Animal microecological preparation with anti-stress function and its preparation method

Abstract

The application provides an animal microecological preparation with stress resistance and a preparation method thereof, and belongs to the technical field of microecological preparations, and comprises the following steps: culturing Bacillus subtilis and lactic acid bacteria to obtain Bacillus subtilis seed liquid and lactic acid bacteria seed liquid, carrying out aerobic fermentation on the Bacillus subtilis seed liquid, and adding the lactic acid bacteria seed liquid to carry out micro-aerobic fermentation; culturing Saccharomyces cerevisiae to obtain yeast seed liquid; after the fermentation product and the yeast seed liquid are mixed, centrifugation and freeze-drying are sequentially carried out to obtain freeze-dried bacterial powder; the freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, yeast beta-glucan and deionized water are mixed to obtain a core solution; after the core solution and a sodium alginate solution are mixed, the mixture is dropped into a calcium chloride solution to carry out cross-linking, the mixture is soaked in a chitosan solution, and then the mixture is soaked in a trisodium phosphate solution to carry out cross-linking, and finally, the mixture is subjected to spray drying. Through the composite embedding of multi-strain synergistic fermentation and functional components, the animal microecological preparation is improved in the stress resistance.

Description

Technical Field

[0001] This invention belongs to the field of microecological preparation technology, and relates to an animal microecological preparation with anti-stress function and its preparation method. Background Technology

[0002] Animals are prone to stress responses during transportation, regrouping, and sudden environmental changes, leading to decreased growth performance, weakened immunity, and even death. Traditional anti-stress methods often rely on chemical drugs or single probiotic preparations. However, chemical drugs pose risks of residue and drug resistance, while conventional probiotic preparations are difficult to effectively regulate animal stress states due to low bacterial activity, limited functionality, or poor intestinal colonization. Current technologies have attempted to enhance product function through synergistic effects of complex microbial communities or metabolite enrichment, but bottlenecks remain in strain compatibility design, activity protection, and large-scale production processes, limiting practical application effectiveness.

[0003] Currently, optimization of the preparation process for microecological preparations mainly focuses on controlling fermentation conditions or improving encapsulation technology. For example, multi-stage fermentation can be used to enhance cell proliferation or metabolite synthesis, or microencapsulation can be employed to improve cell tolerance. However, such methods often face problems such as competitive inhibition of bacterial strains, high loss of viable cells during drying, or insufficient stability of functional components. In addition, existing encapsulation materials and processes have limited adaptability to complex bacterial communities, making it difficult to simultaneously meet the dual requirements of sustained cell release and resistance to gastric acid and bile corrosion, resulting in unsatisfactory targeted release of the preparations in the animal digestive tract.

[0004] With the upgrading of animal health and breeding demands, developing a product that combines highly effective anti-stress functions with long-lasting microecological regulation has become a key focus in the industry. Current technologies have not yet effectively integrated functional strain screening, metabolic pathway optimization, and delivery system design, particularly lacking systematic solutions for the integration of solid-state and liquid-state fermentation processes and the synergistic maintenance of multi-strain activity. Therefore, there is an urgent need to explore a controllable, cost-effective, and scalable preparation method to overcome the current technical barriers to the application of microecological preparations in anti-stress applications. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an animal microecological preparation with anti-stress function and its preparation method. Through multi-strain synergistic fermentation and complex encapsulation of functional components, the anti-stress function of the animal microecological preparation is enhanced. The final animal microecological preparation not only contains highly active complex flora but also integrates multiple anti-stress functional factors. Through multiple mechanisms of action, such as regulating intestinal flora balance, enhancing antioxidant capacity, maintaining mucosal barrier, and activating immune response, it comprehensively improves the animal's resistance and adaptability to stress factors.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing an animal microecological preparation with anti-stress function, the preparation method comprising:

[0008] (I) Bacillus subtilis and lactic acid bacteria were cultured to obtain Bacillus seed liquid and lactic acid bacteria seed liquid. The Bacillus seed liquid was subjected to aerobic fermentation, and then the lactic acid bacteria seed liquid was added for microaerobic fermentation to obtain fermentation products.

[0009] (II) The yeast seed liquid was obtained by culturing the brewing yeast. The fermentation product was mixed with the yeast seed liquid and then centrifuged and freeze-dried in sequence to obtain freeze-dried yeast powder.

[0010] (III) The freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, yeast β-glucan and deionized water were mixed to obtain a core material solution. The core material solution was mixed with sodium alginate solution and then dropped into calcium chloride solution for cross-linking to obtain composite microspheres.

[0011] (IV) The composite microspheres are immersed in chitosan solution, then removed and immersed in sodium tripolyphosphate solution for cross-linking, and spray-dried to obtain the animal microecological preparation.

[0012] The method for preparing animal microecological preparations provided by this invention enhances the anti-stress function of animal microecological preparations through multi-strain synergistic fermentation and complex encapsulation of functional components. The resulting animal microecological preparations not only contain highly active complex flora but also integrate multiple anti-stress functional factors. Through multiple mechanisms of action, such as regulating intestinal flora balance, enhancing antioxidant capacity, maintaining mucosal barrier, and activating immune response, the preparations comprehensively improve the animal's resistance and adaptability to stress factors.

[0013] The composition of different bacterial strains optimized the anti-stress effect of metabolites during the fermentation stage. Bacillus subtilis preferentially utilizes nutrients in the fermentation substrate during aerobic fermentation, rapidly proliferating and secreting various enzymes. The produced proteases and amylases promote the breakdown of large nutrient molecules into smaller molecules, providing an easily usable carbon source for subsequent lactic acid bacteria. Simultaneously, the lactic acid environment produced by lactic acid bacteria metabolism promotes the formation of stress-resistant spores by Bacillus subtilis, resulting in a synergistic effect between the two. Furthermore, lactic acid bacteria produce organic acids and antimicrobial peptides under microaerobic conditions, which not only inhibit the proliferation of intestinal pathogens but also regulate intestinal acid-base balance, improving digestive dysfunction in animals under stress. Through the complementary metabolic effects of Bacillus subtilis and lactic acid bacteria, the fermentation products simultaneously contain active bacteria and multiple metabolites. When animals encounter stress, this allows for the rapid replenishment of beneficial bacteria and direct regulation of the intestinal microenvironment through the metabolites. The introduction of Saccharomyces cerevisiae further enriches the diversity of the microbial community. The active substances in its metabolites can enhance the barrier function of intestinal epithelial cells and reduce the risk of increased intestinal permeability caused by stress. At the same time, the late addition of Saccharomyces cerevisiae avoids competitive inhibition with Bacillus, and its rich B vitamins and amino acids further enhance the nutritional support function of the preparation.

[0014] The synergistic effect of prebiotics in the core material with functional components such as chlorogenic acid, L-glutamine, and yeast β-glucan enhances the anti-stress effect of the animal microecological preparation. Fructooligosaccharides and galactooligosaccharides, as bifidus factors, selectively promote the colonization and proliferation of beneficial bacteria, forming a functional complement to the live bacteria in the freeze-dried bacterial powder. Chlorogenic acid, as a natural antioxidant, effectively scavenges excess free radicals generated in animals due to stress, reducing the impact of oxidative damage on cells. L-glutamine maintains the integrity of the intestinal mucosa structure, preventing stress-induced intestinal mucosal atrophy, while yeast β-glucan enhances the animal's immune response under stress by activating the activity of immune cells. The synergistic effect of these components in the core material produces multiple anti-stress effects, ranging from antioxidant and mucosal protection to immune regulation.

[0015] This invention employs a two-stage cross-linking process using sodium alginate and chitosan. By encapsulating the active ingredients in the core material, a double-layered core-shell structure is formed, significantly improving the stability and targeting of animal microecological preparations. The first cross-linking, through the cross-linking of sodium alginate and calcium ions, forms a three-dimensional network gel structure with controllable pore size, effectively encapsulating the active ingredients in the core material. This protects the activity of heat-sensitive components during freeze-drying and allows for control of the release rate through pore size design, preventing damage to bacteria and functional substances from the acidic gastric environment. The second cross-linking, through the cross-linking of chitosan and sodium tripolyphosphate, not only enhances the mechanical strength of the composite microspheres but also utilizes the mucosal adhesion properties of chitosan to prolong the retention time of the animal microecological preparation in the intestine. The solubility characteristics of the outer layer of chitosan under specific pH conditions in the intestine ensure the precise release of the active ingredients in the core material at the target site. The double-layer encapsulation structure enables the gradient release of active substances. Initially, the outermost chitosan layer slowly dissolves in the weakly alkaline intestinal fluid, releasing some functional components, while the inner calcium alginate layer continuously releases probiotics and active substances, forming a sustained anti-stress effect. This effectively solves the technical problems of traditional microecological preparations being inactivated in the acidic environment of the stomach and insufficiently released in the intestine, allowing the anti-stress components to continue to play a role in the animal's body.

[0016] It should be noted that the cultivation process of Bacillus subtilis and lactic acid bacteria in step (I) is disclosed in the prior art. The present invention does not make specific requirements or special limitations on this. It is sufficient to refer to the commonly used cultivation methods in the field, which generally include: rehydrating and activating the bacterial powder to obtain a bacterial suspension, inoculating the bacterial suspension into a liquid culture medium at a certain volume ratio, placing it in a shaker for heating and shaking culture, and obtaining a seed liquid.

[0017] Optionally, the culture process of Bacillus subtilis includes:

[0018] Bacillus subtilis lyophilized powder was rehydrated and activated in sterile physiological saline to obtain a Bacillus suspension. The Bacillus suspension was then inoculated into LB liquid medium and cultured with shaking to obtain a Bacillus seed culture.

[0019] For example, the ratio of Bacillus subtilis lyophilized powder to sterile physiological saline is 0.1g:(8~12)mL, such as 0.1g:8mL, 0.1g:8.5mL, 0.1g:9mL, 0.1g:9.5mL, 0.1g:10mL, 0.1g:10.5mL, 0.1g:11mL, 0.1g:11.5mL or 0.1g:12mL, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0020] For example, the rehydration activation temperature of Bacillus subtilis lyophilized powder is 36~37℃, such as 36℃, 36.1℃, 36.2℃, 36.3℃, 36.4℃, 36.5℃, 36.6℃, 36.7℃, 36.8℃, 36.9℃ or 37℃, but is not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0021] For example, the standing time for rehydration and activation of Bacillus subtilis lyophilized powder is 10-15 min, such as 10 min, 10.5 min, 11 min, 11.5 min, 12 min, 12.5 min, 13 min, 13.5 min, 14 min, 14.5 min or 15 min, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0022] For example, the volume of the Bacillus subtilis suspension is 2.5 to 3.5% of the volume of LB liquid culture medium, such as 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4% or 3.5%, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0023] For example, the shaking culture temperature of Bacillus subtilis is 36~37℃, such as 36℃, 36.1℃, 36.2℃, 36.3℃, 36.4℃, 36.5℃, 36.6℃, 36.7℃, 36.8℃, 36.9℃ or 37℃, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0024] For example, the shaking culture time of Bacillus subtilis is 18 to 20 hours, such as 18 hours, 18.2 hours, 18.4 hours, 18.6 hours, 18.8 hours, 19 hours, 19.2 hours, 19.4 hours, 19.6 hours, 19.8 hours, or 20 hours, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0025] For example, the shaking rate for Bacillus subtilis shaking culture is 230~250 rpm, such as 230 rpm, 232 rpm, 234 rpm, 236 rpm, 238 rpm, 240 rpm, 242 rpm, 244 rpm, 246 rpm, 248 rpm or 250 rpm, but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0026] Optionally, the cultivation process of lactic acid bacteria includes:

[0027] Lactic acid bacteria lyophilized powder was rehydrated and activated in sterile physiological saline to obtain a lactic acid bacteria suspension. The lactic acid bacteria suspension was then inoculated into MRS liquid medium and cultured with shaking to obtain a lactic acid bacteria seed culture.

[0028] For example, the lyophilized lactic acid bacteria powder includes lyophilized Lactobacillus plantarum powder and lyophilized Lactobacillus casei powder.

