Clostridium butyricum and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to clostridium butyricum and application thereof, and the preservation number of the clostridium butyricum is CGMCC (China General Microbiological Culture Collection Center) No.27853. According to the present invention, with the application of the bacterial strain, the species diversity of the host intestinal flora can be significantly reduced, the Firmicite / Bacteroidetes ratio can be significantly increased, the abundance of harmful bacteria such as Escherichia coli and Salmonella bacteria can be reduced, and the abundance of beneficial bacteria can be improved; in addition, the yield of butyric acid of the clostridium butyricum is larger than or equal to 1.5 g / L, and the activity of amylase in fermentation liquor of the clostridium butyricum is 16.04 + / -0.46 U / mL. When the feed additive is used in feed, intestinal functions of organisms can be effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Clostridium butyricum and application thereof. Background Art

[0002] About 90% of antibiotics are used in food animals every year, leading to increasingly prominent problems such as bacterial resistance and drug residues. According to relevant requirements, all growth-promoting drug feed additives except traditional Chinese medicine will be withdrawn from January 1, 2020, marking the beginning of the implementation of the most stringent antibiotic restriction, ban and no antibiotic policy in my country's agriculture.

[0003] In the past 20 years, as animal husbandry has entered the "post-antibiotic era", a large number of studies have shown that probiotics as new feed additives can effectively reduce or replace the use of feed antibiotics. Probiotics refer to live microbial preparations and their metabolites that can promote the balance of intestinal microorganisms and have a beneficial effect on the host. Lactobacillus and bifidobacteria, as the dominant flora in the normal microbial flora of the animal digestive tract, are the earliest and most widely used probiotics in animal production. However, these two probiotics are not resistant to high temperatures and the storage time of live bacteria at room temperature is short. After being treated at 80°C for 5 minutes, the loss rate of live bacteria is as high as about 75%, which limits their application and application effect as feed additives in animal production. Compared with lactic acid bacteria and bifidobacteria, Clostridium butyricum (also known as Clostridium butyricum) has better stress resistance due to its ability to form endogenous spores, such as high temperature resistance, gastric acid resistance and bile salt resistance. Moreover, Clostridium butyricum can promote the reproduction of probiotics such as lactic acid bacteria and bifidobacteria in the intestines and inhibit the growth of pathogenic bacteria such as Staphylococcus aureus and Escherichia coli. In particular, Clostridium butyricum can secrete a variety of digestive enzymes during its growth, and can produce beneficial substances such as butyric acid and hydrogen through anaerobic fermentation of carbohydrates such as starch and cellulose. On March 24, 2022, under the organization of the National Feed Industry Standardization Technical Committee, the agricultural standard "Feed Additive-Clostridium Butyricum" jointly drafted by five units including the Beijing Animal Husbandry and Veterinary Research Institute of the Chinese Academy of Agricultural Sciences successfully passed the final review.

[0004] Therefore, it is necessary to explore a probiotic that is suitable for adding to livestock and poultry feed and can be effectively utilized by animals. Summary of the invention

[0005] The present invention has discovered a Clostridium butyricum, which can significantly increase the abundance of more than ten plant probiotics mainly composed of Pseudomonas in the intestinal flora, and reduce the abundance of harmful bacteria such as Escherichia coli, Helicobacter typhlonius, Helicobacter hepaticus, Salmonella enterica, Staphylococcus nepalensis and Bacteroides vulgatus; and the butyric acid production of the Clostridium butyricum is not less than 1.5 g / L; it produces cellulase, and the diameter of the hydrolysis transparent zone formed in the cellulose Congo red agar culture medium is not less than 0.8 cm; it produces amylase, and the amylase activity in the fermentation broth reaches 175.98±3.45 IU / OD; it can be used in feed to effectively improve the intestinal function of the body.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides a Clostridium butyricum, whose deposit number is CGMCC No.27853.

[0008] Preferably, the 16S rRNA sequence of the above-mentioned Clostridium butyricum is shown as SEQ ID N0.5.

[0009] Another aspect of the present invention further provides a fermentation broth, which is obtained by fermenting Clostridium butyricum in the present invention.

[0010] In yet another aspect, the present invention provides a feed comprising the Clostridium butyricum of the present invention or the fermentation broth of the present invention.

[0011] It should be noted that the application of Clostridium butyricum and its fermentation liquid in feed can be achieved by inducing the strain to form spores and then adding it to the basic diet (1×10 10 CFU / kg), which can significantly inhibit the growth of intestinal harmful bacteria such as Escherichia coli, Helicobacter and Salmonella, and promote the growth of beneficial bacteria, especially the growth of various plant probiotics in the genus Pseudomonas, thereby providing theoretical support for the use of Clostridium butyricum as a feed additive to replace antibiotics to improve the intestinal health of the host, and for the harmless treatment of mouse and livestock feces.

[0012] It should also be noted that the Clostridium butyricum in the present invention is used in feed, and its application scope is not limited to being used as feed for economic breeding or ornamental animals such as livestock, aquatic products and pets. In addition, feeding Clostridium butyricum of the present invention can significantly improve the biosafety of mouse or other breeding and ornamental animal feces, and can be developed into organic fertilizers that promote plant growth and plant disease biocontrol agents.

[0013] Preferably, the Clostridium butyricum in the above feed is Clostridium butyricum powder.

[0014] It should be noted that Clostridium butyricum can be made into bacterial powder and then added to feed as an additive to prepare feed, which can improve the intestinal function of animals.

[0015] Preferably, the preparation method of the Clostridium butyricum powder in the feed comprises: inducing Clostridium butyricum to form spores, then adding 5%-10% by mass of auxiliary materials to the fermentation liquid, and preparing the Clostridium butyricum powder by spray drying.

[0016] Preferably, the auxiliary materials include one or more of maltodextrin, corn cobs, corn flour, corn starch and light calcium carbonate.

[0017] Preferably, the spore content of the Clostridium butyricum powder is ≥ 2×10 9 CFU / g, the effective amount of spores added in feed is 1-10×10 9 CFU / kg feed.

[0018] In another aspect, the present invention provides a use of the Clostridium butyricum of the present invention or the fermentation broth of the present invention as or in the preparation of a feed additive for promoting the repair and regeneration of intestinal epithelial cells, promoting the development of the intestine of livestock and poultry, and enhancing their immunity and disease resistance.

