Feed additive taking corn DDGS (distillers dried grains with soluble) as main raw material as well as preparation method and application of feed additive

Abstract

The invention provides a feed additive taking corn DDGS as a main raw material as well as a preparation method and application of the feed additive, and belongs to the technical field of feed additives. The feed additive with corn DDGS as the main raw material provided by the invention comprises the following raw materials: a fermentation substrate, a complex microbial inoculant and cellulase, the fermentation substrate comprises dry substances and water, and the dry substances comprise corn DDGS, corn germ meal and bran; the complex microbial inoculants comprise bacillus licheniformis YY-1, lactobacillus plantarum YY-9 and saccharomyces cerevisiae YY-4. According to the feed additive, the additional value of corn DDGS can be increased, the application range of corn DDGS in the breeding field is widened, the raw materials are wide in source and low in cost, and the situation that protein feed is tense at present can be relieved. In addition, the feed additive has various effects of improving animal growth performance, improving animal immunity, increasing feed utilization rate, regulating animal intestinal flora, further improving intestinal micro-ecological environment and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of feed additives, and in particular relates to a feed additive using corn DDGS as a main raw material, and a preparation method and application thereof. Background Art

[0002] One of the main problems currently facing the development of animal husbandry is the low utilization rate of feed raw materials, which leads to a shortage of raw materials, especially protein raw materials. In addition to exploring new sources of feed raw materials, improving the utilization rate of existing feed raw materials is the main way to alleviate this problem. Announcement No. 194 of the Ministry of Agriculture and Rural Affairs stipulates that feed production enterprises will stop producing commercial feed containing growth-promoting drug feed additives (except traditional Chinese medicine) from July 1, 2020. Against the background of "antibiotic ban" in feed, the breeding industry faces severe problems with animal intestinal health. Therefore, the focus of research on livestock and poultry diets to optimize intestinal health and animal production performance has shifted from antibiotics to antibiotic substitutes.

[0003] DDGS is a byproduct of ethanol production from corn kernels, with an annual production of approximately 4 to 5 million tons. It is primarily used as a feed ingredient. DDGS has a protein content of approximately 26%, but it is primarily composed of alcohol-soluble proteins and gluten, making it poorly water-soluble. Furthermore, due to the high processing and drying temperatures, the protein in DDGS is poorly digestible and inefficient, limiting the release of functional amino acids such as glutamine. DDGS also has a high crude fiber content, making it inefficient and unpalatable when used directly as feed. For these reasons, DDGS is often added to animal feed in very small amounts.

[0004] Biofermentation is an effective method for improving the nutritional value of feed ingredients and reducing anti-nutritional factors. Biofermentation can enhance the digestibility and utilization of DDGS nutrients, improving animal growth performance. This is crucial for increasing the added value of corn DDGS and broadening its application in animal husbandry. However, since the fermentable sugars in corn are already utilized in alcohol production, the resulting byproduct, DDGS, often hinders the rapid growth of bacteria, yeast, and other organisms when used as a main culture medium. Summary of the Invention

[0005] In view of this, one of the objects of the present invention is to provide a feed additive with corn DDGS as the main raw material and a preparation method thereof, which has the advantages of reducing feed costs, improving feed utilization, and improving animal growth performance and immunity.

[0006] The second object of the present invention is to provide a feed with the advantages of low feed cost, high feed utilization rate, and improved animal growth performance and immunity.

[0007] The third object of the present invention is to provide the feed additive and the application of the feed.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The invention provides a feed additive with corn DDGS as a main raw material. The raw materials of the feed additive include a fermentation substrate, a composite bacterial agent and cellulase; the fermentation substrate includes dry matter and water, and the dry matter includes corn DDGS, corn germ meal and bran; the mass ratio of the corn DDGS, corn germ meal and bran is (7-9):(0.5-1.5):(0.5-1.5); the composite bacterial agent includes Bacillus licheniformis YY-1 with a preservation number of CGMCC No. 13109, Lactobacillus plantarum YY-9 with a preservation number of CGMCC No. 24693 and Saccharomyces cerevisiae YY-4 with a preservation number of CGMCC No. 21353.

[0010] Preferably, the volume ratio of the seed liquid of Bacillus licheniformis YY-1, Lactobacillus plantarum YY-9 and Saccharomyces cerevisiae YY-4 in the composite bacterial agent is (8-10): (4-5): (3-4); the OD of the seed liquid of Bacillus licheniformis YY-1 is 600nm The value is 0.5~0.6; the seed liquid OD of Lactobacillus plantarum YY-9 600nm The value is 0.5-0.6; the seed liquid OD of Saccharomyces cerevisiae YY-4 600nm The value is 0.5~0.6.

[0011] Preferably, the addition amount of the composite bacterial agent is 8 to 11 mL per 100 g of fermentation substrate.

[0012] Preferably, the mass ratio of the dry matter to water is 100:35-50.

[0013] Preferably, the added amount of the cellulase is 40 to 50 U per 1 g of fermentation substrate.

[0014] The present invention also provides a method for preparing the feed additive, comprising the following steps: mixing a fermentation substrate, a composite bacterial agent and cellulase, and performing anaerobic fermentation to obtain the feed additive.

[0015] Preferably, the temperature of the anaerobic fermentation is 26-32° C., and the time is 3-7 days.