[0029] For example, the mass ratio of Lactobacillus plantarum freeze-dried powder to Lactobacillus casei freeze-dried powder is (2~3):1, for example, it can be 2.0:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1 or 3.0:1, but is not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0030] For example, the ratio of lyophilized lactic acid bacteria powder to sterile physiological saline is 0.1g:(8~12)mL, such as 0.1g:8mL, 0.1g:8.5mL, 0.1g:9mL, 0.1g:9.5mL, 0.1g:10mL, 0.1g:10.5mL, 0.1g:11mL, 0.1g:11.5mL or 0.1g:12mL, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0031] For example, the rehydration activation temperature of the lyophilized lactic acid bacteria powder is 36~37℃, such as 36℃, 36.1℃, 36.2℃, 36.3℃, 36.4℃, 36.5℃, 36.6℃, 36.7℃, 36.8℃, 36.9℃ or 37℃, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0032] For example, the standing time for rehydration and activation of the lyophilized lactic acid bacteria powder is 10-15 min, such as 10 min, 10.5 min, 11 min, 11.5 min, 12 min, 12.5 min, 13 min, 13.5 min, 14 min, 14.5 min or 15 min, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0033] For example, the volume of the lactic acid bacteria suspension is 2 to 3% of the volume of the MRS liquid culture medium, such as 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9% or 3.0%, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0034] For example, the temperature for shaking culture of lactic acid bacteria is 36~37℃, such as 36℃, 36.1℃, 36.2℃, 36.3℃, 36.4℃, 36.5℃, 36.6℃, 36.7℃, 36.8℃, 36.9℃ or 37℃, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0035] For example, the shaking culture time for lactic acid bacteria is 20-22 hours, such as 20 hours, 20.2 hours, 20.4 hours, 20.6 hours, 20.8 hours, 21 hours, 21.2 hours, 21.4 hours, 21.6 hours, 21.8 hours or 22 hours, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0036] For example, the shaking rate for the shaking culture of lactic acid bacteria is 100~120 rpm, such as 100 rpm, 102 rpm, 104 rpm, 106 rpm, 108 rpm, 110 rpm, 112 rpm, 114 rpm, 116 rpm, 118 rpm or 120 rpm, but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0037] As a preferred technical solution of the present invention, in step (I), a fermentation substrate is placed in the fermentation tank, and the Bacillus seed liquid is introduced into the fermentation tank for aerobic fermentation.

[0038] In some alternative examples, the fermentation substrate consists of wheat bran, soybean meal, magnesium sulfate, and deionized water.

[0039] In some optional instances, the amount of magnesium sulfate added is 0.1 to 0.2 wt% of the total mass of the bran and soybean meal, for example, it may be 0.1 wt%, 0.11 wt%, 0.12 wt%, 0.13 wt%, 0.14 wt%, 0.15 wt%, 0.16 wt%, 0.17 wt%, 0.18 wt%, 0.19 wt%, or 0.2 wt%, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0040] This invention incorporates magnesium sulfate into the fermentation substrate. Magnesium sulfate primarily serves as an essential inorganic salt source for microbial growth during fermentation, and its magnesium ions play a crucial regulatory role in microbial metabolic activities. Magnesium is an activator of various enzymes, particularly playing a key role in energy metabolism-related ATPases and nucleic acid synthase systems. In the aerobic fermentation stage of Bacillus subtilis, appropriate amounts of magnesium ions promote cell wall stability and transmembrane transport efficiency, accelerating substrate decomposition and cell proliferation. In the microaerobic fermentation process of lactic acid bacteria, magnesium ions participate in the regulation of lactate dehydrogenase activity, directly affecting the efficiency of organic acid synthesis. Furthermore, sulfate ions, as a sulfur source, participate in the synthesis of sulfur-containing amino acids, providing a basic element for microbial protein metabolism and indirectly influencing the composition of metabolites.

[0041] This invention specifically limits the amount of magnesium sulfate added to 0.1-0.2 wt% of the total mass of wheat bran and soybean meal. When the amount of magnesium sulfate added is less than 0.1 wt%, the supply of magnesium required for bacterial metabolism is insufficient, leading to a decrease in the enzyme activity of Bacillus subtilis and a reduction in the efficiency of organic acid synthesis by lactic acid bacteria. This results in a decrease in the types of functional metabolites in the fermentation products and a slowdown in bacterial proliferation, affecting the fermentation process. When the amount of magnesium sulfate added exceeds 0.2 wt%, excess magnesium ions will combine with phytic acid in the substrate to form insoluble substances, reducing the utilization rate of trace elements. High concentrations of sulfate ions will also interfere with transmembrane transport of bacteria, inhibit the proton gradient maintenance ability of lactic acid bacteria, and cause osmotic pressure imbalance, leading to premature spore formation and a decrease in the proportion of active bacteria.

[0042] It should be noted that the process parameters used in this invention, such as the basic carbon and nitrogen sources of wheat bran and soybean meal as the fermentation substrate, the range of their mass ratio, the moisture content of the fermentation substrate, and the inoculation amount of the seed liquid, are already common choices recognized by those skilled in the art in the field of microbial solid-state fermentation. Therefore, this invention does not limit the technical parameters such as the mass ratio of wheat bran to soybean meal, the moisture content of the fermentation substrate, and the inoculation amount of the seed liquid as essential technical features in the claims.

[0043] For example, the mass ratio of wheat bran to soybean meal is (1~2):1, such as 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1 or 2.0:1, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0044] For example, the moisture content of the fermentation substrate is 50-60 wt%, such as 50 wt%, 51 wt%, 52 wt%, 53 wt%, 54 wt%, 55 wt%, 56 wt%, 57 wt%, 58 wt%, 59 wt%, or 60 wt%, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0045] For example, the inoculation amount of Bacillus seed solution is 0.1 to 0.3 mL of Bacillus seed solution per gram of dry fermentation substrate (total mass of wheat bran, soybean meal, and magnesium sulfate). For example, it can be 0.1 mL, 0.12 mL, 0.14 mL, 0.16 mL, 0.18 mL, 0.2 mL, 0.22 mL, 0.24 mL, 0.26 mL, 0.28 mL, or 0.3 mL, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0046] The mass ratio of wheat bran to soybean meal, the moisture content of the fermentation substrate, and the seed liquid inoculation amount described in the specification are supplementary to the complete implementation of the technical solution. They are intended to ensure that those skilled in the art can fully understand and fully implement the technical solution through exemplary descriptions of specific values, rather than to impose interpretative limitations on the claims or additional constraints on the scope of protection.

[0047] In the specific implementation process, those skilled in the art can make adaptive adjustments to the carbon and nitrogen source ratio, the moisture content of the fermentation substrate, and the seed liquid inoculation amount according to the actual conditions such as strain characteristics, production equipment, and regional resources. For example, the mass ratio of wheat bran to soybean meal can be expanded to a wider range of 0.8:1 to 2.5:1, or the moisture content can be adjusted to a flexible range of 45 to 65 wt% according to the humidity control performance of the fermenter. In fact, other carbon and nitrogen sources such as corn flour and rapeseed meal can be used to partially replace wheat bran or soybean meal, as long as the substitutes can still meet the basic nutritional needs of Bacillus subtilis and lactic acid bacteria for growth and metabolism.

[0048] As a preferred technical solution of the present invention, in step (I), during the aerobic fermentation process, the air flow rate into the fermentation tank is 1~1.5vvm, for example, it can be 1.0vvm, 1.05vvm, 1.1vvm, 1.15vvm, 1.2vvm, 1.25vvm, 1.3vvm, 1.35vvm, 1.4vvm, 1.45vvm or 1.5vvm, but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0049] In some optional instances, the aerobic fermentation stirring rate is 150 to 200 rpm, for example, 150 rpm, 155 rpm, 160 rpm, 165 rpm, 170 rpm, 175 rpm, 180 rpm, 185 rpm, 190 rpm, 195 rpm or 200 rpm, but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0050] In some optional instances, the aerobic fermentation time is 20 to 30 hours, for example, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, or 30 hours, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0051] In some optional instances, during the aerobic fermentation process, the pH of the Bacillus seed liquid is controlled at 6.5 to 6.8, for example, 6.5, 6.55, 6.6, 6.65, 6.7, 6.75 or 6.8, but not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0052] In the aerobic fermentation process of Bacillus, if it is necessary to stabilize the pH value within the range of 6.5 to 6.8, a suitable pH adjuster needs to be selected according to the properties of the metabolites. This invention does not impose specific requirements or limitations on the type and concentration of the pH adjuster. Recommended alkaline adjusters include sodium hydroxide solution with a concentration of 0.1 to 1 mol / L or ammonia solution with a concentration of 5 to 10 wt%. Recommended acidic adjusters include phosphoric acid solution with a concentration of 0.1 to 0.5 mol / L. Of course, those skilled in the art using other types or concentrations of pH adjusters not mentioned above also fall within the scope of protection of this invention.

[0053] In some optional instances, the volume ratio of the lactic acid bacteria seed culture to the Bacillus seed culture is 1:(0.8~1.2), for example, it can be 1:0.8, 1:0.85, 1:0.9, 1:0.95, 1:1.0, 1:1.05, 1:1.1, 1:1.15 or 1:1.2, but is not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0054] As a preferred technical solution of the present invention, in step (I), during the microaerobic fermentation process, the air flow rate into the fermentation tank is 0.2~0.4 vvm, for example, it can be 0.2 vvm, 0.22 vvm, 0.24 vvm, 0.26 vvm, 0.28 vvm, 0.3 vvm, 0.32 vvm, 0.34 vvm, 0.36 vvm, 0.38 vvm or 0.4 vvm, but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0055] In some optional instances, the stirring rate of the microaerobic fermentation is 80 to 100 rpm, for example, 80 rpm, 82 rpm, 84 rpm, 86 rpm, 88 rpm, 90 rpm, 92 rpm, 94 rpm, 96 rpm, 98 rpm or 100 rpm, but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0056] In some optional instances, the microaerobic fermentation time is 30 to 40 hours, for example, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, or 40 hours, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0057] In some optional instances, during the microaerobic fermentation process, the pH value of the mixed seed liquid in the fermenter is controlled at 5.5 to 6.0, for example, it can be 5.5, 5.55, 5.6, 5.65, 5.7, 5.75, 5.8, 5.85, 5.9, 5.95 or 6.0, but is not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0058] In the microaerobic fermentation process of lactic acid bacteria, if it is necessary to stabilize the pH value within the range of 5.5 to 6.0, a suitable pH adjuster needs to be selected according to the properties of the metabolites. This invention does not impose specific requirements or limitations on the type and concentration of the pH adjuster. Recommended alkaline adjusters include sodium hydroxide solution with a concentration of 0.1 to 1 mol / L or ammonia solution with a concentration of 5 to 10 wt%. Recommended acidic adjusters include phosphoric acid solution with a concentration of 0.1 to 0.5 mol / L. Of course, those skilled in the art using other types or concentrations of pH adjusters not mentioned above also fall within the scope of protection of this invention.

[0059] It should be noted that the fermentation temperatures for aerobic and microaerobic fermentation have been disclosed in the prior art, and this invention does not make specific requirements or special limitations on them.

[0060] For example, the temperature for aerobic fermentation is 36~38℃, such as 36℃, 36.2℃, 36.4℃, 36.6℃, 36.8℃, 37℃, 37.2℃, 37.4℃, 37.6℃, 37.8℃ or 38℃, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0061] For example, the temperature for microaerobic fermentation is 36~38℃, such as 36℃, 36.2℃, 36.4℃, 36.6℃, 36.8℃, 37℃, 37.2℃, 37.4℃, 37.6℃, 37.8℃ or 38℃, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0062] It should be noted that the cultivation process of brewing yeast in step (II) is disclosed in the prior art. The present invention does not make specific requirements or special limitations on this. It can refer to the cultivation methods commonly used in the field, which generally include: rehydrating and activating the yeast powder to obtain a bacterial suspension, inoculating the bacterial suspension into a liquid culture medium at a certain volume ratio, placing it in a shaker for heating and shaking culture, and obtaining a seed culture.

[0063] For example, the present invention provides a specific cultivation process for brewing yeast, as follows:

[0064] Saccharomyces cerevisiae powder was added to sterile physiological saline and stirred at low speed to complete rehydration and activation, resulting in a yeast suspension. The yeast suspension was then inoculated into YPD liquid culture medium and cultured by shaking to obtain yeast seed culture.

