CN108094686A - Composite preparation capable of effectively improving disease resistance of aquatic animals and preparation method thereof - Google Patents

Abstract

The invention discloses a compound preparation capable of effectively improving disease resistance of aquatic animals and a preparation method thereof, wherein the compound preparation comprises the following components in parts by mass: 55-65 parts of microbial powder, 10-15 parts of astragalus polysaccharide, 10-15 parts of yeast cell walls, 5-10 parts of plant essential oil and 61-5 parts of vitamin B; the components are matched with each other, so that the structure of intestinal flora can be reformed, the proportion of unsaturated fatty acid in metabolites of acid-producing bacteria in intestinal tracts can be changed, pathogenic bacteria in intestinal tracts and other harmful substances can be inhibited, intestinal mucosa can be repaired, nutrient digestion and absorption can be promoted, meanwhile, the nonspecific immunity of an organism can be enhanced, the stress resistance of the organism can be enhanced, the resistance to foreign viruses, germs, parasites and polluted environment can be stronger, the utilization rate and the survival rate of bait of the organism can be comprehensively improved, residual bait can be reduced, excrement can be purified, and the intensive culture mode can be optimized and standardized.

Description

Composite preparation capable of effectively improving disease resistance of aquatic animals and preparation method thereof

technical field

The invention belongs to the field of applied biotechnology, and in particular relates to a compound preparation which can effectively improve the disease resistance of aquatic animals and a preparation method.

Background technique

With the deepening of the intensive farming model in the industry, high-density farming technology has attracted more and more attention from the aquatic industry. However, due to irregular operations such as high stocking density, excessive feeding of bait, and abuse of fishing medicines, harmful substances such as feces, residual bait, and residual drugs in the water body and sediment continue to accumulate, and various viral diseases, bacterial diseases, parasitic diseases, etc. Insect diseases continue to breed in the breeding environment. In order to prevent and treat various diseases in the water body, many farmers choose to inject antibiotics, which seriously destroys the balance of the micro-ecological system in the natural water body and animal intestines, greatly improving the self-purification ability of the water body and the disease resistance of farmed animals. reduce. At present, in order to better improve animal disease resistance and reduce the use of antibiotics, the method of adding immune enhancers to organisms to improve biological non-specific immunity is adopted, but the immune enhancers added from outside are not easy to digest and absorb in the intestine, so The improvement of disease resistance of aquatic animals is limited.

Contents of the invention

In order to solve the above-mentioned technical problems in the prior art, the present invention provides a compound preparation and a preparation method that can effectively improve the disease resistance of aquatic animals.

The technical solution of the present invention is: a compound preparation that can effectively improve the disease resistance of aquatic animals, which is characterized in that the components and mass ratios are as follows: microbial powder 55-65, astragalus polysaccharide 10-15, yeast cell wall 10 ~15, plant essential oil 5~10, vitamin B6 1~5; the microbial powder is composed of Acremonium terricola powder ( Acremonium terricola ) and Clostridium butyricum powder ( Clostridium butyricum ) according to the mass ratio of 35~50:15~ 30, the yeast cell wall is baker's yeast cell wall containing 30% β-glucan, and the plant essential oil is composed of cinnamon aldehyde, thymol, and lavender essential oil in a mass ratio of 2-4:2-4:1 ~2 mixtures; the active ingredient of Acremonium acremonium powder in the composite preparation is ≥200 mg/g, the number of live bacteria of Clostridium butyricum powder is ≥5×10 ⁸ CFU/g, and the preservation number of the Acremonium acremonium powder is ATCC13215 ( NBRC30538) and was identified by CICC, China Culture Collection Management Center, and the Clostridium butyricum preservation number is CICCNo.M 23847.