[0016] The present invention also provides a feed comprising the above-mentioned feed additive and a basic diet; the mass percentage of the feed additive in the feed is 5% to 15%, and the mass percentage of the basic diet in the feed is 85% to 95%.

[0017] Preferably, the nutritional content of the basal diet complies with the group standard T / CFIAS002-2018 for compound feed for laying hens and broilers.

[0018] The present invention also provides the use of the above-mentioned feed additive or the above-mentioned feed in any of the following: (1) improving animal growth performance; (2) improving animal immunity; (3) improving feed utilization; (4) regulating animal intestinal flora; (5) preparing products for preventing and treating animal diarrhea; (6) preparing products for preventing and treating animal inflammation; and (7) alleviating intestinal damage.

[0019] Beneficial effects of the present invention:

[0020] The feed additive provided by this invention uses corn DDGS, a byproduct of alcohol production, as its primary raw material, increasing the added value of corn DDGS and broadening its application in animal husbandry. Furthermore, the additive uses corn germ meal, a byproduct of corn starch wet processing, and bran, a byproduct of flour processing, as auxiliary raw materials, resulting in a wide range of raw material sources and low costs, helping to alleviate the current shortage of protein feed. Furthermore, the feed additive provided by this invention has multiple benefits, including improving animal growth performance, immunity, and feed utilization, as well as regulating the intestinal flora and thus improving the intestinal microecological environment.

[0021] The feed provided by the present invention uses the feed additive to replace a portion of the basic diet, thereby reducing the amount of the basic diet and thus reducing the cost of the feed. The feed provided by the present invention can improve animal growth performance, enhance animal immunity, and increase feed utilization, and can also regulate the intestinal flora of animals and thus improve the intestinal microecological environment.

[0022] Preservation Instructions

[0023] The Bacillus licheniformis YY-1 of the present invention is deposited in the General Microbiology Center of the China Culture Collection Administration Committee of Microorganisms, and is classified and named Bacillus licheniformis. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a deposit number of CGMCC No. 13109 and a deposit date of October 13, 2016.

[0024] The plant lactobacillus YY-9 of the present invention is deposited in the General Microbiology Center of the China Culture Collection Administration Committee of Microorganisms, classified and named Lactobacillus plantarum, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC No. 24693, and the deposit date is April 18, 2022.

[0025] The brewer's yeast YY-4 of the present invention is deposited in the General Microorganism Center of the China Culture Collection Administration, and is classified and named as brewer's yeast Saccharomyces cerevisiae. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 21353, and the deposit date is December 11, 2020. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The bar graph shows relative abundance of species at the phylum level;

[0027] Figure 2 The bar graph shows relative abundance of species at the genus level;

[0028] Figure 3 The results of Bacillus screening are shown in Figure 2. Different letters on the columns indicate significant differences when comparing different groups (P < 0.05).

[0029] Figure 4 The results of screening of Lactobacillus plantarum are shown. If the letters on the columns are different, it indicates significant difference (P < 0.05).

[0030] Figure 5 The results of screening of Saccharomyces cerevisiae strains are shown in Figure 2. Different letters on the columns indicate significant differences when comparing different groups, with P < 0.05.

[0031] Figure 6 The results are from the cooperative fermentation of multiple bacteria. When different groups are compared, if the letters on the columns are different, it indicates a significant difference, P < 0.05. DETAILED DESCRIPTION

[0032] The invention provides a feed additive with corn DDGS as a main raw material. The raw materials of the feed additive include a fermentation substrate, a composite bacterial agent and cellulase; the fermentation substrate includes dry matter and water, and the dry matter includes corn DDGS, corn germ meal and bran; the mass ratio of the corn DDGS, corn germ meal and bran is (7-9):(0.5-1.5):(0.5-1.5); the composite bacterial agent includes Bacillus licheniformis YY-1 with a preservation number of CGMCC No. 13109, Lactobacillus plantarum YY-9 with a preservation number of CGMCC No. 24693 and Saccharomyces cerevisiae YY-4 with a preservation number of CGMCC No. 21353.

