Preparation method of bacillus solid aerobic fermentation culture with antibacterial function and application of bacillus solid aerobic fermentation culture in livestock and poultry

Abstract

The invention relates to the technical field of microorganisms, in particular to a preparation method of a bacillus solid aerobic fermentation culture with an antibacterial function and application of the bacillus solid aerobic fermentation culture in livestock and poultry. The invention relates to a preparation method of a bacillus solid aerobic fermentation culture with an antibacterial function. The preparation method comprises the following steps: (1) bacillus strain culture: culturing a strain on a plate; picking single colonies, inoculating the single colonies into an NB culture medium, culturing the single colonies for 20-24 hours to obtain a primary seed solution, inoculating the single colonies into the NB culture medium according to the inoculation amount of 1-10% (V / V), and culturing the single colonies for 40-48 hours to obtain a secondary seed solution; (2) preparing a solid-state fermentation culture medium: adding water into fermentation culture medium raw materials, uniformly mixing, sterilizing, and cooling for later use; (3) fermentation culture: transferring the secondary seed solution in the step (1) into the solid-state fermentation culture medium prepared in the step (2), performing aerobic fermentation for 48-96 hours under the conditions that the inoculation amount is 1-10% and the temperature is 30-40 DEG C, and then drying and crushing to obtain a bacillus solid-state fermentation culture with an antibacterial function; wherein the fermentation culture medium is prepared from the following raw materials: 500 to 900g of bran, 300 to 500g of corn and 100 to 300g of soybean meal. According to the preparation method of the bacillus solid aerobic fermentation culture with the antibacterial function, the problems that in an existing bacillus facultative anaerobic fermentation culture, bacillus breeding is limited, bacillus metabolites are few, and the antibacterial activity is poor are solved, a high-concentration viable bacterium preparation and rich metabolites are obtained, and the bacillus solid aerobic fermentation culture with the antibacterial function is obtained. And the bacillus culture has an antibacterial effect.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and more specifically, to a preparation method of a solid aerobic fermentation culture of Bacillus with antibacterial function and its application in livestock and poultry. Background Art

[0002] Modern intensive livestock production restricts animals' contact with the soil and the natural environment around them. Such feeding conditions are not conducive to animals establishing normal gastrointestinal flora. When the external environmental conditions change, it is easy to cause the imbalance of the gastrointestinal microecological flora of animals, thereby reducing the resistance to pathogenic bacteria. Currently, the main methods to reduce this loss are to improve feeding management and add antibiotics to the diet. However, with the widespread use of antibiotics, the resulting drug resistance and residue problems have become increasingly prominent, attracting high attention from scientific workers to ordinary consumers. Probiotics have become the first choice for antibiotic substitutes due to their safety, no residue, and beneficial to the sustainable development of the ecological environment. Bacillus is one of the most widely used probiotics.

[0003] The existing production process of Bacillus is usually liquid high-density fermentation, followed by spray drying after centrifuging to obtain the bacterial cells. The product only contains live bacterial preparations, and the metabolites in the liquid fermentation are not included in the product. The viable count and the yield of Bacillus metabolites are very key indicators for measuring Bacillus agents. In the optimization of the high-density fermentation process, most only improve the viable count, while the formation rate of Bacillus metabolites is relatively low. The low rate of Bacillus metabolites will reduce the activity and stress resistance quality stability of the agent. The solid fermentation culture is usually obtained by facultative anaerobic fermentation (respiration bag mode) of a combination of three or two of Bacillus, yeast, and lactic acid bacteria with different by-product raw materials as carriers. The reproduction of Bacillus in the culture is limited, and the metabolites are mainly lactic acid bacteria metabolites. Lactic acid reduces the environmental pH value, and when the pH value drops to a certain extent, it will inhibit its own growth; moreover, the formation conditions of its spores are extremely harsh, affected by many factors such as nutritional conditions and environmental factors. To achieve high Bacillus metabolites and high spore conversion rate, overall consideration is required, and appropriate culture medium components and fermentation process parameters are indispensable. Summary of the Invention

[0004] Aiming at the problems of limited reproduction of Bacillus, less Bacillus metabolites, and poor antibacterial properties in the existing facultative anaerobic fermentation culture of Bacillus, the present application provides a preparation method of a solid aerobic fermentation culture of Bacillus with antibacterial function to obtain a high-concentration live bacterial preparation and rich metabolites, and a Bacillus culture with antibacterial efficacy.

[0005] In the first aspect, the present application provides a preparation method of a solid aerobic fermentation culture of Bacillus with antibacterial function, adopting the following technical scheme: A preparation method of a solid aerobic fermentation culture of Bacillus with antibacterial function, comprising the following steps: (1) Culturing Bacillus strains: After the strains are cultured on a plate; pick a single colony and inoculate it into NB medium, culture for 20-24 h as the primary seed liquid, and inoculate it into NB medium at an inoculation amount of 1-10% (V / V) and culture for 40-48 h as the secondary seed liquid; (2) Preparation of solid fermentation medium: Add the raw materials of the fermentation medium to water and mix evenly, sterilize and then cool for standby; (3) Fermentation culture: Transfer the secondary seed liquid in step (1) to the solid fermentation medium prepared in step (2), with an inoculation amount of 1-10%, a temperature of 30-40 °C, and perform aerobic fermentation for 48-96 h, then dry and crush to obtain a solid fermentation culture of Bacillus with antibacterial function; wherein, the raw materials of the fermentation medium include 500-900 g of bran, 300-500 g of corn, and 100-300 g of soybean meal.

