Probiotics capable of reducing yew toxicity and application thereof

Abstract

The invention relates to a probiotic capable of reducing the toxicity of Taxus chinensis and an application thereof. The probiotic comprises one or more bacteria with a preservation number of CCTCC NO: M 20231513, CCTCC NO: M 20231514 and CCTCC NO: M 20231515. The probiotic is added into a liquid culture medium containing Taxus chinensis for fermentation to obtain a fermentation liquid. Animal experiments have confirmed that the toxicity of Taxus chinensis is significantly reduced, wherein the strain with a preservation number of CCTCC NO: M 20231514 can reduce the toxicity of Taxus chinensis to the liver, the strain with a preservation number of CCTCC NO: M 20231513 can reduce the toxicity of Taxus chinensis to the kidney, and the strain with a preservation number of CCTCC NO: M 20231515 can reduce the toxicity of Taxus chinensis to the liver and kidney, and the probiotic can be used to reduce the toxic and side effects of Taxus chinensis.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and more specifically to a probiotic capable of alleviating yew toxicity and application thereof. Background Art

[0002] The medicinal part of yew in medicine is the branches and leaves of the yew plant. The prescription name is yew and red cedar pine. It has the effects of dredging menstruation and relieving pain, diuresis, lowering blood sugar and treating kidney disease. According to clinical pharmacology, yew also has anti-cancer effects. Yew contains paclitaxel, which has the effect of inhibiting cancer cells, especially for advanced breast cancer, advanced ovarian cancer, esophageal cancer, bladder cancer, nasopharyngeal cancer, lymphoma, and gastrointestinal cancer. It has a significant auxiliary therapeutic effect; in addition, yew can also improve human immunity. However, yew has a certain toxicity. If it is taken improperly, symptoms of poisoning will occur, such as nausea, vomiting, limb weakness, and difficulty breathing. Because of its slightly toxic characteristics, the direct application of yew is limited to a certain extent. Therefore, studying how to reduce the toxicity of yew is conducive to promoting its application. Summary of the invention

[0003] In view of the above defects or improvement needs of the prior art, the present invention provides a probiotic capable of reducing the toxicity of Taxus chinensis and an application thereof. The purpose is to discover three new strains, all of which have been deposited in the China Center for Type Culture Collection, and are respectively recorded as Saccharomyces cerevisiae CE13-M5 (with a deposit number of CCTCC NO: M 20231513), Lactobacillus paracasei CE12-M3 (with a deposit number of CCTCC NO: M 20231514), and Lactobacillus plantarum CE13-L4 (with a deposit number of CCTCC NO: M20231515). It is also found that these three strains can significantly reduce the toxicity of Taxus chinensis, wherein Lactobacillus paracasei CE12-M3 can reduce the toxicity of Taxus chinensis to the liver, Saccharomyces cerevisiae CE13-M5 can reduce the toxicity of Taxus chinensis to the kidneys, and Lactobacillus plantarum CE13-L4 can reduce the toxicity of Taxus chinensis to the liver and kidneys, thereby solving the technical problem that the existing direct administration of Taxus chinensis has toxic side effects.

[0004] To achieve the above object, according to one aspect of the present invention, a probiotic having the ability to reduce the toxicity of Taxus chinensis is provided, comprising one or more of Saccharomyces cerevisiae CE13-M5, Lactobacillus paracasei CE12-M3, and Lactobacillus plantarum CE13-L4;

[0005] The brewer's yeast CE13-M5 strain is named Saccharomyces cerevisiae CESWGF002, and its preservation number is CCTCC NO: M 20231513; the Lactobacillus paracasei CE12-M3 strain is named Lactobacillus paracasei CESWGF003, and its preservation number is CCTCC NO: M 20231514; the Lactobacillus plantarum CE13-L4 strain is named Lactiplantibacillus plantarum CESWGF004, and its preservation number is CCTCC NO: M20231515; all of them were deposited in the China Center for Type Culture Collection on August 21, 2023.

[0006] Preferably, the probiotics capable of reducing the toxicity of Taxus include one or more strains with the deposit numbers of CCTCC NO: M 20231515 and CCTCC NO: M 20231514.

[0007] Preferably, the probiotics capable of reducing the toxicity of Taxus include one or more strains having the deposit numbers of CCTCC NO: M 20231515 and CCTCC NO: M 20231513.

