TW202135845A - 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

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 product of purine nucleotide metabolism that can be excreted in the 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, the high concentration of uric acid in the blood (that is, hyperuricemia) is the main cause of gout, and the high concentration of uric acid is more likely to cause diabetes and 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 is by inhibiting the proximal tubule. The reabsorption of uric acid can promote the excretion of uric acid, such as allopurinol (allopurinol); the latter inhibits xanthine (xanthine) metabolizing into uric acid by inhibiting the main enzyme-xanthine oxidase (xanthine oxidase). ), preventing 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 the content of uric acid, and apply them to the development of hyperuricemia 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 hyperuricemia drugs.

The Lactobacillus embryonicum BFA-LA04 strain with the ability of lowering 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.

According to this, 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 hyperuricemia drugs. For patients who are administered conventional drugs for promoting uric acid excretion or drugs for inhibiting uric acid production, it is the effect of the present invention to provide a uric acid-lowering option for patients who have side effects.

Based on the same technical concept, the use of the Lactobacillus embryonicum BFA-LA04 strain of the present invention is for the preparation of hyperuricemia drugs, where the Lactobacillus embryonicum BFA-LA04 strain is deposited with the Japanese independent administrative legal person’s product evaluation technology base agency Licensed microbiological deposit center, its deposit number is NITE BP-03110.

Accordingly, because the Lactobacillus embryonicum BFA-LA04 strain of the present invention can be applied to the preparation of hyperuricemia drugs, it can be administered to the desired individual, so that the Lactobacillus embryo BFA-LA04 strain can be placed in the desired individual. Play a role to reduce the content of uric acid in the blood of the required individual, thereby preventing the occurrence of complications such as gout, diabetes, kidney disease, arteriosclerosis, and cerebral cardiovascular disease caused by hyperuricemia, 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 administered to a desired individual to reduce the uric acid content in the blood of the desired individual.

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 desired individual. 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 desired individual, which is the effect of the present invention.

The use of the Lactobacillus plantarum BFA-LA04 strain of the present invention, wherein the Lactobacillus plantarum BFA-LA04 strain is continuously administered to the desired 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.

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 hyperuricemia drug.

The Lactobacillus embryonicum BFA-LA04 strain was 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 in the Licensed Microorganism Depositary (NITE Patent Microorganisms Depositary, NPMD) of the Japan Independent Administrative Legal Person Product Evaluation Technology Foundation Agency (NITE Patent Microorganisms Depositary, NPMD) on January 20, 2020, and its deposit number is NITE BP-03110.

Table 1. The formula of MRS solid medium for culturing Lactobacillus embryonicum BFA-LA04 strain Formula ingredients Content (g) Peptone 10 Beef Extract 10 Yeast Extract 5 Dextrose 20 Ammonium Citrate 2 Magnesium sulfate (MgSO ₄ ‧7H ₂ O) 0.2 Manganese sulfate (MnSO ₄ ‧H ₂ O) 0.05 Dipotassium hydrogen phosphate (K ₂ HPO ₄ ) 2 Sodium Acetate 5 Polysorbate 80 (Tween 80) 1 Calcium carbonate (CaCO ₃ ) 5 Agar 15 RO water Make up to 1 liter

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.

Table 2 Comparison results of 16S rDNA gene sequence of Lactobacillus embryonicum BFA-LA04 strain Description Identity #01 Lactobacillus plantarum strain YLL-03 100% #02 Lactobacillus plantarum strain LLY-606 100% #03 Lactobacillus plantarum strain pc-26 100% #04 Lactobacillus plantarum subsp. plantarum strain BNH17 100% #05 Lactobacillus plantarum strain WGX143 100% #06 Lactobacillus plantarum strain EM 100% #07 Lactobacillus plantarum strain IRG1 100% #08 Lactobacillus plantarum subsp. plantarum strain nF1-FD 100% #09 Lactobacillus plantarum strain KACC 92189 100% #10 Lactobacillus plantarum strain WLPL04 100%

