Acid-resistant cholate-resistant lactobacillus plantarum and application thereof

Abstract

The invention provides acid-resistant and cholate-resistant lactobacillus plantarum and application thereof, and relates to the technical field of microorganisms. The invention provides a strain with the capacity of degrading nucleic acid and inhibiting xanthine oxidase, namely lactobacillus plantarum HXJS3-1, and the lactobacillus plantarum HXJS3-1 has the effects of reducing the uric acid level and preventing and treating hyperuricemia and gout. In addition, the lactobacillus plantarum HXJS3-1 also has a certain anti-inflammatory effect and an effect of enhancing immunity. And a novel probiotic or medicine material is provided for treatment of hyperuricemia and related diseases thereof. The lactobacillus plantarum HXJS3-1 also has the characteristics of acid resistance, cholate resistance and no influence of existence of a carbon source and a nitrogen source, so that the lactobacillus plantarum HXJS3-1 can be widely applied to various scenes.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to an acid-resistant and bile-resistant Lactobacillus plantarum strain and its application. Background Art

[0002] Hyperuricemia (HUA) is a chronic clinical syndrome in which the blood uric acid content significantly exceeds the normal range due to purine metabolism disorder or abnormal uric acid excretion. It generally refers to the fasting blood uric acid level of men higher than 420μmol / L and women higher than 360μmol / L on two different days under a normal purine diet. Gout (Gout) is a crystalline arthritis caused by monosodium urate deposition, which is directly related to hyperuricemia caused by purine metabolism disorder. Hyperuricemia is not only the early stage of gout, but also an independent risk factor for hypertension, diabetes, coronary heart disease, and chronic kidney disease. Gout has become the "fourth high" after diabetes, hypertension, and hyperlipidemia, causing serious harm to people's health.

[0003] Clinically, hyperuricemia is generally prevented and treated in three ways. The first way is to reduce the production of uric acid. The drugs used are xanthine oxidase (XOD) inhibitors, such as allopurinol and febuxostat, which reduce the conversion of xanthine to uric acid by inhibiting the activity of xanthine oxidase. The second way is to increase the excretion of uric acid. The drugs used are URAT1 inhibitors, such as benzbromarone and lesinade, which reduce the reabsorption of uric acid by the renal tubules by inhibiting the uric acid transporter URAT1, thereby increasing the excretion of uric acid and reducing blood uric acid. The third way is to degrade uric acid. The drugs used are urate oxidase drugs, such as rasburicase and purikacil, which degrade uric acid into more easily soluble allantoin and excrete it from the body to reduce blood uric acid. However, xanthine oxidase inhibitors and URAT1 inhibitors are chemical drugs that cause certain damage to liver and kidney functions and have strong toxic side effects. Although urate oxidase drugs have good efficacy, they have strong immunogenicity and are generally used as second-line drugs.

[0004] In recent years, improving traditional metabolic diseases such as hyperuricemia based on intestinal microecology has become a new development direction. Many studies have shown that regulating intestinal microecology through probiotics, prebiotics, etc. plays an important role in regulating serum uric acid and uric acid distribution in the body and helps in the treatment of hyperuricemia and gout. The latest clinical data show that the intestinal flora of patients with hyperuricemia and gout changes, including reduced diversity of intestinal flora and the abundance of conditional pathogens.

[0005] In summary, the current drugs used for hyperuricemia have many problems, have high side effects, and are not suitable for long-term use. More and more probiotics have been shown to have good effects in preventing or relieving hyperuricemia. Supplementation of probiotics can directly reduce the accumulation of uric acid, and can also indirectly reduce uric acid levels by regulating intestinal flora and its metabolites. Therefore, screening and finding new strains that are effective in preventing or relieving hyperuricemia and can tolerate the gastrointestinal environment is the current research focus. Summary of the invention

[0006] The purpose of the present invention is to provide a plant lactobacillus that is acid-resistant, bile-resistant, and unaffected by the presence of carbon sources and nitrogen sources, and its application, so as to obtain probiotics and biomaterials that have the effects of stably reducing blood uric acid levels, relieving hyperuricemia and gout, and enhancing immunity.

[0007] On the one hand, the present application provides Lactobacillus plantarum, wherein the Lactobacillus plantarum is Lactobacillus plantarum HXJS3-1, which is deposited in the General Microbiological Center of China National Committee for the Preservation of Microorganisms with a deposit number of CGMCC NO: 29676.

[0008] In a preferred embodiment, Lactobacillus plantarum is Lactobacillus plantarum HXJS3-1, which is deposited in the General Microbiological Center of China National Committee for the Preservation of Microorganisms with a deposit number of CGMCCNO:29676.

[0009] Preferably, the Lactobacillus plantarum may include Lactobacillus plantarum HXJS3-1, a Lactobacillus plantarum HXJS3-1 bacterial agent, a dead bacteria suspension of Lactobacillus plantarum HXJS3-1, a metabolite of Lactobacillus plantarum HXJS3-1, or an extract of Lactobacillus plantarum HXJS3-1.