A method for preparing the above-mentioned compound preparation that can effectively improve the disease resistance of aquatic animals is characterized in that the specific steps are as follows:

A Preparation of microbial powder

(1) Preparation of Acremonium mold powder

a. Strain activation

Inoculate the lyophilized powder of Acremonium acremonium on the PDA medium, and cultivate it at 25°C for 7 days;

b. Seed culture

The activated strains were transferred to shake flasks with 2% inoculum, and shaken at 25°C for 24-36 hours to obtain seed culture liquid. The mass percentages of liquid medium components were: white granulated sugar 1.4%, silkworm chrysalis Yeast powder 2.3%, yeast powder 1.2%, water balance, pH value is 6.5;

c. Fermenter culture

The seed culture solution is inserted into a 50L fermenter with 20% inoculum, and the filling coefficient is 70%. After 48-60 hours of aerated culture, it can be placed in the tank. The mass percentage of the medium components in the fermenter is: soybean powder 2.8%, white 2.5% sugar, 1.3% yeast powder, 2.5% silkworm chrysalis powder, 0.05% MgSO ₄ , 0.05% KH ₂ PO ₄ , 0.05% defoamer, the rest of water, adjust the pH value to 6.5 with saturated NaOH;

d. Solid fermentation culture

Put the fermentation liquid obtained in step c into the sterilized solid fermentation medium with an inoculation amount of 20% (v/m). The mass percentages of the components of the solid medium are: corn flour 55%, soybean meal 32%, Wheat bran 13%; culture conditions: 25°C, humidity 70%, fermentation time 72h;

e. Collection of Acremonium culture

After the solid fermentation is over, collect the culture, directly dry it at 60~80°C until the water content is less than 10%, then crush it and pass it through a 40-mesh sieve, which contains 94mg/g polysaccharide, 28mg/g polypeptide and 35mg/g biomass Acremonium mold powder with active ingredients such as g.

(2) Preparation of Clostridium butyricum powder

a. Activation of strains

Pick the freeze-dried powder of Clostridium butyricum and insert it into the activation medium for static anaerobic culture; medium composition: peptone 10g, beef extract 10g, yeast extract 3g, glucose 10g, NaCl 5g, NaAc 3g, hydrochloric acid cysteine Amino acid 0.5g, CaCO ₃ 3g, distilled water to 1000mL, pH 7.0, culture at 37°C for 24h, transfer to a new activation medium with 2% inoculum, and culture for 48h under the same conditions;

b. Seed cultivation

Put the bacterial solution into a 10L seed tank with an inoculation amount of 10%, and the filling coefficient is 70%. The composition of the seed medium is: peptone 5~10g, beef extract 5~10g, yeast extract 3~10g, glucose 5~ 10g, NaCl 5g, NaAc 1~3g, CaCO ₃ 1~3g, distilled water to 1000mL, pH 7.0, anaerobic culture at 37℃ for 20h;

c. Fermenter culture

The seed culture solution is put into a 100L fermenter with 10% inoculation amount, and the filling coefficient is 70%. The medium composition of the fermenter is: peptone 5~10g, yeast extract 3~10g, glucose 5~15g, starch 0.5~1g , NaCl 5g, NaAc 1~3g, CaCO ₃ 3~10g, water to 1000mL, pH 7.0, anaerobic culture at 37℃ for 24h;

d. Bacteria collection

Collect the fermentation broth, discard the supernatant after centrifugation, and obtain wet cells; mix the wet cells of Clostridium butyricum and dry starch at a mass ratio of 1:0.5~5, dry at 50°C for 24 hours, pulverize with a pulverizer, and pass through a 60-mesh sieve. Adjust the number of live bacteria of Clostridium butyricum ≥ 1×10 ¹⁰ CFU/g, which is the original bacterial powder of Clostridium butyricum;

B respectively crush astragalus polysaccharide, yeast cell wall and vitamin _B6 and pass through a 60-mesh sieve, then put astragalus polysaccharide, yeast cell wall, vitamin _B6 , acremonium acremonium culture, clostridium butyricum powder and plant essential oil into the mixing equipment , stir evenly.

The present invention is composed of acremonium acremonium, clostridium butyricum, astragalus polysaccharide, yeast cell wall, plant essential oil and vitamin _B6 . The mutual cooperation of each component can restructure the intestinal flora structure and change the intestinal acid-producing bacteria The proportion of unsaturated fatty acids in metabolites can inhibit intestinal pathogenic bacteria and other harmful substances, repair intestinal mucosa, promote nutrient digestion and absorption, and at the same time enhance the body's non-specific immunity, enhance anti-stress, and resist foreign viruses, bacteria, and parasites. and polluted environment have stronger resistance ability, comprehensively improve the bait utilization rate and survival rate of the body, reduce residual bait, purify feces, optimize and standardize the intensive farming model.

