Microecological preparation based on synergistic interaction of traditional Chinese medicines and probiotics and application

Abstract

The invention discloses a microecological preparation based on synergistic interaction of traditional Chinese medicines and probiotics and application, and belongs to the technical field of green prevention and control of livestock and poultry epidemic diseases. The traditional Chinese medicine microecological preparation disclosed by the invention is prepared by adding four traditional Chinese medicines of common andrographis herb, great burdock achene, astragalus membranaceus and liquorice into a fermentation substrate and fermenting with lactobacillus plantarum according to a certain mass ratio. The traditional Chinese medicine microecological preparation prepared by the invention can remarkably prolong the survival time of mice infected by influenza viruses, reduce the death rate of the mice, reduce the lung index and the lung virus titer of the mice, relieve lung pathological damage caused by virus infection and inhibit mRNA and protein level expression of influenza virus NP and M1 genes of the mice. The invention is applied to anti-influenza virus drugs for livestock and poultry breeding.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a microecological preparation and its application based on the synergistic effect of traditional Chinese medicine and probiotics. Background Technology

[0002] Influenza viruses can spread rapidly among livestock and poultry through the respiratory and digestive tracts, causing high fever, respiratory distress, and secondary bacterial infections. This severely reduces livestock and poultry production performance and poses a significant threat to the economic and public health security of the livestock industry. Studies show that the high mutagenicity of the influenza virus hemagglutinin (HA) protein makes it easy to evade traditional vaccine immunization, while the overuse of antibiotics in densely populated farms further exacerbates the risk of cross-infection by drug-resistant strains (such as Escherichia coli and Salmonella). Developing novel, green antiviral agents has become an urgent need for the prevention and control of influenza in livestock and poultry.

[0003] Traditional Chinese veterinary medicine has a long history of application in the antiviral field of livestock and poultry farming; however, traditional Chinese veterinary medicine preparations have problems such as low extraction rate and bioavailability of active ingredients and limited effect of single application, which to some extent restricts their promotion in large-scale farming.

[0004] The Lactobacillus plantarum MⅢ selected in this invention is a high-performance probiotic. This strain has strong acid resistance and gastrointestinal colonization ability. Its secreted β-glucosidase can convert arctiin into arctigenin, which has a stronger activity, thereby improving antiviral efficiency. The extracellular polysaccharides produced during its fermentation process can form a complex with Astragalus polysaccharide, thereby activating the mucosal immune response. Summary of the Invention

[0005] The purpose of this invention is to provide a microecological preparation and its application based on the synergistic effect of traditional Chinese medicine and probiotics. This preparation combines traditional Chinese medicine and probiotics and undergoes special treatment to enable the two to exert a synergistic effect, thereby increasing the activity against influenza virus and having a significant effect on the anti-influenza virus in livestock and poultry. It is a green and novel preparation.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is: a traditional Chinese medicine microecological preparation, which is prepared by the following method: mixing traditional Chinese medicine powder, then adding water and nutrient substrate as fermentation substrate, mixing evenly and then sterilizing under high pressure, cooling and then inoculating the fermentation substrate with Lactobacillus plantarum MⅢ secondary seed liquid, fermenting under closed conditions at 35-39℃ for 20-30 h, freeze drying and pulverizing to obtain the product; The nutrient substrate consists of 2-4 parts corn starch, 2-4 parts brown sugar, 2-4 parts fructooligosaccharides, 2-4 parts lysine, and 1-3 parts talc, all by weight. The nutrient substrate accounts for 5-30% of the total mass of the Chinese herbal powder and water.

[0007] The Lactobacillus plantarum MⅢ is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2019560.

[0008] Preferably, the preparation method of the secondary seed culture of *Lactobacillus plantarum* MⅢ is as follows: a small amount of activated *Lactobacillus plantarum* MⅢ is picked from the solid culture medium with an inoculation loop and inoculated into MRS broth medium. The medium is then placed in a constant temperature shaker at 35-38℃ and shaken at 180-220 rpm for 12-24 hours to obtain the primary seed culture of *Lactobacillus plantarum* MⅢ; the primary seed culture of *Lactobacillus plantarum* MⅢ is then pipetted into a sterile EP tube, MRS broth medium is added to the EP tube, and the tube is then placed in a constant temperature shaker at 35-38℃ and shaken at 180-220 rpm for 10-16 hours to obtain the secondary seed culture of *Lactobacillus plantarum* MⅢ.

[0009] Preferably, the Lactobacillus MⅢ secondary seed culture accounts for 10-50% of the total mass of the fermentation substrate.

[0010] Preferably, the preparation contains a viable bacteria count of not less than 1×10⁻⁶. 9 CFU / g.

[0011] Preferably, the best method for preparing the traditional Chinese medicine microecological preparation is as follows: mix the traditional Chinese medicine powder, then add water and nutrient substrate as fermentation substrate, mix evenly and sterilize under high pressure, cool and then inoculate the fermentation substrate with probiotic secondary seed liquid, ferment under 37°C closed conditions for 24 h, freeze dry, and pulverize to obtain the preparation.

[0012] Preferably, the traditional Chinese medicine consists of 2 parts Andrographis paniculata, 1 part Arctium lappa, 3 parts Astragalus membranaceus, and 1 part Glycyrrhiza uralensis.

[0013] Preferably, the Lactobacillus plantarum MⅢ secondary seed culture accounts for 20% of the total weight of the fermentation substrate.

[0014] Preferably, the nutrient substrate consists of 2 parts corn starch, 2 parts brown sugar, 2 parts fructooligosaccharides, 2 parts lysine, and 1 part talc, and the nutrient substrate accounts for 15% of the total weight of the Chinese medicine powder and water.