[0010] Preferably, the plant lactobacillus has the effects of acid resistance, bile salt resistance, nucleoside degradation, inhibition of xanthine oxidase activity, inhibition of inflammation, lowering uric acid levels, prevention and / or treatment of hyperuricemia, prevention and / or treatment of gout, and enhancement of immunity.

[0011] On the other hand, the present application also provides a composition comprising the Lactobacillus plantarum.

[0012] In a preferred embodiment, the present application also provides a bacterial agent, which includes the Lactobacillus plantarum.

[0013] In a preferred embodiment, the number of viable Lactobacillus plantarum in the composition is 1×10 8 -1×10 12 CFU.

[0014] Preferably, the viable count may be 1×10 8 CFU, 1×10 9 CFU, 1×10 10 CFU, 1×10 11 CFU, 1×10 12 CFU and any value above.

[0015] It will be understood by those skilled in the art that the dosage of Lactobacillus plantarum or the composition can be adjusted according to actual conditions, as long as the total viable count reaches 1×10 8 CFU is sufficient. This application only gives an example and does not impose any specific limitation on it.

[0016] Preferably, the composition comprises an auxiliary material.

[0017] It will be understood by those skilled in the art that known auxiliary materials and solvents may be added to the composition without affecting the activity and efficacy of the strain.

[0018] The excipients may be appropriate solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc.

[0019] Furthermore, the composition can be prepared by a common method, such as directly mixing the strains and auxiliary materials or other components, wherein one or more diluents or pharmaceutically acceptable carriers can also be added.

[0020] In addition, the composition may also contain other known effective ingredients without affecting the activity and efficacy of the bacteria.

[0021] In a preferred embodiment, the composition can be a medicine, probiotic yogurt, soy milk, compressed candy and / or solid beverage.

[0022] On the other hand, the present application also provides the use of the plant lactobacillus in acid resistance and / or bile resistance.

[0023] The pH of the acid may be 1-5.

[0024] In a preferred embodiment, the acid may be hydrochloric acid. It will be understood by those skilled in the art that the acid here only refers to an acidic environment, and different types of acids may be used to create an acidic environment during actual operation, and the specific type of acid has no significant effect on the acid resistance of the plant lactobacillus of the present application.

[0025] The bile salt concentration may be 0.1%-0.5%.

[0026] The pH value may be selected from any value of 1, 2, 3, 4, and 5, and the bile salt concentration may be selected from any value of 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%.

[0027] On the other hand, the present application also provides the use of the Lactobacillus plantarum or the composition in degrading nucleosides and / or preparing degraded nucleoside products.

[0028] Preferably, the nucleoside includes one or more of inosine, guanosine and adenosine; more preferably, the nucleoside includes inosine and / or guanosine.

[0029] In the present application, Lactobacillus plantarum HXJS3-1 is used for the first time to degrade nucleosides.

[0030] Preferably, the process of degrading nucleosides is not affected by carbon sources and / or nitrogen sources, that is, when sugar substances or proteins are contained, the Lactobacillus plantarum described in the present application can still maintain a high efficiency in degrading nucleosides.

[0031] Preferably, the carbon source can be selected from one or more of glucose, sucrose, starch, and glycerol, and the nitrogen source can be selected from one or more of yeast extract powder, peptone, yeast extract, beef extract, and corn steep liquor.

[0032] More preferably, the concentration of the carbon source may be 1-5 g / L, and the concentration of the nitrogen source may be 1-5 g / L.

[0033] The concentration of the carbon source can be 1g / L, 2g / L, 3g / L, 4g / L, 5g / L and any values ​​above, and the concentration of the nitrogen source can be 1g / L, 2g / L, 3g / L, 4g / L, 5g / L and any values ​​above.

[0034] In a preferred embodiment, the carbon source is sucrose and / or glucose, and the nitrogen source is yeast and its extract.

[0035] Furthermore, the experiments of the present application verified that the degradation of nucleosides by strain HXJS3-1 can be accelerated in the presence of other carbon and nitrogen sources.

[0036] On the other hand, the present application also provides the use of the Lactobacillus plantarum or the composition in inhibiting xanthine oxidase (XOD) activity and / or preparing a xanthine oxidase (XOD) inhibition product.

[0037] The present application first uses Lactobacillus plantarum HXJS3-1 to verify its xanthine oxidase inhibition ability. The xanthine oxidase inhibition rate of Lactobacillus plantarum HXJS3-1 can reach 100%. That is, the present application provides a new microbial material for xanthine oxidase inhibitors and related products.

[0038] On the other hand, the present application also provides the use of the plant lactobacillus or the composition in inhibiting inflammation for non-disease diagnosis and treatment purposes and / or in preparing an inflammation-inhibiting product.

