Feed additive produced by using Bacillus subtilis and production method thereof

Abstract

The present invention relates to the technical field of microbial feed additives, and more particularly to a feed additive produced using Bacillus subtilis and a production method thereof. The feed additive disclosed in the present invention comprises a product obtained by lysing vegetative Bacillus subtilis, overcomes the potential safety hazards associated with the addition of live bacteria, is low-cost, beneficial to animal growth, and improves animal production performance.

Description

Technical Field

[0001] The present invention relates to the technical field of feed microbial additives, in particular to a feed additive produced by utilizing Bacillus subtilis and a production method thereof. Background Art

[0002] Bacillus subtilis is an aerobic, endospore-forming Gram-positive bacterium that is environmentally friendly, food-safe, and harmless to humans, animals, and plants. It is a probiotic strain commonly added to animal feed. It is generally added to animal feed in the form of spores after fermentation. However, the addition of live bacteria or spores to feed still has certain safety issues. For example, many natural microecological live bacteria products, including Bacillus subtilis, have resistance factors themselves. Although the probability of transfer of such resistance factors is low, there is still a risk of transfer. At the same time, studies have pointed out that when the intestines of animals are immature, inflamed, or immunocompromised, live microecological preparations can break through the intestinal wall and invade the body, causing inflammation. Therefore, live bacterial preparations can sometimes cause inflammation. Even yeast and lactobacilli that are harmless to animals can also cause this phenomenon (for example, endocarditis). Summary of the Invention

[0003] In order to solve the above problems, the present invention provides a feed additive with higher safety produced by Bacillus subtilis and a production method thereof.

[0004] The purpose of the present invention is to be solved by the following technical solutions:

[0005] One aspect of the present invention provides a feed additive, wherein the feed additive includes a lysis product of Bacillus subtilis vegetative body.

[0006] After entering the animal intestine, the live bacteria of Bacillus subtilis can reproduce rapidly, promote the growth and reproduction of anaerobic beneficial bacteria through biological oxygen deprivation, and competitively inhibit the survival of harmful bacteria, thereby regulating the balance of intestinal microecology. Therefore, it is widely used as a feed additive, such as patents 201310219751.8, 201310719942.0, 201810220641.6, etc. However, those skilled in the art often overlook the role of Bacillus subtilis' own components. For example, Bacillus subtilis is Gram-positive (G +) bacteria, the main component of its cell wall is peptidoglycan, which can reach 80% of the cell wall weight. Peptidoglycan, also known as mucopeptide, murein or mucopeptide complex, has the effect of directly inhibiting bacteria, activating the nonspecific immunity of the intestinal mucosa, stimulating the growth and development of animal immune organs, and has the function of activating T and B lymphocytes, improving the levels of immunoglobulins and antibodies, and enhancing cellular immunity and humoral immunity. At the same time, mycotoxins in feed have always been a difficult problem that has plagued the feed industry. Peptidoglycan is an effective adsorbent for mycotoxins, which can effectively adsorb mycotoxins in feed, making the toxins inactivated, thereby weakening and eliminating the toxic effects. In addition, Bacillus subtilis itself also contains other active metabolites such as protease, amylase, and cellulase. Therefore, adding the cracked Bacillus subtilis to animal feed will also have a beneficial effect on the animals. In addition, adding the product after the cracking of the Bacillus subtilis vegetative body can effectively avoid the above-mentioned safety issues.

[0007] The vegetative body of Bacillus subtilis can be lysed by physical methods such as repeated freezing and thawing, ultrasonic treatment, and osmosis, or by enzymatic hydrolysis using protease.

[0008] Preferably, lysozyme is used to lyse the Bacillus subtilis vegetative cells.

[0009] Lysozyme is a hydrolase that specifically acts on the peptidoglycan of microbial cell walls. It can cut the β-1,4 glycosidic bond between N-acetylglucose and N-acetylmuramic acid in peptidoglycan, destroy the peptidoglycan scaffold, and cause the cells to split under the action of internal osmotic pressure, causing bacterial lysis and death. + The bacterial cell wall is almost entirely composed of peptidoglycan, and - (Gram-negative) bacteria have little effect. Human and animal cells have no cell wall structure and no peptidoglycan, so lysozyme has no destructive effect on human and animal cells. In addition, lysozyme, as a natural protein, can be digested and absorbed as a nutrient in the gastrointestinal tract. At the same time, as an immune factor of animals and microorganisms themselves, it has multiple beneficial effects such as antibacterial and anti-inflammatory, antiviral, hemostatic, analgesic, and accelerates tissue repair, reduces diarrhea, enhances the body's nonspecific immunity, and promotes the proliferation of bifidobacteria. Therefore, by enzymatically lysing the Bacillus subtilis vegetative body with lysozyme, on the one hand, the Bacillus subtilis can be lysed without affecting the active substances such as proteases therein; on the other hand, it can be directly added to the feed after enzymatic lysis and may produce better results.

