Butyric acid producing bacteria and application thereof

Abstract

The application relates to the field of microorganisms, in particular to a butyric acid producing bacterium, which is identified as a new bacterial species under the genus CAG-81 of Lachnospiraceae, has a preservation number of CGMCC NO.41195, is free of virulence related genes and drug resistance genes, has a simple high-density culture method, has a high butyric acid production capacity, is of great significance for establishing a high-quality butyric acid producing bacterial strain resource library, and is expected to become a beneficial bacterium reserve for fermenting butyric acid and preparing a regulation intestinal microecological product.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms, and particularly relates to a butyric acid-producing bacterium and application thereof. BACKGROUND

[0002] Short-chain fatty acids (SCFAs) are saturated fatty acids with 1-6 carbon atoms in the carbon chain, among which acetic acid, propionic acid and butyric acid are the most important SCFAs, accounting for more than 95% of all SCFAs. SCFAs are the main metabolic products 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, is an important energy substance for intestinal epithelial cells, and plays a key role in regulating host metabolism, the immune system and cell proliferation, etc. It reduces oxidative stress and inflammation by controlling pathogens, and reduces cholesterol synthesis.

[0003] Butyric acid-producing bacteria are an important group of bacteria that can ferment carbohydrates to produce butyric acid. As a new generation of probiotics, they have become a frontier and hotspot in recent years in the study of intestinal probiotics. Butyric acid-producing bacteria have high species and functional diversity, but only a small part of the butyric acid-producing strains have been isolated and studied. There are still a large number of unknown new butyric acid-producing strains to be isolated and identified, and more butyric acid-producing strains need to be developed. SUMMARY

[0004] The present application provides a butyric acid-producing bacterium, which is isolated from healthy male feces. The butyric acid-producing bacterium is cultured artificially and fermented, and the butyric acid content in the fermentation broth is detected. The butyric acid yield can reach 716.54 mg / L. Therefore, from the perspective of functional science, it is named butyric acid-producing bacterium, and is numbered as MF06.

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

[0006] The present application also provides the application of the butyric acid-producing bacterium MF06 in the fermentation of butyric acid.

[0007] The prior art shows that short chain fatty acids (SCFAs) are a class of saturated fatty acids containing 1-6 carbon atoms in structure, mainly including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid and isocaproic acid, among which the total content of acetic acid, propionic acid and butyric acid is the highest, and they are the three main SCFAs produced in the intestinal tract, accounting for about 90%-95% of SCFAs, and they are the end products of fermentation of indigestible carbohydrates by cecal and colonic microbial flora. Acetate is produced by a variety of bacteria, including Lactobacillus, Bifidobacterium, Streptococcus, Prevotella, Bacteroides, Clostridium, Streptococcus, etc. Butyrate is produced by some species of Streptococcus, Lactobacillus, Eubacterium, Erysipelotrichaceae and Clostridium. Butyrate can help reduce food intake by promoting the production of satiety hormone peptide YY (PYY); butyrate is also an energy source for colon cells and participates in the repair of intestinal barrier function; butyrate can also reduce the expression and secretion of inflammatory response factors, inhibit the activation of host B-cell nuclear factor kappa light chain enhancer signaling pathway by binding to G protein coupled receptor 41 (GPCR41) and G protein coupled receptor 43 (GPCR43), and reduce the release of pro-inflammatory cytokines, thereby playing an anti-inflammatory role. Acetic acid, propionic acid and butyric acid can promote lipid oxidative metabolism by regulating the expression of PPARγ in mouse liver and adipose tissue. Therefore, the MF06 isolated in the present application can be used as a beneficial intestinal bacterium to prepare a microbial preparation.

[0008] According to the general understanding in the art, all preparations that can promote the growth and reproduction of normal microflora and inhibit the growth and reproduction of pathogenic bacteria are called "microecological preparations". Since 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, the microecological preparation made of butyric acid producing bacteria MF06 and / or its metabolites can be combined with the functions of SCFAs, and the microecological preparation made of MF06 is expected to have the functions of regulating the intestinal tract, restoring the intestinal microecological balance, promoting fat metabolism, etc.

