A microecological preparation for improving intestinal inflammation of pets, and a preparation method and application thereof

Abstract

The application discloses a microecological preparation for improving pet intestinal inflammation and a preparation method and application thereof, and belongs to the technical field of animal feeding feedstuff. The microecological preparation comprises the following raw materials: probiotics, inactivated lactic acid bacteria and metabolites thereof, prebiotics and repair factors, wherein the probiotics comprise Lactobacillus acidophilus LA-14 and Bacillus subtilis, and the viable bacterial count ratio of the two is 1:2-3. The microecological preparation composition can improve cell activity, inhibit inflammatory response and up-regulate the expression of intestinal tight junction proteins, so that the intestinal barrier function of pets can be protected and repaired.

Description

Technical Field

[0001] This invention belongs to the field of animal feeding technology, specifically relating to a microecological preparation for improving intestinal inflammation in pets, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease in pets, especially in felines, is a common digestive system disease in clinical practice. Its etiology is complex, involving multiple factors such as genetics, diet, immune abnormalities, and gut microbiota imbalance. In recent years, with the increase in pet ownership and changes in the living environment, the incidence of pet bowel disease has been on the rise, seriously affecting the quality of life of pets and causing distress to pet owners. Currently, clinical treatment for pet bowel disease mainly relies on antibiotics, immunosuppressants, and glucocorticoids. However, long-term use of antibiotics can easily exacerbate gut microbiota dysbiosis and may also induce bacterial resistance; while immunosuppressants and glucocorticoids can quickly control inflammation, they have certain side effects, such as metabolic disorders and decreased immunity, and relapse is common after discontinuation. Therefore, developing a safe, effective treatment strategy that can fundamentally regulate the gut microbiota and repair the intestinal barrier function has significant clinical importance and market value.

[0003] With the deepening of research in microecology, the role of probiotics and postbiotics in regulating gut health has received increasing attention. Studies have shown that the occurrence and development of intestinal inflammation are closely related to gut microbiota dysbiosis, impaired intestinal barrier function, and abnormal immune responses. Supplementing with probiotics with specific functions can competitively inhibit pathogen colonization, regulate intestinal immune cell function, and alleviate inflammatory responses. However, single probiotics often face problems in practical applications, such as insufficient strain activity, low colonization efficiency, and difficulty in exerting their effects in inflammatory environments.

[0004] To address the shortcomings of existing technologies, this invention aims to provide a microecological preparation capable of synergistic, multi-target intervention for pet intestinal inflammation. This preparation combines functional probiotics with innovatively introduced inactivated lactic acid bacteria and their metabolites, complex prebiotics, and structurally modified repair factors to construct a comprehensive intervention system that integrates regulation of intestinal flora balance, efficient repair of damaged intestinal mucosal barriers, and inhibition of inflammatory factor release. Cellular experiments have demonstrated that this composition exhibits good safety, effectively repairs intestinal barrier damage, significantly upregulates tight junction protein expression, and strongly inhibits the release of pro-inflammatory factors, thus providing a safe, efficient, and stable microecological solution for improving pet intestinal inflammation. Summary of the Invention

[0005] The purpose of this invention is to provide a microecological preparation for improving intestinal inflammation in pets.

[0006] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: A microecological preparation for improving intestinal inflammation in pets includes the following components: a microecological mixture, prebiotics, and repair factors, with a mass ratio of 2:6:0.2. The microecological mixture includes probiotics, inactivated lactic acid bacteria, and their metabolites, with a mass ratio of 2-3:20-25.

[0007] Furthermore, the probiotics include *Lactobacillus acidophilus* LA-14 and *Lactobacillus terrestris*, with a live bacteria ratio of 1:2-3; the *Lactobacillus acidophilus* LA-14 has the preservation number CGMCC No. 31624, and the *Lactobacillus terrestris* has the preservation number CGMCC No. 1.3846; the live bacteria count of the probiotics is ≥1.0 × 10⁻⁶. 10 CFU / g.

[0008] Furthermore, the prebiotics include xylooligosaccharides, fructooligosaccharides, and mannan oligosaccharides, with a mass ratio of 4:1:1.

[0009] Furthermore, the method for preparing the probiotics is as follows: (1) Pretreatment and addition of protective agent: When each cell grows to the late stage of the stable period, centrifuge at 6000-8000 rpm for 10-15 min at 4℃ to harvest the fungal mud. Mix the fungal mud with the sterile freeze-drying protective agent at 4℃ to obtain a mixture. (2) Pre-freezing: Dispense the mixture into freeze-drying bottles and quickly treat it at -80°C to -40°C to solidify the sample completely and form fine ice crystals; (3) Primary drying: The pre-frozen sample is quickly transferred to the pre-cooled freeze dryer chamber, and the temperature of the partition is gradually increased from -40℃ to about -20℃ to -10℃, and maintained in this temperature range for 20-40h. (4) Secondary drying: Slowly increase the temperature of the partition to 25-30℃ and maintain it for 4-8 hours. Keep the vacuum pressure at a low level throughout, and strictly control the residual moisture content to 2-4%. Probiotics are obtained. (5) Post-processing and storage: After drying, the freeze-dried powder is vacuum sealed or sealed after being filled with nitrogen in the freeze dryer chamber or transferred to a drying operation box with relative humidity <20%. After sealing, it is immediately placed in a light-proof and dry condition at -20℃ for long-term storage or 4℃ for short-term storage.

[0010] Furthermore, the mass ratio of the bacterial sludge to the sterile freeze-drying protectant is 1:1.5-2.5; the freeze-drying protectant includes sodium caseinate, sodium octenyl succinate starch, sodium alginate, and monosodium glutamate, with a mass ratio of 3-4:2-3:1-2:0.5-1.

[0011] Furthermore, the inactivated lactic acid bacteria and their metabolites are prepared using the following steps: S1 strain activation and seed culture: After activating the freeze-dried Lactobacillus acidophilus, it was inoculated into MRS liquid medium and activated and cultured for 1-2 generations at 37℃. Lactobacillus terrestris was inoculated into GYP liquid medium and activated and cultured for 1-2 generations at 30℃. The above were then scaled up to seed tanks step by step to obtain seed solutions. S2 high-density fermentation: Inoculate the seed liquid into each fermentation medium, control the temperature at 30-37℃, and ferment in an anaerobic or micro-aerobic environment for 18-24 hours; S3 cell collection and washing: After fermentation, the cell precipitate is collected by centrifugation, washed 2-3 times with physiological saline or buffer to obtain pure bacterial sludge, and then resuspended with buffer to obtain bacterial suspension; S4 inactivation treatment: Moist heat inactivation is used, and the treatment is carried out at 60-80℃ for 10-30 minutes; S5 Drying and Powdering: Add a protective agent to the inactivated bacterial suspension, and make it into powder by spray drying or freeze drying, controlling the moisture content to <5%. After drying, sieve to obtain inactivated lactic acid bacteria and their metabolites.

[0012] Furthermore, the number of inactivated lactic acid bacteria cells in the inactivated lactic acid bacteria and their metabolites is ≥1.0 × 10⁻⁶. 10 / g; in step S4, the mass-to-volume ratio of bacterial sludge to buffer solution is 1kg / 0.3-0.4L; the amount of protective agent added is 100g per liter of bacterial suspension, and the protective agent includes trehalose, skim milk powder, and monosodium glutamate, with a mass ratio of 5:10:2.

[0013] Furthermore, the ratio of Lactobacillus acidophilus LA-14 to Lactobacillus terrestris in the inactivated lactic acid bacteria and their metabolites is 1:1.

