Feed additive capable of replacing fish meal as well as preparation method and application of feed additive

Abstract

The invention relates to the field of animal breeding, in particular to a feed additive capable of replacing fish meal as well as a preparation method and application of the feed additive. The invention provides a feed additive capable of replacing fish meal. The feed additive comprises the following raw materials: a fermentation base material, a microbial agent and an enzyme preparation, the fermentation base material comprises corn protein powder, soybean meal and corn germ meal; on the basis of the mass of the fermentation base material, the inoculation amount of the microbial inoculum is 3-7%, and the dosage of the enzyme preparation is 20-140U / g. The content of soluble protein and polypeptide in the feed additive is high, and 10-30% of fish meal in conventional animal feed can be replaced.

Description

Technical Field

[0001] The invention relates to the field of animal breeding, and in particular to a feed additive capable of replacing fish meal, and a preparation method and application thereof. Background Art

[0002] With the rapid development of pig farming in my country, early weaning technology has been widely promoted. At present, most pig farms adopt weaning at 21 days of age. At this time, the intestinal and immune systems of piglets are not yet fully developed. At the same time, changes in nutrition sources and living environment will also cause piglets to have diarrhea, decreased feed intake, slow growth and even death and other stress symptoms, which are extremely detrimental to the growth and health of piglets. Studies have shown that when the nutrition source of piglets is mainly high-fiber plant raw materials, the villi of the small intestine of piglets are easily worn, causing the villi of the small intestine to atrophy and the crypts to proliferate, thereby reducing the absorption area of ​​the small intestine for nutrients, resulting in a significant decrease in the absorption function of the piglet's digestive tract. Therefore, the feed for weaned piglets needs to meet the characteristics of high nutritional requirements and easy digestion. At present, the feed for weaned piglets is mainly based on the addition of fish meal. This kind of high-quality high-protein feed that is easy to digest and absorb can effectively shorten the stress period of piglets and quickly enter the growth period.

[0003] my country has a high corn production, with a national corn production of 272 million tons, of which 25% is processed. Every 1,000 tons of corn processed produces 60 tons of corn gluten meal, and the by-product output is huge. Corn gluten meal is currently used directly as feed. Although the protein content in corn gluten meal is high, it is mainly alcohol-soluble protein and gluten with poor water solubility, and the digestibility is low, which limits its large-scale application in animal feed. Therefore, it is particularly important to find a feed that can make full use of corn gluten meal. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a feed additive that can replace fish meal, and its preparation method and application. The feed additive of the present invention has high soluble protein and polypeptide content and can replace 10-30% of fish meal in conventional animal feed.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a feed additive capable of replacing fish meal, comprising the following raw materials: fermentation base material, bacterial agent and enzyme;

[0007] The fermentation base material includes corn gluten meal, soybean meal and corn germ meal;

[0008] Based on the mass of the fermentation base material, the inoculation amount of the bacterial agent is 3-7%, and the dosage of the enzyme preparation is 20U / g-140U / g.

[0009] Preferably, the mass ratio of the corn gluten meal, soybean meal and corn germ meal is 5:3:2.

[0010] Preferably, the bacterial agent comprises Bacillus subtilis YY-10, Saccharomyces cerevisiae YY-14 and Lactobacillus plantarum YY-8; the concentration of Bacillus subtilis YY-10 in the bacterial agent reaches 10 7 ~10 8 cfu / g, the concentration of Saccharomyces cerevisiae YY-14 reached 10 7 ~10 8 cfu / g, the concentration of Lactobacillus plantarum YY-8 reached 10 7 ~10 8 cfu / g;

[0011] The deposit number of the Bacillus subtilis YY-10 is CGMCC No.24694;

[0012] The deposit number of the Saccharomyces cerevisiae YY-14 is CGMCC No.24698;

[0013] The deposit number of Lactobacillus plantarum YY-8 is CGMCC No.24692.

[0014] Preferably, the enzyme preparation is a protease.

[0015] The present invention provides a method for preparing the feed additive described in the above technical solution, comprising the following steps:

[0016] After mixing the bacterial agent with water, a diluted bacterial agent is obtained;

[0017] The fermentation base material, diluted bacterial agent and enzyme preparation are mixed and fermented to obtain feed additives.

[0018] Preferably, the fermentation time is 1 to 5 days and the temperature is 20°C to 40°C.

[0019] Preferably, the mass volume ratio of the fermentation base material to the diluted bacterial agent is 1 g: 0.3-1.1 mL.

[0020] The present invention provides application of the feed additive described in the above technical solution in preparing animal feed.

[0021] The present invention also provides the use of the feed additive described in the above technical solution in the preparation of products that improve animal production performance and / or immunity

[0022] Preferably, the animal comprises a mouse and / or a piglet.

[0023] Beneficial effects:

[0024] The present invention provides a feed additive that can replace fish meal, comprising the following raw materials: fermentation base, bacterial agent and enzyme; the fermentation base comprises corn protein powder, soybean meal and corn germ meal; based on the mass of the fermentation base, the inoculation amount of the bacterial agent is 3-7%, and the dosage of the enzyme preparation is 20U / g-140U / g. The feed additive of the present invention has high soluble protein and polypeptide content. The feed additive is used to replace part of the fish meal in the animal diet, which can improve the growth performance of weaned animals. As the animal age increases, the replacement amount gradually increases, and it has a good alleviating effect on DSS-induced animal ulcerative colitis. The feed additive of the present invention can improve the production performance and immune ability of animals in animal breeding. The feed additive of the present invention is used in the feeding of UC rats, which can significantly improve the various pathological indicators, immune function and intestinal morphology of UC rats, and reduce the oxidative stress level and intestinal permeability of the rat body.

[0025] Furthermore, the feed of the present invention can effectively alleviate the weaning stress response of animals in the early stage of weaning, thereby achieving the effect of protecting the intestinal barrier function.

[0026] The present invention uses corn protein powder, soybean meal and corn germ meal as raw materials for bacterial enzyme synergistic fermentation, and obtains a feed additive with high soluble protein and polypeptide content that can replace fish meal by selecting and optimizing fermentation conditions. Compared with unfermented feed additives, the feed additive of the present invention has increased soluble protein and polypeptide content, good nutritional safety, reduced crude fiber content, and increased viable bacteria count. The method provided by the present invention is simple and feasible. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0028] Figure 1 Representative pictures of rat colon; among them, the 10% fish meal replacement group is animal feed group 1, the 20% fish meal replacement group is animal feed group 2, and the 30% fish meal replacement group is animal feed group 3.

[0029] Biological deposit information

[0030] Bacillus subtilis YY-10, biologically classified as Bacillus subtilis, is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 18, 2022, and the deposit number is CGMCC No. 24694.

[0031] Brewer's yeast YY-14, biologically classified as Saccharomyces cerevisiae, is deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 18, 2022, and the deposit number is CGMCC No. 24698.

[0032] Lactobacillus plantarum YY-8, biologically classified as Lactobacillus plantarum, is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 18, 2022, and the deposit number is CGMCC No. 24692. DETAILED DESCRIPTION

[0033] Unless otherwise specified, the components used in the present invention can be purchased by those skilled in the art.

[0034] The invention provides a feed additive capable of replacing fish meal, comprising the following raw materials: a fermentation base material, a bacterial agent and an enzyme; the fermentation base material comprises corn protein powder, soybean meal and corn germ meal; based on the mass of the fermentation base material, the inoculation amount of the bacterial agent is 3-7%, and the dosage of the enzyme preparation is 20U / g-140U / g.

[0035] In the present invention, the mass ratio of the corn gluten meal, soybean meal and corn germ meal is preferably 5:3:2.

