Enzyme preparation for livestock feed as well as preparation method and application of enzyme preparation

Abstract

The invention discloses an enzyme preparation for livestock feed as well as a preparation method and application thereof, belongs to the technical field of agricultural microbial enzymes, and particularly relates to a preparation method of the enzyme preparation for livestock feed, which comprises the following steps: carrying out shaking table fermentation culture on a bacillus licheniformis solution to obtain a bacillus licheniformis fermentation mixed solution, centrifuging, and taking the supernatant to obtain a beta-mannase solution; performing fermentation culture on the bacillus velezensis bacterial liquid to obtain bacillus velezensis fermentation mixed liquid, performing centrifugation, and taking supernatant to obtain cellulase liquid; compounding aspergillus niger liquid, performing fermentation culture to obtain aspergillus niger liquid fermentation mixed liquid, and performing centrifugation to obtain compound enzyme liquid; mixing the beta-mannase liquid, the cellulase liquid and the compound enzyme liquid, and performing freeze drying treatment to obtain mixed enzyme powder; and mixing the mixed enzyme powder with momordica grosvenori glucoside to obtain the enzyme preparation for the livestock feed. The compound enzyme liquid is prepared by selecting specific strains for fermentation, the effect of improving the feed utilization rate of the prepared enzyme preparation is improved, and growth and development of livestock can be effectively promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural microbial enzymes, and in particular relates to an enzyme preparation for livestock feed, a preparation method thereof and an application thereof. Background Art

[0002] Enzymes, as highly efficient biocatalysts, are widely used in industry, medicine, and scientific research. Even enzymes of the same type catalyzing the same reaction can exhibit significant differences in stability and functional properties depending on the source organism (e.g., bacteria, fungi, archaea), the production strain (different strains of the same microorganism), and the fermentation process. These differences directly impact the effectiveness and economic viability of enzyme applications. For example, the amino acid sequences of enzymes derived from thermophiles, acidophiles, alkaliphiles, and halophiles typically contain more ion pairs, salt bridges, hydrophobic interactions, a high proline content, a high number of disulfide bonds, and a more compact hydrophobic core. These structural features confer exceptional stability under extreme environments (high temperature, extreme pH, and high salt). Archaeal enzymes are generally more thermostable than their bacterial and fungal counterparts. Different isoforms of Bacillus subtilis proteases have different hydrolysis site preferences for synthetic substrates or natural proteins. Fungal and bacterial proteases often have different substrate profiles. α-amylases from different sources can differ in their hydrolysis efficiency for amylose / amylopectin and in the distribution of products (maltose, glucose, and limit dextrins). The proportions and synergistic efficiency of cellulase components (endoglucanase, exoglucanase, β-glucosidase) produced by different fungi (such as Trichoderma reesei, Aspergillus niger) or bacteria are different, which affects their ability to degrade different types of cellulose (crystalline region, amorphous region).

[0003] As an external influencing factor, fermentation conditions can also affect the stability and function of enzymes obtained by microbial fermentation. Fermentation temperature and pH can induce microorganisms to produce stress response proteins, which may assist the correct folding of target enzymes or affect the post-translational modification of the enzymes themselves, thereby affecting their final conformation and stability. For example, the components of the culture medium, such as carbon and nitrogen sources, affect the growth rate and metabolic flow of bacteria, indirectly affecting the synthesis efficiency and folding environment of enzyme proteins; the type and concentration of specific inducers (such as lactose-induced β-galactosidase) affect the constitutive or inducible expression of enzymes, and sometimes also affect the structural characteristics of enzymes; metal ions in the culture medium (such as Ca) affect the enzyme's expression and the enzyme's stability. 2+ Critical for α-amylase and protease stability) may be directly integrated into the enzyme structure or act as stabilizers.

[0004] Many industrial enzyme preparations are mixtures of multiple isozymes or synergistic enzyme components. Fermentation conditions and strain differences can lead to changes in the proportions of the components in the mixture, thereby changing the overall functional properties of the enzyme preparation (such as substrate preference and hydrolysis product distribution). Screening specific bacterial strains, discovering their fermentation conditions such as fermentation temperature and culture medium composition, and developing new enzyme preparations that can be used to improve feed utilization and enhance feed effectiveness are crucial to the research and development and production of enzyme preparations. Summary of the Invention

[0005] To solve the above technical problems, the present invention proposes an enzyme preparation for livestock feed, a preparation method and application thereof. The present invention selects specific strains for fermentation to prepare β-mannanase, cellulase and a composite enzyme liquid composed of glucose oxidase, xylanase and phytase, thereby improving the effect of the prepared enzyme preparation on improving feed utilization and effectively promoting the growth and development of livestock.

