Microecological preparation for improving reproductive performance of sows and preparation method thereof

Abstract

The invention discloses a microecological preparation for improving reproductive performance of sows and a preparation method of the microecological preparation, and relates to the technical field of microorganism application. Comprising the following steps: activating and fermenting clostridium butyricum to obtain bacterial sludge; mixing the bacterial sludge with tryptone, glucose, L-cysteine and fructose to obtain an inner-layer mixture, and granulating to obtain inner-layer mixture particles; preparing chitosan-coated colloid, soaking the inner-layer mixture particles in the chitosan-coated colloid, taking out the inner-layer mixture particles, and freeze-drying the inner-layer mixture particles to obtain a barrier film outside the inner-layer mixture; and coating the outer surface of the barrier film with outer-layer powder to obtain the microecological preparation. According to the preparation method, bacterial sludge of clostridium butyricum is coated with a barrier film and outer-layer powder, and the microecological preparation which is used for feeding sows and carrying out clostridium butyricum intervention on pregnant sows is prepared; after the sow is fed with the feed mixed with the microecological preparation prepared by the invention, the reproductive capacity of the sow is obviously improved, and the growth and development of the piglet are also obviously promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial applications, specifically relates to the technical fields of probiotics and livestock and poultry breeding technology. More specifically, the present invention relates to a probiotic preparation for improving the reproductive performance of sows and a preparation method thereof. Background Art

[0002] In pig breeding, the reproductive ability of sows plays an important role. The reproductive ability of sows is mainly reflected in aspects such as backfat thickness, litter size performance, number of live-born piglets, number of stillborn piglets, litter weight at birth, and individual weight at birth. Among them, there is an important correlation between the backfat thickness of sows and their reproductive ability. Generally speaking, the thicker the backfat of sows, the lower their reproductive ability.

[0003] Research shows that the regulatory effect of the intestinal flora in sows has an important impact on the reproductive ability of sows. The dominant phyla in the sow intestine mainly include Firmicutes, Spirochaetes, Bacteroidetes, Actinobacteria, and Proteobacteria. Prevotella, Treponema, Ruminococcus, Streptococcus, and Intestinimonas are also relatively abundant in the intestine of pregnant sows; Clostridium butyricum mainly regulates the immune-metabolism of sows and controls the backfat thickness of sows to achieve the regulation of the reproductive performance of sows. Clostridium butyricum normally grows in the colon and cecum of pigs, and its suitable pH range is 4-10, and it is easily inactivated in gastric acid.

[0004] Therefore, in order to improve the reproductive performance of sows, it is extremely necessary to carry out relevant interventions using Clostridium butyricum. At present, probiotics are usually made into probiotic preparations (such as capsules, coated preparations) and mixed with feed to feed pigs to achieve the regulation of the intestinal flora in pigs.

[0005] Due to the particularity of pregnant sows, the requirements for the types of wall materials of probiotic preparations are relatively strict. Chitosan is an excellent membrane material for probiotic preparations, with good film-forming properties and low cost, and chitosan has broad-spectrum antibacterial properties (such as inhibiting Escherichia coli and Salmonella). When used in combination with probiotics (such as Lactobacillus and Bifidobacterium and Clostridium butyricum), it can inhibit the competitive proliferation of pathogenic bacteria and create a favorable colonization environment for beneficial bacteria. However, chitosan is easily soluble in acetic acid, oxalic acid, and citric acid, and the metabolites of butyric acid include butyric acid, acetic acid, lactic acid, formic acid, etc. Therefore, directly coating Clostridium butyricum with chitosan is relatively easy to damage the chitosan wall material from the inside, and chitosan is also relatively easily dissolved and damaged in gastric acid, which is not conducive to forming anti-gastric acid protection for probiotics; chitosan also has the characteristic of good hygroscopicity, absorbing water and swelling. Therefore, at present, chitosan is usually blended with substances with good film-forming properties such as guar gum, arabic gum, xanthan gum, sodium carboxymethyl cellulose, or konjac glucomannan to overcome the problems of poor hygroscopicity and acid resistance of chitosan. However, after blending various membrane materials, the film-forming property, film homogeneity, and coating property will become worse. Summary of the Invention

[0006] One object of the present invention is to solve at least the above problems and / or deficiencies and provide at least the advantages described hereinafter.