[0036] In the present invention, the inoculation amount of the bacterial agent is 3% to 7%, preferably 3.5% to 6.5%, more preferably 4% to 6%, and most preferably 5%, based on the mass of the fermentation base. In the present invention, the bacterial agent is preferably a bacterial liquid; when the bacterial agent is a bacterial liquid, the inoculation amount is added according to the volume-to-mass ratio, that is, the volume of the bacterial agent mL: the mass of the fermentation base g. In the present invention, the concentration of Bacillus subtilis YY-10 in the bacterial agent is preferably 10 7 ~10 8 cfu / g, the concentration of Saccharomyces cerevisiae YY-14 is preferably 10 7 ~10 8 cfu / g, the concentration of Lactobacillus plantarum YY-8 is preferably 10 7 ~10 8 cfu / g; the preservation number of the Bacillus subtilis YY-10 is CGMCC No.24694; the preservation number of the Saccharomyces cerevisiae YY-14 is CGMCC No.24698; and the preservation number of Lactobacillus plantarum YY-8 is CGMCC No.24692.

[0037] The present invention has no special limitation on the preparation method of the bacterial agent, and a preparation method well known to those skilled in the art can be used; in a specific embodiment of the present invention, the preparation method of the bacterial agent comprises the following steps: activating Bacillus subtilis YY-10, Saccharomyces cerevisiae YY-14 and Lactobacillus plantarum YY-8 respectively, inoculating the activated strains into a liquid culture medium respectively for culturing to a logarithmic growth phase, and mixing the obtained seed liquids to obtain a bacterial agent; the bacterial agent preferably comprises Bacillus subtilis YY-10, Saccharomyces cerevisiae YY-14 and Lactobacillus plantarum YY-8; the volume ratio of the Bacillus subtilis YY-10 seed liquid, the Saccharomyces cerevisiae YY-14 seed liquid and the Lactobacillus plantarum YY-8 seed liquid used for mixing is preferably 3:1:2; the concentration of the Bacillus subtilis YY-10 seed liquid preferably reaches 10 7 ~10 8 cfu / g, preferably 5×10 7 cfu / g; the concentration of the Saccharomyces cerevisiae YY-14 seed solution preferably reaches 10 7 ~10 8 cfu / g, preferably 5×10 7 cfu / g; the concentration of the Lactobacillus plantarum YY-8 seed solution preferably reaches 10 7 ~10 8 cfu / g, preferably 5×10 7 cfu / g.

[0038] The present invention has no limitation on the activation method, and the method known to those skilled in the art can be used. In the present invention, the activation medium used in the activation of Bacillus subtilis YY-10 is preferably LB medium; the LB medium preferably includes the following components in the following concentrations: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride and 15-20 g / L agar; the pH of the LB medium is preferably neutral; the LB medium is preferably sterilized by steam at 121°C for 30 min.

[0039] The activation medium used in the activation of the brewer's yeast YY-14 of the present invention is preferably a PDA medium; the PDA medium preferably includes the following components in concentrations: 200 g / L potato, 20 g / L glucose and 15-20 g / L agar; the pH of the PDA medium is preferably a natural pH value; the PDA medium is preferably sterilized by steam at 121° C. for 30 min.

[0040] The activation medium used in the activation of the plant lactobacillus YY-8 of the present invention is preferably an MRS medium; the MRS medium preferably includes the following components: 10g / L peptone, 10g / L beef extract, 5g / L yeast powder, 2g / L dipotassium hydrogen phosphate, 2g / L diammonium citrate, 5g / L sodium acetate, 20g / L glucose, 1mL / L Tween 80, 0.58g / L magnesium sulfate heptahydrate, 0.25g / L manganese sulfate tetrahydrate and 1-20g / L agar; the pH of the MRS medium is preferably adjusted to 6.2-6.6; the MRS medium is preferably sterilized by steam at 121°C for 30min. The present invention has no special requirements for the method of adjusting the MRS medium, and a method well known to those skilled in the art can be used; in the embodiment of the present invention, hydrochloric acid and sodium hydroxide are preferably used for adjustment.

[0041] In the present invention, the liquid culture medium used for culturing Bacillus subtilis YY-10, Saccharomyces cerevisiae YY-14 and Lactobacillus plantarum YY-8 are all liquid culture mediums obtained by omitting agar from the activated culture medium of the three strains. Taking Bacillus subtilis YY-10 as an example, the culture described in the present invention is preferably carried out in a shaker; the rotation speed is preferably 150 to 250 r / min, more preferably 180 to 220 r / min; the culture temperature is preferably 28 to 32°C, more preferably 30°C. The culture conditions of Saccharomyces cerevisiae YY-14 and Lactobacillus plantarum YY-8 described in the present invention are the same as those of Bacillus subtilis YY-10, so they will not be repeated. The culture is terminated when the strains in the liquid culture medium reach the logarithmic growth phase, and the concentrations of the three bacteria in the liquid culture medium all reach 10 8 cfu / g.

[0042] The strain used in the present invention can significantly degrade corn protein and destroy the structure of corn protein, thereby releasing functional polypeptide sequences in corn protein and improving the biological function of feed additives. The present invention uses three bacteria in a mixed manner, wherein Bacillus subtilis YY-10 and Saccharomyces cerevisiae YY-14 are facultative anaerobic bacteria, which consume a large amount of oxygen during growth, thereby forming a hypoxic or anaerobic environment. In addition, because the fermentation system is acidic and plant lactic acid bacteria produce acid in a hypoxic or anaerobic environment, the pH value of the fermentation system is further reduced, thereby inhibiting the growth and reproduction of harmful microorganisms.

[0043] The present invention uses the enzyme preparation in an amount of 20U / g to 140U / g, more preferably 30U / g to 130U / g, still more preferably 40U / g to 120U / g, more preferably 50U / g to 110U / g, and most preferably 60U / g to 100U / g, based on the weight of the fermentation substrate. In the present invention, the enzyme preparation is preferably a protease, more preferably an acid protease. The present invention utilizes the bacterial enzyme combination technology to release functional polypeptides in corn protein to a greater extent.

[0044] The present invention provides a method for preparing the feed additive described in the above technical solution, comprising the following steps:

[0045] After mixing the bacterial agent with water, a diluted bacterial agent is obtained;

[0046] The fermentation base material, diluted bacterial agent and enzyme preparation are mixed and fermented to obtain feed additives.

[0047] The present invention mixes the bacterial agent with water to obtain a diluted bacterial agent. The present invention has no requirements for the mixing method, and a method well known to those skilled in the art can be used. The volume ratio of the bacterial agent to water in the present invention is preferably 3-7:23-107, more preferably 4-6:24-106, and more preferably 5:25-105.

[0048] The present invention has no limitation on the mixing order of the fermentation base material, the diluted bacterial agent, the enzyme preparation and the water. The present invention has no limitation on the mixing method, and any method known to those skilled in the art can be used.

[0049] The fermentation base material and enzyme preparation of the present invention have been introduced in detail above, and will not be repeated here. In the present invention, the fermentation time is preferably 1 to 5 days, more preferably 2 to 4 days, and more preferably 3 days; the fermentation temperature is preferably 20°C to 40°C, more preferably 25 to 35°C, and more preferably 30°C. In the present invention, the fermentation method is preferably one-way valve bag fermentation; the present invention does not have any limitation on the specific implementation method of the one-way valve bag fermentation method, and the method familiar to those skilled in the art can be adopted. The present invention has no special limitation on the bag turning process during the one-way valve bag fermentation process, and the method familiar to those skilled in the art can be adopted; in the present invention, the effect of the present invention can be achieved without or with bag turning.

