CN112980735B

CN112980735B - Clostridium butyricum, microbial inoculum, application of clostridium butyricum and microbial inoculum and preparation method of microbial inoculum

Info

Publication number: CN112980735B
Application number: CN202110331624.1A
Authority: CN
Inventors: 佟毅; 陈欣怡; 李皓然; 卢宗梅; 杨鑫; 郑晓卫; 金渭武; 陈博; 陈影; 刘利利
Original assignee: Cofco Nutrition and Health Research Institute Co Ltd; Cofco Biotechnology Co Ltd
Current assignee: Cofco Nutrition and Health Research Institute Co Ltd; Cofco Biotechnology Co Ltd
Priority date: 2021-03-29
Filing date: 2021-03-29
Publication date: 2022-08-02
Anticipated expiration: 2041-03-29
Also published as: CN112980735A

Abstract

The invention relates to the field of microorganisms, and discloses clostridium butyricum, a microbial inoculum, application of the clostridium butyricum and the microbial inoculum, and a preparation method of the microbial inoculum. Clostridium butyricum (C)Clostridium butyricum) The preservation number of (2) is CGMCC No. 20417. The strain is derived from caecum contents of healthy pigs; has better antibacterial activity and butyric acid production performance. The microecological preparation prepared from clostridium butyricum has the function of regulating the intestinal tracts of animals, and butyric acid generated by the metabolism of the microecological preparation can reduce the pH value of the intestinal tracts and inhibit the growth of harmful bacteria; when used together with other microorganisms as a water adjusting agent, the water quality can be improved.

Description

Clostridium butyricum, microbial inoculum, application of clostridium butyricum and microbial inoculum and preparation method of microbial inoculum

Technical Field

The invention relates to the field of microorganisms and discloses a clostridium butyricum strainClostridium butyricum) A microbial inoculum, application thereof and a preparation method of the microbial inoculum.

Background

The microbial agent is a live microbial feed additive which can improve the intestinal health of animals and adjust the balance of intestinal flora and has favorable effect on the health of the animals, and has the advantages of no residue, no side effect, no environmental pollution, no drug resistance, low cost, convenient use and the like. 35 microbial strains are recorded in 2013 edition feed additive variety catalog of Ministry of agriculture in China, specifically lactobacillus (22 species), bacillus (6 species), saccharomycete (2 species), photosynthetic bacteria (1 species), mould (2 species), propionibacterium (1 species) and clostridium butyricum (1 species). The probiotics mainly improve the balance of intestinal microorganisms, inhibit the invasion and growth of pathogenic bacteria and stabilize intestinal flora through competitive exclusion, antibacterial substance generation and other ways; short chain fatty acid can be produced, and various digestive enzymes can be secreted, so that the host can directly utilize the short chain fatty acid or promote the metabolic absorption of the animal on nutrient substances; can also be used for secondary animal intestinal mucosa immunoreaction to enhance the animal immune function; can also reduce the ammonia level in animal intestinal tract and blood and improve the culture environment; the probiotics have the functions of purifying water quality and improving water body.

The clostridium butyricum is a strict anaerobic bacterium and mainly exists in cheese, natural yoghourt, healthy animal intestinal tracts, animal excrement and soil, metabolites of the clostridium butyricum mainly comprise butyric acid, acetic acid, lactic acid and the like, cells in PYG broth are straight rods, the two ends of the cells are blunt, the cells move and generate flagella circularly, and the diameter of the cells is 0.5-1.7 mu m multiplied by 2.4-7.6 mu m. Single, paired or short chain, occasionally filamentous, sporulated, mesogenic or subterminal, generally do not cause cell expansion. The clostridium butyricum exists in an endophytic spore state, has the characteristics of strong tolerance to low gastric acid, bile resistance and the like, has strong resistance to the external environment, and can still keep higher activity and probiotic quantity after being treated by high-temperature processing, granulation and the like.

Efficacy of clostridium butyricum: 1. the feed additive can improve the consumption and absorption capacity of animals, improve the growth performance and improve the feed conversion rate. 2. As probiotics, the bacteriostatic active substances generated by the metabolism of the probiotics can inhibit the growth of pathogenic bacteria and putrefying bacteria, reduce the generation of harmful substances such as amines, indoles, hydrogen sulfide and the like, promote the reproduction of intestinal beneficial bacteria and improve the intestinal health of animals. 3. Metabolism produces a probiotic. The clostridium butyricum in the intestinal tract can generate substances such as B vitamins, vitamin K and the like, wherein butyric acid is a main metabolite of the clostridium butyricum, and butyric acid is a main nutrient substance for regeneration and repair of epithelial tissue cells of the intestinal tract. 4. Activating the immune system of the organism, promoting the immunity of the organism to be enhanced, and further maintaining the health of animals. Clostridium butyricum in animal husbandry is mainly applied to feed additives and veterinary drugs, can effectively improve the capability of resisting pathogenic microorganisms of animal organisms, inhibit the generation of intestinal putrefaction products, increase the generation of short-chain fatty acids in intestinal tracts, and improve the immunity, growth performance and product quality of the organisms. In aquaculture, the clostridium butyricum can also decompose organic matters in water, inhibit the growth of harmful bacteria, improve water quality and maintain stable water quality.

