KR20210073823A - A composition for preventing, improving or treating alcoholic hepatitis of the comprising lactobacillus plantarum v135, it culture broth or heat-killed lactobacillus plantarum v135 as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing, alleviating or treating alcoholic hepatitis containing a Lactobacillus plantarum V135 strain, dead cells of a strain, or a metabolite of the strain as an active component. More specifically, the present invention relates to a pharmaceutical composition for preventing, alleviating or treating alcoholic hepatitis, and a food additive composition for preventing or alleviating alcoholic hepatitis, which contain the Lactobacillus plantarum V135 KCTC18796P strain, dead cells of the strain, or the metabolite of the strain as the active component.

Description

Lactobacillus plantarum V135 strain, a dead cell of the strain or a composition for preventing, improving or treating alcoholic hepatitis containing a metabolite of the strain as an active ingredient {A COMPOSITION FOR PREVENTING, IMPROVING OR TREATING ALCOHOLIC HEPATITIS OF THE COMPRISING LACTOBACILLUS PLANTARUM V135, IT CULTURE BROTH OR HEAT-KILLED LACTOBACILLUS PLANTARUM V135 AS AN ACTIVE INGREDIENT}

The present invention relates to a composition for the prevention, improvement or treatment of alcoholic hepatitis containing the Lactobacillus plantarum V135 strain, the dead cells of the strain, or a metabolite of the strain as an active ingredient, and more specifically, Lactobacillus planta Room V135 ( Lactobacillus plantarum V135) KCTC18796P strain, a pharmaceutical composition for the prevention, improvement or treatment of alcoholic hepatitis containing the dead cells of the strain or metabolites of the strain as an active ingredient, and a food additive composition for preventing or improving alcoholic hepatitis is about

Compared to animal lactic acid bacteria, plant lactic acid bacteria lack nutrients and have strong viability in harsh environments such as low pH and high salt concentration, so they have the advantage of being able to reach the intestines stably. In addition, animal lactic acid bacteria use carbohydrates, proteins, fats, etc. as medium nutrients to increase the number of strains that are corrupted by proteins and fats after long-term fermentation, but plant lactic acid bacteria do not decay during fermentation because they use only carbohydrates as medium nutrients. For this reason, in Korea, research results that evaluate the excellence of lactic acid bacteria isolated from fermented vegetable foods such as kimchi are continuously published.

Microorganisms that have various beneficial actions are called probiotics. The definition of probiotics has been gradually expanded. In 1989, Fuller defined probiotics as “probiotics that have a beneficial effect on the host by improving the intestinal flora”. Although established as a microorganism, Salminen et al. in 1999 defined “a microorganism or a component of a microorganism having a beneficial action on the host”, thereby extending the scope of probiotics to dead cells and their components. In recent years, a new concept of postbiotics has emerged from the concept of probiotics, which has been extended to include dead cells and their components. Postbiotics are a form in which live bacteria and metabolic (fermented) products are not allowed to grow by heat treatment, etc. It is known that bacterial components and metabolic (fermented) products directly or indirectly regulate intestinal immunity (Aguilar-Toal￡). JE et al., Trends Food Sci Technol, 2018). Cell body components include cytoplasm, cell wall, lipoteichoic acid, eichoic acid, peptidoglycan, DNA, etc., and metabolic (fermentation) products are organic acids ( organic acids), short chain fatty acids, and polysaccharides. The organic acid, a representative by-product, stimulates the PIE where immune cells are concentrated to release immune substances and suppresses harmful substances in the vicinity. Postbiotics have been reported to be a number of killing methods, physical methods include mechanical disruption, heat treatment, gamma or UV irradiation (gamma- or UV irradiation), high pressure (High hydrostatic pressure), There are freeze-drying and sonication, and chemical methods include acid deactivation, enzymatic treatment, and solvent extract. Postbiotics are called by various terms such as heat-treated probiotics, ghost probiotics, inactivated probiotics, paraprobiotics, lactic acid bacteria dead cells, heat-treated lactic acid bacteria, and lactic acid bacteria extracts.

