CN113999787B

CN113999787B - Clostridium butyricum, microbial inoculum, preparation method and application thereof, and feed

Info

Publication number: CN113999787B
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: 2023-06-06
Anticipated expiration: 2041-08-30
Also published as: CN113999787A

Abstract

The invention relates to the field of microorganisms, and discloses clostridium butyricum Clostridium butyricum which is preserved in China Center for Type Culture Collection (CCTCC) No. M2021607, a microbial inoculum, a preparation method and an application thereof, and a feed. 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 additive can also obviously reduce the feed-to-weight ratio, improve the content of immunoglobulin, replace the use of pharmaceutical additives such as antibiotics and the like and improve the health level of animals.

Description

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

Technical Field

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

Background

In order to improve the benefit of raising livestock and poultry, 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 massive use of antibiotics, a plurality of safety problems such as endogenous infection of livestock and poultry, reduced organism immunity, drug-resistant strains, antibiotic residues and the like appear, and serious threaten to the livestock benefit and human health. Since 7 months and 1 day in 2020, feed manufacturers stop producing commercial feeds containing growth-promoting drug feed additives (except traditional Chinese medicines), however, the feed end disables antibiotics, and challenges various diseases of livestock and poultry in the breeding process. The biological fermentation feed becomes a main feed choice for livestock and poultry cultivation in the future, particularly the microbial fermentation feed, and is one of the solutions with great potential, and the technology is characterized in that the high-activity probiotics are continuously supplemented for the livestock and the poultry, so that the mass reproduction of pathogenic bacteria is inhibited, the intestinal microecological balance of the livestock and the poultry is rebuilt or restored, and the purposes of active disease resistance, growth promotion, drug residue reduction and microenvironment improvement are realized.

Clostridium butyricum is an obligate anaerobic gram-positive bacillus with butyric acid, lactic acid and acetic acid as main metabolites, widely distributed in the intestinal tracts of human and animals, and is a normal flora in the intestinal tracts of human and animals. Clostridium butyricum was approved by the european union as a feed additive for weaned piglets and broilers as early as 2003, and was incorporated in annex two of the chinese feed additive variety catalogue (2013) in China in 2013. Clostridium butyricum has been widely used as a feed additive in practical production, and is mainly characterized by adjusting intestinal microecology balance, reducing enterotoxin production and avoiding intestinal diseases; can produce various growth promoting factors such as amino acid, B vitamins and the like, and improve the digestion and absorption capacity of animals on fat and protein; can tolerate the actions of high acidity and digestive enzymes in vivo, etc. In recent years, clostridium butyricum has shown positive effects in improving livestock and poultry production performance, preventing and treating animal bacterial diseases, improving animal food safety and the like, and development of new clostridium butyricum with excellent antibacterial effect is a main research and development direction in the field.

Disclosure of Invention

The invention aims to provide clostridium butyricum with strong antibacterial capability, and provides 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.

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

In a second aspect the invention provides a bacterial agent comprising clostridium butyricum as described above.

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

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

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

In a sixth aspect the invention provides a feed, characterized in that the feed comprises clostridium butyricum as described above or a bacterial agent as described above.

By the technical scheme, 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 feces of healthy weaned pigs and can be used for feed addition, and has no pathogenicity;

2. the clostridium butyricum provided by the invention has the characteristic of strong antibacterial capacity; when the feed additive is used for producing feeds and baits, the feed additive can also obviously reduce the feed-to-weight ratio, improve the content of immunoglobulin, replace the use of pharmaceutical additives such as antibiotics and the like, and improve the health level of animals; can solve the abuse condition of commercial feed antibiotics and relieve the problems of environmental safety and food safety;

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, the viable bacteria concentration of the microbial inoculum clostridium butyricum is high, and the stability is good.

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

Preservation of organisms

Clostridium butyricum (Clostridium butyricum) provided by the invention is preserved in China center for type culture collection (address: jia mountain of Wuchang, university of Wuhan, postal code: 430072) (abbreviated as CCTCC of preservation unit), and the preservation number is CCTCC NO: m2021607.

Drawings

FIG. 1 shows the microscopic morphology of Clostridium butyricum according to the present invention.

Detailed Description

No endpoints of the ranges and any values disclosed herein are limited to the precise range or value, and such range or value should be understood to encompass values that are close to the range or value. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, and are contemplated as specifically disclosed herein.

The first aspect of the invention provides clostridium butyricum Clostridium butyricum with the preservation number of CCTCC No. M2021607.

The clostridium butyricum CCTCC NO. M2021607 is separated from the feces of healthy weaned pigs.

