A fermented feed additive for antagonizing broiler intestinal damage, and its preparation method and application

Abstract

The present invention provides a fermented feed additive for antagonizing intestinal damage in broilers, and a preparation method and application thereof, belonging to the technical field of feed additives. The present invention combines puffed corn protein powder with other raw materials as a fermentation substrate, and adds acidic protease and composite bacterial agent for synergistic fermentation, and the glutamine content in the obtained fermented feed additive can reach 8.6% of the total soluble protein content. The fermented feed additive of the present invention can significantly improve the growth performance of broilers, increase the relative length of the small intestine, and promote the development of small intestinal villi; alleviate the reduction in the relative length of the small intestine of broilers caused by LPS, the damage to the morphology of small intestinal villi and the increase in the content of jejunal pro-inflammatory factors IL-6 and TNF-α, while increasing the content of anti-inflammatory factor IL-10, which can achieve the effect of antagonizing intestinal damage in broilers.

Description

Technical Field

[0001] The invention belongs to the technical field of feed additives, and in particular relates to a fermented feed additive for antagonizing intestinal damage of broiler chickens, and a preparation method and application thereof. Background Art

[0002] As the largest immune organ of broilers, the intestine plays a critical role in resisting pathogen invasion, promoting nutrient digestion and absorption, and maintaining body health. Once there is a problem with intestinal health, the life and production activities of chickens will be greatly affected. Under the general background of "antibiotic-free feed", broiler farming faces problems such as increased prevalence of intestinal diseases and decreased production performance. Therefore, it is necessary to develop a green, safe, and efficient feed additive that can improve the intestinal integrity of broilers, antagonize the continuous occurrence of intestinal inflammation in broilers, and improve growth performance.

[0003] Glutamine (Gln) and glutamine dipeptide are often used as enteral nutritional supplements to maintain intestinal health. Studies have shown that glutamine-binding peptides have the functions of promoting intestinal development, improving intestinal morphology, protecting intestinal immune barriers and promoting broiler growth. Corn gluten meal is a commonly used protein feed raw material in poultry. Its glutamine content accounts for about one-third of the gluten content and is an excellent carrier of glutamine-binding peptides. Since corn gluten meal is difficult to dissolve in water, the function of glutamine peptides cannot be exerted when used directly as feed. Studies have shown that enzymatic hydrolysis of corn gluten meal can effectively release corn glutamine peptides to maintain intestinal health. Although the yield of enzymatic hydrolysis products is high, the enzymatic hydrolysis conditions are harsh, the amount of enzyme preparations is large, the operation process is complicated, the production cost is high, and it is difficult to promote and apply in actual production. At present, no research has been found on the preparation of glutamine peptide-rich feed additives from puffed corn gluten meal. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a fermented feed additive for antagonizing intestinal damage in broiler chickens, as well as a preparation method and application thereof, which can significantly improve the growth performance of broiler chickens, increase the relative length of the small intestine, promote the development of small intestinal villi, and achieve the effect of antagonizing intestinal damage in broiler chickens.

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

[0006] The invention provides a composite bacterial agent for preparing a fermented feed additive for antagonizing intestinal damage of broilers. The composite bacterial agent is prepared from a bacterial liquid of Bacillus subtilis YY-12, a bacterial liquid of Saccharomyces cerevisiae YY-14 and a bacterial liquid of Lactobacillus plantarum YY-7. The Bacillus subtilis YY-12 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No. 24696; the Saccharomyces cerevisiae YY-14 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No. 24698; and the Lactobacillus plantarum YY-7 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No. 24691.

[0007] Preferably, the preparation method of the Bacillus subtilis YY-12 bacterial liquid comprises: inoculating Bacillus subtilis in LB medium at an inoculation amount of 0.5-1.5%, and fermenting at 35-40° C. and a rotation speed of 150-200 r / min for 14-18 hours; the preparation method of the Saccharomyces cerevisiae YY-14 bacterial liquid comprises: inoculating Saccharomyces cerevisiae YY-14 in YPD medium at an inoculation amount of 0.5-1.5%, and fermenting at 32-38° C. and a rotation speed of 150-200 r / min for 14-18 hours; the preparation method of the Lactobacillus plantarum YY-7 bacterial liquid comprises: inoculating Lactobacillus plantarum YY-7 in MRS medium at an inoculation amount of 0.5-1.5%, and fermenting at 35-40° C. for 18-20 hours.

[0008] More preferably, the number of cells of the Bacillus subtilis YY-12 bacterial solution is (1.0-2.0)×10 8 CFU / mL, the number of cells of the Saccharomyces cerevisiae YY-14 bacterial solution is (1.0-2.0)×10 7 CFU / mL, the bacterial count of the plant lactobacillus YY-7 bacterial liquid is (1.0-3.0)×10 7 CFU / mL; the volume ratio of the Bacillus subtilis YY-12 bacterial solution, the Saccharomyces cerevisiae YY-14 bacterial solution and the Lactobacillus plantarum YY-7 bacterial solution is (1-3):(1-3):(1-3).

