Probiotic-short-chain fatty acid composition, preparation method thereof and application of probiotic-short-chain fatty acid composition in enhancing anti-infection ability of host

Abstract

The invention provides a probiotic-short-chain fatty acid composition as well as a preparation method and application thereof in enhancing the infection resistance of a host. The probiotic-short-chain fatty acid composition comprises the following raw materials in parts by weight: 2-6 parts of butyric acid microsphere powder and 10-14 parts of a probiotic-prebiotic compound. According to the composition disclosed by the invention, pathogenic bacteria are competitively inhibited through colonization of probiotics, and butyric acid directly regulates functions of immune cells (such as regulatory T cells and macrophages), so that'flora-immunity 'dual regulation is realized, and the anti-infection ability of a host is remarkably enhanced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microecological preparations and immunoregulation, and particularly relates to a probiotic-short-chain fatty acid composition, a preparation method thereof and application thereof in enhancing the anti-infection ability of a host. BACKGROUND

[0002] Intestinal flora is the largest microbial community in the human body, which participates in physiological functions such as host barrier function, immune regulation, metabolic support, etc. The barrier function is that the intestinal flora inhibits pathogenic bacteria colonization by competitive occupation and secreting antibacterial peptides (such as lysozyme), thereby maintaining the integrity of the intestinal mucosa. The immune regulation function is that the intestinal flora can secrete bacterial metabolites (such as short-chain fatty acids SCFAs) to promote the differentiation of regulatory T cells (Treg) by activating G protein-coupled receptors (GPR41 / GPR43), thereby inhibiting excessive inflammatory response. The metabolic support function is that the flora ferments dietary fiber to generate SCFAs (butyric acid, propionic acid, acetic acid), which provides energy for colon epithelial cells and participates in lipid metabolism and blood glucose homeostasis.

[0003] Flora imbalance can lead to various serious consequences: when the diversity of flora decreases (such as after antibiotic use), opportunistic pathogenic bacteria such as Clostridium difficile and Salmonella excessively proliferate, which causes diarrhea, enteritis and even systemic infection; it can also lead to a decrease in SCFAs level and thus cause an increase in intestinal barrier permeability ("leaky gut") and the release of pro-inflammatory factors (TNF-α, IL-6). Studies have shown that more than 70% of patients with antibiotic-associated diarrhea (AAD) are accompanied by intestinal flora imbalance.

[0004] Although probiotics are widely used to regulate flora imbalance, their single application has the defect of insufficient colonization competition ability. Most probiotics (such as lactobacilli) rely on specific nutrient substrates (such as fructooligosaccharides) for colonization, while pathogenic bacteria (such as Escherichia coli) can occupy the ecological niche by secreting iron carriers and other mechanisms. Clinical trials have shown that the cure rate of single strain (such as Lactobacillus rhamnosus GG) for Clostridium difficile infection is only 30-40%, which is significantly lower than that of combined therapy (Lancet Gastroenterology & Hepatology, 2020). Secondly, oral probiotics need to pass the test of gastric acid (pH 1.5-3.5) and bile (containing bile salts), and the survival rate of live bacteria is less than 10%. For example, the survival rate of Bifidobacterium in simulated gastrointestinal fluid is only about 30%. In addition, probiotics mainly inhibit pathogenic bacteria by occupying the niche, but lack the ability to directly regulate the immune response of the host. For example, they cannot activate the anti-inflammatory signaling pathway (such as STAT3) like SCFAs.

[0005] Therefore, it is of great significance to provide a composite preparation with high probiotic survival rate, which can effectively inhibit pathogenic bacteria colonization, regulate immune response and significantly enhance the anti-infection ability of the host. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a probiotic-short-chain fatty acid composition, a preparation method thereof and application thereof in enhancing the anti-infection ability of a host. The composition of the present application can inhibit pathogenic bacteria through probiotic colonization competition and directly regulate the function of immune cells (such as regulatory T cells and macrophages) through butyric acid, so as to realize double regulation of "microbiota-immune" and significantly enhance the anti-infection ability of the host.

[0007] The technical scheme of the present application is as follows: A probiotic-short-chain fatty acid composition comprises the following raw materials in parts by weight: 2-6 parts of butyric acid microsphere powder and 10-14 parts of probiotic prebiotic compound.

[0008] According to the present application, preferably, the composition comprises the following raw materials in parts by weight: 4 parts of butyric acid microsphere powder and 12 parts of probiotic prebiotic compound.

[0009] According to the present application, preferably, the preparation method of the butyric acid microsphere powder comprises the following steps: mixing ethyl acetate and a chitosan acetic acid aqueous solution, adding an emulsifier Tween 80, emulsifying to form an oil-in-water emulsion, adding an ethanol solution of butyric acid dropwise, stirring and reacting to form butyric acid microspheres in the oil phase, and centrifuging, washing and drying to obtain butyric acid microsphere powder.