[0006] By adopting the above technical solution, compared with the traditional liquid high-density fermentation and spray-dried product which does not have antibacterial efficacy itself, the solid aerobic fermentation of Bacillus can obtain rich metabolites while obtaining a high viable cell count, making the fermentation product itself have antibacterial efficacy. Compared with the traditional solid anaerobic fermentation, the solid aerobic fermentation effect of Bacillus is greatly improved. The solid aerobic fermentation culture of Bacillus uses the raw materials of the fermentation medium including bran, corn and soybean meal to culture Bacillus with the best effect. The fermentation medium uses bran as the carbon source fermentation substrate, which can improve the oxygen permeability during fermentation, obtain a high concentration of viable bacteria, produce various active enzymes, and enhance the activities of hydrolytic enzymes such as amylase, cellulase, protease, and phytase, destroy the anti-nutritional factors, and significantly improve the antioxidant capacity and free radical scavenging rate of the fermentation culture. Corn can provide easily decomposable carbohydrates (such as starch, etc.) and promote the rapid growth of bacteria as the main carbon source; soybean meal powder is rich in nutrients such as small molecule proteins, polypeptides, amino acids, vitamins, trace elements, and various growth factors, which can eliminate impurities that are not conducive to the absorption and transformation of microorganisms. After mixing it with corn, the content of low molecular peptides and amino acids produced by fermentation increases significantly, maximizing the utilization of the nutrient elements in soybean meal powder, thereby effectively promoting the growth of microorganisms, improving the conversion rate and utilization rate of nitrogen source in the medium, and obtaining rich metabolites; at the same time, the combination of bran, corn and soybean meal shows comprehensive advantages of nutritional promotion, high process efficiency and environmental friendliness in the solid aerobic fermentation of Bacillus, fermenting to produce antibacterial peptides and phenolic acids, inhibiting pathogenic bacteria such as Escherichia coli, and promoting the growth of probiotics at the same time, which can shorten the fermentation cycle, improve the digestibility and polysaccharide content at the same time, making the fermentation product itself have antibacterial efficacy, and obtaining rich metabolites while obtaining a high viable cell count. The medium of this application can also be used to predict the microbial contamination situation in biological preparations, and greatly improve the viable cell count of Bacillus with antibacterial function and the variety of metabolites in the final product.

[0007] The use of solid-state aerobic fermentation improves the efficiency of the preparation process. By further optimizing the composition of the culture medium and the fermentation process conditions adopted in each step, the viable bacteria content of Bacillus in the final product is greatly increased, the antibacterial effect is significantly enhanced, and it can maintain high efficiency and stability in the intestinal environment, laying a foundation for the wide popularization and use of antibacterial functional Bacillus preparations; it has the characteristics of high spore formation rate, high temperature resistance, and being convenient for processing and storage.

[0008] The mass ratio of the bran: corn: soybean meal is (5 - 9):(3 - 5):1.

[0009] By adopting the above technical solution, bran, corn, and soybean meal are used as raw materials for the solid-state aerobic fermentation medium of Bacillus in a certain proportion. While obtaining a high number of viable bacteria, rich metabolites are obtained, making the fermentation product itself have antibacterial effects. The bran medium, as the main carbon source substrate, can enhance the antibacterial effect and oxygen permeability and increase the viable bacteria number of Bacillus. Then, a certain mass of corn is added. Corn is rich in carbohydrates, proteins, amino acids, inorganic salts, etc., which can meet the basic needs of microbial growth and reproduction, is non-toxic and harmless to microorganisms, and its physical and chemical properties such as pH value and osmotic pressure are suitable for the growth of microorganisms; soybean meal can also provide some growth factors and trace elements, further promoting the growth of microorganisms, and can provide the necessary nitrogen source and other nutritional components. By optimizing the culture conditions, the yield and quality of the product are improved, the reproduction of Bacillus is increased, and thus the viable bacteria number and metabolites are increased. If the content of corn is too low, the metabolites in the solid-state aerobic fermentation culture of Bacillus with antibacterial function will decrease, which is not conducive to the growth of microorganisms, reduces the activity of microorganisms, and the viable bacteria number decreases. If the content of bran is too low, the content and activity of hydrolase will decrease, the inhibitory effect on anti-nutritional factors will decline, and the antioxidant capacity and free radical scavenging rate of the fermentation culture will be insufficient, and thus the antibacterial performance against Clostridium perfringens will be insufficient. The sterilization in step (1) is at 100 - 121 °C for 20 - 60 min.

[0010] The bran includes fine bran and coarse bran, and the mass ratio of fine bran to coarse bran is 1:(1.2 - 1.8).

[0011] By adopting the above technical solution, through the combined use of fine bran and coarse bran in a certain proportion, the Bacillus medium has excellent oxygen permeability, meets the conditions of solid-state aerobic fermentation, and improves the reproduction rate and viable bacteria number of Bacillus, obtaining a rich variety of metabolites.

[0012] The raw materials of the fermentation medium also include 5 - 10 g of glucose, 5 - 10 g of maltodextrin, 0 - 1.5 g of D - mannose, and 10 - 16 g of PBS buffer.

[0013] The mass ratio of the maltodextrin to the D-mannose is (4 - 8):1.

[0014] By adopting the above technical solution, adding glucose, maltodextrin, and PBS buffer enriches carbon sources for the growth of microorganisms, enhances biocompatibility, is non-toxic and harmless to microorganisms, and its physical and chemical properties such as pH value and osmotic pressure are suitable for the growth of microorganisms, accelerating the growth cycle of microorganisms and increasing the richness of metabolites. The applicant found that by mixing D-mannose and maltodextrin in a certain proportion, it is beneficial to the fermentation of microorganisms such as bacteria and fungi, the probiotic proliferation ability, increases the activity of solid-state aerobic fermentation of Bacillus, which has antibacterial function in the system, increases the number of viable bacteria. When used as an additive, it can improve the reproduction rate and activity of Bacillus, and further enhance the antibacterial performance of the solid-state aerobic fermentation culture of Bacillus. In the second aspect, the solid-state aerobic fermentation culture of Bacillus with antibacterial function obtained in this application is used as an additive for livestock and poultry feed.

[0015] The solid-state aerobic fermentation culture of Bacillus with antibacterial function is added to livestock and poultry feed at a mass ratio of 0.01 - 0.1%.

[0016] Adding a certain amount of viable bacteria of the solid-state aerobic fermentation culture of Bacillus to livestock and poultry diets can, to a certain extent, have a positive effect on the body condition, reproductive performance, serum antioxidant and immunobiochemical indexes of livestock and poultry, and the growth performance of their offspring. Moreover, adding the solid-state aerobic fermentation culture of Bacillus can increase the abundance of beneficial bacteria and decrease the abundance of harmful bacteria, thus playing a role in regulating the intestinal microecological balance. After the solid-state aerobic fermentation culture of Bacillus enters the animal intestine, it can reduce the number of harmful microorganisms in the intestine, improve the digestion and absorption of nutrients in the intestine, improve the feed conversion utilization rate, promote animal growth, and thus reduce the generation of livestock and poultry waste gas, playing a positive role in environmental improvement.

