CN114181882A

CN114181882A - Method for improving manganese ion content in bacillus subtilis and composite microbial inoculum

Info

Publication number: CN114181882A
Application number: CN202111600041.0A
Authority: CN
Inventors: 王宝维; 杨铸钰; 邹函峪; 王秉翰; 张名爱; 凡文磊; 孔敏
Original assignee: Qingdao Huihe Biotechnology Co ltd; Qingdao Agricultural University
Current assignee: Qingdao Huihe Biotechnology Co ltd; Qingdao Agricultural University
Priority date: 2021-12-24
Filing date: 2021-12-24
Publication date: 2022-03-15
Anticipated expiration: 2041-12-24
Also published as: CN114181882B

Abstract

The invention provides a method for improving manganese ion content in bacillus subtilis, which is to ferment and culture the bacillus subtilis and clostridium butyricum simultaneously in a culture medium rich in manganese ions. The bacillus subtilis strain is bacillus subtilis NT66 strain, which is preserved in No. 3 of West Lu No.1 of the Kyoho, the republic of China Committee for culture Collection of microorganisms, the China academy of sciences, with the preservation number of CGMCC No.23751, at 11 months and 8 days in 2021. According to the invention, the bacillus subtilis strain domesticated by high manganese ions and the clostridium butyricum are jointly cultured according to a certain proportion, so that the quantity of the bacillus subtilis can be increased, the small peptide content in the domesticated bacillus subtilis strain can be effectively increased, the prepared bacillus product can effectively enhance the immunity of animals, promote the growth and development and improve the utilization rate of feed.

Description

Method for improving manganese ion content in bacillus subtilis and composite microbial inoculum

Technical Field

The invention belongs to the technical field of livestock and poultry micro-ecological preparation, and particularly relates to a method for improving manganese ion (Mn) in bacillus subtilis²⁺) Content method and composite microbial inoculum.

Background

In the field of livestock and poultry breeding, proper manganese ions (Mn) are added into feed²⁺) Can promote growth and development of livestock and poultry and enhance the quality of eggshells of eggs. The manganese digestion and absorption utilization rate is reported to be low, and the manganese digestion and absorption utilization rate of the poultry is only 2-5%. Inorganic manganese Mn directly added into feed²⁺Most of the waste water is discharged out of the machine body; particularly in large-scale livestock and poultry breeding, the content of manganese ions discharged into the environment is very high, water eutrophication is easily caused after the manganese ions accumulate for a long time, and Mn in livestock and poultry products is caused²⁺The content is high; because the livestock and poultry manure contains metal Mn²⁺High Mn content in produced agricultural products²⁺Also high residual levels, jeopardizing food safety and the overall ecological environment. Therefore, a method for promoting the growth and development of livestock and poultry, maintaining the intestinal health and improving the eggshell strength of the livestock and poultry and reducing the emission of manganese is researched, and the method has great social value for the nutrition precision and the maintenance of the ecological good cycle of the animal husbandry.

However, Bacillus subtilis, which is not acclimatized, is rich in metalsHas certain inertia, especially to Mn²⁺Enrichment is very limited. The bacillus subtilis cultured by the intensified domestication method can greatly enhance and enrich Mn²⁺Tolerance to high concentrations of Mn²⁺The enrichment of the ions has good adaptability and bearing capacity. The specificity of the domesticated bacteria lays a good foundation for producing the manganese-rich bacillus subtilis feed additive.

Disclosure of Invention

The invention aims to provide a method for improving the content of manganese ions in bacillus subtilis, so that the content of the manganese ions in the bacillus subtilis is effectively improved, and the defects of the prior art are overcome.

The method for improving the manganese ion content in the bacillus subtilis provided by the invention is to simultaneously ferment and culture the bacillus subtilis and clostridium butyricum in a culture medium rich in manganese ions;

the concentration of manganese ions in the culture medium is 15-70.0 mg/L; preferably 40.0 mg/L.

As a concrete description of the examples, a concrete composition of the culture medium is as follows: 5-15 g/L of peptone, 1-5 g/L of beef extract powder, 1-10 g/L of NaCl and Mn²⁺The concentration is 15-70.0 mg/L, and the pH value is 5.5-7.9 + -0.2.

Further, the Bacillus subtilis contains high concentration of Mn²⁺The ionic culture medium is acclimatized.