[0015] Furthermore, the above-mentioned traditional Chinese medicine microecological preparations are applied in the preparation of anti-influenza virus drugs for livestock and poultry; the traditional Chinese medicine microecological preparations prepared by this method have higher viable bacteria counts, higher total lactone, total flavonoid and total saponin content, and better anti-influenza activity.

[0016] The compatibility of the four traditional Chinese medicines described in this invention is based on the following: Andrographis paniculata, Arctium lappa, Astragalus membranaceus, and Glycyrrhiza uralensis all possess antiviral effects. Andrographis paniculata is cold in nature and has the effects of clearing heat and detoxifying, cooling blood and reducing swelling; Arctium lappa is cold in nature and has the effects of dispersing wind-heat, clearing the lungs, detoxifying and relieving sore throat; Astragalus membranaceus is warm in nature and has the effects of tonifying qi and raising yang, and strengthening qi and consolidating the exterior; Glycyrrhiza uralensis is neutral in nature and has the effects of detoxifying, expectorating phlegm, relieving pain, and relieving spasms. When used in combination, their effects are enhanced, with yin and yang complementing each other and stabilizing the body, thus establishing a dynamic balance. Furthermore, Andrographis paniculata (which has anti-influenza virus replication activity) and Arctium lappa (which can block influenza virus adsorption) form a "dual-target" antiviral core, while Astragalus membranaceus (which has immune-activating activity) and Glycyrrhiza uralensis (which has anti-inflammatory and repairing effects) synergistically reduce tissue damage caused by influenza virus infection. The combination of these four traditional Chinese medicines covers the entire cycle of influenza virus infection. Moreover, all four traditional Chinese medicines are food-grade medicinal materials and are safe to use.

[0017] The selection criteria for the probiotics described in this invention are as follows: Lactobacillus plantarum is a type of lactic acid bacteria with good antibacterial and stress-resistant properties. It can survive in the acidic environment of the gastrointestinal tract of livestock and poultry and has antiviral and competitive exclusion and inhibition functions against pathogens (such as Clostridium perfringens, which is one of the common pathogens in pig herds. Its infection can cause a variety of serious diseases, leading to symptoms such as growth retardation, persistent diarrhea, and even death in pigs).

[0018] The selection of the preparation method described in this invention is based on the following: Previous research by the research group has shown that the traditional Chinese medicine selected in this invention has no antagonistic effect on the probiotic Lactobacillus plantarum MⅢ. By fermenting the traditional Chinese medicine together with the probiotic, on the one hand, it can promote the decomposition of macromolecules and the release of active ingredients in the traditional Chinese medicine, thereby improving the efficacy; on the other hand, the traditional Chinese medicine can also provide nutrients for the growth and reproduction of the probiotic, thereby promoting the proliferation of the probiotic and improving its activity.

[0019] The beneficial effects of this invention are as follows: This invention screened out synergistic compound traditional Chinese medicine and probiotics through in vitro experiments, and prepared a traditional Chinese medicine microecological preparation by combined fermentation of the two. The preparation was verified to have anti-influenza effect on livestock and poultry through in vivo mouse infection with WSN influenza virus. The traditional Chinese medicine microecological preparation of this invention fully considers the composition of raw materials and the ratio between each raw material, so that each raw material cooperates and works together under a specific ratio, and has a significant synergistic effect in anti-influenza virus. The four traditional Chinese medicines selected in this invention, namely Andrographis paniculata, Arctium lappa, Astragalus membranaceus, and Glycyrrhiza uralensis, have clear anti-influenza virus activity and synergistic potential. Literature reports that andrographolide, the active ingredient in Andrographis paniculata, can block influenza virus replication by inhibiting the entry of viral nucleoprotein (NP) into the nucleus; arctiin, a component of burdock seed, can target viral HA protein and interfere with the binding of the virus to the sialic acid receptor of host cells; astragalus polysaccharide, the main component of Astragalus membranaceus, can promote the secretion of antiviral cytokines such as IL-6 and TNF-α by macrophages by activating the TLR4 / NF-κB signaling pathway; and glycyrrhizic acid in licorice can inhibit the activity of influenza virus neuraminidase (NA) and reduce virus-induced pulmonary inflammatory damage. Attached Figure Description

[0020] Figure 1 : Curves showing changes in mouse body weight during preventive and therapeutic administration (**) p <0.01, *** p <0.001 vs model, n=6); Figure 2 Mice in the normal group and mice in each group infected with WSN influenza virus; Figure 3 The effects of traditional Chinese medicine microecological preparations on the survival time and survival rate of influenza virus in mice ( ### p <0.001 vs normal, * p <0.05,** p <0.01, *** p <0.001 vs model); Figure 4 H&E staining image of mouse lung tissue; Figure 5 Virus titer in mouse lungs; Figure 6 Effects of traditional Chinese medicine microecological preparations on the expression of NP mRNA, cRNA, vRNA, and MmRNA in the lungs of mice infected with WSN influenza virus (ns) p >0.05, * p <0.05,** p <0.01, *** p <0.001 vsmodel, n=3); Figure 7 Effects of traditional Chinese medicine microecological preparations on the expression of NP and M1 proteins in the lungs of mice infected with WSN influenza virus (ns p >0.05, * p <0.05,** p <0.01, *** p <0.001 vs model, n=3). Detailed Implementation

[0021] The present invention will now be described in further detail with reference to specific embodiments. The embodiments given are only for explaining the present invention and are not intended to limit the scope of the present invention. Several modifications and improvements can be made without departing from the concept of the technical solution of this application, and these all fall within the protection scope of this application.

