TWI788633B - Application of Novel Lactic Acid Bacteria in Preparation of Purine Compound Degrading Agent - Google Patents

Abstract

The present invention discloses a novel lactic acid bacterium and its application, wherein the novel lactic acid bacterium is isolated from cereal sources, has good tolerance in alcohol environment, and can effectively treat or/and prevent hyperuricemia and alcoholic liver disease damage effect.

Description

Application of Novel Lactic Acid Bacteria in Preparation of Purine Compound Degrading Agent

The present invention relates to a novel bacterial strain and its use, in particular to the use of a novel lactic acid bacterium for preparing a purine compound degrading agent.

The liver has the functions of absorption, metabolism and immune protection, and studies have shown that long-term intake of alcohol will increase the incidence of alcoholic liver disease. Specifically, the cause of alcoholic liver disease is that the ethanol formed by alcohol metabolism will change the intestinal The permeability of intestinal epithelial cells increases the amount of bacterial toxins in the intestinal tract, especially the production of endotoxins, which in turn increases the chance of endotoxins being transferred from the intestinal lumen to the blood or lymph fluid in the hepatic hilum, thereby inducing or aggravating alcoholic liver disease; In addition, when the concentration of endotoxin in the body increases, the oxidative stress in the body increases, resulting in the generation of active oxygen free radicals, and induces the macrophages in the liver to produce inflammatory substances, causing inflammation and damage to liver cells.

Uric acid is the metabolite of nucleic acid purine. When eating too much high-purine food or food that increases purine synthesis, the uric acid content in the blood will be too high, which will cause hyperuricemia, which may damage the kidney metabolism. At present, most of the drugs used clinically to treat hyperuricemia and gout are accompanied by side effects, which may even cause death in severe cases, such as allergic syndrome.

The main purpose of the present invention is to provide a novel lactic acid bacterium and its application, wherein the novel lactic acid bacterium is isolated from cereal sources, has good tolerance in alcohol environment, and can effectively treat or/and prevent hyperuricemia And the effect of alcoholic liver damage.

Furthermore, in one embodiment of the present invention, the novel lactic acid bacterium is Lactobacillus plantarum subsp. plantarum TTL 8-16, deposited in Taiwan Hsinchu Food Industry Development Research Institute, and the date of deposit is On February 15, 2019, the deposit number is BCRC 910871.

In another embodiment of the present invention, the novel lactic acid bacteria is Lactobacillus paracasei subsp. paracasei TTL 6-10, deposited in Hsinchu, Taiwan

Food Industry Development Institute, the deposit date is October 30, 2019, and the deposit number is BCRC 910951.

The novel lactic acid bacteria revealed by the present invention can effectively reduce the expression of inflammation-related hormones in cells, reduce the content of ALT, AST and triglyceride in blood and liver, and can degrade purine compounds. Therefore, the novel lactic acid bacteria disclosed by the present invention It has the dual effects of treating or preventing alcoholic liver damage and hyperuricemia at the same time.

In one embodiment disclosed in the present invention, a dual-effect pharmaceutical composition is provided, which comprises an effective amount of the novel lactic acid bacteria and at least one pharmaceutically acceptable carrier. By administering the dual-effect pharmaceutical composition to an individual, it can reduce the content of triglycerides in blood and liver, and can degrade the content of uric acid in blood, so as to effectively treat or/and prevent alcoholic liver disease. Effects of injury and/or hyperuricemia.

Among them, alcoholic liver injury refers to liver cell damage and its symptoms caused by alcohol intake, such as fatty liver or hepatitis.

Figure 1 is the result of microscopic observation of TTL 8-16 strains.

Figure 2 is the result of 16s rDNA sequence analysis of TTL 8-16 strain.

Figure 3 shows the results of the API 50CHL identification system analysis for TTL 8-16 strains.

Figure 4 is the result of microscopic observation of TTL 6-10 strains.

Figure 5 shows the results of 16s rDNA sequence analysis of TTL 6-10 strains.

