A feed additive for preventing perinatal disease of dairy cows and improving production performance, and a preparation method and application thereof

Abstract

This invention provides a feed additive for preventing peripartum diseases in dairy cows and improving production performance, along with its preparation method and application, belonging to the technical field of animal husbandry and feed additives. The preparation method of the feed additive includes: encapsulating *Lactobacillus salivarius* liquid with xylooligosaccharides to obtain co-encapsulated microcapsules; mixing and preheating the co-encapsulated microcapsules, phytase microparticles, and glutamine microparticles, coating them with a starch-based coating solution, and solidifying them to obtain targeted release particles; mixing and pulverizing *Leonurus japonicus*, *Tetrapanax papyriferus*, *Astragalus membranaceus*, and *Taraxacum mongolicum* to obtain a traditional Chinese medicine compound powder; and mixing the targeted release particles, betaine, rumen-protected choline chloride, yeast culture, traditional Chinese medicine compound powder, rosemary extract, and wheat bran. This invention, through a double-layer coating delivery system, precisely delivers probiotics and phytase to the dairy cow's intestines, effectively avoiding degradation and inactivation in the rumen, and through multi-target synergistic effects, effectively preventing peripartum diseases in dairy cows with high safety.

Description

Technical Field

[0001] This invention belongs to the field of livestock breeding and feed additive technology, specifically relating to a feed additive for preventing periparturient diseases in dairy cows and improving their production performance, as well as its preparation method and application. Background Technology

[0002] The peripartum period for dairy cows typically refers to the critical physiological stage from 21 days before calving to 21 days after calving. This is a highly sensitive period for dairy cows as they experience late pregnancy, calving stress, and the onset of lactation. It is a critical stage in the production cycle characterized by significant physiological stress and a high incidence of metabolic diseases. The metabolic homeostasis, immune function, and health status of dairy cows during the peripartum period directly determine their production performance and farm economic benefits throughout the entire lactation cycle. During this period, dairy cows face triple nutritional demands: rapid fetal development, a surge in energy consumption during calving, and the onset of lactation. Simultaneously, they are affected by abdominal compression, hormonal fluctuations, and dietary changes, leading to a significant decrease in dry matter intake and a generally negative energy balance. This results in excessive mobilization of body fat, accumulation of non-esterified fatty acids, and excessive ketone body production, significantly increasing the risk of metabolic diseases such as ketosis and fatty liver. At the same time, the rapid redistribution of blood calcium during the peripartum period, the downregulation of immune cell function, and the exacerbation of oxidative stress and inflammatory response often induce diseases such as hypocalcemia, retained placenta, endometritis, and mastitis. This not only leads to a decrease in milk production, deterioration of milk quality, prolonged reproductive cycle and reduced conception rate, but also increases the culling rate of dairy cows, causing huge economic losses to the dairy farming industry.

[0003] In the existing technology, feed additives used in the peripartum period are mostly of the following types: (1) Single-function additives such as simple probiotic powder, anionic salt, choline chloride, etc., which can only solve a certain problem and lack systematicity; (2) Ordinary compound premixes simply mix multiple functional components physically. Among them, the active ingredients (such as probiotics and enzyme preparations) are easily inactivated in the feed processing and rumen environment, and have low bioavailability; and often contain a large amount of inorganic calcium and phosphorus, which conflicts with the scientific low calcium management strategy in the peripartum period, and even increases the risk of postpartum paralysis; (3) Chinese medicine additives mostly use medicinal powder directly, with low dissolution of effective ingredients, which are degraded by microorganisms in the rumen, and the dosage is huge, affecting palatability and formulation space. In addition, dairy cows are prone to acute diarrhea 1 day before calving or 1-2 days after calving, and a series of symptoms such as body weight change, dehydration and acidosis may occur. If not diagnosed and treated in time, it can cause death in a short period of time. Therefore, the urgent technical problem to be solved in this field is: how to provide a systemic functional additive that can effectively protect the core active ingredients to reach the intestines, has a clear synergistic mechanism of action of each component, and is safe and compatible with perinatal scientific feeding programs. Summary of the Invention

[0004] To address the problems existing in the prior art, the primary objective of this invention is to provide a feed additive for preventing peripartum diseases in dairy cows and improving production performance, as well as a method for preparing the additive. Through a double-layer coating delivery system, the core active ingredients (probiotics, phytase, etc.) are precisely delivered to the dairy cow's intestines, thereby increasing the intestinal colonization rate and effectively avoiding the degradation and inactivation of active ingredients in the rumen.

[0005] The second objective of this invention is to provide the above-mentioned feed additives for the preparation of products that prevent peripartum diseases in dairy cows and improve production performance, or for their application in dairy farming.

[0006] A third objective of this invention is to provide a feed that prevents peripartum diseases in dairy cows and improves their production performance.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a feed additive to prevent peripartum diseases in dairy cows and improve their production performance, comprising the following steps: (1) Lactobacillus salivarius liquid and xylooligosaccharide were cross-linked and encapsulated with sodium alginate-calcium chloride to obtain co-encapsulated microcapsules; the co-encapsulated microcapsules, phytase microparticles and glutamine microparticles were mixed, preheated, coated with starch-based coating solution, and solidified to obtain targeted release particles; (2) Motherwort, Tetrapanax papyriferus, Astragalus membranaceus and Taraxacum mongolicum are mixed and pulverized to obtain a compound powder of traditional Chinese medicine; (3) Targeted release particles, betaine, rumen-protected choline chloride, yeast culture, traditional Chinese medicine compound powder, rosemary extract and wheat bran are mixed to obtain feed additive.

[0008] Preferably, in step (1), the ratio of *Lactobacillus salivarius* solution to xylooligosaccharides is 1-3 mL: 0.5-1.5 g; the viable count of *Lactobacillus salivarius* solution is 1×10⁻⁶. 8 CFU / mL ~9×10 9 CFU / mL, the Lactobacillus salivarius was Lactobacillus salivarius KS1018, preservation number CCTCC NO: M 2025020.

[0009] Preferably, in step (1), the mass ratio of the co-encapsulated microcapsules, phytase microparticles, and glutamine microparticles is (6~8):(5~7):(8~10); and the preheating temperature is 25~35℃.

[0010] Preferably, in step (1), the starch-based coating solution is obtained by dissolving pH-sensitive hydroxypropyl methylcellulose succinate powder in an ethanol solution; the solid content of the starch-based coating solution is 15%~25%, and the pH value is 5.5~6.2; the coating method is bottom spray coating, the spraying rate is 2~4 mL / min, the atomization pressure is 0.1~0.15 MPa, the air inlet temperature is maintained at 35~45℃ and the material temperature is 30~40℃ during the coating process, and the coating weight gain is 20%~25% of the total weight of the mixed particles.

[0011] Preferably, in step (2), the mass ratio of Leonurus japonicus, Tetrapanax papyriferus, Astragalus membranaceus and Taraxacum mongolicum is (2~4):(2~4):(1~3):(0.5~1.5).

[0012] Preferably, in step (3), by mass parts, there are 120-150 parts of targeted release particles, 50-70 parts of betaine, 20-40 parts of rumen-protected choline chloride, 150-250 parts of yeast culture, 300-400 parts of traditional Chinese medicine compound powder, 15-25 parts of rosemary extract, and 350-430 parts of wheat bran.

[0013] The present invention also provides a feed additive prepared by the above preparation method.

[0014] The present invention also provides the application of the above feed additive in any of the following: (1) preparing drugs for preventing peripartum diseases in dairy cows; (2) preparing products to improve production performance; (3) dairy cow farming.

[0015] The present invention also provides a feed for preventing peripartum diseases in dairy cows and improving production performance, the feed comprising the above-mentioned feed additives.

[0016] Preferably, the feed further comprises a total mixed ration (TMR), and the amount of feed additives added is 0.5% to 3% DM based on the dry matter basis of the TMR.

[0017] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: (1) Targeted delivery and efficient colonization. This invention uses an innovative double-layer coating delivery system to precisely deliver the core active ingredients (probiotics, phytase, etc.) to the dairy cow's intestines, effectively avoiding degradation and inactivation in the rumen. Compared with ordinary probiotic powder, the intestinal colonization efficiency is increased by 3 to 5 times. (2) Systematically solve the core contradictions of the peripartum period. This invention targets the syndrome of diarrhea, hypocalcemia, fatty liver and other factors that are common in dairy cows during the peripartum period. It provides a systemic solution through the synergistic effect of multiple targets of "microecological regulation + intestinal repair + metabolic regulation + immune enhancement", rather than single symptom control. (3) High safety and compatibility with scientific diet strategies. The formula of this invention does not contain or strictly controls easily absorbed calcium sources (such as dicalcium phosphate), and achieves "enzyme addition and phosphorus reduction" through phytase, so that the prepared product can be perfectly compatible with the scientific "low calcium diet" or "anion salt diet" strategy during the peripartum period, fundamentally reducing the risk of postpartum hypocalcemia. (4) Good stability and convenient use. In this invention, the active ingredients are coated and protected, which significantly improves their stability during feed processing (granulation) and storage. The product is provided in the form of premix and can be directly added to TMR (Total Mixed Ration) without complicated processing, making it convenient for farm applications.