[0014] Furthermore, the repair factor is prepared using the following steps: (1) Feeding and precooling: 1 mole of L-glutamine was added to 900 mL of purified water. The reaction flask was placed in an ice-water bath and the reaction solution was cooled to 0-5℃. 5 mol / L NaOH solution was added dropwise to adjust the pH of the system to 10-11. The mixture was stirred until L-glutamine was completely dissolved. (2) Acylation reaction: In an ice-water bath, 1.2 moles of propionyl chloride were placed in a constant pressure dropping funnel, and 240 mL of 5 mol / L NaOH solution was placed in another constant pressure dropping funnel. While stirring, propionyl chloride and NaOH solution were added dropwise to the reaction flask of step 1. The pH of the system was maintained at 9.5-10.5 and the temperature at 0-5℃. After the addition was completed, the reaction was stirred at 0-5℃ for 1 hour. (3) Post-treatment and purification: After the reaction is completed, solid sodium chloride is added to the reaction solution under stirring until the sodium chloride no longer dissolves. The system is placed in an ice-water bath to cool to 0-5℃. 6mol / L hydrochloric acid solution is added dropwise under stirring to adjust the pH to 2.5-3.0. The mixture is allowed to stand at room temperature for 1 hour, then filtered. The filter cake is washed with 200mL of ice water, collected, and placed in a vacuum drying oven. It is then dried under reduced pressure at 40℃ to constant weight to obtain the repair factor.

[0015] A method for preparing a probiotic preparation for improving intestinal inflammation in pets includes the following steps: Probiotics, inactivated lactic acid bacteria and their metabolites, and repair factors are prepared separately. The formula amount of prebiotics is mixed for 15-25 minutes to obtain a prebiotic premix. Then, the formula amount of inactivated lactic acid bacteria and their metabolites, repair factors, and probiotics are added, and the mixture is mixed for another 20-30 minutes. The mixture is then passed through a 60-80 mesh sieve to obtain a microecological preparation.

[0016] The present invention also provides the application of the microecological preparation for improving intestinal inflammation in pets, specifically for improving intestinal inflammation in cats and dogs, wherein the amount of microecological preparation added to cat food and dog food is 0.1-0.5%.

[0017] All raw materials used in this invention are commercially available. Lactobacillus acidophilus LA-14 was provided by Inner Mongolia Ketuo Microecological Technology Development Co., Ltd.; Lactobacillus terrestris was purchased from the China General Microbiological Culture Collection Center, with the original preservation date being March 25, 2005.

[0018] This invention combines lactic acid bacteria to obtain probiotic components, which improve intestinal inflammation through multiple mechanisms: (I) Constructing a synergistic defense network, reshaping the intestinal microecological balance, regulating the function of intestinal immune cells, and directly alleviating intestinal inflammation. Unlike other lactobacilli in the formula, *Lactobacillus terrestris* has a spore morphology, which can withstand the strong killing effect of gastric acid and bile salts, ensuring that a high number of live bacteria reach deep into the intestines for release and germination. The absence of this strain will lead to a significant increase in the loss rate of the microecological preparation in the stomach, making it impossible to guarantee that sufficient live bacteria reach the lesion site to exert their effects. (II) In the highly inflammatory intestinal environment, oxygen content often increases. As a microaerophilic bacterium, *Lactobacillus terrestris* can consume oxygen in the intestines, creating a suitable low-oxygen colonization environment for *Lactobacillus acidophilus*, significantly enhancing the synergistic effect of the compound system. Without *Lactobacillus terrestris*, the colonization efficiency of *Lactobacillus acidophilus* will be greatly reduced, its efficacy will be difficult to fully exert, and an effective intestinal microecological balance regulation system cannot be formed, thus failing to effectively alleviate intestinal inflammation. (III) Lactobacillus terrestris can also secrete a variety of active substances, which work synergistically with repair factors to promote the repair of intestinal mucosal damage, enhance the intestinal mucosal barrier function, reduce the recurrence of intestinal inflammation, and at the same time help improve the overall immunity of pets, inhibit the occurrence and development of intestinal inflammation from the immune level, and further consolidate the effect of inflammation improvement.

[0019] This invention innovatively incorporates a repair factor, a novel derivative obtained by modifying L-glutamine with a propionyl group. Compared to unmodified L-glutamine, it exhibits improved stability in the cat's intestines, more efficiently repairing damaged intestinal mucosa, reducing intestinal inflammatory exudation, and is easily absorbed by the pet's intestines without any toxic side effects. The prebiotic in this invention specifically promotes the growth of probiotics and pre-existing beneficial bacteria in the intestines, while simultaneously providing a carrier for the pet's intestinal mucosa repair factor, enhancing its retention time and effectiveness in the intestines.

[0020] Beneficial effects This invention's microecological preparation systematically improves intestinal inflammation in pets through the synergistic effect of four mechanisms: probiotics, inactivated lactic acid bacteria and their metabolites, prebiotics, and repair factors. Specifically, highly active probiotics colonize the gut, restoring microecological balance; high concentrations of inactivated lactic acid bacteria and their metabolites directly regulate immunity and suppress inflammatory responses; prebiotics promote the proliferation of beneficial bacteria; and the repair factor, an L-glutamine derivative, targets and repairs the damaged intestinal mucosal barrier. These four mechanisms work synergistically, forming a closed loop from gut microbiota regulation and immunosuppression to mucosal repair, significantly protecting Caco-2 cells, repairing the intestinal barrier, and exerting anti-inflammatory effects. It offers both preventative and therapeutic benefits and is safe with no side effects. This invention's microecological preparation is suitable for formulations in various pet foods, including but not limited to cat and dog food.

[0021] This invention uses Caco-2 cells as a model. First, the safe dosage of each component of the probiotic preparation was screened using the CCK-8 assay, showing that a single component was non-toxic to cells. Based on this, the cytotoxicity of the compound composition was tested, confirming that the composition was non-toxic to Caco-2 cells. A Caco-2 intestinal epithelial monolayer model was constructed using Transwell chambers. After 20 days of culture, the transmembrane resistance (TEER) was greater than 500 Ω·cm², indicating successful model construction. LPS intervention significantly reduced cellular transmembrane resistance and damaged intestinal barrier function, while the composition of this invention significantly restored TEER values, effectively repairing LPS-induced intestinal barrier damage. ELISA results showed that LPS significantly upregulated the secretion levels of inflammatory factors IL-1β, IL-6, and TNF-α, and the composition of this invention significantly inhibited the release of these pro-inflammatory factors, exerting a potent anti-inflammatory effect. Western blot results further showed that LPS significantly downregulated the expression of tight junction proteins Claudin-1 and ZO-1, and the composition of the present invention could significantly reverse this effect, restoring the protein expression levels of Claudin-1 and ZO-1. In summary, the microecological preparation composition of the present invention achieves the effects of protecting and repairing intestinal barrier function by enhancing cell activity, inhibiting inflammatory responses, and upregulating the expression of intestinal tight junction proteins. Attached Figure Description

[0022] Figure 1 CCK8 assay was used to assess cell viability in Caco-2 cells after different treatments. Figure 2 CCK8 assay was used to assess cell viability in Caco-2 cells after different treatments. Figure 3 Changes in TEER in Caco-2 cells after different treatments; Figure 4 To detect the levels of inflammatory factors IL-1β, IL-6 and TNFα in the lower chamber culture medium of different treatment groups; Figure 5 Figure showing the expression of claudin1 and zo-1 proteins in Caco2 cells after different treatments, detected by Western blot. Figure 6 A statistical graph showing the expression of claudin1 protein in Caco2 cells after different treatments using Western blot analysis. Figure 7 A statistical graph showing the expression of zo-1 protein in Caco2 cells after different treatments, detected by Western blot. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0024] Example 1 A microecological preparation for improving intestinal inflammation in pets includes the following components: a microecological mixture, prebiotics, and repair factors, with a mass ratio of 2:6:0.2. The microecological mixture includes probiotics, inactivated lactic acid bacteria, and their metabolites, with a mass ratio of 3:20.

[0025] The probiotics include Lactobacillus acidophilus LA-14 and Lactobacillus terrestris, with a live bacteria ratio of 1:2; the Lactobacillus acidophilus LA-14 has the preservation number CGMCC No. 31624, and the Lactobacillus terrestris has the preservation number CGMCC No. 1.3846; the live bacteria count of the probiotics is 1.5 × 10⁻⁶. 10 CFU / g.