[0006] To achieve the above object, the present invention provides a method for preparing an enzyme preparation for livestock feed, comprising the following steps:

[0007] 1) Fermenting a Bacillus licheniformis bacterial solution on a shaking platform to obtain a Bacillus licheniformis fermentation mixture, centrifuging it first, and collecting the supernatant to obtain a β-mannanase solution;

[0008] 2) fermenting a Bacillus velezensis liquid to obtain a Bacillus velezensis fermentation mixed liquid, centrifuging it a second time, and collecting the supernatant to obtain a cellulase liquid;

[0009] 3) fermenting the composite Aspergillus niger liquid to obtain a fermentation mixture of the Aspergillus niger liquid, and then centrifuging the mixture to obtain a composite enzyme solution;

[0010] 4) mixing the β-mannanase solution obtained in step 1), the cellulase solution obtained in step 2), and the complex enzyme solution obtained in step 3), and freeze-drying the mixture at -50 to -40°C for 15 to 20 hours to obtain a mixed enzyme powder;

[0011] 5) The mixed enzyme powder obtained in step 4) is mixed with mogroside to obtain an enzyme preparation for livestock feed.

[0012] Preferably, the Bacillus licheniformis culture solution in step 1) is a Bacillus licheniformis CICC 10334 culture solution; the Bacillus Velez culture solution in step 2) is a Bacillus Velez CICC 24257 culture solution; and the composite Aspergillus niger culture solution in step 3) is a mixture of Aspergillus CICC 40277 culture solution, Aspergillus niger CICC 40273 culture solution and Aspergillus niger CICC 41796 culture solution in a volume ratio of 1:1:1.

[0013] Further preferably, the effective viable cell count of the Bacillus licheniformis solution in step 1) is ≥ 1×10 8CFU / mL; the viable cell number of the B. licheniformis bacterial liquid in step 2) is ≥ 1×10 8 CFU / mL; the total viable cell number of the compound A. niger bacterial liquid in step 3) is ≥ 10 8 CFU / mL; the viable cell number of A. niger CICC 40277 in the compound A. niger bacterial liquid is ≥ 10 5 CFU / mL; the viable cell number of A. niger CICC 40273 in the compound A. niger bacterial liquid is ≥ 10 6 CFU / mL; the viable cell number of A. niger CICC 41796 in the compound A. niger bacterial liquid is ≥ 10 6 CFU / mL.

[0014] Preferably, the inoculation amount of the B. licheniformis bacterial liquid in step 1) is 8% to 10% v / v, the temperature of the shake flask fermentation culture in step 1) is 35 to 39℃, the rotation speed of the shake flask fermentation culture in step 1) is 150 to 250 rpm, and the time of the shake flask fermentation culture in step 1) is 48 to 72 h.

[0015] Preferably, the inoculation amount of the B. licheniformis bacterial liquid in step 2) is 2% to 10% v / v, the temperature of the fermentation culture in step 2) is 33 to 37℃, the rotation speed of the fermentation culture in step 2) is 150 to 250 rpm, the time of the fermentation culture in step 2) is 48 to 96 h, and the pH of the fermentation culture in step 2) is 6.5 to 7.5.

[0016] Preferably, the inoculation amount of the compound A. niger bacterial liquid in step 3) is 8% to 10% v / v, the temperature of the fermentation culture in step 3) is 28 to 37℃, the rotation speed of the fermentation culture in step 3) is 400 to 800 rpm, the time of the fermentation culture in step 3) is 72 to 120 h, and the pH of the fermentation culture in step 3) is 5.0 to 5.5.

[0017] Preferably, the mixing volume ratio of the β-mannanase liquid obtained in step 1), the cellulase liquid obtained in step 2), and the compound enzyme liquid obtained in step 3) in step 4) is 1:1:1.

[0018] Preferably, the mass ratio of the mixed enzyme powder obtained in step 4) and the mogroside in step 5) is 8 to 12:1.

[0019] The application also provides an enzyme preparation for poultry feed prepared by the preparation method.