[0033] The present invention is not particularly limited to the specific sources of the raw materials in the feed additive, and conventional commercial products in the art can be used. In the present invention, the addition amount of the composite bacterial agent is preferably 8 to 11 mL per 100 g of fermentation substrate. In some embodiments, 8 mL, 9 mL, 10 mL or 11 mL is added per 100 g of fermentation substrate. In the present invention, the seed liquid volume ratio of Bacillus licheniformis YY-1, Lactobacillus plantarum YY-9 and Saccharomyces cerevisiae YY-4 in the composite bacterial agent is preferably (8 to 10): (4 to 5): (3 to 4). In some embodiments of the present invention, the seed liquid volume ratio of Bacillus licheniformis YY-1, Lactobacillus plantarum YY-9 and Saccharomyces cerevisiae YY-4 is 8:4:3, 8:5:3, 8:5:4, 8:4:4, 9:4:3, 9:4:4, 9:5:3, 9:5:4, 10:4:3, 10:4:4, 10:5:3 or 10:5:4. In the present invention, the OD of the seed solution of Bacillus licheniformis YY-1 is 600nm The value is preferably 0.5 to 0.6, and in some embodiments may be 0.5, 0.55 or 0.6; the OD of the seed solution of Lactobacillus plantarum YY-9 600nm The value is preferably 0.5 to 0.6, and in some embodiments may be 0.5, 0.55 or 0.6; the OD of the seed solution of Saccharomyces cerevisiae YY-4 600nm The value is preferably 0.5 to 0.6, and in some embodiments may be 0.5, 0.55 or 0.6. In the present invention, the method for preparing the seed liquid of Bacillus licheniformis YY-1, Lactobacillus plantarum YY-9 and Saccharomyces cerevisiae YY-4 preferably comprises the following steps: activating Bacillus licheniformis YY-1, Lactobacillus plantarum YY-9 and Saccharomyces cerevisiae YY-4 respectively, inoculating the activated strains into the corresponding liquid culture medium and culturing them until the OD 600nm The value is 0.5 to 0.6. In the present invention, the activation culture medium and liquid culture medium of Bacillus licheniformis YY-1 are preferably LB solid culture medium and LB liquid culture medium, respectively, the activation culture medium and liquid culture medium of Lactobacillus plantarum YY-9 are preferably MRS solid culture medium and MRS liquid culture medium, respectively, and the activation culture medium and liquid culture medium of Saccharomyces cerevisiae YY-4 are preferably YPD solid culture medium and YPD liquid culture medium, respectively. In the present invention, the temperature of the activation culture and seed culture of Bacillus licheniformis YY-1 is preferably 28 to 30°C, and in some embodiments, it can be 28°C, 29°C or 30°C; the temperature of the activation culture and seed culture of Lactobacillus plantarum YY-9 is preferably 28 to 30°C, and in some embodiments, it can be 28°C, 29°C or 30°C; the temperature of the activation culture and seed culture of Saccharomyces cerevisiae YY-4 is preferably 28 to 30°C, and in some embodiments, it can be 28°C, 29°C or 30°C.

[0034] In the present invention, the mass ratio of dry matter to water is preferably 100:35-50, and in some embodiments, it can be 100:35, 100:40, 100:45, or 100:50. In the present invention, the amount of cellulase added is preferably 40-50 U per 1 g of fermentation substrate, and in some embodiments, it can be 40 U, 42 U, 44 U, 46 U, 48 U, or 50 U per 1 g of fermentation substrate.

[0035] The present invention also provides a method for preparing the feed additive, comprising the following steps: mixing a fermentation substrate, a composite bacterial agent and cellulase, and performing anaerobic fermentation to obtain the feed additive.

[0036] In the present invention, the temperature of the anaerobic fermentation is preferably 26-32°C, and in some embodiments, it can be 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, or 32°C; the time of the anaerobic fermentation is preferably 3-7 days, and in some embodiments, it can be 3 days, 4 days, 5 days, 6 days, or 7 days. In some embodiments of the present invention, the anaerobic fermentation is carried out in a 2kg-25kg one-way valve breathing fermentation bag.

[0037] In view of the high fiber content in corn DDGS, the present invention adopts a composite bacterial agent and cellulase synergistic fermentation method to prepare feed additives. Through the action of microorganisms and enzymes, the protein, fat, fiber and other components in the raw materials are degraded, so that the soluble protein content and polypeptide content are significantly increased, the crude fat and crude fiber content are significantly reduced, and the antioxidant activity of the feed additive is significantly improved. The present invention adopts a bagged bacterial enzyme synergistic raw material solid fermentation method to produce feed additives, which can simultaneously exert the synergistic effect of microorganisms and hydrolases. The bagged raw material fermentation does not require heat-consuming culture medium sterilization treatment, and the fermentation method is suitable for feed production enterprises to start production. The feed additive prepared by the present invention is loose and soft in appearance, exudes a pleasant sweet and sour taste, and improves the palatability of the feed. The main technical indicators of the product obtained by the method of the present invention after inspection are as follows: crude protein content of the product: 28.10% to 28.94%; soluble protein content of the product: 74.88 to 77.11 mg / g; polypeptide content of the product: 60.07 to 61.2 mg / g; crude fat content of the product: 7.18% to 8.22%; crude fiber content of the product: 5.25% to 5.81%; ash content of the product: 4.79% to 5.49%; and viable bacterial count in the product: 4.1 to 5.7×10 7 CFU / g.

[0038] The present invention also provides a feed comprising the above-mentioned feed additive and a basic diet; the mass percentage of the feed additive in the feed is 5% to 15%, and the mass percentage of the basic diet in the feed is 85% to 95%.

[0039] The feed of the present invention uses feed additives to replace part of the basic diet, which can reduce feed costs, improve the growth performance and immune function of animals, and improve the intestinal microecological environment. In some embodiments of the present invention, the nutritional component indicators of the basic diet meet the nutritional component index requirements of Table 1 in the group standard T / CFIAS 002-2018 for laying hens and broiler feeds. In some embodiments of the present invention, the basic diet includes the following raw materials in parts by weight: 51 parts of corn, 29 parts of soybean meal, 8 parts of corn DDGS, 1 part of corn germ meal, 1 part of wheat bran, 4.4 parts of soybean oil, 2 parts of stone powder, 1 part of calcium bicarbonate, 1.2 parts of premix, 0.99 parts of DL-methionine, 0.18 parts of lysine, and 0.23 parts of salt.

