Phytobacterium plantarum XD087 and application of astragalus mongholicus fermentation liquor prepared by fermentation of phytobacterium plantarum XD087 in antiviral products

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a plant lactobacillus XD087 and application of astragalus membranaceus fermentation liquor obtained by fermenting astragalus membranaceus in virus resistance. The bacterial strain is lactobacillus plantarum XD087 and is preserved in the China Center for Type Culture Collection, and the preservation number of the bacterial strain is CCTCC NO: M 20241729. The preparation method of the astragalus mongholicus fermentation liquor comprises the following steps: inoculating the lactobacillus plantarum XD087 into astragalus mongholicus enzymatic hydrolysate, and fermenting in a fermentation temperature range suitable for the lactobacillus plantarum XD087 to obtain the astragalus mongholicus fermentation liquor, and the astragalus mongholicus fermentation liquor obtained by fermenting astragalus mongholicus by using the strain can achieve the best antiviral effect.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to application of Lactobacillus plantarum XD087 and astragalus fermentation liquid prepared by fermentation in antiviral products. Background Art

[0002] Currently, the treatments for COVID-19 mainly include vaccination, antiviral drugs and symptomatic treatment. However, due to the continuous mutation of the virus, the effectiveness of existing treatments may be affected. Therefore, it is urgent to explore safer and more effective prevention and auxiliary treatment methods.

[0003] Astragalus, derived from the dried root of Astragalus mongolica or Astragalus capsulatus, has the effects of invigorating qi and strengthening the exterior, strengthening the body and eliminating evil, and has long been used to enhance immunity and improve chronic diseases. Modern research shows that Astragalus contains a variety of bioactive ingredients, including astragaloside, polysaccharide and flavonoid compounds, which have significant immunomodulatory, anti-inflammatory and antiviral effects. Among them, astragaloside IV and flavonoid components are the main components of Astragalus to exert pharmacological effects, and they show antiviral activity against influenza virus, hepatitis virus (HBV, HCV), dengue virus, etc., and are natural antiviral compounds with development potential.

[0004] In recent years, the application of microbial fermentation technology in the research of traditional Chinese medicine has made significant progress. Microbial fermentation can promote the transformation and release of its active ingredients and enhance its efficacy. As a probiotic, Lactobacillus plantarum has unique advantages in metabolic activity, secretion of biological enzymes and immune regulation. Based on this, after fermentation by Lactobacillus plantarum, the content and biological activity of flavonoids in Astragalus membranaceus are significantly improved. At the same time, the fermented product can further enhance the antiviral effect, so that the fermented Astragalus membranaceus liquid has a stronger potential application value in the fight against the new coronavirus.

[0005] Therefore, developing Astragalus fermentation broth based on Lactobacillus plantarum fermentation and studying its application in anti-COVID-19 products not only provides new ideas for the modern utilization of traditional Chinese medicine, but also provides a potential new auxiliary means for the prevention and treatment of COVID-19, which has important social value and market prospects. Summary of the invention

[0006] To this end, the present invention provides a plant lactobacillus XD087, which ferments astragalus with the plant lactobacillus, and can significantly improve the anti-new coronavirus effect of the astragalus fermentation liquid;

[0007] On the other hand, the present invention provides an Astragalus fermentation broth, which has a good anti-new coronavirus effect.

[0008] To solve the above problems, the technical solution of the present invention is:

[0009] A Lactiplantibacillus plantarum (Lactiplantibacillus plantarum) XD087, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241729.

[0010] The application of the above-mentioned Lactobacillus plantarum XD087 in fermenting astragalus or preparing astragalus fermentation liquid.

[0011] The above-mentioned Lactobacillus plantarum XD087 is used in fermenting Astragalus to improve the ability of Astragalus fermentation liquid to resist the new coronavirus.

[0012] The invention discloses an astragalus fermentation liquid, which is prepared by enzymolysis of astragalus and inoculation with the plant lactobacillus XD087 and fermentation.

[0013] Preferably, the enzyme preparation selected for enzymatic hydrolysis of Astragalus membranaceus includes cellulase and pectinase.

