A feed additive, its preparation method and application

Abstract

The application provides a feed additive and a preparation method and application thereof, and belongs to the technical field of fermented feed.The feed additive provided by the application comprises a mixed bacterial agent, acid protease and a base material, 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.The specific mixed bacterial agent is used for synergistic acid protease and base material mixed fermentation, the soluble protein content, small peptide content and viable bacterial count in the feed additive are improved, the prepared feed additive has good nutritional safety and high digestible utilization rate.The fermented feed provided by the application can improve the production performance of animals, reduce the breeding cost and improve the feed utilization rate by adding the feed additive containing the mixed bacterial agent.

Description

Technical Field

[0001] This invention belongs to the field of fermented feed technology, specifically relating to a feed additive, its preparation method, and its application. Background Technology

[0002] In recent years, with the acceleration of industrialization, the production of industrial ethanol has steadily increased, and the production of its industrial processing byproduct, corn DDGS, has also been rising. Dried distillers grains with solubles (DDGS) is a protein feed ingredient with a protein content of 28.0-30.0%, mainly containing corn glutenin and zein. Corn glutenin has a relatively balanced amino acid composition, containing many 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. DDGS also contains a certain proportion of albumin and globulin, and contains relatively high amounts of essential amino acids such as lysine and methionine, which can meet the nutritional needs of animals. This special amino acid composition of corn DDGS gives it physiological functions such as maintaining intestinal function, anti-oxidation, and improving immunity in animals. DDGS can be used as a feed ingredient, and compared with fishmeal and soybean meal commonly used in the feed industry, it has significant resource advantages, is inexpensive, and has high feed value. However, currently, most methods involve directly mixing DDGS into feed. Because DDGS has a high fiber content and contains a certain amount of proteases with poor water solubility, it cannot be effectively absorbed and utilized by animals, resulting in low digestibility and utilization. Therefore, how to increase the proportion of DDGS added to feed while simultaneously improving its protein bioavailability is an urgent problem to be solved. Summary of the Invention

[0003] In view of this, one of the objectives of the present invention is to provide a mixed bacterial agent that can improve the bioavailability of proteins in DDGS.

[0004] The second objective of this invention is to provide a feed additive with DDGS as the main raw material and its preparation method. The feed additive provided by this invention has the characteristic of high DDGS content. By using mixed microbial agents in conjunction with acidic protease, the soluble protein content, small peptide content and viable bacteria count in DDGS are improved.

[0005] The third objective of this invention is to provide a fermented feed that can improve animal production performance and feed utilization while reducing feeding costs.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The present invention provides a mixed microbial agent comprising Bacillus subtilis YY-18 with accession number CGMCC No. 33097, Saccharomyces cerevisiae YY-21 with accession number CGMCC No. 33100, and Enterococcus faecalis YY-20 with accession number 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); and the effective viable counts of Bacillus subtilis YY-18, Saccharomyces cerevisiae YY-21, and Enterococcus faecalis YY-20 per g or per mL of the mixed bacterial agent are ≥3×10⁻⁶. 9 CFU.

[0009] The present invention also provides a feed additive comprising the above-mentioned mixed microbial agent, acidic protease and 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 microbial agent, acidic protease and substrate is (12-18):(1-3):100.

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

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

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

[0014] The present invention also provides a fermented feed, which includes the above-mentioned feed additives and basic feed; the mass ratio of the feed additives to the basic feed is (10-30):(70-90) based on the mass parts of dry matter.

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

[0016] The present invention also provides the application of any one of the following in improving animal production performance and / or improving feed utilization: (1) the above-mentioned mixed microbial 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 the following: liver index, spleen index, abdominal fat percentage, and egg production performance; the egg production performance includes one or more of the following: number of eggs laid, egg weight, number of broken eggs, number of soft-shelled eggs, and egg quality.

[0018] The beneficial effects of this invention are:

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

[0020] The feed additive provided by this invention uses DDGS as the main raw material and employs a specific mixed microbial agent in conjunction with acidic protease and base material for fermentation, which improves the soluble protein content, small peptide content and viable bacteria count in the feed additive, resulting in the feed additive prepared by this invention having good nutritional safety and high digestibility.

[0021] The fermented feed provided by this invention, by adding feed additives containing mixed microbial agents, reduces the amount of soybean meal used in the basic feed, thereby lowering breeding costs. It also improves animal production performance and feed utilization. Compared to unfermented feed, it has higher soluble protein content, higher small peptide content, higher probiotic count, better nutritional safety, and higher digestibility, making it a good feed for animals. Verification has shown that feeding the fermented feed prepared by this invention to Hy-Line Grey laying hens nearing culling in the late laying stage significantly improves their production performance and increases nutrient digestibility. Laying hens fed the fermented feed provided by this invention exhibit significantly improved egg production performance, a significantly lower feed conversion ratio, significantly increased total antioxidant capacity in the ovaries, and significantly higher serum hormone levels, resulting in significantly improved egg quality and increased economic benefits.