[0018] Biological Preservation Instructions Lactobacillus salivarius KS1018, classified and named Ligilactobacillus salivarius KS1018 was deposited on January 6, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China. The accession number is CCTCC NO: M 2025020. Detailed Implementation

[0019] This invention provides a method for preparing a feed additive to prevent peripartum diseases in dairy cows and improve their production performance, comprising the following steps: (1) Lactobacillus salivarius liquid and xylooligosaccharide were cross-linked and encapsulated with sodium alginate-calcium chloride to obtain co-encapsulated microcapsules; the co-encapsulated microcapsules, phytase microparticles and glutamine microparticles were mixed, preheated, coated with starch-based coating solution, and solidified to obtain targeted release particles; (2) Motherwort, Tetrapanax papyriferus, Astragalus membranaceus and Taraxacum mongolicum are mixed and pulverized to obtain a compound powder of traditional Chinese medicine; (3) Targeted release particles, betaine, rumen-protected choline chloride, yeast culture, traditional Chinese medicine compound powder, rosemary extract and wheat bran are mixed to obtain feed additive.

[0020] In step (1) of this invention, the ratio of *Lactobacillus salivarius* liquid to xylooligosaccharides is 1-3 mL: 0.5-1.5 g, preferably 2 mL: 1 g; the viable count of the *Lactobacillus salivarius* liquid is 1×10⁻⁶. 8 CFU / mL ~9×109 CFU / mL, preferably 9×10⁻⁶ 8 CFU / mL ~ 1×10 9 CFU / mL. The *Lactobacillus salivarius* used in this invention is *Lactobacillus salivarius* KS1018, with accession number CCTCCNO: M 2025020.

[0021] In step (1) of this invention, the preparation of co-encapsulated microcapsules is a primary coating process. The method of sodium alginate-calcium chloride cross-linking encapsulation includes: adding Lactobacillus salivarius solution and xylooligosaccharide to sodium alginate solution, stirring and mixing, then adding the mixture dropwise to calcium chloride solution for cross-linking, filtering, washing, and drying to obtain co-encapsulated microcapsules. Preferably, the concentration of the sodium alginate solution is 2%, and the concentration of the calcium chloride solution is 2%; the stirring and mixing forms a homogeneous sodium alginate suspension containing probiotics and prebiotics. The homogeneous sodium alginate suspension is added dropwise to calcium chloride solution through an electrostatic atomization device for cross-linking, and the cross-linking time is 5~15 min, preferably 10 min. After cross-linking, the microspheres are filtered, collected, washed with pure water, and dried to constant weight to obtain co-encapsulated microcapsules; the drying temperature can be selected as 35℃, and the drying time can be selected as 2~6 h.

[0022] In step (1) of this invention, the preparation of the targeted release particles is a two-stage coating process. The mass ratio of the co-encapsulated microcapsules, phytase microparticles, and glutamine microparticles in this invention is (6~8):(5~7):(8~10), preferably 7:6:9; the phytase microparticles are preferably loaded with porous starch, with an enzyme activity of 5000 FTU / g. The preheating temperature in this invention is 25~35℃, preferably 30℃. As an optional embodiment, the co-encapsulated microcapsules, phytase microparticles, and glutamine microparticles in this invention are preheated in a fluidized bed, with a fan frequency of 25 Hz, and preheated for 5~10 min at an inlet air temperature of 40℃, so that the material temperature rises to the preheating temperature.

[0023] The starch-based coating solution of this invention is obtained by dissolving pH-sensitive hydroxypropyl methylcellulose succinate powder in an ethanol solution. It is a pH-sensitive starch-based coating solution. The particle size of the pH-sensitive hydroxypropyl methylcellulose succinate powder is preferably 80 mesh, and the ethanol solution is preferably an 80%~90% ethanol solution (ethanol:water volume ratio = 80:20~90:10). As an optional embodiment, the pH-sensitive hydroxypropyl methylcellulose succinate (HPMCAS) powder is slowly added to the ethanol solution and dispersed until completely dissolved under stirring at 400 rpm to obtain the coating solution. The coating solution of this invention is preferably left to stand for 2~12 hours before use. The solid content of the starch-based coating solution of this invention is 15%~25% (mass-volume ratio g / mL), preferably 20%, and the pH value is 5.5~6.2, preferably 5.8~6.

[0024] The coating method described in this invention is a bottom-spray coating process, which involves coating a mixture of co-encapsulated microcapsules, phytase microparticles, and glutamine microparticles using a bottom-spray process. The spray rate is 2-4 mL / min, preferably 3 mL / min; the atomization pressure is 0.1-0.15 MPa, preferably 0.12 MPa; the inlet air temperature is maintained at 35-45°C, preferably 40°C; the material temperature is maintained at 30-40°C, preferably 35°C; and the coating weight gain is 20%-25% of the total weight of the mixed particles, preferably 22%-23%.

[0025] After the coating process is completed, it is preferable to continue drying with forced air for 20-30 minutes, preferably 25 minutes, and then solidify at 40°C for 3 hours to obtain targeted release particles with intestinal targeted release function, thus completing the secondary coating.

[0026] In step (1) of this invention, two levels of coating are performed. The first-stage coating employs co-encapsulation technology, where *Lactobacillus salivarius* and xylooligosaccharides (XOS) work synergistically. A sodium alginate-calcium chloride gel network physically isolates and protects the probiotics, while simultaneously locking the prebiotic XOS around them, creating an integrated intestinal colonization microenvironment of "probiotics + prebiotics," greatly improving the survival rate and colonization efficiency of the probiotics. The second-stage coating minimizes the loss of probiotics in the rumen, ensuring targeted colonization in the intestine. The pH-sensitive starch-based polymer coating layer is stable in the neutral environment of the rumen (pH 6.0~7.0), completely protecting all internal active ingredients as they pass through the rumen. When the particles enter the abomasum and the acidic environment of the lower intestinal tract (pH < 6.0), the coating layer dissolves, releasing the co-encapsulated probiotics, phytase, glutamine, and other core functional components in one step, achieving a synergistic effect.

[0027] In step (2) of this invention, the mass ratio of Leonurus japonicus, Tetrapanax papyriferus, Astragalus membranaceus, and Taraxacum mongolicum is (2~4):(2~4):(1~3):(0.5~1.5), preferably 3:3:2:1. As an optional embodiment, this invention mixes Leonurus japonicus, Tetrapanax papyriferus, Astragalus membranaceus, and Taraxacum mongolicum in a specific ratio, then pulverizes the mixture using an ultrafine pulverizer and passes it through a 40-mesh sieve to obtain a compound powder of traditional Chinese medicine for later use.

[0028] In step (3) of this invention, the following ingredients are present by weight: 120-150 parts targeted release granules, 50-70 parts betaine, 20-40 parts rumen-protected choline chloride, 150-250 parts yeast culture, 300-400 parts traditional Chinese medicine compound powder, 15-25 parts rosemary extract, and 350-430 parts wheat bran; preferably, the following ingredients are present by weight: 134 parts targeted release granules, 60 parts betaine, 30 parts rumen-protected choline chloride, 200 parts yeast culture, 360 parts traditional Chinese medicine compound powder, 20 parts rosemary extract, and 390 parts wheat bran. As an optional embodiment, the above raw materials are fed into a three-dimensional mixer and mixed at 12 rpm for 12 minutes to obtain the feed additive.

[0029] In the above-mentioned preparation method of the present invention, the most vulnerable probiotics, enzymes and other components are coated and protected by a "regional targeted delivery system"; the risky inorganic phosphorus and calcium are replaced by phytase technology through "nutritional function reconstruction"; and the upgrade from "single supplementation" to "system regulation" is achieved through "multi-target compound design", thereby effectively and safely preventing peripartum syndrome in dairy cows.

[0030] The functions of each component in this invention are described below: Lactobacillus salivarius acts as an immune adjuvant, stimulating the intestinal epithelium to produce more secretory immunoglobulin A (sIgA), which is the first line of defense in mucosal immunity and can effectively neutralize viruses and bacteria.

[0031] Xylooligosaccharides (XOS), as a prebiotic, target and promote the proliferation of Bifidobacteria in the gut. Together with Lactobacillus salivarius, they form a golden combination of "prebiotic + probiotic," synergistically optimizing the gut microbiota.