[0009] The present invention also provides a fermented feed additive for antagonizing intestinal damage in broilers, comprising the following raw materials in parts by weight: 3-5 parts of puffed corn protein powder, 0.5-2 parts of bran, 0.5-2 parts of soybean meal, 3-5 parts of corn germ meal, 0.5-2 parts of corn steep liquor and 0.05-0.15 parts of the composite bacterial agent described in any one of claims 1 to 3.

[0010] The present invention also provides a method for preparing the fermented feed additive, comprising: mixing puffed corn protein powder, bran, soybean meal, corn germ meal and corn steep liquor to obtain pre-fermented feed, inoculating a composite bacterial agent, adding acidic protease, and sealing and fermenting at 30-35° C. for 5-7 days to obtain the fermented feed additive; the method for preparing the puffed corn protein powder comprises: extruding and puffing the corn protein powder at 150-160° C. for 5-10 seconds.

[0011] Preferably, the acidic protease is added according to the mass of the pre-fermented feed, and the added amount of the acidic protease is 20-30 U / g.

[0012] Preferably, the water content of the pre-fermented feed is 40-50%.

[0013] The present invention also provides the use of the fermented feed additive in preparing a product for antagonizing broiler intestinal damage.

[0014] The present invention also provides application of the fermented feed additive in replacing soybean meal feed.

[0015] Preferably, the added amount of the fermented feed additive is 15%-30%.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention puffs corn protein powder to increase the content of glutamine in the feed additive, and the feed is fermented by acidic protease and composite bacterial agent. The addition of acidic protease first partially decomposes the macromolecular protein in the puffed corn protein powder, provides more decomposition sites for the later microbial fermentation and the protease secreted by the microorganism, improves the decomposition and utilization of the protein in the puffed corn protein powder, and releases more small peptides with glutamine. The glutamine content in the obtained fermented feed additive can reach 8.6% of the total soluble protein content. The fermented feed additive of the present invention can significantly improve the growth performance of broilers, increase the relative length of the small intestine, and promote the development of small intestinal villi; alleviate the reduction of the relative length of the small intestine of broilers caused by LPS, the damage of the morphology of the small intestinal villi and the increase of the content of the jejunal pro-inflammatory factors IL-6 and TNF-α, and increase the content of the anti-inflammatory factor IL-10, so as to achieve the effect of antagonizing the intestinal damage of broilers.

[0018] Biodeposit Information:

[0019] The Bacillus subtilis YY-12 described in the present invention is Bacillus subtilis in Latin, deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 24696, a deposit date of April 18, 2022, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0020] The brewer's yeast YY-14 described in the present invention is Saccharomyces cerevisiae in Latin, and is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.24698, a deposit date of April 18, 2022, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0021] The plant lactobacillus YY-7 described in the present invention is Lactobacillus plantarum in Latin, and is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 24691, a deposit date of April 18, 2022, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The effect of different diets on broiler body weight;

[0023] Figure 2 To study the effects of different dietary feeds on the daily weight gain of broiler chickens;

[0024] Figure 3 To study the effects of different dietary feeds on the daily feed intake of broiler chickens;

[0025] Figure 4 The effect of different diets on feed-to-weight ratio of broiler chickens;

[0026] Figure 5 To study the effects of different dietary feeds on the height of small intestinal villi in broiler chickens;

[0027] Figure 6 To study the effects of different dietary feeds on the depth of small intestinal crypts in broiler chickens;

[0028] Figure 7 The effect of different dietary feeds on the intestinal villus to crypt ratio of broiler chickens;

[0029] Figure 8 The effect of different dietary feeds on the relative length of the small intestine of broiler chickens;

[0030] Fig. 9 To study the effects of different dietary feeds on the IL-10 content in the jejunum of broiler chickens;

[0031] Fig.10 To study the effects of different dietary feeds on the IL-6 content in the jejunum of broiler chickens;

[0032] Fig.11 To investigate the effects of different dietary feeds on the TNF-α content in the jejunum of broiler chickens. DETAILED DESCRIPTION

[0033] The invention provides a composite bacterial agent for preparing a fermented feed additive for antagonizing intestinal damage of broilers. The composite bacterial agent is prepared from bacterial liquid of Bacillus subtilis YY-12, bacterial liquid of Saccharomyces cerevisiae YY-14 and bacterial liquid of Lactobacillus plantarum YY-7. The Bacillus subtilis YY-12 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No. 24696; the Saccharomyces cerevisiae YY-14 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No. 24698; and the Lactobacillus plantarum YY-7 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No. 24691.