[0022] Preservation Instructions

[0023] The present invention relates to Bacillus subtilis YY-18, which is deposited at the China General Microbiological Culture Collection Center (CGMCC), classified and named Bacillus subtilis, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33097 and deposit date of December 17, 2024.

[0024] The Saccharomyces cerevisiae of this invention, YY-21, is deposited at the China General Microbiological Culture Collection Center (CGMCC), classified and named Saccharomyces cerevisiae, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo. 33100 and deposit date of December 17, 2024.

[0025] The *Enterococcus faecalis* YY-20 of this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC), classified and named *Enterococcus faecalis*, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33099, and deposited on December 17, 2024. Detailed Implementation

[0026] The present invention provides a mixed microbial agent comprising Bacillus subtilis YY-18 with accession number CGMCC No. 33097, Saccharomyces cerevisiae YY-21 with accession number CGMCC No. 33100, and Enterococcus faecalis YY-20 with accession number CGMCC No. 33099.

[0027] In this invention, the *Bacillus subtilis* YY-18, *Saccharomyces cerevisiae* YY-21, and *Enterococcus faecalis* YY-20 are all isolated, screened, and purified from naturally fermented enzymes. In this invention, the mixed bacterial agent is preferably a mixed bacterial liquid or a mixed bacterial powder. This invention does not have a particular limitation on the preparation method of the mixed bacterial powder; any preparation method well-known to those skilled in the art can be used. In a specific embodiment of this invention, when the mixed bacterial agent is a mixed bacterial liquid, the preparation method is as follows: after activating the *Bacillus subtilis* YY-18, *Saccharomyces cerevisiae* YY-21, and *Enterococcus faecalis* YY-20 respectively, the activated strains are inoculated into their respective liquid culture media and cultured until the logarithmic growth phase. The resulting seed liquids are then mixed to obtain the mixed bacterial agent. In this invention, the activation medium and liquid medium for Bacillus subtilis YY-18 are preferably LB solid medium and LB liquid medium, respectively; the activation medium and liquid medium for Saccharomyces cerevisiae YY-21 are preferably YPD solid medium and YPD liquid medium, respectively; and the activation medium and liquid medium for Enterococcus faecalis YY-20 are preferably MRS solid medium and MRS liquid medium, respectively. The three strains of this invention are preferably cultured in a shaker, with a shaker speed preferably of 180–220 r / min, more preferably 190–210 r / min; the temperature for culturing the strains in LB and MRS media is preferably 37°C; and the temperature for culturing the strains in YPD media is preferably 28°C. In the mixed microbial agent of the present invention, the preferred volume ratio of Bacillus subtilis YY-18, Saccharomyces cerevisiae YY-21, and Enterococcus faecalis YY-20 is (3-5):(1-3):(5-7), more preferably 4:1:6; in the mixed microbial agent of the present invention, the preferred effective viable count of Bacillus subtilis YY-18, Saccharomyces cerevisiae YY-21, and Enterococcus faecalis YY-20 per g or per mL of the mixed microbial agent is ≥3×10⁻⁶. 9 CFU.

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

[0029] This invention does not specifically limit the source of each raw material in the base material. In this invention, the preferred weight proportions of each raw material in the base material are: 8 parts DDGS, 1-3 parts corn germ meal, 0.6-1.0 parts beet molasses, 1-2 parts corn steep liquor, and 0.5 parts lysine mother liquor. In the feed additive of this invention, DDGS mainly provides protein. Through decomposition by the mixed microbial agent and acidic protease, soluble protein and small peptides can be formed, increasing the soluble protein and small peptide content in the feed additive. Corn germ meal not only provides albumin and sour protein but also provides energy substances for the mixed microbial agent. Beet molasses is an inexpensive and readily available industrial fermentation raw material, providing the carbon source required for the growth of the mixed microbial agent. Corn steep liquor is a byproduct formed during the wet starch production process of corn, rich in soluble protein, lactic acid (LA), sugars, and starch, and rich in various amino acids, minerals, trace elements, and vitamins, providing the nitrogen source required for the growth of the mixed microbial agent. Lysine mother liquor is an intermediate or byproduct produced during lysine production, with a high lysine content. DDGS is deficient in lysine. Adding lysine mother liquor can not only compensate for the lysine deficiency in DDGS, but also enable the fermentation system to have a more suitable carbon-nitrogen ratio for the growth of mixed microbial agents. Moreover, lysine mother liquor is inexpensive, which can significantly reduce production costs. In the feed additive described in this invention, the preferred mass ratio of mixed microbial agents, acidic protease, and substrate is (12-18):(1-3):100, more preferably (14-16):2:100; the measured enzyme activity of the acidic protease is preferably 7000 U / g, and this invention does not have a specific limitation on the specific source of the acidic protease.

