Preparation method of functional feed additive for preventing and treating avian necrotic enteritis

Abstract

The present application relates to a high-enzyme-activity protease producing bacillus natto FMN-1 with preservation number of CGMCC No.20659, and relates to the enzyme produced by the strain and the preparation method of the enzyme, and relates to a functional feed additive for preventing and treating avian necrotic enteritis of the strain and the enzyme, and relates to the preparation method of the additive, belonging to the technical field of poultry feed and feed additive. The weight fraction composition is as follows: 20-35 parts of traditional Chinese medicine enzymolysis mixture, 4-9 parts of bacillus natto, 4-10 parts of vitamin C, 4-10 parts of vitamin E, and 40-60 parts of talc. The feed digestion rate, daily weight gain of poultry can be improved, and the feed conversion ratio can be reduced. Taking broiler as an example, the daily weight gain is between 1.37% and 2.48%, and the feed conversion ratio is between 1.64 and 1.67. The immune function of poultry is improved, the disease resistance is enhanced, and the avian necrotic enteritis caused by clostridium perfringens is effectively prevented and treated.

Description

[0001] This application is a divisional application. The original application number is: 202111503213.2, and the application date is: 2021.12.09. The name of the invention is: A functional feed additive for preventing and treating necrotic enteritis in poultry and its preparation method. Technical Field

[0002] The invention relates to a functional feed additive for preventing and treating necrotic enteritis in poultry and a preparation method of the additive, belonging to the technical field of poultry feed and feed additives. Background Art

[0003] Necrotic enteritis, also known as enterotoxemia, is an infectious disease caused by Clostridium perfringens, a conditionally pathogenic bacterium that mainly occurs in poultry. Healthy poultry can also carry this bacterium. Poor ventilation in breeding houses, damp ground, or feeding moldy and spoiled fish meal and soybean meal can all induce this disease. Repeated attacks in the same area or the same animal house, with a long course of disease, not only affect the production performance of poultry but also easily induce other diseases, causing serious losses.

[0004] Necrotic enteritis can occur throughout the year, mainly in summer and autumn, especially when the temperature changes suddenly, which will cause greater stress to poultry, temporarily unbalance the body's physiological functions, and cause the disease. The disease mainly occurs in animals aged 2-6 weeks, mostly in broilers. The disease is mainly transmitted through the digestive tract. The excrement of the chickens contaminates feeding tools, drinking water, bedding, etc., causing the spread of pathogens.

[0005] Necrotic enteritis typically presents suddenly, with the chickens refusing to eat, lying prone, and passing loose, yellow-green or dark feces, sometimes with blood. Cases and deaths are sporadic, and the mortality rate is significantly increased if mixed infections occur. Sick birds exhibit marked depression, close their eyes, become lethargic, have a decreased appetite, suffer from diarrhea, and have matted, shaggy, and easily broken feathers. Affected animals experience stunted growth and development, and pass black or gray feces, sometimes mixed with blood. In some cases, coccidia may also be present, resulting in slightly looser, yellowish, or fleshy feces.

[0006] Traditional treatments rely primarily on clinical medication, using antibiotics. While effective, antibiotics are often associated with residual disease and drug resistance. Currently, antibiotic use is increasingly restricted in China's livestock farming environment. Antibiotics are no longer permitted in feed, leaving the disease to be treated with additional medications containing Western medicines. These medications can also leave residues in livestock products, posing a risk to human health. With increasing public awareness of health and food safety, the development of a functional feed additive that can prevent and treat necrotic enteritis in poultry is essential.

[0007] Currently, there are also some traditional Chinese medicine preparations that treat necrotic enteritis caused by Clostridium perfringens. For example, the following application, Publication No. 105616906A, a Chinese invention entitled "A Chinese medicine composition for treating necrotic enteritis in chickens and its preparation method," discloses a Chinese medicine composition for treating necrotic enteritis in chickens and its preparation method. The components and their weight ratios of the Chinese medicine composition are as follows: 10-15 parts of Houttuynia cordata; 5-10 parts of Artemisia annua; 5-8 parts of Pulsatilla scabra; 5-10 parts of Sanguisorba officinalis; 5-10 parts of Aucklandia lappa; 5-10 parts of Scutellaria baicalensis; 5-10 parts of Anemarrhena asphodeloides; 8-15 parts of Pharbitidis seeds; and 5-10 parts of Rhubarb. The present invention has the effects of antibacterial and anti-inflammatory, clearing away heat and relieving swelling, and is characterized by rapid onset, long duration of action, minimal toxic and side effects, and no drug residue, making it an ideal Chinese medicine composition for treating necrotic enteritis in chickens.

[0008] Publication No. 102846700A, a Chinese invention titled "A Traditional Chinese Medicine Composition for Preventing and Treating Mixed Infection of Coccidiosis and Necrotic Enteritis in Poultry," discloses a traditional Chinese medicine composition for preventing and treating coccidiosis and necrotizing enteritis in poultry. The composition comprises thistle (Euphrasia officinalis), which cools blood and stops bleeding, dispels blood stasis, and reduces swelling, as the main ingredient; Artemisia annua, which clears away heat and clears away heat, cools blood, and removes steam, as the secondary ingredient; astragalus, which invigorates qi and strengthens the exterior, supports sores and promotes tissue regeneration; and white atractylodes, which invigorates the spleen and replenishes qi, eliminates dampness, and promotes diuresis; and liquorice, which invigorates the spleen and replenishes qi, clears heat and detoxifies, harmonizing the various ingredients. Together, these ingredients offer the effects of cooling blood and removing blood stasis, clearing heat and detoxifying, and strengthening the body's foundation. The composition is suitable for the prevention and treatment of coccidiosis and necrotizing enteritis, either alone or in combination, at different stages of the disease. Clinical application of this composition to a large number of poultry diseases has demonstrated its effectiveness, lack of toxic side effects, and absence of drug residue.

[0009] The Chinese invention, Publication No. 101697774A, "A Traditional Chinese Medicine Feed Additive for Treating Chicken Coccidiosis," discloses a traditional Chinese medicine feed additive for treating chicken coccidiosis. The selected raw Chinese medicinal materials and their weight components are: 6 parts of Cimicifuga foetida, 5 parts of Scutellaria baicalensis, 6 parts of Sophora flavescens, 6 parts of Pulsatilla scabra, 3 parts of Brucea javanica, 3 parts of Dichroa striata, 6 parts of Artemisia annua, and 5 parts of Stemona radix; the preparation method comprises: mixing the above raw medicinal materials and then crushing them, or crushing them separately and then mixing them. The raw medicinal materials selected in the present invention have the effects of clearing heat and detoxifying, killing insects and relieving itching. By feeding the feed daily, the purpose of preventing and treating chicken coccidiosis, necrotic enteritis, and white crown disease in chickens can be achieved.

[0010] The Chinese invention, Publication No. 111345401A, titled "A Wheat Grain Composite Antimicrobial Feed Additive," discloses a wheat grain composite antimicrobial feed additive. The feed additive comprises wheat grains, and is supplemented with cerevisiae yeast CCTCCM2019051, and further comprises one or more of Bacillus polymyxa CCTCCM2019024, Bacillus natto CCTCCM2019025, and Lactobacillus plantarum CCTCCM2019026. The cerevisiae yeast CCTCCM2019051 is added in an amount of 0.5-5%, and the total microbial content is 1-8%. This invention not only has a low cost and a simple preparation process, but also effectively enhances the immunity and disease resistance of livestock and poultry, effectively replacing antibiotics and reducing antibiotic usage.

