A Bacillus subtilis composition for improving animal gut health and its preparation method

Abstract

This invention relates to the field of feed additive technology, specifically to a Bacillus subtilis composition for improving animal intestinal health and its preparation method. It comprises the following raw materials: Bacillus subtilis engineered bacterial powder, cinnamaldehyde microcapsules, proanthocyanidins B2, selenium-enriched yeast culture, glycoterpenes, trehalose, and a carrier. The core of this invention lies in using Bacillus subtilis EhrBS-9 (CGMCC No. 24947) as the strain, constructing a dual-function engineered bacterium with tolerance and transformation through gradient cinnamaldehyde domestication and cad gene integration. This, combined with the intestinal-targeted delivery of cinnamaldehyde microcapsules, barrier strengthening of proanthocyanidins B2, and the construction of a multidimensional anti-stress network of selenium-enriched yeast, glycoterpenes, and trehalose, forms a synergistic mechanism of gut microbiota regulation, immune enhancement, and stress buffering. This addresses the industry pain point of insufficient synergistic effects of existing feed additives in terms of high-efficiency antibacterial activity, safe immune regulation, root-cause anti-stress, and ecological safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of feed additives, in particular to a bacillus subtilis composition for improving the intestinal health of animals and a preparation method thereof. BACKGROUND

[0002] Feed additives refer to a small amount or trace amount of substances added in the process of feed production and processing. The amount of feed additives is very small, but the effect is significant. Feed additives are raw materials that must be used in modern feed industry. They have obvious effects on strengthening the nutritional value of basic feed, improving animal production performance, ensuring animal health, saving feed cost, and improving the quality of livestock products.

[0003] While pursuing high antibacterial and high immune enhancement, existing additives face significant challenges in biological safety and functional synergy: On the one hand, antibiotics or chemical antibacterial agents are used as means to inhibit harmful bacteria in the intestinal tract. Although they have basic antibacterial properties, long-term use can disrupt the balance of intestinal flora, weaken the ability of probiotics to colonize, and induce the growth of drug-resistant strains, and the residues in livestock products exceed the standard (such as sulfonamides and tetracyclines). At the same time, such additives have limited stimulating effect on the animal's own immune system, and may even inhibit normal immune responses, making animals more vulnerable to diseases.

[0004] On the other hand, synthetic additives or hormone substances (such as glucocorticoids and immune stimulants) are commonly used to enhance immune function and relieve stress. Although they can improve animal resistance to disease or reduce stress in the short term, excessive or improper use of such substances can lead to immune system disorders and decreased stress adaptation. In addition, existing additives often treat the symptoms rather than the root cause and are difficult to improve the overall stress resistance of animals from the root of intestinal health when relieving common stress such as heat stress and transportation stress in breeding.

[0005] In summary, existing feed additive technology cannot achieve a balance between high-efficiency antibacterial, safe immune enhancement, long-term stress relief, and environmental health, especially in intensive breeding, high-density feeding, and disease-prone production environments. The long-term effect and biological safety are significantly restricted. SUMMARY

[0006] The purpose of the present application is to solve the defects of existing feed additive technology in the synergistic effect of multiple functions such as high-efficiency antibacterial, safe immune regulation, root stress resistance, and ecological safety.

[0007] The purpose of the present application is to provide a bacillus subtilis composition for improving the intestinal health of animals and a preparation method thereof to solve the problems raised in the background art.

[0008] To achieve the above objectives, one objective of this invention is to provide a Bacillus subtilis composition for improving intestinal health in animals, comprising the following components in parts by weight: 15-25 parts of engineered Bacillus subtilis bacterial powder, 1-5 parts of cinnamaldehyde microcapsules, 2-6 parts of proanthocyanidins B2, 5-15 parts of selenium-enriched yeast culture, 3-8 parts of glycoterpenoids, 4-10 parts of trehalose, and 30-50 parts of carrier. The engineered Bacillus subtilis bacterial powder is a recombinant Bacillus subtilis strain that integrates and expresses the cinnamaldehyde degrading enzyme gene. The starting strain is Bacillus subtilis EhrBS-9, with preservation number CGMCC No. 24947. This strain was isolated from the intestines of healthy piglets and exhibits excellent stress resistance and probiotic functions. The starting strain was domesticated and cultured in a medium containing gradient concentrations of cinnamaldehyde to obtain a domesticated strain tolerant to cinnamaldehyde. Then, the cinnamaldehyde dehydrogenase gene (cad gene) from *Pseudomonas putida* was integrated and expressed using homologous recombination technology. After liquid deep fermentation, the powder was adsorbed and dried using corn flour as a carrier.

[0009] As a further improvement to this technical solution, the viable count of the Bacillus subtilis engineered bacterial powder is ≥1.0×10^10 CFU / g, and the spore rate is ≥90%.

[0010] As a further improvement to this technical solution, the cinnamaldehyde microcapsules are microcapsules prepared with β-cyclodextrin as the wall material and cinnamaldehyde as the core material, with a cinnamaldehyde content ≥15%, a release rate ≤20% in simulated gastric juice over 2 hours, and a release rate ≥85% in simulated intestinal juice over 2 hours.

[0011] As a further improvement to this technical solution, the proanthocyanidin B2 is a grape seed extract, which is purified by high-speed countercurrent chromatography with a purity of ≥90%. The selenium-enriched yeast culture was prepared by fermenting Saccharomyces cerevisiae in a selenium-enriched medium, followed by inactivation and drying. Its total selenium content was ≥1000 mg / kg, of which organic selenium accounted for ≥95%. The glycoterpenoids are a mixture of triterpenoid saponins and sugars extracted from plants of the Theaceae family, wherein the content of triterpenoid saponins is ≥30%.