[0010] Preferably, the mass concentration of chitosan in the chitosan acetic acid aqueous solution is 0.1-1.5%; the chitosan acetic acid aqueous solution is prepared by dissolving chitosan in an acetic acid aqueous solution with a mass concentration of 0.5-2%; the mass of the emulsifier Tween 80 is 0.1-1% of the total mass of the ethyl acetate and the chitosan acetic acid aqueous solution; the emulsification condition is 200-600 rpm stirring for 5-15 minutes; the volume concentration of the ethanol solution of butyric acid is 1-10%; and the stirring and reaction temperature is room temperature, and the stirring and reaction time is 1-4 hours.

[0011] Preferably, the mass ratio of ethyl acetate, chitosan acetic acid aqueous solution and butyric acid is 0.5-8:1-8:1-8, preferably 0.5-2:1-5:1-5, and further preferably 1:3:3.

[0012] According to the present application, preferably, the preparation method of the probiotic prebiotic compound comprises the following steps: uniformly mixing Lactobacillus acidophilus bacterial bodies, Bifidobacterium animalissubsp. lactis bacterial bodies, fructo-oligosaccharides (FOS), resistant starch and a freeze-drying protective agent solution, and freeze-drying to obtain the probiotic prebiotic compound.

[0013] Preferably, the specific strain of Lactobacillus acidophilus is Lactobacillus acidophilus CGMCC1.1854; and the specific strain of Bifidobacterium animalis is Bifidobacterium animalis subsp. Animalis CGMCC1.1852.

[0014] Preferably, the method for preparing Lactobacillus acidophilus cells or Bifidobacterium animalis cells includes the following steps: inoculating Lactobacillus acidophilus or Bifidobacterium animalis into MRS medium containing cysteine, anaerobically culturing at 30-40°C for 18-24 hours, and collecting the cells by centrifugation to obtain Lactobacillus acidophilus cells or Bifidobacterium animalis cells. More preferably, the mass of cysteine ​​is 0.05% of the mass of the MRS medium.

[0015] Preferably, the freeze-drying protectant solution is a mixture of trehalose, skim milk powder and deionized water, wherein the total mass concentration of trehalose and skim milk powder is 10-20%, and the mass ratio of trehalose to skim milk powder is 3-15:1-9, preferably 5-9:1-5, and more preferably 7:3.

[0016] Preferably, the mass ratio of fructooligosaccharides, resistant starch, Lactobacillus acidophilus cells, Bifidobacterium animalis cells, and lyophilization protectant solution is 1~10:0.5~10:2~15:2~15:0.5~23, more preferably 1~5:0.5~2:4~8:4~8:4~8, and even more preferably 3:1:6:6:6.

[0017] Preferably, the freeze-drying conditions are as follows: pre-freeze to -20~-40℃ and hold for 1~4 hours, then perform sublimation drying at -10~-20℃ for 4~10 hours and desorption drying at 2~15℃ for 1~6 hours in a vacuum freeze dryer.

[0018] The preparation method of the above-mentioned probiotic-short-chain fatty acid composition includes the following steps: The probiotic-prebiotic complex is mixed evenly with butyric acid microsphere powder to obtain a probiotic-short-chain fatty acid composition.

[0019] According to a preferred embodiment of the present invention, the mixture is mixed under sterile conditions in a three-dimensional mixer at a rate of 350 r / min for 100-150 minutes until a composite mixture with uniform color and uniform particle distribution is obtained.

[0020] According to a preferred embodiment of the present invention, the dosage form of the probiotic-short-chain fatty acid composition may be capsules, tablets, or the like.

[0021] According to a preferred embodiment of the present invention, the preparation method of probiotic-short-chain fatty acid composition tablets is as follows: (1) Mix the probiotic prebiotic complex with butyric acid microspheres thoroughly to obtain a mixture; (2) Add magnesium stearate to the mixture as a lubricant, mix evenly and compress to obtain probiotic short-chain fatty acid composition tablets.

[0022] Preferably, in step (2), the mass of the magnesium stearate is 0.5 to 1.5% of the mass of the mixture.

[0023] Preferably, in step (2), the probiotic short-chain fatty acid composition tablets have a hardness of 6~12 kN, a disintegration time of ≤15 minutes, and a tablet weight of 150-250 mg / tablet.

[0024] The application of the above-mentioned probiotic-short-chain fatty acid composition in the preparation of drugs that enhance the host's ability to fight infection.

[0025] The technical features of this invention are as follows: In this invention, Bifidobacterium animalis, Lactobacillus acidophilus, butyric acid, and fructooligosaccharides serve as core active substances. Through a progressive chain of action—"probiotic colonization - prebiotic proliferation - SCFAs immune regulation"—they enhance the host's anti-infection ability in multiple dimensions. Bifidobacterium animalis and Lactobacillus acidophilus have a competitive exclusionary colonization effect: As the dominant symbiotic bacteria in the human gut, Bifidobacterium animalis and Lactobacillus acidophilus can rapidly colonize the intestinal mucosa by binding to specific receptors on intestinal epithelial cells through surface adhesion proteins (such as pili and lipoteichoic acid), forming a "biological barrier" that competitively inhibits the adhesion sites of pathogenic bacteria such as Escherichia coli and Salmonella, reducing the chance of pathogen invasion.