Fig. 6 is the result of analyzing the API 50CHL identification system for TTL 6-10 strains.

Figure 7 shows the results of detecting the TTL 6-10 strains clearing the AST produced by alcohol stimulation in HepG2 cells.

Fig. 8 is the result of detecting the TTL 6-10 strain to eliminate ALT produced by alcohol stimulation in HepG2 cells.

Figure 9 shows the results of detecting the AST produced by alcohol stimulation in HepG2 cells by the TTL 8-16 strain.

Fig. 10 is the result of detecting the TTL 8-16 strain to eliminate ALT produced by alcohol stimulation in HepG2 cells.

Figure 11 shows the degradation rates of inosine detected by TTL 8-16 strains and TTL 6-10 strains at different time points.

Figure 12 shows the degradation rates of guanosine detected by TTL 8-16 strains and TTL 6-10 strains at different time points.

Figure 13 shows the body weight changes of mice in each group during the experiment.

Figure 14 shows the changes of AST content in the serum of each group of mice during the test period.

Fig. 15 is a graph showing the change of serum triglyceride content in each group of mice during the test period.

Figure 16 shows the changes of triglyceride content in the liver of mice in each group during the test period.

Fig. 17 shows the detection of the changes of GPx activity in the liver of mice in each group during the test period.

Figure 18 shows the results of H&E staining of the mouse liver sections in the blank group.

Figure 19 shows the results of H&E staining of the liver sections of mice in the alcohol group.

Figure 20 shows the results of H&E staining of the liver sections of mice in the TTL 8-16 group.

Figure 21 shows the change of uric acid concentration in serum of rats in each group.

Fig. 22 shows the change of blood urea nitrogen concentration in the rats of each group.

The novel lactic acid bacteria disclosed in the present invention include: Lactobacillus paracasei subsp. paracasei TTL 6-10 (hereinafter referred to as TTL 6-10 strain) was deposited in Hsinchu Food Industry, Taiwan on October 30, 2019 Development Research Institute, the deposit number is BCRC 910951; and Lactobacillus plantarum subsp.plantarum TTL 8-16 (hereinafter referred to as TTL 8-16 strain) was deposited in Taiwan Hsinchu Food Industry on February 15, 2019 Development Institute, deposit number BCRC 910871.

The two strains were isolated from cereal sources and stored in MRS broth containing 15% glycerol at a temperature of -80°C. The culture medium used was Lactobacilli MRS broth containing 0.05% L-cysteine. ), cultivated at 30-37°C for 24 hours.

Furthermore, as shown in Figure 1, the TTL 8-16 strain is identified as a Gram-positive bacillus without catalase, oxidase and motility, and does not produce endospores. grow in an oxygen environment. As shown in Figure 2 and Figure 3, the results of 16s rDNA sequence analysis and API 50CHL identification system analysis showed that the TTL 8-16 strain belonged to Lactobacillius plantarum subsp.plantarum .

As shown in Figure 4, the TTL 6-10 strains are Gram-positive bacilli that do not have catalase, oxidase and motility, do not produce endospores, and can grow in both aerobic and anaerobic environments. As shown in Figure 5 and Figure 6, the results of 16s rDNA sequence analysis and API 50CHL identification system analysis showed that the TTL 6-10 strain belonged to Lactobacillus paracasei subsp.paracasei .

The term "effective amount" disclosed in the present invention refers to the amount of the compound or active ingredient required to produce the desired specific effect, which can be represented by its weight percentage in the composition. As will be understood by those of ordinary skill in the art to which this invention pertains, the effective amount will vary depending on the mode of administration to elicit a particular effect. Generally, the active ingredient or compound is present in the composition in an amount ranging from about 1% to about 100%, preferably from about 30% to about 100%, by weight of the composition.