[0039] In this application, Lactobacillus plantarum HXJS3-1 is used for the first time to inhibit inflammatory factors, reduce the number of inflammatory factors in the sample, and increase the number of anti-inflammatory factors. In a preferred embodiment, the application specifically verifies the content of inflammatory factors and anti-inflammatory factors in the liver and kidney of the sample, proving that Lactobacillus plantarum HXJS3-1 can effectively reduce the content of inflammatory factors in the liver and kidney, and increase the content of anti-inflammatory factors.

[0040] Preferably, the sample is a hyperuricemia sample.

[0041] Preferably, the sample is a liver or kidney sample from an individual with hyperuricemia.

[0042] Preferably, the inflammation-related factors include one or more of IL-1β, IL-6, TNF-α, and IL-11, and the anti-inflammatory factor is IL-10.

[0043] On the other hand, the present application also provides the use of the Lactobacillus plantarum or the composition in the preparation of a product for reducing uric acid levels.

[0044] Preferably, the uric acid level may be the uric acid content in urine or the uric acid content in serum.

[0045] In the present application, Lactobacillus plantarum HXJS3-1 is used for the first time to prepare a product for reducing uric acid levels and for reducing uric acid levels.

[0046] On the other hand, the present application also provides the use of the Lactobacillus plantarum or the composition in the preparation of a product for preventing and / or treating hyperuricemia.

[0047] On the other hand, the present application also provides the use of the Lactobacillus plantarum or the bacterial agent in the preparation of a product for preventing and / or treating gout.

[0048] Among them, prevention and / or treatment of hyperuricemia and gout can be achieved by inhibiting xanthine oxidase (XOD) activity, reducing the content of inflammatory factors or increasing anti-inflammatory factors and reducing uric acid content.

[0049] The Lactobacillus plantarum HXJS3-1 and the composition described in the present application can effectively reduce the probability of further conversion of hyperuricemia into gout while reducing the risk of hyperuricemia, thereby playing a role in preventing and / or treating gout.

[0050] Specifically, since an increase in serum uric acid content can cause an individual to suffer from hyperuricemia, and hyperuricemia can cause an increase in the concentration of inflammatory factors in the body, thereby increasing the risk of gout in an individual. In addition, studies have shown that an increase in the concentration of inflammatory factors may lead to an increase in XOD activity, and XOD can convert xanthine into uric acid and increase the production of uric acid. Therefore, in this application, the production of uric acid is reduced by inhibiting the activity of XOD, and at the same time, the risk of gout in an individual is reduced by reducing the level of inflammatory factors.

[0051] On the other hand, the present application also provides the use of Lactobacillus plantarum or the composition in enhancing immunity.

[0052] On the other hand, the present application also provides use of Lactobacillus plantarum or the composition in the preparation of an IL-11 inhibitor.

[0053] Preferably, the IL-11 inhibitor can be used to prevent or treat a disease selected from anti-atherosclerosis, anti-diabetes, heart disease, anti-cancer effects, diabetic complications, neuropathic pain, microvascular dysfunction, life extension, mitochondrial diseases, mitochondrial myopathy, neurodegenerative diseases (e.g., Alzheimer's disease, amyotrophic sclerosis, Parkinson's disease), chronic obstructive pulmonary disease (COPD) and psoriasis.

[0054] Furthermore, the IL-11 inhibitor can be used in oral preparations and / or external preparations for anti-aging and / or life extension.

[0055] In addition, the Lactobacillus plantarum HXJS3-1 described in the present application is highly safe and has no obvious effect on other indicators of the body after taking it, which proves that it is safe and non-toxic.

[0056] The present invention has the following beneficial effects:

[0057] The present invention provides a strain having the ability to degrade nucleic acids and inhibit xanthine oxidase, namely, Lactobacillus plantarum HXJS3-1, which reduces the conversion of xanthine to uric acid by inhibiting the activity of xanthine oxidase, thereby achieving the effects of reducing uric acid levels, preventing and treating hyperuricemia and gout. In addition, Lactobacillus plantarum HXJS3-1 also has good anti-inflammatory effects and immunity-enhancing effects. A new probiotic or pharmaceutical material is provided for the treatment of hyperuricemia and related diseases.

[0058] Lactobacillus plantarum HXJS3-1 has a good inhibitory effect on IL-11 and can be used as an oral preparation and / or topical preparation for inhibiting organ fibrosis, anti-atherosclerosis, anti-diabetes, heart disease, anti-cancer effects, diabetic complications, neuropathic pain, microvascular dysfunction, life extension, mitochondrial diseases, mitochondrial myopathy, neurodegenerative diseases (for example, Alzheimer's disease, amyotrophic sclerosis, Parkinson's disease), chronic obstructive pulmonary disease (COPD), psoriasis, anti-aging and / or life extension.

[0059] Lactobacillus plantarum HXJS3-1 is also resistant to acid and bile salts and is not affected by the presence of carbon and nitrogen sources, making it widely used in a variety of scenarios.