[0019] It should be noted that butyric acid, the main metabolite of Clostridium butyricum, helps repair and regenerate intestinal epithelial cells, promotes the development of the intestine of livestock and poultry, and enhances their immunity and disease resistance. In particular, butyric acid can provide the main energy for colon mucosal cells and provide an energy basis for the proliferation and maturation of intestinal cells.

[0020] In another aspect, the present invention provides a use of Clostridium butyricum or the fermentation broth of the present invention in reducing harmful bacteria in intestinal flora.

[0021] It should be noted that the Clostridium butyricum in the present invention or the fermentation liquid in the present invention can replace the effect of antibiotics to reduce harmful bacteria in the intestinal flora.

[0022] The Clostridium butyricum provided by the present invention was deposited in the General Microbiology Center of the China Culture Collection Administration on July 10, 2023. The address of the depository is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 27853, and the taxonomic name is Clostridium butyricum.

[0023] The beneficial effects of the present invention include at least:

[0024] (1) The Clostridium butyricum provided by the present invention can significantly increase the abundance of more than ten plant probiotics, mainly Pseudomonas, in the intestinal flora, and reduce the abundance of harmful bacteria such as Escherichia coli, Helicobacter typhlonius, Helicobacterhepaticus, Salmonella enterica, Staphylococcus nepalensis and Bacteroides vulgatus.

[0025] (2) The butyric acid production of Clostridium butyricum provided by the present invention is not less than 1.5 g / L; it produces cellulase, and the diameter of the hydrolysis transparent zone formed in the cellulose Congo red agar medium is not less than 0.8 cm; it produces amylase, and the amylase activity in the fermentation broth reaches 175.98±3.45 IU / OD; and its application in feed can effectively improve the intestinal function of the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A Schematic diagram of initial screening of Clostridium butyricum on TSC double-layer agar plates;

[0027] Figure 1B The situation of Clostridium butyricum further screened by gas production characteristics;

[0028] Figure 1C The specificity of colony PCR for rescreening Clostridium butyricum was tested;

[0029] Figure 1D This is the agarose gel electrophoresis pattern of identifying Clostridium butyricum using 16S rRNA universal primers;

[0030] Figure 2A The activity of amylase in different Clostridium butyricum fermentation broths HADIG-CB003;

[0031] Figure 2B It is the transparent circle map of starch hydrolysis by Clostridium butyricum HADIG-CB003;

[0032] Figure 2C It is the transparent circle map of five strains of Clostridium butyricum hydrolyzing carboxymethyl cellulose;

[0033] Figure 2D The cellulase activity in different Clostridium butyricum fermentation broths;

[0034] Figure 2E This is a single colony map of Clostridium butyricum HADIG-CB003 screened by ten-fold dilution coating on RCM plates;

[0035] Figure 2F The bacterial morphology of Clostridium butyricum HADIG-CB003 was observed under a microscope (100×);

[0036] Figure 2G Phylogenetic tree of Clostridium butyricum HADIG-CB003 based on 16S rRNA gene sequence.

[0037] Figure 2H Gas chromatogram of positive control standard gas (containing H2);

[0038] Fig.2I To analyze the composition of the gas produced by the HADIG-CB003 strain by gas chromatography;

[0039] Figure 2J This is a microscopic image of spore formation by HADIG-CB003 (40×);

[0040] Figure 2K The survival of HADIG-CB003 strain in artificial gastric fluid;

[0041] Figure 2L The survival of HADIG-CB003 strain in artificial intestinal fluid;

[0042] Figure 3A The weight changes of Kunming mice after being fed with Clostridium butyricum for different days;

[0043] Figure 3B The average daily weight gain of Kunming mice fed with Clostridium butyricum at different time periods;

[0044] Figure 3C is the average daily feed intake of Kunming mice fed with Clostridium butyricum at different time periods;

[0045] Figure 3D The feed-to-weight ratio of Kunming mice fed with Clostridium butyricum at different time periods;

[0046] Figure 3E The Chao1 index of microbial communities in the cecal contents of Kunming mice in the experimental group (CB) supplemented with Clostridium butyricum relative to the blank control group (NC) was plotted between groups (p<0.05);

[0047] Figure 3F The box plot of Faith's Phylogenetic Diversity (PD) between groups of the microbial community in the cecal contents of Kunming mice in the experimental group supplemented with Clostridium butyricum relative to the blank control group;

[0048] Figure 3G The Simpson index box plot of the microbial community in the cecal contents of Kunming mice in the experimental group supplemented with Clostridium butyricum relative to the blank control group;

[0049] Figure 3H This is a box plot of the Shannon index difference between the groups of microbial communities in the cecal contents of Kunming mice in the experimental group supplemented with Clostridium butyricum relative to the blank control group;

[0050] Fig. 3I PCoA diagram of microbial communities in the cecal contents of Kunming mice in the experimental group supplemented with Clostridium butyricum relative to the blank control group;

[0051] Figure 3J NMDS graph of microbial communities in the cecal contents of Kunming mice in the experimental group supplemented with Clostridium butyricum relative to the blank control group;

[0052] Figure 4A The composition of intestinal flora at the phylum level;

[0053] Figure 4B is the relative proportion of Firmicutes and Bacteroidetes in the intestinal flora;

[0054] Figure 4C The composition of intestinal flora at the class level;

[0055] Figure 4D It is the composition of intestinal flora at the mesh level;

[0056] Figure 4E The composition of intestinal flora at the family level;

[0057] Figure 4F It is the composition of intestinal flora at the genus level;

[0058] Figure 4G The composition of intestinal flora at the species level;

[0059] Figure 5A Sankey plot of the composition of the microbial community in the cecal contents of Kunming mice fed and not fed with Clostridium butyricum for the high-abundance genera (sorted by total average abundance);

[0060] Figure 5B The Sangkey plot of the composition of the microbial community in the cecal contents of Kunming mice fed and not fed with Clostridium butyricum in terms of high abundance species (sorted by total average abundance);

[0061] Figure 5C Differences in the 40 most abundant genera in the microbial community composition of cecal contents of Kunming mice fed and not fed with Clostridium butyricum;

[0062] Figure 5D The differences in the 40 most abundant species in the microbial community composition of cecal contents of Kunming mice fed and not fed with Clostridium butyricum;

[0063] Fig. 6AThe LDA value distribution bar graph of the differential species analysis (LEfse analysis) of the microbial composition in the cecum contents of Kunming mice fed and not fed with Clostridium butyricum;