[0012] A second objective of this invention is to provide a method for preparing the aforementioned Bacillus subtilis composition for improving animal intestinal health, comprising the following steps: Step S1: Using Bacillus subtilis EhrBS-9 with accession number CGMCC No.24947 as the starting strain, the starting strain was gradually domesticated in a medium containing gradient concentrations of cinnamaldehyde to obtain a domesticated strain tolerant to cinnamaldehyde; a homologous recombination integration vector was constructed to integrate the cinnamaldehyde dehydrogenase gene from Pseudomonas putida into the endonuclease-1,4-β-xylanase gene site of the domesticated strain chromosome to obtain a recombinant engineered strain; The recombinant engineered strain was activated, cultured, and fermented in a liquid deep layer, then adsorbed and dried using corn flour as a carrier to obtain Bacillus subtilis engineered strain powder. Step S2: Prepare a saturated solution of β-cyclodextrin, mix it with cinnamaldehyde, add anhydrous ethanol, filter, dry, grind and sieve to obtain cinnamaldehyde microcapsules; Grape seed crude extract was separated by AB-8 macroporous resin, purified by high-speed countercurrent chromatography, and then purified by preparative HPLC to obtain proanthocyanidin B2. Using Saccharomyces cerevisiae as the inoculum, fermentation was carried out in a liquid culture medium, followed by inactivation and drying to obtain a selenium-enriched yeast culture. Triterpenoid saponins were extracted from plants of the Theaceae family and then mixed with sugars to obtain glycoterpenoids; Step S3: Weigh out the following components by weight: Bacillus subtilis engineered bacterial powder, cinnamaldehyde microcapsules, proanthocyanidins, selenium-enriched yeast culture, glycoterpenes, trehalose, and carrier. The above components are placed in a three-dimensional mixer and mixed to obtain a mixture. Step S4: Divide the mixture into portions and vacuum pack or nitrogen-filled pack using aluminum foil composite film bags to obtain the finished product, and store it in a cool, dry place.

[0013] As a further improvement to this technical solution, in step S1, the specific method for the cinnamaldehyde domestication culture is as follows: Bacillus subtilis EhrBS-9 is inoculated into LB liquid medium containing 1 / 4 MIC concentration of cinnamaldehyde, cultured at 37℃ for 24h, and the culture solution is transferred to a medium containing 1 / 2 MIC concentration of cinnamaldehyde. The concentration of cinnamaldehyde is gradually increased until a domesticated strain that can stably grow at a concentration of 2.5 μmol / mL cinnamaldehyde is obtained. The domestication cycle is 20-30 generations. The recombinant engineered strain was inoculated into LB medium, activated at 37°C, and then gradually expanded to obtain a secondary seed culture. The secondary seed culture was inoculated into a fermenter at a rate of 5%, and fermented for 28-32 hours until the spore rate was ≥95%. After fermentation, the bacterial sludge was collected by centrifugation, mixed with sterilized corn flour at a ratio of 1:1.5-2, dried in a fluidized bed until the moisture content was ≤8%, and then pulverized through a 60-mesh sieve to obtain Bacillus subtilis engineered bacterial powder.

[0014] Furthermore, the fermentation medium in the fermenter consists of 2% corn flour, 1.5% soybean meal, 1% molasses, 0.5% yeast powder, 0.03% MnSO4·H2O, and 0.2% CaCO3, with a pH of 7.0-7.2. The temperature is controlled at 36-38℃, the aeration rate is 1:0.8-1.2, and the stirring speed is 200-400 rpm. The inlet air temperature during fluidized bed drying is 55-60℃, and the outlet air temperature is 35-40℃.

[0015] As a further improvement to this technical solution, in step S2, β-cyclodextrin is prepared into a saturated solution, mixed with cinnamaldehyde at a wall-to-core ratio of 6:1, anhydrous ethanol is added, and the mixture is stirred and embedded at 50°C for 4 hours, then allowed to stand overnight at 4°C. The mixture is then filtered, washed, vacuum dried at 40°C, ground, and sieved to obtain cinnamaldehyde microcapsules. Saccharomyces cerevisiae was fermented in a liquid culture medium containing 30 mg / L of selenium for 40 h, first with aerobic fermentation at 30 °C for 24 h and then with anaerobic fermentation for 24 h. The culture was then dried and pulverized at 50-55 °C to obtain a selenium-enriched yeast culture.

[0016] Furthermore, the liquid culture medium was obtained by mixing fermentation broth and solid culture medium at a ratio of 1:1.5, and the solid culture medium was prepared by mixing wheat bran, soybean meal powder, and corn flour at a ratio of 5:3:2. During fermentation, the yeast culture was first aerobic fermented at 30°C for 24 hours and then anaerobic fermented for 24 hours. The yeast culture was then dried and pulverized at 50-55°C to obtain a selenium-enriched yeast culture. Defatted camellia seed meal was extracted three times by reflux with 85% ethanol at 70-80℃. The extract was concentrated and dried to obtain total camellia saponins. The filter residue was extracted three times with water at 80-90℃. The extract was concentrated and precipitated with alcohol to obtain total camellia sugar. The total camellia saponins and total camellia sugar were compounded to obtain glycoterpenoids.

[0017] As a further improvement to this technical solution, in step S3, the ambient temperature in the three-dimensional mixer is ≤25℃ and the relative humidity is ≤50%, and the mixture is mixed at a speed of 20-30 rpm for 20-30 minutes to obtain a mixed material.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: In the Bacillus subtilis composition for improving animal gut health and its preparation method, Bacillus subtilis EhrBS-9 with preservation number CGMCC No.24947 is used as the chassis strain. Through gradient cinnamaldehyde domestication and cad gene integration, an engineered bacterium with dual functions of tolerance and transformation is constructed, thus solving the problem of mutual inhibition between cinnamaldehyde and probiotics. Simultaneously, β-cyclodextrin microcapsule technology is used to achieve intestinal-targeted delivery of cinnamaldehyde, enabling it to precisely block the quorum sensing system of harmful bacteria at a sub-inhibitory concentration in the intestinal segment without killing the bacteria. Combined with proanthocyanidins B2 to activate the intestinal Nrf2-Keap1 pathway and strengthen the epithelial barrier, selenium-enriched yeast, glycoterpenes, and trehalose construct an organic selenium-cortisol regulation-molecular chaperone multidimensional anti-stress network. Through engineered bacteria transforming cinnamaldehyde into p-hydroxybenzoic acid, it serves as a prebiotic to promote the proliferation of beneficial bacteria, forming a synergistic system of microbial regulation, immune reinforcement, and stress buffering. Ultimately, it achieves multiple synergistic effects of highly efficient antibacterial activity, safe immunity, and root-cause anti-stress, solving the industry pain points of existing additives such as single efficacy, incompatibility, and insufficient ecological safety. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the preparation process of the present invention. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] One objective of this invention is to provide a Bacillus subtilis composition for improving intestinal health in animals, comprising the following components in parts by weight: 15-25 parts of engineered Bacillus subtilis bacterial powder, 1-5 parts of cinnamaldehyde microcapsules, 2-6 parts of proanthocyanidins B2, 5-15 parts of selenium-enriched yeast culture, 3-8 parts of glycoterpenoids, 4-10 parts of trehalose, and 30-50 parts of carrier. The engineered Bacillus subtilis bacterial powder is a recombinant Bacillus subtilis strain integrating and expressing the cinnamaldehyde degrading enzyme gene. The starting strain is Bacillus subtilis EhrBS-9 (CGMCC No. 24947), isolated from the intestines of healthy piglets, which exhibits excellent stress resistance and probiotic functions. The starting strain was domesticated in a medium containing gradient concentrations of cinnamaldehyde to obtain a domesticated strain tolerant to cinnamaldehyde. Then, the cinnamaldehyde dehydrogenase gene (cad gene) from *Pseudomonas putida* was integrated and expressed using homologous recombination technology. After liquid deep fermentation, the powder was adsorbed and dried using corn flour as a carrier. The viable count is ≥1.0 × 10^10 CFU / g, and the spore rate is ≥90%.