[0026] The prebiotics include xylooligosaccharides, fructooligosaccharides, and mannan oligosaccharides, with a mass ratio of 4:1:1.

[0027] The method for preparing the probiotics is as follows: (1) Pretreatment and addition of protective agent: When each cell grows to the late stage of the stable period, centrifuge at 6000-8000 rpm for 10-15 min at 4℃ to harvest the fungal mud. Mix the fungal mud with the sterile freeze-drying protective agent at 4℃ to obtain a mixture. (2) Pre-freezing: Dispense the mixture into freeze-drying bottles and quickly treat it at -80°C to -40°C to solidify the sample completely and form fine ice crystals; (3) Primary drying: The pre-frozen sample is quickly transferred to the pre-cooled freeze dryer chamber, and the temperature of the partition is gradually increased from -40℃ to about -20℃ to -10℃ and maintained in this temperature range for 20 hours. (4) Secondary drying: Slowly increase the temperature of the partition to 25°C and maintain it for 8 hours. Keep the vacuum pressure at a low level throughout, and strictly control the residual moisture content to 2-4%. Probiotics are obtained. (5) Post-processing and storage: After drying, the freeze-dried powder is vacuum sealed or sealed after being filled with nitrogen in the freeze dryer chamber or transferred to a drying operation box with relative humidity <20%. After sealing, it is immediately placed in a light-proof and dry condition at -20℃ for long-term storage or 4℃ for short-term storage.

[0028] The mass ratio of the bacterial sludge to the sterile freeze-drying protectant is 1:2.5; the freeze-drying protectant includes sodium caseinate, sodium octenyl succinate starch, sodium alginate, and monosodium glutamate, with a mass ratio of 3:3:1:0.5.

[0029] The inactivated lactic acid bacteria and their metabolites are prepared using the following steps: S1 strain activation and seed culture: freeze-dried Lactobacillus acidophilus was inoculated into MRS liquid medium and activated and cultured for 1-2 generations at 37℃. Lactobacillus terrestris was inoculated into GYP liquid medium and activated and cultured for 1-2 generations at 30℃. The above were then scaled up to seed tanks step by step to obtain seed solutions. S2 high-density fermentation: Inoculate the seed liquid into each fermentation medium, control the temperature at 30-37℃, and ferment in an anaerobic or micro-aerobic environment for 18-24 hours; S3 cell collection and washing: After fermentation, the cell precipitate is collected by centrifugation, washed 2-3 times with physiological saline or buffer to obtain pure bacterial sludge, and then resuspended with buffer to obtain bacterial suspension; S4 inactivation treatment: Moist heat inactivation was performed at 60℃ for 10 min; S5 Drying and Powdering: Add a protective agent to the inactivated bacterial suspension, and make it into powder by spray drying or freeze drying, controlling the moisture content to <5%. After drying, sieve to obtain inactivated lactic acid bacteria and their metabolites.

[0030] The number of inactivated lactic acid bacteria cells in the inactivated lactic acid bacteria and their metabolites was 1.0 × 10⁻⁶. 10 The number of Lactobacillus acidophilus LA-14 and Lactobacillus aeruginosa in the inactivated lactic acid bacteria and their metabolites is 1:1; the mass-to-volume ratio of bacterial sludge to buffer solution in step S4 is 1 kg / 0.3 L; the amount of protective agent added is 100 g per liter of bacterial suspension, and the protective agent includes trehalose, skim milk powder, and monosodium glutamate in a mass ratio of 5:10:2.

[0031] The repair factor is prepared using the following steps: (1) Feeding and precooling: 1 mole of L-glutamine was added to 900 mL of purified water. The reaction flask was placed in an ice-water bath and the reaction solution was cooled to 0-5℃. 5 mol / L NaOH solution was added dropwise to adjust the pH of the system to 10-11. The mixture was stirred until L-glutamine was completely dissolved. (2) Acylation reaction: In an ice-water bath, 1.2 moles of propionyl chloride were placed in a constant pressure dropping funnel, and 240 mL of 5 mol / L NaOH solution was placed in another constant pressure dropping funnel. While stirring, propionyl chloride and NaOH solution were added dropwise to the reaction flask of step 1. The pH of the system was maintained at 9.5-10.5 and the temperature at 0℃. After the addition was completed, the reaction was stirred at 0℃ for 1 hour. (3) Post-treatment and purification: After the reaction is completed, solid sodium chloride is added to the reaction solution under stirring until the sodium chloride no longer dissolves. The system is placed in an ice-water bath to cool to 0°C. 6 mol / L hydrochloric acid solution is added dropwise under stirring to adjust the pH to 2.5-3.0. The mixture is allowed to stand at room temperature for 1 hour, then filtered. The filter cake is washed with 200 mL of ice water, collected, and placed in a vacuum drying oven. It is then dried under reduced pressure at 40°C to constant weight to obtain the repair factor.

[0032] A method for preparing a probiotic preparation for improving intestinal inflammation in pets includes the following steps: Probiotics, inactivated lactic acid bacteria and their metabolites, and repair factors were prepared separately. The prebiotics were mixed in the formula amount for 25 minutes to obtain a prebiotic premix. Then, the inactivated lactic acid bacteria and their metabolites, repair factors, and probiotics were added in the formula amount, and the mixture was mixed for another 20 minutes. The mixture was then passed through a 60-80 mesh sieve to obtain a microecological preparation.

[0033] Example 2 A microecological preparation for improving intestinal inflammation in pets includes the following components: a microecological mixture, prebiotics, and repair factors, with a mass ratio of 2:6:0.2. The microecological mixture includes probiotics, inactivated lactic acid bacteria, and their metabolites, with a mass ratio of 2:25.

[0034] The probiotics include Lactobacillus acidophilus LA-14 and Lactobacillus aeruginosa, with a live bacteria ratio of 1:3; the Lactobacillus acidophilus LA-14 has the preservation number CGMCC No. 31624, and the Lactobacillus aeruginosa has the preservation number CGMCC No. 1.3846; the live bacteria count of the probiotics is 1.0 × 10⁻⁶. 10 CFU / g.

[0035] The prebiotics include xylooligosaccharides, fructooligosaccharides, and mannan oligosaccharides, with a mass ratio of 4:1:1.

[0036] The method for preparing the probiotics is as follows: (1) Pretreatment and addition of protective agent: When each cell grows to the late stage of the stable period, centrifuge at 6000-8000 rpm for 10-15 min at 4℃ to harvest the fungal mud. Mix the fungal mud with the sterile freeze-drying protective agent at 4℃ to obtain a mixture. (2) Pre-freezing: Dispense the mixture into freeze-drying bottles and quickly treat it at -80°C to -40°C to solidify the sample completely and form fine ice crystals; (3) Primary drying: The pre-frozen sample is quickly transferred to the pre-cooled freeze dryer chamber, and the temperature of the partition is gradually increased from -40℃ to about -20℃ to -10℃, and maintained in this temperature range for 20-40h. (4) Secondary drying: Slowly increase the temperature of the partition to 30°C and maintain it for 4 hours. Keep the vacuum pressure at a low level throughout, and strictly control the residual moisture content to 2-4%. Probiotics are obtained. (5) Post-processing and storage: After drying, the freeze-dried powder is vacuum sealed or sealed after being filled with nitrogen in the freeze dryer chamber or transferred to a drying operation box with relative humidity <20%. After sealing, it is immediately placed in a light-proof and dry condition at -20℃ for long-term storage or 4℃ for short-term storage.

[0037] The mass ratio of the bacterial sludge to the sterile freeze-drying protectant is 1:1.5; the freeze-drying protectant includes sodium caseinate, sodium octenyl succinate starch, sodium alginate, and monosodium glutamate, with a mass ratio of 4:2:2:1.