[0022] 1. Experimental Materials MDCK cells: donated by the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, and preserved by our laboratory.

[0023] 2. Strains Lactobacillus plantarum ( Lactobacillus plantarum MIII was isolated and screened from the cecum of healthy chickens by our research group in the previous stage. It has been deposited in the China Center for Type Culture Collection, with accession number CCTCC NO: M2019560.

[0024] 3. Virus strain A / WSN / 33 (H1N1) influenza virus (WSN): donated by the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, and preserved in our laboratory; the WSN virus was multiplied in 10-day-old chicken embryos at 37°C for 3 days before use, and the median tissue culture infective dose (TCID) of the WSN virus was experimentally determined. 50 ) is 10 -6.5 / 100μL, Median lethal dose (LD50) 50 ) is 10 -5.64 / 50μL; All experiments involving WSN virus were conducted in a biosafety level 2 laboratory; This invention selects WSN as a model virus. Its HA / NA key functional region has 87.6% homology with the currently popular porcine triple recombinant H1N1 strain (such as A / swine / Henan / 127 / 2020) and shares the α-2,6-sialic acid receptor binding characteristics, which can effectively simulate the potential cross-host transmission mechanism of swine influenza virus.

[0025] 4. Experimental animals SPF-grade BALB / c mice, female, 5 - 6 weeks old, were purchased from the Animal Experiment Center of China Three Gorges University, with the license number SCXK (E) 2022 - 0012, and the animal ethics and welfare application review and filing number was 20220055; all experimental mice were housed in the SPF-grade animal room of the School of Life Sciences, Hubei University. The feeding environment was a temperature-controlled animal feeding facility (temperature: 20.0 ± 2.0 °C, humidity: 55 ± 10%), maintaining a 12 h light cycle and individually ventilated cages (IVC), and they could freely access food and water. After 5 days of adaptive feeding before the experiment, the mice were used for the experiment.

[0026] 5. Drugs and reagents Traditional Chinese medicine microecological preparation, traditional Chinese medicine control, and probiotic control: The main components of the traditional Chinese medicine microecological preparation include traditional Chinese medicine (Andrographis paniculata: Arctium lappa: Astragalus membranaceus: Glycyrrhiza uralensis = 2:1:3:1), nutritional substrates (corn starch: brown sugar: fructo-oligosaccharide: lysine: talc powder mass ratio = 2:2:2:2:1), and Lactobacillus plantarum MⅢ. The main components of the traditional Chinese medicine (Traditional Chinese medicine, abbreviated as TCM) control include traditional Chinese medicine, nutritional substrates, and MRS broth medium. The main components of the probiotic control (Probiotics) are nutritional substrates and Lactobacillus plantarum MⅢ.

[0027] Weighed appropriate amounts of the traditional Chinese medicine microecological preparation and the traditional Chinese medicine control, and added 0.5% CMC-Na to prepare 50 mg / mL traditional Chinese medicine microecological preparation and traditional Chinese medicine suspension. Weighed appropriate amounts of the probiotic control, and added 0.5% CMC-Na to prepare 9 a 10

[0028] CFU / mL probiotic suspension.

[0029] Carboxymethyl cellulose sodium (Carboxymethyl Cellulose-Na, abbreviated as CMC-Na), purchased from Sinopharm Chemical Reagent Co., Ltd.; weighed 0.5 g of CMC-Na, added secondary water to 100 mL, placed a rotor, continuously stirred for 12 h, and then placed it in a high-pressure steam sterilizer at 121 °C for 30 min to prepare a 0.5% CMC-Na solution for standby.

[0030] 4% paraformaldehyde fixative, 0.25% EDTA-trypsin: Biosharp.

[0031] 1% chicken red blood cell suspension: purchased from Nanjing Senbeijia Biotechnology Co., Ltd.

[0032] Fetal bovine serum (FBS): PAN (Germany).

[0033] DMEM cell culture medium: Gibco (USA).

[0034] Penicillin and streptomycin: Gino Biomedical Technology Co., Ltd. (Hangzhou).

[0035] TPCK-pancreatin: Sigma-Aldrich, USA.

[0036] TRIzol: Shanghai Aibixin Biotechnology Co., Ltd.

[0037] Reverse transcription kit: Shanghai Toyobo Biotechnology Co., Ltd.

[0038] SYBR Green PCR Kit: QIAGEN GmbH, Germany.

[0039] PCR primers: Shanghai Sangon Biotech Co., Ltd.

[0040] RIPA lysis buffer, protease inhibitor (Cocktail), BCA protein quantification kit, SDS-PAGE protein loading buffer: Shanghai Beyotime Biotechnology Co., Ltd.

[0041] Protein Marker: Beijing Polymer Biotechnology Co., Ltd.

[0042] Skim milk: BD Company, USA.

[0043] Influenza Avirus NP and M1 antibodies: GeneTex, USA.

[0044] β-actin, rabbit-HRP, mouse-HRP: Antibodies from CST Biotechnology, USA.

[0045] Example 1: Preparation and Detection of Traditional Chinese Medicine Microecological Preparations The powders of four Chinese herbs—Andrographis paniculata, Arctium lappa, Astragalus membranaceus, and Glycyrrhiza uralensis—were mixed in a mass ratio of 2:1:3:1. Then, 1.5 times the total mass of the herbs and 15% of the total mass of the mixture of herbs and water were added as a nutrient substrate (corn starch: brown sugar: fructooligosaccharides: lysine: talc in a mass ratio of 2:2:2:2:1) to serve as the fermentation substrate. After mixing thoroughly, the mixture was autoclaved at 121°C for 30 min. Subsequently, 20% of the total mass of the fermentation substrate was inoculated with Lactobacillus plantarum MⅢ secondary seed culture. The mixture was then fermented in a sealed container at 37°C for 24 h. After freeze-drying, the mixture was pulverized and passed through a 60-mesh sieve to obtain the Chinese herbal microecological preparation, which was stored at 4°C for later use.