[0022] Cinnamaldehyde microcapsules are microcapsules prepared with β-cyclodextrin as the wall material and cinnamaldehyde as the core material. The cinnamaldehyde content is ≥15%, and the release rate in simulated gastric fluid is ≤20% in 2 hours and ≥85% in simulated intestinal fluid in 2 hours. Proanthocyanidins B2 are grape seed extracts, purified by high-speed countercurrent chromatography with a purity ≥90%; selenium-enriched yeast culture is prepared by fermenting Saccharomyces cerevisiae in a selenium-enriched medium, followed by inactivation and drying, with a total selenium content ≥1000 mg / kg, of which organic selenium accounts for ≥95%; glycoterpenes are a mixture of triterpenoid saponins and sugars extracted from Camellia plants, with a triterpenoid saponin content ≥30%; Trehalose is produced by fermentation and has a purity of ≥98%.

[0023] Additionally, please see Figure 1 As shown, a second objective of this invention is to provide a method for preparing the above-mentioned Bacillus subtilis composition for improving animal intestinal health, comprising the following steps: Step S1: First, using Bacillus subtilis EhrBS-9 (CGMCC No. 24947) as the starting strain, which was isolated from the intestines of healthy piglets, the starting strain was gradually domesticated in a medium containing gradient concentrations of cinnamaldehyde to obtain a domesticated strain tolerant to cinnamaldehyde. A homologous recombination integration vector was constructed, and the cinnamaldehyde dehydrogenase gene (cad gene) from Pseudomonas putida was integrated into the endonuclease-1,4-β-xylanase gene site on the chromosome of the domesticated strain to obtain a recombinant engineered strain, named Bacillus subtilis BS-CDR1.

[0024] BS-CDR1 was inoculated into LB medium and activated at 37℃, followed by step-by-step scaling-up to obtain a secondary seed culture. The secondary seed culture was then inoculated into a fermenter at a 5% inoculation rate. The fermentation medium consisted of 2% corn flour, 1.5% soybean meal, 1% molasses, 0.5% yeast powder, 0.03% MnSO4·H2O, and 0.2% CaCO3, with a pH of 7.0-7.2. The temperature was controlled at 36-38℃, the aeration rate at 1:0.8-1.2, and the stirring speed at 200-400 rpm. Fermentation was carried out for 28-32 hours until the spore count was ≥95%. After fermentation, the bacterial sludge was collected by centrifugation and mixed with sterilized corn flour at a ratio of 1:1.5-2. The mixture was then dried in a fluidized bed (inlet air 55-60℃, outlet air 35-40℃) until the moisture content was ≤8%. The mixture was then pulverized through a 60-mesh sieve to obtain Bacillus subtilis engineered bacterial powder. The viable count of the finished product was ≥1.0×10^10 CFU / g, and the spore count was ≥90%.

[0025] Step S2: Prepare a saturated solution of β-cyclodextrin and mix it with cinnamaldehyde at a wall-to-core ratio of 6:1. Add anhydrous ethanol (ethanol:water = 1:2), stir and embed at 50°C for 4 hours, then let stand at 4°C overnight. Filter, wash, vacuum dry at 40°C, grind and sieve to obtain cinnamaldehyde microcapsules with a cinnamaldehyde content ≥15%, a 2-hour release rate ≤20% in simulated gastric juice, and a 2-hour release rate ≥85% in simulated intestinal juice. Separate the crude grape seed extract with AB-8 macroporous resin, purify by high-speed countercurrent chromatography (solvent system: hexane:ethyl acetate:methanol:water = 1:5:1:5), and then purify by preparative HPLC to obtain proanthocyanidin B2 with a purity ≥90%.

[0026] Saccharomyces cerevisiae was fermented in a liquid medium containing 30 mg / L selenium for 40 h. The fermentation broth was mixed with a solid medium (wheat bran: soybean meal: corn flour = 5:3:2) at a ratio of 1:1.5. The mixture was first fermented aerobically at 30 °C for 24 h, then anaerobically for 24 h. The mixture was dried and pulverized at 50-55 °C to obtain a selenium-enriched yeast culture with a total selenium content ≥1000 mg / kg and an organic selenium content ≥95%. Defatted camellia seed meal was extracted three times with 85% ethanol under reflux at 70-80 °C. The extract was concentrated and dried to obtain total camellia saponins. The residue was extracted three times with water at 80-90 °C. The extract was concentrated and precipitated with alcohol to obtain total camellia sugar. The total camellia saponins and total camellia sugar were compounded to obtain glycoterpenoids with a triterpenoid saponin content ≥30%. Commercially available fermented trehalose with a purity ≥98% was purchased.

[0027] Step S3: Accurately weigh 15-25 parts of Bacillus subtilis engineered bacterial powder, 1-5 parts of cinnamaldehyde microcapsules, 2-6 parts of proanthocyanidins B2, 5-15 parts of selenium-enriched yeast culture, 3-8 parts of glycoterpenoids, 4-10 parts of trehalose, and 30-50 parts of carrier according to the following weight proportions: Place the above components in a three-dimensional mixer, control the ambient temperature to ≤25℃ and the relative humidity to ≤50%, and mix at a speed of 20-30 rpm for 20-30 minutes to ensure that the components are evenly distributed and obtain a mixture.

[0028] Step S4: Divide the well-mixed materials into smaller portions and vacuum-pack them in aluminum foil composite film bags or nitrogen-filled bags, labeling them with product information; store the finished product in a cool, dry place.