Detailed ways

Example 1:

The composite preparation of the present invention that can effectively improve the disease resistance of aquatic animals has the following component and quality ratios: 65% of microbial powder, 15% of astragalus polysaccharide, 15% of yeast cell wall, 10% of plant essential oil, and 5% of vitamins; the microbial powder is It is made by mixing Acremonium terricola powder and Clostridium butyricum powder ( Clostridium butyricum ) at a mass ratio of 50:30, the yeast cell wall is baker's yeast cell wall containing 30% β-glucan, the The plant essential oil is made by mixing cinnamon aldehyde, thymol, and lavender essential oil in a mass ratio of 2:2:1; the active ingredient of Acremonium acremonium powder in the compound preparation is ≥200mg/g, and the number of live bacteria of Clostridium butyricum powder ≥5×10 ⁸ CFU/g, the Acremonium acremonium has a preservation number ATCC13215 (NBRC30538), and has been identified by the Chinese Culture Collection Center CICC, and the Clostridium butyricum preservation number is CICC No.M23847.

A method for preparing the above-mentioned compound preparation that can effectively improve the disease resistance of aquatic animals is characterized in that the specific steps are as follows:

A Preparation of microbial powder

(1) Preparation of Acremonium mold powder

a. Strain activation

Inoculate the freeze-dried powder of Acremonium acremonium (purchased) on the PDA medium, culture at 25°C for 7 days, and transfer after a large number of spore bundles are produced;

b. Seed culture

The activated strains were transferred to shake flasks with 2% inoculum, and shaken at 25°C for 24-36 hours to obtain seed culture liquid. The mass percentages of liquid medium components were: white granulated sugar 1.4%, silkworm chrysalis Yeast powder 2.3%, yeast powder 1.2%, water balance, pH value is 6.5;

c. Fermenter culture

The seed culture solution is inserted into a 50L fermenter with 20% of the inoculum, and the filling coefficient is 70%. After 48-60 hours of aerated culture, the hyphae grow in large quantities and begin to produce spores. The mass percentages are: soybean powder 2.8%, white sugar 2.5%, yeast powder 1.3%, silkworm chrysalis powder 2.5%, MgSO ₄ 0.05%, KH ₂ PO ₄ 0.05%, defoamer 0.05%, water balance, adjusted with saturated NaOH The pH value is 6.5;

d. Solid fermentation culture

Put the fermentation liquid obtained in step c into the sterilized solid fermentation medium with an inoculation amount of 20% (v/m). The mass percentages of the components of the solid medium are: corn flour 55%, soybean meal 32%, Wheat bran 13%; culture conditions: 25°C, humidity 70%, fermentation time 72h;

e. Collection of Acremonium culture

After the solid fermentation is over, collect the culture, directly dry it at 60~80°C until the water content is less than 10%, then crush it and pass it through a 40-mesh sieve, which contains 94mg/g polysaccharide, 28mg/g polypeptide and 35mg/g biomass Acremonium mold powder with active ingredients such as g.

(2) Preparation of Clostridium butyricum powder

a. Activation of strains

Pick the freeze-dried powder of Clostridium butyricum (purchased) into the activation medium, and let it stand for anaerobic culture; medium composition: peptone 10g, beef extract 10g, yeast extract 3g, glucose 10g, NaCl 5g, NaAc 3g , cysteine hydrochloride 0.5g, CaCO ₃ 3g, distilled water to 1000mL, pH 7.0, culture at 37°C for 24h, transfer to new activation medium with 2% inoculum, culture for 48h under the same conditions, microscopic examination , more than 90% of the bacteria form spores;