[0009] Those skilled in the art can prepare a microecological preparation of MF06 in combination with the prior art, for example, the method for preparing a microecological preparation of MF06 of the present application comprises the following steps:

[0010] 1. Activation of MF06 bacterial cells

[0011] The strain MF06 stored at -80℃ was inoculated into RCM liquid medium, and cultured at 37℃ for 3 days under anaerobic condition for activation. Then the activated strain was inoculated into RCM liquid medium, and cultured at 37℃ for 3 days under anaerobic condition. The MF06 strain was collected by centrifugation.

[0012] 2. Preparation of MF06 strain microecological preparation

[0013] After centrifugation to obtain the MF06 strain, the strain was mixed with a freeze-drying protective agent, and vacuum freeze-dried to obtain a freeze-dried powder of the live strain. The powder was used alone or in combination with other probiotics to prepare a compound probiotic agent.

[0014] The freeze-drying protective agent had the following formulation: 100 mg / g of fructooligosaccharide, 250 mg / g of skim milk powder, 90 mg / g of galactose, 60 mg / g of lactose, 50 mg / g of proline, and 60 mg / g of water-soluble trehalose.

[0015] The vacuum freeze-drying conditions were as follows: 30 g / 100 g of the ratio of the bacterial slurry, -45℃ of the pre-freezing temperature, 4 h of the pre-freezing time, 1.0 cm of the freeze-drying thickness, and 0.2 mbar of the vacuum degree.

[0016] The content of the live strain in the MF06 preparation was not less than 10 8 CFU / g, and the adult dosage was 2-4 g per day.

[0017] Although the safety of probiotics is an important issue in the evaluation of probiotics, the main contents involved are the drug resistance and potential pathogenicity of the strain. The MF06 strain has not yet obtained a food use license, but reference is made to the current domestic and foreign techniques and methods for evaluating the safety of probiotics, which mainly include genome-based virulence-related gene analysis and drug resistance gene analysis, detection of potential toxic substances by high-performance liquid chromatography, drug resistance detection by minimum inhibitory concentration, and animal and human clinical trials. Genome analysis can obtain information of functional genes such as virulence-related genes and drug resistance genes of probiotic strains, which is generally used as the first step of strain safety evaluation to identify the drug resistance and potential pathogenicity of probiotics. The MF06 strain in the present application does not have virulence-related genes and drug resistance genes, is isolated from healthy humans, and produces high yields of acetic acid and butyric acid. Therefore, the MF06 strain has the functions of regulating intestinal flora, stabilizing gastrointestinal function, and improving immunity, and has a high possibility of passing the safety evaluation in the future, so the probability of becoming a beneficial bacterium for human use is extremely high.

[0018] The present application also provides the application of MF06 in the preparation of drugs. The application of MF06 in the preparation of drugs in the present application refers to using MF06 and / or its metabolites as the active ingredients of the drugs. The content of the live strain in the MF06 preparation is not less than 10 8CFU / g, adult dosage 2-4 g per day. The purpose of making drugs from MF06 and / or its metabolites is not limited to the currently known weight loss, colorectal cancer adjuvant therapy, relief of irritable bowel syndrome, Crohn's disease treatment, insulin resistance, anti-inflammatory, etc. or as an auxiliary drug for the above treatment purposes based on its metabolites acetic acid and butyric acid.

[0019] In the above application, the method for obtaining MF06 or its metabolites comprises: inoculating activated butyric acid producing bacteria MF06 into RCM liquid medium, and anaerobic fermentation culture. The RCM liquid medium mainly comprises: 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, and pH value 6.8±0.1. The anaerobic fermentation condition is 37°C anaerobic fermentation for 72 h, and the inoculation amount can be selected by those skilled in the art within the range of 1-10%vol of the conventional amount.

[0020] Beneficial effects:

[0021] 1. The present application isolates and screens a butyric acid producing bacteria strain MF06 from healthy male fecal samples, and the preservation number is CGMCC NO. 41195. The strain MF06 has a simple culture method and high butyric acid production capacity, and has important significance for establishing a high-quality butyric acid producing bacteria strain resource library. At present, the butyric acid production capacity of MF06 is 716.54 mg / L, and as a new strain, the butyric acid production capacity will be higher through strain domestication and culture optimization.