The term "pharmaceutical composition" disclosed in the present invention includes an effective amount of the required lactic acid bacteria to produce a specific effect and at least one pharmaceutically acceptable carrier. As understood by those with ordinary knowledge in the technical field of the present invention, the form of the pharmaceutical composition may vary according to the mode of administration to cause a specific effect, such as lozenges, powders, injections, etc., and the carrier is also Depending on the form of the pharmaceutical composition, it may be solid, semi-solid or liquid. For example, carriers include, but are not limited to, gelatin, emulsifiers, hydrocarbon mixtures, water, glycerin, saline, buffered saline, lanolin, paraffin, beeswax, simethicone, ethanol.

In the following, in order to illustrate the technical features and effects of the present invention, several examples will be given together with drawings for detailed description as follows.

It needs to be further explained that the amount of the novel lactic acid bacteria disclosed by the present invention used in the following examples is above 10 ⁹ CFU/ml, such as 10 ⁹ CFU/ml, 10 ¹⁰ CFU/ml, etc. In order to prove the efficacy of the novel lactic acid bacteria disclosed in the present invention, the following examples take the amount of bacteria as 10 ⁹ CFU/ml as an example, but this dosage does not limit the protection scope of this case.

Example 1: Cell Culture

The human liver cancer cell line HepG2 cell line (registration number: BCRC 60177, hereinafter referred to as HepG2 cells) was purchased from the Bioresources Preservation and Research Center of Food Industry Research Institute, Hsinchu, Taiwan. The medium used was MEM medium supplemented with 10% fetal bovine serum, 1% non-toxic Essential amino acids and 1.5g sodium bicarbonate.

Mouse macrophage RAW264.7 (registration number: BCRC 60001, hereinafter referred to as RAW264.7 cells) was purchased from the Bioresources Preservation and Research Center of Food Industry Research Institute, Hsinchu, Taiwan. The medium used was DMEM medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids and 1.85g sodium bicarbonate.

Example 2: Anti-inflammation test

HepG2 cells (2 X 10 ⁵ cell/well) and RAW264.7 cells (1 X 105 cell/well) were inoculated in 24-well plates, and when the cells grew to 80%, they were washed with phosphate buffer and added fresh 800 μl of cell culture medium, then add 100 μl endotoxin lipopolysaccharide (100ng/ml) and 100 μl lactic acid bacteria solution (10 ⁹ CFU/ml), wherein, the lactic acid bacteria solution is washed with phosphate buffer first, and the supernatant is removed , and then reconstituted with cell culture medium, placed the plate in an environment of 5% carbon dioxide and cultured for 24 hours, collected the cell supernatant, and analyzed TNF-α and IL-6 in the cell supernatant with an ELISA kit (purchased from BioLegend) The content of IL-8 in the cell supernatant was analyzed with an ELISA kit (BD OptEIA Set Human IL-8, purchased from BD Biosciences), and the results are shown in Table 1 below.

From the results in Table 1, it can be seen that both the TTL 8-16 strain and the TTL 6-10 strain disclosed in the present invention can reduce the concentration of intracellular cytokines related to inflammation, which shows that the TTL 8-16 strain and the TTL 6- 10 strains can effectively achieve anti-inflammatory effect.

Example 3: Alcohol tolerance test

The TTL 8-16 strain and the TTL 6-10 strain disclosed in the present invention were activated once. The culture medium was lactic acid bacteria MRS medium containing 0.05% L-cysteine, the culture condition was 37°C, and the culture time was 16 hours. Take 100 μl of the cultured bacteria solution and inoculate it into 4ml of MRS medium without alcohol, and 4ml of MRS medium with final alcohol concentrations of 1%, 5%, 10%, and 15%. The culture temperature is 37°C and the culture time is 16 hours. . After the culture was completed, the bacterial liquid was serially diluted, and the amount of bacteria was calculated by plate counting. The medium was lactic acid bacteria MRS agar medium containing 0.05% L-cysteine, the culture temperature was 37°C, and the culture time was 48 hours. The results are shown in Table 2 shown.

