Animal microecological preparation for promoting intestinal health and preparation method thereof

Abstract

The invention belongs to the technical field of microecological preparations, and provides an animal microecological preparation for promoting intestinal health and a preparation method thereof. The preparation method comprises the following steps: firstly, respectively culturing lactobacillus reuteri and pediococcus acidilactici in an MRS culture medium, culturing bacillus subtilis in an LB culture medium, and culturing saccharomyces cerevisiae in a YPD culture medium to obtain high-activity bacterial liquid; the method comprises the following steps: mixing a multi-strain bacterial liquid with a substrate containing soybean meal hydrolysate, wheat bran and the like, and carrying out staged fermentation, so as to realize metabolism synergy among different strains; then, mixing the fermentation product with skimmed milk powder, maltodextrin and glycerol, and performing freeze drying to prepare a multi-strain probiotic preparation; and finally, mixing the probiotic preparation with nisin, pediococcus acidilactici, fructo-oligosaccharide and xylooligosaccharide, and performing vacuum drying to prepare the microecological preparation, so that the microecological preparation can effectively regulate the microecological balance of animal intestinal tracts, enhance the health level and reduce the use of antibiotics, and has remarkable economic and ecological benefits.

Description

Technical Field

[0001] This invention belongs to the field of microecological preparation technology, and relates to an animal microecological preparation for promoting intestinal health and its preparation method. Background Technology

[0002] The gut is a vital organ in animals, housing a vast and complex community of microorganisms. These microorganisms play a crucial role in the host's health and growth through various mechanisms, including regulating nutrient metabolism, maintaining intestinal barrier function, and resisting pathogen invasion. Gut health directly affects an animal's nutrient absorption efficiency, immunity level, and disease resistance. Therefore, maintaining gut health has become an important goal in animal husbandry for improving production performance and ensuring animal health.

[0003] In poultry farming, the widespread adoption of intensive farming methods presents significant challenges to poultry gut health. High-density rearing environments, monotonous feed compositions, and inadequate management practices easily lead to an imbalance in the gut microbiota, causing digestive disorders, diarrhea, inflammation, and other problems that severely impact poultry health and production performance. To address these issues, antibiotics were once widely used in poultry farming. Antibiotics effectively reduced the incidence of intestinal diseases in poultry in the short term by directly inhibiting the growth of pathogens. However, long-term and excessive use of antibiotics not only increases antibiotic resistance in pathogens but also disrupts the normal balance of the gut microbiota, triggering a series of secondary health problems. Furthermore, antibiotic residues pose a potential threat to food safety and the ecological environment. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention aims to provide an animal microecological preparation for promoting intestinal health and its preparation method. The method involves separately amplifying and culturing *Lactobacillus reuteri*, *Pediococcus acidilactici*, *Bacillus subtilis*, and *Saccharomyces cerevisiae*, mixing them in a specific ratio, and then fermenting them in stages with a composite substrate. During fermentation, aeration and pH control are used to promote synergistic metabolism of the microbial community, yielding functional fermentation products. The fermentation products are then mixed with a preservative and freeze-dried to prepare a multi-strain probiotic preparation. Subsequently, a bacteriocin additive with nisin and pediococcus lactis as its core is prepared and mixed with prebiotics such as fructooligosaccharides and xylooligosaccharides. The final microecological preparation is then prepared through vacuum drying, thus meeting the needs of practical production.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing an animal microecological preparation for promoting intestinal health, the preparation method comprising:

[0007] S1. Lactobacillus reuteri and Pediococcus acidilactici were amplified and cultured in MRS broth medium to obtain bacterial solutions A and B, respectively. Bacillus subtilis was amplified and cultured in LB broth medium to obtain bacterial solution C. Saccharomyces cerevisiae was cultured in YPD liquid medium to obtain bacterial solution D. The mixed bacterial solutions were inoculated into substrates for fermentation to obtain fermented products. The fermented products, skim milk powder, maltodextrin and glycerol were then mixed and freeze-dried to obtain a multi-strain probiotic preparation.

[0008] S2, nisin, penicillin, fructooligosaccharides, xylooligosaccharides and maltodextrin are mixed and vacuum dried to obtain a bacteriocin additive. The multi-strain probiotic preparation is mixed with the bacteriocin additive to obtain an animal microecological preparation for promoting intestinal health.

[0009] Lactobacillus reuteri and Pediococcus acidilactici were cultured statically in MRS medium to simulate the intestinal microaerobic environment, promoting their acid production and antimicrobial substance synthesis. Lactobacillus reuteri secreted reuterin, which specifically inhibited pathogens, while Pediococcus acidilactici enhanced intestinal barrier function through competitive adhesion. Bacillus subtilis, cultured in LB medium using a shaker, activated its aerobic metabolic characteristics, secreting digestive enzymes to break down complex substrates and simultaneously forming tolerant spores to cope with subsequent processing stress. Saccharomyces cerevisiae, cultured in YPD medium using a microaerobic shaker, balanced its metabolic pathways, synthesized immunologically active substances such as β-glucan, and created a survival advantage for anaerobic bacteria through aerobic consumption.