[0020] The application also provides the application of the enzyme preparation for poultry feed prepared by the preparation method in the preparation of feed for promoting the growth and development of poultry.

[0021] Compared with the prior art, the present invention has the following advantages and technical effects:

[0022] The present invention provides an enzyme preparation for livestock feed, a preparation method and an application thereof. Specific strains of Bacillus licheniformis CICC 10334, Bacillus velez CICC 24257, Aspergillus CICC 40277, Aspergillus niger CICC40273 and Aspergillus niger CICC 41796 are selected for fermentation to prepare beta-mannanase, cellulase and a composite enzyme solution composed of glucose oxidase, xylanase and phytase. By optimizing the production strains and the process conditions for fermentation preparation of the enzymes, the effect of the prepared enzyme preparation on improving feed utilization is improved, and the growth and development of livestock can be effectively promoted. In the enzyme preparation provided by the present invention, β-mannanase can degrade mannan in feed, releasing nutrients wrapped by it, thereby improving feed utilization. By degrading mannan, the viscosity of intestinal contents is reduced, making nutrients easier to digest and absorb. The generated mannan oligosaccharides can serve as prebiotics, promote the proliferation of beneficial intestinal microorganisms, inhibit harmful bacteria, and enhance animal immunity. Phytase can decompose phytic acid in feed, release phosphorus and other minerals, and improve the bioavailability of these nutrients. β-mannanase and xylanase are two major hemicellulose-degrading enzymes, and their synergistic effect in feed can significantly improve the digestibility of the diet. The coordinated effect of β-mannanase and glucose oxidase in feed is mainly reflected in promoting the digestion and absorption of nutrients, improving intestinal health, enhancing liver function, and preventing mold growth. The enzyme preparation of the present invention improves feed utilization and animal health through the above-mentioned effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 Statistics of average daily weight gain of experimental pigs in different groups;

[0025] Figure 2 Statistics of feed-to-weight ratio of experimental pigs in different groups;

[0026] Figure 3 Statistics on slaughter rate of experimental pigs in different groups;

[0027] Figure 4 Statistics of ketone body lean meat rate of different groups of experimental pigs. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] Unless otherwise specified, the "parts" in the present invention are all based on mass. Materials not otherwise specified in the present invention are all commercially available.

[0034] Bacillus licheniformis CICC 10334, Bacillus velezensis CICC 24257, Aspergillus sp. CICC 40277, Aspergillus niger CICC 40273, and Aspergillus niger CICC 41796 were all purchased from the China Industrial Microbiological Culture Collection. Mogroside was purchased from Huacheng Biotechnology; β-mannanase (1000 U / g) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; and glucose oxidase (10,000 U / g) was purchased from Merck.

[0035] The experimental feed used in the present invention is commercially available, and its formula is 36% corn, 24% soybean meal, 35% second flour, 2% rice husk, 1.2% calcium hydrogen phosphate, 0.5% stone powder, 0.40% salt, 0.1% lysine, 0.1% methionine, 20g / 50kg of multivitamins, and 0.7% of trace element premix; the nutritional components DE (MCal / kg) are 3.00, DM 86.63%, CP 18.69%, EE 2.46%, CF 4.59%, Ca 0.88%, and P 0.67%.

[0036] Example 1

[0037] 1) Bacillus licheniformis CICC 10334 bacterial solution (effective viable cell count ≥ 1×10 8 CFU / mL), according to the inoculation amount of 9% v / v, 37 ° C, 200 rpm shaker fermentation culture for 60 h to obtain Bacillus licheniformis fermentation mixture, 4 ° C, 12000 rpm first centrifugation for 20 min, the supernatant was taken to obtain β-mannanase solution;

[0038] Culture medium: 17.5 g konjac flour, 17.5 g soybean cake powder, 5 g yeast extract, 3 g (NH4)2SO4, 1 g K2HPO4, 1 g KH2PO4, 0.3 g MgSO4·7H2O, 0.1 g CaCl2, and water to 1 L, pH 7.2.

[0039] 2) Bacillus velezensis CICC 24257 bacterial solution (effective viable cell count ≥ 1×10 8 CFU / mL), according to the inoculation amount of 6% v / v, 35°C, 200 rpm, pH 7, fermentation culture for 72 hours, to obtain the Bacillus velezensis fermentation mixture, 4°C, 12000 rpm second centrifugation for 20 minutes, the supernatant was taken to obtain the cellulase solution;

[0040] Culture medium: CMC-Na 10 g, yeast extract 7.5 g, peptone 7.5 g, KH2PO4 2 g, K2HPO4 2 g, MgSO4·7H2O 0.4 g, CaCl2 0.03 g, and water to 1 L.