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

[0031] In the preparation method of the feed additive of the present invention, the anaerobic fermentation temperature is preferably 28-30℃, more preferably 29℃; the anaerobic fermentation time is preferably 4-5 days. In one embodiment of the present invention, the anaerobic fermentation is carried out in a container, the container preferably including a one-way valve breathing fermentation bag; the fermentation method is preferably raw material fermentation. In one embodiment of the present invention, when using one-way valve breathing fermentation, it is preferable to perform a bag-turning operation, more preferably after 2 days of fermentation. The present invention, by turning the bag, can not only prevent the bottom material from clumping, but also allow the compound microorganisms to mix better with the bottom material, thereby promoting fermentation. In the feed additive of the present invention, the moisture content of the fermented material is preferably 42%-58%, more preferably 45%-50%. This invention utilizes a combination of Bacillus subtilis YY-18, Saccharomyces cerevisiae YY-21, and Enterococcus faecalis YY-20, along with controlled composition and ratio of the fermented substrate, moisture content, inoculum amount, fermentation time, and fermentation temperature, to prepare a feed additive with high soluble protein and small peptide content. Compared to unfermented substrate, it increases the soluble protein and small peptide content, resulting in a feed additive with good nutritional safety and high digestibility.

[0032] The present invention also provides a fermented feed, which includes the above-mentioned feed additives and basic feed; the mass ratio of the feed additives to the basic feed is (10-30):(70-90) based on the mass parts of dry matter.

[0033] In the fermented feed of this invention, the preferred mass ratio of feed additives to basal feed is (15-25):(75-85), more preferably 20:80. In this invention, the dry matter refers to the dried material after natural air drying. For example, the dry matter of feed additives is obtained by naturally air drying after the feed additives are prepared. Similarly, the dry matter of basal feed refers to the dried basal feed. In this invention, the basal feed preferably includes the following components by weight: 367-490 parts corn, 143-220 parts soybean meal, 1-5 parts salt, 20-28 parts soybean oil, 1-5 parts DL-methionine, 60 parts limestone powder, 80 parts dicalcium phosphate, and 20 parts premix; more preferably: 370-434 parts corn, 148-182 parts soybean meal, 2-3 parts salt, 22-26 parts soybean oil, 2-3 parts DL-methionine, 60 parts limestone powder, 80 parts dicalcium phosphate, and 20 parts premix. In the fermented feed described in this invention, the addition of the feed additive described in this invention can reduce the amount of soybean meal added to the basal feed, thereby reducing costs while ensuring excellent breeding results. This invention does not have specific limitations on the specific sources of the raw materials in the basal feed; conventional commercially available products in the field can be used.

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

[0035] In the application described in this invention, the animals preferably include livestock and poultry, and the livestock and poultry preferably include laying hens; the laying hens preferably include Hy-Line Grey laying hens, and the Hy-Line Grey laying hens include those about to be culled in the later stages of egg production. Hy-Line Grey laying hens experience a significant decrease in egg production and a significant increase in the rate of broken eggs in the later stages of egg production. However, feeding them the fermented feed provided by this invention can significantly reduce the number of broken or deformed eggs and also increase the number of eggs produced and egg weight. In this invention, the production performance preferably includes one or more of the following: liver index, spleen index, abdominal fat percentage, and egg production performance; the egg production performance preferably includes one or more of the following: number of eggs produced, egg weight, number of broken or deformed eggs, number of soft-shelled eggs, and egg quality.

[0036] The technical solutions provided by the present invention will be 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.

[0037] Unless otherwise specified, the following embodiments are all conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] In the following examples, DDGS was 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 commercially available, and acidic protease was purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd.

