CN113999787A

CN113999787A - Clostridium butyricum, microbial inoculum, preparation method and application of microbial inoculum and feed

Info

Publication number: CN113999787A
Application number: CN202111007078.2A
Authority: CN
Inventors: 申航; 李胜; 刘杰
Original assignee: Xi'an Haijijiao Biotechnology Research Institute Co ltd
Current assignee: Xi'an Haijijiao Biotechnology Research Institute Co ltd
Priority date: 2021-08-30
Filing date: 2021-08-30
Publication date: 2022-02-01
Anticipated expiration: 2041-08-30
Also published as: CN113999787B

Abstract

The invention relates to the field of microorganisms, and discloses Clostridium butyricum, a microbial inoculum, a preparation method, application and feed thereof, wherein the Clostridium butyricum is preserved in China center for type culture Collection with the preservation number of CCTCC NO. M2021607. The clostridium butyricum provided by the invention has good effect of inhibiting intestinal pathogenic bacteria; when the feed additive is used for producing feed baits, the feed weight ratio can be obviously reduced, the content of immunoglobulin is increased, the feed additive can replace antibiotics and other pharmaceutical additives, and the animal health level is improved.

Description

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

Technical Field

The invention relates to the field of fermentation, and discloses a clostridium butyricum, a microbial inoculum, a preparation method of the microbial inoculum, the microbial inoculum prepared by the method, application of the clostridium butyricum or the microbial inoculum as an antibiotic substitute in the field of livestock and aquaculture, and a feed.

Background

In order to improve the livestock and poultry raising benefits, antibiotics are usually added into the feed to prevent animal diseases, promote the growth of livestock and poultry, improve the feed conversion rate, the quality of livestock products and the like, but with the heavy use of the antibiotics, a plurality of safety problems of endogenous infection of the livestock and poultry, reduction of the immunity of organisms, drug-resistant strains, antibiotic residues and the like occur, and the livestock and poultry raising benefits and the human health are seriously threatened. From 7/1/2020, feed production enterprises stop producing commercial feeds containing growth-promoting drug feed additives (except traditional Chinese medicines), but antibiotics are forbidden in feed ends, which makes a challenge for livestock and poultry to suffer from various diseases in the breeding process. The biological fermentation feed becomes a main feed choice for future livestock and poultry breeding, particularly the microbial fermentation feed, and is one of solutions with great potential, and the core of the technology lies in continuously supplementing high-activity probiotics for livestock and poultry, inhibiting mass propagation of pathogenic bacteria, achieving the purposes of reestablishing or recovering the intestinal microecological balance of the livestock and poultry, realizing active disease resistance, promoting growth, reducing drug residues and improving microenvironment.

Clostridium butyricum is an obligate anaerobic gram-positive bacillus which takes butyric acid, lactic acid and acetic acid as main metabolites, is widely distributed in the intestinal tracts of human beings and animals, and is a normal flora in the intestinal tracts of human beings and animals. Clostridium butyricum is approved as a feed additive for weaned piglets and broilers by the european union as early as 2003, and is included in annex two of the chinese feed additive variety catalog (2013) in 2013. The clostridium butyricum is widely applied to actual production as a feed additive, and is mainly characterized by adjusting the intestinal microecological balance, reducing the generation of enterotoxin and avoiding intestinal diseases; can generate a plurality of growth promoting factors such as amino acid, B vitamins and the like, and improves the digestion and absorption capacity of animals to fat and protein; can resist the action of high acidity and digestive enzyme in vivo. In recent years, clostridium butyricum has positive effects on the aspects of improving the production performance of livestock and poultry, preventing and treating animal bacterial diseases, improving the safety of animal food and the like, and the development of a novel clostridium butyricum with excellent bacteriostatic effect is a main research and development direction in the field.

Disclosure of Invention

The clostridium butyricum has the characteristic of strong bacteriostatic ability, can obviously reduce the material weight ratio, improve the content of immunoglobulin, and can replace the use of pharmaceutical additives such as antibiotics and the like when used for producing feed and bait so as to improve the animal health level.

In order to achieve the above object, the invention provides a Clostridium butyricum with a preservation number of CCTCC No. m 2021607 in a first aspect.