[0008] Preferably, the probiotics capable of reducing the toxicity of Taxus include the strain with the deposit number of CCTCCNO:M 20231515.

[0009] According to another aspect of the present invention, there is also provided a use of the probiotics according to the present invention in the preparation of a medicine containing Taxus chinensis.

[0010] Preferably, the application comprises adding the probiotics of the present invention to a liquid culture medium containing Taxus chinensis according to a preset ratio for fermentation to obtain a fermentation liquid, and using the fermentation liquid as a raw material for preparing a drug.

[0011] Preferably, in the application, the preset proportion is to add the probiotic suspension at 10% to 15% of the volume of the liquid culture medium.

[0012] Preferably, the application and the medicine act on the liver, and the added probiotics include one or more strains of CCTCC NO: M20231515 and CCTCC NO: M 20231514.

[0013] Preferably, the application and the medicine act on the kidney, and the added probiotics include one or more strains of CCTCC NO: M20231515 and CCTCC NO: M 20231513.

[0014] Preferably, the application, the medicine, and the added probiotics include a strain with a preservation number of CCTCCNO:M20231515.

[0015] In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects compared with the prior art:

[0016] The invention discovers three new strains, wherein the strain with the preservation number of CCTCC NO: M 20231514 can reduce the toxicity of yew to the liver, the strain with the preservation number of CCTCC NO: M 20231513 can reduce the toxicity of yew to the kidney, and the strain with the preservation number of CCTCC NO: M20231515 can reduce the toxicity of yew to the liver and kidney, can be used for fermentation to reduce the toxicity of yew, and is applied to the preparation of yew-containing medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the ALT and AST activity test results;

[0018] Figure 2 It is the test result of uric acid and creatinine. DETAILED DESCRIPTION

[0019] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0020] The present invention isolates three new strains, which are respectively recorded as brewer's yeast CE13-M5, Lactobacillus paracasei CE12-M3, and Lactobacillus plantarum CE13-L4, all of which were deposited in China Center for Type Culture Collection on August 21, 2023, and their classification names are Saccharomyces cerevisiae CESWGF002, Lactobacillus paracasei CESWGF003, and Lactiplantibacillus plantarum CESWGF004, respectively; their preservation numbers are CCTCC NO: M 20231513, CCTCC NO: M 20231514, and CCTCC NO: M 20231515, respectively.

[0021] Animal experiments have shown that the above three strains can reduce the toxicity of Taxus. Among them, the brewer's yeast CE13-M5 strain can reduce the toxic effect of Taxus on the kidneys, the Lactobacillus paracasei CE12-M3 strain can reduce the toxic effect of Taxus on the liver, and the Lactobacillus plantarum CE13-L4 strain can reduce the toxic effect of Taxus on the liver and kidneys.

[0022] Based on this discovery, the present invention provides a probiotic with the ability to reduce the toxicity of Taxus chinensis, which comprises one or more of Saccharomyces cerevisiae CE13-M5, Lactobacillus paracasei CE12-M3, and Lactobacillus plantarum CE13-L4;

[0023] The brewer's yeast CE13-M5 strain is named Saccharomyces cerevisiae CESWGF002 and has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: M 20231513;

[0024] The Lactobacillus paracasei CE12-M3 strain is named Lactobacillus paracasei CESWGF003, and has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: M 20231514;

[0025] The Lactobacillus plantarum CE13-L4 strain is named Lactiplantibacillus plantarum CESWGF004 and has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: M 20231515.

[0026] Preferably, the probiotics capable of reducing the toxicity of Taxus chinensis include Lactobacillus plantarum CE13-L4, i.e., Lactiplantibacillus plantarum CESWGF004, with a preservation number of CCTCC NO:M 20231515.

[0027] In some embodiments, the probiotics having the ability to reduce the toxicity of Taxus chinensis are Lactobacillus plantarum CE13-L4 and Lactobacillus paracasei CE12-M3, that is, the preservation numbers are CCTCC NO: M20231515 and CCTCC NO: M20231514.

[0028] In some embodiments, the probiotics having the ability to reduce the toxicity of Taxus chinensis are Lactobacillus plantarum CE13-L4 and Saccharomyces cerevisiae CE13-M5, namely, the preservation numbers of which are CCTCC NO: M20231515 and CCTCC NO: M 20231513.