[0041] 3) Composite Aspergillus niger liquid (total effective viable bacteria ≥10 8 Spores / mL, Aspergillus CICC 40277 culture (effective viable count ≥ 10 5 spores / mL), Aspergillus niger CICC 40273 bacterial solution (effective viable count ≥10 6 spores / mL) and Aspergillus niger CICC41796 culture (effective viable bacteria count ≥10 6 spores / mL) were mixed in a volume ratio of 1:1:1), and the inoculum size was 9% v / v, and the mixture was fermented at 33°C, 600 rpm, and pH 5.3 for 96 h to obtain a fermentation mixture of Aspergillus niger, and the mixture was centrifuged at 4°C, 12000 rpm for 20 min to obtain a complex enzyme solution;

[0042] Culture medium: bran 5 g, glucose 1.25 g, corn steep liquor 2 g, yeast extract 1 g, (NH4)2SO4 0.2 g, KH2PO4 0.2 g, MgSO4·7H2O 0.07 g, CaCl2·2H2O 0.03 g, FeSO4·7H2O 0.003 g.

[0043] 4) mixing the β-mannanase solution, the cellulase solution, and the complex enzyme solution in a volume ratio of 1:1:1, and freeze-drying the solution at -50°C for 15 hours to obtain a mixed enzyme powder;

[0044] 5) The mixed enzyme powder and mogroside are mixed in a mass ratio of 10:1 to obtain an enzyme preparation for livestock feed.

[0045] Example 2

[0046] 1) Bacillus licheniformis CICC 10334 bacterial solution (effective viable cell count ≥ 1×10 8 CFU / mL), according to the inoculation amount of 8% v / v, 35 ° C, 150 rpm shaker fermentation culture for 48 hours to obtain Bacillus licheniformis fermentation mixture, 4 ° C, 12000 rpm first centrifugation for 20 minutes, the supernatant was collected to obtain β-mannanase solution;

[0047] Culture medium: 15g konjac flour, 15g soybean cake powder, 5g yeast extract, 3g (NH4)2SO4, 1g K2HPO4, 1g KH2PO4, 0.3g MgSO4·7H2O, 0.1g CaCl2, and dilute to 1L with water, pH 7.2.

[0048] 2) Bacillus velezensis CICC 24257 bacterial solution (effective viable cell count ≥ 1×10 8 CFU / mL), according to the inoculation amount of 2% v / v, 33 ° C, 150 rpm, pH 6.5, fermentation culture for 48 hours to obtain Bacillus Velez fermentation mixture, 4 ° C, 12000 rpm second centrifugation for 20 minutes, the supernatant was taken to obtain cellulase solution;

[0049] Culture medium: CMC-Na 5 g, yeast extract 5 g, peptone 5 g, KH2PO4 1 g, K2HPO4 1 g, MgSO4·7H2O 0.2 g, CaCl2 0.01 g, and water to 1 L.

[0050] 3) Composite Aspergillus niger liquid (total effective viable bacteria ≥10 8 Spores / mL, Aspergillus CICC 40277 culture (effective viable count ≥ 10 5 spores / mL), Aspergillus niger CICC 40273 bacterial solution (effective viable count ≥10 6 spores / mL) and Aspergillus niger CICC41796 culture (effective viable bacteria count ≥10 6 spores / mL) were mixed in a volume ratio of 1:1:1), and the inoculum size was 8% v / v, and the mixture was fermented at 28° C., 400 rpm, and pH 5.0 for 72 h to obtain a fermentation mixture of Aspergillus niger, and the mixture was centrifuged at 4° C., 12000 rpm for 20 min to obtain a complex enzyme solution;

[0051] Culture medium: bran 3 g, glucose 0.5 g, corn steep liquor 1 g, yeast extract 0.5 g, (NH4)2SO4 0.1 g, KH2PO4 0.1 g, MgSO4·7H2O 0.05 g, CaCl2·2H2O 0.01 g, FeSO4·7H2O 0.001 g.