In a second aspect, the present invention provides a microbial inoculum comprising clostridium butyricum as described above.

The third aspect of the invention provides a preparation method of a microbial inoculum, wherein the microbial inoculum is prepared by fermenting the clostridium butyricum in a fermentation medium.

The fourth aspect of the present invention provides the microbial agent prepared according to the method as described above.

In a fifth aspect, the present invention provides the use of a clostridium butyricum as described above or a microbial inoculum as described above as an antibiotic substitute in the field of livestock and aquaculture.

A sixth aspect of the present invention provides a feed, which is characterized by comprising the clostridium butyricum as described above or the microbial agent as described above.

Through the technical scheme of the invention, the following beneficial effects can be obtained:

1. the clostridium butyricum CCTCC NO. M2021607 provided by the invention is a safe strain which is separated from the excrement of healthy weaned piglets and can be used for feed addition, and has no pathogenicity;

2. the clostridium butyricum provided by the invention has the characteristic of strong bacteriostatic ability; when the feed additive is used for producing feed and bait, the feed-weight ratio can be obviously reduced, the content of immunoglobulin is increased, the feed additive can replace antibiotics and other pharmaceutical additives, and the animal health level is improved; the abuse condition of commercial feed antibiotics can be solved, and the problems of environmental safety and food safety are relieved;

3. when the clostridium butyricum provided by the invention is used for preparing the microbial inoculum, the clostridium butyricum is easy to culture and prepare, and the microbial inoculum, namely clostridium butyricum, has high viable bacteria concentration and good stability.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Biological preservation

The Clostridium butyricum (Clostridium butyricum) provided by the invention is preserved in a China center for type culture collection (address: Wuchang Lojia mountain, Wuhan university, postal code: 430072) (the preservation unit is abbreviated as CCTCC) at 24 days 5 and 24 months in 2021, the preservation number is CCTCC NO: m2021607.

Drawings

FIG. 1 is a microscopic form of Clostridium butyricum according to 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, one or more new ranges of values may be obtained from combinations of values between the endpoints of each range, the endpoints of each range and the individual values, and the individual values of the points, and these ranges of values should be considered as specifically disclosed herein.

The invention provides Clostridium butyricum with the preservation number of CCTCC NO. M2021607 in a first aspect.

The clostridium butyricum of the invention has CCTCC NO. M2021607 separated from the feces of healthy weaned piglets.

Specifically, fresh excrement of healthy weaned piglets is aseptically weighed, is subjected to gradient dilution, is inoculated to a clostridium selective medium (TSN medium), is placed at 37 ℃ for anaerobic culture for 48-72h, and suspected colonies are selected and inoculated to a clostridium multiplication medium (RCM medium) for continuous culture until pure cultures of the suspected colonies are obtained. And further performing physiological and biochemical identification and 16S rDNA molecular identification to determine that the clostridium butyricum is named HJJBC 50096.

The clostridium butyricum has the following properties of CCTCC NO. M2021607:

morphological characteristics: culturing on RCM solid culture medium for 20-24h, wherein the diameter of the surface of the grown colony is 1-3mm, the colony is circular and convex, the edge is irregular, the surface is moist, milky yellow or white, and the colony is not transparent and slightly smells sour; microscopically, the cells were short rods in morphology (see FIG. 1), and were gram-positive in staining.

The physiological and biochemical characteristics accord with the characteristics of clostridium butyricum.

The 16S rDNA identification result is aligned with the sequence of NCBI Clostridium butyricum (Clostridium butyricum), and the strain is identified as Clostridium butyricum (Clostridium butyricum) and named HJJBC 50096.

Experiments show that the clostridium butyricum provided by the invention has good bacteriostatic activity on Escherichia coli, salmonella enteritidis and Shigella dysenteriae, and the bacteriostatic circle can reach more than 25mm, which indicates that the clostridium butyricum provided by the invention has excellent bacteriostatic activity.

The solid microbial inoculum prepared by the clostridium butyricum provided by the invention is preserved in a preservation 2The number of viable bacteria can be maintained at 10 after year⁹The stability of the microbial inoculum is good when cfu/g is higher than that of the microbial inoculum.

