Feed additive as well as preparation method and application thereof

Abstract

The invention provides a feed additive as well as a preparation method and application thereof, and belongs to the technical field of fermented feeds. The feed additive provided by the invention comprises a mixed bacterial agent, acid protease and a base material, and the mixed bacterial agent comprises bacillus subtilis YY-18 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.33097, saccharomyces cerevisiae YY-21 with the preservation number of CGMCC No.33100 and enterococcus faecalis YY-20 with the preservation number of CGMCC No.33099. According to the invention, the specific mixed bacterial agent is adopted to cooperate with the acid protease and the base material for mixed fermentation, so that the soluble protein content, the small peptide content and the viable count in the feed additive are improved, and the prepared feed additive is good in nutrition safety and high in digestible utilization rate. According to the fermented feed provided by the invention, by adding the feed additive containing the mixed microbial agent, the production performance of animals can be improved, the breeding cost is reduced, and the feed utilization rate is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of fermented feed, and specifically relates to a feed additive and a preparation method and application thereof. Background Art

[0002] In recent years, with the acceleration of industrialization, the output of industrial ethanol has steadily increased, and the output of corn DDGS, a byproduct of its industrial processing, has also been rising. Corn dried distillers grain with solubles (DDGS) is a protein feed raw material with a protein content of 28.0-30.0%, mainly containing corn glutenin and zein. The amino acid composition of corn glutenin is relatively balanced, containing a variety of essential amino acids required for animal growth and physiological functions. Zein has a unique amino acid composition, rich in a high proportion of proline and glutamine. There is also a certain proportion of albumin and globulin in DDGS, which contains relatively more essential amino acids such as lysine and methionine, which can meet the nutritional needs of animals. The special amino acid composition of corn DDGS enables it to maintain animal intestinal function, anti-oxidation and improve immunity. DDGS can be used as a feed raw material. Compared with fish meal and soybean meal commonly used in the feed industry, it has obvious resource advantages, low price and high feeding value. However, at present, DDGS is mostly mixed directly into feed. Due to the high fiber content and a certain amount of alcohol-soluble protein in DDGS, it is poorly water-soluble and cannot be effectively absorbed and utilized by animals. The digestibility is low, which reduces the utilization rate of DDGS. How to increase the high proportion of DDGS added to feed and improve the protein bioavailability of DDGS is an urgent problem to be solved. Summary of the invention

[0003] In view of this, one of the objects of the present invention is to provide a mixed bacterial agent capable of improving the bioavailability of protein in DDGS.

[0004] The second object of the present invention is to provide a feed additive with DDGS as the main raw material and a preparation method thereof. The feed additive provided by the present invention has the characteristics of a high DDGS ratio, and the soluble protein content, small peptide content and viable bacteria count in DDGS are increased by mixing bacterial agents and coordinating acid proteases.

[0005] The third object of the present invention is to provide a fermented feed which can improve animal production performance and feed utilization rate while reducing feeding costs.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a mixed bacterial agent, which comprises Bacillus subtilis YY-18 with a preservation number of CGMCC No. 33097, Saccharomyces cerevisiae YY-21 with a preservation number of CGMCC No. 33100 and Enterococcus faecalis YY-20 with a preservation number of CGMCC No. 33099.

[0008] Preferably, the volume ratio of Bacillus subtilis YY-18, Saccharomyces cerevisiae YY-21 and Enterococcus faecalis YY-20 is (3-5): (1-3): (5-7); the effective viable counts of Bacillus subtilis YY-18, Saccharomyces cerevisiae YY-21 and Enterococcus faecalis YY-20 are ≥3×10- 9 CFU.

[0009] The present invention also provides a feed additive, which comprises the mixed bacterial agent, acidic protease and a base material, wherein the base material comprises DDGS, corn germ meal, beet molasses, corn steep liquor and lysine mother liquor.

[0010] Preferably, the mass ratio of the mixed bacterial agent, the acidic protease and the base material is (12-18):(1-3):100.

[0011] Preferably, the enzymatic activity of the acidic protease is 7000 U / g.

[0012] Preferably, the weight proportions of the raw materials in the base material are: 8 parts of DDGS, 1-3 parts of corn germ meal, 0.6-1.0 parts of beet molasses, 1-2 parts of corn steep liquor and 0.5 parts of lysine mother liquor.

[0013] The present invention also provides a method for preparing the above-mentioned feed additive, comprising the following steps: mixing a base material, an acidic protease and water to obtain a fermented material, mixing the fermented material with a mixed bacterial agent, and performing anaerobically fermentation at 27-31° C. for 3-6 days to obtain the feed additive; the water content of the fermented material is 40%-60%.

[0014] The present invention also provides a fermented feed, which comprises the above-mentioned feed additive and basic feed; in terms of mass fraction of dry matter, the mass ratio of the feed additive to the basic feed is (10-30):(70-90).

[0015] Preferably, the basic feed comprises the following raw materials in parts by weight: 367-490 parts of corn, 143-220 parts of soybean meal, 1-5 parts of salt, 20-28 parts of soybean oil, 1-5 parts of DL-methionine, 60 parts of stone powder, 80 parts of calcium hydrogen phosphate and 20 parts of premix.

[0016] The present invention also provides any one of the following uses in improving animal production performance and / or improving feed utilization: (1) the above-mentioned mixed bacterial agent; (2) the above-mentioned feed additive; (3) the above-mentioned preparation method; (4) the above-mentioned fermented feed.

[0017] Preferably, the production performance includes: one or more of liver index, spleen index, abdominal fat rate and egg-laying performance; the egg-laying performance includes: one or more of egg number, egg weight, number of broken and deformed eggs, number of soft-shell eggs and egg quality.

[0018] Beneficial effects of the present invention:

[0019] The mixed bacterial agent provided by the present invention uses Bacillus subtilis YY-18, Saccharomyces cerevisiae YY-21 and Enterococcus faecalis YY-20 in combination, which can increase the soluble protein content, small peptide content and viable bacteria count in DDGS, and improve the digestibility and utilization rate of DDGS.