[0038] The inactivated lactic acid bacteria and their metabolites are prepared using the following steps: S1 strain activation and seed culture: freeze-dried Lactobacillus acidophilus was inoculated into MRS liquid medium and activated and cultured for 1-2 generations at 37℃. Lactobacillus terrestris was inoculated into GYP liquid medium and activated and cultured for 1-2 generations at 30℃. The above were then scaled up to seed tanks step by step to obtain seed solutions. S2 high-density fermentation: Inoculate the seed liquid into each fermentation medium, control the temperature at 30-37℃, and ferment in an anaerobic or micro-aerobic environment for 18-24 hours; S3 cell collection and washing: After fermentation, the cell precipitate is collected by centrifugation, washed 2-3 times with physiological saline or buffer to obtain pure bacterial sludge, and then resuspended with buffer to obtain bacterial suspension; S4 inactivation treatment: Moist heat inactivation was performed at 80℃ for 30 minutes; S5 Drying and Powdering: Add a protective agent to the inactivated bacterial suspension, and make it into powder by spray drying or freeze drying, controlling the moisture content to <5%. After drying, sieve to obtain inactivated lactic acid bacteria and their metabolites.

[0039] The number of inactivated lactic acid bacteria cells in the inactivated lactic acid bacteria and their metabolites was 1.0 × 10⁻⁶. 10 The number of Lactobacillus acidophilus LA-14 and Lactobacillus aeruginosa in the inactivated lactic acid bacteria and their metabolites is 1:1; the mass-to-volume ratio of bacterial sludge to buffer solution in step S4 is 1 kg / 0.4 L; the amount of protective agent added is 100 g per liter of bacterial suspension, and the protective agent includes trehalose, skim milk powder, and monosodium glutamate in a mass ratio of 5:10:2.

[0040] The repair factor is prepared using the following steps: (1) Feeding and pre-cooling: 1 mole of L-glutamine was added to 900 mL of purified water. The reaction flask was placed in an ice-water bath and the reaction solution was cooled to 5°C. 5 mol / L NaOH solution was added dropwise to adjust the pH of the system to 10-11. The mixture was stirred until L-glutamine was completely dissolved. (2) Acylation reaction: In an ice-water bath, 1.2 moles of propionyl chloride were placed in a constant pressure dropping funnel, and 240 mL of 5 mol / L NaOH solution was placed in another constant pressure dropping funnel. While stirring, propionyl chloride and NaOH solution were added dropwise to the reaction flask of step 1. The pH of the system was maintained at 9.5-10.5 and the temperature at 5℃. After the addition was completed, the reaction was stirred at 5℃ for 1 hour. (3) Post-treatment and purification: After the reaction is completed, solid sodium chloride is added to the reaction solution under stirring until the sodium chloride no longer dissolves. The system is placed in an ice-water bath and cooled to 5°C. 6 mol / L hydrochloric acid solution is added dropwise under stirring to adjust the pH to 2.5-3.0. The mixture is allowed to stand at room temperature for 1 hour, then filtered. The filter cake is washed with 200 mL of ice water, collected, and placed in a vacuum drying oven. It is then dried under reduced pressure at 40°C to constant weight to obtain the repair factor.

[0041] A method for preparing a probiotic preparation for improving intestinal inflammation in pets includes the following steps: Probiotics, inactivated lactic acid bacteria and their metabolites, and repair factors were prepared separately. The prebiotics were mixed in the formula amount for 15 minutes to obtain a prebiotic premix. Then, the inactivated lactic acid bacteria and their metabolites, repair factors, and probiotics were added in the formula amount, and the mixture was mixed for another 30 minutes. The mixture was then passed through a 60-80 mesh sieve to obtain a microecological preparation.

[0042] Example 3 A microecological preparation for improving intestinal inflammation in pets includes the following components: a microecological mixture, prebiotics, and repair factors, with a mass ratio of 2:6:0.2. The microecological mixture includes probiotics, inactivated lactic acid bacteria, and their metabolites, with a mass ratio of 2:20.

[0043] The probiotics include Lactobacillus acidophilus LA-14 and Lactobacillus aeruginosa, with a live bacteria ratio of 1:2; the Lactobacillus acidophilus LA-14 has the preservation number CGMCC No. 31624, and the Lactobacillus aeruginosa has the preservation number CGMCC No. 1.3846; the live bacteria count of the probiotics is 1.2 × 10⁻⁶. 10 CFU / g. The prebiotics include xylooligosaccharides, fructooligosaccharides, and mannan oligosaccharides, with a mass ratio of 4:1:1.

[0044] The method for preparing the probiotics is as follows: (1) Pretreatment and addition of protective agent: When each cell grows to the late stage of the stable period, centrifuge at 6000-8000 rpm for 10-15 min at 4℃ to harvest the fungal mud. Mix the fungal mud with the sterile freeze-drying protective agent at 4℃ to obtain a mixture. (2) Pre-freezing: Dispense the mixture into freeze-drying bottles and quickly treat it at -80°C to -40°C to solidify the sample completely and form fine ice crystals; (3) Primary drying: The pre-frozen sample is quickly transferred to the pre-cooled freeze dryer chamber, and the temperature of the partition is gradually increased from -40℃ to about -20℃ to -10℃, and maintained in this temperature range for 20-40h. (4) Secondary drying: Slowly increase the temperature of the partition to 25°C and maintain it for 6 hours. Keep the vacuum pressure at a low level throughout, and strictly control the residual moisture content to 2-4%. Probiotics are obtained. (5) Post-processing and storage: After drying, the freeze-dried powder is vacuum sealed or sealed after being filled with nitrogen in the freeze dryer chamber or transferred to a drying operation box with relative humidity <20%. After sealing, it is immediately placed in a light-proof and dry condition at -20℃ for long-term storage or 4℃ for short-term storage.

[0045] The mass ratio of the bacterial sludge to the sterile freeze-drying protectant is 1:2.0; the freeze-drying protectant includes sodium caseinate, sodium octenyl succinate starch, sodium alginate, and monosodium glutamate, with a mass ratio of 3:2:2:1.

[0046] The inactivated lactic acid bacteria and their metabolites are prepared using the following steps: S1 strain activation and seed culture: freeze-dried Lactobacillus acidophilus was inoculated into MRS liquid medium and activated and cultured for 1-2 generations at 37℃. Lactobacillus terrestris was inoculated into GYP liquid medium and activated and cultured for 1-2 generations at 30℃. The above were then scaled up to seed tanks step by step to obtain seed solutions. S2 high-density fermentation: Inoculate the seed liquid into each fermentation medium, control the temperature at 30-37℃, and ferment in an anaerobic or micro-aerobic environment for 18-24 hours; S3 cell collection and washing: After fermentation, the cell precipitate is collected by centrifugation, washed 2-3 times with physiological saline or buffer to obtain pure bacterial sludge, and then resuspended with buffer to obtain bacterial suspension; S4 inactivation treatment: Moist heat inactivation was performed at 70℃ for 20 minutes; S5 Drying and Powdering: Add a protective agent to the inactivated bacterial suspension, and make it into powder by spray drying or freeze drying, controlling the moisture content to <5%. After drying, sieve to obtain inactivated lactic acid bacteria and their metabolites.

[0047] The number of inactivated lactic acid bacteria cells in the inactivated lactic acid bacteria and their metabolites was 1.0 × 10⁻⁶.10 The number of Lactobacillus acidophilus LA-14 and Lactobacillus aeruginosa in the inactivated lactic acid bacteria and their metabolites is 1:1; the mass-to-volume ratio of bacterial sludge to buffer solution in step S4 is 1 kg / 0.4 L; the amount of protective agent added is 100 g per liter of bacterial suspension, and the protective agent includes trehalose, skim milk powder, and monosodium glutamate in a mass ratio of 5:10:2.