[0029] In this invention, *Bacillus subtilis* EhrBS-9 (CGMCC No. 24947) was used as the starting strain. This strain, isolated from the intestines of healthy piglets, possesses excellent stress resistance and probiotic functions. Through gradient concentration domestication culture, it acquired natural tolerance to cinnamaldehyde, leading to the construction of an engineered *Bacillus subtilis* strain BS-CDR1 that integrates the cinnamaldehyde dehydrogenase gene, endowing it with a unique "tolerance-conversion" dual function. Using the domesticated EhrBS-9 as the starting strain, the engineered bacterium integrated the cad gene from *Pseudomonas putida* through homologous recombination technology, enabling the strain to directionally convert cinnamaldehyde into p-hydroxybenzoic acid. This fundamentally solves the mutual inhibition problem between cinnamaldehyde and *Bacillus subtilis*, allowing the engineered bacterium not only tolerating cinnamaldehyde but also actively converting it into a low-toxicity prebiotic. Simultaneously, cinnamaldehyde was encapsulated using β-cyclodextrin microencapsulation technology, achieving its spatiotemporal release in the gastrointestinal tract. The microcapsules remain stable in the acidic environment of the stomach (release rate ≤20% in 2 hours), ensuring that cinnamaldehyde and engineered bacteria are isolated from each other in the stomach. Upon entering the intestines, they are rapidly released (release rate ≥85% in 2 hours). At this point, cinnamaldehyde acts at a sub-inhibitory concentration on the quorum sensing system (LuxS / AI-2 pathway) of harmful bacteria, effectively reducing the expression of virulence factors and adhesion ability without directly killing the bacteria, thus rendering the harmful bacteria "ineffective." This quorum sensing inhibition strategy differs from the direct killing mode of traditional antibiotics, avoiding the development of drug resistance while preserving the potential value of harmful bacteria as metabolic substrates.

[0030] While cinnamaldehyde completes quorum sensing inhibition, the engineered bacterium BS-CDR1 secretes cinnamaldehyde dehydrogenase to convert free cinnamaldehyde into p-hydroxybenzoic acid, which can selectively promote the proliferation of the engineered bacterium itself and other beneficial bacteria as a prebiotic. The introduction of proanthocyanidins B2 establishes a second synergistic mechanism—it, together with cinnamaldehyde metabolites, activates the Nrf2-Keap1 antioxidant pathway in intestinal epithelial cells, inducing the expression of phase II detoxification enzymes and antioxidant enzyme systems, thereby enhancing intestinal barrier function at the cellular level.

[0031] Furthermore, selenium-enriched yeast culture, along with glycoterpenes and trehalose, constructs a multidimensional anti-stress system. The organic selenium (≥95%) provided by the selenium-enriched yeast participates in the composition of glutathione peroxidase through selenoproteins; glycoterpenes reduce cortisol levels by regulating the hypothalamic-pituitary-adrenal axis; and trehalose acts as a molecular chaperone, protecting the native conformation of proteins under heat stress conditions. The synergistic effect of these three components enables animals to maintain intestinal homeostasis and normal metabolic function under heat stress, transportation stress, and other conditions.

[0032] Ultimately, through the aforementioned multi-component, multi-target synergistic design, this invention constructs a four-in-one anti-stress system integrating engineered bacteria tolerance transformation, cinnamaldehyde quorum sensing inhibition, proanthocyanidin Nrf2 pathway activation, and selenium-enriched yeast / glycoterpenes / trehalose. Each component performs its specific function while also synergizing with others in the intestinal environment: engineered bacteria transform potential inhibitors into synergists; cinnamaldehyde precisely regulates the virulence of harmful bacteria at sub-inhibitory concentrations; proanthocyanidin B2 strengthens the intestinal barrier at the cellular level; and selenium-enriched yeast, along with glycoterpenes and trehalose, buffers stress shocks. This multi-level synergistic mechanism, from gut microbiota regulation and immune enhancement to anti-stress response, fundamentally addresses the shortcomings of existing feed additive technologies in achieving sufficient synergistic effects in high-efficiency antibacterial activity, safe immune regulation, root-cause anti-stress, and ecological safety, thus realizing a systemic improvement in gut health.

[0033] The following specific examples further illustrate the Bacillus subtilis composition for improving animal intestinal health and its preparation method proposed in this invention.

[0034] Example 1 Step S1: Using Bacillus subtilis EhrBS-9 (CGMCC No. 24947) as the starting strain, isolated from the intestines of healthy piglets, the starting strain was gradually acclimated to cinnamaldehyde in culture media containing gradient concentrations to obtain acclimated strains tolerant to cinnamaldehyde (acclimation method: inoculated into LB liquid medium containing 1 / 4 MIC concentration of cinnamaldehyde, cultured at 37℃ for 24 h, transferred to medium containing 1 / 2 MIC concentration, and the concentration was gradually increased to 2.5 μmol / mL, acclimated for 20 generations). A homologous recombination integration vector was constructed, and the cinnamaldehyde dehydrogenase gene (cad gene) from *Pseudomonas putida* was integrated into the endonuclease-1,4-β-xylanase gene site on the chromosome of the acclimated strain to obtain the recombinant engineered strain, named *Bacillus subtilis* BS-CDR1.

[0035] BS-CDR1 was inoculated into LB medium and activated at 37℃, followed by step-by-step scaling-up to obtain a secondary seed culture. The secondary seed culture was then inoculated into a fermenter at a 5% inoculation rate. The fermentation medium consisted of 2% corn flour, 1.5% soybean meal, 1% molasses, 0.5% yeast powder, 0.03% MnSO4·H2O, and 0.2% CaCO3, with a pH of 7.0-7.2. The temperature was controlled at 36℃, the aeration rate at 1:1.2, and the stirring speed at 200 rpm. Fermentation was carried out for 32 hours until the spore count was ≥95%. After fermentation, the bacterial sludge was collected by centrifugation and mixed with sterilized corn flour at a 1:1.5 ratio. The mixture was then dried in a fluidized bed (inlet air 60℃, outlet air 35℃) until the moisture content was ≤8%. The mixture was then pulverized through a 60-mesh sieve to obtain Bacillus subtilis engineered bacterial powder. The viable count of the finished product was ≥1.0×10^10 CFU / g, and the spore count was ≥90%.

[0036] Step S2: Prepare a saturated solution of β-cyclodextrin and mix it with cinnamaldehyde at a wall-to-core ratio of 6:1. Add anhydrous ethanol (ethanol:water = 1:2), stir and embed at 50°C for 4 hours, then let stand at 4°C overnight. Filter, wash, vacuum dry at 40°C, grind and sieve to obtain cinnamaldehyde microcapsules with a cinnamaldehyde content ≥15%, a 2-hour release rate ≤20% in simulated gastric juice, and a 2-hour release rate ≥85% in simulated intestinal juice. Separate the crude grape seed extract with AB-8 macroporous resin, purify by high-speed countercurrent chromatography (solvent system: hexane:ethyl acetate:methanol:water = 1:5:1:5), and then purify by preparative HPLC to obtain proanthocyanidin B2 with a purity ≥90%.