The Bacillus subtilis strain is a manganese-resistant Bacillus subtilis NT66 strain, which is preserved in No. 3 of No.1 Su West Lu of North Chen of the Yangyang district in Beijing at 11 months and 8 days in 2021, and the preservation number is CGMCC No.23751 at the China general microbiological culture Collection center of the institute of microbiology of China academy of sciences.

The invention also provides a composite microbial inoculum which comprises domesticated bacillus subtilis and clostridium butyricum;

preferably, the number ratio of the bacillus subtilis NT66 strain to the clostridium butyricum in the composite microbial inoculum is 1: 1-2.

According to the invention, the bacillus subtilis strain domesticated by high manganese ions is co-cultured with clostridium butyricum, so that the quantity of the bacillus subtilis can be increased, and the small peptide content in the domesticated bacillus subtilis strain is effectively increased. The bacillus subtilis NT66 strain and clostridium butyricum combined microbial inoculum has good use effect, can effectively enhance the immunity of animals, inhibit harmful bacteria, maintain the health of intestinal tracts, promote the growth and development and improve the utilization rate of feed.

Drawings

FIG. 1: a graph of the effect of acclimation on the morphology of bacillus subtilis;

FIG. 2: the fermentation graph of the bacillus subtilis manganese in the liquid culture medium containing the clostridium butyricum supernatant is shown, wherein the control group is that the bacillus subtilis manganese is fermented in a common liquid culture medium, and the experimental group is that the bacillus subtilis manganese is fermented in the liquid culture medium containing the clostridium butyricum supernatant.

FIG. 3: a fermentation pattern of clostridium butyricum in a liquid culture medium containing a bacillus subtilis manganese supernatant, wherein a control group is that clostridium butyricum is fermented in a common liquid culture medium; the experimental group is that clostridium butyricum is fermented in a liquid culture medium containing a bacillus subtilis manganese supernatant.

FIG. 4: protein standard curve graph;

FIG. 5: determining figures of butyric acid standards with different concentrations by liquid chromatography;

FIG. 6: butyric acid standard curve chart.

Detailed Description

The bacillus subtilis is not a resident bacterium of the intestinal tract, only belongs to a passerby, can not be planted and grown in the intestinal tract for a long time, enters the gastrointestinal tract along with food, is finally discharged out of the body together with excrement, and plays a role in a culture environment. The bacillus subtilis belongs to aerobic bacteria, has strong gastric acid and bile salt resistance, can keep higher activity, and inhibits the propagation of harmful bacteria in intestinal tracts. The invention combines the bacillus subtilis and the clostridium butyricum to culture, so that more organic manganese can be enriched in the bacillus subtilis.

The present invention will be described in detail with reference to specific embodiments.

Example 1: culturing and domesticating bacillus subtilis

By gradually increasing Mn in the medium²⁺Domestication is carried out in a mode of increasing the concentration and the culture temperature; after the domesticated strain is continuously cultured for a plurality of generations, the strain is compared with the bacillus subtilis before domestication in terms of thallus morphology, enzyme activity, small peptides, amino acids and the like, the property characteristics of the domesticated bacillus subtilis are further determined, and stable passage can be realized. The effect of acclimation on bacillus subtilis morphology is shown in figure 1. As can be seen from FIG. 1, the manganese-rich Bacillus subtilis strain becomes thicker and longer under the same times of strain, and the surface texture is clear and smooth. The appearance of the bacillus subtilis is obviously changed under the domestication effect of the manganese ions.

Wherein the domesticated and cultured Bacillus subtilis NT66 strain capable of tolerating high-concentration manganese sulfate (300mg/L) is preserved in No. 3 Xilu No.1 Beijing, the sunward area, 11.8 days in 2021, and the China general microbiological culture Collection center of the institute of microbiology, China, with the preservation number of CGMCC 23751.

The manganese ion enrichment fermentation is respectively carried out by adopting domesticated copper-rich bacillus subtilis (the preservation number is CGMCC No.15329), zinc-rich bacillus subtilis (the preservation number is CGMCC No.15330) and manganese-rich bacillus subtilis (the preservation number is CGMCC No. 23751). Respectively inoculating copper-resistant, zinc-resistant and manganese-resistant bacillus subtilis strains in a culture medium containing 40mg/L manganese ions, culturing at 37 ℃ for 24 hours, centrifuging, eluting and drying bacterial liquid, and determining the content of thalli and the content of organic manganese in the thalli.