[0040] The present invention also provides the use of the above-mentioned feed additive or the above-mentioned feed in any of the following: (1) improving animal growth performance; (2) improving animal immunity; (3) improving feed utilization; (4) regulating animal intestinal flora; (5) preparing products for preventing and treating animal diarrhea; (6) preparing products for preventing and treating animal inflammation; and (7) alleviating intestinal damage.

[0041] In the present invention, the animal preferably includes broilers. In some embodiments, the broilers are white-feathered broilers. In the present invention, the growth performance preferably includes body weight and feed-to-weight ratio. In some embodiments, the animal diarrhea refers to loose stools caused by intestinal oxidative stress, preferably LPS-induced intestinal oxidative stress.

[0042] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0043] In the following examples, unless otherwise specified, all methods are conventional.

[0044] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0045] In the following examples, DDGS (a byproduct of alcohol production) was purchased from COFCO Biochemical Energy (Zhaodong) Co., Ltd., and cellulase was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd. Corn germ meal (a byproduct of corn starch wet processing) and bran (a byproduct of flour processing) were commercially available.

[0046] The culture medium used in the following examples was prepared as follows:

[0047] LB solid medium was composed of 10 g of tryptone, 5 g of yeast powder, 10 g of sodium chloride, and 20 g of agar powder, which were dissolved in distilled water and then made up to 1000 mL. The pH was adjusted to 6.8, and the medium was sterilized at 121°C for 30 min.

[0048] LB liquid medium was composed of 10 g of tryptone, 5 g of yeast powder, and 10 g of sodium chloride, which was dissolved in distilled water and then made up to 1000 mL. The pH was adjusted to 6.8 and sterilized at 121°C for 30 min.

[0049] MRS solid medium consists of 10 g of peptone, 5 g of yeast extract, 20 g of glucose, 5 g of sodium acetate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate, 2 g of dipotassium hydrogen phosphate, 1 mL of Tween 80, 2 g of diammonium hydrogen citrate, 10 g of beef extract, and 20 g of agar. The medium is dissolved in water to a volume of 1000 mL, the pH is adjusted to 6.2, and the medium is sterilized at 121°C for 30 min.

[0050] MRS liquid medium consists of 10 g of peptone, 5 g of yeast extract, 20 g of glucose, 5 g of sodium acetate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate, 2 g of dipotassium hydrogen phosphate, 1 mL of Tween 80, 2 g of diammonium hydrogen citrate, and 10 g of beef extract. The medium is dissolved in water to a volume of 1000 mL, the pH is adjusted to 6.2, and the medium is sterilized at 121°C for 30 min.

[0051] YPD solid medium is composed of 10 g yeast extract, 20 g tryptone, 20 g glucose, and 20 g agar powder, dissolved in water to a volume of 1000 mL, with natural pH, and sterilized at 121°C for 30 min.

[0052] YPD liquid medium is composed of 10 g of yeast extract, 20 g of tryptone, and 20 g of glucose, dissolved in water to a volume of 1000 mL, with a natural pH, and sterilized at 121°C for 30 min.

[0053] Example 1

[0054] A feed additive with corn DDGS as the main raw material, which is made by fermentation of fermentation substrate, compound bacterial agent and cellulase;

[0055] The fermentation substrate consists of dry matter and water, the dry matter consists of corn DDGS, corn germ meal and bran in a mass ratio of 9:1.5:1.5, and the mass ratio of water to dry matter is 50g:100g;

[0056] The volume mass ratio of the composite bacterial agent to the fermentation substrate is 11 mL:100 g;

[0057] The amount of cellulase added is 50U per 1g of fermentation substrate;

[0058] The composite bacterial agent is composed of Bacillus licheniformis YY-1 seed liquid, Lactobacillus plantarum YY-9 seed liquid and Saccharomyces cerevisiae YY-4 seed liquid in a volume ratio of 8:5:4; the OD of the Bacillus licheniformis YY-1 seed liquid is 0.01477 W / m. 600nmThe value is 0.5, the OD of Lactobacillus plantarum YY-9 seed solution 600nm The value is 0.5, the OD of the seed solution of Saccharomyces cerevisiae YY-4 600nm The value is 0.5;

[0059] OD 600nm The preparation process of the seed solution of Bacillus licheniformis YY-1 with an OD value of 0.5 is as follows: first, Bacillus licheniformis YY-1 is inoculated onto LB solid medium for slant culture; the slant culture conditions are: culture at 35°C for 2 days. Then, the slant cultured Bacillus licheniformis YY-1 is picked and inoculated into LB liquid medium for seed culture; the seed culture conditions are: culture at 35°C until the seed solution has an OD value of 0.5. 600nm The value is 0.5, and the seed solution of Bacillus licheniformis YY-1 is obtained;

[0060] OD 600nm The preparation process of the seed liquid of Lactobacillus plantarum YY-9 with an OD value of 0.5 is as follows: first, Lactobacillus plantarum YY-9 is inoculated onto MRS solid medium for slant culture; the slant culture condition is cultured at 35°C for 1 day. Then, the slant cultured Lactobacillus plantarum YY-9 is picked and inoculated into MRS liquid medium for seed culture; the seed culture condition is cultured at 35°C until the seed liquid reaches an OD value of 0.5. 600nm The value is 0.5, and the seed liquid of Lactobacillus plantarum YY-9 is obtained.