Table 3 Comparison of the clpP gene sequence of Lactobacillus embryonicum BFA-LA04 strain Description Identity #01 Lactobacillus plantarum strain ATG-K6 98.52% #02 Lactobacillus plantarum strain ATG-K8 98.52% #03 Lactobacillus plantarum strain DR7 98.52% #04 Lactobacillus plantarum SN35N 98.52% #05 Lactobacillus plantarum strain HAC01 98.52% #06 Lactobacillus plantarum strain plantarum 98.52% #07 Lactobacillus plantarum strain SRCM102022 98.52% #08 Lactobacillus plantarum strain CLP0611 98.52% #09 Lactobacillus plantarum strain SRCM103357 97.45% #10 Lactobacillus plantarum strain JBE245 97.45%

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 embryonicum BFA-LA04 strain can be administered to a desired individual, for example, at ^{a dose of 1.25×10 5} ～2.5×10 ⁵ CFU/kg/day, continuously administered to the desired individual for 7-30 days , So that the active ingredients contained in the Lactobacillus embryonicum BFA-LA04 strain can act in the body of the desired 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 resistance and bile salt resistance 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 to cultivate, and the serial dilution method was continued. After obtaining the appropriate dilution ratio, 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%, and 0.4% bile salts, after 3 hours, the number of viable bacteria can still reach ^{more than 10 5} CFU/mL, indicating that the 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 plantarum strains of the same genus and species include Lactobacillus plantarum strains purchased from the Bioresource Conservation and Research Center of the Food Industry Development Institute, and their numbers are BCRC 10069 and BCRC 910787, respectively.

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 embryos of the same species (BCRC 910787). , BCRC 10069, p ≦0.001), showing that compared with other Lactobacillus embryos of the same species, the Lactobacillus embryonica 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 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 applied to the preparation of hyperuricemia. The drug provides a uric acid-lowering option for patients who are given conventional drugs for promoting uric acid excretion or drugs for inhibiting uric acid production to induce side effects. This is the effect of the present invention.

Furthermore, because the Lactobacillus embryonicum BFA-LA04 strain of the present invention can be applied to the preparation of hyperuricemia drugs, it can be administered to the desired individual, so that the Lactobacillus embryonicum BFA-LA04 strain can be placed in the desired individual. Play a role to reduce the content of uric acid in the blood of the required individual, thereby preventing the occurrence of complications such as gout, diabetes, kidney disease, arteriosclerosis, and cerebral cardiovascular disease caused by hyperuricemia, which is the effect of the present invention.

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.

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[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. [Picture 2] In the experiment (A), the survival rate of Lactobacillus embryonicum BFA-LA04 strain changes histogram in different environments. [Picture 3] In the experiment (B), the xanthine decomposition rate change of each group of Lactobacillus embryonicum strains is a bar graph. [Picture 4] In the experiment (C), the bar graph of the xanthine oxidase inhibition rate of each group of Lactobacillus embryonicum strains.

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

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 Lactobacillus embryonicum BFA-LA04 strain is used to prepare hyperuricemia drugs, where the Lactobacillus embryonicum BFA-LA04 strain is deposited in the Licensed Microorganism Deposit Center of the Japanese Independent Administrative Legal Person Product Evaluation Technology Foundation Agency, and its deposit The number is NITE BP-03110. The use of the Lactobacillus plantarum BFA-LA04 strain of claim 2, wherein the Lactobacillus plantarum BFA-LA04 strain is administered to a desired individual to reduce the uric acid content in the blood of the desired individual. The use of the Lactobacillus plantarum BFA-LA04 strain of claim 3, wherein the Lactobacillus plantarum BFA-LA04 strain is administered orally to the desired individual. Such as the use of the Lactobacillus embryonicum BFA-LA04 strain of claim 3, wherein the Lactobacillus embryonicum BFA-LA04 strain is continuously administered at a dose of ^{1.25×10 5} to 2.5×10 ^{5 CFU/kg/day} Individual 7-30 days.

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