[0017] In summary, this application has the following beneficial effects: 1. The culture medium raw materials for the solid-state aerobic fermentation of Bacillus spp. are best when using wheat bran, corn, and soybean meal to culture Bacillus spp. The combination of wheat bran, corn, and soybean meal exhibits comprehensive advantages of nutritional promotion, high process efficiency, and environmental friendliness in the solid-state aerobic fermentation of Bacillus spp. It ferments to produce antibacterial peptides and phenolic acids, inhibits pathogenic bacteria such as Escherichia coli, and promotes the growth of probiotics at the same time. It can shorten the fermentation cycle, improve the digestibility and polysaccharide content, making the fermentation product itself have antibacterial effects, obtaining a high viable count of bacteria while obtaining rich metabolites. Therefore, wheat bran, corn, and soybean meal are used as culture medium raw materials in a certain proportion through the solid-state aerobic fermentation process of this application. By further optimizing the composition of the culture medium and the fermentation process conditions used in each step, the Bacillus spp. culture with antibacterial effects is used as a livestock and poultry feed additive, which greatly increases the viable count of Bacillus spp. in the final product, significantly enhances the antibacterial effect, and can maintain high efficiency and stability in the intestinal environment of livestock and poultry. It can selectively enrich microorganisms, increase the abundance of beneficial bacteria, and reduce the abundance of harmful bacteria, thus playing a role in regulating the intestinal microecological balance and being used to prevent diseases related to the intestinal flora, such as intestinal infections and irritable bowel syndrome. The culture medium of this application greatly increases the viable count of Bacillus spp. with antibacterial functions and the variety of metabolites in the final product, laying a foundation for the wide promotion and use of Bacillus spp. preparations with antibacterial functions. It has the characteristics of a high spore formation rate, high temperature resistance, and being convenient for processing and storage.

[0018] 1. Wheat bran, corn, and soybean meal are used as raw materials for the solid-state aerobic fermentation medium of Bacillus spp. in a certain proportion, making the fermentation product itself have antibacterial effects, obtaining a high viable count of bacteria while obtaining rich metabolites. The wheat bran medium, as the main carbon source substrate, can enhance the antibacterial effect and oxygen permeability and increase the viable count of Bacillus spp. Adding a certain quality of corn, which is rich in carbohydrates, proteins, amino acids, inorganic salts, etc., can meet the basic needs of microbial growth and reproduction, is non-toxic and harmless to microorganisms, and its physical and chemical properties such as pH value and osmotic pressure are suitable for the growth of microorganisms. Soybean meal can also provide some growth factors and trace elements, further promoting the growth of microorganisms, being able to provide the necessary nitrogen source and other nutritional components. By optimizing the culture conditions, the yield and quality of the product can be improved, the reproduction of Bacillus spp. can be increased, and then the viable count of bacteria and metabolites can be increased. If the content of corn is too low, the metabolites in the solid-state aerobic fermentation culture of Bacillus spp. with antibacterial functions will decrease, which is not conducive to the growth of microorganisms, reduces the activity of microorganisms, and the viable count of bacteria decreases. If the content of wheat bran is too low, the content and activity of hydrolase will decrease, the inhibitory effect on anti-nutritional factors will decline, and the antioxidant capacity and free radical scavenging rate of the fermentation culture will be insufficient, and then the antibacterial performance against Clostridium perfringens will be insufficient.

[0019] 3. Adding a certain amount of viable Bacillus solid aerobic fermentation culture to livestock and poultry diets can, to a certain extent, have a positive effect on the body condition, reproductive performance, serum antioxidant and immunobiochemical indices of livestock and poultry, as well as the growth performance of their offspring. Moreover, adding the Bacillus solid aerobic fermentation culture can increase the abundance of beneficial bacteria and decrease the abundance of harmful bacteria, thereby playing a role in regulating the intestinal microecological balance. After the Bacillus solid aerobic fermentation culture enters the animal intestine, it can reduce the number of harmful microorganisms in the intestine, improve the digestion and absorption of nutrients by the intestine, increase the feed conversion utilization rate, promote animal growth, and thus reduce the generation of livestock and poultry waste gas, playing a positive role in environmental improvement. Detailed implementation manners

[0020] The following further elaborates on the present application in conjunction with the examples.

[0021] The relevant raw materials used in the examples and comparative examples are all conventional products that can be obtained through market purchase.

[0022] Example 1 A preparation method of a Bacillus solid aerobic fermentation culture with antibacterial function includes the following steps: (1) Bacillus strain culture: After the strain is cultured on an NA plate, a single colony is picked and inoculated into an NB medium, cultured at 36 °C for 24 h as the primary seed liquid, and then inoculated into the NB medium at an inoculation amount of 10% (V / V) and cultured for 48 h as the secondary seed liquid, and inoculation is carried out to make the moisture of the mixed material close to 38%; (2) Preparation of solid fermentation medium: Add the fermentation medium raw materials to 600 g of water and mix evenly, sterilize at 121 °C for 20 min, and then cool for standby; (3) Fermentation culture: Transfer the secondary seed liquid in step (1) to the solid fermentation medium prepared in step (2), mix evenly, with an inoculation amount of 5%, put it into a conical flask or a container with holes, control the loading thickness, carry out aerobic fermentation at 37 °C for 72 h, and dry and crush at 65 °C to obtain a Bacillus solid fermentation culture with antibacterial function; wherein, the fermentation medium raw materials include 800 g of wheat bran, 400 g of corn, and 100 g of soybean meal; the wheat bran is fine wheat bran and coarse wheat bran with a mass ratio of 1:1.2.

