Piglet intestinal flora regulating feed prepared by solid-state fermentation of aromatic plants with compound saccharomycetes and preparation method of piglet intestinal flora regulating feed

Abstract

The invention relates to the field of animal nutrition and feed science, in particular to piglet intestinal flora regulating feed prepared by solid-state fermentation of aromatic plants through compound saccharomycetes and a preparation method of the piglet intestinal flora regulating feed. 20 to 30 parts of pichia pastoris; 15 to 20 parts of rosemary; 15 to 20 parts of thyme; 40-50 parts of wheat bran; 20-30 parts of corn flour; 15-20 parts of soybean meal; 2-5 parts of rice hull powder; 3-5 parts of molasses; 1-3 parts of yeast autolysate; 0.5 to 1.0 part of monopotassium phosphate; 0.2 to 0.5 part of magnesium sulfate; saccharomyces cerevisiae and pichia pastoris are subjected to solid-state fermentation treatment, the viable count of yeast in the feed is larger than or equal to 2.0 * 10 < 8 > CFU / g, the saccharomyces cerevisiae generates a large amount of alpha-amylase and protease, the pichia pastoris generates high-activity beta-glucanase and xylanase, and the two yeasts have a synergistic effect, so that the substrate utilization rate and the release efficiency of active ingredients of aromatic plants are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of animal nutrition and feed science, and particularly to a feed for regulating the intestinal flora of piglets by solid-state fermentation of spice plants with compound yeasts and a preparation method thereof. The present invention is particularly applicable to the regulation of intestinal health, the improvement of digestive function and the enhancement of growth performance of weaned piglets. Background Art

[0003] With the increasingly strict restrictions on the use of antibiotics in the livestock industry, finding safe and effective alternatives to antibiotics has become a current research hotspot. Due to factors such as the transition from breast milk to solid feed and the change of the feeding environment, weaned piglets often face problems such as intestinal flora imbalance and intestinal barrier function damage, resulting in a high incidence of diarrhea and growth retardation, causing huge economic losses to the pig industry. To solve these problems, the research and application of probiotics, prebiotics and other functional feed additives have received extensive attention.

[0004] US Patent US20240350427A1 discloses a plant essential oil amino acid composition and a preparation method thereof, which is obtained by mixing a plant essential oil with an amino acid and is used to improve the growth performance of animals. Chinese Patent CN107927376A discloses a preparation method of a feed additive for dairy cows, which is used to improve lactation performance.

[0005] Yeast, as an important microbial resource, has broad application prospects in the feed field. Chinese Patent CN103074241A discloses a Saccharomyces cerevisiae engineering bacterium, its application and a feed additive. By constructing a Saccharomyces cerevisiae engineering bacterium and transferring a pig defensin gene with antibacterial function into Saccharomyces cerevisiae, Saccharomyces cerevisiae also has antibacterial function; Chinese Patent CN109645241A discloses a plant essential oil feed additive, a preparation method and an application thereof, including a core material and a coating material coated outside the core material. The core material includes thymol, carvacrol, microcrystalline cellulose and silicon dioxide; fluidized bed is used for coating thymol and carvacrol, reducing the volatilization loss of thymol and carvacrol, ensuring the effectiveness of the active ingredients in the additive for edible animals, and improving the effect of the additive.

[0006] In summary, the prior art has the following deficiencies: 1) Most technologies only use single strains, and the synergistic effect of multiple strains cannot be achieved; 2) The active ingredients of spice plants are not effectively transformed, and the bioavailability is low; 3) The precision of temperature and humidity control in the solid-state fermentation process is insufficient, and the product quality stability is poor; 4) A multi-component synergistic action system of microorganism-plant active ingredient-matrix cannot be formed.

[0007] Therefore, there is an urgent need to develop a feed for regulating the intestinal flora of piglets prepared by solid-state fermentation of spice plants with compound yeast, so as to effectively improve the intestinal health of weaned piglets, improve growth performance, and solve the problems existing in the prior art. Summary of the Invention

[0008] The object of the present invention is to provide a feed for regulating the intestinal flora of piglets prepared by solid-state fermentation of spice plants with compound yeast and its preparation method, aiming to solve the technical problems in the prior art such as limited effects of single strains, low bioavailability of active ingredients in spice plants, and insufficient precision control of solid-state fermentation process. Through the compound fermentation system of Saccharomyces cerevisiae and Pichia pastoris, combined with the functional plant components of rosemary and thyme, and adopting precise temperature and humidity control technology for solid-state fermentation, the present invention realizes the multi-element synergistic effect of microorganisms - plant active ingredients - matrix, thereby significantly improving the intestinal flora regulation function of the product.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] A feed for regulating the intestinal flora of piglets prepared by solid-state fermentation of spice plants with compound yeast, by weight, comprises the following components: 60 - 75 parts of Saccharomyces cerevisiae, 20 - 30 parts of Pichia pastoris, 15 - 20 parts of rosemary, 15 - 20 parts of thyme, 40 - 50 parts of wheat bran, 20 - 30 parts of corn flour, 15 - 20 parts of soybean meal, 2 - 5 parts of rice hull powder, 3 - 5 parts of molasses, 1 - 3 parts of yeast autolysate, 0.5 - 1.0 part of potassium dihydrogen phosphate, 0.2 - 0.5 part of magnesium sulfate; the Saccharomyces cerevisiae and Pichia pastoris are subjected to solid-state fermentation treatment, and the viable yeast count in the feed ≥ 2.0×10 8 CFU / g.

[0011] Preferably, the Saccharomyces cerevisiae is ATCC 9080 strain, the Pichia pastoris is GS115 or X-33 strain, and the weight ratio of Saccharomyces cerevisiae to Pichia pastoris is 3:1.

[0012] Preferably, the total phenol content in the rosemary extract ≥ 8%, the rosmarinic acid content ≥ 3.5%, and the rosmaridol content ≥ 2.0%; the total phenol content in the thyme extract ≥ 10%, the thymol content ≥ 4.0%, and the carvacrol content ≥ 3.0%.

[0013] Preferably, the feed further comprises the following components: 2 - 4 parts of microcrystalline cellulose, 3 - 5 parts of maltodextrin, 0.2 - 0.5 part of mixed tocopherols, and 0.3 - 0.8 part of citric acid.

[0014] Preferably, the feed has the following characteristics: moisture content ≤ 14%, pH value 4.5 - 5.5, total phenol content ≥ 2.5%, α - amylase activity ≥ 400 U / g, protease activity ≥ 250 U / g, β - glucanase activity ≥ 320 U / g.

[0015] The present invention also provides a method for preparing a feed for regulating the intestinal flora of piglets by solid - state fermentation of the above - mentioned composite yeast and spice plants, comprising the following steps:

[0016] (1) Yeast activation and expansion: Saccharomyces cerevisiae and Pichia pastoris are respectively inoculated into a liquid medium and cultured at 28 - 30 °C for 16 - 36 hours. After collecting the thalli, they are mixed in a ratio of 3:1 to prepare a composite bacterial liquid;

[0017] (2) Pretreatment of spice plants: Rosemary and thyme are dried and crushed to 80 - 120 meshes, mixed enzymes are added, and they are treated at 45 ± 2 °C for 60 - 90 minutes, then ultrasonically treated for 15 - 20 minutes, and dried to a moisture content ≤ 8%;

[0018] (3) Preparation of solid - state fermentation substrate: Wheat bran, corn flour, soybean meal, and rice husk powder are mixed, 5 - 8% of the treated spice plants are added, molasses, yeast autolysate, potassium dihydrogen phosphate, and magnesium sulfate are added, the pH is adjusted to 6.0 - 6.5, and the water content is adjusted to 60 - 65%. It is sterilized at 121 °C for 20 minutes;

[0019] (4) Solid - state fermentation: The composite bacterial liquid prepared in step (1) is inoculated into the sterilized substrate in step (3) at 5 - 8% (v / w), and fermented for 120 ± 4 hours under temperature - controlled and humidity - controlled conditions;

[0020] (5) Post - fermentation treatment: The fermentation product is dried at 45 - 55 °C to a moisture content ≤ 14% and crushed to 60 - 80 meshes;

[0021] (6) Stabilization treatment: Microcrystalline cellulose, maltodextrin, mixed tocopherols, and citric acid are added to the crushed fermentation product, and after mixing evenly, it is sealed and packaged.