Postbiotics (dead cells) have various advantages over probiotics (live cells) and are already being commercialized in various forms in Japan, the United States, and Europe. Most probiotics are killed by the physical and chemical (gastric acid, bile, digestive enzymes) digestion process in the gastrointestinal tract, so their stability due to the external environment is low and they are unstable to heat, so they need to be stored at a low temperature in the distribution process. As a result, the production cost increases. In addition, there is a disadvantage that it is impossible to apply to products including a sterilization process because it is unstable to heat. In the case of postbiotics, they are not affected by the physical and chemical digestion processes in the gastrointestinal tract in a form that has already prevented the growth of bacteria, are stable to heat, show certain functionality for each storage period, and produce costs as low-temperature storage and low-temperature delivery are unnecessary. can save In addition, it has thermal stability, so it can be applied to products including sterilization process, so it has a variety of applied products. Due to these characteristics, postbiotics have greater material stability than probiotics, so they have a wider range of industrial applications, are easy to handle in the distribution process, and are added to general foods to enhance the functionality of food and provide the same effect as probiotics in immunomodulatory functions. In addition to the fields where lactic acid bacteria have been applied, such as , food additives, pharmaceuticals, and animal feed, the market is expanding as it is also applied as a cosmetic raw material. In addition, as regulations on the use of antibiotics are being strengthened, it can be said that there are only a handful of companies that have entered into full-fledged production of dead cells and products as an alternative, so the market and growth potential are large. In particular, in Japan, many dead cell products have already been released and are being promoted based on immune-related effects such as pollen allergy. However, in Korea, there is no material recognized as an individually recognized material, and related products are also dependent on imports.

According to the studies reported on the physiological activity of the lactic acid bacteria dead cells, in the case of mice suffering from DSS (dextran sulfate sodium)-induced colitis, DSS was alleviated in mice fed the lactic acid bacteria dead cells, and transplanted sarcoma cancer cells (Sarcoma). -180) was reported to decrease proliferation and activation of NK cells (Tadano et al., J. Japan Mibyou System association, 2011). In addition, with respect to the inhibition of harmful bacteria and intestinal action, the effect of rapidly proliferating beneficial bacteria and inhibiting harmful bacteria in mice administered with antibiotics compared to the intestinal control group has been disclosed (Simohashi et al., Medicine and biology, 2002). In addition, the various physiological activities of the dead cells of lactic acid bacteria are not affected by heat and pH due to the characteristics of the dead cells, so there is an advantage that they can be processed into various types of preparations (Kan, Food industry, 2001).

On the other hand, Republic of Korea Patent No. 10-2028744 discloses a Lactobacillus plantarum HY7717 strain having immune enhancing activity, antioxidant activity and digestive juice resistance, and pharmaceutical compositions and food compositions containing the same.

Korean Patent Registration No. 10-1919938 discloses a novel Lactobacillus plantarum LM1004 and Although the contents related to immune enhancing compositions, pharmaceutical compositions, health functional foods, feed additives, functional drinks, and fermented products containing it as an active ingredient are disclosed, Lactobacillus plantarum V135 ( Lactobacillus plantarum V135) KCTC18796P strain, There is no disclosure of a pharmaceutical composition for preventing, improving or treating alcoholic hepatitis and a food additive composition for preventing or improving alcoholic hepatitis containing the dead cells of the strain or metabolites of the strain as an active ingredient.

Republic of Korea Patent Registration No. 10-2028744 Republic of Korea Patent Registration No. 10-1919938

The present invention has been devised to solve the problems as described above and provide the necessary technology,

The present invention is Lactobacillus plantarum V135 (L actobacillus plantarum V135) KCTC18796P strain, the dead cells of the strain or the metabolite of the strain as an active ingredient To provide a pharmaceutical composition for preventing, improving or treating alcoholic hepatitis There is a purpose.

In addition, the present invention Lactobacillus plantarum V135 (L actobacillus plantarum V135) KCTC18796P strain, the dead cells of the strain or a metabolite of the strain as an active ingredient To provide a food additive composition for preventing or improving alcoholic hepatitis There is another purpose.

As an embodiment of the present invention for achieving the above object,

The present invention provides a pharmaceutical composition for preventing, improving or treating alcoholic hepatitis, which contains the Lactobacillus plantarum V135 KCTC18796P strain, the dead cells of the strain or a metabolite of the strain as an active ingredient.

