TWI730679B - Lactobacillus plantarum bfa-la04 able to lower uric acid and use of lactobacillus plantarum bfa-la04 - Google Patents

Abstract

Lactobacillus plantarum BFA-LA04 which can lower uric acid has a 16S rDNA gene sequence set forth as SEQ ID NO: 1, and a clpP gene sequence set forth as SEQ ID NO: 2. The Lactobacillus plantarum BFA-LA04 is deposited at NITE Patent Microorganisms Depositary (NPMD) of Japan with a deposit number NITE BP-03110. The use of the Lactobacillus plantarum BFA-LA04 is also disclosed.

Description

Lactobacillus embryo BFA-LA04 strain capable of lowering uric acid and its use way

The present invention relates to a Lactobacillus embryonicum strain, especially a Lactobacillus embryonicum BFA-LA04 strain with the ability to reduce uric acid. The present invention also relates to the use of the Lactobacillus embryonica BFA-LA04 strain.

Uric acid is a heterocyclic compound containing carbon, nitrogen, oxygen, and hydrogen. It is a metabolic decomposition product of purine nucleotides and can be excreted in urine.

If uric acid cannot be excreted normally, it will flow to the connective tissue through the blood and deposit in the joints, capillaries, skin and other tissues in the form of needle-like crystals. At this time, the white blood cells will be treated as As foreign matter, swallow these needle-like crystals and cause local inflammation, which is gout. Generally speaking, high blood uric acid concentration (ie, hyperuricemia) is the main cause of gout, and high uric acid concentration is more likely to lead to diabetes (diabetes) and kidney disease (kidney disease) , Arteriosclerosis (arteriosclerosis) and cardiovascular disease (cardiovascular disease) and other complications.

Clinically, doctors can use conventional uricosuric agents or xanthine oxidase inhibitors to control the concentration of uric acid in the blood. The former promotes the excretion of uric acid by inhibiting the reabsorption of uric acid by the proximal tubule, such as allopurinol; the latter inhibits xanthine (xanthine) excretion. ) The main enzyme that metabolizes uric acid-xanthine oxidase (xanthine oxidase), prevents the formation of uric acid, such as probenecid. However, the incidence of side effects of allopurinol is low, but some patients will have fatal serious adverse drug reactions to allopurinol, and when the patient’s creatinine clearance rate (CCR) is too low, probenecid will be lost Effective, and there is a risk of urinary calculi and uric acid nephropathy.

In view of this, if we can further find active ingredients that have significant effects on reducing uric acid content and apply them to the development of uric acid-lowering drugs, it will be the expectation of all walks of life to effectively improve the health problems of Chinese people.

In order to solve the above-mentioned problems, the purpose of the present invention is to provide a Lactobacillus embryonicum BFA-LA04 strain, which has good uric acid-lowering ability.

The second purpose of the present invention is to provide a use of Lactobacillus embryonicum BFA-LA04 strain for preparing uric acid-lowering drugs.

The Lactobacillus embryonicum BFA-LA04 strain with the ability to lower uric acid of the present invention has the 16S rDNA gene sequence shown in SEQ ID NO: 1 and the clpP gene sequence shown in SEQ ID NO: 2, and the Lactobacillus embryonicum The BFA-LA04 strain is deposited in the Licensed Microorganism Deposit Center of Japan's Independent Administrative Legal Person Product Evaluation Technology Base Agency, and its deposit number is NITE BP-03110.

Accordingly, the Lactobacillus embryonicum BFA-LA04 strain of the present invention can not only decompose xanthine, but also inhibit the activity of xanthine oxidase, so it can be used as an active ingredient for lowering uric acid, and can be used to prepare uric acid lowering drugs. Pre-learning drugs or learning to promote uric acid excretion The efficacy of the present invention is to provide a uric acid-lowering option for patients whose side effects are induced by drugs that inhibit uric acid production.

Based on the same technical concept, the use of the Lactobacillus plantarum BFA-LA04 strain of the present invention is to prepare uric acid-lowering drugs, where the Lactobacillus plantarum BFA-LA04 strain is deposited with a licensed microorganism from the Japanese independent administrative legal person’s product evaluation technology base agency The deposit center, its deposit number is NITE BP-03110.