As can be seen from the results in Table 2, when the TTL 8-16 bacterial strain and the TTL 6-10 bacterial strain disclosed by the present invention are at an alcohol concentration of 15%, the number of bacteria can still be maintained at about 7.4-7.8 Log CFU/ml, which shows that they have good alcohol concentration. tolerance.

Example 4: Detection of liver function biochemical values of HepG2 cells

HepG2 cells (2 X 10 ⁵ cell/well) were inoculated in a 24-well plate. When the cells grew to 80%, they were washed with phosphate buffer, and then the medium was replaced with MEM medium without fetal bovine serum. And divided into four groups, wherein: blank group: add 1ml medium; alcohol group: add 900μl medium and 100μl alcohol, the final concentration of alcohol is 100mM; silymarin group: add 800μl medium, 100μl alcohol and 100μl silymarin (30μg/mL); experimental group : Add 800 μl of culture medium, 100 μl of alcohol and 100 μl of the bacteria solution of TTL 8-16 strain or TTL 6-10 strain disclosed in the present invention.

Each group was left for 24 hours, and the supernatant and cells were collected, and then the liver function biochemical indicators were analyzed with commercially available liver function test kits (Aspartate Aminotransferase Activity Colorimetric Assay Kit and Alanine Aminotransferase Colorimetric Assay Kit). The results are shown in Figure 7 to Figure 10 shown.

As can be seen from Figure 7 and Figure 8, the TTL 6-10 bacterial strain revealed by the present invention has a clearance rate of more than 100% for AST (Aspartate aminotransferase) and ALT (Alanine aminotransferase) produced by HepG2 cells stimulated by alcohol; and from Figure 9 and Figure 10 shows that the 8-16 strains disclosed in the present invention have a clearance rate of more than 100% for the AST produced by HepG2 cells stimulated by alcohol.

Based on the fact that ALT and AST are mostly used clinically to evaluate the degree of liver cell damage and liver disease classification, when the liver cells are damaged and ruptured, the concentrations of ALT and AST in the blood will increase, so by detecting the levels of ALT and AST in liver cells can be As an indicator to judge whether the liver cells are healthy. The above results show that the TTL 8-16 strains and TTL 6-10 strains disclosed in the present invention can indeed reduce the ALT and/or AST produced by alcohol stimulation in liver cells, so as to achieve the purpose of preventing or treating alcoholic liver injury. Efficacy, among them, the effect of TTL 6-10 strain is better.

Example 5: Uric acid lowering test

After activating TTL 8-16 strains and TTL 6-10 strains respectively once, take 100 μl of bacterial liquid and inoculate into 4ml MRS medium without purine compound, culture condition is 37℃, culture time is 16~18 hours, and then culture The subsequent bacterial solution was serially diluted, and the amount of bacteria was calculated by plate counting, as The number of bacteria in normal state. In the experimental group, inosine and guanosine were added to 4ml of MRS medium to make the final concentrations 0.1% and 1%, and then TTL 8-16 strains and TTL 6-10 strains activated once were inoculated into them in an amount of 100 μl. , and after culturing under the above conditions, count the number of bacteria. The results are shown in Table 3.

After activating TTL 8-16 strains and TTL 6-10 strains, centrifuge at 8000xg/10 minutes to collect the bacteria, wash the bacteria with phosphate buffer, and then add 1ml of inosine (1.25mM) Mix well with guanosine (1.25mM), put it in an incubator at 37°C/140rpm for cultivation, and take out the co-cultured bacteria at 0, 1, and 2 hours after cultivation, and centrifuge at 4°C/6000rpm. The supernatants were obtained and analyzed by HPLC to calculate the degradation rates of inosine and guanosine for TTL 8-16 strains and TTL 6-10 strains, respectively. The results are shown in Figure 11 and Figure 12 .