[0011] However, the aforementioned preparation process involves complex Chinese herbal ingredients and primarily relies on pure Chinese herbs. In reality, the primary role of Chinese herbs lies in their active ingredients, including flavonoids, phenols, amino acids, and minerals. The preparation process disclosed in the aforementioned literature fails to effectively extract these active ingredients from Chinese herbs. Furthermore, the formulations based on pure Chinese herbal combinations are relatively simple and lack the ability to combine with other ingredients to achieve enhanced preventive and therapeutic effects. Therefore, comprehensive consideration of both safety and efficacy during feeding is insufficient.

[0012] Meanwhile, in the above preparation process, if multiple microorganisms are mixed, each strain needs to be activated and cultured separately, which is cumbersome and does not have a particularly specific and efficient inhibitory effect on Clostridium perfringens that causes necrotic enteritis in poultry. Summary of the Invention

[0013] Natural plants, Chinese herbal medicines, or plant-derived compounds have excellent in vitro antioxidant activity and are widely used as feed additives in animal studies to improve animal growth performance and antioxidant capacity. However, plant cell walls restrict the dissolution of intracellular bioactive substances, hindering the digestion, absorption, and full utilization of the natural functional components within plant cells in animals. Enzymatic hydrolysis technology, which uses cellulases, hemicellulases, and pectinases, either alone or in combination, to promote the dissolution of bioactive substances within plant cells, is an environmentally friendly and effective method for destroying plant cell walls. Enzymes destroy the plant cell wall matrix to promote the release of intracellular bioactive components or their more efficient extraction. Enzymatic hydrolysis technology has also been studied in animal feed raw materials and has achieved good results.

[0014] At the same time, microorganisms have a good regulatory effect on the intestinal health of animals, and the metabolites produced by the bacteria themselves have varying degrees of inhibitory effects on some pathogens. The combination of traditional Chinese medicine and microorganisms can achieve better synergistic effects.

[0015] The first object of the present invention is to provide a functional feed additive for preventing and treating necrotic enteritis in poultry. The raw materials are composed of the following parts by weight:

[0016] 20-35 parts of traditional Chinese medicine enzymatic hydrolysis mixture, 4-9 parts of Bacillus natto, 4-10 parts of vitamin C, 4-10 parts of vitamin E, and 40-60 parts of talc.

[0017] Furthermore, the traditional Chinese medicine enzymatic hydrolysis mixture is composed of the following parts by weight: 30-40 parts of Artemisia annua, 15-25 parts of Rhubarb, 20-30 parts of Artemisia capillaris, 25-35 parts of Andrographis paniculata, 4-10 parts of pectinase, 4-10 parts of cellulase, and 3-8 parts of protease.

[0018] Furthermore, the Chinese medicine enzymatic hydrolysis mixture is composed of the following parts by weight: 30-35 parts of Artemisia annua, 15-20 parts of Rhubarb, 20-25 parts of Artemisia capillaris, 25-30 parts of Andrographis paniculata, 4-8 parts of pectinase, 4-8 parts of cellulase, and 3-6 parts of protease.

[0019] Furthermore, the natto Bacillus subtilis is prepared by the following method:

[0020] Bacillus natto was inoculated into a liquid culture medium of beef extract peptone and incubated at 37°C for 24 h for activation. The number of viable bacteria in the seed liquid of Bacillus natto FMN-1 after activation was detected to be (2.0-5.0)×10 8 CFU / mL-(2.0~5.0)×10 9 CFU / mL. In order to be applied to large-scale production applications, it is necessary to expand the culture. The seed liquid is inoculated into a beef extract peptone liquid culture medium at a 1% inoculum volume. The expansion culture temperature is 37°C and the culture is statically cultured for 24 hours.

[0021] Furthermore, the natto Bacillus subtilis is FMN-1, with a deposit number of CGMCC No. 20659;

[0022] Furthermore, the protease in the traditional Chinese medicine enzymatic hydrolysis mixture is provided by a purified strain of Bacillus natto FMN-1 that produces high-activity protease, and the strain collection number of the Bacillus natto FMN-1 is CGMCC No.20659.

[0023] The Bacillus natto FMN-1 was deposited in the General Microbiology Center of the China Culture Collection Administration on September 16, 2020. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 20659. The strain was screened from Japanese natto by Jiang Dan of Shenyang Fengmei Biotechnology Co., Ltd. in the biological laboratory of Shenyang Fengmei Biotechnology Co., Ltd. between July 30 and August 30, 2020.

[0024] Bacillus natto, also known as Bacillus natto, belongs to the Bacteriaceae family and the genus Bacillus. It is a subspecies of Bacillus subtilis. It is typically 0.7-0.8 μm x 0.2-0.3 μm in size and is aerobic, Gram-positive. Its spores are oval or columnar, mesophytic or semi-mesophytic. Even if the spore swells, it is not noticeable. It has flagella and is motile. The maximum growth temperature for Bacillus natto is 45-55°C, while the minimum is 5-20°C. The optimal growth temperature is 37°C. The pH range is 7.0-7.5, with an optimal pH of 7.3. The spores are heat-resistant and can germinate in the intestines. Natto bacteria have strong resistance, and their spores are highly stable with resistance to acid, alkali, high temperature (100°C), and extrusion. They are easy to preserve and can be stored for a long time in a refrigerator at -70°C in the laboratory. The survival rate after 4 hours in gastric acid is 100%. At the same time, it has a strong ability to inhibit pathogens. After entering the small intestine, it can change the ecology of the animal's intestinal flora, regulate the intestinal flora, and the antimicrobial peptides it produces have a broad-spectrum antibacterial effect.

[0025] The preparation method of the functional feed additive is as follows:

[0026] The feed additive is obtained by uniformly mixing all raw materials of the feed additive in proportion.

[0027] Application of the functional feed additive of the present invention in poultry compound feed: the feed additive is added to the poultry compound feed at a ratio of 0.2-0.5‰.

[0028] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a functional feed additive for preventing and treating necrotic enteritis in poultry. The functional feed additive of the present invention can significantly reduce the incidence of necrotic enteritis in poultry and at the same time improve the production performance and immune function of poultry.

[0029] Beneficial effects

[0030] 1. The functional feed additive provided by the present invention has the following characteristics:

[0031] (1) Improving the digestibility of feed in poultry can increase the daily weight gain of poultry and reduce the feed-to-meat ratio. Taking broilers as an example, the daily weight gain is between 1.37% and 2.48%, and the feed-to-meat ratio is between 1.64 and 1.67.

[0032] (2) Improve the immune function of poultry, enhance their disease resistance, and effectively prevent and treat necrotic enteritis caused by Clostridium perfringens.

[0033] (3) The Bacillus natto FMN-1 of the present invention can produce a relatively good antibacterial effect against Clostridium perfringens. At the same time, a highly efficient protease is prepared for the enzymatic hydrolysis of traditional Chinese medicine. Various enzymes act on the cell walls of plants, which is more conducive to the dissolution of the active ingredients in traditional Chinese medicine.

[0034] 2. The functional feed additive provided by the present invention is easy to prepare, convenient to operate and control, and can be easily industrialized.

