Application of lactobacillus reuteri B1-10 in preparation of uric acid reducing product

Abstract

The invention provides an application of Lactobacillus reuteri B1-10 in preparation of uric acid reducing products, the Lactobacillus reuteri B1-10 has been preserved in China General Microbiological Culture Collection Center (CGMCC) on December 8, 2022, and the preservation number is CGMCC NO.26166. The Lactobacillus reuteri B1-10 has been preserved in the China General Microbiological Culture Collection Center (CGMCC). The lactobacillus reuteri B1-10 disclosed by the invention has a good effect of reducing uric acid, and has a bright prospect in development and application of products.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to application of a strain of Lactobacillus reuteri B1-10 derived from breast milk in the preparation of a uric acid-lowering product. Background Art

[0002] As the intake of protein, sugar, fat and other foods in people's diet has increased significantly, the prevalence of hyperuricemia (HUA) and gout has gradually increased. Hyperuricemia is caused by excessive production of uric acid in the body or blocked excretion. Uric acid is a product of purine metabolism, and purine comes from nucleotides and nucleic acids. The origin of purine nucleotides is glucose, ATP and many amino acids; starting from 5-phosphoribose, the atoms provided by amino acids are used to assemble the mother nucleus skeleton of the purine molecule, and after multiple steps of reaction, the most important substance, inosine nucleotide, IMP, is synthesized, which is also called inosinic acid. IMP can be converted into adenylic acid and guanylic acid, or it can be converted into hypoxanthine, and then oxidized into xanthine and uric acid.

[0003] About 90% of clinical hyperuricemia is caused by uric acid excretion disorders, and 10% is caused by excessive uric acid production. Uric acid excretion disorders are caused by changes in the levels of transporters involved in uric acid excretion and reabsorption, which cause serum uric acid excretion disorders. Excessive uric acid synthesis is mainly due to single gene mutations, high-purine diets, alcohol and fructose intake, etc., which directly or indirectly affect purine anabolism, leading to excessive uric acid production. Adjusting dietary structure and drug treatment are common treatments for HUA. However, it is not easy to maintain strict dietary restrictions and it is difficult to achieve long-term compliance. The side effects of uric acid-lowering drugs limit their clinical application. In recent years, studies have been conducted to alleviate HUA through the multi-target synergistic effects of plant active ingredients, but the cost of targeted preparation of extracts is high and the effect is limited. Currently, only a few varieties of uric acid-lowering animal and plant extracts are on the market.

[0004] The intestine is the second largest organ for uric acid excretion. Promoting uric acid excretion from the intestine through probiotic intervention can be used as a new direction for the treatment of gout and hyperuricemia. The increase in uric acid levels in systemic metabolism will cause changes in the intestinal environment, thereby causing changes in the intestinal flora. Changes in the intestinal flora will cause changes in its metabolites, which in turn further affect HUA.

[0005] However, there are many types of probiotics, and there are few studies on probiotics from breast milk, and even fewer studies on their potential functions. It is of great significance to explore more functions of probiotics from breast milk. Summary of the invention

[0006] The purpose of the present invention is to provide an application of a strain of Lactobacillus reuteri B1-10 in the preparation of a uric acid-lowering product. The Lactobacillus reuteri B1-10 (CGMCC No. 26166) derived from breast milk of the present invention has a good uric acid-lowering effect, has potential probiotic development potential, and fills the research gap of lactic acid bacteria derived from breast milk. The Lactobacillus reuteri B1-10 derived from breast milk of the present invention has a bright prospect for the development and application of products.

[0007] Solution In order to achieve the above-mentioned purpose, the present invention provides an application of Limosilactobacillus reuteri B1-10 derived from breast milk in the preparation of uric acid-lowering products. The Lactobacillus reuteri B1-10 has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on December 8, 2022, with the deposit number CGMCC No.26166.

[0008] Furthermore, the product is a medicine, a food or a functional food.