At this time, the pharmaceutical composition for the prevention, improvement or treatment of alcoholic hepatitis, Lactobacillus plantarum V135 (Lactobacillus plantarum V135) KCTC18796P strain, dead cells of the strain or metabolites of the strain are 1 × 10 ^{5 per 1 g of the composition} It may be characterized in that it is included in cfu to 1×10 ^{12 cfu.}

In addition, the dead cells of the strain contained in the pharmaceutical composition for the prevention, improvement or treatment of alcoholic hepatitis, Lactobacillus plantarum V135 (Lactobacillus plantarum V135) KCTC18796P strain is inoculated into an edible medium to prepare a culture solution, and the culture solution It may be characterized in that the heat treatment is carried out by repeating the process of heating to a temperature of 80°C or higher and then rapidly cooling it to a temperature of 60°C or lower, repeated one or more times.

The present invention provides a food additive composition for the prevention or improvement of alcoholic hepatitis containing Lactobacillus plantarum V135 (Lactobacillus plantarum V135) KCTC18796P strain, a dead cell of the strain or a metabolite of the strain as an active ingredient.

At this time, the food additive composition for the prevention or improvement of alcoholic hepatitis, Lactobacillus plantarum V135 (Lactobacillus plantarum V135) KCTC18796P strain, dead cells of the strain or metabolites of the strain 1 × 10 ⁵ cfu per 1 g of the composition It may be characterized as included as 1×10 ^{12 cfu.}

In addition, the dead cells of the strain contained in the food additive composition for the prevention or improvement of alcoholic hepatitis are inoculated with Lactobacillus plantarum V135 (Lactobacillus plantarum V135) KCTC18796P strain into an edible medium to prepare a culture solution, and then the culture solution is 80 It may be characterized in that it is heat-treated by repeating the process of heating to a temperature of ℃ or higher and then rapidly cooling to a temperature of 60 ℃ or lower, repeated one or more times.

The composition for preventing, improving or treating alcoholic hepatitis containing the Lactobacillus plantarum V135 strain, the dead cells of the strain or the metabolites of the strain as an active ingredient according to an embodiment of the present invention, as the heat treatment process is performed Since it has no cytotoxicity and exhibits a therapeutic effect of hepatitis by preventing and improving liver damage caused by alcoholic hepatitis, it is effective as an active ingredient of a composition for preventing or treating liver damage due to hepatitis, which can be used as a pharmaceutical composition or food additive composition. can

In other words, killing the Lactobacillus plantarum V135 (L actobacillus plantarum V135) KCTC18796P strain through heat treatment improves the disadvantages of live cells (stability, product applicability, etc.) It has the advantage of being able to provide a material with equivalent or higher functionality.

In addition, the dead cells treated with Lactobacillus plantarum V135 (L actobacillus plantarum V135) KCTC18796P strain showed gross and histological abnormalities (inflammatory cell infiltration, hepatocyte infiltration, It has the effect of preventing, improving or treating hepatitis by reducing sinusoidal vasodilation), reducing AST and ALT levels, which are hepatocellular damage indicators, inhibiting the production of inflammatory cytokines, and reducing the expression of hepatitis inducing factors.

1 is a plate culture photograph of the Lactobacillus plantarum V135 strain isolated in the present invention.
2 is a photograph evaluating the antibacterial activity of Lactobacillus plantarum V135 dead cells against Escherichia coli (A), Salmonella typhimurium (B), and Bacillus cereus (C).
3 is a graph evaluating the antioxidant activity of Lactobacillus plantarum V135 dead cells.
Figure 4 is a photograph and graph showing the pathological changes in the liver tissue by the Lactobacillus plantarum V135 strain and dead cells in mice induced by alcohol administration.
5 is a graph showing the changes in the values of AST and ALT, hepatocellular damage indicators, by the Lactobacillus plantarum V135 strain and dead cells in mice induced by alcohol administration.
6 is a graph showing changes in the expression of inflammatory cytokines in liver tissue by Lactobacillus plantarum V135 strain and dead cells in mice induced by alcohol administration.
7 is a photograph and graph showing the protein expression change of the hepatitis inducer by the Lactobacillus plantarum V135 strain and dead cells in mice induced by alcohol administration.
8 is a photograph and graph showing changes in the expression of inflammatory proteins in the liver tissue by the Lactobacillus plantarum V135 strain and dead cells in mice induced by alcohol administration.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art to which the present invention pertains can easily carry out the present invention. The embodiments of the present invention are provided in order to more completely explain the present invention to those of ordinary skill in the art. Accordingly, the embodiment of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

Throughout the specification of the present invention, when a part "includes" a certain component, it does not exclude other components unless otherwise stated, but further includes other components.