[0007] To achieve these objects and other advantages in accordance with the present invention, there is provided a method for preparing a probiotic preparation for improving the reproductive performance of sows, comprising the following steps: Step 1: Activate and ferment Clostridium butyricum to obtain a bacterial sludge; Step 2: Mix the bacterial sludge with tryptone, glucose, L-cysteine, and fructose to obtain an inner mixture, and granulate to obtain inner mixture particles; Step 3: Prepare a chitosan-coated colloid, soak the inner mixture particles in the chitosan-coated colloid, take them out and freeze-dry to obtain a barrier film on the outside of the inner mixture; Step 4: Coat an outer powder on the outer surface of the barrier film to obtain the probiotic preparation.

[0008] Preferably, in Step 1, the specific method for activating and fermenting Clostridium butyricum to obtain a bacterial sludge includes: S11: Dissolve Clostridium butyricum to obtain a bacterial solution, inoculate the bacterial solution into a sterilized RCM medium, and culture at 37 °C under anaerobic conditions on a shaker at 160-200 r / min for 12-24 h to obtain a Clostridium butyricum bacterial solution; S12: Inoculate the Clostridium butyricum seed solution into a fermentation tank and culture at 37 °C under anaerobic conditions for 12-24 h; centrifuge at 3000-6000 rpm for 20-40 min using a tubular centrifuge, and discard the supernatant to obtain a bacterial sludge.

[0009] Preferably, in Step 2, the mass ratio of the bacterial sludge, tryptone, glucose, and L-cysteine is 5-10:0.05-1:0.5-2:0.1-0.2:0.2-0.5.

[0010] Preferably, in Step 3, the specific method for preparing the chitosan-coated colloid includes: S31: Dissolve chitosan in an acetic acid solution to obtain a chitosan solution; add nano-silica powder and polyethylene glycol-1000 to the chitosan solution, and perform ultrasonic dispersion at 50-80 kHz for 30-60 min to obtain a dispersion system; S32: Add a 4 wt% genipin-ethanol solution to the dispersion system, heat and stir in a water bath to raise the temperature to 40-60 °C, keep warm for 6-12 h, and let it stand and cool to obtain a crude colloid solution; S33: Add sodium alginate to thicken the crude colloid solution, heat at 60-80 °C and evaporate for 1-3 h to obtain the chitosan-coated colloid.

[0011] Preferably, the nano-silica powder in S31 is poly(ethylene glycol)-400 modified nano-silica powder. The specific modification method includes: ultrasonically dispersing 60 - 80 kHz of nano-silica powder with a particle size of 50 - 120 nm in deionized water to obtain a dispersion liquid, with a dispersion time of 5 - 20 min; adding poly(ethylene glycol)-400 to the dispersion liquid, stirring at 600 - 1200 rpm and heating to 80 - 90 °C, holding for 1 - 3 h, cooling to room temperature and then standing for 12 - 24 h, centrifuging and washing the powder, and drying it in vacuum at 50 - 60 °C for 20 - 50 min to obtain poly(ethylene glycol)-400 modified nano-silica powder; the dosage ratio of nano-silica powder, poly(ethylene glycol)-400 and deionized water is 5 - 20 g: 0.5 - 5 g: 300 - 1000 mL.

[0012] Preferably, in S31, the degree of acetylation of the chitosan is greater than 80%, and the viscosity-average molecular weight is 500 - 2500 kDa; the concentration of acetic acid is 2 - 5 vol%; the dosage ratio of chitosan to acetic acid solution is 1 - 5 g: 100 - 200 mL.

[0013] Preferably, in S31, the mass ratio of chitosan, nano-silica powder and poly(ethylene glycol)-1000 is 1 - 5: 0.02 - 1: 0.05 - 1.

[0014] Preferably, in S32, the dosage of genipin-ethanol solution is 1% - 20% of the volume of the acetic acid solution; In S33, the mass ratio of sodium alginate to chitosan is 0.5: 1 - 5.

[0015] Preferably, in the fourth step, the outer layer powder by weight includes: 1 - 5 parts of sodium carboxymethylcellulose, 2 - 5 parts of starch, 5 - 10 parts of soybean powder and 1 - 2 parts of xanthan gum.

[0016] A probiotic preparation for improving the reproductive performance of sows, which is prepared by the preparation method of the probiotic preparation for improving the reproductive performance of sows described above.

[0017] The present invention has at least the following beneficial effects: By using a barrier film and an outer layer powder to coat the bacterial sludge of Clostridium butyricum, the present invention prepares a probiotic preparation that can be fed to sows; after feeding the feed mixed with the probiotic preparation prepared by the present invention, the reproductive ability of sows has been significantly improved, indicating that the probiotic preparation of the present invention can ensure that most Clostridium butyricum are released at a fixed point in the sow intestine and become dominant strains, intervening in the intestinal flora of sows. Clostridium butyricum regulates the backfat thickness of sows, regulates the metabolism of sows, and improves the reproductive performance of sows.