[0035] 3. The raw Chinese medicines in the present invention can produce a variety of active substances after mixed enzymatic hydrolysis, which can work together to achieve better effects in animal production applications.

[0036] Artemisia annua is an annual herb belonging to the genus Artemisia of the Asteraceae family. In traditional medicine, it has antiparasitic and antipyretic effects. In addition to the antimalarial ingredient artemisinin, Artemisia annua also contains volatile oils, phenols, flavonoids, as well as amino acids, vitamins, minerals and other nutrients, and has multiple functions such as antioxidant, antibacterial, anti-inflammatory, and immune regulation.

[0037] Artemisia capillaris, also known as cotton artemisia, white wormwood, velvet wormwood, and pine-hair wormwood, is a perennial herb or shrub with a slightly cold nature, a pungent and bitter taste, and the ability to clear dampness and heat. Its main active ingredients are flavonoids and chlorogenic acid. Flavonoids have various biological activities, including anti-allergic, antibacterial, anti-inflammatory, and antioxidant properties.

[0038] Andrographis paniculata, also known as Andrographis paniculata and Andrographis paniculata, is a plant of the Acanthaceae family. It has a bitter taste and cold properties, and possesses heat-clearing and detoxifying, swelling-relieving and analgesic, anti-inflammatory, and antibacterial properties. Andrographis paniculata exhibits varying degrees of sensitivity to both Gram-positive and Gram-negative bacteria, with varying degrees of inhibitory activity against both. The lactones in Andrographis paniculata exhibit significant anti-inflammatory and nonspecific immune-regulating properties.

[0039] Rhubarb is also known as brocade pattern and yellow grain. After enzymatic hydrolysis, its active ingredients are anthraquinone derivatives, of which rhein, emodin, and aloe-emodin possess the strongest antibacterial properties, offering heat-clearing, detoxifying, platelet-increasing, and antibacterial benefits. Its antibacterial properties are strong and broad, encompassing most Gram-positive and Gram-negative bacteria.

[0040] Vitamin C can directly participate in the humoral immunity of the animal body, mainly by promoting the production of antibodies in the animal body through promoting the production of lymphocytes, further improving the immunity of the animal body. In addition, it can improve the immune function of the animal body, participate in the detoxification process of various toxic substances, and play a vital role in the activity of various enzymes in cells.

[0041] Vitamin E cannot generally be synthesized in the body and must be supplied by feed. It can improve the animal's antioxidant capacity, immunity and reproductive performance; it enhances the body's humoral immunity by preventing peroxidation reactions and free radicals from damaging lymphocytes. It can not only enhance humoral immunity, but also improve cellular immunity and the function of neutrophils. After feeding animals with an appropriate amount of vitamin E, the antibody level in the body increases, the number of phagocytes increases, and the phagocytic function is strengthened.

[0042] In the present invention, the Chinese medicine is dried after enzymatic hydrolysis and then mixed with vitamins C, E and natto bacillus and then applied to poultry breeding, which has a significant synergistic improvement effect on the immune function, production performance and prevention and treatment of necrotic enteritis of poultry.

[0043] 4. The Bacillus natto FMN-1 in the present invention can produce a protease with high enzyme activity. It was deposited on September 16, 2020 at the General Microbiology Center of the China Culture Collection Administration (referred to as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), with a preservation number of CGMCC No. 20659 and a classification name of Bacillus natto. DETAILED DESCRIPTION

[0044] A functional feed additive for preventing and treating necrotic enteritis in poultry, the raw materials are composed of the following parts by weight:

[0045] 20-35 parts of traditional Chinese medicine enzymatic hydrolysis mixture, 4-9 parts of natto Bacillus subtilis FMN-1, 4-10 parts of vitamin C, 4-10 parts of vitamin E, and 40-60 parts of talc.

[0046] A preferred embodiment of the present invention is as follows: the Chinese medicine enzymatic hydrolysis mixture is 25-28 parts, natto bacillus is 4-7 parts, vitamin C is 5-8 parts, vitamin E is 5-8 parts, and talc is 40-60 parts.

[0047] As a preferred embodiment of the present invention, the Chinese medicine enzymatic hydrolysis mixture can be 25 parts, 26 parts, 27 parts, or 28 parts; the natto Bacillus subtilis FMN-1 can be 4 parts, 5 parts, 6 parts, or 7 parts; the vitamin C can be 5 parts, 6 parts, 7 parts, or 8 parts; and the vitamin E can be 5 parts, 6 parts, 7 parts, or 8 parts.

[0048] A more preferred embodiment of the present invention is as follows: the Chinese medicine enzymatic hydrolysis mixture is 26 parts, Bacillus natto is 6 parts, vitamin C is 6 parts, vitamin E is 6 parts, and talc is 50 parts.

[0049] The effective components are released after the artemisia, rhubarb, capillaris, and andrographis are enzymolyzed by cellulase, pectinase and protease, and are uniformly mixed with bacillus natto FMN-1, vitamin C and vitamin E, and are applied in the poultry compound feed, and have the effects of antibacterial, antioxidation, and regulating the immune function of the body, and can improve the production performance of animals and prevent and treat necrotic enteritis of poultry.

[0050] In the embodiment of the application, the protease used in the traditional Chinese medicine enzymolysis mixture is produced by bacillus natto, and the bacillus natto is bacillus natto FMN-1 with the preservation number of CGMCC No.20659, and the strain is screened from Japanese natto.

[0051] The screening method of the bacillus natto FMN-1 is as follows:

[0052] The natto is weighed in 0.85% sterile normal saline, and is shaken, and the bacterial suspension is heated in a constant temperature water bath at 80 DEG C for 10 min, and is quickly cooled to room temperature, and the bacterial suspension is the original bacterial liquid.

[0053] The original bacterial liquid is diluted with normal saline to prepare 10 -1 ~10 -8 concentration diluents, 100 μL of the 10 -5 ~10 -8 concentration dilution bacterial suspension is added to the beef extract protein peptone culture medium plate, the diluent is evenly coated, and the plate is placed in a 37 DEG C constant temperature incubator and cultured for 24 h.

[0054] The single colonies with different colony morphologies are picked for gram staining, and are observed under a microscope, and according to the description about bacillus morphology in the common bacterial system identification manual, the strain with bacillus cell morphology is picked, and the bacteriostatic test on clostridium perfringens is carried out, and the strain with strong bacteriostatic effect on clostridium perfringens is repeatedly purified, and the strain shape of the bacillus natto is round, the edge is irregular, the middle is convex, the surface is wrinkled, the color is white, and the shape is opaque and dense.

[0055] The screening method of the strain with strong bacteriostatic effect on clostridium perfringens is as follows:

[0056] The type C clostridium perfringens is recovered on tryptone-sulfite-cycloserine agar culture medium, and a single black colony is picked to liquid sulfite culture medium, and is cultured at 37 DEG C for 10 h in anaerobic conditions. Meanwhile, a test tube is prepared, and is filled with sterilized liquid sulfite culture medium for standby.

[0057] Ten strains of Bacillus natto which have been identified and selected single colony after culture were selected for further screening. Bacillus natto and Clostridium perfringens were added into test tubes containing liquid thioethanolate medium, and cultured at 37℃ for 24h under anaerobic condition. The growth of bacteria was observed by naked eyes. If the solution in the test tube was clear and transparent, it indicated that no bacteria grew. If there were flocculent bacteria or turbidity in the test tube, it indicated that bacteria grew.