[0029] In addition, the present invention also provides a use of the probiotics of the present invention in the preparation of a drug containing Taxus chinensis;

[0030] Preferably, the drug comprises Lactiplantibacillus plantarum CESWGF004.

[0031] In some embodiments, the drug acts on the liver and includes Lactiplantibacillus plantarum CESWGF004 and Lactobacillus paracasei CESWGF003, i.e., the deposit number is CCTCC NO: M 20231515 and the deposit number is CCTCC NO: M 20231514.

[0032] In some embodiments, the drug acts on the kidney and includes Lactiplantibacillus plantarum CESWGF004 and Saccharomyces cerevisiae CESWGF002, i.e., the deposit number is CCTCC NO: M 20231515 and the deposit number is CCTCC NO: M 20231513.

[0033] The following are examples

[0034] Example 1 Isolation and purification of strains

[0035] (1) Brewer's yeast

[0036] Sample: Aged Chinese herbal extract from the Savage Valley

[0037] MRS medium: 10g protein Chen, 5g beef powder, 4g yeast powder, 2g glucose, 1ml Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 15g agar powder, 1000ml distilled water.

[0038] Specific implementation steps:

[0039] a. Diluted sample: 1 ml of the extract was added to 10 ml of phosphate buffer solution and mixed well to obtain a dilution. The dilution was spread on an MRS medium plate and cultured anaerobically at 30°C for 3 days.

[0040] b Single colony purification: single colonies are selected according to colony morphology, color and size and three lines are drawn on the MRS culture medium plate. After multiple selections of hyphae at the edge of single colonies for purification, pure bacteria are obtained.

[0041] After 16s identification and preservation, the strain was identified as brewer's yeast, and the strain was recorded as Saccharomyces cerevisiae CE13-M5 (brewer yeast CE13-M5). On August 21, 2023, the isolated new brewer's yeast strain was sent to the China Type Culture Collection Center on the campus of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province for preservation (Wuhan University Collection Center), with the preservation number CCTCC NO: M 20231513, the preservation date being August 21, 2023, and the classification name being Saccharomyces cerevisiae CESWGF002.

[0042] (2) Lactobacillus paracasei

[0043] Sample: Medicinal wine soaked in aged yew and other Chinese herbal medicines from the Yeren Valley area

[0044] MRS medium: 10g protein Chen, 5g beef powder, 4g yeast powder, 2g glucose, 1ml Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 15g agar powder, 1000ml distilled water.

[0045] Specific implementation steps:

[0046] a. Diluted sample: 1 ml of the extract was added to 10 ml of phosphate buffer solution and mixed well to obtain a dilution. The dilution was spread on an MRS medium plate and cultured anaerobically at 30°C for 3 days.

[0047] b Single colony purification: single colonies were selected according to colony morphology, color and size, and three lines were drawn on the MRS medium plate. After multiple selections of single colony edge hyphae for purification, pure bacteria were obtained. After 16s identification and preservation, the strain was identified as Lactobacillus paracasei CE12-M3 (Lactobacillus paracasei CE12-M3), and the isolated new strain of Lactobacillus paracasei was sent to the China Type Culture Collection Center on the campus of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province on August 21, 2023 for preservation (Wuhan University Collection Center), with a preservation number of CCTCC NO: M 20231514, a preservation date of August 21, 2023, and a classification name of Lactobacillus paracasei CESWGF003.

[0048] (3) Lactobacillus plantarum

[0049] Sample: Aged Chinese herbal extract from the Savage Valley

[0050] MRS medium: 10g protein Chen, 5g beef powder, 4g yeast powder, 2g glucose, 1ml Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 15g agar powder, 1000ml distilled water.

[0051] Specific implementation steps:

[0052] a. Diluted sample: 1 ml of the extract was added to 10 ml of phosphate buffer solution and mixed well to obtain a dilution. The dilution was spread on an MRS medium plate and cultured anaerobically at 30°C for 3 days.

[0053] b Single colony purification: single colonies are selected according to colony morphology, color and size and three lines are drawn on the MRS culture medium plate. After multiple selections of hyphae at the edge of single colonies for purification, pure bacteria are obtained.