[0014] Preferably, the added amount of the cellulase is 0.05-0.3%, and the added amount of the pectinase is 0.2-0.5%.

[0015] More preferably, the added amount of the cellulase is 0.1%, and the added amount of the pectinase is 0.25%.

[0016] More preferably, the enzymatic activity of the cellulase is 10000 U / g, and the enzymatic activity of the pectinase is 5000 U / g.

[0017] Preferably, the viable count of Lactobacillus plantarum XD087 is 1.0×10 8 CFU / mL and above.

[0018] Preferably, the activation step of Lactobacillus plantarum XD087 is as follows: inoculating the strain into MRS medium, culturing at 35°C for 24 hours, and continuously subculturing for 3 times to fully activate the strain until the viable count is 1.0×10 8 CFU / mL or more, wherein the composition and preparation method of the MRS medium are as follows: the medium formula is peptone 10.0 g / L, beef extract powder 5.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium hydrogen citrate 2g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween-80 1g / L. Take the above MRS medium and add 1L of pure water, sterilize it with high pressure steam at 121℃ for 15min for standby use.

[0019] Preferably, the inoculation amount of Lactobacillus plantarum XD087 is 1-5%. Preferably, the inoculation amount of Lactobacillus plantarum XD087 is 2%.

[0020] Preferably, the weight ratio of the astragalus powder to water is 1:(5-10), more preferably 1:7.

[0021] A method for preparing the above-mentioned astragalus fermentation liquid comprises the following steps: (1) adding an enzyme preparation to a mixture of astragalus and water for enzymolysis, sterilizing after the enzymolysis is completed, and obtaining an astragalus hydrolyzate; (2) inoculating Lactobacillus plantarum XD087 into the astragalus hydrolyzate for fermentation, separating the solid from the liquid, and obtaining the supernatant as the astragalus fermentation liquid.

[0022] Preferably, the enzymatic hydrolysis temperature is 40-60°C and the time is 40-80 min.

[0023] Further preferably, the enzymatic hydrolysis is carried out in a shaker at a rotation speed of 150-200 rpm.

[0024] More preferably, the enzymatic hydrolysis further includes a homogenization step, and the homogenization time is 3-10 minutes.

[0025] Preferably, the sterilization temperature is 100-120°C and the time is 10-30 minutes.

[0026] Preferably, the fermentation temperature is 30-40°C and the fermentation time is 2-4 days.

[0027] Further preferably, the fermentation temperature is 37° C. and the fermentation time is 72 h.

[0028] The astragalus fermented liquid and the astragalus fermented liquid obtained by the preparation method are used in antiviral products.

[0029] The technical solution of the present invention has the following advantages:

[0030] 1. The present invention provides a plant lactobacillus XD087, which can improve the anti-new coronavirus effect of astragalus fermentation liquid by fermenting astragalus.

[0031] 2. The present invention provides an Astragalus fermented liquid, which is prepared by fermenting Lactobacillus plantarum XD087 and has a good anti-new coronavirus effect.

[0032] 3. The present invention provides a method for preparing an Astragalus fermentation broth. Through the process of enzymatic hydrolysis followed by fermentation, and after fermentation with Lactobacillus plantarum XD087, the fermentation broth can achieve better anti-new coronavirus efficacy, and the process is simple and suitable for industrial production.

[0033] Instructions for strain preservation

[0034] Lactobacillus plantarum XD117, Lactobacillus plantarum XD117 was deposited in the China Center for Type Culture Collection on August 2, 2024, with the address at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M20241730. On August 25, 2022, the updated “List of Bacteria That Can Be Used in Food” changed the name of Lactobacillus plantarum to Lactiplantibacillus plantarum. Lactobacillus plantarum XD117 and Lactiplantibacillus plantarum XD117 are the same strain.

[0035] Lactobacillus plantarum XD087, Lactobacillus plantarum XD087 was deposited in the China Center for Type Culture Collection on August 2, 2024, with the address at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 20241729. On August 25, 2022, the updated “List of Bacteria That Can Be Used in Food” changed the name of Lactobacillus plantarum to Lactiplantibacillus plantarum. Lactobacillus plantarum XD087 and Lactiplantibacillus plantarum XD087 are the same strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a graph showing the antiviral ability of Astragalus enzymatic hydrolysis and fermentation broth. DETAILED DESCRIPTION

[0037] To make the purpose, technical solutions and advantages of the present invention clearer, the embodiments of the present invention are described in further detail below. Based on the embodiments disclosed in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present disclosure.