[0018] Among them, the barrier film used in the present invention uses chitosan as the main raw material. Since chitosan is easily soluble in acid, the present invention incorporates nano-silica and polyethylene glycol-1000, and then uses genipin for cross-linking, significantly reducing the acid solubility, improving the acid resistance of the barrier film and the probiotic preparation, effectively protecting the Clostridium butyricum inside the barrier film, preventing most of the Clostridium butyricum from being inactivated by gastric acid, and thus being released into the sow intestine, increasing the viable count of Clostridium butyricum in the sow intestine, enabling Clostridium butyricum to become one of the dominant bacterial species in the sow intestine, and regulating the metabolism of pregnant sows through Clostridium butyricum.

[0019] At the same time, after incorporating nano-silica modified by polyethylene glycol-400, the present invention significantly reduces the moisture absorption performance of chitosan, avoiding a large amount of water absorption during the storage of the probiotic preparation and after entering the sow digestive tract, thereby swelling and causing damage to the wall material of the probiotic preparation, and can significantly extend the storage time of the probiotic preparation, ensuring the integrity of the wall material structure of the probiotic preparation. Among them, polyethylene glycol-400 is coated on the surface of the nano-silica particles, and a large number of hydroxyl groups are modified on the surface of the nano-silica particles, improving the compatibility of nano-silica with chitosan and polyethylene glycol-1000, enabling the three to cross-link to obtain a dense and stable network structure, reducing the number of moisture absorption channels formed by the colloidal film layer, and reducing the water absorption rate of the chitosan colloidal film layer. At the same time, nano-silica also has the effect of enhancing the overall strength of the barrier film and reducing the swelling property of the barrier film.

[0020] Other advantages, objectives, and features of the present invention will be partially reflected by the following description and partially understood by those skilled in the art through the research and practice of the present invention. Detailed Description of the Invention

[0021] The following further detailed description of the present invention is provided so that those skilled in the art can implement it with reference to the text of the specification.

[0022] It should be understood that the terms such as "having", "comprising", and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0023] The preparation method of the bacterial sludge used in the following examples includes: S11. Take 10 mg of Clostridium butyricum original powder (purchased from Shandong Ruiyuan Biotechnology Co., Ltd., EINECS registration number 231-821-4, viable count 2.5×10 9Dissolve (CFU / g) in 0.5 mL of sterile water to obtain a bacterial solution. Inoculate the bacterial solution into 1000 mL of RCM medium (peptone 10 g / L, beef extract 10 g / L, yeast extract 3 g / L, glucose 5 g / L, starch 1 g / L, sodium chloride 5 g / L, agar 0.5 g / L, pH value 6.5) sterilized at 121 °C for 15 minutes. Incubate at 37 °C under anaerobic conditions on a shaker at 160 r / min for 12 h to obtain a Clostridium butyricum bacterial solution; S12: Inoculate the Clostridium butyricum seed solution into a 5 L fermenter (peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 5 g / L, starch 1 g / L, sodium chloride 2 g / L, agar 1 g / L, pH value 6.5). Incubate at 37 °C under anaerobic conditions for 24 h; Centrifuge at 6000 rpm for 30 min using a tubular centrifuge, and discard the supernatant to obtain bacterial sludge.

[0024] Example 1

[0025] A preparation method of a microecological preparation for improving the reproductive performance of sows, comprising the following steps: Step 1: Mix 50 g of bacterial sludge with 5 g of tryptone, 5 g of glucose, 2 g of L-cysteine, and 2 g of fructose to obtain an inner mixture; Granulate the inner mixture through fluidized bed granulation (inlet air temperature 30 °C, outlet temperature 35 °C) to obtain inner mixture particles.

