Lactobacillus rhamnosus +21 for antiviral use and application thereof

Abstract

The present invention belongs to the technical field of probiotics, and specifically relates to a Lactobacillus rhamnosus +21 used for antiviral and its application. The strain has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 8, 2024, with the deposit number GDMCC No: 64839. The Lactobacillus rhamnosus ( Lactobacillus rhamnosus )+21 is a probiotic strain approved for use by the National Health Commission, which has a significant effect on fighting viruses. Daily administration of 9 billion strains of the present invention and 130 mg of metabolites can sustainably provide the necessary endogenous amounts of oleanolic acid, α-spinasterol, β-glucan, and L-rhamnose during the intestinal reproduction and growth process, and has broad application prospects in fighting viruses and maintaining a healthy nervous system.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics, and particularly relates to an antiviral Lactobacillus rhamnosus +21 and an application thereof. Background Art

[0002] 1. Probiotics can regulate physical barriers to fight viruses

[0003] The physical barrier includes the epidermis and the mucosa. Probiotics enhance the tight junction signals (TJs) between intestinal epithelial cells (IECs) to form a barrier to defend against viral invasion. Studies have shown that different types of probiotics can protect the host from viral infection by regulating the expression of tight junction signaling-related genes and proteins in intestinal epithelial cells, such as plant lactic acid bacteria, Lactobacillus sibaculus or Lactobacillus saké, which can protect intestinal epithelial cells from viral infection by maintaining the activity of tight junction proteins. Therefore, tight junction proteins between epidermal cells are necessary to maintain the integrity of the mucosal barrier.

[0004] 2. Probiotics can regulate chemical barriers to fight viruses

[0005] The chemical barrier is the first barrier encountered by pathogenic microorganisms after invading the intestines, mainly including mucin (MUC), antimicrobial peptides (AMP), digestive juices, lysozymes, mucopolysaccharides, other chemicals and antibacterial substances. Many studies have shown that probiotics can regulate the expression of mucin, affect the formation of the mucus layer, and maintain the intestinal barrier function. Short-chain fatty acids such as lactic acid, acetic acid, and butyric acid are not only the products of probiotic fermentation, but also potential regulators of intestinal epithelial cell proliferation. Among them, lactic acid can not only bind to viruses and affect the function of viruses, thereby limiting the invasion of viruses into host cells, but also destroy the envelope of viruses and dissolve the viral particles of enveloped viruses. Butyric acid can enhance the host's resistance to influenza virus infection. In summary, probiotics enhance the chemical barrier that maintains host health through antiviral metabolites or stimulating cells to produce mucin.

[0006] 3. Probiotics can regulate the immune barrier to fight viruses

[0007] The intestinal immune barrier is the last line of defense for the host to inhibit viral infection, including the gut-associated lymphoid tissue (GALT) and immune cells in the intestine. Probiotics can directly or indirectly regulate immune and anti-inflammatory functions. For example, Bifidobacterium infantis promotes the maturation of dendritic cells in the gut-associated lymphoid tissue and the accumulation of tolerant CD103 dendritic cells, thereby further regulating the differentiation of lymphocytes, inducing the expression of anti-inflammatory factors, improving the intestinal mucosal immune response, and enhancing the immune barrier to fight viruses. Probiotic metabolites, such as short-chain fatty acids, can act on immune cells, such as mononuclear phagocytes and lymphocytes, affect the release of inflammatory factors and immune chemotaxis, and inhibit the proliferation of immune effector cells. In addition, they can also participate in the immune regulation of the intestine.

[0008] Probiotics form a microbial barrier against viruses

[0009] The intestinal microbial barrier is a bacterial membrane barrier formed by the symbiotic intestinal microbiota tightly adhering to the surface of the intestinal epithelial mucosa. Intestinal probiotics are an important component of the intestinal microbial barrier and help regulate the balance of the number and structure of intestinal flora. Probiotics can compete with pathogens for nutrients and competitively inhibit the attachment sites of target cells or the spread of microcolonies through spatial barriers to resist the invasion of pathogens. Lactobacillus acidophilus and Lactobacillus rhamnosus can competitively attach to the adhesion sites of laryngeal epidermoid carcinoma cells and lung metastatic cells of human colon adenocarcinoma, reduce the adhesion sites on the cell surface, and prevent the invasion of pathogenic microorganisms such as enteropathogenic Escherichia coli. Probiotics can secrete non-bacteriocin components to act on pathogenic microorganisms or host cells, weakening the adhesion of pathogenic microorganisms.

[0010] However, the exploration of probiotics with antiviral functions and their active metabolites is still very limited, and the effective ingredients and mechanisms of action need to be elucidated. Summary of the invention

[0011] In view of the deficiencies in the prior art, the present invention aims to provide a Lactobacillus rhamnosus +21 for antiviral use and its application. The strain is isolated from fresh American ginseng in Changchun City, Jilin Province, and is identified as Lactobacillus rhamnosus by 16S rRNA method (16S ribosomal RNA gene sequencing technology). The strain has the effect of regulating the balance of intestinal flora and promoting human digestion and absorption, and also has antiviral effects.

[0012] In order to achieve the above technical objectives, the technical solution provided by the present invention is as follows:

[0013] In a first aspect of the present invention, a strain of Lactobacillus rhamnosus ( Lactobacillus rhamnosus )+21, the strain was deposited in the Guangdong Microbiological Culture Collection Center (address: 5th Floor, Laboratory Building, Guangdong Institute of Microbiology, No. 100, Xianlie Middle Road, Guangzhou) on July 8, 2024, with the deposit number GDMCC No: 64836.

[0014] A second aspect of the present invention provides the above-mentioned Lactobacillus rhamnosus ( Lactobacillus rhamnosus )+21, the culture method comprising: expansion culture, primary seed culture, seed tank culture and fermentation culture.

[0015] Preferably, the culture medium for the expanded culture contains: 15-20 g stachyose, 1-3 g sodium phosphate, 1-3 g beef extract powder, 0.2-0.6 g sodium acetate, 0.3-0.6 mL Tween-80, and 0.3-0.5 g potassium dihydrogen phosphate per 1000 mL water, and the pH value of the culture medium for the expanded culture is adjusted to 7.0 (alkaline solution adjustment).

[0016] Preferably, the culture conditions for the expanded culture are: culture at 36-38° C. for 10-20 h.

[0017] Further preferably, the culture medium for the expanded culture contains: 17 g stachyose, 2 g sodium phosphate, 2 g beef extract powder, 0.5 g sodium acetate, 0.5 mL Tween-80, and 0.4 g potassium dihydrogen phosphate per 1000 mL water, and the pH value of the culture medium for the expanded culture is adjusted to 7.0 (alkaline solution adjustment).

[0018] Preferably, the culture conditions for the expanded culture are: culture at 37° C. for 16 h.

[0019] Preferably, the culture medium for primary seed culture contains: 2-5 g D-galactose, 1-3 g peptone, 0.5-1.5 g beef extract powder, 0.5-1 g sodium acetate, 1-3 g leucine, and 0.5-1.5 mL Tween-80 per 1000 mL water, and the pH value of the culture medium for primary seed culture is natural.

[0020] Preferably, the culture conditions of the primary seed culture are: culture at 36-38° C. for 20-30 h; and the inoculation amount of the primary seed culture is 0.3%-0.6%, calculated by volume ratio.

[0021] Further preferably, the culture medium for the primary seed culture contains: 3 g D-galactose, 2 g peptone, 1 g beef extract powder, 0.8 g sodium acetate, 2 g leucine, and 1 mL Tween-80 per 1000 mL water, and the pH value of the culture medium for the primary seed culture is natural.