[0060] In addition, consumption of this strain has no obvious effect on the sample's body indicators, proving that it is highly safe and can be widely used in the fields of medicine and food. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0062] Figure 1 is a schematic diagram of the morphology of Lactobacillus plantarum HXJS3-1;

[0063] Figure 2 is a statistical graph of the inhibition rate of XOD activity in mouse liver and serum in Example 7;

[0064] Figure 3 is a statistical graph of serum uric acid levels of mice on days 0, 7, 14, and 21 in Example 7;

[0065] Figure 4 is a statistical graph of uric acid content in mouse urine in Example 7;

[0066] Figure 5 is a statistical graph of IL-1β content in the liver and kidney of mice in Example 7;

[0067] Figure 6 is a statistical graph of TNF-α content in the liver and kidney of mice in Example 7;

[0068] Figure 7 is a statistical graph of IL-6 content in the liver and kidney of mice in Example 7;

[0069] Figure 8 is a statistical graph of IL-11 content in the liver and kidney of mice in Example 7;

[0070] Fig. 9is a statistical graph of IL-10 content in the liver and kidney of mice in Example 7;

[0071] A strain of Lactobacillus plantarum HXJS3-1 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 19, 2024, with the deposit number CGMCC NO: 29676, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences. DETAILED DESCRIPTION

[0072] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0073] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0074] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0075] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in microbiology, biochemistry, analytical chemistry, cell culture, and related fields conventional in the technical field.

[0076] The culture medium and reagent components involved in the following examples are as follows:

[0077] MRS solid medium: 10g peptone, 5g beef powder, 20g glucose, 4g yeast powder, 5g sodium acetate, 2g potassium dihydrogen phosphate, 0.2g magnesium sulfate, 2g ammonium citrate, 0.05g manganese sulfate, 1mL Tween 80, 15g agar powder, add to 1000mL distilled water, stir evenly to dissolve, divide into portions, and sterilize at 121℃ high temperature and high pressure for 15min. This medium is mainly used for the separation and cultivation of lactic acid bacteria.

[0078] MRS broth medium: 10g peptone, 5g beef powder, 20g glucose, 4g yeast powder, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 0.2g magnesium sulfate, 2g triammonium citrate, 0.05g manganese sulfate, 1mL Tween 80, add to 1000mL distilled water, heat and boil to dissolve, divide into portions, and sterilize at 121℃ for 15min. Mainly used for the culture of lactic acid bacteria.

[0079] PCA medium: 5g tryptone, 2g yeast extract powder, 1g glucose, 15g agar, dissolved in 1000mL distilled water, sterilized at 121℃ for 20min. Mainly used for lactic acid bacteria colony counting.

[0080] Nucleoside degradation solution: Dissolve 33.7 mg of inosine and 35.7 mg of guanosine in 100 mM K3PO4 solution to prepare inosine-guanosine degradation solution. The final concentration is about 1.26 mM.

[0081] HPLC mobile phase: Weigh 3.4 g KH2PO4 and dissolve it in 950 mL ultrapure water, then add 50 mL methanol to prepare 1 L of mobile phase.

[0082] Xanthine oxidase solution: Dissolve xanthine oxidase (100 U) in 0.2 mol / L phosphate buffer (pH 7.5) to 0.5 U / mL and store at 4°C.

[0083] 2 mM xanthine substrate: Accurately weigh 30.422 mg of xanthine standard, first dissolve it with 0.1 mol / L NaOH solution by ultrasonication, and then dilute to 100 mL with 0.2 mol / L phosphate buffer (pH 7.5) to prepare a 2 mM xanthine substrate solution.

[0084] 100mM K3PO4 buffer: weigh 21.23g K3PO4, add 995mL ultrapure water, add about 5mL phosphoric acid after dissolving, adjust pH to 7.0, and place at room temperature for use.

[0085] In addition, the "water" mentioned in the present invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion exchange water, double distilled water, high-purity water, and purified water.

[0086] In the following examples, unless otherwise specified, % means wt %, i.e. weight percentage.

[0087] Example 1 Initial screening of strains

[0088] The commercially available slurry solution was diluted 10 times, 100 times, and 1000 times, respectively, and applied to MRS solid medium containing 1% CaCO3, three parallels per group, and cultured at 37°C. After three days of culture, 25 colonies of different morphologies were picked from the single colonies with obvious transparent circles on the plate, inoculated into MRS broth medium, and cultured at 37°C and 220rpm for 24h. Then transferred to new MRS broth medium, cultured for 16h, and the nucleoside degradation ability was tested after the bacterial solution became turbid.

[0089] Example 2 Determination of Nucleoside Degradation Ability

[0090] In this example, the nucleoside degradation ability of the strain obtained in Example 1 was determined. At the same time, Lactobacillus plantarum BNCC336943 purchased from Beina Chuanglian Biotechnology Co., Ltd. was used as a control group for detection.