[0010] After the four bacterial strains are mixed in a specific volume ratio, *Bacillus subtilis* dominates substrate decomposition in the early stages of fermentation, using its secreted amylases and proteases to convert soybean meal protein and corn starch into small-molecule nutrients, providing metabolic precursors for lactic acid bacteria. *Lactobacillus reuteri* and *Pediococcus acidilactici* subsequently lower the pH of the environment by producing acid, inhibiting the proliferation of other bacteria and activating their own antimicrobial peptide expression, forming an acidic microecological barrier. *Saccharomyces cerevisiae* maintains a microaerobic environment in the middle and later stages of fermentation by consuming residual oxygen, and its synthesized antioxidants such as glutathione protect the bacterial community from oxidative damage. Soybean meal hydrolysate provides easily absorbed small peptides and amino acids, wheat bran acts as a prebiotic to stimulate bacterial proliferation, gelatinized corn starch increases the content of fermentable sugars, and yeast extract activates the bacterial metabolic enzyme system through B vitamins. In the staged fermentation process, the initial high-temperature forced ventilation accelerates substrate decomposition by Bacillus subtilis, while turning the compost evenly distributes nutrients and prevents localized overheating. The subsequent low-temperature static fermentation regulates gas exchange through a permeable membrane, promoting lactic acid bacteria biofilm formation to prolong the active metabolic period. Simultaneously, the antioxidants of Saccharomyces cerevisiae stabilize the fermentation system. In the protective agent system, skim milk powder encapsulates the cells through casein micelles to reduce freezing damage, maltodextrin forms a glassy matrix to fix cell structure, and glycerol regulates osmotic pressure to maintain cell membrane integrity.

[0011] In stage S2, functional additives are constructed by integrating antimicrobial active substances and prebiotics, forming a synergistic system with the probiotic preparations prepared in stage S1 to achieve multi-dimensional regulation of the intestinal microecology. Nisin and pechocin, as bacteriocinoid antimicrobial peptides, selectively inhibit the proliferation of harmful bacteria such as Salmonella by targeting and disrupting the cell membrane integrity of pathogenic bacteria, while not affecting the activity of beneficial bacteria such as lactic acid bacteria. Fructooligosaccharides and xylooligosaccharides, as bifidus factors, resist the degradation by host digestive enzymes through β-glycosidic bonds in their molecular structure. Upon reaching the intestines, they are preferentially metabolized and utilized by probiotics, stimulating the secretion of short-chain fatty acids and enhancing bacterial adhesion, thus creating a positive feedback probiotic effect.

[0012] Maltodextrin plays a dual role at this stage: firstly, as an excipient, it adsorbs bacteriocins and prebiotics, forming a uniform dispersion system through intermolecular hydrogen bonds to avoid local aggregation and inactivation of active ingredients; secondly, during vacuum drying, it forms a porous carrier structure, reducing water activity to inhibit the thermal denaturation of bacteriocins and minimizing oxidative damage to sensitive components caused by oxygen permeation. Vacuum drying employs a gentle temperature and time-controlled strategy to ensure efficient water removal while maintaining the spatial conformational stability of bacteriocins, avoiding protein denaturation caused by high temperatures. Finally, when the bacteriocin additive and probiotic preparation are mixed in a specific ratio, the bacteriocin forms an antibacterial barrier in the intestinal environment, inhibiting the competitive rejection of probiotics by pathogens; prebiotics continuously activate the metabolic activity of probiotics, extending their colonization period; and the organic acids produced by probiotic metabolism further enhance the antibacterial effect of bacteriocins, forming a closed-loop regulation of "antibacterial inhibition-proliferation promotion-metabolic enhancement."

[0013] As a preferred embodiment of the present invention, in step S1, *Lactobacillus reuteri* and *Pediococcus acidilactici* are amplified and cultured separately in MRS broth medium under the following conditions: temperature 37±1℃, for example, 36.0℃, 36.2℃, 36.4℃, 36.6℃, 36.8℃, 37.0℃, 37.2℃, 37.4℃, etc. Incubate at 37.6℃, 37.8℃, or 38.0℃, pH 6±0.2 (e.g., 5.8, 5.9, 6.0, 6.1, or 6.2), with static incubation for 16-17 hours (e.g., 16.0h, 16.1h, 16.2h, 16.3h, 16.4h, 16.5h, 16.6h, 16.7h, 16.8h, 16.9h, or 17.0h). The target values ​​are: OD600 ≥ 2.0, and a viable Lactobacillus reuteri concentration ≥ 1 × 10⁻⁶. 9 CFU / mL, viable concentration of Pediococcus acidilactici ≥5×10⁻⁶ 8 The value is CFU / mL, but not limited to the listed values; other unlisted values ​​within this range also apply. The *Lactobacillus reuteri* is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 15062, and the *Pediococcus acidilactici* is deposited at the same center with accession number CGMCC No. 30078.