[0058] The test tubes which could make the solution clear and transparent were selected. The protease produced by Bacillus natto was a kind of neutral serine protease, which could hydrolyze casein plate to form transparent circle. Single colonies of Bacillus natto which had been identified and screened by bacteriostatic screening were selected and inoculated on casein medium plate for continuous streaking and separation. The plate was placed in a 37℃ constant temperature incubator and cultured for 24h. Strains which produced hydrolysis circle around the colonies and had the largest ratio of hydrolysis circle diameter to colony diameter were selected for further separation and purification to obtain Bacillus natto producing high-activity protease, which was named as Bacillus natto FMN-1, and preserved with the preservation number of CGMCC No.20659.

[0059] The composition of the beef extract proteose peptone medium is as follows: agar powder 20.0g / L, proteose peptone 10.0g / L, beef extract 5.0g / L, sodium chloride 5.0g / L, and the rest is water, with pH value of 7.0-7.2.

[0060] The composition of the trypsin-sulfite-cycloserine agar medium is as follows: trypsin 15.0g / L, soybean peptone 5.0g / L, yeast powder 5.0g / L, sodium pyrosulfite 1.0g / L, ferric ammonium citrate 1.0g / L, 0.5% D-cycloserine solution 10%, agar 15.0g / L, and the rest is water, with pH value of 7.6±0.2.

[0061] The composition of the liquid thioethanolate medium is as follows: trypsin-digested casein peptone 15.0g / L, L-cystine 0.5g / L, glucose 5.0g / L, yeast paste 5.0g / L, sodium chloride 2.5g / L, sodium thioethanolate 0.5g / L, resazurin 0.001g / L, and the rest is water, with pH value of 7.1±0.2.

[0062] The composition of the casein medium is as follows: agar powder 20.0g / L, casein 5.0g / L, glucose 1.0g / L, yeast paste 1.0g / L, dipotassium hydrogen phosphate 1.0g / L, potassium dihydrogen phosphate 0.5g / L, magnesium sulfate 0.1g / L, and the rest is water, with pH value of 7.0-7.2.

[0063] The preparation method of the protease for traditional Chinese medicine enzymolysis mixture is as follows:

[0064] Bacillus natto FMN-1 was inoculated into a beef extract peptone liquid culture medium at a 12% inoculation ratio and cultured at 37°C and 180 rpm for 24 h until the viable count reached (4.0-6.0)×10 8 ~(4.0~6.0)×10 9 cfu / g, inoculated into liquid fermentation medium at a rate of 2%, cultured at 37°C and 180 rpm for 48 h, then the fermentation liquid was taken out and centrifuged at 5000 rpm for 10 min. The supernatant was the crude protease solution.

[0065] The beef extract peptone liquid culture medium comprises: 10.0 g / L peptone, 5.0 g / L beef extract, 5.0 g / L sodium chloride, and the remainder is water, with a pH value of 7.0-7.2.

[0066] The liquid fermentation medium is composed of: 10.0 g / L glucose, 10.0 g / L sucrose, 20.0 g / L soy peptone, 4.0 g / L dipotassium hydrogen phosphate, 2.0 g / L potassium dihydrogen phosphate, 0.2 g / L calcium chloride, 0.5 g / L magnesium sulfate, and the rest is water, with a pH value of 7.0.

[0067] The enzymatic activity characteristics and stability of the protease in the crude enzyme solution produced by Bacillus natto FMN-1 provided by the present invention are shown in Table 1 below:

[0068] Table 1 Stability test of protease activity

[0069]

[0070] The strain was continuously propagated for 10 generations on beef extract peptone medium. The results of protease activity determination in the fermentation broth of each generation of strain after 48 hours showed that the characteristics of the produced protease could still remain stable within 6 generations, reaching 26.23U / mL in the 4th generation, with high enzyme activity and good enzyme production.

[0071] Slowly add ammonium sulfate to the crude enzyme solution until it reaches 25% saturation. Incubate at 4-8°C overnight. Centrifuge at 8000 rpm for 30 minutes. Collect the supernatant and add ammonium sulfate to the supernatant until the final saturation reaches 60%. Incubate at 4-8°C overnight. Centrifuge at 8000 rpm for 30 minutes. Collect the precipitate to remove contaminants and polysaccharides from the supernatant. Redissolve 100 mg of the precipitate in 10 mL of 20 mm PB buffer. Perform gel chromatography and cation exchange chromatography on the reconstituted protein solution.

[0072] To determine the optimal buffer concentration for gel chromatography, 10 mL of the reconstituted protein solution was first subjected to gel chromatography. The chromatography column used was XK 26 / 100, and 10, 20, and 30 mm PB buffer solutions were selected, respectively. The flow rate was 3 mL / min. After the sample was eluted through the gel chromatography column, all filtrates were collected and dried to obtain protease powder. The activity and specific activity of the purified protease are shown in Table 2. It can be concluded that 20 mm PB buffer solution provides the best elution effect for gel chromatography column elution, confirming that the buffer concentration for gel chromatography column elution is 20 mm PB.

[0073] Table 2 Activity of proteases after gel chromatography purification

[0074]

[0075] The filtrate eluted by the gel chromatography column with 20 mm PB buffer solution was collected and further subjected to cation exchange chromatography. The cation exchange chromatography column was Hitrap SP FF. Elution was optimized using 20 mm PB buffer solutions with different pH values, namely 6.5, 7.0, and 7.5. The flow rate was 2 mL / min. After elution, the filtrate was collected and dried to obtain protease powder. The activity and specific activity of the purified protease are shown in Table 3.

[0076] The preparation process of 10mmPB solution is as follows: take 8g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4, and adjust the volume to 1000mL.

[0077] The preparation process of 20mmPB solution is as follows: take 16g NaCl, 0.4g KCl, 2.88g Na2HPO4, 0.48g KH2PO4, and adjust the volume to 1000mL.

[0078] The preparation process of 30mmPB solution is as follows: take 24g NaCl, 0.6g KCl, 4.32g Na2HPO4, 0.72g KH2PO4, and adjust the volume to 1000mL.

[0079] The preparation process of 20mmPB solution with different pH values ​​is as follows: take 16g NaCl, 0.4g KCl, 2.88g Na2HPO4, and 0.48g KH2PO4, dilute to 1000mL, prepare three portions respectively, and then adjust the pH values ​​to 6.5, 7.0, and 7.5 with sodium hydroxide and glacial acetic acid.

[0080] Table 3 Activity of protease after cationic chromatography purification

[0081]

[0082] In gel chromatography, elution using different concentrations of PB buffer solutions revealed that 20 mm PB buffer provided the best elution results. In cation exchange chromatography, elution using 20 mm PB buffer solutions at different pH values ​​revealed that the pH of 7.0 in 20 mm PB buffer provided the most optimal results for total protein quality, protease activity, and specific activity.