[0026] Step 2: Prepare a chitosan-coated colloid. The specific method includes: S31: Dissolve 20 g of chitosan (degree of acetylation greater than 80%, viscosity-average molecular weight 2000 kDa) in 1000 mL of 3 vol% acetic acid solution to obtain a chitosan solution; Add 5 g of nano-silica powder and 5 g of polyethylene glycol-1000 to the chitosan solution, and perform ultrasonic dispersion at 60 kHz for 60 min to obtain a dispersion system; S32: Add 50 mL of 4 wt% genipin-ethanol solution to the dispersion system, heat and stir in a water bath to raise the temperature to 50 °C, keep warm for 6 h, and let it stand and cool to obtain a crude colloid solution; S33: Add 5 g of sodium alginate to the crude colloid solution for thickening, heat at 60 °C and evaporate for 1 h to obtain a chitosan-coated colloid; Immerse the inner mixture particles in the chitosan-coated colloid for 3 h, take them out and freeze-dry at -40 °C for 30 min to obtain a barrier film on the outside of the inner mixture particles; Step 3: Coat the outer powder on the outer surface of the barrier film. The outer powder includes, by weight: 20 g of sodium carboxymethylcellulose, 20 g of starch, 50 g of soybean powder, and 10 g of xanthan gum. Mix the above raw materials and add them to 400 mL of sterile water, and coat them on the outer surface of the barrier film (coat 50 g of inner mixture particles with every 100 g of outer powder). After freeze-drying at -40 °C for 1 h, a microecological preparation is obtained.

[0027] Example 2

[0028] A preparation method of a microecological preparation for improving the reproductive performance of sows, comprising the following steps: Step 1: Mix 50 g of bacterial sludge with 5 g of tryptone, 5 g of glucose, 2 g of L-cysteine, and 2 g of fructose to obtain an inner mixture; granulate the inner mixture through a fluidized bed (inlet air temperature 30°C, outlet temperature 35°C) to obtain inner mixture particles.

[0029] Step 2: Prepare a chitosan-coated colloid, and the specific method includes: S31: Dissolve 30 g of chitosan (degree of acetylation greater than 80%, viscosity-average molecular weight of 2200 kDa) in 1000 mL of 3 vol% acetic acid solution to obtain a chitosan solution; add 8 g of nano-silica powder, 5 g of polyethylene glycol-1000 to the chitosan solution, and after ultrasonic dispersion at 60 kHz for 60 min, obtain a dispersion system; S32: Add 30 mL of 4 wt% genipin-ethanol solution to the dispersion system, heat and stir in a water bath to 50°C, keep warm for 6 h, and obtain a crude colloid solution after standing and cooling; S33: Add 5 g of sodium alginate to the crude colloid solution for thickening, heat at 60°C and evaporate for 1 h to obtain a chitosan-coated colloid; soak the inner mixture particles in the chitosan-coated colloid for 3 h, take them out and freeze-dry at -40°C for 30 min to obtain a barrier film on the outside of the inner mixture particles; Step 3: Coat the outer powder on the outer surface of the barrier film. The outer powder includes, by weight: 20 g of sodium carboxymethylcellulose, 20 g of starch, 50 g of soybean powder, and 10 g of xanthan gum. Mix the above raw materials and add them to 400 mL of sterile water, and coat them on the outer surface of the barrier film (50 g of inner mixture particles are coated with 100 g of outer powder), and obtain the microecological preparation after freeze-drying at -40°C for 1 h.

[0030] Example 3

[0031] A preparation method of a microecological preparation for improving the reproductive performance of sows, comprising the following steps: Step 1: Mix 50 g of bacterial sludge with 5 g of tryptone, 5 g of glucose, 2 g of L-cysteine, and 2 g of fructose to obtain an inner mixture; granulate the inner mixture through a fluidized bed (inlet air temperature 30°C, outlet temperature 35°C) to obtain inner mixture particles.

[0032] Step 2: Prepare a chitosan-coated colloid, and the specific method includes: S31. Dissolve 20 g of chitosan (with a degree of acetylation greater than 80% and a viscosity-average molecular weight of 1500 kDa) in 1000 mL of 3 vol% acetic acid solution to obtain a chitosan solution; add 5 g of nano-silica powder and 4 g of polyethylene glycol-1000 to the chitosan solution, and after ultrasonic dispersion at 60 kHz for 60 min, obtain a dispersion system; S32. Add 20 mL of 4 wt% genipin-ethanol solution to the dispersion system, heat and stir in a water bath to raise the temperature to 50 °C, keep warm for 6 h, and after standing and cooling, obtain a crude colloidal solution; S33. Add 5 g of sodium alginate to thicken the crude colloidal solution, heat at 60 °C and evaporate for 1 h to obtain a chitosan-coated colloid; soak the inner layer mixture particles in the chitosan-coated colloid for 3 h, take them out and freeze-dry at -40 °C for 30 min to obtain a barrier film on the outside of the inner layer mixture particles; Step 3. Coat the outer layer powder on the outer surface of the barrier film. The outer layer powder includes, by weight: 20 g of sodium carboxymethylcellulose, 20 g of starch, 50 g of soybean powder, and 10 g of xanthan gum. Mix the above raw materials and put them into 400 mL of sterile water, and coat them on the outer surface of the barrier film (coat 50 g of inner layer mixture particles with every 100 g of outer layer powder), and after freeze-drying at -40 °C for 1 h, obtain a probiotic preparation.