[0009] Furthermore, the product comprises Lactobacillus reuteri B1-10 and a pharmaceutically acceptable carrier.

[0010] Furthermore, the product is a probiotic.

[0011] Furthermore, the dosage form of the product is one or more of a solution, a suspension, an emulsion, a powder, a lozenge, a pill, a syrup, a tablet and a capsule.

[0012] Furthermore, the tablet comprises an oral tablet.

[0013] Furthermore, the product is an oral medicine.

[0014] Furthermore, the product is bacterial powder, and the number of viable bacteria is 0.1~100×10 10 cfu / g bacterial powder.

[0015] Furthermore, the product is bacterial powder, and the number of viable bacteria is 1~100×10 10 cfu / g bacterial powder.

[0016] Furthermore, the product is bacterial powder, and the number of viable bacteria is 1~15×10 10 cfu / g bacterial powder.

[0017] Beneficial Effects The Lactobacillus reuteri B1-10 (CGMCC No. 26166) derived from breast milk of the present invention has a good uric acid-lowering effect, has potential probiotic development potential, and fills the research gap of lactic acid bacteria derived from breast milk. The Lactobacillus reuteri B1-10 derived from breast milk of the present invention has a bright prospect for product development and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0019] Figure 1 The identification results of strain B1-10 in Example 2 of the present invention, wherein (A) is the Gram staining of strain B1-10, which is blue-purple and rod-shaped; (B) is the colony morphology of strain B1-10; and (C) is a species map of the top 20 similarity comparisons of the whole genome sequencing of strain B1-10.

[0020] Figure 2 The results of the hemolymph uric acid content on the 12th day of the uric acid-lowering bee model of test example 1, wherein CV is the blank control group, HUA is the high uric acid group (uric acid-lowering bee model), and B110 is the intervention group B110 (Lactobacillus reuteri B1-10).

[0021] Figure 3 It is the expression level of the inflammatory factor MAPK-phl on the 12th day of the uric acid-lowering bee model of test example 2, wherein CV is the blank control group, HUA is the high uric acid group (uric acid-lowering bee model), and B110 is the intervention group B110 (Lactobacillus reuteri B1-10).

[0022] Figure 4 It is the expression level of the inflammatory factor Wnt-Rho1 on the 12th day of the uric acid-lowering bee model of test example 2, wherein CV is the blank control group, HUA is the high uric acid group (uric acid-lowering bee model), and B110 is the intervention group B110 (Lactobacillus reuteri B1-10).

[0023] Figure 5 It is the expression level of the inflammatory factor PGRP-LC on the 12th day of the uric acid-lowering bee model of test example 2, wherein CV is the blank control group, HUA is the high uric acid group (uric acid-lowering bee model), and B110 is the intervention group B110 (Lactobacillus reuteri B1-10).

[0024] Figure 6It is the expression level of the inflammatory factor Atg1 on the 12th day of the uric acid-lowering bee model of test example 2, wherein CV is the blank control group, HUA is the high uric acid group (uric acid-lowering bee model), and B110 is the intervention group B110 (Lactobacillus reuteri B1-10).

[0025] Figure 7 It is the expression level of the inflammatory factor Toll on the 12th day of the uric acid-lowering bee model of test example 2, wherein CV is the blank control group, HUA is the high uric acid group (uric acid-lowering bee model), and B110 is the intervention group B110 (Lactobacillus reuteri B1-10). DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of 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 invention.

[0027] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0030] Lactobacillus reuteri of the present invention ( Limosilactobacillus reuteri ) B1-10, was deposited on December 8, 2022 in the General Microbiology Center of China Microorganism Culture Collection (referred to as CGMCC, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101), with the deposit number: CGMCC NO.26166.

[0031] Lactobacillus reuteri, also known as Lactobacillus reuteri.

[0032] In the following examples, all the raw materials used are commercially available.

