A complex yeast culture for lamb growth and a method for preparing the same

Abstract

The present application relates to the technical fields of feed additive chemical synthesis, and discloses a kind of compound yeast culture for raising sheep and a preparation method thereof, wherein the compound yeast culture for raising sheep includes selenium-containing composite core material and double-wall material structure.The selenium-containing composite core material is prepared from fermentation dry powder, gelatin-coated nano selenium powder and sodium bicarbonate powder.The double-wall material is composed of calcium alginate gel network and stearic acid palmitic acid glyceride lipid barrier layer.The preparation method includes six steps of gelatin-coated nano selenium powder preparation, solid fermentation, vacuum drying, core material preparation, wall material coating, spray granulation and cross-linking solidification.

Description

Technical Field

[0001] This invention relates to the field of chemical synthesis technology of feed additives, and more specifically, to a sheep-rearing compound yeast culture and its preparation method. Background Technology

[0002] Large-scale fattening sheep farms employ intensive feeding using high-concentrate diets combined with automated feeding systems. The basal diet is processed into pelleted feed through ring die pelleting, with the pelleting and conditioning temperature typically between 65 and 80°C. Under the high-concentrate feeding model, starch in the rumen ferments rapidly, producing large amounts of organic acids. The rumen pH frequently drops to the 5.0-5.5 range, triggering subacute rumen acidosis. Damage to the rumen epithelial barrier leads to increased oxidative stress. Furthermore, the low serum selenium levels in fattening sheep result in insufficient selenium-dependent glutathione peroxidase activity, making it difficult for the endogenous antioxidant defense system to effectively cope.

[0003] Existing sheep-feeding compound yeast cultures are usually in powder form, with materials such as sodium alginate as the encapsulation wall material, and nano-selenium is directly added to supplement the selenium requirements of fattening sheep.

[0004] Existing technologies have the following drawbacks: Live bacteria in powdered cultures cannot tolerate the high-temperature steam (65-80°C) during granulation and conditioning, resulting in insufficient effective live bacteria counts upon arrival in the rumen; when nano-selenium is directly added to the culture, the nano-selenium particles coordinate with the carboxyl groups in the sodium alginate wall material, causing the nano-selenium to aggregate to the micron level, leading to a sharp decline in bioavailability; and there is a lack of synergistic release mechanisms among live bacteria, selenium source, and buffering components, making it impossible to match the time window of high incidence of rumen acidosis after high-concentrate feed intake. These drawbacks mean that existing sheep-rearing compound yeast cultures cannot simultaneously meet the triple functional requirements of live bacteria protection, effective delivery of nano-selenium, and rumen acid buffering under the dual conditions of granulation processing and the acidic environment of the rumen. Summary of the Invention

[0005] This invention provides a sheep-rearing compound yeast culture and its preparation method, solving the technical problems of poor stability of nano-selenium, easy loss of active ingredients in yeast fermentation products, and uncontrollable release of functional components in the digestive tract in related technologies.

[0006] This invention discloses a method for preparing a sheep-rearing compound yeast culture, comprising the following steps:

[0007] Sodium selenite was reduced with ascorbic acid to obtain nano-selenium colloid, which was then coated with gelatin and spray-dried to obtain gelatin-coated nano-selenium powder.

[0008] After mixing and adjusting the water content of flaked corn, corn, soybean meal, corn alcohol residue and beet meal, the mixture is inoculated with brewer's yeast liquid and Lactobacillus plantarum liquid for anaerobic fermentation to obtain solid fermentation product. The solid fermentation product is then mixed with liquid yeast metabolite, vacuum dried and pulverized to obtain fermented dry powder.

[0009] A selenium-containing composite core material is obtained by mixing fermented dry powder with gelatin-coated nano-selenium powder and sodium bicarbonate powder.

[0010] Sodium alginate and glyceryl stearate palmitate were emulsified and dispersed to form a wall material emulsion. After cooling, a selenium-containing composite core material was added and stirred to obtain a coating slurry.

[0011] The coating slurry is spray-granulated to obtain microencapsulated particles, which are then cross-linked and cured with calcium chloride solution and dried to obtain the finished product.

[0012] Further, 0.5 to 1.2 parts by weight of sodium selenite are dissolved in 100 parts by weight of deionized water, and 4 to 6 parts by weight of ascorbic acid are added and stirred at room temperature for 40 to 60 minutes. 2 to 4 parts by weight of gelatin are added to the nano-selenium colloid, and the mixture is stirred and coated at 40 to 48°C for 1 to 2 hours. The spray drying process is carried out at an inlet air temperature of 120 to 140°C and an outlet air temperature of 60 to 70°C, with the instantaneous residence time of the material not exceeding 5 seconds. The resulting gelatin-coated nano-selenium powder has a particle size of 1 to 10 μm.

[0013] Furthermore, the gelatin is food-grade gelatin with a gel strength of 150 to 250 g and a viscosity of 2 to 5 mPa·s (measured as a 6.67% aqueous solution at 60°C); the stirring speed during the reduction reaction stage is 200 to 400 rpm, and the stirring speed during the coating stage is 150 to 300 rpm.

[0014] Furthermore, in the anaerobic fermentation, 50 to 60 parts by weight of flaked corn and 40 to 50 parts by weight of corn are mixed, crushed, and sieved through a 40 to 60 mesh sieve. 18 to 22 parts by weight of soybean meal, 15 to 20 parts by weight of corn distillers' grains, and 10 to 15 parts by weight of beet pulp are added. The moisture content is adjusted to 34 to 38 parts by weight. The volume ratio of brewer's yeast liquid to Lactobacillus plantarum liquid is 1:2 to 1:2.5. The total inoculum amount is 8 to 15 parts by weight of the total mass of the mixed culture medium. The mixture is then sealed and anaerobic fermented at 30 to 35°C for 40 to 56 hours.

[0015] Furthermore, in the vacuum drying process, 65 to 75 parts by weight of solid fermentation product are mixed with 25 to 35 parts by weight of liquid yeast metabolite, and dried for 4 to 7 hours under vacuum conditions of -0.06 to -0.09 MPa and temperature of 42 to 50°C until the moisture content drops to 8 to 11 parts by weight. The dried product is then pulverized through a 60 to 80 mesh sieve.

[0016] Furthermore, in the selenium-containing composite core material, based on 100 parts by weight of fermented dry powder, the gelatin-coated nano-selenium powder is 0.5 to 1.5 parts by weight, and the sodium bicarbonate powder is 2 to 4 parts by weight, wherein the particle size of the sodium bicarbonate powder is 60 to 100 mesh.

[0017] Furthermore, in the wall material emulsion, based on 100 parts by weight of selenium-containing composite core material, 2 to 3.5 parts by weight of sodium alginate are dissolved in 40 to 55 parts by weight of deionized water, and 3 to 5 parts by weight of glyceryl stearate palmitate are added. The mixture is emulsified and dispersed at 58 to 65°C for 20 to 40 minutes with a stirring speed of 800 to 1200 rpm. After cooling to 35 to 40°C, the selenium-containing composite core material is added and stirred and dispersed for 20 to 40 minutes.

[0018] Furthermore, the inlet air temperature of the spray granulation is 36 to 43°C, the atomization pressure is 0.15 to 0.3 MPa, and the microencapsulated particle size is 0.5 to 1.5 mm; the calcium chloride solution concentration is 2 to 3.5 parts by weight of calcium chloride per 100 parts by weight of water, the crosslinking and curing time is 10 to 20 min, the crosslinking and curing temperature is 20 to 25°C, the airflow drying temperature is 35 to 42°C, and the moisture content of the finished product does not exceed 9 parts by weight.

[0019] Furthermore, before immersing the microencapsulated particles into the calcium chloride solution, the surface of the microencapsulated particles is pre-sprayed and wetted with a calcium chloride solution with a mass fraction of 0.5 to 1%, the spray amount is 3 to 5 parts by weight of the microencapsulated particles, the wetting time is 1 to 2 minutes, and then the particles are immersed in the calcium chloride solution for cross-linking and curing.