[0091] The method for detecting the nucleoside degradation ability comprises: using a blank MRS broth medium to measure the OD of the fermentation liquid of Example 1 or the OD of Lactobacillus plantarum BNCC336943; 600 Adjust to 4.0. Take 1mL of fermentation broth and centrifuge at 4℃, 8000rpm for 2min, discard the supernatant and collect the bacteria. Add 750μL of saline to the bacteria to wash the bacteria, repeat the washing twice, and collect the washed bacterial cells. Add 750μL of nucleoside degradation solution to the above-collected bacterial cells, and use equal volumes of saline and MRS broth culture medium as blank controls. Set the bacterial cells inactivated at 100℃ for 10min as the heat inactivated control. Incubate the prepared nucleoside degradation system at 37℃ and 120rpm for 30min, 60min, 90min, and 120min. After the reaction is completed, inactivate the system at 100℃ for 10min and centrifuge at 12000rpm for 2min. Use a 0.22μm filter membrane to filter the supernatant into a liquid phase vial for detection.

[0092] The nucleoside content was determined by high performance liquid chromatography, with the mobile phase being 25 mM KH2PO4 containing 5% methanol. An ODS-3 (4.6×250 mm, 5 μm) chromatographic column was used. Detection conditions: PDA detector, detection wavelength 254 nm, column temperature 35°C, flow rate 1 mL / min, injection volume 20 μL, retention time 20 min.

[0093] The calculation formula of nucleoside degradation rate is: (initial nucleoside concentration-remaining nucleoside concentration after reaction) / initial nucleoside concentration×100%.

[0094] Finally, a lactic acid bacterium (HXJS3-1) with a high nucleoside degradation ability was screened out, and its nucleoside degradation ability is shown in Table 1 below.

[0095] Table 1 Nucleoside degradation ability

[0096]

[0097] As shown in Table 1, half of the nucleosides of the screened HXJS3-1 strain were degraded after 1 hour of reaction, while the nucleosides of the purchased Lactobacillus plantarum BNCC336943 in the control group were almost not degraded within 1 hour of reaction. After 2 hours of reaction, the degradation rate of inosine and guanosine of strain HXJS3-1 reached about 80%, and it had a strong ability to degrade nucleosides.

[0098] Example 3 Identification of strains

[0099] (1) Colony morphology characteristics

[0100] Example 2 The strain HXJS3-1 obtained by screening is a Gram-positive bacterium, which grows well in MRS medium, with an optimal growth temperature of 37°C and an optimal growth pH of 6.5. It does not form spores and has no flagella. It enters the stable phase after 16 hours of shaking culture. The plaques on the MRS solid plate are slightly yellow, round, with neat edges, opaque, and smooth surface. Figure 1 shown.

[0101] (2) Molecular Biological Identification

[0102] In this embodiment, 16S rDNA sequencing was performed on strain HXJS3-1: a few colonies on the MRS solid plate were picked and added to 20 μL of the prepared PCR reaction solution. The composition of the reaction solution is shown in Table 2. PCR amplification was performed according to the pre-set program (as shown in Table 3), and then 4 μL was taken to run DNA gel electrophoresis for verification, and a band was found at about 1400 bp. The primer sequences involved in the amplification were: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-TA CGGYTACCTTGTTACGACTT-3'.

[0103] The remaining PCR reaction solution was sent for sequencing, and the sequencing work was completed by Qingke Biotechnology Co., Ltd. The obtained 16SrDNA sequence (as shown in SEQ ID No. 1) was BLAST compared on NCBI, and homology was compared with the sequence information of known strains to identify the strain, and the final strain was identified as Lactobacillus plantarum. The present invention named the strain HXJS3-1. The plant lactobacillus (Lactobacillus plantarum) HXJS3-1 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on January 19, 2024, with a deposit number of CGMCCNO: 29676, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences.

[0104] Table 2

[0105]

[0106] Table 3 PCR reaction program:

[0107]

[0108] 16S rDNA sequence of Lactobacillus plantarum HXJS3-1 (SEQ ID No. 1):

[0109]

[0110] Example 4 Determination of nucleoside degradation ability in the presence of carbon source / nitrogen source

[0111] Considering that the food people eat contains not only nucleosides but also many sugar substances and proteins, the degradation rate of nucleosides by Lactobacillus plantarum HXJS3-1 in the presence of different sugar substrates was tested. The specific substrates and detection methods are as follows:

[0112] Substrate 1: 1.26 mM inosine-1.26 mM guanosine-2 g / L sucrose-2 g / L yeast extract;

[0113] Substrate 2: 1.26 mM inosine-1.26 mM guanosine-2 g / L glucose-2 g / L yeast extract;

[0114] Substrate 3: 1.26 mM inosine-1.26 mM guanosine.

[0115] Take OD 600 The value of plant lactobacillus HXJS3-1 bacterial liquid was 4.0, and the purchased plant lactobacillus BNCC336943 was used as the control. After washing with physiological saline, equal volumes of substrate 1, 2, and 3 solutions were added respectively and incubated for 2 hours. Physiological saline and nucleoside degradation solution were used as blank controls. The determination method was the same as in Example 2. The nucleoside degradation abilities of strains HXJS3-1 and BNCC336943 under different conditions are shown in Table 4 below.

