Composition for inhibiting Clostridium perfringens and preparation method and application thereof

Abstract

The present invention provides a composition for inhibiting Clostridium perfringens, its preparation method and application, relating to the fields of microorganisms and fermentation. The composition is a co-fermentation product of Coptis chinensis and Propionibacterium. The co-fermentation product of Propionibacterium and Coptis chinensis can inhibit the infection of Clostridium perfringens and has a good prevention and treatment effect on intestinal damage caused by Clostridium perfringens infection. Experimental results show that the co-fermentation product can significantly increase the secretion of IFN-β and IL-10 in mice, reduce the production of IL-1β and TNF-α, increase the proportions of CD4+IL-4 and CD8+IFN-γ T cells, and increase the expression of ZO-1 and Occludin in the small intestine. Compared with the单独使用黄连或丙酸杆菌相比,共发酵产物表现出更强的协同增效作用,可有效预防和抑制魏氏梭菌感染,且不易产生耐药性。本发明制备方法简单,原料易得,适用于开发抗菌药物和饲料添加剂。 It should be noted that there seems to be some inaccuracy or incompleteness in the original Chinese text where it says "单独使用黄连或丙酸杆菌相比", which might need to be corrected in the original for a more accurate translation. The above translation is based on the existing text as much as possible.

Description

Technical Field

[0001] The present application relates to the field of microorganisms and fermentation, and specifically to a composition for inhibiting Clostridium perfringens and a preparation method and application thereof. Background Art

[0002] The information disclosed in the background of the present invention is intended to increase the understanding of the overall background of the present invention, and the disclosure should not necessarily be regarded as an admission or any form of suggestion that the information has become the prior art known to ordinary technicians in the field.

[0003] Clostridium perfringens Clostridium perfringens ), also known as Clostridium perfringens, is a Gram-positive anaerobic bacillus widely found in nature and in the intestines of humans and animals. It can produce a variety of potent exotoxins, such as α, β, ε, ι, etc. These toxins can cause a variety of serious diseases in humans and animals, causing huge economic losses to animal husbandry, and also attracted widespread attention in the field of public health. In animal breeding, Clostridium perfringens can cause enterotoxemia, necrotizing enteritis, gas gangrene and other diseases in livestock (such as pigs, cattle, sheep, etc.). Acute infection often causes sudden death of animals, while chronic infection slows down animal growth, reduces feed conversion rate, and increases breeding costs. According to statistics, in some areas, the incidence of intestinal diseases in pigs caused by Clostridium perfringens can reach 20%-30%, and the mortality rate is as high as more than 50%; the incidence of Clostridium perfringens disease in sheep is between 10%-20%, and the mortality rate is also quite high. In the field of human medicine, Clostridium perfringens is one of the main pathogens that cause gas gangrene. This disease develops rapidly and can cause tissue necrosis and systemic poisoning symptoms. If not treated in time, the mortality rate is extremely high. In addition, Clostridium perfringens is also closely related to food poisoning. After people eat food contaminated by it, they will experience symptoms such as vomiting, diarrhea, and abdominal pain, which seriously affects their health.

[0004] At present, the treatment of Clostridium perfringens infection mainly adopts antibiotic therapy, such as penicillin, cephalosporin, etc. However, with the widespread use of antibiotics, the problem of drug resistance of Clostridium perfringens is becoming increasingly serious. Studies have found that Clostridium perfringens isolates in some areas have developed resistance to a variety of commonly used antibiotics, and the resistance rate has been increasing year by year. This greatly reduces the effectiveness of traditional antibiotic treatment and increases the difficulty and cost of treatment. Moreover, the abuse of antibiotics will also destroy the normal balance of microbial flora in animals and humans, cause other secondary infections and adverse reactions, and pose a potential threat to the health of the body. Therefore, it is urgent to find a new anti-Clostridium perfringens drug or treatment method that is safe, effective, and not prone to drug resistance. Summary of the invention

[0005] The present invention provides a composition for inhibiting Clostridium perfringens and a preparation method and application thereof, wherein the composition is a co-fermentation composition of Propionibacterium and Coptis chinensis. The composition significantly improves the prevention and inhibition effect of Clostridium perfringens infection through the synergistic effect of probiotics and Coptis chinensis, enhances the body's immune function, protects the integrity of the intestinal barrier, and provides a new technical solution for solving the problems in the prior art.

[0006] In a first aspect of the present invention, a composition for inhibiting Clostridium perfringens is provided, wherein the composition is a co-fermentation product of Coptis chinensis and Propionibacterium.

[0007] In some embodiments of the present invention, the Clostridium perfringens ( Clostridium perfringens ) The strain is preferably Clostridium perfringens. For example, in one embodiment, the Clostridium perfringens is a strain purchased from Beina Bio-Henan Industrial Microbial Strain Engineering Technology Research Center (BNCC) and numbered BNCC185933.

[0008] In some embodiments of the present invention, the Propionibacterium ( Propionibacterium ) is preferably Propionibacterium freudenreichii ( Propionibacterium freudenreichii. ), for example, in one embodiment, the Propionibacterium is a strain purchased from Beina Bio-Henan Industrial Microbial Strain Engineering Technology Research Center (BNCC) and numbered BNCC336447.

[0009] In some embodiments of the present invention, the Coptis chinensis is a decoction of Coptis chinensis, and the Coptis chinensis meets the standards of the Chinese Pharmacopoeia.

[0010] In some embodiments of the present invention, the fermentation product is a whole fermentation broth.

[0011] In the embodiment of the present invention, it has been verified that the composition has the following beneficial effects: immunomodulatory effect, including: increasing the secretion of IFN-β and IL-10, reducing the production of IL-1β and TNF-α. Enhance immune function, including: increasing CD4 + IL-4 T cell ratio, maintain CD8 + IFN-γ T cell levels. Protect the intestinal barrier, including: increasing the expression of ZO-1 and Occludin, maintaining intestinal tissue integrity, and reducing inflammatory responses.

[0012] In an embodiment of the present invention, in in vivo experiments, the composition exhibits: preventive effects, including maintaining animal weight, improving survival rate, and improving diarrhea; safety, including no adverse reactions during administration, no organ toxicity, and good tolerance.

[0013] In a second aspect of the present invention, a method for preparing the composition described in the first aspect is provided, the method comprising: co-fermenting Coptis chinensis and Propionibacterium to obtain a fermentation product.

[0014] In some embodiments of the present invention, the method comprises: preparing a water decoction of Coptis chinensis, mixing the water decoction of Coptis chinensis with a liquid culture medium, inoculating Propionibacterium and then co-fermenting.

[0015] In a preferred embodiment of the present invention, the method specifically comprises: preparing a water decoction of Coptis chinensis, mixing the water decoction of Coptis chinensis with a liquid culture medium, sterilizing after adjusting the pH value, and co-fermenting after inoculating Propionibacterium. The liquid culture medium is preferably an MRS culture medium.