[0020] This invention discloses a sheep-rearing compound yeast culture, which is prepared by the above-described preparation method.

[0021] The beneficial effects of this invention are as follows:

[0022] This invention addresses the technical problem of agglomeration caused by coordination complexation between selenium nanoparticles and carboxyl groups on sodium alginate molecular chains by pre-coating the surface of selenium nanoparticles with a gelatin protein layer. This prevents the selenium nanoparticles from forming a complex with the sodium alginate wall material, thus maintaining the nanoscale dispersion of selenium nanoparticles in the finished sheep-rearing yeast culture. Furthermore, this invention employs a calcium alginate gel network and a glyceryl stearate palmitate lipid barrier layer to form a dual wall material structure. This solves the technical problem of decreased survival rate of live bacteria under high-temperature conditions during granulation and conditioning, achieving a reduced actual temperature rise within the selenium-containing composite core material and maintaining a certain survival rate of live bacteria after granulation. Finally, this invention co-formulates and embeds sodium bicarbonate microparticles, fermentation dry powder, and gelatin-coated selenium nanoparticles into a selenium-containing composite core material. This solves the technical problem of the lack of synergistic release mechanisms for live bacteria, selenium source, and buffer components, achieving a technical effect where the release time windows of the three functional components match the time window of increased rumen acid load after high-concentrate feed intake. Attached Figure Description

[0023] Figure 1 This is a flowchart of the preparation process of sheep-rearing compound yeast culture provided in the embodiments of the present invention;

[0024] Figure 2 This is a schematic diagram comparing the D90 values ​​of the nano-selenium particles of the released components in each sample provided in the embodiments of the present invention;

[0025] Figure 3 This is a schematic diagram comparing the survival rates of live bacteria in various samples after heat treatment, provided in the embodiments of the present invention.

[0026] Figure 4 This is a schematic diagram comparing the simulated rumen pH buffering capacity of various samples provided in the embodiments of the present invention;

[0027] Figure 5 This is a schematic diagram of the rumen pH change curves of samples 2 and 8 provided in the embodiments of the present invention over time;

[0028] Figure 6 This is a schematic diagram of the TEM cross-sectional morphology of gelatin-coated selenium nanoparticles provided in an embodiment of the present invention;

[0029] Figure 7 This is a schematic diagram of a TEM image of nano-selenium agglomerates with gelatin coating (Comparative Example 1) provided in an embodiment of the present invention;

[0030] Figure 8 This is a schematic diagram of the SEM morphology of microencapsulated particles of sheep-rearing compound yeast culture provided in an embodiment of the present invention. Detailed Implementation

[0031] The subject matter described herein will now be discussed with reference to exemplary embodiments. It should be understood that these embodiments are discussed only to enable those skilled in the art to better understand and implement the subject matter described herein, and changes may be made to the function and arrangement of the elements discussed without departing from the scope of this specification. Various processes or components may be omitted, substituted, or added as needed in the examples. Furthermore, some features described in the examples may be combined in other examples.

[0032] See Figure 1 This invention discloses a method for preparing a sheep-rearing compound yeast culture, comprising:

[0033] Step 1, Preparation of gelatin-coated selenium nanopowder

[0034] Dissolve 0.5 to 1.2 parts by weight of sodium selenite in 100 parts by weight of deionized water, and add 4 to 6 parts by weight of ascorbic acid as a reducing agent. Stir and react at room temperature for 40 to 60 minutes. The ascorbic acid reduces the tetravalent selenium in selenite to zero-valent elemental selenium, resulting in a red, transparent nano-selenium colloidal solution. Add 2 to 4 parts by weight of gelatin to the nano-selenium colloidal solution and stir at 40 to 48°C for 1 to 2 hours to coat the nano-selenium particles, allowing the gelatin molecules to be uniformly adsorbed onto the surface of the nano-selenium particles, forming a protein coating layer. Spray dry the resulting coating solution at an inlet air temperature of 120 to 140°C and an outlet air temperature of 60 to 70°C, with a material instantaneous residence time not exceeding 5 seconds, to obtain gelatin-coated nano-selenium powder with a particle size of 1 to 10 μm.

[0035] Furthermore, the particle size of the gelatin-coated selenium nanoparticles was determined using a laser diffraction particle size analyzer.

[0036] Furthermore, after the reduction reaction is complete, the residual ascorbic acid and selenite ions in the resulting nano-selenium colloidal solution must be confirmed before the coating step. The color of the nano-selenium colloidal solution is tested. If the solution is uniformly red and free of colorless or gray precipitate, the reduction reaction is complete. If the solution exhibits localized turbidity or gray precipitate, the stirring time must be extended until the reaction is complete before proceeding with the coating step to prevent residual selenite ions from triggering uncontrollable secondary reactions in subsequent steps.

[0037] Furthermore, sodium selenite is a toxic chemical; protective gloves and masks must be worn during handling. Weighing and dissolving operations should be carried out in a fume hood to avoid dust inhalation or skin contact. Waste liquid containing sodium selenite and cleaning wastewater must be collected separately and disposed of by a professional waste liquid treatment company; direct discharge into sewers is prohibited.

[0038] It should be noted that the gelatin used is food-grade gelatin with a gel strength of 150 to 250 g, a viscosity of 2 to 5 mPa·s (measured as a 6.67% aqueous solution at 60°C), and a moisture content not exceeding 14%. The gel strength and viscosity of the gelatin determine the density of the film formed on the surface of the selenium nanoparticles. When the gel strength is below 150 g, the strength of the formed protein film is insufficient, and it cannot effectively prevent the carboxyl groups from penetrating and complexing to the surface of the selenium nanoparticles when in contact with the sodium alginate wall material. When the gel strength is above 250 g, the viscosity of the gelatin solution is too high, resulting in uneven dispersion during the coating process, with some areas of the selenium nanoparticles being excessively coated while others are exposed.

[0039] It should be noted that the mass ratio of sodium selenite to ascorbic acid ranges from 1:3.3 to 1:12, with a preferred ratio of 1:5. This mass ratio range corresponds to the ratio of 0.5 to 1.2 parts by weight of sodium selenite and 4 to 6 parts by weight of ascorbic acid in step 1. Ascorbic acid, as a reducing agent, needs to be in excess to ensure sufficient reduction of sodium selenite. When the amount of ascorbic acid is insufficient, the formation of zero-valent selenium is incomplete, and the residual selenite ions in the nano-selenium colloidal solution may trigger uncontrollable secondary reactions in subsequent steps.

[0040] It should be noted that the instantaneous residence time of the material in the spray drying process shall not exceed 5 seconds. The thermal denaturation temperature of gelatin is approximately 70 to 80°C, while the inlet air temperature is 120 to 140°C. By controlling the instantaneous residence time of the material to within 5 seconds, the actual temperature of the material is maintained within the range of 60 to 70°C by utilizing the endothermic effect of droplet evaporation. This prevents the gelatin from undergoing irreversible thermal denaturation during the drying process and losing its film-forming function.

[0041] Furthermore, the stirring speed during the reduction reaction stage is controlled at 200 to 400 rpm, and the stirring speed during the coating stage is controlled at 150 to 300 rpm. When the stirring speed is below 200 rpm during the reduction reaction stage, the system is not sufficiently mixed, and the concentration of selenium nanoparticles is too high in local areas, leading to agglomeration and uneven particle size distribution in the resulting selenium nanoparticle colloidal solution. When the stirring speed is above 400 rpm, the shear force is too strong, causing the already formed primary selenium nanoparticles to be mechanically broken and then re-agglomerated, resulting in an increased particle size. The stirring speed during the coating stage is controlled at 150 to 300 rpm to ensure uniform adsorption of gelatin molecules on the surface of the selenium nanoparticles, while avoiding excessive shearing that could damage the already formed selenium nanoparticle colloidal structure.