[0065] In some optional instances, the ratio of brewer's yeast powder to sterile physiological saline is 0.1g:(18~22)mL, for example, it can be 0.1g:18mL, 0.1g:18.5mL, 0.1g:19mL, 0.1g:19.5mL, 0.1g:20mL, 0.1g:20.5mL, 0.1g:21mL, 0.1g:21.5mL or 0.1g:22mL, but is not limited to the values ​​listed, other unlisted values ​​within this range are also applicable.

[0066] In some optional instances, the rehydration activation temperature of brewing yeast powder is 30~32℃, for example, 30℃, 30.2℃, 30.4℃, 30.6℃, 30.8℃, 31℃, 31.2℃, 31.4℃, 31.6℃, 31.8℃ or 32℃, but is not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0067] In some optional instances, the brewing yeast powder and sterile saline are stirred at a low speed of 80 to 120 rpm, for example, 80 rpm, 85 rpm, 90 rpm, 95 rpm, 100 rpm, 105 rpm, 110 rpm, 115 rpm or 120 rpm, but not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0068] In some optional instances, the brewing yeast powder is stirred at low speed with sterile physiological saline for 10 to 15 minutes, for example, 10 minutes, 10.5 minutes, 11 minutes, 11.5 minutes, 12 minutes, 12.5 minutes, 13 minutes, 13.5 minutes, 14 minutes, 14.5 minutes or 15 minutes, but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0069] In some optional instances, the inoculum of yeast suspension is 3.5 to 4.5% of the volume of YPD liquid medium, for example, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4% or 4.5%, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0070] In some optional instances, the temperature for shaking culture of Saccharomyces cerevisiae is 30–32°C, for example, 30°C, 30.2°C, 30.4°C, 30.6°C, 30.8°C, 31°C, 31.2°C, 31.4°C, 31.6°C, 31.8°C, or 32°C, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0071] In some optional instances, the shaking culture time for Saccharomyces cerevisiae is 28 to 30 hours, for example, 28 hours, 28.2 hours, 28.4 hours, 28.6 hours, 28.8 hours, 29 hours, 29.2 hours, 29.4 hours, 29.6 hours, 29.8 hours, or 30 hours, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0072] In some optional instances, the shaking rate for the shaking culture of Saccharomyces cerevisiae is 100 to 150 rpm, for example, 100 rpm, 105 rpm, 110 rpm, 115 rpm, 120 rpm, 125 rpm, 130 rpm, 135 rpm, 140 rpm, 145 rpm or 150 rpm, but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0073] It should be noted that the inventors have provided specific operational steps for the cultivation processes of lactic acid bacteria, Bacillus, and Saccharomyces cerevisiae, including culture medium preparation, strain activation, and fermentation condition control, as well as detailed descriptions of multiple process parameters such as temperature, pH value, rotation speed, and inoculation ratio. These contents serve as supporting descriptions of the technical solutions, and their purpose is to fully demonstrate the complete implementation path of the technical solutions to those skilled in the art, ensuring that the sufficiency requirements of patent disclosure are met.

[0074] In practice, those skilled in the art can adapt the culture parameters according to the characteristics of the strain, equipment conditions, production scale, and other actual needs. For example, they can use a gradient heating strategy to optimize the accumulation of metabolites, adjust the carbon-nitrogen ratio to increase cell density, or even use novel bioreactors to change the dissolved oxygen control method. As long as the culture scheme after such technical adjustments still contains the core features explicitly defined in the claims, the resulting technical solution, regardless of whether it fully adopts the operating parameters described in the specification, should be considered to fall within the protection scope of this invention.

[0075] As a preferred technical solution of the present invention, in step (II), the volume ratio of the fermentation product to the yeast seed liquid is 1:(0.2~0.4), for example, it can be 1:0.2, 1:0.22, 1:0.24, 1:0.26, 1:0.28, 1:0.3, 1:0.32, 1:0.34, 1:0.36, 1:0.38 or 1:0.4, but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0076] It should be noted that, preferably, before mixing the fermentation product with the yeast seed culture, a phosphoric acid solution is added dropwise to adjust the pH value to 5.3-5.7. This pH range is suitable for the optimal metabolic activity of Saccharomyces cerevisiae in a slightly acidic environment, improving the survival rate of subsequent freeze-drying by maintaining cell membrane integrity and energy metabolism efficiency. It also buffers the acid shock introduced by the lactic acid bacteria fermentation product, avoiding the inhibition of yeast function by the system pH being too low. In addition, this pH range can meet the pH requirements of the subsequent sodium alginate cross-linking process, preventing the gel strength of sodium alginate from decreasing at too low a pH and avoiding the loss of control over the calcium ion cross-linking rate at too high a pH. This ensures the uniformity of the microsphere structure and the sustained-release characteristics of the functional components, ultimately achieving synergistic effects between the anti-stress components and the probiotics in the animal intestinal environment.

[0077] In some optional instances, trehalose is added to the mixture of the fermentation product and the yeast seed culture, wherein the amount of trehalose added is 1 to 2 wt% of the total mass of the fermentation product and the yeast seed culture, for example, 1.0 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, or 2.0 wt%, but is not limited to the values ​​listed, and other unlisted values ​​within this range are also applicable.

[0078] In some optional instances, the centrifugation speed is 3000~4000 rpm, for example, it can be 3000 rpm, 3100 rpm, 3200 rpm, 3300 rpm, 3400 rpm, 3500 rpm, 3600 rpm, 3700 rpm, 3800 rpm, 3900 rpm or 4000 rpm, but is not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0079] In some optional instances, the centrifugation time is 30 to 40 minutes, for example, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes or 40 minutes, but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0080] In some optional instances, the freeze-drying process includes sequential pre-freezing and sublimation drying.

[0081] In some alternative instances, the pre-freezing temperature is -35 to -40°C, for example, it can be -35°C, -35.5°C, -36°C, -36.5°C, -37°C, -37.5°C, -38°C, -38.5°C, -39°C, -39.5°C, or -40°C, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0082] In some optional instances, the pre-freezing time is 4 to 6 hours, for example, 4.0 hours, 4.2 hours, 4.4 hours, 4.6 hours, 4.8 hours, 5.0 hours, 5.2 hours, 5.4 hours, 5.6 hours, 5.8 hours, or 6.0 hours, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0083] In some alternative instances, the sublimation drying temperature is -25 to -30°C, for example, -25°C, -25.5°C, -26°C, -26.5°C, -27°C, -27.5°C, -28°C, -28.5°C, -29°C, -29.5°C, or -30°C, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0084] In some optional instances, the sublimation drying time is 18 to 24 hours, for example, 18 hours, 18.5 hours, 19 hours, 19.5 hours, 20 hours, 20.5 hours, 21 hours, 21.5 hours, 22 hours, 22.5 hours, 23 hours, 23.5 hours, or 24 hours, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0085] As a preferred embodiment of the present invention, in step (III), the mass ratio of the freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, and yeast β-glucan is 100:(14~16):(0.8~1.2):(1.8~2):(1.3~1.5), for example, 100:14:0.8:1.8:1.3, 100:14.2:0.85:1.82:1.32, 100:14.4:0.9:1.84:1.34, 100:14.6:0.95:1.86: 1.36, 100:14.8:1:1.88:1.38, 100:15:1.05:1.9:1.4, 100:15.2:1.1:1.92:1.42, 100:15.4:1.15:1.94:1.44, 100:15.6:1.2:1.95:1.46, 100:15.8:1:1.98:1.48, or 100:16:1.1:2:1.5, but not limited to the listed values; other unlisted values ​​within this range also apply.

[0086] As a carrier of active microorganisms, freeze-dried probiotic powder exerts a fundamental anti-stress effect by maintaining the balance of intestinal flora. The Bacillus subtilis, lactic acid bacteria, and yeast it contains form complementary functions: Bacillus subtilis produces digestive enzymes to promote nutrient absorption, lactic acid bacteria inhibit the excessive proliferation of pathogenic bacteria, and yeast metabolites enhance the intestinal barrier. The fructooligosaccharides and galactooligosaccharides in the prebiotics can stimulate the specific proliferation of beneficial bacteria, activating the active flora in the freeze-dried powder. Chlorogenic acid reduces oxidative stress damage to the intestinal mucosa by scavenging free radicals, while its moderate antibacterial activity can regulate the flora structure without affecting the activity of probiotics. L-glutamine, as the main energy source for intestinal epithelial cells, works with the metabolites of probiotics to maintain the intestinal physical barrier. Yeast β-glucan enhances the systemic immune response by activating macrophages and lymphocytes, forming an immune-microbial regulatory mechanism with local flora regulation.

[0087] To achieve the ideal anti-stress effect, this invention specifically limits the mass ratio of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, and yeast β-glucan to 100:(14~16):(0.8~1.2):(1.8~2):(1.3~1.5). When the mass ratio of these active components is within this range, it can ensure the colonization advantage of probiotics and balance the functions of antioxidation, mucosal repair, and immune regulation, avoiding the metabolic burden caused by excessive action of a single function.

[0088] When the proportion of prebiotics is below the lower limit defined in this invention, it is difficult to provide sufficient specific carbon source to support the activation and proliferation of the bacterial community in the freeze-dried bacterial powder. In particular, Lactobacillus and Bifidobacterium, which are sensitive to oligosaccharides, will experience a decrease in metabolic activity, causing the rate of bacterial community reconstruction to lag behind the stress response process. When the proportion of prebiotics exceeds the upper limit defined in this invention, excessive undigested oligosaccharides will change the osmotic pressure of the intestinal contents, triggering osmotic diarrhea. At the same time, high concentrations of oligosaccharides will competitively inhibit the activity of some enzymes, thus weakening the synergistic effect between the various strains in the freeze-dried bacterial powder.

[0089] When the proportion of chlorogenic acid is below the lower limit of the range defined in this invention, its antioxidant capacity cannot effectively neutralize the excess free radicals generated by stress. The permeability of intestinal epithelial cells increases under oxidative damage, indirectly affecting the colonization environment of probiotics. When the proportion of chlorogenic acid exceeds the upper limit of the range defined in this invention, the concentration of phenolic substances reaches the antibacterial threshold, particularly inhibiting the metabolic activity of lactic acid bacteria, resulting in a decrease in the production of short-chain fatty acids in the fermentation products. Furthermore, high concentrations of chlorogenic acid can also complex with metal ions, reducing the bioavailability of components such as L-glutamine.

[0090] When the proportion of L-glutamine is below the lower limit of the range defined in this invention, the repair rate of intestinal villi is lower than the rate of mucosal damage caused by stress, affecting the adhesion and colonization efficiency of probiotics on the intestinal wall. When the proportion of L-glutamine exceeds the upper limit of the range defined in this invention, excess glutamine is decomposed by microorganisms to produce byproducts such as ammonia, increasing the metabolic burden on the liver; at the same time, excessive L-glutamine will over-activate cell proliferation through the mTOR pathway, thereby inducing abnormal proliferation of intestinal epithelium and weakening the selective permeability regulation function of the intestinal barrier.

[0091] When the proportion of yeast β-glucan is below the lower limit of the range defined in this invention, the immunomodulatory effect is insufficient to effectively activate innate immune cells, resulting in a disconnect between systemic immunity and local intestinal immune response. When the proportion of yeast β-glucan exceeds the upper limit of the range defined in this invention, it overstimulates the immune system, triggering excessive secretion of pro-inflammatory factors, which exacerbates the inflammatory response during periods of stress sensitivity.

[0092] In some optional instances, the prebiotic includes fructooligosaccharides and galactooligosaccharides in a mass ratio of (0.8~1.2):1, for example, 0.8:1, 0.85:1, 0.9:1, 0.95:1, 1.0:1, 1.05:1, 1.1:1, 1.15:1 or 1.2:1, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0093] In some optional instances, the total mass fraction of the freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, and yeast β-glucan in the core material solution is 10 to 12 wt%, for example, it can be 10 wt%, 10.2 wt%, 10.4 wt%, 10.6 wt%, 10.8 wt%, 11 wt%, 11.2 wt%, 11.4 wt%, 11.6 wt%, 11.8 wt%, or 12 wt%, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0094] In some optional instances, the core material solution also includes a thermal protectant, which includes skim milk powder, trehalose, and monosodium glutamate.