[0083] The final gel chromatography and cation exchange chromatography were determined as follows: 10 mL of the above-mentioned re-dissolved protein solution was taken for gel chromatography, and the chromatography column selected was XK 26 / 100, 20 mm buffer solution, with a flow rate of 3 mL / min. After the sample was eluted through the gel chromatography column, the filtrate was collected, and then the collected filtrate was further subjected to cation exchange chromatography. The cation exchange chromatography column was Hitrap SP FF, and the elution was optimized with 20 mm PB buffer solution with a pH value of 7.0. The flow rate was 2 mL / min. After elution, the filtrate was collected and dried to obtain protease powder.

[0084] This protease is stable within the pH range of 5.0-11.0; after 6-7 hours at 50°C, it retains 20% residual activity. After 48 hours at 37°C, it maintains approximately 80% activity. Therefore, when preparing enzymatic hydrolysis mixtures of traditional Chinese medicines, even at a hydrolysis temperature of 40°C, this protease can still effectively decompose proteins. Working in synergy with cellulase and pectinase, it can accelerate the enzymatic hydrolysis and filtration of traditional Chinese medicines and increase the concentration of effective substances in the medicines.

[0085] The functional additive for preventing and treating necrotic enteritis in poultry is obtained by uniformly mixing a mixture of traditional Chinese medicine enzymatic hydrolysis and vitamins C and E under the effective antibacterial effect of Bacillus natto.

[0086] The preparation method of the Chinese medicine enzymatic hydrolysis mixture is as follows:

[0087] 30-40 parts of Artemisia annua, 15-25 parts of Rhubarb, 20-30 parts of Artemisia capillaris, 25-35 parts of Andrographis paniculata, 4-10 parts of pectinase, 4-10 parts of cellulase, and 3-8 parts of protease are uniformly mixed, and 40% of the total weight of the mixture is added with water for enzymatic hydrolysis at a temperature of 40°C and a pH of 6.0 for 48 hours. After enzymatic hydrolysis, the mixture is dried and pulverized through a 40-mesh sieve for later use;

[0088] The protease is produced by Bacillus natto, and the specific preparation method is as described above;

[0089] Furthermore, the Chinese medicine enzymatic hydrolysis mixture is composed of the following parts by weight: 30-35 parts of Artemisia annua, 15-20 parts of Rhubarb, 20-25 parts of Artemisia capillaris, 25-30 parts of Andrographis paniculata, 4-8 parts of pectinase, 4-8 parts of cellulase, and 3-6 parts of protease.

[0090] The added amount of the functional feed additive in the basic diet is 0.2-0.5‰ (mass ratio).

[0091] Example 1

[0092] A functional feed additive for preventing and treating necrotic enteritis in poultry is prepared by a method comprising the following steps:

[0093] 1. Preparation of Chinese medicine enzymatic hydrolysis mixture

[0094] 30 parts of Artemisia annua, 15 parts of Rhubarb, 20 parts of Artemisia capillaris, 25 parts of Andrographis paniculata, 4 parts of pectinase, 4 parts of cellulase, and 3 parts of protease are uniformly mixed, and 40% of the total weight of the mixture is added with water for enzymatic hydrolysis at 40°C and pH 6.0 for 48 hours. After enzymatic hydrolysis, the mixture is dried and pulverized through a 40-mesh sieve for later use;

[0095] 2. Preparation of Bacillus natto

[0096] Bacillus natto FMN-1 (CGMCC No. 20659) was inoculated into a beef extract peptone liquid culture medium and incubated at 37°C for 24 h for activation. The viable bacterial count of the activated Bacillus natto FMN-1 seed liquid was (2.0-5.0)×10 8 CFU / mL-(2.0~5.0)×10 9 CFU / mL. In order to be applied to large-scale production applications, it is necessary to expand the culture. The seed liquid is inoculated into the liquid medium of beef extract peptone at a 1% inoculum volume. The temperature of the expanded culture is 37°C and the culture is static for 24 hours. The bacterial activity in the bacterial liquid is 5.2×10 9 CFU / ml.

[0097] The beef extract peptone liquid culture medium is composed of: 10.0 g / L peptone, 5.0 g / L beef extract, 5.0 g / L sodium chloride, and the rest is water.

[0098] 3. Mix

[0099] 25 parts of the traditional Chinese medicine enzymatic hydrolysis mixture, 4 parts of Bacillus natto, 5 parts of vitamin C, 5 parts of vitamin E and 60 parts of talc are uniformly mixed to obtain the product.

[0100] The functional feed additive can be used to prevent and treat necrotic enteritis in poultry, and the addition amount in the basic diet is 0.3‰ by mass.

[0101] Example 2

[0102] 1. Preparation of Chinese medicine enzymatic hydrolysis mixture

[0103] 33 parts of Artemisia annua, 18 parts of rhubarb, 22 parts of capillaris, 27 parts of andrographis paniculata, 6 parts of pectinase, 6 parts of cellulase and 4 parts of protease are uniformly mixed, and 40% of water of the total weight of the mixture is added for enzymolysis. The enzymolysis temperature is 40°C, the pH value is 6.0, and the hydrolysis is carried out for 48 hours. After enzymolysis, it is dried and pulverized through a 40-mesh sieve for later use.

[0104] 2. Preparation of Bacillus natto is the same as in Example 1

[0105] 3. Mix

[0106] 26 parts of the traditional Chinese medicine enzymatic hydrolysis mixture, 6 parts of natto bacillus, 6 parts of vitamin C, 6 parts of vitamin E and 50 parts of talc are uniformly mixed to obtain a product.

[0107] The functional feed additive can be used to prevent and treat necrotic enteritis in poultry, and the addition amount in the basic diet is 0.3‰ by mass.

[0108] Example 3

[0109] 1. Preparation of Chinese medicine enzymatic hydrolysis mixture

[0110] 35 parts of Artemisia annua, 20 parts of rhubarb, 25 parts of capillaris, 30 parts of andrographis paniculata, 8 parts of pectinase, 8 parts of cellulase and 6 parts of protease are uniformly mixed, and 40% of water of the total weight of the mixture is added for enzymolysis. The enzymolysis temperature is 40°C, the pH value is 6.0, and the hydrolysis is carried out for 48 hours. After enzymolysis, it is dried and pulverized through a 40-mesh sieve for later use.

[0111] 2. Preparation of Bacillus natto is the same as in Example 1

[0112] 3. Mix

[0113] 28 parts of the traditional Chinese medicine enzymatic hydrolysis mixture, 7 parts of Bacillus natto, 8 parts of vitamin C, 8 parts of vitamin E and 40 parts of talc are uniformly mixed to obtain a product.

[0114] The functional feed additive can be used to prevent and treat necrotic enteritis in poultry, and the addition amount in the basic diet is 0.3‰ by mass.

[0115] Example 4

[0116] 1. Preparation of Chinese medicine enzymatic hydrolysis mixture

[0117] 38 parts of Artemisia annua, 23 parts of rhubarb, 28 parts of capillaries, 32 parts of andrographis paniculata, 9 parts of pectinase, 9 parts of cellulase and 7 parts of protease are uniformly mixed, and 40% of water of the total weight of the mixture is added for enzymolysis. The enzymolysis temperature is 40°C, the pH value is 6.0, and the hydrolysis is carried out for 48 hours. After enzymolysis, it is dried and pulverized through a 40-mesh sieve for later use.

[0118] 2. Preparation of Bacillus natto is the same as in Example 1

[0119] 3. Mixing

[0120] Mixing 20 parts of the Chinese medicine enzymatic mixture, 4 parts of Bacillus natto, 4 parts of vitamin C, 4 parts of vitamin E, and 40 parts of talc to obtain a mixture.