[0022] Preferably, the culture conditions of the primary seed culture are: culture at 37° C. for 24 h; and the inoculation amount of the primary seed culture is 0.5% by volume.

[0023] Preferably, the culture medium for the seed tank culture includes, by weight percentage: 2-5% D-galactose, 1-1.5% casein peptone, 0.2-0.4% dipotassium hydrogen phosphate, 0.8-1.2% serine, 0.2-0.5% sodium acetate, 0.8-1.2% phenylalanine, 0.08-0.12% Tween-80, 0.08-0.12% polyether defoamer, and 90.5-93.5% tap water, and the pH value of the culture medium for the seed tank culture is natural.

[0024] Preferably, the culture conditions of the seed tank culture are: tank pressure 0.04~0.06 MPa, culturing at 36~38°C for 10~20h; the inoculation amount of the seed tank culture is 3%~6%, calculated by volume ratio.

[0025] Further preferably, the culture medium for seed tank culture includes, by weight percentage, 3% D-galactose, 1.2% casein peptone, 0.3% dipotassium hydrogen phosphate, 1% serine, 0.4% sodium acetate, 1% phenylalanine, 0.1% Tween-80, 0.1% polyether defoamer, and 92.9% tap water, and the pH value of the culture medium for seed tank culture is natural.

[0026] Further preferably, the culture conditions of the seed tank culture are: tank pressure 0.05 MPa, culturing at 37°C for 18 h; and the inoculation amount of the seed tank culture is 5%, calculated by volume.

[0027] Preferably, the culture medium for fermentation and culture comprises, by weight percentage: 2-5% D-galactose, 1.2-2.5% casein peptone, 1-3% glucose, 0.3-0.6% stachyose, 0.8-1.2% bovine colostrum, 1.2-1.8% yeast extract, 1.5-1.8% sodium acetate, 0.1-0.3% Tween-80, 0.5-0.7% leucine, 0.8-1.2% dipotassium hydrogen phosphate, 0.05-0.12% polyether defoamer, and 80-88% tap water, and the pH value of the culture medium for fermentation and culture is adjusted to 6.5.

[0028] Preferably, the culture conditions of the fermentation culture are: tank pressure 0.04-0.06 MPa, culturing at 36-38° C. for 15-20 h; and the inoculation amount of the fermentation culture is 4%-6%, calculated by volume ratio.

[0029] Further preferably, the culture medium for fermentation culture includes, by weight percentage, 4% D-galactose, 1.5% casein peptone, 2% glucose, 0.5% stachyose, 1% bovine colostrum, 1.5% yeast extract powder, 1.6% sodium acetate, 0.2% Tween-80, 0.6% leucine, 1% dipotassium hydrogen phosphate, 0.1% polyether defoamer, and 86% tap water, and the pH value of the culture medium for fermentation culture is adjusted to 6.5.

[0030] Further preferably, the culture conditions of the fermentation culture are: tank pressure 0.05 MPa, culturing at 37° C. for 18 h; and the inoculation amount of the fermentation culture is 5%, calculated by volume.

[0031] The third aspect of the present invention provides a microbial agent comprising an active ingredient, wherein the active ingredient comprises the Lactobacillus rhamnosus or a fermentation broth thereof as described in the first aspect.

[0032] Preferably, the microbial agent comprises inactivated Lactobacillus rhamnosus and its fermentation broth.

[0033] A fourth aspect of the present invention provides the above-mentioned Lactobacillus rhamnosus ( Lactobacillus rhamnosus )+21 or the use of the above-mentioned bacterial agents in the preparation of food.

[0034] A fifth aspect of the present invention provides the above-mentioned Lactobacillus rhamnosus ( Lactobacillus rhamnosus )+21 or the use of the above-mentioned bacterial agents in the preparation of health foods that help regulate intestinal flora or aid digestion.

[0035] A sixth aspect of the present invention provides the above-mentioned Lactobacillus rhamnosus ( Lactobacillus rhamnosus )+21 or the use of the above-mentioned bacterial agents in the preparation of antiviral products.

[0036] Preferably, the virus types are novel coronavirus, herpes simplex virus and hepatitis B virus.

[0037] Preferably, the product is a medicine.

[0038] Preferably, the product is in the form of freeze-dried bacterial powder.

[0039] The seventh aspect of the present invention provides a method for preparing an antiviral freeze-dried bacterial powder, comprising: inactivating and centrifuging the fermented bacterial liquid obtained after fermentation and cultivation in the second aspect to obtain inactivated wet bacterial mud metabolites, and freeze-drying the inactivated wet bacterial mud metabolites, and then mixing them with auxiliary materials to obtain a suspension, and freeze-drying the suspension.

[0040] Preferably, the inactivation refers to maintaining the fermentation broth at 100-140°C for 5-8 s for UHT inactivation treatment; further preferably, the inactivation refers to maintaining the fermentation broth at 120°C for 6 s for UHT inactivation treatment.

[0041] Preferably, the centrifugal rotation speed is 8000 r / min~12000 r / min, specifically centrifuging at a feed rate of 2~5 kg / min.

[0042] Preferably, the drying conditions of the freeze-drying are: vacuum degree 8-12 Pa, temperature 25-28° C., and drying time 8-12 h; further preferably, the drying conditions of the freeze-drying are: vacuum degree 10 Pa, temperature 25° C., and drying time 10 h.

[0043] Preferably, the moisture content of the freeze-dried bacterial powder is ≤4% by mass.

[0044] Preferably, the auxiliary materials include soybean peptide powder, oligofructose, magnesium sulfate and sodium gluconate; further preferably, the mass addition ratio of the inactivated wet bacterial mud metabolites to the auxiliary materials is 1:2.5~3.5; further preferably, the mass addition ratio of the inactivated wet bacterial mud metabolites to the auxiliary materials is 1:1.31.

[0045] Beneficial technical effects of one or more of the above technical solutions:

[0046] (1) Lactobacillus rhamnosus provided by the present invention ( Lactobacillus rhamnosus )+21 is a probiotic and a major component of the normal intestinal flora in the human body. It is non-toxic, effective, safe, and green, and can play an important role in human health.

[0047] (2) Studies have shown that the Lactobacillus rhamnosus provided by the present invention ( Lactobacillus rhamnosus ) + 21 to make a finished product, which not only regulates the balance of intestinal flora and promotes human digestion and absorption, but also has antiviral effects. Daily administration of 9 billion strains of the present invention and 130 mg of metabolites can sustainably provide the necessary endogenous amounts of oleanolic acid, α-spinasterol, β-glucan, and L-rhamnose during the intestinal reproduction and growth process. It has a high application prospect in anti-virus and maintaining the health of the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings required for use in the description of the embodiment will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0049] Figure 1 The weight change of hamsters after SARS-CoV-2 infection in the experiment of Experimental Example 1 of the present invention;

[0050] Figure 2 is the number of virus copies in the lung tissue of mice on the 5th day after SARS-CoV-2 infection in Experimental Example 1 of the present invention;

[0051] FIG3 (a) is a lung pathological image of hamster 1 in the control group of Experimental Example 1 of the present invention;

[0052] FIG3( b ) is a lung pathology image of hamster 2 in the control group of Experimental Example 1 of the present invention;

[0053] FIG3 (c) is a lung pathology image of hamster 3 in the control group of Experimental Example 1 of the present invention;