[0095] In some optional instances, the mass ratio of the skim milk powder, trehalose, and monosodium glutamate is (2~3):(1~2):1, for example, it can be 2:1:1, 2.1:1.1:1, 2.2:1.2:1, 2.3:1.3:1, 2.4:1.4:1, 2.5:1.5:1, 2.6:1.6:1, 2.7:1.7:1, 2.8:1.8:1, 2.9:1.9:1, or 3:2:1, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0096] This invention incorporates a thermal protectant into the core material solution. The thermal protectant includes skim milk powder, trehalose, and monosodium glutamate (MSG). The skim milk powder primarily acts as a protein matrix support; its whey and casein proteins form a network structure during drying, providing a physical protective barrier for the bacteria and active ingredients. The hydrophobic regions of the milk proteins adhere to the bacterial surface through intermolecular forces, reducing the mechanical stress on the cell membrane during water evaporation. Simultaneously, the lactose in the skim milk powder works synergistically with trehalose to buffer the high-temperature shock in the subsequent spray drying stage, absorbing some heat energy through a reversible thermal denaturation process, thus preventing irreversible damage to the bacterial proteins. The sodium ions of MSG regulate the osmotic pressure gradient, helping the bacteria maintain a gradual shrinkage of cell volume during dehydration, preventing membrane rupture caused by sudden osmotic pressure changes. Furthermore, the glutamate anions form ionic interactions with the amino groups of the milk proteins, preventing bacterial aggregation due to surface charge imbalance during drying.

[0097] In some optional examples, the amount of heat protectant added is 25 to 35 wt% of the total mass of the freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine and yeast β-glucan, for example, it can be 25 wt%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, 30 wt%, 31 wt%, 32 wt%, 33 wt%, 34 wt% or 35 wt%, but is not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0098] As a preferred technical solution of the present invention, in step (III), the core material solution is mixed with sodium alginate solution to obtain a mixed solution, sucrose ester is added to the mixed solution, and then high-speed stirring and emulsification are carried out to obtain a mixed emulsion. The mixed emulsion is dropped into the calcium chloride solution to form gel balls, and the gel balls are soaked in calcium chloride solution for cross-linking to obtain the composite microspheres.

[0099] In some optional examples, the amount of sucrose ester added is 0.3 to 0.8 wt% of the mass of the mixed solution, for example, 0.3 wt%, 0.35 wt%, 0.4 wt%, 0.45 wt%, 0.5 wt%, 0.55 wt%, 0.6 wt%, 0.65 wt%, 0.7 wt%, 0.75 wt%, or 0.8 wt%, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0100] In some alternative examples, the volume ratio of the core material solution to the sodium alginate solution is 1:(1.8~2.3), for example, it can be 1:1.8, 1:1.85, 1:1.9, 1:1.95, 1:2.0, 1:2.05, 1:2.1, 1:2.15, 1:2.2, 1:2.25 or 1:2.3, but is not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0101] This invention specifically limits the volume ratio of the core material solution to the sodium alginate solution to 1:(1.8~2.3). Within this range, the sodium alginate solution provides appropriate viscosity, and the resulting mixture with the core material solution exhibits good fluidity, facilitating the subsequent gelation process. Furthermore, the sodium alginate molecular chains can form a uniform three-dimensional network structure upon calcium ion crosslinking. Within this range, the porosity of the gel shell is moderate, ensuring both the mechanical strength of the composite microsphere shell, resisting the erosion of the gastric acid environment and protecting the core material components, and maintaining appropriate swelling characteristics in the alkaline environment of the intestine, without excessively hindering the subsequent release of internal active substances.

[0102] When the proportion of sodium alginate solution is below the lower limit defined in this invention, the viscosity of the mixed solution is insufficient, making it difficult to form composite microspheres. The resulting gel shell is too thin and loosely structured, easily disintegrating prematurely in the acidic environment of the stomach, causing premature exposure and inactivation of the bacteria and active ingredients in the core material. Simultaneously, the surface roughness of the composite microspheres increases, making them prone to adhesion during spray drying. When the proportion of sodium alginate solution exceeds the upper limit defined in this invention, the resulting gel shell structure becomes too dense, severely hindering effective release into the intestinal environment. Furthermore, excessive sodium alginate residue interferes with the uniform adhesion of subsequent chitosan encapsulation, and the high viscosity of the mixed solution increases the difficulty of atomization during spray drying, resulting in inconsistent particle sizes in the final animal microecological preparation.

[0103] In some optional instances, the sodium alginate solution has a mass fraction of 1 to 5 wt%, for example, it may be 1.0 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, 3.0 wt%, 3.5 wt%, 4.0 wt%, 4.5 wt%, or 5.0 wt%, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0104] In some optional instances, the stirring speed is 6000~8000 rpm, for example, it can be 6000 rpm, 6200 rpm, 6400 rpm, 6600 rpm, 6800 rpm, 7000 rpm, 7200 rpm, 7400 rpm, 7600 rpm, 7800 rpm or 8000 rpm, but is not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0105] In some optional instances, the stirring time is 5 to 10 minutes, for example, 5.0 minutes, 5.5 minutes, 6.0 minutes, 6.5 minutes, 7.0 minutes, 7.5 minutes, 8.0 minutes, 8.5 minutes, 9.0 minutes, 9.5 minutes, or 10.0 minutes, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0106] In some optional instances, the calcium chloride solution has a mass fraction of 0.5 to 1.5 wt%, for example, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, or 1.5 wt%, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0107] In some optional instances, the gel balls are soaked in the calcium chloride solution for 30 to 40 minutes, for example, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes or 40 minutes, but are not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0108] As a preferred technical solution of the present invention, in step (IV), the mass fraction of chitosan in the chitosan solution is 0.5~1.5wt%, for example, it can be 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1.0wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt% or 1.5wt%, but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0109] It should be noted that, in this invention, chitosan is preferably dissolved in a 0.5-1.5 wt% acetic acid solution and mixed evenly to obtain a chitosan solution. The purpose of using acetic acid solution to dissolve chitosan is to promote the protonation of amino groups in the chitosan molecular chain to form cationic properties through a weak acid environment, effectively destroying its crystal structure to achieve full dissolution, while avoiding excessive hydrolysis of molecular chains or damage to cross-linking functional sites caused by strong acid.

[0110] This invention specifically limits the chitosan mass fraction in the chitosan solution to 0.5~1.5wt%. The concentration of the chitosan solution directly determines the adsorption amount of chitosan molecules on the surface of the microspheres and the compactness of the coating structure. When the concentration of the chitosan solution is within the range specified in this invention, the chitosan molecules can stably bind to the composite microspheres through electrostatic interaction to form a moderately loose primary coating, providing sufficient and uniformly distributed amino active sites for the subsequent cross-linking reaction of sodium tripolyphosphate, thereby realizing the construction of an ionic cross-linking network. This structure can ensure the strong binding between the chitosan layer and the calcium alginate layer, and maintain the strength and sustained-release performance of the animal microecological preparation.

[0111] When the chitosan solution has a mass fraction of less than 0.5 wt%, the number of chitosan segments in the solution is insufficient, making it difficult to form a complete and continuous coating layer on the surface of the composite microspheres. This results in some areas lacking effective reaction sites during subsequent sodium tripolyphosphate crosslinking, leading to local defects in the final bilayer embedded structure, which is prone to premature disintegration in the intestinal environment. When the chitosan solution has a mass fraction of more than 1.5 wt%, the high viscosity of the solution hinders the uniform spreading of chitosan molecules on the surface of the composite microspheres. The resulting chitosan coating is too thick and dense, which not only restricts the penetration and crosslinking of sodium tripolyphosphate ions but also excessively seals the surface pores of the composite microspheres, delaying the release rate of the embedded active ingredients in the intestine.

[0112] In some optional instances, the composite microspheres are soaked in the chitosan solution for 0.5 to 1.5 hours, for example, 0.5 hours, 0.6 hours, 0.7 hours, 0.8 hours, 0.9 hours, 1.0 hours, 1.1 hours, 1.2 hours, 1.3 hours, 1.4 hours, or 1.5 hours, but are not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0113] In some optional instances, the sodium tripolyphosphate solution has a mass fraction of 0.3 to 0.8 wt%, for example, 0.3 wt%, 0.35 wt%, 0.4 wt%, 0.45 wt%, 0.5 wt%, 0.55 wt%, 0.6 wt%, 0.65 wt%, 0.7 wt%, 0.75 wt%, or 0.8 wt%, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0114] In some optional instances, the composite microspheres are soaked in the sodium tripolyphosphate solution for 15 to 25 minutes, for example, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes or 25 minutes, but are not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0115] The bilayer encapsulation of sodium alginate and chitosan significantly improves the anti-stress function of the probiotic preparation. The inner sodium alginate layer, through a gel network formed by calcium ion cross-linking, creates a physical barrier in the acidic gastric environment, effectively protecting the active bacteria and heat-sensitive components in the freeze-dried bacterial powder. This significantly improves the survival rate of probiotics under acidic conditions, ensuring a sufficient number of live bacteria enter the intestines to exert their effects. The outer chitosan layer utilizes its cationic properties to electrostatically adsorb the anionic components of the intestinal mucus layer, prolonging the residence time of the probiotic preparation in the intestines and creating more opportunities for probiotic colonization and the release of functional components.

[0116] As a preferred technical solution of the present invention, in step (IV), the inlet air temperature of the spray drying is 40~45℃, for example, it can be 40℃, 40.5℃, 41℃, 41.5℃, 42℃, 42.5℃, 43℃, 43.5℃, 44℃, 44.5℃ or 45℃, but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0117] In some optional instances, the outlet air temperature of the spray dryer is 30~35°C, for example, it can be 30°C, 30.5°C, 31°C, 31.5°C, 32°C, 32.5°C, 33°C, 33.5°C, 34°C, 34.5°C or 35°C, but is not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0118] Secondly, the present invention provides an animal microecological preparation with anti-stress function prepared by the preparation method described in the first aspect.

[0119] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0120] The method for preparing animal microecological preparations provided by this invention enhances the anti-stress function of animal microecological preparations through multi-strain synergistic fermentation and complex encapsulation of functional components. The resulting animal microecological preparations not only contain highly active complex flora but also integrate multiple anti-stress functional factors. Through multiple mechanisms of action, such as regulating intestinal flora balance, enhancing antioxidant capacity, maintaining mucosal barrier, and activating immune response, the preparations comprehensively improve the animal's resistance and adaptability to stress factors. Attached Figure Description

[0121] Figure 1 This is a process flow diagram of the preparation of animal microecological preparations with anti-stress function provided in Examples 1-5 of the present invention;

[0122] Figure 2 Infrared curves of sodium alginate, chitosan, and the animal microecological preparation obtained in Example 1 of this invention;

[0123] Figure 3 This is a graph showing the cumulative release rate of the active ingredients of the animal microecological preparation obtained in Example 1 of the present invention over time under different pH conditions. Detailed Implementation

[0124] The technical solutions of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings. The embodiments described herein are specific implementations of the present invention, used to illustrate the concept of the present invention; these descriptions are explanatory and exemplary, and should not be construed as limiting the implementation methods or the scope of protection of the present invention. In addition to the embodiments described herein, those skilled in the art can employ other obvious technical solutions based on the content disclosed in the claims and specification of this application. These technical solutions include those that make any obvious substitutions and modifications to the embodiments described herein.

[0125] Example 1

[0126] This embodiment provides a method for preparing an animal microecological preparation with anti-stress function, such as... Figure 1 As shown, the preparation method specifically includes the following steps:

[0127] (1) Add Bacillus subtilis lyophilized powder to sterile physiological saline. The ratio of Bacillus subtilis lyophilized powder to sterile physiological saline is 0.1g:8mL. After standing at 36℃ for 15min to complete rehydration and activation, a Bacillus suspension is obtained. The Bacillus suspension is inoculated into LB liquid medium. The volume of the Bacillus subtilis suspension is 2.5% of the volume of LB liquid medium. Then, it is placed in a shaker and cultured at a culture temperature of 36℃ and a shaking rate of 230rpm for 20h to obtain Bacillus seed liquid.