[0033] Example 4

[0034] A preparation method of a probiotic preparation for improving the reproductive performance of sows, comprising the following steps: Step 1. Mix 50 g of bacterial sludge with 5 g of tryptone, 5 g of glucose, 2 g of L-cysteine, and 2 g of fructose to obtain an inner layer mixture; granulate the inner layer mixture by fluidized bed granulation (inlet air temperature 30 °C, outlet temperature 35 °C) to obtain inner layer mixture particles.

[0035] Step 2. Prepare a chitosan-coated colloid, and the specific method includes: S31. Dissolve 20 g of chitosan (with a degree of acetylation greater than 80% and a viscosity-average molecular weight of 2000 kDa) in 1000 mL of 3 vol% acetic acid solution to obtain a chitosan solution; add 5 g of nano-silica powder modified by polyethylene glycol-400 and 5 g of polyethylene glycol-1000 to the chitosan solution, and after ultrasonic dispersion at 60 kHz for 60 min, obtain a dispersion system; Among them, the preparation method of the nano-silica powder modified by polyethylene glycol-400 includes: Disperse 10 g of nano-silica powder with a particle size of 50 - 120 nm in 500 mL of deionized water by ultrasonic dispersion at 80 kHz to obtain a dispersion liquid, with a dispersion time of 20 min; add 1 g of polyethylene glycol - 400 to the dispersion liquid, stir and heat it to 80 °C at 600 rpm, keep it warm for 2 h, cool it to room temperature and then let it stand for 24 h, centrifuge and wash the powder, and dry it in vacuum at 60 °C for 30 min to obtain polyethylene glycol - 400 modified nano-silica powder; S32. Add 50 mL of 4 wt% genipin - ethanol solution to the dispersion system, heat it with stirring in a water bath to 50 °C, keep it warm for 6 h, and let it stand and cool to obtain a crude colloidal solution; S33. Add 5 g of sodium alginate to thicken the crude colloidal solution, heat it to 60 °C and evaporate for 1 h to obtain a chitosan - coated colloid; soak the inner layer mixture particles in the chitosan - coated colloid for 3 h, take them out and freeze - dry them at - 40 °C for 30 min to obtain a barrier film on the outside of the inner layer mixture particles; Step 3. Coat the outer layer powder on the outer surface of the barrier film. The outer layer powder includes, by weight: 20 g of sodium carboxymethylcellulose, 20 g of starch, 50 g of soybean powder, and 10 g of xanthan gum. Mix the above raw materials and put them into 400 mL of sterile water, and coat them on the outer surface of the barrier film (coat 50 g of inner layer mixture particles with every 100 g of outer layer powder), and freeze - dry them at - 40 °C for 1 h to obtain the micro - ecological preparation.

[0036] Example 5

[0037] A preparation method of a micro - ecological preparation for improving the reproductive performance of sows, comprising the following steps: Step 1. Mix 50 g of bacterial sludge with 5 g of tryptone, 5 g of glucose, 2 g of L - cysteine, and 2 g of fructose to obtain an inner layer mixture; granulate the inner layer mixture through a fluidized bed (inlet air temperature 30 °C, outlet temperature 35 °C) to obtain inner layer mixture particles.