[0032] Glutamine serves as an energy source for intestinal epithelial cells. During perinatal stress, supplementing with exogenous glutamine can prevent intestinal wall atrophy, maintain barrier integrity, and thus reduce the risk of systemic inflammation.

[0033] Phytase (activity ≥ 5000 FTU / g), as a biocatalyst, releases phosphorus, calcium, zinc, and other minerals chelated by phytic acid in feed, improving their utilization rate. This not only saves on the addition of inorganic phosphorus, but more importantly, sufficient active calcium and phosphorus are crucial for preventing postpartum paralysis and ensuring mammary gland health in peripartum dairy cows.

[0034] Yeast cultures, as rumen microecological regulators, optimize rumen function and improve overall nutrient absorption and health.

[0035] Betaine, as an osmotic regulator, has a hepatoprotective effect by regulating osmotic pressure and reducing fatty liver caused by high-concentrate diets during the perinatal period. Simultaneously, as a methyl donor, it participates in protein and DNA synthesis, improving overall metabolic efficiency.

[0036] Rumen-exposed choline chloride acts as a functional metabolic regulator, ensuring hepatic lipid transport and promoting intrahepatic fat output by supporting the synthesis of PC and VLDL. Simultaneously, it maintains methyl metabolism homeostasis and, synergistically with betaine, supports anabolism.

[0037] Rosemary extract, as an antioxidant, can replace traditional antioxidants with a much stronger antioxidant capacity than vitamin E, and it also has unique anti-inflammatory properties, inhibiting inflammatory pathways such as NF-κB. Simultaneously, it can regulate rumen function, improve rumen fermentation patterns, and promote propionic acid production.

[0038] Motherwort is used for postpartum recovery, promoting blood circulation and regulating menstruation, and reducing swelling; Tetrapanax papyriferus promotes lactation and prevents inflammation, promotes downward flow of qi, clears heat and dampness, unclogs mammary glands, and prevents mastitis; Astragalus membranaceus enhances immunity, replenishes qi and strengthens the exterior, improves immunity, and resists stress; Taraxacum mongolicum clears heat and detoxifies, and reduces inflammation and swelling.

[0039] Wheat bran, as a filler, is a key auxiliary ingredient.

[0040] In this invention, the uncoated components exert a synergistic effect: choline chloride and betaine synergistically promote hepatic fat transport and prevent fatty liver; yeast culture optimizes rumen function; specific traditional Chinese medicine formulations promote postpartum uterine involution, lactation, and anti-inflammation; the overall formula, without adding risky calcium sources, improves endogenous calcium and phosphorus utilization through phytase, safely supporting calcium metabolism needs. Furthermore, astragalus polysaccharides abundant in astragalus and yeast β-glucan can synergistically activate immunity; chlorogenic acid and rosmarinic acid in dandelion can synergistically exert anti-inflammatory effects.

[0041] This invention also provides a feed additive prepared by the above-mentioned method, which offers a systemic solution through the synergistic effect of multiple targets including "microecological regulation + intestinal repair + metabolic regulation + immune enhancement." It has a good preventive effect on syndromes involving multiple factors, such as diarrhea, hypocalcemia, and fatty liver, which are common in peripartum dairy cows. It is perfectly compatible with scientific peripartum "low-calcium diets" or "anion salt diets," fundamentally reducing the risk of postpartum hypocalcemia. The feed additive described in this invention exhibits high stability during feed processing (pelleting) and storage.

[0042] The present invention also provides the application of the above-mentioned feed additives in any of the following: (1) preparing drugs for the prevention of peripartum diseases in dairy cows; wherein the peripartum diseases in dairy cows include diarrhea, hypocalcemia, fatty liver, ketosis and the resulting metritis, mastitis and other diseases that are interconnected and form a "peripartum syndrome". (2) preparing products to improve production performance; wherein the products include feed or drugs. (3) dairy cow farming.

[0043] This invention also provides a feed for preventing peripartum diseases in dairy cows and improving production performance, comprising the above-mentioned feed additive. As an optional embodiment, the feed of this invention is obtained by directly adding the feed additive to a TMR (total mixed ration); the amount of the feed additive added is 0.5-3% DM (DM refers to dry matter basis), preferably 1.5% DM.

[0044] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0045] In a specific embodiment of the present invention, *Lactobacillus salivarius* was *Lactobacillus salivarius* KS1018, with accession number CCTCC NO: M2025020; xylooligosaccharide was purchased from Lanzhou Waterles Biotechnology Co., Ltd.; phytase microparticles (loaded in porous starch, enzyme activity 5000) were used. FTU / g was purchased from Beijing Xindimei Feed Technology Co., Ltd.; glutamine microparticles were purchased from Shandong Huiheng Biotechnology Co., Ltd.; pH-sensitive hydroxypropyl methylcellulose succinate was purchased from Xi'an Tianzheng Pharmaceutical Excipients Co., Ltd.; betaine was purchased from Zhejiang Yicun Biotechnology Co., Ltd.; rumen-protected choline chloride was purchased from Henan Banglai Industry Co., Ltd.; yeast culture was purchased from Hebei Zunmu Biotechnology Co., Ltd.; rosemary extract was purchased from Lanzhou Waterles Biotechnology Co., Ltd.; wheat bran was purchased from Lingshou County Xinde Agricultural Products Co., Ltd.; the basal diet TMR was provided by the farm, referring to the NRC (2001) peripartum dairy cow nutrition standards, with corn silage, alfalfa hay, flaked corn, soybean meal, cottonseed meal and other main raw materials, and the nutritional levels were: net energy for milk production 1.45~1.55 Mcal / kg, crude protein 14%~16%, neutral detergent fiber 28%~32%, starch 22%~26%. The control group and the experimental group were fed the same batch of TMR.

[0046] Unless otherwise specified, the following embodiments are all conventional methods.

[0047] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0048] Example 1 Preparation of feed additives for preventing peripartum diseases and improving production performance in dairy cows: (1) Lactobacillus salivarius solution (1×10 9CFU / mL and xylooligosaccharides were added to a 2% sodium alginate solution at a ratio of 2 mL: 1 g and stirred to form a homogeneous sodium alginate suspension containing probiotics and prebiotics. The mixture was then added dropwise to a 2% calcium chloride solution using an electrostatic nebulizer (10 kV) for 10 min to crosslink. The microspheres were collected by filtration, washed with pure water, and gently dried at 35°C to constant weight to obtain co-encapsulated microcapsules.

[0049] (2) The co-encapsulated microcapsules, phytase microparticles and glutamine microparticles are mixed in a mass ratio of 7:6:9 and fed into a fluidized bed. The fan frequency is set to 25 Hz and the material is preheated at an inlet air temperature of 40℃ to raise the material temperature to 30℃.

[0050] (3) Preparation of pH-sensitive starch-based coating solution: pH-sensitive hydroxypropyl methylcellulose succinate (HPMCAS) powder (passed through an 80-mesh sieve) was slowly added to an ethanol-water mixed solvent (ethanol:water volume ratio = 80:20), and dispersed until completely dissolved under stirring at 400 rpm to obtain a coating solution with a solid content of 20% and a pH of 6.0. It was left to stand for 12 h before use.

[0051] Bottom spraying was used to coat the mixed particles. The spraying parameters were: spray rate 3 mL / min, atomization pressure 0.12 MPa. The coating weight gain was controlled to be 22% of the total weight of the mixed particles. During the coating process, the inlet air temperature was maintained at 40℃ and the material temperature at 30~40℃.

[0052] After coating, continue drying with forced air for 25 minutes, and then cure at 40℃ for 3 hours to obtain targeted release particles.

[0053] (4) Mix motherwort, tetrapanax papyriferus, astragalus membranaceus and dandelion in a ratio of 3:3:2:1, then pulverize them with an ultra-fine pulverizer and pass them through a 40-mesh sieve to obtain the Chinese herbal compound powder for later use.

[0054] (5) By mass fraction, 134 parts of targeted release granules, 60 parts of betaine, 30 parts of rumen-protected choline chloride, 200 parts of yeast culture, 360 parts of traditional Chinese medicine compound powder, 20 parts of rosemary extract, and 390 parts of wheat bran were put into a three-dimensional mixer and mixed at 12 rpm for 12 min to obtain the feed additive.

[0055] Example 2 Preparation of feed additives for preventing peripartum diseases and improving production performance in dairy cows: (1) Lactobacillus salivarius solution (1×10 8CFU / mL and xylooligosaccharides were added to a 2% sodium alginate solution at a ratio of 1 mL:1.5 g and stirred to form a homogeneous sodium alginate suspension containing probiotics and prebiotics. The mixture was then added dropwise to a 2% calcium chloride solution using an electrostatic nebulizer (10 kV) for 10 min to crosslink. The microspheres were collected by filtration, washed with pure water, and gently dried at 35°C to constant weight to obtain co-encapsulated microcapsules.