[0050] In the present invention, the mass volume ratio of the fermentation base material to the diluted bacterial agent is preferably 1g: 0.3-1.1mL, recorded as a material-liquid ratio of 1: 0.3-1.1, further preferably 1g: 0.3-0.9mL, recorded as a material-liquid ratio of 1: 0.3-0.9, more preferably 1: 0.5g-0.7mL, recorded as a material-liquid ratio of 1: 0.5-0.7. The use of water in the diluted bacterial agent of the present invention can well avoid the loss of soluble carbohydrates, the reproduction of undesirable microorganisms, the production of a large amount of butyric acid, the odor of the feed, the stickiness, the roughness, the difficulty in compacting, the presence of some air between the feeds will cause heat generation, the large-scale reproduction of aerobic bacteria, the moldy deterioration and freezing of the feed due to excessive or low water content; and provides a good environment for the development of microorganisms in the solid fermentation system of the present invention.

[0051] When feeding animals with feed materials such as corn gluten meal, there are problems such as low digestibility, poor growth performance and unbalanced amino acid composition. The fermentation base material is fermented by the bacterial enzyme of the present invention to make it easy to digest. At the same time, the corn gluten meal is used in combination with soybean meal and corn germ meal to alleviate the problem of amino acid imbalance in the feed additive. At the same time, the feed additive obtained by the combination of the three contains high protein, and can also replace high-protein fish meal and fermented soybean meal in feed, and has the characteristics of low price and good biological activity. Therefore, the feed additive of the present invention can be used to prepare animal feed.

[0052] The present invention provides the use of the above-mentioned feed additive in the preparation of animal feed. In the present invention, the animals preferably include rats, weaned piglets, etc. The animal feed of the present invention can improve the production performance and immune ability of animals in animal breeding, especially animals in the early stage of weaning. The feed additive of the present invention has a high content of soluble protein and polypeptide. The use of the feed additive to replace part of the fish meal in the animal diet can improve the growth performance of weaned animals. As the age of the animal increases, the replacement amount also gradually increases, and it has a good alleviating effect on DSS-induced animal ulcerative colitis. In an embodiment of the present invention, the animal feed of the present invention is used in the feeding of UC rats, which alleviates the various pathological indicators of UC rats and the damage to the kidneys and spleen, reduces the oxidative stress level of the rat body, inhibits the inflammatory response of the rat body, improves the intestinal permeability and colon morphology of the rat, and increases the content of immunoglobulins in the serum and intestinal mucosa of the rat.

[0053] In the application, the animal feed preferably includes the following components in percentage by weight: 54.32-55.72% corn, 24.40-25.60% soybean meal, 5.29-8.50% wheat bran, 5.60-7.20% fish meal, 1.03-3.07% feed additive of the present invention, 2.05-2.20% bone meal, 0.90-1.10% soybean oil, 0.80% salt, 0.20% premix, 0.1% Choline chloride, 0.11-0.12% DL-methionine and 0.14-0.20% lysine; more preferably 54.57% corn, 24.40% soybean meal, 8.50% wheat bran, 7.20% fish meal, 1.03% feed additives, 2.05% bone meal, 0.90% soybean oil, 0.80% salt, 0.20% premix, 0.1% choline chloride, 0.11% DL-methionine and 0.14% lysine;

[0054] Or further preferably, 55.32% corn, 24.80% soybean meal, 6.93% wheat bran, 6.40% fish meal, 2.05% feed additives, 2.10% bone meal, 1.00% soybean oil, 0.80% salt, 0.20% premix, 0.1% choline chloride, 0.12% DL-methionine and 0.18% lysine;

[0055] Or further preferably, it is 55.72% corn, 25.60% soybean meal, 5.29% wheat bran, 5.60% fish meal, 3.07% feed additives, 2.10% bone meal, 1.10% soybean oil, 0.80% salt, 0.20% premix, 0.1% choline chloride, 0.12% DL-methionine, and 0.20% lysine.

[0056] The present invention preferably includes the following components by mass per kg of premix: vitamin A 8000 IU, vitamin D 31500 IU, vitamin E 20.0 IU, vitamin K 32.0 mg, vitamin B1 11.5 mg, vitamin B2 24.0 mg, vitamin B6 1.5 mg, vitamin B12 20.0 μg, niacin 30.0 mg, pantothenic acid 15.0 mg, folic acid 0.6 mg and biotin 0.1 mg.

[0057] The feed of the present invention includes the feed additive of the present invention, corn, soybean meal, wheat bran, fish meal and other components. In other words, the feed additive of the present invention can be combined with the diet of weaned animals to form a complete feed for feeding animals. It can be seen that the feed additive fermented feed of the present invention can be directly added to the diet or replace part of the original diet, such as replacing part of soybean meal, part of fish meal, etc., and can be used to feed animals at all stages, but is particularly suitable for animals in the early stage of weaning.

[0058] According to the advantages of the above feed additives, the present invention also provides the use of the feed additives described in the above technical solution in the preparation of products that improve animal production performance and / or immunity. In the present invention, the animals preferably include mice and / or piglets, and further preferably include rats and / or weaned piglets.

[0059] In order to further illustrate the present invention, a feed additive, animal feed, preparation method and application thereof provided by the present invention are described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0060] Preparation Example

[0061] Bacillus subtilis YY-10, Saccharomyces cerevisiae YY-14 and Lactobacillus plantarum YY-8 were activated respectively, and then the activated strains were inoculated into liquid culture medium and cultured on a shaker until the logarithmic growth phase to obtain seed liquid, wherein the concentration of Bacillus subtilis YY-10 seed liquid was 5×10 7 cfu / g, the concentration of Saccharomyces cerevisiae YY-14 seed solution was 5×10 7 cfu / g, the concentration of Lactobacillus plantarum YY-8 seed solution was 5×10 7 cfu / g; the seed liquid of Bacillus subtilis YY-10, the seed liquid of Saccharomyces cerevisiae YY-14 and the seed liquid of Lactobacillus plantarum YY-8 were mixed in a volume ratio of 3:1:2 to obtain a bacterial solution. Therefore, the concentration of Bacillus subtilis YY-10 in the obtained bacterial solution reached 10 7 cfu / g, the concentration of Saccharomyces cerevisiae YY-14 reached 10 7 cfu / g, the concentration of Lactobacillus plantarum YY-8 reached 10 7 cfu / g;

[0062] Among them, the activation medium for Bacillus subtilis YY-10 is LB medium, specifically 10g tryptone, 5g yeast extract, 10g sodium chloride, pH neutral, 15-20g agar, fixed to 1000mL, and steam sterilized at 121°C for 30min;

[0063] The base of the liquid culture medium for Bacillus subtilis YY-10 is LB liquid culture medium, specifically 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, neutral pH, fixed to 1000 mL, and steam sterilized at 121°C for 30 min;

[0064] The activation medium for Saccharomyces cerevisiae YY-14 is PDA medium: specifically, 200 g potato, 20 g glucose, natural pH, 15-20 g agar, fixed to 1000 mL, steam sterilized at 121°C for 30 min;

[0065] The liquid culture medium for Saccharomyces cerevisiae YY-14 is PDA medium: specifically, 200 g potato, 20 g glucose, natural pH, 15-20 g agar, fixed to 1000 mL, steam sterilized at 121°C for 30 min;

[0066] The activation medium of Lactobacillus plantarum YY-8 is MRS medium: specifically, peptone 10g, beef extract 10g, yeast powder 5g, dipotassium hydrogen phosphate, diammonium citrate 2g each, sodium acetate 5g, glucose 20g, Tween 80 1mL, magnesium sulfate heptahydrate 0.58g, manganese sulfate tetrahydrate 0.25g, agar 1-20g, fixed volume to 1000mL; pH adjusted to 6.2-6.6, 121℃ steam sterilization for 30min;

[0067] The liquid culture medium of Lactobacillus plantarum YY-8 is MRS culture medium: specifically, peptone 10g, beef extract 10g, yeast powder 5g, dipotassium hydrogen phosphate, diammonium citrate 2g each, sodium acetate 5g, glucose 20g, Tween 80 1mL, magnesium sulfate heptahydrate 0.58g, manganese sulfate tetrahydrate 0.25g, fixed volume to 1000mL; pH adjusted to 6.2-6.6, 121℃ steam sterilization for 30min.