When the microbial inoculum prepared by the clostridium butyricum provided by the invention is used in the field of livestock and aquaculture, the material-weight ratio can be obviously reduced, and the content of immunoglobulin is improved.

In a second aspect, the present invention provides a microbial inoculum comprising Clostridium butyricum (Clostridium butyricum) as described above.

According to the present invention, the form of the microbial inoculum may not be particularly limited, and may be, for example, a liquid microbial inoculum, a semi-liquid microbial inoculum, a concentrated microbial inoculum, or a solid microbial inoculum. The solid microbial inoculum can be a dry microbial inoculum, for example, a freeze-dried microbial inoculum, a spray-dried microbial inoculum, and the like.

The semi-liquid microbial inoculum refers to a concentrated fermentation liquid or a fermentation liquid mixed with a fermentation substrate, and the solid microbial inoculum can be a microbial inoculum prepared by drying, such as a freeze-dried microbial inoculum, a spray-dried microbial inoculum or a vacuum-dried microbial inoculum.

Preferably, the number of viable bacteria in the microbial inoculum is 10⁸cfu/mL or more, more preferably 10⁹cfu/mL or more.

The product form of the microbial inoculum can comprise but is not limited to capsules, tablets, oral liquid and powder.

The third aspect of the invention provides a preparation method of a microbial inoculum, wherein the microbial inoculum is prepared by carrying out fermentation culture on the clostridium butyricum in a fermentation culture medium.

The clostridium butyricum can be stored through a cryopreservation tube, and before the clostridium butyricum in the cryopreservation tube is used, the clostridium butyricum can be cultured through a slant so as to recover the activity of the clostridium butyricum. The slant medium may be a medium commonly used in the art, and may be, for example, RCM solid medium.

The conditions for slant culture may be those conventional in the art, for example, anaerobic culture at 30-38 deg.C for 2-3 days. The clostridium butyricum cultured by the inclined plane can recover the activity and is used for the subsequent culture and fermentation.

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

The seed solution is obtained by liquid culture of clostridium butyricum provided by the invention, the culture method is not particularly required, as long as the clostridium butyricum can proliferate, for example, the seed solution can be obtained by inoculating clostridium butyricum thallus into a clostridium butyricum culture medium, and performing anaerobic culture for 2-3 days at the temperature of 30-38 ℃ under anaerobic conditions.

The clostridium butyricum medium can be various media suitable for culturing clostridium butyricum known in the art, and can be RCM medium, for example.

Among them, the RCM medium preferably includes: 1-5g/L of yeast extract powder, 5-15g/L of beef extract, 5-15g/L of peptone, 0.5-1.5g/L of soluble starch, 2-8g/L of glucose, 0.2-0.8 g/L of cysteine hydrochloride, 1-5g/L of sodium chloride and 1-5g/L of sodium acetate, and the pH is natural. Autoclaving at 121 deg.C for 15-20 min. When the culture medium is a solid medium, it preferably further contains agar 15-20 g/L.

And inoculating the seed liquid into a fermentation culture medium for fermentation to obtain a fermentation liquid. Preferably, the inoculation amount of the seed liquid is 5-15%.

According to the present invention, the fermentation medium may be a fermentation medium conventional in the art, and preferably, the fermentation medium includes a carbon source, a nitrogen source, and inorganic salts; wherein the carbon source is selected from at least one of starch, glucose, glycerol, sucrose and molasses; wherein the nitrogen source is yeast extract powder and/or ammonium sulfate; wherein the inorganic salt is at least one selected from the group consisting of dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride and manganese sulfate.

In a preferred aspect of the present invention, the fermentation medium includes starch, yeast extract powder, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, and manganese sulfate; wherein in the fermentation medium, the content of starch is 15-25g/L, the content of yeast extract powder is 5-15g/L, the content of dipotassium hydrogen phosphate is 1-3g/L, the content of magnesium sulfate is 2-4g/L, the content of calcium chloride is 0.1-1g/L, and the content of manganese sulfate is 0.5-1.5 g/L.

According to the present invention, the fermentation conditions may be conventional conditions for fermentation of clostridium butyricum well known in the art, and preferably, the fermentation is performed under anaerobic conditions. Preferably, the conditions of the fermentation comprise: the temperature is 30-37 deg.C, pH is 6-7, and the time is 60-120 h.