[0128] Lyophilized lactic acid bacteria powder (including lyophilized Lactobacillus plantarum powder and lyophilized Lactobacillus casei powder in a mass ratio of 2:1) was added to sterile physiological saline at a ratio of 0.1 g: 8 mL. The mixture was allowed to stand at 36 °C for 15 min to complete rehydration and activation, resulting in a lactic acid bacteria suspension. The lactic acid bacteria suspension was then inoculated into MRS liquid medium at a volume of 2% of the MRS liquid medium. The medium was then placed in a shaker and cultured at 36 °C and 100 rpm for 22 h to obtain a lactic acid bacteria seed culture.

[0129] A fermentation substrate consisting of wheat bran, soybean meal, magnesium sulfate, and deionized water was placed in a fermentation tank. The mass ratio of wheat bran to soybean meal was 1:1, and the amount of magnesium sulfate added was 0.1 wt% of the total mass of wheat bran and soybean meal. The moisture content of the fermentation substrate was 50 wt%.

[0130] Bacillus seed culture was introduced into the fermenter. 0.1 mL of Bacillus seed culture was inoculated per gram of dry fermentation substrate (total mass of bran, soybean meal, and magnesium sulfate). Air was introduced into the fermenter for aerobic fermentation at a flow rate of 1 vvm, a fermentation temperature of 36℃, a fermentation time of 30 h, and a stirring rate of 200 rpm. During the aerobic fermentation process, the pH of the Bacillus seed culture was controlled at 6.5.

[0131] Subsequently, lactic acid bacteria seed liquid was added to the fermenter, with a volume ratio of lactic acid bacteria seed liquid to Bacillus seed liquid of 1:0.8. Air was introduced into the fermenter for micro-aerobic fermentation at a flow rate of 0.2 vvm, a fermentation temperature of 36℃, a fermentation time of 40 h, and a stirring rate of 100 rpm. During the micro-aerobic fermentation, the pH value of the mixed seed liquid in the fermenter was controlled at 5.5. After the fermentation was completed, the fermentation product was obtained.

[0132] (2) Add the brewing yeast powder to sterile physiological saline. The ratio of brewing yeast powder to sterile physiological saline is 0.1g:18mL. Stir for 15min at 30℃ and 80rpm to complete the rehydration and activation, and obtain the yeast suspension.

[0133] Yeast suspension was inoculated into YPD liquid medium at a volume of 3.5% of the YPD liquid medium. The medium was then placed in a shaker and cultured at 30°C and 100 rpm for 30 h to obtain yeast seed culture.

[0134] Phosphoric acid solution was added dropwise to the yeast seed culture to adjust its pH to 5.3. Then, the fermentation product and the pH-adjusted yeast seed culture were mixed evenly at a volume ratio of 1:0.2 to obtain a mixture. Trehalose was added to the mixture at a rate of 1 wt% of the total mass of the fermentation product and the yeast seed culture. The mixture was then centrifuged at 3000 rpm for 40 min. The precipitate after centrifugation was pre-frozen at -35℃ for 6 h, and then sublimated and dried at -25℃ for 24 h to obtain freeze-dried yeast powder.

[0135] (3) A core material solution was obtained by mixing freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, yeast β-glucan and deionized water. The mass ratio of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine and yeast β-glucan was 100:14:0.8:1.8:1.3. The prebiotics included fructooligosaccharides and galactooligosaccharides in a mass ratio of 0.8:1. The total mass fraction of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine and yeast β-glucan in the core material solution was 10 wt%.

[0136] The core material solution also includes a heat protectant, which is composed of skim milk powder, trehalose, and monosodium glutamate in a mass ratio of 2:1:1. The amount of heat protectant added is 25 wt% of the total mass of the freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, and yeast β-glucan.

[0137] A mixed solution was obtained by mixing the core material solution with a 1 wt% sodium alginate solution, with a volume ratio of 1:2.3. Sucrose ester was added to the mixed solution at a mass of 0.3 wt%. The mixture was then stirred and emulsified at 6000 rpm for 10 min to obtain a mixed emulsion. The mixed emulsion was then dropped into a 0.5 wt% calcium chloride solution to form gel spheres. The gel spheres were then soaked in the calcium chloride solution for 40 min to crosslink, resulting in composite microspheres.

[0138] (4) Chitosan was dissolved in a 0.5 wt% acetic acid solution and mixed evenly to obtain a chitosan solution with a chitosan mass fraction of 0.5 wt%. The composite microspheres were soaked in the chitosan solution for 1.5 h, then taken out and soaked in a 0.3 wt% sodium tripolyphosphate solution for 25 min. Finally, they were spray dried. The inlet air temperature of the spray drying was 40 ℃ and the outlet air temperature was 30 ℃. The animal microecological preparation was obtained after spray drying.

[0139] Figure 2 The figures show the infrared curves of sodium alginate, chitosan, and the animal microecological preparation obtained in this example. As can be seen from the figures, in the infrared curve of sodium alginate, 1598 cm⁻¹... -1 and 1407cm -1 The characteristic peaks at 1100 cm⁻¹ belong to the symmetric and asymmetric stretching vibrations of -COO, respectively. -1 ~1000cm -1 The characteristic peaks within this range belong to the stretching vibration of C=O. In the infrared curve of chitosan, the peak at 1640 cm⁻¹... -1 and 1586cm -1 The characteristic peaks at 1378 cm⁻¹ belong to the tensile vibration of C=O and the bending vibration of NH₄⁺ in the amide, respectively. -1 and 1066cm -1 The characteristic peaks at 1151 cm⁻¹ belong to the tensile vibrations of CN and COC, respectively. -1 The characteristic peak at 1538 cm⁻¹ belongs to the bending vibration of NH in secondary amines. In the infrared curves of animal microecological preparations, not only characteristic peaks of sodium alginate and chitosan appeared, but also those at 1538 cm⁻¹. -1 New absorption peaks also appeared, mainly originating from protonated amino groups (-NH3). + The bending vibration of sodium alginate and the vibration frequency shift of carboxylate (-COO-) indicate that sodium alginate and chitosan form a stable composite structure through ionic cross-linking, effectively encapsulating the active components of the core material.

[0140] Figure 3The graph shows the cumulative release rate of the active ingredients in the animal microecological preparation prepared in this embodiment under different pH environments over time. As can be seen from the graph, in an environment of pH=1.2 (mimicking gastric acid), the active ingredients in the core material are rapidly released within 0-2 hours. This is because, during the preparation of the microecological preparation, a small amount of the active ingredients in the core material were not completely encapsulated and remained on the surface of the microspheres. These free components rapidly dissolved and diffused upon contact with gastric acid, leading to a sharp increase in the initial release rate. Within 3-8 hours, the release rate of the active ingredients in the core material decreased significantly. This is because the outer layer of chitosan protonated and contracted, and the pores of calcium alginate closed, limiting the further release of the active ingredients in the core material. After 8 hours, the release rate of the active ingredients in the core material reached a plateau. This is because the sodium alginate-chitosan double-layer encapsulation structure was completely sealed, with only a very small amount of small molecules leaking out.

[0141] In an environment with pH=6.8 (simulating intestinal fluid), the release rate of the active ingredients in the core material is low within 0-2 hours, due to the partial dissolution of the outer chitosan layer, while the calcium alginate has not yet swelled. Within 3-6 hours, the active ingredients in the core material are in a burst release phase, with pH triggering the disintegration of the calcium alginate network, and probiotics and functional components rapidly escaping. Within 7-12 hours, the release rate of the active ingredients in the core material decreases again, entering a slow-release phase, with the residual components of the core material released through a dissolution mechanism. After 12 hours, the release of the active ingredients in the core material ceases, and the outer encapsulation material is completely degraded.

[0142] Example 2

[0143] This embodiment provides a method for preparing an animal microecological preparation with anti-stress function, such as... Figure 1 As shown, the preparation method specifically includes the following steps:

[0144] (1) Add Bacillus subtilis lyophilized powder to sterile physiological saline. The ratio of Bacillus subtilis lyophilized powder to sterile physiological saline is 0.1g:9mL. After standing at 36.2℃ for 13min to complete rehydration and activation, a Bacillus suspension is obtained. The Bacillus suspension is inoculated into LB liquid medium. The volume of Bacillus subtilis suspension is 2.8% of the volume of LB liquid medium. Then, it is placed in a shaker and cultured at a culture temperature of 36.2℃ and a shaking rate of 235rpm for 19.5h to obtain Bacillus seed liquid.

[0145] Lyophilized lactic acid bacteria powder (including lyophilized Lactobacillus plantarum powder and lyophilized Lactobacillus casei powder in a mass ratio of 2.2:1) was added to sterile physiological saline at a ratio of 0.1 g: 9 mL. The mixture was allowed to stand at 36.2 °C for 13 min to complete rehydration and activation, resulting in a lactic acid bacteria suspension. The lactic acid bacteria suspension was then inoculated into MRS liquid medium at a volume of 2.2% of the MRS liquid medium volume. The medium was then placed in a shaker and cultured at 36.2 °C and a shaking rate of 105 rpm for 21.5 h to obtain a lactic acid bacteria seed culture.

[0146] A fermentation substrate was placed in the fermentation tank. The fermentation substrate consisted of wheat bran, soybean meal, magnesium sulfate, and deionized water. The mass ratio of wheat bran to soybean meal was 1.2:1. The amount of magnesium sulfate added was 0.12 wt% of the total mass of wheat bran and soybean meal. The moisture content of the fermentation substrate was 52 wt%.

[0147] Bacillus seed culture was introduced into the fermenter. 0.15 mL of Bacillus seed culture was inoculated per gram of dry fermentation substrate (total mass of bran, soybean meal, and magnesium sulfate). Air was introduced into the fermenter for aerobic fermentation at a flow rate of 1.2 vvm, a fermentation temperature of 36.5℃, a fermentation time of 28 h, and a stirring rate of 180 rpm. During the aerobic fermentation process, the pH of the Bacillus seed culture was controlled at 6.6.

[0148] Subsequently, lactic acid bacteria seed liquid was added to the fermenter, with a volume ratio of lactic acid bacteria seed liquid to Bacillus seed liquid of 1:0.9. Air was introduced into the fermenter for microaerobic fermentation at a flow rate of 0.25 vvm, a fermentation temperature of 36.5℃, a fermentation time of 38 h, and a stirring rate of 95 rpm. During the microaerobic fermentation process, the pH value of the mixed seed liquid in the fermenter was controlled at 5.6. After the fermentation was completed, the fermentation product was obtained.

[0149] (2) Add the brewing yeast powder to sterile physiological saline. The ratio of brewing yeast powder to sterile physiological saline is 0.1g:19mL. Stir for 13min at 30.5℃ and 90rpm to complete the rehydration and activation, and obtain the yeast suspension.

[0150] Yeast suspension was inoculated into YPD liquid medium at a volume of 3.8% of the YPD liquid medium. The medium was then placed in a shaker and cultured at a temperature of 30.5°C and a shaking rate of 110 rpm for 29.5 h to obtain yeast seed culture.

[0151] Phosphoric acid solution was added dropwise to the yeast seed culture to adjust its pH to 5.4. Then, the fermentation product and the pH-adjusted yeast seed culture were mixed evenly at a volume ratio of 1:0.25 to obtain a mixture. Trehalose was added to the mixture at an amount of 1.2 wt% of the total mass of the fermentation product and the yeast seed culture. The mixture was then centrifuged at 3200 rpm for 38 min. The precipitate after centrifugation was pre-frozen at -36℃ for 5.5 h, and then sublimated and dried at -26℃ for 22 h to obtain freeze-dried yeast powder.

[0152] (3) A core material solution was obtained by mixing freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, yeast β-glucan, and deionized water. The mass ratio of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, and yeast β-glucan was 100:14.5:0.9:1.85:1.35. The prebiotics included fructooligosaccharides and galactooligosaccharides in a mass ratio of 0.9:1. The total mass fraction of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, and yeast β-glucan in the core material solution was 10.5 wt%.

[0153] The core material solution also includes a heat protectant, which consists of skim milk powder, trehalose, and monosodium glutamate in a mass ratio of 2.2:1.2:1. The amount of heat protectant added is 28 wt% of the total mass of the freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, and yeast β-glucan.