[0068] Example 1

[0069] Mix corn gluten powder, soybean meal and corn germ meal evenly, wherein the mass ratio of corn gluten powder, soybean meal and corn germ meal is 5:3:2, to obtain a fermentation base material;

[0070] Mix water and the bacterial agent prepared in the preparation example in a volume ratio of 47:3 to obtain a diluted bacterial agent;

[0071] The fermentation base material, diluted bacterial agent and acidic protease are mixed and fermented in a one-way valve fermentation bag to obtain a feed additive, wherein the mass volume ratio of the fermentation base material and the diluted bacterial agent is 1g:0.5mL, the amount of enzyme added is 100U / g based on the mass of the fermentation base material, the inoculation amount (the percentage of the volume of the bacterial agent before dilution (mL) to the mass of the fermentation base material (g), v / w) is 3%, the fermentation temperature is 30°C, and the fermentation time is 2d.

[0072] Example 2

[0073] The steps are the same as those in Example 1, except that water and the bacterial agent prepared in the preparation example are mixed in a volume ratio of 46:4 to obtain a diluted bacterial agent; and the inoculation amount (v / w) is 4%.

[0074] Example 3

[0075] The steps are the same as those in Example 1, except that water and the bacterial agent prepared in the preparation example are mixed in a volume ratio of 45:5 to obtain a diluted bacterial agent; the inoculation amount (the volume of the bacterial agent before dilution (mL) accounts for the percentage of the mass (g) of the fermentation base material, v / w) is 5%.

[0076] Example 4

[0077] The steps are the same as those in Example 1, except that water and the bacterial agent prepared in the preparation example are mixed in a volume ratio of 44:6 to obtain a diluted bacterial agent; the inoculation amount (the volume of the bacterial agent before dilution (mL) as a percentage of the mass (g) of the fermentation base material, v / w) is 6%.

[0078] Example 5

[0079] The steps are the same as those in Example 1, except that water and the bacterial agent prepared in the preparation example are mixed in a volume ratio of 43:7 to obtain a diluted bacterial agent; the inoculation amount (the volume of the bacterial agent before dilution (mL) accounts for the percentage of the mass (g) of the fermentation base material, v / w) is 7%.

[0080] Comparative Example 1

[0081] Corn gluten meal, soybean meal and corn germ meal were mixed and stirred evenly, wherein the mass ratio of corn gluten meal, soybean meal and corn germ meal was 5:3:2, and recorded as the unfermented group.

[0082] The feed additives prepared in Examples 1 to 5 and Comparative Example 1 were taken out and dried at 60°C to test the soluble protein and polypeptide contents. The soluble protein content was determined by the Folin-phenol method, and the polypeptide content was determined by the TCA precipitation method. The test data are shown in Table 1.

[0083] Table 1 Soluble protein and polypeptide contents of fermentation products with different inoculum amounts of bacterial species

[0084] Inoculation amount (%) Unfermented group 3 4 5 6 7 Soluble protein content (mg / g) <![CDATA[44.12 d ]]> <![CDATA[106.77 c ]]> <![CDATA[103.43 c ]]> <![CDATA[126.00 a ]]> <![CDATA[116.02 b ]]> <![CDATA[113.35 b ]]> Peptide content (mg / g) <![CDATA[19.86 e ]]> <![CDATA[82.41 d ]]> <![CDATA[82.07 d ]]> <![CDATA[102.74 a ]]> <![CDATA[96.42 b ]]> <![CDATA[87.98 c ]]>

[0085] Note: Different capital letters in the same row indicate extremely significant differences (P<0.01), and the same letters indicate insignificant differences (P>0.05), the same below.

[0086] As shown in Table 1, compared with the non-fermented group, the soluble protein and polypeptide contents of different experimental groups were significantly increased (P<0.05). Within the range of the inoculation amount designed in the experiment, with the increase of the inoculation amount, the soluble protein and polypeptide contents in the feed additives generally showed a trend of first increasing and then decreasing. When the inoculation amount was 5%, the soluble protein and polypeptide contents in the feed additives reached the highest, 126.00 mg / g and 102.74 mg / g, respectively. When the inoculation amount was low (3%-4%) or high (6%-7%), the soluble protein and polypeptide contents in the feed additives were significantly lower than those in the treatment of 5% inoculation amount (P<0.05). This may be because when the inoculation amount was low, the strain biomass was insufficient, resulting in an extension of the fermentation cycle, and the microorganisms did not fully play a role during the experimental period; when the inoculation amount was high, the microbial density was too large, and the competition for space, oxygen and nutrients between each other increased, resulting in the inhibition of their growth and reproduction. Therefore, the inoculation amount of 5% was selected as the optimal inoculation amount.

[0087] Example 6

[0088] Mix corn gluten meal: soybean meal: corn germ meal in a mass ratio of 5:3:2 and mix well to obtain a fermentation base material;

[0089] Mix water and the bacterial agent prepared in the preparation example in a volume ratio of 45:5 to obtain a diluted bacterial agent;

[0090] The fermentation base material, diluted bacterial agent and acidic protease are mixed and fermented in a one-way valve fermentation bag to obtain a feed additive, wherein the mass volume ratio of the fermentation base material and the diluted bacterial agent is 1g:0.5mL, the amount of enzyme added is 20U / g based on the mass of the fermentation base material, the inoculation amount (the percentage of the volume of the bacterial agent before dilution (mL) to the mass of the fermentation base material (g), v / w) is 5%, the fermentation temperature is 30°C, and the fermentation time is 2d.

[0091] Example 7

[0092] The steps are the same as those in Example 6, except that the amount of enzyme added is 60 U / g.

[0093] Example 8

[0094] The steps are the same as those in Example 6, except that the amount of enzyme added is 120 U / g.

[0095] Example 9

[0096] The steps are the same as those in Example 6, except that the amount of enzyme added is 140 U / g.

[0097] The feed additives prepared in Example 3, Examples 6 to 9 and Comparative Example 1 were taken out and dried at 60°C, and the soluble protein and polypeptide contents were tested. The soluble protein content was determined by the Folin-phenol method, and the polypeptide content was determined by the TCA precipitation method. The test data are shown in Table 2.

[0098] Table 2 Soluble protein and polypeptide content of fermentation products with different enzyme addition amounts

[0099] Enzyme dosage (U / g) Unfermented group 20 60 100 120 140 Soluble protein content (mg / g) <![CDATA[44.12 e ]]> <![CDATA[96.02 d ]]> <![CDATA[110.60 c ]]> <![CDATA[126.00 a ]]> <![CDATA[130.93 a ]]> <![CDATA[120.35 b ]]> Peptide content (mg / g) <![CDATA[19.86 e ]]> <![CDATA[74.58 d ]]> <![CDATA[85.45 c ]]> <![CDATA[102.74 a ]]> <![CDATA[103.56 a ]]> <![CDATA[99.69 b ]]>

[0100] It can be seen from Table 2 that compared with the non-fermented group, the soluble protein and polypeptide contents in the test groups with different enzyme addition amounts were significantly increased (P<0.05). Within the enzyme addition amount range designed in the experiment, with the increase of enzyme addition amount, the soluble protein and polypeptide contents in the feed additives showed a trend of first increasing and then decreasing. When the enzyme addition amount was 120U / g, the soluble protein and polypeptide contents in the feed additives were the highest, reaching 130.93mg / g and 103.56mg / g, respectively. When the amount of enzyme added was less (20U / g and 60U / g) or more (120U / g and 140U / g), the content of soluble protein and polypeptide in feed additives was lower than that in the group with 120U / g enzyme addition (P<0.05). This may be because when the amount of enzyme added was less, the substrate concentration in the fermentation matrix was much greater than the enzyme concentration, and the enzymatic efficiency was low during the test period; when the amount of enzyme added was more, the enzymatic efficiency was high, and after the substrate was fermented to a certain extent, the small molecular protein was over-utilized by microorganisms, resulting in a decrease in the content of soluble protein and polypeptide. When the amount of enzyme added was 100U / g and 120U / g, there was no significant difference in the content of soluble protein and polypeptide in feed additives (P<0.05). Taking all factors into consideration, 100U / g was selected as the optimal amount of enzyme added.