[0056] (2) Co-encapsulated microcapsules, phytase microparticles and glutamine microparticles are mixed in a mass ratio of 6:5:8 and fed into a fluidized bed. The fan frequency is set to 25 Hz and the material is preheated at an inlet air temperature of 40℃ to raise the material temperature to 25℃.

[0057] (3) Preparation of pH-sensitive starch-based coating solution: pH-sensitive hydroxypropyl methylcellulose succinate (HPMCAS) powder (passed through an 80-mesh sieve) was slowly added to an ethanol-water mixed solvent (ethanol:water volume ratio = 90:10), and dispersed until completely dissolved under stirring at 400 rpm to obtain a coating solution with a solid content of 15% and a pH of 5.5. It was left to stand for 12 h before use.

[0058] Bottom spraying was used to coat the mixed particles. The spraying parameters were: spray rate 2 mL / min, atomization pressure 0.1 MPa. The coating weight gain was controlled to be 20% of the total weight of the mixed particles. During the coating process, the inlet air temperature was maintained at 35℃ and the material temperature at 30℃.

[0059] After coating, continue drying with forced air for 25 minutes, and then cure at 40℃ for 3 hours to obtain targeted release particles.

[0060] (4) Mix motherwort, tetrapanax papyriferus, astragalus membranaceus and dandelion in a ratio of 2:2:1:0.5, then pulverize them with an ultra-fine pulverizer and pass them through a 40-mesh sieve to obtain the Chinese herbal compound powder for later use.

[0061] (5) By mass fraction, 120 parts of targeted release granules, 50 parts of betaine, 40 parts of rumen-protected choline chloride, 250 parts of yeast culture, 300 parts of traditional Chinese medicine compound powder, 25 parts of rosemary extract, and 430 parts of wheat bran were put into a three-dimensional mixer and mixed at 12 rpm for 12 min to obtain the feed additive.

[0062] Example 3 Preparation of feed additives for preventing peripartum diseases and improving production performance in dairy cows: (1) Lactobacillus salivarius solution (9×10) 9CFU / mL and xylooligosaccharides were added to a 2% sodium alginate solution at a ratio of 3 mL: 0.5 g, and stirred to form a homogeneous sodium alginate suspension containing probiotics and prebiotics. The mixture was then added dropwise to a 2% calcium chloride solution using an electrostatic nebulizer (10 kV) for 10 min for cross-linking. The microspheres were collected by filtration, washed with pure water, and gently dried at 35°C to constant weight to obtain co-encapsulated microcapsules.

[0063] (2) Co-encapsulated microcapsules, phytase microparticles and glutamine microparticles are mixed in a mass ratio of 8:7:10 and fed into a fluidized bed. The fan frequency is set to 25 Hz and the material is preheated at an inlet air temperature of 40℃ to raise the material temperature to 35℃.

[0064] (3) Preparation of pH-sensitive starch-based coating solution: pH-sensitive hydroxypropyl methylcellulose succinate (HPMCAS) powder (passed through an 80-mesh sieve) was slowly added to an ethanol-water mixed solvent (ethanol:water volume ratio = 90:10), and dispersed until completely dissolved under stirring at 400 rpm to obtain a coating solution with a solid content of 25% and a pH of 6.2. It was left to stand for 12 h before use.

[0065] Bottom spraying was used to coat the mixed particles. The spraying parameters were: spray rate 4 mL / min, atomization pressure 0.15 MPa. The coating weight gain was controlled to be 25% of the total weight of the mixed particles. During the coating process, the inlet air temperature was maintained at 45℃ and the material temperature at 40℃.

[0066] After coating, continue drying with forced air for 25 minutes, and then cure at 40℃ for 3 hours to obtain targeted release particles.

[0067] (4) Mix motherwort, tetrapanax papyriferus, astragalus membranaceus and dandelion in a ratio of 4:4:3:1, then pulverize them with an ultra-fine pulverizer and pass them through a 40-mesh sieve to obtain the Chinese herbal compound powder for later use.

[0068] (5) By mass fraction, 150 parts of targeted release granules, 70 parts of betaine, 20 parts of rumen-protected choline chloride, 150 parts of yeast culture, 400 parts of traditional Chinese medicine compound powder, 15 parts of rosemary extract, and 350 parts of wheat bran are put into a three-dimensional mixer and mixed at 12 rpm for 12 min to obtain the feed additive.

[0069] Experimental Example 1: In vitro efficacy verification (1) Rumen passage and release experiment The feed additive prepared in Example 1 was incubated in simulated rumen fluid (pH 6.5, 39°C) for 2 h, and the survival rate of live bacteria was measured. After being transferred to simulated intestinal fluid (pH 6.0), the release rate of probiotics and the release of phytase activity were measured within 2 h.

[0070] Fermentation broth of *Lactobacillus salivarius* KS1018 was taken without coating treatment. Maltodextrin was added directly as a protectant, and the mixture was spray-dried (inlet air temperature controlled at 160-180℃, outlet air temperature at 70-80℃). The uncoated bacterial powder was collected and labeled as ordinary probiotic powder. Testing showed that the initial viable count of this powder was not less than 1×10⁻⁶. 10 CFU / g.

[0071] Rumen survival rate was determined by comparing it with ordinary probiotic powder. The method for calculating rumen survival rate was as follows: The product from Example 1 and the control (ordinary probiotic powder) were diluted with sterile PBS to the same initial viable bacteria concentration (1×10⁻⁶). 8 (CFU / mL). Add 1 mL of the diluent to artificial rumen fluid, incubate at 39°C for 2 h, then plate the sample onto an MRS plate and anaerobically incubate at 39°C for 48 h for counting. Viability (%) = (number of viable bacteria after treatment / initial number of viable bacteria) × 100%. The initial number of viable bacteria was determined as follows: 1 mL of the diluent was serially diluted and plated, anaerobically incubated at 37°C for 48 h, and the count was taken as the initial number of viable bacteria.

[0072] Intestinal release rate calculation method: Microcapsules or samples treated with rumen fluid for 2 h were transferred to artificial intestinal fluid (pH 6.0, 39℃). Samples were taken at 0, 0.5, 1, and 2 h. After appropriate dilution, the samples were plated on MRS plates and anaerobically incubated at 39℃ for 48 h for counting. Cumulative release rate (%) = (Initial total number of viable bacteria embedded / Number of viable bacteria released at each time point) × 100%. Wherein, the initial total number of viable bacteria embedded refers to the total number of viable bacteria measured after thorough disruption of the same sample that had not undergone rumen fluid treatment.

[0073] Phytase activity retention calculation method: Untreated Example 1 product was dissolved / extracted with sterile PBS, and the initial enzyme activity (U / g) was determined according to the standard phytase assay method (phosphomolybdic blue colorimetric method). Example 1 product was treated with artificial rumen fluid (pH 6.5, 39℃) for 2 h, and the residual enzyme activity was also extracted and determined. Residual enzyme activity (%) = (Initial enzyme activity / Enzyme activity after treatment) × 100%.

[0074] The experimental results are shown in the table below.

[0075] Table 1 Results of rumen- and intestinal release experiments

[0076] Under the same conditions, the rumen survival rate of uncoated ordinary probiotic powder was less than 20% (data not shown), indicating that the coating process of the present invention significantly improves the rumen-passing ability of probiotics.

[0077] (2) Stability test Under different storage conditions, the decrease in the number of viable probiotics and the retention rate of phytase activity in the feed additive prepared in Example 1 were measured.

[0078] The calculation method for probiotic live bacteria retention rate is as follows: Live bacteria retention rate (%) = (Initial live bacteria count / Live bacteria count after a certain storage time) × 100%. Wherein, the initial live bacteria count is the number of live bacteria measured on storage day 0, and the live bacteria count after storage is the number of live bacteria measured after storage under the corresponding conditions for a certain period of time.

[0079] The calculation method for phytase activity retention rate is as follows: Enzyme activity retention rate (%) = (initial enzyme activity / enzyme activity after storage for a certain period of time) × 100%. Wherein, the initial enzyme activity is the enzyme activity measured on day 0 of storage, and the enzyme activity after storage is the enzyme activity measured after storage for a certain period of time under the corresponding conditions.

[0080] The experimental results are shown in the table below.

[0081] Table 2 Stability test results

[0082] Experimental results show that the feed additive prepared in Example 1 has high stability.

