isolated autobacteria to be used as an autoprobiotic for restoring the intestinal microflora of humans and valuable animal and bird specimens.Autostrains of bifidobacteria and lactobacilli are isolated simultaneously as natural intestinal complexes from humans and / or animals by decontaminating the intestinal contents from extraneous microflora through repeated dilutions of the test material with a liquid nutrient medium containing selective agents suitable for the isolation and cultivation of bifidobacteria and lactobacilli, followed by removal of allochthonous microflora by adding blood serum from the same individual from whom the intestinal sample was collected to the biomaterial diluted to 10-6, followed by cultivation for 48 hours at 38°C, and subsequent subculture of the resulting biomass onto liquid nutrient media for culturing bifidobacteria and lactobacilli without the addition of selective agents. A significant drawback of this method is the use of blood serum from the test individuals for subsequent use in isolating the combined microbiocenosis of bifidobacteria and lactobacilli.It's common knowledge that many people experience severe stress during the procedure of having blood drawn from a vein. This very fact discourages them from isolating bacterial strains of the autobiotic preparation (or its creation) and, ultimately, from using it to correct imbalances in their own microflora. Furthermore, this method doesn't take into account the circadian rhythms of the autobiotic strains' biological activity.

[0012] The closest approach to the claimed invention is a method for producing an autoprobiotic containing live bifidobacteria and lactobacilli (Patent RU 2580002 C1). The method involves isolating bifidobacteria and lactobacilli from the host's feces. After isolating isolated colonies of bifidobacteria and lactobacilli, they are subcultured in liquid nutrient media and supplemented with 1⋅10 Bacillus bacteria biomass. 8 -1⋅10 10Live cells per 1.0 L of nutrient medium. The invention allows for the production of an autoprobiotic with enhanced physiological activity. However, this method does not take into account the circadian rhythms of the autoprobiotic strains' biological activity.

[0013] Technical task: development of a method for increasing the antagonistic activity and survival rate of an autoprobiotic containing live bifidobacteria and lactobacilli.

[0014] DISCLOSURE OF THE ESSENCE OF THE METHOD

[0015] The technical result of the method is to increase the antagonistic activity and survival rate of an autoprobiotic containing live bifidobacteria and lactobacilli.

[0016] The essence of the method

[0017] The patient submits the test material (stool) in a sterile container 72 hours prior to collection. They must not take laxatives, probiotics, or fermented milk products, and they must not be taking antibiotics. Delivery time for the sample is up to 2 hours from collection.

[0018] Next, cultivation is carried out on nutrient media, biomass is sown to isolate autostrains, autostrains are reseeded, and autostrains are washed off to prepare an autoprobiotic, which is carried out in the period from 10:00 to 12:00 local time.

[0019] The developed method for preparing an autoprobiotic based on bacteria was developed and studied in laboratory conditions at the Tyumen State Medical University.

[0020] The antagonistic activity of Bifidobacterium bifidum, Bifidobacterium longum and Lactobacillus acidophilus, Lactobacillus crispatus in relation to the growth and reproduction of pathogenic and opportunistic microorganisms, their persistent and pathogenic properties was studied using the chronobiological method; the maximum activity of the cultures was proven in the period from 10.00 to 12.00.

[0021] At the next stage, the efficacy of autoprobiotic cultures isolated from one patient was assessed. They were obtained using two methods: the method proposed by the authors (n=30) and the method (n=30) proposed by Kuznetsov O. Yu. et al., 2010, "A Method for Obtaining an Autoprobiotic Containing Live Bifidobacteria and Lactobacilli." It was proven that the autoprobiotic prepared using the proposed method at a specific time of day, taking into account the daily rhythms of biological activity of autostrains, contains pure cultures of one or more strains with maximum antagonistic activity in the morning and afternoon. Normalization of the patient's intestinal microbiota required 2-4 weeks less (p=0.05) than the autoprobiotic obtained using the method of Kuznetsov O. Yu.) and the survival rate was on average 15% higher (p=0.05).

[0022] Therefore, it is recommended to take one's own bifido- and lactobacilli, isolated from the intestines of an individual and propagated in laboratory conditions (autoflora), between 10:00 and 12:00 local time, since normobiota microbes take root more quickly in the intestines of a given patient.

[0023] EXAMPLES OF IMPLEMENTING THE METHOD

[0024] Example 1

[0025] Patient K., 32, with an intestinal microflora disorder, was prescribed autologous probiotic strains. The patient had not taken laxatives, probiotics, or fermented milk products for 72 hours prior to sampling, and had avoided antibiotic therapy.

[0026] The feces were transported in a sterile container; the delivery time of the material to the microbiological laboratory was 1 hour from the moment of collection.

[0027] To prepare a personal autoprobiotic, the biomass was diluted with saline solution by serial dilutions up to 10 10 CFU / ml.