b. Seed cultivation

Put the bacterial solution into a 10L seed tank with an inoculation amount of 10%, and the filling coefficient is 70%. The composition of the seed medium is: peptone 5~10g, beef extract 5~10g, yeast extract 3~10g, glucose 5~ 10g, NaCl 5g, NaAc 1~3g, CaCO ₃ 1~3g, distilled water to 1000mL, pH 7.0, anaerobic culture at 37℃ for 20h;

c. Fermenter culture

The seed culture solution is put into a 100L fermenter with 10% inoculation amount, and the filling coefficient is 70%. The medium composition of the fermenter is: peptone 5~10g, yeast extract 3~10g, glucose 5~15g, starch 0.5~1g , NaCl 5g, NaAc 1~3g, CaCO ₃ 3~10g, water to 1000mL, pH 7.0, anaerobic culture at 37°C for 24 hours, microscopic examination, more than 90% of the bacteria formed spores, that is, put the tank to end the culture;

d. Bacteria collection

Collect the fermentation broth, discard the supernatant after centrifugation, and obtain wet cells;

Mix the wet bacteria of Clostridium butyricum and dry starch at a mass ratio of 1:0.5~5, dry at 50°C for 24 hours, pulverize with a pulverizer, pass through a 60-mesh sieve, and adjust the number of viable bacteria of Clostridium butyricum to ≥1×10 ¹⁰ CFU /g, which is the original bacteria powder of Clostridium butyricum;

B respectively crush astragalus polysaccharide, yeast cell wall and vitamin _B6 and pass through a 60-mesh sieve, then put astragalus polysaccharide, yeast cell wall, vitamin _B6 , acremonium acremonium culture, clostridium butyricum powder and plant essential oil into the mixing equipment , stir evenly.

Example 2:

The composite preparation of the present invention that can effectively improve the disease resistance of aquatic animals is characterized in that the components and the mass ratio are as follows: microbial bacterial powder 55, astragalus polysaccharide 10, yeast cell wall 10, plant essential oil 5, vitamin B6 1; The microbial powder is made by mixing Acremonium terricola powder and Clostridium butyricum powder ( Clostridium butyricum ) at a mass ratio of 35:15, and the yeast cell wall is baker's yeast containing 30% β-glucan Cell wall, the plant essential oil is formed by mixing cinnamon aldehyde, thymol, and lavender essential oil in a mass ratio of 2:2:1; the active ingredient of Acremonium acremonium powder in the composite preparation is ≥200 mg/g, Clostridium butyricum The number of live bacteria in the powder is ≥5×10 ⁸ CFU/g, and the acremonium acremonium has a preservation number ATCC13215 (NBRC30538), and has been identified by the China Culture Collection Center CICC, and the Clostridium butyricum preservation number is CICC No.M23847.

The preparation method is the same as in Example 1.

Example 3:

The composite preparation that can effectively improve the disease resistance of aquatic animals of the present invention is characterized in that each component and the mass ratio are as follows: microbial bacterial powder 60, astragalus polysaccharide 12, yeast cell wall 13, plant essential oil 8, vitamin B6 3; The microbial powder is made by mixing Acremonium terricola and Clostridium butyricum at a mass ratio of 40:20, and the yeast cell wall is baker's yeast containing 30% β-glucan Cell wall, the plant essential oil is formed by mixing cinnamon aldehyde, thymol, and lavender essential oil in a mass ratio of 3:3:2; the active ingredient of Acremonium acremonium powder in the composite preparation is ≥200 mg/g, Clostridium butyricum The number of live bacteria in the powder is ≥5×10 ⁸ CFU/g, and the acremonium acremonium has a preservation number ATCC13215 (NBRC30538), and has been identified by the China Culture Collection Center CICC, and the Clostridium butyricum preservation number is CICC No.M23847.

The preparation method is the same as in Example 1.

The composite preparation of the present invention is used as an aquatic feed additive, and the method of use is as follows: the addition amount is 0.05% to 0.3% of the total mass of the feed, and it can be directly added to the aquatic animal feed, or mixed with a carrier to make a premix; or mixed with other Feed additives or feed materials are mixed to make premixes and concentrates to feed aquatic animals.