[0026] Bifidobacterium animalis and Lactobacillus acidophilus have the functions of producing acid and secreting antibacterial substances: these two types of probiotics produce organic acids such as lactic acid and acetic acid by fermenting dietary fiber, which lowers the local pH of the intestine to 4.5-5.5 and inhibits the growth of aerobic pathogens (such as Staphylococcus aureus) and putrefactive bacteria (such as Clostridium perfringens); at the same time, Bifidobacterium animalis can secrete bacteriocins (such as bifidobacterialin), and Lactobacillus acidophilus can produce hydrogen peroxide (H2O2), which directly destroys the cell membrane of pathogens and enhances the mucosal resistance to infection.

[0027] Butyric acid participates in intestinal epithelial energy supply and barrier repair: Butyric acid is the preferred energy source for colonic epithelial cells (accounting for 60%-70% of the energy requirements of intestinal cells). It can promote the proliferation of intestinal epithelial cells, upregulate the expression of tight junction proteins (such as occludin and claudin-1) and adhesion junction proteins (such as E-cadherin), repair defects in the intestinal mucosal barrier, and reduce the transepithelial penetration of pathogens and their toxins (such as endotoxin LPS) ("leakage in the gut").

[0028] Butyric acid participates in the inhibition of inflammatory pathways and the regulation of immune balance: By inhibiting the NF-κB signaling pathway, butyric acid reduces the release of pro-inflammatory factors (TNF-α, IL-6, IL-1β), while activating G protein-coupled receptors (GPR43 / GPR109A), promoting the differentiation of regulatory T cells (Treg) and the secretion of anti-inflammatory factors (IL-10), balancing the Th1 / Th2 immune response, and avoiding tissue damage caused by excessive inflammatory response.

[0029] Fructooligosaccharides (FOS) selectively proliferate beneficial bacteria: As a prebiotic, fructooligosaccharides (FOS) are not broken down by the host's digestive enzymes and can selectively proliferate beneficial bacteria such as Bifidobacterium animalis and Lactobacillus acidophilus (the proliferation rate is 2-3 times higher than that of the control group), forming a synergistic proliferation effect of "probiotics-prebiotics" and amplifying the colonization and barrier protection functions of probiotics.

[0030] Fructooligosaccharides synergistically produce SCFAs: After fermentation by probiotics, fructooligosaccharides produce acetic acid, propionic acid and a small amount of butyric acid (forming a concentration gradient with exogenous butyric acid). Among them, propionic acid can inhibit liver lipase (such as ACC), indirectly reducing the production of inflammatory mediators; acetic acid, on the other hand, diffuses throughout the body and participates in cholesterol metabolism regulation, indirectly enhancing the host's overall anti-infection status.

[0031] The beneficial effects of this invention are as follows: This invention, through an innovative design of "microsphere encapsulation + prebiotic matrix," achieves a synergistic effect between probiotics and short-chain fatty acids (SCFAs). Compared with traditional single-component formulations or simple mixed formulations, it exhibits significant advantages in terms of technical performance, application effects, and industrialization value, as detailed below: 1. Core mechanisms work together to overcome traditional technological bottlenecks. Traditional probiotic preparations often suffer from limitations in efficacy due to low survival rates (easily inactivated in the gastrointestinal environment) and singular effects (relying solely on the activity of the strain itself). This invention achieves synergistic optimization through the following mechanism: (1) Prebiotic matrix carrier: using prebiotics such as resistant starch and fructooligosaccharides as the matrix, on the one hand, it provides a continuous fermentation substrate for probiotics, extending their colonization cycle in the intestine; on the other hand, it generates endogenous SCFAs (such as acetic acid and propionic acid) through prebiotic fermentation, forming a concentration gradient with the exogenously added butyric acid, which synergistically strengthens the intestinal mucosal barrier repair and immune regulation function. (2) SCFAs support probiotic colonization: Probiotics (such as Bifidobacterium and butyric acid-producing bacteria) produce SCFAs by fermenting dietary fiber. Combined with exogenous SCFAs, they can reduce the local pH value of the intestine (to 4.5-5.5), inhibit the adhesion of pathogenic bacteria such as Escherichia coli, and provide a more suitable slightly acidic living environment for probiotics (such as Bifidobacterium and Lactobacillus). SCFAs, in turn, support probiotic colonization by promoting goblet cell differentiation and increasing the thickness of the mucus layer, forming a positive cycle of "SCFAs promote colonization - probiotics produce SCFAs", which further consolidates the synergistic effect.

[0032] 2. Through process optimization and ingredient compatibility, this invention achieves the following improvement in probiotic survival rate: verified by simulated gastrointestinal fluid digestion experiments, the survival rate of probiotics (Bifidobacterium animalis and Lactobacillus acidophilus) in the preparation is ≥60%.