[0083] Experiment Example 2: Dairy Cow Feeding Trial I. Experimental Design: Forty Holstein dairy cows with similar parities (2-3 parities), expected calving dates, and body condition scores (BCS) between 3.0 and 3.5 were selected and randomly divided into a control group and an experimental group. The experimental period was from 21 days before calving to 21 days after calving.

[0084] Control group: 20 animals (basal diet TMR), divided into 4 groups of 5 animals each.

[0085] Experimental group: 20 heads (basal diet TMR + feed additive prepared in Example 1, 1.5% DM), divided into 4 groups of 5 heads each.

[0086] Dosage Explanation: In Example 1, the phytase activity concentration in the final product is approximately 125.6 FTU / g. Based on a dairy cow diet supplementation of 1.5% DM, the actual phytase intake is approximately 1884 FTU / kg DM (125.6 FTU / g × 15 g / kg), which is within the effective dosage range reported in the literature. The rosemary extract accounts for approximately 1.67% of the final product by mass. Based on a diet supplementation of 1.5% DM, the actual rosemary extract intake of the dairy cow is approximately 250 mg / kg DM.

[0087] II. Feeding and Management: Before the trial began, the barn was thoroughly disinfected. All dairy cows were kept in the same barn and fed the same TMR (Total Mixed Ration) feeding schedule (twice daily at 10:00 AM and 6:00 PM) with free access to water. Feed additives were in powder form and were mixed evenly with the first feed in the morning.

[0088] III. Detection Indicators: (1) Production performance indicators: Dry matter intake of dairy cows was recorded daily during the experimental period, milk yield was recorded daily after calving, milk composition and somatic cell count were recorded on days 7, 14, and 21 postpartum, and cow body weight was recorded weekly before calving. All data were analyzed using SPSS 26.0 for independent samples t-tests or repeated measures ANOVA. P <0.05 indicates a significant difference. P <0.01 indicates a highly significant difference.

[0089] Table 3 Production Performance Indicators

[0090] Table 4 Weekly Weight Before Delivery

[0091] (2) Health status indicators: The incidence of diarrhea in dairy cows was recorded daily, and a 5-point scoring system was adopted, referring to the Manure scoring system proposed by Michigan State University.

[0092] Table 5. 5-point scoring criteria

[0093] Diarrhea diagnosis criteria: A fecal score ≥3 is considered normal, and a score ≥4 is considered constipation / excessive roughage. When calculating the diarrhea rate, a score of 1-2 is considered diarrhea. Scoring method: Fecal characteristics are observed and recorded daily before morning feeding (at a fixed time); the scores are independently scored by at least two trained feeders, and the average is taken; daily fecal scores are continuously recorded throughout the entire trial period (21 days before farrowing to 21 days after farrowing), with a focus on analyzing the incidence of diarrhea within 14 days postpartum, a high-incidence window for peripartum diarrhea.

[0094] Daily records of clinical disease occurrences, including: postpartum paralysis (clinical hypocalcemia), ketosis, retained placenta, metritis, and mastitis, and calculation of morbidity rates; daily records of dairy cow culling and mortality.

[0095] Table 6 Health Status Indicators

[0096] There was no significant difference in prenatal weight between the two groups. P>0.05), the experimental group recovered weight faster postpartum, and their weight gain was significantly higher than that of the control group from 14 days postpartum. P <0.05).

[0097] Table 7 Elimination and Death Statistics

[0098] The total culling rate in the experimental group was 66.7% lower than that in the control group, indicating that feed additives help reduce the risk of culling during the perinatal period.

[0099] (3) Metabolic and immune indicators: Blood samples were collected from dairy cows 21 days before calving, 7 days before calving, on the day of calving, 7 days after calving, 14 days after calving, and 21 days after calving to test β-hydroxybutyrate, blood glucose, blood phosphorus, blood magnesium, and malondialdehyde (MDA). The indicators were relatively stable before calving, with the greatest stress and most significant changes in indicators on the day of calving and 7 days after calving, and gradually recovered from 14 to 21 days after calving.

[0100] Table 8 Metabolic and Immune Indicators

[0101] Table 9. β-Hydroxybutyrate (mmol / L) Index

[0102] The peak value of β-hydroxybutyrate in the experimental group was significantly lower than that in the control group, indicating that the additive effectively reduced the risk of perinatal fat mobilization and ketosis.

[0103] Table 10 Blood Glucose (mg / dL) Indicators

[0104] The experimental group experienced a smaller drop in blood glucose during the perinatal period and a faster postpartum recovery, indicating improved energy metabolism.

[0105] Table 11. Serum phosphorus (mg / dL) levels

[0106] The experimental group maintained better postpartum blood phosphorus levels, which may be related to phytase improving phosphorus utilization.

[0107] Table 12 Serum magnesium (mg / dL) levels

[0108] Table 13 Malondialdehyde (MDA, nmol / mL) Index

[0109] The perinatal MDA level in the experimental group was significantly lower than that in the control group, indicating that the additive effectively alleviated oxidative stress.

[0110] The overall results showed that, compared with the control group, the experimental group of dairy cows had: a lower incidence of diarrhea within 14 days postpartum; a lower incidence of clinical hypocalcemia postpartum; a significantly lower concentration of β-hydroxybutyrate (ketosis marker) in the blood; and a 28.5% increase in colostrum IgG, indicating that the additive improved passive immune transmission.

[0111] Comparative Example 1 The only difference between this comparative example and Example 1 is that bran is not added, and in step (5), the targeted release particles, betaine, rumen-protected choline chloride, yeast culture, traditional Chinese medicine compound powder, and rosemary extract are directly mixed. The rest of the preparation process is exactly the same as in Example 1.

[0112] Experiment 3 verifies the role of "wheat bran" as a carrier. The feed additives prepared in Example 1 and Comparative Example 1 were used for testing respectively: I. Experimental Design: Twenty Holstein dairy cows with parity (2-3 parities), similar expected calving dates, and body condition scores (BCS) between 3.0 and 3.5 were selected and randomly divided into a control group and an experimental group. The experimental period was from 21 days before calving to 21 days after calving.

[0113] Experimental group: 10 heads (basal diet TMR + feed additive from Example 1, 1.5% DM), divided into 2 groups of 5 heads each.

[0114] Control group: 10 animals (basal diet TMR + comparative example 1 feed additive, 1.5% DM), divided into 2 groups of 5 animals each.

[0115] II. Feeding and Management: Before the trial began, the barn was thoroughly disinfected. All dairy cows were kept in the same barn and fed the same TMR (Total Mixed Ration) feeding schedule (twice daily at 10:00 AM and 6:00 PM) with free access to water. Feed additives were in powder form and were mixed evenly with the first feed in the morning.

[0116] The mixing uniformity (coefficient of variation, CV) of the two product groups was compared using the tracer method, flowability (angle of repose), storage stability (changes in the uniformity of distribution of active ingredients after 30 days at 40°C), and feeding palatability (first-feeding rate of dairy cows to TMR). The results are shown in the table below.

[0117] Table 14 Results verifying the role of the "bran" carrier

[0118] The results showed that the experimental group had significantly better mixing uniformity (CV < 5%) than the control group (CV > 15%), better flowability, and was less prone to component stratification during storage. The control group product was prone to clumping, affecting feeding uniformity and palatability. This demonstrates that bran as a carrier is crucial for ensuring product uniformity, stability, and performance.

[0119] Comparative Example 2 The only difference between this comparative example and Example 1 is that the herbal compound powder is replaced with a qi-tonifying and blood-nourishing formula. The ratio of Astragalus membranaceus: Codonopsis pilosula: Angelica sinensis: Rehmannia glutinosa is 3:3:2:1, then pulverized using an ultrafine pulverizer and passed through a 40-mesh sieve to obtain the herbal compound powder. All other preparation processes are exactly the same as in Example 1.

[0120] Comparative Example 3 The only difference between this comparative example and Example 1 is that the addition of Tetrapanax papyriferus, Astragalus membranaceus, and Taraxacum mongolicum was omitted, and Leonurus japonicus powder was used in place of the compound powder of traditional Chinese medicine in an equal amount. The rest of the preparation process is exactly the same as that of Example 1.

[0121] Comparative Example 4 The only difference between this comparative example and Example 1 is that the addition of the traditional Chinese medicine compound powder is omitted, while the rest of the preparation process is exactly the same as that of Example 1.

[0122] Experiment 4 verifies the effect of "traditional Chinese medicine compound". The feed additives prepared in Example 1 and Comparative Examples 2-4 were tested respectively: I. Experimental Design: Forty Holstein dairy cows with similar parities (2-3 parities), expected calving dates, and body condition scores (BCS) between 3.0 and 3.5 were selected and randomly divided into a control group and an experimental group. The experimental period was from 21 days before calving to 21 days after calving.