[0101] Example 10

[0102] Mix corn gluten meal: soybean meal: corn germ meal in a mass ratio of 5:3:2 and mix well to obtain a fermentation base material;

[0103] Mix water and the bacterial agent prepared in the preparation example in a volume ratio of 25:5 to obtain a diluted bacterial agent;

[0104] The fermentation base material, the diluted bacterial agent and the acidic protease are mixed and fermented in a one-way valve fermentation bag to obtain a feed additive, wherein the material-liquid ratio of the fermentation base material and the diluted bacterial agent is 1:0.3, the amount of enzyme added is 100U / g based on the mass of the fermentation base material, the inoculation amount (the percentage of the volume (mL) of the bacterial agent before dilution to the mass (g) of the fermentation base material, v / w) is 5%, the fermentation temperature is 30°C, and the fermentation time is 2d.

[0105] Embodiment 11

[0106] The steps are the same as those in Example 10, except that water and the bacterial agent prepared in the preparation example are mixed in a volume ratio of 45:5 to obtain a diluted bacterial agent; and the material-liquid ratio of the fermentation base material to the diluted bacterial agent is 1:0.5.

[0107] Example 12

[0108] The steps are the same as those in Example 10, except that water and the bacterial agent prepared in the preparation example are mixed in a volume ratio of 65:5 to obtain a diluted bacterial agent; and the ratio of the fermentation base material to the diluted bacterial agent is 1:0.7.

[0109] Example 13

[0110] The steps are the same as those in Example 10, except that water and the bacterial agent prepared in the preparation example are mixed in a volume ratio of 85:5 to obtain a diluted bacterial agent; and the ratio of the fermentation base material to the diluted bacterial agent is 1:0.9.

[0111] Embodiment 14

[0112] The steps are the same as those in Example 10, except that water and the bacterial agent prepared in the preparation example are mixed in a volume ratio of 105:5 to obtain a diluted bacterial agent; and the material-liquid ratio of the fermentation base material and the diluted bacterial agent is 1:1.1.

[0113] The feed additives prepared in Examples 10 to 14 and Comparative Example 1 were taken out and dried at 60°C, and then the soluble protein and polypeptide contents were tested. The soluble protein content was determined by the Folin-phenol method, and the polypeptide content was determined by the TCA precipitation method. The test data are shown in Table 3.

[0114] Table 3 Soluble protein and polypeptide content of fermentation products with different feed-water ratios

[0115] Material-water ratio (U / g) Unfermented group 1：0.3 1：0.5 1：0.7 1：0.9 1：1.1 Soluble protein content (mg / g) <![CDATA[44.12 d ]]> <![CDATA[92.68 c ]]> <![CDATA[125.60 a ]]> <![CDATA[127.18 a ]]> <![CDATA[120.10 b ]]> <![CDATA[91.02 c ]]> Peptide content (mg / g) <![CDATA[19.86 d ]]> <![CDATA[71.48 c ]]> <![CDATA[97.95 a ]]> <![CDATA[99.53 a ]]> <![CDATA[95.89 b ]]> <![CDATA[72.57 c ]]>

[0116] As shown in Table 3, compared with the non-fermented group, the soluble protein and polypeptide contents of the experimental groups with different feed-water ratios were significantly increased (P<0.05). Within the feed-water ratio range of the experimental design, with the increase of the feed-water ratio, the soluble protein and polypeptide contents of the feed additives showed a trend of first increasing and then decreasing. When the feed-water ratio was 1:0.7, the bacterial enzyme synergistic fermentation effect was the best, and the soluble protein and polypeptide contents in the feed additives reached 127.18 mg / g and 99.53 mg / g. When the feed-water ratio was small (1:0.3 and 1:0.5) or large (1:0.9 and 1:1.1), the soluble protein and polypeptide content in the feed additives were lower than that in the test group with a feed-water ratio of 1:0.7. This may be because when the water content in the fermentation matrix is ​​low, the internal structure of the feed is loose and there is a lot of air, which makes aerobic harmful bacteria proliferate vigorously. At the same time, the amount of dissolved nutrients is small, resulting in the inhibition of microbial growth; when the water content is high, the feed is easy to form lumps, mass transfer and heat transfer are difficult, and water-soluble nutrients are easy to lose, affecting the normal fermentation of the feed. There was no significant difference in the soluble protein and polypeptide content in the feed additives of the test groups with a feed-water ratio of 1:0.5 and 1:0.7 (P>0.05). Considering that the fermentation system is too high in water content and is not easy to store and transport, 1:0.5 was selected as the optimal feed-water ratio.

[0117] Embodiment 15

[0118] Mix corn gluten meal: soybean meal: corn germ meal in a mass ratio of 5:3:2 and mix well to obtain a fermentation base material;

[0119] Mix water and the bacterial agent prepared in the preparation example in a volume ratio of 45:5 to obtain a diluted bacterial agent;

[0120] The fermentation base material, the diluted bacterial agent and the acidic protease are mixed and fermented in a one-way valve fermentation bag to obtain a feed additive, wherein the material-liquid ratio of the fermentation base material to the diluted bacterial agent is 1:0.5 (w / v), the amount of enzyme added is 100U / g based on the mass of the fermentation base material, the inoculation amount (the percentage of the volume (mL) of the bacterial agent before dilution to the mass (g) of the fermentation base material, v / w) is 5%, the fermentation temperature is 20°C, and the fermentation time is 2d.

[0121] Example 16

[0122] The steps are the same as those in Example 15, except that the fermentation temperature is 25°C.

[0123] Embodiment 17

[0124] The steps are the same as those in Example 15, except that the fermentation temperature is 35°C.

[0125] Embodiment 18

[0126] The steps are the same as those in Example 15, except that the fermentation temperature is 40°C.

[0127] The feed additives prepared in Examples 16 to 18 and Comparative Example 1 were taken out and dried at 60°C, and then the soluble protein and polypeptide contents were tested. The soluble protein content was determined by the Folin-phenol method, and the polypeptide content was determined by the TCA precipitation method. The test data are shown in Table 4.

[0128] Table 4 Soluble protein and polypeptide content of fermentation products at different fermentation temperatures

[0129] Fermentation temperature (℃) Unfermented group 20 25 30 35 40 Soluble protein content (mg / g) <![CDATA[44.12 d ]]> <![CDATA[80.35 c ]]> <![CDATA[102.10 c ]]> <![CDATA[125.60 a ]]> <![CDATA[110.35 b ]]> <![CDATA[106.36 b ]]> Peptide content (mg / g) <![CDATA[19.86 e ]]> <![CDATA[62.35 d ]]> <![CDATA[79.28 c ]]> <![CDATA[97.95 a ]]> <![CDATA[90.67 b ]]> <![CDATA[79.05 c ]]>

[0130] As shown in Table 4, compared with the non-fermented group, the soluble protein and polypeptide contents of the experimental groups at different fermentation temperatures were significantly increased (P<0.05). Within the fermentation temperature range of the experimental design, with the increase of fermentation temperature, the soluble protein and polypeptide contents in the feed additives increased first and then decreased. When the fermentation temperature was 30°C, the soluble protein and polypeptide contents of the feed additives were the highest, reaching 136.10 mg / g and 109.68 mg / g, respectively. When the fermentation temperature was low (fermentation temperature was 20℃ and 25℃) or high (fermentation temperature was 35℃ and 40℃), the soluble protein and polypeptide contents in feed additives were significantly lower than those in the experimental group with a fermentation temperature of 30℃ (P<0.05). This may be because when the fermentation temperature is low, the activity of microorganisms and or acidic proteases is low and cannot play a role; when the fermentation temperature is high, although the activity of acidic proteases is high, the growth and reproduction of microorganisms are inhibited, and the raw material fermentation layer is thicker, the temperature in the center of the layer is higher than the temperature in the outer layer, and the water loss is serious, which further hinders the growth and reproduction of microorganisms. Therefore, 30℃ is selected as the optimal fermentation temperature.