[0010] Another aspect of the present invention provides a method for producing a feed additive using Bacillus subtilis, the method comprising the following steps:

[0011] Step (1) inoculating the Bacillus subtilis mother strain into a triangular flask seed culture medium, and culturing the culture medium in a shaking table at 37° C. and 180-200 rpm for 12-16 hours to obtain triangular flask seeds.

[0012] Step (2) inoculating the triangular flask seeds into the culture medium of the first-level seed tank at an inoculation rate of 0.1-0.5%, and culturing for 8-12 hours at 37° C., 180-200 rpm, and a ventilation rate of 1:0.4-0.6 to obtain first-level seed tank seeds.

[0013] Step (3) inoculates the seeds from the primary seed tank into the culture medium of the main fermentation tank at a 1-3% inoculation rate and cultures them for 16-20 hours under the same conditions as step (2). After this step, the vegetative cells of Bacillus subtilis reach more than 6 billion cfu / mL.

[0014] Step (4) dissolving the solid powder lysozyme with a small amount of sterile water and putting it into the main fermentation tank of step (3), adding 1000 to 2000 units of lysozyme per liter of fermentation liquid, stirring evenly, maintaining the temperature at 35 to 37 ° C, standing for 4 to 8 hours, standing and stirring 1 to 2 times during the period, and adsorbing with an adsorbent or spray drying to obtain the feed additive. After the lysozyme in this step cleaves the Bacillus subtilis vegetative body in the main fermentation tank, no complete vegetative body can be seen by microscopic examination, indicating that the lysozyme can completely cleave the Bacillus subtilis vegetative body. In addition, the protease activity of the cleavage product is measured. Compared with before cleavage, the protease activity per milliliter increases by more than 200 units, indicating that adding lysozyme to cleave the Bacillus subtilis vegetative body can enhance the activity of Bacillus subtilis metabolites (i.e., proteases).

[0015] Preferably, the culture medium is composed of 30-45 g / L soybean meal, 20-30 g / L corn flour, 6.0-10.0 g / L dipotassium hydrogen phosphate, 4-6 g / L ammonium sulfate, 1.0-1.5 g / L calcium chloride, 0.5-1.0 g / L magnesium sulfate and water, and is prepared by sterilization at 121° C. for 20 min.

[0016] In the prior art, when Bacillus subtilis is used as a feed additive, it plays a role in the form of live bacteria, and adding Bacillus subtilis in the form of a vegetative body to the feed is not conducive to maintaining the number of live bacteria of Bacillus subtilis. In order to ensure that Bacillus subtilis can exist in the form of live bacteria when entering the intestine, a higher spore ratio is required. Therefore, the prior art is mostly aimed at improving the spores of Bacillus subtilis. For example, patent 201711033875.1 provides a culture medium that can promote Bacillus subtilis spore production, and patent 201410162370.5 provides a method for efficiently improving the germination rate of Bacillus subtilis. The product after the cracking of Bacillus subtilis vegetative body is added to the feed additive of the present invention, and the fermentation process of Bacillus subtilis is aimed at producing Bacillus subtilis vegetative body. The above-mentioned steps (1) to step (3) and culture medium are the optimal parameters obtained by the inventor after a large number of experimental optimizations. Under this condition, the spores of Bacillus subtilis will not be produced, but a large amount of Bacillus subtilis vegetative bodies will be obtained. Furthermore, the present invention's vegetative fermentation of Bacillus subtilis eliminates the need for spore formation, significantly shortening fermentation time. Energy consumption associated with ventilation and high-speed stirring during fermentation is also minimized. Furthermore, the soybean meal and corn flour used as the main components of the culture medium are both inexpensive and readily available agricultural byproducts. Therefore, the present method offers the advantages of low cost, minimal energy consumption, and a short fermentation time.

