Butyric acid producing strain and application thereof

Abstract

The invention relates to the field of microorganisms, in particular to a butyric acid producing strain, which is identified as a new strain under the genus of Hispiraceae (Lacnospiraceae) CAG-81, has the preservation number of CGMCC NO.41195, has no toxicity-related genes and drug-resistant genes, is simple in high-density culture method, has high butyric acid yield capacity, has important significance for establishing a high-quality butyric acid producing strain resource library, and can be used for preparing a high-quality butyric acid producing strain. The strain is hopeful to become fermented butyric acid, preparation of products for regulating intestinal micro-ecology, and beneficial bacterium reserve for food fermentation.

Description

Technical Field

[0001] The present application belongs to the field of microorganisms, and specifically relates to a high-yielding butyrate bacterium and its application. Background Art

[0002] Short-chain fatty acids (SCFAs) are saturated fatty acids with 1 to 6 carbon atoms in the carbon chain. Among them, acetic acid, propionic acid and butyric acid are the main SCFAs, accounting for more than 95% of all SCFAs. SCFAs are the most important metabolites of intestinal flora and play an important role in maintaining the homeostasis of the intestinal environment. They can participate in maintaining the energy balance of the host body by regulating various signal transduction processes and are closely related to the occurrence and development of various immune and inflammatory diseases. Butyric acid has many important functions and is an important energy substance for intestinal epithelial cells. It plays a key role in regulating host metabolism, immune system and cell proliferation. It reduces oxidative stress and inflammation by controlling pathogens and reduces cholesterol synthesis.

[0003] Butyrate-producing bacteria are an important group of bacteria that can ferment carbohydrates to produce butyrate. As a new generation of probiotics, they have become a frontier and hot topic in intestinal probiotic research in recent years. The species and functional diversity of butyrate-producing bacteria is very high, but only a small part of the butyrate-producing strains have been isolated and studied. There are still a large number of unknown new butyrate-producing strains to be isolated and identified, and there is an urgent need to develop more butyrate-producing strains. Summary of the invention

[0004] The present invention first provides a butyric acid-producing bacterium, which is screened and separated from the feces of healthy male people. After artificial cultivation and fermentation and detection of the butyric acid content in the fermentation liquid, the butyric acid production can reach 716.54 mg / L. Therefore, it is named as butyric acid-producing bacteria from a functional point of view and is numbered as MF06.

[0005] The butyric acid-producing bacteria MF06 described in this application was deposited in the China General Microbiological Culture Collection Center (CGMCC) on June 24, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The name is butyric acid-producing bacteria MF06 and the deposit number is CGMCC NO.41195.

[0006] Secondly, the present application also provides the application of butyric acid producing bacteria MF06 in fermentation to produce butyric acid.

[0007] Existing technologies show that short chain fatty acids (SCFAs) are a class of saturated fatty acids with 1 to 6 carbon atoms in their structure, mainly including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid and isocaproic acid. Among them, acetic acid, propionic acid and butyric acid have the highest total content and are the three main SCFAs produced in the intestine, accounting for about 90% to 95% of SCFAs. They are the end products of indigestible carbohydrates fermented by cecal and colonic microbial flora. Acetate is produced by a variety of bacteria, including lactobacilli, bifidobacteria, rumenococci, Prevotella, Bacteroides, Clostridium, Streptococcus, etc. Butyrate is produced by some species of Ruminococcaceae, Lactobacillaceae, Eubacteraceae, Erysipelothrixaceae and Clostridiaceae. Butyric acid can help reduce food intake by promoting the production of satiety hormone peptide YY (peptide YY, PYY); butyric acid is also an energy source for colon cells and participates in the repair of intestinal barrier function; butyric acid can also reduce the expression and secretion of inflammatory response factors, and inhibit the activation of the host B cell nuclear factor κ light chain enhancer signaling pathway by binding to G protein coupled receptor 41 (G protein coupled receptor 41, GPCR41) and G protein coupled receptor 43 (G protein coupled receptor 43, GPCR43), thereby reducing the release of proinflammatory cytokines and playing an anti-inflammatory role. Acetic acid, propionic acid and butyric acid can promote lipid oxidation metabolism by regulating the expression of PPARγ in mouse liver and adipose tissue. Therefore, MF06 isolated in this application can be used as a beneficial intestinal bacterium for the preparation of microbial preparations.