Accordingly, the Lactobacillus embryonicum BFA-LA04 strain of the present invention can be applied to the preparation of uric acid-lowering drugs, so it can be administered to the hyperuric acid individual, so that the Lactobacillus embryonicum BFA-LA04 strain can function in the hyperuric acid individual. It is the effect of the present invention to reduce the uric acid content in the blood of the hyperuric acid individual, thereby preventing the occurrence of complications such as gout, diabetes, kidney disease, arteriosclerosis, and cerebral cardiovascular disease caused by hyperuricemia.

The use of the Lactobacillus embryonicum BFA-LA04 strain of the present invention, wherein the Lactobacillus embryonicum BFA-LA04 strain is administered to an individual with high uric acid to reduce the uric acid content in the blood of the individual with high uric acid.

The use of the Lactobacillus plantarum BFA-LA04 strain of the present invention, wherein the Lactobacillus plantarum BFA-LA04 strain is administered orally to the subject with high uric acid. In this way, the user can ingest the Lactobacillus embryonicum BFA-LA04 strain in a simple manner, so that the Lactobacillus embryonicum BFA-LA04 strain can function in the body of the hyperuric acid individual, which is the effect of the present invention.

The use of the Lactobacillus embryonicum BFA-LA04 strain of the present invention, wherein the Lactobacillus embryonicum BFA-LA04 strain is continuously administered to the high uric acid individual at a dose of ^{1.25×10 5} to 2.5×10 ^{5 CFU/kg/day 7} ~30 days. In this way, by adjusting the dosage, the Lactobacillus embryonicum BFA-LA04 strain can effectively exert its biological activity.

[Figure 1a] The evolutionary relationship diagram of the 16S rDNA gene of the Lactobacillus embryonicum BFA-LA04 strain of the present invention and other Lactobacillus embryonicum strains.

[Figure 1b] The evolutionary relationship diagram of the clpP gene of the Lactobacillus embryonicum BFA-LA04 strain of the present invention and other Lactobacillus embryonicum strains.

[Figure 2] In the experiment (A), the survival rate of Lactobacillus embryonicum BFA-LA04 strain changes histogram in different environments.

[Figure 3] In the experiment (B), the xanthine decomposition rate changes of each group of Lactobacillus embryonicum strains are bar graphs.

[Figure 4] In the test (C), the xanthine oxidase inhibition rate change of each group of Lactobacillus embryonicum strains is a bar graph.

In order to make the above and other objectives, features and advantages of the present invention more comprehensible, the following describes the preferred embodiments of the present invention in conjunction with the accompanying drawings in detail as follows:

The Lactobacillus plantarum BFA-LA04 strain of the present invention can decompose xanthine and can also inhibit the activity of xanthine oxidase, thereby inhibiting the production of uric acid, so it can be used as a uric acid-lowering drug.

The Lactobacillus embryonicum BFA-LA04 strain is obtained from the muskmelon fruit. In detail, after the muskmelon fruit is squeezed, the muskmelon fruit juice is coated on a calcium carbonate-containing MRS solid medium (the formula is shown in the first As shown in the table above), after selecting a single colony, the Lactobacillus embryonicum BFA-LA04 strain was further selected from it. The Lactobacillus embryonicum BFA-LA04 strain has been deposited at the Licensed Microorganism Depositary (NITE Patent Microorganisms Depositary, NPMD) of the Japanese Independent Administrative Legal Person Product Evaluation Technology Foundation Agency (NITE Patent Microorganisms Depositary, NPMD) on January 20, 2020. The number is NITE BP-03110.

In addition, the 16S rDNA and clpP genes of the Lactobacillus embryonicum BFA-LA04 strain were molecularly identified. The Lactobacillus embryonicum BFA-LA04 strain has the 16S rDNA gene sequence shown in SEQ ID NO:1 and has the 16S rDNA gene sequence shown in SEQ ID NO:1. : The clpP gene sequence shown in 2, the 16S rDNA gene sequence and the clpP gene sequence were compared with the sequence alignment database (BLAST) of the National Center for Biotechnology Information (National Center for Biotechnology; NCBI), respectively, The results are shown in Tables 2 and 3, respectively.

In addition, the 16S rDNA gene sequence and the clpP gene sequence can draw the kinship tree diagrams as shown in Figures 1a and 1b, respectively, showing that the Lactobacillus embryonicum BFA-LA04 strain is a novel strain belonging to Lactobacillus embryonicum.