[0123] A1 (Example 1 Group): 10 heads (basal diet TMR + Example 1 feed additive, 1.5% DM), divided into 2 groups of 5 heads each.

[0124] A2 (Qi-boosting and blood-tonifying group): 10 heads (basal diet TMR + comparative ratio 2 feed additive, 1.5% DM), divided into 2 groups of 5 heads each.

[0125] A3 (Single Function Group): 10 heads (basal diet TMR + comparative ratio 3 feed additive, 1.5% DM), divided into 2 groups of 5 heads each.

[0126] A4 (No Traditional Chinese Medicine Group): 10 heads (basal diet TMR + comparative ratio 4 feed additive, 1.5% DM), divided into 2 groups of 5 heads each.

[0127] II. Feeding and Management: Before the trial began, the barn was thoroughly disinfected. All dairy cows were kept in the same barn and fed the same TMR (Total Mixed Ration) feeding schedule (twice daily at 10:00 AM and 6:00 PM) with free access to water. Feed additives were in powder form and were mixed evenly with the first feed in the morning.

[0128] Measurement indicators: The focus was on postpartum uterine involution, which is directly related to the function of traditional Chinese medicine (the criteria for uterine involution were: ultrasound monitoring showed that the diameter of the uterine horn had decreased to ≤3.5 cm and there was no obvious fluid or pus accumulation in the uterine cavity, indicating that involution was complete. The number of postpartum days required for each dairy cow to reach this standard was recorded as the uterine involution time), lochia discharge time, mammary gland health (soy cell count in milk), and inflammation level (serum IL-1β and TNF-α). The results are shown in the table below.

[0129] Table 15 Results of verifying the effects of "traditional Chinese medicine compound".

[0130] Table Note: Compared with Group A1, P <0.05, P <0.01.

[0131] The results showed that A1 (Example 1 group) performed best overall in promoting uterine involution, reducing SCC, and alleviating systemic inflammation, significantly outperforming A2, A3, and A4. A2 group may have improved blood circulation but lacked sufficient flow, resulting in slow uterine involution; A3 group promoted uterine contractions but lacked immune support and anti-inflammatory effects, leading to poor SCC control; A4 group was inferior in all indicators. This directly demonstrates the inventiveness of the herbal compound formula in this invention in terms of compatibility and its irreplaceable technical effects.

[0132] Comparative Example 5 The only difference between this comparative example and Example 1 is that the coating process using the coating solution is omitted, and the co-encapsulated microcapsules, phytase microparticles, and glutamine microparticles are directly mixed with betaine, rumen-exposed choline chloride, yeast culture, traditional Chinese medicine compound powder, rosemary extract, and wheat bran. The remaining preparation process is exactly the same as in Example 1.

[0133] Comparative Example 6 The only difference between this comparative example and Example 1 is that the co-encapsulation treatment was omitted, and the *Lactobacillus salivarius* liquid, xylooligosaccharide, phytase microparticles, glutamine microparticles, betaine, rumen-protected choline chloride, yeast culture, traditional Chinese medicine compound powder, rosemary extract, and wheat bran were directly mixed. The remaining preparation process was exactly the same as in Example 1.

[0134] Experiment 5 verifies the necessity of the "two-stage coating" process. Referring to the in vitro simulation experiment of Experiment Example 1, the feed additives prepared in Example 1 and Comparative Examples 5-6 were tested respectively. The rumen survival rate and intestinal timed release rate of probiotics in the three groups of products were compared. The results are shown in the table below.

[0135] Table 16 Results verifying the effectiveness of "secondary embedding"

[0136] The results showed that group B1 had the highest rumen survival rate and intestinal timed release rate; group B2 had a relatively good rumen survival rate, but the probiotics were released too quickly after entering the intestine and could not release other components simultaneously; the active ingredients in group B3 were almost completely inactivated in the rumen. This demonstrates that the "two-stage coating" system is necessary and optimized for achieving synergistic targeted delivery of multiple components.

[0137] Comparative Example 7 The only difference between this comparative example and Example 1 is that *Lactobacillus salivarius* is replaced with *Enterococcus faecalis*, a commonly used probiotic in animal husbandry (*Enterococcus faecalis* freeze-dried powder, purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product batch number B81365). The remaining preparation process is exactly the same as in Example 1.

[0138] Comparative Example 8 The only difference between this comparative example and Example 1 is that *Lactobacillus salivarius* is replaced with a heat-resistant probiotic—*Bacillus subtilis* (a *Bacillus subtilis* preparation purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product batch number B98052). The remaining preparation process is exactly the same as in Example 1.

[0139] Comparative Example 9 The only difference between this comparative example and Example 1 is that the addition of Lactobacillus salivarius was omitted; the rest of the preparation process is exactly the same as in Example 1.

[0140] Experiment 6 verifies the criticality of selecting the "Lactobacillus salivarius" strain. The feed additives prepared in Example 1 and Comparative Examples 7-9 were tested respectively: I. In vitro evaluation 1. Mucosal immune stimulation capacity: Co-culture with bovine intestinal epithelial cells and measure the amount of sIgA secreted in the culture supernatant.

[0141] (1) Tissue collection and preprocessing Fresh jejunal tissue (approximately 10-15 cm) from healthy dairy cows was collected from a local slaughterhouse and placed in sterile PBS at 4°C containing 2% penicillin (200 U / mL, streptomycin 200 μg / mL). The tissue was transported back to the laboratory within one hour. In a laminar flow hood, the intestinal segment was repeatedly rinsed 3-5 times with PBS containing 2% penicillin and streptomycin until the rinsing solution was clear. The intestinal segment was longitudinally cut with sterile surgical scissors, and the intestinal contents and mucus layer were gently scraped away with a sterile glass slide. The intestinal tissue was then cut into pieces approximately 1-2 mm in size. 3 Small pieces.

[0142] (2) Tissue digestion Prepare the digestion solution: 0.2% type I collagenase + 0.1% neutral protease (dispase) dissolved in serum-free DMEM medium. Place the shredded tissue pieces in the digestion solution (tissue to digestion solution ratio approximately 1:5) and incubate at 37°C with shaking for 30 min. Every 10 min, gently disperse the tissue pieces with a pipette to promote digestion.

[0143] (3) Cell isolation and purification After digestion, filter the solution through a 100-mesh sieve and collect the filtrate. Centrifuge the filtrate at 800×g for 5 min and discard the supernatant. Resuspend the precipitate in PBS and gently pipette to mix. Add 5 mL of 2% sorbitol solution to a 15 mL centrifuge tube as the bottom layer, and carefully add the cell suspension to the sorbitol layer. Centrifuge at 400×g for 10 min and collect the interfacial cells (this step removes monocytes, lymphocytes, and muscle cells). Wash the collected cells twice with PBS (800×g, 5 min).

[0144] (4) Cell seeding and culture The isolated cells were resuspended in DMEM / F12 complete medium (containing 10% fetal bovine serum and 1% penicillin-dextrose antibody). The cell density was adjusted to 2 × 10⁶ cells / year. 5 Cells were seeded at a density of 1 / mL into cell culture flasks and incubated at 37°C in a 5% CO2 incubator. The medium was changed for the first time after 48 hours of culture to remove non-adherent cells. The medium was then changed every 2-3 days thereafter. Cells were passaged when the confluence reached 80%-90%.

[0145] (5) Cell identification Morphological identification: Under an inverted microscope, the epithelial cells exhibited a typical paving stone morphology.

[0146] Immunofluorescence identification: Immunofluorescence staining was performed using anti-cytokeratin 18 antibody; positive cells emitted green fluorescence.

[0147] (6) Cell plating Logarithmic growth phase bovine small intestinal epithelial cells were collected and digested with 0.25% trypsin to prepare a single-cell suspension. Cells were counted using a hemocytometer, and the cell density was adjusted to 2 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of 1 mL / mL into 24-well cell culture plates and incubated at 37°C with 5% CO2 for 24 hours to allow the cells to adhere and form a monolayer.

[0148] (7) Preparation of bacterial culture Take 10 g of each of the feed additives prepared in Example 1 and Comparative Examples 7-9, add 90 mL of sterile PBS, shake to mix, and prepare a 10-fold dilution.

[0149] Example 1 and Comparative Example 7: Take 1 mL of the above diluted solution, add 9 mL of MRS liquid culture medium, and anaerobic culture at 37°C for 24 h.

[0150] Comparative Example 8: Take 1 mL of the above diluted solution, add 9 mL of LB liquid medium, and incubate at 37°C with aerobic shaking (180 rpm) for 24 hours.

[0151] Comparative Example 9: Take 1 mL of the above diluted solution and add 9 mL of sterile PBS as a negative control.