[0048] The repair factor is prepared using the following steps: (1) Feeding and pre-cooling: 1 mole of L-glutamine was added to 900 mL of purified water. The reaction flask was placed in an ice-water bath and the reaction solution was cooled to 0°C. 5 mol / L NaOH solution was added dropwise to adjust the pH of the system to 10-11. The mixture was stirred until L-glutamine was completely dissolved. (2) Acylation reaction: In an ice-water bath, 1.2 moles of propionyl chloride were placed in a constant pressure dropping funnel, and 240 mL of 5 mol / L NaOH solution was placed in another constant pressure dropping funnel. While stirring, propionyl chloride and NaOH solution were added dropwise to the reaction flask of step 1. The pH of the system was maintained at 9.5-10.5 and the temperature at 0℃. After the addition was completed, the reaction was stirred at 0℃ for 1 hour. (3) Post-treatment and purification: After the reaction is completed, solid sodium chloride is added to the reaction solution under stirring until the sodium chloride no longer dissolves. The system is placed in an ice-water bath to cool to 0°C. 6 mol / L hydrochloric acid solution is added dropwise under stirring to adjust the pH to 2.5-3.0. The mixture is allowed to stand at room temperature for 1 hour, then filtered. The filter cake is washed with 200 mL of ice water, collected, and placed in a vacuum drying oven. It is then dried under reduced pressure at 40°C to constant weight to obtain the repair factor.

[0049] A method for preparing a probiotic preparation for improving intestinal inflammation in pets includes the following steps: Probiotics, inactivated lactic acid bacteria and their metabolites, and repair factors were prepared separately. The prebiotics were mixed in the formula amount for 20 minutes to obtain a prebiotic premix. Then, the inactivated lactic acid bacteria and their metabolites, repair factors, and probiotics were added in the formula amount, and the mixture was mixed for another 25 minutes. The mixture was then passed through a 60-80 mesh sieve to obtain a microecological preparation.

[0050] Comparative Example 1 Compared with Example 1, this comparative example is identical to Example 1 except that no repair factor is added. All other raw materials and steps are the same as in Example 1.

[0051] Comparative Example 2 Compared with Example 1, this comparative example is identical to Example 1 except that Bacillus aureus is not added to the probiotics, inactivated lactic acid bacteria and their metabolites.

[0052] Performance testing I. CCK8 Screening for Non-Toxic Doses 1. Caco-2 cells were cultured in DMEM medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) in a cell culture incubator at 37°C containing 5% CO2. 2. Cell passage culture: When the cells reach 80-90% confluence, remove the culture medium, wash with PBS 1-2 times, remove the PBS, add 1 mL of trypsin to digest for 1-3 min, add 3 mL of complete culture medium to neutralize the trypsin and stop the digestion, transfer the digested cells to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 3 mL of culture medium to resuspend the cells, and passage them 1:3 into culture dishes for culture. 3. Digest the Caco-2 cells that have grown to 80-90% in the culture dish with trypsin, centrifuge at 1000 rpm, remove the supernatant, resuspend the cells in complete culture medium, and seed the cells into 96-well plates, 5000 cells per well. 4. After the cells have adhered to the wall, add solutions of each component at different concentrations as required, and incubate for 24 hours; 5. Add CCK8 diluted according to the instructions of the cell counting kit to the culture medium, incubate in an incubator for 2 hours, and measure the absorbance at 450 nm using a microplate reader.

[0053] Experimental results are as follows Figure 1 As shown, none of the components in the microecological preparation of the present invention have significant toxicity to Caco-2 cells. This result determined the safe usage range of each component for subsequent experiments as follows: xylooligosaccharide < 0.05 mg / mL, fructooligosaccharide < 0.05 mg / mL, mannan oligosaccharide < 0.05 mg / mL, the microecological mixture prepared in Example 1 < 0.05 mg / mL, and the repair factor < 0.005 mg / mL.

[0054] II. Cytotoxicity Test of Co-culture System 1. Caco-2 cells were cultured in DMEM medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) in a cell culture incubator at 37°C containing 5% CO2. 2. Cell passage culture: When the cells reach 80-90% confluence, remove the culture medium, wash with PBS 1-2 times, remove the PBS, add 1 mL of trypsin to digest for 1-3 min, add 3 mL of complete culture medium to neutralize the trypsin and stop the digestion, transfer the digested cells to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 3 mL of culture medium to resuspend the cells, and passage them 1:3 into culture dishes for culture. 3. Digest the Caco-2 cells that have grown to 80-90% in the culture dish with trypsin, centrifuge at 1000 rpm, remove the supernatant, resuspend the cells in complete culture medium, and seed the cells into 96-well plates, 5000 cells per well. 4. After the cells adhered to the wall, add 0.05 mg / mL xylooligosaccharide, 0.0125 mg / mL fructooligosaccharide, 0.0125 mg / mL mannan oligosaccharide, 0.025 mg / mL microecological mixture, and 0.0025 mg / mL repair factor prepared in Example 1, and incubate for 24 h. 5. Add CCK8 diluted according to the instructions of the cell counting kit to the culture medium, incubate in an incubator for 2 hours, and measure the absorbance at 450 nm using a microplate reader.

[0055] Experimental results are as follows Figure 2 As shown, the microecological preparation of the present invention (labeled as a mixed drug in the figure) is non-toxic to Caco-2 cells.

[0056] III. Transwell Single-Layer Model Co-Cultivation Experiment (one) 1. Caco-2 cells were cultured in MEM medium (containing 10% fetal bovine serum, 1% NEAA, 100 U / mL penicillin, and 100 μg / mL streptomycin) and in a cell culture incubator at 37°C containing 5% CO2. 2. Cell passage and expansion culture: When the cells reach 70-80% confluence, remove the culture medium, wash 1-2 times with PBS, remove the PBS, and add 1 ml of trypsin for 1-2 minutes of digestion. Observe under a microscope until the cells change from an extended shape to a round shape, then quickly add about 3-6 mL of culture medium to stop the digestion (shake the culture dish). Use a pipette to remove the cells, ensuring there are no large cell clumps. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 3 minutes. Prepare a culture flask containing 6 mL of complete culture medium for centrifugation. After centrifugation, discard the liquid, add 3 mL of complete culture medium to the centrifuge tube, and repeatedly pipette to count the cells. 2. 5×10 4 The cells were seeded in a non-contact co-culture chamber of a 12-well plate. 500 μL of culture medium was added to the upper chamber and 1.5 mL of culture medium was added to the lower chamber. The cells were incubated at 37°C for 20 days, with the medium changed every 2 days. 3. Before measuring transmembrane resistance, immerse the electrode in the culture medium for 24 hours to equilibrate, then immerse it in 75% alcohol for 30 minutes to sterilize, remove it and let it dry at room temperature for 1 minute to allow the electrode to dry naturally, then place it in the buffer solution for 15 minutes to equilibrate. 4. Insert both ends of the electrode into the upper and lower chambers of each well of the Transwell culture plate in sequence and measure the resistance value. Measure the resistance value at any position in each well. The test results are shown in Table 1.