Degradation rate (%)=[1.25(mM)-concentration after reaction of inosine and guanosine(mM)]/1.25(mM) x 100%

From the results of the above experiments, it was found that compared with the normal bacterial count, the TTL 8-16 strain and the TTL 6-10 strain disclosed in the present invention had an increase in the bacterial count when the final concentration of the purine compound was 0.1% and 1%. , the increase range is about 0.74~0.99Log CFU/ml, and the increase rate of TTL 8-16 strains is relatively high. The results show that the TTL 8-16 strains and TTL 6-10 strains disclosed in the present invention can use purine compounds as nutrients for growth and can degrade purine compounds, and can reduce the concentration of purine compounds in cells to achieve treatment or prevention of high blood pressure. The effect of uric acidemia.

Furthermore, through the results of HPLC analysis, it can be seen that the degradation rate of TTL 8-16 strain and TTL 6-10 strain disclosed in the present invention at each time point can reach 99% for inosine, and the degradation rate for guanosine can reach 98%. The above results show that the TTL 8-16 strains and TTL 6-10 strains disclosed in the present invention can indeed degrade the purine compounds in cells, so as to effectively achieve the effect of treating or preventing hyperuricemia.

Example 6: Animal experiment (1)

A plurality of 7-week-old C57BL/6N male mice (purchased from Lesco Biological Co., Ltd.) were randomly divided into three groups, and the test period was 8 weeks. They were treated with the following different conditions: blank group: fed with liquid Feed; Alcohol group: fed with liquid ethanol feed; TTL 8-16 group: fed with liquid ethanol feed and 9 Log CFU/ml TTL 8-16 strain every day.

The mice in each group were raised in an environment with a temperature of 22±2°C, a relative humidity of 55±5%, and a 12-hour light cycle; the feed was changed every day; the body weight was recorded and the growth status was observed every week (as shown in Figure 13). Eye socket blood was collected at 0, 2, 4, 6, and 8 weeks. After the serum was separated, a testing company was entrusted to analyze the biochemical index of liver function. The results are shown in Figures 14 and 15. After the experiment, the mice were sacrificed and their livers were harvested. H&E staining analysis was performed after tissue sectioning, and triglyceride content and GPx (Glutathione peroxidase) activity in the liver were detected. The results are shown in FIGS. 16 to 20 .

From the results in Figure 14, it can be seen that the AST content in the serum of the mice in the blank group was almost unchanged, and the AST content in the serum of the mice in the alcohol group was significantly increased (53%). The content of AST in the serum of the mice increased, but compared with the alcohol group, the content of AST in the serum of the mice in the TTL 8-16 group decreased significantly.

As can be seen from the results in Figure 15, the triglyceride content in the serum of the mice in the blank group was almost unchanged, and the triglyceride content in the serum of the mice in the alcohol group increased as the feeding time increased, while The content of triglycerides in the serum of mice in the TTL 8-16 group began to decrease significantly at the 4th week. Compared with the mice in the alcohol group, it decreased by about 44.8%. The effect of medium triglyceride content can be maintained continuously.

From the results in Figure 16, it can be seen that the triglyceride content in the liver of mice in the alcohol group increased significantly, indicating that long-term intake of alcohol can damage liver cells and may cause fatty liver; The liver triglyceride content of the mice with the TTL 8-16 strain disclosed in the present invention was significantly reduced, which was about 45.9% lower than that of the mice in the alcohol group.

From the results in Figure 17, it can be seen that compared with the mice in the blank group, the activity of GPx in the liver of the mice in the alcohol group decreased by about 31.9%. The activity of GPx in the liver of the mice was increased, which means that compared with the mice in the alcohol group, the activity of GPx in the liver of the mice in the TTL 8-16 group increased by about 16.9%.

From the results in Figure 18 to Figure 20, it can be seen that there is almost no obvious accumulation of fat oil droplets in the liver of the mice in the blank group and the TTL 8-16 group, and the nucleus is very complete, while the liver of the mice in the alcohol group has obvious fat accumulation , showing that long-term alcohol intake can damage liver cells and cause liver diseases such as fatty liver.