The clostridium butyricum often causes the lag phase of strain growth at the early stage of fermentation due to the problems of unstable fermentation conditions and the like in the fermentation process, and in addition, the strains often have the problems of less bacteria number of fermentation liquor, lower strain activity, lower spore rate and the like in the fermentation process.

Disclosure of Invention

The invention aims to provide a strain of clostridium butyricum (Clostridium butyricum) (III)Clostridium butyricum) The clostridium butyricum has the advantages of high activity and high spore rate in the fermentation process, and the fermentation process is simple, so that the fermentation contamination is effectively reduced, the cost of fermentation raw materials and time is reduced, and the stability of the microbial inoculum is obviously improved.

One aspect of the invention provides a strain of Clostridium butyricumClostridium butyricum) The preservation number of the clostridium butyricum is CGMCC number 20417.

In a second aspect, the present invention provides a microbial agent comprising Clostridium butyricum (Clostridium butyricum) (described above)Clostridium butyricum）。

The third aspect of the invention provides a preparation method of a microbial inoculum, which comprises the following steps: inoculating clostridium butyricum into a fermentation medium for fermentation to obtain fermentation liquor;

optionally, mixing the fermentation liquor with a protective agent and/or a solid carrier to obtain a microbial inoculum;

wherein the clostridium butyricum is the clostridium butyricum and/or the microbial inoculum.

In a fourth aspect, the present invention provides a microbial inoculum prepared by the method as described above.

In a fifth aspect, the invention provides applications of the clostridium butyricum described above and/or the microbial inoculum described above in production of butyric acid, preparation of microecologics, water adjusting agents and feeds.

The invention can obtain the following beneficial effects:

the preservation number of the clostridium butyricum is CGMCC number 20417. The strain is derived from caecum contents of healthy pigs; has good antibacterial activity and thallus activity.

The clostridium butyricum is nontoxic and nonpathogenic, can be added into a microorganism list in feed, and is safe to human and livestock and environment-friendly.

The microecological preparation prepared from clostridium butyricum has the function of regulating the intestinal tracts of animals, and butyric acid generated by the metabolism of the microecological preparation can reduce the pH value of the intestinal tracts and inhibit the growth of harmful bacteria; the strain is used together with other microorganisms as a water adjusting agent, and can improve water quality.

The preparation method of the microecological preparation provided by the invention has the advantages that the process is simple, the fermentation contamination is effectively reduced, the cost of fermentation raw materials and time is reduced, and the quality stability of the product is obviously improved.

Biological preservation

The strain of the invention is clostridium butyricum (Clostridium butyricum) (III)Clostridium butyricum) And was deposited in the general microbiological culture collection center of the China Committee for culture Collection of microorganisms (address: west road No. 1, north west of the township, beijing, ministry of sciences, china, institute of microbiology, zip code: 100101) (abbreviated as CGMCC for preservation unit), and the preservation number is CGMCC No. 20417.

Drawings

FIG. 1 is a diagram showing the form of a microorganism of Clostridium butyricum of the present invention under a microscope;

FIG. 2 is a graph showing the cell concentration and butyric acid production in the fermentation broth in example 6 of the present invention.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

One aspect of the invention provides a strain of Clostridium butyricumClostridium butyricum) The preservation number of the clostridium butyricum is CGMCC No. 20417。

Screening of clostridium butyricum of the invention: collecting caecum content of healthy pig, weighing 5.0 g, dissolving in 25 mL sterile normal saline, and shaking to obtain 10 ^-1 Bacterial suspension; diluting to 10 degrees in stages ^-6 And (3) coating the bacterial suspensions with different dilution gradients on a reinforced clostridium culture medium (RCM) plate in an anaerobic workstation, culturing for 24 hours, picking typical bacterial colonies on the plate for microscopic examination (see figure 1), and primarily judging the type of the bacterial strain. The strain with the fusiform shape is streaked and purified.

And (3) selecting the separated single bacterial colony, inoculating the single bacterial colony in a centrifuge tube in a clostridium culture medium, carrying out anaerobic culture at 37 ℃ for 24 hours, and taking a small amount of bacterial liquid for gram staining microscopic observation.

Similar strains were screened for subsequent molecular characterization by gram-stained microscopy and by reference to Bergey's Manual of Systematic Bacteriology (second edition).

16srDNA sequencing and identifying to obtain the strain and clostridium butyricum: (Clostridium butyricum) The strain has the highest homology, and the 16S rDNA complete sequence is shown as SEQ ID NO: 1:

TGCAAGTCGAGCGATGAAGCTCCTTCGGGAGTGGATTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTCATAGAGGGGAATAGCCTTTCGAAAGGAAGATTAATACCGCATAAGATTGTAGTACCGCATGGTACAGCAATTAAAGGAGTAATCCGCTATGAGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCAACGCCGCGTGAGTGATGACGGTCTTCGGATTGTAAAGCTCTGTCTTTAGGGACGATAATGACGGTACCTAAGGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTACTGGGCGTAAAGGGAGCGTAGGTGGATATTTAAGTGGGATGTGAAATACCCGGGCTTAACCTGGGTGCTGCATTCCAAACTGGATATCTAGAGTGCAGGAGAGGAAAGGAGAATTCCTAGTGTAGCGGTGAAATGCGTAGAGATTAGGAAGAATACCAGTGGCGAAGGCGCCTTTCTGGACTGTAACTGACACTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTAGGGGTTGTCATGACCTCTGTGCCGCCGCTAACGCATTAAGTATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTAGACTTGACATCTCCTGAATTACTCTGTAATGGAGGAAGCCACTTCGGTGGCAGGAAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTAAGTCCCGCACGAGCGCACCCTTATTGTAGTTGCTCCAT。

the strain of the invention is clostridium butyricum (Clostridium butyricum) (III)Clostridium butyricum) And was deposited in China general microbiological culture Collection center (address: west road No. 1, north west of the township, beijing, ministry of sciences, china, institute of microbiology, zip code: 100101) (CGMCC as short as preservation organization), and the preservation number is CGMCC No. 20417.

Experiments show that the clostridium butyricum (Clostridium butyricum) provided by the inventionClostridium butyricum) Has better bacteriostatic activity on conditional pathogenic bacteria staphylococcus aureus and escherichia coli, and has stronger inhibiting effect on gram-negative (G-) bacteria than gram-positive (G +) bacteria.

The microbial inoculum prepared from clostridium butyricum has an adjusting effect on animal intestinal tracts, and butyric acid generated by the metabolism of the microbial inoculum can reduce the pH value of the intestinal tracts and inhibit the growth of harmful bacteria; the strain is used together with other microorganisms as a water adjusting agent, and can improve water quality.

In the present invention, the composition of the RCM medium preferably includes: 1-5g of yeast extract, 5-15g of beef extract, 0.5-1.5g of soluble starch, 3-7g of glucose, 0.1-1g of cysteine hydrochloride, 1-5g of NaCl, 1-5g of NaAc and 1-5mg/L of resazurin. Adjusting pH to 8-9.

Preferably, the microbial agent contains a live bacterium of the clostridium butyricum.

Preferably, the number of the live clostridium butyricum in the microbial inoculum is more than or equal to 2 hundred million cfu/g.

The microbial inoculum can endure dry heat treatment at 85 ℃ for 3 min.

The microbial inoculum may be in the form of a solid, liquid or semi-liquid, etc.

That is, the microbial inoculum can be prepared by directly preparing fermentation liquor or mixing with other substances.

Preferably, the clostridium butyricum is inoculated in the form of seed liquid.

The seed solution can be prepared by a conventional preparation method in the field, for example, clostridium butyricum as described above or a microbial inoculum as described above can be inoculated into a seed culture medium for propagation to obtain the seed solution.

The clostridium butyricum or the microbial inoculum can be subjected to activation treatment before being expanded in a seed culture medium, and the activated culture medium can be the RCM culture medium of the first aspect.

Preferably, the activation conditions of clostridium butyricum comprise: the culture temperature is 30-37 deg.C, pH is 6.0-7.0, culture time is 10-24 hr, and anaerobic condition.

In the present invention, the culture may be performed in a seed culture medium conventionally used in the art, and may be a culture medium conventionally used in the art (e.g., RCM medium). Preferably, the seed medium comprises a carbon source, a nitrogen source, potassium dihydrogen phosphate, calcium carbonate, and optionally magnesium sulfate.

In the present invention, the carbon source may be a carbon source commonly used in the art, including but not limited to at least one of starch, glucose, glycerol, sucrose and molasses.

The starch may include, among other things, soluble starch.

In the present invention, the nitrogen source may be a nitrogen source commonly used in the art, including but not limited to yeast extract, beef extract, peptone, urea, ammonium sulfate, ammonium nitrate, etc., and preferably, the nitrogen source is yeast extract and/or ammonium sulfate.

In a preferred aspect of the invention, the seed medium comprises a carbon source, yeast extract, ammonium sulfate, dipotassium hydrogen phosphate, calcium carbonate, and optionally magnesium sulfate heptahydrate; preferably, the carbon source is selected from at least one of starch, glycerol and glucose; preferably, in the seed culture medium, the content of the carbon source is 15-30g/L, the content of the yeast extract powder is 5-15 g/L, the content of ammonium sulfate is 1-5 g/L, the content of dipotassium hydrogen phosphate is 1-3g/L, the content of magnesium sulfate heptahydrate is 0-0.3 g/L, and the content of calcium carbonate is 1-3 g/L.

In the present invention, resazurin may also be contained in the seed culture medium, and resazurin may be provided in the form of a resazurin solution, which may be at any concentration, such as 0.1 to 1 wt%.

Preferably, the content of the resazurin in the seed culture medium is 1-5 mg/L.

Preferably, the culture conditions of clostridium butyricum comprise: the culture temperature is 30-37 deg.C, pH is 6-7, culture time is 18-24 h, and anaerobic condition is adopted.

Wherein the anaerobic condition may be provided by nitrogen blowing or paraffin liquid sealing.

The culture may be at least one stage of culture as long as a seed solution can be obtained.