[0023] Example 2 A preparation method of a Bacillus solid aerobic fermentation culture with antibacterial function includes the following steps: (1) Bacillus strain culture: After the strain is cultured on an NA plate, a single colony is picked and inoculated into an NB medium, cultured at 36 °C for 24 h as the primary seed liquid, and then inoculated into the NB medium at an inoculation amount of 10% (V / V) and cultured for 48 h as the secondary seed liquid, and inoculation is carried out to make the moisture of the mixed material close to 38%; (2) Preparation of solid-state fermentation medium: Add the raw materials of the fermentation medium to 600 g of water and mix evenly. Sterilize at 121 °C for 20 min and then cool for standby. (3) Fermentation culture: Transfer the secondary seed liquid in step (1) to the solid-state fermentation medium prepared in step (2) and mix evenly. The inoculation amount is 5%. Put it into a conical flask or a perforated container, control the loading thickness, and carry out aerobic fermentation at 37 °C for 72 h. Dry and crush at 65 °C to obtain a solid-state fermentation product of Bacillus with antibacterial function. Among them, the raw materials of the fermentation medium include 800 g of wheat bran, 400 g of corn, and 100 g of soybean meal; the wheat bran is fine wheat bran.

[0024] Example 3 A preparation method of a solid-state aerobic fermentation product of Bacillus with antibacterial function includes the following steps: (1) Cultivation of Bacillus strains: After the strains are cultured on an NA plate, pick a single colony and inoculate it into an NB medium. Culture at 36 °C for 24 h as the primary seed liquid, and inoculate into the NB medium at an inoculation amount of 10% (V / V) and culture for 48 h as the secondary seed liquid, and inoculate to make the moisture of the mixed material close to 38%; (2) Preparation of solid-state fermentation medium: Add the raw materials of the fermentation medium to 600 g of water and mix evenly. Sterilize at 121 °C for 20 min and then cool for standby. (3) Fermentation culture: Transfer the secondary seed liquid in step (1) to the solid-state fermentation medium prepared in step (2) and mix evenly. The inoculation amount is 5%. Put it into a conical flask or a perforated container, control the loading thickness, and carry out aerobic fermentation at 37 °C for 72 h. Dry and crush at 65 °C to obtain a solid-state fermentation product of Bacillus with antibacterial function. Among them, the raw materials of the fermentation medium include 400 g of wheat bran, 600 g of corn, and 300 g of soybean meal; the wheat bran is a mixture of fine wheat bran and coarse wheat bran with a mass ratio of 1:1.2.

[0025] Example 4 A preparation method of a solid-state aerobic fermentation product of Bacillus with antibacterial function includes the following steps: (1) Cultivation of Bacillus strains: After the strains are cultured on an NA plate, pick a single colony and inoculate it into an NB medium. Culture at 36 °C for 24 h as the primary seed liquid, and inoculate into the NB medium at an inoculation amount of 10% (V / V) and culture for 48 h as the secondary seed liquid, and inoculate to make the moisture of the mixed material close to 38%; (2) Preparation of solid-state fermentation medium: Add the raw materials of the fermentation medium to 600 g of water and mix evenly. Sterilize at 121 °C for 20 min and then cool for standby. (3) Fermentation culture: Transfer the secondary seed liquid in step (1) to the solid-state fermentation medium prepared in step (2), mix well, with an inoculation amount of 5%, place it in an Erlenmeyer flask or a perforated container, control the loading thickness, and perform aerobic fermentation at 37°C for 72 h, then dry and crush at 65°C to obtain a solid-state fermentation culture of Bacillus with antibacterial function; among them, the raw materials of the fermentation medium include 800 g of wheat bran, 100 g of corn, and 300 g of soybean meal; the wheat bran is fine wheat bran and coarse wheat bran with a mass ratio of 1:1.2.

[0026] Example 5 A preparation method of a solid-state aerobic fermentation culture of Bacillus with antibacterial function, comprising the following steps: (1) Bacillus strain culture: After the strain is cultured on an NA plate; pick a single colony and inoculate it into NB medium, culture at 36°C for 24 h as the primary seed liquid, and inoculate into NB medium at an inoculation amount of 10% (V / V) and culture for 48 h as the secondary seed liquid, and inoculate to make the moisture of the mixed material close to 38%; (2) Preparation of solid-state fermentation medium: Add the raw materials of the fermentation medium to 600 g of water, mix well, sterilize at 121°C for 20 min, and then cool for standby; (3) Fermentation culture: Transfer the secondary seed liquid in step (1) to the solid-state fermentation medium prepared in step (2), mix well, with an inoculation amount of 5%, place it in an Erlenmeyer flask or a perforated container, control the loading thickness, and perform aerobic fermentation at 37°C for 72 h, then dry and crush at 65°C to obtain a solid-state fermentation culture of Bacillus with antibacterial function; among them, the raw materials of the fermentation medium include 800 g of wheat bran, 400 g of corn, 100 g of soybean meal, 100 g of glucose, 80 g of maltodextrin, 10 g of D-mannose, and 100 g of PBS buffer; the wheat bran is fine wheat bran and coarse wheat bran with a mass ratio of 1:1.2.

[0027] Example 6 A preparation method of a solid-state aerobic fermentation culture of Bacillus with antibacterial function, comprising the following steps: (1) Bacillus strain culture: After the strain is cultured on an NA plate; pick a single colony and inoculate it into NB medium, culture at 36°C for 24 h as the primary seed liquid, and inoculate into NB medium at an inoculation amount of 10% (V / V) and culture for 48 h as the secondary seed liquid, and inoculate to make the moisture of the mixed material close to 38%; (2) Preparation of solid-state fermentation medium: Add the raw materials of the fermentation medium to 600 g of water, mix well, sterilize at 121°C for 20 min, and then cool for standby; (3) Fermentation culture: Transfer the secondary seed liquid in step (1) to the solid-state fermentation medium prepared in step (2), mix well, with an inoculation amount of 5%, put it into a conical flask or a perforated container, control the loading thickness, and perform aerobic fermentation at 37°C for 72 h, then dry and crush at 65°C to obtain a solid-state fermentation culture of Bacillus with antibacterial function; among them, the raw materials of the fermentation medium include 800 g of bran, 400 g of corn, 100 g of soybean meal, 100 g of glucose, 40 g of maltodextrin, 10 g of D-mannose, and 100 g of PBS buffer; the bran is fine bran and coarse bran with a mass ratio of 1:1.2.