[0020] The feed additive provided by the present invention uses DDGS as the main raw material, adopts a specific mixed bacterial agent to coordinate acid protease and base material for mixed fermentation, thereby increasing the soluble protein content, small peptide content and viable bacteria count in the feed additive, so that the feed additive prepared by the present invention has good nutritional safety and high digestibility and utilization rate.

[0021] The fermented feed provided by the present invention reduces the amount of soybean meal in the basic feed and the breeding cost by adding a feed additive containing a mixed bacterial agent, and can also improve the production performance of animals and improve the feed utilization rate; relative to unfermented feed, it has a high soluble protein content, a high small peptide content, a high number of probiotics, good nutritional safety, and a high digestibility and utilization rate, and can be used as a good feed for feeding animals. It has been verified that feeding the fermented feed prepared by the present invention to the Hailan gray laying hens that are about to be eliminated in the late egg-laying period can significantly improve the production performance of the laying hens and improve the digestibility of nutrients; the laying hens fed with the fermented feed provided by the present invention have significantly improved egg-laying performance, significantly reduced feed-egg ratio, significantly improved total ovarian antioxidant capacity, significantly improved serum hormone levels, significantly improved egg quality of laying hens, and improved economic benefits.

[0022] Collection Instructions

[0023] The Bacillus subtilis YY-18 of the present invention is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, classified and named Bacillus subtilis, 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. 33097, and the deposit date is December 17, 2024.

[0024] The brewer's yeast YY-21 of the present invention is deposited in the General Microbiology Center of the China Culture Collection Administration Committee for Microbiological Cultures, 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. 33100, and the deposit date is December 17, 2024.

[0025] The Enterococcus faecalis YY-20 of the present invention is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration Committee, classified and named Enterococcus faecalis, 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.33099, and the deposit date is December 17, 2024. DETAILED DESCRIPTION

[0026] The invention provides a mixed bacterial agent, which comprises Bacillus subtilis YY-18 with a preservation number of CGMCC No. 33097, Saccharomyces cerevisiae YY-21 with a preservation number of CGMCC No. 33100 and Enterococcus faecalis YY-20 with a preservation number of CGMCC No. 33099.

[0027] In the present invention, the Bacillus subtilis YY-18, Saccharomyces cerevisiae YY-21 and Enterococcus faecalis YY-20 are all obtained by separation, screening and purification from natural fermentation enzymes. In the present invention, the mixed bacterial agent is preferably a mixed bacterial liquid or a mixed bacterial powder. The present invention does not specifically limit the preparation method of the mixed bacterial powder, and the preparation method well known to those skilled in the art can be adopted. In a specific embodiment of the present invention, when the mixed bacterial agent is a mixed bacterial liquid, the preparation method is: after activating the Bacillus subtilis YY-18, Saccharomyces cerevisiae YY-21 and Enterococcus faecalis YY-20 respectively, the activated strains are inoculated into the corresponding liquid culture medium respectively and cultured to the logarithmic growth phase, and the obtained seed liquids are mixed to obtain a mixed bacterial agent. In the present invention, the activation medium and liquid medium of Bacillus subtilis YY-18 are preferably LB solid medium and LB liquid medium, respectively; the activation medium and liquid medium of Saccharomyces cerevisiae YY-21 are preferably YPD solid medium and YPD liquid medium, respectively; the activation medium and liquid medium of Enterococcus faecalis YY-20 are preferably MRS solid medium and MRS liquid medium, respectively. The three bacteria of the present invention are preferably expanded and cultured in a shaking table, and the shaking table speed is preferably 180 to 220 r / min, more preferably 190 to 210 r / min; the temperature of culturing strains in LB and MRS medium is preferably 37°C; the temperature of culturing strains in YPD medium is preferably 28°C. In the mixed bacterial agent of the present invention, the volume ratio of Bacillus subtilis YY-18, Saccharomyces cerevisiae YY-21 and Enterococcus faecalis YY-20 is preferably (3-5): (1-3): (5-7), more preferably 4:1:6; in the mixed bacterial agent of the present invention, the effective viable counts of Bacillus subtilis YY-18, Saccharomyces cerevisiae YY-21 and Enterococcus faecalis YY-20 are preferably ≥3×10 9 CFU.

[0028] The present invention also provides a feed additive, which comprises the mixed bacterial agent, acidic protease and a base material, wherein the base material comprises DDGS, corn germ meal, beet molasses, corn steep liquor and lysine mother liquor.

[0029] The present invention does not specifically limit the specific sources of each raw material in the base material. In the present invention, the weight proportions of each raw material in the base material are preferably: 8 parts of DDGS, 1-3 parts of corn germ meal, 0.6-1.0 parts of beet molasses, 1-2 parts of corn steep liquor and 0.5 parts of lysine mother liquor. In the feed additive of the present invention, DDGS mainly provides protein, which can form soluble protein and small peptides through the decomposition of mixed bacterial agents and acid proteases, thereby increasing the soluble protein content and small peptide content in the feed additive; corn germ meal can not only provide albumin and albumin, but also provide energy substances for mixed bacterial agents; beet molasses is a cheap and readily available industrial fermentation raw material, which can provide the carbon source required for the growth of mixed bacterial agents; corn steep liquor is a by-product formed in the process of corn wet production of starch, which is rich in soluble protein, lactic acid (LA), sugars and starch, and rich in various amino acids, minerals, trace elements and vitamins, which can provide the nitrogen source required for the growth of mixed bacterial agents; lysine mother liquor is an intermediate or by-product produced in the process of lysine production, and its rich lysine content is high. DDGS lacks lysine. Adding lysine mother liquor can not only make up for the lack of lysine in DDGS, but also enable the fermentation system to have a carbon-nitrogen ratio that is more suitable for the growth of mixed bacterial agents. Lysine mother liquor is cheap, which can significantly reduce production costs. In the feed additive of the present invention, the mass ratio of mixed bacterial agents, acid protease and base material is preferably (12-18): (1-3): 100, more preferably (14-16): 2: 100; the measured enzyme activity of the acid protease is preferably 7000 U / g. The present invention does not specifically limit the specific source of the acid protease.