[0008] According to the common understanding in the art, all preparations that can promote the growth and reproduction of normal microorganisms and inhibit the growth and reproduction of pathogenic bacteria are called "microecological preparations". In view of the fact that MF06 has the ability to produce high concentrations of acetic acid and butyric acid, and has the safety of virulence gene detection and drug resistance gene detection, microecological preparations made from butyrate-producing bacteria MF06 and / or its metabolites can be used. In combination with the function of SCFAs, it is expected that the microecological preparations made from MF06 will have the effect of regulating the intestine, restoring the balance of intestinal microecology, and promoting fat metabolism.

[0009] Those skilled in the art can prepare microecological preparations using the MF06 of the present application in combination with the prior art. For example, the method for preparing microecological preparations using the MF06 of the present application includes the following steps: 1. Activation of MF06 bacteria Use an inoculation loop to pick up the strain MF06 stored in -80℃, inoculate it into RCM liquid culture medium, and culture it anaerobically at 37℃ for 3 days to activate the strain. Then inoculate the activated strain into RCM liquid culture medium, culture it anaerobically at 37℃ for 3 days, and collect MF06 bacteria by centrifugation.

[0010] 2. Preparation of MF06 bacterial microecological preparation After obtaining MF06 bacterial bodies by centrifugation, they are compounded with freeze-drying protective agents and vacuum freeze-dried to obtain live bacteria freeze-dried powder, which is then compounded alone or with other probiotics to prepare a composite probiotic agent.

[0011] The freeze-drying protective agent formula is: oligofructose 100 mg / g, skim milk powder 250 mg / g, galactose 90 mg / g, lactose 60 mg / g, proline 50 mg / g, and water-soluble trehalose 60 mg / g.

[0012] The vacuum freeze-drying conditions were as follows: bacterial sludge ratio 30 g / 100 g, pre-freezing temperature -45 °C, pre-freezing time 4 h, freeze-dried thickness 1.0 cm, and vacuum degree 0.2 mbar.

[0013] The content of live bacteria in the preparation prepared by MF06 is not less than 10 8 CFU / g, adult dosage is 2-4g per day.

[0014] Although the safety of probiotics is an important issue in the evaluation of probiotics, the content involved is mainly the drug resistance and potential pathogenicity of the strain. MF06 bacteria has not yet obtained a food use license, but the reference to the current technology and method for the safety assessment of probiotics at home and abroad mainly includes virulence-related gene analysis and drug resistance gene analysis based on genomics, the detection of potential toxic substances by high performance liquid chromatography, the use of minimum inhibitory concentration for drug resistance detection and animal and human clinical trials, etc. Genomic analysis can obtain the information of functional genes such as virulence-related genes and drug resistance genes of probiotic strains, which is generally used as the first step in the safety assessment of strains to identify the drug resistance and potential pathogenicity information of probiotics. The present application MF06 does not have independent related genes and drug resistance genes after testing, and is separated from healthy human bodies, and produces high-yield acetic acid and butyric acid, so MF06 has the effect of regulating intestinal flora, stabilizing gastrointestinal function, and improving immunity, and the possibility of passing the safety evaluation in the future is extremely high, so the probability of becoming a beneficial bacteria that can be used by the human body is extremely high.

[0015] The present application also provides an application in food, containing the above-mentioned butyric acid-producing bacteria MF06 and / or its metabolites as active ingredients, and the active ingredients are not limited to the currently clear active ingredients such as acetic acid and butyric acid. The application of butyric acid-producing bacteria MF06 in food mainly refers to its addition to food as a beneficial bacteria; the application of butyric acid-producing bacteria MF06 metabolites in food mainly refers to the addition of butyric acid produced by MF06 metabolism in food, which plays a role in eliminating intestinal inflammation and improving intestinal flora.