[0033] 3. The combination of short-chain fatty acids (SCFAs) and probiotics in this invention exhibits synergistic advantages. After colonization, the probiotics continuously produce endogenous SCFAs, which, when used in combination with exogenous SCFAs, prolong their intestinal retention time and amplify the immunomodulatory effect. Through a dual pathway of "intestinal microecological regulation - enhanced immune function," it demonstrates a faster anti-infection speed in practical applications: animal experiments show that mice administered the preparation of this invention by gavage experienced a shortened symptom disappearance time of 10 days after infection with pathogenic Escherichia coli. Attached Figure Description

[0034] Figure 1 The graphs show the bacterial activity detection at 0h and 4h for the control group and the experimental group. Detailed Implementation

[0035] The present invention will be further illustrated below with specific embodiments, but is not limited thereto. Examples 1-3 and Comparative Examples 1-2 have different raw material ratios; Comparative Examples 3-4 have different raw material compositions; Experimental Examples 1-3 are specific experiments to explore the optimal raw material dosage and composition through the examples and comparative examples.

[0036] Lactobacillus acidophilus CGMCC1.1854 and Bifidobacterium animalis subsp. Animalis CGMCC1.1852 were purchased from the China General Microbiological Culture Collection Center. Escherichia coli ETEC O25:K19 was purchased from Shanghai Lianmai Biotechnology Co., Ltd.

[0037] Example 1 A probiotic-short-chain fatty acid composition comprises the following raw materials in parts by weight: 4 parts butyrate microsphere powder and 12 parts probiotic prebiotic complex.

[0038] The butyric acid microsphere powder was prepared as follows: Ethyl acetate (analytical grade) and 1 wt% chitosan solution (dissolved in 1 wt% acetic acid aqueous solution) were thoroughly mixed. Then, Tween 80 was added as an emulsifier (the mass of Tween 80 was 0.5% of the mass of the ethyl acetate and chitosan-acetic acid mixture), and emulsification was carried out at 400 rpm for 10 minutes to form an oil-in-water emulsion. Subsequently, an ethanol solution of butyric acid (5% butyric acid concentration, v / v) was added dropwise, and stirring continued for 2 hours to allow butyric acid to form microspheres in the oil phase. The microspheres were then collected by centrifugation (8000×g, 15 minutes), washed three times with deionized water, and vacuum dried at 40℃ for 6 hours to obtain butyric acid microsphere powder. The mass ratio of ethyl acetate, chitosan solution, and butyric acid was 1:3:3.

[0039] The freeze-drying protectant solution is prepared as follows: trehalose and skim milk powder are dissolved in sterile deionized water to prepare a freeze-drying protectant solution with a total mass concentration of 15% for both trehalose and skim milk powder. The mass ratio of trehalose to skim milk powder is 7:3.

[0040] The probiotic prebiotic complex was prepared as follows: *Lactobacillus acidophilus* and *Bifidobacterium animalis* were selected and inoculated into MRS medium (containing 0.05 wt% cysteine), respectively, and cultured anaerobically at 37°C for 24 hours until the logarithmic growth phase. The cells were collected by centrifugation to obtain *Lactobacillus acidophilus* cells and *Bifidobacterium animalis* cells. The *Lactobacillus acidophilus* cells, *Bifidobacterium animalis* cells, fructooligosaccharides (FOS), resistant starch, and lyophilization protectant solution were thoroughly mixed and placed in a lyophilization tray. The mixture was pre-frozen at -40°C for 2 hours, and then subjected to sublimation drying (-20°C / 8 hours) and desorption drying (10°C / 4 hours) sequentially in a vacuum freeze dryer to obtain the probiotic prebiotic complex powder. The mass ratio of fructooligosaccharides, resistant starch, *Lactobacillus acidophilus* cells, *Bifidobacterium animalis* cells, and lyophilization protectant solution was 3:1:6:6:6.

[0041] The preparation method of the above-mentioned probiotic-short-chain fatty acid composition tablets includes the following steps: (1) The probiotic prebiotic complex and butyric acid microsphere powder were mixed in the above proportion under sterile conditions using a three-dimensional mixer at a rate of 350 r / min for 130 minutes until a composite mixture with uniform color and uniform particle distribution was obtained.

[0042] (2) Add magnesium stearate to the mixture as a lubricant (the mass of magnesium stearate is 1% of the mass of the mixture), mix evenly, and then compress into tablets (200 mg / tablet) using a rotary tablet press. Control the hardness to 8~10 kN and the disintegration time to ≤15 minutes to finally obtain probiotic-short chain fatty acid composition tablets.

[0043] The above-described probiotic-short-chain fatty acid composition can be used to formulate capsules, tablets, etc. This embodiment uses tablets as an example for illustration.

[0044] Example 2 A probiotic-short-chain fatty acid composition comprises the following raw materials in parts by weight: 2 parts butyrate microsphere powder and 10 parts probiotic prebiotic complex.

[0045] The butyric acid microsphere powder was prepared in the same manner as in Example 1, except that the mass ratio of ethyl acetate, chitosan solution and butyric acid was 0.5:5:5.