[0037] Saccharomyces cerevisiae was fermented in a liquid medium containing 30 mg / L selenium for 40 h. The fermentation broth was mixed with a solid medium (wheat bran: soybean meal: corn flour = 5:3:2) at a ratio of 1:1.5. The mixture was first fermented aerobically at 30 °C for 24 h and then anaerobically for 24 h. The mixture was then dried and pulverized at 55 °C to obtain a selenium-enriched yeast culture with a total selenium content ≥1000 mg / kg and an organic selenium content ≥95%. Defatted camellia seed meal was extracted three times with 85% ethanol under reflux at 70 °C. The extract was concentrated and dried to obtain total camellia saponins. The residue was extracted three times with water at 90 °C. The extract was concentrated and precipitated with alcohol to obtain total camellia sugar. The total camellia saponins and total camellia sugar were compounded to obtain glycoterpenoids with a triterpenoid saponin content ≥30%. Commercially available fermented trehalose with a purity ≥98% was purchased.

[0038] Step S3: Accurately weigh 15 parts by weight of Bacillus subtilis engineered bacterial powder, 5 parts by weight of cinnamaldehyde microcapsules, 2 parts by weight of proanthocyanidin B2, 15 parts by weight of selenium-enriched yeast culture, 3 parts by weight of glycoterpenoids, 10 parts by weight of trehalose, and 30 parts by weight of carrier; place the above components in a three-dimensional mixer, control the ambient temperature to ≤25℃ and the relative humidity to ≤50%, mix at 30 rpm for 20 minutes to ensure that each component is evenly distributed, and obtain a mixture.

[0039] Step S4: Divide the well-mixed materials into smaller portions and vacuum-pack them in aluminum foil composite film bags or nitrogen-filled bags, labeling them with product information; store the finished product in a cool, dry place.

[0040] Example 2 Step S1: Using Bacillus subtilis EhrBS-9 (CGMCC No. 24947) as the starting strain, isolated from the intestines of healthy piglets, the starting strain was gradually acclimated to cinnamaldehyde in culture media containing gradient concentrations to obtain acclimated strains tolerant to cinnamaldehyde (acclimation method: inoculated into LB liquid medium containing 1 / 4 MIC concentration of cinnamaldehyde, cultured at 37℃ for 24 h, transferred to medium containing 1 / 2 MIC concentration, and the concentration was gradually increased to 2.5 μmol / mL, acclimation for 25 generations). A homologous recombination integration vector was constructed, and the cinnamaldehyde dehydrogenase gene (cad gene) from *Pseudomonas putida* was integrated into the endonuclease-1,4-β-xylanase gene site on the chromosome of the acclimated strain to obtain the recombinant engineered strain, named *Bacillus subtilis* BS-CDR1.

[0041] BS-CDR1 was inoculated into LB medium and activated at 37℃, followed by step-by-step scaling-up to obtain a secondary seed culture. The secondary seed culture was then inoculated into a fermenter at a 5% inoculation rate. The fermentation medium consisted of 2% corn flour, 1.5% soybean meal, 1% molasses, 0.5% yeast powder, 0.03% MnSO4·H2O, and 0.2% CaCO3, with a pH of 7.0-7.2. The temperature was controlled at 38℃, the aeration rate at 1:1, and the stirring speed at 300 rpm. Fermentation was carried out for 30 hours until the spore count was ≥95%. After fermentation, the bacterial sludge was collected by centrifugation and mixed with sterilized corn flour at a ratio of 1:1.8. The mixture was then dried in a fluidized bed (inlet air 60℃, outlet air 40℃) until the moisture content was ≤8%. The mixture was then pulverized through a 60-mesh sieve to obtain Bacillus subtilis engineered bacterial powder. The viable count of the finished product was ≥1.0×10^10 CFU / g, and the spore count was ≥90%.

[0042] Step S2: Prepare a saturated solution of β-cyclodextrin and mix it with cinnamaldehyde at a wall-to-core ratio of 6:1. Add anhydrous ethanol (ethanol:water = 1:2), stir and embed at 50°C for 4 hours, then let stand at 4°C overnight. Filter, wash, vacuum dry at 40°C, grind and sieve to obtain cinnamaldehyde microcapsules with a cinnamaldehyde content ≥15%, a 2-hour release rate ≤20% in simulated gastric juice, and a 2-hour release rate ≥85% in simulated intestinal juice. Separate the crude grape seed extract with AB-8 macroporous resin, purify by high-speed countercurrent chromatography (solvent system: hexane:ethyl acetate:methanol:water = 1:5:1:5), and then purify by preparative HPLC to obtain proanthocyanidin B2 with a purity ≥90%.

[0043] Saccharomyces cerevisiae was fermented in a liquid medium containing 30 mg / L selenium for 40 h. The fermentation broth was mixed with a solid medium (wheat bran: soybean meal: corn flour = 5:3:2) at a ratio of 1:1.5. The mixture was first fermented aerobically at 30 °C for 24 h and then anaerobically for 24 h. The mixture was then dried and pulverized at 50 °C to obtain a selenium-enriched yeast culture with a total selenium content ≥1000 mg / kg and an organic selenium content ≥95%. Defatted camellia seed meal was extracted three times with 85% ethanol under reflux at 75 °C. The extract was concentrated and dried to obtain total camellia saponins. The residue was extracted three times with water at 85 °C. The extract was concentrated and precipitated with alcohol to obtain total camellia sugar. The total camellia saponins and total camellia sugar were compounded to obtain glycoterpenoids with a triterpenoid saponin content ≥30%. Commercially available fermented trehalose with a purity ≥98% was purchased.

[0044] Step S3: Accurately weigh 20 parts by weight of Bacillus subtilis engineered bacterial powder, 3 parts by weight of cinnamaldehyde microcapsules, 4 parts by weight of proanthocyanidins B2, 10 parts by weight of selenium-enriched yeast culture, 6 parts by weight of glycoterpenoids, 7 parts by weight of trehalose, and 40 parts by weight of carrier; place the above components in a three-dimensional mixer, control the ambient temperature to ≤25℃ and the relative humidity to ≤50%, mix at 25 rpm for 25 minutes to ensure that the components are evenly distributed and obtain a mixture.