[0016] The present application also provides the use of MF06 in drug preparation. The use of MF06 in drugs described in the present application refers to using MF06 and / or its metabolites as active ingredients of drugs, and the content of live bacteria in the preparation prepared by MF06 is not less than 10 8 CFU / g, the daily dosage for adults is 2-4g. The purpose of making drugs from MF06 and / or its metabolites is not limited to the currently known weight loss, auxiliary treatment of colorectal cancer, relief of irritable bowel syndrome, treatment of Crohn's disease, combating insulin resistance, anti-inflammatory, etc. based on its metabolites acetic acid and butyric acid, or as auxiliary drugs for the above treatment purposes.

[0017] In the above application, the method for obtaining MF06 or its metabolites includes: inoculating the activated butyric acid-producing bacteria MF06 into RCM liquid culture medium for anaerobic fermentation. Wherein, the RCM liquid culture medium mainly includes: peptone 10g / L, beef powder 10g / L, yeast powder 3g / L, glucose 5g / L, soluble starch 1g / L, sodium chloride 5g / L, sodium acetate 3g / L, L-cysteine ​​hydrochloride 0.5g / L, pH value 6.8±0.1; wherein, the anaerobic fermentation conditions are: anaerobic fermentation at 37°C for 72h; wherein, the inoculation amount can be selected by the technicians in this field within the range of 1 to 10%vol of the conventional amount.

[0018] Beneficial effects: 1. The present invention isolates and screens a butyrate-producing bacterial strain MF06 from a healthy male fecal sample, and the deposit number is CGMCC NO.41195. The strain MF06 has a simple culture method and a high butyrate-producing ability, which is of great significance for establishing a high-quality butyrate-producing bacterial strain resource library. At present, the butyrate-producing ability of MF06 is 716.54 mg / L. As a new strain, the butyrate-producing ability will be higher through strain domestication and culture optimization.

[0019] 2. It has been detected that MF06 not only produces high levels of butyrate, but also high levels of acetic acid, and no virulence-related genes or drug-resistance genes have been detected, that is, the safety evaluation is safe, and it is isolated from healthy humans. Therefore, it has great potential for use in the preparation of drugs for weight loss, adjuvant treatment of colorectal cancer, relief of irritable bowel syndrome, treatment of Crohn's disease, combating insulin resistance, anti-inflammatory, or as an auxiliary drug for the above treatment purposes.

[0020] Description of the drawings Figure 1 This is the colony morphology of MF06 cultured for 3 days.

[0021] Figure 2 This is the fastani alignment result of MF06.

[0022] Figure 3 This is the butyric acid gas chromatography-mass spectrometry detection chart of MF06. DETAILED DESCRIPTION

[0023] The specific embodiments of this application are illustrated by cases in the experimental process, rather than the experimental process of the creative work of this application, and it does not mean that this is the entire work of this application.

[0024] The culture medium formula involved in the embodiment is: PBS buffer: NaCl 8 g / L, KCl 0.2 g / L, Na2HPO4 1.42 g / L, KH2PO4 0.24 g / L, pH 7.4±0.1.

[0025] MWC liquid medium: tryptone 10 g / L, gelatin peptone 10 g / L, yeast extract 5 g / L, glucose 1 g / L, NaCl 5 g / L, arginine 1 g / L, sodium pyruvate 1 g / L, Tween 80 1 mL / L, glacial acetic acid 1 mL / L, hemin 5 mg / L, mupirocin 0.1 g / L, L-cysteine ​​hydrochloride 0.5 g / L, pH 5.6 ± 0.1. MWC solid medium: add 1.8% (w / v) agar to MWC liquid medium and pour in an anaerobic workstation.

[0026] MWC is a separation medium. The use of mupirocin and L-cysteine ​​hydrochloride in the medium is beneficial to the separation of Gram-positive bacilli and anaerobic bacteria, that is, it is beneficial to the separation of butyrate-producing bacteria. Mupirocin inhibits Gram-positive cocci and some Gram-negative bacteria.