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

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.

According to the invention, 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.

According to the present invention, the protective agent may be any of various protective agents conventionally used in the art, and for example, may be at least one of skim milk powder, maltodextrin, trehalose, dextran, glycerin, and the like. The amount of the protective agent can be selected and adjusted by those skilled in the art according to the needs, and the amount of the protective agent is 15-20 wt%. The protective agent is generally used for preparing freeze-dried bacterial powder with higher purity.

According to the invention, the solid carrier of the microbial inoculum comprises but is not limited to bran, soybean meal and corn meal.

According to the present invention, preferably, before adding the protective agent to the concentrated microbial inoculum, the method further comprises washing the concentrated microbial inoculum with a buffer. The buffer may be a buffer conventionally used in the art for washing the cells, and may be, for example, physiological saline or PBS buffer.

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, and the like.

In one 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 (frozen) dried to prepare the microbial inoculum.

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

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

(1) inoculating the seed solution of clostridium butyricum into a fermentation culture medium for fermentation to obtain fermentation liquor;

(2) centrifuging the fermentation liquor obtained in the step (1) to obtain bacterial sludge;

(3) mixing the bacterial sludge with protectant, and adjusting viable bacteria concentration to 10⁹Selecting bran as a carrier, uniformly stirring the bran and the clostridium butyricum bacterial mud (for example, in a ratio of 1: 1-10: 1), and drying in an oven at 40-45 ℃ for 1-2 days to obtain the microbial inoculum.

According to the invention, the clostridium butyricum can replace antibiotics, is mixed with basic feed or basic bait, and is fed to livestock or aquatic products.

A sixth aspect of the present invention provides a feed comprising clostridium butyricum as described above or a microbial agent as described above.

The addition amount of the clostridium butyricum or the microbial inoculum can be selected in a wide range, and a person skilled in the art can adjust the addition amount according to actual needs, for example, when the feed contains the microbial inoculum, the addition amount of the microbial inoculum can be 0.01-1 wt%.

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

The clostridium selection medium (TSN medium) is used for the separation culture of clostridium butyricum, and the formula of the clostridium selection medium is as follows: 17g/L tryptone, 3g/L soyabean peptone, 5g/L sodium chloride, 2.5g/L potassium dihydrogen phosphate and 2.5g/L glucose;

the RCM culture medium is used for pure culture of clostridium butyricum and has the formula as follows: 3g/L of yeast extract powder, 10g/L of beef extract, 10g/L of peptone, 1g/L of soluble starch, 5g/L of glucose, 0.5g/L of cysteine hydrochloride, 3g/L of sodium chloride and 3g/L of sodium acetate;

nutrient Broth (NB) medium: 10g/L of peptone, 5g/L of beef extract and 5g/L of sodium chloride.

The culture mediums are respectively added with 2 percent of agar to prepare corresponding solid culture mediums.

Unless otherwise specified, reagents or materials used are commercially available and procedures conventional in the art are performed.

The control strain is clostridium butyricum CICC 10390 purchased from China center for culture Collection of industrial microorganisms.

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, purification and identification of the Clostridium butyricum HJJBC 50096 strain according to the present invention.

Aseptically weighing 50g of fresh feces of healthy weaned piglets, adding into 450g of aseptic normal saline or phosphate buffer solution, fully beating and uniformly mixing to obtain bacterial suspension, diluting the bacterial suspension to 10%^-4、10^-5、 10^-6、10^-7、10^-8And 5 gradients, inoculating 100 mu L of feces diluent into a TSN culture medium in a pouring mode, placing the TSN culture medium in a constant temperature incubator at 37 ℃ for standing anaerobic culture for 48-72h, and observing the morphological characteristics of bacterial colonies. And selecting the colony meeting the morphological characteristics of the clostridium butyricum, streaking and inoculating the colony to an RCM culture medium, and continuing culturing until a pure culture of the suspicious colony is obtained. The separated strain is subjected to physiological and biochemical identification according to Bergey's handbook, and is subjected to molecular biological identification by using a PCR identification method. The specific identification results are as follows:

(1) and (3) morphological observation: culturing on an RCM solid culture medium for 48-72h, wherein the surface of a growing colony is 1-3mm in morphological diameter, is round and convex, is irregular in edge, is moist, milky yellow or white in surface, is opaque and slightly smells sour; microscopic cell morphology was short rod-like (see FIG. 1), gram-positive.