[0052] 4) mixing the β-mannanase solution, the cellulase solution, and the complex enzyme solution in a volume ratio of 1:1:1, and freeze-drying the solution at -40°C for 15 hours to obtain a mixed enzyme powder;

[0053] 5) The mixed enzyme powder and mogroside are mixed in a mass ratio of 8:1 to obtain an enzyme preparation for livestock feed.

[0054] Example 3

[0055] 1) Bacillus licheniformis CICC 10334 bacterial solution (effective viable cell count ≥ 1×10 8 CFU / mL), according to the inoculation amount of 10% v / v, 39 ° C, 250 rpm shaker fermentation culture for 72 hours to obtain Bacillus licheniformis fermentation mixture, 4 ° C, 12000 rpm first centrifugation for 20 minutes, the supernatant was collected to obtain β-mannanase solution;

[0056] Culture medium: 20 g konjac flour, 20 g soybean cake powder, 5 g yeast extract, 3 g (NH4)2SO4, 1 g K2HPO4, 1 g KH2PO4, 0.3 g MgSO4·7H2O, 0.1 g CaCl2, and water to 1 L, pH 7.2.

[0057] 2) Bacillus velezensis CICC 24257 bacterial solution (effective viable cell count ≥ 1×10 8 CFU / mL), according to the inoculum size of 10% v / v, 37°C, 250 rpm, pH 7.5, fermentation culture for 96 hours to obtain the Bacillus Velez fermentation mixture, 4°C, 12000 rpm for the second centrifugation for 20 minutes, and the supernatant was taken to obtain the cellulase solution;

[0058] Culture medium: CMC-Na 15 g, yeast extract 10 g, peptone 10 g, KH2PO4 3 g, K2HPO4 3 g, MgSO4·7H2O 0.5 g, CaCl2 0.05 g, and water to 1 L.

[0059] 3) Composite Aspergillus niger liquid (total effective viable bacteria ≥10 8 Spores / mL, Aspergillus CICC 40277 culture (effective viable count ≥ 10 5 spores / mL), Aspergillus niger CICC 40273 bacterial solution (effective viable count ≥10 6 spores / mL) and Aspergillus niger CICC41796 culture (effective viable bacteria count ≥10 6 spores / mL) were mixed in a volume ratio of 1:1:1), the inoculum size was 10% v / v, and the mixture was fermented at 37°C, 800 rpm, and pH 5.5 for 120 h to obtain a fermentation mixture of Aspergillus niger, and the mixture was centrifuged at 4°C, 12000 rpm for 20 min to obtain a complex enzyme solution;

[0060] Culture medium: bran 8 g, glucose 2 g, corn steep liquor 3 g, yeast extract 1.5 g, (NH4)2SO4 0.3 g, KH2PO4 0.3 g, MgSO4·7H2O 0.1 g, CaCl2·2H2O 0.05 g, FeSO4·7H2O 0.005 g.

[0061] 4) mixing the β-mannanase solution, the cellulase solution, and the complex enzyme solution in a volume ratio of 1:1:1, and freeze-drying the solution at -50°C for 20 hours to obtain a mixed enzyme powder;

[0062] 5) The mixed enzyme powder and mogroside are mixed in a mass ratio of 12:1 to obtain an enzyme preparation for livestock feed.

[0063] Comparative Example 1

[0064] 1) Bacillus velezensis CICC 24257 bacterial solution (effective viable cell count ≥ 1×10 8 CFU / mL), according to the inoculation amount of 6% v / v, 35°C, 200 rpm, pH 7, fermentation culture for 72 hours, to obtain the Bacillus velezensis fermentation mixture, 4°C, 12000 rpm second centrifugation for 20 minutes, the supernatant was taken to obtain the cellulase solution;

[0065] Culture medium: CMC-Na 10 g, yeast extract 7.5 g, peptone 7.5 g, KH2PO4 2 g, K2HPO4 2 g, MgSO4·7H2O 0.4 g, CaCl2 0.03 g, and water to 1 L.

[0066] 2) Composite Aspergillus niger liquid (total effective viable bacteria count ≥ 10 8 Spores / mL, Aspergillus CICC 40277 culture (effective viable count ≥ 10 5 spores / mL), Aspergillus niger CICC 40273 bacterial solution (effective viable count ≥10 6 spores / mL) and Aspergillus niger CICC41796 culture (effective viable bacteria count ≥10 6 spores / mL) were mixed in a volume ratio of 1:1:1), and the inoculum size was 9% v / v, and the mixture was fermented at 33°C, 600 rpm, and pH 5.3 for 96 h to obtain a fermentation mixture of Aspergillus niger, and the mixture was centrifuged at 4°C, 12000 rpm for 20 min to obtain a complex enzyme solution;

[0067] Culture medium: bran 5 g, glucose 1.25 g, corn steep liquor 2 g, yeast extract 1 g, (NH4)2SO4 0.2 g, KH2PO4 0.2 g, MgSO4·7H2O 0.07 g, CaCl2·2H2O 0.03 g, FeSO4·7H2O 0.003 g.