[0016] In some specific embodiments of the present invention, the mixing ratio of the Coptis chinensis decoction to the liquid culture medium is a volume ratio of 1:4-20, and preferably 6% of the Coptis chinensis decoction is added to the culture medium.

[0017] In some specific embodiments of the present invention, the inoculation conditions of Propionibacterium are as follows: the inoculation amount is 5%-10%, preferably the inoculation amount is 5%. The inoculated bacterial solution is preferably a bacterial solution in the logarithmic growth phase activated for 24-36 hours.

[0018] In some specific embodiments of the present invention, the fermentation process parameters are as follows: pH range: 6.5-7.2, preferably 6.8-7.0; fermentation temperature: 30-40°C, preferably 37°C; fermentation time: 24-96 hours, preferably 72 hours; and the fermentation method is preferably anaerobic static fermentation.

[0019] In some embodiments of the present invention, the preparation method of the Coptis chinensis water decoction includes: crushing the Coptis chinensis into coarse powder, sieving it, adding distilled water, soaking it and placing it in an extraction tank, heating it to boiling, keeping it in a slightly boiling state for decocting, and after the decoction is completed, filtering it with gauze to collect the filtrate; repeatedly decocting the filter residue according to the above method, and combining the filtrate; decompressing and concentrating the combined filtrate to obtain a Chinese medicine concentrate, which is the Coptis chinensis water decoction.

[0020] In a preferred embodiment of the present invention, the method for preparing the coptis chinensis decoction comprises:

[0021] 1. Raw material pretreatment:

[0022] Grind Rhizoma Coptidis into coarse powder and pass through a 40-mesh sieve;

[0023] Add distilled water at a solid-liquid ratio of 1:10 (g / mL) and soak for more than 12 hours;

[0024] 2. Extraction process:

[0025] Place the soaking liquid in an extraction tank;

[0026] Heat to boiling and keep it at a simmer;

[0027] The first decoction is 1.5-2 hours;

[0028] Filter through 4 layers of gauze and collect the filtrate;

[0029] Add water to the residue and boil it repeatedly 1-2 times, and combine the filtrate;

[0030] 3. Concentration treatment:

[0031] The combined filtrate was concentrated under reduced pressure, the temperature was controlled at 60°C-65°C, and the relative density was 1.1-1.2;

[0032] Sterilize at 121℃ for 20 minutes, cool to room temperature and set aside.

[0033] In the third aspect of the present invention, a composite formulation is provided, wherein the composite formulation comprises the composition for inhibiting Clostridium perfringens described in the first aspect.

[0034] In some embodiments of the present invention, the compound formulation is a medicine or a feed additive.

[0035] In some embodiments of the present invention, the composite preparation includes liquid preparations, such as fermentation broth, oral solution, etc., solid preparations, such as lyophilized powder, granules, etc.; suitable auxiliary materials can be added during preparation.

[0036] In some embodiments of the present invention, the composition of the preparation can be added with appropriate auxiliary materials according to the type of preparation. For example, a protective agent (such as skimmed milk powder) can be added as needed, and an excipient and a stabilizer can be added as needed. When using auxiliary materials, the content of the active ingredient (co-fermentation product) can be adjusted as needed.

[0037] In some embodiments of the present invention, the compound preparation can be a drug, such as veterinary drugs, anti-infective drugs, intestinal drugs, immunomodulators, etc.

[0038] In some embodiments of the present invention, the compound preparation can be a feed additive, such as a probiotic preparation, a preventive additive, a nutritional enhancer, an immunopotentiator, and the like.

[0039] In the fourth aspect of the present invention, there is provided the use of the composition for inhibiting Clostridium perfringens described in the first aspect or the composite formulation described in the third aspect in the preparation of a drug or feed additive for preventing and / or alleviating infectious diseases caused by Clostridium perfringens infection.

[0040] In some embodiments of the present invention, the composition or compound preparation has preventive applications, which is manifested in preventing Clostridium perfringens infection, enhancing animal immunity, improving intestinal health, improving production performance, etc.

[0041] In some embodiments of the present invention, the composition or compound preparation has therapeutic applications, which are manifested in relieving infection symptoms, reducing inflammatory response, repairing intestinal damage, improving clinical prognosis, etc.

[0042] In a fifth aspect of the present invention, there is provided a use of the composition for inhibiting Clostridium perfringens described in the first aspect or the composite preparation described in the third aspect in the preparation of an antibacterial agent, wherein the antibacterial agent has an effect of inhibiting Clostridium perfringens. In particular, the Clostridium perfringens is Clostridium perfringens ovis.

[0043] Through one or more of the above technical means, the present invention can achieve the following beneficial effects:

[0044] The co-fermentation composition of Propionibacterium and Coptis chinensis of the present invention exhibits significant technical effects and excellent immunomodulatory effects. In terms of enhancing the body's immune function, the present invention can significantly increase the secretion of antiviral factor IFN-β and anti-inflammatory factor IL-10, and reduce the production of pro-inflammatory factors IL-1β and TNF-α, and this effect is significantly better than using Coptis chinensis or Propionibacterium alone. In terms of immune cell regulation, the present invention can significantly increase the secretion of antiviral factor IFN-β and anti-inflammatory factor IL-10, and reduce the production of pro-inflammatory factors IL-1β and TNF-α. + IL-4 + T cell ratio enhances humoral immunity and maintains CD8 + IFN-γT cell level enhances cellular immunity, showing stronger immunomodulatory ability than using any component alone. In terms of protecting the intestinal barrier, the present invention can significantly increase the expression of tight junction proteins ZO-1 and Occludin, maintain the morphological integrity of intestinal tissue, reduce congestion, edema and mucosal damage, and the protective effect is better than using any component alone.

[0045] Compared with the prior art, the present invention achieves a synergistic effect through the co-fermentation of traditional Chinese medicine and probiotics, overcoming the problem of limited effect of using them alone; compared with traditional antibiotics, the present invention is not easy to produce drug resistance, has no residue problem, and has little effect on intestinal flora. In addition, the present invention has the characteristics of simple and feasible preparation method and wide source of raw materials, which can be used for drug development and the development of feed additives. The above technical effects and excellent effects have been fully verified by multiple experimental indicators such as weight change, survival rate, immune index, tissue pathology, etc., indicating that the present invention has significant practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The drawings constituting part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. The implementation scheme of the present application is described in detail below in conjunction with the drawings, wherein:

[0047] Figure 1The body weight change curve of mice is shown.

[0048] Figure 2 Mouse fecal diarrhea scores are shown.

[0049] Figure 3 Mouse survival curves are shown.

[0050] Figure 4 The figure shows the changes of cytokines in mouse serum after infection with Clostridium perfringens, A: IFN-β content in mouse serum; B: IL-1β content in mouse serum; C: IL-10 content in mouse serum; D: TNF-α in mouse serum; Among them, **** P <0.0001;*** P <0.001;** P <0.01;* P <0.05; ns (no significance) P >0.05.