[0042] Furthermore, after the gelatin-coated selenium nanoparticles obtained from spray drying are collected, the dispersion state of the selenium nanoparticles is observed using a transmission electron microscope to confirm the integrity of the gelatin coating layer on the surface of the selenium nanoparticles; at the same time, the total selenium content in the gelatin-coated selenium nanoparticles is determined by inductively coupled plasma mass spectrometry to calculate the conversion rate of sodium selenite and the selenium recovery rate in step 1.

[0043] Step 2, Preparation of solid-state fermentation product

[0044] Mix 50 to 60 parts by weight of flaked corn with 40 to 50 parts by weight of corn, and pulverize through a 40 to 60 mesh sieve to obtain mixed corn flour. Add 18 to 22 parts by weight of soybean meal, 15 to 20 parts by weight of corn distillers grains, and 10 to 15 parts by weight of beet pulp to the mixed corn flour, mix thoroughly, and then add deionized water to adjust the moisture content to 34 to 38 parts by weight (based on 100 parts by weight of dry matter). Inoculate with brewer's yeast broth and Lactobacillus plantarum broth at a volume ratio of 1:2 to 1:2.5, with a total inoculation amount of 8 to 15 parts by weight of the total mass of the mixed culture medium (based on 100 parts by weight of the total mass of the mixed culture medium). Seal and anaerobic ferment at 30 to 35°C for 40 to 56 hours to obtain solid fermentation product.

[0045] It should be noted that the number of viable cells in the brewer's yeast culture is not less than 1×10⁻⁶. 9 CFU / mL, where CFU is colony-forming units; the viable count in the *Lactobacillus plantarum* culture is not less than 5 × 10⁻⁶. 9 CFU / mL. The preferred volume ratio of Saccharomyces cerevisiae culture to Lactobacillus plantarum culture is 1:2. This ratio ensures that the acid production rate of Lactobacillus plantarum in the fermentation system matches the alcohol production and metabolite generation rate of Saccharomyces cerevisiae, avoiding excessive Lactobacillus plantarum leading to an excessively low pH in the fermentation system and inhibiting the activity of Saccharomyces cerevisiae.

[0046] It should be noted that *Lactobacillus plantarum* is highly acid-resistant and can maintain its activity at pH 3.5 to 4.0, making it suitable for co-fermentation systems with *Saccharomyces cerevisiae*.

[0047] It should be noted that the mixing ratio of flaked corn to corn is 50 to 60 parts by weight and 40 to 50 parts by weight. The flaked corn undergoes increased starch gelatinization after steam pressing, providing a more readily available carbon source for brewer's yeast. Beet meal provides soluble fiber as a fermentation substrate for Lactobacillus plantarum, while corn distillers grains provide crude protein and B vitamins to maintain the nitrogen source and cofactors required for the growth and metabolism of the microbial community.

[0048] Furthermore, the Saccharomyces cerevisiae and Lactobacillus plantarum cultures are premixed at a volume ratio of 1:2 to 1:2.5 and then inoculated into the mixed culture medium all at once to ensure the initial uniformity of the two cultures in the mixed culture medium and to avoid local imbalances in the proportion of microorganisms when added in batches.

[0049] Furthermore, the final pH and viable cell count of the solid-state fermentation product must be tested after fermentation. The final pH should be between 3.8 and 4.5. If the pH is higher than 4.5, it indicates insufficient acid production by *Lactobacillus plantarum*, and the fermentation time must be extended. If the pH is lower than 3.8, it indicates that the system acidity is too high, and the inoculation ratio of the bacterial solution must be checked to ensure it meets the requirements. The viable count of *Saccharomyces cerevisiae* in the solid-state fermentation product at the final fermentation point should not be less than 1 × 10⁻⁶. 8CFU / g, viable count of Lactobacillus plantarum not less than 1×10⁻⁶ 9 CFU / g is used to ensure that the viable bacteria count meets the product's functional requirements in subsequent steps.

[0050] Step 3, Preparation of fermented dry powder

[0051] Add 65 to 75 parts by weight of the solid fermentation product obtained in step 2 and 25 to 35 parts by weight of the liquid yeast metabolite to a mixer and mix at room temperature for 15 to 30 minutes until homogeneous. Vacuum dry at a vacuum degree of -0.06 to -0.09 MPa and a temperature of 42 to 50°C for 4 to 7 hours until the moisture content is reduced to 8 to 11 parts by weight (based on 100 parts by weight on a dry basis). Pulverize the dried product through a 60 to 80 mesh sieve to obtain fermentation powder.

[0052] It should be noted that liquid yeast metabolites are the supernatant obtained after centrifugation to remove the cell bodies following liquid fermentation of Saccharomyces cerevisiae. They are rich in small peptides, amino acids, B vitamins, and organic acids, among other metabolites. The dry matter content of the liquid yeast metabolites is 15 to 25 parts by weight (based on 100 parts by weight of the total liquid yeast metabolites).

[0053] It should be noted that the vacuum drying temperature is controlled between 42 and 50°C to maintain the survival rate of live bacteria in the fermentation powder while removing moisture. When the drying temperature exceeds 50°C, the survival rate of Lactobacillus plantarum and Saccharomyces cerevisiae will decrease significantly; when the temperature is below 42°C, the rate of moisture evaporation is too low under the same vacuum conditions, and extending the drying time will increase the exposure time of the bacteria in a low moisture activity environment, which is also detrimental to the survival of live bacteria.

[0054] Furthermore, the moisture content of the fermented powder was determined using the loss on drying method to confirm whether the vacuum drying endpoint met the requirement of 8 to 11 parts by weight (based on 100 parts by weight on a dry basis). The viable counts of Saccharomyces cerevisiae and Lactobacillus plantarum in the fermented powder were determined using the plate count method to verify the impact of the drying step on the viable cell survival rate.

[0055] Step 4, Preparation of selenium-containing composite core material

[0056] Add 100 parts by weight of the fermented dry powder obtained in step 3, 0.5 to 1.5 parts by weight of the gelatin-coated nano-selenium powder obtained in step 1, and 2 to 4 parts by weight of sodium bicarbonate powder to a mixer and mix at room temperature for 10 to 20 minutes until uniform to obtain a selenium-containing composite core material.

[0057] It should be noted that the particle size of the sodium bicarbonate micron powder is 60 to 100 mesh. The amount of sodium bicarbonate added to the selenium-containing composite core material is 2 to 4 parts by weight (based on 100 parts by weight of fermentation dry powder), preferably 3 parts by weight. As a buffering component, sodium bicarbonate reacts with organic acids in the rumen when the microencapsulated particles are subsequently released into the rumen, providing a local pH buffering effect. The range of sodium bicarbonate addition is determined based on the rumen acid load level under the single feed intake conditions of fattening sheep, and can provide effective acid neutralization protection for the rumen epithelial barrier area without excessively interfering with the normal fermentation environment of the rumen.

[0058] It should be noted that the amount of gelatin-coated nano-selenium powder added is 0.5 to 1.5 parts by weight (based on 100 parts by weight of fermented dry powder), preferably 1.0 part by weight. This amount translates to an elemental selenium content of approximately 0.1 to 0.5 mg / kg (calculated based on the recommended addition ratio of sheep-rearing compound yeast culture product in the diet), meeting the safety limit requirements for selenium supplementation in fattening sheep diets.

[0059] Furthermore, sodium bicarbonate powder must be stored under dry conditions and confirmed to be free from moisture and clumping before being added to the mixer. If clumping occurs, the sodium bicarbonate powder must be ground and re-sieved through a 60-100 mesh sieve before use to ensure uniform dispersion of the sodium bicarbonate powder in the selenium-containing composite core material. A slight gas-generating reaction may occur between sodium bicarbonate and residual organic acids in the fermentation powder during the mixing stage; therefore, the mixer must be kept open at atmospheric pressure or equipped with an exhaust vent to prevent pressure buildup within the sealed container.