The Lactobacillus plantarum BFA-LA04 strain is combined with a pharmaceutically acceptable carrier or excipient to form a pharmaceutical composition or a food composition. It can also be added to the pharmaceutical composition or the food composition. Active ingredients that contribute to the metabolism of uric acid (for example, green tea polyphenols, hawthorn, etc.) and/or other active ingredients that help inhibit xanthine oxidase (for example, anthocyanins, curcumin, etc.), and can be prepared In any convenient edible form, such as lozenges, capsules, powders, granules or liquids, etc., in a form suitable for consumption, the organism can be taken orally.

In addition, the Lactobacillus plantarum BFA-LA04 strain can be administered to an individual with high uric acid, for example, at ^{a dose of 1.25×10 5} ~2.5×10 ⁵ CFU/kg/day, continuously administered to the individual with high uric acid for 7 to 30 days , So that the active ingredients contained in the Lactobacillus embryonicum BFA-LA04 strain can act in the body of the hyperuric acid individual.

In order to verify that the Lactobacillus embryonicum BFA-LA04 strain can indeed decompose xanthine and inhibit xanthine oxidase, the following tests were carried out:

(A) Acid and bile salt tolerance test of Lactobacillus embryonicum BFA-LA04 strain

In order to confirm that the Lactobacillus embryonicum BFA-LA04 strain of the present invention can survive in a strong acid environment, this test system will cultivate the Lactobacillus embryonicum BFA-LA04 strain in the stationary phase, with a pH of 6.5 and a pH of 3.0, respectively. MRS liquid culture medium was allowed to stand for 3 hours, and then continuous dilution method was used. After obtaining the appropriate dilution factor, spread it to the MRS solid medium. After culturing at 37°C for 24 hours, count the number of bacteria.

Please refer to Figure 2, in an environment with a pH of 3.0, after 3 hours, about 10 ⁵ CFU/mL of Lactobacillus embryonicum BFA-LA04 strain survived, indicating that the Lactobacillus embryonicum BFA-LA04 strain can Living in a strong acid environment, it can pass through the gastric juice and enter the intestinal tract to exert its effects.

Next, in order to verify that the Lactobacillus embryonicum BFA-LA04 strain of the present invention can survive in an environment with bile salts, this experiment will also cultivate the Lactobacillus embryonicum BFA-LA04 strain to contain 0.2%, 0.3% and respectively. MRS medium containing 0.4% bile salt was allowed to stand for 3 hours, and then the serial dilution method was continued. After obtaining the appropriate dilution ratio, it was spread into the MRS solid medium, and the number of bacteria was counted after culturing at 37°C for 24 hours.

Please refer to Figure 2, in an environment containing 0.2%, 0.3%, 0.4% bile salts, after 3 hours, the number of surviving bacteria can still reach ^{more than 10 5} CFU/mL, showing that Lactobacillus embryonicum BFA-LA04 Strains can also survive in an environment containing bile salts, so they can survive in the intestines for a long time to exert their effects.

(B) Xanthine decomposition ability of Lactobacillus embryonicum BFA-LA04 strain

This test system compares the xanthine-decomposing ability of a novel Lactobacillus embryonicum strain (ie, the Lactobacillus embryonicum BFA-LA04 strain) selected from the fruit of melon and other Lactobacillus embryonicum strains of the same genus and species. In detail, the other Lactobacillus embryo strains of the same genus and species include Lactobacillus embryonica strains purchased from the Bioresource Conservation and Research Center of the Food Industry Development Institute, The serial numbers are BCRC 10069 and BCRC 910787.

After culturing the aforementioned Lactobacillus plantarum strain to the stagnation period, add the bacteria to the 10-fold diluted MRS liquid medium, and add xanthine and glucose. After culturing for 2, 4 hours, the supernatant was obtained, and the HPLC analysis was continued. The xanthine content is then converted into the xanthine decomposition rate of each Lactobacillus embryonicum strain.

Please refer to Figure 3, after 2 hours of culture, the xanthine decomposition rate of the Lactobacillus embryonicum BFA-LA04 strain is higher than that of other Lactobacillus embryonicum strains of the same species (BCRC 910787, BCRC 10069, p <0.05) ), and after 4 hours of culture, the xanthine decomposition rate of the Lactobacillus embryonicum BFA-LA04 strain is higher than that of other Lactobacillus embryonicum strains of the same species (BCRC 910787, BCRC 10069, p <0.02), showing that it is relatively Compared with other strains of Lactobacillus plantarum of the same species, the Lactobacillus plantarum BFA-LA04 strain does have better xanthine decomposition ability.