[0040] Example 1

[0041] A mixed microbial agent, wherein the mixed microbial agent comprises 4 parts by volume of Bacillus subtilis YY-18 with accession number CGMCC No.33097, 1 part by volume of Saccharomyces cerevisiae YY-21 with accession number CGMCC No.330100, and 6 parts by volume of Enterococcus faecalis YY-20 with accession number 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℃. The activated Bacillus subtilis YY-18 was then inoculated into LB liquid medium and cultured at 37℃ with a shaker at 180 rpm until the logarithmic growth phase, yielding a Bacillus subtilis YY-18 seed culture. The effective viable count of the Bacillus subtilis YY-18 seed culture was 3 × 10⁻⁶. 9 CFU / mL;

[0044] Saccharomyces cerevisiae YY-21 was inoculated into YPD solid medium and activated at 28℃. The activated YY-21 was then inoculated into YPD liquid medium and cultured at 28℃ with a shaker at 180 rpm until the logarithmic growth phase, yielding a seed culture of Saccharomyces cerevisiae YY-21. The effective viable count of the YY-21 seed culture was 3 × 10⁻⁶. 9 CFU / mL;

[0045] Enterococcus faecalis YY-20 was inoculated into MRS solid medium and activated at 37℃. The activated Enterococcus faecalis YY-20 was then inoculated into MRS liquid medium and cultured at 37℃ with a shaker at 180 rpm until the logarithmic growth phase was reached, yielding the Enterococcus faecalis YY-20 seed culture. The effective viable count of the Enterococcus faecalis YY-20 seed culture was 3 × 10⁻⁶. 9 CFU / mL;

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

[0047] The LB solid medium consists of 10g tryptone, 5g yeast extract, 10g sodium chloride, pH neutral, and 20g agar, diluted to 1000mL, and sterilized by steam at 121℃ for 30min. The LB liquid medium differs from the LB solid medium in that it does not contain agar, but is otherwise the same as the LB solid medium.

[0048] The YPD solid medium consists of 10g yeast extract, 20g peptone, 20g glucose, and 15g agar, diluted to 1000mL and sterilized by steam at 121℃ for 30min. The YPD liquid medium differs from the YPD solid medium in that it does not contain agar, but is otherwise identical to the YPD solid medium.

[0049] The MRS solid medium consists of 10g peptone, 10g beef extract, 5g yeast powder, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 5g sodium acetate, 20g glucose, 1mL Tween 80, 0.58g magnesium sulfate heptahydrate, 0.25g manganese sulfate tetrahydrate, and 20g agar, diluted to 1000mL, pH adjusted to 6.4, and sterilized by steam at 121℃ for 30min. The MRS liquid medium differs from the MRS solid medium in that it does not contain agar; otherwise, it is the same as the MRS solid medium.

[0050] Example 2

[0051] A feed additive is composed of the following raw materials in parts by weight: 1.9 parts of the mixed microbial agent obtained in Example 1, 1 part of acidic protease (the measured enzyme activity of 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 DDGS, 2 parts corn germ meal, 1 part beet molasses, 1 part corn steep liquor and 0.5 parts lysine mother liquor.

[0053] The preparation method of the feed additive is as follows: the base material, acidic protease and water are mixed to obtain fermentation material, the water content of the fermentation material is 50%, the fermentation material is mixed and stirred evenly with the mixed bacterial agent obtained in Example 1, and then put into a one-way valve fermentation bag and sealed. Anaerobic fermentation is carried out at 28°C for 4 days. On the second day of fermentation, the bag is turned over once. After the fermentation is completed, the feed additive is obtained.

[0054] Example 3

[0055] A feed additive is composed of the following raw materials in parts by weight: 2 parts of the mixed microbial agent obtained in Example 1, 1 part of acidic protease (the measured enzyme activity of acidic 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 DDGS, 2 parts corn germ meal, 1 part beet molasses, 2 parts corn steep liquor and 0.5 parts lysine mother liquor.

[0057] The preparation method of the feed additive is as follows: the base material, acidic protease and water are mixed to obtain fermentation material with a water content of 50%. The fermentation material is mixed with the mixed microbial agent and stirred evenly, then put into a one-way valve fermentation bag and sealed. Anaerobic fermentation is carried out at 30°C for 5 days. On the second day of fermentation, the bag is turned over once. After the fermentation is completed, the feed additive is obtained.

[0058] Example 4

[0059] The nutrient content and active ingredients of the substrate before and after fermentation in Example 2 were determined, and the fermentation products were analyzed.

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

[0061] Table 1. Results of determination of basic nutrient content of substrate 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: Data in the table are expressed as mean ± standard deviation. No letter on the superscript indicates no significant difference among peers, while different lowercase letters indicate statistically significant differences between samples (P<0.05).