[0022] 2. At present, it is detected that MF06 not only has high butyric acid production capacity, but also has high acetic acid production capacity, and no virulence related genes and drug resistance genes are detected, that is, the safety evaluation is safe, and it is isolated from healthy humans, so it has great potential for application in preparing weight loss, colorectal cancer adjuvant therapy, relief of irritable bowel syndrome, Crohn's disease treatment, insulin resistance, anti-inflammatory or as an auxiliary drug for the above treatment purposes. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the colony morphology of MF06 cultured for 3 days.

[0024] Figure 2 is the fastani comparison result of MF06.

[0025] Figure 3 is the butyric acid gas chromatography-mass spectrometry detection diagram of MF06. DETAILED DESCRIPTION

[0026] The specific embodiments of the present application are exemplified by the experimental process, and are not the experimental process of the creative labor of the present application, nor represent the entire work of the present application.

[0027] Culture medium formula involved in the embodiments:

[0028] PBS buffer: NaCl 8 g / L, KCl 0.2 g / L, Na2HPO41.42 g / L, KH2PO40.24 g / L, pH value 7.4±0.1.

[0029] 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 chloride 5 mg / L, Mupirocin 0.1 g / L, L-cysteine hydrochloride 0.5 g / L, pH value 5.6±0.1. MWC solid medium: 1.8% (w / v) agar is added to the MWC liquid medium and poured in an anaerobic workstation.

[0030] MWC is a separation medium, and the application of Mupirocin and L-cysteine hydrochloride in the medium is beneficial to the separation of Gram-positive bacilli and anaerobes, i.e. beneficial to the separation of butyric acid-producing bacteria, and Mupirocin inhibits Gram-positive cocci and part of Gram-negative bacteria.

[0031] RCM liquid medium: Tryptone 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 value 6.8±0.1. RCM solid medium: 1.8% (w / v) agar is added to the RCM liquid medium and poured in an anaerobic workstation. Embodiment

[0032] Test Example 1 Screening and identification of butyric acid-producing bacterial strain MF06

[0033] MF06 is confirmed as Firmicutes, Clostridia, Eubacteriales, Lachnospiraceae sp. through isolation, preliminary screening, 16S rRNA and whole genome determination, and is named as butyric acid-producing bacteria MF06 in combination with its function of producing butyric acid. The preservation agency recommends the classification and naming as Lachnospiraceae sp.

[0034] 1. Isolation and culture of butyric acid-producing bacterial strain MF06

[0035] (1) Sample collection: The sample was isolated from the fecal sample of a healthy male in Beijing. The strain was isolated at the Institute of Microbiology, Chinese Academy of Sciences.

[0036] (2) Isolation and purification of the strain: The isolation process was strictly anaerobic. In an anaerobic workstation, 0.5 g of the sample was suspended in sterilized and deoxygenated PBS buffer, mixed thoroughly, and then gradient diluted. 10 5 , 10 6 , 10 7 Gradient diluted suspension 100 μL was plated on MWC plates, incubated at 37°C for 3 days, and single colonies were picked for streak purification. The isolated pure culture strain was preserved, but gene (acetyl-CoA transferase gene) screening, and 16S rRNA gene identification.

[0037] 2, Butyric acid-producing strain MF06 screening

[0038] (1) Genomic extraction: The isolated strain was cultured using the same culture medium and culture conditions as during isolation. When the strain concentration reached the order of 10 8 cfu / mL, 2 mL of bacterial solution was taken for genomic DNA extraction.

[0039] (2) PCR amplification of the but gene of the strain: Using primers BCoATscr-F (5'-GCNGANCATTTCACNTGGAAYWSNTGGCAYATG-3') and BCoATscr-R (5'-CCTGCCTTTGCAATRTCNACRAANGC-3'), the but gene was amplified using DNA as a template.

[0040] The PCR amplification program was: 95°C for 5 min; 95°C for 30 s, 50°C for 30 s, 72°C for 60 s, 35 cycles; 72°C for 5 min.

[0041] (3) Electrophoresis detection: The obtained PCR product was detected by 2% (w / v) agarose electrophoresis. The band position of the but gene was about 500 bp, and the butyric acid-producing strain MF06 was preliminarily obtained.