[0027] RCM liquid medium: peptone 10 g / L, beef powder 10 g / L, yeast powder 3 g / L, glucose 5 g / L, soluble starch 1 g / L, sodium chloride 5 g / L, sodium acetate 3 g / L, L-cysteine ​​hydrochloride 0.5 g / L, pH 6.8±0.1. RCM solid medium: add 1.8% (w / v) agar to RCM liquid medium and pour in an anaerobic workstation. Example

[0028] Experimental Example 1 Screening and Identification of Butyric Acid Producing Bacterial Strain MF06 MF06 was confirmed to be a Firmicutes, Clostridium, and Lachnospiraceae of the Eubacteriales through isolation, primary screening, 16SrRNA, and whole genome sequencing. It has not been classified into a specific species, but was named butyrate-producing bacteria MF06 based on its function of producing butyrate. The preservation agency recommended the classification name of Lachnospiraceae sp.

[0029] 1. Isolation and culture of butyrate-producing bacterial strain MF06 (1) Sample collection: The isolated sample came from a healthy male stool sample in Beijing. The strain was isolated at the Institute of Microbiology, Chinese Academy of Sciences.

[0030] (2) Isolation and purification of strains: The isolation process is strictly anaerobic. In the anaerobic workstation, 0.5 g of sample was suspended in sterilized and deoxygenated PBS buffer, mixed thoroughly, and then gradient diluted. 10 5 , 10 6 , 10 7 100 μL of the gradient diluted suspension was spread on MWC plates and cultured at 37°C for 3 days. Single colonies were picked for streaking purification. The isolated pure culture strains were preserved, preliminarily screened for but gene (acetyl-CoA transferase gene) and identified for 16S rRNA gene.

[0031] 2. Initial screening of butyrate-producing strain MF06 (1) Genome extraction: Cultivate the isolated strain using the same culture medium and culture conditions as used for isolation until the strain concentration reaches 10 8 When the number of bacterial cultures was at the cfu / mL level, 2 mL of bacterial culture was taken for genomic DNA extraction.

[0032] (2) PCR amplification of the but gene of the strain: Use primers BCoATscr-F (5'-GCNGANCATTTCACNTGGAAYWSNTGGCAYATG-3') and BCoATscr-R (5'-CCTGCCTTTGCAATRTCNACRAANGC-3') to amplify the but gene using DNA as a template.

[0033] The PCR amplification program was: 95°C for 5 min; 95°C for 30 s, 50°C for 30 s, and 72°C for 60 s, 35 cycles; 72°C for 5 min.

[0034] (3) Electrophoresis detection: The PCR products were subjected to 2% (w / v) agarose electrophoresis. The band position of the but gene was approximately 500 bp, and the butyrate-producing bacteria strain MF06 was preliminarily obtained.

[0035] 3. Identification of the 16S rRNA gene of butyrate-producing strain MF06 (1) Genomic DNA extraction: Cultivate the isolated strain using the same culture medium and culture conditions as used for isolation until the strain concentration reaches 10 8 When the number of bacterial cultures was at the cfu / mL level, 2 mL of bacterial culture was taken for genomic DNA extraction.

[0036] (2) PCR amplification of the 16S rRNA gene of the strain: The universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGYTACCTTGTTACGACTT-3') were used to amplify the 16S rRNA gene using DNA as a template.

[0037] The PCR amplification program was: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, and 72°C for 90 s, 30 cycles; 72°C for 5 min.

[0038] (3) Purification and sequencing: The PCR product was subjected to 1.5% (w / v) agarose electrophoresis. The band position of the 16S rRNA gene amplification product was approximately 1500 bp. The purified product was then sequenced.

[0039] (4) Strain 16S rRNA gene sequence comparison: The obtained sequences were compared by BLAST in the NCBI database. The results showed that MF06 had the highest similarity with the butyrate-producing bacterium GM2 / 1 of the Firmicutes, Clostridium, and Eubacteriales, at 99.42%. The internationally recommended threshold for 16S rRNA gene sequence similarity for bacterial species classification is 98.7%. This result supports that strain MF06 is a butyrate-producing bacterium.