[0064] As shown in Table 1, the feed additive prepared by this invention has increased the soluble protein content and small peptide content by 181% and 211% respectively compared with the unfermented base material, and reduced the crude fiber content by 5.76%. This indicates that the feed additive prepared by this invention has good nutritional safety, improved digestibility and utilization, and reduced fiber content, which is beneficial to the digestion of animal intestines and more beneficial to the digestion and absorption of laying hens.

[0065] In vitro simulation of livestock and poultry digestion

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

[0067] Weigh 5g of fermented feed sample and unfermented feed sample that have passed through a 100-mesh sieve, and place them in a 500mL Erlenmeyer flask. Set up three parallel samples and one blank for each sample. Add 125mL of phosphate buffer solution (0.1mol / L), stir until well mixed, then add 100mL of hydrochloric acid solution (0.2mol / L), and adjust the pH value to 2.0 with 1mol / L hydrochloric acid or 1mol / L sodium hydroxide solution.

[0068] Simulated gastric digestion: Add 50 mL of prepared porcine pepsin gastric juice to the solution from the previous step, mix well, seal, and place in a 37℃ constant temperature shaking incubator for 4 hours of shaking digestion at 120 rpm. Measure the pH of the digestive fluid. 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 fluid, stir well, and adjust the pH to 6.8.

[0069] Simulated small intestinal digestion: 50 mL of prepared simulated pig small intestinal fluid was added to the digestive juice after gastric digestion, mixed well, sealed and placed in a 37℃ constant temperature shaking incubator for 12 h of digestion. The shaking frequency of the incubator was 120 r / min, and the pH of the digested solution was measured.

[0070] Crude protein and dry matter digestibility: The residue after simulated small intestinal digestion was filtered and then rinsed with deionized water until no feed sample residue remained in the Erlenmeyer flask. 25-50 mL of 95% ethanol solution was used to rinse the filtered undigested residue, followed by 25-50 mL of 99.5% acetone solution. After the feed sample was completely filtered through the filter paper, the filter paper was removed and placed in a prepared petri dish. The dish was then dried in a 65℃ oven for 72 hours, followed by rehydration for >48 hours. A second drying cycle was performed for 24 hours, followed by rehydration for >24 hours (if data variation is large, a third drying cycle of 24 hours and rehydration for 48 hours was performed). After cooling, the contents were weighed and recorded; the difference in weight was the dry matter weight. Crude protein content was determined using the Dumas combustion method.

[0071] The viable cell count was determined by the plate count method. The results of the 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 pH of gastric digestive juices 2.26±0.01 2.23±0.01 pH of intestinal digestive fluids <![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 beneficial bacteria count (cfu / g) / <![CDATA[1.6×10 9 ]]>

[0074] Note: Data in the table are expressed as mean ± standard deviation. No letter on the superscript indicates no significant difference among peers, while different lowercase letters indicate statistically significant differences between samples (P<0.05).

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

[0076] The above results demonstrate that the soluble proteins and small peptides produced by the synergistic fermentation of mixed microbial agents and acidic proteases ensure the nutritional safety and high digestibility of the feed additives prepared in this invention.

[0077] Example 5

[0078] A fermented feed is composed of the following raw materials in parts by dry matter: 100 parts of the feed additive obtained in Example 2 and 900 parts of the basic feed.

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

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

[0081] Example 6

[0082] A fermented feed is composed of the following raw materials in parts by dry matter: 200 parts of the feed additive obtained in Example 2 and 800 parts of the basic feed.

[0083] The basic feed consists of the following ingredients by weight: 434 parts corn (dry weight), 182 parts soybean meal (dry weight), 2 parts salt, 20 parts soybean oil, 2 parts DL-methionine, 60 parts limestone powder, 80 parts dicalcium phosphate, and 20 parts premix (dry weight).

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

[0085] Example 7

[0086] A fermented feed is composed of the following raw materials in parts by dry matter weight: 300 parts of feed additive obtained in Example 2 and 700 parts of basic feed.

[0087] The basic feed consists of the following ingredients by weight: 367 parts corn (dry weight), 143 parts soybean meal (dry weight), 1 part salt, 28 parts soybean oil, 1 part DL-methionine, 60 parts limestone powder, 80 parts dicalcium phosphate, and 20 parts premix (dry weight).

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

[0089] Comparative Example 1

[0090] A basic feed consists of the following ingredients by weight: 540 parts corn (dry matter mass), 260 parts soybean meal (dry matter mass), 10 parts salt, 20 parts soybean oil, 10 parts DL-methionine, 60 parts limestone powder, 80 parts dicalcium phosphate, and 20 parts premix (dry matter mass).

[0091] The preparation method is as follows: mix all the raw materials.