[0034] In the present invention, the preparation method of the Bacillus subtilis YY-12 bacterial liquid preferably comprises: inoculating Bacillus subtilis in an LB medium at an inoculum amount of 0.5-1.5%, and fermenting for 14-18 hours at 35-40°C and a rotation speed of 150-200 r / min; the inoculum amount is more preferably 1%; the fermentation temperature is more preferably 37°C, and the rotation speed is more preferably 180 r / min; the number of cells of the Bacillus subtilis YY-12 bacterial liquid is (1.0-2.0)×10 8 CFU / mL, more preferably 1.5×10 8 CFU / mL; the LB medium preferably includes: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, distilled water to 1000mL, and steam sterilized at 121°C for 15min.

[0035] In the present invention, the preparation method of the yeast YY-14 bacterial liquid preferably comprises: inoculating the yeast YY-14 in a YPD medium at an inoculation rate of 0.5-1.5%, and fermenting for 14-18 hours at 32-38°C and a rotation speed of 150-200 r / min; the inoculation rate is more preferably 1%; the fermentation temperature is more preferably 35°C, and the rotation speed is more preferably 180 r / min; the number of cells of the yeast YY-14 bacterial liquid is (1.0-2.0)×10 7 CFU / mL, more preferably 1.6×107 CFU / mL; the YPD liquid culture medium preferably comprises: 10 g of yeast extract and 20 g of peptone dissolved in 900 mL of distilled water, and steam sterilized at 121°C for 15 min; 20 g of glucose dissolved in 100 mL of distilled water, and steam sterilized at 121°C for 15 min; the two sterilized solutions are mixed evenly.

[0036] In the present invention, the preparation method of the plant lactobacillus YY-7 bacterial liquid preferably comprises: inoculating plant lactobacillus YY-7 in an MRS medium at an inoculation amount of 0.5-1.5%, and fermenting at 35-40°C for 18-20h; the inoculation amount is more preferably 1%; the fermentation temperature is more preferably 37°C; the number of cells of the plant lactobacillus YY-7 bacterial liquid is 2.0-3.0×10 7 CFU / mL, more preferably 2.2×10 7 CFU / mL. The MRS liquid culture medium preferably includes: 10g of peptone, 10g of beef extract, 5g of yeast powder, 2g of dipotassium hydrogen phosphate, 2g of diammonium citrate, 5g of sodium acetate, 20g of glucose, 1mL of Tween 80, 0.58g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate tetrahydrate, the pH value is adjusted to 6.2-6.6, the volume is made up to 1000mL with distilled water, and steam sterilized at 121°C for 15min.

[0037] In the present invention, the number of cells of the Bacillus subtilis YY-12 bacterial solution is preferably (1.0-2.0)×10 8 CFU / mL, the number of cells of the Saccharomyces cerevisiae YY-14 bacterial solution is preferably (1.0-2.0)×10 7 CFU / mL, the number of cells of the Lactobacillus plantarum YY-7 bacterial solution is preferably (1.0-3.0)×10 7 CFU / mL; the volume ratio of the Bacillus subtilis YY-12 bacterial solution, the Saccharomyces cerevisiae YY-14 bacterial solution and the Lactobacillus plantarum YY-7 bacterial solution is preferably (1-3):(1-3):(1-3), and more preferably 2:2:2.

[0038] In the present invention, the Bacillus subtilis YY-12 has a strong enzyme production ability, and can produce a variety of digestive enzymes such as protease, lipase, amylase, etc. to increase the digestive enzyme activity in feed and promote the absorption of nutrients by animals; the Saccharomyces cerevisiae YY-14 has a complete intracellular and extracellular enzyme system, and the high protein digestion utilization rate is conducive to improving the feed conversion rate, and at the same time, it produces aromatic substances and improves the palatability of animals; the Lactobacillus plantarum YY-7 secretes a variety of acids, such as lactic acid, etc., and has a strong bactericidal ability, while reducing the pH of the feed and extending the shelf life of the feed. In addition, the Bacillus subtilis YY-12 and yeast YY-14 have a fast reproduction speed, consume oxygen in the feed, and cause a low oxygen environment in the feed, which is conducive to the reproduction of Lactobacillus plantarum YY-7; Lactobacillus plantarum YY-7 can utilize other substances such as amino acids, vitamins and pyruvate produced by Bacillus subtilis YY-12 and yeast YY-14, and the metabolites of Lactobacillus plantarum YY-7 provide an energy source for Saccharomyces cerevisiae YY-14. The three strains work synergistically to improve the utilization efficiency of the substrate and increase the protein content and nutritional function of the product.