(C) Xanthine oxidase inhibitory ability of Lactobacillus embryonicum BFA-LA04 strain

In this experiment, the aforementioned Lactobacillus plantarum strains were also cultured to the stagnation period, then new MRS liquid medium was added, and after 2, 4, and 6 hours of culture, the supernatant was obtained, and xanthine oxidase was added to the supernatant. Reacted at 37°C for 10 minutes, added xanthine, and reacted at 37°C for 30 minutes. Finally, the reaction was terminated with hydrochloric acid, and the uric acid content was analyzed by HPLC to calculate the xanthine oxidase inhibition rate of each Lactobacillus embryonica strain.

Please refer to Figure 4, whether after 2, 4, or 6 hours of culture, the inhibition rate of Xanthine oxidase of Lactobacillus embryonicum BFA-LA04 is significantly higher than that of other Lactobacillus embryonica strains (BCRC 910787). , BCRC 10069, p ≦0.001), showing that compared with other Lactobacillus embryonicum strains of the same species, the Lactobacillus embryonicum BFA-LA04 strain does have better xanthine oxidase inhibitory ability.

In summary, the Lactobacillus embryonicum BFA-LA04 strain of the present invention can not only Decomposing xanthine can also inhibit the activity of xanthine oxidase, so it can be used as an active ingredient for lowering uric acid, and can be applied to the preparation of uric acid lowering drugs, for the administration of conventional drugs to promote uric acid excretion or conventional drugs to inhibit uric acid production. For patients with side effects, it is the effect of the present invention to provide a uric acid lowering option.

Furthermore, because the Lactobacillus embryonicum BFA-LA04 strain of the present invention can be used to prepare uric acid-lowering drugs, it can be administered to the hyperuric acid individual, so that the Lactobacillus embryonicum BFA-LA04 strain can function in the hyperuric acid individual. It is the effect of the present invention to reduce the uric acid content in the blood of the hyperuric acid individual, thereby preventing the occurrence of complications such as gout, diabetes, kidney disease, arteriosclerosis, and cerebral cardiovascular disease caused by hyperuricemia.

Although the present invention has been disclosed using the above-mentioned preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with the art without departing from the spirit and scope of the present invention may make various changes and modifications relative to the above-mentioned embodiments. The technical scope of the invention is protected. Therefore, the scope of protection of the invention shall be subject to the scope of the attached patent application.

【Biological Material Deposit】

Foreign hosting information

Lactobacillus embryonicum BFA-LA04 strain: JP Japan, Independent Administrative Agency Product Evaluation Technology Foundation Agency Licensed Microorganism Deposit Center, January 20, 2020, NITE BP-03110

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Claims (5)

A Lactobacillus embryonicum BFA-LA04 strain with the ability to lower uric acid, which has the 16S rDNA gene sequence shown in SEQ ID NO: 1 and the clpP gene sequence shown in SEQ ID NO: 2, and the Lactobacillus embryonicum BFA- The LA04 strain is deposited in the Licensed Microorganism Deposit Center of the Japan Independent Administrative Legal Person Product Evaluation Technology Foundation Agency, and its deposit number is NITE BP-03110. A use of the Lactobacillus embryonicum BFA-LA04 strain as in claim 1 is for preparing a uric acid-lowering drug. The use of the Lactobacillus embryonicum BFA-LA04 strain of claim 2, wherein the Lactobacillus embryonicum BFA-LA04 strain is administered to an individual with high uric acid to reduce the uric acid content in the blood of the individual with high uric acid. The use of the Lactobacillus plantarum BFA-LA04 strain of claim 3, wherein the Lactobacillus plantarum BFA-LA04 strain is administered orally to the subject with high uric acid. Such as the use of the Lactobacillus embryonicum BFA-LA04 strain of claim 3, wherein the Lactobacillus embryonicum BFA-LA04 strain is continuously administered to the high uric acid at a dose of ^{1.25×10 5} to 2.5×10 ^{5 CFU/kg/day} Individual 7-30 days.

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