(2) The physiological and biochemical identification results are shown in table 1:

TABLE 1

Glucose

+

Mannitol

+^w

Lactose

+

Sucrose

+

Maltose

+

Salix alcohol

+

Xylose

+

Arabinose

+

Glycerol

-

Cellobiose

+

Mannose

+

Songsansan candy

-

Cotton seed candy

+

Sorbitol

-

Rhamnose

-

Trehalose

+

Indole formation

-

Hydrolysis of urea

-

Hydrolysis of gelatin

-

Hydrolysis of esculin

+

Catalase enzyme

-

Growth in GMB Medium

+

(3)16S rDNA identification: the genome of clostridium butyricum is extracted by using a bacterial genome deoxyribonucleic acid rapid extraction kit, and sequence determination is completed by a biological engineering (Shanghai) company Limited. The 16S rDNA identification result is aligned with the sequence of NCBI Clostridium butyricum (Clostridium butyricum), and the strain is identified as Clostridium butyricum (Clostridium butyricum).

(4) And (3) identification result: the 16S rDNA sequencing result is combined with the physiological and biochemical identification result, and the strain is determined to be clostridium butyricum and is named HJJBC 50096.

Example 2

This example illustrates the ability of Clostridium butyricum (Clostridium butyricum) to inhibit enteropathogenic bacteria.

The clostridium butyricum CCTCC NO. M2021607 and the control strain CICC 10390 provided by the invention are respectively used for evaluating the bacteriostatic performance according to the following methods.

Escherichia coli (Escherichia coli), Salmonella enterica (Salmonella enterica) and Shigella dysenteriae (Shigella dysenteriae) were cultured at 37 ℃ for 24h using NB liquid medium to obtain a bacterial solution. Wherein the Escherichia coli, Salmonella enteritidis and Shigella dysenteriae are purchased from China Industrial microorganism culture Collection management center and are respectively numbered as CICC 10389, CICC 21513 and CICC 10983.

The clostridium butyricum is cultured for 48-72h at 37 ℃ by using an RCM liquid culture medium to obtain a bacterial liquid.

(1) Oxford cup method

Pouring about 2-3mm of agar culture medium which is cooled to 55-60 ℃ into the culture dish, and standing until the agar culture medium is completely solidified. Placing 4 aseptic oxford cups at equal intervals by using tweezers, adding 250 mu L of correspondingly prepared escherichia coli, salmonella enteritidis and shigella dysenteriae bacterial liquid when 100mL of RCM culture medium is cooled to about 50 ℃, fully mixing uniformly, pouring into the culture medium with the oxford cups, and horizontally placing until the coagulation is complete. After the solution is completely solidified, taking out the oxford cup by using a sterile forceps to form a round hole, adding 150 mu L of clostridium butyricum bacterial solution supernatant into the round hole, and sealing the periphery of the culture dish by using a sealing film. The petri dish was placed horizontally in a refrigerator at 4 ℃ and diffused for 2-3 h. Transferring to 37 ℃ for anaerobic culture for 24-48h after the outer ring of the liquid in the hole diffuses, and measuring the diameter of the inhibition zone of each hole, wherein the results are shown in Table 2.

(2) Viable bacteria counting method

Adjusting the concentration of Escherichia coli, Salmonella enteritidis and Shigella dysenteriae to 1 × 10⁷CFU/mL, at a concentration of 1X 10⁷The bacterial liquid of the Clostridium butyricum with the concentration of 1 multiplied by 10 is CFU/mL respectively⁷CFU/mL bacterial solutions of Escherichia coli, Salmonella enteritidis and Shigella dysenteriae were mixed in equal volumes, incubated at 37 ℃ under anaerobic conditions for 1h, 2h and 3h, respectively, 100. mu.L of the mixed bacterial solution was applied to NB solid medium, incubated at 37 ℃ under aerobic conditions for 24h, and colony counts of Escherichia coli, Salmonella enteritidis and Shigella dysenteriae were performed, as shown in Table 3.