[0046] The preparation method of Lactobacillus plantarum MⅢ secondary seed culture is as follows: a small amount of activated Lactobacillus plantarum MⅢ is picked from the solid culture medium with an inoculation loop and inoculated into MRS broth medium. The medium is then placed in a 37℃ constant temperature shaker and cultured at 220 rpm for 24 h to obtain Lactobacillus plantarum MⅢ primary seed culture. 2 mL of Lactobacillus plantarum MⅢ primary seed culture is pipetted into a sterile 50 mL EP tube (inoculation amount is 5%). MRS broth medium is added to the EP tube to make up to 40 mL. The tube is then placed in a 37℃ constant temperature shaker and cultured at 220 rpm for 14 h to obtain Lactobacillus plantarum MⅢ secondary seed culture.

[0047] The comparative test process for the above Example 1 is as follows.

[0048] Traditional Chinese medicine control: Take an equal amount of traditional Chinese medicine powder, replace the Lactobacillus plantarum MⅢ secondary seed liquid with an equal amount of sterile MRS broth, and follow the same steps as the preparation process of traditional Chinese medicine microecological preparations. The resulting sample is used as the traditional Chinese medicine control.

[0049] Probiotic control: No traditional Chinese medicine was added, and the rest of the preparation process was the same as that of traditional Chinese medicine microecological preparations. The resulting sample was used as a probiotic control.

[0050] The total number of viable bacteria in the traditional Chinese medicine microecological preparation prepared in Example 1 was detected, and the contents of total lactones, total flavonoids, and total saponins in the preparation were also detected. The specific detection methods are as follows: (1) Detection of total lactone content ① Construction of standard curve Accurately weigh 10 mg of andrographolide reference standard dried to constant weight, dissolve it in ethanol in a 25 mL volumetric flask to obtain a 0.4 mg / mL reference solution; accurately measure 0, 0.5, 1.0, 1.5, 2.0, and 2.5 mL of the reference solution into 10 mL volumetric flasks, add 2 mL of 60% ethanol, 1 mL of 2% 3,5-dinitrobenzoic acid, and 1 mL of 2% KOH reagent to each flask, shake well, and use the first tube as a blank. Use a UV-Vis spectrophotometer to measure the absorbance at a wavelength of 540 nm, plot the absorbance on the ordinate and the total lactone concentration on the abscissa to construct a standard curve; ② Extraction and determination of total lactones in samples Accurately weigh 3.0 g of the sample to be tested and place it in a 200 mL Soxhlet extraction flask. Add 90 mL of 70% ethanol and extract at 80℃ for 3 h. Make up the volume to 100 mL in a volumetric flask. Accurately pipette 1 mL of the sample extract and place it in a 10 mL volumetric flask. Add 2 mL of 60% ethanol, 1 mL of 2% 3,5-dinitrobenzic acid solution, and 1 mL of 2% KOH solution. Shake well. Measure the absorbance at a wavelength of 540 nm. Calculate the total lactone content in the sample based on the fitted standard curve.

[0051] (2) Detection of total flavonoid content ① Preparation of the standard curve: Accurately weigh 10.0 mg of rutin reference standard and dilute to 10 mL with 30% ethanol to obtain a 1.0 mg / mL standard solution; add 0, 0.4, 0.8, 1.2, 1.6 and 2.0 mL of the standard solution to 10 mL volumetric flasks respectively, then add 2.0, 1.6, 1.2, 0.8, 0.4 and 0 mL of 30% ethanol solution respectively, then add 0.5 mL of 5% sodium nitrite and shake well; after standing for 6 min, add 0.5 mL of 10% aluminum nitrate solution, after standing for 6 min, add 4.0 mL of 4% sodium hydroxide solution, and dilute to the mark with distilled water; after shaking well and standing for 15 min, measure the absorbance at 510 nm. Plot the absorbance as the ordinate and the total flavonoid concentration as the ordinate to prepare a standard curve; ② Extraction and determination of total flavonoids in samples Accurately weigh 3.0 g of the sample to be tested and place it in a 200 mL Soxhlet extraction flask. Add 90 mL of 70% ethanol and extract at 80℃ for 3 h. Make up the volume to 100 mL in a volumetric flask. Accurately pipette 1 mL of the sample extract into a 10 mL volumetric flask, add 0.5 mL of 5% sodium nitrite solution, shake well, and let stand for 6 min. Add 0.5 mL of 10% aluminum nitrate solution and let stand for 6 min. Add 4.0 mL of 4% sodium hydroxide solution and shake well. Add 30% ethanol to the mark and let stand for 15 min. Measure the absorbance at a wavelength of 510 nm. Calculate the total flavonoid content in the sample based on the fitted standard curve.