[0014] In some optional examples, the amplification culture of Bacillus subtilis in LB broth medium is carried out under the following conditions: temperature 37±1℃, for example, 36.0℃, 36.2℃, 36.4℃, 36.6℃, 36.8℃, 37.0℃, 37.2℃, 37.4℃, 37.6℃, 37.8℃, or 38.0℃; pH 7±0.2, for example, 6.8, 6.9, 7.0, 7.1, or 7.2; and shaking culture for 18-19 h, for example, 18.0 h, 18.1 h, or 18.2 h. The shaking time is 18.3h, 18.4h, 18.5h, 18.6h, 18.7h, 18.8h, 18.9h, or 19.0h, with a shaking speed of 130-150 rpm (e.g., 130 rpm, 132 rpm, 134 rpm, 136 rpm, 138 rpm, 140 rpm, 142 rpm, 144 rpm, 146 rpm, 148 rpm, or 150 rpm). The target values ​​are: OD600 ≥ 1.5 and Bacillus subtilis viable cell concentration ≥ 1 × 10⁻⁶. 9 The value is CFU / mL, but not limited to the listed values. Other unlisted values ​​within this range also apply. The Bacillus subtilis is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 24947.

[0015] In some optional instances, the *Saccharomyces cerevisiae* is cultured in YPD liquid medium under the following conditions: 30±1℃, for example, 29.0℃, 29.2℃, 29.4℃, 29.6℃, 29.8℃, 30.0℃, 30.2℃, 30.4℃, 30.6℃, 30.8℃, or 31.0℃; pH 5.8±0.2, for example, 5.6, 5.7, 5.8, 5.9, or 6.0; and cultured on a shaker for 20-21 hours, for example, 20.0h, 20.1h, 20.2h, 20.3h, 20.4h, 20.5h, 20.6h, 20.7h, 20.8h, 20.9h, or 21.0h, with the target being: OD600 ≥ 3.0 and viable cell concentration ≥ 5 × 10⁻⁶. 8 The value is CFU / mL, but not limited to the listed values. Other unlisted values ​​within this range also apply. The Saccharomyces cerevisiae is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 13133.

[0016] In some optional instances, the volume ratio of bacterial solution A, bacterial solution B, bacterial solution C to bacterial solution D in the mixed bacterial solution is 4:4:1:1.

[0017] In some alternative instances, the mass ratio of the mixed bacterial culture to the substrate is (10-11):100, for example, (10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9 or 11.0):100, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0018] In some optional examples, the mass ratio of soybean meal hydrolysate, wheat bran, corn starch, maltodextrin, and yeast extract in the substrate is (5-5.5):2:(1.5-1.7):(1-1.2):0.5, for example, it can be (5.00, 5.05, 5.10, 5.15, 5.20, 5.25, 5.30, 5.35, 5.40, 5.45, or 5.50):2:(1.50, 1.52, 1.54, 1.56, 1.58, 1... 0.60, 1.62, 1.64, 1.66, 1.68 or 1.70): (1.00, 1.02, 1.04, 1.06, 1.08, 1.10, 1.12, 1.14, 1.16, 1.18 or 1.20): 0.5. Soybean meal hydrolysate is hydrolyzed with neutral protease at 55℃ for 6 hours. Wheat bran is passed through an 80-mesh sieve. Corn starch is gelatinized at 90℃ for 30 minutes. However, this is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0019] In some optional examples, the fermentation is as follows: Stage 1, 0-12h, temperature 37±1℃, for example, 36.0℃, 36.2℃, 36.4℃, 36.6℃, 36.8℃, 37.0℃, 37.2℃, 37.4℃, 37.6℃, 37.8℃, or 38.0℃, forced ventilation 1L / min / kg, turning the pile every 4h, adjusting the pH with 5wt.% NaHCO3 when it is ≥5.5; Stage 2, 12-36h, temperature 30±1℃, for example, 29.0℃, 29.2℃, 29.4℃, 29.6℃, 29.8℃, 30.0℃, 30.2℃, 30.4℃, 30.6℃, 30.8℃, or 31.0℃, covered with a breathable membrane for static fermentation, turning the pile every 8h, with a final total viable count ≥1.5×10⁻⁶. 9 CFU / g, wet basis, but not limited to the listed values; other unlisted values ​​within this range also apply.

[0020] In some alternative instances, the mass ratio of the fermented product, skim milk powder, maltodextrin, and glycerol is 100:(8-9):50:(2-3), for example, it can be 100:(8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0):50:(2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0), but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0021] As a preferred embodiment of the present invention, in S2, the mass ratio of nisin, penicillin, fructooligosaccharides, xylooligosaccharides, and maltodextrin is (15-20):5:(25-28):(15-17):40, for example, it can be (15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, or 20.0):5: (25.0, 25.3, 25.6, 25.9, 26.2, 26.5, 26.8, 27.1, 27.4, 27.7 or 28.0): (15.0, 15.2, 15.4, 15.6, 15.8, 16.0, 16.2, 16.4, 16.6, 16.8 or 17.0): 40, but not limited to the listed values; other unlisted values ​​within this range also apply.