[0064] Figure 6B The species branch evolution diagram in the differential species analysis (LEfse analysis) of the microbial composition in the cecal contents of Kunming mice fed and not fed with Clostridium butyricum;

[0065] in, Figure 1C In the figure, lane M is DL2000 Plus DNAMarker, lane 1: negative control using genomic DNA of Lactobacillus reuteri as template, lane 2: positive control using Clostridium butyricum strain screened from Clostridium butyricum live bacterial tablets of Japan Miyarisan Pharmaceutical Co., Ltd., and the strain was named JCM 1391, lanes 3-6: PCR amplification products using genomic DNA of suspected Clostridium butyricum strain as template;

[0066] Figure 1D In the figure, lane M is DL2000 Plus DNAMarker, lane 1 is the PCR product amplified using genomic DNA of strain JCM 1391 as template, and lanes 2-5 are the PCR amplification products using genomic DNA of different Clostridium butyricum as templates;

[0067] FIG. 3E to FIG. 3H In the figure, the horizontal axis represents the group alias, and the vertical axis represents the value of the α diversity index. The box line close to the lower side of the horizontal axis represents the lower quartile, which is equal to the 25% number after all the values ​​in the sample are arranged from small to large; the vertical line in the next box is the median, which is equal to the 50% number after all the values ​​in the sample are arranged from small to large, and the box line on the upper side represents the upper quartile, which is equal to the 75% number after all the values ​​in the sample are arranged from small to large. The lower and upper ends of the extension line represent the lower and upper edges respectively. The dots represent outliers;

[0068] Fig. 3I In the PCoA dot diagram, the horizontal and vertical axes represent the two principal coordinates that contribute most to the difference and their contribution rates to the sample differences, respectively. Each point represents a sample. The distance between the sample points represents the similarity of the microbial communities in the samples. The smaller the distance, the higher the similarity. Samples in the same group are represented by the same color and shape.

[0069] Figure 3JIn the NMDS dot plot, each dot represents a sample, the distance between the dots indicates the degree of difference, and the samples in the same group are represented by the same color and shape. When Stress < 0.2, it means that NMDS can accurately reflect the degree of difference between samples. NC (Normal control) represents the normal control group without Clostridium butyricum added to the diet, and CB (Clostridium butyricum) represents the experimental group with Clostridium butyricum added to the diet;

[0070] Figure 4A , 4C to Figure 4G In the figure, the horizontal axis is the group name, the vertical axis is the relative abundance, different colors represent different groups, Others represents the sum of the relative abundances of all species other than those ranked 10 and species without annotation information; NC (Normal control) represents the normal control group without Clostridium butyricum added to the diet, and CB (Clostridium butyricum) represents the experimental group with Clostridium butyricum added to the diet;

[0071] FIG. 5A to FIG. 5B In the figure, the horizontal axis is the sample name, the vertical axis is the relative abundance, and different colors represent different groups;

[0072] FIG. 5C to FIG. 5D In the figure, the horizontal axis represents sequence abundance, and the vertical axis represents classification attributes, which are phylum and genus / species, respectively. The vertical axis is arranged from large to small according to the total average abundance of genus / species. Colors represent different groups. The box line close to the left side of the vertical axis represents the lower quartile, which is equal to the 25% number after all the values ​​in the sample are arranged from small to large. The next vertical line in the box is the median, which is equal to the 50% number after all the values ​​in the sample are arranged from small to large, and the box line on the right represents the upper quartile, which is equal to the 75% number after all the values ​​in the sample are arranged from small to large. The left and right ends of the extension line represent the lower and upper edges, respectively. The dots represent outliers. NC (Normal control) represents the normal control group without Clostridium butyricum added to the diet, and CB (Clostridium butyricum) represents the experimental group with Clostridium butyricum added to the diet.

[0073] Fig. 6A In the figure, the horizontal axis represents the size of the LDA value, that is, the longer the column, the more important the group. The colors represent different groups, indicating that these groups have significant enrichment characteristics in this group, that is, the average abundance is the highest and the difference between groups is significant;

[0074] Figure 6BIn the figure, a circular dendrogram was constructed for the groups with significant test results according to the hierarchical structure of the classification data. NC (Normal control) represents the normal control group without Clostridium butyricum added to the diet, and CB (Clostridium butyricum) represents the experimental group with Clostridium butyricum added to the diet. DETAILED DESCRIPTION

[0075] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0076] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0077] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0078] 1. Isolation and identification of Clostridium butyricum from pigs

[0079] (I) Screening of Clostridium butyricum on TSC double-layer agar plates

[0080] Weigh 5 g of fresh feces from a healthy three-way pig and dilute it to 10% with sterile saline. -5 , 10 -6 and 10 -7 There are 3 gradients in total. Take 500 μL of fecal dilution and add it to the bottom of the dish. Pour the TSC lower culture medium by pouring method. After the lower culture medium solidifies, pour the upper TSC culture medium containing gelatin (20 g / L) to the lid of the dish and cover the dish. After the culture medium solidifies, place it in a 37°C constant temperature incubator overnight and count the number of black colonies.

[0081] In addition, the same method was used to select a Clostridium butyricum strain from the Clostridium butyricum live bacteria tablets of Japan Miyarisan Pharmaceutical Co., Ltd. as a positive control, and the strain was named JCM 1391.

[0082] (II) PCR specific rescreening of Clostridium butyricum

[0083] Use the tip of the pipette to pick up the large black colonies (such as Figure 1A As shown, it shows that the black single colony of Clostridium was successfully screened from pig manure and inoculated into the reinforced Clostridium liquid medium (RCM) (as shown in Figure 1B ), cultured overnight at 37°C under aerobic conditions, wherein dense white bubbles can be accumulated on the culture medium (for excluding Clostridium perfringens), and the bacterial characteristics and Gram staining characteristics of different strains are further observed with an oil lens under a microscope, and strains that meet the morphological characteristics of Clostridium butyricum are selected. Then, PCR amplification is performed using primers that specifically identify Clostridium butyricum (primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.2); finally, the strain with the correct PCR product size is serially diluted 10 times and then applied to RCM culture medium for continued culture until a pure culture of Clostridium butyricum is obtained.