In a preferred embodiment of the present invention, the clostridium butyricum is inoculated in the form of a seed solution prepared by a method comprising: and respectively inoculating clostridium butyricum into a seed culture medium for primary seed culture and secondary seed culture to obtain seed solutions.

It is understood that the conditions for the primary seed culture and the secondary seed culture may be the same or different.

In the present invention, the method of fermentation preferably comprises: and inoculating the clostridium butyricum seed liquid into a fermentation culture medium for fermentation to obtain fermentation liquid.

Preferably, the number of viable bacteria in the seed liquid is 1 × 10 ⁸ -5×10 ⁸ cfu/mL。

Preferably, the amount of inoculation is 1-10 volume%.

In the present invention, preferably, the composition of the fermentation medium includes: the fermentation medium comprises a carbon source, a nitrogen source, monopotassium phosphate, magnesium sulfate and calcium carbonate.

The types of carbon source and nitrogen source are described above, and are not described herein again.

In a preferred embodiment of the invention, the fermentation medium comprises glucose, yeast extract, ammonium sulfate, dipotassium phosphate, magnesium sulfate heptahydrate and calcium carbonate.

Preferably, in the fermentation medium, the content of glucose is 15-20 g/L, the content of yeast extract powder is 10-15 g/L, the content of ammonium sulfate is 2-5 g/L, the content of dipotassium hydrogen phosphate is 1-3g/L, the content of magnesium sulfate heptahydrate is 0.1-0.2 g/L, and the content of calcium carbonate is 2-3 g/L.

In the invention, resazurin may be contained in the fermentation medium, and preferably, the content of resazurin in the fermentation medium is 1-5 mg/L.

Preferably, the fermentation conditions of the clostridium butyricum comprise: the temperature is 30-37 deg.C, pH is 6-7, and the time is 10-24 h. The stirring speed in the fermentation process is determined according to the volume of the fermentation tank, and can be adjusted by a person skilled in the art according to actual conditions.

The fermentation is preferably carried out under anaerobic conditions, which may be provided by nitrogen blowing or paraffin liquid encapsulation.

The clostridium butyricum provided by the invention can be metabolized to generate butyric acid in a fermentation process, and preferably, a neutralizing agent is also added in the fermentation process. Neutralizing butyric acid with the neutralizing agent to form butyrate. The resulting fermentation supernatant (e.g., from solid-liquid separation) can be processed to produce butyrate (e.g., sodium or calcium salt) for use as a feed additive.

The neutralizing agent may be an alkaline material conventionally used in the art, and includes, but is not limited to, sodium carbonate, sodium hydroxide, calcium carbonate, and the like. Preferably, the neutralising agent is sodium carbonate.

The sodium carbonate may be added in the form of a solution, preferably at a concentration of 100-250 g/L.

The fermentation liquor obtained by fermentation can be directly used as a microbial inoculum, and the microbial inoculum can also be prepared by mixing the fermentation liquor with an optional protective agent and/or a solid carrier and drying the mixture.

The drying treatment may be a preparation method conventional in the art, and for example, may be air drying, freeze drying, spray drying, oven drying, vacuum drying, etc., and preferably spray drying.

Preferably, the conditions of the spray drying include: the spraying pressure is 0.008-0.012MPa, the inlet temperature is 120-140 ℃, and the outlet temperature is 40-60 ℃.

In the present invention, the air volume and the liquid feeding speed of the spray drying can be adjusted according to the size of the spray drying equipment, and preferably, the air volume is 0.3-0.5 m ³ Min, liquid feeding speed is 10-12 mL/min.

In an embodiment of the invention, the fermentation broth of clostridium butyricum obtained by fermentation is concentrated to obtain clostridium butyricum bacterial sludge, and the bacterial sludge is mixed with a protective agent and dried to prepare the microbial inoculum.

The concentration method may be a concentration method conventional in the art, and may be, for example, a method of centrifugation or filtration, as long as the clostridium butyricum puree can be obtained.

Preferably, the protective agent is selected from at least one of starch, sucrose, lactose, trehalose, sodium citrate, skim milk powder and maltodextrin.

Preferably, the protectant is added in the form of a protectant solution. More preferably, the content of the protecting agent in the protecting agent solution is 3-8 wt%.

In a preferred embodiment of the invention, the protective agent comprises a water-soluble starch and maltodextrin. Preferably, the weight ratio of the water-soluble starch and the maltodextrin is 1: 0.5-15.

Preferably, the protectant is present in an amount of 3 to 5 weight percent, based on the total weight of the protectant solution and the clostridium butyricum puree.

In a preferred embodiment of the invention, the fermentation broth of clostridium butyricum obtained by fermentation is mixed with a solid carrier and dried to prepare the microbial inoculum.

It should be understood that the fermentation broth may be concentrated or diluted as long as the cells and the solid carrier are uniformly mixed.

The solid carrier includes, but is not limited to, bran, soybean meal, and corn meal.

The drying may be carried out in a fluidised bed, the particular conditions being adjusted as required.

The microbial inoculum prepared by spray drying can endure 85 ℃ and 3 min dry heat treatment, and the survival rate of the microbial inoculum is more than 90 percent.