[0054] After 16s identification and preservation, the strain was identified as Lactobacillus plantarum, and the strain was recorded as Lactiplantibacillus plantarum CE13-L4 (Lactobacillus plantarum CE13-L4). On August 21, 2023, the isolated new strain of Lactobacillus plantarum was sent to the China Type Culture Collection Center on the campus of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province for preservation (Wuhan University Collection Center), with a preservation number of CCTCC NO: M 20231515, a preservation date of August 21, 2023, and a classification name of Lactiplantibacillus plantarum CESWGF004;

[0055] The three strains obtained above were stored in a -80°C refrigerator.

[0056] Example 2 Animal Experiment on Taxus Chinensis's Extreme Toxicity

[0057] Experimental animals: Kunming male healthy mice, weighing 26.0 g to 31.0 g. In this example, 50 Kunming male healthy mice were selected.

[0058] Experimental strain: Saccharomyces cerevisiae CE13-M5, its deposit number is: CCTCCNO: M20231513, classification name: Saccharomyces cerevisiae CESWGF002;

[0059] Lactobacillus paracasei CE12-M3 (M3-1), with a deposit number of CCTCCNO: M20231514 and a classification name of Lactobacillus paracasei CESWGF003;

[0060] Lactiplantibacillus plantarum CE13-L4, its deposit number is: CCTCCNO: M20231515, and its classification name is: Lactiplantibacillus plantarum CESWGF004.

[0061] Experimental culture medium:

[0062] (1) Strain activation: The three strains obtained in Example 1 were taken out from a -80°C refrigerator, and were placed in a liquid culture medium constant temperature incubator at 37°C for 24 h at an inoculum size of 5% to obtain a first-generation bacterial suspension; the first-generation bacterial suspension was inoculated into MRS broth medium at an inoculum size of 5% and cultured at 37°C for 18 h to obtain a second-generation bacterial suspension, thereby obtaining the corresponding activated strains.

[0063] (2) Preparation of culture medium for fermentation of Taxus by strains: 10% of the activated strains were inoculated into Taxus + MRS culture medium, mixed and allowed to stand, cultured at 37°C for 48 h, and then centrifuged at 6000 rpm for 20 min. The supernatant was collected and stored at 4°C to obtain culture medium for fermentation of Taxus by strain CE12-M3, culture medium for fermentation of Taxus by strain CE13-L4, and culture medium for fermentation of Taxus by strain CE13-M5, respectively, for later gavage.

[0064] The Taxus chinensis + MRS medium was prepared as follows:

[0065] The yew branches and leaves were placed in an oven and dried at 60° C., and crushed into yew powder. 2.4 g of yew powder was added to every 40 ml of MRS culture medium, and the mixture was sterilized to obtain yew + MRS culture medium.

[0066] Fifty healthy male Kunming mice were selected. The mice were adaptively raised for one week before the experiment to make them familiar with the environment and restore their original rhythm. The mice were randomly divided into five groups according to their initial weight, namely, normal control group, yew group, CE12-M3 fermented yew group, CE13-L4 fermented yew group, and CE13-M5 fermented yew group. The mice in each group were gavaged at a dose of 300 mg / kg, as follows:

[0067] Group 1, the normal control group, had free access to drinking water. From day 1 to day 10, mice were intragastrically administered with MRS liquid culture medium at a dose of 300 mg / kg per day.

[0068] The second group, the Taxus group, had free access to drinking water. From day 1 to day 10, mice were intragastrically administered with Taxus + MRS liquid culture medium at a dose of 300 mg / kg per day.

[0069] The third group, CE12-M3 fermented yew group, had free access to drinking water. From day 1 to day 10, mice were intragastrically administered with 300 mg / kg of CE12-M3 fermented yew liquid culture medium every day.

[0070] The fourth group, CE13-L4 fermented yew group, had free access to drinking water. From day 1 to day 10, mice were intragastrically administered with 300 mg / kg of CE13-L4 fermented yew liquid culture medium every day.

[0071] The fifth group, CE13-M5 fermented yew group, had free access to drinking water. From day 1 to day 10, mice were intragastrically administered with 300 mg / kg of CE13-M5 fermented yew liquid culture medium every day.

[0072] After the gavage time was over, the mice in each group were fasted for 24 hours and blood was collected from their eyes. The blood was placed in clean EP tubes that had been marked and placed at 4°C for the detection of serum transaminases (ALT and AST), uric acid, and creatinine.