[0054] FIG3( d ) is a lung pathological image of hamster 1 in the model group in Experimental Example 1 of the present invention;

[0055] FIG3 (e) is a lung pathological image of hamster 2 in the model group in Experimental Example 1 of the present invention;

[0056] FIG3( f ) is a lung pathological image of hamster 3 in the model group in Experimental Example 1 of the present invention;

[0057] FIG3( g ) is a lung pathological image of hamster 1 in the experimental group fed with 1.5% probiotics in Experimental Example 1 of the present invention;

[0058] FIG3(h) is a lung pathological image of hamster 2 in the experimental group fed with 1.5% probiotics in Experimental Example 1 of the present invention;

[0059] FIG3 (i) is a lung pathology image of hamster 3 in the experimental group fed with 1.5% probiotics in Experimental Example 1 of the present invention;

[0060] Figure 4 The 14-day survival rate of mice in Experimental Example 1 of the present invention;

[0061] Figure 5 is the viral load in the mouse brain tissue in Experimental Example 1 of the present invention;

[0062] Figure 6 The levels of IL-1β, TNF-α, and IFN-α in the brain tissue of mice in Experimental Example 1 of the present invention;

[0063] Figure 7 Comparison of liver function indicators of rats in each group in Experimental Example 1 of the present invention;

[0064] Figure 8 Comparison of liver fibrosis indexes of rats in each group in Experimental Example 1 of the present invention;

[0065] Fig. 9 Comparison of inflammatory factors in rats of each group in Experimental Example 1 of the present invention;

[0066] Fig.10 Comparison of HBsAg levels in the serum of rats in each group in Test Example 1 of the present invention;

[0067] Fig.11 Comparison of HBsAg levels in the serum of rats in each group in Test Example 1 of the present invention;

[0068] Fig.12Comparison of the oxidative stress SOD index of rats in each group in Experimental Example 1 of the present invention;

[0069] Fig.13 Comparison of the oxidative stress GSH-Px and MDA indicators of rats in each group in Experimental Example 1 of the present invention;

[0070] Fig.14 The expression of Fas, FasL and Caspase-3 proteins in rat tissues in Experimental Example 1 of the present invention;

[0071] Fig.15 This is a diagram of the original LC-MS mass spectrum data information obtained from the main metabolite information of the present invention. DETAILED DESCRIPTION

[0072] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0073] Method for determining the content of oleanolic acid: high performance liquid chromatography; method for determining the content of α-spinasterol: liquid chromatography tandem mass spectrometry; method for determining the content of β-glucan: enzyme-colorimetry; method for determining the content of L-rhamnose: high performance liquid chromatography.

[0074] Equipment used in production: strain incubator, clean workbench, constant temperature incubator, various test tubes, triangular flasks, seed tanks, fermentation tanks, vacuum freeze dryer, three-dimensional mixer, low-temperature crusher, vacuum packaging machine.

[0075] Considering that maintaining the balance of intestinal microorganisms is helpful to fight against viruses, the present invention proposes a Lactobacillus rhamnosus +21 for antiviral application.

[0076] Specifically, in a first typical embodiment of the present invention, a strain of Lactobacillus rhamnosus ( Lactobacillus rhamnosus )+21, the strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center (address: 5th Floor, Laboratory Building, Guangdong Provincial Institute of Microbiology, No. 100, Xianlie Middle Road, Guangzhou) on July 8, 2024, with the deposit number GDMCCNo: 64836.

[0077] In a second typical embodiment of the present invention, the above-mentioned Lactobacillus rhamnosus ( Lactobacillus rhamnosus )+21, the culture method comprising: expansion culture, primary seed culture, seed tank culture and fermentation culture.

[0078] In some embodiments, the culture medium for the expanded culture contains, per 1000 mL of water: 15-20 g stachyose, 1-3 g sodium phosphate, 1-3 g beef extract powder, 0.2-0.6 g sodium acetate, 0.3-0.6 mL Tween-80, and 0.3-0.5 g potassium dihydrogen phosphate, and the pH value of the culture medium for the expanded culture is adjusted to 7.0 (alkaline solution adjustment).

[0079] In some embodiments, the culture conditions for the expanded culture are: culturing at 36-38° C. for 10-20 h.

[0080] In some embodiments, the culture medium for the expanded culture contains the following: 17 g stachyose, 2 g sodium phosphate, 2 g beef extract powder, 0.5 g sodium acetate, 0.5 mL Tween-80, and 0.4 g potassium dihydrogen phosphate per 1000 mL of water, and the pH value of the culture medium for the expanded culture is adjusted to 7.0 (alkaline solution adjustment).

[0081] In some embodiments, the culture conditions for the expanded culture are: culture at 37° C. for 16 h.

[0082] In some embodiments, the culture medium for the primary seed culture contains: 2-5 g D-galactose, 1-3 g peptone, 0.5-1.5 g beef extract powder, 0.5-1 g sodium acetate, 1-3 g leucine, and 0.5-1.5 mL Tween-80 per 1000 mL water, and the pH value of the culture medium for the primary seed culture is natural.

[0083] In some embodiments, the culture conditions of the primary seed culture are: culture at 36-38° C. for 20-30 h; and the inoculation amount of the primary seed culture is 0.5%-0.6%, by volume.

[0084] In some embodiments, the culture medium for the primary seed culture contains: 3 g D-galactose, 2 g peptone, 1 g beef extract powder, 0.8 g sodium acetate, 2 g leucine, and 1 mL Tween-80 per 1000 mL water, and the pH value of the culture medium for the primary seed culture is natural.

[0085] In some embodiments, the culture conditions of the primary seed culture are: culture at 37° C. for 24 h; and the inoculation amount of the primary seed culture is 0.5%, by volume.

[0086] In some embodiments, the culture medium for seed tank culture includes, by weight percentage: 2-5% D-galactose, 1-1.5% casein peptone, 0.2-0.4% dipotassium hydrogen phosphate, 0.8-1.2% serine, 0.2-0.5% sodium acetate, 0.8-1.2% phenylalanine, 0.08-0.12% Tween-80, 0.08-0.12% polyether defoamer, and 90.5-93.5% tap water, and the pH value of the culture medium for seed tank culture is natural.

[0087] In some embodiments, the culture conditions of the seed tank culture are: tank pressure 0.04~0.06 MPa, 36~38°C culture for 10~20 h; the inoculation amount of the seed tank culture is 3%~6%, calculated by volume ratio.

[0088] In some embodiments, the culture medium for seed tank culture includes, by weight percentage: 3% D-galactose, 1.2% casein peptone, 0.3% dipotassium hydrogen phosphate, 1% serine, 0.4% sodium acetate, 1% phenylalanine, 0.1% Tween-80, 0.1% polyether defoamer, and 92.9% tap water, and the pH value of the culture medium for seed tank culture is natural.

[0089] In some embodiments, the culture conditions of the seed tank culture are: tank pressure 0.05 MPa, culturing at 37° C. for 18 h; the inoculation amount of the seed tank culture is 5%, calculated by volume.

[0090] In some embodiments, the fermentation culture medium includes, by weight percentage: 2-5% D-galactose, 1.2-2.5% casein peptone, 1-3% glucose, 0.3-0.6% stachyose, 0.8-1.2% bovine colostrum, 1.2-1.8% yeast extract, 1.5-1.8% sodium acetate, 0.1-0.3% Tween-80, 0.5-0.7% leucine, 0.8-1.2% dipotassium hydrogen phosphate, 0.05-0.12% polyether defoamer, and 80-88% tap water, and the pH value of the fermentation culture medium is adjusted to 6.5.