[0040] Among them, the 16S rRNA gene sequence of strain MF06 is: AGGTAAATAAGATGAAGTTTTCGGATGGATTTTTATTTACCGAGTGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTCATACAGGGGGATAACGGTTAGAAATGACTGCTAATACCGCATAAGCGCACAGTACCGCATGGTACGGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGATTAGCTAGTTGGTGAGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGACCTGAGAGGGTGACCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGGATATTGCACAATGGAGGAAACTCTGATGCAGCGACGCCGCGTGAGTGAAGAAGTATTTCGGTATGTAAAGCTCTATCAGCAGGGAAGAAAATGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGCGACGCAAGTCTGAAGTGAAATACCCGGGCTCAACCTGGGAACTGCTTTGGAAACTGTGTTGCTAGAGTGCTGGAGAGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAAGAACACCAGTGGCGAAGGCGGCTTACTGGACAGTAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAA 4. Whole-genome identification of butyric acid-producing bacterium strain MF06 Because the 16S rRNA gene sequence comparison failed to identify the MF06 strain to the species and genus, the whole genome sequence was uploaded to JSpecies (https: / / jspecies.ribohost.com / jspeciesws / ) for average nucleotide similarity (cANI) analysis. MF06 had the highest similarity with butyrate-producing bacterium SS3 / 4 of Firmicutes, Clostridia, and Eubacteriales, which was 99.32%. The internationally recommended threshold for genome ANI classification of bacterial species is 95-96%, and this result supports that strain MF06 is a butyrate-producing bacterium.

[0041] Further analysis by fastani yields the following results: Figure 2 The results showed that the similarity between MF06 and f__Lachnospiraceae;g__CAG-81;s__CAG-81 sp900066055 was 98.3%, so the strain was named CAG-81 MF06.

[0042] It is well known to those skilled in the art that through statistical analysis of thousands of genomes and 16S rRNA gene sequences, it was found that when the similarity between the 16S rRNA gene sequences of two strains is less than ≈ 98.65%, it can be judged that they belong to different species; when it is higher than ≈ 98.65%, they may belong to the same species or different species. At this time, it is necessary to calculate the average nucleotide identity (ANI) of the whole genome based on its genome sequence to obtain accurate species identification results. When the ANI value of the genome of the two strains is 95-96%, it is equivalent to a DNA-DNA hybridization value of 70%. Therefore, when the ANI value of the genome of the two strains is greater than 96%, they are identified as the same species; when it is less than 95%, they are identified as different species. Therefore, through the identification of the 16S rRNA gene and genome of MF06, it was finally determined that strain MF06 was a butyrate-producing bacterium, a new bacterium under the genus CAG-81 of the Lachnospiraceae family, and was deposited in the China General Microbiological Culture Collection Center with the preservation number CGMCC NO.41195.

[0043] 5. Microbiological characterization of butyrate-producing strain MF06 Morphological characteristics: The isolated pure strain MF06 was cultured on RCM solid medium at 37°C in an anaerobic environment for 3 days. The colonies were yellow-white, round, opaque, and convex, with a diameter of about 2 mm ( Figure 1 ).

[0044] Microscopic characteristics: Under 10,000x microscope magnification, the bacteria of MF06 are rod-shaped, Gram-positive, with a diameter of about 1 μm and a length of 2~4 μm.

[0045] Test Example 2 Safety Evaluation of Butyrate-Producing Bacterial Strain MF06 1. Virulence-related gene analysis Using DIAMOND software, the amino acid sequence of each genome was compared with the Virulence Factor Database (VFDB) to analyze virulence-related genes. The protein sequence similarity ≥ 95% and coverage ≥ 95% were selected as positive results.

[0046] Analysis results: No virulence-related genes were detected.

[0047] 2. Analysis of drug resistance genes The genome was analyzed for resistance genes using the resistance gene sequences and resistance gene identification tool RGI v5.2.0 provided by the Comprehensive Antibiotic Resistance Database (CARD) according to default parameters. Mobile resistance genes were analyzed using the software ResFinder v4.0 according to default parameters. Alignment results with protein sequence similarity ≥95% and coverage ≥95% were screened as positive results.