In a preferred embodiment of the present invention, the preparation method of the microbial inoculum comprises:

(1) preparing a seed solution: respectively carrying out primary seed culture and secondary seed culture on clostridium butyricum to obtain seed solutions;

(2) fermentation culture: inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 1-10 vol%, and culturing for 10-24h at 30-37 deg.C and pH of 6-7 under anaerobic condition to obtain fermentation broth;

(3) and mixing the fermentation liquor with a protective agent and/or a solid carrier, and performing spray drying to obtain the microbial inoculum.

The clostridium butyricum microbial inoculum can also comprise sodium butyrate or calcium butyrate and the like under exogenous conditions to be mixed as a feed additive.

The properties of the microbial inoculum have been described in detail in the second aspect, and are not described in detail herein.

In a fifth aspect, the invention provides applications of clostridium butyricum as described above and/or microbial inoculum as described above in production of butyric acid, preparation of microecologics, water regulating agents and feeds.

The present invention will be described in detail below by way of examples.

The composition of the RCM medium was (1L): 3g of yeast extract, 10g of beef extract, 1g of soluble starch, 5g of glucose, 0.5g of cysteine hydrochloride, 3g of NaCl, 3g of NaAc and 3mg/L of resazurin. Adjusting pH to 8.5, and performing moist heat sterilization at 121 ℃ for 30 min.

The seed culture medium comprises the following components: 20 g/L of glucose, 10 g/L of yeast extract powder, 3g/L of ammonium sulfate, 2 g/L of sodium chloride, 0.2 g/L of magnesium sulfate and 1.5 g/L of dipotassium phosphate.

The fermentation medium comprises the following components: 20 g/L of glucose, 10 g/L of yeast extract powder, 3g/L of ammonium sulfate, 2 g/L of sodium chloride, 0.2 g/L of magnesium sulfate and 1.5 g/L of dipotassium phosphate.

In the following examples, significance analysis was performed using single factor method analysis using SPSS17.0 (SPSS inc., USA) statistical software, and Dunan's multiple comparison analysis was performed for significant differences. Data are expressed as mean ± sem.

Example 1

This example illustrates the isolation of Clostridium butyricum.

Collecting caecum content of healthy pig, weighing 5.0 g, dissolving in 25 mL sterile normal saline, and shaking to obtain 10 ^-1 Bacterial suspension; diluting to 10 degrees in stages ^-6 Coating bacterial suspensions with different dilution gradients on a reinforced clostridium culture medium (RCM) plate in an anaerobic workstation, culturing for 24h, selecting typical bacterial colonies on the plate for microscopic examination (see figure 1), primarily judging the type of the bacterial strain, and performing plate streaking purification on the bacterial strain with the fusiform thallus form to obtain a target bacterial strain.

Example 2

This example illustrates the identification of strains.

1. Cell morphological characterization

The single colony separated in the example 1 is selected and inoculated in a centrifuge tube in a clostridium culture medium, anaerobic culture is carried out at 37 ℃ for 24h, and then a small amount of bacterial liquid is taken for gram staining microscopic observation.

Gram stain test: and (3) dropwise adding an appropriate amount of bacteria onto the glass slide, fixing for 2-3 times by flame, dropwise adding ammonium oxalate crystal violet solution, and dyeing the fixed smear for 1 min. The staining solution on the smear is slowly rinsed with water and blotted dry with absorbent paper. Mordant dyeing: dripping 1 drop of iodine solution, dyeing for 1min, and washing with water. Absorbing residual water, continuously dropwise adding 95% ethanol for decolorizing for 20-30s until effluent liquid is purple, and immediately washing with water. Absorbing residual water, continuously dropwise adding 95% ethanol for decolorizing for 20-30s until effluent liquid is purple, and immediately washing with water.

2. 16S rDNA gene sequence analysis and identification:

and (3) picking the strain separated in the cell morphological characteristic identification, inoculating the strain into an RCM culture medium, carrying out anaerobic culture at 37 ℃ for 24h, and then taking a small amount of sample to carry out PCR amplification on a 16S rDNA sequence. With 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2) as a forward primer, using 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 3) as reverse primer, the PCR reaction system (25. mu.L) was: takara PrimerSTARmix 12.5. mu.L; ddH ₂ O9.5 mu L; 1 mu L of forward and reverse primers respectively; bacterial suspension 1. mu.L. PCR amplification procedure: pre-denaturation at 98 deg.C for 10min, denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 5s, extension at 72 deg.C for 2min, performing 30 cycles, extension at 72 deg.C for 10min, and storing at 4 deg.C. The PCR product is detected by 1% agarose gel electrophoresis, and the positive product with the fragment of about 1500bp is purified and then subjected to sequence determination.

The sequencing result shows that the length of the 16SrRNA gene sequence of the selected strain is 1,047bp, and the nucleotide sequence is shown as SEQ ID NO: 1:

strains selected using BLAST on NCBI and the use thereofClostridium butyricumThe strain has the highest homology.