[0022] Preferably, in step (1), the culture medium composition for activating Saccharomyces cerevisiae is: glucose 20 ± 1 g / L, peptone 20 ± 1 g / L, yeast extract 10 ± 0.5 g / L, pH value 5.5 ± 0.2; the culture medium composition for activating Pichia pastoris is: glucose 10 ± 0.5 g / L, glycerol 10 ± 0.5 g / L, peptone 20 ± 1 g / L, yeast extract 10 ± 0.5 g / L, pH value 6.0 ± 0.2.

[0023] Preferably, in step (2), the mixed enzyme is composed of cellulase, pectinase and hemicellulase in a ratio of 3:2:1, and the enzyme dosage is 0.2-0.5% of the substrate; the power of the ultrasonic treatment is 300-500 W, and the temperature is controlled at <50°C.

[0024] Preferably, the solid-state fermentation in step (4) adopts a staged temperature and humidity control strategy: the first stage (0-24 h): the temperature is 28±0.5°C, and the relative humidity is 75-80%; the second stage (24-72 h): the temperature is 30±0.5°C, and the relative humidity is 75-80%, and the material is turned over every 24 hours; the third stage (72-120 h): the temperature is 26±0.5°C, and the relative humidity is 70-75%.

[0025] Preferably, the determination criteria for the fermentation end point in step (4) are: the fermentation time is 120±4 hours, the pH value is 4.5-5.5, the viable yeast count ≥ 2.0×10 8 CFU / g, the α-amylase activity ≥ 400 U / g, the protease activity ≥ 250 U / g, and the β-glucanase activity ≥ 320 U / g.

[0026] Advantages of the present invention:

[0027] 1. Synergistic effect of composite yeasts: The present invention innovatively adopts a 3:1 composite system of Saccharomyces cerevisiae and Pichia pastoris to form a complementary enzyme system and metabolic network. Saccharomyces cerevisiae produces a large amount of α-amylase and protease, while Pichia pastoris produces highly active β-glucanase and xylanase. The two yeasts act synergistically, significantly improving the substrate utilization rate and the release efficiency of active ingredients in spice plants. Compared with a single strain, the fermentation efficiency of the composite yeast system is increased by 40-60%.

[0028] 2. Efficient conversion of active ingredients in spice plants: The present invention realizes the efficient conversion and release of polyphenolic substances in spice plants through enzyme-assisted ultrasonic pretreatment technology combined with solid-state fermentation biotransformation. Hydrolytic enzymes such as β-glucanase produced by yeast can effectively break the cell wall to release plant active ingredients, convert plant glycosides into more easily absorbable aglycone forms, and the biological activity is increased by 30-50%. In addition, yeast metabolites such as glutathione can protect plant polyphenolic antioxidant components and improve their stability by 2-3 times.

[0029] 3. Precise three-stage temperature and humidity control: The present invention develops a three-stage precise temperature and humidity control technology, with a temperature control accuracy of ±0.5°C and a humidity control accuracy of ±3%. Through the control of temperature gradient (28°C → 30°C → 26°C) and humidity gradient (80% → 75% → 70%), the yeast metabolic pathway is precisely regulated, and the active ingredients in spice plants are retained and transformed to the greatest extent, and the product batch consistency is increased to more than 92%.

[0030] 4. Multiple intestinal regulatory mechanisms: The product of the present invention regulates the intestinal flora and health of piglets through multiple mechanisms, including: (1) Yeast β-glucan and mannan oligosaccharide, as high-quality prebiotics, selectively promote the growth of beneficial bacteria; (2) Plant polyphenolic substances (such as rosmarinic acid and thymol) have selective antibacterial effects and inhibit the reproduction of harmful bacteria; (3) Organic acids produced by yeast reduce the intestinal pH value and create an environment conducive to the growth of beneficial bacteria; (4) Multiple yeast metabolites and plant active ingredients synergistically enhance the intestinal barrier function. Experimental results show that the use of the product of the present invention can reduce the diarrhea rate of piglets by 40-60%, increase the feed conversion rate by 7-12%, and increase the daily weight gain by 8-15%.

[0031] 5. Environment-friendly benefits: The product of the present invention also has obvious environment-friendly benefits. By promoting the digestion and absorption of nutrients in feed and improving the intestinal flora structure, it significantly reduces the emissions of ammonia nitrogen (reduced by 25-35%) and hydrogen sulfide (reduced by 30-40%) in feces, and reduces the environmental pollution caused by the pig industry. Detailed implementation manners

[0032] The present invention will be further described in detail below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.

[0033] During the implementation process of the present invention, the specific information of the raw materials used is as follows:

[0034] 1. Saccharomyces cerevisiae

[0035] The Saccharomyces cerevisiae strain used in the present invention is the ATCC 9080 strain. The characteristics of this strain include: high enzyme activity, β-glucan content > 20%, strong heat resistance (tolerant temperature range 25-38°C), α-amylase activity > 500 U / g, and protease activity > 300 U / g. After purchase, it is resuscitated and cultured according to the method provided by ATCC. Commercially available Angel high-activity Saccharomyces cerevisiae or Angel brand Saccharomyces cerevisiae (active dry yeast) can be selected instead, but they need to be activated and pre-cultured.

[0036] 2. Pichia pastoris

[0037] The Pichia pastoris GS115 or X-33 strains used in the present invention can be purchased from the China Center of Industrial Culture Collection. The characteristics of the strains include: high ability to express heterologous proteins, resistance to low pH values (3.0 - 6.5), β-glucanase activity > 400 U / g, xylanase activity > 350 U / g, and they can be directly purchased. Pichia pastoris is a methanol-nutritional yeast that can use methanol as the sole carbon source and has the following characteristics compared with Saccharomyces cerevisiae:

[0038] It can achieve a higher cell density (> 100 g / L dry cell mass); has a shorter and less immunogenic glycosylation pattern; the ability to express heterologous proteins is 10 - 100 times higher than that of Saccharomyces cerevisiae; has the characteristics of high-efficiency expression of heterologous proteins and high β-glucanase activity; can perform correct post-translational modifications, such as disulfide bond formation and glycosylation.

[0039] 3. Rosemary and thyme raw materials

[0040] The raw materials of rosemary (Rosmarinus officinalis) and thyme (Thymus vulgaris) adopt medicinal-grade dried plant materials and can be purchased from professional traditional Chinese medicine or plant extract suppliers. Raw materials meeting the standards of the Chinese Pharmacopoeia should be selected, with volatile oil content not less than 1.2% (rosemary) and 1.0% (thyme), and total ash content not exceeding 9% and 12% respectively.

[0041] 4. Wheat bran

[0042] Fresh, dry, and mildew-free wheat bran is selected, with starch content > 45%, protein content > 15%, and moisture < 12%. It can be purchased from flour mills or professional feed raw material suppliers. Before use, it is screened through a 100-mesh sieve to remove impurities. As the main carbon source and matrix for solid-state fermentation, the loose and porous structure of wheat bran is conducive to the growth of yeast and oxygen diffusion.

[0043] 5. Corn flour

[0044] Food-grade corn flour is used, with starch content > 70%, fat content < 4%, and moisture < 13%. It can be purchased from food raw material or feed raw material suppliers. Corn flour provides a rich carbon source and promotes the growth and metabolism of yeast.

[0045] 6. Soybean meal

[0046] Select high-quality defatted soybean meal with a protein content of >42%, soluble non-starch polysaccharides >8%, and moisture <12%. It can be purchased from feed processing plants or professional feed raw material suppliers. Soybean meal provides rich protein and is a nitrogen source for yeast growth.

[0047] 7. Rice Husk Powder

[0048] Use high-quality crushed rice husks with a cellulose content of >35%, ash content of <20%, and moisture content of <10%. You can buy raw rice husks from rice processing plants and crush them to 60-80 mesh by yourself. In solid-state fermentation, rice husk powder is mainly used as a loosening agent to improve the air permeability of the substrate.

[0049] 8. Molasses

[0050] Use sugarcane molasses or beet molasses with a total sugar content of >45%, which can be purchased from sugar factories or food additive suppliers. Molasses serves as an easily available carbon source to promote rapid proliferation and metabolism of yeast.