[0028] Then at 10:00 local time, 0.1 ml of biomass was sown at a dilution of 10 2 CFU / ml; 10 3 CFU / ml; 10 4 CFU / ml were cultured on solid MRS medium, modification 1, and semi-liquid MRS medium, modification 2, to isolate Lactobacillus spp. and placed in a thermostat for 48 hours at 37°C. The grown colonies were examined microscopically and replated on MRS, modification 1, for further species identification.

[0029] After 48 hours at 10:00 local time, the biomass was sown at a dilution of 10 6 CFU / ml; 10 7 CFU / ml; 10 9 CFU / ml 0.1 ml and 10 10 1 ml of semi-liquid bifidum agar modification 2, placed in a thermostat at 37°C for 2 days to isolate Bifidumbacterium spp. The grown isolated colonies were transferred to dense Scheddler agar, placed in a thermostat for 48 hours for species identification of bacteria.

[0030] Species identification of Lactobacillus spp. and Bifidumbacterium spp. was carried out using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDITOF MS) based on protein profiles. According to the protein profile results, opportunistic and pathogenic bacteria were absent, and the number of bifidobacteria and lactobacilli was determined to be at least 10 9 CFU per 1 ml. An autoprobiotic was prepared and used to correct dysbiosis in the patient.

[0031] The finished product was given to the patient in the amount of 10.0 ml of a specific species or a mixture of species: Lactobacillus spp. (5.0 ml) and Bifidumbacterium spp. (5.0 ml) by rinsing with saline from MPC1 and Scheddler media. The rinsing was performed at 10:00 a.m.

[0032] The patient took his own bifido- and lactobacilli isolated from the intestines (autoflora) at 10 am local time for a month.

[0033] After one month, the effectiveness of the autoprobiotic cultures was assessed. The patient underwent repeat bacterioscopic examination. The sample predominantly yielded Lactobacillus spp. and Bifidumbacterium spp. The patient experienced a persistent reduction in dysbiosis symptoms.

[0034] Example 2

[0035] Patient A., 45, with an intestinal microflora disorder, was prescribed autologous probiotic strains. The patient had not taken laxatives, probiotics, or fermented milk products for 72 hours prior to sampling, and had avoided antibiotic therapy.

[0036] The feces were transported in a sterile container; the delivery time of the material to the microbiological laboratory was 1 hour from the moment of collection.

[0037] To prepare a personal autoprobiotic, the biomass was diluted with saline solution by serial dilutions up to 10 10 CFU / ml.

[0038] Then at 12:00 local time, 0.1 ml of biomass was sown at a dilution of 10 2CFU / ml; 10 3 CFU / ml; 10 4 CFU / ml were cultured on solid MRS medium, modification 1, and semi-liquid MRS medium, modification 2, to isolate Lactobacillus spp. and placed in a thermostat for 48 hours at 37°C. The grown colonies were examined microscopically and replated on MRS, modification 1, for further species identification.

[0039] After 48 hours at 12:00 local time, the biomass was sown at a dilution of 10 6 CFU / ml; 10 7 CFU / ml; 10 9 CFU / ml 0.1 ml and 10 10 1 ml of semi-liquid bifidum agar modification 2, placed in a thermostat at 37°C for 2 days to isolate Bifidumbacterium spp. The grown isolated colonies were transferred to dense Scheddler agar, placed in a thermostat for 48 hours for species identification of bacteria.

[0040] Species identification of Lactobacillus spp. and Bifidumbacterium spp. was carried out using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDITOF MS) based on protein profiles. According to the protein profile results, opportunistic and pathogenic bacteria were absent, and the number of bifidobacteria and lactobacilli was determined to be at least 10 9 CFU per 1 ml. An autoprobiotic was prepared and used to correct dysbiosis in the patient.

[0041] The finished product was given to the patient in the amount of 10.0 ml of a specific species or mixture of species: Lactobacillus spp. (5.0 ml) and Bifidumbacterium spp. (5.0 ml) by rinsing with saline from MPC1 and Scheddler media. The rinsing was performed at 12:00 p.m.

[0042] The patient took his own bifido- and lactobacilli isolated from the intestines (autoflora) at 12 o'clock local time for a month.

[0043] After one month, the effectiveness of the autoprobiotic cultures was assessed. The patient underwent repeat bacterioscopic examination. The sample predominantly yielded Lactobacillus spp. and Bifidumbacterium spp. The patient experienced a persistent reduction in dysbiosis symptoms.

Claims

A method for increasing the antagonistic activity and survival rate of autoprobiotic strains, characterized by isolating bifido- and lactobacilli from the host's feces, culturing them on nutrient media, and using them as autostrains for treating dysbacteriosis, characterized in that the sowing of biomass for isolating autostrains, reseeding of autostrains, and washing of autostrains for preparing an autoprobiotic are carried out between 10:00 and 12:00 local time.