That is, the identified strain should be Clostridium butyricum: (C.butyricum)Clostridium butyricum）。

Example 3

This example illustrates the high temperature detection of Clostridium butyricum according to the invention

0.1 mL of each clostridium butyricum strain (CGMCC number 20417 and CICC 23847) preserved in a glycerinum tube is inoculated into 10 mL of liquid RCM culture medium, and anaerobic culture is carried out for 48h to form spores. Respectively placing 2 strains of bacteria in oven at 80 deg.C, 85 deg.C, 90 deg.C and 95 deg.C for 3 min, immediately cooling, and detecting viable count. Adding 5g of bacterial liquid to be detected into 45 mL of physiological saline, fully and uniformly mixing, absorbing 1 mL of the bacterial liquid, transferring the bacterial liquid into 9 mL of physiological saline, and uniformly mixing to form 10 ^-1 Diluting the solution, sucking 10 ^-1 1 mL of the dilution was transferred to 9 mL of physiological saline to form 10 ^-2 Dilution of the resulting mixture to form 10 ^-3 ，10 ^-4 ，10 ^-5 ，10 ^-6 The diluted solutions are respectively absorbed by 10 ^-4 ，10 ^-5 ，10 ^-6 Each 100. mu.L of the diluent was coated on a Clostridium butyricum medium, and subjected to inverted anaerobic culture at 37 ℃ for 48 hours, viable bacteria were counted and the survival rate was calculated, and the results are shown in Table 1.

Survival rate = (number of viable bacteria in control group-number of viable bacteria in test group)/number of viable bacteria in control group x 100%.

The result shows that the survival rate of the clostridium butyricum CGMCC number 20417 is obviously higher than that of the clostridium butyricum CICC23847, and the clostridium butyricum CGMCC number 20417 has good high-temperature resistance.

TABLE 1

Example 4

This example illustrates the acid tolerance detection of Clostridium butyricum according to the invention

0.1 mL of the Clostridium butyricum strain preserved in the glycerinum tube was inoculated into 10 mL of liquid RCM medium and anaerobically cultured for 18 hours. The activated strain was inoculated at 2% to RCM medium at pH 1.5, pH 2.5, pH 3.5, pH4.5, and cultured by static culture at 37 ℃. The inoculated culture solution for 1h was taken, viable bacteria were counted by plate dilution and the survival rate was calculated, and the results are shown in table 2.

The result shows that the survival rate of the clostridium butyricum CGMCC number 20417 is obviously higher than that of the clostridium butyricum CICC23847, the clostridium butyricum CGMCC number 20417 has good acid resistance and higher survival rate in a low pH environment, and more clostridium butyricum can reach intestinal tracts through a stomach acid environment.

TABLE 2

Example 5

This example illustrates the detection of the bacteriostatic activity of Clostridium butyricum according to the invention

0.1 mL of the Clostridium butyricum strain preserved in the glycerinum tube was inoculated into 10 mL of liquid RCM medium and anaerobically cultured for 18 hours. Inoculating the activated strain to an RCM culture medium, carrying out anaerobic culture for 24h, then inoculating to a seed culture medium according to the inoculation amount of 5%, and carrying out anaerobic culture for 48h at 37 ℃ to obtain a seed solution. Centrifuging the seed solution at 14000 r/min for 5 min, collecting supernatant, and filtering with 0.2 μm filter membrane for sterilization.

Inoculating Escherichia coli and Staphylococcus aureus on LB solid medium, culturing at 37 deg.C for 24 hr, sequentially eluting thallus with sterile normal saline to obtain bacterial suspension, and diluting the bacterial concentration of the bacterial suspension to 10 ⁶ CFU/mL，And (5) standby.

The target bacteria plate is prepared by adopting a double-layer plate method. Adding 15 ml of LRCM culture medium into a culture dish, and taking the culture dish as a lower layer after solidification; and adding 5 mL of RCM culture medium containing 2% of different target bacteria suspensions to the solidified lower culture medium to serve as an upper layer for later use.

The antimicrobial activity was determined by the oxford cup method. Vertically placing the Oxford cup on prepared different target bacteria flat plates, enabling the Oxford cup to have no gap with the surface of a culture medium, adding 150 mu L of clostridium butyricum fermentation supernatant into the Oxford cup, culturing for 16h at 37 ℃, and observing and measuring the diameter of a bacteriostatic circle. Kanamycin was used as a positive control and sterile water as a negative control, and two replicates were assayed per group. The larger the inhibition zone, the stronger the antibacterial activity. The results are shown in Table 3 below.

The bacteriostatic effect of the clostridium butyricum strain (CICC 23847) was measured according to the above-described method, and the results are shown in table 3.

TABLE 3

The clostridium butyricum has better bacteriostatic activity on conditional pathogenic bacteria (staphylococcus aureus and escherichia coli), and has stronger inhibiting effect on gram-negative (G-) bacteria than gram-positive (G +) bacteria.

Example 6

This example illustrates the fermentation performance of Clostridium butyricum according to the present invention.

The clostridium butyricum is amplified in a 2L fermentation tank, and the spore generation condition in the culture process is detected.

Seed liquid was prepared according to the method described in example 5, inoculated into a fermentation medium and cultured, with a liquid loading of 60%, under the culture conditions: the initial rotation speed is 200 r/min at 37 ℃, the anaerobic environment is controlled by adopting a nitrogen replacement method, and the initial pH is 6.0.