[0061] OD 600nm The preparation process of the seed liquid of Saccharomyces cerevisiae YY-4 with an OD value of 0.5 is as follows: Saccharomyces cerevisiae YY-4 is inoculated onto YPD solid medium for slant culture; the slant culture condition is incubated at 30°C for 1 day. Then, the slant cultured Saccharomyces cerevisiae YY-4 is picked and inoculated into YPD liquid medium for seed culture; the seed culture condition is incubated at 30°C and 180r / min until the seed liquid reaches an OD value of 0.5. 600nm The value is 0.5, and the seed liquid of brewer's yeast YY-4 is obtained.

[0062] The preparation method of the feed additive is:

[0063] The fermentation substrate, compound bacterial agent and cellulase were mixed and placed into a 10 kg one-way valve breathing bag, and anaerobically fermented at 26°C for 7 days to obtain the feed additive.

[0064] Example 2

[0065] A feed additive with corn DDGS as the main raw material, which is made by fermentation of fermentation substrate, compound bacterial agent and cellulase;

[0066] The fermentation substrate consists of dry matter and water, the dry matter consists of corn DDGS, corn germ meal and bran in a mass ratio of 7:0.5:0.5, and the mass ratio of water to dry matter is 35g:100g;

[0067] The volume mass ratio of the composite bacterial agent to the fermentation substrate is 8 mL:100 g;

[0068] The amount of cellulase added is 40U per 1g of fermentation substrate;

[0069] The composite bacterial agent is composed of Bacillus licheniformis YY-1 seed liquid, Lactobacillus plantarum YY-9 seed liquid and Saccharomyces cerevisiae YY-4 seed liquid in a volume ratio of 9:5:3; the OD of the Bacillus licheniformis YY-1 seed liquid is 0. 600nm The value is 0.5, the OD of Lactobacillus plantarum YY-9 seed solution 600nm The value is 0.5, the OD of the seed solution of Saccharomyces cerevisiae YY-4 600nm The preparation method of various sub-liquids is the same as that in Example 1.

[0070] The preparation method of the feed additive is:

[0071] The fermentation substrate, water, compound bacterial agent and cellulase were mixed and placed into a 2 kg one-way valve breathing bag, and anaerobically fermented at 32°C for 3 days to obtain the feed additive.

[0072] Example 3

[0073] A feed additive with corn DDGS as the main raw material, which is made by fermentation of fermentation substrate, compound bacterial agent and cellulase;

[0074] The fermentation substrate consists of dry matter and water, the dry matter consists of corn DDGS, corn germ meal and bran in a mass ratio of 8:1.5:1.5, and the mass ratio of water to dry matter is 40g:100g;

[0075] The volume mass ratio of the composite bacterial agent to the fermentation substrate is 9 mL:100 g;

[0076] The amount of cellulase added is 45U per 1g of fermentation substrate;

[0077] The composite bacterial agent is composed of Bacillus licheniformis YY-1 seed liquid, Lactobacillus plantarum YY-9 seed liquid and Saccharomyces cerevisiae YY-4 seed liquid in a volume ratio of 9:4:4; the OD of the Bacillus licheniformis YY-1 seed liquid is 0.01477 W / m. 600nm The value is 0.5, the OD of Lactobacillus plantarum YY-9 seed solution 600nm The value is 0.5, the OD of the seed solution of Saccharomyces cerevisiae YY-4 600nm The preparation method of various sub-liquids is the same as that in Example 1.

[0078] The preparation method of the feed additive is:

[0079] The fermentation substrate, compound bacterial agent and cellulase were mixed and placed into a 15 kg one-way valve breathing bag, and anaerobically fermented at 30°C for 4 days to obtain the feed additive.

[0080] Example 4

[0081] A feed additive with corn DDGS as the main raw material, which is made by fermentation of fermentation substrate, compound bacterial agent and cellulase;

[0082] The fermentation substrate consists of dry matter and water, the dry matter consists of corn DDGS, corn germ meal and bran in a mass ratio of 8:1:1, and the mass ratio of water to dry matter is 45g:100g;

[0083] The volume mass ratio of the composite bacterial agent to the fermentation substrate is 10 mL:100 g;

[0084] The amount of cellulase added is 42U per 1g of fermentation substrate;

[0085] The composite bacterial agent is composed of Bacillus licheniformis YY-1 seed liquid, Lactobacillus plantarum YY-9 seed liquid and Saccharomyces cerevisiae YY-4 seed liquid in a volume ratio of 10:4:3; the OD of the Bacillus licheniformis YY-1 seed liquid is 0.01477 W / m. 600nm The value is 0.5, the OD of Lactobacillus plantarum YY-9 seed solution 600nm The value is 0.5, the OD of the seed solution of Saccharomyces cerevisiae YY-4 600nm The preparation method of various sub-liquids is the same as that in Example 1.

[0086] The preparation method of the feed additive is:

[0087] The fermentation substrate, compound bacterial agent and cellulase were mixed and placed into a 25 kg one-way valve breathing bag, and anaerobically fermented at 28°C for 5 days to obtain the feed additive.

[0088] The nutritional components in the feed additives obtained in Examples 1 to 4 were detected respectively, and the results are shown in Table 1.