The result shows that when the addition amount of manganese is 40.0mg/L, the thallus content is 0.46g/L, the organic manganese content of the thallus is 2521.26mg/kg, which is far greater than the manganese content of copper-rich bacillus subtilis and zinc-rich bacillus subtilis, and the domesticated strain has a better manganese-rich effect. The manganese-rich effect of the strain can not be achieved by the domesticated copper-rich bacillus subtilis or zinc-rich bacillus subtilis.

Moreover, since high-temperature tolerance acclimation is also performed in manganese-rich acclimation, the screened acclimated copper-rich bacillus subtilis has higher temperature tolerance than that of the acclimated conventional bacillus subtilis. The temperature of 30-40 ℃ is the proper growth temperature of the bacillus subtilis, and the bacillus subtilis breeds one generation every 20-30 minutes under the temperature condition. And the temperature exceeds 50 ℃, the growth speed of the manganese-rich bacillus subtilis is inhibited, but spores can be formed to resist high-temperature environment. The manganese-rich bacillus subtilis domesticated by the invention can survive at a high temperature of more than 60 ℃, and the survival rate is greatly higher than that of the common bacillus subtilis strain which is not domesticated. The yeast can not survive at the temperature of over 60 ℃, the bifidobacterium can not survive at the temperature of over 70 ℃, and the manganese-rich bacillus subtilis screened and domesticated by the method can also survive for about 10 minutes at the temperature of 100 ℃. The domesticated manganese-rich bacillus subtilis has obvious high temperature resistance, so that the requirement that probiotics added in the livestock and poultry pellet feed have the high temperature resistance can be met.

Example 2: symbiotic effect of bacillus subtilis NT66 strain and clostridium butyricum

1. Growth promoting effect of bacillus subtilis NT66 strain and clostridium butyricum cell-free supernatant

Clostridium butyricum (Clostridium butyricum) used in the test was purchased from Koehania bioengineering, Inc., Shandong Su.

1) Preparation of two sterile cell-free supernatants: respectively selecting a ring of strains from test tube inclined planes of the two bacteria, inoculating the strains into corresponding 250ml of culture medium, repeating 6 strains for each strain, culturing at 37 ℃ for 24 hours (clostridium butyricum anaerobic culture), centrifuging at 6000/min for 10min, and then carrying out autoclaving to obtain two bacteria cell-free supernatants.

Respectively selecting a ring of strains from test tube inclined planes of the two strains, inoculating the strains into corresponding 250ml of culture medium, repeating the steps for 3 strains respectively, culturing the strains at 37 ℃ for 24 hours (clostridium butyricum anaerobic culture) to obtain seed solutions of the two strains, and performing microscopic examination and counting respectively.

(2) The symbiotic effect of two bacteria has influence on the growth of the bacteria: the fermentation broth was sampled every 4 hours and measured for OD600, and the growth curves of Bacillus subtilis NT66 strain were recorded, as shown in FIGS. 2 and 3, respectively. FIG. 2 shows the growth promoting effect of the fermentation product of Clostridium butyricum on Bacillus subtilis NT66 strain, and the experimental group shows that the cell-free supernatant of Clostridium butyricum is added into the culture medium of Bacillus subtilis NT66 strain after acclimation, and the OD600 value of the final culture solution is 1.49 at most; the control group was Bacillus subtilis NT66 strain without addition of Clostridium butyricum cell-free supernatant, and the OD600 value of the final culture solution was 1.23 at the maximum. FIG. 3 demonstrates the growth promoting effect of the fermentation product of the acclimated Bacillus subtilis on Clostridium butyricum, and the experimental group shows that the culture medium of Clostridium butyricum contains the acclimated Bacillus subtilis supernatant, and the final culture medium has an OD600 value of 0.88 at most; the control group showed growth of C.butyricum without acclimatized B.subtilis supernatant, and the final culture had an OD600 of 0.58 at the maximum.

The results show that the domesticated bacillus subtilis NT66 strain can form a synergistic effect with clostridium butyricum, so that the growth of the bacillus subtilis NT66 strain is promoted; meanwhile, the bacillus subtilis NT66 strain also has the capability of promoting the growth of clostridium butyricum.