[0154] A mixed solution was obtained by mixing the core material solution with a sodium alginate solution of 2 wt% by mass, with a volume ratio of 1:2.2. Sucrose ester was added to the mixed solution at a mass of 0.4 wt%. The mixture was then stirred and emulsified at 6500 rpm for 8 min to obtain a mixed emulsion. The mixed emulsion was then dropped into a calcium chloride solution of 0.8 wt% by mass to form gel spheres. The gel spheres were then soaked in the calcium chloride solution for 38 min to crosslink and obtain composite microspheres.

[0155] (4) Chitosan was dissolved in 0.8 wt% acetic acid solution and mixed evenly to obtain chitosan solution. The mass fraction of chitosan in chitosan solution was 0.8 wt%. The composite microspheres were soaked in chitosan solution for 1.2 h, then taken out and soaked in sodium tripolyphosphate solution with a mass fraction of 0.4 wt% for 22 min. Finally, they were spray dried. The inlet air temperature of spray drying was 42 ℃ and the outlet air temperature was 32 ℃. The animal microecological preparation was obtained after spray drying.

[0156] Example 3

[0157] This embodiment provides a method for preparing an animal microecological preparation with anti-stress function, such as... Figure 1As shown, the preparation method specifically includes the following steps:

[0158] (1) Add Bacillus subtilis lyophilized powder to sterile physiological saline. The ratio of Bacillus subtilis lyophilized powder to sterile physiological saline is 0.1g:10mL. After standing at 36.5℃ for 12min to complete rehydration and activation, a Bacillus suspension is obtained. The Bacillus suspension is inoculated into LB liquid medium. The volume of Bacillus subtilis suspension is 3% of the volume of LB liquid medium. Then, it is placed in a shaker and cultured at a culture temperature of 36.5℃ and a shaking rate of 240rpm for 19h to obtain Bacillus seed liquid.

[0159] The lyophilized lactic acid bacteria powder (including lyophilized Lactobacillus plantarum powder and lyophilized Lactobacillus casei powder in a mass ratio of 2.5:1) was added to sterile physiological saline at a ratio of 0.1 g:10 mL. The mixture was allowed to stand at 36.5 °C for 12 min to complete rehydration and activation, resulting in a lactic acid bacteria suspension. The lactic acid bacteria suspension was then inoculated into MRS liquid medium at a volume of 2.5% of the MRS liquid medium volume. The medium was then placed in a shaker and cultured at 36.5 °C and 110 rpm for 21 h to obtain a lactic acid bacteria seed culture.

[0160] A fermentation substrate consisting of wheat bran, soybean meal, magnesium sulfate, and deionized water was placed in a fermentation tank. The mass ratio of wheat bran to soybean meal was 1.5:1, and the amount of magnesium sulfate added was 0.15 wt% of the total mass of wheat bran and soybean meal. The moisture content of the fermentation substrate was 55 wt%.

[0161] Bacillus seed culture was introduced into the fermenter. 0.2 mL of Bacillus seed culture was inoculated per gram of dry fermentation substrate (total mass of bran, soybean meal, and magnesium sulfate). Air was introduced into the fermenter for aerobic fermentation at a flow rate of 1.3 vvm, a fermentation temperature of 37℃, a fermentation time of 25 h, and a stirring rate of 170 rpm. During the aerobic fermentation process, the pH of the Bacillus seed culture was controlled at 6.6.

[0162] Subsequently, lactic acid bacteria seed liquid was added to the fermenter, with a volume ratio of lactic acid bacteria seed liquid to Bacillus seed liquid of 1:1. Air was introduced into the fermenter for micro-aerobic fermentation at a flow rate of 0.3 vvm, a fermentation temperature of 37℃, a fermentation time of 35 h, and a stirring rate of 90 rpm. During the micro-aerobic fermentation process, the pH value of the mixed seed liquid in the fermenter was controlled at 5.7. After the fermentation was completed, the fermentation product was obtained.

[0163] (2) Add the brewing yeast powder to sterile physiological saline. The ratio of brewing yeast powder to sterile physiological saline is 0.1g:20mL. Stir for 12min at 31℃ and 100rpm to complete the rehydration and activation, and obtain the yeast suspension.

[0164] Yeast suspension was inoculated into YPD liquid medium at a volume of 4% of the YPD liquid medium. The medium was then placed in a shaker and cultured at 31°C and 120 rpm for 29 hours to obtain yeast seed culture.

[0165] Phosphoric acid solution was added dropwise to the yeast seed culture to adjust its pH to 5.5. Then, the fermentation product and the pH-adjusted yeast seed culture were mixed evenly at a volume ratio of 1:0.3 to obtain a mixture. Trehalose was added to the mixture at an amount of 1.5 wt% of the total mass of the fermentation product and the yeast seed culture. The mixture was then centrifuged at 3500 rpm for 35 min. The precipitate after centrifugation was pre-frozen at -37℃ for 5 h, and then sublimated and dried at -27℃ for 22 h to obtain freeze-dried yeast powder.

[0166] (3) A core material solution was obtained by mixing freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, yeast β-glucan and deionized water. The mass ratio of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine and yeast β-glucan was 100:15:1:1.9:1.4. The prebiotics included fructooligosaccharides and galactooligosaccharides in a mass ratio of 1:1. The total mass fraction of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine and yeast β-glucan in the core material solution was 11 wt%.

[0167] The core material solution also includes a heat protectant, which consists of skim milk powder, trehalose, and monosodium glutamate in a mass ratio of 2.5:1.5:1. The amount of heat protectant added is 30 wt% of the total mass of the freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, and yeast β-glucan.

[0168] A mixed solution was obtained by mixing the core material solution with a 3 wt% sodium alginate solution, with a volume ratio of 1:2. Sucrose ester was added to the mixed solution at a mass of 0.5 wt%. The mixture was then emulsified by high-speed stirring at 7000 rpm for 7 min to obtain a mixed emulsion. The mixed emulsion was then dropped into a 1 wt% calcium chloride solution to form gel spheres. The gel spheres were then soaked in the calcium chloride solution for 35 min to crosslink and obtain composite microspheres.

[0169] (4) Chitosan is dissolved in 1 wt% acetic acid solution and mixed evenly to obtain chitosan solution. The mass fraction of chitosan in chitosan solution is 1 wt%. The composite microspheres are soaked in chitosan solution for 1 h, then taken out and soaked in sodium tripolyphosphate solution with a mass fraction of 0.5 wt% for 20 min. Finally, they are spray dried. The inlet air temperature of spray drying is 43 ℃ and the outlet air temperature is 33 ℃. The animal microecological preparation is obtained after spray drying.

[0170] Example 4

[0171] This embodiment provides a method for preparing an animal microecological preparation with anti-stress function, such as... Figure 1 As shown, the preparation method specifically includes the following steps:

[0172] (1) Add Bacillus subtilis lyophilized powder to sterile physiological saline. The ratio of Bacillus subtilis lyophilized powder to sterile physiological saline is 0.1g:11mL. After standing at 36.8℃ for 11min to complete rehydration and activation, a Bacillus suspension is obtained. The Bacillus suspension is inoculated into LB liquid medium. The volume of the Bacillus subtilis suspension is 3.2% of the volume of LB liquid medium. Then, it is placed in a shaker and cultured at a culture temperature of 36.8℃ and a shaking rate of 245rpm for 18.5h to obtain Bacillus seed liquid.

[0173] Lyophilized lactic acid bacteria powder (including lyophilized Lactobacillus plantarum powder and lyophilized Lactobacillus casei powder in a mass ratio of 2.8:1) was added to sterile physiological saline at a ratio of 0.1 g:11 mL. The mixture was allowed to stand at 36.8 °C for 11 min to complete rehydration and activation, resulting in a lactic acid bacteria suspension. The lactic acid bacteria suspension was then inoculated into MRS liquid medium at a volume of 2.8% of the MRS liquid medium volume. The medium was then placed in a shaker and cultured at 36.8 °C and a shaking rate of 115 rpm for 20.5 h to obtain a lactic acid bacteria seed culture.

[0174] A fermentation substrate consisting of wheat bran, soybean meal, magnesium sulfate, and deionized water was placed in a fermentation tank. The mass ratio of wheat bran to soybean meal was 1.8:1, and the amount of magnesium sulfate added was 0.18 wt% of the total mass of wheat bran and soybean meal. The moisture content of the fermentation substrate was 58 wt%.

[0175] Bacillus seed culture was introduced into the fermenter. 0.25 mL of Bacillus seed culture was inoculated per gram of dry fermentation substrate (total mass of bran, soybean meal, and magnesium sulfate). Air was introduced into the fermenter for aerobic fermentation at a flow rate of 1.4 vvm, a fermentation temperature of 37.5℃, a fermentation time of 22 h, and a stirring rate of 160 rpm. During the aerobic fermentation process, the pH of the Bacillus seed culture was controlled at 6.7.

[0176] Subsequently, lactic acid bacteria seed liquid was added to the fermenter, with a volume ratio of lactic acid bacteria seed liquid to Bacillus seed liquid of 1:1.1. Air was introduced into the fermenter for micro-aerobic fermentation at a flow rate of 0.35 vvm, a fermentation temperature of 37.5℃, a fermentation time of 32 h, and a stirring rate of 85 rpm. During the micro-aerobic fermentation, the pH value of the mixed seed liquid in the fermenter was controlled at 5.8. After the fermentation was completed, the fermentation product was obtained.

[0177] (2) Add the brewing yeast powder to sterile physiological saline. The ratio of brewing yeast powder to sterile physiological saline is 0.1g:21mL. Stir for 11min at 31.5℃ and 110rpm to complete the rehydration and activation, and obtain the yeast suspension.

[0178] The yeast suspension was inoculated into YPD liquid medium at a volume of 4.2% of the YPD liquid medium. The medium was then placed in a shaker and cultured at a temperature of 31.5℃ and a shaking rate of 130 rpm for 28.5 h to obtain the yeast seed culture.

[0179] Phosphoric acid solution was added dropwise to the yeast seed culture to adjust its pH to 5.6. Then, the fermentation product and the pH-adjusted yeast seed culture were mixed evenly at a volume ratio of 1:0.35 to obtain a mixture. Trehalose was added to the mixture at an amount of 1.8 wt% of the total mass of the fermentation product and the yeast seed culture. The mixture was then centrifuged at 3800 rpm for 32 min. The precipitate after centrifugation was pre-frozen at -38℃ for 4.5 h, and then sublimated and dried at -28℃ for 20 h to obtain freeze-dried yeast powder.

[0180] (3) A core material solution was obtained by mixing freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, yeast β-glucan, and deionized water. The mass ratio of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, and yeast β-glucan was 100:15.5:1.1:1.95:1.45. The prebiotics included fructooligosaccharides and galactooligosaccharides in a mass ratio of 1.2:1. The total mass fraction of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, and yeast β-glucan in the core material solution was 11.5 wt%.

[0181] The core material solution also includes a heat protectant, which consists of skim milk powder, trehalose, and monosodium glutamate in a mass ratio of 2.8:1.8:1. The amount of heat protectant added is 32 wt% of the total mass of the freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, and yeast β-glucan.

[0182] A mixed solution was obtained by mixing the core material solution with a sodium alginate solution with a mass fraction of 4 wt%, and the volume ratio of the core material solution to the sodium alginate solution was 1:1.9. Sucrose ester was added to the mixed solution at a mass fraction of 0.6 wt%. The mixture was then stirred and emulsified at 7500 rpm for 6 min to obtain a mixed emulsion. The mixed emulsion was then dropped into a calcium chloride solution with a mass fraction of 1.2 wt% to form gel spheres. The gel spheres were then soaked in the calcium chloride solution for 32 min to crosslink and obtain composite microspheres.

[0183] (4) Chitosan was dissolved in a 1.2 wt% acetic acid solution and mixed evenly to obtain a chitosan solution with a chitosan mass fraction of 1.2 wt%. The composite microspheres were soaked in the chitosan solution for 0.8 h, then taken out and soaked in a 0.6 wt% sodium tripolyphosphate solution for 18 min. Finally, they were spray dried. The inlet air temperature of the spray drying was 44 ℃ and the outlet air temperature was 34 ℃. The animal microecological preparation was obtained after spray drying.