[0052] (3) Detection of total saponin content ① Construction of standard curve Accurately weigh 5.0 mg of astragaloside A reference standard and place it in a beaker. Dissolve it in methanol and transfer it to a 10 mL volumetric flask. Add methanol to the mark and dilute to obtain a 0.5 mg / mL standard solution. Accurately pipette 0.0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL into test tubes numbered 1-6, add methanol to each to make up to 0.5 mL, then add 0.5 mL of 8% vanillin anhydrous ethanol test solution to each, shake well, add 5.0 mL of 72% (volume fraction) sulfuric acid, shake well, and immediately place in a 62℃ constant temperature water bath for 20 min. Quickly remove and place in a cold water bath for 10 min. Within 30 min, using the test solution in test tube 1 as a blank reference, measure the absorbance value at a wavelength of 540 nm using a UV-Vis spectrophotometer. Plot a standard curve with absorbance as the ordinate and total saponin concentration as the ordinate. ② Extraction and determination of total saponins in the sample: Accurately weigh 3.0 g of the sample to be tested and place it in a 200 mL Soxhlet extraction flask. Add 90 mL of 70% ethanol and extract at 80℃ for 3 h. Make up the volume to 100 mL in a volumetric flask. Accurately pipette 0.2 mL of the sample solution into a stoppered test tube, add methanol to make up to 0.5 mL, add 5 mL of 8% vanillin anhydrous ethanol solution, and shake well. Add 5.0 mL of 72% sulfuric acid and shake well. Immediately place it in a 62℃ constant temperature water bath and keep it warm for 20 min. Quickly remove it and place it in a cold water bath for 10 min, and shake well. Measure the absorbance value at a wavelength of 540 nm. Calculate the total saponin content in the sample according to the fitted standard curve.

[0053] The test results are shown in Table 1 below:

[0054] The results in Table 1 show that, compared with the probiotic control, the total number of live bacteria in the traditional Chinese medicine microecological preparation was significantly increased, and the total number of live bacteria was not less than 1×10⁹ CFU / g. At the same time, the fermentation by probiotics significantly increased the content of total lactones, total flavonoids and total saponins of the effective components of traditional Chinese medicine, indicating that Lactobacillus plantarum has the effect of promoting the transformation and release of traditional Chinese medicine components.

[0055] Example 2: Preliminary investigation of the protective effect of traditional Chinese medicine microecological preparations against influenza virus infection in mice. To preliminarily evaluate whether the traditional Chinese medicine probiotic preparation prepared in Example 1 has a protective effect against influenza virus infection, this experiment involved challenging experimental mice with the virus and administering different types and doses of the test drug. The specific methods are as follows: 1. Experimental Grouping Forty-eight female BALB / c mice aged 5-6 weeks, weighing 15.0 ± 2.0 g, were randomly divided into eight groups of six mice each. The eight groups were: normal control group, virus control group, traditional Chinese medicine control group, probiotic control group, low- and high-dose traditional Chinese medicine probiotic preparation groups (preventive and treatment), high-dose traditional Chinese medicine probiotic preparation treatment group, and positive control group. The grouping and gavage dosage are as follows: ① Normal control group [CMC-Na + PBS + CMC-Na] ② Virus control group [CMC-Na+H1N1(WSN)+CMC-Na] ③ Traditional Chinese medicine control group [1000mg / kg / d TCM+H1N1(WSN)+1000mg / kg / d TCM] ④ Probiotic control group [10⁸ CFU / d Probiotics + H1N1 (WSN) + 10⁸ CFU / d Probiotics] ⑤ Low-dose prophylactic and therapeutic administration group of traditional Chinese medicine probiotics [500mg / kg / d CMMP + H1N1 (WSN) + 500mg / kg / d CMMP] ⑥ High-dose prophylactic and therapeutic administration group of traditional Chinese medicine probiotics [1000mg / kg / d CMMP + H1N1 (WSN) + 1000mg / kg / d CMMP] ⑦ High-dose treatment group of traditional Chinese medicine probiotics [H1N1 (WSN) + 1000mg / kg / d CMMP] ⑧ Positive control group [H1N1(WSN) + 40 mg / kg / day oselt]; 2. Infections caused by medication and nasal drops Throughout the experiment, mice were allowed unrestricted food and water. Groups ①-⑥ underwent pre-treatment by gavage with sterile CMC-Na solution, sterile CMC-Na solution, 1000 mg / kg TCM, Probiotics containing 10⁸ CFU of live bacteria, and 500 and 1000 mg / kg CMMP, respectively, at a volume of 0.2 mL / 10g, for 5 consecutive days. After pre-treatment, group ① mice were intranasally injected with 50 μL of sterile PBS solution, and groups ②-⑧ mice were intranasally infected with 50 μL of 5LD50 WSN virus. Two hours after infection, groups ①-⑥ mice continued the same administration regimen, while groups ⑦ and ⑧ mice were gavage with 1000 mg / kg CMMP and 40 mg / kg oselt, respectively, at a volume of 0.2 mL / 10g, for 7 consecutive days, with each administration occurring 24 hours apart. From 5 days before infection to 14 days after infection, the mice's condition and weight were monitored daily. 3. Observation Indicators The mice were observed and their weight was recorded daily until day 14 post-infection. Calculate the body mass index and survival rate of mice for each group according to the following formula, and plot the body mass index curve and survival rate curve of mice. Body mass index = average daily weight of mice / average weight of mice before infection Survival rate (%) = Number of mice surviving each day / Total number of mice before infection × 100%; 4. Statistical Methods The unpaired student's t-test was performed using GraphPad Prism 8 statistical software, and the survival rate of mice in each group was calculated using the log-rank (Mantel-Cox) test. 5. Experimental Results The experimental results are shown in Table 2:

[0056] During the pre-drug administration period, clinically, the mice in all groups showed normal hair, appetite, mental state, and activity levels; Figure 1 As shown, during the pre-drug administration period, the body weight of mice in each group was not significantly affected, indicating that at this dosage, traditional Chinese medicine, probiotics, traditional Chinese medicine microecological preparations and oseltamivir had no obvious toxicity to mice; During the 14-day observation period after infection, clinically, the normal control group mice exhibited normal appetite, were active, responsive, and in good spirits; from the second day after WSN virus infection, the virus-infected mice all showed rough, dull fur, lethargy, and listlessness; normal mice and mice in each WSN virus-infected group showed... Figure 2As the infection time increased, compared with the virus control group, the symptoms of mice in the low- and high-dose preventive treatment groups of traditional Chinese medicine (TCM) microecological preparations, the treatment group of TCM microecological preparations, and the oseltamivir group were all reduced to varying degrees. At the same time, on the 3rd day after the mice were infected with WSN virus, the body weight of mice in the treatment group of TCM microecological preparations and the oseltamivir group was significantly increased compared with the virus control group, indicating that TCM microecological preparations and oseltamivir have the effect of alleviating the weight loss of mice infected with WSN influenza virus.

[0057] like Figure 3 As shown in Table 2, compared with the virus control group, both the low-dose and high-dose preventive treatment groups of traditional Chinese medicine (TCM) microecological preparations significantly improved the survival rate and average survival time of mice. The high-dose preventive treatment group of TCM microecological preparations was comparable to the positive control group (oseltamivir). The TCM microecological preparation treatment group significantly improved the average survival time of mice. The results indicate that TCM microecological preparations have a significant protective effect against death in mice infected with WSN virus and have a certain anti-WSN influenza virus effect.

[0058] Example 3: Efficacy evaluation experiment of traditional Chinese medicine microecological preparations against influenza virus-infected mice. 1. Experimental Grouping Thirty-nine female BALB / c mice aged 5-6 weeks, weighing 15.0 ± 2.0 g, were randomly divided into 5 groups after weighing. Except for a normal control group of 3 mice, the other 4 groups each contained 9 mice. The 5 groups were: normal control group, virus control group, low- and high-dose prophylactic treatment groups of traditional Chinese medicine probiotics, and positive control group. The grouping and gavage dosage are as follows: Normal control group [CMC-Na+PBS+CMC-Na]; Virus control group [CMC-Na+H1N1(WSN)+CMC-Na]; Low-dose prophylactic and therapeutic administration group of traditional Chinese medicine microecological preparations [500mg / kg / d CMMP + H1N1 (WSN) + 500mg / kg / d CMMP]; High-dose prophylactic and therapeutic administration group of traditional Chinese medicine microecological preparations [1000mg / kg / d CMMP + H1N1 (WSN) + 1000mg / kg / d CMMP]; Positive control group [H1N1(WSN) + 40 mg / kg / day]; 2. Infections caused by medication and nasal drops Throughout the experiment, mice were allowed unrestricted food and water. Groups ①-④ underwent pre-treatment by gavage with sterile CMC-Na solution, sterile CMC-Na solution, and 500 and 1000 mg / kg CMMP, respectively, at a volume of 0.2 mL / 10g, for 5 consecutive days. After the pre-treatment, group ① mice were intranasally instilled with 50 μL of sterile PBS solution, and groups ②-⑤ mice were intranasally infected with 50 μL of 5LD50 WSN virus. Two hours after infection, groups ①-④ mice continued to be administered the same medication as above, and group ⑤ mice were gavage with 40 mg / kg oselt at a volume of 0.2 mL / 10g, for 5 consecutive days, with each administration occurring at 24-hour intervals. 3. Weigh and calculate the lung index and lung index inhibition rate. On day 6 after mice were infected with WSN virus, the mice were weighed and dissected to collect lungs. The lung index and lung index inhibition rate were calculated according to the following formula. Lung index = lung wet weight (mg) / body weight (g) × 100%; Lung index inhibition rate = (lung index of model control group - lung index of drug treatment group) / (lung index of model control group - lung index of normal control group) × 100%; 4. Detection of pathological changes in mouse lung tissue using H&E staining method Six days after mice were infected with WSN virus, the lungs of the mice were dissected and collected. The lung tissue was fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, stained with H&E, and then the pathological changes of the lung tissue were observed under an optical microscope (200×) and photographed. 5. TCID50 detection of viral titer in lung tissue On days 2, 4, and 6 after mice were infected with WSN virus, a portion of the mouse lung was taken and placed in a homogenization tube. 400 μL of PBS solution containing 10% penicillin antibody was added to the tube, and the mixture was allowed to stand at room temperature for 1 hour. Then, the mixture was homogenized (3 times, 20 s / time, 5 min interval). The homogenate was stored in a freezer at -80°C. After repeated freeze-thaw cycles twice, the mixture was centrifuged at 104 rpm for 5 min, and 300 μL of the supernatant was collected. The virus titer was determined immediately or after being stored in a freezer at -80°C and then thawed. The TCID50 method was used to determine the viral titer. 6. qRT-PCR detection of NP mRNA, cRNA, vRNA and M mRNA expression in mouse lungs (1) Extraction of total RNA from tissues using the TRIzol method ① Sample preparation: Take about 50 mg of lung tissue and put it into a 2 mL EP tube. Add magnetic beads and 1 mL TRIzol to the tube and grind it vigorously with a grinder. After standing at room temperature for 5 minutes, put it in a -80℃ refrigerator for later use. ② RNA isolation: Add 200 μL of chloroform to the EP tube, shake vigorously for 15 seconds, let stand at room temperature for 15 minutes, centrifuge at 4℃ and 1.2 × 104 rpm for 15 minutes (RNA is in the upper aqueous phase), and carefully aspirate the supernatant into a new EP tube; ③Precipitate RNA: Add an equal volume of isopropanol to the new EP tube, invert the EP tube to mix thoroughly, let stand on ice for 10 min, then centrifuge at 4℃ and 1.2×104 rpm for 10 min, and discard the liquid in the tube. ④ Washing RNA: Add 1 mL of pre-cooled 75% ethanol (prepared with DEPC water) to the substrate precipitate in the EP tube, gently invert and wash, centrifuge at 7600 rpm for 5 min, discard the liquid in the tube, continue centrifuging at 7600 rpm for 1 min, and then use a pipette tip to discard the liquid in the tube. ⑤ Dissolve RNA: Dry the tube on ice for about 3 minutes until there are no obvious water droplets in the tube, add 20 μL of DEPC water, and repeatedly pipette to dissolve the RNA precipitate; (2) Identification of total RNA integrity and determination of its concentration ① Gel preparation: Weigh 0.5g agarose, add 25mL 1×TAE buffer, seal with PE gloves and poke 2-3 small holes, microwave and boil 3 times, add 2.5μL Gelred nucleic acid dye when the temperature drops below 60℃, mix well and pour into a gel tank, let it solidify at room temperature for about 30 minutes to make 2% agarose gel. ②RNA electrophoresis: 1×TAE was poured into the electrophoresis tank, 2μL of total RNA and 2μL of 6×DNA loading buffer were mixed at a ratio of 1:1 and then the mixture was loaded. Electrophoresis was performed at a constant voltage of 90V and 55mA for 15min. ③ Gel imaging detection: RNA is imaged using a gel imager, including 28S, 18S and 5S (from top to bottom). If the brightness of the 28S band is 1.0 to 2.0 times that of the 18S band, and the 5S band is lighter, it indicates that the RNA has good integrity. ④ RNA concentration determination: The concentration and purity of RNA were detected using an ultra-micro biological detector (zeroed with DEPC water); an OD260 / 280 ratio between 1.8 and 2.0 is acceptable, indicating that the RNA purity is high and can be used for subsequent experiments; (3) Removal of gDNA reverse transcription reaction The gDNA removal reaction system was prepared on ice. The total RNA volume was 3 μg. Based on the measured RNA sample concentrations of each group, the required RNA volume V for each group was calculated, as shown in Table 3.