[0033] Example 1, strain isolation Fresh breast milk samples were selected, gradiently diluted with sterile PBS buffer solution, and the appropriate dilution concentration was selected. The samples were spread on MRS solid culture medium containing 1% CaCO3, and cultured at 37°C anaerobic conditions for 48 hours. Single white or milky white colonies with obvious transparent circles were picked for Gram staining and microscopic examination. Rod-shaped and Gram-positive bacterial strains were selected. A lactobacillus strain with good growth was selected from many strains and named B1-10. The selected strains were enriched and cultured in a 37°C anaerobic incubator; the bacterial solution was mixed with sterilized 30% glycerol in a volume ratio of 1:1 and stored in a -80°C refrigerator for use.

[0034] Example 2: Morphological identification and full genome analysis of strain B1-10 Morphological identification results The identification results are as follows Figure 1 (A) Figure 1 (B) The results showed that the strain Lactobacillus mucosa B1-10 was blue-purple and rod-shaped after Gram staining. Its colony morphology was round and smooth.

[0035] Whole genome analysis The results of whole genome analysis are as follows Figure 1 As shown in (C), the complete gene sequence of Lactobacillus B1-10 has 97.6% homology with Lactobacillus reuteri. Therefore, combined with the morphological characteristics of strain B1-10, strain B1-10 was identified as Lactobacillus reuteri ( Limosilactobacillus reuteri ) (also known as Lactobacillus reuteri), named Lactobacillus reuteri B1-10.

[0036] Example 3: Establishment of uric acid lowering model Method for establishing a high uric acid bee model: A high uric acid bee model was constructed by using pollen (rapeseed pollen, Qinghai) as the base diet (containing 1% yeast RNA). The yeast RNA was purchased from Sigma (Cat. No.: 10109223001). The results showed that compared with the blank control group, the uric acid content in the hemolymph of the bees in the model group was significantly increased, indicating that the model was successfully established. Specifically: Bees are divided into three groups: Blank control group CV: fed with 50% sucrose water + pollen for 12 days; Hyperuric acid group HUA: fed with 50% sucrose water + 1% yeast RNA + pollen for 12 days; Intervention group B110: 50% sucrose water + 1% yeast RNA + pollen, fed for 7 days, 50% sucrose water + 1% yeast RNA + pollen mixture (containing 10 10CFU / g B1-10) were fed for 5 days, probiotic powder and pollen were added to bee feed at a ratio of 1:20, and new diet was replaced every 24 hours to maintain the activity of probiotics.

[0037] Test Example 1: Detection of uric acid content in hemolymph On the 12th day of the establishment of the uric acid-lowering and hyperuric acid-producing bee model in Example 3, the uric acid content in the hemolymph was detected using a uric acid content detection kit (boxbio, Beijing). The sampling and detection methods are as follows: Hemolymph sampling: Take the experimental bee, clamp the thorax with tweezers to fix it, insert a 10ul pipette into the white spot on the bee's neck towards the thoracic cavity, slowly extract the hemolymph, put it into a centrifuge tube, freeze it with liquid nitrogen, and store it in a -80℃ refrigerator.

[0038] Uric acid test: Beijing Boxbio AKAO014M uric acid test kit was used for testing. Due to the demand for test sample volume, the hemolymph of 12 bees was mixed into one test sample and diluted 10 times with deionized water for testing.

[0039] The results are as follows Figure 2 , Figure 2 The results showed that compared with the HUA group, the hemolymph uric acid content in the B110 group was extremely significantly decreased, and compared with the CV group, there was no significant change in the hemolymph uric acid content in the B110 group.