[0060] Step 5, Preparation of wall material emulsion and core material coating

[0061] Dissolve 2 to 3.5 parts by weight of sodium alginate in 40 to 55 parts by weight of deionized water, add 3 to 5 parts by weight of glyceryl stearate palmitate, and emulsify and disperse at 58 to 65°C for 20 to 40 minutes until homogeneous to form a wall material emulsion; the amounts of each component are based on 100 parts by weight of selenium-containing composite core material. After cooling the wall material emulsion to 35 to 40°C, add 100 parts by weight of the selenium-containing composite core material obtained in step 4, and stir and disperse for 20 to 40 minutes until homogeneous to obtain a coating slurry.

[0062] It should be noted that the viscosity of sodium alginate is 200 to 400 mPa·s (measured as a 1% aqueous solution at 25°C), the weight-average molecular weight is 120,000 to 220,000 Da, and the guluronic acid content accounts for no less than 55% of the total uronic acid content. The guluronic acid content in sodium alginate directly determines the density and strength of the gel network formed by subsequent calcium ion crosslinking. When the guluronic acid molar fraction is less than 55%, the crosslinked gel network has excessively large pores, resulting in insufficient thermal insulation protection of the wall material under high-temperature granulation conditions.

[0063] It should be noted that glyceryl stearate palmitate has a melting point of 52 to 58°C, a hydrophilic-lipophilic balance value of 2 to 4, and an acid value not exceeding 3 mg KOH / g. In wall material emulsions, glyceryl stearate palmitate acts as a hydrophobic lipid barrier layer within the sodium alginate gel network. At emulsification temperatures of 58 to 65°C, glyceryl stearate palmitate is in a molten state and can uniformly disperse in the sodium alginate aqueous solution to form lipid droplets. After the wall material emulsion cools to 35 to 40°C, the lipid droplets solidify and embed themselves within the sodium alginate matrix, forming a lipid microdomain distribution. When the microencapsulated particles are heated during granulation and conditioning, the hydrophobic lipid barrier layer impedes the penetration of water vapor into the selenium-containing composite core material, thereby reducing the actual temperature rise of the selenium-containing composite core material and protecting the live bacteria from high-temperature damage.

[0064] It should be noted that the wall material emulsion is cooled to 35 to 40°C before the selenium-containing composite core material is added and stirred. This temperature control is to avoid the high-temperature wall material emulsion directly contacting the live bacteria in the fermented dry powder, which would lead to heat inactivation. At the same time, the temperature range of 35 to 40°C can maintain the appropriate fluidity of the wall material emulsion, ensuring that the selenium-containing composite core material is evenly dispersed in the wall material emulsion.

[0065] Furthermore, the stirring speed during the emulsification and dispersion step is controlled between 800 and 1200 rpm. When the speed is below 800 rpm, there is insufficient shear between the molten phase of stearic acid palmitate and the aqueous phase of sodium alginate, resulting in larger and more widely distributed lipid droplets. After cooling and solidification, the lipid microdomains are unevenly distributed in the sodium alginate matrix, affecting the consistency of the thermal insulation performance of the wall material. When the speed is above 1200 rpm, the high-speed shear causes local heating of the system, which may lead to partial degradation of the sodium alginate molecular chains and reduce the strength of the gel network formed by subsequent cross-linking.

[0066] Furthermore, before proceeding to step 6 (spray granulation), the coating slurry must be visually inspected to ensure it is uniform in appearance and free from obvious particle settling or stratification. If stratification occurs, it must be stirred continuously at 35 to 40°C until homogeneous before use. The coating slurry should not be used for more than 2 hours from the time of preparation; after this period, the uniformity of the coating slurry must be reassessed.

[0067] Step 6, spray granulation and cross-linking curing

[0068] The coating slurry obtained in step 5 is spray-granulated in a fluidized bed coating equipment with an inlet air temperature of 36 to 43°C and an atomization pressure of 0.15 to 0.3 MPa to obtain microencapsulated particles with a particle size of 0.5 to 1.5 mm.

[0069] Furthermore, the particle size of the microencapsulated particles was determined by standard sieving method.

[0070] The microencapsulated particles were immersed in a calcium chloride solution for cross-linking and curing for 10 to 20 minutes. After filtration, they were air-dried at 35 to 42°C until the moisture content did not exceed 9 parts by weight (based on 100 parts by weight on a dry basis) to obtain the finished sheep breeding compound yeast culture product.

[0071] It should be noted that the concentration of the calcium chloride solution is 2 to 3.5 parts by weight of calcium chloride per 100 parts by weight of water, preferably 2.5 parts by weight of calcium chloride per 100 parts by weight of water. Calcium ions in the calcium chloride solution undergo ionic cross-linking with the guluronic acid segments in sodium alginate, forming an "egg-box" structured calcium alginate gel network. When the concentration of the calcium chloride solution is less than 2 parts by weight per 100 parts by weight of water, the degree of cross-linking is insufficient, the pores of the gel network are too large, and the heat insulation effect of the wall material is weakened during high-temperature granulation. When the concentration of the calcium chloride solution is greater than 3.5 parts by weight per 100 parts by weight of water, the cross-linking rate is too fast, the surface of the microencapsulated particles becomes excessively dense, resulting in insufficient internal calcium ion penetration and uneven cross-linking in the thickness direction of the wall material.

[0072] It should be noted that the inlet air temperature for fluidized bed spray granulation is 36 to 43°C. This temperature range represents the equilibrium between the survival of live bacteria and the quality of the wall material forming. When the inlet air temperature is below 36°C, the drying rate of the atomized coating slurry droplets is too low, and the particles are prone to adhesion and aggregation. When the inlet air temperature is above 43°C, the heat accumulation effect on the live bacteria in the selenium-containing composite core material during granulation increases, resulting in a decrease in survival rate.

[0073] Furthermore, the temperature of the calcium chloride solution used for cross-linking curing is controlled between 20 and 25°C. When the temperature is below 20°C, the diffusion rate of calcium ions into the microencapsulated particles slows down, resulting in insufficient cross-linking in the thickness direction of the wall material within the same curing time. When the temperature is above 25°C, the diffusion rate of calcium ions accelerates, and the cross-linking rate of the surface layer is significantly faster than that of the interior, exacerbating the unevenness of the cross-linking degree of the wall material.

[0074] Furthermore, to improve the crosslinking uniformity of the wall material gel network, before immersing the microencapsulated particles in the calcium chloride solution, the surface of the microencapsulated particles can be pre-wetted with a 0.5 to 1% (w / w) calcium chloride solution. The spraying amount is 3 to 5 parts by weight of the microencapsulated particles (based on a total mass of 100 parts by weight of microencapsulated particles), and the wetting time is 1 to 2 minutes. Then, the particles are immersed in the calcium chloride solution of the aforementioned concentration for crosslinking and curing. Pre-wetting allows the sodium alginate layer on the surface of the microencapsulated particles to come into contact with low-concentration calcium ions, forming a preliminary, slightly crosslinked surface layer. This prevents the surface layer from densifying too quickly when directly immersed in a higher concentration of calcium chloride solution, thus hindering the penetration of calcium ions into the interior and improving the uniformity of crosslinking throughout the thickness of the wall material.

[0075] Furthermore, after the airflow drying process, the following tests were performed on the finished sheep-rearing compound yeast culture: the moisture content of the finished sheep-rearing compound yeast culture was determined by the loss on drying method, confirming that it did not exceed 9 parts by weight (based on 100 parts by weight on a dry basis); the viable counts of Saccharomyces cerevisiae and Lactobacillus plantarum in the finished sheep-rearing compound yeast culture were determined by the plate count method to evaluate the viable cell retention rate after the entire process; the total selenium content in the finished sheep-rearing compound yeast culture was determined by inductively coupled plasma mass spectrometry to calculate the selenium recovery rate in the entire process; and the particle size distribution of the finished sheep-rearing compound yeast culture was determined by the standard sieving method, confirming that the particle size was concentrated in the range of 0.5 to 1.5 mm.