[0068] 3) β-mannanase (1000 U / g, purchased from Shanghai Yuanye Biotechnology Co., Ltd.), cellulase solution, and complex enzyme solution were mixed at a volume ratio of 1:1:1, and the solution was freeze-dried at -50°C for 15 h to obtain mixed enzyme powder;

[0069] 5) The mixed enzyme powder and mogroside were mixed in a mass ratio of 10:1 to obtain a commercial mixed enzyme preparation 1.

[0070] Comparative Example 2

[0071] 1) Bacillus licheniformis CICC 10334 bacterial solution (effective viable cell count ≥ 1×10 8CFU / mL), according to the inoculation amount of 9% v / v, 37 ° C, 200 rpm shaker fermentation culture for 60 h to obtain Bacillus licheniformis fermentation mixture, 4 ° C, 12000 rpm first centrifugation for 20 min, the supernatant was taken to obtain β-mannanase solution;

[0072] Culture medium: 17.5 g konjac flour, 17.5 g soybean cake powder, 5 g yeast extract, 3 g (NH4)2SO4, 1 g K2HPO4, 1 g KH2PO4, 0.3 g MgSO4·7H2O, 0.1 g CaCl2, and water to 1 L, pH 7.2.

[0073] 2) Bacillus velezensis CICC 24257 bacterial solution (effective viable cell count ≥ 1×10 8 CFU / mL), according to the inoculation amount of 6% v / v, 35°C, 200 rpm, pH 7, fermentation culture for 72 hours, to obtain the Bacillus velezensis fermentation mixture, 4°C, 12000 rpm second centrifugation for 20 minutes, the supernatant was taken to obtain the cellulase solution;

[0074] Culture medium: CMC-Na 10 g, yeast extract 7.5 g, peptone 7.5 g, KH2PO4 2 g, K2HPO4 2 g, MgSO4·7H2O 0.4 g, CaCl2 0.03 g, and water to 1 L.

[0075] 3) Composite Aspergillus niger liquid (total effective viable bacteria ≥10 8 Spores / mL, Aspergillus niger CICC 40273 bacterial solution (effective viable count ≥ 10 6 spores / mL) and Aspergillus niger CICC 41796 culture (effective viable count ≥10 6 The mixture was inoculated with 9% v / v of spores / mL) and cultured at 33° C., 600 rpm, and pH 5.3 for 96 h to obtain a fermentation mixture of Aspergillus niger. The mixture was then centrifuged at 4° C., 12,000 rpm, and 20 min to obtain a complex enzyme solution. The complex enzyme solution was then mixed with glucose oxidase (10,000 U / g, purchased from Merck) at a volume ratio of 2:1 to obtain a premixed enzyme solution.

[0076] Culture medium: bran 5 g, glucose 1.25 g, corn steep liquor 2 g, yeast extract 1 g, (NH4)2SO4 0.2 g, KH2PO4 0.2 g, MgSO4·7H2O 0.07 g, CaCl2·2H2O 0.03 g, FeSO4·7H2O 0.003 g.

[0077] 4) mixing the β-mannanase solution, the cellulase solution, and the premixed enzyme solution in a volume ratio of 1:1:1, and freeze-drying the solution at -50°C for 15 hours to obtain a mixed enzyme powder;

[0078] 5) The mixed enzyme powder and mogroside were mixed in a mass ratio of 10:1 to obtain a commercial mixed enzyme preparation II.

[0079] Experimental Example 1

[0080] 75 test pigs (weighing 18 kg ± 1 kg) were selected and divided into three groups. They were fed the enzyme preparation prepared in Example 1, the commercial mixed enzyme preparation 1 prepared in Comparative Example 1, and the commercial mixed enzyme preparation 2 prepared in Comparative Example 2, respectively. A pre-feeding period was conducted, with the test diets fed for 7 days. Afterwards, the formal test began. The enzyme preparation prepared in Example 1, the commercial mixed enzyme preparation 1 prepared in Comparative Example 1, or the commercial mixed enzyme preparation 2 prepared in Comparative Example 2 were mixed into the feed diets at a mass fraction of 1.5%. The three groups of test pigs were fed four times daily, until full each time, for 30 days, with free access to water.