[0051] Figure 5 The CD3 + CD4 + The number of IL-4T cells; among them, **** P <0.0001;*** P <0.001;** P <0.01;* P <0.05; ns (no significance) P >0.05.

[0052] Figure 6 The CD3 + CD8 + The number of IFN-γT cells; among them, **** P <0.0001;*** P <0.001;** P <0.01;* P <0.05; ns (no significance) P >0.05.

[0053] Figure 7 The relative expression of tight junction proteins in the intestine of each group of mice in the experiment is shown; A: relative expression of ZO-1 in the small intestine; B: relative expression of Occludin in the small intestine; where **** P <0.0001;*** P <0.001;** P <0.01;* P<0.05; ns (no significance) P >0.05.

[0054] Figure 8 Pathological tissue sections of the small intestine of mice in each group are shown, where A: PBS group; B: Coptis chinensis group; C: Propionibacterium group; D: co-fermentation group; E: control group; scale bar 200 μm. DETAILED DESCRIPTION

[0055] The present application is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0056] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents or raw materials used in this application can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in this application are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the content described herein can be applied to the method of this application. The preferred implementation methods and materials described in the text are for demonstration purposes only.

[0057] Example 1 Screening of compositions for inhibiting the growth of Clostridium perfringens

[0058] 1. Experimental materials: This example selected 8 kinds of Chinese medicines, including coptis root, scutellaria root, forsythia suspensa, honeysuckle, isatis root, angelica root, white peony root, and panax notoginseng, as research objects. These Chinese medicines were purchased from local regular Chinese medicine pharmacies and were identified to meet the relevant standards of the Chinese Pharmacopoeia.

[0059] Bacterial strain: Clostridium perfringens ( Clostridium perfringens ) strain is Clostridium perfringens, purchased from Beina Bio-Henan Industrial Microbial Strain Engineering Technology Research Center (BNCC), numbered BNCC185933, and referred to as Clostridium perfringens in subsequent experiments. The strain was stored in cooked meat culture medium, placed in a 4°C refrigerator, and subcultured every 1-2 weeks to maintain its activity. Before the experiment, Clostridium perfringens was inoculated on anaerobic blood agar plates and incubated in an anaerobic incubator (37°C, 5% CO 2 , 10% H 2 , 85% N 2 ) for 18-24 hours, pick a single colony and inoculate it into thioglycollate fluid culture medium, incubate it at 37℃ for 6-8 hours, prepare a bacterial suspension, and adjust the bacterial concentration to 1×10 6 -1×10 7 CFU / mL, for future use.

[0060] Propionibacterium Propionibacterium freudenreichii. ) The strain is Propionibacterium freudenreichii, purchased from Beina Bio-Henan Industrial Microbial Strain Engineering Technology Research Center (BNCC), numbered BNCC336447, and referred to as Propionibacterium in subsequent experiments. The strain was stored on an MRS medium slant, stored in a refrigerator at 4°C, and passaged once a month. When used, Propionibacterium was inoculated into MRS liquid culture medium, cultured at 30°C for 24-36 hours, and then transferred to fresh MRS liquid culture medium at an inoculation rate of 2%-3%, and continued to be cultured until the logarithmic growth phase for fermentation experiments.

[0061] 2. Main reagents: MRS medium, cooked meat medium, anaerobic blood agar plate, thioglycollate fluid medium, glucose, yeast extract powder, peptone, sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, calcium carbonate, ethanol, ethyl acetate, n-butanol, petroleum ether, hydrochloric acid, sodium hydroxide, chloroform, methanol, acetonitrile, etc., all of which were analytically pure reagents and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0062] 3. Main instruments: HZQ-F160 full-temperature oscillating incubator (Harbin Donglian Electronic Technology Development Co., Ltd.), SPX-25B biochemical incubator (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory), SW-CJ-2FD double-person single-sided clean bench (Suzhou Purification Equipment Co., Ltd.), LDZX-50KBS vertical pressure steam sterilizer (Shanghai Shen'an Medical Equipment Factory), TDL-5-A centrifuge (Shanghai Anting Scientific Instrument Factory), RE-52AA rotary evaporator (Shanghai Yarong Biochemical Instrument Factory), SHZ-D(III) circulating water vacuum pump (Gongyi Yuhua Instrument Co., Ltd.), Agilent 1260 Infinity high-performance liquid chromatograph (Agilent Technologies, USA), Thermo Scientific Q Exactive high-resolution mass spectrometer (Thermo Fisher Scientific, USA), JEM-2100 transmission electron microscope (Japan Electron Co., Ltd.), Nicolet iS50 Fourier transform infrared spectrometer (Thermo Fisher Scientific, USA), Bruker AVANCE III 400MHz NMR spectrometer (Bruker, Germany), etc.

[0063] 4. Experimental methods

[0064] 1) Preparation of Chinese medicine solution

[0065] Eight kinds of Chinese medicines, including Coptis chinensis, Scutellaria baicalensis, Forsythia suspensa, Honeysuckle, Radix Isatidis, Angelica sinensis, White Peony Root, and Panax notoginseng, were crushed into coarse powders by a grinder and passed through a 40-mesh sieve. The following operations were performed for each coarse Chinese medicine powder: a certain amount of coarse Chinese medicine powder was weighed, distilled water was added according to a solid-liquid ratio of 1:10 (g / mL), and soaked for 12 hours. The soaking liquid was then placed in a multifunctional extraction tank, heated to boiling, and kept boiling for 1.5 hours. After the decoction was completed, it was filtered with 4 layers of gauze to collect the filtrate. The filter residue was decocted again according to the above method once, and the filtrate was combined. The combined filtrate was concentrated under reduced pressure to a relative density of 1.1 (60°C), which was about 1 / 5 of the original volume, to obtain a Chinese medicine concentrate (referred to as Chinese medicine liquid in subsequent experiments). Finally, the Chinese medicine concentrate was sterilized at 121°C for 20 minutes, cooled to room temperature, and set aside for co-fermentation with Propionibacterium and in vitro antibacterial experiments in this embodiment.

[0066] 2) Cultivation of Propionibacterium and co-fermentation with traditional Chinese medicine

[0067] The propionibacterium stored on the MRS medium slant was inoculated into the MRS liquid medium and cultured at 30°C for 24 hours for activation. The activated propionibacterium was transferred to the fresh MRS liquid medium at a 2% volume inoculation amount and continued to be cultured until the logarithmic growth phase as the propionibacterium seed liquid. The prepared Chinese medicine liquid was mixed evenly with the MRS liquid medium, wherein the amount of Chinese medicine liquid added was 6% (volume) of the MRS liquid medium, and the pH value was adjusted to 6.8. Then, the propionibacterium seed liquid was inoculated at a 5% volume inoculation amount and anaerobically fermented at 37°C for 72 hours. During the fermentation process, samples were taken every 24 hours to determine the number of bacteria and pH value in the fermentation broth to monitor the fermentation progress.