The terms "about", "substantially", etc. to the extent used throughout the specification of the present invention are used in or close to the numerical value when manufacturing and material tolerances inherent in the stated meaning are presented, and the present invention It is used to prevent unfair use by unconscionable infringers of the disclosure in which exact or absolute figures are mentioned to help understand.

The present invention provides a Lactobacillus plantarum V135 (L actobacillus plantarum V135) KCTC18796P strain, a dead cell of the strain, or a pharmaceutical composition for the prevention, improvement or treatment of alcoholic hepatitis containing the metabolite of the strain as an active ingredient (hereinafter, Also referred to as a 'pharmaceutical composition').

In addition, the present invention is Lactobacillus plantarum V135 (L actobacillus plantarum V135) KCTC18796P strain, the dead cells of the strain or a food additive composition for the prevention or improvement of alcoholic hepatitis containing a metabolite of the strain as an active ingredient (hereinafter, Also referred to as 'food additive composition').

The pharmaceutical composition and food additive composition according to the present invention contain Lactobacillus plantarum V135 (L actobacillus plantarum V1 35) KCTC18796P strain, dead cells of the strain or metabolites of the strain as an active ingredient. Gross findings of histological liver tissue, reduction of histological abnormalities (inflammatory cell infiltration, hepatocyte infiltration, sinusoidal vasodilation), reduction of AST and ALT levels, which are indicators of hepatocyte damage, inhibition of inflammatory cytokine production and It is expected that the effect of preventing, improving, or treating hepatitis is high by causing a decrease in protein expression of hepatitis inducing factors.

According to one embodiment of the present invention, the pharmaceutical composition for the prevention, improvement or treatment of alcoholic hepatitis and the food additive composition for the prevention or improvement of the alcoholic hepatitis are Lactobacillus plantarum V135 (L actobacillus plantarum V135) KCTC18796P strain, It may be characterized in that the dead cells of the strain or the metabolites of the strain ^{are included in an amount of 1×10 5} cfu to 1×10 ^{12 cfu per 1 g of the composition.}

This is Lactobacillus plantarum V135 (L actobacillus plantarum V135) KCTC18796P strain, the dead cells of the strain or metabolites of the strain per 1 g of the composition 1 × 10 ⁵ cfu When included at a concentration of less than 5 cfu prevention, improvement or This is because there is a fear that the treatment effect cannot be described, and the range (concentration) of the number of bacteria is 1×10 ¹² cfu by calculating the preclinical intake concentration against the body surface area for the human application test and the adult body weight (60 kg). was set.

In addition, according to one embodiment of the present invention, the dead cells of the strain contained in the pharmaceutical composition for the prevention, improvement or treatment of alcoholic hepatitis and the food additive composition for the prevention or improvement of the alcoholic hepatitis, Lactobacillus plantarum The process of preparing a culture solution by inoculating V135 (L actobacillus plantarum V135) KCTC18796P strain into an edible medium, heating the culture solution to a temperature of 80°C or higher to induce the death of microorganisms, and then rapidly cooling it to a temperature of 60°C or lower. It may be characterized in that it is heat-treated by repeating one or more times.

Lactobacillus plantarum V135 (L actobacillus plantarum V135) KCTC18796P strain of the present invention, the dead cells of the strain or a pharmaceutical composition for the prevention, improvement or treatment of alcoholic hepatitis containing the metabolite of the strain as an active ingredient as an active ingredient is alcoholic hepatitis Gross findings of histological liver tissue, reduction of histological abnormalities (inflammatory cell infiltration, hepatocyte infiltration, sinusoidal vasodilation), reduction of AST and ALT levels, hepatocyte damage indicators, inhibition of inflammatory cytokine production And it can be used as a pharmaceutical composition for preventing, improving or treating hepatitis by causing a decrease in protein expression of hepatitis inducers.

Diseases or diseases that can use the pharmaceutical composition of the present invention are not limited thereto, and all diseases or diseases caused by alcoholic hepatitis may be included.