[0028] Example 7 A preparation method of a solid-state aerobic fermentation culture of Bacillus with antibacterial function, comprising the following steps: (1) Bacillus strain culture: After the strain is cultured on an NA plate; pick a single colony and inoculate it into an NB medium, culture at 36°C for 24 h as the primary seed liquid, and inoculate into the NB medium at an inoculation amount of 10% (V / V) and culture for 48 h as the secondary seed liquid, and inoculate to make the moisture of the mixed material close to 38%; (2) Preparation of solid-state fermentation medium: Add the raw materials of the fermentation medium to 600 g of water, mix well, sterilize at 121°C for 20 min, and then cool for standby; (3) Fermentation culture: Transfer the secondary seed liquid in step (1) to the solid-state fermentation medium prepared in step (2), mix well, with an inoculation amount of 5%, put it into a conical flask or a perforated container, control the loading thickness, and perform aerobic fermentation at 37°C for 72 h, then dry and crush at 65°C to obtain a solid-state fermentation culture of Bacillus with antibacterial function; among them, the raw materials of the fermentation medium include 800 g of bran, 400 g of corn, 100 g of soybean meal, 100 g of glucose, 80 g of maltodextrin, and 100 g of PBS buffer; the bran is fine bran and coarse bran with a mass ratio of 1:1.2.

[0029] Comparative Example 1 A preparation method of a solid-state aerobic fermentation culture of Bacillus with antibacterial function, comprising the following steps: (1) Bacillus strain culture: After the strain is cultured on an NA plate; pick a single colony and inoculate it into an NB medium, culture at 36°C for 24 h as the primary seed liquid, and inoculate into the NB medium at an inoculation amount of 10% (V / V) and culture for 48 h as the secondary seed liquid, and inoculate to make the moisture of the mixed material close to 38%; (2) Fermentation culture: Transfer the secondary seed liquid in step (1) to a 1 kg breathing bag at an inoculation amount of 5%, ferment at 37°C for 72 h, then dry and crush at 65°C to obtain a solid-state fermentation culture of Bacillus with antibacterial function; among them, the raw materials of the fermentation medium include 800 g of bran, 400 g of corn, 100 g of soybean meal; the bran is fine bran and coarse bran with a mass ratio of 1:1.2.

[0030] Comparative Example 2 A preparation method of a solid aerobic fermentation culture of Bacillus with antibacterial function, comprising the following steps: (1) Culturing of Bacillus strains: After culturing the strains on an NA plate, picking single colonies and inoculating them into an NB medium, culturing at 36 °C for 24 h as the first-stage seed liquid, inoculating into the NB medium at an inoculation amount of 10% (V / V) and culturing for 48 h as the second-stage seed liquid, and inoculating to make the moisture of the mixed material close to 38%; (2) Preparation of solid fermentation medium: Adding the raw materials of the fermentation medium to 600 g of water, mixing evenly, and cooling for standby; (3) Fermentation culture: Transferring the second-stage seed liquid in step (1) to the solid fermentation medium prepared in step (2), mixing evenly, with an inoculation amount of 5%, putting it into a conical flask or a perforated container, controlling the loading thickness, carrying out aerobic fermentation at 37 °C for 72 h, drying and pulverizing at 65 °C to obtain a solid fermentation culture of Bacillus with antibacterial function; wherein, the raw materials of the fermentation medium include 800 g of bran, 400 g of corn, and 100 g of soybean meal; the bran is fine bran and coarse bran with a mass ratio of 1:1.2.

[0031] Comparative Example 3 A preparation method of a solid aerobic fermentation culture of Bacillus with antibacterial function, comprising the following steps: (1) Culturing of Bacillus strains: After culturing the strains on an NA plate, picking single colonies and inoculating them into an NB medium, culturing at 36 °C for 24 h as the first-stage seed liquid, inoculating into the NB medium at an inoculation amount of 10% (V / V) and culturing for 48 h as the second-stage seed liquid, and inoculating to make the moisture of the mixed material close to 38%; (2) Preparation of solid fermentation medium: Adding the raw materials of the fermentation medium to 600 g of water, mixing evenly, sterilizing at 121 °C for 20 min, and then cooling for standby; (3) Fermentation culture: Transferring the second-stage seed liquid in step (1) to the solid fermentation medium prepared in step (2), mixing evenly, with an inoculation amount of 5%, putting it into a conical flask or a perforated container, controlling the loading thickness, carrying out aerobic fermentation at 37 °C for 72 h, drying and pulverizing at 65 °C to obtain a solid fermentation culture of Bacillus with antibacterial function; wherein, the raw materials of the fermentation medium include 1200 g of bran and 100 g of soybean meal; the bran is fine bran and coarse bran with a mass ratio of 1:1.2.

[0032] Comparative Example 4 A preparation method of a Bacillus solid aerobic fermentation culture with antibacterial function, comprising the following steps: (1) Bacillus strain culture: After the strain is cultured on an NA plate, a single colony is picked and inoculated into an NB medium, cultured at 36 °C for 24 h as the primary seed liquid, and inoculated into the NB medium at an inoculation amount of 10% (V / V) and cultured for 48 h as the secondary seed liquid, and the inoculation is carried out to make the moisture of the mixed material close to 38%; (2) Preparation of solid fermentation medium: Add the raw materials of the fermentation medium to 600 g of water and mix evenly, sterilize at 121 °C for 20 min, and then cool for standby; (3) Fermentation culture: Transfer the secondary seed liquid in step (1) to the solid fermentation medium prepared in step (2), mix evenly, the inoculation amount is 5%, put it into a conical flask or a container with holes, control the loading thickness, and carry out aerobic fermentation at 37 °C for 72 h, dry and crush at 65 °C to obtain a Bacillus solid fermentation culture with antibacterial function; wherein, the raw materials of the fermentation medium include 900 g of bran and 400 g of corn; the bran is fine bran and coarse bran with a mass ratio of 1:1.2.

[0033] Control Example 5 A preparation method of a Bacillus solid aerobic fermentation culture with antibacterial function, comprising the following steps: (1) Bacillus strain culture: After the strain is cultured on an NA plate, a single colony is picked and inoculated into an NB medium, cultured at 36 °C for 24 h as the primary seed liquid, and inoculated into the NB medium at an inoculation amount of 10% (V / V) and cultured for 48 h as the secondary seed liquid, and the inoculation is carried out to make the moisture of the mixed material close to 38%; (2) Preparation of solid fermentation medium: Add the raw materials of the fermentation medium to 600 g of water and mix evenly, sterilize at 121 °C for 20 min, and then cool for standby; (3) Fermentation culture: Transfer the secondary seed liquid in step (1) to the solid fermentation medium prepared in step (2), mix evenly, the inoculation amount is 5%, put it into a conical flask or a container with holes, control the loading thickness, and carry out aerobic fermentation at 37 °C for 72 h, dry and crush at 65 °C to obtain a Bacillus solid fermentation culture with antibacterial function; wherein, the raw materials of the fermentation medium include 1300 g of bran; the bran is fine bran and coarse bran with a mass ratio of 1:1.2.