[0051] 9. Yeast autolysate

[0052] Yeast autolysate can be purchased directly from biochemical reagents or fermentation industry raw material suppliers as commercial products, with an amino nitrogen content of >3.5% and a B vitamin content of >0.5%. It can also be made by yourself using the following method:

[0053] Preparation of yeast autolysate:

[0054] 1) Take fresh Saccharomyces cerevisiae (containing 20-25% dry matter) and adjust the pH to 5.0-5.5;

[0055] 2) adding 0.5% papain and autolyzing at 45-50°C for 12-16 hours;

[0056] 3) Heat at 85-90°C for 15 minutes to terminate the reaction;

[0057] 4) Centrifugation (5000 g, 20 min) to remove insoluble matter;

[0058] 5) The supernatant is spray-dried or vacuum-dried to obtain yeast autolysate powder.

[0059] Yeast autolysate is rich in amino acids, short peptides, nucleotides and B vitamins, and is a high-quality nitrogen source and growth factor for yeast growth.

[0060] 10. Potassium dihydrogen phosphate and magnesium sulfate

[0061] Analytical reagent grade or food-grade potassium dihydrogen phosphate (KH2PO4) and magnesium sulfate (MgSO4·7H2O) are used and can be purchased from chemical reagent suppliers. These two compounds provide the phosphorus source and magnesium ions required for solid-state fermentation respectively. Phosphorus is an essential element for yeast energy metabolism and nucleic acid synthesis, and magnesium ions are cofactors for various enzymes.

[0062] 11. Microcrystalline cellulose

[0063] Microcrystalline cellulose meeting the pharmacopoeia standards (CP / USP / EP) with an average particle size of 50 - 100 μm is used and can be purchased from pharmaceutical excipient or food additive suppliers. In the present invention, microcrystalline cellulose serves as a dispersant and stabilizer to improve the fluidity and dispersibility of the product.

[0064] 12. Maltodextrin

[0065] Food-grade maltodextrin with a DE value (dextrin equivalent) of 8 - 12 is used and can be purchased from food additive suppliers. Maltodextrin serves as an encapsulating agent in the present invention to form a microcapsule structure with the active ingredient, improving stability.

[0066] 13. Mixed tocopherols

[0067] Food-grade mixed tocopherols (vitamin E) with an α-tocopherol content > 50% are used and can be purchased from food additive or nutritional fortifier suppliers. As a potent antioxidant, it protects unsaturated fatty acids and polyphenolic compounds in the product from oxidation.

[0068] 14. Citric acid

[0069] Food-grade anhydrous citric acid with a purity > 99.5% is used and can be purchased from food additive suppliers. Citric acid serves as a pH regulator and preservative in the present invention to maintain the acidic environment of the product and inhibit microbial contamination.

[0070] In the field of biological fermentation, the synergistic effect between Saccharomyces cerevisiae and Pichia pastoris shows significant advantages. Saccharomyces cerevisiae produces α-amylase and protease, while Pichia pastoris produces β-glucanase and xylanase. Their synergistic effect can achieve a comprehensive and efficient decomposition of carbohydrates and proteins, increasing the synergistic degradation efficiency by 35 - 50%, far exceeding the efficiency of a single strain. In addition, the metabolite mutual benefit is significant. Amino acids produced by Saccharomyces cerevisiae can promote the growth of Pichia pastoris, while vitamins produced by Pichia pastoris promote the metabolism of Saccharomyces cerevisiae, and the two-way promotion increases the growth efficiency by 20 - 30%. In terms of probiotic effects, β-glucan in the cell wall of Saccharomyces cerevisiae can activate intestinal macrophages, and mannan oligosaccharides produced by Pichia pastoris stimulate the growth of intestinal Bifidobacterium. The combined use can enhance the immunomodulatory effect by 40 - 60%.

[0071] The synergistic mechanism between spice plants and yeast also deserves attention. Rosmarinic acid in rosemary can promote the fluidity of yeast cell membranes and increase the enzyme secretion efficiency by 15-25%; thymol in thyme selectively inhibits miscellaneous bacteria, ensuring the dominant fermentation of yeast and increasing the purity by more than 95%. In terms of biotransformation, yeast β-glucanase can hydrolyze plant cell walls to release more active ingredients, with the extraction efficiency increased by 40-60%; yeast glycosidase converts plant glycosides into active aglycones, with the bioavailability increased by 30-50%. In addition, yeast metabolites such as glutathione can protect plant polyphenolic antioxidants, with the stability increased by 2-3 times; the complex formed by plant polyphenols and yeast cell walls can prolong the intestinal residence time, with the sustained-release effect increased by 1.5-2 times.

[0072] The synergistic effect among substrates, strains, and plants is more complex. Substrates provide basic nutrients, yeast metabolism produces B vitamins, and plants provide secondary metabolites. The nutritional spectra of the three complement each other, covering vitamins, minerals, antioxidants, enzyme systems, and prebiotics. In terms of functional polysaccharides, yeast β-glucan, plant pectin, and substrate arabinoxylan form a triple prebiotic system, synergistically promoting the growth of intestinal lactic acid bacteria and bifidobacteria and inhibiting Escherichia coli and Salmonella, with the effect improved by 65-85% compared with single components. In terms of intestinal environment regulation, organic acids produced by yeast fermentation can lower the intestinal pH, plant polyphenolic substances selectively inhibit bacteria, and fermentable fibers provided by substrates promote the production of short-chain fatty acids. The triple synergy improves the intestinal microecology and reduces the incidence of diarrhea by 40-60%.

[0073] The intestinal flora regulation function of the product of the present invention is based on the following several synergistic mechanisms:

[0074] 1. Immunomodulatory effect of yeast cell wall components:

[0075] β-1,3 / 1,6-glucan in the cell wall of Saccharomyces cerevisiae binds to the Dectin-1 receptor on the surface of intestinal macrophages and dendritic cells, activating the innate immune system and enhancing the intestinal immune barrier function. Mannooligosaccharides produced by Pichia pastoris can specifically bind to the lectins on the surface of intestinal pathogens (such as Escherichia coli and Salmonella), preventing the adhesion of pathogens to intestinal epithelial cells, thereby reducing the risk of infection. The two yeasts act synergistically to form a double protection barrier.

[0076] 2. Synergistic antibacterial effect of plant polyphenols and yeast metabolites:

[0077] The main active components in rosemary, rosmarinic acid and carnosol, have fatty acid esterase inhibitory activity and can inhibit the growth of Gram-negative bacteria; thymol and carvacrol in thyme play an antibacterial role by disrupting the bacterial cell membrane structure and are particularly effective against Gram-positive bacteria. Organic acids (such as lactic acid and succinic acid) produced during yeast fermentation can lower the intestinal pH value and form a synergistic antibacterial effect with plant polyphenols, enhancing the inhibitory effect on pathogenic bacteria.

[0078] 3. Multiple prebiotics synergistically promote the growth of beneficial bacteria:

[0079] During the fermentation process, various oligosaccharide substances such as β-glucan, mannan oligosaccharide, arabinoxylan, and fructooligosaccharide produced act as high-quality prebiotics, selectively promoting the growth of beneficial bacteria such as Bifidobacterium, Lactobacillus, and Butyricicoccus. These beneficial bacteria inhibit the colonization of pathogenic bacteria through the principle of competitive exclusion and produce short-chain fatty acids (such as acetic acid, propionic acid, and butyric acid), providing energy for intestinal epithelial cells, promoting the expression of tight junction proteins, and enhancing the intestinal barrier function.

[0080] 4. The synergistic action of enzyme systems improves feed digestibility:

[0081] Multiple enzymes such as α-amylase, protease, β-glucanase, and xylanase produced by complex yeast fermentation can degrade difficult-to-digest components such as starch, protein, and non-starch polysaccharides in feed, improving the digestibility and absorption rate of nutrients. These enzymes continuously function in the small intestine of piglets, making up for the insufficient secretion of their own digestive enzymes after weaning, reducing the digestive burden, and improving feed utilization efficiency.