[0040] Test Example 2: Detection of inflammatory factor expression On the 12th day of the establishment of the uric acid-lowering and hyperuricemia-producing bee model in Example 3, Taq Pro Universal SYBRqPCR Master Mix (Vazyme Biotech, Nanjing) was used to detect the expression levels of genes related to intestinal inflammatory factors (Atg1, MAPK-phl, PGRP-LC, Toll, Wnt-Rho1, etc.). The detection method is as follows: One intestine was used as one test sample. RNA was extracted from the intestine (Novizan RNA-easy Isolation Reagent R701 kit), reverse transcribed into cDNA (Novizan HiScript III All-in-one RT SuperMix Perfectfor qPCR R333 kit), and then qPCR relative quantitative detection was performed (Novizan Taq Pro Universal SYBR qPCR Master Mix Q712 kit). Primers were synthesized by Shanghai Bio-Technology, and the instrument used was the Thermo Fisher QuantStudio™ 1 real-time fluorescence quantitative PCR system.

[0041] The results are as follows Figures 3 to 7As shown in the figure, the results showed that compared with the CV group, the expression levels of five inflammatory factors, Atg1, MAPK-phl, PGRP-LC, Toll, and Wnt-Rho1, in the intestinal tract of honey bees in the HUA group were significantly increased. Compared with the HUA group, the expression levels of Atg1, PGRP-LC, and Wnt-Rho1 genes in the B110 group fed with probiotics were significantly decreased, and there was no significant difference between the expression levels of Atg1, MAPK-phl, PGRP-LC, Toll, and Wnt-Rho1 genes and the CV group.

[0042] In this experiment, a high uric acid bee model was first constructed, and the model bees were fed with the test probiotic strains. After the treatment, the hemolymph and intestinal tissues were taken to detect the uric acid content and the relative expression of inflammatory factors, respectively, to evaluate the regulatory effect of the test strains on the hemolymph uric acid of the bees. The uric acid test results showed that the hemolymph uric acid content of the bees in the probiotic B110 group was significantly lower than that in the model group; the inflammatory factor test results showed that the expression of five inflammatory factors in the model group was significantly or significantly higher than that in the blank control group, while the inflammatory factors Atg1, PGRP-LC and Wnt-Rho1 of the bees colonized with B110 probiotics were significantly decreased to varying degrees compared with the model group. This shows that B110 bacteria can effectively reduce the hemolymph content of the hyperuric acid model bees and alleviate the inflammatory response caused by hyperuricemia.

[0043] In summary, Lactobacillus reuteri B1-10 has the effect of effectively reducing uric acid, and the bacteria has the potential for functional and probiotic development of medicines, functional foods, etc., filling the research gap of lactic acid bacteria derived from breast milk. The Lactobacillus reuteri B1-10 derived from breast milk of the present invention has a bright prospect for the development and application of products.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Lactobacillus reuteri ( Limosilactobacillus reuteri ) B1-10 is used in the preparation of uric acid-lowering products. The Lactobacillus reuteri B1-10 has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on December 8, 2022, with the deposit number: CGMCC NO.26166.

2. The use according to claim 1, characterized in that: The product is a medicine, a food or a functional food.

3. The use according to claim 2, characterized in that: The product comprises Lactobacillus reuteri B1-10 and a pharmaceutically acceptable carrier.

4. The use according to claim 1, characterized in that: The product is a probiotic.

5. The use according to any one of claims 1 to 4, characterized in that: The dosage form of the product is one or more of solution, suspension, emulsion, powder, lozenge, pill, syrup, tablet and capsule.

6. The use according to claim 5, characterized in that: The lozenges include oral lozenges.

7. The use according to claim 5, characterized in that: The product described is an oral medication.

8. The use according to any one of claims 1 to 4, characterized in that: The product is bacterial powder with a viable bacterial count of 0.1~100×10 10 cfu / g bacterial powder.

9. The use according to any one of claims 1 to 4, characterized in that: The product is bacterial powder with a viable bacterial count of 1~100×10 10 cfu / g bacterial powder.

10. The use according to claim 7, characterized in that: The product is bacterial powder with a live bacterial count of 1~15×10 10 cfu / g bacterial powder.

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