[0030] The present invention also provides a method for preparing the above-mentioned feed additive, comprising the following steps: mixing a base material, an acidic protease and water to obtain a fermented material, mixing the fermented material with a mixed bacterial agent, and performing anaerobically fermentation at 27-31° C. for 3-6 days to obtain the feed additive; the water content of the fermented material is 40%-60%.

[0031] In the preparation method of the feed additive of the present invention, the temperature of the anaerobic fermentation is preferably 28-30°C, more preferably 29°C; the time of the anaerobic fermentation is preferably 4-5 days. In one embodiment of the present invention, the anaerobic fermentation is carried out in a container, and the container preferably includes a one-way valve respiratory fermentation bag; the fermentation method is preferably raw material fermentation. In one embodiment of the present invention, when fermentation is carried out by one-way valve respiratory fermentation, it is preferably necessary to turn the bag over, and more preferably turn the bag over after 2 days of fermentation. The present invention can not only avoid the agglomeration of the base material by turning the bag, but also make the composite bacteria and the base material better mixed, thereby promoting the fermentation. In the feed additive of the present invention, the water content of the fermented material is preferably 42% to 58%, more preferably 45% to 50%. The invention prepares a feed additive with high soluble protein content and small peptide content by compounding Bacillus subtilis YY-18, Saccharomyces cerevisiae YY-21 and Enterococcus faecalis YY-20, and controlling the components and proportions of a fermentation base material, the water content, the inoculation amount, the fermentation time and the fermentation temperature. Compared with an unfermented base material, the soluble protein content and the small peptide content are increased, so that the feed additive prepared by the invention has good nutritional safety and high digestibility and utilization rate.

[0032] The present invention also provides a fermented feed, which comprises the above-mentioned feed additive and basic feed; in terms of mass fraction of dry matter, the mass ratio of the feed additive to the basic feed is (10-30):(70-90).

[0033] In the fermented feed of the present invention, the mass ratio of the feed additive to the basic feed is preferably (15-25): (75-85), more preferably 20:80. In the present invention, the dry matter refers to the dry material after the material is naturally air-dried, such as the dry matter of the feed additive is obtained by naturally air-drying after the feed additive is prepared. Similarly, the dry matter of the basic feed refers to the dry basic feed. In the present invention, the basic feed preferably includes the following components in parts by weight: 367-490 parts of corn, 143-220 parts of soybean meal, 1-5 parts of salt, 20-28 parts of soybean oil, 1-5 parts of DL-methionine, 60 parts of stone powder, 80 parts of calcium hydrogen phosphate and 20 parts of premix; further preferably: 370-434 parts of corn, 148-182 parts of soybean meal, 2-3 parts of salt, 22-26 parts of soybean oil, 2-3 parts of DL-methionine, 60 parts of stone powder, 80 parts of calcium hydrogen phosphate and 20 parts of premix. In the fermented feed of the present invention, adding the feed additive of the present invention can reduce the amount of soybean meal added in the basic feed, thereby reducing the cost and ensuring excellent breeding effect. The present invention has no special restrictions on the specific sources of the raw materials in the basic feed, and conventional commercial products in the field can be used.

[0034] The present invention also provides any one of the following uses in improving animal production performance and / or improving feed utilization: (1) the above-mentioned mixed bacterial agent; (2) the above-mentioned feed additive; (3) the above-mentioned preparation method; (4) the above-mentioned fermented feed.

[0035] In the application of the present invention, the animals preferably include livestock and poultry, and the livestock and poultry preferably include laying hens; the laying hens preferably include Hy-Line gray laying hens, and the Hy-Line gray laying hens include Hy-Line gray laying hens that are about to be eliminated in the late egg-laying period. The egg production of Hy-Line gray laying hens in the late egg-laying period drops significantly, and the egg breakage rate increases significantly, but after feeding the fermented feed provided by the present invention, the number of broken and deformed eggs can be significantly reduced, and the number of eggs laid, the egg weight, etc. can also be increased. In the present invention, the production performance preferably includes: one or more of liver index, spleen index, abdominal fat rate and egg-laying performance; the egg-laying performance preferably includes: one or more of the number of eggs laid, the egg weight, the number of broken and deformed eggs, the number of soft-shell eggs and egg quality.

[0036] 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.

[0037] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] The DDGS in the following examples were purchased from COFCO (Zhaodong) Biochemical Technology Co., Ltd., corn germ meal, corn steep liquor and lysine mother liquor were purchased from Qiqihar Longjiang Fufeng Biotechnology Co., Ltd., beet molasses was conventionally commercially available, and acidic protease was purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd.

[0040] Example 1

[0041] A mixed bacterial agent, the mixed bacterial agent consisting of 4 parts by volume of Bacillus subtilis YY-18 with a preservation number of CGMCC No.33097, 1 part by volume of Saccharomyces cerevisiae YY-21 with a preservation number of CGMCC No.330100, and 6 parts by volume of Enterococcus faecalis YY-20 with a preservation number of CGMCC No.33099;

[0042] The preparation method of the mixed bacterial agent is as follows:

[0043] Bacillus subtilis YY-18 was inoculated into LB solid medium and activated at 37°C. The activated Bacillus subtilis YY-18 was inoculated into LB liquid medium and cultured in a shaking incubator at 37°C and 180 r / min until the logarithmic growth phase to obtain Bacillus subtilis YY-18 seed liquid. The effective viable count of Bacillus subtilis YY-18 seed liquid was 3×10 9 CFU / mL;

[0044] Saccharomyces cerevisiae YY-21 was inoculated into YPD solid medium and activated at 28°C. The activated Saccharomyces cerevisiae YY-21 was inoculated into YPD liquid medium and cultured in a shaking incubator at 28°C and 180 r / min until the logarithmic growth phase to obtain the Saccharomyces cerevisiae YY-21 seed liquid. The effective viable count of the Saccharomyces cerevisiae YY-21 seed liquid was 3×10 9 CFU / mL;

[0045] Enterococcus faecalis YY-20 was inoculated into MRS solid medium and activated at 37°C. The activated Enterococcus faecalis YY-20 was inoculated into MRS liquid medium and cultured in a shaking incubator at 37°C and 180 r / min until the logarithmic growth phase to obtain the seed solution of Enterococcus faecalis YY-20. The effective viable count of the seed solution of Enterococcus faecalis YY-20 was 3×10 9 CFU / mL;

[0046] Finally, 4 parts by volume of Bacillus subtilis YY-18 seed solution, 1 part by volume of Saccharomyces cerevisiae YY-21 seed solution and 6 parts by volume of Enterococcus faecalis YY-20 seed solution were mixed to obtain a mixed bacterial agent.