[0039] The present invention also provides a fermented feed additive for antagonizing intestinal damage in broilers, comprising the following raw materials in parts by weight: 3-5 parts of puffed corn protein powder, 0.5-2 parts of bran, 0.5-2 parts of soybean meal, 3-5 parts of corn germ meal, 0.5-2 parts of corn steep liquor and 0.05-0.15 parts of the above-mentioned composite bacterial agent.

[0040] The present invention also provides a preparation method of the fermented feed additive, comprising: mixing puffed corn protein powder, bran, soybean meal, corn germ meal and corn steep liquor to obtain pre-fermented feed, inoculating a composite bacterial agent, adding acidic protease, and sealing and fermenting at 30-35° C. for 5-7 days to obtain the fermented feed additive.

[0041] In the present invention, the preparation method of the puffed corn gluten powder preferably comprises: extruding and puffing the corn gluten powder at 150-160°C for 5-10s, the temperature is more preferably 155°C, and the time is more preferably 5s. After the extrusion and puffing, it is also preferably crushed; the particle size of the crushed is preferably 30-50 mesh, more preferably 40 mesh. After the extrusion and puffing process of the corn gluten powder, the molecular structure of the protein changes, the hydrogen bonds and disulfide bonds between the molecules are partially broken, resulting in protein denaturation, the digestibility of the protein is significantly improved, and the glutamine peptide is easily released.

[0042] In the present invention, the acidic protease is added according to the mass of the pre-fermented feed, and the addition amount of the acidic protease is preferably 20-30 U / g, more preferably 25 U / g. The acidic protease of the present invention is purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd.

[0043] In the present invention, the water content of the pre-fermented feed is preferably 40-50%, more preferably 45%; the pH value of the fermentation is natural.

[0044] The sealed fermentation of the present invention is a solid-state fermentation of raw materials. The present invention has no special restrictions on the equipment for sealed fermentation, and conventional equipment in the art can be used. As an practicable method, the sealed fermentation uses a one-way valve breathable bag.

[0045] The addition of the acidic protease of the present invention first partially decomposes the macromolecular protein in the puffed corn gluten powder, provides more decomposition sites for the subsequent microbial fermentation utilization and the protease secreted by the microorganisms, improves the decomposition and utilization of the protein in the puffed corn gluten powder, and releases more small peptides with glutamine.

[0046] The present invention adopts bacterial enzyme synergistic fermentation to organically combine enzyme preparation and microbial efficacy, and the two work together to efficiently degrade feed raw materials, increase the content of effective ingredients in feed, and have the advantages of high product yield, simple operation, and low price. Therefore, the bacterial enzyme synergistic method is used to prepare corn protein powder fermented feed, which can not only degrade the macromolecular protein of corn protein powder to release glutamine peptides, but also its product can be directly fed to animals to improve feed utilization. In addition, bacterial enzyme synergistic fermentation can reduce the amount of enzyme preparation, thereby solving the problem of high production cost of enzymatic preparation of glutamine peptides, and its product is a green, environmentally friendly, economical and new product.

[0047] The present invention also provides the use of the fermented feed additive in the preparation of a product for antagonizing intestinal damage in broilers. The fermented feed additive of the present invention can significantly improve the growth performance of broilers, increase the relative length of the small intestine, and promote the development of small intestinal villi; alleviate the reduction in the relative length of the small intestine of broilers caused by LPS, the damage to the morphology of small intestinal villi, and the increase in the content of jejunal pro-inflammatory factors IL-6 and TNF-α, while increasing the content of the anti-inflammatory factor IL-10, so as to achieve the effect of antagonizing intestinal damage in broilers.

[0048] The present invention also provides application of the feed additive obtained by the preparation method in replacing soybean meal feed.

[0049] In the present invention, the added amount of the fermented feed additive is preferably 15%-30%.

[0050] The present invention has no particular limitation on the sources of the raw materials not mentioned, and conventional commercial products known to those skilled in the art may be used.

[0051] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0052] Example 1

[0053] Screening of bacterial strains

[0054] 1. Screening of Bacillus subtilis YY-12 (screening strains with strong ability to degrade corn gluten powder)

[0055] The Bacillus strains isolated and preserved in the laboratory were diluted in series and 10 -3 ,10 -4 and 10 -5 Spread on skim milk plate, incubate at 37℃ for 48h for primary screening. Inoculate strains with larger hydrolysis transparent zone on corn gluten powder screening medium for secondary screening, and identify strains with larger hydrolysis transparent zone on corn gluten powder screening medium.

[0056] Formula of skim milk screening medium: 10% skim milk powder, 2% agar;

[0057] Corn gluten meal screening medium formula: corn gluten meal 3%, agar 2%.