[0091] In some embodiments, the culture conditions of the fermentation culture are: tank pressure 0.04~0.06 MPa, 36~38°C culture for 15~20 h; the inoculation amount of the fermentation culture is 4%~6%, calculated by volume ratio.

[0092] In some embodiments, the fermentation culture medium includes, by weight percentage: 4% D-galactose, 1.5% casein peptone, 2% glucose, 0.5% stachyose, 1% bovine colostrum, 1.5% yeast extract powder, 1.6% sodium acetate, 0.2% Tween-80, 0.6% leucine, 1% dipotassium hydrogen phosphate, 0.1% polyether defoamer, and 86% tap water, and the pH value of the fermentation culture medium is adjusted to 6.5.

[0093] In some embodiments, the culture conditions of the fermentation culture are: tank pressure 0.05 MPa, culturing at 37° C. for 18 h; the inoculation amount of the fermentation culture is 5%, calculated by volume.

[0094] In a third typical embodiment of the present invention, a microbial agent is provided, comprising an active ingredient, wherein the active ingredient comprises the Lactobacillus rhamnosus or a fermentation broth thereof as described in the first aspect.

[0095] In some embodiments, the microbial agent comprises inactivated Lactobacillus rhamnosus and its fermentation broth.

[0096] In a fourth typical embodiment of the present invention, the above-mentioned Lactobacillus rhamnosus ( Lactobacillus rhamnosus )+21 or the use of the above-mentioned bacterial agents in the preparation of food.

[0097] In a fifth typical embodiment of the present invention, the above-mentioned Lactobacillus rhamnosus ( Lactobacillus rhamnosus )+21 or the use of the above-mentioned bacterial agents in the preparation of health foods that help regulate intestinal flora or aid digestion.

[0098] In a sixth typical embodiment of the present invention, the above-mentioned Lactobacillus rhamnosus ( Lactobacillus rhamnosus )+21 or the use of the above-mentioned bacterial agents in the preparation of antiviral products.

[0099] In some embodiments, the virus types are novel coronavirus, herpes simplex virus and hepatitis B virus.

[0100] In some embodiments, the product is a pharmaceutical product.

[0101] In some embodiments, the product is in the form of freeze-dried bacterial powder.

[0102] The seventh aspect of the present invention provides a method for preparing an antiviral freeze-dried bacterial powder, comprising: inactivating and centrifuging the fermented bacterial liquid obtained after fermentation and cultivation in the second aspect to obtain inactivated wet bacterial mud metabolites, and freeze-drying the inactivated wet bacterial mud metabolites, and then mixing them with auxiliary materials to obtain a suspension, and freeze-drying the suspension.

[0103] In some embodiments, the inactivation refers to maintaining the fermentation broth at 100-140° C. for 5-8 s for UHT inactivation treatment.

[0104] In some embodiments, the inactivation refers to maintaining the fermentation broth at 120° C. for 6 s for UHT inactivation treatment.

[0105] In some embodiments, the centrifugal rotation speed is 8000 r / min~12000 r / min, specifically centrifuging at a feed rate of 2~5 kg / min.

[0106] In some embodiments, the drying conditions of the freeze-drying are: vacuum degree 8-12 Pa, temperature 25-28° C., and drying time 8-12 h.

[0107] In some embodiments, the freeze-drying conditions are: vacuum degree 10 Pa, temperature 25° C., and drying time 10 h.

[0108] In some embodiments, the moisture content of the freeze-dried bacterial powder is ≤4% by mass.

[0109] In some embodiments, the auxiliary materials include soybean peptide powder, oligofructose, magnesium sulfate, and sodium gluconate.

[0110] In some embodiments, the mass addition ratio of the inactivated wet bacterial mud metabolites to the auxiliary materials is 1:2.5~3.5.

[0111] In some embodiments, the mass addition ratio of the inactivated wet bacterial mud metabolites to the auxiliary materials is 1:1.31.

[0112] The present invention is further described below in conjunction with examples. The present invention is further described below by way of examples, but the present invention is not limited to the scope of the embodiments described. Based on the embodiments in the present invention, any variation of the present invention by those skilled in the art without making a creative premise all belongs to the protection scope of the present invention. Meanwhile, in the embodiments of the present invention, unless otherwise specified, all preparation raw materials are commercially available products well known to those skilled in the art.

[0113] Example 1: Strain Identification

[0114] A Lactobacillus rhamnosus strain was isolated from fresh American ginseng in Changchun City, Jilin Province and was identified as Lactobacillus rhamnosus +21 by 16S rRNA method. The 16S rDNA sequence of the strain is shown in SEQ ID NO:1.

[0115] The above-screened Lactobacillus rhamnosus ( Lactobacillus rhamnosus)+21, the strain was deposited in the Guangdong Microbiological Culture Collection Center (address: 5th Floor, Laboratory Building, Guangdong Institute of Microbiology, No. 100, Xianlie Middle Road, Guangzhou) on July 8, 2024, with the deposit number GDMCC No: 64836.

[0116] Example 2: Cultivation of strains

[0117] 1. Expand bacterial culture:

[0118] The Lactobacillus rhamnosus +21 obtained in Example 1 and stored in a -80°C refrigerator was expanded and cultured. The culture medium was prepared as follows: 17 g stachyose, 2 g sodium phosphate, 2 g beef extract powder, 0.5 g sodium acetate, 0.5 mL Tween-80, 0.4 g potassium dihydrogen phosphate, 1000 mL tap water, and the pH was adjusted to 7.0 (alkaline solution adjustment).

[0119] Operation: Add the stachyose and other raw materials in the above formula into 1000 mL of water in the formula without any particular order, stir for 10 minutes to dissolve, and then divide into test tubes, each test tube contains 5 mL, seal with kraft paper stopper, and place in a medical sterilizer for sterilization at 0.1-0.12 MPa for 20 minutes. The culture medium is ready.

[0120] Inoculation: Under sterile conditions (clean bench), open the -80℃ low-temperature freezer to store the frozen tube of bacteria, and dissolve it at room temperature. Then take one sterilized test tube culture medium, add 0.5 mL of the dissolved bacterial solution into the test tube, seal the tube mouth with a rubber stopper and kraft paper, shake it gently by hand, and place it in a constant temperature incubator at 37℃ for 16 hours. This is called expansion culture medium.

[0121] 2. Primary Seed Cultivation

[0122] Primary seed culture formula: D-galactose 3 g, peptone 2 g, beef extract powder 1 g, sodium acetate 0.8 g, leucine 2 g, Tween-80 1 mL, tap water 1000 mL, pH natural.

[0123] Operation: Add the above raw materials into 1000 mL of tap water without any distinction, start the blender, and stir for 10 minutes to dissolve them all. This is called the primary seed culture solution.

[0124] Add the stirred and dissolved first-level seed culture solution into the conical flask according to the dosage, then wrap the bottle mouth with 6 layers of gauze and one layer of kraft paper, and put it into the sterilizer for sterilization. Before sterilization, first open the exhaust valve on the sterilizer and close it when a small amount of steam is discharged. When the pressure on the sterilizer reaches 0.05 MPa, open the exhaust valve on the sterilizer to exhaust for 6-8 minutes, then close the exhaust valve and continue heating. When the steam pressure in the sterilizer reaches 0.1-0.12 MPa, start timing. The sterilization time is 20 minutes. After completion, leave the heat source and let it cool down naturally. When the pressure gauge on the sterilizer returns to zero, open the sterilizer, take out the sterilized culture medium and put it into the clean bench for natural cooling. When the temperature drops to 37°C, transfer the above cultured expansion culture solution to the sterilized culture solution in the conical flask. The inoculation amount is 0.5%, that is, 100 mL of first-level seed culture solution and 0.5 mL of expansion culture solution. mL. After completion, seal the bottle mouth with the original gauze and kraft paper, shake it slightly by hand to mix the inoculated bacteria and culture solution evenly, and then place it in a constant temperature box and culture it at 37℃ for 24 hours. This is called the primary culture solution.