[0047] The LB solid medium is composed of 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, neutral pH and 20 g agar, fixed to 1000 mL, and steam sterilized at 121° C. for 30 min; the difference between the LB liquid medium and the LB solid medium is that agar is not added, and the rest is the same as the LB solid medium.

[0048] The YPD solid culture medium is composed of 10 g yeast extract powder, 20 g peptone, 20 g glucose and 15 g agar, which is fixed to 1000 mL and sterilized by steam at 121° C. for 30 min. The difference between the YPD liquid culture medium and the YPD solid culture medium is that agar is not added, and the rest is the same as the YPD solid culture medium.

[0049] The MRS solid culture medium is prepared by diluting the volume to 1000 mL with 10 g of peptone, 10 g of beef extract, 5 g of yeast powder, 2 g of dipotassium hydrogen phosphate, 2 g of diammonium citrate, 5 g of sodium acetate, 20 g of glucose, 1 mL of Tween 80, 0.58 g of magnesium sulfate heptahydrate, 0.25 g of manganese sulfate tetrahydrate and 20 g of agar, adjusting the pH to 6.4, and steam sterilizing at 121° C. for 30 min. The difference between the MRS liquid culture medium and the MRS solid culture medium is that agar is not added, and the rest are the same as the MRS solid culture medium.

[0050] Example 2

[0051] A feed additive, comprising the following raw materials in parts by weight: 1.9 parts of the mixed bacterial agent obtained in Example 1, 1 part of acidic protease (the measured enzyme activity of the acidic protease is 7000 U / g) and 12.5 parts of base material;

[0052] The base material is composed of the following raw materials in parts by weight: 8 parts of DDGS, 2 parts of corn germ meal, 1 part of beet molasses, 1 part of corn steep liquor and 0.5 parts of lysine mother solution.

[0053] The preparation method of the feed additive is as follows: a base material, an acidic protease and water are mixed to obtain a fermentation material, wherein the water content of the fermentation material is 50%, the fermentation material is mixed with the mixed bacterial agent obtained in Example 1, stirred evenly, and then put into a fermentation bag with a one-way valve and sealed, and anaerobically fermented at 28° C. for 4 days, and a bag turning operation is performed on the second day of fermentation, and the feed additive is obtained after the fermentation is completed.

[0054] Example 3

[0055] A feed additive, comprising the following raw materials in parts by weight: 2 parts of the mixed bacterial agent obtained in Example 1, 1 part of acid protease (the measured enzyme activity of the acid protease is 7000 U / g) and 13.5 parts of base material;

[0056] The base material is composed of the following raw materials in parts by weight: 8 parts of DDGS, 2 parts of corn germ meal, 1 part of beet molasses, 2 parts of corn steep liquor and 0.5 parts of lysine mother solution.

[0057] The preparation method of the feed additive is as follows: mixing a base material, an acidic protease and water to obtain a fermentation material, wherein the water content of the fermentation material is 50%, mixing the fermentation material and the mixed bacterial agent evenly, and then putting the fermentation material into a one-way valve fermentation bag and sealing it, and performing anaerobically fermentation at 30° C. for 5 days, performing a bag turning operation on the second day of fermentation, and obtaining the feed additive after the fermentation is completed.

[0058] Example 4

[0059] The nutrient content and active ingredients of the base material before and after fermentation of Example 2 were measured, and the components of the fermentation product were analyzed.

[0060] Determination items and methods: Crude protein content, crude fat content, crude fiber content, crude ash content, refer to GB / T6432-1994, GB / T 6433-1994, GB / T 5009-2003 and GB / T 6438-1992 respectively. The soluble protein content was determined by the Folin-phenol method. The small peptide content was determined by the trichloroacetic acid (TCA) precipitation method. The results of the determination of the basic nutrient content of the base material before and after fermentation are shown in Table 1.

[0061] Table 1 Determination results of basic nutrient content of base material before and after fermentation

[0062] Element Before fermentation After fermentation Crude protein content (%) 30.25±0.32 29.30±0.61 Crude fat content (%) 8.21±0.14 7.96±0.25 Crude fiber content (%) <![CDATA[6.42±0.21 a ]]> <![CDATA[6.05±0.13 b ]]> Crude ash content (%) 5.02±0.52 4.98±0.62 Soluble protein content (mg / g) <![CDATA[47.86±0.58 b ]]> <![CDATA[134.25±0.01 a ]]> Small peptide content (mg / g) <![CDATA[40.67±0.32 b ]]> <![CDATA[126.35±0.01 a ]]>

[0063] Note: The data in the table are expressed as "mean ± SD". No letters in the shoulder indicate no significant difference in the same industry, and different lowercase letters indicate significant difference between samples (P<0.05).

[0064] As can be seen from Table 1, the soluble protein content and small peptide content of the feed additive prepared by the present invention are respectively increased by 181% and 211% compared with the unfermented base material, and the crude fiber content is reduced by 5.76%, indicating that the feed additive prepared by the present invention has good nutritional safety, improved digestibility and utilization, and reduced fiber content, which is beneficial to the digestion of the animal intestine and more beneficial to the digestion and absorption of laying hens.