Adding Bacillus subtilis to the culture medium containing Mn 40.0mg/kg at an inoculation amount of 3%²⁺The fermentation was completed after culturing in a shaking incubator at a constant temperature of 150r/min at 37 ℃ for 24 h. Centrifuging the fermentation liquor at 6000r/min and 4 ℃ to obtain wet thalli, washing the wet thalli for 2-3 times by using deionized water, and drying the wet thalli at 55 ℃ to obtain bacillus subtilis powder. Weighing a certain amount of bacillus subtilis powder, and measuring the content of organic manganese in the bacteria. Under the condition, the thallus content is 0.40g/L, and the content of the organic manganese in the thallus is 1337.55 mg/kg. Compared with the bacillus subtilis cultured by the domesticated bacillus subtilis NT66 strain and clostridium butyricum in a combined way, the content of the bacillus subtilis is 15 percent lower, and the content of the manganese in the bacillus subtilis is reduced by 46.76 percent.

2. Effect of synergistic Effect on Small peptide content

Taking 20ml of two-bacterium liquid fermentation liquor, centrifuging at 4 ℃ and 6000r/min for 10min, taking 5ml of supernate, adding 0.4mol/L of TCA solution with the same volume, standing for 0.5h, centrifuging at 4000r/min for 10min, and removing insoluble protein and long-chain peptide. And centrifuging for 5min at 4000r/min, and taking a certain amount of supernatant to perform biuret method determination.

(1) The preparation method of the reagent used in the test comprises the following steps:standard protein solution: accurately weighing 0.4g of bovine serum albumin, dissolving with distilled water to a constant volume of 100mL, and storing in a refrigerator at 4 ℃; biuret reagent: weighed 1.50g copper sulfate (CuSO)₄:5H₂O) and 6.0g of sodium potassium tartrate (KNaC)₄H₄O₆·4H₂O), dissolving with 500mL of water, adding 300mL of 10% NaOH solution under stirring, diluting to 1L with water, and storing in a plastic bottle; 10% trichloroacetic acid (TCA): 1mL of TCA was weighed out accurately and dissolved in distilled water to a constant volume of 10 mL.

(2) Calibration of protein standard curve: accurately weighing 1.0g of sample, adding 9mL of water, mixing uniformly, adding 10mL of 10% TCA solution, shaking, mixing uniformly, standing for 30min, and finally centrifuging to obtain supernatant for later use.

Adding the above reagents respectively as shown in Table 1 below, mixing, reacting in dark for 30min, measuring absorbance at 540nm wavelength after reaction, and taking protein concentration as abscissa and absorbance as ordinate as standard curve. The results of the standard curve are shown in FIG. 4.

(3) And (3) measuring the content of the small peptide in the fermentation liquor: accurately transferring 1mL of supernatant into a test tube by using a pipette, adding 4mL of distilled water, uniformly mixing, adding 5mL of biuret reagent, uniformly mixing, reacting for 30min in a dark place, measuring the absorbance value at a wavelength of 540nm after the reaction is finished, and substituting the absorbance value into a standard curve to calculate the small peptide content of the sample. The protein standard curve is plotted in table 1. The small peptide content results are shown in table 2.

Table 1: drawing table of protein standard curve

Table 2: influence table of symbiotic effect of bacillus subtilis manganese and clostridium butyricum on small peptide content

Note: BS is domesticated bacillus subtilis NT66 strain; the CB supernatant is the supernatant obtained after fermentation of clostridium butyricum; CB is clostridium butyricum; the BS supernatant is the supernatant obtained after fermentation of the Bacillus subtilis NT66 strain.

The CB supernatant liquid BS fermentation promotion test verifies that the clostridium butyricum fermentation liquor has the effect of promoting the bacillus subtilis NT66 to generate small peptides, the BS group is the small peptide content in the normal bacillus subtilis NT66 fermentation liquor, and the BS + CB supernatant liquid group is the small peptide content in the bacillus subtilis NT66 fermentation liquor based on the clostridium butyricum supernatant liquid. The test result shows that the content of the small peptide produced by the bacillus subtilis NT66 strain is obviously increased under the influence of the clostridium butyricum fermentation liquor (p is less than 0.01).

The BS supernatant CB promotion fermentation test verifies that the fermentation liquor of the bacillus subtilis NT66 strain has the effect of promoting the clostridium butyricum to generate small peptides, the CB group is the content of the small peptides in the normal clostridium butyricum fermentation liquor, and the CB + BS supernatant group is the content of the small peptides in the clostridium butyricum fermentation liquor based on the bacillus subtilis NT66 strain supernatant; the test result shows that the content of the small peptide produced by the clostridium butyricum is obviously increased (p is less than 0.01) under the influence of the fermentation liquid of the bacillus subtilis NT66 strain.