[0048] Analysis results: No drug-resistant genes were detected.

[0049] In summary, referring to the current technologies and methods for safety assessment of probiotics at home and abroad, the functional genes of MF06 such as virulence-related genes and drug-resistant genes were detected, and it was developed as a beneficial bacteria for food and medicine. It was preliminarily determined to be safe and isolated from a healthy human body. Therefore, the inventors infer that its application in microbial preparations, functional foods, and medicines is highly likely to be safe and effective.

[0050] Example 1 Application of butyrate-producing bacterial strain MF06 to produce butyrate in culture medium The strain MF06 stored in -80°C was picked up with an inoculation loop and inoculated into RCM liquid culture medium. The strain was activated by anaerobically culturing at 37°C for 3 days. The activated strain was then inoculated into RCM liquid culture medium and anaerobically cultured at 37°C for 3 days. The fermentation broth was taken for butyric acid detection.

[0051] Quantitative analysis of butyric acid: Using 2-methylvaleric acid as the internal standard, a standard curve was prepared using the concentrations and relative peak areas of a series of butyric acid standards to calculate the butyric acid content in the sample.

[0052] The specific operations are as follows: (1) Pretreatment: Take 0.1 mL of fermentation broth, add 0.05 mL of 15% phosphoric acid, and then add 0.2 mL of extract (containing internal standard 2-methylpentanoic acid, 25 mg / L, methyl tert-butyl ether), vortex for 30 seconds, shake for 10 minutes, ultrasonicate for 10 minutes (ice water bath), centrifuge at 10,000 rpm at 4°C for 10 minutes, let stand at -20°C for 30 minutes, and take the supernatant for testing.

[0053] (2) On-machine detection: A Shimadzu GC2030-QP2020 NX gas chromatograph-mass spectrometer was used, and the chromatographic column was an Agilent HP-FFAP capillary column (30m×250μm×0.25μm, J&W Scientific, Folsom, CA, USA); split injection, injection volume 1 μL, split ratio 5:1. Inlet temperature 220℃; ion source temperature 240℃; transfer line temperature 240℃. The starting temperature was 50℃, maintained for 1 min; the temperature was increased to 150℃ at 50℃ / min and maintained for 1 min; then the temperature was increased to 170℃ at 10℃ / min; the temperature was increased to 225℃ at 25℃ / min and maintained for 1 min; finally, the temperature was increased to 240℃ at 40℃ / min and maintained for 1 min. The carrier gas (Helium) was used at a carrier gas flow rate of 1.2 mL / min. The ionization mode was EI (electron ionization), and the electron energy was 70 eV.

[0054] (3) Test results such as Figure 3 The results show that the butyric acid content in the fermentation broth is 716.54 mg / L. It is well known to those skilled in the art that after domestication or cultivation optimization of such a high-yield strain, the butyric acid production capacity will be higher.

[0055] Existing technology ("Screening of Clostridium butyricum for Alleviating Obesity and Research on Its Mechanism of Action", Master's Thesis of Jiangnan University in 2023, author Liao Jingyi, majoring in Food Industry and Engineering) shows that the relationship between intestinal flora and obesity is constantly being revealed and is identified as one of the environmental factors that cause the onset of obesity. Compared with individuals with normal weight, obese individuals have significant differences in the diversity and composition of intestinal flora, and the abundance of butyric acid-producing Clostridium in the intestinal flora of obese individuals is significantly reduced. Clostridium butyricum is one of the clostridia that produce butyrate in the intestine, which can play a prebiotic role by regulating the functions of tissues such as the intestine, adipose tissue, liver and brain. Studies have shown that the abundance of Clostridium butyricum shows a downward trend in patients with type 2 diabetes, and an increase in its abundance is associated with a reduced susceptibility to obesity. In addition, supplementation with Clostridium butyricum can alleviate obesity-related phenotypes.