[0082] 5. Antioxidant protection system:

[0083] Antioxidant substances such as glutathione and superoxide dismutase produced by yeast fermentation, combined with polyphenolic antioxidants (such as rosmarinic acid, caffeic acid, thymol, etc.) in rosemary and thyme, form a multi-level antioxidant protection network, effectively scavenging free radicals and reactive oxygen species in the intestine, reducing oxidative stress, and protecting intestinal epithelial cells from damage.

[0084] Through the synergistic action of the above multiple mechanisms, the product of the present invention can comprehensively regulate the balance of the intestinal flora of piglets, promote the growth of beneficial bacteria, inhibit the reproduction of harmful bacteria, enhance the intestinal barrier function and immune function, thereby effectively improving the intestinal health of weaned piglets, reducing diarrhea occurrence, and improving growth performance.

[0085] Example 1: Preparation of a feed for regulating the intestinal flora of piglets by solid-state fermentation of spice plants with a standard formula of complex yeast

[0086] The piglet intestinal flora-regulating feed for solid-state fermentation of spice plants by compound yeast comprises the following components in parts by weight: 68 parts of Saccharomyces cerevisiae (ATCC 9080), 22 parts of Pichia pastoris (GS115 or X-33), 17 parts of rosemary extract, 17 parts of thyme extract, 45 parts of wheat bran, 25 parts of corn flour, 18 parts of soybean meal, 3 parts of rice husk powder, 4 parts of molasses, 2 parts of yeast autolysate, 0.8 part of potassium dihydrogen phosphate, 0.4 part of magnesium sulfate, 3 parts of microcrystalline cellulose, 4 parts of maltodextrin, 0.3 part of mixed tocopherols and 0.5 part of citric acid.

[0087] The preparation method comprises the following steps:

[0088] (1) Activation and expansion culture of yeast: Inoculate Saccharomyces cerevisiae ATCC 9080 into a medium (20 g / L of glucose, 20 g / L of peptone, 10 g / L of yeast extract, pH 5.5), and culture at 28 °C and 180 rpm for 20 hours; inoculate Pichia pastoris GS115 or X-33 into a medium (10 g / L of glucose, 10 g / L of glycerol, 20 g / L of peptone, 10 g / L of yeast extract, pH 6.0), and culture at 30 °C and 200 rpm for 28 hours. Centrifuge the two yeast culture solutions respectively (5000 g, 10 minutes, 4 °C) to collect the thalli, wash them 2 times with sterile 0.9% physiological saline, resuspend them in sterile water until OD600 = 22, add 0.8% trehalose as a protective agent, and finally mix them in a ratio of 3:1 (v / v) to prepare a compound bacterial solution.

[0089] (2) Pretreatment of spice plants: Crush the dried samples of rosemary and thyme to 100 meshes and mix them in a ratio of 1:1. Add a mixed enzyme composed of cellulase, pectinase and hemicellulase in a ratio of 3:2:1 (the enzyme dosage is 0.35% of the substrate), the substrate concentration is 18% (w / v), pH 4.8, and treat at 45 °C for 75 minutes. Then carry out ultrasonic treatment (power 400 W, time 18 minutes, temperature controlled below 45 °C). The treated mixture is vacuum-dried at 50 °C for 3 hours until the moisture content is 7%.

[0090] (3) Preparation of solid fermentation substrate: Mix wheat bran, corn flour, soybean meal and rice husk powder evenly, and add the spice plant mixture treated in step (2) (accounting for 7% of the total weight of the substrate). Then add the following components: 4 parts of molasses (dissolved in an appropriate amount of water), 2 parts of yeast autolysate, 0.8 part of potassium dihydrogen phosphate, 0.4 part of magnesium sulfate, and a trace element mixture (0.03 g / kg of zinc sulfate, 0.02 g / kg of manganese sulfate, 0.008 g / kg of copper sulfate, 0.02 g / kg of ferrous sulfate, 0.002 g / kg of cobalt chloride, 0.0015 g / kg of sodium molybdate). Adjust the pH of the mixture to 6.2 and the water content to 62%. After mixing evenly, pack it into heat-resistant bags and sterilize at 121 °C for 20 minutes, then cool to room temperature.

[0091] (4) Solid fermentation: Inoculate the compound bacterial liquid prepared in step (1) into the sterilized substrate in step (3) at 6% (v / w), mix well and then pack it into a fermentation bag with a breathable membrane. The fermentation process adopts a three-stage temperature and humidity control strategy: The first stage (0 - 24 h): temperature 28 °C, relative humidity 78%; The second stage (24 - 72 h): temperature 30 °C, relative humidity 78%. Turn the material at 24 h, 48 h and 72 h, and each turning lasts for 5 minutes; The third stage (72 - 120 h): temperature 26 °C, relative humidity 72%. After 120 hours of fermentation, measure the pH value (4.8), viable yeast count (2.5×10 8 CFU / g), α-amylase activity (450 U / g), protease activity (280 U / g) and β-glucanase activity (360 U / g) of the fermentation product to confirm the completion of fermentation.

[0092] (5) Post-fermentation treatment: Spread the fermentation product on a stainless steel tray with a layer thickness of 4 cm and dry it at 50 °C for 6 hours until the moisture content is 12%. Use a hammer mill to crush the dried product to 70 mesh.

[0093] (6) Stabilization treatment: Add 3 parts of microcrystalline cellulose, 4 parts of maltodextrin, 0.3 part of mixed tocopherols and 0.5 part of citric acid to the crushed fermentation product, and mix in a V-type mixer for 18 minutes. Finally, pack the product into an aluminum foil composite bag, fill it with nitrogen and seal it, and store it at 2 - 8 °C.

[0094] This technology adopts a phased precise fermentation control technology, and precisely controls the yeast metabolic pathway through a three-stage temperature gradient control (28°C → 30°C → 26°C). In the first stage, it promotes the rapid proliferation and adaptation of yeast at 28°C. In the second stage, it increases the enzyme activity and metabolic rate at 30°C. In the third stage, it delays the metabolism at 26°C to promote the accumulation of secondary metabolites. The innovation lies in the precise temperature control window, which improves the yeast metabolite spectrum by 45 - 60%. At the same time, it adopts a precise humidity control strategy. In the early stage of fermentation (0 - 72h), it maintains a high humidity (75 - 80%) to ensure yeast growth. In the later stage of fermentation (72 - 120h), it controls the humidity (70 - 75%) to balance growth and the accumulation of active substances, and is equipped with an automatic humidity compensation system, which automatically adjusts the ventilation volume according to the changes in CO2 concentration and temperature. The innovation lies in high-precision humidity sensing and automatic regulation, which improves the product batch consistency to over 92%.

[0095] In addition, it adopts an enzyme-assisted ultrasonic synergistic extraction technology. First, a mixed enzyme (cellulase:pectinase:hemicellulase = 3:2:1) degrades part of the cell wall, and then ultrasonic waves (300 - 500W, 15 - 20min) are used to generate cavitation effects to further break the wall. The two-step synergistic effect increases the extraction rate of plant active substances by 75 - 90%. The innovation lies in that compared with traditional solvent extraction, the extraction rate of active substances is increased by 2 - 3 times, and there is no solvent residue. The whole process adopts low-temperature gradient extraction. The enzymatic hydrolysis temperature is controlled at 45 ± 2°C, the ultrasonic temperature is controlled at <50°C, and the drying temperature is controlled at 45 - 55°C. The innovation lies in that the whole process temperature is <55°C, and the retention rate of thermosensitive substances (such as volatile oils) is >85%.

[0096] Finally, it adopts a biological protectant combined stabilization technology to construct a triple antioxidant system, including endogenous antioxidants (such as glutathione in yeast metabolites), plant antioxidants (rosemary and thyme polyphenols), and additive antioxidants (mixed tocopherols (0.2 - 0.5%) and ascorbic acid (0.2 - 0.4%)). The innovation lies in multi-level antioxidant protection, which increases the product shelf life by 50 - 80%. At the same time, it adopts a microencapsulation protection technology to form microcapsules with sensitive active ingredients and maltodextrin (3 - 5%). The average particle size of the microcapsules is 8 - 12μm, which improves the taste and masks the bitterness of plant extracts. The innovation lies in intestinal-targeted release, which increases the bioavailability by 35 - 50%.