[0068] 3) In vitro antibacterial test method

[0069] The Oxford cup method was used to determine the diameter of the inhibition zone of Clostridium perfringens. First, the Clostridium perfringens suspension was adjusted to a concentration of 1×10 7 CFU / mL. Take 100μL of bacterial suspension and spread it evenly on the anaerobic blood agar plate. Place Oxford cups (inner diameter 6mm, outer diameter 8mm, height 10mm). Place 3 Oxford cups on each plate in an equilateral triangle arrangement with a distance of no less than 25mm between cups. Add 200μL of the test substance to the Oxford cup. Place the plate in an anaerobic incubator (37℃, 5%CO 2 、10%H 2 , 85%N 2 ) for 18 h, observe the formation of inhibition zone, and measure the diameter of inhibition zone with vernier caliper.

[0070] 4) Optimization of fermentation conditions

[0071] Chinese medicinal materials were fermented with Propionibacterium, and the fermentation time, fermentation temperature and strain inoculation amount were optimized to obtain the best fermentation method. The fermentation broth of the strain obtained was subjected to in vitro antibacterial experiments (Oxford cup method, the same method as above). At the same time, the Propionibacterium fermentation group without adding Chinese medicinal liquid was used as a control.

[0072] 1) Fermentation time optimization:

[0073] Add 6% volume of Coptis chinensis Chinese medicine solution to 50mL MRS liquid medium, adjust the pH value to 6.8, sterilize at 121℃ for 25min, and inoculate 5% bacterial solution. Ferment at 37℃ for 24h, 48h, 72h, and 96h. Determine the size of the inhibition zone of Clostridium perfringens after fermentation, and select the optimal fermentation time by evaluation index.

[0074] 2) Fermentation temperature optimization:

[0075] Add 6% volume of Coptis chinensis Chinese medicine solution to 50mL MRS liquid medium, adjust the pH value to 6.8, sterilize at 121℃ for 25min, inoculate 5% bacterial solution, and adjust the pH value to 6.8. According to the optimal fermentation time screened out, place it at 30℃, 35℃, 37℃, 40℃, and 42℃ for static culture. Determine the size of the inhibition zone of Clostridium perfringens after fermentation, and select the optimal fermentation temperature by evaluation index.

[0076] 3) Optimization of bacterial inoculation amount:

[0077] Add 6% volume of Coptis chinensis Chinese medicine solution to 50mL MRS liquid medium, adjust the pH value to 6.8, sterilize at 121℃ for 25min, and inoculate 0.05%, 0.5%, 5%, and 50% of the bacterial solution into the fermentation broth. According to the optimal fermentation time and optimal fermentation temperature screened out, measure the size of the inhibition zone of Clostridium perfringens after fermentation, and evaluate the index to select the inoculation amount of the optimal strain.

[0078] Results 1) Propionibacterium and Chinese medicine pre-fermentation test

[0079] Table 1 Monitoring of bacterial count changes in co-fermentation of Propionibacterium and traditional Chinese medicine (10 7 CFU / mL)

[0080]

[0081] Table 2 Monitoring pH changes in co-fermentation of Propionibacterium and traditional Chinese medicine

[0082]

[0083] 2) In vitro inhibitory effect of Chinese herbal medicine on Clostridium perfringens

[0084] In vitro antibacterial experiments were conducted with Chinese herbal medicine liquid as the test object. By measuring the size of each inhibition zone, the antibacterial effects of various Chinese herbal medicine liquids were compared. The results showed that Coptis chinensis had the best inhibition effect, and the diameter of its inhibition zone was 16.2±1.1 mm. The results are shown in Table 3. Therefore, Coptis chinensis was co-fermented with Propionibacterium in the subsequent optimization experiment.

[0085] Table 3 Antibacterial effect of various Chinese medicine solutions on Clostridium perfringens

[0086]

[0087] 3) Optimization of Propionibacterium fermented Chinese medicine

[0088] Optimization of fermentation time

[0089] In vitro antibacterial experiments were carried out with Propionibacterium fermentation broth and Propionibacterium and Coptis chinensis co-fermentation broth as test objects, and the inhibition zones of Clostridium perfringens at different fermentation times were determined. The results are shown in Table 4. The results show that with the extension of fermentation time, the diameter of the inhibition zone first increases and then tends to stabilize. At 48h of fermentation, the diameter of the inhibition zone is (17.2±1.2) mm; at 72h of fermentation, the diameter of the inhibition zone reaches the maximum, which is (21.5±1.5) mm; after continuing to ferment for 96h, the diameter of the inhibition zone decreases to (20.1±0.3) mm. This shows that the fermentation time has a significant effect on the antibacterial effect of the co-fermentation broth, and the strongest inhibitory effect on Clostridium perfringens is achieved at around 72h.

[0090] Table 4 Diameter of inhibition zone against Clostridium perfringens at different fermentation times (mm)

[0091]

[0092] Optimization of fermentation temperature

[0093] In vitro antibacterial experiments were carried out with Propionibacterium fermentation broth and Propionibacterium and Coptis chinensis co-fermentation broth as test objects, and the inhibition zones of Clostridium perfringens at different fermentation temperatures were measured. The results are shown in Table 5. The results show that with the increase of fermentation temperature, the diameter of the inhibition zone showed a trend of first increasing and then decreasing. When the fermentation temperature was 37°C, the diameter of the inhibition zone was (21.5±1.8) mm, and the diameter of the inhibition zone reached the maximum. When the fermentation temperature was increased to 42°C, the diameter of the inhibition zone decreased to (15.8±0.3) mm. This shows that the fermentation temperature has a significant effect on the antibacterial effect of the co-fermentation broth, and the inhibition effect on Clostridium perfringens is the strongest at around 37°C.

[0094] Table 5 Diameters of inhibition zones against Clostridium perfringens at different fermentation temperatures (mm)

[0095]

[0096] Screening of bacterial inoculum volume

[0097] In vitro antibacterial experiments were carried out with Propionibacterium fermentation broth and Propionibacterium and Coptis chinensis co-fermentation broth as test objects, and the diameters of the inhibition zones of Clostridium perfringens with different Propionibacterium inoculation amounts were measured. The results are shown in Table 6. The results show that with the increase of the inoculation amount of the bacterial solution, the diameter of the inhibition zone showed a trend of first increasing and then decreasing. When the inoculation amount of the bacterial solution was 5%, the diameter of the inhibition zone was (21.4±1.4) mm, and the diameter of the inhibition zone reached the maximum. When the inoculation amount of the bacterial solution was 50%, the diameter of the inhibition zone was (17.2±0.9) mm. This shows that the inoculation amount of the bacterial solution has a significant effect on the antibacterial effect of the co-fermentation broth, and the inhibition effect on Clostridium perfringens is the strongest when it is about 5%.