[0057] Table 1. Results of transmembrane resistance measurement of Caco-2 cells after 20 days of culture. The Caco-2 cell Transwell monolayer model constructed in this experiment was successfully cultured. The cells formed a structurally complete and tightly connected epithelial monolayer with normal intestinal barrier function. The TEER values ​​of the 12 wells showed good uniformity, with overall stable values ​​and no abnormal wells with significantly low values, indicating that the model construction has high reproducibility and can be used for subsequent LPS injury and drug intervention experiments, thus eliminating the interference of "model itself being unqualified" on subsequent experimental results. (two) 1. Caco-2 cells were cultured in MEM complete medium containing 20% ​​fetal bovine serum (with 1% NEAA added) and cultured in a cell culture incubator containing 5% CO2 at 37°C. When the cells proliferated to 80%-90%, they were digested with trypsin and passaged at a ratio of 1:3. 2. 5×10 4 The cells were seeded in a non-contact co-culture chamber of a 12-well plate. 500 μL of culture medium was added to the upper chamber and 1.5 mL of culture medium was added to the lower chamber. The cells were incubated at 37°C for 20 days, with the medium changed every 2 days. 3. Before measuring transmembrane resistance, immerse the electrode in the culture medium for 24 hours to equilibrate, then immerse it in 75% alcohol for 30 minutes to sterilize, remove it and let it dry at room temperature for 1 minute to allow the electrode to dry naturally, then place it in the buffer solution for 15 minutes to equilibrate. 4. Insert both ends of the electrode into the upper and lower chambers of each well of the Transwell culture plate in sequence and measure the resistance value. Measure the resistance value at any position in each well to verify whether the model has been successfully constructed. 5. Experimental Grouping and Treatment: All groups were configured with 3 technical replicates. Three biological replicates were performed. Specific groupings were as follows: Control group: Only complete culture medium was added, without any test substance or LPS modeling, followed by incubation; Model group: 1 μg / mL LPS was added for modeling, without any test substance, followed by incubation; Experimental group: 1 μg / mL LPS was added for modeling, and simultaneously, the test substance prepared in Example 1 (denoted as med in the figure) was added, dissolved in complete culture medium, and diluted to a final concentration of 0.05 mg / mL xylooligosaccharide, 0.0125 mg / mL fructooligosaccharide, 0.0125 mg / mL mannan oligosaccharide, 0.025 mg / mL microecological mixture, and 0.0025 mg / mL repair factor; all were incubated together; resistivity values ​​were measured at 24 h and 48 h, and the results are shown in [Figure Number]. Figure 3 .

[0059] Depend on Figure 3 It can be seen that, compared with the control group at the same time point, the TEER value of the model group was significantly reduced, indicating that LPS successfully disrupted the tight junctions of Caco-2 cells, causing damage to the intestinal barrier function, and the model was successfully established. Compared with the model group at the same time point, the TEER value of the experimental group was significantly increased, indicating that the mixed drug of the microecological preparation of the present invention has a significant repair and protective effect on improving the damage of tight junctions of intestinal epithelium caused by LPS and on the damaged intestinal barrier, and continues to play a role within 48 hours.

[0060] IV. ELISA Detection Experiment Add 1 ml of sample diluent to a standard tube and incubate at room temperature for 10 min, then mix thoroughly. Arrange seven 1.5 ml centrifuge tubes sequentially, adding 500 µl of sample diluent to each. Pipette 500 µl of standard into the first centrifuge tube and gently mix. Pipette 500 µl of standard from the first centrifuge tube into the second EP tube and gently mix. Continue this serial dilution of the standard, with the last tube containing the sample diluent. Add 100 µL of standard and 100 µL of the sample to be tested to each well, cover the plate, and incubate at 37 °C for 90 min. Discard the liquid in the wells, add 100 µL of biotinylated antibody working solution to each well, cover the plate, and incubate at 37 °C for 1 h. Discard the liquid in the wells, add 350 µL of washing buffer, and wash the plate four times, soaking for 1 minute each time, then shake dry. Add 100 μL of HRP enzyme conjugate working solution to each well, cover with a new membrane, and incubate at 37°C for 30 min. Discard the liquid in the wells, add 350 μL of washing buffer, and wash the plate 4 times, soaking for 1 minute each time, then shake dry. Add 90 μL of TMB substrate solution to each well, cover with a membrane, and incubate at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well to terminate the reaction. Within 5 minutes after the reaction is terminated, measure the optical density (OD value) of each well sequentially at a wavelength of 450 nm using a microplate reader. The experimental groups and treatments are the same as in Experiment III (II). The detection results are shown in [Figure 1]. Figure 4 .

[0061] The levels of inflammatory factors IL-1β, IL-6, and TNFα in the lower chamber culture medium of the Transwell co-culture system were detected by ELISA. These three factors are core pro-inflammatory factors in the intestinal inflammatory response; higher levels indicate more severe inflammatory damage to the intestinal epithelium. The experimental groups were consistent with the Transwell experiment (ctrl control group, LPS model group, LPS+med mixed group with the probiotic preparation from Example 1), and measurements were taken at 24h and 48h. IL-1β is a key pro-inflammatory factor initiating the intestinal inflammatory response and a core indicator of early inflammatory activation. Figure 4 As shown in Figure A, compared with the control group at the same time point (ctrl-24h / ctrl-48h), the IL-1β content in the LPS model group (LPS-24h / LPS-48h) increased significantly, indicating that LPS not only disrupted the intestinal barrier but also successfully induced an inflammatory response in Caco-2 cells, demonstrating the effectiveness of the inflammatory model construction. Compared with the LPS model group at the same time point, the IL-1β content in the microecological preparation mixture group (LPS+med-24h / LPS+med-48h) decreased significantly, and the inhibitory effect of the drugs on IL-1β remained significant at 48h, indicating that the mixed drugs can continuously inhibit the release of inflammatory initiating factors and alleviate intestinal inflammation from the source of the inflammatory response.

[0062] IL-6 is a core pro-inflammatory factor that mediates amplified intestinal inflammation and maintains the inflammatory response. It also participates in further damage to the intestinal barrier. Figure 4 As shown in Figure B, at the same time point, the IL-6 content in the LPS model group was much higher than that in the control group, and the IL-6 content in the LPS group remained high at 48h, indicating that the LPS-induced intestinal inflammation showed a continuous amplification trend and did not spontaneously resolve. The IL-6 content in the microecological preparation group decreased sharply compared with the LPS model group, indicating that the microecological preparation of the present invention can effectively block the amplification process of the inflammatory response, avoid the cascade reaction of inflammatory factors from causing more serious damage to the intestinal epithelium, and at the same time alleviate the inhibitory effect of inflammation on intestinal barrier repair.

[0063] TNFα is a highly cytotoxic pro-inflammatory cytokine that directly damages intestinal epithelial cells and disrupts tight junctions. It also promotes the secretion of other pro-inflammatory cytokines and is an important cofactor in intestinal inflammation and barrier damage. Figure 4As shown in Figure C, the TNFα content in the LPS model group was significantly higher than that in the control group, and remained high at both 24h and 48h. This indicates that LPS directly aggravates intestinal epithelial cell damage and barrier disruption by inducing TNFα release. The TNFα content in the microecological preparation mixture group was significantly lower than that in the LPS model group, and the inhibitory effect was more obvious over time (24h→48h). These findings suggest that microecological preparations can reduce the release of highly toxic pro-inflammatory factors, directly reduce the damage of inflammation to intestinal epithelial cells, create a favorable microenvironment for intestinal barrier repair, and block the synergistic damaging effects of TNFα and other inflammatory factors.

[0064] V. Western Blot Experiment Collect the processed tissue, wash it thoroughly with pre-chilled PBS, weigh 50 mg, homogenize it at low temperature, add 200 μl of RIPA lysis buffer, and lyse on ice for 30 min. After lysis, transfer the lysis buffer to a pre-chilled 1.5 ml centrifuge tube (operate on ice). Centrifuge at 12000 rpm for 10 min at 4 °C. Transfer the supernatant to a 1.5 ml centrifuge tube and store at -20 °C. Prepare an appropriate amount of BCA working solution according to the sample quantity, using 50 volumes of BCA reagent A and 1 volume of BCA reagent B (50:1), and mix thoroughly. The BCA working solution is stable at room temperature for 24 hours. Completely dissolve the protein standard, take 10 μl and dilute with PBS to 100 μl, making a final concentration of 0.5 mg / ml. Add standards at concentrations of 0, 1, 2, 4, 8, 12, 16, and 20 μl to the standard wells of a 96-well plate, and bring the total volume to 20 μl with dilution solution. Add an appropriate volume of sample to the sample wells of the 96-well plate, and bring the total volume to 20 μl with dilution solution. Add 200 μl of BCA working solution to each well and incubate at 37°C for 30 minutes. Measure the protein concentration at A562 and calculate the concentration based on the standard curve.