[0097] The product prepared in this example is in the form of light brown fine powder, has a slight aromatic smell of spice plants, and has no off-flavors. The viable yeast count in the product is 2.5×10 8 CFU / g, the pH value is 4.8, the total phenol content is 3.2%, the α-amylase activity is 450U / g, the protease activity is 280U / g, and the β-glucanase activity is 360U / g.

[0098] Example 2: Preparation of Piglet Intestinal Flora-Regulating Feed by Solid-State Fermentation of Spice Plants with a High Proportion of Saccharomyces cerevisiae in Complex Yeast

[0099] The piglet intestinal flora-regulating feed prepared by solid-state fermentation of spice plants with complex yeast in this example comprises the following components by weight: 75 parts of Saccharomyces cerevisiae (ATCC 9080), 20 parts of Pichia pastoris (GS115 or X-33), 16 parts of rosemary extract, 16 parts of thyme extract, 48 parts of wheat bran, 22 parts of corn flour, 16 parts of soybean meal, 4 parts of rice husk powder, 3.5 parts of molasses, 1.5 parts of yeast autolysate, 0.6 part of potassium dihydrogen phosphate, 0.3 part of magnesium sulfate, 2.5 parts of microcrystalline cellulose, 3.5 parts of maltodextrin, 0.25 part of mixed tocopherols, and 0.4 part of citric acid.

[0100] The preparation method is similar to that of Example 1, but with the following differences:

[0101] (1) Activation and expansion culture of yeast: The culture time of Saccharomyces cerevisiae is 18 hours, the culture time of Pichia pastoris is 26 hours, the trehalose concentration in the complex bacterial liquid is 0.6%, and the final ratio of Saccharomyces cerevisiae to Pichia pastoris is 3.75:1 (v / v).

[0102] (2) Pretreatment of spice plants: Ground to 90 mesh, the dosage of mixed enzyme is 0.25% of the substrate, the substrate concentration is 16% (w / v), the treatment time is 65 minutes, the ultrasonic power is 350 W, and the time is 16 minutes.

[0103] (3) Preparation of solid-state fermentation substrate: The addition amount of the spice plant mixture is 6% of the total weight of the substrate, the pH is adjusted to 6.4, and the water content is adjusted to 64%.

[0104] (4) Solid-state fermentation: The inoculation amount is 5.5% (v / w), the temperature in the first stage is 28.5 °C, and the relative humidity is 80%; the temperature in the second stage is 30.5 °C, and the relative humidity is 80%; the temperature in the third stage is 26.5 °C, and the relative humidity is 74%. The fermentation time is 124 hours.

[0105] (5) Post-fermentation treatment: The drying temperature is 48 °C, the drying time is 7 hours, the final moisture content is 11%, and it is ground to 65 mesh.

[0106] The final product is in the form of light brown fine powder, the viable yeast count is 2.3×10 8 CFU / g, the pH value is 4.7, the total phenol content is 2.9%, the α-amylase activity is 520 U / g, the protease activity is 300 U / g, and the β-glucanase activity is 330 U / g.

[0107] Example 3: Preparation of Piglet Intestinal Flora-Regulating Feed by Solid-State Fermentation of Spice Plants with a High Proportion of Pichia pastoris in Complex Yeast

[0108] The piglet intestinal flora-regulating feed prepared by solid-state fermentation of spice plants with compound yeast in this example comprises the following components by weight: 60 parts of Saccharomyces cerevisiae (ATCC 9080), 30 parts of Pichia pastoris (GS115 or X-33), 18 parts of rosemary extract, 18 parts of thyme extract, 42 parts of wheat bran, 28 parts of corn flour, 20 parts of soybean meal, 2 parts of rice hull powder, 4.5 parts of molasses, 2.5 parts of yeast autolysate, 1.0 part of potassium dihydrogen phosphate, 0.5 part of magnesium sulfate, 3.5 parts of microcrystalline cellulose, 4.5 parts of maltodextrin, 0.4 part of mixed tocopherols, and 0.7 part of citric acid.

[0109] The preparation method is similar to that of Example 1, but there are the following differences:

[0110] (1) Activation and expansion culture of yeast: The culture time of Saccharomyces cerevisiae is 22 hours, the culture time of Pichia pastoris is 30 hours, the trehalose concentration in the compound bacterial liquid is 1.0%, and the final ratio of Saccharomyces cerevisiae to Pichia pastoris is 2:1 (v / v).

[0111] (2) Pretreatment of spice plants: Ground to 110 meshes, the dosage of the mixed enzyme is 0.45% of the substrate, the substrate concentration is 20% (w / v), the treatment time is 85 minutes, the ultrasonic power is 450 W, and the time is 20 minutes.

[0112] (3) Preparation of solid-state fermentation substrate: The addition amount of the spice plant mixture is 8% of the total weight of the substrate, the pH is adjusted to 6.0, and the water content is adjusted to 60%.

[0113] (4) Solid-state fermentation: The inoculation amount is 7% (v / w), the temperature in the first stage is 27.5 °C, and the relative humidity is 76%; the temperature in the second stage is 29.5 °C, and the relative humidity is 76%; the temperature in the third stage is 25.5 °C, and the relative humidity is 70%. The fermentation time is 116 hours.

[0114] (5) Post-fermentation treatment: The drying temperature is 52 °C, the drying time is 5 hours, the final moisture content is 13%, and it is ground to 75 meshes.

[0115] The final product is in the form of brown fine powder, the viable yeast count is 2.8×10 8 CFU / g, the pH value is 5.0, the total phenol content is 3.5%, the α-amylase activity is 420 U / g, the protease activity is 260 U / g, and the β-glucanase activity is 390 U / g.

[0116] Example 4: Preparation of Piglet Intestinal Flora-Regulating Feed by Solid-State Fermentation of Spice Plants with Compound Yeast with High Spice Plant Content

[0117] The piglet intestinal flora-regulating feed prepared by solid-state fermentation of spice plants with compound yeast according to this example comprises the following components by weight: 65 parts of Saccharomyces cerevisiae (ATCC 9080), 25 parts of Pichia pastoris (GS115 or X-33), 20 parts of rosemary extract, 20 parts of thyme extract, 40 parts of wheat bran, 30 parts of corn flour, 19 parts of soybean meal, 2.5 parts of rice hull powder, 5 parts of molasses, 3 parts of yeast autolysate, 0.9 part of potassium dihydrogen phosphate, 0.45 part of magnesium sulfate, 4 parts of microcrystalline cellulose, 5 parts of maltodextrin, 0.5 part of mixed tocopherols, and 0.8 part of citric acid.

[0118] The preparation method is similar to that of Example 1, but with the following differences:

[0119] (1) Activation and expansion culture of yeast: The OD600 in the compound bacterial liquid is adjusted to 24, the trehalose concentration is 0.9%, and the final ratio of Saccharomyces cerevisiae to Pichia pastoris is 2.6:1 (v / v).

[0120] (2) Pretreatment of spice plants: Ground to 120 mesh, the dosage of the mixed enzyme is 0.5% of the substrate, the substrate concentration is 19% (w / v), the pH value is 4.5, the ultrasonic power is 500 W, and the time is 15 minutes.

[0121] (3) Preparation of solid-state fermentation substrate: The addition amount of the trace element mixture is increased by 20%.

[0122] (4) Solid-state fermentation: The temperature in the first stage is 28 °C and the relative humidity is 78%; the temperature in the second stage is 30 °C and the relative humidity is 78%; the temperature in the third stage is 26 °C and the relative humidity is 70%. The fermentation time is 118 hours.

[0123] The final product is in the form of a dark brown fine powder, with an obvious aromatic odor of spice plants. The viable yeast count is 2.6×10 8 CFU / g, the pH value is 4.9, the total phenol content is 4.0%, the α-amylase activity is 430 U / g, the protease activity is 270 U / g, and the β-glucanase activity is 350 U / g.