[0098] Table 6 Diameter of inhibition zone of Clostridium perfringens at different bacterial inoculation amounts (mm)

[0099]

[0100] Example 2 Investigation of the protective effect of co-fermentation of Propionibacterium and Coptis chinensis on mice infected with Clostridium perfringens

[0101] In this example, Propionibacterium and Coptis chinensis were co-fermented (co-fermentation group), and mice were fed with PBS group, Propionibacterium group, and Coptis chinensis group, and challenge protection test was carried out. The weight change of mice, the content of IFN-β, TNF-α, IL-1β, IL-10 in serum, the activation of immune organs and immune cells, and the pathological changes of small intestine of mice were detected and analyzed by ELISA, Flow Cytometry and H&E staining, etc., to study the protective effect of the co-fermentation product of Coptis chinensis and Propionibacterium on mice infected with Clostridium perfringens.

[0102] 1. Experimental strains:

[0103] Propionibacterium Propionibacterium sp. ) strain is Propionibacterium freudenreichii, purchased from Beina Bio-Henan Industrial Microbial Strain Engineering Technology Research Center (BNCC), with the number BNCC336447, and referred to as Propionibacterium in subsequent experiments.

[0104] Clostridium perfringens Clostridium perfringens ) strain is Clostridium perfringens, purchased from Beina Bio-Henan Industrial Microbial Strain Engineering Technology Research Center (BNCC), with the number BNCC185933, and referred to as Clostridium perfringens in subsequent experiments.

[0105] 2. Experimental Animals

[0106] Fifty healthy 30-day-old mice were selected and randomly divided into 5 groups, with 10 mice in each group. The mice were kept in isolation and fed with food and water at 8:00, 14:00, and 20:00 every day.

[0107] 3. Reagents

[0108] Mouse interleukin 1β (IL-1β), mouse interferon β (IFN-β), mouse tumor necrosis factor α (TNF-α), and mouse interleukin 10 (IL-10) ELISA kits were purchased from ELISA (Jiangsu) Industrial Co., Ltd. Mouse CD3-PE-Cy7 antibody, CD4-PE antibody, CD8-FITC antibody (BD Company); 1640 cell culture medium (Hyclone); blocked mouse serum was stored by the laboratory; antibody diluent: 1% BSA; red blood cell lysis solution, Fluor 488 labeled goat anti-rabbit IgG (H + L) (Biyuntian Biotechnology Co., Ltd.); FACS solution (1000mL PBS for cell culture, 10mL FBS, 0.9g sodium azide); AXYGENPCR STRIP TUBES (Corning Incorporated, USA); Mini BEST Viral RNA / DNA Extraction Kit Ver5.0 (Code No.9766 Takara); goat serum for blocking; DAPI staining solution; antifade mounting medium; 4% paraformaldehyde solution; sodium citrate antigen retrieval solution.

[0109] 4. Main experimental equipment:

[0110] ABI Prism7500QT-qPCR instrument (ABI, USA); sodium heparin anticoagulant tube and procoagulant tube were purchased from (Jiangsu) Kangjian Medical Supplies Co., Ltd.; tissue embedding machine, paraffin slicer, DMi8 fluorescence inverted microscope were purchased from (Germany) Leica Instrument Co., Ltd.; tissue homogenizer was domestically produced.

[0111] 5. Experimental methods:

[0112] 1) Experimental plan

[0113] The experimental animals were divided into groups: PBS group (n=10), Propionibacterium group (n=10), Coptis chinensis group (n=10), co-fermentation group (n=10), and control group (n=10). Male and female animals were randomly assigned. The groups are shown in Table 7 below.

[0114] Table 7 Animal test groups

[0115]

[0116] Immunization and virus attack procedures:

[0117] Each group of mice was immunized on d1, d3, d5, d7, d9, d11, and d13. Except for the PBS group and the control group, which were gavaged with 0.5 mL PBS, the other groups were gavaged with corresponding drugs. Each mouse in the Propionibacterium group was gavaged with 1×10 9CFU / mL of Propionibacterium 0.5 mL, each mouse in the Coptis chinensis group was gavaged with 0.5 mL of Coptis chinensis Chinese medicine solution, and each mouse in the co-fermentation group was gavaged with 0.5 mL of Propionibacterium and Coptis chinensis co-fermentation solution.

[0118] The preparation method of Propionibacterium fermentation broth is as follows: 5% Propionibacterium (2.5 mL) was inoculated into 50 mL MRS liquid medium and fermented at 37 °C for 72 hours. After the fermentation, the whole fermentation broth was taken after being fully shaken to ensure that the number of viable bacteria remained at 1 × 10 9 CFU / mL, shake well before use.

[0119] Preparation method of Chinese herbal medicine liquid of Coptis chinensis: Take Coptis chinensis and grind it into coarse powder with a grinder, pass it through a 40-mesh sieve, weigh the coarse powder and add it to distilled water according to the solid-liquid ratio of 1:10 (g / mL) (for example: 25g of Coptis chinensis coarse powder is added to 250mL of distilled water), and soak it for 12 hours. Then place the soaking liquid in a multifunctional extraction tank, heat it to boiling, and keep it in a slightly boiling state for 1.5 hours. After the decoction is completed, filter it with 4 layers of gauze and collect the filtrate. The filter residue is decocted again according to the above method once, and the filtrate is combined. The combined filtrate is concentrated under reduced pressure to a relative density of 1.1 (60℃), which is about 1 / 5 of the original volume, to obtain 50mL of Chinese herbal medicine concentrate. Finally, the Chinese herbal medicine concentrate is sterilized at 121℃ for 20 minutes, cooled to room temperature, and then shaken thoroughly before use.

[0120] Preparation method of Propionibacterium and Coptis chinensis co-fermentation liquid: add 6% of the aforementioned Coptis chinensis Chinese medicine liquid (i.e., 3 mL) to 50 mL of MRS liquid culture medium, adjust the pH to 6.8, sterilize at 121°C for 20 min, inoculate 5% Propionibacterium (2.5 mL) and ferment at 37°C for 72 hours. After the fermentation is completed, shake well and take the whole fermentation liquid to ensure that the number of viable bacteria remains at 1×10 9 CFU / mL, shake well before use.

[0121] After immunization, the mice were weighed once a day, and the data were recorded to calculate the average daily weight gain. On day 14, except for the PBS group, which was intraperitoneally injected with 0.5 mL PBS, the other groups were intraperitoneally injected with Clostridium perfringens suspension (concentration of 1×10 9 CFU / mL, injection volume was 0.5mL).

[0122] 2) Growth performance index detection

[0123] Body weight: The mice were weighed once a day, and the weight change data were recorded and a weight change curve was drawn. The feces of the mice were recorded and scored every day. The feces scoring method: The rectal feces of each mouse were collected and evaluated, and the professional veterinarian used these photos to score the consistency of the feces. The feces consistency score ranged from 1 to 4, where 1 = normal; 2 = mild diarrhea; 3 = moderate diarrhea; 4 = severe watery diarrhea.