[0125] 3. Seed tank cultivation

[0126] Sterilization of fermentation equipment, pipelines and sterile filtration systems:

[0127] First, open the valves of all inlet and outlet pipelines and sterile air pipelines, pass 0.12-0.14 MPa steam, allow the steam to connect with the pipeline valve and discharge a small amount of steam, ventilate for 40 minutes, then close the valves of all inlet and outlet pipelines and set aside.

[0128] Sterilization of empty seed tanks and fermentation tanks:

[0129] Close all valves, open the drain valve at the bottom of the tank and the drain valve in the tank interlayer, open the direct steam valve to introduce 0.12-0.14 MPa steam into the pipe, start timing when the temperature in the tank reaches 121°C, the sterilization time is 40 min, then close the drain valve at the bottom of the tank and the drain valve in the interlayer, and the temperature in the tank will naturally drop to 37°C for standby use.

[0130] Seed tank culture formula: D-galactose 3%, casein peptone 1.2%, dipotassium hydrogen phosphate 0.3%, serine 1%, sodium acetate 0.4%, phenylalanine 1%, Tween-80 0.1%, polyether defoamer 0.1%, tap water 92.9%, natural pH.

[0131] Specific operation: First, pour the tap water used in the formula into the sterilized seed tank, start the mixer, add the raw materials required in the formula respectively (the order of addition is not important), then stir continuously and pass steam for heating. First, use the steam in the tank interlayer to heat to 95°C, then switch to direct steam heating. When the temperature in the tank reaches 121°C, maintain it for 20 minutes to achieve the sterilization effect, then turn off the steam, and start cooling through the tap water in the tank interlayer. When the temperature in the tank reaches 37°C, it is ready for use. This is called seed culture medium.

[0132] Then inoculate the above-cultured first-level bacterial culture solution into the seed tank under sterile conditions. The inoculation amount is 5% of the culture medium in the tank. 100 kg of seed tank culture medium can be inoculated with 5 kg of first-level bacterial solution. Then cover the inoculation cap on the tank and start stirring and culturing. The culture conditions are: temperature 37°C, natural pH value, stirring speed 140 r / min, tank pressure 0.05MPa, and culture time 18 h to meet the requirements. If the tank body is lowered, sterile air can be introduced to maintain the tank pressure. If the tank pressure is too high, the air release valve on the tank top can be used to adjust it. This is called seed tank culture.

[0133] 4. Fermentation

[0134] Fermentation culture formula: D-galactose 4%, casein peptone 1.5%, glucose 2%, stachyose 0.5%, bovine colostrum 1%, yeast extract powder 1.5%, sodium acetate 1.6%, Tween-80 0.2%, leucine 0.6%, dipotassium hydrogen phosphate 1%, polyether defoamer 0.1%, tap water 86%.

[0135] Specific operation: First, add the tap water used in the formula into the sterilized fermentation tank, start the mixer, add the raw materials used in the formula into the fermentation tank (the order of addition is not important), and then pass steam for heating. First, use the interlayer steam to heat to 95°C, and then switch to direct steam heating. When the temperature in the tank reaches 121°C, start timing, and keep it for 30 minutes to achieve the sterilization effect, and then cool it down with the tap water in the interlayer. When the temperature in the tank drops to 37°C, start to inoculate the strain. Before inoculation, first increase the tank pressure of the seed tank to 0.1 MPa, and keep the tank pressure of the fermentation tank at 0.05MPa, then open the inoculation pipeline valve, and transport the seeds cultured in the seed tank to the fermentation tank by pressure difference, and then close the inoculation channel valve.

[0136] The inoculation amount is 5 kg of bacteria cultured in the seed tank for every 100 kg of culture medium in the fermentation tank. The culture conditions are: culture temperature 37°C, stirring speed 140 r / min, tank pressure maintained at 0.05 MPa, and the acid and alkali automatic control system is used to maintain the pH value at 6.5 during the fermentation process. If the tank pressure drops, sterile air can be introduced to maintain the tank pressure until the fermentation is completed. The total fermentation time is 18 hours, and then the temperature is lowered to 20°C. This is the fermentation liquid.

[0137] 5. Inactivation and freeze drying of Lactobacillus fermentation broth

[0138] 1. The fermentation broth obtained by fermentation culture was subjected to UHT inactivation treatment at 120°C for 6 s.

[0139] 2. Pump the fermentation liquid into a storage tank for centrifugation. Operation: Start the centrifuge, adjust the speed to 10,000 r / min, and centrifuge at a feed rate of 3 kg / min. After centrifugation, collect the inactivated wet bacterial sludge metabolites, and freeze-dry the collected inactivated wet bacterial sludge metabolites.

[0140] 3. Freeze drying of inactivated bacterial sludge metabolites

[0141] Preparation of suspension: Take 1000 g of inactivated wet bacterial sludge metabolites, add 2000 mL of sterile saline, 600 g of soybean peptide powder, 2000 g of oligofructose, 20 g of magnesium sulfate, and 5 g of sodium gluconate.

[0142] Operation: First, take 1500 mL of normal saline in the formula, add oligofructose and stir to dissolve, then add 1000 g of inactivated wet bacterial mud metabolites, and then add the remaining normal saline and soybean peptide powder and auxiliary materials in the formula and stir thoroughly to obtain a suspension. The suspension is freeze-dried (place the suspension in a freeze-drying tray, start the freeze dryer, cool to -50°C in 120 min, keep for 1 h, turn on the vacuum pump, heat to 25°C at 4°C / h, keep for 10 h, the vacuum degree reaches 10 Pa, the moisture content is 3%, and the water activity is 0.18), and the moisture content is finally controlled to 4%, which becomes the freeze-dried bacterial powder of inactivated bacterial metabolites.

[0143] The freeze-dried bacterial powder product can be used in food or medicine; the food includes health food, baked food, solid beverage, liquid beverage, compressed candy and the like.

[0144] Test Example 1: Animal Experimental Research

[0145] 1. Antiviral Animal Experiments - COVID-19:

[0146] According to the experimental design in Table 1, 50 golden hamsters were divided into 5 groups and fed for 10 days. On the 11th day, the virus was infected by intranasal drops, and each hamster was infected with 1×10 4TCID 50 The prototype strain of SARS-CoV-2 was used to observe the condition of the hamsters after infection and record their weight. Figure 1 ; After 5 days of feeding, the hamsters were killed, and then the lung tissue of the hamsters was extracted. After grinding, the homogenate was taken to extract RNA, and the tissue viral load was measured. The results are shown in Figure 2 The hamsters with the best feeding quality ratio were selected, and their left lungs were fixed with formaldehyde for histopathological examination. Three hamsters were selected from each group for result analysis, namely Hamster 1, Hamster 2, and Hamster 3. The results are shown in Figures 3 (a) to 3 (i).

[0147] Table 1 Experimental design

[0148]

[0149] Table 2 Lung viral load in rats on the 5th day after SARS-CoV-2 infection (log 10 copies / g)

[0150]

[0151] according to Figure 1 It can be seen that the weight of hamsters decreased significantly on the second day after infection, and they began to show symptoms of lethargy and hunchback. Their weight began to increase on the third day. The weight of hamsters fed with probiotics increased significantly in a dose-dependent manner.