[0124] Example 5: Preparation of piglet intestinal flora-regulating feed by solid-state fermentation of spice plants with compound yeast with a high wheat bran ratio

[0125] The piglet intestinal flora-regulating feed prepared by solid-state fermentation of compound yeast with spice plants in this example comprises the following components by weight: 64 parts of Saccharomyces cerevisiae (ATCC 9080), 26 parts of Pichia pastoris (GS115 or X-33), 16 parts of rosemary extract, 16 parts of thyme extract, 50 parts of wheat bran, 20 parts of corn flour, 15 parts of soybean meal, 5 parts of rice husk powder, 4 parts of molasses, 2 parts of yeast autolysate, 0.7 part of potassium dihydrogen phosphate, 0.35 part of magnesium sulfate, 2.8 parts of microcrystalline cellulose, 3.8 parts of maltodextrin, 0.35 part of mixed tocopherols, and 0.6 part of citric acid.

[0126] The preparation method is similar to that of Example 1, but there are the following differences:

[0127] (1) Activation and expansion culture of yeast: The final ratio of Saccharomyces cerevisiae to Pichia pastoris in the compound bacterial liquid is 2.5:1 (v / v).

[0128] (2) Pretreatment of spice plants: The substrate concentration is 17% (w / v), and the ultrasonic treatment time is 17 minutes.

[0129] (3) Preparation of solid-state fermentation substrate: The addition amount of the spice plant mixture is 6.5% of the total weight of the substrate, and the water content is adjusted to 61%.

[0130] (4) Solid-state fermentation: The inoculation amount is 6.5% (v / w), the relative humidity in the first stage is 79%; the relative humidity in the second stage is 79%; the relative humidity in the third stage is 73%. The turning frequency of the material is adjusted to once every 36 hours.

[0131] The viable yeast count of the final product is 2.4×10 8 CFU / g, the pH value is 4.7, the total phenol content is 2.8%, the α-amylase activity is 480 U / g, the protease activity is 290 U / g, and the β-glucanase activity is 340 U / g.

[0132] Example 6: Preparation of piglet intestinal flora-regulating feed by solid-state fermentation of compound yeast with spice plants at low temperature

[0133] The composition of the piglet intestinal flora-regulating feed prepared by solid-state fermentation of compound yeast with spice plants in this example is the same as that of Example 1, but in the preparation process, the solid-state fermentation temperature is adjusted to: the temperature in the first stage (0 - 24 h) is 27 °C, the temperature in the second stage (24 - 72 h) is 29 °C, and the temperature in the third stage (72 - 120 h) is 25 °C. In addition, the fermentation time is extended to 128 hours.

[0134] The viable yeast count of the final product is 2.2×10 8 CFU / g, the pH value is 5.1, the total phenol content is 3.0%, the α-amylase activity is 410 U / g, the protease activity is 250 U / g, and the β-glucanase activity is 340 U / g.

[0135] Comparative Example 1: Piglet Intestinal Flora-Regulating Feed of Compound Yeast Solid-State Fermented Spice Plants by Single Saccharomyces cerevisiae Fermentation

[0136] The piglet intestinal flora-regulating feed of compound yeast solid-state fermented spice plants in this comparative example, calculated by weight, comprises the following components: 90 parts of Saccharomyces cerevisiae (ATCC 9080), 17 parts of rosemary extract, 17 parts of thyme extract, and the remaining components are the same as those in Example 1. During the preparation process, only Saccharomyces cerevisiae is used for fermentation, and Pichia pastoris is not added. Other preparation conditions are the same as those in Example 1.

[0137] The viable yeast count of the final product is 2.1×10 8 CFU / g, pH value is 4.9, total phenol content is 2.6%, α-amylase activity is 460 U / g, protease activity is 300 U / g, and β-glucanase activity is 150 U / g (significantly lower than that in Example 1).

[0138] Comparative Example 2: Piglet Intestinal Flora-Regulating Feed of Compound Yeast Solid-State Fermented Spice Plants by Single Pichia pastoris Fermentation

[0139] The piglet intestinal flora-regulating feed of compound yeast solid-state fermented spice plants in this comparative example, calculated by weight, comprises the following components: 90 parts of Pichia pastoris (GS115 or X-33), 17 parts of rosemary extract, 17 parts of thyme extract, and the remaining components are the same as those in Example 1. During the preparation process, only Pichia pastoris is used for fermentation, and Saccharomyces cerevisiae is not added. Other preparation conditions are the same as those in Example 1.

[0140] The viable yeast count of the final product is 1.9×10 8 CFU / g, pH value is 5.2, total phenol content is 2.8%, α-amylase activity is 180 U / g (significantly lower than that in Example 1), protease activity is 210 U / g, and β-glucanase activity is 380 U / g.

[0141] Comparative Example 3: Piglet Intestinal Flora-Regulating Feed of Compound Yeast Solid-State Fermented Spice Plants without Pretreatment of Spice Plants

[0142] The composition of the piglet intestinal flora-regulating feed of compound yeast solid-state fermented spice plants in this comparative example is the same as that in Example 1, but during the preparation process, the spice plants are not pretreated by enzyme-assisted ultrasonic treatment and are directly crushed to 100 meshes and then added to the fermentation substrate. Other preparation conditions are the same as those in Example 1.

[0143] The viable yeast count of the final product is 2.3×10 8CFU / g, pH value 4.8, total phenol content 1.8% (significantly lower than that in Example 1), α-amylase activity 430 U / g, protease activity 270 U / g, β-glucanase activity 350 U / g.

[0144] Comparative Example 4: Compound yeast solid-state fermented spice plant feed for regulating intestinal flora of piglets without spice plants

[0145] The compound yeast solid-state fermented spice plant feed for regulating intestinal flora of piglets in this comparative example does not add rosemary extract and thyme extract, and other components are the same as those in Example 1. During the preparation process, the same compound yeast fermentation process as in Example 1 is adopted.

[0146] The viable yeast count of the final product is 2.6×10 8 CFU / g, pH value 4.7, total phenol content 0.6% (extremely significantly lower than that in Example 1), α-amylase activity 440 U / g, protease activity 275 U / g, β-glucanase activity 355 U / g.

[0147] Comparative Example 5: Compound yeast solid-state fermented spice plant feed for regulating intestinal flora of piglets with constant temperature fermentation

[0148] The composition of the compound yeast solid-state fermented spice plant feed for regulating intestinal flora of piglets in this comparative example is the same as that in Example 1, but during the preparation process, the solid-state fermentation adopts a constant temperature strategy, maintaining at 28°C throughout the process, and the relative humidity is maintained at 75%. Other preparation conditions are the same as those in Example 1.

[0149] The viable yeast count of the final product is 2.0×10 8 CFU / g, pH value 5.0, total phenol content 2.5%, α-amylase activity 400 U / g, protease activity 260 U / g, β-glucanase activity 330 U / g. The batch consistency evaluation shows that the batch coefficient of variation of the constant temperature fermentation product is 12.5%, significantly higher than 5.8% of the three-stage temperature control strategy.

[0150] To evaluate the intestinal flora regulation function of the product of the present invention, the following series of experiments were carried out:

[0151] Experiment 1: In vitro antibacterial activity evaluation

[0152] The agar diffusion method was used to evaluate the antibacterial activities of products of different examples and comparative examples against common intestinal pathogenic bacteria. 1 g of the sample was dissolved in 10 ml of sterile water, thoroughly mixed, centrifuged (10000 g, 15 minutes) after extraction at 4°C for 12 hours, and the supernatant was taken for testing. The test strains included: Escherichia coli ATCC 25922, Salmonella ATCC 13076, Staphylococcus aureus ATCC 25923, and Clostridium ATCC 9714. The results are shown in Table 1.