[0116] Table 4 Nucleoside degradation ability of strains in the presence of carbon and nitrogen sources

[0117]

[0118]

[0119] The experimental results in Table 4 prove that the presence of sugar substrates does not affect the degradation of nucleosides by strain HXJS3-1, and can even accelerate the degradation of nucleosides by strain HXJS3-1 in the presence of other carbon and nitrogen sources.

[0120] Example 5 XOD inhibition ability determination

[0121] In this embodiment, the xanthine oxidase (XOD) inhibition ability was determined for Lactobacillus plantarum HXJS3-1, and the purchased strain BNCC336943 was used as a control during the determination. The specific determination method is as follows:

[0122] Take 0.75mL of xanthine substrate solution and mix with an equal volume of fermentation broth (OD 600The bacterial solution of Lactobacillus plantarum HXJS3-1 or BNCC336943 with a value of 4.0) was mixed, and finally 0.5 mL of xanthine oxidase solution (0.5 U / mL) pre-warmed at 25°C for 20 min was added. The reaction system was placed at 25°C for 30 min and the absorbance was detected at a wavelength of 293 nm. Three parallels were used for each group.

[0123] Among them, control sample ①: 0.75mL xanthine + 0.5mL xanthine oxidase + 0.75mL PBS;

[0124] Control sample ②: 0.75 mL xanthine + 0.5 mL PBS + 0.75 mL fermentation liquid;

[0125] Test sample ③: 0.75 mL xanthine + 0.5 mL xanthine oxidase + 0.75 mL fermentation liquid;

[0126] Control sample ④: 0.75 mL xanthine + 1.25 mL PBS.

[0127] The above reaction system was mixed and reacted at 25°C for 30 minutes, and then the XOD inhibition ability of the plant lactobacillus HXJS3-1 bacterial solution was determined, and the xanthine oxidase activity was detected. Since xanthine oxidase can catalyze xanthine to produce uric acid, the product uric acid has a characteristic absorption peak at 293nm. The change in the absorbance value of the reaction system within a certain period of time, that is, the amount of uric acid generated, can be measured by using an ELISA instrument to measure the change in xanthine oxidase activity. The XOD inhibition rate is shown in Table 5 below.

[0128] XOD inhibition rate calculation formula: ((absorbance value of control sample ① - absorbance value of control sample ④) - (absorbance value of test sample ③ - absorbance value of control sample ②)) / (control sample ① - control sample ④) × 100%

[0129] Table 5 XOD inhibition rate

[0130] Strain name XOD inhibition rate HXJS3-1 100％ BNCC336943 45.7％

[0131] From the results in Table 5, it can be seen that the inhibition rate of xanthine oxidase by Lactobacillus plantarum HXJS3-1 screened and obtained in the present application can reach 100%, while the inhibition rate of xanthine oxidase by the control bacterium BNCC336943 is only 45.7%.

[0132] Example 6 Acid and bile salt resistance test

[0133] (1) Acid resistance test

[0134] Take the second generation activated bacterial solution and change the initial OD 600The bacterial liquid of Lactobacillus plantarum HXJS3-1 with a value of 1.0 was inoculated into MRS broth medium with a pH of 3, and the cells were counted after culturing at 37°C and 100 rpm for 0 h, 2 h, and 4 h. The survival rate was calculated using MRS broth medium as a blank control.

[0135] Survival rate (%) = Nt / N0×100, where N0 is the initial number of viable bacteria and Nt is the number of viable bacteria after culturing for different time periods.

[0136] The survival rates of the strains under acidic conditions are shown in Table 6 below.

[0137] Table 6 Survival rate of strains under acidic conditions

[0138]

[0139] As can be seen from Table 6, Lactobacillus plantarum HXJS3-1 has good acid resistance and can grow in an acidic environment.

[0140] (2) Bile salt tolerance test

[0141] Take the second generation activated bacterial solution and change the initial OD 600 The bacterial solution of Lactobacillus plantarum HXJS3-1 with a value of 1.0 was inoculated into MRS broth medium with pig bile salt concentrations of 0%, 0.1%, and 0.3%, and counted after culturing at 37°C and 100 rpm for 0h, 2h, and 4h, and the fresh MRS medium was used as a blank control to calculate the survival rate, and the calculation method was the same as above. The bile salt tolerance of the strain is shown in Table 7 below.

[0142] Table 7 Survival rate of strain HXJS3-1 at different bile salt concentrations

[0143]

[0144] As can be seen from Table 7, Lactobacillus plantarum HXJS3-1 has good bile salt tolerance and can survive in bile salt environments of different concentrations.

[0145] Example 7 Mouse Experiment

[0146] 1. Modeling and feeding of hyperuricemia mice

[0147] Twenty-four male mice, 4 weeks old and weighing 16-20 g, were purchased and allowed to acclimate for one week before the experiment. The mice were housed under standard laboratory conditions of 22°C ± 1°C and 60% relative humidity, 12 h light / 12 h dark cycle (7:00-19:00 light), and fed with standard commercial mouse food. The probiotic feeding amount was 1×10 9 CFU / pc. Divided into the following groups equally:

[0148] (1) Blank group: The mice were fed with basal feed and gavaged with 0.5 mL of sterile skim milk every day.