[0034] Control Example 6 A preparation method of a Bacillus solid aerobic fermentation culture with antibacterial function, comprising the following steps: (1) Bacillus strain culture: After the strain is cultured on an NA plate, a single colony is picked and inoculated into an NB medium, cultured at 36 °C for 24 h as the primary seed liquid, and inoculated into the NB medium at an inoculation amount of 10% (V / V) and cultured for 48 h as the secondary seed liquid, and the inoculation is carried out to make the moisture of the mixed material close to 38%; (2) Preparation of solid-state fermentation medium: Add the raw materials of the fermentation medium to 600 g of water and mix evenly. Sterilize at 121 °C for 20 min and then cool for standby. (3) Fermentation culture: Transfer the secondary seed liquid in step (1) to the solid-state fermentation medium prepared in step (2) and mix evenly. The inoculation amount is 5%. Put it into a conical flask or a perforated container, control the loading thickness, and carry out aerobic fermentation at 37 °C for 72 h. Dry and crush at 65 °C to obtain a solid-state fermentation product of Bacillus with antibacterial function; among them, the raw materials of the fermentation medium include 1300 g of corn.

[0035] Control Example 7 A preparation method of a solid-state aerobic fermentation product of Bacillus with antibacterial function, comprising the following steps: (1) Cultivation of Bacillus strains: After the strains are cultured on NA plates; pick single colonies and inoculate them into NB medium, and culture at 36 °C for 24 h as the primary seed liquid. Inoculate according to the inoculation amount of 10% (V / V) into NB medium and culture for 48 h as the secondary seed liquid, and inoculate to make the moisture of the mixed material close to 38%; (2) Preparation of solid-state fermentation medium: Add the raw materials of the fermentation medium to 600 g of water and mix evenly. Sterilize at 121 °C for 20 min and then cool for standby. (3) Fermentation culture: Transfer the secondary seed liquid in step (1) to the solid-state fermentation medium prepared in step (2) and mix evenly. The inoculation amount is 5%. Put it into a conical flask or a perforated container, control the loading thickness, and carry out aerobic fermentation at 37 °C for 72 h. Dry and crush at 65 °C to obtain a solid-state fermentation product of Bacillus with antibacterial function; among them, the raw materials of the fermentation medium include 1300 g of soybean meal.

[0036] Performance detection 1. Carry out antibacterial tests on the solid-state aerobic fermentation products of Bacillus with antibacterial function prepared in the examples and control examples, using the following method: Weigh 10 g of each sample of the examples and control examples respectively, add 40 mL of sterile water to dissolve and soak, perform ultrasonic treatment for 15 min, filter, take 10 mL of the filtrate, sterilize at 121 °C by high-pressure steam for 20 min, and centrifuge at 10000 rpm for 10 min after cooling to obtain an antibacterial test sample with a concentration of 20%. The control group is an antibacterial test sample with a concentration of 0.625% obtained by gradient dilution of the sample of Example 1 with sterile water as needed.

[0037] Preparation of antibacterial test indicator bacteria liquid: Clostridium perfringens: Take a glycerol tube (about 1.5 mL), transfer all of it to a 10 mL FTG test tube, seal it with paraffin liquid, and place it at 36 °C for static culture for 24 h for standby.

[0038] Antibacterial test: Take 0.1 mL of the cultured Clostridium perfringens and spread it on a solidified 20 mL MHA plate; ② Use a sterilized punch with a diameter of 8 mm to punch holes in the agar and pick out the agar pieces; ③ Add the antibacterial test samples of each example and each comparative example, with at least 2 replicates for each sample; ④ Place the plate upright in an anaerobic incubator at 36 °C for 18 h, observe and record the diameter of the antibacterial zone, and the test results are shown in Table 1 below, where "×" indicates no antibacterial effect: Table 1 Performance test results The antibacterial effects of fermentation products with different inoculation amounts on Clostridium perfringens at different concentrations are basically the same. The antibacterial zone of Clostridium perfringens at an addition amount of 0.625% is still higher than 15 mm, and the viable cell count is higher than 1.0×10 10 CFU / g; The antibacterial effect and viable cell count of the solid-state aerobic fermentation sample after overnight drying and pulverization at 42 °C change little. The antibacterial effect on Clostridium perfringens is different. For the respiration bag fermentation, the viable cell count is greatly reduced and the antibacterial effect is also worse. Comparing Example 1 with Examples 3-4, it can be seen that when the corn content is too low, the metabolites in the solid-state aerobic fermentation culture of Bacillus with antibacterial function formed are reduced, which is not conducive to the growth of microorganisms, thereby reducing the microbial diversity, reducing the microbial activity, reducing the antibacterial property, and reducing the viable cell count. When the bran content is too low, the content and activity of hydrolase are reduced, and the inhibitory effect on anti-nutritional factors decreases, resulting in insufficient antioxidant capacity and free radical scavenging rate of the fermentation culture, and thus insufficient antibacterial performance against Clostridium perfringens.

[0039] Comparing Example 1 with Examples 5-7, it can be seen that when D-mannose and maltodextrin are mixed as additives in a certain proportion, the reproduction rate and activity of Bacillus can be improved, and the antibacterial performance of the solid-state aerobic fermentation culture of Bacillus can be further enhanced.

[0040] 2. Test the application effect of the solid-state aerobic fermentation culture of Bacillus with antibacterial function prepared in Example 1 and Comparative Example 1 on regulating the intestinal flora of sows; Test animals: Pregnant sows 1 month before parturition (all 2 parity), a total of 30 heads.