[0056] Similar to the above-mentioned prior art, the prior art "Research Progress on the Regulatory Mechanism of Short-chain Fatty Acids on Obesity" (author Xue Yili et al.), published in the June 2024 issue of Volume 25 of the Chinese Journal of Preventive Medicine, reviewed the mechanism of short-chain fatty acids in alleviating obesity. The existing research results show that: SCFAs bind to GPCR43 and GPCR41 to trigger cell-specific cascade signals, promote the secretion of intestinal hormones, and can also participate in gene expression regulation, intestinal barrier function regulation, immune regulation, oxidative stress, cell differentiation, etc. by inhibiting histone deacetylase (HDAC). SCFAs also enhance triglyceride hydrolysis and free fatty acid oxidation in adipose tissue by affecting the composition of intestinal flora, promoting beige fat production and mitochondrial biogenesis.

[0057] GPCR41 and GPCR43 can be effectively activated by acetate, propionate, butyrate and other SCFAs, and their affinity varies from species to species. Longer butyrate is more selective for GPR41, shorter acetate is more selective for GPR43, and propionate binds to both receptors. Propionate activates GPCR41 and promotes the secretion of gastrointestinal hormones GLP-1 and PYY. GLP-1 can promote insulin secretion, and PYY is involved in appetite regulation. GLP-1 and PYY work together to promote the proliferation and differentiation of pancreatic β cells, improve the sensitivity of pancreatic cells to glucose and insulin resistance, and play an important role in regulating glucose homeostasis. Butyrate can directly induce gluconeogenesis in intestinal cells, and propionate activates GPCR41 in the periportal afferent nervous system to induce gluconeogenesis. Increased gluconeogenesis in intestinal epithelial cells leads to decreased hepatic glucose production and improved energy homeostasis.

[0058] Therefore, combining the prior art and the detection results of MF06 metabolites in this application, those skilled in the art can expect that MF06 in this application has the effects of regulating intestinal flora, stabilizing gastrointestinal function, improving immunity, reducing insulin resistance, and reducing fat.

[0059] Example 2 Application of MF06 in the preparation of microecological preparations (1) Activation of MF06 bacteria Use an inoculation loop to pick up the strain MF06 stored in -80℃, inoculate it into RCM liquid culture medium, and culture it anaerobically at 37℃ for 3 days to activate the strain. Then inoculate the activated strain into RCM liquid culture medium, culture it anaerobically at 37℃ for 3 days, and collect MF06 bacteria by centrifugation.

[0060] (2) Preparation of MF06 bacterial microecological preparation After obtaining MF06 bacterial bodies by centrifugation, they are compounded with freeze-drying protective agents and vacuum freeze-dried to obtain live bacteria freeze-dried powder, which is then compounded alone or with other probiotics to prepare a composite probiotic agent.

[0061] The freeze-drying protective agent formula is: oligofructose 100 mg / g, skim milk powder 250 mg / g, galactose 90 mg / g, lactose 60 mg / g, proline 50 mg / g, and water-soluble trehalose 60 mg / g.

[0062] The vacuum freeze-drying conditions were as follows: bacterial sludge ratio 30 g / 100 g, pre-freezing temperature -45 °C, pre-freezing time 4 h, freeze-dried thickness 1.0 cm, and vacuum degree 0.2 mbar.

[0063] The content of live bacteria in the preparation prepared by MF06 is not less than 10 8 CFU / g, adult dosage is 2-4g per day.

[0064] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A butyric acid producing bacterium, characterized in that The deposit number is CGMCC NO.41195.

2. The method for culturing butyrate-producing bacteria according to claim 1, characterized in that: The butyrate-producing bacteria are inoculated into RCM medium for anaerobic culture, and the inoculation concentration is 1-10% vol.

3. Use of the butyric acid-producing bacteria according to claim 1 in producing butyric acid.

4. Use of the butyric acid producing bacteria and / or its metabolites according to claim 1 in the prepared microecological preparation.

5. Use of the butyric acid producing bacteria and / or its metabolites according to claim 1 in food.

6. Use of the butyrate-producing bacteria and / or its metabolites according to claim 1 in the preparation of drugs for regulating intestinal flora, lowering blood sugar, reducing fat, and adjuvant treatment of irritable bowel syndrome, enteritis, Crohn's disease, and colorectal cancer.

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