[0152] according to Figure 2 As shown in Table 3, the lung viral load of hamsters fed with probiotics was significantly reduced in a dose-dependent manner.

[0153] According to Figure 3 (a) to Figure 3 (i), the lung tissue structure of the hamsters in the model group was severely abnormal, with thickening of the alveolar wall, vascular congestion, perivascular lymphocyte infiltration, pulmonary edema, hemorrhage, alveolar dilation, and perivascular edema. Compared with the model group, the lung tissue structure of the hamsters in the experimental group fed with 1.5% probiotics was slightly abnormal, and the lesions were significantly alleviated. There was no pulmonary edema, perivascular lymphocyte infiltration, and perivascular edema, indicating that the probiotics can significantly reduce the lung tissue structure lesions caused by the invasion of the new coronavirus into the lung tissue.

[0154] Specific analysis:

[0155] Figure 3 (a), (b), (c) are the lung pathology images of hamsters 1 to 3 in the control group; Figure 3 (d), (e), (f) are the lung pathology images of hamsters 1 to 3 in the model group; Figure 3 (g), (h), (i) are the lung pathology images of hamsters 1 to 3 in the experimental group fed with 1.5% probiotics;

[0156] Figure 3 (a) The lung tissue structure is mildly abnormal, with a small area of ​​mild thickening of the alveolar wall accompanied by a small amount of inflammatory cell infiltration (black arrow); a small number of bronchial epithelial cells are arranged irregularly, and a small number of necrotic cells and eosinophilic substances can be seen in the cavity (blue arrow); and congestion can be seen in many blood vessels (purple arrow).

[0157] Figure 3 (b) The lung tissue structure is moderately abnormal, with mild thickening of the alveolar wall in a large area, accompanied by a large number of inflammatory cell infiltration (black arrows); bleeding can be seen in a small area of ​​the alveoli (orange arrows); and congestion can be seen in many blood vessels (purple arrows).

[0158] Figure 3 (c) The lung tissue structure is mildly abnormal, with a small area of ​​mild thickening of the alveolar wall accompanied by a small amount of inflammatory cell infiltration (black arrow); congestion can be seen in many blood vessels (purple arrow).

[0159] Figure 3 (d) Severe abnormalities in lung tissue structure, with moderate area of ​​lung consolidation, disappearance of alveolar structure, and a large number of inflammatory cell infiltration (black arrow); a small number of bronchial epithelial cells are arranged irregularly, and a small number of necrotic cells and eosinophilic substances can be seen in the cavity (blue arrow); a small number of lymphocytes can be seen in cuff-like infiltration around a small number of blood vessels (green arrow), a small number of blood vessels are edematous, the connective tissue is sparsely arranged, and a small number of lymphocytes are infiltrated (yellow arrow); a small area of ​​pulmonary edema can be seen, and tissue exudate can be seen in a small number of alveoli (red arrow); a small number of alveolar dilation (orange arrow).

[0160] Figure 3 (e) The lung tissue structure is severely abnormal, with diffuse severe thickening of the alveolar wall and a large number of inflammatory cell infiltration (black arrow); a small number of bronchial epithelial cells are arranged irregularly, and a small number of necrotic cells and hemorrhage can be seen in the cavity (blue arrow); a small number of lymphocytes can be seen in cuff-like infiltration around a small number of blood vessels (green arrow); a small area of ​​pulmonary edema can be seen, and tissue exudate can be seen in a small number of alveoli (red arrow); hemorrhage can be seen in a small area of ​​alveoli (orange arrow); congestion can be seen in many blood vessels (purple arrow); a small number of alveolar dilation (green arrow).

[0161] Figure 3 (f) Abnormal lung tissue structure, with mild thickening of the alveolar wall in a large area, accompanied by a large number of inflammatory cell infiltration (black arrow); a small number of lymphocytes can be seen in cuff-like infiltration around a small number of blood vessels (green arrow); a large area of ​​pulmonary edema can be seen, and tissue exudate can be seen in a large number of alveoli (red arrow); bleeding can be seen in a small area of ​​alveoli (orange arrow); congestion can be seen in many blood vessels (purple arrow); a small number of alveoli are dilated (green arrow).

[0162] Figure 3 (g) The lung tissue structure is mildly abnormal, with a small area of ​​mild thickening of the alveolar wall accompanied by a small amount of inflammatory cell infiltration (black arrow); a small number of alveolar dilation (green arrow).

[0163] Figure 3 (h) The lung tissue structure was mildly abnormal, with moderately thickened alveolar walls and a large number of inflammatory cell infiltrations (black arrows).

[0164] Figure 3 (i) The lung tissue structure is moderately abnormal, with large areas of severe thickening of the alveolar wall and a large number of inflammatory cell infiltration (black arrows); congestion can be seen in many blood vessels (purple arrows).

[0165] 2. Antiviral Animal Experiment - Herpes Simplex Virus Encephalitis

[0166] According to the experimental design in Table 3, a total of 50 mice were divided into 5 groups and pre-fed for 10 days. On the 11th day, the mice were anesthetized with sodium pentobarbital and infected with the virus through nasal drops. 50 μL of virus was dripped into each mouse. The mice were fed continuously for 14 days, and the clinical symptoms and deaths of the mice were recorded. The results are shown in Figure 4 The control group was dripped with an equal amount of saline. After 3 days of feeding, some mice were killed, and then the brain tissue of the mice was extracted. After grinding, the homogenate was taken to extract RNA, and the tissue viral load was measured. The results are shown in Figure 5 After 6 days of feeding, the remaining mice were killed, and the left temporal lobe tissue of the brain was taken. It was homogenized with 10% PBS and divided into a single use amount. The brain tissue of the dead mice was immediately taken and frozen in a -80℃ refrigerator. The levels of IL-1β, TNF-α, and IFN-α in the mouse brain tissue were determined according to the instructions of the ELISA detection kit. The results are shown in Figure 6 .

[0167] Table 3 Experimental design

[0168]

[0169] according to Figure 4 It can be seen that 3 days after being infected with the virus, the mice began to show symptoms of decreased appetite, slow movement, inverted hair, and hunched backs, while the mice that were gavaged with probiotics did not show these symptoms. The mice in the model group began to die on the 2nd day after being infected with the virus, and the survival rate was 0 on the 14th day. After being gavaged with probiotics, the survival rate of the mice increased significantly.

[0170] according to Figure 5 It can be seen that the viral load in the brain tissue of mice fed with probiotics was significantly reduced in a dose-dependent manner.

[0171] according to Figure 6 It can be seen that compared with the control group, the levels of IL-1β, TNF-α and IFN-α in the model group were significantly increased, and the levels of IL-1β, TNF-α and IFN-α in mice fed with probiotics were significantly reduced in a dose-dependent manner, indicating that probiotics can effectively reduce the body's inflammation level.

[0172] 3. Antiviral Animal Experiment - Hepatitis B Virus

[0173] According to the experimental design in Table 4, the hepatitis B virus was diluted with saline, mixed and transferred to a syringe, and injected into the tail vein of SD rats once a week for 3 consecutive weeks, and then the ALT, AST, HBsAg and HBeAg levels in the rat serum were detected to determine whether the model was successfully established. Another 10 rats were injected with an equal dose of saline through the tail vein and recorded as the control group. The rats with successful modeling were divided into 4 groups and fed for 7 days.