[0092] Example 8

[0093] Two hundred and forty Hy-Line Grey laying hens of similar weight, laying rate, and egg weight, and in good health, aged 75 weeks and nearing culling, were randomly divided into four groups (Example 5, Example 6, Example 7, and Comparative Example 1), with four replicates per group and 15 hens per replicate. The feeding trial employed a conventional multi-layer cage system. Feeding management and routine immunization followed the technical standards for laying hen feeding management. Ventilation in the chicken house utilized a combination of natural and mechanical ventilation. Natural and artificial lighting were combined to ensure 16 hours of illumination. Free access to water was provided. Chickens were fed twice daily (8:00 AM and 5:00 PM), with consistent feed amounts across all groups. Eggs were collected daily at 4:30 PM, the cages were cleaned, and the eggs were weighed and stored repeatedly after collection. Feed formulation was carried out in accordance with the "Group Standard for Compound Feed for Layer Hens and Broilers" (T / CFIAS 002-2018) and the "Hy-Line Brown Layer Hen Feeding and Management Manual 2018". The composition of feed for different groups is shown in Table 3.

[0094] Table 3. Composition of feeds in different groups

[0095]

[0096] Determination of egg production performance

[0097] During the formal trial, egg weight, number of eggs laid, number of broken eggs, number of soft-shelled eggs, and culling status were recorded daily in replicates. The remaining feed was weighed weekly, and the average egg weight, laying rate, average daily feed intake, average daily egg production, feed conversion ratio, and rate of broken or deformed eggs were calculated for the early and late stages of the trial. The formulas for each indicator are as follows:

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

[0099] Average daily egg production [g / (hens·d)] = Total egg weight (g) / (Total number of laying hens (hens) × Number of days in the experiment (d));

[0100] Feed conversion ratio = Total feed intake (g) / Total egg weight (g);

[0101] Egg production rate (%) = Total number of eggs (chickens) / [Total number of laying hens (birds) × Number of days in the experiment (d)] × 100%;

[0102] Average egg weight (g) = Total egg weight (g) / Total number of eggs laid;

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

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

[0105] Table 4 Egg production performance of laying hens

[0106]

[0107] Note: Data in the table are expressed as mean ± standard deviation. No letter on the superscript indicates no significant difference among peers, while different lowercase letters indicate statistically significant differences between samples (P<0.05).

[0108] As shown in Table 4, by the end of feeding at 79 weeks, the average egg weight of Examples 5, 6, and 7 was not significantly different from that of Comparative Example 1 (P>0.05); the egg production rate of Examples 5, 6, and 7 was significantly higher than that of Comparative Example 1 (P<0.05), while the egg production 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); and the feed conversion ratio and the rate of broken eggs of Examples 5, 6, and 7 were significantly lower than those of Comparative Example 1 (P<0.05). By the end of 82 weeks of feeding, the average egg weight of Examples 5, 6, and 7 was not significantly different from that of Comparative Example 1 (P>0.05); the egg production rate of Examples 6 and 7 was significantly higher than that of Comparative Example 1 (P<0.05), while the egg production rate of Example 5 was not significantly different from that of Comparative Example 1 and Example 6 (P>0.05), and the egg production 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, 6, and 1 was the same. There was no significant difference in the quantity of eggs produced (P>0.05); the average daily egg production of Examples 6 and 7 was significantly higher than that of Comparative Example 1 (P<0.05), while there was no significant difference in the average daily egg production of Examples 5 and Comparative Example 1 (P>0.05); except that the feed conversion ratio of Example 6 was significantly lower than that of Comparative Example 1 (P<0.05), there was no significant difference in the feed conversion ratio of Examples 6, 7 and Comparative Example 1 (P>0.05); the rate of broken eggs in Examples 5, 6 and 7 was significantly lower than that in Comparative Example 1 (P<0.05).

[0109] In summary, adding the feed additive of this invention to the basic feed (daily ration) can significantly improve the egg production performance of laying hens. The advantages of adding the fermented feed of this invention to the basic feed (daily ration) of laying hens in the later stage of egg production are even more obvious compared with ordinary diets. The fermented feed prepared in Example 6 is more suitable for laying hens.

[0110] Determination of egg quality indicators

[0111] One day prior to sampling, three eggs were randomly selected from each group for egg quality testing. Egg shape index, shell strength, Haugh unit (HU), and yolk color were measured using a digital egg analyzer (DET-6000, NABEL, Japan). The Dumas combustion method was used to determine the yolk and whole egg albumen content. Total cholesterol (T-CHO) and triglyceride (TG) content in the yolk were determined using appropriate kits from the Nanjing Jiancheng Bioengineering Institute.