[0045] Step S4: Divide the well-mixed materials into smaller portions and vacuum-pack them in aluminum foil composite film bags or nitrogen-filled bags, labeling them with product information; store the finished product in a cool, dry place.

[0046] Example 3 Step S1: Using Bacillus subtilis EhrBS-9 (CGMCC No. 24947) as the starting strain, isolated from the intestines of healthy piglets, the starting strain was gradually domesticated in culture media containing gradient concentrations of cinnamaldehyde to obtain domesticated strains tolerant to cinnamaldehyde (domestication method: inoculated into LB liquid medium containing 1 / 4 MIC concentration of cinnamaldehyde, cultured at 37℃ for 24 h, transferred to medium containing 1 / 2 MIC concentration, and the concentration was gradually increased to 2.5 μmol / mL, domesticated for 30 generations). A homologous recombination integration vector was constructed, and the cinnamaldehyde dehydrogenase gene (cad gene) from *Pseudomonas putida* was integrated into the endonuclease-1,4-β-xylanase gene site on the chromosome of the domesticated strain to obtain the recombinant engineered strain, named *Bacillus subtilis* BS-CDR1.

[0047] BS-CDR1 was inoculated into LB medium and activated at 37℃, followed by step-by-step scaling-up to obtain a secondary seed culture. The secondary seed culture was then inoculated into a fermenter at a 5% inoculation rate. The fermentation medium consisted of 2% corn flour, 1.5% soybean meal, 1% molasses, 0.5% yeast powder, 0.03% MnSO4·H2O, and 0.2% CaCO3, with a pH of 7.0-7.2. The temperature was controlled at 38℃, the aeration ratio at 1:0.8, and the stirring speed at 400 rpm. Fermentation was carried out for 28 hours until the spore count was ≥95%. After fermentation, the bacterial sludge was collected by centrifugation and mixed with sterilized corn flour at a 1:2 ratio. The mixture was then dried in a fluidized bed (inlet air 55℃, outlet air 40℃) until the moisture content was ≤8%. The mixture was then pulverized through a 60-mesh sieve to obtain Bacillus subtilis engineered bacterial powder. The viable count of the finished product was ≥1.0×10^10 CFU / g, and the spore count was ≥90%.

[0048] Step S2: Prepare a saturated solution of β-cyclodextrin and mix it with cinnamaldehyde at a wall-to-core ratio of 6:1. Add anhydrous ethanol (ethanol:water = 1:2), stir and embed at 50°C for 4 hours, then let stand at 4°C overnight. Filter, wash, vacuum dry at 40°C, grind and sieve to obtain cinnamaldehyde microcapsules with a cinnamaldehyde content ≥15%, a 2-hour release rate ≤20% in simulated gastric juice, and a 2-hour release rate ≥85% in simulated intestinal juice. Separate the crude grape seed extract with AB-8 macroporous resin, purify by high-speed countercurrent chromatography (solvent system: hexane:ethyl acetate:methanol:water = 1:5:1:5), and then purify by preparative HPLC to obtain proanthocyanidin B2 with a purity ≥90%.

[0049] Saccharomyces cerevisiae was fermented in a liquid medium containing 30 mg / L selenium for 40 h. The fermentation broth was mixed with a solid medium (wheat bran: soybean meal: corn flour = 5:3:2) at a ratio of 1:1.5. The mixture was first fermented aerobically at 30 °C for 24 h and then anaerobically for 24 h. The mixture was then dried and pulverized at 50 °C to obtain a selenium-enriched yeast culture with a total selenium content ≥1000 mg / kg and an organic selenium content ≥95%. Defatted camellia seed meal was extracted three times with 85% ethanol under reflux at 80 °C. The extract was concentrated and dried to obtain total camellia saponins. The residue was extracted three times with water at 80 °C. The extract was concentrated and precipitated with alcohol to obtain total camellia sugar. The total camellia saponins and total camellia sugar were compounded to obtain glycoterpenoids with a triterpenoid saponin content ≥30%. Commercially available fermented trehalose with a purity ≥98% was purchased.

[0050] Step S3: Accurately weigh 25 parts of Bacillus subtilis engineered bacterial powder, 1 part of cinnamaldehyde microcapsules, 6 parts of proanthocyanidins B2, 5 parts of selenium-enriched yeast culture, 8 parts of glycoterpenoids, 4 parts of trehalose, and 50 parts of carrier according to the weight ratio; place the above components in a three-dimensional mixer, control the ambient temperature ≤25℃ and relative humidity ≤50%, mix at a speed of 20 rpm for 30 minutes to ensure that each component is evenly distributed and obtain a mixture.

[0051] Step S4: Divide the well-mixed materials into smaller portions and vacuum-pack them in aluminum foil composite film bags or nitrogen-filled bags, labeling them with product information; store the finished product in a cool, dry place.

[0052] The Bacillus subtilis compositions were prepared according to Examples 1-3, and then the compositions were subjected to the following performance tests: Growth performance test: Ninety healthy piglets of similar weight at 28 days of age were randomly divided into a control group, Example 1 group, Example 2 group, Example 3 group, and a positive control group, with 6 replicates per group and 3 piglets per replicate. The control group was fed a basal diet, while Example 1-3 groups were fed a basal diet supplemented with 0.1% of the Bacillus subtilis composition prepared in Example 1-3, respectively. The positive control group was fed a basal diet supplemented with 500 mg / kg of ordinary Bacillus subtilis powder. The pre-trial period was 7 days, and the formal trial period was 28 days. Piglets were weighed at the beginning and end of the trial, and feed consumption was recorded. Average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (F / G) were calculated.

[0053] Diarrhea rate test: Observe and record the number of piglets with diarrhea every day, and calculate the diarrhea rate (diarrhea rate = total number of piglets with diarrhea / (number of days in the test × total number of piglets) × 100%).

[0054] Antibacterial effect test: At the end of the experiment, fresh fecal samples were collected for each repetition, and the number of Escherichia coli and Salmonella in the feces was detected by plate counting method. The results are expressed as lg (CFU / g).

[0055] Immunological marker testing: At the end of the experiment, one piglet was randomly selected from each replicate, blood was collected from the anterior vena cava, serum was separated, and the levels of immunoglobulin A (IgA), immunoglobulin G (IgG), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the serum were measured using an ELISA kit.

[0056] Antioxidant index test: The activities of superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) in serum were measured using a kit.