[0184] Example 5

[0185] This embodiment provides a method for preparing an animal microecological preparation with anti-stress function, such as... Figure 1 As shown, the preparation method specifically includes the following steps:

[0186] (1) Add Bacillus subtilis lyophilized powder to sterile physiological saline. The ratio of Bacillus subtilis lyophilized powder to sterile physiological saline is 0.1g:12mL. After standing at 37℃ for 10min to complete rehydration and activation, a Bacillus suspension is obtained. The Bacillus suspension is inoculated into LB liquid medium. The volume of the Bacillus subtilis suspension is 3.5% of the volume of LB liquid medium. Then, it is placed in a shaker and cultured at 37℃ and 250rpm for 18h to obtain Bacillus seed liquid.

[0187] The lyophilized lactic acid bacteria powder (including lyophilized Lactobacillus plantarum powder and lyophilized Lactobacillus casei powder in a mass ratio of 3:1) was added to sterile physiological saline at a ratio of 0.1 g: 12 mL. The mixture was allowed to stand at 37 °C for 10 min to complete rehydration and activation, resulting in a lactic acid bacteria suspension. The lactic acid bacteria suspension was then inoculated into MRS liquid medium at a volume of 3% of the MRS liquid medium volume. The medium was then placed in a shaker and cultured at 37 °C and a shaking rate of 120 rpm for 20 h to obtain a lactic acid bacteria seed culture.

[0188] A fermentation substrate consisting of wheat bran, soybean meal, magnesium sulfate, and deionized water was placed in a fermentation tank. The mass ratio of wheat bran to soybean meal was 2:1, and the amount of magnesium sulfate added was 0.2 wt% of the total mass of wheat bran and soybean meal. The moisture content of the fermentation substrate was 60 wt%.

[0189] Bacillus seed culture was introduced into the fermenter. 0.3 mL of Bacillus seed culture was inoculated per gram of dry fermentation substrate (total mass of bran, soybean meal, and magnesium sulfate). Air was introduced into the fermenter for aerobic fermentation at a flow rate of 1.5 vvm, a fermentation temperature of 38℃, a fermentation time of 20 h, and a stirring rate of 150 rpm. During the aerobic fermentation process, the pH of the Bacillus seed culture was controlled at 6.8.

[0190] Subsequently, lactic acid bacteria seed liquid was added to the fermenter, with a volume ratio of lactic acid bacteria seed liquid to Bacillus seed liquid of 1:1.2. Air was introduced into the fermenter for micro-aerobic fermentation at a flow rate of 0.4 vvm, a fermentation temperature of 38℃, a fermentation time of 30 h, and a stirring rate of 80 rpm. During the micro-aerobic fermentation, the pH value of the mixed seed liquid in the fermenter was controlled at 6.0. After the fermentation was completed, the fermentation product was obtained.

[0191] (2) Add the brewing yeast powder to sterile physiological saline. The ratio of brewing yeast powder to sterile physiological saline is 0.1g:22mL. Stir for 10min at 32℃ and 120rpm to complete the rehydration and activation, and obtain the yeast suspension.

[0192] Yeast suspension was inoculated into YPD liquid medium at a volume of 4.5% of the YPD liquid medium volume. The medium was then placed in a shaker and cultured at 32°C and 150 rpm for 28 hours to obtain yeast seed culture.

[0193] Phosphoric acid solution was added dropwise to the yeast seed culture to adjust its pH to 5.7. Then, the fermentation product and the pH-adjusted yeast seed culture were mixed evenly at a volume ratio of 1:0.4 to obtain a mixture. Trehalose was added to the mixture at an amount of 2 wt% of the total mass of the fermentation product and the yeast seed culture. The mixture was then centrifuged at 4000 rpm for 30 min. The precipitate after centrifugation was pre-frozen at -40℃ for 4 h, and then sublimated and dried at -30℃ for 18 h to obtain freeze-dried yeast powder.

[0194] (3) A core material solution was obtained by mixing freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, yeast β-glucan and deionized water. The mass ratio of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine and yeast β-glucan was 100:16:1.2:2:1.5. The prebiotics included fructooligosaccharides and galactooligosaccharides in a mass ratio of 1.2:1. The total mass fraction of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine and yeast β-glucan in the core material solution was 12 wt%.

[0195] The core material solution also includes a heat protectant, which is composed of skim milk powder, trehalose, and monosodium glutamate in a mass ratio of 3:2:1. The amount of heat protectant added is 35 wt% of the total mass of the freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, and yeast β-glucan.

[0196] A mixed solution was obtained by mixing the core material solution with a 5 wt% sodium alginate solution, with a volume ratio of 1:1.8. Sucrose ester was added to the mixed solution at a mass of 0.8 wt%. The mixture was then stirred and emulsified at 8000 rpm for 5 min to obtain a mixed emulsion. The mixed emulsion was then dropped into a 1.5 wt% calcium chloride solution to form gel spheres. The gel spheres were then soaked in the calcium chloride solution for 30 min to crosslink and obtain composite microspheres.

[0197] (4) Chitosan was dissolved in a 1.5 wt% acetic acid solution and mixed evenly to obtain a chitosan solution with a chitosan mass fraction of 1.5 wt%. The composite microspheres were soaked in the chitosan solution for 0.5 h, then taken out and soaked in a 0.8 wt% sodium tripolyphosphate solution for 15 min. Finally, they were spray dried. The inlet air temperature of the spray drying was 45 ℃ and the outlet air temperature was 35 ℃. The animal microecological preparation was obtained after spray drying.

[0198] Comparative Example 1

[0199] This comparative example provides a method for preparing an animal microecological preparation with anti-stress function. The difference from Example 1 is that the mass ratio of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine and yeast β-glucan is adjusted to 100:10:0.8:1.8:1.3. Other process parameters and operating steps are exactly the same as in Example 1.

[0200] Comparative Example 2

[0201] This comparative example provides a method for preparing an animal microecological preparation with anti-stress function. The difference from Example 1 is that the mass ratio of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine and yeast β-glucan is adjusted to 100:20:0.8:1.8:1.3. Other process parameters and operating steps are exactly the same as in Example 1.

[0202] Comparative Example 3

[0203] This comparative example provides a method for preparing an animal microecological preparation with anti-stress function. The difference from Example 1 is that the mass ratio of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine and yeast β-glucan is adjusted to 100:14:0.5:1.8:1.3. Other process parameters and operating steps are exactly the same as in Example 1.

[0204] Comparative Example 4

[0205] This comparative example provides a method for preparing an animal microecological preparation with anti-stress function. The difference from Example 1 is that the mass ratio of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine and yeast β-glucan is adjusted to 100:14:1.5:1.8:1.3. Other process parameters and operating steps are exactly the same as in Example 1.

[0206] Comparative Example 5

[0207] This comparative example provides a method for preparing an animal microecological preparation with anti-stress function. The difference from Example 1 is that the mass ratio of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine and yeast β-glucan is adjusted to 100:14:0.8:1.5:1.3. Other process parameters and operating steps are exactly the same as in Example 1.

[0208] Comparative Example 6

[0209] This comparative example provides a method for preparing an animal microecological preparation with anti-stress function. The difference from Example 1 is that the mass ratio of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine and yeast β-glucan is adjusted to 100:14:0.8:2.5:1.3. Other process parameters and operating steps are exactly the same as in Example 1.

[0210] Comparative Example 7

[0211] This comparative example provides a method for preparing an animal microecological preparation with anti-stress function. The difference from Example 1 is that the mass ratio of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine and yeast β-glucan is adjusted to 100:14:0.8:1.8:1. Other process parameters and operating steps are exactly the same as in Example 1.

[0212] Comparative Example 8

[0213] This comparative example provides a method for preparing an animal microecological preparation with anti-stress function. The difference from Example 1 is that the mass ratio of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine and yeast β-glucan is adjusted to 100:14:0.8:1.8:2. Other process parameters and operating steps are exactly the same as in Example 1.

[0214] Comparative Example 9

[0215] This comparative example provides a method for preparing an animal microecological preparation with anti-stress function. The difference from Example 1 is that the volume ratio of the core material solution to the sodium alginate solution is adjusted to 1:1.5, while other process parameters and operating steps are exactly the same as in Example 1.

[0216] Comparative Example 10

[0217] This comparative example provides a method for preparing an animal microecological preparation with anti-stress function. The difference from Example 1 is that the volume ratio of the core material solution to the sodium alginate solution is adjusted to 1:2.5, while other process parameters and operating steps are exactly the same as in Example 1.

[0218] Comparative Example 11

[0219] This comparative example provides a method for preparing an animal microecological preparation with anti-stress function. The difference from Example 1 is that the mass fraction of chitosan in the chitosan solution is adjusted to 0.1 wt%, while other process parameters and operating steps are exactly the same as in Example 1.

[0220] Comparative Example 12

[0221] This comparative example provides a method for preparing an animal microecological preparation with anti-stress function. The difference from Example 1 is that the mass fraction of chitosan in the chitosan solution is adjusted to 2 wt%, while other process parameters and operating steps are exactly the same as in Example 1.

[0222] The anti-stress effects of the animal microecological preparations with anti-stress functions prepared in Examples 1-5 and Comparative Examples 1-12 were tested, and the testing process is as follows:

[0223] (1) Measurement of serum cortisol concentration

[0224] This experiment used SPF-grade rats as model animals, which were randomly divided into three groups: a blank control group (treated with physiological saline), a positive control group (treated with commercially available anti-stress agent glutamine), and an experimental group (treated with animal microecological preparations obtained in Examples 1-5 and Comparative Examples 1-12 of this invention). The administration was oral, at a rate of 0.5-1.0 g / kg body weight mixed into the feed daily for 7 consecutive days. After administration, a high-temperature stress model (ambient temperature 40℃ for 6 hours) was used to induce stress response in the rats, followed by sample collection.

[0225] At 0 h (baseline) and 6 h after stress induction, 5 mL of venous blood was collected from rats. After standing for 30 min, the serum was separated by centrifugation at 3000 rpm for 10 min. 100 μL of serum sample was taken, acetonitrile was added to precipitate proteins, the mixture was vortexed and centrifuged to collect the supernatant, dried under nitrogen, reconstituted in methanol, and filtered through a 0.22 μm filter membrane.

[0226] Serum cortisol concentration was detected using liquid chromatography-tandem mass spectrometry (LC-MS / MS). A C18 column (2.1 mm × 50 mm, 1.7 μm) was used, with a mobile phase of 0.1% formic acid aqueous solution and acetonitrile. The gradient elution program was 0–2 min, increasing the acetonitrile concentration from 20% to 95%. Mass spectrometry employed an electrospray ionization source (ESI negative ion mode) to monitor the characteristic ion pairs of cortisol at m / z 363.2→121.1 (quantitative ion) and m / z 363.2→327.2 (qualitative ion). Concentrations were calculated using a standard curve (1–100 ng / mL). Data are expressed as ng / mL. Differences between the experimental and control groups were verified using a t-test (P < 0.05).

[0227] (2) Determination of the levels of immunoglobulins IgA, IgG, and IgM

[0228] On day 7 of drug administration and 24 hours after stress induction, rat serum samples were collected and centrifuged at 3000 rpm for 10 min to remove impurities. The serum was diluted 1:1000 and added to a 96-well plate pre-coated with antibodies (anti-IgA, IgG, and IgM monoclonal antibodies). After incubation at 37°C for 1 h, the plate was washed, and horseradish peroxidase (HRP)-labeled secondary antibody was added and incubated for 1 h. The plate was washed again and TMB chromogenic solution was added and reacted in the dark for 15 min. The reaction was terminated with 2M sulfuric acid.

[0229] The absorbance (OD value) was read at a wavelength of 450 nm using an ELISA reader. A curve was plotted based on the concentration of the standard, and the contents of IgA (unit: μg / mL), IgG (unit: mg / mL), and IgM (unit: μg / mL) were calculated.

[0230] The test results are shown in Table 1.