From the above results, it can be seen that the TTL 8-16 bacterial strain disclosed by the present invention can indeed treat or prevent liver disease or liver damage caused by alcohol, such as fatty liver, and can effectively reduce the content of triglycerides in the blood, and increase The ability of liver cells to resist oxidation.

Example 7: Animal experiment (2)

The Wistar male rats purchased at the age of 30 days (about 4 weeks) were randomly divided into four groups and raised under the following conditions: the temperature was 23 ± 1°C, the relative humidity was maintained at 40-60%, and 12 hours The environment of the light-dark cycle includes: blank group: normal rats; hyperuric acid-induced group: hyperuricemia rats; TTL 8-16 group: hyperuricemia rats, administered TTL 8- 16 strains with a dose of 10 ⁹ CFU/mL; TTL 6-10 group: rats with hyperuricemia, administered the TTL 6-10 strain disclosed in the present invention with a dose of 10 ⁹ CFU/mL.

Among them, the rat model of hyperuricemia is Wistar male rats injected intraperitoneally with Potassium oxonate (0.35mg/100g BW/day) and yeast extract (15g/kg BW/day) to induce hyperuricemia The administered lactic acid bacteria are first activated, then centrifuged, the bacteria are collected, the supernatant is removed, the bacteria are washed with phosphate buffer, and the bacteria are relyed with phosphate buffer.

At the 0th, 1st and 2nd week of the experiment, the venous blood of the rats in each group was collected, and the concentrations of uric acid and urea nitrogen in the serum of the rats in each group were tested. The results are shown in Fig. 21 and Fig. 22 .

As can be seen from the results in Figure 21, the concentration of uric acid in the serum of rats in the high uric acid induction group was significantly higher than that in the blank group, indicating that Potassium oxonate and yeast extract can indeed cause hyperuricemia in rats; In the uric acid induction group, the rats administered the TTL 8-16 strain and the TTL 6-10 strain disclosed in the present invention, the uric acid concentration in the serum decreased by 16.43% and 20% respectively.

As can be seen from the results in Figure 22, at the second week, the concentration of urea nitrogen in the serum of rats in the high uric acid induction group was about 13.64% higher than that in the blank group; while in the high uric acid induction group, TTL 8- 16 strains and TTL 6-10 strains of rats, the concentration of urea nitrogen in serum decreased significantly, the rate of decline was about 22.42% and 27.27%.

The above results show that the TTL 8-16 strain and the TTL 6-10 strain disclosed in the present invention have the ability to promote the degradation of uric acid and urea nitrogen in the blood, so they can be effectively used to treat or prevent hyperuricemia caused by high purine syndrome, and can achieve the effect of preventing kidney disease.

【Biological Material Storage】

Plant Lactobacillus subspecies TTL 8-16, the deposit date is February 15, 2019, and the deposit number is BCRC 910871.

Lactobacillus casei subspecies TTL 6-10, the deposit date is October 30, 2019, and the deposit number is BCRC 910951.

Claims (3)

A use of lactic acid bacteria for the preparation of a purine compound degradation agent, wherein: the lactic acid bacteria are alcohol-tolerant and are Lactobacillus plantarum subspecies TTL 8-16 or/and Lactobacillus paracasei subspecies TTL 6 -10, of which: the Lactobacillus plantarum subspecies TTL 8-16 was deposited in Taiwan Hsinchu Food Industry Development Institute on February 15, 2019, and the deposit number was BCRC 910871; the Lactobacillus paracasei paracasei Subspecies TTL 6-10, deposited in Taiwan Hsinchu Food Industry Development Research Institute, the date of deposit is October 30, 2019, and the deposit number is BCRC 910951. The use as described in Claim 1, wherein the purine compound degradation agent is used for treating or/and preventing alcohol-induced liver damage or/and hyperuricemia. The use as described in Claim 2, wherein the alcohol-induced liver damage is hepatitis or fatty liver.

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