[0131] Embodiment 19

[0132] Mix corn gluten meal: soybean meal: corn germ meal in a mass ratio of 5:3:2 and mix well to obtain a fermentation base material;

[0133] Mix water and the bacterial agent prepared in the preparation example in a volume ratio of 45:5 to obtain a diluted bacterial agent;

[0134] The fermentation base material, the diluted bacterial agent and the acidic protease are mixed and fermented in a one-way valve fermentation bag to obtain a feed additive, wherein the material-liquid ratio of the fermentation base material to the diluted bacterial agent is 1:0.5 (w / v), the amount of enzyme added is 100U / g based on the mass of the fermentation base material, the inoculation amount (the percentage of the volume (mL) of the bacterial agent before dilution to the mass (g) of the fermentation base material, v / w) is 5%, the fermentation temperature is 30°C, and the fermentation time is 1 day.

[0135] Embodiment 20

[0136] The steps are the same as those in Example 19, except that the fermentation time is 3 days.

[0137] Embodiment 21

[0138] The steps are the same as those in Example 19, except that the fermentation time is 4 days.

[0139] Embodiment 22

[0140] The steps are the same as those in Example 19, except that the fermentation time is 5 days.

[0141] The feed additives prepared in Examples 19 to 22 and Comparative Example 1 were taken out and dried at 60°C, and then the soluble protein and polypeptide contents were tested. The soluble protein content was determined by the Folin-phenol method, and the polypeptide content was determined by the TCA precipitation method. The test data are shown in Table 5.

[0142] Table 5 Soluble protein content and polypeptide content of fermentation products at different fermentation times

[0143] Fermentation time (d) Unfermented group 1 2 3 4 5 Soluble protein content (mg / g) <![CDATA[44.12 e ]]> <![CDATA[89.25 d ]]> <![CDATA[126.00 a ]]> <![CDATA[132.85 a ]]> <![CDATA[124.62 b ]]> <![CDATA[120.65 c ]]> Peptide content (mg / g) <![CDATA[19.86 d ]]> <![CDATA[50.23 c ]]> <![CDATA[102.74 a ]]> <![CDATA[105.87 a ]]> <![CDATA[100.56 a ]]> <![CDATA[93.33 b ]]>

[0144] As can be seen from Table 5, compared with the non-fermented group, the soluble protein and polypeptide contents of the experimental groups at different fermentation temperatures were significantly increased (P<0.05). Within the fermentation time range of the experimental design, with the extension of fermentation time, the soluble protein and polypeptide contents in the feed additives showed a trend of first increasing and then decreasing. When the fermentation time was 2 days, the polypeptide content in the feed additive was the highest, reaching 101.67 mg / g. When the fermentation time was 3 days, the soluble protein content in the feed additive was the highest, reaching 132.85 mg / g. This may be because when the fermentation time is short, the exogenous enzyme and microorganism action time is short, and the feed quality cannot be fully improved; when the fermentation time is long, most of the nutrients in the fermentation system are consumed by microorganisms, and with the extension of fermentation time, lactic acid, bioactive peptides and flavor substances in the feed accumulate more, so 3 days is selected as the optimal fermentation time.

[0145] Embodiment 23

[0146] Mix corn gluten powder, soybean meal and corn germ meal evenly, wherein the mass ratio of corn gluten powder, soybean meal and corn germ meal is 5:3:2, to obtain a fermentation base material;

[0147] Mix water and the bacterial agent prepared in the preparation example in a volume ratio of 45:5 to obtain a diluted bacterial agent;

[0148] The fermentation base material, the diluted bacterial agent and the acidic protease are mixed and fermented in a one-way valve fermentation bag to obtain a feed additive, wherein the material-liquid ratio of the fermentation base material and the diluted bacterial agent is 1g:0.5mL (w / v), the amount of enzyme added is 100U / g based on the mass of the fermentation base material, the inoculation amount (the percentage of the volume (mL) of the bacterial agent before dilution to the mass (g) of the fermentation base material, v / w) is 5%, the fermentation temperature is 20°C, the fermentation time is 3d, and the bag is turned over once on the first and second days of fermentation.

[0149] The nutritional content of the material before and after fermentation in Example 23 was measured, and the nutritional safety of the feed additive was evaluated. The test results are shown in Table 6.

[0150] Determination items and methods: The soluble protein content was determined by the Folin-phenol method; the peptide content was determined by the TCA precipitation method; the conventional nutrients crude protein, crude fiber, crude fat, crude ash, calcium, phosphorus and viable bacteria count were determined by the agricultural industry standards of the People's Republic of China GB / T 6432-1994, GB / T 6434-2006, Soxhlet extraction method, GB / T6438-2007, GB / T6436-2002, GB / T 6437-2002 and GB 4789.2-2016, respectively.

[0151] Table 6 Basic nutrient content of materials before and after fermentation

[0152]

[0153] As shown in Table 6, the soluble protein, polypeptide, crude fat, crude protein, crude ash, calcium and viable bacteria count in the feed additive obtained after fermentation were increased.

[0154] Embodiment 24

[0155] The feed additive prepared in Example 23 was added to the animal diet in the form of replacing part of the fish meal in the weaned animal diet, and the replacement doses were 10%, 20% and 30%, respectively, and were recorded as animal feed 1, animal feed 2 and animal feed 3, respectively. The specific components and dosages are shown in Table 7. 40 healthy SD male weaned rats aged 23±2 days were randomly divided into 4 groups according to the principle of similar body weight, namely a blank group (feed was recorded as weaned animal diet), a 10% fish meal replacement group (feed was recorded as animal feed 1), a 20% fish meal replacement group (feed was recorded as animal feed 2), and a 30% fish meal replacement group (feed was recorded as animal feed 3), with 10 replicates in each group. The test started after 7 days of pre-feeding, and the test lasted for a total of 21 days.

[0156] Table 7 Composition and dosage of different feeds

[0157]

[0158] Note: per kg premix: Vitamin A 8000 IU; Vitamin D 31500 IU; Vitamin E 20.0 IU; Vitamin K 32.0 mg; Vitamin B1 11.5 mg; Vitamin B2 24.0 mg; Vitamin B6 1.5 mg; Vitamin B12 20.0 μg; Niacin 30.0 mg; Pantothenic acid 15.0 mg; Folic acid 0.6 mg; Biotin 0.1 mg;

[0159] The crude protein and crude ash content in the nutritional levels are measured values, and the rest are calculated values.

[0160] (1) Growth performance measurement

[0161] The body weight and feed intake of rats were recorded every day in the experiment, and the average daily weight gain (ADG) and average feed intake (ADFI) were calculated. The feed-to-weight ratio (F / G) was calculated based on the feed intake and daily weight gain. The results are shown in Table 8.

[0162] Among them, ADG = (average weight at the end of the experiment - average weight at the beginning of the experiment) / number of experimental days; ADFI = total feed intake / number of experimental days; F / G = total feed intake of rats / (final weight of rats - initial weight of rats)

[0163] Table 8 Growth performance of rats fed different diets

[0164]

[0165]

[0166] Note: The average initial weight is the average weight of rats on the first day of the experiment, and the average final weight is the average weight of rats on the last day of the experiment; the data in the table are average values, * indicates significant difference compared with the blank group, P<0.05; ** indicates extremely significant difference compared with the blank group, P<0.01.