[0152] After incubation, adjust the concentration of each bacterial culture to 1×10⁻⁶ using sterile PBS. 7 CFU / mL (except for Comparative Example 9). Take 100 μL of the above suspension for serial dilution and plate it. After incubation at 37°C for 48 h, count the number of viable bacteria.

[0153] Table 17 Viable Bacterial Count Results

[0154] (8) Co-culture treatment Discard the original culture medium in the 24-well plate.

[0155] Blank control group: Add 1 mL of DMEM / F12 complete culture medium.

[0156] Example 1 group: Add 1 mL of DMEM / F12 complete culture medium containing Lactobacillus salivarius (1×10⁻⁶) 7 (CFU / mL).

[0157] Comparative Example 7: Add 1 mL of DMEM / F12 complete medium containing Enterococcus faecalis (1×10⁻⁶) 7 (CFU / mL).

[0158] Comparative Example 8: Add 1 mL of DMEM / F12 complete medium containing Bacillus subtilis (1×10⁻⁶) 7 (CFU / mL).

[0159] Comparative Example 9: Add 1 mL of DMEM / F12 complete culture medium (without probiotics) containing the Comparative Example 9 sample.

[0160] Each group was divided into 3 replicates, with a final volume of 1 mL per well, and was incubated at 37℃ in a 5% CO2 incubator for 24 h.

[0161] (9) Sample collection After co-culture, collect the cell culture supernatant from each well into a 1.5 mL centrifuge tube, centrifuge at 1000×g for 10 min, take the supernatant (to remove any possible residual bacteria), aliquot it into new centrifuge tubes, and store at -20℃ for later use.

[0162] 2. sIgA content detection (ELISA method) The sIgA content in cell culture supernatant was detected using a double-antibody sandwich ELISA method (ELISA kit: Cusabio CSB-E11192b): The ELISA kit was removed from 4℃ and allowed to equilibrate to room temperature for 30 min. Washing buffer was prepared according to the manufacturer's instructions, and 20× concentrated washing buffer was diluted 1:20 with distilled water. Standards were prepared according to the manufacturer's instructions, and the standards were serially diluted according to the manufacturer's instructions, setting up 8 concentration gradients.

[0163] A standard curve was plotted with the standard concentration on the x-axis and the OD value on the y-axis, and the regression equation was calculated. The sample OD value was then substituted into the regression equation to calculate the sIgA concentration in the sample. The results are shown in the table below.

[0164] Table 18 sIgA Concentration Detection Results

[0165] 2. Inhibition ability against pathogens: The diameter of the inhibition zone against enterotoxigenic Escherichia coli (ETEC) was determined.

[0166] (1) Activation of ETEC strain Remove the ETEC frozen bacterial culture (purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product batch number B00156) from the -80℃ freezer and thaw at room temperature. Use an inoculation loop to pick up the bacterial culture and streak it onto LB solid medium, and incubate at 37℃ aerobically for 20 h. Pick a single colony and inoculate it into 5 mL of LB liquid medium, and incubate at 37℃ with shaking (180 rpm) for 12 h.

[0167] (2) Preparation of ETEC bacterial suspension Take 1 mL of the activated ETEC bacterial culture and add 9 mL of sterile PBS to prepare 10 -1 Diluent. Continue serial dilution to 10. -6 ~10 -7Count the bacteria on the coating. Adjust the bacterial concentration to 1×10⁻⁶. 6 CFU / mL (approximately OD) 600 =0.1~0.2), for backup.

[0168] (3) Preparation of test bacterial suspension Take 10 g of each of the feed additives prepared in Example 1 and Comparative Examples 7-9, add 90 mL of sterile PBS, and shake to mix.

[0169] Example 1 and Comparative Example 7: Take 1 mL of the above suspension, add 9 mL of MRS liquid culture medium, and anaerobic culture at 37°C for 24 h.

[0170] Comparative Example 8: Take 1 mL of the above suspension, add 9 mL of LB liquid culture medium, and incubate at 37°C with aerobic shaking (180 rpm) for 24 h.

[0171] Comparative Example 9: Take 1 mL of the above suspension and add 9 mL of sterile PBS as a negative control.

[0172] After incubation, adjust the concentration of each bacterial culture to 1×10⁻⁶ using sterile PBS. 8 CFU / mL (except for Comparative Example 9), for later use.

[0173] (4) Preparation of indicator bacteria plates Take sterilized LB solid culture medium, heat to melt, and then cool to approximately 50°C. Under aseptic conditions, pour the medium into sterile 90 mm diameter petri dishes, 15–20 mL per dish, and allow to solidify. Take 1×10 6 100 μL of ETEC bacterial suspension (CFU / mL) was evenly spread on the surface of an LB agar plate and allowed to stand for 10 min to allow the bacterial suspension to be fully absorbed.

[0174] (5) Placement of Oxford cup Using sterile forceps, place the Oxford cups vertically on an LB agar plate coated with ETEC, with three Oxford cups on each plate (arranged in an equilateral triangle). Gently press the Oxford cups to ensure close contact with the culture medium surface.

[0175] (6) Sample addition and incubation Take 1×10⁻⁶ bacterial suspensions from Example 1 and Comparative Examples 7-9 respectively. 8 Add 200 μL of each of the CFU / mL samples to an Oxford cup. Make three parallel plates for each sample and incubate the plates in a 37°C aerobic incubator for 24 h.

[0176] (7) Measurement of inhibition zone After incubation, observe whether a transparent inhibition zone appears around the Oxford cup. Measure the diameter of the inhibition zone (including the outer diameter of the Oxford cup) using calipers. Measure each inhibition zone three times from different directions and record the average result in mm. (The Oxford cup must be autoclaved at 121℃ for 20 minutes before the experiment; when spreading the bacterial solution, spread it evenly across the entire plate surface, avoiding areas that are too thick or too thin; avoid air bubbles during sample addition to prevent affecting inhibition zone formation; select inhibition zones with clearly defined edges for measurement.) Table 19 Results of Antibacterial Zone Detection

[0177] 3. Synergy with XOS: The maximum specific growth rate of different bacterial species was determined in a culture medium using XOS as the sole carbon source. The method and steps are as follows: (1) Preparation of basal culture medium (without carbon source) Prepare a carbon-free basal culture medium (pH 6.5) according to the following formula, dissolve it in deionized water, and autoclave it at 121°C for 15 min: Table 20 Basic Culture Medium Formulation

[0178] Note: Vitamin solutions and trace element solutions need to be filtered through a 0.22 μm filter membrane for sterilization and added after the basal culture medium has been sterilized and cooled to 50°C.

[0179] (2) Preparation of XOS carbon source culture medium Xylooligosaccharides (XOS, purity ≥95%, degree of polymerization 2-4) were dissolved in deionized water to prepare a 20% stock solution, which was then sterilized by filtration through a 0.22 μm filter membrane. The solution was added to a final concentration of 10 g / L to the sterilized carbon-free basal culture medium cooled to 50°C, mixed thoroughly, and dispensed into sterile Erlenmeyer flasks (50 mL / 250 mL Erlenmeyer flasks).

[0180] (3) Preparation of bacterial suspension Take 10 g each of the feed additives prepared in Example 1 and Comparative Examples 7-9, and prepare bacterial suspensions according to the following method: Example 1 and Comparative Example 7: Take 10 g of additive, add 90 mL of sterile PBS, shake to mix, then take 1 mL and add it to 9 mL of MRS liquid culture medium, and anaerobic culture at 37℃ for 24 h.

[0181] Comparative Example 8: Take 10 g of additive, add 90 mL of sterile PBS, shake to mix, then take 1 mL and add it to 9 mL of LB liquid medium, and incubate at 37℃ with aerobic shaking (180 rpm) for 24 h.

[0182] Comparative control group 9 (no probiotic control group): 10 g of additive was added to 90 mL of sterile PBS, and the mixture was shaken and mixed. This was used as a negative control and no growth curve was measured.

[0183] After incubation, wash the bacterial cells twice with sterile PBS (8000×g, 10 min, 4℃), resuspend in sterile PBS, and adjust OD. 600 Up to 0.5 ± 0.05 (approximately 1 × 10⁻⁵) 8 (CFU / mL), for later use.

[0184] (4) Inoculation and culture Take the above bacterial suspension and inoculate it into XOS carbon source medium at an inoculation rate of 2% (v / v) (i.e., inoculate 1 mL of bacterial suspension per 50 mL of medium), and mix well. Each group should have 3 replicates.

[0185] Culture conditions: Example 1 and Comparative Example 7: anaerobic culture at 37℃; Comparative Example 8: aerobic shaking culture at 37℃ (150 rpm).

[0186] (5) Growth curve determination Starting from inoculation, samples were taken every 2 hours until 24 hours of culture (a total of 13 time points: 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 hours). Sampling method: Anaerobic culture group: Samples were taken inside an anaerobic operating table to avoid oxygen exposure.