[0089] Table 1 Nutritional components of the feed additives obtained in Examples 1 to 4

[0090]

[0091] Example 5

[0092] A feed, comprising 10% by weight of the feed additive obtained in Example 1 and 90% by weight of a basic diet; the basic diet comprises the following raw materials in parts by weight: 51 parts of corn, 29 parts of soybean meal, 8 parts of corn DDGS, 1 part of corn germ meal, 1 part of wheat bran, 4.4 parts of soybean oil, 2 parts of stone powder (main component: calcium carbonate), 1 part of calcium bicarbonate, 1.2 parts of a premix (purchased from Zhuozhou Medbold Biotechnology Development Co., Ltd.), 0.99 parts of DL-methionine, 0.18 parts of lysine, and 0.23 parts of salt.

[0093] Example 6

[0094] A feed comprising 5% by mass of the feed additive obtained in Example 2 and 95% by mass of a basic diet; the composition of the basic diet is the same as that of Example 5.

[0095] Example 7

[0096] A feed comprising 15% by mass of the feed additive obtained in Example 3 and 85% by mass of a basic diet; the composition of the basic diet is the same as that of Example 5.

[0097] Example 8

[0098] A feed comprising 5% by mass of the feed additive obtained in Example 4 and 95% by mass of a basic diet; the composition of the basic diet is the same as that of Example 5.

[0099] Example 9

[0100] A feed comprising 10% by mass of the feed additive obtained in Example 4 and 90% by mass of a basic diet; the composition of the basic diet is the same as that of Example 5.

[0101] Example 10

[0102] A feed comprising 15% by mass of the feed additive obtained in Example 4 and 85% by mass of a basic diet; the composition of the basic diet is the same as that of Example 5.

[0103] Example 11

[0104] White broiler chickens were used as breeding objects, and the feed additives in Example 8 (5% of the basic diet, denoted as F 5Y Group), Example 9 (feed additives replaced 10% of the basic diet, recorded as F 10Y Group) or Example 10 (feed additive replaced 15% of the basic diet, recorded as F 15YThe growth performance, immune indexes and cecal intestinal flora structure of white-feathered broilers were tested after feeding with the feed of the group) for 42 days. The control group (CN) was fed with only the basic diet (without feed additives). The effects of different groups on the growth performance of white-feathered broilers are shown in Table 2. The results showed that F 10Y The group of animals (using the feed additive obtained in Example 4 to replace 10% of the soybean meal in the basic diet) can significantly increase the daily weight gain of broiler chickens and reduce the feed-to-gain ratio (P<0.05).

[0105] Table 2 Effects of different groups on the growth performance of white-feathered broilers

[0106] project CN <![CDATA[F 5Y ]]> <![CDATA[F 10Y ]]> <![CDATA[F 15Y ]]> 1 day old weight / g1dBW 39.63±0.99 39.57±1.27 39.8±1.51 40.1±0.46 21-day-old weight / g21dBW <![CDATA[611.00±0.94 b ]]> <![CDATA[603.00±0.43 d ]]> <![CDATA[617.6±0.61 a ]]> <![CDATA[607.6±0.35 c ]]> Weight at 42 days old / g42dBW <![CDATA[2051±0.04 a ]]> <![CDATA[2120±0.11 a ]]> <![CDATA[2123±0.11 a ]]> <![CDATA[1857±0.02 b ]]> Average daily gain / gADG <![CDATA[47.89±1.04 a ]]> <![CDATA[49.54±2.51 a ]]> <![CDATA[49.61±2.58 a ]]> <![CDATA[43.25±0.37 b ]]> Average daily feed intake / gADFI <![CDATA[63.77 a ]]> <![CDATA[62.19 b ]]> <![CDATA[61.36 c ]]> <![CDATA[60.05 d ]]> Feed weight ratio F / G <![CDATA[1.33 b ]]> <![CDATA[1.26 c ]]> <![CDATA[1.23 d ]]> <![CDATA[1.39 a ]]> Mortality / %Mortality 0 0 0 0

[0107] Note: Data within the same row with different letters indicate significant differences (P < 0.05)

[0108] The effects of different groups on the immune indicators of white-feathered broilers are shown in Tables 3 and 4. The results showed that with the increase in the amount of feed additives added, the levels of IgA, IgM, IgG and sIgA in the serum, jejunum and ileum of broilers were significantly increased (P<0.05) (Table 3); with the increase in the amount of feed additives added, the levels of TNF-α, DAO and IL-2 in the serum, jejunum and ileum were significantly decreased, and the level of IL-4 was significantly increased (P<0.05) (Table 4).

[0109] Table 3 Effects of different groups on immune function of white-feathered broiler chickens

[0110]

[0111]

[0112] Note: Data within a row with different letters in the shoulder indicate significant differences (P<0.05).

[0113] Table 4 Effects of different groups on the levels of inflammatory factors in white-feathered broilers

[0114]

[0115] Note: Data within a row with different letters in the shoulder indicate significant differences (P<0.05).

[0116] The effects of different groups on the structure of cecal intestinal flora in white-feathered broiler chickens are shown in Figure 1 and Figure 2 The results showed that the richness of the cecal microbial flora was significantly increased, mainly composed of Firmicutes and Bacteroidetes, and the Firmicutes / Bacteroidetes (F / B) ratio was significantly higher than that of the unsubstituted soybean meal group (P<0.05).

[0117] Example 12

[0118] White-feathered broiler chickens in an oxidative stress state induced by lipopolysaccharide (LPS) were fed with the feeds of Examples 8 to 10, respectively.