[0124] 3) Detection methods of immune indicators IFN-β, TNF-α, IL-1β, IL-10

[0125] After infection with Clostridium perfringens, blood was collected from the eyeballs of mice in each group. 1 mL of blood was collected and centrifuged in a centrifuge tube to separate the serum and store it at -80°C. The levels of IFN-β, TNF-α, IL-1β, and IL-10 in the serum were detected by ELISA.

[0126] 4) Preparation of single cell suspension

[0127] Preparation of spleen single cell suspension: Take the spleen part and grind it gently to obtain single cell suspension, pass it through a 200 mesh filter into the corresponding 15mL centrifuge tube, balance it, and centrifuge it at 1650rpm, 4℃, for 5min. Discard the supernatant, resuspend the cells in 1mL red blood cell lysis buffer, lyse on ice for 10min, take out the cells at 5min in the middle, and shake them for 30s. Add 10mL PBS buffer to stop, balance it, and centrifuge it at 1650rpm, 4℃, for 5min. Discard the supernatant, resuspend the cells in 1mL FACS buffer, and count them.

[0128] Preparation of single cell suspension of mesenteric lymph nodes: Carefully and gently grind all lymph nodes into single cell suspension, filter through a 200-mesh filter into a corresponding 15 mL centrifuge tube, balance, and centrifuge at 1650 rpm, 4°C, for 5 min. Discard the supernatant, resuspend the cells in 0.2 mL FACS buffer, and count.

[0129] 5) Flow cytometry

[0130] Flow cytometry antibody staining

[0131] (1) Divide the single cell suspensions into tubes, ensuring that each tube contains 1×10 6 cells (peripheral blood guaranteed to be 5×10 5 cells), with a total volume of 100 μL;

[0132] (2) Add the corresponding volume of flow cytometry antibody for cell surface markers according to the antibody titer.

[0133] (3) After oscillation and mixing, place at 4°C for 30 min away from light.

[0134] (4) After labeling, add 3.5 ml of ice-cold FACS buffer to the tube, centrifuge at 1650 rpm and 4°C for 5 min, discard the supernatant, and resuspend the cells with a small amount of FACS buffer.

[0135] (5) Repeat step (4) once to fully remove the unbound antibodies and reduce nonspecific staining on the cell surface. The samples were detected using a BD flow cytometer, and the data analysis and graphics processing were performed using FlowJo_v10.6.2 software.

[0136] 6) Detection of intestinal tight junction proteins by fluorescence quantitative PCR

[0137] Extracting RNA from sample tissues or cells includes the following steps: First, the sample is added to a lysis buffer and broken to release RNA. Next, the RNA is separated from the supernatant by a phenol / chloroform mixture, and the RNA is precipitated with isopropanol or ethanol. Subsequently, the RNA precipitate is washed to remove impurities, and finally the RNA is dissolved with RNase-free water or buffer. The extracted RNA can be tested for concentration and purity by colorimetry or fluorescence analyzer and stored in a -80°C refrigerator to prevent degradation. These steps may be adjusted depending on the specific experimental purpose and sample type. The relative quantitative q-PCR method was used to detect the transcription levels of two barrier proteins, ZO-1 and Occludin, in the mouse small intestine

[0138] 7) H&E staining to detect intestinal pathological changes

[0139] In order to further observe the pathological changes of the mouse small intestine, the middle section of the mouse small intestine was obtained, and the same part of the intestine was cut in each group and fixed in 4% paraformaldehyde solution for more than two days. The fixed tissue was then cut neatly, embedded, sliced, and stained with H&E. The steps are as follows:

[0140] (1) Dehydration: Place the samples in 70% alcohol, 80% alcohol, and 85% alcohol in order for 2 h each, dehydrate in 90% alcohol overnight, dehydrate in 95% I alcohol and 95% II alcohol for 1 h each, and dehydrate in 100% I alcohol and 100% II alcohol for 1 h each.

[0141] (2) Transparency: Place the sample into xylene I liquid and xylene II liquid in sequence for 5 minutes each, until the red skin-like substance can be seen with the naked eye.

[0142] (3) Wax immersion: Place the tissue blocks in wax I, wax II, and wax III in order. After immersion in wax at 56°C for 1 hour, place the tissue blocks in an embedding box for embedding.

[0143] (4) Sectioning: Cut each embedded block into 3.5 μm thick tissue slices and spread them on clean slides in a 41°C water bath. Place the slides in a drying oven at 80°C for 1 hour and perform H&E staining.

[0144] (5) The H&E staining procedure is as follows: Place the sample in xylene I and xylene II for 8 minutes, then place it in 100% alcohol I, 100% alcohol II, 95% alcohol, 80% alcohol and 70% alcohol for 1 minute, and rinse the part exceeding the alcohol in ultrapure water. Then stain with hematoxylin, rinse with ultrapure water, differentiate with 0.5% hydrochloric acid in an alcohol bath for 5 seconds, then rinse, then place in light ammonia water for 2 minutes, then rinse with water, place in 0.5% eosin aqueous solution for staining for 5 minutes, rinse with water, rinse in 80% alcohol bath, observe eosin staining, extract the sample in 95%, 100% I, 100% II alcohol baths for 5 times in sequence, then place in xylene I and xylene II for 2 minutes in sequence, and seal with neutral gum.

[0145] 6. Experimental results:

[0146] 1) Growth performance index test results

[0147] Body weight changes were recorded during the experiment, and the results showed that the co-fermentation liquid of Propionibacterium and Coptis chinensis could protect mice from infection with Clostridium perfringens and reduce the loss of body weight.

[0148] Specifically, during the immunization period from day 0 to day 14, the weight of mice in all groups showed a steady growth trend, and the growth conditions of each group were similar. After the 14th day of the virus attack (intraperitoneal infection with Clostridium perfringens), the growth of the PBS group was basically unaffected, and the weight loss of the control group was larger, reflecting the negative impact of Clostridium perfringens infection. The Coptis chinensis group and the Propionibacterium group could alleviate the sudden drop in weight to a certain extent, and the weight changes of mice in the co-fermentation group were significantly different. The above weight changes indicate that mice fed with the co-fermentation liquid of Propionibacterium and Coptis chinensis can resist the infection of Clostridium perfringens to a certain extent, and play a preventive and protective role. The results are shown in Figure 1 .

[0149] When scoring the diarrhea of ​​each group of mice, the results showed that the PBS group occasionally increased, but basically maintained at around 1 point, indicating that the feces were normal; the score of the control group gradually increased to 2-3 points, indicating that obvious diarrhea symptoms appeared, reaching a more serious level on the 6th to 7th day; the score of the Coptis chinensis group was basically maintained below 2, with mild diarrhea; the score of the Propionibacterium group was basically maintained at around 1-1.5 points, with occasional mild diarrhea; this shows that the Coptis chinensis group and the Propionibacterium group can alleviate the degree of diarrhea to a certain extent compared with the control group, but the score of the co-fermentation group has been maintained at 1 point, indicating that the feces are normal, which shows that the co-fermentation liquid of Propionibacterium and Coptis chinensis can alleviate the diarrhea of ​​mice caused by Clostridium perfringens infection. The results are shown in Figure 2 .