[0017] Preferably, the lysozyme is hen egg white lysozyme, which is commercially available and meets the standards of food-grade enzyme preparations and is highly safe.

[0018] The advantages of the present invention are: (1) The feed additive provided by the present invention uses enzymatically hydrolyzed Bacillus subtilis vegetative bodies, overcoming the safety issues that may arise from the addition of live bacteria. (2) The present invention uses lysozyme to enzymatically hydrolyze and cleave the Bacillus subtilis vegetative bodies, and while cleaving the vegetative bodies, the activity of active substances such as proteases and amylases produced by metabolism during fermentation and released from the vegetative bodies after cleavage is not impaired, and lysozyme itself is also beneficial to animal growth. (3) The feed additive provided by the present invention can improve the production performance of animals, reduce diarrhea, and enhance immunity. DETAILED DESCRIPTION

[0019] The present invention will be further described in detail below through specific examples.

[0020] The Bacillus subtilis strain 10260 used in the present invention was introduced by the applicant from the China Industrial Culture Collection Center (CICC) and can also be obtained by the public through other public channels.

[0021] Example 1

[0022] (1) Preparation of Bacillus subtilis vegetative cells

[0023] Bacillus subtilis mother stock spores were inoculated into seed culture medium in a triangular flask and incubated in a shaker at 37°C, 180-200 rpm for 14 hours to produce triangular flask seeds. The triangular flask seeds were inoculated into the culture medium of a primary seed tank at a 0.1% inoculum and incubated at 37°C, 180-200 rpm, with an aeration ratio of 1:0.4-0.6 for 12 hours to produce primary seed tank seeds. The primary seed tank seeds were inoculated into the culture medium of the main fermentation tank at a 3% inoculum and incubated under the same conditions for 16 hours to obtain a fermentation culture containing a large number of Bacillus subtilis vegetative cells, reaching 6.1 billion cfu / mL, which was set aside. The culture medium for the triangular flask, primary seed tank, and main fermentation tank consisted of 30 g / L soybean meal, 30 g / L corn flour, 10.0 g / L potassium dihydrogen phosphate, 6 g / L ammonium sulfate, 1.0 g / L calcium chloride, 0.5 g / L magnesium sulfate, and water, and sterilized at 121°C for 20 minutes.

[0024] (2) Crushing and lysis of Bacillus subtilis vegetative cells

[0025] Dissolve the solid powdered lysozyme in a small amount of sterile water and add it directly to the main fermentation tank in (1). The amount of lysozyme added is 1000 units per liter of fermentation liquid. Stir evenly, maintain the temperature at 35-37°C, and let it stand for 8 hours. Stir 1-2 times during the standing period. No complete vegetative body can be seen under microscopic examination. The protease activity increases from 202 units per milliliter before enzymolysis to 419 units per milliliter. After completion, directly adsorb it with an adsorbent or spray dry it to form a product. The lysozyme is commercially available egg white lysozyme, which meets the standards of food-grade enzyme preparations.

[0026] Example 2

[0027] (1) Preparation of Bacillus subtilis vegetative cells

[0028] The preparation of triangular flask seeds is the same as in Example 1. The triangular flask seeds are inoculated into the culture medium of the first-stage seed tank at a 0.3% inoculum and cultured for 10 hours at 37°C, 180-200 rpm, and an aeration ratio of 1:0.4-0.6 to obtain first-stage seed tank seeds. The first-stage seed tank seeds are inoculated into the culture medium of the main fermentation tank at a 2% inoculum and cultured for 18 hours under the same conditions to obtain a fermentation culture containing a large number of Bacillus subtilis vegetative bodies, with a total of 6.6 billion cfu / mL of Bacillus subtilis vegetative bodies, which are set aside. The culture medium for the triangular flask, first-stage seed tank, and main fermentation tank is composed of 40g / L soybean meal, 25g / L corn flour, 8.0g / L potassium dihydrogen phosphate, 5g / L ammonium sulfate, 1.25g / L calcium chloride, 0.75g / L magnesium sulfate, and water, and are sterilized at 121°C for 20 minutes.

[0029] (2) Crushing and lysis of Bacillus subtilis vegetative cells

[0030] Dissolve the solid powdered lysozyme in a small amount of sterile water and add it directly to the main fermentation tank in (1). Add 1500 units of lysozyme per liter of fermentation liquid. Stir evenly, maintain the temperature at 35-37°C, and let it stand for 6 hours, stirring 1-2 times during the standing period. Microscopic examination shows no intact vegetative bodies. The protease activity increases from 227 units per milliliter before enzymolysis to 440 units per milliliter. The rest is the same as in Example 1.