TABLE 2

TABLE 3

Note: the unit of viable count is 10⁷cfu/mL。

The data in table 3 show that the clostridium butyricum of the invention can inhibit the growth of intestinal pathogenic bacteria such as escherichia coli, salmonella enteritidis, shigella dysenteriae and the like to different degrees, and the bacteriostatic effect is better than that of a control strain.

Example 3

This example illustrates the storage stability of Clostridium butyricum (Clostridium butyricum) inoculum.

The clostridium butyricum CCTCC No. m 2021607 and the control strain provided by the present invention were used to prepare microbial agents and evaluate the storage stability as follows.

Inoculating clostridium butyricum stored at-80 ℃ in a frozen tube form into an RCM slant culture medium in a serpentine streak mode, culturing for 48-72h under an anaerobic condition at 37 ℃, and collecting test tube slant strains; selecting an inoculating loop slant lawn to 10mL RCM liquid culture medium, culturing for 48-72h at 37 ℃ under anaerobic condition to ensure that the strain completely recovers activity, and then performing amplification culture to obtain seed liquid; inoculating the seed solution into a culture medium of a liquid fermentation tank according to the inoculation amount of 10%, and then performing fermentation amplification culture for 72h under the anaerobic condition of 30-32 ℃ to obtain fermentation liquor.

The number of the viable bacteria reaches 10⁹-10¹⁰And (3) centrifugally separating the clostridium butyricum fermentation liquor with the concentration of more than CFU/mL to obtain bacterial sludge. Washing the bacterial sludge by using normal saline, then adding bran into the bacterial sludge in a ratio of 1:1, drying for 2 days at 45 ℃, and crushing to obtain the clostridium butyricum microbial inoculum.

The prepared microbial inoculum is stored in a dry environment at 15-18 ℃, an RCM culture medium is used periodically, and the recovery culture is carried out under the anaerobic condition, so that the storage stability of the microbial inoculum is detected, and the result of viable count is shown in Table 4.

TABLE 4

Storage time (moon)	Clostridium butyricum cfu/g of the present invention	Control Strain cfu/g
			0	5.0×10⁹	6.1×10⁹
24	7.5×10⁸	8.7×10⁶

As can be seen from Table 4, the solid microbial inoculum prepared by the clostridium butyricum has good stability, and the viable count can still reach 10 after the solid microbial inoculum is stored for 2 years⁸cfu/g is better than that of the control strain.

Example 4

This example illustrates the effect of Clostridium butyricum (Clostridium butyricum) on the growth status and immunity of broiler chickens.

Two clostridium butyricum inocula prepared in example 3 were used respectively, and the influence of the growth state and the immunity of broilers was evaluated.

200 broiler chicks of 1 day old are selected as experimental objects and randomly divided into 5 groups, wherein the group settings are as follows: the feed comprises a basic feed group (No. 1), a clostridium butyricum microbial inoculum feed group (No. 2) added with 0.01 percent of the invention, a clostridium butyricum microbial inoculum feed group (No. 3) added with 0.1 percent of the invention, a clostridium butyricum microbial inoculum feed group (No. 4) added with 1 percent of the invention, a chlortetracycline feed group (No. 5) added with 150mg/kg and a contrast strain microbial inoculum feed group (No. 6), and chicks are raised in cages with feeders and nipple-type drinking water fountains, so that the feeding experiments of the 42 days of the behavior period are carried out in a common way. Wherein:

the basic feed for 1 to 21 days comprises the following components: 53.78% of corn flour, 38.75% of bean cake powder, 3.15% of soybean oil, 1.98% of calcium hydrophosphate, 1.05% of lime powder, 0.35% of sodium chloride, 0.18% of DL-methionine, 0.04% of lysine, 0.30% of choline, 0.03% of vitamin complex and 0.30% of mineral substances.

The basic feed for 22-42 days comprises the following components: 61.28% of corn flour, 32.04% of bean cake powder, 2.85% of soybean oil, 1.69% of calcium hydrophosphate, 1.08% of lime powder, 0.35% of sodium chloride, 0.12% of DL-methionine, 0.02% of lysine, 0.25% of choline, 0.02% of vitamin complex and 0.30% of mineral substances.