[0084] (III) Identification of 16S rRNA of Clostridium butyricum

[0085] The pure culture of the isolates cultured to the logarithmic growth phase was collected by centrifugation, and the genomic DNA was extracted using the FastPure Microbiome DNA Isolation Kit of Nanjing Novozyme Biotechnology Co., Ltd., which was used as a template to perform PCR amplification on the 16S rRNA of 5 Clostridium butyricum using universal bacterial identification primers (primer sequences are shown in SEQ ID N0.3 and SEQ ID N0.4); the PCR products were then sent to BGI for strain identification; finally, the sequencing results were subjected to homology analysis using the BLAST tool in the NCBI database to obtain the 10 homologous sequences with the greatest similarity to the sequences of the species to be tested, and the phylogenetic tree was constructed using the neighbor-joining (NJ) method in MEGA7.0 software to determine the species relationship of the strains.

[0086] The results are as follows Figure 1C As shown in the figure, the size of the PCR products is about 1022 bp, indicating that the five strains screened are all Clostridium butyricum, and are named JCM 1391, HADIG-CB001-001, HADIG-CB001-002, HADIG-CB001-003 and HADIG-CB001-004, respectively. Figure 1D As shown, agarose gel electrophoresis analysis showed that the PCR products of the five strains were all positive bands of about 1500 bp. These PCR products were sent to BGI for strain identification, and BLAST sequencing results further proved that all five strains were Clostridium butyricum.

[0087] 2. Analysis of biological characteristics of strains

[0088] (I) Comparison of acid production performance

[0089] The content of butyric acid and acetic acid in the fermentation broth of 5 strains of Clostridium butyricum was determined by gas chromatography. The specific steps are as follows: 5% of the activated seed liquid of 5 strains of Clostridium butyricum was inoculated into fresh sterile RCM medium, and the fermentation broth was cultured at 37°C for 24 hours to obtain the fermentation broth; then the fermentation broth was centrifuged at 8000rpm for 5 minutes, 900μL of the supernatant was taken, 100μL of 25% metaphosphoric acid solution was added, and the reaction was allowed to stand at room temperature for 3-4 hours, and then centrifuged at 15000×g for 10 minutes, the supernatant was filtered with a 45μm microporous filter membrane, and added to the upper machine bottle for testing. Gas chromatography (GC) conditions: Agilent 6890N gas chromatograph was used, equipped with a flame ionization detector and a DB-FFAP (30m×0.25mm×0.25μm) chromatographic column for analysis. The heating program is as follows: initial temperature 60℃, rise to 220℃ at a rate of 20℃ / min, maintain for 1min, detector FID temperature is 280℃, injection port temperature is 250℃, injection volume is 1L. High-purity nitrogen is used as carrier gas, flow rate is 0.8mL / min; split ratio is: 20:1. The measurement results are shown in Table 1.

[0090] Table 1 Comparative analysis of the acetic acid and butyric acid production capabilities of five strains of Clostridium butyricum

[0091]

[0092]

[0093] As shown in Table 1 above, the butyric acid and acetic acid contents in the fermentation broth of Clostridium butyricum are about 1.6 g / L and 3.2 g / L, respectively. Among them, the acid production value of the HADIG-CB001 strain is the largest, but there is no significant difference in the acid production capacity between different Clostridium butyricum. Surprisingly, the test results show that the acetic acid content in the fermentation broth is significantly higher than the butyric acid content (Table 1). We speculate that this may be related to the fact that the RCM medium contains 3 g / L sodium acetate (mainly used to prevent contamination of Gram-negative bacteria).

[0094] (II) Comparison of amylase activity

[0095] The activated seed liquid of 5 strains of Clostridium butyricum was inoculated into fresh sterile RCM medium at a ratio of 5%, and cultured at 37°C for 12 hours; then the OD of each bottle of bacterial liquid was measured. 600 The fermentation broth was centrifuged at 4°C and 8000 rpm for 5 min, and 1 mL of the supernatant (crude enzyme solution) was taken to detect the amylase activity according to the following steps:

[0096] Preparation of 1% DNS color developing solution: 1g 3,5-dinitrosalicylic acid is dissolved in 20mL 2mol / L NaOH, 50mL of deionized water is added, and then 30g of potassium sodium tartrate tetrahydrate is added, the volume is made up to 100mL, and the brown bottle is sealed for storage.

[0097] Preparation of pH 6.0 citric acid-sodium citrate buffer: prepare 0.1 mol / L citric acid and sodium citrate solutions respectively, then mix 3.8 mL of citric acid with 16.2 mL of sodium citrate solution, shake well and seal for storage.

[0098] Maltose standard concentrations: 1, 0.75, 0.5, 0.25, 0.125, and 0 mg / mL.

[0099] Determination of amylase activity: Take 1mL of 1% soluble starch solution and mix it with 0.25mL of pH 6.0 citric acid-sodium citrate buffer, preheat at 50℃ for 5min, add 0.2mL of crude enzyme solution (add an equal volume of deionized water to the blank tube), continue incubating at 50℃ for 10min, and immediately add 0.1mL of HCl solution (0.1mol / L) to terminate the reaction. Then add 0.35mL of DNS colorimetric solution, boil in water bath for 5min, cool rapidly with running water, and dilute and mix with 2.1mL of deionized water. Use a spectrophotometer to measure the absorbance of the reaction solution at a wavelength of 540nm. One unit of amylase activity (U) is defined as: the amount of enzyme required to generate 1mg of maltose every 10min under the above reaction conditions. In addition, 100 μL of HADIG-CB003 seed solution was taken and dropped in the center of the RCM agar plate containing 0.5% soluble starch. After the fermentation liquid was absorbed by the culture medium, it was placed in an anaerobic tank and cultured at 37°C for 48 hours. Then a layer of iodine solution was covered on the surface of the culture medium, and the diameter of the transparent circle was observed and measured. The results are as follows: Figure 2A -B.

[0100] Depend on Figure 2A It can be seen that among the five strains of Clostridium butyricum, the fermentation broth of HADIG-CB003 strain has the strongest amylase activity, reaching 16.04±0.46U / mL. Figure 2B As shown, HADIG-CB003 can form an obvious hydrolysis transparent zone in the starch plate, indicating that the HADIG-CB003 strain has strong amylase activity.

[0101] (III) Comparison of cellulase activity

[0102] (1) Evaluation of the extracellular cellulase activity of each Clostridium butyricum strain by hydrolysis circle method

[0103] Take the same OD amount of seed droplets of the above five strains of Clostridium butyricum and inoculate them onto cellulose Congo red medium plates (CMC-Na 0.2%, peptone 0.5%, yeast extract 0.4%, sodium citrate 0.5%, MgSO4·7H2O 0.05%, K2HPO4 0.1%, MnSO4·H2O 0.025%, Congo red 0.02%, agar 2.0%, pH 7.0, 121°C, sterilized for 20 min for use), cultured anaerobically for 48 h, and measured the diameter of the transparent zone produced.