[0076] Furthermore, the waste calcium chloride solution obtained after cross-linking and curing contains trace amounts of selenium and sodium alginate leaching, which must be collected separately and treated by a professional waste liquid treatment agency, and must not be discharged directly. The exhaust gas containing trace amounts of dust generated during the airflow drying process must be filtered by a bag filter before being discharged to prevent selenium-containing dust from escaping into the workshop environment.

[0077] To address the issue of aggregation caused by coordination complexation between nano-selenium and sodium alginate wall material, this invention pre-coats the surface of nano-selenium particles with a gelatin protein layer, introducing a physical barrier between the nano-selenium particles and the sodium alginate wall material. The gelatin protein layer covers the active sites on the surface of the nano-selenium particles, preventing coordination complexation between the nano-selenium particles and the carboxyl groups on the sodium alginate molecular chain. This avoids the aggregation of nano-selenium particles to the micron scale in the microencapsulated particle system, maintaining the nano-scale dispersion of nano-selenium in the finished sheep-rearing yeast culture. After the finished sheep-rearing yeast culture enters the rumen, the gelatin protein layer is gradually degraded by rumen proteases, releasing the dispersed nano-selenium particles. This facilitates absorption by the rumen epithelium, thereby providing selenium source support for maintaining the activity of selenium-dependent glutathione peroxidase.

[0078] To address the issue of decreased survival rate of live bacteria under high-temperature granulation and conditioning conditions, this invention employs a dual-wall structure consisting of a gel network formed by sodium alginate cross-linking with calcium chloride and a stearate-palmitate lipid barrier layer. The gel network provides a structural framework and some thermal insulation, while the lipid barrier layer impedes water vapor permeation and heat transfer. At ring die granulation temperatures of 65 to 80°C, the dual-wall structure reduces the actual temperature rise within the selenium-containing composite core material, allowing *Lactobacillus plantarum* and *Saccharomyces cerevisiae* embedded in the selenium-containing composite core material to maintain a certain survival rate after granulation. This overcomes the defect of significant inactivation of live bacteria in powdered cultures due to direct exposure to high-temperature steam.

[0079] To address the lack of a synergistic release mechanism between live bacteria, selenium source, and buffering components, and the inability to match the high-incidence time window of rumen acidosis, this invention co-formulates sodium bicarbonate micropowder, fermentation dry powder, and gelatin-coated nano-selenium micropowder into a selenium-containing composite core material, which is then embedded in microencapsulated particles. After the sheep-feeding compound yeast culture enters the rumen, the calcium alginate gel network gradually swells under rumen pH conditions of 5.5 to 6.8, releasing the contents of the selenium-containing composite core material. Sodium bicarbonate reacts with organic acids in the rumen to exert a local pH buffering effect. Live bacteria are released simultaneously and continuously regulate the rumen microecological balance. Nano-selenium is dispersed and released after protease degradation of the gelatin protein layer. The three functional components achieve simultaneous release through co-encapsulation, with their release time window matching the time window of increased rumen acid load after high-concentrate feed intake. This provides a triple function of microecological regulation, antioxidant protection, and acid buffering during the high-incidence stage of rumen acidosis, meeting the application needs of large-scale fattening sheep farms under the dual conditions of pelleting processing and the acidic environment of the rumen.

[0080] Example 1

[0081] Step 1: Dissolve 0.5 parts by weight of sodium selenite in 100 parts by weight of deionized water, add 4 parts by weight of ascorbic acid (sodium selenite to ascorbic acid mass ratio of 1:8), and stir at 200 rpm for 40 min to obtain a uniform red nano-selenium colloidal solution. After visually confirming that the solution is uniformly red and free of gray precipitate, add 2 parts by weight of gelatin (freezing strength 150 g, viscosity 2 mPa·s), and stir at 150 rpm for 1 h at 40℃ to coat the solution. Spray dry the coating solution with an inlet air temperature of 120℃ and an outlet air temperature of 60℃, ensuring the instantaneous residence time of the material does not exceed 5 s. Collect the gelatin-coated nano-selenium powder with a particle size of 1 μm.

[0082] Step 2: Mix 50 parts by weight of flaked corn with 40 parts by weight of corn, grind them through a 40-mesh sieve to obtain mixed corn flour. Add 18 parts by weight of soybean meal, 15 parts by weight of corn distillers grains, and 10 parts by weight of beet pulp, mix well, and then add deionized water to adjust the moisture content to 34 parts by weight (based on 100 parts by weight of dry matter). Premix brewer's yeast liquid and Lactobacillus plantarum liquid at a volume ratio of 1:2 and inoculate at once, with a total inoculation amount of 8 parts by weight (based on 100 parts by weight of the total mass of the mixed culture medium). Seal and anaerobic ferment at 30℃ for 40 hours. The pH at the end of fermentation is measured to be 3.8, yielding solid fermented product.

[0083] Step 3: Add 65 parts by weight of solid fermentation product and 25 parts by weight of liquid yeast metabolite (15 parts by weight of dry matter) to a mixer and mix at room temperature for 15 minutes until homogeneous. Vacuum dry at -0.06 MPa and 42°C for 4 hours until the moisture content drops to 8 parts by weight (based on 100 parts by weight on a dry basis). Pulverize the dried product through a 60-mesh sieve to obtain fermentation powder.

[0084] Step 4: Add 100 parts by weight of fermented dry powder, 0.5 parts by weight of gelatin-coated nano-selenium powder, and 2 parts by weight of sodium bicarbonate powder (particle size 60 mesh) to a mixer and mix at room temperature for 10 minutes until uniform to obtain selenium-containing composite core material.

[0085] Step 5: Dissolve 2 parts by weight of sodium alginate (viscosity 200 mPa·s, weight-average molecular weight 120,000 Da) in 40 parts by weight of deionized water, add 3 parts by weight of glyceryl stearate palmitate, and emulsify and disperse at 58℃ and 800 rpm for 20 min to form a wall material emulsion. After cooling the wall material emulsion to 35℃, add 100 parts by weight of selenium-containing composite core material, stir and disperse for 20 min until uniform, to obtain the coating slurry.

[0086] Step 6: Spray granulation of the coating slurry in a fluidized bed coating equipment with an inlet air temperature of 36℃ and an atomization pressure of 0.15MPa to obtain microencapsulated particles with a particle size of 0.5mm. First, pre-wet the surface of the microencapsulated particles with a 0.5% calcium chloride solution (3 parts by weight, based on 100 parts by weight of the total mass of the microencapsulated particles) for 1 minute. Then, immerse the microencapsulated particles in a calcium chloride solution with a concentration of 2 parts by weight per 100 parts by weight of water at 20℃ for cross-linking and curing for 10 minutes. After filtration, air dry at 35℃ until the moisture content does not exceed 9 parts by weight (based on 100 parts by weight of dry basis) to obtain the finished sheep breeding compound yeast culture product, designated as Sample 1.

[0087] Example 2

[0088] Step 1: Dissolve 0.85 parts by weight of sodium selenite in 100 parts by weight of deionized water, add 5 parts by weight of ascorbic acid (the mass ratio of sodium selenite to ascorbic acid is 1:5.9, taking the median value), and stir at 300 rpm for 50 min to obtain a uniform red nano-selenium colloidal solution. After confirming that the solution appearance is qualified, add 3 parts by weight of gelatin (freezing power 200 g, viscosity 3.5 mPa·s), and stir at 225 rpm for 1.5 h at 44℃ to coat the solution. Spray dry with an inlet air temperature of 130℃ and an outlet air temperature of 65℃, ensuring the instantaneous residence time of the material does not exceed 5 s. Collect the gelatin-coated nano-selenium powder with a particle size of 5 μm.

[0089] Step 2: Mix 55 parts by weight of flaked corn with 45 parts by weight of corn, and pulverize through a 50-mesh sieve. Add 20 parts by weight of soybean meal, 17.5 parts by weight of corn distillers grains, and 12.5 parts by weight of beet pulp, mix thoroughly, and adjust the moisture content to 36 parts by weight (based on 100 parts by weight of dry matter). Inoculate with a premix of brewer's yeast and Lactobacillus plantarum culture at a volume ratio of 1:2.25, with a total inoculation amount of 11.5 parts by weight. Seal and anaerobic ferment at 32.5℃ for 48 hours. The final pH of the fermentation is measured to be 4.2, yielding a solid-state fermented product.