[0031] Example 3

[0032] (1) Preparation of Bacillus subtilis vegetative cells

[0033] The preparation of triangular flask seeds was the same as in Example 1. The triangular flask seeds were inoculated into the culture medium of the primary seed tank at a 0.5% inoculum and cultured for 8 hours at 37°C, 180-200 rpm, and an aeration ratio of 1:0.4-0.6 to produce the primary seed tank seeds. The primary seed tank seeds were inoculated into the culture medium of the main fermentation tank at a 3% inoculum and cultured for 16 hours under the same conditions to produce a fermentation culture containing a large number of Bacillus subtilis vegetative bodies, with a total of 6.9 billion cfu / mL of Bacillus subtilis vegetative bodies, which were set aside. The culture medium for the triangular flask, primary seed tank, and main fermentation tank all consisted of 45 g / L soybean meal, 20 g / L corn flour, 6.0 g / L dipotassium hydrogen phosphate, 4 g / L ammonium sulfate, 1.5 g / L calcium chloride, 1.0 g / L magnesium sulfate, and water, and were sterilized at 121°C for 20 minutes.

[0034] (2) Crushing and lysis of Bacillus subtilis vegetative cells

[0035] Dissolve the solid powdered lysozyme in a small amount of sterile water and add it directly to the main fermentation tank in (1). Add 2000 units of lysozyme per liter of fermentation liquid. Stir evenly, maintain the temperature at 35-37°C, and let it stand for 4 hours, stirring 1-2 times during the standing period. Microscopic examination shows no intact vegetative bodies. The protease activity increases from 240 units per milliliter before enzymolysis to 455 units per milliliter. The rest is the same as in Example 1.

[0036] Comparative Example 1

[0037] (1) Preparation of Bacillus subtilis vegetative cells

[0038] The culture medium for the flask, primary seed tank, and main fermentation tank was a complete lysing medium consisting of 10.0 g / L peptone, 5.0 g / L yeast extract, 5.0 g / L sodium chloride, 8.0 g / L glucose, and water. The pH was adjusted to 7.2. All other conditions were the same as in Example 1. A fermentation culture containing vegetative cells and spores of Bacillus subtilis was obtained, with a vegetative cell count of 3.5 billion cfu / mL and a spore count of 600 million cfu / mL.

[0039] (2) Crushing and lysis of Bacillus subtilis vegetative cells

[0040] The enzymatic hydrolysis was carried out in the same manner as in Example 1. After the enzymatic hydrolysis, no complete vegetative bodies were observed under microscopic examination, and the protease activity increased from 31 units per milliliter before the enzymatic hydrolysis to 107 units per milliliter.

[0041] Comparative Example 2

[0042] (1) Preparation of Bacillus subtilis vegetative cells

[0043] Bacillus subtilis mother strain spores were inoculated into a seed culture medium in a triangular flask and incubated in a shaker at 37°C, 140-150 rpm for 14 hours to produce triangular flask seeds. The triangular flask seeds were inoculated into the culture medium of a primary seed tank at a 0.1% inoculum and incubated at 37°C, 140-150 rpm, and an aeration ratio of 1:0.3-0.4 for 12 hours to produce primary seed tank seeds. The primary seed tank seeds were inoculated into the culture medium of a main fermentation tank at a 3% inoculum and incubated under the same conditions for 30 hours to obtain a fermentation culture containing a large number of Bacillus subtilis vegetative cells, with a vegetative cell count of 4 billion cfu / mL. No spores were observed under microscopic examination.

[0044] (2) Crushing and lysis of Bacillus subtilis vegetative cells

[0045] The same enzymatic hydrolysis as in Example 1 was carried out. After enzymatic hydrolysis, no complete vegetative bodies were observed under microscopic examination, and the protease activity increased from 87 units per milliliter before enzymatic hydrolysis to 203 units per milliliter.

[0046] Example 4

[0047] The fermentation cultures of Bacillus subtilis in the Examples and Comparative Examples were observed and counted under a microscope, and the viable bacteria content (vegetative bodies) and spore content were determined as shown in the following table.