[0065] In vitro simulated livestock digestion

[0066] Prepare 0.1mol / L phosphate buffer solution (pH 6.0), 0.2mol / L phosphate buffer solution (pH 6.8), 0.2mol / L hydrochloric acid solution, 0.6mol / L sodium hydroxide solution, 1mol / L hydrochloric acid solution, and 1mol / L sodium hydroxide solution. Simulated porcine gastric juice: weigh 40mg of pepsin (1:3000) and add it to 300mL of hydrochloric acid solution (0.2mol / L), stirring until dissolved. Simulated porcine small intestinal juice: accurately weigh 60mg of trypsin (1:2500) and add it to 300mL of sodium hydroxide solution (0.6mol / L), stirring until dissolved.

[0067] Weigh 5 g of fermented feed sample and unfermented feed sample passed through a 100-mesh sieve respectively and put them into a 500 mL conical flask. Set up three parallel samples and one blank for each sample. Then add 125 mL of phosphate buffer solution (0.1 mol / L). After stirring until mixed, add 100 mL of hydrochloric acid solution (0.2 mol / L) and adjust the pH value to 2.0 with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution.

[0068] Simulate gastric digestion: Add 50 mL of prepared porcine pepsin gastric juice to the solution in the previous step, mix well and seal, place in a 37°C constant temperature shaking incubator, and shake at 120 r / min for 4 hours. Determine the pH value of the digestive juice. Add 100 mL of phosphate buffer solution (0.2 mol / L) and 50 mL of sodium hydroxide solution (0.6 mol / L) to the gastric digestive juice, stir well and adjust the pH to 6.8.

[0069] Simulated small intestinal digestion: Add 50 mL of prepared simulated porcine small intestinal fluid to the digestive fluid after gastric digestion, mix well, seal the tube and place in a 37°C constant temperature shaking incubator for digestion for 12 hours with an shaking frequency of 120 r / min. Measure the pH of the digested solution.

[0070] Crude protein and dry matter digestibility: Filter the residue after simulated small intestinal digestion, and then rinse with deionized water until there is no feed sample residue in the triangular flask, measure 25-50mL ethanol solution (95%) to rinse the undigested residue filtered out, and then measure 25-50mL acetone solution (99.5%) to rinse again. After the feed sample in the filter paper is completely filtered, remove the filter paper and put it in the prepared culture dish; place it in a 65℃ oven for the first drying for 72h, and the moisture regain is >48h. The second drying is 24h, and the moisture regain is >24h (if the data variation is large, the third drying is 24h, and the moisture regain is 48h), and after cooling, weigh and record. The difference between the weight before and after is the dry matter weight. The crude protein content is determined using the Dumas combustion method.

[0071] The number of viable bacteria was determined by the spread plate counting method. The results of in vitro simulated digestion of the substrate before and after fermentation are shown in Table 2.

[0072] Table 2 Results of in vitro simulated digestion of substrate before and after fermentation

[0073] Element Before fermentation After fermentation Gastric digestive juice pH 2.26±0.01 2.23±0.01 pH of intestinal digestive juice <![CDATA[7.39±0.04 a ]]> <![CDATA[7.31±0.14 b ]]> Dry matter digestibility (%) <![CDATA[52.88±0.57 b ]]> <![CDATA[69.14±0.28 a ]]> Crude protein digestibility (%) <![CDATA[44.93±0.85 b ]]> <![CDATA[66.94±1.04 a ]]> Total number of beneficial bacteria cfu / g / <![CDATA[1.6×10 9 ]]>

[0074] Note: The data in the table are expressed as "mean ± SD". No letters in the shoulder indicate no significant difference in the same industry, and different lowercase letters indicate significant difference between samples (P<0.05).

[0075] As shown in Table 2, after gastrointestinal digestion, the pH of the intestinal digestive juice was significantly lower than that before fermentation, and the dry matter digestibility and crude protein digestibility were significantly increased compared with before fermentation, with the dry matter digestibility increasing by 30.75% and the crude protein digestibility increasing by 48.99%.

[0076] The above results show that the soluble protein and small peptide produced by the synergistic fermentation of the mixed bacterial agent and the acidic protease in the present invention ensure that the feed additive prepared by the present invention has good nutritional safety and high digestibility and utilization rate.

[0077] Example 5

[0078] A fermented feed, comprising the following raw materials in terms of dry matter weight: 100 parts of the feed additive obtained in Example 2 and 900 parts of a basic feed;

[0079] The basic feed is composed of the following raw materials by weight: 490 parts of corn (dry matter weight), 220 parts of soybean meal (dry matter weight), 5 parts of salt, 20 parts of soybean oil, 5 parts of DL-methionine, 60 parts of stone powder, 80 parts of calcium hydrogen phosphate and 20 parts of premix (dry matter weight). The premix was purchased from Shijiazhuang Guonong Feed Co., Ltd.

[0080] The method for preparing the fermented feed is as follows: mixing the feed additive obtained in Example 2 with the basic feed.

[0081] Example 6

[0082] A fermented feed, comprising the following raw materials in terms of dry matter weight: 200 parts of the feed additive obtained in Example 2 and 800 parts of a basic feed;

[0083] The basic feed is composed of the following raw materials in parts by weight: 434 parts of corn (dry matter weight), 182 parts of soybean meal (dry matter weight), 2 parts of salt, 20 parts of soybean oil, 2 parts of DL-methionine, 60 parts of stone powder, 80 parts of calcium hydrogen phosphate and 20 parts of premix (dry matter weight).

[0084] The method for preparing the fermented feed is as follows: mixing the feed additive obtained in Example 2 with the basic feed.

[0085] Example 7

[0086] A fermented feed, comprising the following raw materials in terms of dry matter weight: 300 parts of the feed additive obtained in Example 2 and 700 parts of a basic feed;

[0087] The basic feed is composed of the following raw materials in parts by weight: 367 parts of corn (dry matter weight), 143 parts of soybean meal (dry matter weight), 1 part of salt, 28 parts of soybean oil, 1 part of DL-methionine, 60 parts of stone powder, 80 parts of calcium hydrogen phosphate and 20 parts of premix (dry matter weight).