[0057] Intestinal morphology test: At the end of the experiment, one piglet was randomly selected from each replicate and slaughtered. Jejunum and ileum tissues were taken, paraffin sections were prepared, villus height and crypt depth were measured, and the villus height / crypt depth ratio (V / C) was calculated.

[0058] Digestive enzyme activity test: Small intestinal contents were collected, and the activities of protease and amylase were determined using a kit.

[0059] Stress resistance test: On day 21 of the experiment, piglets were subjected to transportation stress treatment (transportation for 2 hours). Blood was collected 2 hours after transportation to measure the levels of cortisol (COR) and heat shock protein 70 (HSP70) in serum.

[0060] The values ​​obtained from the above performance tests are recorded in Table 1-7.

[0061] Table 1. Growth performance of the Bacillus subtilis compositions prepared in Examples 1-3 Table 2. Antibacterial effect of the Bacillus subtilis compositions prepared in Examples 1-3 (feces, 1g CFU / g) Table 3. Immunological indicators of the Bacillus subtilis compositions prepared in Examples 1-3 Table 4. Antioxidant indices of the Bacillus subtilis compositions prepared in Examples 1-3 Table 5. Intestinal morphology of the Bacillus subtilis compositions prepared in Examples 1-3 Table 6. Digestive enzyme activities of the Bacillus subtilis compositions prepared in Examples 1-3 Table 7. Stress resistance of the Bacillus subtilis compositions prepared in Examples 1-3 According to Table 1-7, Regarding growth performance: The average daily weight gain of groups 1-3 was higher than that of the control group and higher than that of the positive control group; the feed conversion ratio was lower than that of the control group and lower than that of the positive control group; the diarrhea rate was lower than that of the control group and lower than that of the positive control group. This indicates that the composition of the present invention can significantly promote the growth of piglets and reduce the incidence of diarrhea.

[0062] Regarding antibacterial effects: In Examples 1-3, the number of Escherichia coli in feces was lower than that in the control group and lower than that in the positive control group; the number of Salmonella was also lower than that in the control group and lower than that in the positive control group. This indicates that the composition of the present invention can effectively inhibit harmful intestinal bacteria.

[0063] Regarding immune function: The serum IgA content in groups 1-3 was higher than that in the control group and higher than that in the positive control group; the IgG content was higher than that in the control group and higher than that in the positive control group; the inflammatory factors IL-6 and TNF-α were lower than those in the control group, indicating that the composition of the present invention can significantly enhance the body's immune function and reduce the inflammatory response.

[0064] Regarding antioxidant capacity: In Examples 1-3, serum SOD activity was higher than that in the control group and higher than that in the positive control group; CAT activity was higher than that in the control group and higher than that in the positive control group; MDA content was lower than that in the control group and lower than that in the positive control group. This indicates that the composition of the present invention has significant antioxidant effects.

[0065] Regarding intestinal morphology: The height of jejunal villi in groups 1-3 was higher than that in the control group and also higher than that in the positive control group; the V / C ratio was higher than that in the control group and also higher than that in the positive control group. This indicates that the composition of the present invention can significantly improve intestinal morphology and enhance intestinal absorption function.

[0066] Regarding digestive enzyme activity: In Examples 1-3, protease activity was increased compared to the control group and the positive control group; amylase activity was also increased compared to the control group and the positive control group. This indicates that the composition of the present invention can promote digestive enzyme secretion and improve feed digestibility and utilization.

[0067] Regarding stress resistance: After transport stress, the serum cortisol levels in groups 1-3 were lower than those in the control group and lower than those in the positive control group; the levels of heat shock protein 70 were higher than those in the control group and higher than those in the positive control group. This indicates that the composition of the present invention can significantly enhance the stress resistance of animals.

[0068] The above data show that the Bacillus subtilis compositions prepared in Examples 1-3 are significantly superior to the control group and the positive control group in terms of improving piglet growth performance, reducing diarrhea rate, inhibiting harmful bacteria, enhancing immune function and antioxidant capacity, improving intestinal morphology, increasing digestive enzyme activity, and enhancing stress resistance.

[0069] In summary, the Bacillus subtilis composition for improving animal gut health and its preparation method provided by this invention use the authentic preserved strain Bacillus subtilis EhrBS-9 (CGMCC No. 24947) as the starting strain. Through domestication culture and genetic engineering, engineered bacteria with cinnamaldehyde tolerance-conversion function are obtained. This is then scientifically compounded with functional components such as cinnamaldehyde microcapsules, proanthocyanidins B2, selenium-enriched yeast culture, glycoterpenes, and trehalose. This allows for the preparation of a Bacillus subtilis composition with multiple synergistic effects, significantly improving animal gut health, enhancing immune function, increasing antioxidant capacity, and promoting growth performance. This effectively solves the shortcomings of existing feed additive technologies in terms of insufficient synergistic effects in multiple functions such as high-efficiency antibacterial activity, safe immune regulation, root-cause stress resistance, and ecological safety.

[0070] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a Bacillus subtilis composition for improving intestinal health in animals, characterized in that, Includes the following steps: Step S1: Using Bacillus subtilis EhrBS-9 with accession number CGMCC No.24947 as the starting strain, the starting strain was gradually domesticated in a medium containing gradient concentrations of cinnamaldehyde to obtain a domesticated strain tolerant to cinnamaldehyde; a homologous recombination integration vector was constructed to integrate the cinnamaldehyde dehydrogenase gene from Pseudomonas putida into the endonuclease-1,4-β-xylanase gene site of the domesticated strain chromosome to obtain a recombinant engineered strain; The recombinant engineered strain was activated, cultured, and fermented in a liquid deep layer, then adsorbed and dried using corn flour as a carrier to obtain Bacillus subtilis engineered strain powder. Step S2: Prepare a saturated solution of β-cyclodextrin, mix it with cinnamaldehyde, add anhydrous ethanol, filter, dry, grind and sieve to obtain cinnamaldehyde microcapsules; Using Saccharomyces cerevisiae as the inoculum, fermentation was carried out in a liquid culture medium, followed by inactivation and drying to obtain a selenium-enriched yeast culture. Triterpenoid saponins were extracted from plants of the Theaceae family and then mixed with sugars to obtain glycoterpenoids; Step S3: Weigh out 15-25 parts of Bacillus subtilis engineered bacterial powder, 1-5 parts of cinnamaldehyde microcapsules, 2-6 parts of proanthocyanidins B2, 5-15 parts of selenium-enriched yeast culture, 3-8 parts of glycoterpenoids, 4-10 parts of trehalose, and 30-50 parts of carrier by weight. The above components are placed in a three-dimensional mixer and mixed to obtain a mixture. Step S4: Divide the mixture into portions and vacuum pack or nitrogen-filled pack using aluminum foil composite film bags to obtain the finished product, and store it in a cool, dry place.