[0048] Table 1. Results of live bacteria and spore counts

[0049]

[0050] The test results show that the conditions for preparing Bacillus subtilis vegetative cells in Examples 1-3 produced a large number of Bacillus subtilis vegetative cells without producing spores. The fermentation conditions in Comparative Example 1 produced both vegetative cells and spores, with the number of vegetative cells produced significantly lower than those in Examples 1-3. Although no spores were produced in Comparative Example 2, the number of vegetative cells produced was significantly lower than in Examples 1-3. In summary, Examples 1-3 represent the optimal conditions for preparing Bacillus subtilis vegetative cells.

[0051] It should be noted that the fermentation purpose (for example, the purpose of producing spores or the purpose of producing trophoblasts) is different, and the fermentation conditions are very different. In actual production, there are differences between the fermentation conditions of different strains. For example, some strains grow in higher numbers when using soybean meal and / or corn as the main raw materials of the culture medium, while some grow in higher numbers when using beef extract and / or peptone. In the animal husbandry industry, the effect of feeding animals with fermented products with high fermentation metabolites is generally better, and using soybean meal and / or corn as the main raw materials will produce more metabolites than using beef extract and / or peptone as the main raw materials. Therefore, soybean meal and / or corn are usually used as the main raw materials in fermentation production to produce more metabolites.

[0052] Beef extract and peptone are known to facilitate spore formation. When spore count is the goal, particularly in the pharmaceutical industry, and to facilitate subsequent concentration steps, completely soluble beef extract and peptone are often used as primary raw materials. Unlike spore production, the inventors optimized the culture medium with vegetative count as the goal. The resulting optimized culture medium (such as the culture medium in Examples 1-3) exhibits a low likelihood of spore formation even with extended fermentation times.

[0053] Furthermore, Bacillus (including Bacillus subtilis) does not produce spores while multiplying and dividing. Instead, it stops growing or begins spore formation when the vegetative body reaches a certain size and the fermentation environment becomes unsuitable for growth. Therefore, by controlling the fermentation conditions of Bacillus, it is possible to produce only vegetative bodies without spores.

[0054] In Examples 1-3, fermentation was performed using a culture medium containing soybean meal and corn as the main raw materials, which produced more beneficial metabolites. In addition, the culture medium composition and specific fermentation conditions (such as temperature, ventilation volume, and time) were all designed to produce vegetative bodies. Therefore, a large number of vegetative bodies of Bacillus subtilis were obtained in the final fermentation without producing spores.

[0055] Example 5

[0056] Comparative experiment on the effect of feeding weaned piglets with enzymatic hydrolysis products of Bacillus subtilis vegetative bodies

[0057] This study selected 96 28-day-old "Du Da Chang" weaned piglets (half male and half female). All piglets were individually weighed and divided into two treatments, a control group and an experimental group, based on weight and sex. Each treatment had eight replicates, with six piglets per replicate. The control group was fed a basal diet consisting of: 60.8% corn, 15.0% soybean meal, 10.0% puffed soybeans, 5.0% fish meal (Peruvian), 6.0% whey powder, 1.2% calcium hydrogen phosphate, 0.6% rock dust, 0.3% salt, 0.1% lysine, and 1.0% premix. The experimental group received a basal diet supplemented with 0.2% defatted rice bran adsorbent (equivalent to 2 L of Bacillus subtilis fermentation lysate, the feed additive described in Example 1).

[0058] The experimental period was 28 days. The piglets were weighed on day 0 and day 28 of the experiment, and feed consumption was recorded. The average daily growth, average daily feed intake, and feed-to-weight ratio of the piglets were calculated. The frequency of piglet diarrhea was recorded daily, and the piglet diarrhea rate was calculated (Table 2):

[0059] Diarrhea rate (%) = the sum of the number of diarrheal piglets in the experimental period / (number of experimental days × total number of experimental piglets) × 100%.

[0060] On the 28th day of the experiment, one piglet from each pen was selected for blood sampling from the anterior vena cava to measure its immune-related indicators; the results are shown in Table 3.

[0061] As can be seen from Table 2, after the Bacillus subtilis vegetative lysate produced in Example 1 was added to the feed, the average daily weight gain of the piglets was significantly increased (p < 0.05), the feed-to-weight ratio was significantly reduced (p < 0.05), and the diarrhea rate of the piglets was also significantly reduced (p < 0.05). As can be seen from Table 3, after the Bacillus subtilis produced in Example 1 was added to the feed, the IgA and IgG levels in the piglet serum were significantly increased (p < 0.05). The above animal test results show that the addition of the Bacillus subtilis vegetative lysate produced in Example 1 to the feed can improve the production performance of weaned piglets, reduce diarrhea in piglets, and improve the immunity of piglets.