[0041] Experimental design: There were 3 treatment groups in the experiment, with 10 replicates for each treatment and 1 pig for each replicate. Group O was the 102 / 911 feed without the solid aerobic fermentation culture of Bacillus with antibacterial function; Group K was the 102 / 911 feed in Group O supplemented with 0.08% of the solid aerobic fermentation culture of Bacillus with antibacterial function in Comparative Example 1 by mass fraction; Group Y was the 102 / 911 feed in Group O supplemented with 0.08% of the solid aerobic fermentation culture of Bacillus with antibacterial function in Example 1 by mass fraction.

[0042] Feeding management: The feeding mode was 2 times a day, with quantitative restricted feeding (not exceeding 3.5 kg / head per day), and sows had free access to water during the experiment. The feeding and production procedures in the farrowing house were carried out according to the normal process of the pig farm. If a sow got sick during the experiment, it was treated in time and records were made.

[0043] Table 2 lists the four dominant bacterial communities ranked in descending order of abundance at the phylum level in each treatment group, namely Firmicutes, Bacteroidota, Euryarchaeota, and Spirochaetota. Data statistics and analysis: The experimental data were sorted out using Excel software, and one-way ANOVA was performed using SPSS 26.0 software. The data were expressed as "mean ± standard error". P < 0.05 indicated significant differences, and P > 0.05 indicated no significant differences. The data and analysis results are as follows: Firmicutes can promote the body's absorption of energy. An increase in the abundance of Firmicutes is beneficial to the body's health. A decrease in the abundance of Bacteroidota can reduce the risk of the body being infected by pathogens. In animal production, a high ratio of Firmicutes / Bacteroidota (F / B) can improve the production performance of animals to a certain extent. Euryarchaeota can form the bacterial community that produces methane in the animal gastrointestinal tract, and this bacterial community is related to the generation of waste gas. Spirochaetota can reduce the occurrence of diarrhea.

[0044] Table 2 Relative abundances of the main dominant bacterial communities at the phylum level (n = 7) Combined with the data in Table 2, it can be seen that there were no significant differences in the relative abundances of the main dominant bacteria in the feces of sows in each group (P>0.05). However, compared with the O group and the K group, the relative abundances in the Y group tended to increase; compared with the O group and the K group, the relative abundances of Euryarchaeota in the Y group decreased, indicating that adding Bacillus preparations can reduce the generation of sows' exhaust gas, thus playing a positive role in environmental improvement. Compared with the O group and the K group, the relative abundances of Spirochaetes in the Y group increased (P>0.05), while the relative abundances of Spirochaetes in the K group decreased (P>0.05). From the above results, it can be preliminarily concluded that adding Bacillus preparations to sows' diets can increase the abundance of beneficial bacteria and decrease the abundance of harmful bacteria, thereby playing a role in regulating the intestinal microecological balance. Among the Bacillus products produced by different fermentation processes, the Bacillus solid-state aerobic fermentation culture has better effects; in the Bacillus preparation product of this application, the Bacillus solid-state aerobic fermentation culture has the best effects. Adding the Bacillus solid-state aerobic fermentation culture Bacillus preparation to sows' diets can, to a certain extent, have a positive effect on the body condition, reproductive performance, serum antioxidant and immunobiochemical indexes of sows and the growth performance of their offspring piglets. Moreover, adding Bacillus preparations can increase the abundance of beneficial bacteria and decrease the abundance of harmful bacteria, thereby playing a role in regulating the intestinal microecological balance, and further reducing the generation of sows' exhaust gas, playing a positive role in environmental improvement.

[0045] 3. Test on the application effect of the Bacillus solid-state aerobic fermentation culture with antibacterial function prepared in Example 1 and Comparative Example 1 in improving the growth performance of piglets; Experimental animals and experimental design: Select 75 pigs with the same breed, healthy body condition and similar body weight. There are a total of 3 treatment groups, with 5 replicates in each treatment and 5 pigs in each replicate. The control group is 102 / 911; the Comparative Example 1 group is added with 0.08% of the Bacillus solid-state aerobic fermentation culture with antibacterial function of Comparative Example 1 on the basis of the control group; the Example 1 group is added with 0.08% of the Bacillus solid-state aerobic fermentation culture with antibacterial function of Example 1 on the basis of the control group, and the experimental period is 42 days.

[0046] Feeding management: Keep the temperature in the pen at 27°C during the experimental period, ensure sufficient drinking water and light for the pigs every day; feed the pigs 2 times a day at regular intervals to meet the free feeding of the pigs. During the feeding period, pay attention to the health status of the pigs at any time, treat them in time and make good records.

[0047] Growth performance: Weigh the pigs on the 1st day, 14th day and 42nd day of the formal experiment, and the pigs should be in a fasting state for more than 12 hours before weighing; record the consumption of the basal diet of the pigs during the formal experiment period; the calculation formulas for the average daily feed intake, average daily weight gain and feed-to-weight ratio of the pigs are as follows: Average daily gain (g / head / day) = (Final litter weight kg - Initial litter weight kg + Weight at culling or death kg) / (Number of pigs at the end * Number of experimental days + Number of culled / dead pigs * Number of days the culled or dead piglets participated in the experiment) × 1000 Average daily feed intake (g / head / day) = Total feed intake / (Number of pigs at the end × Number of experimental days + Number of culled pigs × Number of days the culled pigs participated in the experiment) × 1000 Feed to gain ratio = Average daily feed intake / Average daily gain Diarrhea rate of piglets: Record the diarrhea situation of piglets in each group and calculate the diarrhea rate. The calculation formula is as follows: Diarrhea rate / % = (Number of piglets with diarrhea × Number of diarrhea days) / (Number of experimental days × Number of experimental piglets).