Experimental example one:

Select carps with similar specifications and randomly divide them into four groups: compound group, bacteria powder group, immune agent group and blank control group. They are fed in an indoor aquarium at a water temperature of 20-25°C, 100 in each group, and the feeding cycle is 100 sky. Each group was fed the same basic commercial feed. The compound group added 0.06% of the compound preparation of the embodiment of the present invention, the bacterial powder group added 0.06% of the microbial bacterial powder in Example 1 of the present invention, the immune agent group added 0.025% of Astragalus membranaceus, 0.025% of yeast cell wall, and 0.01% of plant essential oil, blank The control group only added basic commercial feed, and other feeding conditions were the same. Record fish growth and water quality, see Table 1.

Table 1

group Fish weight gain rate (%) Fish survival rate (%) Feed coefficient Fish vitality water color Compound group 80 99 2.21 active refreshing Bacteria powder group 70 95 2.30 more active refreshing immune booster 65 94 2.52 active turbid Blank control group 60 90 2.55 sluggish turbid

The test results show that the weight gain and survival rate of the fish in the compound group added with the compound preparation of the embodiment of the present invention are significantly higher than those in other groups (bacteria powder group, immune enhancement group and blank control group), and the bait coefficient is significantly lower than that in the control group. It shows that the feed additive of the present invention can promote the digestion of carp, which is beneficial to eating, thereby promoting body growth. Compared with the control group, the water quality of the experimental group was better, the water color was stable,

Experimental example two:

Select the similar gibel carp of specification, be divided into four groups at random: compound group, bacteria powder group, immune agent group and blank control group, 200 of every group, culture condition is with experimental example one. Each group adds the same basic commodity feed, the composite group adds 0.1% of the compound preparation of the embodiment of the present invention 1, the bacterial powder group adds 0.1% of the microbial bacterial powder in the embodiment of the present invention 1, and the immune agent group adds 0.045% of Radix Astragali, yeast Cell wall 0.045%, plant essential oil 0.01%, the blank control group only added basic commercial feed, and other feeding conditions were the same. Record the fish growth and water quality, and further test and analyze the quality of the gibel crucian carp. The results are shown in Table 2.

Table 2

The test results showed that: compared with the control group, the daily gain and survival rate of gibel crucian carp in the composite group were increased to varying degrees, the crude fat content in the meat was significantly reduced, and the superoxide dismutase activity in the serum was greatly increased compared with the control group. This shows that the compound preparation of the present invention can effectively promote the feeding and digestion ability of gibel crucian carp, increase the survival rate, and can significantly improve immunity.

Experimental example three:

400 rainbow trout with uniform size and weight were randomly put into 4 breeding tanks, 100 per tank, and the 4 tanks were randomly divided into four groups: compound group, bacterial powder group, immune agent group and blank control group. The compound group added 0.1% of the compound preparation of Example 2 of the present invention, and the bacteria powder group, immune agent group and blank control group were the same as in Experimental Example 2. The food intake was recorded in detail, the water temperature was controlled at 16-18°C, and continuous oxygenation was maintained. After 35 days of breeding, they were fasted for 24 hours, weighed one by one, and measured their body length. The results are shown in Table 3.

table 3

group Fish weight gain rate (%) Fish survival rate (%) Feed coefficient Fish vitality water color Compound group 85 100 1.87 active refreshing Bacteria powder group 72 97 1.89 more active refreshing immune booster 66 95 2.11 active turbid control group 59 89 2.32 sluggish turbid

The test results show that the weight gain and survival rate of rainbow trout in the compound group are higher than those in the control group in varying degrees, indicating that the invented compound preparation can effectively improve the growth performance of rainbow trout, and the feed coefficient of the compound group is lower than that of the control group, indicating that this The compound preparation provided by the invention can better improve feed utilization.

Experimental example four:

According to the ratio of 0.3% and 0.1%, the compound preparation of Example 2 of the present invention was added to the feed of Penaeus vannamei, and the feeding period was 100 days. The experimental results showed that the survival rate of the compound group added with 0.3% increased by 18.9% and the feed coefficient decreased by 0.16 compared with the control group; the survival rate of the group added with 0.1% feed additive increased by 13.2% compared with the control group, and the feed coefficient decreased by 0.12. The experiment was analyzed by variance, and the difference between the composite group and the control group reached a significant level (P<0.05). The experimental results are shown in Table 4.

