Application of *Lactobacillus plantarum* XD087 and its fermented Astragalus membranaceus broth in antiviral products

Abstract

This invention belongs to the field of microbial fermentation technology, specifically relating to the application of *Lactiplantibacillus plantarum* XD087 and the astragalus fermentation broth obtained from fermenting astragalus in antiviral applications. The strain is *Lactiplantibacillus plantarum* XD087, deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20241729. The preparation method of the astragalus fermentation broth in this invention involves inoculating *Lactiplantibacillus plantarum* XD087 into astragalus enzymatic hydrolysate and fermenting within the suitable fermentation temperature range of *Lactiplantibacillus plantarum* XD087 to obtain the astragalus fermentation broth. The astragalus fermentation broth obtained by fermenting astragalus with this strain achieves the best antiviral efficacy.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to the application of *Lactobacillus plantarum* XD087 and its fermented Astragalus membranaceus broth in antiviral products. Background Technology

[0002] Currently, treatments for COVID-19 primarily 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, exploring safer and more effective prevention and supportive treatments has become an urgent priority.

[0003] Astragalus, derived from the dried root of *Astragalus mongholicus* or *Astragalus capsulatum*, is a legume with the effects of invigorating qi, strengthening the exterior, and dispelling pathogens. It has long been used to enhance immunity and improve chronic diseases. Modern research shows that astragalus contains various bioactive components, including astragaloside, polysaccharides, and flavonoids, which have significant immunomodulatory, anti-inflammatory, and antiviral effects. Astragaloside A and flavonoids are the main components responsible for the pharmacological effects of astragalus, exhibiting antiviral activity against influenza viruses, hepatitis viruses (HBV, HCV), dengue fever viruses, etc., and are natural antiviral compounds with development potential.

[0004] In recent years, significant progress has been made in the application of microbial fermentation technology in the research of traditional Chinese medicine. Microbial fermentation can promote the transformation and release of active ingredients, thereby enhancing their efficacy. Lactobacillus plantarum, as a probiotic, possesses unique advantages in metabolic activity, enzyme secretion, and immune regulation. Based on this, the content and bioactivity of flavonoids in Astragalus membranaceus are significantly increased after fermentation with Lactobacillus plantarum. Simultaneously, the fermented product can further enhance antiviral effects, thus giving fermented Astragalus membranaceus extract a stronger potential application value in combating the novel 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 modernization of traditional Chinese medicine, but also provides a potential new auxiliary means for the prevention and control 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 membranaceus and can significantly improve the anti-COVID-19 effect of Astragalus membranaceus fermentation broth;

[0007] On the other hand, the present invention provides an Astragalus fermentation liquid, which has a good anti-COVID-19 effect.

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

[0009] A species of Lactiplantibacillus plantarum, XD087, is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20241729.

[0010] The application of the above-mentioned Lactobacillus plantarum XD087 in the fermentation of Astragalus membranaceus or the preparation of Astragalus membranaceus fermentation broth.

[0011] The aforementioned *Lactobacillus plantarum* XD087 was used in the fermentation of *Astragalus membranaceus* to enhance the anti-COVID-19 ability of the fermentation broth.

[0012] A fermentation broth of Astragalus membranaceus is prepared by enzymatic hydrolysis of Astragalus membranaceus and fermentation with Lactobacillus plantarum XD087.

[0013] Preferably, the enzyme preparations used for the enzymatic hydrolysis of Astragalus include cellulase and pectinase.

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

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

[0016] More preferably, the cellulase has an activity of 10,000 U / g and the pectinase has an activity of 5,000 U / g.

[0017] Preferably, the viable count of the *Lactobacillus plantarum* XD087 is 1.0 × 10⁻⁶. 8 CFU / mL or higher.

[0018] Preferably, the activation steps for *Lactobacillus plantarum* XD087 are as follows: the strain is inoculated into MRS medium and cultured at 35°C for 24 hours, followed by three consecutive subcultures to fully activate the strain to a viable count of 1.0 × 10⁻⁶. 8 The concentration of CFU / mL or higher was specified. The composition and preparation method of the MRS medium were as follows: the medium formula was 10.0 g / L peptone, 5.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2 g / L triammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1 g / L Tween-80. The above MRS medium was added to 1 L of pure water and autoclaved at 121℃ for 15 min.