[0059] After preparing the gDNA removal reaction system, incubate at 37°C for 5 min to obtain the gDNA removal reaction solution; Prepare the reverse transcription reaction system according to the reverse transcription kit, as shown in Table 4:

[0060] After preparing the reverse transcription reaction system, cDNA was obtained by reverse transcription at 37℃ for 15 min, 98℃ for 5 min, and 4℃ for 20 min. (4) Real-time quantitative PCR: The mRNA levels of specific genes in cells were detected using the SYBR Green PCR kit. The reaction system is shown in Table 5.

[0061] After preparing the above reaction system, cover all reaction wells with a sealing film and smooth the film before instrumental analysis. The reaction conditions were: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 5 s, 60℃ annealing for 10 s, and 72℃ for 45 s, for 40 cycles. The 2-ΔΔCq method was used for relative quantification of mRNA levels. Primer sequences are as follows:

[0062] 7. Western blot analysis of NP and M1 protein expression in mouse lungs ① Gel preparation: Prepare a 10% separating gel using 5.9 mL sterile water, 5.0 mL 30% Acr-Bis, 3.8 mL 1.5 M Tris-HCl (pH 6.8), 0.15 mL 10% SDS, 0.15 mL 10% APS, and 0.006 mL TEMED. Add 1 mL isopropanol for sealing and gel at room temperature for 40 min. Prepare a 5% stacking gel using 5.5 mL sterile water, 1.3 mL 30% Acr-Bis, 1.0 mL 1.0 M Tris-HCl (pH 6.8), 0.080 mL 10% SDS, 0.080 mL 10% APS, and 0.008 mL TEMED. Gel at room temperature for 40 min. ② Electrophoresis: After loading the sample, electrophoresis at a constant voltage of 80V for about 30 minutes. When the protein markers begin to separate, change the voltage to 120V and continue electrophoresis until the loading buffer is about to run out of the lower gel. Then stop electrophoresis. ③ Transfer membrane: Activate the 0.22μm PVDF membrane with methanol for 1 min. Assemble the transfer device in the order of "1 layer of sponge - 2 layers of filter paper - gel - membrane - 2 layers of filter paper - 1 layer of sponge" and place it in an ice-water mixture. Transfer the membrane at a constant current of 200mA for about 90 min. ④ Sealing: Wash the PVDF membrane three times with TBST for 10 minutes each time, cut off the upper left corner to mark it, and transfer it to a petri dish containing sealing solution (5% skim milk powder) with plastic tweezers. Seale at room temperature for 2 hours. ⑤ Incubation with primary antibody: Dilute the primary antibody with diluent at a certain dilution ratio (1:1000), wash the blocked membrane with TBST, and then place the protein membrane in the antibody solution and incubate overnight at 4°C. ⑥ Membrane washing: Wash the membrane 3 times with TBST, 10 minutes each time; ⑦ Incubate the secondary antibody: Prepare a secondary antibody dilution buffer (1:10000) and block it with a protein membrane at room temperature for 1 hour; ⑧ Membrane washing: Wash the membrane 3 times with TBST, 10 minutes each time; ⑨ Luminescence and Development: Prepare a chemiluminescent mixture in a dark room and add it to the PVDF membrane. After reacting at room temperature for half a minute, develop the membrane using a gel imaging system. ⑩ Results Analysis: ImageJ software was used for protein grayscale scanning, Excel spreadsheets were used for data processing and calculation, and GraphPad Prism 8 was used for graphing and analysis. 8. Experimental Results (1) Lung index and lung index inhibition rate

[0063] As shown in Table 7, the results indicated that infection of mice with the WSN virus strain significantly increased the lung index compared to the normal control group (p < 0.001). After administration of the corresponding drugs to each treatment group, the lung index values ​​exhibited different trends. Both the low- and high-dose groups of the traditional Chinese medicine probiotic preparation showed a decreasing trend in lung index, with significant differences compared to the model control group (p < 0.05). The high-dose group of the traditional Chinese medicine probiotic preparation demonstrated comparable inhibitory effects on the lung index to the positive control drug oseltamivir phosphate group.