[0104] (2) The extracellular cellulase activity of each Clostridium butyricum strain was determined by 3,5-dinitrosalicylic acid method

[0105] Preparation of 3,5-dinitrosalicylic acid colorimetric solution: Weigh 6.3g 3,5-dinitrosalicylic acid, add 500mL water, gradually add 262mL 2mol / L NaOH in a 45℃ water bath, stirring at the same time. Then gradually add 185g potassium sodium tartrate tetrahydrate, 5g phenol (heat to dissolve) and 5g anhydrous sodium sulfite, and continue stirring until completely dissolved. After cooling, dilute the solution to 1L with distilled water and store in a brown reagent bottle for later use;

[0106] Preparation of 0.1mol / L pH 4.5 acetic acid-sodium acetate buffer: Add 3.86g glacial acetic acid and 2.93g sodium acetate to nearly 1L of distilled water, stir thoroughly and dilute to 1L;

[0107] 0.5% sodium carboxymethyl cellulose (CMC) aqueous solution: weigh 0.5g sodium carboxymethyl cellulose, dissolve it in 100mL 0.1mol / L acetic acid-sodium acetate buffer with a pH value of 4.5, and stir well;

[0108] Glucose standard stock solution (10 mg / ml): weigh 1 g of anhydrous glucose to an accuracy of 0.1 mg, dissolve it in water and make up to 100 mL;

[0109] 1 mg / mL glucose standard solution: Add 10 mL of glucose standard stock solution into a 100 mL volumetric flask, dilute to 100 mL with distilled water, cover the bottle cap and shake well for later use.

[0110] (3) Determination of cellulase activity produced by Clostridium butyricum

[0111] The method for preparing the crude enzyme solution from the fermentation of Clostridium butyricum is the same as the determination of the amylase activity mentioned above;

[0112] Drawing of glucose standard curve: Take 7 25mL graduated colorimetric tubes and process them according to the operating steps in Table 2. After shaking each tube, boil it in a boiling water bath for 10 minutes and cool it to room temperature. Then add distilled water to make up to 25mL, plug it tightly and mix it upside down. Use a No. 0 tube to adjust the zero point, perform colorimetry at 550nm on a spectrophotometer, and measure the optical density (OD) values ​​of tubes 1-6. Finally, use the glucose optical density of each tube as the horizontal coordinate and the glucose concentration as the vertical coordinate to draw the standard curve and calculate the regression equation.

[0113] Table 2 Method for drawing glucose standard curve

[0114]

[0115] Determination of blank tube: Take 0.5ml crude enzyme solution and 1.5ml distilled water and put them into a 25ml graduated colorimetric tube. Heat it in a boiling water bath for 15min to inactivate the enzyme, then cool it. Then add 3ml 0.5% CMC and put it into a 40℃ water bath together with the sample tube for reaction for 30min. Other operations are the same as the sample tube.

[0116] Sample determination: Take 18 25 mL graduated colorimetric tubes and mark them as blank tubes and sample tubes. For details, see Table 3. Calculation: Calculate the OD of each sample tube. 550 Substitute the value into the regression equation of the glucose standard curve to calculate the glucose concentration in the sample tube. Cellulase activity is defined as the amount of enzyme required to produce 1 mg of glucose every 30 minutes under the above reaction conditions.

[0117] Table 3 Determination method of cellulase activity produced by Clostridium butyricum

[0118]

[0119] The results are as follows Figure 2C As shown in the figure, although the five strains of Clostridium butyricum can form obvious hydrolysis transparent zones in the cellulose Congo red medium plate, there is no significant difference in their activities. Based on this, the 3,5-dinitrosalicylic acid method was further used to determine the extracellular cellulase activity of each Clostridium butyricum strain. Figure 2D As shown, among the five strains of Clostridium butyricum, HADIG-CB003 strain had the strongest extracellular cellulase activity, reaching 20.88±1.27U / mL. And compared with JCM 1391 strain, the extracellular cellulase activity produced by HADIG-CB003 strain was 31.71% stronger (p<0.01).

[0120] (IV) Identification of HADIG-CB003 strain

[0121] The HADIG-CB003 strain was further identified. The single colony morphology of the HADIG-CB003 strain was as follows: Figure 2E In addition, the microscopic examination, bacterial morphology and phylogenetic tree are shown in Figure 2F The sequencing result of its strain identification is shown in SEQ ID No. 5, and it has been stored in the GenBank database with the accession number OP984128.

[0122] (V) Fermentation characteristics of HADIG-CB003 strain for hydrogen production

[0123] First, the gas produced by Clostridium butyricum was collected in a gas sampling bag (Dalian Haide Technology Co., Ltd.), and its volume was measured with a glass syringe. The gas composition was analyzed using a gas chromatograph (GC456) equipped with a TCD detector. The carrier gas was nitrogen with a flow rate of 30 mL / min. The column temperature was set to 50 ° C, the inlet was 120 ° C, and the detector outlet was 175 ° C. The standard gas mixture components used for calibration include methane (74.600% by volume), ethylene (0.993%), oxygen (0.501%), carbon dioxide (5.210%), ethane (1.020%), acetylene (0.101%), hydrogen (1.000%), and the remaining balance gas is nitrogen.

[0124] The experimental results showed that the volume of gas produced by Clostridium butyricum HADIG-CB003 was about 2.5 times the volume of the culture medium. Gas chromatography detected that hydrogen accounted for 56.97% of the total gas volume, oxygen accounted for 0.77%, and carbon dioxide accounted for 6.56% ( Fig.2I ). The above experimental results show that HADIG-CB003 has a hydrogen production capacity similar to other Clostridium butyricum strains, even when there is a trace amount of oxygen in the culture system.

[0125] (VI) Fermentation to prepare Clostridium butyricum with high spore rate

[0126] The formula of the spore-forming fermentation medium of Clostridium butyricum HADIG-CB003 is: 1%-2% fish peptone, 0.5%-1% yeast powder, 0.3%-0.5% lactose, 1-2.5% g / L corn starch (pre-gelatinized in a microwave oven); 0.1%-0.15% cysteine ​​hydrochloride: 0.2%-0.5% sodium citrate; 0.1%-0.3% dipotassium hydrogen phosphate; 0.1%-0.15% potassium dihydrogen phosphate; 0.02%-0.04% MgSO4·7H2O; 0.5ml / L-1ml / L Tween-80, pH 7.0. The inoculation amount is 1%-3%. After culturing at 37°C and 80rpm for 12h, 0.03%-0.05% MnSO4·H2O is added, and the pH value of the fermentation liquid is adjusted to about 6.5 with NaOH. The culture is continued at 37°C and 80rpm for 12h.