[0022] In some alternative instances, the vacuum drying temperature is 40-45°C, for example, 40.0°C, 40.5°C, 41.0°C, 41.5°C, 42.0°C, 42.5°C, 43.0°C, 43.5°C, 44.0°C, 44.5°C, or 45.0°C, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0023] In some optional instances, the vacuum drying time is 4-5 hours, for example, 4.0 hours, 4.1 hours, 4.2 hours, 4.3 hours, 4.4 hours, 4.5 hours, 4.6 hours, 4.7 hours, 4.8 hours, 4.9 hours, or 5.0 hours, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0024] In some optional examples, the nisin activity is ≥30000 IU / g, measured by agar diffusion, with Staphylococcus aureus as the indicator bacterium.

[0025] In some alternative instances, the mass ratio of the multi-strain probiotic preparation to the bacteriocin additive is 2:(1-1.2), for example, it can be 2:(1.00, 1.02, 1.04, 1.06, 1.08, 1.10, 1.12, 1.14, 1.16, 1.18 or 1.20), but is not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0026] Secondly, the present invention provides an animal microecological preparation for promoting intestinal health, prepared by the preparation method described in the first aspect.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Through the scientific ratio and staged fermentation process of Lactobacillus reuteri, Pediococcus acidilactici, Bacillus subtilis and Saccharomyces cerevisiae, metabolic synergy among different probiotic species is achieved. The synergistic effect of lactic acid bacteria regulating the acidity of the fermentation environment, Bacillus subtilis secreting digestive enzymes, and yeast producing functional metabolites significantly improves the functionality of probiotic preparations; (2) The probiotic preparations are combined with bacteriocins such as nisin and pediococcin, as well as prebiotics such as fructooligosaccharides and xylooligosaccharides to form a multi-level functional system. Bacteriocins directly kill pathogens through broad-spectrum antibacterial effects, and prebiotics promote the proliferation of probiotics and the generation of metabolites. The two work together to strengthen the regulation of the intestinal microecological balance, which is significantly better than single probiotic or prebiotic preparations. Detailed Implementation

[0028] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The embodiments described herein are specific implementations of the present invention and are used to illustrate the concept of the present invention; these descriptions are explanatory and exemplary and should not be construed as limiting the implementation of the present invention or the scope of protection of the present invention. In addition to the embodiments described herein, those skilled in the art can also adopt other obvious technical solutions based on the content disclosed in the claims and the specification of this application. These technical solutions include those that make any obvious substitutions and modifications to the embodiments described herein.

[0029] The chemical reagents used in the embodiments and comparative examples of this invention are all commercially available products and have not undergone any further purification treatment.

[0030] Example 1

[0031] This embodiment provides an animal microecological preparation for promoting gut health and its preparation method, the preparation method specifically including the following steps:

[0032] S1. *Lactobacillus reuteri* and *Pediococcus acidilactici* were amplified and cultured separately in MRS broth medium under the following conditions: temperature 36℃, pH 5.8, and static culture for 16 h, yielding bacterial suspensions A and B, respectively. Both suspensions had a BOD600 ≥ 2.0, and suspension A contained a viable *Lactobacillus reuteri* concentration ≥ 1 × 10⁻⁶. 9 CFU / mL, viable concentration of Pediococcus acidilactici in bacterial culture ≥5×10⁻⁶ 8 Bacillus subtilis was amplified and cultured in LB broth at CFU / mL under the following conditions: temperature 36℃, pH 6.8, shake incubation for 18 h at a shaker speed of 130 rpm, yielding bacterial culture C. The bacterial culture had a COD600 ≥ 1.5 and a viable Bacillus subtilis concentration ≥ 1 × 10⁻⁶ CFU / mL. 9 Saccharomyces cerevisiae was cultured in YPD liquid medium at CFU / mL under the following conditions: 30℃, pH 5.8, and shake incubation for 20 h to obtain bacterial culture solution D. The OD600 of bacterial culture solution D was ≥3.0, and the viable cell concentration was ≥5×10⁻⁶. 8 CFU / mL, 1 kg of mixed bacterial solution was inoculated onto 10 kg of substrate for fermentation to obtain fermented product. The volume ratio of bacterial solution A, B, C and D in the mixed bacterial solution was 4:4:1:1. The mass ratio of soybean meal hydrolysate, wheat bran, corn starch, maltodextrin and yeast extract in the substrate was 5:2:1.5:1:0.5. The soybean meal hydrolysate was hydrolyzed with neutral protease at 55℃ for 6 h, the wheat bran was sieved through an 80-mesh sieve, and the corn starch was gelatinized at 90℃ for 30 min. The fermentation was carried out as follows: Stage 1, 0-12 h, temperature 37℃, forced ventilation 1 L / min / kg, turning the pile every 4 h, adjusting the pH with 5 wt.% NaHCO3 when it was ≥5.5; Stage 2, 12-36 h, temperature 30℃, covered with a breathable membrane and allowed to ferment statically, turning the pile every 8 h, with a final total viable count ≥1.5 × 10⁻⁶. 9 CFU / g, wet basis, then mix 1000g fermentation product, 80g skim milk powder, 500g maltodextrin and 20g glycerol, freeze dry to obtain multi-strain probiotic preparation;