[0088] The method for preparing the fermented feed is as follows: mixing the feed additive obtained in Example 2 with the basic feed.

[0089] Comparative Example 1

[0090] A basic feed is composed of the following raw materials in parts by weight: 540 parts of corn (dry matter mass), 260 parts of soybean meal (dry matter mass), 10 parts of salt, 20 parts of soybean oil, 10 parts of DL-methionine, 60 parts of stone powder, 80 parts of calcium hydrogen phosphate and 20 parts of premix (dry matter mass).

[0091] The preparation method is as follows: mixing various raw materials.

[0092] Example 8

[0093] Select body weight, egg production rate and egg weight close and healthy same batch 75 week-old egg-laying late stage about to be eliminated 240 Hailan gray laying hens, randomly divided into 4 groups (4 groups are respectively embodiment 5 groups, embodiment 6 groups, embodiment 7 groups and comparative example 1 group), 4 repetitions in each group, each repetition 15 laying hens.Feeding experiment adopts conventional multi-layer cage culture mode, feeding management and conventional immunity are carried out according to laying hen feeding management technical standard, ventilation adopts natural ventilation and mechanical ventilation exchange, natural light and artificial light combination method, ensure illumination 16h, free drinking water, daily feeding 2 times (early 8:00 and afternoon 17:00), each feeding keeps each group of feeding amount consistent, chicken keeps free feeding state.Pick up eggs at 16:30 every day, clean chicken coop, repeat weighing and storage after picking up eggs. The feed was formulated in accordance with the Group Standard for Compound Feeds for Layers and Broilers (T / CFIAS 002-2018) and the Hy-Line Brown Layer Feeding and Management Manual 2018. The ingredients of feeds for different groups are shown in Table 3.

[0094] Table 3 Composition of feeds in different groups

[0095]

[0096] Determination of egg laying performance

[0097] During the formal experiment, the egg weight, number of eggs, number of broken eggs, number of soft-shelled eggs and dead eggs were recorded every day in units of repetition. The remaining feed was weighed every week to calculate the average egg weight, egg production rate, average daily feed intake, average daily egg production, feed-egg ratio and broken and deformed egg rate in the early and late stages of the experiment. The calculation formulas for each index are as follows:

[0098] Average daily feed intake [g / (hen·d)] = {[total weight of feed (g) - final weight of feed (g)] / total number of laying hens (hens)} / number of experimental days (d);

[0099] Average daily egg production [g / (hen·d)] = total egg weight (g) / (total number of laying hens (hens) × number of experimental days (d));

[0100] Feed-egg ratio = total feed intake (g) / total egg weight (g);

[0101] Egg production rate (%) = total number of eggs laid / [total number of laying hens × number of experimental days] × 100%;

[0102] Average egg weight (g) = total egg weight (g) / total number of eggs produced (pieces);

[0103] Deformed egg rate (%) = total number of deformed eggs (pieces) / total number of eggs produced (pieces);

[0104] The egg production results of Hy-Line Grey laying hens are shown in Table 4.

[0105] Table 4 Egg laying performance of laying hens

[0106]

[0107] Note: The data in the table are expressed as "mean ± SD". No letters in the shoulder indicate no significant difference in the same industry, and different lowercase letters indicate significant difference between samples (P<0.05).

[0108] As shown in Table 4, at the end of feeding to 79wk, there was no significant difference in the average egg weight of Examples 5, 6, and 7 and the average egg weight of Comparative Example 1 (P>0.05); the egg laying rate of Examples 5, 6, and 7 was significantly higher than that of Comparative Example 1 (P<0.05), and at the same time, the egg laying rate of Example 7 was significantly lower than that of Examples 5 and 6 (P<0.05); the average daily feed intake of Examples 5 and 6 was significantly higher than that of Comparative Example 1 and Example 7 (P<0.05); the average daily egg production of Examples 5, 6, and 7 was significantly higher than that of Comparative Example 1 (P<0.05); the feed-egg ratio and the rate of broken and deformed eggs of Examples 5, 6, and 7 were significantly lower than those of Comparative Example 1 (P<0.05). At the end of feeding to 82wk, there was no significant difference in the average egg weight of Examples 5, 6, and 7 compared with the average egg weight of Comparative Example 1 (P>0.05); the egg laying rate of Examples 6 and 7 was significantly higher than that of Comparative Example 1 (P<0.05), and the egg laying rate of Example 5 was not significantly different from that of Comparative Example 1 and Example 6 (P>0.05). At the same time, the egg laying rate of Example 5 was significantly lower than that of Example 6 (P<0.05); except that the average daily feed intake of Example 7 was higher than that of Comparative Example 1, the average daily feed intake of Examples 5, Example 6, and Comparative Example 1 was There was no significant difference in the amount of eggs per day (P>0.05); the average daily egg production of Example 6 and Example 7 was significantly higher than that of Comparative Example 1 (P<0.05), and there was no significant difference in the average daily egg production of Example 5 and that of Comparative Example 1 (P>0.05); except that the feed-to-egg ratio of Example 6 was significantly lower than that of Comparative Example 1 (P<0.05), there was no significant difference in the feed-to-egg ratio of Example 6, Example 7 and Comparative Example 1 (P>0.05); the malformed egg rate of Examples 5, 6, and 7 was significantly lower than that of Comparative Example 1 (P<0.05).

[0109] In summary, adding the feed additive of the present invention to the basal feed (daily diet) can significantly improve the egg-laying performance of laying hens. The advantage of adding the fermented feed of the present invention to the basal feed (daily diet) for laying hens in the late egg-laying period is more obvious than that of the ordinary diet. The fermented feed prepared in Example 6 is more suitable for laying hens.

[0110] Determination of egg quality indicators

[0111] Three eggs were randomly selected from each group for egg quality determination one day before sampling. Egg shape index, eggshell strength, Haugh unit (HU), and yolk color were determined using a digital egg tester (DET-6000, NABEL, Japan). Yolk and whole egg protein were determined using the Dumas combustion method, and yolk total cholesterol (T-CHO) and triglyceride (TG) content were determined using the corresponding kits of Nanjing Jiancheng Bioengineering Institute.