[0187] Aerobic culture group: direct sampling.

[0188] Measurement of OD 600 Values: Using the corresponding uninoculated XOS medium as a blank control, the OD values ​​of each sample were measured using a UV spectrophotometer. 600 value.

[0189] (6) Maximum specific growth rate (μ) max )calculate Determination of logarithmic growth phase: Using culture time (h) as the x-axis, OD... 600 The logarithm of (lnOD) 600 Plot a growth curve with OD as the ordinate. 600 The period between 0.2 and 0.8, during which the curve rises linearly, is considered the logarithmic growth period.

[0190] Linear regression: The lnOD during the logarithmic growth phase... 600 Perform a linear regression with time (t) to fit the linear equation: lnOD t =ln OD0+μ max t. Among them, OD t =OD at time t600 Value, OD0 = OD at the start of the logarithmic growth phase 600 Value, μ max =Maximum specific growth rate (h) -1 Ri, that is, the slope of the regression line. Judgment criterion: Ri 2 Only values ​​≥0.98 can be included in the calculation.

[0191] (7) Data processing Each group had three replicates, and each replicate was measured three times. Results are expressed as mean ± standard deviation. One-way ANOVA was used for inter-group comparisons.

[0192] Table 21 Results of maximum specific growth rate detection

[0193] II. Animal feeding verification: Dairy cows under peripartum stress, exhibiting decreased feed intake, abnormal fecal characteristics, or altered mental state, were selected. Ten cows were grouped into two separate pens (five cows per pen), serving as two independent experimental replicates. A short-term feeding trial lasting 14 days (from 7 days before calving to 7 days after calving) was conducted. This experiment employed a short-term stress model (7 days before calving to 7 days after calving, a total of 14 days) to rapidly evaluate the mucosal immune modulation and diarrhea prevention effects of different bacterial strains under peripartum stress conditions.

[0194] Forty Holstein dairy cows with parity (2-3 parities), similar expected calving dates, and body condition scores (BCS) between 3.0 and 3.5 during the peripartum period were selected and randomly divided into a control group and an experimental group.

[0195] C1 (Invention Group): 10 heads (basal diet TMR + feed additive of Example 1, 1.5% DM), divided into 2 groups of 5 heads each.

[0196] C2 (alternative group A): 10 heads (basal diet TMR + comparative ratio 7 feed additive, 1.5% DM), divided into 2 groups of 5 heads each.

[0197] C3 (Alternative Group B): 10 heads (basal diet TMR + comparative ratio 8 feed additive, 1.5% DM), divided into 2 groups of 5 heads each.

[0198] C4 (blank group): 10 heads (basal diet TMR + comparative ratio 9 feed additive, 1.5% DM), divided into 2 groups of 5 heads each.

[0199] The fecal scoring and diarrhea assessment criteria were the same as in Experiment 2. The diarrhea rate was calculated as follows: (1) Fecal scoring was performed on each dairy cow daily, and a score ≤2 was recorded as "diarrhea occurred". (2) The number of days each dairy cow experienced diarrhea during the entire experiment was counted. (3) The number of days of diarrhea for all dairy cows was added together to obtain the "total number of days of diarrhea". (4) The "total number of days of experiment" was calculated as: Total number of dairy cows × Number of days of experiment. (5) The diarrhea rate was calculated using the following formula: Diarrhea rate (%) = (Total number of days of diarrhea / Total number of days of experiment) × 100%.

[0200] The content of sIgA in intestinal mucosal samples was detected by double antibody sandwich ELISA (ELISA kit: CusabioCSB-E11192b): (1) Take about 0.5 g of rectal mucosal tissue and rinse with pre-cooled PBS (0.01 M, pH 7.4) to remove blood; (2) Cut the tissue into small pieces and add 5 times the volume of pre-cooled PBS (containing protease inhibitor); (3) Homogenize the tissue in an ice bath (10000 rpm, 30 s × 3 times, 30 s interval); (4) Centrifuge the homogenate at 10000 × g for 10 min at 4℃; (5) Take the supernatant, aliquot it and store it at -80℃ for later use. The detection was performed according to the ELISA kit instructions. Calculate the average OD value of each standard and sample, subtract the OD value of the blank well as the corrected OD value, plot the four-parameter logic function standard curve with the standard concentration (pg / mL or μg / mL) as the x-axis and the OD value as the y-axis, substitute the corrected OD value of the sample into the standard curve equation, calculate the sIgA concentration in the sample, and calculate the sIgA content in the original tissue according to the sample dilution factor.

[0201] The relative abundance of Escherichia coli in feces was detected by quantitative real-time PCR (qPCR): (1) Take about 0.5 g of fecal sample and operate according to the instructions of the DNA extraction kit (QIAamp Fast DNA Stool Mini Kit); (2) Determine the purity and concentration (OD) of the extracted DNA using a UV spectrophotometer. 260 / 280 (3) Store DNA samples at -20℃ for later use; (4) Detect using quantitative real-time PCR. The results are shown in the table below.

[0202] Table 22 Results of verifying the effects of different bacterial species

[0203] The results showed that group C1 (Lactobacillus salivarius) was significantly superior to groups C2 and C3 in terms of sIgA-stimulated secretion and inhibitory ability against ETEC, and exhibited the highest growth rate in XOS medium, demonstrating its optimal synergy with XOS. Group C1 (Lactobacillus salivarius) showed the lowest diarrhea rate, the highest intestinal sIgA level, and the most significant decrease in pathogenic bacteria abundance, with its overall effect significantly better than other bacterial replacement groups and the control group. This demonstrates that the specific biological function of Lactobacillus salivarius is key to its core effect of "enhancing mucosal immunity and preventing diarrhea," and other bacterial species cannot achieve the same or better overall effect under the same conditions.

[0204] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a feed additive to improve the peripartum production performance of dairy cows, characterized in that, Includes the following steps: (1) Lactobacillus salivarius liquid and xylooligosaccharide were cross-linked and encapsulated with sodium alginate-calcium chloride to obtain co-encapsulated microcapsules; the co-encapsulated microcapsules, phytase microparticles and glutamine microparticles were mixed, preheated, coated with coating solution, and solidified to obtain targeted release particles; (2) Motherwort, Tetrapanax papyriferus, Astragalus membranaceus and Taraxacum mongolicum are mixed and pulverized to obtain a compound powder of traditional Chinese medicine; (3) Targeted release particles, betaine, rumen-protected choline chloride, yeast culture, traditional Chinese medicine compound powder, rosemary extract and wheat bran are mixed to obtain feed additive; In step (1), the ratio of Lactobacillus salivarius solution to xylooligosaccharides is 1-3 mL: 0.5-1.5 g; the viable count of Lactobacillus salivarius solution is 1×10⁻⁶. 8 CFU / mL ~9×10 9 CFU / mL, Lactobacillus salivarius is Lactobacillus salivarius KS1018, preservation number is CCTCC NO: M 2025020; the mass ratio of co-encapsulated microcapsules, phytase microparticles and glutamine microparticles is (6~8):(5~7):(8~10); the preheating temperature is 25~35 ℃; the coating solution is obtained by dissolving pH-sensitive hydroxypropyl methylcellulose succinate powder in ethanol solution; the solid content of the coating solution is 15%~25%, the pH value is 5.5~6.2; the coating method is bottom spray coating, the spraying rate is 2~4 mL / min, the atomization pressure is 0.1~0.15 MPa, the inlet air temperature is maintained at 35~45 ℃ and the material temperature is 30~40 ℃ during the coating process, and the coating weight gain is 20%~25% of the total weight of the mixed particles; In step (2), the mass ratio of Leonurus japonicus, Tetrapanax papyriferus, Astragalus membranaceus, and Taraxacum mongolicum is (2~4):(2~4):(1~3):(0.5~1.5). In step (3), by mass fraction, there are 120-150 parts of targeted release particles, 50-70 parts of betaine, 20-40 parts of rumen-protected choline chloride, 150-250 parts of yeast culture, 300-400 parts of traditional Chinese medicine compound powder, 15-25 parts of rosemary extract, and 350-430 parts of wheat bran.

2. The feed additive prepared by the preparation method according to claim 1.

3. The use of the feed additive of claim 2 in any of the following: (1) preparing a drug for preventing peripartum diseases in dairy cows; (2) preparing a product for improving peripartum production performance in dairy cows.

4. A feed for improving peripartum production performance in dairy cows, characterized in that, The feed contains the feed additive as described in claim 2.

5. The feed according to claim 4, characterized in that, The feed also includes a total mixed ration (TMR), and the amount of feed additives added is 0.5% to 3% DM based on the dry matter of the TMR.

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