[0150] Figure 3The survival of each group is shown. During the 0-14 day immunization period, the survival rate of each group was 100%, indicating that there was no death during the immunization period and the drug administration was safe. After the 14th day of infection (peritoneal infection with Clostridium perfringens), the PBS group and the co-fermentation group maintained a 100% survival rate, indicating that the mice fed with the co-fermentation liquid of Propionibacterium and Coptis chinensis showed excellent preventive and protective effects against the infection of Clostridium perfringens; the survival rate of the Coptis chinensis group and the Propionibacterium group began to decline from the 15th day, and dropped to 40% on the 16th day, but was still better than the control group. The survival rate of the control group dropped significantly after the infection and dropped to 0 on the 16th day.

[0151] 2) Test results of immune indicators IFN-β, TNF-α, IL-1β, IL-10

[0152] The collected serum was used to detect the secretion of cytokines IFN-β, TNF-α, IL-1β, and IL-10 using EILSA. The results are shown in Figure 4 .

[0153] After immunization, mice in each group were infected with Clostridium perfringens. The IFN-β content in the control group decreased significantly compared with the PBS group (** P <0.01), indicating that infection with Clostridium perfringens would inhibit the secretion of IFN-β in mice; there was no significant difference between the Rhizoma Coptidis, Propionibacterium and control groups (ns), indicating that the Rhizoma Coptidis and Propionibacterium groups did not significantly improve the reduction of IFN-β caused by Clostridium perfringens infection; the difference in IFN-β between the co-fermentation group and the control group was particularly significant (*** P <0.001), indicating that the co-fermentation liquid can significantly increase the secretion of IFN-β, and the secretion amount is also higher than that of the PBS group, which indicates that the co-fermentation liquid can significantly improve the immunity of mice and resist the infection of Clostridium perfringens. Figure 4 As shown in A.

[0154] After immunization, mice in each group were infected with Clostridium perfringens. Compared with the PBS group, the IL-1β content in the control group increased significantly (**** P <0.0001), indicating that infection with Clostridium perfringens would induce the production of a large amount of pro-inflammatory factor IL-1β in mice; compared with the control group, the IL-1β level in the Coptis chinensis group and the Propionibacterium group was significantly reduced (** P <0.01, but still significantly higher than the PBS group, indicating that both treatments can inhibit inflammatory response to a certain extent; the difference in IL-1β between the co-fermentation group and the control group was extremely significant (**** P <0.0001), and its IL-1β level was closest to that of the PBS group, indicating that the co-fermentation liquid could significantly inhibit the production of IL-1β, which showed that the co-fermentation liquid had the strongest anti-inflammatory effect and could effectively inhibit the inflammatory response caused by Clostridium perfringens infection. Figure 4 As shown in B.

[0155] After immunization, mice in each group were infected with Clostridium perfringens. The IL-10 content in the control group decreased significantly compared with the PBS group (** P <0.01), indicating that infection with Clostridium perfringens would inhibit the secretion of anti-inflammatory factor IL-10 in mice; there was no significant difference in IL-10 levels between the Rhizoma Coptidis and Propionibacterium groups and the control group (ns), indicating that the two treatments had limited effects on maintaining IL-10 levels; there was a significant difference in IL-10 between the co-fermentation group and the control group (** P <0.01), the IL-10 level was significantly higher than that of the control group and close to that of the PBS group, indicating that the co-fermentation liquid can maintain a high level of IL-10 secretion, which means that the co-fermentation liquid can effectively maintain the body's anti-inflammatory ability and help to fight against the inflammatory response caused by Clostridium perfringens infection. Figure 4 As shown in C.

[0156] After immunization, mice in each group were infected with Clostridium perfringens. Compared with the PBS group, the TNF-α content in the control group increased significantly (**** P <0.0001), indicating that infection with Clostridium perfringens would induce the production of a large amount of pro-inflammatory factor TNF-α in mice; the TNF-α level in the Rhizoma Coptidis group was slightly lower than that in the control group (* P <0.05, while the Propionibacterium group showed a more significant decrease (*** P <0.001), indicating that both treatments have the effect of inhibiting inflammation, and the effect of Propionibacterium is better than that of Coptis chinensis, but the difference between the two groups compared with the PBS group is still significant; the difference in TNF-α between the co-fermentation group and the control group is extremely significant (**** P <0.0001), the TNF-α level was the lowest, close to the level of the PBS group, indicating that the co-fermentation liquid had the strongest anti-inflammatory effect and could significantly inhibit the production of TNF-α induced by Clostridium perfringens infection, further confirming its excellent anti-inflammatory effect. Figure 4 As shown in D.

[0157] 3) Effects of co-fermentation products of Propionibacterium and Coptis chinensis on TH2 response

[0158] TH2 cell immunity participates in humoral immunity by secreting IL-4, so the activation level of TH2 cells in the spleen was detected. The results showed that after infection with Clostridium perfringens, Propionibacterium, Coptis chinensis Chinese medicine liquid and Propionibacterium and Coptis chinensis co-fermentation liquid could activate CD4 + T cells secrete IL-4. Importantly, the co-fermentation liquid of Propionibacterium and Coptis chinensis can stimulate higher levels of IL-4 and the synergistic fermentation has the best immune protection effect. Figure 5 shown.

[0159] according to Figure 5It can be seen that compared with the PBS group, the control group had a higher number of CD4 + The proportion of IL-4T cells decreased significantly (**** P <0.0001), indicating that infection with Clostridium perfringens significantly inhibited the Th2 immune response in mice; compared with the control group, CD4 + The proportion of IL-4T cells increased significantly (** P <0.01, indicating that both treatments can maintain Th2 immune response to a certain extent; the difference between the co-fermentation group and the control group was extremely significant (**** P <0.0001), whose CD4 + The proportion of IL-4T cells was close to that of the PBS group, indicating that the co-fermentation solution could significantly enhance the Th2 immune response, which means that the co-fermentation solution could effectively maintain the body's immune balance and enhance the defense against Clostridium perfringens infection.

[0160] 4) Effects of co-fermentation products of Propionibacterium and Coptis chinensis on TH1 response

[0161] Cytotoxic T cells produce TH1 immune response by secreting IFN-γ, so the secretion level of IFN-γ in the spleen was detected. The results showed that after infection with Clostridium perfringens, Propionibacterium, Coptis chinensis Chinese medicine liquid and Propionibacterium and Coptis chinensis co-fermentation liquid could activate CD3 + CD8 + T cells secrete higher levels of IFN-γ, improving cellular immune response, and the co-fermentation liquid of acidobacillus and coptis chinensis has a more significant effect. The results are as follows Figure 6 shown.