[0119] 320 one-day-old Arbor Echo white broiler chickens were weighed one by one and divided into 5 groups according to the principle of uniform average weight, with four replicates in each group and 16 chickens in each replicate. The 5 groups were CNY group (not injected with LPS and fed with basal diet), LPS group (injected with LPS and fed with basal diet), F 5L Group (Example 8 feed, LPS injection), F 10L Group (Example 9 feed, LPS injection), F 15L Group (Example 10 feed, LPS injection).

[0120] White-feathered broilers were used as breeding subjects. When they were raised to 35 days old, LPS was injected once every two days at a dose of 500 μg / kg. The feed composition of each group remained unchanged during the injection period. A total of 3 injections were given. When they were raised to 42 days old, they were fasted for 12 hours and killed by bleeding from the jugular vein. The serum, jejunum, and ileum of each group of animals were collected to detect immune indicators and inflammatory factors.

[0121] The results are shown in Tables 5 and 6. Compared with the CNY group and LPS group, F 5L Group, F 10L The levels of IgA, IgG, IgM and sIgA in the serum, jejunum and ileum of the animals in group F were significantly increased (P<0.05) (Table 5); 10L Group, F 15L The levels of TNF-α, IL-2 and DAO in the serum of the animals in the F group were significantly decreased, while the level of IL-4 was significantly increased (P<0.05). 5L Group, F 10L The levels of TNF-α, IL-2 and DAO in the jejunum and ileum of the animals in the control group were significantly decreased, and the level of IL-4 was significantly increased (P<0.05) (Table 6).

[0122] Table 5 Effects of different groups on immunoglobulin in lipopolysaccharide-stressed white broiler chicken model

[0123]

[0124] Note: Data within a row with different letters in the shoulder indicate significant differences (P<0.05).

[0125] Table 6 Effects of different groups on the levels of inflammatory factors in lipopolysaccharide-stressed white-feather broiler chicken models

[0126]

[0127]

[0128] Note: Data with different letters in the same row indicate significant differences (P < 0.05)

[0129] Example 13

[0130] Screening of Bacillus:

[0131] The difference from Example 1 is that separate Bacillus bacterial solutions are used for fermentation. The separate Bacillus bacterial solutions refer to the cultivation of subtilis 01003, licheniformis 07008, licheniformis 13109 (Bacillus licheniformis YY-1), subtilis 07009 and Bacillus coagulans to OD 600nm The results were the same as those in Example 1. The unfermented group was used as a control (the bacterial solution and seed solution were replaced by an equal volume of sterile water). The soluble protein and polypeptide contents of the different groups were tested. The results were as follows: Figure 3 As shown, the content of soluble protein and polypeptide produced by Bacillus coagulans was significantly higher than that of other strains (P < 0.05), followed by Bacillus licheniformis 13109 strain (Bacillus licheniformis YY-1).

[0132] Example 14

[0133] Screening of Lactobacillus plantarum:

[0134] The difference from Example 1 is that a separate Lactobacillus plantarum bacterial solution is used for fermentation, wherein the separate Lactobacillus plantarum bacterial solution refers to Lactobacillus plantarum 05008, Lactobacillus plantarum 01002, Lactobacillus plantarum 09002 and Lactobacillus plantarum 13110 (Lactobacillus plantarum YY-9) being cultured to OD 600nm The results were the same as those in Example 1. The unfermented group was used as a control (the bacterial solution and seed solution were replaced by an equal volume of sterile water). The soluble protein and polypeptide contents of the different groups were tested. The results were as follows: Figure 4 As shown, the content of soluble protein and polypeptide produced by Lactobacillus plantarum 13110 (Lactobacillus plantarum YY-9) was significantly higher than that of other strains (P < 0.05).

[0135] Example 15

[0136] Screening of brewer's yeast:

[0137] The difference from Example 1 is that separate Saccharomyces cerevisiae bacterial liquids are used for fermentation, wherein the separate Saccharomyces cerevisiae bacterial liquids refer to Saccharomyces cerevisiae A20, Saccharomyces cerevisiae B20, Saccharomyces cerevisiae 2606 and Saccharomyces cerevisiae 2607 (Saccharomyces cerevisiae YY-4) cultured to OD 600nm The results were the same as those in Example 1. The unfermented group was used as a control (the bacterial solution and seed solution were replaced by an equal volume of sterile water). The soluble protein and polypeptide contents of the different groups were tested. The results were as follows: Figure 5As shown, the content of soluble protein and polypeptide produced by Saccharomyces cerevisiae 2607 (Saccharomyces cerevisiae YY-4) was significantly higher than that of other strains (P < 0.05).

[0138] Example 16

[0139] Determination of the cooperative fermentation ability of strains:

[0140] The four strains (Bacillus coagulans, Bacillus licheniformis 13109 strain (i.e., Bacillus licheniformis YY-1 of the present invention), Lactobacillus plantarum 13110 (i.e., Lactobacillus plantarum YY-9 of the present invention) and Saccharomyces cerevisiae 2607 (i.e., Saccharomyces cerevisiae YY-4 of the present invention)) after the initial screening of Examples 13 to 15 were arranged and combined according to their roles in fermentation, wherein ① Bacillus licheniformis 13109 and Bacillus coagulans were mixed in a volume ratio of 1:1, ② Bacillus licheniformis 13109, Lactobacillus plantarum 13110 and Saccharomyces cerevisiae 2607 were mixed in a volume ratio of 1:1:1, ③ Bacillus coagulans, Lactobacillus plantarum 13110 and Saccharomyces cerevisiae 2607 were mixed in a volume ratio of 1:1:1, and ④ Bacillus licheniformis 1309, Bacillus coagulans, Lactobacillus plantarum 13110 and Saccharomyces cerevisiae 2607 were mixed in a volume ratio of 1:1:1:1. All the above bacteria were cultured to OD 600nm is 0.5, and then mixed according to the corresponding volume ratio.