[0062] Table 2. Results of production performance and diarrhea rate of piglets in different treatment groups

[0063]

[0064]

[0065] Note: a a indicates a significant increase compared with the control group, p < 0.05; b indicates a significant decrease compared with the control group, p < 0.05.

[0066] Table 3. Results of serum immunoglobulin detection in piglets in different treatment groups

[0067]

[0068] Note: a Indicates a significant increase compared with the control group, p < 0.05.

[0069] The above embodiments are intended only to illustrate the technical concepts and features of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications based on the technical concepts and technical solutions proposed by the present invention are within the scope of protection of the present invention. Technologies not covered by the present invention can be implemented using existing technologies. Unless otherwise specified, the materials used in the present invention can be obtained through commercial channels.

Claims

1. A method for producing a spore-free feed additive with a live bacterial content of 6.9 billion cfu / mL before enzymatic hydrolysis using Bacillus subtilis, characterized in that: The specific steps of the method are: Step (1) inoculating the Bacillus subtilis mother strain into a triangular flask seed culture medium, and culturing the culture medium in a shaking table at 37° C. and 180-200 rpm for 14 h to obtain triangular flask seeds; Step (2) inoculating the triangular flask seeds into the culture medium of the first-level seed tank at a 0.5% inoculation rate, and culturing for 8 hours at 37° C., 180-200 rpm, and a ventilation rate of 1:0.4-0.6 to obtain first-level seed tank seeds; Step (3) inoculating the seeds from the primary seed tank into the culture medium of the main fermentation tank at a 3% inoculum amount, and culturing for 16 hours under the same conditions as step (2); the culture medium is composed of 45 g / L soybean meal powder, 20 g / L corn flour, 6.0 g / L dipotassium hydrogen phosphate, 4 g / L ammonium sulfate, 1.5 g / L calcium chloride, 1.0 g / L magnesium sulfate and water, and is prepared by sterilizing at 121° C. for 20 minutes; Step (4) dissolving the solid powder lysozyme with a small amount of sterile water and adding the solid powder lysozyme to the main fermentation tank of step (3) at an amount of 2000 units of lysozyme per liter of fermentation liquid, stirring evenly, maintaining the temperature at 35-37°C, standing for 4 hours, stirring 1-2 times during the standing period, and adsorbing with an adsorbent or spray drying to obtain the feed additive, wherein the lysozyme is hen egg white lysozyme.

2. A feed additive having a live bacterial content of 6.9 billion cfu / mL before enzymatic hydrolysis and containing no spores, characterized in that: The feed additive comprises a lysis product of a Bacillus subtilis vegetative body, and the lysis method is an enzymatic hydrolysis method, wherein the Bacillus subtilis vegetative body is lysed by using lysozyme; the preparation process of the feed additive is as follows: Step (1) inoculating the Bacillus subtilis mother strain into a triangular flask seed culture medium, and culturing the culture medium in a shaking table at 37° C. and 180-200 rpm for 14 h to obtain triangular flask seeds; Step (2) inoculating the triangular flask seeds into the culture medium of the first-level seed tank at a 0.5% inoculation rate, and culturing for 8 hours at 37° C., 180-200 rpm, and a ventilation rate of 1:0.4-0.6 to obtain first-level seed tank seeds; Step (3) inoculating the seeds from the first-level seed tank into the culture medium of the main fermentation tank at a 3% inoculation rate, and culturing for 16 hours under the same conditions as step (2); the culture medium is composed of 45g / L soybean meal powder, 20g / L corn flour, 6.0g / L dipotassium hydrogen phosphate, 4g / L ammonium sulfate, 1.5g / L calcium chloride and 1.0g / L magnesium sulfate, and is sterilized at 121°C for 20 minutes; step (4) dissolving the solid powder lysozyme with a small amount of sterile water, and adding the solid powder lysozyme to the main fermentation tank of step (3), adding 2000 units of lysozyme per liter of fermentation liquid, stirring evenly, maintaining the temperature at 35-37°C, standing for 4 hours, stirring 1-2 times during the standing period, and adsorbing with an adsorbent or spray drying to obtain the feed additive; the lysozyme is egg white lysozyme.

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