[0058] 2. Screening of Saccharomyces cerevisiae YY-14 (screening strains with large bacterial protein)

[0059] The Saccharomyces cerevisiae strains isolated and preserved in the laboratory were diluted in series and 10 -3 ,10 -4 and 10 -5 Spread on YPD medium and culture at 37℃ for 48h. Inoculate the bacteria that can grow well on YPD solid medium into YPD liquid medium, culture at 37℃, 180r / min for 24h, centrifuge at 12000r / min, remove the supernatant, resuspend the bacteria with sterile water three times, dry and weigh the obtained bacteria, and identify the strains with larger dry weight.

[0060] 3. Screening of Lactobacillus plantarum YY-7 (screening strains with strong acid production ability)

[0061] The lactic acid bacteria strains isolated and preserved in the laboratory were diluted in gradients and 10 -3 ,10 -4 and 10 -5 Spread on MRS-CaCO3 solid medium and incubate at 37℃ for 48h. Take the strain with the largest hydrolysis transparent zone for identification.

[0062] MRS-CaCO3 solid culture medium formula: MRS medium, 1% CaCO3, 2% agar.

[0063] Example 2

[0064] Preparation of compound bacterial agent

[0065] (1) Cultivation of Bacillus subtilis YY-12

[0066] LB liquid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, natural pH, 1000 mL of distilled water, steam sterilized at 121°C for 15 min.

[0067] Bacillus subtilis YY-12 was inoculated into LB medium at 1% inoculation amount, fermentation temperature was 37°C, shaker speed was 180 r / min, and fermentation time was 18 h. The number of bacteria was 1.5×10 8 CFU / mL, and obtain the bacterial solution.

[0068] (2) Cultivation of Saccharomyces cerevisiae YY-14

[0069] YPD liquid culture medium: 10 g yeast extract and 20 g peptone were dissolved in 900 mL distilled water and sterilized by steam at 121°C for 15 min; 20 g glucose was dissolved in 100 mL distilled water and sterilized by steam at 121°C for 15 min; the two sterilized solutions were mixed evenly.

[0070] Saccharomyces cerevisiae YY-14 was inoculated into YPD medium at 1% inoculation rate, fermented at 35°C, shaker speed 180 r / min, and fermented for 16 h. The number of cells was 1.6×10 7 CFU / mL, and obtain the bacterial solution.

[0071] (3) Cultivation of Lactobacillus plantarum YY-7

[0072] MRS liquid culture medium: peptone 10g, beef extract 10g, yeast powder 5g, dipotassium hydrogen phosphate 2g, diammonium citrate 2g, sodium acetate 5g, glucose 20g, Tween 80 1mL, magnesium sulfate heptahydrate 0.58g, manganese sulfate tetrahydrate 0.25g, pH adjusted to 6.2-6.6, volume adjusted to 1000mL, steam sterilized at 121℃ for 15min.

[0073] Lactobacillus plantarum YY-7 was inoculated into MRS medium at a 1% inoculum volume, and the fermentation temperature was 37°C and the culture was statically cultured for 18 h. The bacterial count was 2.2×10 7 CFU / mL, and obtain the bacterial solution.

[0074] (4) Mixing

[0075] The Bacillus subtilis YY-12 bacterial solution, the Saccharomyces cerevisiae YY-14 bacterial solution and the Lactobacillus plantarum YY-7 bacterial solution in steps (1) to (3) are mixed in equal volumes in a ratio of 1:1:1 to obtain a composite bacterial solution.

[0076] Example 3

[0077] Preparation of expanded corn gluten powder

[0078] The corn gluten powder was extruded and puffed at 155° C. for 8 seconds, and then crushed and passed through a 40-mesh sieve to obtain puffed corn gluten powder.

[0079] Example 4

[0080] 4 kg of puffed corn protein powder of Example 3, 1 kg of bran, 1 kg of soybean meal, 4 kg of corn germ and 1 kg of corn steep liquor were mixed and hydrated to a moisture content of 40%, and 100 g of the composite bacterial agent obtained in Example 2 was inoculated. Acid protease was added according to the mass of the pre-fermented feed in an amount of 25 U / g. The mixture was placed in a one-way valve breathable bag, sealed and fermented at 32° C. for 6 days to obtain a fermented feed additive.

[0081] Example 5

[0082] 5 kg of puffed corn protein powder in Example 3, 2 kg of bran, 2 kg of soybean meal, 5 kg of corn germ and 2 kg of corn steep liquor were mixed and hydrated to a moisture content of 50%, and 150 g of the composite bacterial agent obtained in Example 2 was inoculated. Acid protease was added according to the mass of the pre-fermented feed in an amount of 25 U / g. The mixture was placed in a one-way valve breathable bag, sealed and fermented at 35° C. for 5 days to obtain a fermented feed additive.