[0127] Take 1mL of fermentation liquid in a sterile 10mL centrifuge tube, place in a water bath at 80℃ for 10min, dilute it 10-fold with sterilized saline and count it on a TSC double-layer agar plate. Select the dilution with a colony count between 30-300 for calculation (colony count is the number of black dots). Number of spores in fermentation medium = number of colonies × 10 n+1 , where n is the dilution multiple when the bacterial solution is applied.

[0128] According to calculations, the maximum number of fermentation spores of strain HADIG-CB003 was 2.3×10 8 CFU / mL, spore rate was 63.2% ( Figure 2J ).

[0129] (VII) Experimental study on the tolerance of HADIG-CB003 strain to artificial gastric juice, artificial intestinal juice and bile salts

[0130] Preparation method of artificial gastric juice: Take 1 mol / mL dilute hydrochloric acid, dilute with water, and adjust the pH to 2.5. Add 1 g of pepsin to every 100 mL of liquid, mix well, and filter with a 0.2 μm sterile filter for later use.

[0131] Preparation method of artificial intestinal fluid: Take 6.8g KH2PO4, dissolve it in water, adjust the pH to 6.8 with 0.4% NaOH. Add 1g trypsin to every 100mL liquid, mix well, and filter with a 0.2μm sterile filter for later use.

[0132] 1. Artificial gastric juice tolerance study: The culture at the end of growth was centrifuged at 3,000 rpm for 10 min, washed twice with PBS and resuspended. 7 CFU / mL (spore count) was inoculated into artificial gastric fluid, shaken and mixed, incubated in a 37°C water bath, and samples were taken every 1 hour for viable bacteria count, 3 replicates / sample, for a total of 3 hours;

[0133] 2. Artificial intestinal fluid tolerance study: The bacteria treated with artificial gastric fluid were centrifuged at 3,000 rpm for 10 min, washed twice with PBS and resuspended. 7 CFU / mL (spore count) was inoculated in artificial intestinal fluid, and the experimental procedures were the same as above.

[0134] 3. Bile salt tolerance study: Add pig bile salt to RCM liquid culture medium to make the mass fraction NC, 0.10%, 0.20%, 0.30%, 0.40%, respectively, sterilize at 121℃ for 15min. Inoculate the strain at 1%, culture in an anaerobic incubator, observe the growth of the bacteria, 3 replicates / sample.

[0135] like Figure 2K and 2LAs shown, within 3 hours of incubation, artificial gastric juice and artificial intestinal juice had little effect on the survival rate of HADIG-CB003 strain spores. In addition, as shown in Table 4, 0.1% bile salt had little effect on the growth of HADIG-CB003 strain, while 0.2% bile salt significantly inhibited the growth of HADIG-CB003 strain, and 0.3% and 0.4% bile salt severely inhibited the growth of HADIG-CB003 strain.

[0136] Table 4 Tolerance of Clostridium butyricum HADIG-CB003 to different concentrations of bile salts

[0137] Strain name Strain number Inoculation volume Bile salt concentration Growth Clostridium butyricum HADIG-CB003 5％ NC +++ Clostridium butyricum HADIG-CB003 5％ 0.1％ +++ Clostridium butyricum HADIG-CB003 5％ 0.2％ ++ Clostridium butyricum HADIG-CB003 5％ 0.3％ + Clostridium butyricum HADIG-CB003 5％ 0.4％ +

[0138] Effects of Clostridium butyricum HADIG-CB003 on growth performance and intestinal flora of Kunming mice

[0139] After the mouse basal diet was crushed with a grinder, Clostridium butyricum spores and their fermentation liquid were added until the spore content was 1×10 10 cfu / kg basal diet, and add 10% sterile water, and mix well with a blender. Add an equal volume of sterile water to the basal diet of the blank group. Then use a 20mL sterile syringe to make the stirred powder into a compact feed strip of about 10cm in length, bake it in a 65℃ oven overnight, and store it at room temperature for feeding mice.

[0140] (1) Experimental animals: Twelve male Kunming mice (2 weeks old), weighing about 10 g, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. The animal breeding environment was a 12-hour day and night cycle, the indoor temperature was about 27°C, and all animals were free to eat and drink water. This study protocol was approved by the Animal Management Ethics Committee of Hunan Xiangnong Animal Pharmaceutical Co., Ltd., and all animals were handled in strict accordance with animal ethical procedures and regulations.

[0141] (2) Animal grouping and treatment: The experimental mice were randomly divided into a blank control group (NC) and a Clostridium butyricum experimental group (CB), with 6 mice in each group, and were housed separately in boxes. Both groups were fed a commercial basal diet, in which the Clostridium butyricum spore content in the basal diet of the Clostridium butyricum group was 1×10 10 cfu / kg, and the mice were fed continuously for 12 days, and the weight, food intake and water intake of the mice were recorded on time every four days.

[0142] Record data such as Figures 3A-3D As shown, the body weight (BW) of Kunming mice fed with Clostridium butyricum (CB) ( Figure 3A ), average daily weight gain ( Figure 3B ), average daily feed intake ( Figure 3C ) and feed-to-weight ratio (F / G)( Figure 3D ) had no significant difference compared with the blank group (NC).

[0143] (3) Obtaining intestinal flora samples: During the feeding period, after fasting for 12 h, the cecal contents of the mice in the control group and the experimental group were dissected and collected on the 12th day, placed in a disposable sampler for storage, and sent to Chengdu Luoning Biotechnology Co., Ltd. for third-generation sequencing analysis.

[0144] (4) Analysis of intestinal flora sequencing data: Basecalling: The data from the sequencing machine is in fast5 format. Guppy software is used to convert the electrical signal data recorded in the fast5 file into sequences and quality information recorded in fastq format. Data quality control: Use NanoFilt to remove: filter out sequences with an average quality lower than 10; remove sequences with a length of less than 1400bp and greater than 1600bp. Use the Uchime algorithm and gold database to remove chimeras and obtain valid data. Species annotation and evolutionary tree construction: Use Kraken2 to annotate each sequence with species, and count the number of species to construct an OTU table. For each species, select a sequence with the best quality as a representative sequence, and use muscle and FastTree to construct an evolutionary tree. Perform homogenization on each sample, and resample based on the sample with the least amount of data. Community composition analysis: Use R language for various data conversions, and ggplot2 package for graphics. In addition, data analysis software and database version information: NanoFilt: v2.7.1; Usearch: 10.0.240; R language: 4.0.3; Python: 3.7.4; SILVA database: 138.