[0141] The difference from Example 1 is that the composite bacterial agent in Example 1 is replaced by the four groups in this example, and the rest are the same as Example 1. The unfermented group is used as a comparative example (the same volume of sterile water as when the three bacteria are compounded is used instead of the bacterial solution seed liquid). The soluble protein and polypeptide contents of different groups are tested respectively. The results are as follows Figure 6 As shown in the figure, compared with the non-fermented group, mixed fermentation can significantly increase the content of soluble protein and polypeptide (P < 0.05), among which the yield of the product after mixed fermentation in group ② was significantly higher than that in other groups.

[0142] Example 17

[0143] Determination of the ratio of mixed bacteria fermentation seed liquid:

[0144] The difference from Example 1 is that the volume ratios of Bacillus licheniformis YY-1 seed solution, Lactobacillus plantarum YY-9 seed solution, and Saccharomyces cerevisiae YY-4 seed solution in the composite bacterial agent are shown in Table 7, and the rest are the same as in Example 1. The soluble protein content of different groups was tested, and the results are shown in Tables 7 and 8.

[0145] Table 7 Intuitive analysis of orthogonal test of inoculation ratio

[0146]

[0147] Table 7

[0148]

[0149]

[0150] Table 8 Variance analysis table of orthogonal test of inoculation ratio

[0151] factor Sum of Squared Deviations degrees of freedom F ratio F critical value Bacillus licheniformis 2.768 2 1.381 5.140 Lactobacillus plantarum 1.080 2 0.539 5.140 Saccharomyces cerevisiae 2.166 2 1.080 5.140 error 6.01 6

[0152] Note: F 0.05 (2,6)=5.14

[0153] Through orthogonal test analysis of inoculation ratio, the optimal inoculation ratio was determined to be Bacillus licheniformis YY-1 seed solution: Lactobacillus plantarum YY-9 seed solution: Saccharomyces cerevisiae YY-4 seed solution = 10:4:3.

[0154] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A feed additive with corn DDGS as main raw material, characterized in that: The raw materials of the feed additive include a fermentation substrate, a composite bacterial agent and a cellulase; the fermentation substrate includes dry matter and water, and the dry matter includes corn DDGS, corn germ meal and bran; the mass ratio of the corn DDGS, corn germ meal and bran is (7-9):(0.5-1.5):(0.5-1.5); the composite bacterial agent includes Bacillus licheniformis YY-1 with a preservation number of CGMCC No. 13109, Lactobacillus plantarum YY-9 with a preservation number of CGMCC No. 24693 and Saccharomyces cerevisiae YY-4 with a preservation number of CGMCC No. 21353.

2. The feed additive according to claim 1, characterized in that The volume ratio of the seed liquid of Bacillus licheniformis YY-1, Lactobacillus plantarum YY-9 and Saccharomyces cerevisiae YY-4 in the composite bacterial agent is (8-10): (4-5): (3-4); the OD of the seed liquid of Bacillus licheniformis YY-1 is 600nm The value is 0.5~0.6; the seed liquid OD of Lactobacillus plantarum YY-9 600nm The value is 0.5-0.6; the seed liquid OD of Saccharomyces cerevisiae YY-4 600nm The value is 0.5~0.

6.

3. The feed additive according to claim 1, characterized in that The addition amount of the composite bacterial agent is 8 to 11 mL per 100 g of fermentation substrate.

4. The feed additive according to claim 1, characterized in that The mass ratio of the dry matter to water is 100:35-50.

5. The feed additive according to claim 1, characterized in that The added amount of the cellulase is 40 to 50 U per 1 g of fermentation substrate.

6. The method for preparing the feed additive according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: mixing a fermentation substrate, a composite bacterial agent and a cellulase, and performing anaerobically fermentation to obtain a feed additive.

7. The preparation method according to claim 6, characterized in that The temperature of the anaerobic fermentation is 26-32° C., and the time is 3-7 days.

8. A feed, characterized in that The feed comprises the feed additive according to any one of claims 1 to 5 and a basic diet; the mass percentage of the feed additive in the feed is 5% to 15%, and the mass percentage of the basic diet in the feed is 85% to 95%.

9. The feed according to claim 8, characterized in that The nutritional content of the basic diet complies with the group standard T / CFIAS 002-2018 for compound feeds for laying hens and broilers.

10. Use of the feed additive according to any one of claims 1 to 5 or the feed according to any one of claims 8 to 9 in any of the following items, characterized in that: (1) Improve animal growth performance; (2) Improve animal immunity; (3) Improve feed utilization; (4) Regulate animal intestinal flora; (5) Prepare products for preventing and treating animal diarrhea; (6) Prepare products for preventing and treating animal inflammation; (7) Alleviate intestinal damage.

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