[0112] The results of the egg quality indexes are shown in Table 5.

[0113] Table 5 Effect of fermented feed on egg quality

[0114]

[0115] Note: The data in the table are expressed as "mean ± SD". No letters in the shoulder indicate no significant difference in the same industry, and different lowercase letters indicate significant difference between samples (P<0.05).

[0116] As shown in Table 5, after feeding to the end of 79wk, the eggshell strength of Example 6 was significantly higher than that of Examples 5, 7 and Comparative Example 1 (P<0.05), and there was no significant difference in the eggshell strength of Example 5, Example 7 and Comparative Example 1 (P>0.05); the egg shape index of Example 6 was significantly higher than that of Comparative Example 1 (P<0.05), and there was no significant difference in the egg shape index of Example 5, Example 7 and Comparative Example 1 (P>0.05); there was no significant difference in the yolk color of each group (P>0.05); the Haugh unit of Example 7 was significantly higher than that of Comparative Example 1 (P<0.05), and there was no significant difference in the Haugh unit of Examples 5, 6 and Comparative Example 1 (P>0.05); the yolk protein content of each group There was no significant difference in the amount of whole egg protein between Example 5 and Comparative Example 1 (P>0.05); except that Example 5 was significantly lower than Comparative Example 1 (P<0.05), there was no significant difference in the whole egg protein between Examples 6 and 7 and the whole egg protein between Comparative Example 1 (P>0.05); except that Example 5 was significantly higher than Comparative Example 1 (P<0.05), there was no significant difference in the egg yolk triglyceride between Examples 6 and 7 and the egg yolk triglyceride between Comparative Example 1 (P>0.05); there was no significant difference in the egg yolk total cholesterol between Example 7 and the egg yolk total cholesterol between Comparative Example 1 (P>0.05), and the egg yolk total cholesterol in Examples 5 and 6 was significantly lower than the egg yolk total cholesterol in Comparative Example 1 (P<0.05). After feeding to the end of 82wk, compared with Comparative Example 1, the eggshell strength and yolk triglyceride of Examples 5, 6 and 7 were significantly higher than those of Comparative Example 1; there were no significant differences in yolk color and yolk protein between Examples 5, 6 and 7 and Comparative Example 1 (P>0.05); there was no significant difference in egg shape index between Examples 5 and 7 and Comparative Example 1 (P>0.05); the Haugh units of Examples 6 and 7 were significantly higher than those of Comparative Example 1 (P<0.05), and there was no significant difference in Haugh units between Example 5 and Comparative Example 1 (P>0.05); the whole egg protein of Examples 6 and 7 was significantly higher than that of Comparative Example 1 (P<0.05), and there was no significant difference in whole egg protein between Example 5 and Comparative Example 1 (P>0.05); the yolk total cholesterol of Examples 5, 6 and 7 was significantly lower than that of Comparative Example 1 (P<0.05), and there was no significant difference among the example groups (P>0.05).

[0117] In summary, compared with comparative example 1, adding the feed additive of the present invention into the laying hen diet can significantly increase the whole egg protein content and the whole egg triglyceride content in the eggs, and at the same time, can effectively reduce the cholesterol in the eggs.

[0118] Organ index and follicle development of laying hens

[0119] Laying hens were fed to the end of 79 weeks and 82 weeks, and their ovaries were removed after dissection. The number of follicles in the ovaries was counted according to the size of the follicle diameter (2-5mm, 5-10mm, >10mm). At the same time, the spleen, liver, and abdominal fat were taken, and the surrounding tissues and fat were removed and weighed. The calculation formula is:

[0120] Liver index (g / kg) = liver weight (g) / live weight (kg);

[0121] Spleen index (g / kg) = spleen weight (g) / live weight (kg);

[0122] Abdominal fat rate (g / kg) = abdominal fat weight (g) / live weight (kg);

[0123] The results are shown in Table 6. Compared with Comparative Example 1, there were no significant differences in the liver index and spleen index of Examples 5, 6, and 7 in the early and late stages of the experiment (P>0.05). At the end of feeding to 79 weeks, the abdominal fat rate of Example 6 was significantly higher than that of Examples 5, 7, and Comparative Example 1 (P<0.05), and there was no significant difference in the abdominal fat rate of Examples 5, 7, and Comparative Example 1 (P>0.05); there was no significant difference in the follicles (≥10 mm) of Examples 5, 6, 7, and Comparative Example 1 (P>0.05); the follicles (5-10 mm) of Example 6 were significantly higher than those of Comparative Example 1. Feeding to the end of 82wk, there was no significant difference in the abdominal fat rate of Examples 5, 6, and 7 (P>0.05); the follicles (2-5mm) of Examples 5, 6, and 7 were significantly higher than those of Comparative Example 1 (P<0.05), and there was no significant difference between the Example groups (P>0.05); the follicles (5-10mm) of Examples 5 and 6 were significantly higher than those of Comparative Example 1, and the follicles (5-10mm) of Example 7 were not significantly different from those of Comparative Example 1 (P>0.05); the follicles (>10mm) of Examples 5 and 6 were significantly higher than those of Comparative Example 1, and the follicles (>10mm) of Example 7 were not significantly different from those of Comparative Example 1 (P>0.05). It can be seen that compared with Comparative Example 1, adding the feed additive of the present invention to the laying hen diet had no significant effect on the liver index, spleen index, and abdominal fat rate of laying hens, but significantly increased the number of follicles produced in laying hens.

[0124] Table 6 Organ index and follicle development of laying hens

[0125]

[0126] Note: The data in the table are expressed as "mean ± SD". No letters in the shoulder indicate no significant difference in the same industry, and different lowercase letters indicate significant difference between samples (P<0.05).