[0145] 1) Alpha diversity analysis

[0146] After obtaining the OTU abundance matrix, the Alpha diversity analysis of the intestinal flora of the two groups of Kunming mice was performed, using the Chao1 index to measure species abundance, and the PD, Shannon, and Simpson indices to measure species diversity. The analysis was performed using the R language. The PD index was calculated using the package Picante, and the other indices were calculated using the package Vegan. The Wilcoxon rank sum test used the wilcox test function of the stats package, and the Kruskal test function was used for the Kruaksl-Wallis rank sum test of the two groups. Multiple comparisons were performed using the agricolae package.

[0147] The results are as follows Figure 3E , Figure 3F , Figure 3G and Figure 3HAs shown in the figure, the Chao1 index of the group fed with Clostridium butyricum was significantly lower than that of the blank group (p<0.05). Studies have shown that under the same species abundance, the larger the Chao1 value, the more species there are. Therefore, the above results indicate that feeding Clostridium butyricum significantly reduces the species diversity of the intestinal flora of Kunming mice.

[0148] 2) Beta diversity analysis

[0149] The analysis was performed using R. Unifrac distance was calculated using the GuniFrac package, and Bray-Curtis and Jaccard distances were calculated using the vegdits function of the Vegan package. PCoA analysis was performed using the ape package. PCA and NMDS analyses were performed using the vegan package. Cluster analysis was performed using the hclust function of the stas package. Anosim and PerMANOVA were calculated using the anosim function and adonis function of the vegan package, respectively.

[0150] In addition, PCoA analysis and NMDS statistics were performed using Bray-Curtis distance and Unweighted UniFrac algorithm, respectively. Fig. 3I and Figure 3J As shown in the figure, PCo1 accounted for 49.5% of the total variance, PCo2 accounted for 21.8% of the total variance, and the distance between the Clostridium butyricum group and the blank group was significantly separated, indicating that the principal components of the flora structure of the two groups of samples were significantly different. In the non-metric multi-dimensional scaling (NMDS) analysis, the stress value (Stress) = 0.08, which is less than 0.2, indicating that the grouping and sampling methods are reliable.

[0151] 3) Analysis of intestinal flora composition and relative abundance of dominant flora

[0152] In order to obtain the abundance information of each group at each classification level, based on the OTU abundance table and the annotated classification information table, this patent converts the data at each classification level such as Phylum, Class, Order, Family, Genus, and Species to obtain the relative abundance table of each sample at each classification level. In addition, in order to intuitively display the average abundance of the high-abundance groups (Top20) of each group of samples, the grouped data are averaged and displayed using a bar chart.

[0153] Among them, at the door level, such as Figure 4AAs shown in the results, after 12 days of adding Clostridium butyricum to the basal diet of Kunming mice, the abundance percentage of Proteobacteria increased by nearly 36% in the Clostridium butyricum group compared with the blank group, and the abundance percentages of Firmicutes, Bacteroidetes and Tenericutes decreased by nearly 34%, 70% and 72%, respectively. Therefore, the above research results show that at the phylum level, feeding Clostridium butyricum increased the abundance of Proteobacteria and reduced the abundance of Firmicutes, Bacteroidetes and Tenericutes. According to the Proceedings of the National Academy of Sciences, the intestinal microbiome of obese patients showed a decrease in the number of Bacteroidetes and an increase in the number of Firmicutes compared with normal weight individuals. This shows that the F / B ratio is an important biomarker for assessing the trend of individual obesity. Therefore, the relative proportions of Firmicutes and Bacteroidetes in the intestinal flora were further analyzed. Figure 4B As shown, compared with the blank control group, the F / B ratio in the intestinal flora of Kunming mice fed with Clostridium butyricum was significantly increased (p<0.05), suggesting that if the feeding period of Clostridium butyricum is extended, it may have the potential to promote the weight gain of economic animals.

[0154] At the level of Figure 4C As shown, compared with the blank group, the abundance percentages of Gammaproteobacteria and Betaproteobacteria in the Clostridium butyricum group were upregulated by nearly 125% (p<0.05) and 181% (p<0.05), respectively, while the abundance percentages of Alphaproteobacteria, Clostridia, Bacteroidia, Epsilonproteobacteria, Bacilli and Mollicutes were downregulated by nearly 31%, 27%, 70%, 55%, 63% (p<0.05) and 71%, respectively.

[0155] At the target level, Figure 4DAs shown, relative to the blank group, in the Clostridium butyricum group, Pseudomonadales, Rhizobiales and Burkholderiales were upregulated by nearly 317% (p<0.01), 27% and 217% (p<0.01), respectively, while Clostridiales, Bacteroidales, Sphingomonadales, Enterobacterales, Campylobacterales, Bacillales and Mycoplasmatales were downregulated by nearly 25%, 70%, 62%, 82%, 55%, 75% (p<0.05) and 70%, respectively.

[0156] At the family level, compared with the blank group, the Pseudomonadaceae and Rhizobiaceae families in the Clostridium butyricum group were up-regulated by nearly 317% (p<0.01) and 166%, respectively, while the Sphingomonadacea, Lachnospiracea, Enterobacteriacea, Helicobacteraceae, Bradyrhizobiaceae, and Bacteroidaceae families were down-regulated by nearly 62%, 36%, 83%, 55%, 14%, and 77%, respectively (P<0.01). Figure 4E shown).

[0157] At the genus level, Figure 4F As shown in the figure, the abundance percentage of Pseudomonas in the Clostridium butyricum group was upregulated by nearly 317% (p<0.01) compared with the blank group, and its relative abundance was the highest in the cecum of Kunming mice fed with Clostridium butyricum, reaching 44%. However, Sphingomonas, Helicobacter, Bacteroides, Escherichia, Blautia, Mycoplasma, Lachnoclostridium and Salmonella were downregulated by nearly 63%, 55%, 77%, 90%, 55%, 70%, 47% and 72%, respectively, and other genera decreased by nearly 13%.