[0167] As shown in Table 8, there were no significant differences in ADG and ADFI of rats in different test groups compared with the blank group when the rats were tested 0-7 days. The feed-to-weight ratio of weaned rats fed with animal feed 1 was significantly lower than that of the blank group, and the other two groups were higher than the blank group. When the rats were tested 7-14 days, the feed-to-weight ratio of rats in the fish meal groups replaced by different doses of feed additives was reduced compared with the blank group, among which the effect of feeding animal feed 2 was the best. When the rats were tested 14-21 days, the feed-to-weight ratio of rats in different test groups was significantly reduced compared with the blank group, among which the effect of feeding animal feed 3 was the best. When the rats were tested 0-21 days, the feed-to-weight ratio of rats in each test group was significantly reduced compared with the blank group, and there was no significant difference between the test groups. It can be seen that the feed additive of the present invention can replace 10-30% fish meal in the basic daily feed.

[0168] Embodiment 28

[0169] Fifty healthy SD male rats aged 23±2 days were randomly divided into 5 groups according to the principle of weight proximity, namely, blank group, model group, animal feed group 1, animal feed group 2 and animal feed group 3 (specific components are shown in Table 7). DSS powder was dissolved in sterile water to prepare a DSS solution with a concentration of 5wt.%, which was freely given to the rats in the test group and the model group for drinking for 7 consecutive days, with the DSS solution replaced once a day, and the blank group was free to drink sterile water.

[0170] Table 9 Feeding table for rats

[0171]

[0172] All rats were fasted for 12 h before sampling, and 2 wt.% sodium pentobarbital (50 mg / kg) was injected intraperitoneally into the rats before blood collection. After anesthesia took effect, the rats were fixed on the table, and non-anticoagulation negative pressure blood collection tubes were used to collect blood from the heart. Serum was centrifuged to measure serum biochemical, antioxidant and immune indicators, including malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) content and secretory immunoglobulin A (sIgA) content in colonic mucosa.

[0173] The specific operation method was carried out according to the instructions in the kit. The kits used were: rat tumor necrosis factor α (TNFα) ELISA detection kit, rat interleukin-1β (IL-1β) ELISA detection kit, rat interleukin-6 (IL-6) ELISA detection kit, rat interleukin-10 (IL-10) ELISA detection kit, rat immunoglobulin G (IgG) ELISA detection kit, rat immunoglobulin M (IgM) ELISA detection kit, rat immunoglobulin A (IgA) ELISA detection kit, rat secretory immunoglobulin A (IgA) ELISA detection kit, rat alanine aminotransferase (ALT) ELISA detection kit, rat aspartate aminotransferase (AST) ELISA detection kit, rat malondialdehyde (MDA) ELISA detection kit and rat superoxide dismutase (SOD) ELISA detection kit; the kits were purchased from Jianglai Biotechnology Co., Ltd.

[0174] After the rats were killed, the colon between the end of the ileocecal part and the anus was removed to check the appearance of the rat colon and measure its length. The measurement results are shown in Tables 10 and Figure 1 .

[0175] Table 10 Colon length of rats

[0176]

[0177]

[0178] Note: * indicates significant difference compared with the blank group, P<0.05; ** indicates extremely significant difference compared with the blank group, P<0.01; # indicates significant difference compared with the model group, P<0.05; ## indicates extremely significant difference compared with the model group, P<0.01.

[0179] From Table 10 and Figure 1 It can be seen that the colon tissue of the blank group rats was intact, the entire intestinal segment was uniform in thickness, formed feces were visible in the intestinal cavity, and there was no bleeding in the intestine. Compared with the blank group, the colon tissue of the model group rats was damaged and shortened, the entire intestinal segment was uneven in thickness, part of the intestinal wall was thin and transparent, and it was very easy to break when torn, and the feces in the intestinal cavity were thinner. Compared with the model group, the colon tissue of each test group was relatively intact, the feces in the intestinal cavity were basically formed and the bleeding phenomenon was reduced, among which the animal feed group 2 had the best effect. Compared with the blank group, the colon length of the model group rats was extremely significantly shortened (P<0.01). Compared with the model group, the colon length of the rats in each test group was extremely significantly increased (P<0.01), among which the animal feed group 2 had the most obvious effect, increasing by 5.2cm, indicating that the feed additive provided by the present invention can alleviate the symptoms of DSS-induced shortening of the rat colon.

[0180] Table 11 Oxidative stress level indicators in rats

[0181]

[0182] Note: * indicates significant difference compared with the blank group, P<0.05; ** indicates extremely significant difference compared with the blank group, P<0.01; # indicates significant difference compared with the model group, P<0.05; ## indicates extremely significant difference compared with the model group, P<0.01.

[0183] Oxidative stress is one of the important signs of ulcerative colitis. When the body is under adverse stimulation, excessive reactive oxygen species (ROS) can be detected in the intestinal mucosa. The accumulation of ROS will destroy the body's redox balance and cause tissue damage. MDA is one of the most important products of membrane lipid peroxidation. Its cytotoxicity is an important manifestation of the degree of lipid peroxidation in the body, and its content can indirectly reflect the degree of cell damage. SOD is a major antioxidant enzyme in the body that can effectively inhibit the formation of lipid peroxides, scavenge superoxide anion free radicals, and protect tissues from oxidative damage. Reduced MDA content and increased SOD activity in serum are important signs of oxidative stress in animals.

[0184] As shown in Table 11, compared with the model group, the SOD enzyme activity in the serum of the rats in the test groups increased, among which the animal feed group 3 had the best effect, increasing by 66.59%. Compared with the model group, the MDA content in the serum of the rats in each test group was significantly reduced (P<0.01), among which the animal feed group 3 had the best effect, decreasing by 40.99%. This shows that feeding intervention with feed additives can effectively reduce the oxidative stress level of DSS-induced colitis.

[0185] Table 12 Serum inflammatory indexes

[0186]

[0187]

[0188] Note: The data in the table are all mean values, * indicates significant difference compared with the blank group, P<0.05; ** indicates extremely significant difference compared with the blank group, P<0.01; # indicates significant difference compared with the model group, P<0.05; ## indicates extremely significant difference compared with the model group, P<0.01.

[0189] As shown in Table 12, compared with the model group, the MPO enzyme activity in the colon tissue of rats in animal feed group 2 and animal feed group 3 was significantly reduced, among which animal feed group 3 had the best effect, and animal feed group 1 had no significant change, indicating that the medium and high doses of feed additive replacement groups can significantly reduce the MPO enzyme activity in the colon tissue of rats. Compared with the blank group, the levels of IL-1β, IL-6 and TNF-α in the serum of rats in the model group were significantly increased, indicating that DSS can induce inflammatory response in animals and promote the expression of inflammatory factors. Compared with the blank group, the content of IL-10 in the serum of rats in the model group was significantly reduced (P<0.01). Compared with the model group, the content of IL-10 in the serum of rats in each test group was increased, among which animal group 3 had the best effect, and there was no significant difference with the control group.

[0190] Table 13 Serum biochemical indexes

[0191] Group Blank group Model Group Animal feed 1 group Animal feed 2 groups Animal feed 3 groups ALT (ng / mL) 26.76 <![CDATA[46.15 ** ]]> <![CDATA[42.20 ** ]]> <![CDATA[40.32 ** ]]> <![CDATA[38.04 *## ]]> AST (ng / mL) 22.58 <![CDATA[40.25 ** ]]> <![CDATA[36.04 **# ]]> <![CDATA[35.22 **# ]]> <![CDATA[33.33 **# ]]>

[0192] Note: The data in the table are all mean values, * indicates significant difference compared with the blank group, P<0.05; ** indicates extremely significant difference compared with the blank group, P<0.01; # indicates significant difference compared with the model group, P<0.05; ## indicates extremely significant difference compared with the model group, P<0.01.