[0149] (2) Model group: mice were fed a basal diet and gavaged with 300 mg / kg / d of potassium oxonate (OP) and 10 g / kg / d of yeast extract powder in sterile skim milk.

[0150] (3) HUA+HXJS3-1 treatment group: The mice were fed a basal diet containing 300 mg / kg / d oxonoate potassium (OP) and 10 g / kg yeast extract powder, and an equal volume and concentration of sterile skim milk containing the probiotic HXJS3-1 was administered by gavage.

[0151] (4) HUA+drug control group: The mice were fed a basal diet containing 300 mg / kg / d oxonate potassium (OP) and 10 g / kg yeast extract powder, and an equal volume and concentration of sterile skim milk containing 5 mg / kg / d allopurinol was administered by gavage.

[0152] Feed according to the above standards for 21 days.

[0153] 2. Detection indicators

[0154] (1) Uric acid content detection

[0155] A Urine

[0156] During the 21-day feeding period, mice in each group were moved into clean empty cages every three days for 1 h to collect urine samples. The fresh urine of mice was diluted 10 times with distilled water and placed in a 60°C water bath to ensure that there was no urate deposition in the urine sample. Then, the uric acid content of all samples was tested according to the instructions provided by the UA detection kit (Jiancheng, Nanjing, China). The uric acid content was tested immediately after the sample was collected. The effect of Lactobacillus plantarum HXJS3-1 on the uric acid content in the urine of hyperuricemic mice is shown in Figure 4 It can be seen that Lactobacillus plantarum HXJS3-1 can effectively inhibit the uric acid content in the urine of hyperuricemia mice.

[0157] B Blood

[0158] Blood was collected from each group of mice on days 0, 7, 14, and 21 and allowed to stand for 30 min. The collected blood samples were centrifuged at 4000 rpm for 20 min to obtain serum, and the uric acid content was detected according to the instructions provided by the UA detection kit (Jiancheng, Nanjing, China).

[0159] Effect of Lactobacillus plantarum HXJS3-1 on serum uric acid content in hyperuricemia mice Figure 3 It can be seen that Lactobacillus plantarum HXJS3-1 can effectively inhibit the uric acid content in the serum of hyperuricemia mice, and the inhibition rate was 35.13% on the 21st day.

[0160] (2) XOD activity index detection

[0161] On the 21st day of feeding, liver and blood samples of mice were collected, the collected liver samples were rinsed with physiological saline, collected in pre-weighed test tubes with 1 mL of PBS diluent, and stored at -20°C for use, fresh liver was ground on ice with a handheld grinder, and then liver sample suspension was obtained under low-speed centrifugation (8000 rpm, 10 min). XOD activity was detected in the liver (according to the instructions provided by Nanjing Jiancheng XOD detection kit). The collected blood samples were centrifuged at 4000 rpm for 20 min to obtain serum, and XOD activity in the serum was detected according to the instructions provided by Nanjing Jiancheng XOD detection kit.

[0162] The inhibition of Lactobacillus plantarum HXJS3-1 on XOD activity in the liver and serum of hyperuricemia mice is shown in Figure 2 It can be seen that Lactobacillus plantarum HXJS3-1 has a certain inhibitory effect on the XOD activity in the liver and serum of hyperuricemia mice, with inhibition rates of 41.65% and 36.88%, respectively, which can effectively inhibit the production of uric acid from purine.

[0163] (3) Detection of inflammatory factor-related indicators

[0164] On the 21st day of feeding, liver and kidney samples of mice were collected, washed with saline, collected in pre-weighed test tubes with 1 mL of PBS diluent, and stored at -20 °C for use. Fresh livers were ground on ice with a handheld grinder, and then obtained under low-speed centrifugation (8000 rpm, 10 min). Interleukin-1β (IL-1β), IL-6, tumor necrosis factor TNF-α, and IL-11 levels in liver and kidney were detected according to the instructions of the detection kit provided by Beijing Biosci.

[0165] Test results are shown in Figure 5-8 ,according to Figure 5 It can be seen that the inhibition rates of Lactobacillus plantarum HXJS3-1 on the inflammatory factor IL-1β in the liver and kidney of hyperuricemia mice were 47.12% and 47.21%, respectively. Figure 6 It can be seen that the inhibition rates of Lactobacillus plantarum HXJS3-1 on the inflammatory factor TNF-α in the liver and kidney of hyperuricemia mice were 37.81% and 35.28%, respectively. Figure 7 It can be seen that the inhibition rates of Lactobacillus plantarum HXJS3-1 on the inflammatory factor IL-6 in the liver and kidney of hyperuricemia mice were 62.75% and 57.41%, respectively. Figure 8 It can be seen that the inhibition rates of Lactobacillus plantarum HXJS3-1 on the inflammatory factor IL-11 in the liver and kidney of hyperuricemia mice were 38.5% and 64.28%, respectively.