[0042] 3, 16S rRNA gene identification of butyric acid-producing strain MF06

[0043] (1) Genomic DNA extraction: The isolated strain was cultured using the same culture medium and culture conditions as during isolation. When the strain concentration reached the order of 10 8 cfu / mL, 2 mL of bacterial solution was taken for genomic DNA extraction.

[0044] (2) PCR amplification of 16S rRNA gene of the strain: using universal primers 27F (5'- AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGYTACCTTGTTACGACTT-3'), 16S rRNA gene amplification was performed with DNA as a template.

[0045] The PCR amplification procedure was as follows: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 90 s, 30 cycles; 72°C for 5 min.

[0046] (3) Purification and sequencing: the obtained PCR product was detected by 1.5% (w / v) agarose electrophoresis, and the band position of the 16S rRNA gene amplification product was about 1500 bp, and then the purified product was sequenced.

[0047] (4) Sequence alignment of 16S rRNA gene of the strain: the obtained sequence was subjected to BLAST alignment in the NCBI database, and the results showed that MF06 had the highest similarity of 99.42% with the strain butyrate-producing bacterium GM2 / 1 of the phylum Firmicutes, class Clostridia and order Eubacteriales. The international recommended threshold value for classification of bacterial species based on 16S rRNA gene sequence similarity is 98.7%, and this result supports that the strain MF06 is a butyrate-producing bacterium.

[0048] AGGTAAATAAGATGAAGTTTTCGGATGGATTTTTATTTACCGAGTGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTCATACAGGGGGATAACGGTTAGAAATGACTGCTAATACCGCATAAGCGCACAGTACCGCATGGTACGGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGATTAGCTAGTTGGTGAGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGACCTGAGAGGGTGACCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGGATATTGCACAATGGAGGAAACTCTGATGCAGCGACGCCGCGTGAGTGAAGAAGTATTTCGGTATGTAAAGCTCTATCAGCAGGGAAGAAAATGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGCGACGCAAGTCTGAAGTGAAATACCCGGGCTCAACCTGGGAACTGCTTTGGAAACTGTGTTGCTAGAGTGCTGGAGAGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAAGAACACCAGTGGCGAAGGCGGCTTACTGGACAGTAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAA

[0049] 4. Whole genome identification of butyric acid producing bacterial strain MF06

[0050] Because 16S rRNA gene sequence alignment failed to identify the species and genus of MF06, the whole genome sequence was uploaded to JSpecies (https: / / jspecies.ribohost.com / jspeciesws / ) for average nucleotide identity (cANI) analysis. MF06 showed the highest similarity (99.32%) to *Butyrate-producing bacterium* SS3 / 4 (Phyllostachys, Clostridium, Eubacteria). International recommendations suggest a threshold of 95-96% for genome-wide ANI classification of bacteria, supporting the identification of strain MF06 as a butyrate-producing bacterium.

[0051] Further results from fastani are as follows Figure 2 The results showed that MF06 had a 98.3% similarity to f__Lachnospiraceae;g__CAG-81;s__CAG-81 sp900066055, so the strain was named CAG-81 MF06.

[0052] As is well known to those skilled in the art, statistical analysis of thousands of genomes and 16S rRNA gene sequences reveals that when the similarity between the 16S rRNA gene sequences of two strains is less than approximately 98.65%, they can be identified as belonging to different species. However, when the similarity is greater than approximately 98.65%, they may belong to the same species or different species. In such cases, it is necessary to calculate the average nucleotide identity (ANI) of the entire genome based on their genome sequences to obtain an accurate species identification result. A genome ANI value of 95-96% for two strains is equivalent to a DNA-DNA hybridization value of 70%. Therefore, when the genome ANI value of 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, strain MF06 was finally identified as a butyrate-producing bacterium, a new bacterium belonging to the genus CAG-81 of the family Lachnospiraceae, and was deposited at the China General Microbiological Culture Collection Center with the accession number CGMCC NO.41195.

[0053] 5. Microbiological characterization of butyric acid-producing strain MF06

[0054] Morphological characteristics: the isolated pure strain MF06 was cultured on RCM solid medium under anaerobic conditions at 37°C for 3 days, and the colony was yellowish white, round, opaque, and raised, with a diameter of about 2 mm. Figure 1 ).