[0162] like Figure 6 As shown in the figure, compared with the PBS group, the control group had a higher CD8 + The proportion of IFN-γ T cells was significantly reduced (**** P <0.0001), indicating that Clostridium perfringens infection significantly inhibited the cellular immune response; compared with the PBS group, CD8 + The proportion of IFN-γT cells also decreased significantly (**** P <0.0001), indicating that the use of these two treatments alone had limited effects on maintaining cellular immunity; while the CD8 + The level of IFN-γT cells was not significantly different from that in the PBS group (ns), but was significantly higher than that in the control group (**** P <0.0001), indicating that the co-fermentation solution can effectively maintain CD8 + The level of IFN-γT cells showed a significant difference, which indicated that the co-fermentation liquid had the strongest immune protection effect and could effectively prevent the decline of cellular immune function caused by Clostridium perfringens infection.

[0163] 5) Quantitative analysis results of tight junction proteins in mouse small intestine

[0164] The tight junction proteins ZO-1 (Zonula occludens-1) and Occludin in the small intestine of each group of mice were quantified. Among them, ZO-1 is a scaffold protein located on the inner side of the cell, which is mainly responsible for connecting other proteins to the cytoskeleton. Occludin is a transmembrane protein that crosses the cell membrane to interact with the ocludin of adjacent cells. Together, these two proteins constitute the intestinal barrier. They work together to maintain tight junctions between cells and control the selectivity of substances passing through the intercellular gap, thereby protecting the intestine from harmful substances. When the expression of these proteins is reduced, it usually means that the intestinal barrier function is impaired. Therefore, by detecting the expression levels of ZO-1 and Occludin, the integrity and functional status of the intestinal barrier can be evaluated.

[0165] After mice were immunized with Propionibacterium, Coptis chinensis Chinese medicine liquid and Propionibacterium and Coptis chinensis co-fermentation liquid, the relative expression of ZO-1 and Occludin were significantly increased compared with the control group. The results showed that Clostridium perfringens can damage the small intestine and reduce the expression of tight junction proteins in the small intestine. Figure 7 .

[0166] Specifically, Figure 7 As shown in A, the relative expression of ZO-1 mRNA in the small intestine of the control group decreased significantly compared with the PBS group (**** P <0.0001, indicating that infection with Clostridium perfringens significantly disrupts the expression of small intestinal tight junction protein ZO-1; compared with the control group, the expression of ZO-1 in the Coptis chinensis group and the Propionibacterium group was slightly (* P <0.05) and significant (** P <0.01), indicating that these two treatments have a certain effect on maintaining the integrity of the intestinal barrier, but still significantly different from the PBS group; the difference between the co-fermentation group and the control group is extremely significant (**** P <0.0001), and its ZO-1 expression level was closest to that of the PBS group, indicating that the co-fermentation liquid can effectively maintain the expression of small intestinal tight junction protein ZO-1, which means that the co-fermentation liquid has the strongest intestinal barrier protection effect.

[0167] like Figure 7 As shown in B, the relative expression of Occludin mRNA in the small intestine decreased significantly in the control group compared with the PBS group (**** P <0.0001, indicating that infection with Clostridium perfringens significantly disrupts the expression of the intestinal tight junction protein Occludin; compared with the control group, the expression of Occludin in the Coptis chinensis group was slightly increased (* P<0.05), while there was no significant difference between the Propionibacterium group and the control group (ns), indicating that the use of these two treatments alone has limited effect on maintaining the integrity of the intestinal barrier, and both groups are significantly different from the PBS group; the difference between the co-fermentation group and the control group is extremely significant (**** P <0.0001), and its occludin expression level was closest to that of the PBS group, indicating that the co-fermentation liquid can effectively maintain the expression of the small intestinal tight junction protein occludin, which means that the co-fermentation liquid has the strongest intestinal barrier protection effect.

[0168] 6) Small intestinal tissue pathology results

[0169] Observation of intestinal lesions revealed that the PBS group (A) showed normal intestinal tissue structure, neatly arranged villi, intact mucosal layer, and no obvious pathological changes. The control group (E) mice showed obvious lesions such as congestion, edema, and mucosal necrosis in the intestine, the intestinal wall became thinner, and the intestine was filled with gas and bloody contents. The intestinal tissues of the Coptis chinensis group (B) and the Propionibacterium group (C) showed a certain degree of lesions, but the degree was milder than that of the control group, and mild tissue damage and structural changes were observed. In contrast, the intestinal lesions of mice in the co-fermentation group (D) were milder, the intestinal mucosa was relatively intact, the congestion and edema were not obvious, and there was less gas and bloody contents in the intestine. These pathological results indicate that Clostridium perfringens infection can cause severe damage to the intestinal tissue of mice, and the co-fermentation liquid of Propionibacterium and Coptis chinensis can significantly reduce this damage and has a significant protective effect on the intestinal tissue. Its effect is better than that of using Coptis chinensis or Propionibacterium alone. The results are shown in Figure 8 .

[0170] The above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A composition for inhibiting Clostridium perfringens, characterized in that: The composition is a co-fermentation product of coptis chinensis and propionibacterium; wherein the Clostridium perfringens is Clostridium perfringens of sheep, the propionibacterium is Propionibacterium freudenreichii BNCC336447, and the coptis chinensis is a water decoction of coptis chinensis; the volume ratio of the water decoction of coptis chinensis to the liquid culture medium is 1:4-20; the inoculation amount of propionibacterium is 5%-10%; the co-fermentation conditions are: fermentation and culture at 30-40°C for 24-96 hours under pH 6.5-7.

2.

2. The composition for inhibiting Clostridium perfringens according to claim 1, characterized in that: The method for preparing a composition for inhibiting Clostridium perfringens comprises: preparing a coptis chinensis water decoction, mixing the coptis chinensis water decoction with a liquid culture medium, inoculating propionibacterium and then co-fermenting.

3. The composition for inhibiting Clostridium perfringens according to claim 1, characterized in that: The preparation method of the coptis chinensis water decoction comprises: crushing coptis chinensis into coarse powder, sieving it, adding distilled water, soaking it and placing it in an extraction tank, heating it to boiling, decocting it in a slightly boiling state, filtering it with gauze after the decocting is completed, and collecting the filtrate; repeatedly decocting the filter residue according to the above method, and combining the filtrate; decompressing and concentrating the combined filtrate to obtain a Chinese medicine concentrated solution, namely the coptis chinensis water decoction.

4. A composite preparation, characterized in that The composite formulation comprises the composition for inhibiting Clostridium perfringens according to any one of claims 1 to 3.

5. The composite formulation according to claim 4, characterized in that The compound preparation is a medicine or a feed additive.

6. Use of the composition for inhibiting Clostridium perfringens according to any one of claims 1 to 3 or the composite formulation according to claim 4 or 5 in the preparation of a medicament or feed additive for preventing and / or alleviating infectious diseases caused by Clostridium perfringens infection.

7. Use of the composition for inhibiting Clostridium perfringens according to any one of claims 1 to 3 or the composite formulation according to claim 4 or 5 in the preparation of an antibacterial agent, wherein the antibacterial agent has the effect of inhibiting Clostridium perfringens.

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