[0083] Example 6

[0084] 3 kg of puffed corn protein powder of Example 3, 0.5 kg of bran, 0.5 kg of soybean meal, 3 kg of corn germ and 0.5 kg of corn syrup were mixed and hydrated to a moisture content of 30%, and 80 g of the composite bacterial agent obtained in Example 2 was inoculated. Acid protease was added according to the mass of the pre-fermented feed in an amount of 25 U / g. The mixture was placed in a one-way valve breathable bag, sealed and fermented at 30° C. for 7 days to obtain a fermented feed additive.

[0085] Example 7

[0086] Detection of Fermented Feed Additives

[0087] The contents of soluble protein and glutamine in Example 4 were detected before and after fermentation.

[0088] The soluble protein content was determined by the Folin-phenol method, and the glutamine content was determined by the BTI ([bis(trifluoroacetoxy)iodo]benzene) protection method.

[0089] After testing, the fermented feed additive prepared in Example 4 of the present invention has a soluble protein content increased from 81.2 mg / g to 140.5 mg / g compared to before fermentation; the glutamine content is increased from 1.2 mg / g to 12.1 mg / g, and the glutamine content in the fermented feed additive can reach 8.6% of the total soluble protein content.

[0090] Example 8

[0091] Animal Experiments on Fermented Feed Additives

[0092] 1. Animal Experiment Design

[0093] The experimental design was divided into 6 groups, including blank control LPS-group (normal diet), blank control + LPS group, 15% soybean meal replaced LPS-group, 15% soybean meal replaced + LPS group, 30% soybean meal replaced LPS-group, and 30% soybean meal replaced + LPS group. 144 1-day-old Arbor Acres (AA) broilers were selected and divided into 6 treatments, with 4 replicates in each treatment and 6 chickens in each replicate.

[0094] The commercial diet (Zhengda 510H broiler feed approval number: Q / HFCT01-2019) was fed for 7 days, and pre-fed according to the group diet after the 8th day, and formal feeding began on the 15th day. On the 14th, 16th, 18th, and 20th days of the experiment, 12 broilers were selected from each group and injected with LPS solution at a dose of 500μg / kg body weight LPS. In addition, 12 chickens were selected from each group and injected with an equal amount of saline. The feeding experiment was carried out in the chicken house of the South Campus of Qiqihar University for 28 days, which was divided into a non-LPS injection period (non-stress period, 1-14 days), an LPS injection period (stress period, 15-21 days), and a recovery period (22-28 days). The broilers were raised in cages, with free access to food and water. The temperature of the chicken house was maintained at 33°C for the first three days of the experiment, and then lowered by 3°C every week, and the light was controlled according to daylight.

[0095] Table 1 Animal experiment design and grouping

[0096]

[0097] Note: The feeds of each group during the 15-21d stress period are shown in Table 2; the fermented feed additive is the fermented feed additive prepared in Example 4 of the present invention.

[0098] 2. Diet composition and nutritional level in animal experiments

[0099] The energy and protein content of broiler diets was formulated according to the NRC (1994) standard of the United States and the NY / T 33-2004 chicken feeding standard of the agricultural industry standard of the People's Republic of China. The composition and nutritional level of each nutrient in the diet are shown in Table 2.

[0100] Table 2 Diet composition and nutritional level of each group (air-dried basis)

[0101]

[0102] Note: The premix provides per kilogram of diet: VA6141.5 IU; VD31789 U; VE7.80 mg; VB10.66 mg; VB23.95 mg; VB62.01 mg; VB120.01 mg; niacin 18.06 mg; calcium pantothenate 6.75 mg; folic acid 0.59 mg; biotin 0.07 mg; choline 332.28 mg; iron 60.91 mg; copper 6.0 mg; zinc 65.8 mg; manganese 62.5 mg; iodine 0.6 mg; selenium 0.20 mg.

[0103] 3. Determination of growth performance of Arbor Acres (AA) broilers

[0104] The initial body weight at day 1 was recorded, and feed was collected for each treatment group on days 14, 21, and 28, and the broilers were weighed.

[0105] The average daily gain (ADG), average daily feed intake (ADFI) and feed: gain ratio (F / G) were calculated.

[0106] Among them, ADG = (final weight - initial weight) / days; ADFI = (feed consumption / number of broilers) / days; F / G = ADFI / ADG.

[0107] The specific results are shown in Table 3 and Figure 1-Figure 4 .