[0046] The preparation of the freeze-drying protectant solution is the same as in Example 1, except that the mass ratio of trehalose to skim milk powder is 9:1.

[0047] The preparation of the probiotic-prebiotic complex is the same as in Example 1, except that the mass ratio of fructooligosaccharides, resistant starch, Lactobacillus acidophilus animal cells, Bifidobacterium animalis cells, and lyophilization protectant solution is 5:0.5:8:4:8.

[0048] The preparation method of the above-mentioned probiotic-short-chain fatty acid composition tablets is the same as that in Example 1.

[0049] Example 3 A probiotic-short-chain fatty acid composition comprises the following raw materials in parts by weight: 6 parts butyrate microsphere powder and 14 parts probiotic prebiotic complex.

[0050] The preparation of the butyric acid microsphere powder is the same as in Example 1, except that the mass ratio of ethyl acetate, chitosan solution and butyric acid is 2:1:1.

[0051] The preparation of the freeze-drying protectant solution is the same as in Example 1, except that the mass ratio of trehalose to skim milk powder is 5:5.

[0052] The preparation of the probiotic prebiotic complex is the same as in Example 1, except that the mass ratio of fructooligosaccharides, resistant starch, Lactobacillus acidophilus animal cells, Bifidobacterium animalis cells, and lyophilization protectant solution is 1:2:4:8:4.

[0053] The preparation method of the above-mentioned probiotic-short-chain fatty acid composition tablets is the same as that in Example 1.

[0054] Comparative Example 1 A probiotic-short-chain fatty acid composition comprises the following raw materials in parts by weight: 10 parts butyrate microsphere powder and 15 parts probiotic prebiotic complex.

[0055] The preparation of the butyric acid microsphere powder is the same as in Example 1, except that the mass ratio of ethyl acetate, chitosan solution and butyric acid is 4:6:6.

[0056] The preparation of the freeze-drying protectant solution is the same as in Example 1, except that the mass ratio of trehalose to skim milk powder is 15:6.

[0057] The preparation of the probiotic-prebiotic complex is the same as in Example 1, except that the mass ratio of fructooligosaccharides, resistant starch, Lactobacillus acidophilus animal cells, Bifidobacterium animalis cells, and lyophilization protectant solution is 7:4:15:15:23.

[0058] The preparation method of the above-mentioned probiotic-short-chain fatty acid composition tablets is the same as that in Example 1.

[0059] Comparative Example 2 A probiotic-short-chain fatty acid composition comprises the following raw materials in parts by weight: 1 part butyrate microsphere powder and 6 parts probiotic prebiotic complex.

[0060] The butyric acid microsphere powder was prepared in the same manner as in Example 1, except that the mass ratio of ethyl acetate, chitosan solution and butyric acid was 0.5:1:1.

[0061] The preparation of the freeze-drying protectant solution is the same as in Example 1, except that the mass ratio of trehalose to skim milk powder is 3:1.

[0062] The preparation of the probiotic-prebiotic complex is the same as in Example 1, except that the mass ratio of fructooligosaccharides, resistant starch, Lactobacillus acidophilus animal cells, Bifidobacterium animalis cells, and lyophilization protectant solution is 1:0.5:2:2:0.9.

[0063] The preparation method of the above-mentioned probiotic-short-chain fatty acid composition tablets is the same as that in Example 1.

[0064] Comparative Example 3 A probiotic-short-chain fatty acid composition, with the same raw material composition as in Example 1, except that fructooligosaccharides and resistant starch are not added to the probiotic-prebiotic complex. The specific preparation method is as follows: Lactobacillus acidophilus and Bifidobacterium animalis were selected and inoculated into MRS medium (containing 0.05 wt% cysteine), respectively. They were anaerobically cultured at 37°C for 24 hours until the logarithmic growth phase. The cells were collected by centrifugation to obtain Lactobacillus acidophilus and Bifidobacterium animalis cells. The Lactobacillus acidophilus cells, Bifidobacterium animalis cells, and lyophilization protectant solution were thoroughly mixed and placed in a lyophilization tray. The mixture was pre-frozen at -40°C for 2 hours, followed by sublimation drying (-20°C / 8 hours) and desorption drying (10°C / 4 hours) in a vacuum freeze dryer to obtain a probiotic prebiotic complex powder. The mass ratio of Lactobacillus acidophilus animal cells, Bifidobacterium animalis cells, and lyophilization protectant solution was 6:6:6.

[0065] The preparation method of the above-mentioned probiotic-short-chain fatty acid composition tablets is the same as that in Example 1.

[0066] Comparative Example 4 A probiotic preparation, as described in Example 1, except that butyrate microsphere powder is not added; the other raw material composition is the same as in Example 1.

[0067] The preparation method of the above-mentioned probiotic preparation includes the following steps: Magnesium stearate was added as a lubricant to the probiotic prebiotic complex (the mass of magnesium stearate was 1% of the mass of the probiotic prebiotic complex). After mixing evenly, the mixture was compressed into tablets (200 mg / tablet) using a rotary tablet press. The hardness was controlled at 8-10 kN and the disintegration time was ≤15 minutes, thus obtaining the probiotic preparation.