[0065] After aligning the glass plates, place them in the clamps and secure them. Then, vertically clamp them onto the rack, ready for gel pouring. Prepare the required concentration of separating gel (10%), add TEMED, and immediately shake well before pouring. When pouring, use a pipette to draw 3.5-4 ml of gel and release it along the glass. Wait until the gel level reaches the middle line of the green band. Then, add a layer of water on top of the gel and wait for it to solidify. Prepare a 4% stacking gel, add TEMED, and immediately shake well before pouring. After filling the remaining space with stacking gel, immediately insert the comb into the stacking gel. When pouring, ensure the gel flows down the glass plate to avoid air bubbles. Keep the comb horizontal when inserting it. Because the gel shrinks in volume as it solidifies, reducing the sample loading volume in the wells, make sure to press the comb firmly. After the stacking gel solidifies, hold both sides of the comb vertically upwards and gently pull it out. Rinse the wells of the stacking gel with distilled water and place it in the electrophoresis tank. After measuring and quantifying the protein content, transfer the sample to a 0.5 ml centrifuge tube, add 5× SDS loading buffer to a final concentration of 1×, and boil in water for 5 min to denature the protein. Add sufficient electrophoresis buffer and begin loading. Use a microsyringe to aspirate the sample along the tube wall, ensuring no air bubbles are introduced. Insert the pipette needle into the well and slowly add the sample. For the stacking gel, use 80V for 30 min; after the sample enters the separating gel, switch to 120V for 90 min.

[0066] Electrophoresis should be stopped as soon as the bromophenol blue appears, and then the membrane can be transferred. For one membrane transfer, prepare four sheets of filter paper slightly smaller than or the same size as the membrane and one PVDF membrane. Activate the cut PVDF membrane in methanol solution for 1 minute before use. Remove the small glass plate and gently scrape off the stacking gel, being careful not to tear the separating gel. Carefully peel off the separating gel. Soak the peeled gel in transfer buffer. On the transfer apparatus, first stack the two soaked filter papers on the transfer platform and roll them back and forth several times with a glass rod to remove air bubbles. Then place the PVDF membrane on the filter paper. Cover the membrane with the peeled separating gel, adjusting it by hand to align with the filter paper, and gently roll it with a glass rod to remove air bubbles. Cover the gel with two more layers of filter paper and repeat the process to remove air bubbles. The filter papers on both sides of the membrane must not touch each other, otherwise a short circuit will occur. Close the transfer apparatus lid. Adjust the voltage and time to start the transfer at 90V for 1 hour. After the transfer is complete, remove the membrane and seal it. After wetting the membrane with TBS from bottom to top, transfer it to a petri dish containing blocking solution (5% skim milk powder TBST solution) and block on a decolorizing shaker at room temperature for 1 h. Dilute the primary antibody with TBST solution containing 1% BSA and incubate overnight at 4°C. Then wash three times with TBST on a decolorizing shaker at room temperature for 10 min each time. The claudin1 and zo-1 antibodies were diluted 1:1500, and the GAPDH antibody was diluted 1:5000. Dilute the secondary antibody with TBST at 1:5000, incubate for 2 h at room temperature, and then wash three times with TBST on a decolorizing shaker at room temperature for 10 min each time; perform chemiluminescence reaction. Detection results are shown in [Figure number missing]. Figure 5 , Figure 6 and Figure 7 .

[0067] Claudin-1 and ZO-1 are key intestinal tight junction proteins, composed of... Figure 5 The results for Claudin-1 protein showed that, compared to the control group, the Claudin-1 band was significantly lighter and less dense in the LPS model group, indicating that LPS severely disrupted tight junctions, significantly reduced protein expression, and damaged the cell barrier. In the LPS + microecological preparation group, compared to the LPS model group, the Claudin-1 band significantly recovered, becoming darker and thicker, indicating that the microecological preparation of this invention can significantly increase Claudin-1 expression. Similarly, the ZO-1 protein results showed that the ZO-1 band was significantly weakened in the LPS model group, indicating that LPS caused the collapse of intercellular support structures; while in the LPS + microecological preparation group, compared to the LPS model group, ZO-1 protein significantly increased and the band recovered, indicating that the microecological preparation can significantly upregulate ZO-1 expression.

[0068] also, Figure 6 and Figure 7The data also showed that LPS significantly reduced the expression of two tight junction proteins, Claudin-1 and ZO-1, damaging the intestinal barrier; while the addition of microbial agents could significantly reverse this damage, restoring the expression of the two key proteins and thus repairing the intestinal barrier.

[0069] A breeding evaluation trial was conducted on pets using feed supplemented with probiotic preparations from each treatment group. Apparent digestibility test: (a) Subjects: 48 domestic cats aged 43±5 days were divided into Examples 1-3, Comparative Examples 1-2 and Control Group (blank control, no microecological preparations were added to the baked food), with 8 cats in each group, half male and half female.

[0070] Experimental methods: The microecological preparations prepared in Examples 1-3 and Comparative Examples 1-2 were added to the baked food, and the amount of microecological preparation added to each treatment group was 0.1% of the cat food addition amount. The experiment lasted for three weeks, with the baked food added at 0.1 kg / day per cat. The apparent digestibility of crude protein and crude fat of all experimental cats was measured on day 0 and day 21 of the experiment. The experimental results are shown in Table 1.

[0071] (II) Experimental subjects: 42 German Shepherds aged 4±1 months were divided into Examples 1-3, Comparative Examples 1-2 and Control Group (blank control, no microecological preparations were added to the extruded food), with 7 dogs in each group, 5 males and 2 females in each group.

[0072] Experimental methods: The microecological preparations prepared in Examples 1-3 and Comparative Examples 1-2 were added to the extruded food. The amount of microecological preparation added to each treatment group was 0.1% of the amount of extruded food added. The experiment lasted for 30 days. The amount of extruded food was 0.11 kg / day per dog. The apparent digestibility of crude protein and crude fat of the experimental dogs was measured on day 0 and day 30 of the experiment. The experimental results are shown in Table 1.

[0073] Table 1. Results of the apparent digestibility test As shown in Table 1, after adding the microecological preparations of Examples 1-3 prepared according to the present invention, the apparent digestibility of crude protein and crude fat in cats and dogs was higher than that in the control group, indicating that the microecological preparations of the present invention can promote an increasing trend in the digestibility of nutrients in pets. Furthermore, after changing the composition of the microecological preparations of the present invention, the increase in apparent digestibility of crude fat and crude protein in Comparative Examples 1 and 2 decreased, indicating that the repair factors and Bacillus thuringiensis added to the microecological preparations of the present invention are indispensable.

[0074] Gut health, gut stress test: Experimental subjects: 4±1 day old Teddy dogs, 14 in each group, males and females were not counted, and they were divided into 3 groups: group 1, group 2 and group 3.

[0075] Experimental Methods: The microecological preparation prepared in Example 1 was added to two types of extruded food, A and B, at a ratio of 1 kg / ton to obtain A+ and B+, respectively. Group 1 was fed extruded food A for 15 consecutive days and then switched to extruded food B; Group 2 was fed extruded food A for 15 consecutive days and then switched to extruded food B+; Group 3 was fed extruded food A+ for 15 consecutive days and then switched to extruded food B+. The levels of cortisol and malondialdehyde in the serum of Teddy dogs in the three treatment groups were measured 3 days before, 1 day after, 3 days after, and 7 days after the food switch. The data are shown in Table 2.

[0076] Table 2. Results of serum cortisol and malondialdehyde measurements Serum cortisol is an adrenal glucocorticoid that reflects an animal's stress level and is commonly used to detect the physiological response level of animals under stress. As shown in Table 2, the cortisol levels in groups 2 and 3 were significantly lower than those in group 1 at 1, 3, and 7 days after the feed change. Furthermore, the fluctuation in cortisol levels among the Teddy dogs in each treatment group during the entire feed change process was: group 3 < group 2 < group 1, indicating that the stress levels in the three treatment groups during the feed change were: group 3 < group 2 < group 1. Therefore, adding the microecological preparation of this invention to extruded food effectively reduces feed change stress.