[0038] If no specific experimental steps or conditions are specified in the examples, the experiments can be carried out according to the conventional steps or conditions described in the literature in the field. All reagents without manufacturer specified are conventional reagent products that can be obtained through commercial purchase.

[0039] In the following examples, the source information of some products is as follows:

[0040] Cellulase was purchased from Xiasheng Food Grade Cellulase Plant Extract Special Enzyme SPE-017L with an enzyme activity of 10000U / g; pectinase was Xiasheng Food Grade Pectinase SPE-010 with an enzyme activity of 5000U / g.

[0041] Activation and cultivation of bacterial strains in preparation examples

[0042] Activation of lactic acid bacteria: Lactobacillus plantarum XD117 and Lactobacillus plantarum XD were inoculated into MRS medium and cultured at 35°C for 24 h. The culture was subcultured three times to fully activate the strains until the viable count was 1.0×10 8 CFU / mL and above.

[0043] The composition and preparation method of MRS medium are as follows: peptone 10.0 g / L, beef extract powder 5.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium hydrogen citrate 2 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween-80 1 g / L. Add 1 L of pure water to the above MRS medium, sterilize with high pressure steam at 121°C for 15 min, and set aside.

[0044] Example Preparation of Astragalus Fermentation Broth

[0045] The present embodiment provides a method for preparing an astragalus fermentation liquid, comprising the following steps: (1) grinding the astragalus into powder, passing through a 200-mesh sieve, and obtaining an astragalus powder; (2) mixing the astragalus powder with water in a ratio of 1:7, adding 0.1% cellulase and 0.25% pectinase, homogenizing for 5 minutes, performing enzymolysis at 160 rpm in a 50°C shaker for 1 hour, and sterilizing at 115°C for 30 minutes after enzymolysis to obtain an astragalus hydrolyzate; (3) inoculating 2% (2% volume ratio, such as 100 mL of astragalus hydrolyzate has a volume of 100 mL, adding 2 mL of bacterial liquid) of activated plant lactobacillus seed liquid into the astragalus hydrolyzate, fermenting at 37°C for 3 days, centrifuging at 8000 rpm for 10 minutes after the fermentation, and collecting the supernatant to obtain the astragalus fermentation liquid. The plant lactobacillus are respectively plant lactobacillus XD081 and plant lactobacillus XD117, and the number of viable bacteria in the two bacterial liquids is kept the same during inoculation.

[0046] Comparative Example Astragalus enzymatic hydrolysis supernatant

[0047] The present comparative example provides an astragalus enzymatic hydrolysis supernatant, and the preparation method thereof is as follows: after mixing astragalus powder and water in a ratio of 1:7, 0.1% cellulase and 0.25% pectinase are added, homogenized for 5 minutes, and enzymolysis is performed at 160 rpm in a 50°C shaker for 1 hour. After enzymolysis, the mixture is sterilized at 115°C for 30 minutes to obtain an astragalus enzymatic hydrolysis solution, and the mixture is placed at 37°C for 3 days and then centrifuged at 8000 rpm for 10 minutes to collect the supernatant. The supernatant is filtered through a 0.45 μm filter membrane to obtain an astragalus enzymatic hydrolysis supernatant.

[0048] Experimental Example 1 Antiviral Ability Detection

[0049] Antiviral ability testing method:

[0050] (1) Resuscitate cells; co-transfect SARS-CoV-2 S protein expression plasmid, pHIV-luciferase and pSPAX2 plasmid into HEK293T-hACE2 cells. Collect cell culture supernatant 48 hours after transfection, filter with a 0.45μm filter, and centrifuge at 4°C, 3000g for 10 minutes. Transfer the supernatant to a new tube and aliquot it, and store it in a -80°C refrigerator for later use. Dilute it to an equal volume with pH 7.4 PBS buffer before use.