[0068] Experimental Example 1 1. Laboratory animals: (1) SPF grade C57BL / 6 female mice, 6-8 weeks old, weighing 18-22 g.

[0069] (2) They were kept in a barrier environment animal house (temperature 22±1℃, humidity 50±5%, 12 h day and night cycle) with free access to food and water.

[0070] 2. Establishment of infection model: (1) After 7 days of acclimatization, the mice were fasted for 12 hours and then administered 1×10⁻⁶ gavage. 8 CFU Escherichia coli ETEC O25:K19 (suspended in 100 μL PBS).

[0071] (2) Continue to monitor the mice after infection. The model is successful when diarrhea symptoms appear.

[0072] 3. Grouping and Processing (1) The model mice were randomly divided into 6 groups (n=10 / group), including a control group and experimental groups 1-5. Experimental groups 1-3 were given the formulations of Examples 1-3 by gavage, and experimental groups 4-5 were given the formulations of Comparative Examples 1-2 by gavage. The recommended human dose is 2.0g / 60kg.bw per day. The medium dose group was given 333.3mg / kg.bw (equivalent to 10 times the recommended human intake). The solution was prepared with purified water to the required concentration. Each mouse was given 0.6mL / 100g.bw by gavage. The control group was given purified water by gavage.

[0073] (2) Administration method: Gavage at 9:00 am every day for 7 consecutive days.

[0074] 4. Detection Indicators and Methods (1) Record the time when the symptoms of each mouse are completely relieved, and the criteria for complete symptom relief: ① Diarrhea stops (stool is formed, no loose / watery stool, and no abnormalities are observed in two consecutive observations); ② Activity returned to normal (no curling up, ruffled fur, and no difference in activity level compared to normal mice); ③ Weight recovers to more than 90% of the level before infection. (2) After the mice's symptoms were completely relieved, the mice were euthanized and the colonic slgA and serum TNF-α were measured. The specific procedures are as follows: ① Before the operation, the mice were fasted for 2-4 hours but allowed to drink water. The mice were then sacrificed by cervical dislocation. ② Serum collection: a. Blood is drawn via cardiac puncture; b. Let the blood sample stand at room temperature for 30 minutes until it coagulates; c. Centrifuge (3000 rpm, 10 min, 4℃), and collect the upper layer of pale yellow transparent liquid, which is the serum; d. Dispense into smaller portions and store at -80℃ for later use.

[0075] ③ Dissect the large intestine and collect its contents: a. Open the mouse's abdomen and locate the colonic segment between the cecum and rectum; b. Carefully separate the complete colon segment using sterile forceps (avoid pulling and tearing); c. Gently rinse the intestinal lumen 1-2 times with pre-cooled PBS (pH 7.4) and collect the effluent (containing intestinal contents). d. Place all samples on ice immediately and centrifuge as soon as possible.

[0076] e. Centrifugation: 12,000 rpm, 4℃, 10 min, collect the supernatant and store at -80℃.

[0077] 5. Experimental Results Survival rate and clinical symptoms: Data were processed using SPSS statistical software. t-tests were used to analyze significant differences between groups. P < 0.05 was considered statistically significant, P < 0.01 was considered highly statistically significant, and P < 0.01 was considered extremely statistically significant. All data for the tested indicators are expressed as mean ± standard deviation. The test results are shown in Table 1.

[0078] Table 1: Time to complete symptom relief and levels of colonic slgA and serum TNF-α after complete symptom relief in experimental groups 1-5 and control mice. Note: * indicates that compared with the control group, * P < 0.05, *** P < 0.01, *** P < 0.001.

[0079] Experiment 1 showed that different ingredient ratios resulted in different symptom complete relief times, serum TNF-α levels, and colonic slgA levels. Experimental group 1 had the shortest symptom complete relief time (10±1 days) and the lowest serum TNF-α and colonic slgA levels. Except for the control group, experimental group 4 had the longest symptom complete relief time (15.5±3 days), the highest colonic slgA level, and the second highest serum TNF-α level after experimental group 5. This further illustrates that there is an optimal ratio range for different ingredients. The experimental results indicate that the ingredient ratio in Example 1 was the most effective in relieving diarrhea symptoms in mice and is the optimal ingredient ratio.

[0080] Experimental Example 2 Based on the experimental results of Example 1, the composition in Example 1 was selected as the experimental sample. The recommended human dose is 2.0 g / 60 kg bw per day. Three dosage groups were set up: low, medium, and high, at 166.7 mg / kg bw, 333.3 mg / kg bw, and 1000.0 mg / kg bw, respectively. These dosages were equivalent to 5 times, 10 times, and 30 times the recommended human intake, respectively. Evaluation was still conducted using mouse experiments. The experimental method was the same as in Example 1. Mice were randomly divided into 6 groups: a control group and experimental groups 1-5. Experimental groups 1-3 were administered the formulation from Example 1 at the low, medium, and high doses, respectively. Experimental groups 4-5 were administered the composition from Comparative Examples 3-4 at the high dose. The compound preparation was diluted to the required concentration using purified water as a solvent. Each mouse was administered 0.6 mL / 100 g bw by gavage, while the control group was administered purified water by gavage. The determination and data processing of mouse indicators were the same as in Example 1. The experimental indicators for each group are shown in Table 2.