[0073] Take out the mouse eyeball blood from the 4℃ refrigerator, centrifuge it at 4℃, 3000rpm for 30min in a low-temperature high-speed centrifuge; after the centrifugation, take the supernatant into a new 1.5ml EP, and use the aspartate aminotransferase (AST / GOT) test kit, alanine aminotransferase (ALT / GPT) test kit, uric acid test kit and creatinine test kit purchased from Nanjing Jiancheng Bioengineering Research Institute for detection. The results are as follows: Figure 1 and Figure 2 shown.

[0074] Depend on Figure 1 It can be seen that compared with the control group, the ALT and AST of the mice in the yew group were significantly increased, indicating that taking yew has a certain toxic effect on the mouse liver, while the ALT and AST of the mice in the yew fermentation group of strain CE12-M3 and the yew fermentation group of strain CE13-L4 were significantly lower than those in the yew group, indicating that strains CE12-M3 and CE13-L4 can reduce the toxic effects of yew on the liver.

[0075] Depend on Figure 2 It can be seen that compared with the control group, the uric acid and creatinine of mice in the yew group were significantly increased, indicating that taking yew has a certain toxic effect on the mouse kidneys, while the ALT and AST of mice in the yew fermentation group of strain CE13-L4 and the yew fermentation group of strain CE13-M5 were significantly lower than those of mice in the yew group, indicating that strains CE13-L4 and CE13-M5 can reduce the toxic effects of yew on the kidneys.

[0076] In summary, strains CE12-M3, CE13-L4 and CE13-M5 can all reduce the toxic effects of Taxus, among which strain CE12-M3 can reduce the toxic effects of Taxus on the liver, strain CE13-M5 can reduce the toxic effects of Taxus on the kidneys, and strain CE13-L4 can reduce the toxic effects of Taxus on the liver and kidneys. They can be used in combination with strain CE12-M3 or CE13-M5, and the combined strain will be more effective in reducing the toxic effects of Taxus.

[0077] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A probiotic capable of reducing the toxicity of Taxus chinensis, characterized in that: Including one or more of brewer's yeast CE13-M5, Lactobacillus paracasei CE12-M3, and Lactobacillus plantarum CE13-L4; The brewer's yeast CE13-M5 strain is named Saccharomyces cerevisiae CESWGF002, and its preservation number is CCTCC NO: M 20231513; the Lactobacillus paracasei CE12-M3 strain is named Lactobacillus paracasei CESWGF003, and its preservation number is CCTCC NO: M 20231514; the Lactobacillus plantarum CE13-L4 strain is named Lactiplantibacillus plantarum CESWGF004, and its preservation number is CCTCC NO: M 20231515; all of them were deposited in the China Center for Type Culture Collection on August 21, 2023.

2. The probiotics for reducing toxicity of Taxus chinensis according to claim 1, characterized in that: The probiotics include one or more strains with the preservation numbers of CCTCC NO:M 20231515 and CCTCC NO:M 20231514.

3. The probiotics for reducing toxicity of Taxus chinensis according to claim 1, characterized in that: The probiotics include one or more strains with the preservation numbers of CCTCC NO: M 20231515 and CCTCC NO: M 20231513.

4. The probiotics having the ability to reduce toxicity of Taxus chinensis according to claim 2 or 3, characterized in that: The probiotics include strains with a preservation number of CCTCC NO:M 20231515.

5. Use of the probiotics according to any one of claims 1 to 4 in the preparation of a medicament containing a fermented product of Taxus chinensis, characterized in that: The Taxus fermented product is obtained by fermenting Taxus with the probiotics as claimed in any one of claims 1 to 4.

6. The use according to claim 5, characterized in that The probiotics according to any one of claims 1 to 4 are added to a liquid culture medium containing Taxus chinensis according to a preset ratio for fermentation to obtain a fermentation liquid, which is used as a raw material for preparing a drug.

7. The use according to claim 6, characterized in that The preset ratio is to add the probiotic suspension at 10% to 15% of the volume of the liquid culture medium.

8. The use according to claim 7, characterized in that The medicine acts on the liver, and the added probiotics include one or more strains of CCTCC NO:M 20231515 and CCTCC NO:M 20231514.

9. The use according to claim 7, characterized in that The medicine acts on the kidney, and the added probiotics include one or more strains of CCTCC NO:M 20231515 and CCTCC NO:M 20231513.

10. The use according to claim 8 or 9, characterized in that: The probiotics added to the medicine include a strain with a preservation number of CCTCC NO: M 20231515.

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