When the pharmaceutical composition of the present invention is used, it may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

In addition, the pharmaceutical composition according to the present invention is formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods. can be used

The Lactobacillus plantarum V135 (L actobacillus plantarum V135) KCTC18796P strain of the present invention, the dead cells of the strain or the metabolite of the strain as an active ingredient for the prevention or improvement of alcoholic hepatitis food additive composition for Histological gross findings of liver tissue, histological abnormalities (inflammatory cell infiltration, hepatocyte infiltration, sinusoidal vasodilation) decrease, hepatocyte damage index AST and ALT levels decrease, inflammatory cytokine production inhibition and hepatitis It has an effect of preventing and improving hepatitis by inducing a decrease in protein expression of a triggering factor, and may be added to food for this purpose. Although not limited thereto, the food additive composition according to the present invention may be used as a main raw material of food, an auxiliary raw material, a food additive, a functional food or a beverage.

In the present invention, the term 'food' means a natural product or processed product containing one or more nutrients, and preferably means a state that can be directly eaten through a certain processing process, and has a conventional meaning As such, it includes all foods, food additives, health functional foods, health supplements and beverages.

Foods to which the food additive composition of the present invention can be added include various foods, for example, beverages, gums, tea, vitamin complexes, health supplements, and the like, and include pills, powders, granules, needles, tablets, capsules, or beverages. can be formulated in the form.

Hereinafter, the present invention will be described in detail according to specific examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Lactobacillus plantarum V135 (L actobacillus plantarum V135) KCTC18796P strain

1. Securing lactic acid bacteria with high antibacterial activity

To isolate lactic acid bacteria, kimchi samples made at home were used. Aseptically chopped various kinds of kimchi, 1 g of it was suspended in 9 ml of sterile physiological saline (0.85% NaCl), diluted stepwise, and mixed solution 1 ㎖ was taken, diluted by the decimal dilution method, smeared on MRS solid medium (Difco, MI, USA), and cultured at 37° C. for 48 hours. Single colonies formed in each sample medium were smeared on MRS solid medium to which bromocresol purple was added, and colonies showing yellow color were finally selected. A photograph of plate culture of the selected strain is shown in FIG. 1 .

The selected strain was evaluated for antibacterial activity and one type of lactic acid bacteria with the best antibacterial activity was selected and stored at -70°C with 20% glycerol stock.

2. Genetic identification of lactic acid bacteria isolated from kimchi

For the genetic identification of the isolated strain, it was inoculated in MRS broth and then subcultured at 37° C. for 24 hours, and 2 ml of the culture solution was centrifuged at 4,000 rpm for 10 minutes. The cell precipitate obtained by centrifugation was washed 3 times with 0.85% NaCl. After washing, 0.5 ml of lysozyme (10 mg/ml) was added, followed by treatment at 37° C. for 1 hour. After adding 20 μl of protease K and 25 μl of 10% sodium dodecyl sulfate (SDS), the enzyme was treated in a water bath at 60° C. for 30 minutes, and then 1 μl of RNase, an RNA-degrading enzyme, was added. was added and treated at 37°C for 1 hour. The same amount of phenol-chloroform-isoamyl alcohol was added at a ratio of 25:24:1 and suspended, then centrifuged at 14,000rpm for 5 minutes at 4℃ to take only the supernatant. Then, this process was repeated twice. 1/2 times the amount of the supernatant 3M ammonium acetate (ammonium acetate, pH 4.8) and 2 times the amount of 100% alcohol were added and allowed to stand at -20°C for 1 hour. After centrifugation at 3,000rpm at 4℃ for 5 minutes to check the cell sediment, and after completely removing the supernatant, 70% ethanol 1ml/L was added and centrifuged again at 3,000rpm for 5 minutes at 4℃. After removal of the supernatant, the DNA was isolated by drying and suspending in TE buffer or distilled water. Forward primer (27f): (5'-AGA GTT TGA TCM TGG CTC AG -3'; SEQ ID NO: 2) and reverse primer (1492r): (5'- GGT TAC CTT TGT TAC GAC) to amplify 16S rRNA of microorganisms TT-3'; SEQ ID NO: 3) was used. 17 μl of distilled water, 1 μl of forward primer, 1 μl of reverse primer, and 1 μl of DNA were added to a PCR premix (Bioneer, Cat No. K-2012) and mixed, followed by PCR. PCR conditions were repeated 30 times at 94°C for 5 minutes, 1 minute at 94°C, 40 seconds at 62°C, and 40 seconds at 72°C, and the reaction was terminated at 72°C for 7 minutes. The PCR reaction product was confirmed by electrophoresis on 0.8% agarose gel. As a result of analyzing the final nucleotide sequence and comparing it with the database to identify the lactic acid strain, the strain isolated by the present invention was found to be Lactobacillus plantarum, and the lactic acid strain was sent to the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center in December 2019. It was deposited on March 03 (KCTC18796P).