[0081] Table 2: Time to complete symptom relief and levels of colonic slgA and serum TNF-α after complete symptom relief in experimental groups 1-5 and control mice. Note: * indicates that compared with the control group, * P < 0.05, *** P < 0.01, *** P < 0.001.

[0082] Experiment 2 shows that, in terms of raw material composition, experimental groups 1, 2, and 3 consist of a single raw material combination, while experimental groups 4 and 5 each have different raw material compositions. The complete symptom relief time, serum TNF-α level, and colonic slgA levels in experimental groups 4 and 5 were not significantly different from the control group, and were even higher than those in experimental groups 1, 2, and 3. This indicates that the symptom relief effect of the raw material composition formula in Example 1 was superior to that in Comparative Examples 3 and 4, suggesting a synergistic effect among the various raw material compositions. The raw material composition in Example 1 represents the optimal ratio. In the investigation of the effects of different dosages of the formula in Example 1 (experimental groups 1-3), it was found that experimental group 2 had the shortest symptom relief time and the lowest serum TNF-α level and colonic slgA level. This indicates that the optimal dosage of the formula in Example 1 is most effective when used at a medium dose.

[0083] Experimental Example 3 Microbial activity assay (simulating the gastrointestinal environment) 1. Simulate the gastrointestinal environment settings (1) Simulating the stomach environment ① Take 16.4 mL of 0.1 M HCl, add about 800 mL of deionized water, and stir to mix well. Add 0.2 g of NaCl, dissolve, and then bring the volume to 1 L to obtain the basic gastric juice (pH≈1.5).

[0084] ②pH calibration: Adjust the pH to 2.0 using 0.1 M NaOH or 0.1 M HCl.

[0085] ③ Add pepsin: Weigh 20 mg of pepsin, dissolve it in 10 mL of pre-cooled sterile water, and slowly add it to the basal gastric fluid, avoiding local overheating (temperature ≤40℃).

[0086] ④ Sterilization treatment: Filtration was performed using a 0.22 μm sterile filter membrane.

[0087] ⑤ A peristaltic pump is used to simulate gastric contractions (3 times / minute), combined with a real-time pH monitoring and automatic adjustment system.

[0088] (2) Simulating the intestinal environment Add trypsin (1%) and bile salts (0.3%) to potassium dihydrogen phosphate buffer (pH 6.8), sterilize, and use to simulate the intestinal environment.

[0089] 2. Experimental grouping and treatment: (1) A control group and experimental groups 1-3 were set up. Experimental group 1 used the compound preparation of Example 1, and experimental groups 2-3 used the compound preparations of Comparative Examples 3-4, respectively. The recommended human dose is 2.0 g / 60 kg bw per day. The medium dose group was administered at 333.3 mg / kg bw (equivalent to 10 times the recommended human intake), and purified water was used as the solvent to prepare the required volume of 3.0 mL. The control group contained equal amounts of Bifidobacterium animalis and Lactobacillus acidophilus.

[0090] (2) The experimental group and control group solutions were mixed with simulated gastric juice and incubated at 37°C for 2 hours. Then they were transferred to the intestinal environment simulation device and incubated for 6 hours. Samples were taken every 2 hours during the period.

[0091] 3. Fluorescent staining: (1) Take 0.5 mL of sample, add 5 μM of SYTO 9 (live bacteria labeling dye) and 5 μM of propidium iodide (PI, dead bacteria labeling dye) to the final concentration, and incubate in the dark for 15 minutes.

[0092] (2) After staining, wash twice with PBS buffer to remove unbound dye.

[0093] 4. Flow cytometry analysis: (1) Using a BD Accuri C6 flow cytometer, the laser wavelength was set to 488 nm (excitation light) to detect SYTO 9 (green fluorescence, FL1 channel) and PI (red fluorescence, FL3 channel).

[0094] (2) Bacterial gating is set by forward scattering (FSC) and side scattering (SSC) to eliminate interference from cell debris.

[0095] (3) Live bacteria are defined as SYTO 9 + / PI - (Strong green fluorescence, weak red fluorescence), dead bacteria are defined as SYTO 9. + / PI + (Strong red fluorescence).

[0096] 4. Data Analysis: 5. Experimental Results Table 3: Bacterial survival rate (%) in experimental groups 1-3 and control group at different time points Note: * indicates that compared with the model control group, * P < 0.05, *** P < 0.01, *** * P < 0.001.

[0097] Partial graphs of bacterial activity detection at 0h and 4h for the control and experimental groups are shown below. Figure 1As shown.