The results of average daily feed intake, average daily weight gain and feed-to-weight ratio are shown in table 5.

TABLE 5

From the feed weight ratio of the broilers within 1-42 days in table 5, the group fed with 0.01% of clostridium butyricum microbial inoculum of the invention was slightly lower than the group fed with antibiotic, and the two groups were significantly lower than the basal feed group and the 1% control strain microbial inoculum feed group (P < 0.05).

The immunity of the broiler chickens was characterized by measuring the IgG content in the serum, and the results are shown in Table 6.

TABLE 6

Time	Number 1	Number 2	No. 3	Number 4	Number 5	Number 6
							21 days	594	672	728	730	670	631
42 days	564	640	696	706	628	593

As can be seen from Table 6, the content of Ig in the broiler serum of the group of the feed added with 0.01% of the Clostridium butyricum microbial inoculum of the invention is slightly higher than that of the group of the feed added with antibiotics, and is obviously higher than that of the group of the basal feed and the group of the feed added with 1% of the microbial inoculum of the control strain (P is less than 0.05).

Example 5

This example illustrates the effect of Clostridium butyricum (Clostridium butyricum) on the growth status and immunity of piglets.

Two clostridium butyricum inocula prepared in example 3 were used respectively, and the influence on the growth and immunity of piglets was evaluated.

Selecting 50 weaned piglets of about 21 days old (6.2kg), randomly dividing into 5 groups, and respectively setting the groups as follows: the feed is prepared by a basic feed group (No. 1), a feed group (No. 2) added with 0.01% of clostridium butyricum fungicide, a feed group (No. 3) added with 0.1% of clostridium butyricum fungicide, a feed group (No. 4) added with 1% of clostridium butyricum fungicide, a feed group (No. 5) added with 150mg/kg of aureomycin and a feed group (No. 6) added with 1% of contrast strain fungicide, wherein a funnel-type feed trough is adopted for free feeding, sufficient water is supplied, and the daily management is carried out according to the convention, and the feeding experiment of the behavior period of 30 days is carried out. Wherein:

the basic feed comprises the following components: 60.1 percent of corn flour, 28.75 percent of bean cake powder, 4 percent of bran, 2.72 percent of soybean oil, 1.98 percent of calcium hydrophosphate, 1.25 percent of lime powder, 0.35 percent of sodium chloride, 0.18 percent of DL-methionine, 0.04 percent of lysine, 0.30 percent of choline, 0.03 percent of vitamin complex and 0.30 percent of mineral substances.

The results of average daily feed intake, average daily weight gain and feed-to-weight ratio are shown in table 7, and the serum IgG levels (ng/mL) of piglets are shown in table 8.

TABLE 7

Detecting the index	Number 1	Number 2	No. 3	Number 4	Number 5	Number 6
							Average daily food intake (g/d)	579	580	583	581	582	582
Average daily gain (g/d)	244	257	264	267	262	250
							Material to weight ratio	2.37	2.26	2.21	2.18	2.22	2.32

TABLE 8

Group of	Number 1	Number 2	No. 3	Number 4	Number 5	Number 6
							IgG content	229	262	320	331	257	239

As can be seen from tables 7 and 8, the group of the clostridium butyricum microbial inoculum feed added with 0.01 percent of the invention is slightly higher than the group of the feed added with antibiotics, but the two groups are obviously lower than the basal feed group and the 1 percent control strain microbial inoculum feed group (P is less than 0.05); the content of Ig in piglet serum of the feed group added with 0.01 percent of clostridium butyricum microbial inoculum of the invention is slightly higher than that of the feed group added with antibiotics and is obviously higher than that of a basic feed group and a 1 percent of control strain microbial inoculum feed group (P is less than 0.05).

Example 6

This example illustrates the effect of Clostridium butyricum (Clostridium butyricum) on the growth status and immunity of grass carp.

Two clostridium butyricum inocula prepared in example 3 were used respectively, and the influence of the growth state and immunity of freshwater grass carp was evaluated.