Specifically, fresh feces of healthy weaned pigs are aseptically weighed, subjected to gradient dilution, inoculated into a clostridium selective culture medium (TSN culture medium), placed at 37 ℃ for anaerobic culture for 48-72 hours, and selected suspected bacterial colonies are inoculated into a clostridium proliferation culture medium (RCM culture medium) for continuous culture until pure cultures of the suspected bacterial colonies are obtained. Further carrying out physiological and biochemical identification and 16S rDNA molecular identification, determining the clostridium butyricum, and naming the clostridium butyricum as HJBC 50096.

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

morphological features: culturing on RCM solid culture medium for 20-24 hr, wherein the surface morphology of the grown colony has a diameter of 1-3mm, is round and convex, has irregular edge, and has wet, creamy yellow or white surface, is opaque and slightly sour and odorous; the cell morphology under the microscope was short bar-like (see FIG. 1), gram staining positive.

The physiological and biochemical characteristics are in accordance with the characteristics of clostridium butyricum.

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

Experiments show that the clostridium butyricum provided by the invention has good antibacterial activity on escherichia coli, salmonella enteritidis and shigella dysenteriae, and the antibacterial circle can reach more than 25mm, so that the clostridium butyricum provided by the invention has excellent antibacterial activity.

The solid microbial inoculum prepared by the clostridium butyricum provided by the invention can keep the viable count at 10 after being stored for 2 years ⁹ Above cfu/g, the stability of the microbial inoculum is good.

The microbial inoculum prepared from clostridium butyricum can obviously reduce the feed-to-weight ratio and improve the content of immunoglobulin when being used in the field of livestock and aquaculture.

In a second aspect the invention provides a bacterial agent comprising clostridium butyricum (Clostridium butyricum) as described above.

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

Preferably, the viable count in the microbial inoculum is 10 ⁸ cfu/mL or more, more preferably 10 ⁹ cfu/mL or more.

Product forms of the microbial inoculum may include, but are not limited to, capsules, tablets, oral liquids, and powders.

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

The clostridium butyricum can be stored by a freezing tube, and before the clostridium butyricum in the freezing tube is used, the clostridium butyricum can be cultured by an inclined plane to restore the activity. The slant medium may be a medium commonly used in the art, for example, may be an RCM solid medium.

The conditions for the slant culture may be conventional in the art, for example, the temperature may be 30-38℃and the time may be 2-3 days, anaerobic culture. Clostridium butyricum subjected to slant culture can be recovered for subsequent culture and fermentation.

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

The clostridium butyricum provided by the invention is subjected to liquid culture to obtain a seed solution, and the culture method is not particularly required, so long as the clostridium butyricum can be proliferated, for example, clostridium butyricum can be inoculated into clostridium butyricum culture medium, and the clostridium butyricum is subjected to anaerobic culture at the temperature of 30-38 ℃ for 2-3 days under anaerobic conditions to obtain the seed solution.

The clostridium butyricum medium may be any of various media known in the art suitable for clostridium butyricum culture, and may be, for example, RCM medium.

Wherein the RCM medium preferably comprises: 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 g/L of cysteine hydrochloride, 1-5g/L of sodium chloride and 1-5g/L of sodium acetate, and natural pH. Autoclaving at 121deg.C for 15-20min. When the medium is a solid medium, it preferably further comprises agar 15-20g/L.

Inoculating the seed liquid into a fermentation medium for fermentation to obtain fermentation liquor. Preferably, the seed solution is inoculated in an amount of 5-15%.

According to the present invention, the fermentation medium may be a fermentation medium conventional in the art, preferably, the fermentation medium includes a carbon source, a nitrogen source, and an inorganic salt; 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 dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride and manganese sulfate.

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

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

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

The stirring rotation speed in the fermentation process is determined according to the volume of the fermentation tank, and can be adjusted according to actual conditions by a person skilled in the art.

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

According to the present invention, the protective agent may be various protective agents conventional 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 used can be selected and adjusted as desired by the person skilled in the art, said amount being 15-20% by weight. The protective agent is generally used for preparing freeze-dried bacterial powder with higher purity.

According to the invention, the solid carriers of the microbial inoculum include, but are not limited to, bran, soybean meal and corn meal.

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

The drying treatment may be performed in a manner conventional in the art, for example, air drying, freeze drying, spray drying, vacuum drying, and the like.

In one embodiment of the present invention, the bacterial agent is prepared by concentrating the fermented liquid of clostridium butyricum obtained by fermentation to obtain clostridium butyricum bacterial sludge, mixing the bacterial sludge with a protective agent, and (freeze) drying the mixture.