[0166] (4) Detection of anti-inflammatory factor IL-10

[0167] The anti-inflammatory factor IL-10 in the liver and kidney of hyperuricemia mice was detected using the inflammatory factor index detection method.

[0168] Test results are shown in Fig. 9 It can be seen that Lactobacillus plantarum HXJS3-1 has a good recovery effect on the anti-inflammatory factor IL-10 in the liver and kidney of hyperuricemia mice, with recovery rates of 23.17% and 51.74%, respectively.

[0169] In summary, Lactobacillus plantarum HXJS3-1 can effectively reduce the blood uric acid, urine uric acid, XOD activity in the liver and blood, and the levels of inflammatory factors in the liver and kidneys of hyperuricemia mice, and can increase the level of anti-inflammatory factor IL-10 in the liver and kidneys, and has a good effect on the prevention and treatment of hyperuricemia.

[0170] Example 8 Application Example

[0171] (1) Preparation of probiotic yogurt containing Lactobacillus plantarum HXJS3-1

[0172] Lactobacillus plantarum HXJS3-1 was activated in MRS culture medium and OD was detected. 600 When the pH value is 0.8-1.2, take 1 mL of bacterial solution and centrifuge it at 8000 rpm in a low-temperature centrifuge for 5 minutes, wash it three times with sterile saline, add the cleaned strain into 100 mL of sterile cow's milk and ferment it at 42°C for 8-10 hours, then inoculate thermophilic Streptococcus and Bulgarian Lactobacillus for secondary fermentation to form probiotic yogurt.

[0173] (2) Preparation of soy milk containing Lactobacillus plantarum HXJS3-1

[0174] Select high-quality soybeans, wash them three times with clean water, soak them until the soybean hulls can be removed, add boiling water to grind them, then keep them warm at 80°C or above for 10 minutes, filter and centrifuge to obtain the supernatant to obtain crude soy milk, add Lactobacillus plantarum HXJS3-1 when the temperature drops to 37°C, and add the Lactobacillus plantarum HXJS3-1 in an amount corresponding to the amount in Example 8 (1). Store them in a refrigerator at 4°C to obtain soy milk containing Lactobacillus plantarum HXJS3-1.

[0175] (3) Preparation of Tablet Candies Containing Lactobacillus plantarum HXJS3-1

[0176] According to weight proportions, 50 parts of sorbitol, 15 parts of microcrystalline cellulose, 10 parts of Lactobacillus plantarum HXJS3-1 and 5 parts of magnesium stearate are mixed, and the mixture is subjected to mixed dry granulation and tableting to obtain a tablet having a specification of 0.5 g / tablet and a viable bacterial count of 10 billion CFU.

[0177] (4) Preparation of solid beverage containing Lactobacillus plantarum HXJS3-1

[0178] The solid beverage comprises 50 parts of glucose, 15 parts of maltodextrin, 10 parts of plant lactobacillus HXJS3-1, 10 parts of soybean peptide powder, 10 parts of resistant dextrin and 8 parts of isomaltooligosaccharide in parts by weight, which are fully mixed and packaged.

[0179] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. Lactobacillus plantarum, characterized in that The Lactobacillus plantarum is Lactobacillus plantarum HXJS3-1, which is deposited in the General Microbiological Center of China National Committee for the Preservation of Microbiological Cultures, with a deposit number of CGMCCNO: 29676.

2. A composition comprising the plant lactobacillus according to claim 1; preferably, the composition comprises an auxiliary material.

3. Application of the plant lactobacillus as claimed in claim 1 or the composition as claimed in claim 2 in acid resistance and / or bile resistance.

4. Use of the plant lactobacillus as claimed in claim 1 or the composition as claimed in claim 2 in degrading nucleosides and / or preparing degraded nucleoside products.

5. Use of the plant lactobacillus as claimed in claim 1 or the composition as claimed in claim 2 in inhibiting xanthine oxidase activity and / or preparing a xanthine oxidase inhibition product.

6. Use of the plant lactobacillus as claimed in claim 1 or the composition as claimed in claim 2 in suppressing inflammation for non-disease diagnosis and treatment purposes and / or in preparing products suppressing inflammation.

7. Use of the plant lactobacillus according to claim 1 or the composition according to claim 2 in the preparation of a product for reducing uric acid levels.

8. Use of the plant lactobacillus according to claim 1 or the composition according to claim 2 in preparing a product for preventing and / or treating hyperuricemia.

9. Use of the plant lactobacillus according to claim 1 or the composition according to claim 2 in preparing a product for preventing and / or treating gout.

10. Use of the Lactobacillus plantarum according to claim 1 or the composition according to claim 2 in the preparation of an IL-11 inhibitor, preferably, the IL-11 inhibitor can be used in oral preparations and / or external preparations for anti-aging and / or life extension.

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