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

[0113] Table 5. Effects of fermented feed on egg quality

[0114]

[0115] Note: Data in the table are expressed as mean ± standard deviation. No letter on the superscript indicates no significant difference among peers, while different lowercase letters indicate statistically significant differences between samples (P<0.05).

[0116] As shown in Table 5, by the end of 79 weeks of feeding, the eggshell strength of Example 6 was significantly higher than that of Examples 5, 7, and Comparative Example 1 (P<0.05), while there was no significant difference in eggshell strength among Examples 5, 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), while there was no significant difference in egg shape index among Examples 5, 7, and Comparative Example 1 (P>0.05); there was no significant difference in yolk color among the groups (P>0.05); the Haugh unit of Example 7 was significantly higher than that of Comparative Example 1 (P<0.05), while there was no significant difference in Haugh unit among 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 compared to Comparative Example 1 (P>0.05); except for Example 5, where the whole egg protein was significantly lower than Comparative Example 1 (P<0.05), there was no significant difference in the whole egg protein of Examples 6 and 7 compared to Comparative Example 1 (P>0.05); except for Example 5, where the whole egg protein was significantly higher than Comparative Example 1 (P<0.05), there was no significant difference in the yolk triglycerides of Examples 6 and 7 compared to Comparative Example 1 (P>0.05); there was no significant difference in the total cholesterol of the yolk of Example 7 compared to Comparative Example 1 (P>0.05), while the total cholesterol of the yolk of Examples 5 and 6 was significantly lower than that of Comparative Example 1 (P<0.05). By the end of 82 weeks of feeding, compared with Comparative Example 1, the eggshell strength and yolk triglycerides 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 among Examples 5, 6, 7 and Comparative Example 1 (P>0.05); the egg shape index of Examples 5 and 7 was not significantly different from that of 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), while there was no significant difference in Haugh units between Examples 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), while there was no significant difference in whole egg protein between Examples 5 and Comparative Example 1 (P>0.05); the total cholesterol in the yolks of Examples 5, 6, and 7 was significantly lower than that of Comparative Example 1 (P<0.05), and there were no significant differences among the example groups (P>0.05).

[0117] In summary, compared with Comparative Example 1, adding the feed additive of this invention to the diet of laying hens can significantly increase the whole egg protein content and whole egg triglyceride content, while effectively reducing the cholesterol content of eggs.

[0118] Organ indices and follicle development in laying hens

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

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

[0121] Spleen index (g / kg) = Spleen weight (g) / Live body weight (kg);

[0122] Abdominal fat percentage (g / kg) = Abdominal fat weight (g) / Live body weight (kg);

[0123] The results are shown in Table 6. Compared with Comparative Example 1, there were no significant differences in liver and spleen indices in Examples 5, 6, and 7 during the early and late stages of the experiment (P>0.05). At the end of 79 weeks of feeding, the abdominal fat percentage in Example 6 was significantly higher than that in Examples 5, 7, and Comparative Example 1 (P<0.05), while there was no significant difference in abdominal fat percentage between Examples 5, 7, and Comparative Example 1 (P>0.05). There were no significant differences in follicle size (≥10 mm) between Examples 5, 6, 7, and Comparative Example 1 (P>0.05); however, the follicle size (5-10 mm) in Example 6 was significantly higher than that in Comparative Example 1. By the end of 82 weeks of feeding, there was no significant difference in abdominal fat percentage among Examples 5, 6, and 7 (P>0.05); the number of follicles (2-5 mm) in Examples 5, 6, and 7 was significantly higher than that in Comparative Example 1 (P<0.05), and there was no significant difference between the Example groups (P>0.05); the number of follicles (5-10 mm) in Examples 5 and 6 was significantly higher than that in Comparative Example 1, while the number of follicles (5-10 mm) in Example 7 was not significantly different from that in Comparative Example 1 (P>0.05); the number of follicles (>10 mm) in Examples 5 and 6 was significantly higher than that in Comparative Example 1, while the number of follicles (>10 mm) in Example 7 was not significantly different from that in Comparative Example 1 (P>0.05). Therefore, compared with Comparative Example 1, the addition of the feed additive of this invention to the diet of laying hens had no significant effect on the liver index, spleen index, and abdominal fat percentage of laying hens, but significantly increased the number of follicles generated in laying hens.

[0124] Table 6. Organ indices and follicle development in laying hens

[0125]

[0126] Note: Data in the table are expressed as mean ± standard deviation. No letter on the superscript indicates no significant difference among peers, while different lowercase letters indicate statistically significant differences between samples (P<0.05).