Selecting about 50g of 200 grass carps, randomly dividing the grass carps into 5 groups, and respectively setting the groups as follows: a basal feed group (No. 1), a feed group (No. 2) added with 0.01% of clostridium butyricum fungicide, a feed group (No. 3) added with 0.1% of clostridium butyricum fungicide, a feed group (No. 4) added with 1% of clostridium butyricum fungicide, a feed group (No. 5) added with 150mg/kg of aureomycin and a feed group (No. 6) added with 1% of control strain fungicide. Cultured in a culture medium with a volume of 1.5m³Feeding twice a day in a cleaned and sterilized cement pond, wherein the feeding amount is about 2-3% of the weight of the fish, and changing one third of the water in the pond every day, so that the feeding experiment is carried out for 30 days.

The basic feed comprises the following components: 36% of soybean meal, 25% of fish meal, 20% of wheat flour, 15% of corn flour, 1% of alpha-starch, 0.5% of mineral substances, 0.05% of vitamin complex and 2% of soybean oil.

The results of the average daily feed intake, average daily weight gain and feed-to-weight ratio are shown in Table 9, and the serum IgG content (ng/mL) of grass carp is shown in Table 10.

TABLE 9

Detecting the index	Number 1	Number 2	No. 3	Number 4	Number 5	Number 6
							Average daily food intake (g/d)	1.61	1.58	1.59	1.62	1.62	1.61
Average daily gain (g/d)	1.43	1.49	1.54	1.58	1.53	1.48
							Material to weight ratio	1.12	1.06	1.03	1.02	1.06	1.09

Watch 10

Group of	Number 1	Number 2	No. 3	Number 4	Number 5	Number 6
							IgG content	178	202	218.4	219	201	183

As can be seen from tables 9 and 10, the feed group added with 0.01% of the clostridium butyricum microbial inoculum of the invention has no obvious difference with the feed group added with antibiotics, but the feed group and the feed group added with the clostridium butyricum microbial inoculum are obviously lower than the basal feed group and the feed group added with 1% of the control strain microbial inoculum (P is less than 0.05); the content of Ig in the serum of grass carp added with 0.01 percent of clostridium butyricum microbial inoculum feed group is slightly higher than that of the grass carp added with antibiotic feed group, and is obviously higher than that of a basic feed group and 1 percent of control strain microbial inoculum feed group (P is less than 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.

Claims

1. The Clostridium butyricum is characterized in that the preservation number of the Clostridium butyricum is CCTCC NO. M2021607.

2. A microbial agent comprising the clostridium butyricum of claim 1.

3. The microbial agent according to claim 2, wherein the number of viable bacteria in the microbial agent is 10⁸cfu/mL or more.

4. A method for preparing a microbial inoculum, which is characterized in that the microbial inoculum is prepared by fermenting the clostridium butyricum of claim 1 in a fermentation culture medium.

5. The method of claim 4, wherein the fermentation is conducted under anaerobic conditions;

the fermentation conditions include: the temperature is 30-37 deg.C, pH is 6-7, and the time is 60-120 h.

6. The method of claim 4, wherein the fermentation medium comprises a carbon source, a nitrogen source, and inorganic salts;

wherein the carbon source is selected from at least one of starch, glucose, glycerol, sucrose and molasses;

wherein the nitrogen source is yeast extract powder and/or ammonium sulfate;

wherein the inorganic salt is at least one selected from the group consisting of dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride and manganese sulfate.

7. The method of claim 6, wherein the fermentation medium comprises starch, yeast extract, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, and manganese sulfate;

wherein in the fermentation medium, the content of starch is 15-25g/L, the content of yeast extract powder is 5-15g/L, the content of dipotassium hydrogen phosphate is 1-3g/L, the content of magnesium sulfate is 2-4g/L, the content of calcium chloride is 0.1-1g/L, and the content of manganese sulfate is 0.5-1.5 g/L.

8. The microbial inoculum prepared by the method according to any one of claims 4 to 7.

9. Use of clostridium butyricum according to claim 1 or a bacterial agent according to any one of claims 2, 3 and 9 as an antibiotic substitute in the field of livestock and aquaculture.

10. A feed comprising clostridium butyricum of claim 1 or the microbial agent of any one of claims 2, 3 and 9.