[0127] Determination of Antioxidant Capacity of Laying Hen Ovaries

[0128] The total antioxidant capacity T-AOC, catalase CAT, malondialdehyde MDA, superoxide dismutase SOD and glutathione peroxidase GSH-PX were determined using the corresponding kits produced by Nanjing Jiancheng Biological Research Institute. The results are shown in Table 7.

[0129] Table 7 Effects of different feeds on antioxidant indexes of laying hens' ovaries

[0130]

[0131]

[0132] Note: The data in the table are expressed as "mean ± SD". No letters in the shoulder indicate no significant difference in the same industry, and different lowercase letters indicate significant difference between samples (P<0.05).

[0133] As shown in Table 7, when fed to the end of 79wk, compared with Comparative Example 1, there were no significant differences in the total antioxidant capacity T-AOC, superoxide dismutase SOD and catalase CAT of Examples 5, 6, 7 and Comparative Example 1 (P>0.05); the malondialdehyde content of Example 5 was significantly lower than that of Examples 6, 7 and Comparative Example 1 (P<0.05), and there was no significant difference in the malondialdehyde content of Examples 6, 7 and Comparative Example 1 (P>0.05); compared with Comparative Example 1, except that there was no significant difference in glutathione peroxidase GSH-PX in Example 7 (P>0.05), the glutathione peroxidase GSH-PX in Examples 5 and 6 were significantly higher than that in Comparative Example 1 (P<0.05); fed to the end of 82wk, the total antioxidant capacity of Examples 5 and 6 was significantly higher than that of Comparative Example 1 (P<0 .05), the total antioxidant capacity of Example 7 had no significant difference with that of Comparative Example 1 (P>0.05); the catalase CAT of Examples 5, 6, and 7 had no significant difference with that of Comparative Example 1 (P>0.05); the malondialdehyde content of Example 6 was significantly lower than that of Examples 5, 7, and Comparative Example 1 (P<0.05), and there was no significant difference in the malondialdehyde content of Examples 5, 7, and Comparative Example 1 (P>0.05); the superoxide dismutase SOD of Examples 6 and 7 was significantly lower than that of Comparative Example 1 (P<0.05), and there was no significant difference in the superoxide dismutase SOD of Example 5 with that of Comparative Example 1 (P>0.05); the glutathione peroxidase GSH-PX of Examples 5 and 6 was significantly lower than that of Comparative Example 1, and there was no significant difference in the glutathione peroxidase GSH-PX of Example 7 with that of Comparative Example 1. It can be seen that compared with Comparative Example 1, adding the feed additive of the present invention to the laying hen diet has a significant improvement effect on the ovarian antioxidant capacity of laying hens.

[0134] The above results show that the fermented feed provided by the present invention can improve the production performance of animals, reduce breeding costs, and improve feed utilization by adding feed additives. By using the fermented feed prepared by the present invention in the feeding of Hy-Line gray laying hens, the egg-laying performance and egg quality of the laying hens can be significantly improved, and the digestibility of nutrients can be improved; the laying hens fed with the fermented feed prepared by the present invention have significantly improved egg-laying performance, significantly reduced feed-egg ratio, significantly improved total ovarian antioxidant capacity, significantly improved serum hormone levels, significantly improved egg quality of laying hens, and improved economic benefits.

[0135] 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 mixed bacterial agent, characterized in that: The mixed bacterial agent comprises Bacillus subtilis YY-18 with a preservation number of CGMCC No. 33097, Saccharomyces cerevisiae YY-21 with a preservation number of CGMCC No. 33100 and Enterococcus faecalis YY-20 with a preservation number of CGMCC No. 33099.

2. The mixed bacterial agent according to claim 1, characterized in that: The volume ratio of Bacillus subtilis YY-18, Saccharomyces cerevisiae YY-21 and Enterococcus faecalis YY-20 is (3-5): (1-3): (5-7); the effective viable counts of Bacillus subtilis YY-18, Saccharomyces cerevisiae YY-21 and Enterococcus faecalis YY-20 are ≥3×10-1 per gram or per mL of the mixed bacterial agent. 9 CFU.

3. A feed additive, characterized in that: The feed additive comprises the mixed bacterial agent according to claim 1 or 2, acidic protease and base material, and the base material comprises DDGS, corn germ meal, beet molasses, corn steep liquor and lysine mother liquor.

4. The feed additive according to claim 3, characterized in that The mass ratio of the mixed bacterial agent, acidic protease and base material is (12-18):(1-3):

100.

5. The feed additive according to claim 3, characterized in that The enzymatic activity of the acidic protease is 7000 U / g.

6. The feed additive according to claim 3, characterized in that The weight proportions of the raw materials in the base material are: 8 parts of DDGS, 1-3 parts of corn germ meal, 0.6-1.0 parts of beet molasses, 1-2 parts of corn steep liquor and 0.5 parts of lysine mother liquor.

7. The method for preparing the feed additive according to any one of claims 3 to 6, characterized in that: The method comprises the following steps: mixing base material, acidic protease and water to obtain fermentation material, mixing the fermentation material with mixed bacterial agent, and performing anaerobic fermentation at 27-31°C for 3-6 days to obtain feed additive; the water content of the fermentation material is 40%-60%.

8. A fermented feed, characterized in that: The fermented feed comprises the feed additive according to any one of claims 3 to 6 and a basic feed; in terms of mass fraction of dry matter, the mass ratio of the feed additive to the basic feed is (10 to 30): (70 to 90).

9. The fermented feed according to claim 8, characterized in that The basic feed comprises the following raw materials in parts by weight: 367-490 parts of corn, 143-220 parts of soybean meal, 1-5 parts of salt, 20-28 parts of soybean oil, 1-5 parts of DL-methionine, 60 parts of stone powder, 80 parts of calcium hydrogen phosphate and 20 parts of premix.

10. Use of any one of the following in improving animal production performance and / or improving feed utilization, characterized in that: (1) The mixed bacterial agent according to claim 1 or 2; (2) The feed additive according to any one of claims 3 to 6; (3) The preparation method according to claim 7; (4) The fermented feed according to claim 8 or 9.

Images