[0033] S2, 150g of nisin, 50g of pechosin, 250g of fructooligosaccharides, 150g of xylooligosaccharides and 400g of maltodextrin are mixed and vacuum dried at 40℃ for 4.0h to obtain a bacteriocin additive. The nisin has an activity ≥30000IU / g. 200g of multi-strain probiotic preparation is mixed with 100g of bacteriocin additive to obtain an animal microecological preparation for promoting intestinal health.

[0034] Example 2

[0035] This embodiment provides an animal microecological preparation for promoting gut health and its preparation method, the preparation method specifically including the following steps:

[0036] S1. *Lactobacillus reuteri* and *Pediococcus acidilactici* were amplified and cultured separately in MRS broth medium under the following conditions: temperature 38℃, pH 6.2, and static culture for 16.3 h, yielding bacterial suspensions A and B, respectively. Both suspensions had a BOD600 ≥ 2.0, and suspension A contained a viable *Lactobacillus reuteri* concentration ≥ 1 × 10⁻⁶. 9 CFU / mL, viable concentration of Pediococcus acidilactici in bacterial culture ≥5×10⁻⁶ 8 Bacillus subtilis was amplified and cultured in LB broth at CFU / mL under the following conditions: temperature 37.3℃, pH 7, shake incubation for 18.4 h at a shaker speed of 140 rpm, yielding bacterial culture C. The bacterial culture had a COD600 ≥ 1.5 and a viable Bacillus subtilis concentration ≥ 1 × 10⁻⁶ CFU / mL. 9 Saccharomyces cerevisiae was cultured in YPD liquid medium at CFU / mL under the following conditions: 29℃, pH 5.6, and shake incubation for 20.3 h to obtain bacterial culture solution D. The OD600 of bacterial culture solution D was ≥3.0, and the viable cell concentration was ≥5×10⁻⁶. 8CFU / mL, 1.25 kg of mixed bacterial solution was inoculated onto 12 kg of substrate for fermentation to obtain fermented product. The volume ratio of bacterial solution A, B, C and D in the mixed bacterial solution was 4:4:1:1. The mass ratio of soybean meal hydrolysate, wheat bran, corn starch, maltodextrin and yeast extract in the substrate was 5.5:2:1.6:1.2:0.5. The soybean meal hydrolysate was hydrolyzed with neutral protease at 55℃ for 6 h, the wheat bran was sieved through an 80-mesh sieve, and the corn starch was gelatinized at 90℃ for 30 min. The fermentation was carried out as follows: Stage 1, 0-12 h, temperature 36℃, forced ventilation 1 L / min / kg, turning the pile every 4 h, adjusting the pH with 5 wt.% NaHCO3 when it was ≥5.5; Stage 2, 12-36 h, temperature 29℃, covered with a breathable membrane and allowed to ferment statically, turning the pile every 8 h, with a final total viable count ≥1.5 × 10⁻⁶. 9 CFU / g, wet basis, then mix 1000g fermentation product, 90g skim milk powder, 500g maltodextrin and 30g glycerol, freeze dry to obtain multi-strain probiotic preparation;

[0037] S2, 200g of nisin, 50g of pechosin, 280g of fructooligosaccharides, 170g of xylooligosaccharides and 400g of maltodextrin are mixed and vacuum dried at 45℃ for 5.0h to obtain a bacteriocin additive. The nisin has an activity ≥3000IU / g. 200g of multi-strain probiotic preparation is mixed with 120g of bacteriocin additive to obtain an animal microecological preparation for promoting intestinal health.

[0038] Example 3

[0039] This embodiment provides an animal microecological preparation for promoting gut health and its preparation method, the preparation method specifically including the following steps:

[0040] S1. *Lactobacillus reuteri* and *Pediococcus acidilactici* were amplified and cultured separately in MRS broth medium under the following conditions: temperature 37.4℃, pH 6.1, and static culture for 17 h, yielding bacterial suspensions A and B, respectively. Both suspensions had a BOD600 ≥ 2.0, and suspension A contained a viable *Lactobacillus reuteri* concentration ≥ 1 × 10⁻⁶. 9 CFU / mL, viable concentration of Pediococcus acidilactici in bacterial culture ≥5×10⁻⁶ 8Bacillus subtilis was amplified and cultured in LB broth at CFU / mL under the following conditions: temperature 38℃, pH 7.2, shake incubation for 19 h at a shaker speed of 150 rpm, yielding bacterial culture C. The bacterial culture had a COD600 ≥ 1.5 and a viable Bacillus subtilis concentration ≥ 1 × 10⁻⁶ CFU / mL. 9 Saccharomyces cerevisiae was cultured in YPD liquid medium at CFU / mL under the following conditions: 31℃, pH 6.0, and shake incubation for 21 h to obtain bacterial culture solution D. The OD600 of bacterial culture solution D was ≥3.0, and the viable cell concentration was ≥5×10⁻⁶. 8 CFU / mL, 1.65 kg of mixed bacterial solution was inoculated onto 15 kg of substrate for fermentation to obtain the fermented product. The volume ratio of bacterial solution A, B, C and D in the mixed bacterial solution was 4:4:1:1. The mass ratio of soybean meal hydrolysate, wheat bran, corn starch, maltodextrin and yeast extract in the substrate was 5.1:2:1.7:1.1:0.5. The soybean meal hydrolysate was hydrolyzed with neutral protease at 55℃ for 6 h, the wheat bran was sieved through an 80-mesh sieve, and the corn starch was gelatinized at 90℃ for 30 min. The fermentation was carried out as follows: Stage 1, 0-12 h, temperature 38℃, forced ventilation 1 L / min / kg, turning the pile every 4 h, adjusting the pH with 5 wt.% NaHCO3 when it was ≥5.5; Stage 2, 12-36 h, temperature 31℃, covered with a breathable membrane and allowed to ferment statically, turning the pile every 8 h, with a final total viable count ≥1.5 × 10⁻⁶. 9 CFU / g, wet basis, then 1000g fermentation product, 83g skim milk powder, 500g maltodextrin and 24g glycerol are mixed and freeze-dried to obtain a multi-strain probiotic preparation;

[0041] S2, 180g of nisin, 50g of pechosin, 270g of fructooligosaccharides, 160g of xylooligosaccharides and 400g of maltodextrin are mixed and vacuum dried at 44℃ for 4.3h to obtain a bacteriocin additive. The nisin has an activity ≥30000IU / g. 200g of multi-strain probiotic preparation is mixed with 110g of bacteriocin additive to obtain an animal microecological preparation for promoting intestinal health.

[0042] Example 4

[0043] This embodiment provides an animal microecological preparation for promoting gut health and its preparation method, the preparation method specifically including the following steps:

[0044] S1. *Lactobacillus reuteri* and *Pediococcus acidilactici* were amplified and cultured separately in MRS broth medium under the following conditions: temperature 36.6℃, pH 5.9, and static incubation for 16.7 h, yielding bacterial suspensions A and B, respectively. Both suspensions had a BOD600 ≥ 2.0, and suspension A contained a viable *Lactobacillus reuteri* concentration ≥ 1 × 10⁻⁶. 9 CFU / mL, viable concentration of Pediococcus acidilactici in bacterial culture ≥5×10⁻⁶ 8 Bacillus subtilis was amplified and cultured in LB broth at CFU / mL under the following conditions: temperature 36.8℃, pH 6.9, shake incubation for 18.7 h at a shaker speed of 135 rpm, yielding bacterial culture C. The bacterial culture had a COD600 ≥ 1.5 and a viable Bacillus subtilis concentration ≥ 1 × 10⁻⁶ CFU / mL. 9 Saccharomyces cerevisiae was cultured in YPD liquid medium at CFU / mL under the following conditions: 30.5℃, pH 5.9, and shake incubation for 20.8 h to obtain bacterial culture solution D. The OD600 of bacterial culture solution D was ≥3.0, and the viable cell concentration was ≥5×10⁻⁶. 8 CFU / mL, 1.4 kg of mixed bacterial solution was inoculated onto 13 kg of substrate for fermentation to obtain fermented product. The volume ratio of bacterial solution A, B, C and D in the mixed bacterial solution was 4:4:1:1. The mass ratio of soybean meal hydrolysate, wheat bran, corn starch, maltodextrin and yeast extract in the substrate was 5.3:2:1.5:1.2:0.5. The soybean meal hydrolysate was hydrolyzed with neutral protease at 55℃ for 6 h, the wheat bran was sieved through an 80-mesh sieve, and the corn starch was gelatinized at 90℃ for 30 min. The fermentation was carried out as follows: Stage 1, 0-12 h, temperature 36.5℃, forced ventilation 1 L / min / kg, turning the pile every 4 h, adjusting the pH with 5 wt.% NaHCO3 when it was ≥5.5; Stage 2, 12-36 h, temperature 30.5℃, covered with a breathable membrane and allowed to ferment statically, turning the pile every 8 h, with a final total viable count ≥1.5 × 10⁻⁶. 9 CFU / g, wet basis, then 1000g fermentation product, 87g skim milk powder, 500g maltodextrin and 28g glycerol are mixed and freeze-dried to obtain a multi-strain probiotic preparation;

[0045] S2, 160g of nisin, 50g of pechosin, 260g of fructooligosaccharides, 155g of xylooligosaccharides and 400g of maltodextrin are mixed and vacuum dried at 41℃ for 4.8h to obtain a bacteriocin additive. The nisin has an activity ≥30000IU / g. 200g of multi-strain probiotic preparation is mixed with 105g of bacteriocin additive to obtain an animal microecological preparation for promoting intestinal health.