[0038] Step 2. Prepare a chitosan - coated colloid, and the specific method includes: S31. Dissolve 30 g of chitosan (degree of acetylation greater than 80%, viscosity - average molecular weight 2200 kDa) in 1000 mL of 3 vol% acetic acid solution to obtain a chitosan solution; add 8 g of polyethylene glycol - 400 modified nano - silica powder and 5 g of polyethylene glycol - 1000 to the chitosan solution, and after ultrasonic dispersion at 60 kHz for 60 min, obtain a dispersion system; Among them, the preparation method of the polyethylene glycol - 400 modified nano - silica powder includes: Disperse 20 g of nano-silica powder with a particle size of 50 - 120 nm in 600 mL of deionized water by ultrasonic dispersion at 80 kHz for 20 min to obtain a dispersion; add 2 g of polyethylene glycol - 400 to the dispersion, stir at 600 rpm and heat up to 80 °C, keep warm for 3 h, cool to room temperature and then stand for 24 h, centrifuge and wash the powder, and dry it in vacuum at 60 °C for 30 min to obtain polyethylene glycol - 400 modified nano-silica powder; S32: Add 30 mL of 4 wt% genipin - ethanol solution to the dispersion system, heat and stir in a water bath to 50 °C, keep warm for 6 h, and obtain a crude colloidal solution after standing and cooling; S33: Add 5 g of sodium alginate to thicken the crude colloidal solution, heat at 60 °C and evaporate for 1 h to obtain a chitosan - coated colloid; soak the inner layer mixture particles in the chitosan - coated colloid for 3 h, take them out and freeze - dry at - 40 °C for 30 min to obtain a barrier film on the outside of the inner layer mixture particles; Step 3: Coat the outer layer powder on the outer surface of the barrier film. The outer layer powder includes, by weight: 20 g of sodium carboxymethylcellulose, 20 g of starch, 50 g of soybean powder, and 10 g of xanthan gum. Mix the above raw materials and put them into 400 mL of sterile water, coat them on the outer surface of the barrier film (coat 50 g of inner layer mixture particles with every 100 g of outer layer powder), and obtain the microecological preparation after freeze - drying at - 40 °C for 1 h.

[0039] Comparative Example 1 A preparation method of a microecological preparation for improving the reproductive performance of sows, comprising the following steps: Step 1: Mix 50 g of bacterial sludge with 5 g of tryptone, 5 g of glucose, 2 g of L - cysteine, and 2 g of fructose to obtain an inner layer mixture; granulate the inner layer mixture by fluidized bed granulation (inlet air temperature 30 °C, outlet temperature 35 °C) to obtain inner layer mixture particles.

[0040] Step 2: Prepare a chitosan - coated colloid. The specific method includes: S31: Dissolve 20 g of chitosan (degree of acetylation greater than 80%, viscosity - average molecular weight 2000 kDa) in 1000 mL of 3 vol% acetic acid solution to obtain a chitosan solution; S32: Add 50 mL of 4 wt% genipin - ethanol solution to the chitosan solution, heat and stir in a water bath to 50 °C, keep warm for 6 h, and obtain a crude colloidal solution after standing and cooling; S33: Add 5 g of sodium alginate to thicken the crude colloidal solution, heat at 60 °C and evaporate for 1 h to obtain a chitosan - coated colloid; soak the inner layer mixture particles in the chitosan - coated colloid for 3 h, take them out and freeze - dry at - 40 °C for 30 min to obtain a barrier film on the outside of the inner layer mixture particles; Step 3. Coat the outer surface of the barrier film with an outer layer of powder. The outer layer of powder includes, by weight: 20 g of sodium carboxymethylcellulose, 20 g of starch, 50 g of soybean powder, and 10 g of xanthan gum. Mix the above raw materials and put them into 400 mL of sterile water, and coat them on the outer surface of the barrier film (coat 50 g of inner mixture particles with every 100 g of the outer layer of powder). After freeze-drying at -40°C for 1 h, a probiotic preparation is obtained.

[0041] Comparative Example 2 A method for preparing a probiotic preparation for improving the reproductive performance of sows, comprising the following steps: Step 1. Mix 50 g of bacterial sludge with 5 g of tryptone, 5 g of glucose, 2 g of L-cysteine, and 2 g of fructose to obtain an inner mixture; granulate the inner mixture through a fluidized bed (inlet air temperature 30°C, outlet temperature 35°C) to obtain inner mixture particles.

[0042] Step 2. Prepare a chitosan-coated colloid. The specific method includes: S31. Dissolve 30 g of chitosan (degree of acetylation greater than 80%, viscosity-average molecular weight 2200 kDa) in 1000 mL of 3 vol% acetic acid solution to obtain a chitosan solution; S32. Add 30 mL of 4 wt% genipin-ethanol solution to the chitosan solution, heat and stir in a water bath to raise the temperature to 50°C, keep warm for 6 h, and let it stand and cool to obtain a crude colloid solution; S33. Add 5 g of sodium alginate to thicken the crude colloid solution, heat at 60°C and evaporate for 1 h to obtain a chitosan-coated colloid; soak the inner mixture particles in the chitosan-coated colloid for 3 h, take them out and freeze-dry at -40°C for 30 min to obtain a barrier film on the outside of the inner mixture particles; Step 3. Coat the outer surface of the barrier film with an outer layer of powder. The outer layer of powder includes, by weight: 20 g of sodium carboxymethylcellulose, 20 g of starch, 50 g of soybean powder, and 10 g of xanthan gum. Mix the above raw materials and put them into 400 mL of sterile water, and coat them on the outer surface of the barrier film (coat 50 g of inner mixture particles with every 100 g of the outer layer of powder). After freeze-drying at -40°C for 1 h, a probiotic preparation is obtained.