[0193] As shown in Table 13, compared with the blank group, the ALT and AST enzyme activities in the serum of the rats in the model group were significantly increased (P<0.01). Compared with the model group, the ALT and AST enzyme activities in the serum of the rats in the different doses of feed additive replacement groups were decreased, among which the animal feed group 3 had the best effect.

[0194] Table 14 Serum and colon mucosal immunoglobulins

[0195] Group Blank group Model Group Animal feed 1 group Animal feed 2 groups Animal feed 3 groups IgG (mg / mL) 14.19 <![CDATA[7.71 ** ]]> <![CDATA[11.52 **## ]]> <![CDATA[11.65 **## ]]> <![CDATA[7.94 ** ]]> IgM (mg / mL) 1.56 <![CDATA[0.79 ** ]]> <![CDATA[1.33 *## ]]> <![CDATA[1.30 *## ]]> <![CDATA[1.04 **## ]]> IgA (mg / mL) 0.30 <![CDATA[0.18 ** ]]> <![CDATA[0.28 ## ]]> <![CDATA[0.25 *## ]]> <![CDATA[0.22 **# ]]> sIgA (mg / mL) 0.02 <![CDATA[0.01 ** ]]> <![CDATA[0.02 *## ]]> <![CDATA[0.02 *## ]]> <![CDATA[0.01 ** ]]>

[0196] Note: The data in the table are all mean values, * indicates significant difference compared with the blank group, P<0.05; ** indicates extremely significant difference compared with the blank group, P<0.01; # indicates significant difference compared with the model group, P<0.05; ## indicates extremely significant difference compared with the model group, P<0.01.

[0197] As shown in Table 14, compared with the blank group, the levels of IgG, IgM and IgA in the serum of the rats in the model group and the content of sIgA in the colon mucosa were extremely significantly decreased. Compared with the model group, the levels of IgG, IgM and IgA in the serum of the rats in each experimental group and the content of sIgA in the colon mucosa were increased, among which the IgG content increased the most in the 20% fish meal replacement group, and the IgM, IgA and sIgA increased the most in the animal feed group 3, and there was no significant difference in the effects between the animal feed group 1 and the animal feed group 2.

[0198] Organ index

[0199] After blood collection, the rats were killed by cervical dislocation. After death, the kidneys, spleen and liver were collected. The mass of the fresh kidneys, spleen and liver of the rats was weighed to calculate the organ coefficient. Organ coefficient (%) = (organ mass / g) / (final body weight / g) × 100. The survey results are shown in Table 15.

[0200] Table 15 Organ Index

[0201] Group Blank group Model Group Animal feed 1 group Animal feed 2 groups Animal feed 3 groups Renal index (%) 0.80 <![CDATA[0.94 ** ]]> <![CDATA[0.80 # ]]> 0.86 <![CDATA[0.85 # ]]> Liver index (%) 0.24 0.24 0.23 0.26 0.24 Spleen index (%) 3.55 <![CDATA[4.06 * ]]> 3.82 <![CDATA[3.55 ## ]]> 3.85

[0202] Note: The data in the table are all mean values, * indicates significant difference compared with the blank group, P<0.05; ** indicates extremely significant difference compared with the blank group, P<0.01; # indicates significant difference compared with the model group, P<0.05; ## indicates extremely significant difference compared with the model group, P<0.01.

[0203] As shown in Table 15, compared with the blank group, the kidney and spleen indexes of the rats in the model group were significantly increased. Compared with the model group, different doses of feed additive replacement groups could reduce the kidney and spleen indexes of rats with DSS-induced colitis, and the 20% fish meal replacement group and 10% fish meal replacement group had the best effects.

[0204] Intestinal indicators

[0205] After blood collection, the rats were killed by cervical dislocation. After killing, the colon tissue was collected for use. The diamine oxidase (DAO) content in the serum was determined by a rat diamine oxidase (DAO) ELISA detection kit. The colon intestinal morphology of the rats was observed by hematoxylin and eosin (H&E) staining, and the results are shown in Table 16.

[0206] Table 16 Intestinal Permeability

[0207] Group Blank group Model Group Animal feed 1 group Animal feed 2 groups Animal feed 3 groups DAO(U / mL) 76.14 <![CDATA[205.06 ** ]]> <![CDATA[165.32 **## ]]> <![CDATA[153.12 **## ]]> <![CDATA[90.03 ## ]]>

[0208] Note: The data in the table are all mean values, * indicates significant difference compared with the blank group, P<0.05; ** indicates extremely significant difference compared with the blank group, P<0.01; # indicates significant difference compared with the model group, P<0.05; ## indicates extremely significant difference compared with the model group, P<0.01.

[0209] As shown in Table 16, compared with the blank group, the DAO enzyme activity in the serum of the rats in the model group was extremely significantly increased. Compared with the model group, the DAO enzyme activity in the serum of the rats in the different doses of feed additive replacement groups was extremely significantly decreased, among which the animal feed group 3 had the best effect and had no significant difference with the blank group.

[0210] In summary, the feed additive of the present invention has a high content of soluble protein and polypeptide. Using the feed additive to replace part of the fish meal in the animal diet can improve the growth performance of weaned animals. As the age of the animal increases, the replacement amount gradually increases, and it has a good alleviating effect on DSS-induced ulcerative colitis in animals.

[0211] Although the present invention has been disclosed as above in the preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A feed additive that can replace fish meal, characterized in that: Including the following raw materials: fermentation base, bacterial agent and enzyme preparation; The fermentation base material includes corn gluten meal, soybean meal and corn germ meal; Based on the mass of the fermentation base material, the inoculation amount of the bacterial agent is 3% to 7%; the dosage of the enzyme preparation is 20U / g to 140U / g.

2. The feed additive according to claim 1, characterized in that The mass ratio of the corn gluten meal, soybean meal and corn germ meal is 5:3:

2.

3. The feed additive according to claim 1 or 2, characterized in that: The bacterial agent comprises Bacillus subtilis YY-10, Saccharomyces cerevisiae YY-14 and Lactobacillus plantarum YY-8; the concentration of Bacillus subtilis YY-10 in the bacterial agent reaches 10 7 ~10 8 cfu / g, the concentration of Saccharomyces cerevisiae YY-14 reached 10 7 ~10 8 cfu / g, the concentration of Lactobacillus plantarum YY-8 reached 10 7 ~10 8 cfu / g; The deposit number of the Bacillus subtilis YY-10 is CGMCC No.24694; The deposit number of the Saccharomyces cerevisiae YY-14 is CGMCC No.24698; The deposit number of Lactobacillus plantarum YY-8 is CGMCC No.24692.

4. The feed additive according to claim 1 or 2, characterized in that The enzyme preparation is a protease.

5. The method for preparing the feed additive according to any one of claims 1 to 4, characterized in that: The following steps are involved: After mixing the bacterial agent with water, a diluted bacterial agent is obtained; The fermentation base material, diluted bacterial agent and enzyme preparation are mixed and fermented to obtain feed additives.

6. The preparation method according to claim 5, characterized in that: The fermentation time is 1 to 5 days, and the temperature is 20°C to 40°C.

7. The preparation method according to claim 5 or 6, characterized in that: The mass volume ratio of the fermentation base material to the diluted bacterial agent is 1g:0.3-1.1mL.

8. Use of the feed additive according to any one of claims 1 to 4 or the feed additive obtained by the preparation method according to any one of claims 5 to 7 in preparing animal feed.

9. Use of the feed additive according to any one of claims 1 to 4 or the feed additive obtained by the preparation method according to any one of claims 5 to 7 in the preparation of products that improve animal production performance and / or immunity.

10. The use according to claim 8 or 9, characterized in that: The animals include mice and / or piglets.

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