[0090] Step 3: Mix 70 parts by weight of solid fermentation material with 30 parts by weight of liquid yeast metabolite (20 parts by weight of dry matter) for 22.5 min. Vacuum dry at -0.075 MPa and 46°C for 5.5 h until the moisture content drops to 9.5 parts by weight (based on 100 parts by weight on a dry basis). Grind through a 70-mesh sieve to obtain fermented dry powder.

[0091] Step 4: Mix 100 parts by weight of fermented dry powder with 1.0 part by weight of gelatin-coated nano-selenium powder and 3 parts by weight of sodium bicarbonate powder (particle size 80 mesh) for 15 minutes to obtain selenium-containing composite core material.

[0092] Step 5: Dissolve 2.75 parts by weight of sodium alginate (viscosity 300 mPa·s, weight-average molecular weight 170,000 Da) in 47.5 parts by weight of deionized water, add 4 parts by weight of glyceryl stearate palmitate, and emulsify and disperse at 61.5℃ and 1000 rpm for 30 min to form a wall material emulsion. After cooling the wall material emulsion to 37.5℃, add 100 parts by weight of selenium-containing composite core material, stir and disperse for 30 min to obtain the coating slurry.

[0093] Step 6: Fluidized bed spray granulation with an inlet air temperature of 39.5℃ and an atomization pressure of 0.225MPa yields microencapsulated particles with a particle size of 1.0mm. These particles are pre-wetted with a 0.75% calcium chloride solution (4 parts by weight) for 1.5 minutes. They are then immersed in a calcium chloride solution containing 2.5 parts by weight of calcium chloride per 100 parts by weight of water at 22.5℃ for 15 minutes to cross-link and solidify. After filtration, the solution is air-dried at 38.5℃ until the moisture content does not exceed 9 parts by weight (based on 100 parts by weight on a dry basis), yielding the finished sheep-rearing compound yeast culture, designated as Sample 2.

[0094] Example 3

[0095] Step 1: Dissolve 1.2 parts by weight of sodium selenite in 100 parts by weight of deionized water, add 6 parts by weight of ascorbic acid (sodium selenite to ascorbic acid mass ratio of 1:5), and stir at 400 rpm for 60 min to obtain a uniform red nano-selenium colloidal solution. After confirming that the solution appearance is qualified, add 4 parts by weight of gelatin (gel strength 250 g, viscosity 5 mPa·s), and stir at 300 rpm for 2 h at 48℃ to coat the solution. Spray dry with an inlet air temperature of 140℃ and an outlet air temperature of 70℃, ensuring the instantaneous residence time of the material does not exceed 5 s. Collect the gelatin-coated nano-selenium powder with a particle size of 10 μm.

[0096] Step 2: Mix 60 parts by weight of flaked corn with 50 parts by weight of corn, and pulverize through a 60-mesh sieve. Add 22 parts by weight of soybean meal, 20 parts by weight of corn alcohol residue, and 15 parts by weight of beet pulp, mix thoroughly, and adjust the moisture content to 38 parts by weight (based on 100 parts by weight of dry matter). Inoculate with a premix of brewer's yeast and Lactobacillus plantarum culture at a volume ratio of 1:2.5, for a total inoculation amount of 15 parts by weight. Seal and anaerobic ferment at 35°C for 56 hours. The final pH of the fermentation is measured to be 4.5, yielding a solid-state fermented product.

[0097] Step 3: Mix 75 parts by weight of solid fermentation material with 35 parts by weight of liquid yeast metabolite (25 parts by weight of dry matter) for 30 minutes. Vacuum dry at -0.09 MPa and 50°C for 7 hours until the moisture content drops to 11 parts by weight (based on 100 parts by weight on a dry basis). Grind through an 80-mesh sieve to obtain fermented dry powder.

[0098] Step 4: Mix 100 parts by weight of fermented dry powder with 1.5 parts by weight of gelatin-coated nano-selenium powder and 4 parts by weight of sodium bicarbonate powder (particle size 100 mesh) for 20 minutes to obtain selenium-containing composite core material.

[0099] Step 5: Dissolve 3.5 parts by weight of sodium alginate (viscosity 400 mPa·s, weight-average molecular weight 220,000 Da) in 55 parts by weight of deionized water, add 5 parts by weight of glyceryl stearate palmitate, and emulsify and disperse at 65℃ and 1200 rpm for 40 min to form a wall material emulsion. After cooling the wall material emulsion to 40℃, add 100 parts by weight of selenium-containing composite core material, stir and disperse for 40 min to obtain the coating slurry.

[0100] Step 6: Fluidized bed spray granulation with an inlet air temperature of 43℃ and an atomization pressure of 0.3MPa yields microencapsulated particles with a particle size of 1.5mm. The particles are pre-wetted with a 1% calcium chloride solution (5 parts by weight) for 2 minutes; then immersed in a calcium chloride solution containing 3.5 parts by weight of calcium chloride per 100 parts by weight of water at 25℃ for cross-linking and curing for 20 minutes; after filtration, the particles are air-dried at 42℃ until the moisture content does not exceed 9 parts by weight (based on 100 parts by weight on a dry basis) to obtain the finished sheep-rearing compound yeast culture, designated as Sample 3.

[0101] Example 4

[0102] All parameters for steps 1 to 6 are the same as in Example 2, except that the mass ratio of sodium selenite to ascorbic acid in step 1 is adjusted to 1:3 (minimum endpoint value). Specifically, 0.85 parts by weight of sodium selenite are added to 2.55 parts by weight of ascorbic acid (0.85 × 3 = 2.55 parts by weight), and the mixture is stirred at 300 rpm for 50 min. All other parameters for step 1 and all parameters for steps 2 to 6 are consistent with those in Example 2. The resulting sheep-rearing compound yeast culture product is designated as Sample 4.

[0103] Example 5

[0104] All parameters for steps 1 to 6 are the same as in Example 2, except that the mass ratio of sodium selenite to ascorbic acid in step 1 is adjusted to 1:12 (maximum endpoint value). Specifically, 0.85 parts by weight of sodium selenite are added to 10.2 parts by weight of ascorbic acid (0.85 × 12 = 10.2 parts by weight), and the mixture is stirred at 300 rpm for 50 min. All other parameters for step 1 and steps 2 to 6 are consistent with those in Example 2. The resulting sheep-rearing compound yeast culture product is designated as Sample 5.

[0105] Comparative Example 1

[0106] The gelatin coating step is omitted in step 1: 0.85 parts by weight of sodium selenite are dissolved in 100 parts by weight of deionized water, and 5 parts by weight of ascorbic acid are added. The mixture is stirred at 300 rpm for 50 min to obtain a nano-selenium colloidal solution, which is then directly spray-dried (inlet air temperature 130℃, outlet air temperature 65℃) to obtain uncoated nano-selenium powder. All parameters in steps 2 to 6 are completely consistent with those in Example 2. In step 4, an equal amount of uncoated nano-selenium powder is used to replace gelatin-coated nano-selenium powder in the selenium-containing composite core material. The resulting product is designated as Sample 6.

[0107] Comparative Example 2

[0108] In step 5, glyceryl stearate palmitate was omitted, and only sodium alginate was used as the wall material: 2.75 parts by weight of sodium alginate was dissolved in 47.5 parts by weight of deionized water, and stirred at 61.5°C and 1000 rpm for 30 minutes to form a single wall material solution; after cooling to 37.5°C, 100 parts by weight of selenium-containing composite core material was added, and the mixture was stirred and dispersed for 30 minutes to obtain the coating slurry. All parameters in steps 1 to 4 and step 6 were completely consistent with those in Example 2. The resulting product was designated as Sample 7.