[0081] The average daily weight gain of the three groups of experimental pigs was counted and the feed-to-weight ratio was calculated using feed-to-weight ratio = feed consumption / weight gain, which indicates the growth performance and feed utilization rate of the pigs.

[0082] Five experimental pigs were selected from each group. After the feeding experiment, they were slaughtered and the head, hooves, tail and internal organs (except kidneys) were removed. The slaughter rate was calculated using the formula: slaughter rate (%) = carcass weight / live weight of livestock and poultry × 100%. The pigs were divided into three parts according to skin fat, muscle and bone, and the lean meat rate was calculated using the formula: carcass lean meat rate (%) = lean meat weight / (lean meat weight + fat weight + skin weight + bone weight) × 100%.

[0083] Table 1 Statistics of average daily weight gain and feed-to-weight ratio of experimental pigs in different groups

[0084] Group Average daily weight gain (g) Average daily feed intake (g) Material-to-weight ratio Example 1 group 625.13±88.14 1337.78±25.43 2.14±0.02 Comparative Example 1 541.09±121.06 1271.56±30.19 2.35±0.14 Comparative Example 2 560.28±137.71 1428.71±41.25 2.55±0.11

[0085] According to Table 1 and Figure 1 and Figure 2As shown, it can be seen that the enzyme preparation prepared in Example 1 can significantly improve the growth performance of pigs and their average daily weight gain after being mixed with the feed, and the feed-to-weight ratio is significantly better than that of Comparative Example 1 and Comparative Example 2, thereby improving the utilization rate of feed and truly reducing the amount of food consumed by pigs while increasing their daily weight gain. The enzyme preparation mixed with the feed in Comparative Example 1 is commercial β-mannanase. According to the above comparison, it can be seen that the performance of β-mannanases from different sources is different, which further affects the nutritional composition of the mixed feed and the enzymatic hydrolysis of the effective nutrients. This proves that the enzyme preparation prepared by the specific strain of the present invention has superior performance and is suitable for feed addition. In Comparative Example 2, the enzyme preparation mixed into the feed used commercial glucose oxidase. The above results demonstrate that the performance of glucose oxidases from different sources varies, further affecting the enzymatic hydrolysis of the active ingredients in the mixed feed. This demonstrates that the enzyme preparations prepared using the bacterial suspensions of specific strains of Bacillus licheniformis CICC 10334, Bacillus velezensis CICC 24257, Aspergillus CICC 40277, Aspergillus niger CICC 40273, and Aspergillus niger CICC 41796 of the present invention have superior enzymatic hydrolysis effects and are suitable for use as feed additives.

[0086] Table 2 Statistics of slaughter rate and ketone lean meat rate of different groups of experimental pigs

[0087] Group Slaughter rate (%) Ketone body lean meat rate (%) Example 1 group 75.12±1.21 67.75±0.55 Comparative Example 1 71.44±1.78 62.22±0.89 Comparative Example 2 70.26±1.88 61.01±1.05

[0088] According to Table 2 and Figure 3 and Figure 4 It can be seen from the records that the enzyme preparation prepared in Example 1 of the present invention is mixed with the feed, which can significantly improve the slaughter rate of the test pigs, and the ketone body lean meat rate is also improved. This further proves that the enzyme preparation prepared in Example 1 of the present invention can effectively enzymatically hydrolyze the high-utilization nutrients in the feed, and can effectively promote the muscle growth of the test pigs while promoting their weight gain, not only promoting their fattening. This effect may be achieved by combining β-mannanase and glucose oxidase in the feed to promote the digestion and absorption of nutrients, improve intestinal health, and enhance liver function. The enzyme preparations prepared in Comparative Examples 1 and 2 replaced the β-mannanase and glucose oxidase prepared by specific strains, respectively, resulting in a decrease in the slaughter rate and lean meat rate of pigs after mixing the enzyme preparations. This proves that the enzyme preparations prepared by the preparation method of the present invention using the bacterial liquid of specific strains Bacillus licheniformis CICC 10334, Bacillus velezensis CICC 24257, Aspergillus CICC 40277, Aspergillus niger CICC40273, and Aspergillus niger CICC 41796 have excellent enzymatic hydrolysis effects and are suitable for use as feed additives.