[0108] Table 3 Effects of each feed group on broiler growth performance

[0109]

[0110]

[0111] From Table 3 and Figure 1-Figure 4 It can be seen that adding 15% and 30% fermented feed additives to the diet can improve the growth performance of broilers. Compared with the ordinary diet group, the body weight, daily weight gain and daily feed intake of broilers in the 15% and 30% soybean meal replacement groups increased significantly during the entire growth period (1-28d), and the body weight, daily weight gain and daily feed intake of the 15% soybean meal replacement group were higher than those of the 30% soybean meal replacement group. The above results show that adding the fermented feed additive of the present invention can improve the growth performance of broilers.

[0112] 4. Determination of the villi morphology of the small intestine of AA broiler chickens

[0113] The integrity of the animal intestinal barrier is the basis for the normal physiological function of the intestine. The height of intestinal villi and the depth of crypts are important indicators for measuring the health of the intestinal barrier.

[0114] The intestinal villus height and crypt depth of each group of broiler chickens were tested respectively. The specific results are shown in Table 4 and Figure 5-Figure 7 .

[0115] The specific detection method is: separate the intestines, take a small piece of the duodenum, jejunum and middle ileum, and fix it in 4% paraformaldehyde solution for the detection of villus height and crypt depth in the broiler intestine.

[0116] Table 4 Effects of each feed group on intestinal morphology of broiler chickens

[0117]

[0118]

[0119] From Table 4 and Figure 5-Figure 7 It can be seen that compared with the normal diet group, the height of the duodenal villi of broilers in the 15% and 30% soybean meal replacement groups increased by 141.1μm and 79.5μm, the height of the jejunal villi increased by 134.5μm and 55.5μm, the depth of the crypts in the duodenum decreased by 21.9μm and 30.0μm, the depth of the crypts in the jejunum decreased by 8.0μm and 21.9μm, the crypt ratio of the duodenum increased by 3.0 and 3.4, and the crypt ratio of the jejunum increased by 1.6 and 3.6. The above research results show that the addition of 15% and 30% fermented feed additives to the diet can promote the development of small intestinal villi in broilers. Injection of LPS leads to a decrease in the height of the villi and the villus-crypt ratio of the duodenum, jejunum, and ileum of broilers, and an increase in the depth of the crypts in the duodenum and jejunum. After LPS treatment, the depth of duodenal crypts in the blank control group increased by 53.2 μm, the 30% soybean meal replacement group increased by 17.5 μm, and the 15% replacement group did not change; the duodenal villus-crypt ratio of broilers in the blank control group decreased by 4.1, and the 15% replacement group and 30% replacement group decreased by 1.3 and 3.4, respectively. The above results show that the addition of 15% and 30% fermented feed additives to the diet can alleviate the damage to the intestinal villi morphology of broilers caused by LPS.

[0120] 5. Determination of relative length of small intestine of AA broiler chickens

[0121] The relative length of the small intestine of each group of broiler chickens was tested respectively. The specific results are shown in Table 5 and Figure 8 .

[0122] The method for detecting the relative length of the small intestine is: separate the intestine and measure the length of the broiler's intestine with a tape measure; the relative length of the broiler's small intestine (cm / 100g BW) = small intestine length (cm) / body weight (100g).

[0123] Table 5 Effects of each feed group on the relative length of the small intestine of broiler chickens

[0124]

[0125] From Table 5 and Figure 8 It can be seen that compared with the normal diet group, the relative length of the small intestine of broilers in the 15% and 30% soybean meal replacement groups increased by 1.9 and 2.1 cm / 100g BW at 28 days, respectively, and there was no significant difference between the 15% and 30% soybean meal replacement groups. The injection of LPS caused a decrease in the relative length of the small intestine of broilers. The relative length of the small intestine of broilers in the normal diet group, 15% soybean meal replacement group and 30% soybean meal replacement group decreased by 1.6, 0.9 and 0.2 cm / 100g BW, respectively. Compared with the normal diet group, the 15% and 30% soybean meal replacement groups decreased less, and the 30% soybean meal replacement group was better than the 15% soybean meal replacement group. The above results show that feeding 15% and 30% fermented feed additives can increase the relative length of the small intestine of broilers, promote the development of the small intestine of broilers, and alleviate the reduction in the length of the small intestine caused by LPS.

[0126] 6. Determination of inflammatory factors in jejunal tissue of AA broiler chickens

[0127] Inflammatory factors are important signaling molecules of the body's inflammatory response, and the severity of enteritis is closely related to their expression levels.

[0128] The contents of inflammatory factors in the jejunum tissue of broiler chickens in each group were detected. The specific results are shown in Table 6 and Figure 9-11 .

[0129] The method for detecting the content of inflammatory factors in jejunal tissue is as follows: take the middle section of jejunum of broiler chickens, remove the intestinal chyme with normal saline, grind it and use ELISA quantitative kit to determine the content of inflammatory factors.