[0231] Table 1. Performance test results of animal probiotic preparations in Examples 1-5 and Comparative Examples 1-12

[0232] Serum cortisol concentration (ng / mL) Immunoglobulin IgA content (μg / mL) Immunoglobulin IgG level (mg / mL) Immunoglobulin IgM content (μg / mL) Example 1 86.3 248.7 6.4 196.2 Example 2 89.1 242.5 6.2 192.8 Example 3 83.7 253.4 6.5 198.5 Example 4 87.9 239.8 6.1 189.3 Example 5 91.4 236.1 6.0 187.6 Comparative Example 1 134.7 204.3 5.3 168.9 Comparative Example 2 142.5 189.6 4.9 154.2 Comparative Example 3 157.8 175.4 4.5 142.7 Comparative Example 4 148.2 182.9 4.7 148.5 Comparative Example 5 163.4 166.3 4.3 132.8 Comparative Example 6 139.6 193.5 4.8 157.4 Comparative Example 7 152.9 178.2 4.6 146.1 Comparative Example 8 145.7 185.1 4.7 149.3 Comparative Example 9 138.2 198.7 5.1 162.4 Comparative Example 10 131.5 206.8 5.4 171.9 Comparative Example 11 155.3 172.6 4.4 138.7 Comparative Example 12 149.8 179.5 4.6 143.2 Positive control group 178.6 149.8 3.9 121.4 Blank control group 228.4 98.5 2.7 89.3

[0233] As shown in Table 1, Examples 1-5 exhibited the lowest serum cortisol concentrations and the highest levels of immunoglobulins IgA, IgG, and IgM, demonstrating excellent anti-stress and immune-enhancing effects. This is because the present invention optimizes the release rate and protective effect of the core material's active ingredients through a double-layer encapsulation technology, significantly reducing stress hormones (cortisol) and increasing immunoglobulins. In contrast, the key process parameters in Comparative Examples 1-12 exceeded the preferred range defined in this invention, resulting in weakened synergistic effects of the gut microbiota and decreased antioxidant and immunomodulatory capabilities.

[0234] The test data from Example 1, Comparative Example 1, and Comparative Example 2 show that the serum cortisol concentration in Comparative Example 1 and Comparative Example 2 is higher than that in Example 1, while the contents of immunoglobulins IgA, IgG, and IgM are lower than those in Example 1. This is because the proportion of prebiotics in the core material solution was adjusted in Comparative Example 1 and Comparative Example 2. In Comparative Example 1, the proportion of prebiotics was too low, which resulted in the inability of lactic acid bacteria and Bacillus in the freeze-dried bacterial powder to colonize effectively, a decrease in the production of short-chain fatty acids, failure of intestinal pH regulation, and failure to suppress stress signals. In Comparative Example 2, the proportion of prebiotics was too high, and excessive oligosaccharides caused an increase in intestinal osmotic pressure. Probiotics became inactive due to dehydration, their acid production capacity decreased, pathogens proliferated, and the intestinal barrier was damaged.

[0235] The test data from Examples 1, 3, and 4 show that the serum cortisol concentrations in Examples 3 and 4 are higher than those in Example 1, while the levels of immunoglobulins IgA, IgG, and IgM are lower. This is because the proportion of chlorogenic acid in the core material solution was adjusted in Examples 3 and 4. In Example 3, the proportion of chlorogenic acid was too low, resulting in decreased free radical scavenging efficiency, collapse of mitochondrial membrane potential in intestinal epithelial cells, increased release of pro-inflammatory factors, activation of the hypothalamus-pituitary-adrenal axis, and increased cortisol concentration. In Example 4, the proportion of chlorogenic acid was too high. High concentrations of chlorogenic acid can damage the integrity of lactic acid bacteria cell membranes, reduce lactic acid production, increase intestinal pH, and promote the proliferation of pathogens, leading to inflammation.

[0236] The test data from Examples 1, 5, and 6 show that the serum cortisol concentrations in Examples 5 and 6 were higher than those in Example 1, while the levels of immunoglobulins IgA, IgG, and IgM were lower. This is because the proportion of L-glutamine in the core material solution was adjusted in Examples 5 and 6. In Example 5, the proportion of L-glutamine was too low, resulting in insufficient energy (ATP) supply to intestinal epithelial cells, downregulation of tight junction protein expression, increased intestinal permeability, and endotoxin entry into the bloodstream, activating systemic inflammation. In Example 6, the proportion of L-glutamine was too high, and excess L-glutamine was decomposed into ammonia by intestinal flora, leading to elevated blood ammonia levels, inhibition of the TCA cycle, overload of hepatocyte detoxification capacity, and increased release of pro-inflammatory factors.

[0237] The test data from Example 1, Comparative Example 7, and Comparative Example 8 show that the serum cortisol concentration in Comparative Example 7 and Comparative Example 8 was higher than that in Example 1, while the levels of immunoglobulins IgA, IgG, and IgM were lower than those in Example 1. This is because Comparative Example 7 and Comparative Example 8 adjusted the proportion of yeast β-glucan in the core material solution. In Comparative Example 7, the proportion of yeast β-glucan was too low, resulting in decreased macrophage phagocytic activity, reduced antigen presentation efficiency, and hindered IgG affinity maturation. In Comparative Example 8, the proportion of yeast β-glucan was too high, causing local intestinal inflammation and inhibiting probiotic colonization.

[0238] The test data from Examples 1, 9, and 10 show that the serum cortisol concentrations in Examples 9 and 10 were higher than in Example 1, while the contents of immunoglobulins IgA, IgG, and IgM were lower. This is because in Example 9, the volume ratio of the core material solution to the sodium alginate solution was adjusted to 1:1.5, resulting in insufficient viscosity of the mixed solution, an overly thin and loosely structured composite microsphere shell, which easily disintegrated prematurely in the acidic environment of the stomach, causing premature exposure and inactivation of the active ingredients in the core material, leading to an increased serum cortisol concentration, indicating that the stress response was not effectively suppressed. Simultaneously, the decreased contents of immunoglobulins IgA, IgG, and IgM reflect poor intestinal colonization, decreased probiotic survival rate, and weakened immune regulation. In Example 10, the volume ratio of the core material solution to the sodium alginate solution was adjusted to 1:2.5, resulting in an excessively high proportion of sodium alginate solution, forming an overly thick and dense gel shell, severely hindering the release rate in the intestinal environment, and insufficient release of the active ingredients in the intestine, preventing them from exerting their anti-stress effect in a timely manner.

[0239] The test data from Examples 1, 11, and 12 show that the serum cortisol concentrations in Examples 11 and 12 were higher than those in Example 1, while the levels of immunoglobulins IgA, IgG, and IgM were lower. This is because the concentrations of the chitosan solution in Examples 11 and 12 were adjusted. In Example 11, the chitosan solution concentration was too low, resulting in incomplete outer coating. The low concentration of chitosan could not form a dense network, leading to premature release of the active ingredients in the core material in gastric acid and a decrease in the survival rate of live bacteria. In Example 12, the chitosan solution concentration was too high. The dense chitosan layer hindered the swelling of the calcium alginate layer, resulting in insufficient release rate of functional ingredients in the intestine. Furthermore, the high concentration of chitosan solution reacted excessively with sodium tripolyphosphate during cross-linking, forming a hard shell on the surface of the microspheres and hindering internal water exchange.

[0240] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for preparing an animal microecological preparation with anti-stress function, characterized in that, The preparation method includes: (I) Bacillus subtilis and lactic acid bacteria are cultured to obtain Bacillus seed liquid and lactic acid bacteria seed liquid. The Bacillus seed liquid is subjected to aerobic fermentation. A fermentation substrate is placed in a fermentation tank, and the Bacillus seed liquid is introduced into the fermentation tank for aerobic fermentation. The fermentation substrate consists of wheat bran, soybean meal, magnesium sulfate and deionized water. The amount of magnesium sulfate added is 0.1~0.2wt% of the total mass of wheat bran and soybean meal. Then, the lactic acid bacteria seed liquid is added for microaerobic fermentation to obtain the fermentation product. The volume ratio of the lactic acid bacteria seed liquid to the Bacillus seed liquid is 1:(0.8~1.2). (II) The yeast is cultured to obtain yeast seed liquid. The fermentation product and the yeast seed liquid are mixed and then centrifuged and freeze-dried to obtain freeze-dried yeast powder. The volume ratio of the fermentation product to the yeast seed liquid is 1:(0.2~0.4). (III) A core material solution is obtained by mixing freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, yeast β-glucan, and deionized water. The prebiotics include fructooligosaccharides and galactooligosaccharides, with a mass ratio of (0.8~1.2):

1. The mass ratio of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, and yeast β-glucan is 100:(14~16):(0.8~1.2):(1.8~2):(1.3~1.5). The total mass fraction of freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine, and yeast β-glucan in the core material solution is 10~12wt%. The core material solution is mixed with sodium alginate solution and then dropped into calcium chloride solution for cross-linking to obtain composite microspheres. The volume ratio of the core material solution to the sodium alginate solution is 1:(1.8~2.3). (IV) The composite microspheres are immersed in a chitosan solution with a chitosan mass fraction of 0.5~1.5wt%, and then removed and immersed in a sodium tripolyphosphate solution for cross-linking. After spray drying, the animal microecological preparation is obtained.

2. The method for preparing the animal microecological preparation with anti-stress function according to claim 1, characterized in that, In step (I), during the aerobic fermentation process, the air flow rate into the fermenter is 1~1.5 vvm; The stirring rate for the aerobic fermentation is 150-200 rpm; The aerobic fermentation time is 20-30 hours; During the aerobic fermentation process, the pH value of the Bacillus seed liquid is controlled at 6.5~6.

8.

3. The method for preparing the animal microecological preparation with anti-stress function according to claim 2, characterized in that, In step (I), during the microaerobic fermentation process, the air flow rate into the fermenter is 0.2~0.4 vvm; The stirring rate for the microaerobic fermentation is 80-100 rpm; The microaerobic fermentation time is 30-40 hours; During the microaerobic fermentation process, the pH value of the mixed seed liquid in the fermenter is controlled at 5.5~6.

0.

4. The method for preparing the animal microecological preparation with anti-stress function according to claim 1, characterized in that, In step (II), trehalose is added to the mixture of the fermentation product and the yeast seed liquid, wherein the amount of trehalose added is 1-2 wt% of the total mass of the fermentation product and the yeast seed liquid. The centrifuge speed is 3000~4000 rpm; The centrifugation time is 30-40 minutes; The freeze-drying process includes pre-freezing and sublimation drying performed sequentially. The pre-freezing temperature is -35~-40℃; The pre-freezing time is 4-6 hours; The temperature for the sublimation drying is -25~-30℃; The sublimation drying time is 18-24 hours.

5. The method for preparing the animal microecological preparation with anti-stress function according to claim 1, characterized in that, In step (III), the core material solution also includes a heat protectant, which includes skim milk powder, trehalose and monosodium glutamate; The mass ratio of the skim milk powder, trehalose, and monosodium glutamate is (2~3):(1~2):1; The amount of the heat protectant added is 25-35 wt% of the total mass of the freeze-dried bacterial powder, prebiotics, chlorogenic acid, L-glutamine and yeast β-glucan.

6. The method for preparing the animal microecological preparation with anti-stress function according to claim 1, characterized in that, In step (III), the core material solution is mixed with sodium alginate solution to obtain a mixed solution. Sucrose ester is added to the mixed solution, followed by high-speed stirring and emulsification to obtain a mixed emulsion. The mixed emulsion is dropped into the calcium chloride solution to form gel spheres. The gel spheres are soaked in calcium chloride solution for cross-linking to obtain the composite microspheres. The amount of sucrose ester added is 0.3~0.8 wt% of the mass of the mixed solution; The sodium alginate solution has a mass fraction of 1-5 wt%. The stirring speed is 6000~8000 rpm; The stirring time is 5-10 minutes; The calcium chloride solution has a mass fraction of 0.5~1.5 wt%. The gel balls are soaked in the calcium chloride solution for 30-40 minutes.

7. The method for preparing the animal microecological preparation with anti-stress function according to claim 1, characterized in that, In step (IV), the composite microspheres are soaked in the chitosan solution for 0.5 to 1.5 hours. The sodium tripolyphosphate solution has a mass fraction of 0.3~0.8 wt%. The composite microspheres are immersed in the sodium tripolyphosphate solution for 15-25 minutes.

8. The method for preparing the animal microecological preparation with anti-stress function according to claim 1, characterized in that, In step (IV), the inlet air temperature for the spray drying is 40~45℃; The outlet air temperature of the spray dryer is 30~35℃.

9. An animal microecological preparation with anti-stress function prepared by the preparation method according to any one of claims 1 to 8.

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