[0055] Microscopic characteristics: under 10000 times microscopic magnification, the MF06 cell body was rod-shaped, and the gram reaction was positive, with a cell body diameter of about 1 μm and a length of 2-4 μm.

[0056] Safety evaluation of butyric acid-producing bacterial strain MF06

[0057] 1. Analysis of virulence-related genes

[0058] The DIAMOND software was used to align the amino acid sequences of each genome with the Virulence Factor Database (VFDB) to analyze virulence-related genes. The alignment results with a protein sequence similarity of ≥95% and a coverage of ≥95% were selected as positive results.

[0059] Analysis result: no virulence-related genes were detected.

[0060] 2. Analysis of drug resistance genes

[0061] The drug resistance gene sequences and drug resistance gene recognition tool RGI v5.2.0 provided by the Comprehensive Antibiotic Resistance Database (CARD) were used to analyze the drug resistance genes of the genome according to the default parameters. The mobile drug resistance genes were analyzed using the software ResFinder v4.0 according to the default parameters. The alignment results with a protein sequence similarity of ≥95% and a coverage of ≥95% were selected as positive results.

[0062] Analysis result: no drug resistance genes were detected.

[0063] Based on the above, according to the current domestic and foreign technical and method for safety evaluation of probiotics, the functional genes such as virulence-related genes and drug resistance genes of MF06 were detected, and it was isolated from healthy human body, so the inventor deduces that it is safe to develop it as a human beneficial bacteria, and it is preliminarily determined to be safe, and it has a high possibility of being safe and effective in the application of microbial preparations or drugs.

[0064] Example 1 Application of butyric acid-producing bacterial strain MF06 in producing butyric acid in a culture medium

[0065] The -80°C frozen preserved strain MF06 was picked up with a inoculation loop, inoculated into RCM liquid medium, and cultured anaerobically at 37°C for 3 days for strain activation. The activated strain was inoculated into RCM liquid medium, and cultured anaerobically at 37°C for 3 days. The fermentation broth was taken for butyric acid detection.

[0066] Butyric acid quantitative analysis: 2-methylvaleric acid was used as an internal standard. A standard curve was prepared using butyric acid standard series concentration and relative peak area, and the butyric acid content in the sample was calculated.

[0067] The specific operation is as follows:

[0068] (1) Pretreatment: 0.1 mL of fermentation broth was taken, 0.05 mL of 15% phosphoric acid was added, and 0.2 mL of extraction solution (containing internal standard 2-methylvaleric acid, 25 mg / L, methyl tert-butyl ether) was added. Vortex for 30 s, shake for 10 min, ultrasonic for 10 min (ice water bath), centrifuge at 10000 rpm for 10 min at 4°C, stand at -20°C for 30 min, and take the supernatant for testing.

[0069] (2) Machine detection: Shimadzu GC2030-QP2020 NX gas chromatograph mass spectrometer was used, the chromatographic column was Agilent HP-FFAP capillary column (30 m x 250 μm x 0.25 μm, J&W Scientific, Folsom, CA, USA); split injection, injection volume 1 μL, split ratio 5:1. Injection port temperature 220°C; ion source temperature 240°C; transfer line temperature 240°C. Initial temperature 50°C, maintain for 1 min; increase to 150°C at 50°C / min, maintain for 1 min; increase to 170°C at 10°C / min; increase to 225°C at 25°C / min, maintain for 1 min; finally increase to 240°C at 40°C / min, maintain for 1 min. Carrier gas (Helium), carrier gas flow rate 1.2 mL / min. Ionization mode EI (electron ionization), electron energy 70 eV.

[0070] (3) The detection results are as shown in Table 1. Figure 3 The butyric acid content in the fermentation broth was 716.54 mg / L. It is well known to those skilled in the art that the butyric acid production capacity will be higher after domestication or culture optimization of such high-yield strains.