Lactobacillus plantarum V135 (L actobacillus plantarum V1 35) Analysis of utilization characteristics of KCTC18796P strain as a food additive composition

1. Acid resistance evaluation of Lactobacillus plantarum V135 strain

In order for lactic acid bacteria to reach the intestines, they must pass through the stomach with a pH of 2 after ingestion. In order to investigate the acid resistance to withstand these extreme conditions, the Lactobacillus plantarum V135 strain was inoculated in MRS broth medium (MRS broth medium; Difco, USA) and then cultured at 37° C. for 24 hours. After centrifugation (4,000×g, 4℃, 5min), it was washed twice with phosphate-buffered saline (PBS, pH7), and the bacteria _{were adjusted to OD 600} = 1.0 (10 ⁸ ~ 10 ⁹ cfu/ml) with PBS and used. did. 1ml of the lactic acid bacteria dilution was added to 9ml PBS (pH 2 and 7), followed by shaking and incubation at 37°C. After sampling by time (0hour, 30min, 1hour, 3hour) from the beginning of culture, diluted with MRS liquid medium, spread on MRS solid medium, incubated for 48 hours at 37°C, count the number of colonies on the plate medium to measure the number of viable cells and the results are shown in Table 1 below.

As shown in Table 1 below, it was confirmed that the Lactobacillus plantarum V135 strain had a small decrease in the number of viable bacteria even at pH 2 lower than the physiological activity and excellent acid resistance.

division Number of viable cells (cfu/ml) 0 h 30 min 1 h 3 h pH 7 6.30 × 10 ⁷ 8.10 × 10 ⁷ 5.37 × 10 ⁷ 5.20 × 10 ⁷ pH 2 6.50 × 10 ⁷ 4.64 × 10 ⁷ 4.46 × 10 ⁷ 4.91 × 10 ⁶

2. Evaluation of bile resistance of Lactobacillus plantarum V135 strain

Bile resistance of the Lactobacillus plantarum V135 strain according to the present invention was evaluated. Considering that the concentration of bile salts in the intestinal tract is around 0.1%, MRS liquid medium was prepared so that the concentrations of porcine bile salts were 0, 1, and 3%, respectively. The Lactobacillus plantarum V135 strain according to the present invention was inoculated into a sterile MRS broth and cultured at 37° C. for 24 hours, and then inoculated at 0.1% (v/v) each in an MRS broth containing bile salts. After inoculation, samples were collected 0, 0.5, 1, and 3 hours after incubation at 37°C, diluted in sterilized MRS liquid medium, spread on MRS plate medium, incubated at 37°C for 48 hours, and placed on the plate medium. The number of viable cells was determined by counting the number of colonies. The results are shown in Table 2 below, and since the Lactobacillus plantarum V135 strain maintained an appropriate number of bacteria not only at 1% similar to the actual concentration in the intestine, but also at a higher 3%, the Lactobacillus plantarum V135 strain was killed by bile. Without this, it can be a basis for confirming that it can sufficiently survive in the intestines of humans or animals.

division Number of viable cells (cfu/ml) 0 h 30 min 1 h 3 h Oxgall 0% 5.54 × 10 ⁶ 5.04 × 10 ⁶ 3.68 × 10 ⁶ 3.19 × 10 ⁷ Oxgall 1% 4.91 × 10 ⁶ 4.72 × 10 ⁶ 5.31 × 10 ⁶ 1.80 × 10 ⁷ Oxgall 3% 5.41 × 10 ⁶ 7.30 × 10 ⁶ 5.55 × 10 ⁶ 1.99 × 10 ⁷