The concentration treatment may be a concentration method which is conventional in the art, for example, concentration may be performed by centrifugation or filtration, as long as clostridium butyricum paste 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 clostridium butyricum seed liquid into a fermentation medium for fermentation to obtain fermentation liquid;

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

(3) Mixing the bacterial mud with a protective agent, and adjusting the concentration of living bacteria to 10 ⁹ Selecting bran as carrier above cfu/g, stirring bran and clostridium butyricum bacterial mud (such as at a ratio of 1:1-10:1), mixing well, and drying in an oven at 40-45deg.C (such as 1-2 days) to obtain the bacterial agent.

According to the invention, clostridium butyricum can replace antibiotics to be mixed with basic feed or basic bait for feeding livestock or aquatic products.

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

The amount of clostridium butyricum or bacterial agent added can be selected within a wide range, and can be adjusted according to actual needs by a person skilled in the art, for example, when bacterial agent is contained in the feed, the amount of bacterial agent added can be 0.01-1 wt%.

The present invention will be described in detail by examples.

Clostridium selection medium (TSN medium) for isolated culture of clostridium butyricum, the formulation of which is: 17g/L of tryptone, 3g/L of soybean peptone, 5g/L of sodium chloride, 2.5g/L of monopotassium phosphate and 2.5g/L of glucose;

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

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

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

The reagents or materials used, unless otherwise specified, are all commercially available and operate as is conventional in the art.

The control strain was clostridium butyricum CICC 10390 purchased from the chinese industrial microorganism strain collection management center.

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

Example 1

This example is for illustrating the isolation, purification and identification of Clostridium butyricum (Clostridium butyricum) HJBC 50096 strain according to the present invention.

Sterile weighing 50g of fresh feces of healthy weaned pigs, adding into 450g of sterile physiological saline or phosphate buffer solution, fully beating and uniformly mixing to prepare bacterial suspension, and diluting the bacterial suspension to 10 ^-4 、10 ^-5 、 10 ^-6 、10 ^-7 、10 ^-8 5 gradients, respectively taking 100 mu L of fecal dilution liquid, inoculating the fecal dilution liquid into a TSN culture medium in a pouring mode, placing the culture medium in a constant temperature incubator at 37 ℃ for standing anaerobic culture for 48-72 hours, and observing colony morphology characteristics. And selecting a colony conforming to the morphological characteristics of clostridium butyricum, streaking and inoculating the RCM culture medium, and continuously culturing until a pure culture of the suspicious colony is obtained. The isolated strain was subjected to physiological and biochemical identification by reference to "Berger's handbook", and molecular biology identification was performed by using a PCR identification method. The specific identification results are as follows:

(1) Morphological observation: culturing on RCM solid culture medium for 48-72 hr, to form colony with surface diameter of 1-3mm, circular raised edge, moist surface, milky yellow or white surface, no transparency and slightly acidic smell; the cell morphology was short bar-like under the microscope (see FIG. 1), gram-positive.

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

TABLE 1

Glucose

+

Mannitol (mannitol)

+ ^w

Lactose and lactose

+

Sucrose

+

Maltose

+

Liu Chun

+

Xylose

+

Arabinose (Arabic sugar)

+

Glycerol

-

Cellobiose

+

Mannose

+

Songsan sugar

-

Raffinose

+

Sorbitol

-

Rhamnose (rhamnose)

-

Trehalose

+

Indole formation

-

Urea hydrolysis

-

Gelatin hydrolysis

-

Hydrolysis of esculin

+

Catalase enzyme

-

GMB Medium growth

+

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

(4) Identification result: the 16S rDNA sequencing result is combined with the physiological and biochemical identification result, and the strain is identified as clostridium butyricum and named as HJBC 50096.

Example 2

This example illustrates the pathogenic effects of clostridium butyricum (Clostridium butyricum) on the inhibition of intestinal pathogens.

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

The bacterial solutions were obtained by culturing Escherichia coli (Escherichia coli), salmonella enteritidis (Salmonella enterica) and Shigella dysenteriae (Shigella dysenteriae) at 37℃for 24 hours using NB liquid medium. Wherein the escherichia coli, the salmonella enteritidis and the shigella dysenteriae are purchased from China center for type culture Collection of microorganisms, and are numbered as CICC 10389, CICC 21513 and CICC 10983 respectively.

Culturing clostridium butyricum at 37 ℃ for 48-72h by using RCM liquid culture medium to obtain bacterial liquid.