[0089] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing an enzyme preparation for livestock feed, characterized in that: The following steps are involved: 1) Fermenting a Bacillus licheniformis bacterial solution on a shaking platform to obtain a Bacillus licheniformis fermentation mixture, centrifuging it first, and collecting the supernatant to obtain a β-mannanase solution; 2) fermenting a Bacillus velezensis liquid to obtain a Bacillus velezensis fermentation mixed liquid, centrifuging it a second time, and collecting the supernatant to obtain a cellulase liquid; 3) fermenting the composite Aspergillus niger liquid to obtain a fermentation mixture of the Aspergillus niger liquid, and then centrifuging the mixture to obtain a composite enzyme solution; 4) mixing the β-mannanase solution obtained in step 1), the cellulase solution obtained in step 2), and the complex enzyme solution obtained in step 3), and freeze-drying the mixture at -50 to -40°C for 15 to 20 hours to obtain a mixed enzyme powder; 5) The mixed enzyme powder obtained in step 4) is mixed with mogroside to obtain an enzyme preparation for livestock feed.

2. The preparation method according to claim 1, characterized in that The Bacillus licheniformis solution in step 1) is a Bacillus licheniformis CICC 10334 solution; the Bacillus Velez bacteria solution in step 2) is a Bacillus Velez bacteria CICC24257 solution; and the composite Aspergillus niger solution in step 3) is a mixture of Aspergillus CICC 40277 solution, Aspergillus niger CICC 40273 solution, and Aspergillus niger CICC 41796 solution in a volume ratio of 1:1:

1.

3. The preparation method according to claim 2, characterized in that: The effective viable count of the Bacillus licheniformis solution in step 1) is ≥ 1×10 8 CFU / mL; the effective viable count of the Bacillus Velezii liquid in step 2) is ≥1×10 8 CFU / mL; the total effective viable count of the composite Aspergillus niger liquid in step 3) is ≥10 8 spores / mL, the effective viable count of Aspergillus CICC 40277 in the composite black Aspergillus liquid is ≥10 5 spores / mL, the effective viable count of Aspergillus niger CICC40273 in the composite Aspergillus niger bacterial solution is ≥10 6 spores / mL, the effective viable count of Aspergillus niger CICC 41796 in the composite Aspergillus niger bacterial solution is ≥10 6 Spores / mL.

4. The preparation method according to claim 1, characterized in that The inoculation amount of the Bacillus licheniformis solution in step 1) is 8% to 10% v / v, the temperature of the shaking fermentation culture in step 1) is 35 to 39° C., the rotation speed of the shaking fermentation culture in step 1) is 150 to 250 rpm, and the time of the shaking fermentation culture in step 1) is 48 to 72 hours.

5. The preparation method according to claim 1, characterized in that: The inoculation amount of the Bacillus velezensis liquid in step 2) is 2% to 10% v / v, the temperature of the fermentation culture in step 2) is 33 to 37° C., the rotation speed of the fermentation culture in step 2) is 150 to 250 rpm, the fermentation culture time in step 2) is 48 to 96 h, and the pH of the fermentation culture in step 2) is 6.5 to 7.

5.

6. The preparation method according to claim 1, characterized in that: The inoculation amount of the composite Aspergillus niger liquid in step 3) is 8% to 10% v / v, the fermentation culture temperature in step 3) is 28 to 37° C., the fermentation culture speed in step 3) is 400 to 800 rpm, the fermentation culture time in step 3) is 72 to 120 h, and the pH of the fermentation culture in step 3) is 5.0 to 5.

5.

7. The preparation method according to claim 1, characterized in that: In step 4), the mixing volume ratio of the β-mannanase solution obtained in step 1), the cellulase solution obtained in step 2), and the complex enzyme solution obtained in step 3) is 1:1:

1.

8. The preparation method according to claim 1, characterized in that: In step 5), the mass ratio of the mixed enzyme powder obtained in step 4) to mogroside is 8 to 12:

1.

9. The enzyme preparation for livestock feed prepared by the method according to any one of claims 1 to 8.

10. Use of the enzyme preparation for livestock feed prepared by the preparation method according to any one of claims 1 to 8 in preparing feed for promoting the growth and development of livestock.

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