[0153] Table 1 Inhibitory zone diameters (mm) of different products against intestinal pathogenic bacteria

[0154] Product Escherichia coli Salmonella Staphylococcus aureus Clostridium Example 1 16.5±0.8 15.8±0.6 18.2±0.9 14.6±0.7 Example 2 15.8±0.7 15.2±0.5 17.6±0.8 14.0±0.6 Example 3 17.2±0.9 16.5±0.7 19.0±1.0 15.2±0.8 Example 4 18.5±1.0 17.8±0.8 20.2±1.1 16.4±0.9 Example 5 16.0±0.7 15.5±0.6 17.8±0.8 14.2±0.6 Example 6 16.2±0.8 15.6±0.6 18.0±0.9 14.4±0.7 Control Example 1 12.3±0.6 11.5±0.5 14.2±0.7 10.5±0.5 Control Example 2 13.8±0.7 12.8±0.6 15.5±0.8 11.8±0.6 Control Example 3 11.2±0.5 10.6±0.4 12.8±0.6 9.5±0.4 Control Example 4 8.5±0.4 7.8±0.3 9.6±0.5 6.2±0.3 Control Example 5 15.5±0.7 14.8±0.6 17.2±0.8 13.5±0.6

[0155] As can be seen from Table 1, all the products of the examples showed strong antibacterial activities, among which the product of Example 4 (high content of aromatic plants) had the strongest antibacterial activity. The antibacterial activities of Comparative Example 1 and Comparative Example 2 (single yeast fermentation) were significantly lower than those of the products fermented by compound yeasts. The antibacterial activity of Comparative Example 3 (without pretreatment with aromatic plants) was further reduced, while the product of Comparative Example 4 (without aromatic plants) had the weakest antibacterial activity. This indicates that compound yeast fermentation and pretreatment with aromatic plants have important effects on the antibacterial activity of the products.

[0156] Experiment 2: Evaluation of in vitro probiotic-promoting activities

[0157] The promoting effects of different products on the growth of probiotics were evaluated through in vitro fermentation tests. 1 g of the sample was added to 100 ml of sterile anaerobic medium, inoculated with Lactobacillus (Lactobacillus acidophilus ATCC 4356) or Bifidobacterium (Bifidobacterium longum ATCC 15707), and anaerobically cultured at 37°C for 24 hours, and the colony counts were determined. The results are shown in Table 2.

[0158] Table 2 Promoting effects of different products on the growth of probiotics (log10 CFU / ml)

[0159] Product Lactobacillus acidophilus Bifidobacterium longum Blank control 7.32±0.18 6.85±0.15 Example 1 8.95±0.22 8.62±0.20 Example 2 8.82±0.21 8.48±0.19 Example 3 9.08±0.23 8.75±0.21 Example 4 9.15±0.24 8.82±0.22 Example 5 8.90±0.21 8.55±0.19 Example 6 8.88±0.20 8.50±0.18 Control Example 1 8.35±0.18 7.92±0.17 Control Example 2 8.52±0.19 8.10±0.18 Control Example 3 8.28±0.17 7.85±0.16 Control Example 4 8.05±0.16 7.60±0.15 Control Example 5 8.80±0.20 8.45±0.18

[0160] As can be seen from Table 2, all the products could promote the growth of probiotics to varying degrees, and the probiotic-promoting activities of the products of the examples were significantly better than those of the comparative examples. Among them, Example 3 (high proportion of Pichia pastoris) and Example 4 (high content of aromatic plants) showed the strongest probiotic-promoting activities, which may be related to the mannan oligosaccharides produced by Pichia pastoris and the polyphenolic substances in the aromatic plants. In contrast, the product of Comparative Example 4 (without aromatic plants) had the weakest probiotic-promoting activity, indicating that the aromatic plant components have an important promoting effect on the growth of probiotics.

[0161] Experiment 3: Piglet feeding trial

[0162] Two hundred and forty healthy Duroc×Landrace crossbred piglets at 28 days old just weaned, with an average weight of 7.0±0.3 kg, were randomly divided into 6 groups, with 40 piglets in each group. Each group was divided into 4 replicates, with 10 piglets in each replicate. The 6 experimental groups were: control group (basal diet), Example 1 group (basal diet + 1.0% of the product of Example 1), Example 4 group (basal diet + 1.0% of the product of Example 4), Comparative Example 1 group (basal diet + 1.0% of the product of Comparative Example 1), Comparative Example 3 group (basal diet + 1.0% of the product of Comparative Example 3), and Comparative Example 4 group (basal diet + 1.0% of the product of Comparative Example 4). The experimental period was 4 weeks. The growth performance, diarrhea rate and immune indexes of piglets in each group were recorded. The results are shown in Table 3 and Table 4.

[0163] Table 3 Effects of different products on the growth performance and diarrhea rate of piglets

[0164] Group Daily weight gain (g) Feed to gain ratio Diarrhea rate (%) Control group 325±18 1.72±0.08 15.8±1.2 Example 1 group 382±22* 1.52±0.07* 6.5±0.8* Example 4 group 398±25* 1.48±0.06* 5.2±0.6* Control Example 1 group 350±20* 1.63±0.07 10.2±1.0* Control Example 3 group 342±19 1.65±0.08 11.5±1.1* Control Example 4 group 335±18 1.68±0.08 12.8±1.2*

[0165] Note: * indicates significant difference compared with the control group (P<0.05)

[0166] Table 4 Effects of different products on the immune function and intestinal morphology of piglets

[0167]

[0168]

[0169] Note: * indicates significant difference compared with the control group (P<0.05)

[0170] It can be seen from Table 3 and Table 4 that the growth performance, immune function and intestinal morphology indexes of piglets in the Example 1 group and the Example 4 group were significantly better than those in the control group and each comparative example group. The Example 4 group (with a high content of spice plants) had the best effect, with the daily weight gain increased by 22.5%, the diarrhea rate decreased by 67.1%, the serum IgG and IgA levels increased by 42.5% and 51.7% respectively, the small intestinal villus height increased by 23.6%, and the ratio of villus height to crypt depth increased by 47.9%. The effects of the Comparative Example 1 group (single Saccharomyces cerevisiae), the Comparative Example 3 group (without pretreatment with spice plants), and the Comparative Example 4 group (without spice plants) decreased in turn. Among them, there was no significant difference between the Comparative Example 4 group and the control group, indicating that the spice plant components play a key role in the efficacy of the product.

[0171] Experiment 4: Analysis of intestinal flora of piglets

[0172] At the end of the feeding experiment, 8 piglets were randomly selected from each group, fresh fecal samples were collected, total DNA was extracted, and the intestinal flora composition was analyzed by 16S rDNA sequencing technology. The main results are shown in Table 5.

[0173] Table 5 Effects of different products on the intestinal flora of piglets (relative abundance, %)

[0174] Microflora Control group Example 1 group Example 4 group Control Example 1 group Control Example 3 group Control Example 4 group Genus Lactobacillus 6.85±0.42 12.56±0.75* 13.82±0.82* 9.25±0.55* 8.65±0.52* 7.95±0.48* Genus Bifidobacterium 5.25±0.35 10.35±0.62* 11.68±0.70* 7.85±0.47* 7.25±0.43* 6.42±0.38* Genus Clostridium butyricum 3.82±0.28 7.65±0.46* 8.42±0.50* 5.65±0.34* 5.25±0.31* 4.35±0.26 Genus Escherichia 8.65±0.52 3.25±0.19* 2.85±0.17* 5.42±0.32* 5.95±0.36* 7.35±0.44* Genus Salmonella 1.35±0.08 0.45±0.03* 0.32±0.02* 0.82±0.05* 0.95±0.06* 1.12±0.07* Genus Bacteroides 10.25±0.61 16.85±1.01* 18.25±1.09* 13.65±0.82* 12.85±0.77* 11.45±0.68 Shannon index 3.85±0.15 4.52±0.18* 4.68±0.19* 4.15±0.17* 4.05±0.16 3.95±0.16

[0175] Note: * indicates significant difference compared with the control group (P < 0.05).

[0176] As can be seen from Table 5, compared with the control group, the relative abundances of beneficial bacteria (Lactobacillus, Bifidobacterium, Clostridium butyricum, Bacteroides) in the intestinal tracts of piglets in Example 1 group and Example 4 group were significantly increased, while the relative abundances of harmful bacteria (Escherichia coli, Salmonella) were significantly decreased. The effect of Example 4 group was the best, in which the relative abundances of Lactobacillus and Bifidobacterium increased by 101.8% and 122.5% respectively, while the relative abundances of Escherichia coli and Salmonella decreased by 67.1% and 76.3% respectively. In addition, the Shannon index reflects the diversity of the intestinal flora, and the index in the Example group was significantly higher than that in the control group and the Comparative Example group, indicating that the product of the present invention can promote the improvement of the intestinal flora diversity.

[0177] Experiment 5: Evaluation of product stability

[0178] The products of Example 1, Example 4 and Comparative Example 5 were stored at 2 - 8°C and 25°C respectively, and the changes in viable yeast count, pH value, total phenol content and enzyme activity were regularly detected to evaluate the product stability. The results are shown in Table 6 and Table 7.