(1) Oxford cup method

Pouring agar culture medium cooled to 55-60 ℃ about 2-3mm into a culture dish, and standing until solidification is complete. Placing 4 sterile oxford cups with forceps at equal distance, adding 250 μl of corresponding prepared Escherichia coli, salmonella enteritidis and Shigella dysenteriae when 100mL of RCM culture medium is cooled to about 50deg.C, mixing thoroughly, pouring into the culture medium with oxford cups, and standing horizontally until solidification is complete. After the coagulation is completed, the oxford cup is taken out by sterile forceps to form a round hole, 150 mu L of clostridium butyricum bacterial liquid supernatant is added into the round hole, and the periphery of the culture dish is sealed by a sealing film. The dishes were placed horizontally in a refrigerator at 4℃and spread for 2-3h. After the liquid outer ring in the hole is diffused, the liquid outer ring is transferred to anaerobic cultivation at 37 ℃ for 24-48 hours, and the diameter of each bacteriostasis ring in the hole is measured, and the result is shown in Table 2.

(2) Viable bacteria counting method

Regulating the concentration of the bacterial solutions of Escherichia coli, salmonella enteritidis and Shigella dysenteriae to 1×10 ⁷ CFU/mL, concentration was 1X 10 ⁷ CFU/mL clostridium butyricum bacterial liquid and concentration are respectively 1 multiplied by 10 ⁷ CFU/mL of the bacterial liquids of Escherichia coli, salmonella enteritidis and shigella dysenteriae were mixed in equal volumes, and after being placed in anaerobic conditions at 37 ℃ for 1h, 2h and 3h, 100 mu L of the mixed bacterial liquid was coated on NB solid medium and cultured for 24h under aerobic conditions at 37 ℃ to perform colony counting of Escherichia coli, salmonella enteritidis and shigella dysenteriae, and the results are shown in Table 3.

TABLE 2

TABLE 3 Table 3

Note that: the number of viable bacteria is 10 ⁷ cfu/mL。

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

Example 3

This example is a description of the storage stability of clostridium butyricum (Clostridium butyricum) inoculant.

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

Inoculating clostridium butyricum stored in a frozen tube form at-80 ℃ to RCM slant culture medium in a serpentine streaking mode, culturing for 48-72h under anaerobic condition at 37 ℃, and collecting test tube slant strain; selecting an inoculating loop inclined plane 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 performing expanded culture to obtain seed liquid; inoculating the seed solution into a liquid fermentation tank culture medium according to the inoculation amount of 10%, and then fermenting and enlarging and culturing for 72 hours under the anaerobic condition of 30-32 ℃ to obtain fermentation liquor.

The number of the viable bacteria is up to 10 ⁹ -10 ¹⁰ And (3) separating clostridium butyricum fermentation liquor with the concentration of CFU/mL or more through centrifugation to obtain bacterial sludge. Washing bacterial mud by using physiological saline, adding bran into the bacterial mud in a ratio of 1:1, drying at 45 ℃ for 2 days, and crushing to obtain clostridium butyricum bacterial agent.

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

TABLE 4 Table 4

Storage time (month)	Clostridium butyricum cfu/g of the invention	Control strain cfu/g
			0	5.0×10 ⁹	6.1×10 ⁹
24	7.5×10 ⁸	8.7×10 ⁶

As can be seen from Table 4, the solid microbial inoculum prepared from clostridium butyricum has good stability, and the viable count can still reach 10 after 2 years of storage ⁸ cfu/g or more, is better than that of the control strain.

Example 4

The example is used for explaining the influence of clostridium butyricum (Clostridium butyricum) microbial inoculum on the growth state and immunity of broilers.

Two clostridium butyricum bacterial agents prepared in example 3 were used, and the effect of the growth state and immunity of broilers was evaluated.

200 broiler chickens of 1 day old are selected as experimental objects, randomly divided into 5 groups, and the group settings are as follows: the feed is characterized in that a basic feed group (No. 1), a clostridium butyricum feed group (No. 2) added with 0.01% of the feed, a clostridium butyricum feed group (No. 3) added with 0.1% of the feed, a clostridium butyricum feed group (No. 4) added with 1% of the feed, a chlortetracycline feed group (No. 5) added with 150mg/kg and a control strain bacterium feed group (No. 6) added with 1% are fed into cages with feeders and nipple drinkers, free feeding is realized, water supply is sufficient, daily management is conventional, and feeding experiments for 42 days are carried out. Wherein:

the basic feed comprises the following components in 1-21 days: 53.78 percent of corn meal, 38.75 percent of bean cake meal, 3.15 percent of soybean oil, 1.98 percent of calcium hydrophosphate, 1.05 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 compound vitamin and 0.30 percent of mineral.