The above results indicate that the Bacillus subtilis NT66 strain has synergistic effect with Clostridium butyricum.

Example 3: influence of synergistic fermentation of manganese bacillus subtilis NT66 strain and clostridium butyricum on butyric acid content

(1) Drawing a butyric acid standard curve: the concentrations of prepared butyric acid standard substances are respectively 25ppm, 50ppm, 100ppm, 500ppm, 1000ppm, 2500ppm and 5000ppm, and all the butyric acid standard substances with the concentrations pass through a 0.22 mu m water system film and are processed. The measurement of butyric acid standard and butyric acid standard curve are shown in fig. 5 and fig. 6.

(2) Determination of butyric acid content in sample: the test is divided into two parts, the CB supernatant fluid BS fermentation promotion test is to study the change of butyric acid content of clostridium butyricum fermentation liquor on the production of bacillus subtilis NT66 strain in the growth process, two groups of BS, BS + CB supernatant fluid are arranged, and the BS is the fermentation of bacillus subtilis NT66 strain in a basic liquid culture medium; the BS + CB supernatant was the bacillus subtilis NT66 strain fermented in minimal medium supplemented with clostridium butyricum supernatant. The BS supernatant fluid CB promotion fermentation test is to research the change of the content of butyric acid generated by the clostridium butyricum in the growth process of the bacillus subtilis manganese fermentation liquid, two groups of CB, CB + BS supernatant fluid are arranged, and the CB group is that the clostridium butyricum is fermented in a basic liquid culture medium; the group of CB + BS supernatants is a fermentation of Clostridium butyricum in a basal medium supplemented with the supernatant of Bacillus subtilis NT66 strain. The results are detailed in Table 3.

Table 3: table for influence of symbiotic effect of bacillus subtilis NT66 strain and clostridium butyricum on butyric acid content

Note: BS is domesticated bacillus subtilis manganese; the CB supernatant is the supernatant obtained after fermentation of clostridium butyricum; CB is clostridium butyricum; the BS supernatant is the supernatant obtained after the manganese fermentation of the bacillus subtilis, and the contrast groups are respectively the fermentation of the BS and the CB in a common culture medium; the experimental groups were fermented with BS supernatant and CB supernatant added, respectively.

The results in Table 3 show that when the Bacillus subtilis NT66 strain is fermented in a culture medium containing Clostridium butyricum supernatant, the content of butyric acid produced by the strain is obviously improved compared with that of a control group (p is less than 0.01); when clostridium butyricum is fermented in a culture medium containing a supernatant of bacillus subtilis NT66 strain, the content of butyric acid produced by the clostridium butyricum is also remarkably improved compared with that of a control group (p is less than 0.01). This indicates that the Bacillus subtilis NT66 strain can have synergistic effect with Clostridium butyricum fermentation, and increase the content of butyric acid produced during fermentation. The characteristics of the bacteria have important reference value for preparing probiotic preparations.

FIG. 5 shows measured values of butyric acid standards at different concentrations. The butyric acid standard curve was obtained by plotting the butyric acid concentration gradient on the abscissa and the measured value on the ordinate (see FIG. 6). The result of fig. 5 shows that the peak-off time of the sample to be measured is consistent with that of the standard substance, and the content of the substance measured in the sample to be measured is proved to be the content of butyric acid; the peak area size of the graph represents the measured content of butyric acid, and different peak areas are obtained according to the measured samples. The result shows that the domesticated bacillus subtilis and clostridium butyricum can produce higher content of butyric acid through synergistic fermentation.

Example 4: influence of bacillus subtilis NT66 strain on growth and development of meat ducks