[0064] (2) H&E staining results like Figure 4 As shown, in lung sections of normal mice, alveoli were uniformly evacuated and similar in size, with uniform dispersion and no obvious inflammatory cell infiltration. The bronchial walls were normal and intact. In the model group, obvious inflammatory cell infiltration (red arrow), alveolar wall thickening (green arrow), diaphragmatic rupture, and irregular shape of large cysts were observed in the mouse lungs. The pathological conditions such as inflammatory cell infiltration and alveolar wall thickening were significantly improved in the various traditional Chinese medicine probiotic preparations and the positive drug oseltamivir phosphate group. This indicates that traditional Chinese medicine probiotic preparations can reduce lung inflammation caused by viral infection and have a certain anti-WSN virus effect.

[0065] (3) Virus titer in mouse lungs like Figure 5 As shown, on days 4 and 6 after mice were infected with WSN influenza virus, the low- and high-dose groups of traditional Chinese medicine probiotics and the positive control drug oseltamivir phosphate group significantly reduced the viral load in lung tissue, showing a significant difference compared with the model group.

[0066] (4) Effects of traditional Chinese medicine microecological preparations on the mRNA levels of WSN NP and M genes in mouse lungs like Figure 6 As shown, treatment with traditional Chinese medicine (TCM) microecological preparations significantly inhibited the expression of NP mRNA, cRNA, vRNA, and M mRNA in the lungs of mice at 2, 4, and 6 days post-infection, showing significant differences compared to the model group. With increasing concentration of TCM microecological preparations, the inhibitory effect on NP and M gradually increased, exhibiting a certain dose-dependent relationship.

[0067] (5) Effects of traditional Chinese medicine microecological preparations on WSN NP and M1 proteins in mouse lungs like Figure 7 As shown, compared with the virus control group, the treatment with traditional Chinese medicine (TCM) microecological preparations significantly inhibited the expression of NP and M1 proteins in the lungs of mice on days 2, 4, and 6 after infection. The high-dose TCM microecological preparation group showed an inhibitory effect on NP or M1 proteins on day 4 that was close to or stronger than that of the positive control drug oseltamivir group. The results indicate that TCM microecological preparations can effectively inhibit the expression of NP and M1 proteins and inhibit the proliferation of influenza virus, thereby achieving an anti-influenza virus effect.

Claims

1. A microecological preparation based on the synergistic effect of traditional Chinese medicine and probiotics, characterized in that... It is prepared by the following method: Mixing Chinese herbal powders, wherein the Chinese herbal powder consists of 2-4 parts of Andrographis paniculata, 1-3 parts of Arctium lappa, 3-5 parts of Astragalus membranaceus, and 1-3 parts of Glycyrrhiza uralensis, all by weight; then adding water and nutrient substrate as a fermentation matrix, mixing evenly, and then autoclaving, cooling, and inoculating the fermentation matrix with Lactobacillus plantarum MⅢ secondary seed liquid, fermenting under sealed conditions at 35-39℃ for 20-30 h, freeze-drying, and pulverizing to obtain the final product; The nutrient substrate consists of 2-4 parts corn starch, 2-4 parts brown sugar, 2-4 parts fructooligosaccharides, 2-4 parts lysine, and 1-3 parts talc, all by weight; the nutrient substrate accounts for 5-30% of the total weight of the Chinese herbal powder and water.

2. The traditional Chinese medicine microecological preparation according to claim 1, characterized in that: The Lactobacillus plantarum MⅢ secondary seed culture accounts for 10-50% of the total weight of the fermentation substrate.

3. The traditional Chinese medicine microecological preparation according to claim 1, characterized in that: The preparation method of the Lactobacillus plantarum MⅢ secondary seed culture is as follows: a small amount of activated Lactobacillus plantarum MⅢ is picked from MRS solid medium with an inoculation loop and inoculated into MRS broth medium. The medium is then placed in a constant temperature shaker at 35-38℃ and shaken at 180-220 rpm for 12-24 hours to obtain Lactobacillus plantarum MⅢ primary seed culture. The primary seed culture of Lactobacillus plantarum MⅢ is then pipetted into a sterile EP tube. MRS broth medium is added to the EP tube, and the tube is then placed in a constant temperature shaker at 35-38℃ and shaken at 180-220 rpm for 10-16 hours to obtain Lactobacillus plantarum MⅢ secondary seed culture.

4. The traditional Chinese medicine microecological preparation according to claim 1, characterized in that: The fermentation temperature was 37°C and the time was 24 hours.

5. The traditional Chinese medicine microecological preparation according to claim 1, characterized in that: The number of live bacteria in this traditional Chinese medicine microecological preparation is no less than 1×10⁻⁶. 9 CFU / g.

6. The use of the traditional Chinese medicine microecological preparation according to any one of claims 1-5 in the preparation of anti-influenza virus drugs for livestock and poultry.

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