Table 4

group Shrimp body weight gain rate (%) Shrimp survival rate (%) Feed coefficient Shrimp vitality water color 0.3% group 81 99.9 2.01 active refreshing 0.1% group 76 94.2 2.15 more active refreshing control group 68 81.0 2.17 sluggish turbid

Experimental example five

Four pools of a farm were selected to raise rice fish with similar weight and size, and they were divided into four groups: compound group, bacterial powder group, immune agent group and blank control group. The compound group added 0.1% of the compound preparation of Example 3 of the present invention, and the bacteria powder group, immune agent group and blank control group were the same as in Example 2. The breeding conditions of the four groups are exactly the same, and the test period is 100 days. The survival rate and average weight of the rice fish in each group are counted and the water quality is recorded. At the same time, the quality of the rice fish is further tested and analyzed. The results are shown in Table 5.

table 5

group Fish weight gain rate (%) Fish survival rate (%) fat content Fish vitality water color Compound group 87 100 14.34 active refreshing Bacteria powder group 71 95 14.44 more active refreshing immunostimulant group 66 90 15.56 active turbid control group 51 69 15.79 sluggish turbid

The test results showed that the survival rate and average body weight of rice fish in the compound group were significantly higher than that of the control group, and the fat content was significantly lower than that of the control group. There was no fry death in the compound group during the breeding process, and the bacteria agent had a great effect on the cleaning ability of the bottom of the pond. Stronger, reduce the amount of water change, and the water quality is relatively stable. However, in the later stage of culture in the control pond, the fry had gastrointestinal problems and a large number of fry died. Thereby it is illustrated that the compound preparation of the present invention is beneficial to the growth of rice fish, can effectively improve the survival rate, and significantly improve disease resistance.

Experimental example six:

In a field test in a farm, adding the compound preparation of Example 3 of the present invention is used for the treatment of eel vibriosis infected by rainbow trout. The diseased fish shows: fins and gills are congested and red, and the muscle tissue has diffuse or punctate Bleeding, yellow viscous ascites, intestinal mucosal tissue damage, and liver necrosis in some fish. The fishpond mortality rate was 5%. The compound system of Example 3 of the present invention was added to the fish basic feed at an additive amount of 0.3%, fed 3 times a day, and used continuously for 30 days. The effective rate of treatment reached 70-85%, and no dead fish appeared.

Claims (2)

1. a kind of compound formulation for effectively improving aquatic livestock disease resistance, it is characterised in that each component and quality proportioning are such as Under：Microbial germ powder 55 ~ 65, astragalus polyose 10 ~ 15, yeast cell wall 10 ~ 15, plants essential oil 5 ~ 10, vitamin B6 1 ~ 5；Institute It is by mortierella Diding bacterium powder to state microbial germ powder（Acremonium terricola）With clostridium butyricum powder（Clostridium butyricum）In mass ratio 35 ~ 50：15 ~ 30 mix, and the yeast cell wall is the bread ferment containing 30% beta glucan Mother cell wall, the plants essential oil is by cinnaldehydrum, Thymol, lavender essential oil in mass ratio 2 ~ 4：2~4：1 ~ 2 mixes； Mortierella Diding bacterium powder active ingredient >=200mg/g in the compound formulation, clostridium butyricum powder viable count >=5 × 10⁸CFU/g, institute State mortierella Diding bacterium deposit number ATCC13215（NBRC30538）And it is identified through Chinese culture presevation administrative center CICC, institute Clostridium butyricum deposit number is stated as CICC No.M 23847.