[0046] The animal microecological preparations obtained in Examples 1-4 were mixed with basic feed and used for broiler chicken feeding. The amount of animal microecological preparations added to the basic feed was 3 kg / ton.

[0047] Comparative Example 1

[0048] This comparative example only uses basal feed to feed broilers.

[0049] Comparative Example 2

[0050] This comparative example provides an animal microecological preparation for promoting gut health and its preparation method. The difference between this and Example 1 is that the mass of the multi-strain probiotic preparation in step S2 is 0, while other process parameters and operating conditions are exactly the same as in Example 1. The animal microecological preparation prepared in this comparative example is mixed with basal feed for broiler chicken feeding. The amount of animal microecological preparation added to the basal feed should be 3 kg / ton minus the amount corresponding to the multi-strain probiotic preparation.

[0051] Comparative Example 3

[0052] This comparative example provides an animal microecological preparation for promoting intestinal health and its preparation method. The difference between this example and Example 1 is that the mass of the bacteriocin additive in step S2 is 0, while other process parameters and operating conditions are exactly the same as in Example 1. The animal microecological preparation prepared in this comparative example is mixed with basal feed for broiler chicken feeding. The amount of animal microecological preparation added to the basal feed should be 3 kg / ton minus the corresponding amount of bacteriocin additive.

[0053] Basic feed: Corn and soybean meal are the main raw materials, and the feed formula is designed with reference to the People's Republic of China Agricultural Industry Standard for Broiler Chicken Feeding Standard (NY / T33-2004).

[0054] Experimental chickens: 240 one-day-old Ross 308 broilers, with 6 replicates per group and 10 chickens per replicate. Experimental period: 42 days (12 days in the early stage and 30 days in the later stage). Environmental control: Temperature 32℃ in the early stage, decreasing by 2℃ per week; relative humidity 60%–70%; 24-hour light (20 lx) in the early stage, reduced to 18-hour light (10 lx) in the later stage; routine immunization program was maintained, no additional drugs were used, and no antibiotics were added throughout the entire process. Good ventilation in the chicken house was maintained, and the pens were inspected twice daily. The weight of dead chickens was recorded in a timely manner, and the feed consumption of that cage was deducted.

[0055] Average weight gain was measured by weighing the chickens in their cages at 1 day, 21 days, and 42 days of age. Average weight = cage weight / number of live chickens. Average weight gain = (average weight at 42 days - average weight at 1 day) / number of live chickens / number of days. Feed conversion ratio (FCR) was measured using the cumulative method (total feed consumption / total weight gain). Diarrhea rate was determined by fecal score (0-3 points). Diarrhea rate = number of days with a score ≥2 / total number of days × 100%. Fecal score criteria: 0 points: normally formed (cylindrical); 1 point: soft feces (slightly deformed); 2 points: loose feces (moisture content >80%); 3 points: watery feces (anorexia). Extreme value handling: data from dead chickens were excluded (survival rate was counted separately).

[0056] The test results are shown in Table 1.

[0057] Table 1. Test results of Examples 1-4 and Comparative Examples 1-3

[0058] Average weight gain (g / animal / day) Meat-to-fat ratio Diarrhea rate (%) Survival rate (%) Example 1 52.3 1.62 8.3 96 Example 2 54.7 1.60 9.6 97 Example 3 53.6 1.64 6.3 98 Example 4 54.1 1.63 7.5 96 Comparative Example 1 42 1.86 21.7 84 Comparative Example 2 48.7 1.75 12.6 91 Comparative Example 3 47.3 1.78 16.4 90

[0059] As shown in Table 1, compared to Example 1, Comparative Example 1 showed a decrease in average weight gain, an increase in feed conversion ratio, an increase in diarrhea rate, and a decrease in survival rate; Comparative Example 2 showed a decrease in average weight gain, an increase in feed conversion ratio, an increase in diarrhea rate, and a decrease in survival rate; and Comparative Example 3 showed a decrease in average weight gain, an increase in feed conversion ratio, an increase in diarrhea rate, and a decrease in survival rate. This is because Comparative Example 1 was fed only a basic feed without animal microecological preparations, lacking the effect of multi-strain probiotic preparations to promote nutrient absorption and bacteriocin additives to reduce pathogens. Comparative Example 2 lacked multi-strain probiotic preparations, thus lacking the effect of probiotics promoting nutrient absorption. Comparative Example 3 lacked bacteriocin additives, failing to achieve the effect of bacteriocin destroying the pathogen membrane structure, thus increasing the diarrhea rate.