[0174] Table 4 Experimental design

[0175]

[0176] (1) 24 hours after intragastric administration, blood was collected from the tail vein and the supernatant was collected by centrifugation. ELISA kits were used to detect liver function indicators (ALT, AST, TBIL), muscle fibrosis indicators (LN, HA), inflammatory factors (TNF-α, IL-1β), HBsAg and HBeAg levels in rat serum. The results are shown in Table 1. Figure 7 , Figure 8 , Fig. 9 , Fig.10 , Fig.11 .

[0177] according to Figure 7 , Figure 8 , Fig. 9 , Fig.10 , Fig.11 It can be seen that the indicators of the model group rats were significantly increased compared with the control group, indicating that the modeling was successful. The liver function indicators (ALT, AST, TBIL), muscle fibrosis indicators (LN, HA), inflammatory factors (TNF-α, IL-1β), HBsAg and HBeAg levels of rats fed with probiotics were significantly reduced compared with the indicators of the model group, indicating that probiotics can alleviate inflammatory damage and protect the liver.

[0178] (2) After blood was drawn from the tail vein, the rats were killed and the liver tissue was obtained by dissection. An appropriate amount was taken for washing, fixed in fixative, dehydrated with ethanol, transparentized with xylene, and embedded in paraffin. Serial 4 μm sections were cut and stained according to the HE staining instructions after dewaxing. The pathological morphology of the liver tissue was observed under an optical microscope. The results are shown in Table 5.

[0179] Table 5 Pathological changes of liver tissues in rats in each group

[0180]

[0181] (3) Take an appropriate amount of liver tissue for washing, grind and homogenize with liquid nitrogen, take the supernatant after centrifugation, and add the working solution using the corresponding kits for SOD, GSH, and MDA. The results are shown in Fig.12 , Fig.13 .

[0182] according to Fig.12 , Fig.13 It can be seen that compared with the control group, the MDA content in the liver tissue of the rats in the model group increased, and the activities of SOD and GSH decreased. Compared with the model group, the MDA content in the liver tissue of the rats in the experimental group decreased, and the activities of SOD and GSH increased. This shows that probiotics can inhibit the body's oxidative stress.

[0183] (4) The relative expression levels of Fas, FasL, and Caspase-3 proteins in the remaining liver tissue were detected by Western blot. The results are shown in Fig.14 .

[0184] according to Fig.14 It can be seen that the expression of Fas, FasL and Caspase-3 proteins in the tissues of rats fed with probiotics were significantly reduced, indicating that probiotics can inhibit the expression of hepatitis B virus in the body and reduce the replication of the virus.

[0185] Experimental Example 2: Metabolomics analysis of mouse feces was performed using ultra-performance liquid chromatography-tandem mass spectrometry.

[0186] Experimental plan: The mice were divided into a control group and an experimental group. The control group was fed normally, while the experimental group was supplemented with the above-mentioned freeze-dried powder of the inactivated bacterial metabolites (containing only 900 million inactivated bacterial cells and 13 mg of metabolites) in their food. After 30 days of feeding, the fecal samples of the mice in the control group and the experimental group were placed in 400 μL of extraction solution (acetonitrile: methanol = 1: 1), vortexed for 30 seconds, and extracted with low-temperature ultrasound for 30 minutes (5°C, 40 KHz), allowed to stand at -20°C for 30 minutes, placed in a 4°C centrifuge, and centrifuged at 13000 r / min for 15 minutes. The supernatant was taken, dried with nitrogen, and re-dissolved with 120 μL of the reconstitution solution (acetonitrile: water = 1: 1). After low-temperature ultrasonic extraction for 5 minutes (5°C, 40 KHz), the sample was placed in a 4°C centrifuge, and centrifuged at 13000 r / min for 5 minutes. An appropriate amount of supernatant was taken for analysis. 10 uL of sample supernatant was separated by BEH C18 column (100 mm ×2.1 mm id, 1.8µm) and entered mass spectrometry detection. The separation flow rate was 0.40 mL / min and the column temperature was 40°C. After completion, the LC-MS raw data was transferred to the computer for processing and analysis. The MS mass spectrum information was matched with the public database, and the main metabolite information was obtained as shown in Table 6.

[0187] Table 6 Main metabolomics analysis of mice

[0188]

[0189] It can be seen from Table 6 that daily administration of 9 billion strains of the present invention and 130 mg of metabolites of the probiotic product (i.e., the freeze-dried powder prepared in Example 2) can significantly increase the contents of oleanolic acid, α-spinasterol, β-glucan, and L-rhamnose in mouse feces, thereby indicating that the probiotic product provided by the present invention can sustainably provide the endogenously required amount of oleanolic acid, α-spinasterol, β-glucan, and L-rhamnose during the intestinal reproduction and growth process.

[0190] Application Example 1: Anti-virus Application

[0191] The recruitment targets are virus-infected people with an average age of 45 in 8 communities in Jinan City. As of June 2024, a total of 60 people will be recruited to participate in the experiment. All of the above-mentioned persons have signed a written informed consent form. If the participant is unable to sign, an agent (usually a family member) will sign it.

[0192] Table 7 Antiviral application effect

[0193]

[0194] The experimental results in Table 7 show that:

[0195] Coronavirus: 20 people started to collect the results after taking it for 2 days. After taking it for 2 days, 4 people felt that fever, dry cough, fatigue, loss of smell and taste, nasal congestion, runny nose, sore throat, conjunctivitis, myalgia and diarrhea improved. After taking it for 4 days, 8 people felt that fever, dry cough, fatigue, loss of smell and taste, nasal congestion, runny nose, sore throat, conjunctivitis, myalgia and diarrhea were significantly improved. After taking it for 6 days, 12 people felt that fever, dry cough, fatigue, loss of smell and taste, nasal congestion, runny nose, sore throat, conjunctivitis, myalgia and diarrhea improved extremely significantly. After taking it for 7 days, 19 people felt that fever, dry cough, fatigue, loss of smell and taste, nasal congestion, runny nose, sore throat, conjunctivitis, myalgia and diarrhea were completely improved and lung infection disappeared; a total of 1 person did not improve after taking Lactobacillus rhamnosus.

[0196] Herpes simplex virus: 20 people started to collect the results after taking it for 8 days. After taking it for 8 days, 6 people felt that the tingling, itching, burning fever, swollen lymph nodes, repeated blisters or ulcers and other conditions improved. After taking it for 16 days, 10 people felt that the tingling, itching, burning fever, swollen lymph nodes, repeated blisters or ulcers were significantly improved. After taking it for 24 days, 13 people felt that the tingling, itching, burning fever, swollen lymph nodes, repeated blisters or ulcers improved extremely significantly. After taking it for 30 days, 18 people's tingling, itching, burning fever, swollen lymph nodes, repeated blisters or ulcers were completely improved and their immunity improved. A total of 2 people did not improve after taking Lactobacillus rhamnosus.

[0197] Hepatitis B virus: 20 people started to collect the results after taking it for 8 days. After taking it for 8 days, 7 people felt that their physical fatigue, fatigue, loss of appetite, nausea, aversion to oil, upper abdominal discomfort, abdominal distension, yellow urine, or mild fever had improved. After taking it for 16 days, 10 people felt that their physical fatigue, fatigue, loss of appetite, nausea, aversion to oil, upper abdominal discomfort, abdominal distension, yellow urine, or mild fever had improved significantly. After taking it for 24 days, 14 people felt that their physical fatigue, fatigue, loss of appetite, nausea, aversion to oil, upper abdominal discomfort, abdominal distension, yellow urine, or mild fever had improved significantly. After taking it for 30 days, 17 people's physical fatigue, fatigue, loss of appetite, nausea, aversion to oil, upper abdominal discomfort, abdominal distension, yellow urine, or mild fever had completely improved. A total of 3 people did not improve after taking Lactobacillus rhamnosus.