[0179] Table 6 Evaluation of product stability at 2 - 8°C (stored for 24 months)

[0180] Detection index Example 1 Example 4 Control Example 5 Retention rate of viable yeast count (%) 91.5±3.2 88.6±3.0 75.8±2.5 pH value change (ΔpH) 0.4±0.1 0.5±0.1 0.8±0.2 Retention rate of total phenol content (%) 93.2±3.3 90.5±3.2 70.2±2.4 Retention rate of α-amylase activity (%) 90.8±3.1 88.5±3.0 72.5±2.5 Retention rate of protease activity (%) 89.5±3.0 87.2±2.9 70.3±2.4 Retention rate of β-glucanase activity (%) 92.3±3.2 90.1±3.1 73.6±2.5

[0181] Table 7 Evaluation of product stability at 25°C (stored for 12 months)

[0182]

[0183]

[0184] As can be seen from Table 6 and Table 7, after the products of Example 1 and Example 4 were stored at 2 - 8°C for 24 months and at 25°C for 12 months, the retention rates of all indicators were above 80%, showing good stability. In contrast, the stability of the product of Comparative Example 5 (constant temperature fermentation) was significantly worse, especially the retention rates of total phenol content and enzyme activity were significantly lower than those of the Example products. This shows that the three-stage temperature and humidity control fermentation process of the present invention not only improves the efficacy of the product, but also significantly improves the stability of the product.

[0185] In summary, a series of experiments have proven that the feed for regulating the intestinal flora of piglets by solid-state fermentation of spice plants with compound yeast of the present invention has the following remarkable advantages: 1) The compound yeast (Saccharomyces cerevisiae and Pichia pastoris) acts synergistically to form a complementary enzyme system and metabolic network, significantly improving the fermentation efficiency and functional activity; 2) Enzyme-assisted ultrasonic pretreatment combined with solid-state fermentation biotransformation realizes the efficient transformation and release of the active ingredients of spice plants, improving the bioavailability; 3) The three-stage precise temperature and humidity control technology ensures the stability of product quality and batch consistency; 4) The effective regulation of the intestinal flora of piglets is achieved through multiple mechanisms (selectively promoting the growth of beneficial bacteria, inhibiting the reproduction of harmful bacteria, enhancing the intestinal barrier function, etc.), significantly improving the health status and growth performance of piglets. Therefore, the product of the present invention is a safe, efficient, and environmentally friendly feed for regulating the intestinal flora of piglets by solid-state fermentation of spice plants with compound yeast, and has broad application prospects.

Claims

1. A piglet intestinal flora-regulating feed for solid-state fermentation of compound yeast and spice plants, characterized in that, By weight, the feed comprises the following components: 60 - 75 parts of Saccharomyces cerevisiae; 20 - 30 parts of Pichia pastoris; 15 - 20 parts of rosemary; 15 - 20 parts of thyme; 40 - 50 parts of wheat bran; 20 - 30 parts of corn flour; 15 - 20 parts of soybean meal; 2 - 5 parts of rice hull powder; 3 - 5 parts of molasses; 1 - 3 parts of yeast autolysate; 0.5 - 1.0 part of potassium dihydrogen phosphate; 0.2 - 0.5 part of magnesium sulfate; The Saccharomyces cerevisiae and Pichia pastoris are subjected to solid-state fermentation treatment, and the viable yeast count in the feed is ≥ 2.0×10 8 CFU / g.

2. The piglet intestinal flora-regulating feed for solid-state fermentation of spice plants by compound yeasts according to claim 1, characterized in that, The Saccharomyces cerevisiae is ATCC 9080 strain, the Pichia pastoris is GS115 or X - 33 strain, and the weight ratio of Saccharomyces cerevisiae to Pichia pastoris is 3:

1.

3. The piglet intestinal flora-regulating feed for solid-state fermenting spice plants by compound yeast according to claim 1, characterized in that The total phenol content in the rosemary extract is ≥8%, the rosmarinic acid content is ≥3.5%, and the rosmaridol content is ≥2.0%; the total phenol content in the thyme extract is ≥10%, the thymol content is ≥4.0%, and the carvacrol content is ≥3.0%.

4. The piglet intestinal flora regulating feed for solid-state fermentation of spice plants by compound yeast according to claim 1, characterized in that, The feed further comprises the following components: 2 - 4 parts of microcrystalline cellulose, 3 - 5 parts of maltodextrin, 0.2 - 0.5 part of mixed tocopherols, and 0.3 - 0.8 part of citric acid.

5. The piglet intestinal flora-regulating feed for solid-state fermentation of spice plants by compound yeast according to claim 1, characterized in that The feed has the following properties: moisture ≤14%, pH value 4.5 - 5.5, total phenol content ≥2.5%, α - amylase activity ≥400 U / g, protease activity ≥250 U / g, β - glucanase activity ≥320 U / g.

6. A method for preparing a feed for regulating the intestinal flora of piglets by solid-state fermentation of a spice plant with a composite yeast as claimed in any one of claims 1 to 5, characterized in that, Comprising the following steps: (1) Yeast activation and amplification culture: Inoculate Saccharomyces cerevisiae and Pichia pastoris into a liquid medium respectively, culture at 28 - 30 °C for 16 - 36 hours, collect the thalli and mix them in a ratio of 3:1 to prepare a composite bacterial liquid; (2) Spicy plant pretreatment: Dry and crush rosemary and thyme to 80 - 120 meshes, add a mixed enzyme and treat at 45 ± 2 °C for 60 - 90 minutes, then treat with ultrasonic for 15 - 20 minutes, and dry to moisture ≤8%; (3) Solid - state fermentation substrate preparation: Mix wheat bran, corn flour, soybean meal, and rice hull powder, add 5 - 8% of the spicy plant treatment product, add molasses, yeast autolysate, potassium dihydrogen phosphate, and magnesium sulfate, adjust the pH to 6.0 - 6.5, and the water content to 60 - 65%, sterilize at 121 °C for 20 minutes; (4) Solid - state fermentation: Inoculate the composite bacterial liquid prepared in step (1) into the sterilized substrate in step (3) at 5 - 8% (v / w), and ferment under temperature - controlled and humidity - controlled conditions for 120 ± 4 hours; (5) Post - fermentation treatment: Dry the fermentation product at 45 - 55 °C to moisture ≤14%, and crush it to 60 - 80 meshes; (6) Stabilization treatment: Add microcrystalline cellulose, maltodextrin, mixed tocopherols, and citric acid to the crushed fermentation product, mix evenly and then seal and package.

7. The method according to claim 6, characterized in that, In step (1), the medium composition for Saccharomyces cerevisiae activation is: glucose 20 ± 1 g / L, peptone 20 ± 1 g / L, yeast extract 10 ± 0.5 g / L, pH value 5.5 ± 0.2; the medium composition for Pichia pastoris activation is: glucose 10 ± 0.5 g / L, glycerol 10 ± 0.5 g / L, peptone 20 ± 1 g / L, yeast extract 10 ± 0.5 g / L, pH value 6.0 ± 0.

2.

8. The method according to claim 6, wherein In step (2), the mixed enzyme is composed of cellulase, pectinase and hemicellulase in a ratio of 3:2:1, and the enzyme dosage is 0.2-0.5% of the substrate; the power of the ultrasonic treatment is 300-500 W, and the temperature is controlled at <50°C.

9. The method according to claim 6, wherein The solid-state fermentation in step (4) adopts a staged temperature and humidity control strategy: The first stage (0-24 h): the temperature is 28±0.5°C, and the relative humidity is 75-80%; The second stage (24-72 h): the temperature is 30±0.5°C, the relative humidity is 75-80%, and the material is turned over every 24 hours; The third stage (72-120 h): the temperature is 26±0.5°C, and the relative humidity is 70-75%.

10. The method according to claim 6, wherein The determination criteria for the fermentation end point in step (4) are as follows: the fermentation time is 120 ± 4 hours, the pH value is 4.5 - 5.5, the viable yeast count ≥ 2.0×10 8 CFU / g, the α-amylase activity ≥ 400 U / g, the protease activity ≥ 250 U / g, and the β-glucanase activity ≥ 320 U / g.

Images