[0127] Determination of antioxidant capacity of laying hen ovaries

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

[0129] Table 7 Effects of different feeds on antioxidant indices of laying hen ovaries

[0130]

[0131]

[0132] Note: Data in the table are expressed as mean ± standard deviation. No letter on the superscript indicates no significant difference among peers, while different lowercase letters indicate statistically significant differences between samples (P<0.05).

[0133] As shown in Table 7, at the end of feeding (79 weeks), 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, and 7 compared to Comparative Example 1 (P>0.05). The malondialdehyde (MDA) content of Example 5 was significantly lower than that of Examples 6, 7, and Comparative Example 1 (P<0.05), while there were no significant differences in the MDA content of Examples 6, 7, and Comparative Example 1 (P>0.05). Compared with Comparative Example 1, except for the lack of significant difference in glutathione peroxidase (GSH-PX) in Example 7 (P>0.05), the glutathione peroxidase (GSH-PX) of Examples 5 and 6 was significantly higher than that of Comparative Example 1 (P<0.05). At the end of feeding (82 weeks), 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 was not significantly different from that of Comparative Example 1 (P>0.05); the catalase (CAT) content of Examples 5, 6, and 7 was not significantly different from that of Comparative Example 1 (P>0.05); the malondialdehyde (MDA) content of Example 6 was significantly lower than that of Examples 5, 7, and Comparative Example 1 (P<0.05), and the MDA content of Examples 5, 7, and Comparative Example 1 was not significantly different (P>0.05); the superoxide dismutase (SOD) content of Examples 6 and 7 was significantly lower than that of Comparative Example 1 (P<0.05), while the SOD content of Example 5 was not significantly different from that of Comparative Example 1 (P>0.05); the glutathione peroxidase (GSH-PX) content of Examples 5 and 6 was significantly lower than that of Comparative Example 1, while the GSH-PX content of Example 7 was not significantly different from that of Comparative Example 1. Therefore, compared with Comparative Example 1, the addition of the feed additive of this invention to the diet of laying hens has a significant effect on improving the ovarian antioxidant capacity of laying hens.

[0134] The above results indicate that the fermented feed provided by this invention, by adding feed additives, can improve animal production performance, reduce breeding costs, and increase feed utilization. By using the fermented feed prepared by this invention to feed Hy-Line Grey laying hens, it can significantly improve the laying performance and egg quality of the hens, and increase the digestibility of nutrients. Laying hens fed the fermented feed prepared by this invention show significantly improved laying performance, significantly reduced feed conversion ratio, significantly increased total antioxidant capacity of the ovaries, significantly increased serum hormone levels, and significantly improved egg quality, thus improving economic benefits.

[0135] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A feed additive, characterized in that, The feed additive comprises a mixed bacterial agent, an acidic protease and a base material, wherein the base material comprises DDGS, corn germ meal, beet molasses, corn steep liquor and lysine mother liquor; The mass ratio of the mixed bacterial agent, the acidic protease and the base material is (12-18):(1-3):100; The base material comprises 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; The mixed bacterial agent comprises Bacillus subtilis YY-18 with the preservation number of CGMCC 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; 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 cell number of Bacillus subtilis YY-18, Saccharomyces cerevisiae YY-21 and Enterococcus faecalis YY-20 is ≥3×10 9 CFU / g or mL of the mixed bacterial agent, respectively. CFU The preparation method of the feed additive comprises the following steps: mixing the base material, the acidic protease and water to obtain fermentation material, mixing the fermentation material with the mixed bacterial agent, and carrying out anaerobic fermentation at 27-31℃ for 3-6 days to obtain the feed additive; and the water content of the fermentation material is 40%-60%.

2. The feed additive according to claim 1, characterized in that, The enzyme activity of the acidic protease is 7000 U / g.

3. A fermented feed, characterized in that, The fermented feed comprises the feed additive of claim 1 or 2 and a basic feed; and the mass ratio of the feed additive and the basic feed is (10-30):(70-90) in terms of the mass fraction of dry matter.

4. The fermented feed according to claim 3, characterized in that, The basic feed comprises the following raw materials in terms of the mass fraction: corn 367-490 parts, soybean meal 143-220 parts, salt 1-5 parts, soybean oil 20-28 parts, DL-methionine 1-5 parts, stone powder 60 parts, calcium hydrogen phosphate 80 parts and premix 20 parts.

5. The use of any one of the following for improving the production performance of animals and / or for improving the feed utilization, characterized in that, (1) the feed additive of claim 1 or 2; and (2) the fermented feed of claim 3 or 4.

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