[0071] Prior art (Obesity-relieving butyrate-producing Clostridium screening and mechanism of action, Jiangnan University 2023 Master's thesis, author Food Industry and Engineering major Liao Jingyi) shows that the relationship between gut flora and obesity is being revealed and is recognized as one of the environmental factors that cause the onset of obesity. Obese individuals have a significant difference in gut flora diversity and composition compared to individuals with normal body weight, and the abundance of butyrate-producing Clostridium in the gut flora of obese individuals is significantly reduced, and butyrate-producing Clostridium is one of the butyrate-producing Clostridium in the gut, which can exert a probiotic effect by regulating the function of tissues such as the gut, adipose tissue, liver, and brain. Studies have shown that the abundance of butyrate-producing Clostridium decreases in type 2 diabetes patients, and an increase in its abundance is associated with a decrease in obesity susceptibility. In addition, supplementing butyrate-producing Clostridium can relieve obesity-related phenotypes.

[0072] Similar to the above prior art, the prior art "Research Progress on the Regulation Mechanism of Short-chain Fatty Acids on Obesity" (published in Chinese Journal of Preventive Medicine, Vol. 25, No. 6, 2024, authors Xue Yili et al.) reviews the mechanism of short-chain fatty acids in relieving obesity, and existing research results show that:

[0073] SCFAs binding to GPCR43 and GPCR41 can trigger cell-specific cascades of signals, promote the secretion of gut hormones, and also participate in gene expression regulation, intestinal barrier function regulation, immune regulation, oxidative stress, and cell differentiation by inhibiting histone deacetylase (HDAC). SCFAs also enhance triacylglycerol hydrolysis and free fatty acid oxidation in adipose tissue by affecting gut flora composition, promoting beige adipogenesis and mitochondrial biogenesis.

[0074] GPCR41 and GPCR43 can be effectively activated by acetate, propionate, butyrate, and other SCFAs, with affinities varying by species. Longer butyrate is more selective for GPR41, shorter acetic acid is more selective for GPR43, and propionic acid binds to both receptors. Propionic acid activates GPCR41, promoting the secretion of gut 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 beta cells, improve the sensitivity of islet 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 perivascular afferent nervous system to induce gluconeogenesis. Increased intestinal epithelial cell gluconeogenesis leads to decreased liver glucose production and improved energy homeostasis.

[0075] Therefore, in combination with the prior art and the detection results of MF06 metabolites in the present application, those skilled in the art can expect that the MF06 of the present application has the effects of regulating intestinal flora, stabilizing gastrointestinal function, improving immunity, reducing insulin resistance, and reducing fat.

[0076] Example 2 Application of MF06 in preparation of microecological preparation

[0077] (1) Activation of MF06 bacterial cells

[0078] The MF06 strain preserved at -80°C was picked up with an inoculation loop and inoculated into RCM liquid medium, and then cultured anaerobically at 37°C for 3 days for strain activation. The activated strain was then inoculated into RCM liquid medium and cultured anaerobically at 37°C for 3 days, and then centrifuged to collect the MF06 bacterial cells.

[0079] (2) Preparation of MF06 bacterial cell microecological preparation

[0080] After centrifugation to obtain the MF06 bacterial cells, vacuum freeze-drying was performed by compounding with a freeze-drying protective agent to obtain live bacteria freeze-dried powder, which was prepared into a compound probiotic bacterial agent alone or in combination with other probiotics.

[0081] The freeze-drying protective agent formula is: fructooligosaccharide 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.

[0082] The vacuum freeze-drying conditions are: bacteria slurry ratio 30 g / 100 g, pre-freezing temperature -45°C, pre-freezing time 4 h, freeze-drying thickness 1.0 cm, and vacuum degree 0.2 mbar.

[0083] The content of live bacteria in the MF06 preparation is not less than 10 8 CFU / g, and the adult dosage is 2-4 g per day.

[0084] This specific embodiment is only an explanation of the present application and is not a limitation of the present application. Those skilled in the art can make modifications to this embodiment without creative contribution after reading the present specification, but as long as it is within the scope of the claims of the present application, it is protected by the Patent Law.

Claims

1. A butyric acid-producing bacterium, characterized in that, Butyric acid producing bacteria Lachnospiraceae The accession number for sp. is CGMCC NO.41195.

2. A method for culturing butyric acid-producing bacteria, characterized in that, The butyric acid-producing bacteria of claim 1 were inoculated into RCM medium for anaerobic culture at an inoculation concentration of 1-10% vol.

3. The application of the butyric acid-producing bacteria according to claim 1 in the production of butyric acid.

4. The application of the butyric acid-producing bacteria according to claim 1 in the preparation of microecological preparations.

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