[0043] The moisture absorption properties of the chitosan-coated colloids prepared in Examples 1-5 and Comparative Examples 1-2 were measured by the gravimetric method. The test method was as follows: The chitosan-coated colloids obtained in Examples 1-5 and Comparative Examples 1-2 were cast into films on glass plates and freeze-dried at -40°C for 30 min to obtain test samples. The test samples were cut into 10 cm × 10 cm pieces, and each piece was placed in a sealed constant-temperature box with a relative humidity of 81%RH. The measurement temperature was set at 30°C, and the measurement time was 72 h. The moisture absorption rate of each piece was calculated using the following formula: (m1 - m0) / m0 × 100%, where m1 was the weight of the sample after moisture absorption and m0 was the weight of the sample before moisture absorption. The results are shown in Table 1: Table 1 Moisture absorption rate of each sample

[0044] The acid resistance of the probiotics prepared in Examples 1-5 and Comparative Examples 1-2 was determined by gastric acid. The determination method was as follows: 16.4 mL of 0.1 kg / L hydrochloric acid solution was dissolved in 800 mL of water, 10 g of pepsin (1000 NFU / mg) was added, the pH was adjusted to 2.0, and the volume was fixed to 1000 mL; it was filtered and sterilized with a 0.22 μm sterile filter for standby.

[0045] 0.5 g of the probiotic was added to sterile simulated gastric juice and anaerobically cultured at 37°C for 2 h. Dilution coating and counting were performed at 0 and 2 h to calculate the survival rate.

[0046] The initial viable counts of each strain in the probiotics of each example and comparative example were detected by viable count method. After storing at 4°C for 100 days, the viable counts of each strain after 100 days were detected again. The results are shown in Table 2: Table 2 Viable counts of the probiotics of each example and comparative example

[0047] The probiotics prepared in Examples 1 - 5 and Comparative Examples 1 - 2 were respectively mixed with feed and fed to pregnant sows; wherein, the feed included: 65% corn, 10% wheat bran, 14% soybean meal, 5% cottonseed meal, 5% fish meal, 0.5% calcium hydrogen phosphate, and 0.5% salt. Each raw material was ground into powder to obtain the feed. The probiotic complex was mixed and formulated according to a ratio of 2 kg:1 t, and at the same time, the boar feed not formulated with the probiotic complex was used as a blank control. Pregnant sows at 50 days of pregnancy with a thick back fat and low reproductive ability were used as the research objects, with a back fat thickness of more than 3.5 cm. 800 sows were selected and evenly divided into 8 groups, that is, 100 sows in each group. They were respectively fed the feed mixed with the probiotic and the blank control feed. The feeding time started from 50 days of pregnancy of the sows and ended at the weaning of the suckling piglets at 21 days of age. The total number of piglets born, the number of live-born piglets, the number of stillborn piglets, the litter weight at birth, the individual weight at birth of each sow in each group were respectively measured, as well as the individual weight at weaning, average daily gain, diarrhea rate, and weaning survival rate of the piglets. The above data were all averaged. Table 3 and Table 4 were obtained: Table 3 Reproductive performance data of sows in each group

[0048] Table 4 Data of weaned piglets in each group

[0049] It can be seen from Table 3 and Table 4 that after feeding the feed mixed with the probiotics prepared in Examples 1 - 5, the reproductive performance of the sows was improved significantly, which was significantly better than that of Comparative Example 1, Comparative Example 2, and the blank control group, and the growth and development advantages of the piglets in the corresponding groups were also more obvious.

[0050] The equipment quantities and processing scales described here are used to simplify the description of the present invention. The applications, modifications, and variations of the present invention are obvious to those skilled in the art.

[0051] Although the embodiments of the present invention have been disclosed as above, it is not limited to only the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the examples shown and described here.

Claims

1. A preparation method of a microecological preparation for improving the reproductive performance of sows, characterized in that, It includes the following steps: Step 1: Activate and ferment Clostridium butyricum to obtain bacterial sludge; Step 2: Mix the bacterial sludge with tryptone, glucose, L-cysteine, and fructose to obtain an inner-layer mixture, and granulate it to obtain inner-layer mixture particles; Step 3: Prepare a chitosan-coated colloid, soak the inner-layer mixture particles in the chitosan-coated colloid, take them out and freeze-dry to obtain a barrier film outside the inner-layer mixture; Step 4: Coat the outer surface of the barrier film with an outer-layer powder to obtain a probiotic preparation.