[0019] More preferably, the inoculum size of *Lactobacillus plantarum* XD087 is 1-5%. Even more preferably, the inoculum size 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 broth includes the following steps: (1) After Astragalus and water are mixed evenly, an enzyme preparation is added for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the mixture is sterilized to obtain Astragalus hydrolysate; (2) Lactobacillus plantarum XD087 is inoculated into the Astragalus hydrolysate for fermentation. Solid-liquid separation is performed, and the supernatant is Astragalus fermentation broth.

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

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

[0024] More preferably, the enzymatic hydrolysis is preceded by a homogenization step, with a homogenization time of 3-10 minutes.

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

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

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

[0028] The application of the above-mentioned Astragalus fermentation broth and the Astragalus fermentation broth obtained by the above preparation method in antiviral products.

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

[0030] 1. This invention provides a strain of *Lactobacillus plantarum* XD087, which can enhance the anti-COVID-19 effect of *Astragalus membranaceus* fermentation broth by fermenting it.

[0031] 2. This invention provides an Astragalus fermentation broth, which is prepared by fermentation of Lactobacillus plantarum XD087 and has a good anti-COVID-19 effect.

[0032] 3. This invention provides a method for preparing Astragalus fermentation broth. Through a process of enzymatic hydrolysis followed by fermentation, and after fermentation with Lactobacillus plantarum XD087, the fermentation broth can achieve better anti-COVID-19 efficacy. Moreover, the process is simple and suitable for industrial production.

[0033] Instructions for Preservation of Microbial Strains

[0034] Lactobacillus plantarum XD117 was deposited on August 2, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M20241730. On August 25, 2022, the updated "List of Microbial Strains that Can Be Used in Food" renamed Lactobacillus plantarum to Lactiplantibacillus plantarum. Lactobacillus plantarum XD117 and Lactiplantibacillus plantarum XD117 are the same strain.

[0035] Lactobacillus plantarum XD087 was deposited on August 2, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20241729. On August 25, 2022, the updated "List of Microbial Strains that Can Be Used in Food" renamed Lactobacillus plantarum to Lactiplantibacillus plantarum. Lactobacillus plantarum XD087 and Lactiplantibacillus plantarum XD087 are the same strain. Attached Figure Description

[0036] Figure 1 The graph shows the antiviral activity of Astragalus membranaceus enzymatic hydrolysis and fermentation broth. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the embodiments of this invention will be described in further detail below. All other embodiments obtained by those skilled in the art based on the embodiments disclosed in this invention without inventive effort are within the scope of protection of this disclosure.

[0038] For any experimental steps or conditions not specified in the examples, the procedures or conditions described in the literature in this field can be followed. All reagents without specified manufacturers are commercially available standard reagent products.

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

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

[0041] Activation and culture of prepared bacterial strains

[0042] Activation of lactic acid bacteria: *Lactobacillus plantarum* XD117 and *Lactobacillus plantarum* XD strains were inoculated into MRS medium and cultured at 35℃ for 24 hours. This was repeated three times to fully activate the strains until the viable count reached 1.0 × 10⁻⁶. 8 CFU / mL or higher.

[0043] The composition and preparation method of MRS medium are as follows: peptone 10.0 g / L, beef extract 5.0 g / L, yeast extract 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, and Tween-80 1 g / L. Add 1 L of pure water to the above MRS medium, autoclave at 121℃ for 15 min, and set aside.

[0044] Example: Preparation of Astragalus Fermentation Broth

[0045] This embodiment provides a method for preparing Astragalus fermentation broth, including the following steps: (1) Grind Astragalus into powder, pass through a 200-mesh sieve to obtain Astragalus powder; (2) Mix Astragalus powder and water in a 1:7 ratio, add 0.1% cellulase and 0.25% pectinase, homogenize for 5 minutes, and enzymatically hydrolyze at 160 rpm for 1 hour in a shaker at 50℃. After enzymatic hydrolysis, sterilize at 115℃ for 30 minutes to obtain Astragalus enzymatic hydrolysate; (3) Inoculate the Astragalus enzymatic hydrolysate with 2% (2% volume ratio, such as 100mL Astragalus enzymatic hydrolysate volume is 100mL, add 2mL bacterial solution) activated Bacillus plantarum seed liquid and ferment at 37℃ for 3 days. After fermentation, centrifuge at 8000rpm for 10 minutes and collect the supernatant to obtain Astragalus fermentation broth. Among them, Bacillus plantarum are Bacillus plantarum XD081 and Bacillus plantarum XD117, and the viable count of the two bacterial solutions is kept the same during inoculation.