[0130] Table 6 Effects of each group of feed on the content of inflammatory factors in broiler chickens

[0131]

[0132] From Table 6 and Figure 9-11 It can be seen that compared with the LPS treatment group, the IL-6 of the normal diet group, the 15% soybean meal replacement group and the 30% soybean meal replacement group increased by 7.2, 4.6 and 4.4 pg / mL, respectively, and TNF-α increased by 20.5, 10.9 and 15.1 pg / mL, respectively; after LPS treatment, the anti-inflammatory factor IL-10 of the normal diet group was significantly reduced, and the anti-inflammatory factor of the 15% and 30% soybean meal replacement groups was significantly increased; the above results show that adding 15% and 30% fermented feed additives to the diet can increase the content of anti-inflammatory factors in the jejunal tissue of broiler chickens and alleviate the increase in the content of pro-inflammatory factors in the jejunal tissue caused by LPS. The research results show that feeding 15% and 30% fermented feed additives can alleviate the jejunal inflammatory response caused by LPS at the cellular level and maintain the health of the small intestine.

[0133] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composite bacterial agent for preparing a fermented feed additive for antagonizing intestinal damage in broiler chickens, characterized in that: The composite bacterial agent is prepared from a bacterial solution of Bacillus subtilis YY-12, a bacterial solution of Saccharomyces cerevisiae YY-14 and a bacterial solution of Lactobacillus plantarum YY-7; The Bacillus subtilis YY-12 is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.24696; The cerevisiae yeast YY-14 is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number of CGMCC No.24698; The plant lactobacillus YY-7 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, with the deposit number being CGMCC No.24691.

2. The composite bacterial agent according to claim 1, characterized in that: The preparation method of the Bacillus subtilis YY-12 bacterial liquid comprises: inoculating the Bacillus subtilis in an LB culture medium at an inoculum amount of 0.5-1.5%, and fermenting for 14-18 hours at 35-40° C. and a rotation speed of 150-200 r / min; The preparation method of the yeast YY-14 bacterial liquid comprises: inoculating the yeast YY-14 in a YPD medium at an inoculation rate of 0.5-1.5%, and fermenting for 14-18 hours at 32-38° C. and a rotation speed of 150-200 r / min; The preparation method of the plant lactobacillus YY-7 bacterial liquid comprises: inoculating the plant lactobacillus YY-7 in an MRS culture medium at an inoculation rate of 0.5-1.5%, and fermenting at 35-40° C. for 18-20 hours.

3. The composite bacterial agent according to claim 1 or 2, characterized in that: The number of cells of the Bacillus subtilis YY-12 bacterial liquid is (1.0-2.0)×10 8 CFU / mL, the number of cells of the Saccharomyces cerevisiae YY-14 bacterial solution is (1.0-2.0)×10 7 CFU / mL, the bacterial count of the plant lactobacillus YY-7 bacterial liquid is (1.0-3.0)×10 7 CFU / mL; the volume ratio of the Bacillus subtilis YY-12 bacterial solution, the Saccharomyces cerevisiae YY-14 bacterial solution and the Lactobacillus plantarum YY-7 bacterial solution is (1-3):(1-3):(1-3).

4. A fermented feed additive for antagonizing intestinal damage in broiler chickens, characterized in that: The invention comprises the following raw materials in parts by weight: 3-5 parts of puffed corn protein powder, 0.5-2 parts of bran, 0.5-2 parts of soybean meal, 3-5 parts of corn germ meal, 0.5-2 parts of corn steep liquor and 0.05-0.15 parts of the composite bacterial agent according to any one of claims 1 to 3.

5. The method for preparing the fermented feed additive according to claim 4, characterized in that: include: The pre-fermented feed is obtained by mixing puffed corn protein powder, bran, soybean meal, corn germ meal and corn steep liquor, inoculating a composite bacterial agent, adding acidic protease, and sealing and fermenting at 30-35° C. for 5-7 days to obtain the fermented feed additive; The preparation method of the puffed corn gluten powder comprises: extruding and puffing the corn gluten powder at 150-160° C. for 5-10 seconds.

6. The preparation method according to claim 5, characterized in that: The acidic protease is added according to the mass of the pre-fermented feed, and the added amount of the acidic protease is 20-30 U / g.

7. The preparation method according to claim 5, characterized in that: The water content of the pre-fermented feed is 40-50%.

8. Use of the fermented feed additive according to claim 4 or the fermented feed additive obtained by the preparation method according to any one of claims 5 to 7 in the preparation of a product for antagonizing intestinal damage in broiler chickens.

9. Use of the fermented feed additive according to claim 4 or the fermented feed additive prepared by any one of claims 5 to 7 in replacing soybean meal feed.

10. The use according to claim 8 or 9, characterized in that: The added amount of the fermented feed additive is 15%-30%.

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