[0048] Data statistics and analysis: The experimental data were sorted out using Excel software, and one-way analysis of variance was performed using SPSS 26.0 software. The data were expressed as "mean ± standard error". P < 0.05 indicates significant difference, and P > 0.05 indicates no significant difference. The results are shown in Table 3 below Table 3 Effects of different Bacillus preparations on the growth performance and diarrhea rate of nursery pigs during the whole period (n = 5) Result analysis: As can be seen from the data in Table 3, compared with the control group, there were no significant differences in the litter weight at 28 days of age and the average individual weight at 28 days of age in each experimental group (P>0.05), and the initial individual weight of the experimental pigs was about 7.2 kg; compared with the control group, the litter weight and average individual weight at 43 days of age in the comparative example 1 group and the example 1 group increased respectively; compared with the control group, the average daily feed intake and average daily weight gain in the example 1 group were the highest at each stage, the average daily feed intake in the comparative example 1 group was the worst, and compared with the control group, the diarrhea rate in the example 1 group was the lowest at each stage, followed by the comparative example 1 group; therefore, growth performance can reflect the health status and nutritional status of pigs. Through this experiment, by studying the effects of different bacillus preparations on the growth performance and diarrhea rate of nursery pigs from 28 to 43 days of age, 43 to 70 days of age, and 28 to 70 days of age, it was found that compared with the control group, the bacillus solid aerobic fermentation culture in the example 1 was the best in improving the final weight, average daily feed intake, and average daily weight gain of pigs, followed by the bacillus in the comparative example 1, but the average daily feed intake in the comparative example 1 group was still relatively low. The feed-to-gain ratio in the example 1 group was the lowest in the early stage of the experiment, that is, from 28 to 43 days of age, and increased in the later stage of the experiment, that is, from 43 to 70 days of age. The above research results all show that an appropriate amount of bacillus can improve the growth performance of pigs, reduce the diarrhea rate, and improve the utilization rate of feed nutrients by the body. Its mechanism of action may be that after being eaten by pigs, Bacillus subtilis can colonize in the intestine, help the intestine establish a stable and healthy internal environment, improve the intestinal morphology, promote the healthy development of the intestine, improve the digestion and absorption function, and thus improve the growth performance.

[0049] 4. Test the application effect of the bacillus solid aerobic fermentation culture with antibacterial function prepared in Example 1 and Comparative Example 1 on improving the production performance of broilers; Experimental animals and experimental design 210 white - feather broilers were randomly divided into 3 groups, with 5 replicates in each group and 14 chickens in each replicate. The broilers in the control group were fed a basal diet. The comparative example 1 group was added with 0.08% by mass of the bacillus solid aerobic fermentation culture with antibacterial function in the comparative example 1 on the basis of the control group; the example 1 group was added with 0.08% by mass of the bacillus solid aerobic fermentation culture with antibacterial function in the example 1 on the basis of the control group.

[0050] Growth performance: During the experiment, the feed consumption of broilers was recorded daily. The broilers were weighed on the 1st day and 21st day of the experiment respectively, and the average daily feed intake, average daily weight gain, and feed - to - gain ratio were calculated. The results were expressed as mean ± standard error.

[0051] Average daily feed intake = total feed consumption / (number of experimental days × number of chickens) Average daily weight gain = (final weight - initial weight) / (number of experimental days × number of chickens) Feed - to - gain ratio = average daily feed intake / average daily weight gain The results are shown in Table 4 below: Table 4 Effects of different Bacillus subtilis preparations on the growth performance of white - feather broilers For data in the same row, different superscript letters indicate significant differences (P < 0.05), the same letter or no letter indicates no significant difference. The same applies hereinafter.

[0052] For the white - feather broilers in the Example 1 group, the final weight and average daily gain from 1 to 21 days of age were significantly higher than those in the control group (P < 0.05). There was a tendency for the average daily feed intake to increase in the experimental groups supplemented with the Bacillus cultures of Comparative Example 1 and Example 1 in the diet, indicating that adding Bacillus preparations to the diet can promote the feed intake of broilers. After Bacillus enters the animal intestine, it can reduce the number of harmful microorganisms in the intestine, improve the digestion and absorption of nutrients in the intestine, increase the feed conversion rate, promote animal growth, and the Bacillus preparation can improve the slaughter performance of broilers and increase the economic benefits of broiler farming.

[0053] The above are all preferred embodiments of the present application. The protection scope of the present application is not limited thereby. Therefore, all equivalent changes made according to the structure, shape, and principle of the present application should be covered within the protection scope of the present application.

Claims

1. A method for preparing a solid aerobic fermentation culture of Bacillus with antibacterial function, characterized in that: The method comprises the following steps: (1) culturing Bacillus subtilis strains: after culturing the strains on a plate, a single colony is picked and inoculated into NB culture medium, and cultured for 20-24 hours as a primary seed liquid, and then inoculated into NB culture medium at an inoculation amount of 1-10% (V / V) and cultured for 40-48 hours as a secondary seed liquid; (2) preparing a solid-state fermentation culture medium: adding fermentation culture medium raw materials into water and mixing evenly, sterilizing and cooling for standby use; (3) fermentation culture: transferring the secondary seed liquid in step (1) into the solid-state fermentation culture medium prepared in step (2), with an inoculation amount of 1-10%, a temperature of 30-40°C, aerobic fermentation for 48-96 hours, and then drying and crushing to obtain a Bacillus subtilis solid-state fermentation culture with antibacterial function; wherein the fermentation culture medium raw materials include 500-900g bran, 300-500g corn, and 100-300g soybean meal.

2. The method for preparing a solid-state aerobic fermentation culture of Bacillus with antibacterial function according to claim 1, characterized in that: The mass ratio of bran: corn: soybean meal is (5-9): (3-5):

1.

3. The method for preparing a solid-state aerobic fermentation culture of Bacillus with antibacterial function according to claim 1, characterized in that: The sterilization in step (1) is performed at 100-121° C. for 20-60 min.

4. The method for preparing a solid aerobic fermentation culture of Bacillus with antibacterial function according to claim 1, characterized in that: The bran includes fine bran and coarse bran, and the mass ratio of the fine bran to the coarse bran is 1:(1.2-1.8).

5. The method for preparing a solid-state aerobic fermentation culture of Bacillus with antibacterial function according to claim 1, characterized in that: The fermentation medium raw materials also include 5-10g glucose, 5-10g maltodextrin, 0-1.5g D-mannose, and 10-16g PBS buffer.

6. The method for preparing a solid-state aerobic fermentation culture of Bacillus with antibacterial function according to claim 5, characterized in that: The mass ratio of maltodextrin to D-mannose is (4-8):

1.

7. Use of the solid-state aerobic fermentation culture of Bacillus with antibacterial function prepared according to any one of claims 1 to 6 as a livestock and poultry feed additive.

8. The use according to claim 7, characterized in that: The solid aerobic fermentation culture of Bacillus with antibacterial function is added to livestock and poultry feed at a mass ratio of 0.01-0.1%.

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