[0046] Comparative Astragalus enzymatic hydrolysis supernatant

[0047] This comparative example provides an enzymatic hydrolysis supernatant of Astragalus membranaceus, which is prepared by mixing Astragalus membranaceus powder and water in a 1:7 ratio, adding 0.1% cellulase and 0.25% pectinase, homogenizing for 5 minutes, and then enzymatically hydrolyzing at 160 rpm for 1 hour in a shaker at 50°C. After enzymatic hydrolysis, sterilize at 115°C for 30 minutes to obtain the Astragalus membranaceus hydrolysate. After standing at 37°C for 3 days, centrifuge at 8000 rpm for 10 minutes, collect the supernatant, and filter the supernatant through a 0.45 μm filter membrane to obtain the Astragalus membranaceus hydrolysis supernatant.

[0048] Experiment Example 1: Antiviral Capacity Detection

[0049] Antiviral ability testing methods:

[0050] (1) Cell resuscitation: The SARS-CoV-2 S protein expression plasmid, pHIV-luciferase, and pSPAX2 plasmid were co-transfected into HEK293T-hACE2 cells. 48 h after transfection, the cell culture supernatant was collected, filtered through a 0.45 μm filter, and centrifuged at 3000 g for 10 min at 4 °C. The supernatant was transferred to new tubes, aliquoted, and stored at -80 °C for later use. Before use, the supernatant was diluted with an equal volume of PBS buffer to pH 7.4.

[0051] (2) Test samples: Astragalus fermentation broth obtained in the example and Astragalus enzymatic hydrolysis supernatant obtained in the comparative example.

[0052] (3) Testing process

[0053] Day 1: 96-well plates were prepared using HEK293t-hACE2 cells.

[0054] Day 2: ① Co-incubation of fermentation broth and supernatant with virus: Take 100 μL of diluted virus solution + 2 μL of test sample as a mixture and incubate at 37℃ for 20 min. ② Cell infection: Discard the culture supernatant of pre-coated 96-well 293t-hACE2 cells, transfer 100 μL of the mixture from ① to 293t-hACE2 cells for infection, and continue to incubate at 37℃ for about 24 h.

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

[0056] Table 1 Antiviral Capacity

[0057]

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

[0059] Results analysis:

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

[0061] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

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

2. The application of *Lactobacillus plantarum* XD087 in the fermentation of *Astragalus membranaceus* or the preparation of *Astragalus membranaceus* fermentation broth, characterized in that... The preservation number of the *Lactobacillus plantarum* XD087 is CCTCC NO: M 20241729.

3. An Astragalus fermentation liquid, characterized in that, Astragalus membranaceus was prepared by enzymatic hydrolysis and fermentation with Lactobacillus plantarum XD087, preservation number CCTCCNO: M 20241729.

4. The Astragalus fermentation broth according to claim 3, characterized in that, The enzyme preparations used for the enzymatic hydrolysis of Astragalus include cellulase and pectinase.

5. The Astragalus fermentation broth according to claim 4, characterized in that, The amount of cellulase added is 0.05-0.3%, and the amount of pectinase added is 0.2-0.5%.

6. The Astragalus fermentation broth according to claim 3, characterized in that, The inoculum size of *Lactobacillus plantarum* XD087 was 1-5%, and the viable count was 1.0 × 10⁻⁶. 8 CFU / mL or higher.

7. The method for preparing Astragalus fermentation broth according to any one of claims 3-6, characterized in that, The steps include: (1) Mixing Astragalus powder and water evenly, adding an enzyme preparation for enzymatic hydrolysis, sterilizing after enzymatic hydrolysis, and obtaining Astragalus hydrolysate; (2) Inoculating the Astragalus hydrolysate with a fermenting agent for fermentation, separating the solid and liquid, and obtaining the supernatant as Astragalus fermentation liquid.

8. The method according to claim 7, characterized in that, The enzymatic hydrolysis is performed at a temperature of 40-60℃ for 40-80 minutes; the sterilization is performed at a temperature of 100-120℃ for 10-30 minutes; and the fermentation is performed at a temperature of 30-40℃ for 2-4 days.

9. The application of Astragalus fermentation broth according to any one of claims 3-6 in the preparation of anti-COVID-19 products.

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