[0077] Serum malondialdehyde (MDA) is an important indicator reflecting the level of free radicals and lipid peroxidation metabolism in the body. As shown in Table 2, the serum MDA levels in groups 2 and 3 were significantly lower than those in group 1 at 1, 3, and 7 days after the feed change. Furthermore, the fluctuation in serum MDA levels among the Teddy dogs in each treatment group during the entire feed change process was: group 3 < group 2 < group 1, indicating that the antioxidant capacity of the three treatment groups during the feed change was: group 3 > group 2 > group 1. Therefore, adding the microecological preparation of this invention to extruded food effectively improves the body's peroxide scavenging capacity, rapidly clears excess oxygen free radicals generated by stress, and thus reduces adverse reactions caused by stress.

[0078] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A probiotic preparation for improving intestinal inflammation in pets, characterized in that, It includes the following components: a microecological mixture, prebiotics, and repair factors, with a mass ratio of 2:6:0.

2. The microecological mixture includes probiotics, inactivated lactic acid bacteria and their metabolites, with a mass ratio of 2-3:20-25.

2. The microecological preparation for improving intestinal inflammation in pets according to claim 1, characterized in that, The probiotics include Lactobacillus acidophilus LA-14 and Lactobacillus aeruginosa, with a live bacteria ratio of 1:2-3; the Lactobacillus acidophilus LA-14 has the preservation number CGMCC No. 31624, and the Lactobacillus aeruginosa has the preservation number CGMCC No. 1.3846; the live bacteria count of the probiotics is ≥1.0 × 10⁻⁶. 10 CFU / g.

3. The microecological preparation for improving intestinal inflammation in pets according to claim 1, characterized in that, The prebiotics include xylooligosaccharides, fructooligosaccharides, and mannan oligosaccharides, with a mass ratio of 4:1:

1.

4. The microecological preparation for improving intestinal inflammation in pets according to claim 1, characterized in that, The method for preparing the probiotics is as follows: (1) Pretreatment and addition of protective agent: When each cell grows to the late stage of the stable period, centrifuge at 6000-8000 rpm for 10-15 min at 4℃ to harvest the fungal mud. Mix the fungal mud with the sterile freeze-drying protective agent at 4℃ to obtain a mixture. (2) Pre-freezing: Dispense the mixture into freeze-drying bottles and quickly treat it at -80°C to -40°C to solidify the sample completely and form fine ice crystals; (3) Primary drying: The pre-frozen sample is quickly transferred to the pre-cooled freeze dryer chamber, and the temperature of the partition is gradually increased from -40℃ to about -20℃ to -10℃, and maintained in this temperature range for 20-40h. (4) Secondary drying: Slowly increase the temperature of the partition to 25-30℃ and maintain it for 4-8 hours. Keep the vacuum pressure at a low level throughout, and strictly control the residual moisture content to 2-4%. Probiotics are obtained. (5) Post-processing and storage: After drying, the freeze-dried powder is vacuum sealed or sealed after being filled with nitrogen in the freeze dryer chamber or transferred to a drying operation box with relative humidity <20%. After sealing, it is immediately placed in a light-proof and dry condition at -20℃ for long-term storage or 4℃ for short-term storage.

5. The microecological preparation for improving intestinal inflammation in pets according to claim 4, characterized in that, The mass ratio of the bacterial sludge to the sterile freeze-drying protectant is 1:1.5-2.5; the freeze-drying protectant includes sodium caseinate, sodium octenyl succinate starch, sodium alginate, and monosodium glutamate, with a mass ratio of 3-4:2-3:1-2:0.5-1.

6. The microecological preparation for improving intestinal inflammation in pets according to claim 1, characterized in that, The inactivated lactic acid bacteria and their metabolites are prepared using the following steps: S1 strain activation and seed culture: freeze-dried Lactobacillus acidophilus was inoculated into MRS liquid medium and activated and cultured for 1-2 generations at 37℃. Lactobacillus terrestris was inoculated into GYP liquid medium and activated and cultured for 1-2 generations at 30℃. The above were then scaled up to seed tanks step by step to obtain seed solutions. S2 high-density fermentation: Inoculate the seed liquid into each fermentation medium, control the temperature at 30-37℃, and ferment in an anaerobic or micro-aerobic environment for 18-24 hours; S3 cell collection and washing: After fermentation, the cell precipitate is collected by centrifugation, washed 2-3 times with physiological saline or buffer to obtain pure bacterial sludge, and then resuspended with buffer to obtain bacterial suspension; S4 inactivation treatment: Moist heat inactivation is used, and the treatment is carried out at 60-80℃ for 10-30 minutes; S5 Drying and Powdering: Add a protective agent to the inactivated bacterial suspension, and make it into powder by spray drying or freeze drying, controlling the moisture content to <5%. After drying, sieve to obtain inactivated lactic acid bacteria and their metabolites.

7. The microecological preparation for improving intestinal inflammation in pets according to claim 6, characterized in that, The number of inactivated lactic acid bacteria cells in the inactivated lactic acid bacteria and their metabolites is ≥1.0×10⁻⁶. 10 / g; in step S4, the mass-to-volume ratio of bacterial sludge to buffer solution is 1kg / 0.3-0.4L; the amount of protective agent added is 100g per liter of bacterial suspension, and the protective agent includes trehalose, skim milk powder, and monosodium glutamate, with a mass ratio of 5:10:

2.

8. The microecological preparation for improving intestinal inflammation in pets according to claim 1, characterized in that, The repair factor is prepared using the following steps: (1) Feeding and precooling: 1 mole of L-glutamine was added to 900 mL of purified water. The reaction flask was placed in an ice-water bath and the reaction solution was cooled to 0-5℃. 5 mol / L NaOH solution was added dropwise to adjust the pH of the system to 10-11. The mixture was stirred until L-glutamine was completely dissolved. (2) Acylation reaction: In an ice-water bath, 1.2 moles of propionyl chloride were placed in a constant pressure dropping funnel, and 240 mL of 5 mol / L NaOH solution was placed in another constant pressure dropping funnel. While stirring, propionyl chloride and NaOH solution were added dropwise to the reaction flask of step 1. The pH of the system was maintained at 9.5-10.5 and the temperature at 0-5℃. After the addition was completed, the reaction was stirred at 0-5℃ for 1 hour. (3) Post-treatment and purification: After the reaction is completed, solid sodium chloride is added to the reaction solution under stirring until the sodium chloride no longer dissolves. The system is placed in an ice-water bath to cool to 0-5℃. 6mol / L hydrochloric acid solution is added dropwise under stirring to adjust the pH to 2.5-3.

0. The mixture is allowed to stand at room temperature for 1 hour, then filtered. The filter cake is washed with 200mL of ice water, collected, and placed in a vacuum drying oven. It is then dried under reduced pressure at 40℃ to constant weight to obtain the repair factor.

9. A method for preparing a microecological preparation for improving intestinal inflammation in pets as described in any one of claims 1-8, characterized in that, Preparation includes the following steps: Probiotics, inactivated lactic acid bacteria and their metabolites, and repair factors are prepared separately. The formula amount of prebiotics is mixed for 15-25 minutes to obtain a prebiotic premix. Then, the formula amount of inactivated lactic acid bacteria and their metabolites, repair factors, and probiotics are added, and the mixture is mixed for another 20-30 minutes. The mixture is then passed through a 60-80 mesh sieve to obtain a microecological preparation.

10. The application of a microecological preparation for improving intestinal inflammation in pets as described in any one of claims 1-8, characterized in that, The specific application is to improve intestinal inflammation in pet cats and dogs, with the amount of probiotic added to cat and dog food being 0.1-0.5%.

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