[0198] The 16S rDNA sequence of the strain is as follows:

[0199]

[0200] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) + 21, the rhamnosus Lactobacillus ( Lactobacillus rhamnosus )+21 was deposited in Guangdong Provincial Microbiological Culture Collection Center on July 8, 2024, with the deposit number GDMCC No: 64836.

2. A strain of Lactobacillus rhamnosus according to claim 1 ( Lactobacillus rhamnosus )+21, characterized in that, The culture method includes: expansion culture, primary seed culture, seed tank culture and fermentation culture.

3. The culture method according to claim 2, characterized in that The culture medium for the expanded culture contains: 15-20 g of stachyose, 1-3 g of sodium phosphate, 1-3 g of beef extract powder, 0.2-0.6 g of sodium acetate, 0.3-0.6 mL of Tween-80, and 0.3-0.5 g of potassium dihydrogen phosphate per 1000 mL of water. The pH value of the culture medium for the expanded culture is adjusted to 7.

0. The culture conditions for the expanded culture are: culture at 36-38° C. for 10-20 h. The culture medium for the primary seed culture contains: 2-5 g of D-galactose, 1-3 g of peptone, 0.5-1.5 g of beef extract powder, 0.5-1 g of sodium acetate, 1-3 g of leucine, and 0.5-1.5 mL of Tween-80 per 1000 mL of water. The pH value of the culture medium for the primary seed culture is natural. The culture conditions for the primary seed culture are: culture at 36-38° C. for 20-30 h. The inoculation amount of the primary seed culture is 0.3%-0.6%, calculated by volume ratio. The culture medium for the seed tank culture comprises, by weight percentage: 2-5% D-galactose, 1-1.5% casein peptone, 0.2-0.4% dipotassium hydrogen phosphate, 0.8-1.2% serine, 0.2-0.5% sodium acetate, 0.8-1.2% phenylalanine, 0.08-0.12% Tween-80, 0.08-0.12% polyether defoamer, and 90.5-93.5% tap water. The pH value of the culture medium for the seed tank culture is natural. The culture conditions for the seed tank culture are: a tank pressure of 0.04-0.06 MPa, and culture at 36-38° C. for 10-20 h. The inoculation amount of the seed tank culture is 3%-6%, by volume ratio. The culture medium for fermentation culture comprises, by weight percentage, 2-5% D-galactose, 1.2-2.5% casein peptone, 1-3% glucose, 0.3-0.6% stachyose, 0.8-1.2% bovine colostrum, 1.2-1.8% yeast extract, 1.5-1.8% sodium acetate, 0.1-0.3% Tween-80, 0.5-0.7% leucine, 0.8-1.2% dipotassium hydrogen phosphate, 0.05-0.12% polyether defoamer, and 80-88% tap water. The pH value of the culture medium for fermentation culture is adjusted to 6.

5. The culture conditions for fermentation culture are: tank pressure of 0.04-0.06 MPa, culture at 36-38° C. for 15-20 h; and the inoculation amount of the fermentation culture is 4%-6%, by volume ratio.

4. The culture method according to claim 3, characterized in that The culture medium for the expanded culture contains: 17 g stachyose, 2 g sodium phosphate, 2 g beef extract powder, 0.5 g sodium acetate, 0.5 mL Tween-80, and 0.4 g potassium dihydrogen phosphate per 1000 mL water. The pH value of the culture medium for the expanded culture is adjusted to 7.

0. The culture conditions for the expanded culture are: culture at 37° C. for 16 hours. The culture medium for the primary seed culture contains: 3 g of D-galactose, 2 g of peptone, 1 g of beef extract powder, 0.8 g of sodium acetate, 2 g of leucine, and 1 mL of Tween-80 per 1000 mL of water. The pH value of the culture medium for the primary seed culture is natural. The culture conditions for the primary seed culture are: culture at 37° C. for 24 h. The inoculation amount of the primary seed culture is 0.5%, calculated by volume ratio. The culture medium for the seed tank culture comprises, by weight percentage: 3% D-galactose, 1.2% casein peptone, 0.3% dipotassium hydrogen phosphate, 1% serine, 0.4% sodium acetate, 1% phenylalanine, 0.1% Tween-80, 0.1% polyether defoamer, and 92.9% tap water. The pH value of the culture medium for the seed tank culture is natural. The culture conditions for the seed tank culture are: a tank pressure of 0.05 MPa and culture at 37° C. for 18 h. The inoculation amount for the seed tank culture is 5%, by volume ratio. The culture medium for fermentation culture includes, by weight percentage, 4% D-galactose, 1.5% casein peptone, 2% glucose, 0.5% stachyose, 1% bovine colostrum, 1.5% yeast extract powder, 1.6% sodium acetate, 0.2% Tween-80, 0.6% leucine, 1% dipotassium hydrogen phosphate, 0.1% polyether defoamer, and 86% tap water. The pH value of the culture medium for fermentation culture is adjusted to 6.

5. The culture conditions for fermentation culture are: tank pressure of 0.05 MPa, culture at 37°C for 18 h; the inoculation amount of the fermentation culture is 5%, by volume ratio.

5. A microbial agent, characterized in that: The invention comprises an active ingredient, wherein the active ingredient comprises the Lactobacillus rhamnosus or a fermentation liquid thereof according to claim 1; The microbial agent comprises inactivated Lactobacillus rhamnosus and fermentation liquid thereof.

6. The Lactobacillus rhamnosus according to claim 1 ( Lactobacillus rhamnosus )+21 or use of the microbial agent described in claim 5 in preparing food.

7. The Lactobacillus rhamnosus according to claim 1 ( Lactobacillus rhamnosus )+21 or the use of the microbial agent described in claim 5 in the preparation of health foods that help regulate intestinal flora or aid digestion.

8. The Lactobacillus rhamnosus according to claim 1 ( Lactobacillus rhamnosus ) + 21 or use of the microbial agent according to claim 5 in the preparation of antiviral products; The virus types are novel coronavirus, herpes simplex virus and hepatitis B virus; The product described is a pharmaceutical product.

9. A method for preparing an antiviral freeze-dried bacterial powder, characterized in that: include: The fermented bacterial liquid obtained after fermentation and cultivation in the method of claim 2 is inactivated and centrifuged to obtain inactivated wet bacterial mud metabolites, and the inactivated wet bacterial mud metabolites are freeze-dried, and then mixed with auxiliary materials to obtain a suspension, and the suspension is freeze-dried to obtain; The inactivation refers to maintaining the fermentation liquid at 100-140°C for 5-8 seconds for UHT inactivation treatment; The centrifugal speed is 8000 r / min~12000 r / min, specifically centrifuged at a feed rate of 2~5 kg / min; The drying conditions of the freeze drying are: vacuum degree 8-12 Pa, temperature 25-28° C., and drying time 8-12 h; The moisture content of the freeze-dried bacterial powder is ≤4% by mass.

10. The preparation method according to claim 9, characterized in that: The auxiliary materials include soybean peptide powder, oligofructose, magnesium sulfate and sodium gluconate; The mass addition ratio of the inactivated wet bacterial mud metabolites to the auxiliary materials is 1:2.5-3.5.

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