[0158] At the species level, Figure 4GAs shown in the figure, Pseudomonas syringae had the highest abundance (9%) in the cecum of Kunming mice fed with Clostridium butyricum, followed by Pseudomonas chlororaphis and Sphingomonas hengshuiensis. Compared with the blank group, in the Clostridium butyricum group, the abundance percentages of Pseudomonas syringae, Pseudomonas chlororaphis, Pseudomonas putida and Pseudomonas sp.CC6-YY-74 were up-regulated by nearly 324% (p<0.01), 313% (p<0.01), 257% (p<0.01) and 298% (p<0.01), respectively, while those of Sphingomonas, Escherichia coli, Helicobacter typhlonius, Helicobacter hepaticus, Sphingomonas sp.LK11, Bacteroides vulgatus, Salmonella enterica enterica and Blautia sp. N6H1-15 were down-regulated by nearly 69%, 90%, 59%, 51%, 41%, 91%, 72% and 66% respectively, and the abundance of other species was up-regulated by nearly 6.4%. In addition, more than 90% of the bacteria at the species level have not been paid attention to and need further analysis.

[0159] In addition, this patent also uses a Sankey diagram, which is respectively in the category ( Figure 5A ) and species ( Figure 5B ) level more intuitively shows the changing trend of high-abundance species (Top10) in the Clostridium butyricum group relative to the blank group.

[0160] In addition, in order to show more species groups (Top 40) and data distribution characteristics of grouped samples, the present invention also uses box plots to perform community composition analysis. Figure 5C As shown, it was further found that relative to the blank group, the relative abundance of Parabacteroides, Staphylococcus and Alistipes in the butyric Clostridium group was significantly downregulated by nearly 78% (p<0.05), 97% (p<0.05) and 62% (p<0.05), respectively, while the relative abundance of Variovorax was significantly upregulated by nearly 465% (p<0.01).

[0161] At the species level, Figure 5DAs shown in the figure, compared with the blank group, the butyric Clostridium group further found that Pseudomonas protegens, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas koreensis, Pseudomonas sp.M30-35, Pseudomonas stutzeri, Pseudomonas mandelii, Pseudomonas sp.K2W31S-8 and Variovorax PMC12 sp.PMC12) were significantly upregulated by nearly 400% (p<0.05), 324% (p<0.05), 262% (p<0.05), 400% (p<0.05), 278% (p<0.05), 350% (p<0.05), 407% (p<0.05), 338% (p<0.05) and 467% (p<0.01), respectively, while the abundance percentage of Staphylococcus nepalensis was significantly downregulated by nearly 99% (p<0.05).

[0162] 4) Differential species analysis

[0163] In order to find important species with significant differences between groups, the present embodiment uses the LEfse tool (https: / / bitbucket.org / biobakery / biobakery / wiki / Home) to perform LEfSe analysis and screen species with statistical differences between the Clostridium butyricum group and the blank group (LDA value>4). The random forest analysis uses the randomForest package of R language. Metastas analysis uses R language scripts, and the calculation steps can be found in ( https: / / journals.plos.org / ploscompbiol / article?id=10.1371 / journal.pcbi.1000352 )).

[0164] The results are as follows Fig. 6A and Figure 6BAs shown, a total of 20 species contributed greatly to the difference between the two groups and were statistically significant, of which 11 species were significantly enriched in the blank group, including Enterobacterales, Enterobacteriaceae and Escherichia; 9 species were significantly enriched in the Clostridium butyricum group, mainly Pseudomonadales, Pseudomonadaceae and Pseudomonas. Therefore, the above results suggest that feeding Clostridium butyricum can significantly inhibit the growth of intestinal harmful bacteria. In summary, feeding Clostridium butyricum can effectively reduce the abundance of common harmful bacteria such as Escherichia coli, Helicobacter cecum, Helicobacter hepaticus and Salmonella in the cecal contents of Kunming mice, thereby providing theoretical support for the Clostridium butyricum HADIG-CB003 described in this patent to produce a probiotic effect on the host.

[0165] In addition, several patents reported Pseudomonas syringae (CN111286477B and CN104886152B), Pseudomonas chlororaphis (CN112725241B and CN107099474B), Pseudomonas putida (CN110144305B and CN108531425B), Pseudomonas defense (CN110643538A and CN110004087A), Pseudomonas fluorescens (CN102 168030B and CN101864376B), Korean Pseudomonas (CN112920965B and CN106916764B), Stuttgart Pseudomonas (CN101603022B and CN101096644B) and Pseudomonas CC6-YY-74 (CN106148227B) have great development potential in the application fields of plant disease biocontrol and degradation of autotoxic substances and organic pollutants in crop root soil. Therefore, the above research results show that feeding Clostridium butyricum may be beneficial to improve the biosafety of animal manure and its harmless treatment.

[0166] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A Clostridium butyricum, whose deposit number is CGMCC No.27853.

2. The Clostridium butyricum according to claim 1, characterized in that Its 16S rRNA sequence is shown in SEQ ID N0.

5.

3. A fermentation broth, characterized in that: The product is prepared by fermentation of Clostridium butyricum according to claim 1 or 2.

4. A feed, characterized in that: The method comprises the Clostridium butyricum according to claim 1 or 2 or the fermentation broth according to claim 3.

5. The feed according to claim 4, characterized in that The Clostridium butyricum is Clostridium butyricum powder.

6. The feed according to claim 5, characterized in that The preparation method of Clostridium butyricum powder comprises: inducing Clostridium butyricum to form spores, then adding auxiliary materials with a mass fraction of 5%-10% into the fermentation liquid, and preparing the Clostridium butyricum powder by spray drying.

7. The feed according to claim 6, characterized in that The auxiliary materials include one or more of maltodextrin, corn cobs, corn flour, corn starch and light calcium carbonate.

8. The feed according to any one of claims 4 to 7, characterized in that The spore content of Clostridium butyricum powder is ≥2×10 9 CFU / g, the effective amount of spores added in feed is 1-10×10 9 CFU / kg feed.

9. Use of the Clostridium butyricum according to claim 1 or 2 or the fermentation liquid according to claim 3 as or in the preparation of a feed additive for promoting the repair and regeneration of intestinal epithelial cells, promoting the development of the intestine of livestock and poultry, and enhancing their immunity and disease resistance.

10. Use of the Clostridium butyricum according to claim 1 or 2 or the fermentation liquid according to claim 3 in reducing harmful bacteria in intestinal flora.

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