[0060] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for the preparation of an animal microecological preparation for promoting intestinal health, characterized in that, The preparation method comprises: S1, Lactobacillus reuteri and Pediococcus acidilactici are respectively amplified and cultured in MRS broth medium, to obtain bacterial liquid A and bacterial liquid B respectively, Bacillus subtilis is amplified and cultured in LB broth medium to obtain bacterial liquid C, Saccharomyces cerevisiae is cultured in YPD liquid medium to obtain bacterial liquid D, a mixed bacterial liquid comprising the bacterial liquid A, the bacterial liquid B, the bacterial liquid C and the bacterial liquid D is inoculated into a substrate to ferment to obtain a fermentation product, and the fermentation product, skimmed milk powder, malt dextrin and glycerol are mixed and freeze-dried to obtain a multi-strain probiotic preparation; S2, nisin, pediocin, fructooligosaccharide, xylooligosaccharide and malt dextrin are mixed and vacuum dried to obtain a bacteriocin additive, and the multi-strain probiotic preparation and the bacteriocin additive are mixed to obtain an animal microecological preparation for promoting intestinal health.

2. The method for preparing an animal microecological preparation for promoting intestinal health according to claim 1, characterized in that, In S1: Said Lactobacillus reuteri and Pediococcus acidilactici are respectively amplified in MRS broth medium, with the conditions of temperature 37±1℃, pH=6±0.2, static culture for 16-17h, and the target of OD600≥2.0, Lactobacillus reuteri viable concentration ≥1×10 9 CFU / mL, and Pediococcus acidilactici viable concentration ≥5×10 8 CFU / mL.

3. The method of claim 1, wherein the animal microecological preparation for promoting intestinal health is prepared by the steps of: In S1: The Bacillus subtilis is amplified in LB broth culture, with the conditions of temperature 37±1℃, pH=7±0.2, 18-19h of shaker culture, the speed of shaker 130-150rpm, and the target of OD600≥1.5, the live Bacillus subtilis concentration ≥1×10 9 CFU / mL. The Bacillus subtilis is amplified in LB broth culture, with the conditions of temperature 37±1℃, pH=7±0.2, 18-19h of shaker culture, the speed of shaker 130-150rpm, and the target of OD600≥1.5, the live Bacillus subtilis concentration ≥1×10 9 CFU / mL.

4. The method of claim 1, wherein the animal microecological preparation for promoting intestinal health is prepared by the steps of: In S1: The Saccharomyces cerevisiae is cultured in YPD liquid medium under the conditions of 30±1℃, pH=5.8±0.2, and shaking culture for 20-21 h, with the target of OD600≥3.0 and viable cell concentration≥5×10 8 CFU / mL. The Saccharomyces cerevisiae is cultured in YPD liquid medium under the conditions of 30±1℃, pH=5.8±0.2, and shaking culture for 20-21 h, with the target of OD600≥3.0 and viable cell concentration≥5×10 8 CFU / mL.

5. The method of claim 1, wherein the animal microecological preparation for promoting intestinal health is prepared by the steps of: In S1: In the mixed bacterial liquid, the volume ratio of the bacterial liquid A, the bacterial liquid B, the bacterial liquid C and the bacterial liquid D is 4:4:1:

1. The mass ratio of the mixed bacterial liquid to the substrate is (10-11):

100.

6. The method of claim 1, wherein the animal microecological preparation for promoting intestinal health is prepared by the steps of: In S1: In the substrate, the mass ratio of soybean meal hydrolysate, wheat bran, corn starch, malt dextrin and yeast extract powder is (5-5.5):2:(1.5-1.7):(1-1.2):0.5, the soybean meal hydrolysate is hydrolyzed by neutral protease at 55°C for 6h, the wheat bran is sieved to 80 meshes, and the corn starch is gelatinized at 90°C for 30min.

7. The method of claim 1, wherein the animal microecological preparation for promoting intestinal health is prepared by the steps of: In S1: The fermentation is: phase one, 0-12h, temperature 37±1℃, forced ventilation 1L / min / kg, pile turning every 4h, pH≥5.5 is adjusted with 5wt.% NaHCO3, phase two, 12-36h, temperature 30±1℃, static fermentation with breathable film covering, pile turning every 8h, end point total viable bacterial count≥1.5×10 9 CFU / g, wet basis CFU / g, wet basis The mass ratio of the fermentation product, skimmed milk powder, malt dextrin and glycerol is 100:(8-9):50:(2-3).

8. The method of claim 1, wherein the animal microecological preparation for promoting intestinal health is prepared by the steps of: In S2: The mass ratio of nisin, pediocin, fructooligosaccharide, xylooligosaccharide and malt dextrin is (15-20):5:(25-28):(15-17):

40. The activity of nisin is ≥30000IU / g, and the agar diffusion method is used with Staphylococcus aureus as the indicator bacteria.

9. The method of claim 1, wherein the animal microecological preparation for promoting intestinal health is prepared by the steps of: In S2: The mass ratio of the multi-strain probiotic preparation to the bacteriocin additive is 2:(1-1.2). The temperature of vacuum drying is 40-45°C. The time of vacuum drying is 4-5h.

10. An animal microecological preparation for promoting intestinal health, characterized in that, According to any one of claims 1-9.