2. The preparation method of the microecological preparation for improving the reproductive performance of sows according to claim 1, characterized in that, In the above Step 1, the specific method for activating and fermenting Clostridium butyricum to obtain bacterial sludge includes: S11: Dissolve Clostridium butyricum to obtain a bacterial solution, inoculate the bacterial solution into a sterilized RCM medium, and culture it at 37°C under anaerobic conditions on a shaker at 160 - 200 r / min for 12 - 24 h to obtain a Clostridium butyricum bacterial solution; S12: Inoculate the Clostridium butyricum seed solution into a fermenter and culture it at 37°C under anaerobic conditions for 12 - 24 h; centrifuge it at 3000 - 6000 rpm for 20 - 40 min using a tubular centrifuge, and discard the supernatant to obtain bacterial sludge.

3. The preparation method of the probiotic preparation for improving the reproductive performance of sows according to claim 1, characterized in that, In the above Step 2, the mass ratio of the bacterial sludge, tryptone, glucose, L-cysteine is 5 - 10:0.05 - 1:0.5 - 2:0.1 - 0.2:0.2 - 0.

5.

4. The preparation method of the microecological preparation for improving the reproductive performance of sows according to claim 1, characterized in that, In the above Step 3, the specific method for preparing the chitosan-coated colloid includes: S31: Dissolve chitosan in an acetic acid solution to obtain a chitosan solution; add nano-silica powder and polyethylene glycol-1000 to the chitosan solution, and after ultrasonic dispersion at 50 - 80 kHz for 30 - 60 min, obtain a dispersion system; S32: Add a 4 wt% genipin-ethanol solution to the dispersion system, heat it in a water bath with stirring to 40 - 60°C, keep it warm for 6 - 12 h, and let it stand and cool to obtain a crude colloid solution; S33: Add sodium alginate to thicken the crude colloid solution, heat it to 60 - 80°C and evaporate for 1 - 3 h to obtain a chitosan-coated colloid.

5. The preparation method of the microecological preparation for improving the reproductive performance of sows according to claim 4, characterized in that, The nano-silica powder in the above S31 is nano-silica powder modified by polyethylene glycol-400. The specific modification method includes: ultrasonically disperse nano-silica powder with a particle size of 50 - 120 nm in deionized water at 60 - 80 kHz to obtain a dispersion liquid, and the dispersion time is 5 - 20 min; add polyethylene glycol-400 to the dispersion liquid, stir and heat it to 80 - 90°C at 600 - 1200 rpm, keep it warm for 1 - 3 h, cool it to room temperature and let it stand for 12 - 24 h, centrifuge and wash the powder, and vacuum-dry it at 50 - 60°C for 20 - 50 min to obtain nano-silica powder modified by polyethylene glycol-400; the dosage ratio of nano-silica powder, polyethylene glycol-400, and deionized water is 5 - 20 g:0.5 - 5 g:300 - 1000 mL.

6. The preparation method of the microecological preparation for improving the reproductive performance of sows according to claim 4, characterized in that, In the above S31, the degree of acetylation of the chitosan is greater than 80%, the viscosity-average molecular weight is 500 - 2500 kDa; the concentration of acetic acid is 2 - 5 vol%; the dosage ratio of chitosan to the acetic acid solution is 1 - 5 g:100 - 200 mL.

7. The preparation method of the microecological preparation for improving the reproductive performance of sows according to claim 4, characterized in that, In S31, the mass ratio of chitosan, nano-silica powder, and polyethylene glycol-1000 is 1-5:0.02-1:0.05-1.

8. The preparation method of the microecological preparation for improving the reproductive performance of sows according to claim 1, characterized in that, In S32, the dosage of genipin-ethanol solution is 1% to 20% of the volume of the acetic acid solution; In S33, the mass ratio of sodium alginate to chitosan is 0.5:1-5.

9. The preparation method of the microecological preparation for improving the reproductive performance of sows according to claim 1, characterized in that, In the fourth step, the outer layer powder by weight includes: 1-5 parts of sodium carboxymethylcellulose, 2-5 parts of starch, 5-10 parts of soybean powder, and 1-2 parts of xanthan gum.

10. A probiotic preparation for improving the reproductive performance of sows, characterized in that, The microecological preparation is prepared by the preparation method of the microecological preparation for improving the reproductive performance of sows according to any one of claims 1-9.

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