2. a kind of preparation method for the compound formulation that can effectively improve aquatic livestock disease resistance as described in claim 1, special Sign is to be as follows：

A prepares microbial germ powder

（1）Prepare mortierella Diding bacterium powder

A. actication of culture

Mortierella Diding bacterium freeze-dried powder is seeded in PDA culture medium, 25 DEG C are cultivated 7 days；

B. seed culture

Strain after activation is transferred with 2% inoculum concentration in shaking flask, 25 DEG C of 24 ~ 36h of constant-temperature table shaken cultivation obtain seed Culture solution, the mass percent of fluid nutrient medium component are：White granulated sugar 1.4%, dried silkworm chrysalis meal 2.3%, dusty yeast 1.2%, more than water Amount, pH value 6.5；

C. fermentation tank culture

Seed culture fluid accesses 50L fermentation tanks with 20% inoculum concentration, and loading amount coefficient is 70%, ventilates and cultivates through 48 ~ 60h Tank is put, the mass percent of Fermenter Medium Component is：Analysis for soybean powder 2.8%, white granulated sugar 2.5%, dusty yeast 1.3%, dried silkworm chrysalis meal 2.5%、MgSO₄ 0.05%、KH₂PO₄0.05%th, antifoaming agent 0.05%, water surplus are 6.5 with saturation NaOH tune pH value；

D. solid fermentation culture

The zymocyte liquid that step c is obtained is with 20%（v/m）Inoculum concentration access sterilizing after solid fermentation culture medium on, solid The constituent mass percentage of culture medium is：Corn flour 55%, dregs of beans 32%, wheat bran 13%；Condition of culture is：25 DEG C, humidity 70%, Fermentation time 72h；

E. the collection of mortierella Diding culture

After treating solid fermentation, culture is collected, direct 60 ~ 80 DEG C are dried to moisture ＜ 10%, are then crushed and mistake The mortierella Diding bacterium powder of the active ingredients such as 40 mesh sieves, as 94mg/g containing polysaccharide, polypeptide 28mg/g and biomass 35mg/g；

（2）Prepare clostridium butyricum bacterium powder

A actication of culture

Picking clostridium butyricum freeze-dried powder accesses activation medium, stands Anaerobic culturel；Culture medium forms：Peptone 10g, beef leaching Cream 10g, yeast extract 3g, glucose 10g, NaCl 5g, NaAc 3g, cysteine hydrochloride 0.5g, CaCO₃3g, distilled water are fixed Hold to 1000mL, 7.0,37 DEG C of cultures of pH for 24 hours, are cultivated with 2% inoculum concentration switching under new activation medium, the same terms 48h；

B. seed culture

Bacterium solution is accessed into 10L seeding tanks with 10% inoculum concentration, loading amount coefficient is 70%, and seed culture medium composition is：Peptone 5 ~ 10g, 5 ~ 10g of beef extract, 3 ~ 10g of yeast extract, glucose 5 ~ 10g, NaCl 5g, NaAc1 ~ 3g, CaCO₃ 1 ~ 3g, distillation Water is settled to 1000mL, and 7.0,37 DEG C of pH stands Anaerobic culturel 20h；

C. fermentation tank culture

Seed culture fluid accesses 100L fermentation tanks with 10% inoculum concentration, and loading amount coefficient is 70%, and fermentation tank culture medium composition is： 5 ~ 10g of peptone, 3 ~ 10g of yeast extract, 5 ~ 15g of glucose, starch 0.5 ~ 1g, NaCl 5g, NaAc 1 ~ 3g, CaCO₃ 3~ 10g, water are settled to 1000mL, and 7.0,37 DEG C of pH stands Anaerobic culturel for 24 hours；

D. microorganism collection

Zymotic fluid is collected, supernatant is abandoned after centrifugation, obtains wet thallus；By clostridium butyricum wet thallus and dried starch with mass ratio 1: 0.5 ~ 5 mixing, for 24 hours, pulverizer crushes for 50 DEG C of dryings, crosses 60 mesh sieves, adjusts viable count >=1 × 10 of clostridium butyricum¹⁰CFU/g, as Clostridium butyricum opportunistic pathogen powder；

B is respectively by astragalus polyose, yeast cell wall, vitamin B₆60 mesh sieves are smashed it through, afterwards by astragalus polyose, yeast cells Wall, vitamin B₆, mortierella Diding culture, clostridium butyricum powder and plants essential oil be put into mixing plant, stir evenly.