Sampling and detecting the thallus concentration OD of the fermentation liquor ₆₀₀ The values and butyric acid production results are shown in FIG. 2.

Detecting the yield of butyric acid in the fermentation process, wherein the detection method comprises the following steps: using a biorad aminex HPX-87H chromatographic column, mobile phase: 0.005 mol/L sulfuric acid solution, flow rate: 0.5 mL/min, column temperature: 55 ℃, detector: RID, sample size: 20 μ L.

Detecting the spore generation rate by microscopic examination and judging the fermentation end time. After fermenting for 20h, the spore generation rate of clostridium butyricum is more than 98%, and the viable count is 2.9 multiplied by 10 ⁹ CFU/mL, end fermentation. OD of fermentation broth at fermentation end ₆₀₀ The value was 9.98, and the butyric acid yield was 11.57 mg/mL.

The fermentation performance of CICC23847 was measured according to the method described above, and after 20h of fermentation, the sporulation rate was over 94% and the viable count was 1.1X 10 ⁹ CFU/mL, OD of fermentation broth ₆₀₀ The value was 8.50 and the butyric acid yield was 10.24 mg/mL.

Example 7

This example illustrates a method for preparing a Clostridium butyricum preparation.

And (3) centrifuging 6000 r/min of clostridium butyricum fermentation liquor obtained in the embodiment 6 at 4 ℃ for 10min to obtain clostridium butyricum bacterial sludge. Adding a protective agent solution (wherein the content of soluble starch is 3 wt% and the content of maltodextrin is 2 wt%) 5 times of the weight of the bacteria into the clostridium butyricum bacterial sludge, fully dissolving to enable the bacteria to be uniformly suspended, and balancing for 60 min when the pH value is detected to be maintained at about 6.0. The microbial inoculum is prepared by spray drying, and the spray conditions comprise: the inlet temperature is 120 ℃, the outlet temperature is 60 ℃, and the air quantity is 0.4m ³ And/min, the liquid feeding speed is 12 mL/min, and the spraying pressure is 0.01 MPa, so that the clostridium butyricum microbial inoculum is prepared.

The obtained microbial inoculum has viable count of 2 × 10 ¹⁰ CFU/g。

Standing at room temperature for 10 months with viable count of 1.3 × 10 ¹⁰ CFU/g。

The obtained microbial inoculum is subjected to dry heat treatment (oven 85 deg.C, 3 min) and wet heat treatment (water bath 85 deg.C, 3 min), and the change of viable count before and after treatment is detected.

The survival rate of the microbial inoculum after dry heat treatment is 95 percent, and the survival rate of the microbial inoculum after wet heat treatment is 74 percent.

The preparation method of CICC23847 microbial inoculum is characterized in that the culture time is prolonged to 22h to make the spore rate more than 98%, and the viable count is 2.3 × 10 ⁹ CFU/mL. The survival rate is determined, and the number of viable bacteria is 1.2 multiplied by 10 ⁹ CFU/g. Standing at room temperature for 10 months with viable count of 5.7 × 10 ⁸ CFU/g. The survival rate of the microbial inoculum after dry heat treatment is 92 percent, and the survival rate of the microbial inoculum after wet heat treatment is 56 percent.

Example 8

This example illustrates the use of C.butyricum in breeding pigs.

In order to evaluate the influence of clostridium butyricum on the growth performance of weaned piglets, 120 healthy weaned boars with the weight of about 7.33kg are selected and divided into 4 groups (a positive control group, an experimental group, a comparative strain group and a negative control group) according to a completely random group of the weights, each treatment is carried out for 5 times, and each treatment is carried out for 6 weaned piglets. The composition of the basal ration is shown in table 4 below. The nutritional levels of crude protein, calcium, total phosphorus, etc. were kept consistent in each treatment group, with the nutritional levels being referenced to NRC (2012) and chinese swine feeding standards (2004).

TABLE 4

The processing mode of the positive control group is as follows: basal diet +2000ppm zinc oxide.

The treatment mode of the negative control group is as follows: basal diet.

The treatment mode of the experimental group is to add the microbial inoculum (prepared by the method of example 7) of clostridium butyricum CGMCC number 20417 of the invention into basic daily ration, and the addition amount is 0.02 weight percent.

The treatment method of the comparative strain group was to add the microbial inoculum of the comparative strain cic 23847 (prepared according to the method of example 7) to the basal diet in an amount of 0.02 wt%.

At the beginning (day 0), 7 days, 21 days and the end (day 35) of the experiment, the individual weight of weaned piglets and the feed weight were weighed, the number of piglets with diarrhea was counted at 9 am every day, and the Average Daily Gain (ADG), Average Daily Feed Intake (ADFI) and feed-weight ratio (feed: gain, F/G) were calculated, and the results are shown in Table 5.

Average Daily Gain (ADG) = (end-trial weight-initial-trial weight)/number of trial days;

average Daily Feed Intake (ADFI) = (feed amount during test period-remaining amount of feed during test period)/test days.

Feed weight ratio = average daily feed intake/average daily gain.