[0109] Comparative Example 3

[0110] In step 4, sodium bicarbonate micropowder was omitted: 100 parts by weight of fermented dry powder and 1.0 part by weight of gelatin-coated nano-selenium micropowder were added to a mixer and mixed at room temperature for 15 minutes until homogeneous, resulting in a selenium-containing composite core material without buffer components. All parameters in steps 1 to 3 and steps 5 to 6 were completely consistent with those in Example 2. The resulting product was designated as Sample 8.

[0111] Experimental verification:

[0112] This experiment conducted three core performance tests on the sheep-rearing compound yeast cultures prepared in Examples 1 to 5 and Comparative Examples 1 to 3: First, the dispersion state of nano-selenium in the finished product was characterized to verify the effect of the gelatin coating step on preventing the nano-selenium from coordinating and agglomerating with the sodium alginate wall material; second, the viability of live bacteria in the finished product after simulated granulation, conditioning, and heat treatment was determined to verify the thermal protection effect of the dual wall material structure on live bacteria; and third, the pH buffering capacity of the finished product under simulated rumen acidic environment was determined to verify the functional contribution of the sodium bicarbonate buffer component.

[0113] Experimental sample preparation:

[0114] Sheep-rearing compound yeast cultures were prepared according to the methods described in Examples 1 to 5 and Comparative Examples 1 to 3, and were designated as Samples 1 to 8 respectively. After the preparation of each sample, the moisture content of the finished product was confirmed to be no more than 9 parts by weight (based on 100 parts by weight on a dry basis) by the loss on drying method, the particle size was confirmed to be concentrated within the range specified in each example by the standard sieving method, and the total selenium content was determined by inductively coupled plasma mass spectrometry. Only after the above three indicators were qualified could the samples be used for subsequent experiments.

[0115] Experimental conditions:

[0116] Characterization of the dispersion state of nano-selenium: The morphology and distribution of nano-selenium particles in the cross-section of each sample were observed using transmission electron microscopy (accelerating voltage 80kV). Simultaneously, the particle size distribution of selenium particles in the released fraction after soaking in phosphate buffer (pH 6.5, simulating rumen fluid) for 30 min was determined using a laser diffraction particle size analyzer. The value (particle size at 90% of the volumetric cumulative distribution) characterizes the degree of aggregation.

[0117] Simulated granulation and conditioning heat treatment: Referring to the ring die granulation and conditioning process of large-scale fattening sheep farms, each sample was placed in a sealed container and treated with 75℃ steam for 3 minutes to simulate the hot steam exposure conditions during conditioning, followed by cooling to room temperature. The viable counts of *Saccharomyces cerevisiae* (YPD agar medium, 30℃ for 48 h) and *Lactobacillus plantarum* (MRS agar medium, 37℃ for 48 h) were determined using the plate count method before and after heat treatment, respectively. The viable cell survival rate after heat treatment was calculated using the following formula:

[0118]

[0119] in, The survival rate of live bacteria (%). The number of viable bacteria after heat treatment (CFU / g) The number of viable bacteria (CFU / g) before heat treatment.

[0120] Assay for simulated rumen pH buffering capacity: Weigh 2.0 g of each sample and add 100 mL of acetate-sodium acetate buffer (pH 5.2, simulating rumen acidosis after high concentrate feed intake). Incubate at 39℃ and 100 rpm for 60 min. Record the pH value every 10 min. The endpoint pH value and the magnitude of pH increase at 60 min are recorded. Characterizing buffering capacity, where:

[0121]

[0122] in, The endpoint pH value after 60 minutes of incubation. The initial pH value at the start of incubation (5.2).

[0123] Experimental steps:

[0124] Step 1: Prepare samples 1 to 8 according to the above experimental sample preparation method, and complete the quality confirmation test of moisture, particle size and total selenium content.

[0125] Step 2: Prepare ultrathin cross-sectional sections for transmission electron microscopy observation of samples 1 to 8. Simultaneously, disperse 1.0 g of each sample in 10 mL of pH 6.5 phosphate buffer, shake and soak for 30 min, then centrifuge. Collect the supernatant for laser diffraction particle size analysis to determine the released selenium particles. value.

[0126] Step 3: Take 20g of each of samples 1 to 8. Before heat treatment, determine the viable counts of *Saccharomyces cerevisiae* and *Lactobacillus plantarum* using the plate count method, and record them as the viable counts before heat treatment. Then, treat each sample with 75℃ steam for 3 minutes under the simulated granulation and conditioning heat treatment conditions described above. After cooling, determine the viable counts again using the plate count method, and record them according to the formula... Calculate the survival rates of Saccharomyces cerevisiae and Lactobacillus plantarum.

[0127] Step 4: Take 2.0 g of each of samples 1 to 8, add 100 mL of pH 5.2 acetate-sodium acetate buffer, and incubate at 39℃ and 100 rpm for 60 min with shaking. Record the pH values ​​at each time point (0 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min), and calculate... (60 min final pH minus initial pH 5.2).

[0128] Step 5: Summarize all test data from Steps 2 to 4, fill in the results table, and conduct inter-group comparative analysis.

[0129] Experimental or test results such as Figures 2-8 And as shown in the table below:

[0130] Table 1 Key preparation parameters for each sample from step 1 to step 4

[0131]

[0132] Table 2 Summary of characterization results of nano-selenium dispersion state, viable bacterial survival rate, and pH buffering capacity

[0133]

[0134] As shown in Table 2, the following three conclusions are supported by experimental data.

[0135] Firstly, regarding the dispersion state of nano-selenium, the selenium particles that release the components from samples 1 to 5 (Examples 1 to 5) and samples 7 and 8... The values ​​were all within the range of 175 to 210 nm. Transmission electron microscopy showed that the selenium nanoparticles were uniformly dispersed and the gelatin coating layer was intact, indicating that within the parameter range specified in this invention, the gelatin coating step can effectively prevent the coordination complexation of the selenium nanoparticles with the carboxyl groups in the sodium alginate wall material, thus maintaining the nanoscale dispersion of selenium nanoparticles in the finished product. Sample 6 (Comparative Example 1) omitted the gelatin coating step, releasing the selenium particles. The value reached 3850 nm, which is about 22 times higher than that of sample 2. A large number of micron-sized aggregates were observed under transmission electron microscopy, which directly proves that the gelatin coating step is a necessary condition to prevent the coordination complexation and aggregation of nano-selenium and sodium alginate wall material.

[0136] Secondly, regarding the heat protection effect on live bacteria, after heat treatment with steam at 75℃ for 3 minutes, samples 1 to 6 and sample 8, containing dual wall materials, showed survival rates of 68.4% to 71.4% for *Saccharomyces cerevisiae* and 65.5% to 72.7% for *Lactobacillus plantarum*, both remaining at relatively high levels. In sample 7 (Comparative Example 2), after omitting the stearate palmitate lipid barrier layer, the survival rate of *Saccharomyces cerevisiae* decreased to 31.0%, and the survival rate of *Lactobacillus plantarum* decreased to 29.7%, representing decreases of approximately 56.6% and 59.2% respectively compared to sample 2. This indicates that relying solely on the calcium alginate gel network as a single wall material, the heat transfer barrier effect of high-temperature steam to the core material is insufficient, significantly exacerbating heat damage to live bacteria. The comparison between sample 2 and sample 7 directly demonstrates that the dual wall material structure, composed of the stearate palmitate lipid barrier layer and the calcium alginate gel network, is a necessary condition for protecting live bacteria to withstand the high temperatures of granulation and conditioning.

[0137] Thirdly, regarding rumen acid buffering capacity, samples 1 to 7, containing sodium bicarbonate buffer components, after incubation for 60 minutes in a simulated rumen acidic environment (initial pH 5.2), showed an endpoint... The pH increase ranged from 0.48 to 0.73, with the increase in sodium bicarbonate content being greater than that, indicating that sodium bicarbonate can effectively neutralize organic acids under rumen acidic conditions and provide local pH buffering protection. Sample 8 (Comparative Example 3), after omitting sodium bicarbonate, was incubated for 60 min... Only 0.04, compared to sample 2 ( The concentration decreased by about 93.5%, indicating almost no buffering capacity, which directly proves that sodium bicarbonate buffering components are a necessary condition for realizing the buffering and regulation function of rumen acidosis.