[0051] (2) Test samples: Astragalus fermentation broth obtained in Example and Astragalus enzymatic hydrolysis supernatant obtained in Comparative Example

[0052] (3) Testing process

[0053] Day 1: HEK293t-hACE2 cells were plated in 96-well plates.

[0054] The next day: ① Incubate the fermentation liquid and supernatant with the virus: Take 100 μL of the diluted virus liquid + 2 μL of the test sample as a mixture and incubate it in a 37°C cell culture incubator for 20 minutes. ② Infect cells: Discard the culture supernatant of the pre-plated 96-well 293t-hACE2 cells, transfer 100 μL of the mixture in ① to 293t-hACE2 cells for infection, and continue to culture in a 37°C cell culture incubator for about 24 hours.

[0055] Day 3: ① Take out the 96-well plate, discard the supernatant, and gently rinse with 100 μL PBS; ② Prepare lysis buffer (5x passive lysis buffer) in advance, add 50 μL lysis buffer to each well, and shake and lyse at room temperature for 30 min; ③ Transfer 20 μL of lysis buffer to an opaque white 96-well plate; ④ Prepare luciferase substrate solution in advance, add 100 μL substrate solution to each well, and use a multifunctional microplate reader to detect luciferase activity in real time. Specific results are shown in Table 1 and Figure 1 .

[0056] Table 1 Antiviral ability

[0057]

[0058] Note: Different letters in the same column represent significant differences, p < 0.05.

[0059] Result analysis:

[0060] The lower the luminescence, the better the antiviral effect. As shown in Table 1, the fermentation liquid of Lactobacillus plantarum XD087 has better antiviral ability than the fermentation liquid of Lactobacillus plantarum XD117 and the supernatant of Astragalus enzymatic hydrolysis.

[0061] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A Lactobacillus plantarum XD087, characterized in that The deposit number of the Lactobacillus plantarum XD087 is CCTCC NO: M 20241729.

2. Application of Lactobacillus plantarum XD087 in fermenting Astragalus or preparing Astragalus fermentation liquid, characterized in that: The deposit number of the Lactobacillus plantarum XD087 is CCTCC NO: M 20241729.

3. The use according to claim 2, characterized in that: The plant lactobacillus XD087 is used in fermenting astragalus to improve the ability of astragalus fermentation liquid to resist the new coronavirus.

4. An Astragalus fermentation broth, characterized in that: The astragalus is prepared by enzymatic hydrolysis and inoculation with Lactobacillus plantarum XD087 with a preservation number of CCTCC NO: M 20241729.

5. The astragalus fermentation liquid according to claim 4, characterized in that The enzyme preparations selected for enzymolysis of Astragalus include cellulase and pectinase.

6. The astragalus fermentation broth according to claim 5, characterized in that The added amount of the cellulase is 0.05-0.3%, and the added amount of the pectinase is 0.2-0.5%.

7. The astragalus fermentation liquid according to claim 4, characterized in that The inoculation amount of Lactobacillus plantarum XD087 is 1-5%, and the number of viable bacteria is 1.0×10 8 CFU / mL and above.

8. The method for preparing the astragalus fermentation broth according to any one of claims 4 to 7, characterized in that: The method comprises the following steps: (1) mixing astragalus powder and water evenly, adding an enzyme preparation for enzymolysis, sterilizing after the enzymolysis is completed, and obtaining an astragalus enzymolysis liquid; and (2) inoculating a fermentation agent into the astragalus enzymolysis liquid for fermentation, separating the solid from the liquid, and obtaining the supernatant, which is the astragalus fermentation liquid.

9. The method according to claim 8, characterized in that The temperature of the enzymolysis is 40-60°C, and the time is 40-80 minutes; the temperature of the sterilization is 100-120°C, and the time is 10-30 minutes; the temperature of the fermentation is 30-40°C, and the fermentation time is 2-4 days.

10. Use of the astragalus fermented liquid according to any one of claims 4 to 7 and the astragalus fermented liquid obtained by the preparation method according to any one of claims 8 to 9 in the preparation of antiviral products.

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