In the experiment, 180 male Beijing cherry valley duck ducklings of 7 days old are selected and fed adaptively for a period of time. The weight was randomly divided into 6 groups of 5 replicates each, with 6 replicates each. This experiment used 5 treatment groups and 1 control group, NP group: feeding basal diet (the ingredients of the basal diet are shown in Table 4) to the blank control group; BS group: adding a bacillus subtilis NT66 strain acclimatized by manganese on the basis of basic daily ration; group CB: adding clostridium butyricum on the basis of basic ration; BS + CB (1: 1) group: adding mixed probiotic preparation (mixed and added by manganese domesticated bacillus subtilis NT66 strain and clostridium butyricum in a ratio of 1: 1) on the basis of basic daily ration; BS + CB (1: 2) group: adding mixed probiotic preparation (mixed and added by manganese domesticated bacillus subtilis NT66 strain and clostridium butyricum in a ratio of 1: 2) on the basis of basic daily ration; BS + CB (2:1) group: adding mixed probiotic preparation (mixed with manganese acclimatized Bacillus subtilis NT66 strain and Clostridium butyricum at ratio of 2:1) on the basis of basic daily ration, wherein the added probiotic preparation ensures that the bacteria concentration is 1 × 10⁸CFU/ml. The experimental design is shown in table 5. The feeding period was 6 weeks, and slaughter sampling was performed after 6 weeks.

Table 4: ingredient table of basic feed for meat duck

¹The premix provides the following micronutrients (complete food per kg): VA 12000 IU, VD ₃ 2 500IU，VE 20mg，VK₃ 3mg，VB₁ 3mg，VB₂ 8mg，VB₆ 7mg，VB₁₂0.03mg, 20mg of D-pantothenic acid, 50mg of nicotinic acid, 1.5mg of folic acid, 0.1mg of biotin, 500mg of choline, 9mg of copper (as copper sulfate), 110mg of zinc (as zinc sulfate), 100mg of iron (as ferrous sulfate), 100mg of manganese (as manganese sulfate), 0.16mg of selenium (as sodium selenite), and 0.6mg of iodine (as potassium iodide).

²The contents of nutrient components are calculated values

Table 5: meat duck feeding test design table

The test result shows that the weight of the meat duck fed by the bacillus subtilis Mn NT66 strain or the bacillus subtilis NT66 strain and clostridium butyricum in a synergistic manner is obviously higher than that of a control group (p is less than 0.01) when the meat duck is fed by the bacillus subtilis Mn NT66 strain at the age of 35 days; for feed intake, except for the BS + CB (2:1) group, the meat ducks fed daily have large feed intake, and the other treatment test groups have no obvious difference (p is more than 0.05) from the control group. In addition, the feed is added with a mixed preparation of the bacillus subtilis NT66 strain or the bacillus subtilis NT66 strain and clostridium butyricum, so that the feed conversion rate (p is less than 0.01) can be remarkably reduced, and the conversion rate of the meat duck feed can be improved by the domesticated manganese ion-rich bacillus subtilis (Table 6).

Table 6: growth performance table for 35-day-old meat ducks

In conclusion, the invention adopts the gradual increase of Mn in the culture medium²⁺The bacillus subtilis NT66 strain obtained by domesticating and screening by a concentration and culture temperature method not only has the advantages of bacillus subtilis, but also has the capability of remarkably improving the capability of converting inorganic manganese into thallus organic manganese, greatly improves the utilization rate of trace elements and the feed conversion rate of livestock and poultry, promotes growth and development, and better exerts the biological function of the organic manganese.

Claims

1. A method for improving the content of manganese ions in bacillus subtilis is characterized in that the bacillus subtilis and clostridium butyricum are simultaneously fermented and cultured in a manganese ion-rich culture medium.

2. The method according to claim 1, wherein the concentration of manganese ions in the medium is 15 to 70.0 mg/L.

3. The method of claim 2, wherein the concentration of manganese ions in the medium is 40.0 mg/L.

4. The method of claim 1, wherein the composition of the culture medium is as follows: 5-15 g/L of peptone, 1-5 g/L of beef extract powder, 1-10 g/L of NaCl and Mn²⁺The concentration is 15-70.0 mg/L, and the pH value is 5.5-7.9 + -0.2.

5. The method of claim 1, wherein said bacillus subtilis is a bacillus subtilis strain containing a high concentration of Mn²⁺The ionic culture medium is acclimatized.

6. The method of claim 1, wherein the bacillus subtilis has a accession number of CGMCC No. 23751.

7. A composite strain is characterized by comprising bacillus subtilis and clostridium butyricum.

8. The composite strain of claim 7, wherein the ratio of the bacillus subtilis strain to the clostridium butyricum is 1: 1-2.

9. The complex strain of claim 7 or 8, wherein the Bacillus subtilis strain contains a high concentration of Mn²⁺The ionic culture medium is acclimatized.

10. The composite strain of claim 9, wherein the bacillus subtilis strain has a accession number of CGMCC No. 23751.