[0138] Based on the above three sets of experimental results, the sheep-rearing compound yeast culture prepared by this invention is supported by experimental data in the following three aspects.

[0139] First, the necessity of the gelatin coating step. Selenium particles releasing the gelatinous component in Examples 1 to 5 (samples 1 to 5) The values ​​were all below 210 nm, while Comparative Example 1 (Sample 6) omitted the gelatin coating. The value increased to 3850nm, and the degree of aggregation increased by about 22 times, proving that the gelatin protein layer effectively prevented the coordination complexation reaction between the nano-selenium and the carboxyl groups of the sodium alginate wall material through physical isolation, so that the nano-selenium maintained nanoscale dispersion in the finished product, which is conducive to its absorption by the epithelium after being degraded by proteases in the rumen.

[0140] Secondly, the dual-wall structure provides thermal protection for live bacteria. After steam heat treatment at 75°C, the survival rate of live bacteria in the example samples containing the dual-wall structure remained between 65% and 73%, while the survival rate in Comparative Example 2 (sample 7), which omitted the lipid barrier layer, dropped to below 30%, with a live bacteria loss rate approximately 2.3 times higher. This demonstrates that the dual-wall structure, synergistically formed by the stearate palmitate lipid barrier layer and the calcium alginate gel network, provides significant thermal insulation protection for live bacteria in the selenium-containing composite core material under granulation and conditioning temperature conditions.

[0141] Thirdly, the rumen acid buffering function of the sodium bicarbonate buffer component. The example sample containing sodium bicarbonate was tested in a simulated rumen acidic environment for 60 minutes. The values ​​ranged from 0.48 to 0.73, while Comparative Example 3 (Sample 8) omitted sodium bicarbonate. With a concentration of only 0.04, the buffering capacity decreased by approximately 93.5%, demonstrating that the simultaneous release of sodium bicarbonate micropowder, live bacteria, and nano-selenium after co-encapsulation can provide effective local pH buffering protection during the time window of increased rumen acid load after high-concentrate feed intake, achieving synergistic release and functional complementarity of the three functional components.

[0142] The embodiments of the present invention have been described above. However, the embodiments are not limited to the specific implementation methods described above. The specific implementation methods described above are merely illustrative and not restrictive. Those skilled in the art can make more equivalent embodiments under the guidance of the present embodiments, and all of them are within the protection scope of the present embodiments.

Claims

1. A method for preparing a sheep-rearing compound yeast culture, characterized in that, Includes the following steps: Sodium selenite was reduced with ascorbic acid to obtain nano-selenium colloid, which was then coated with gelatin and spray-dried to obtain gelatin-coated nano-selenium powder. After mixing and adjusting the water content of flaked corn, corn, soybean meal, corn alcohol residue and beet meal, the mixture is inoculated with brewer's yeast liquid and Lactobacillus plantarum liquid for anaerobic fermentation to obtain solid fermentation product. The solid fermentation product is then mixed with liquid yeast metabolite, vacuum dried and pulverized to obtain fermented dry powder. A selenium-containing composite core material is obtained by mixing fermented dry powder with gelatin-coated nano-selenium powder and sodium bicarbonate powder. Sodium alginate and glyceryl stearate palmitate were emulsified and dispersed to form a wall material emulsion. After cooling, a selenium-containing composite core material was added and stirred to obtain a coating slurry. The coating slurry is spray-granulated to obtain microencapsulated particles, which are then cross-linked and cured with calcium chloride solution and dried to obtain the finished product.

2. The preparation method according to claim 1, characterized in that, 0.5 to 1.2 parts by weight of sodium selenite are dissolved in 100 parts by weight of deionized water, and 4 to 6 parts by weight of ascorbic acid are added and stirred at room temperature for 40 to 60 minutes. 2 to 4 parts by weight of gelatin are added to the nano-selenium colloid, and the mixture is stirred and coated at 40 to 48°C for 1 to 2 hours. The spray drying process is carried out at an inlet air temperature of 120 to 140°C and an outlet air temperature of 60 to 70°C, with the instantaneous residence time of the material not exceeding 5 seconds. The resulting gelatin-coated nano-selenium powder has a particle size of 1 to 10 μm.

3. The preparation method according to claim 2, characterized in that, The gelatin is food-grade gelatin with a gel strength of 150 to 250 g and a viscosity of 2 to 5 mPa·s, measured as a 6.67% aqueous solution at 60°C. The stirring speed during the reduction reaction stage is 200 to 400 rpm, and the stirring speed during the coating stage is 150 to 300 rpm.

4. The preparation method according to claim 1, characterized in that, In the anaerobic fermentation, 50 to 60 parts by weight of flaked corn and 40 to 50 parts by weight of corn are mixed, crushed, and sieved through a 40 to 60 mesh sieve. 18 to 22 parts by weight of soybean meal, 15 to 20 parts by weight of corn distillers grains, and 10 to 15 parts by weight of beet pulp are added. The moisture content is adjusted to 34 to 38 parts by weight. The volume ratio of brewer's yeast liquid to Lactobacillus plantarum liquid is 1:2 to 1:2.

5. The total inoculum amount is 8 to 15 parts by weight of the total mass of the mixed culture medium. The mixture is then sealed and anaerobic fermented at 30 to 35°C for 40 to 56 hours.

5. The preparation method according to claim 1, characterized in that, In the vacuum drying process, 65 to 75 parts by weight of solid fermentation product and 25 to 35 parts by weight of liquid yeast metabolite are mixed and dried for 4 to 7 hours under vacuum conditions of -0.06 to -0.09 MPa and temperature of 42 to 50°C until the moisture content drops to 8 to 11 parts by weight. The dried product is then pulverized through a 60 to 80 mesh sieve.

6. The preparation method according to claim 1, characterized in that, In the selenium-containing composite core material, based on 100 parts by weight of fermented dry powder, gelatin-coated nano-selenium powder is 0.5 to 1.5 parts by weight, sodium bicarbonate powder is 2 to 4 parts by weight, and the particle size of the sodium bicarbonate powder is 60 to 100 mesh.

7. The preparation method according to claim 1, characterized in that, In the wall material emulsion, based on 100 parts by weight of selenium-containing composite core material, 2 to 3.5 parts by weight of sodium alginate are dissolved in 40 to 55 parts by weight of deionized water, and 3 to 5 parts by weight of glyceryl stearate palmitate are added. The mixture is emulsified and dispersed at 58 to 65°C for 20 to 40 minutes with a stirring speed of 800 to 1200 rpm. After cooling to 35 to 40°C, the selenium-containing composite core material is added and stirred and dispersed for 20 to 40 minutes.

8. The preparation method according to claim 1, characterized in that, The inlet air temperature for spray granulation is 36 to 43°C, the atomization pressure is 0.15 to 0.3 MPa, and the microencapsulated particle size is 0.5 to 1.5 mm. The concentration of the calcium chloride solution is 2 to 3.5 parts by weight of calcium chloride per 100 parts by weight of water. The crosslinking and curing time is 10 to 20 min, the crosslinking and curing temperature is 20 to 25°C, the airflow drying temperature is 35 to 42°C, and the moisture content of the finished product does not exceed 9 parts by weight.

9. The preparation method according to claim 8, characterized in that, Before immersing the microencapsulated particles into the calcium chloride solution, the surface of the microencapsulated particles is pre-sprayed and wetted with a calcium chloride solution with a mass fraction of 0.5 to 1%. The spraying amount is 3 to 5 parts by weight of the microencapsulated particles, and the wetting time is 1 to 2 minutes. Then, the particles are immersed in the calcium chloride solution for cross-linking and curing.

10. A sheep-rearing compound yeast culture prepared by the preparation method according to any one of claims 1 to 9.

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