[0098] Experiment 3 shows that experimental group 1 had the best bacterial survival rate and was significantly different from the control group, while experimental groups 2 and 3 had poorer bacterial survival rates and were not significantly different from the control group. This indicates that there is a synergistic effect among the raw material components. This synergy has a certain protective effect on probiotics, improves their tolerance to various environments in the digestive tract, and results in a higher proportion of live bacteria among the probiotics reaching the intestines, which is more conducive to the probiotics exerting their effects. Therefore, the optimal raw material composition is as listed in Example 1.

[0099] As can be seen from the experiments 1-3, the formula listed in Example 1 has a good effect on relieving diarrhea symptoms in mice and a high survival rate of probiotics. It is the best formula in terms of both raw material composition and ratio.

Claims

1. A probiotic-short-chain fatty acid composition, characterized in that, The ingredients include the following parts by weight: 2-6 parts butyrate microsphere powder and 10-14 parts probiotic prebiotic complex.

2. The probiotic-short-chain fatty acid composition according to claim 1, characterized in that, The composition comprises the following raw materials in parts by weight: 4 parts butyric acid microsphere powder and 12 parts probiotic prebiotic complex.

3. The probiotic-short-chain fatty acid composition according to claim 1, characterized in that, The preparation method of the butyric acid microsphere powder includes the following steps: thoroughly mixing ethyl acetate and chitosan acetic acid aqueous solution, adding emulsifier Tween 80, emulsifying to form an oil-in-water emulsion; adding butyric acid ethanol solution dropwise, stirring and reacting to form microspheres of butyric acid in the oil phase, centrifuging, washing and drying to obtain butyric acid microsphere powder.

4. The probiotic-short-chain fatty acid composition according to claim 3, characterized in that, The chitosan-acetic acid aqueous solution contains 0.1-1.5% chitosan by mass concentration; the chitosan-acetic acid aqueous solution is prepared by dissolving chitosan in an acetic acid aqueous solution with a mass concentration of 0.5-2%; the mass of the emulsifier Tween 80 is 0.1-1% of the total mass of ethyl acetate and the chitosan-acetic acid aqueous solution; the emulsification conditions are: stirring and emulsifying at 200-600 rpm for 5-15 minutes; the volume concentration of the butyric acid ethanol solution is 1-10%; the stirring reaction temperature is room temperature, and the stirring reaction time is 1-4 hours.

5. The probiotic-short-chain fatty acid composition according to claim 3, characterized in that, The mass ratio of ethyl acetate, chitosan acetic acid aqueous solution and butyric acid is 0.5~8:1~8:1~8, preferably 0.5~2:1~5:1~5, and more preferably 1:3:

3.

6. The probiotic-short-chain fatty acid composition according to claim 1, characterized in that, The preparation method of the probiotic prebiotic complex includes the following steps: thoroughly mixing Lactobacillus acidophilus cells, Bifidobacterium animalis subsp. lactis cells, fructooligosaccharide (FOS), resistant starch, and a freeze-drying protectant solution, and then freeze-drying to obtain the probiotic prebiotic complex.

7. The probiotic-short-chain fatty acid composition according to claim 6, characterized in that, Includes one or more of the following conditions: i. A method for preparing Lactobacillus acidophilus cells or Bifidobacterium animalis cells, comprising the steps of: inoculating Lactobacillus acidophilus or Bifidobacterium animalis into MRS medium containing cysteine, anaerobically culturing at 30-40°C for 18-24 h, and collecting the cells by centrifugation to obtain Lactobacillus acidophilus cells or Bifidobacterium animalis cells; preferably, the mass of cysteine ​​is 0.05% of the mass of the MRS medium; ii. The freeze-drying protectant solution is a mixture of trehalose, skim milk powder and deionized water, wherein the total mass concentration of trehalose and skim milk powder is 10-20%, and the mass ratio of trehalose to skim milk powder is 3-15:1-9, preferably 5-9:1-5, and more preferably 7:3; iii. Freeze-drying conditions are as follows: Pre-freeze to -20~-40℃ and hold for 1~4 hours, then perform sublimation drying at -10~-20℃ for 4~10 hours and desorption drying at 2~15℃ for 1~6 hours in a vacuum freeze dryer.

8. The probiotic-short-chain fatty acid composition according to claim 6, characterized in that, The mass ratio of fructooligosaccharides, resistant starch, Lactobacillus acidophilus cells, Bifidobacterium animalis cells, and lyophilization protectant solution is 1~10:0.5~10:2~15:2~15:0.5~23, preferably 1~5:0.5~2:4~8:4~8:4~8, and more preferably 3:1:6:6:

6.

9. A method for preparing the probiotic-short-chain fatty acid composition according to any one of claims 1-8, comprising the following steps: The probiotic-short-chain fatty acid composition is obtained by uniformly mixing the probiotic prebiotic complex with butyric acid microsphere powder. Preferably, the mixture is mixed under sterile conditions in a three-dimensional mixer at a rate of 350 r / min for 100-150 minutes.

10. The use of the probiotic-short-chain fatty acid composition according to any one of claims 1-8 in the preparation of a medicament for enhancing the host's ability to fight infection.

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