[0121] The functional feed additive can be used for preventing and treating necrotic enteritis of poultry, and the addition amount in the basic daily ration is 0.2 ‰ mass ratio.

[0122] Example 5

[0123] 1. Preparation of the Chinese medicine enzymatic mixture

[0124] Mixing 40 parts of Artemisia annua, 25 parts of Rheum officinale, 30 parts of Herba Artemisiae Scopariae, 35 parts of Andrographis paniculata and 10 parts of pectinase, 10 parts of cellulase and 8 parts of protease, adding 40% of water of the total weight of the mixture to perform enzymatic hydrolysis, the enzymatic hydrolysis temperature is 40℃, the pH value is 6.0, the hydrolysis time is 48 h, and after the enzymatic hydrolysis, drying treatment is performed, and the mixture is crushed to pass through a 40-mesh sieve for use.

[0125] 2. Preparation of Bacillus natto, which is the same as in Example 1

[0126] 3. Mixing

[0127] Mixing 35 parts of the Chinese medicine enzymatic mixture, 9 parts of Bacillus natto, 10 parts of vitamin C, 10 parts of vitamin E and 60 parts of talc to obtain a mixture.

[0128] The functional feed additive can be used for preventing and treating necrotic enteritis of poultry, and the addition amount in the basic daily ration is 0.5 ‰ mass ratio.

[0129] Experimental Example 1: Application of the functional feed additive in feed

[0130] In poultry, the incidence of necrotic enteritis is the highest in broilers, so the test animals in the present application are all broilers. 480 white-feather broilers of 28 days old are randomly divided into 4 groups, 12 replicates per group, 10 broilers per replicate (1 / 2 male and 1 / 2 female). The control group is fed with a corn-soybean meal type daily ration; the test groups are added with the feed additive of the present application on the basis of the control group, and the test groups 1, 2 and 3 are added with the feed additives of Examples 1, 2 and 3 respectively, and the addition amount is 0.3 ‰ (mass percentage). The composition and nutritional level of the test feed are shown in Table 4.

[0131] Table 4: Feed formula and nutritional level of broilers

[0132]

[0133] Note: The nutritional levels of the diets fed to the control group and the experimental group were the same. All experimental feeds and feed additives in the present invention were provided by Shenyang Fengmei Biotechnology Co., Ltd.

[0134] The trial was conducted at a broiler chicken farm in Liaoning Province. Broilers were housed in multi-layered cages within a single shed, with free access to feed and water. Vaccinations and pen disinfection were performed as per standard procedures. The trial lasted 15 days. Feed intake and residual feed were recorded for each replicate group during the trial, from which average daily gain, daily feed intake, and feed-to-gain ratio were calculated.

[0135] Table 5 Effect of the functional feed additive of the present invention on the production performance of broiler chickens

[0136]

[0137] The weight, feed intake, and feed-to-meat ratio of each group of experimental broilers are shown in Table 5. Compared with the experimental results of the control group, the daily weight gain of experimental groups 1, 2, and 3 increased by 1.37%, 2.48%, and 1.40% respectively compared with the control group, and the feed-to-meat ratio decreased by 1.01%, 2.20%, and 0.95% respectively compared with the control group. The results were significantly different from those of the control group (P<0.01). The effect of the present invention is reflected in that: on the one hand, after the combined enzyme hydrolyzes the traditional Chinese medicine, the effective ingredients in the traditional Chinese medicine are decomposed to the greatest extent. These effective ingredients have high biological activity functions, can help improve the health of poultry, promote the growth of poultry, increase daily weight gain, and reduce the feed-to-meat ratio. On the other hand, the natto Bacillus FMN-1 in the present invention can produce protease, which can degrade anti-nutritional factors in feed, improve the utilization rate of feed, and also correspondingly supplement the deficiency of endogenous enzymes in the animal body, improve the digestibility and utilization rate of feed in animals, and thus improve the production performance of animals.

[0138] Experimental Example 2 Application test of functional feed additives in animals

[0139] In order to illustrate the effect of the functional feed additive of the present invention on the immune organ index of broiler chickens, immune organ index = immune organ weight (mg) / body weight (g), 80 28-day-old white-feathered broiler chickens were selected for each group and the experiment was divided into the following groups:

[0140] Control group: 80 healthy white broilers were selected and fed a corn-soybean meal diet (composition is the same as in Table 4).

[0141] Infection group: 80 white-feathered broilers with symptoms of necrotic enteritis were selected and fed a corn-soybean meal diet (composition is the same as Table 4).

[0142] Antibiotic group: 80 white-feathered broilers with symptoms of necrotic enteritis were selected and fed a corn-soybean meal diet (composition as shown in Table 4) with bacitracin zinc added at a concentration of 0.3‰ (mass ratio).

[0143] Functional additive group: 80 white-feathered broilers with symptoms of necrotic enteritis were selected and fed a corn-soybean meal diet (composition is the same as in Table 4) with functional feed additives added at a concentration of 0.3‰ (mass ratio).

[0144] The functional feed additive of Example 2 was used in the experiment.

[0145] After 15 days of feeding, the chickens in each experimental group were weighed, and then the liver, spleen, bursa of Fabricius, pancreas and thymus were taken and weighed, and the index of each organ was calculated. The results are shown in Table 6.

[0146] Table 6: Effects of the functional feed additive of the present invention on broiler immune organ indexes

[0147]

[0148] As shown in Table 6, the bursa of Fabricius, liver index, spleen index, and thymus index were all increased in the antibiotic and functional feed additive groups compared to the control and infection groups. The antibiotic and additive groups showed significant increases compared to the control group (P < 0.05). The liver, spleen, bursa of Fabricius, and thymus indexes were significantly higher in the additive and antibiotic groups than in the control and infection groups (P < 0.01). The bursa of Fabricius is a unique and crucial central immune organ in poultry. It primarily induces bone marrow stem cells to differentiate into B cells, playing a crucial role in the formation and function of humoral immunity. The thymus, a primary lymphoid organ in poultry, participates in cellular immunity. The spleen is a key peripheral immune organ. Both the antibiotic and functional feed additive groups significantly increased the immune organ indices of broiler chickens. The functional feed additive group showed significantly higher liver, bursa of Fabricius, and spleen indices than the antibiotic group, with no significant differences (P > 0.05). Under normal physiological conditions, immune organs refer to organs or tissues that carry out immune functions and are the sites where immune-active cells develop and proliferate. The functional status and developmental state of immune organs directly determine the overall immune level of poultry. The growth of immune organs is a sign of rapid growth and development, while an increase in the immune organ index indicates a faster maturation of the immune system. The faster the growth, development, and maturation of immune organs, the stronger the bird's overall immune function, and its ability to resist various pathogenic microorganisms and various stresses is also improved. Therefore, measuring the weight of immune organs is one of the main methods for studying the body's immune status. The spleen, thymus, bursa of Fabricius, and liver are the main immune organs of chickens, and their weights can be used to assess the immune status of poultry. The above data fully demonstrate that the functional feed additive of the present invention can significantly improve the immune organ index of broiler chickens.