2. The method for preparing the Bacillus subtilis composition for improving animal intestinal health according to claim 1, characterized in that: In step S1, the specific method for cinnamaldehyde domestication culture is as follows: Bacillus subtilis EhrBS-9 is inoculated into LB liquid medium containing 1 / 4 MIC concentration of cinnamaldehyde, cultured at 37℃ for 24h, and the culture solution is transferred to medium containing 1 / 2 MIC concentration of cinnamaldehyde. The concentration of cinnamaldehyde is gradually increased until a domesticated strain that can grow stably at a concentration of 2.5 μmol / mL cinnamaldehyde is obtained. The domestication cycle is 20-30 generations. The recombinant engineered strain was inoculated into LB medium, activated at 37°C, and then scaled up stepwise to obtain a secondary seed culture. Inoculate the secondary seed culture into the fermenter at a 5% inoculation rate and ferment for 28-32 hours until the spore rate is ≥95%. After fermentation, the bacterial sludge was collected by centrifugation and mixed with sterilized corn flour at a ratio of 1:1.5-2. The mixture was then dried in a fluidized bed until the moisture content was ≤8%, and then pulverized through a 60-mesh sieve to obtain engineered Bacillus subtilis bacterial powder.

3. The method for preparing the Bacillus subtilis composition for improving animal intestinal health according to claim 2, characterized in that: The fermentation medium in the fermenter consists of 2% corn flour, 1.5% soybean meal, 1% molasses, 0.5% yeast powder, 0.03% MnSO4·H2O, and 0.2% CaCO3, with a pH of 7.0-7.

2. The temperature is controlled at 36-38℃, the aeration rate is 1:0.8-1.2, and the stirring speed is 200-400 rpm. The inlet air temperature during fluidized bed drying is 55-60℃, and the outlet air temperature is 35-40℃.

4. The method for preparing the Bacillus subtilis composition for improving animal intestinal health according to claim 1, characterized in that: In step S1, the viable count of the engineered Bacillus subtilis bacterial powder is ≥1.0×10^10 CFU / g, and the spore rate is ≥90%.

5. The method for preparing the Bacillus subtilis composition for improving animal intestinal health according to claim 1, characterized in that: In step S2, β-cyclodextrin is prepared into a saturated solution, mixed with cinnamaldehyde at a wall-to-core ratio of 6:1, anhydrous ethanol is added, and the mixture is stirred and embedded at 50°C for 4 hours, then allowed to stand overnight at 4°C. The mixture is then filtered, washed, vacuum dried at 40°C, ground and sieved to obtain cinnamaldehyde microcapsules. Saccharomyces cerevisiae was fermented in a liquid culture medium containing 30 mg / L of selenium for 40 h, first with aerobic fermentation at 30 °C for 24 h and then with anaerobic fermentation for 24 h. The culture was then dried and pulverized at 50-55 °C to obtain a selenium-enriched yeast culture.

6. The method for preparing the Bacillus subtilis composition for improving animal intestinal health according to claim 5, characterized in that: The liquid culture medium is obtained by mixing fermentation broth and solid culture medium at a ratio of 1:1.

5. The solid culture medium is prepared by mixing wheat bran, soybean meal powder and corn flour at a ratio of 5:3:

2. During fermentation, the mixture is first fermented aerobically at 30°C for 24 hours and then anaerobically fermented for 24 hours. The mixture is then dried and pulverized at 50-55°C to obtain a selenium-enriched yeast culture. Defatted camellia seed meal was extracted three times by reflux with 85% ethanol at 70-80℃. The extract was concentrated and dried to obtain total camellia saponins. The filter residue was extracted three times with water at 80-90℃. The extract was concentrated and precipitated with alcohol to obtain total camellia sugar. The total camellia saponins and total camellia sugar were compounded to obtain glycoterpenoids.

7. The method for preparing the Bacillus subtilis composition for improving animal intestinal health according to claim 1, characterized in that: The cinnamaldehyde microcapsules contain ≥15% cinnamaldehyde, with a 2-hour release rate ≤20% in simulated gastric fluid and a 2-hour release rate ≥85% in simulated intestinal fluid. The total selenium content of the selenium-enriched yeast culture is ≥1000 mg / kg, of which organic selenium accounts for ≥95%; The content of triterpenoid saponins in the glycoterpenoids is ≥30%.

8. The method for preparing the Bacillus subtilis composition for improving animal intestinal health according to claim 1, characterized in that: In step S3, proanthocyanidin B2 is a grape seed extract, which is purified by high-speed countercurrent chromatography with a purity of ≥90%.

9. The method for preparing the Bacillus subtilis composition for improving animal intestinal health according to claim 1, characterized in that: In step S3, the ambient temperature in the three-dimensional mixer is ≤25℃ and the relative humidity is ≤50%. The mixture is stirred at a speed of 20-30 rpm for 20-30 minutes to obtain a mixed material.

10. A Bacillus subtilis composition for improving animal intestinal health prepared by the preparation method according to any one of claims 1-9, characterized in that, The raw materials include: Bacillus subtilis engineered bacterial powder, cinnamaldehyde microcapsules, proanthocyanidins B2, selenium-enriched yeast culture, glycoterpenes, trehalose, and a carrier; among which: The Bacillus subtilis engineered bacterial powder, as the core probiotic, was constructed by integrating the cinnamaldehyde gradient domestication and cad gene of Bacillus subtilis EhrBS-9 with preservation number CGMCC No.24947. The cinnamaldehyde microcapsules use β-cyclodextrin wall material to encapsulate the cinnamaldehyde core material, achieving its stability in the gastric acid environment and targeted release in the intestine; The proanthocyanidin B2, as an activator of the Nrf2-Keap1 pathway, synergistically strengthens the intestinal epithelial barrier with cinnamaldehyde metabolites; the selenium-enriched yeast culture provides highly bioavailable organic selenium, which participates in the composition of glutathione peroxidase in the form of selenoproteins; the glycoterpenoids regulate the hypothalamic-pituitary-adrenal axis to reduce cortisol levels; and the trehalose, as a molecular chaperone, protects the native conformation of proteins under stress conditions. Together, these three components construct a multidimensional anti-stress network of organic selenium-cortisol regulation-molecular chaperones.

Images