Diarrhea rate = number of diarrhea pigs/(number of test pigs × total days) × 100%

TABLE 5

Note: labeling with different lower case letters indicated significant differences between groups (P < 0.05).

As can be seen from Table 5, the addition of Clostridium butyricum, especially Clostridium butyricum of the present invention, can reduce the feed-weight ratio and diarrhea rate of weaned piglets, and has significant statistical significance.

Example 9

This example illustrates the use of clostridium butyricum in broiler production.

In order to evaluate the influence of clostridium butyricum on the growth performance of broiler chickens, healthy white feather broiler chickens of 1 day old are selected, 576 broiler chickens are randomly divided into 4 treatment groups, each group comprises 144 broiler chickens, the interior of each treatment group is randomly divided into 8 repeated columns, each group comprises 18 repeated chickens, all the broiler chickens are cock chickens, and the production test period is 42 days. The 4 treatment groups were a positive control group (basal diet +20 g/t fast manure), a negative control group (basal diet), a control strain group (basal diet + the microbial inoculum of cic 23847 prepared in example 7), and an experimental group (basal diet + the microbial inoculum of clostridium butyricum of the present invention prepared in example 7), respectively, wherein the addition amount of the microbial inoculum was 0.02%. The composition and nutritional level of the basal diet of the chicken feed are shown in table 6. The fast manure is a fast manure available from the american huibao ltd.

The average daily feed intake ADFI, average daily gain ADG and feed-weight ratio of the broilers in each group during the test were measured, and the results are shown in table 7.

TABLE 6

TABLE 7

As can be seen from table 7, addition of clostridium butyricum in the basic ration, especially clostridium butyricum of the present invention can significantly reduce the feed-to-weight ratio of broiler chickens compared with the negative control group, has significant statistical significance, is equivalent to the positive control group (fast fattening), has the alternative resistance potential, and improves the livestock and poultry productivity.

Example 10

This example is used to illustrate the application of clostridium butyricum of the present invention in crucian production

In order to evaluate the influence of clostridium butyricum on the growth performance of crucian carps, the initial body mass of healthy cottonrose hibiscus crucian carps which are incubated in the same batch and have consistent sizes is 2.0 +/-0.05 g. The fish were randomly divided into 4 groups, namely a positive control group (basal diet + commercial probiotic product), a negative control group (basal diet), a control strain group (basal diet + bacterial preparation of CICC23847 prepared in example 7) and an experimental group (basal diet + bacterial preparation of Clostridium butyricum of the present invention prepared in example 7), each treatment group was repeated 3 times, each time 20 fish were repeated, and the production test period was 56 days. The basal feed composition is shown in table 8. The commercial probiotic product is a voronoi complex probiotic product purchased from voronoi nutrition ltd.

Feeding every 4 hours every morning, noon and evening, wherein the feeding amount is 3-5% of the fish body mass. The test aquaria were continuously aerated with oxygen, the temperature was controlled at 25 ℃, the pH was 6-7, the initial and final fish mass was recorded, and the rate of weight gain was determined, the results are shown in table 9.

Weight gain = (final tail average weight-initial tail average weight)/initial tail average weight × 100%.

TABLE 8

TABLE 9

As can be seen from table 9, the addition of clostridium butyricum to the basal feed, especially the clostridium butyricum of the present invention has an obvious growth promoting effect on crucian, and significantly increases the weight gain rate of crucian (P < 0.05).

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Sequence listing

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Claims

1. The Clostridium butyricum is characterized in that the preservation number of the Clostridium butyricum is CGMCC No. 20417.

2. A microbial agent comprising Clostridium butyricum according to claim 1.

3. A preparation method of a microbial inoculum is characterized by comprising the following steps: inoculating clostridium butyricum into a fermentation medium for fermentation to obtain fermentation liquor;

mixing the fermentation liquor with a protective agent and/or a solid carrier to obtain a microbial inoculum;

wherein the clostridium butyricum is clostridium butyricum of claim 1 and/or microbial inoculum of claim 2.

4. The method of claim 3, wherein the fermentation is performed under anaerobic conditions;

the fermentation conditions include: the temperature is 30-37 deg.C, pH is 6-7, and the time is 10-24 h.

5. A method according to claim 3, wherein the clostridium butyricum is inoculated in the form of a seed liquor, the seed liquor being prepared by a method comprising: and inoculating clostridium butyricum into a seed culture medium for propagation culture to obtain a seed solution.

6. The method of claim 5, wherein the number of viable bacteria in the seed solution is 1 x 10 ⁸ -5×10 ⁸ cfu/mL。

7. The method of claim 5, wherein the seed liquid is inoculated in an amount of 1-10 vol%.

8. The method of claim 3, wherein the protectant is selected from at least one of starch, sucrose, lactose, trehalose, sodium citrate, skim milk powder, and maltodextrin.

9. The method of claim 3, wherein the solid support is selected from at least one of bran, soybean meal, and corn meal.

10. The microbial inoculum prepared by the method according to any one of claims 3-9.

11. Use of clostridium butyricum according to claim 1 and/or a microbial inoculum according to claim 2 or 10 for the production of butyric acid, for the preparation of microbial inoculum and for feed.