[0149] Experimental Example 3 Application test of functional feed additives in animals

[0150] In order to illustrate the effects of using common Bacillus natto and the purified Bacillus natto FMN-1 (CGMCC No. 20659) in functional feed additives on the intestinal flora of broiler chickens, the experiment was divided into the following groups, each group using chickens infected with necrotic enteritis as the test subjects:

[0151] Control group: fed a corn-soybean meal diet (composition same as in Table 4).

[0152] Experimental group 1: fed with corn-soybean meal diet (composition is the same as in Table 4) + 0.3‰ (mass ratio) additive combination (without Bacillus neri).

[0153] The preparation method of the additive combination is as follows:

[0154] (1) Preparation of the Chinese medicine enzymatic hydrolysis mixture is the same as step (1) of Example 2 of the present invention

[0155] (2) 26 parts of the Chinese herbal medicine enzymatic hydrolysis mixture, 6 parts of vitamin C, 6 parts of vitamin E, and 50 parts of talcum powder were uniformly mixed to obtain a product.

[0156] Experimental group 2: fed a corn-soybean meal diet (same composition as in Table 4) + 0.3‰ (mass ratio) additive combination (Bacillus natto, extracted from natto, provided by the Microbiology Laboratory of Shenyang Fengmei Biotechnology Co., Ltd., an unpurified but identified strain).

[0157] The preparation method of the additive combination is as follows:

[0158] (1) Preparation of Bacillus natto:

[0159] Weigh natto in 0.85% sterile saline, shake, heat the bacterial suspension in a constant temperature water bath at 80°C for 10 minutes, and then quickly cool to room temperature. This bacterial suspension is the original bacterial solution.

[0160] The original bacterial solution was diluted with physiological saline to prepare 10 -1 ~10 -8 Concentration of dilution, take 10 -8 , 10 -7 , 10 -6 , 10 -5 100 μL of each diluted bacterial suspension of different concentrations were added to the beef extract peptone medium plate, the diluted liquid was evenly spread, and the above plates were placed in a 37°C constant temperature incubator for inversion culture for 24 hours.

[0161] Single colonies with different colony morphologies were picked for Gram staining and observed under a microscope. According to the description of Bacillus morphology in the common bacterial system identification manual, strains with Bacillus cell morphology were picked and purified to obtain pure strains for preservation.

[0162] The activation step of Bacillus subtilis natto was prepared in the same manner as in Example 2 (2).

[0163] (2) Raw materials: the composition is the same as step (1) in Example 2 of the present invention

[0164] (3) Mixing, same as step (3) in Example 2 of the present invention.

[0165] Experimental group 3: fed with corn-soybean meal type diet (composition is the same as Table 4) + 0.3‰ (mass ratio) of the functional feed additive combination of Example 2.

[0166] After the experiment, the feces of the chickens in the control group and each experimental group were taken respectively, and a certain amount of chicken feces samples were weighed and diluted with sterile water. 0.1 mL of three appropriate gradient dilution solutions were taken and evenly spread on lactic acid plate culture medium, Escherichia coli plate culture medium and tryptone-sulfite-cycloserine agar culture medium, respectively. The dilutions were placed in an incubator at 37°C and inverted for 24 to 48 hours. The total number of viable lactic acid bacteria, Escherichia coli and Clostridium perfringens were calculated respectively. The results are shown in Table 7.

[0167] Table 7 Effect of the functional feed additive of the present invention on the number of intestinal flora in broiler chickens

[0168]

[0169] As can be seen from the result, test group 3 can significantly reduce the quantity of Clostridium perfringens and Escherichia coli in broiler intestinal tract, and has increased the viable bacteria count of lactobacillus, compared with the result of test group 2, the data of test group 3 still have very obvious advantages, explanation: the bacillus natto FMN-1 limited by the present invention and common bacillus natto, in the effect of improving broiler intestinal flora structure, have significant differences. Bacillus natto can regulate the microbial circulation in animal intestinal tract, because the beneficial bacteria and pathogens in animal intestinal tract have a competitive relationship, in animal intestinal tract, lactobacillus can compete with some pathogens to utilize the nutrients in animal intestinal tract, and compete for common adhesion sites in intestinal epithelium and mucosa, lactobacillus can also reduce the pH value of intestinal tract by the product such as organic acids secreted by itself simultaneously, so also can suppress the growth of some pathogens, can reach good inhibitory effect to Clostridium perfringens, also can suppress other pathogenic bacteria simultaneously, therefore improve broiler intestinal flora structure, just equal to improving the intestinal tract of broiler. Feed additive of the present invention has obvious advantages in this respect.

[0170] Experimental Example 4 Application test of functional feed additives in animals

[0171] In order to illustrate the effect of this functional feed additive on the prevention and treatment of necrotic enteritis in broiler chickens, the incidence, mortality and cure rate of necrotic enteritis in the chickens were recorded respectively, and the experiment was divided into the following groups:

[0172] Control group: fed a corn-soybean meal diet (composition is the same as in Table 4)

[0173] Experimental group 1: fed a corn-soybean meal diet (same composition as in Table 4). If antibiotics (bacitracin zinc) were added for treatment after the disease, the added amount was 0.3‰ (mass ratio).

[0174] Experimental Group 2: fed a corn-soybean meal diet (same composition as in Table 4). If additional functional feed additives were added to the formula after the onset of the disease,

[0175] The preparation of the functional feed additive combination is the same as in Example 2, and the addition amount is 0.3‰ (mass ratio).

[0176] Experimental group 3: fed with corn-soybean meal diet (composition is the same as in Table 4) + 0.3‰ (mass ratio) functional feed additives.

[0177] The preparation of the functional feed additive combination is the same as that in Example 2.

[0178] Experimental group 4: fed with corn-soybean meal diet (composition is the same as in Table 4) + 0.3‰ (mass ratio) functional feed additives.

[0179] The preparation of the functional feed additive combination is as follows:

[0180] (1) 33 parts of Artemisia annua, 18 parts of Rhubarb, 22 parts of Artemisia capillaris, 27 parts of Andrographis paniculata, 6 parts of pectinase, 6 parts of cellulase, and 4 parts of protease (purchased from Xingtai Sibet Biotechnology Co., Ltd., enzyme activity 50,000 U / g) were uniformly mixed, and 40% of the total weight of the mixture was added with water for enzymolysis. The enzymolysis temperature was 40°C, the pH value was 6.0, and the hydrolysis was carried out for 48 hours. After enzymolysis, the mixture was dried and crushed through a 40-mesh sieve for later use.

[0181] (2) The activation step of Bacillus subtilis natto is the same as that of Example 2 (2).

[0182] (3) Mix (1) (2) and other raw materials in the same manner as step (3) of Example 2 of the present invention.