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

The results of the average daily feed intake, average daily gain and the feed weight ratio are shown in Table 5.

TABLE 5

From the feed weight ratio of broilers in days 1-42 of Table 5, the feed group with 0.01% clostridium butyricum of the invention was slightly lower than the feed group with antibiotics, both of which were significantly lower than the basal feed group and the 1% control strain microbial inoculum feed group (P < 0.05).

The immunity is characterized by measuring the content of IgG in the serum of the broiler chicken, and the result is shown in Table 6.

TABLE 6

Time	No. 1	No. 2	No. 3	No. 4	No. 5	No. 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 Ig content in the serum of broiler chickens added with 0.01% of clostridium butyricum microbial inoculum feed group of the invention is slightly higher than that of the feed group added with antibiotics, and is obviously higher than that of the basal feed group and the 1% control strain microbial inoculum feed group (P is less than 0.05).

Example 5

This example is for explaining the effect of clostridium butyricum (Clostridium butyricum) inoculant on the growth status and immunity of piglets.

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

50 weaned pigs of about 21 days old (6.2 kg) are selected and randomly divided into 5 groups, and the group settings are as follows: the basic feed group (No. 1), the clostridium butyricum bacterial agent feed group (No. 2) added with 0.01 percent, the clostridium butyricum bacterial agent feed group (No. 3) added with 0.1 percent, the clostridium butyricum bacterial agent feed group (No. 4) added with 1 percent, the aureomycin feed group (No. 5) added with 150mg/kg and the bacterial agent feed group (No. 6) of the 1 percent control strain are adopted to freely feed by adopting a funnel type feeding trough, the water supply is sufficient, and the daily management is carried out according to the routine, and the feeding experiment for 30 days is carried out. Wherein:

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

The results of average daily feed intake, average daily gain and feed weight ratio are shown in Table 7, and the serum IgG content (ng/mL) of piglets is shown in Table 8.

TABLE 7

Detection index	No. 1	No. 2	No. 3	No. 4	No. 5	No. 6
							Average daily feed intake (g/d)	579	580	583	581	582	582
Average daily gain (g/d)	244	257	264	267	262	250
							Ratio of material to weight	2.37	2.26	2.21	2.18	2.22	2.32

TABLE 8

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

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

Example 6

The example is used for explaining the influence of clostridium butyricum (Clostridium butyricum) bacterial agent on the growth state and immunity of freshwater grass carp.

Two clostridium butyricum bacterial agents prepared in example 3 were used, and the effects of the growth state and immunity of freshwater grass carp were evaluated.

About 50g of grass carp 200 are selected, randomly divided into 5 groups, and the group settings are as follows: foundationFeed group (No. 1), clostridium butyricum 0.01% feed group (No. 2), clostridium butyricum 0.1% feed group (No. 3), clostridium butyricum 1% feed group (No. 4), aureomycin 150mg/kg feed group (No. 5) and control strain 1% feed group (No. 6). Cultivated in a volume of 1.5m ³ In the cleaned and sterilized cement pond, feeding is carried out twice a day, the feeding amount is about 2-3% of the weight of the fish, one third of water in the pond is changed every day, and 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 compound vitamins and 2% of soybean oil.

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

TABLE 9

Detection index	No. 1	No. 2	No. 3	No. 4	No. 5	No. 6
							Average daily feed 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
							Ratio of material to weight	1.12	1.06	1.03	1.02	1.06	1.09

Table 10

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

As can be seen from tables 9 and 10, the feed ratio of the feed group added with 0.01% clostridium butyricum of the present invention to the feed group added with antibiotics was not significantly different, but both were significantly lower than the feed group fed with the basal feed group and the feed group fed with 1% control strain microbial inoculum (P < 0.05); the Ig content in grass carp serum added with 0.01% of clostridium butyricum microbial inoculum feed group is slightly higher than that of feed group added with antibiotics, and is obviously higher than that of a basic feed group and a 1% control bacterial strain microbial inoculum feed group (P is less than 0.05).

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including that the individual technical features are combined in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.

Claims

1. Clostridium butyricumClostridium butyricumThe clostridium butyricum has a preservation number of 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 10 ⁸ cfu/mL or more.

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

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

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

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

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

8. A microbial agent prepared according to the method of any one of claims 4-7.

9. Use of clostridium butyricum as claimed in claim 1 or the microbial inoculum as claimed in any one of claims 2, 3 and 8 for the preparation of an antibiotic substitute in the field of livestock and aquaculture.

10. A feed comprising the clostridium butyricum of claim 1 or the microbial inoculum of any one of claims 2, 3 and 8.