[0183] Table 8 The preventive and therapeutic effects of the functional feed additives of the present invention on necrotic enteritis in broiler chickens

[0184]

[0185] The morbidity and mortality rates of test group 3 were lower than those of the other groups, with significant differences. At the same time, the cure rate was also higher than that of the control group, test group 2, and test group 4, and there was no significant difference in the cure rate with the antibiotic group. This shows that the functional feed additive of the present invention has a good preventive and curative effect on necrotic enteritis in broilers, and is safer and more stable than antibiotics, and does not have the problem of drug residues and drug resistance. At the same time, the effect of test group 4 is higher than that of the control group, but lower than that of test group 3, which shows that when the Chinese medicine combination is enzymatically hydrolyzed, the enzyme produced by the preserved strains of the present application has a very obvious advantage, and more biologically active substances will be released during the enzymatic hydrolysis of the Chinese medicine, and synergistically with other raw materials such as Bacillus natto, and can be applied to animal breeding, especially the prevention and treatment of necrotic enteritis in broilers, to achieve more significant effects. The fact that test group 3 is significantly better than test group 2 also shows that the prevention and treatment effect of the functional feed additive of the present invention is significant, far better than the therapeutic effect after the onset of the disease. Currently, the use of antibiotics in feed is prohibited, so breeding terminals add additional antibiotics after animals become ill. In this way, drug residue problems and drug resistance gradually emerge. Therefore, the present invention enzymatically hydrolyzes traditional Chinese medicine, screens strains in a targeted manner and comprehensively utilizes them, which can effectively treat and prevent diseases while also eliminating the problems of drug resistance and drug residues.

[0186] The effects of the present invention are as follows: On the one hand, Bacillus natto has significant antibacterial effects and can produce a variety of antibiotics, such as bacitracin and polymyxin, which have broad-spectrum antibacterial effects and certain antibacterial activity against various bacteria, including Gram-positive and Gram-negative bacteria. It can also promote the growth of beneficial bacteria, which can compete with some pathogens to utilize nutrients in the animal's intestines, thereby maintaining the animal's intestinal health. On the other hand, after the Chinese medicine composition of the present invention is hydrolyzed by a specific enzyme complex, it can better release the biologically active substances in the Chinese medicine, thereby exerting excellent antioxidant, anti-inflammatory, and antibacterial effects in the animal. Furthermore, the present invention also contains additional vitamins C and E, both of which have excellent antioxidant and immune-enhancing functions. These factors work synergistically, effectively regulating the intestinal health of animals. At the same time, combined with the effects of the Chinese medicine combination and the effects of the biologically active substances in the Chinese medicine after enzymatic hydrolysis on the animal, it can achieve a significant antibacterial effect and enhance the body's immune function, showing a good preventive and therapeutic effect against necrotic enteritis caused by Clostridium perfringens in poultry.

[0187] It should be noted that: in order to unify the experimental conditions, although the feed additive of Example 2 was used in all experimental examples, the additives prepared in other examples can also achieve comparable effects, but the effect of Example 2 is more prominent.

[0188] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments can still be modified, or some or all of the technical features therein can be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a functional feed additive for preventing and treating necrotic enteritis in poultry, comprising uniformly mixing the feed additive raw materials according to a certain proportion; The weight parts of the feed additive raw materials are as follows: 20-35 parts of Chinese herbal medicine enzymatic hydrolysis mixture, 4-9 parts of Bacillus natto, 4-10 parts of vitamin C, 4-10 parts of vitamin E, and 40-60 parts of talc; It is characterized in that The Bacillus natto ( Bacillus natto ) The strain deposit number is CGMCC No.20659; The preparation method of the Chinese medicine enzymatic hydrolysis mixture is as follows: 30-40 parts of Artemisia annua, 15-25 parts of Rhubarb, 20-30 parts of Artemisia capillaris, 25-35 parts of Andrographis paniculata, 4-10 parts of pectinase, 4-10 parts of cellulase, and 3-8 parts of protease are uniformly mixed, and 40% of the total weight of the mixture is added with water for enzymatic hydrolysis at a temperature of 40° C. and a pH of 6.0 for 48 hours. After enzymatic hydrolysis, the mixture is dried and pulverized through a 40-mesh sieve for later use; The protease is prepared from Bacillus natto CGMCC No.20659.

2. The method for preparing the feed additive according to claim 1, wherein The feed additive raw materials are composed of the following parts by weight: 25-28 parts of a traditional Chinese medicine enzymatic hydrolysis mixture, 4-7 parts of natto bacillus subtilis, 5-8 parts of vitamin C, 5-8 parts of vitamin E, and 40-60 parts of talc.

3. The method for preparing the feed additive according to claim 1, wherein The feed additive raw materials are composed of the following components in parts by weight: 26 parts of a traditional Chinese medicine enzymatic hydrolysis mixture, 6 parts of natto bacillus subtilis, 6 parts of vitamin C, 6 parts of vitamin E, and 50 parts of talc.

4. The method for preparing the feed additive according to any one of claims 1 to 3, characterized in that: The traditional Chinese medicine enzymatic hydrolysis mixture is composed of the following parts by weight: 30-35 parts of Artemisia annua, 15-20 parts of Rhubarb, 20-25 parts of Artemisia capillaris, 25-30 parts of Andrographis paniculata, 4-8 parts of pectinase, 4-8 parts of cellulase and 3-6 parts of protease.

5. The method for preparing the feed additive according to any one of claims 1 to 3, characterized in that: The traditional Chinese medicine enzymatic hydrolysis mixture is composed of the following parts by weight: 30 parts of Artemisia annua, 15 parts of Rhubarb, 20 parts of Artemisia capillaris, 25 parts of Andrographis paniculata, 4 parts of pectinase, 4 parts of cellulase, and 3 parts of protease.

6. The method for preparing the feed additive according to any one of claims 1 to 3, characterized in that: The traditional Chinese medicine enzymatic hydrolysis mixture is composed of the following parts by weight: 33 parts of Artemisia annua, 18 parts of Rhubarb, 22 parts of Artemisia capillaris, 27 parts of Andrographis paniculata, 6 parts of pectinase, 6 parts of cellulase, and 4 parts of protease.

7. The method for preparing the feed additive according to any one of claims 1 to 3, characterized in that: The traditional Chinese medicine enzymatic hydrolysis mixture is composed of the following parts by weight: 35 parts of Artemisia annua, 20 parts of Rhubarb, 25 parts of Artemisia capillaris, 30 parts of Andrographis paniculata, 8 parts of pectinase, 8 parts of cellulase, and 6 parts of protease.

8. The method for preparing the feed additive according to any one of claims 1 to 3, characterized in that: The preparation method of the protease comprises the following steps: Bacillus natto was inoculated into a beef extract peptone liquid culture medium at a 12% inoculation ratio and cultured at 37°C and 180 rpm for 24 h until the viable count reached 4.0-6.0 × 10 8 ~4.0~6.0×10 9 cfu / g, inoculated into liquid fermentation medium at 2% inoculum, cultured at 37℃ and 180r / min for 48h, then the fermentation liquid was collected and centrifuged at 5000r / min for 10min. The supernatant was the crude protease solution; Ammonium sulfate was slowly added to the above crude enzyme solution until the saturation reached 25%, the mixture was incubated at 4-8°C overnight, and the supernatant was collected by centrifugation at 8000 rpm for 30 min. Ammonium sulfate was added to the supernatant until the final saturation reached 60%, the mixture was incubated at 4-8°C overnight, and the supernatant was collected by centrifugation at 8000 rpm for 30 min. 100 mg of the precipitate was redissolved with 10 mL of 20 mm PB buffer solution and then subjected to gel chromatography using an XK 26 / 100 column and a 20 mm PB buffer solution at a flow rate of 3 mL / min. The collected filtrate was then subjected to cation exchange chromatography using a Hitrap SP FF cation exchange column optimized for elution using a 20 mm PB buffer solution at pH 7.0 at a flow rate of 2 mL / min. After elution, the filtrate was collected and dried to obtain protease powder.

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