3. Evaluation of co-aggregation ability of Lactobacillus plantarum V135 strain

In the anti-inflammatory action of lactic acid bacteria, cohesion with pathogenic harmful bacteria in the intestine is a very important factor while the lactic acid bacteria adhere to the intestine. In the present invention, E. coli ( Escherichia coli ), Escherichia coli ( Escherichia coli O157 ), Staphylococcus aureus , and typhimurium ( Salmonella typhimurium ) strains were used as indicator bacteria. As a culture medium, MRS liquid medium and Nutrient liquid medium (Difco; USA) were used for Lactobacillus plantarum V135 strain and Escherichia coli, respectively. The pre-culture of the Lactobacillus plantarum V135 strain was adjusted to OD ₆₀₀ = 1.0 (10 ⁸ ~ 10 ⁹ cfu/ml) and 1% inoculated in a new 5ml MRS medium, cultured for 24 hours and centrifuged (4,000 ×g, 4 ℃, 5 min) to recover the cells. Pathogenic bacteria were pre-cultured with a culture medium, _{adjusted to OD 600} = 0.1, inoculated, and cultured at 37° C. for 24 hours. This was resuspended in PBS buffer to adjust the number of viable cells to 1.0×10 ⁸ cfu/ml. After mixing 2 ml of each of the Lactobacillus plantarum V135 strain and the harmful bacteria suspension, stirring for 10 seconds, and keeping stationary at 37° C. for 5 hours, the coaggregation ability was investigated for each hour (0hour, 5hour). Coagulation ability was calculated by [Equation 1] below according to the method of Kos et al. (2003), and the results are shown in Table 3 below.

[Formula 1]

division Escherichia
coli Escherichia
coli O157 Salmonella
typhimurium Staphylococcus
aureus cohesive capacity (%) 74% 56% 41% 72%

As shown in Table 3 above, all of the harmful bacteria used in the experiment had a high aggregation rate with the Lactobacillus plantarum V135 strain. In particular, the aggregation rate of Lactobacillus plantarum V135 lactic acid bacteria and Escherichia coli was 74%, showing excellent intestinal adsorption capacity.

Lactobacillus plantarum V135 (L actobacillus plantarum V1 35) Dead cells of KCTC18796P strain

The production method of the Lactobacillus plantarum V135 strain dead cells is as follows.

The Lactobacillus plantarum V135 strain was inoculated into an edible medium and cultured at 37° C. for 24 hours to prepare a culture solution. In the stationary phase, when cell proliferation reached the maximum and contained a large amount of metabolites, the culture medium was first heated (sterilized) to 100° C. and rapidly cooled to 50° C. or less. For complete sterilization, second heating (sterilization) to 100°C and rapid cooling to 50°C or less were performed to prepare heat-treated lactic acid bacteria (hereinafter referred to as ‘dead cells’). Thereafter, excipients (or protective agents) such as dextrin, maltodextrin, and glucose were mixed with the dead cells as excipients, and then spray-dried and powdered. At this time, the inlet temperature of the spray drying was 180 ℃, the outlet temperature was 100 ℃ or less, and spray drying was performed.

Lactobacillus plantarum V135 (L actobacillus plantarum V1 35) Analysis of utilization characteristics of KCTC18796P strain and dead cells as pharmaceutical compositions

Lactobacillus plantarum V135 strain and harmful bacteria antibacterial activity, antioxidant effect, and alcoholic hepatitis improvement effect against the dead cells prepared in Example 3 were evaluated.

1. Antibacterial activity of Lactobacillus plantarum V135 dead cells

Antibacterial activity against dead cells of the novel Lactobacillus plantarum V135 strain was confirmed using an agar well diffusion assay. Escherichia coli O157, Salmonella typhimurium , and Bacillus cereus , which are food harmful bacteria, were cultured in NB (Nutrient broth) medium for more than 24 hours, and then 1 ml of the bacterial solution was dispensed on a nutrient agar medium and dried. Thereafter, wells were made using a sterile biopsy punch (Stiefel Biopsy Punch; Stiefel Laboratories, Research Triangle Park, NC, USA). Control (a) and Lactobacillus plantarum V135 dead cells (b) were dispensed into each well by 100 μl per well and incubated at 37° C. for 24 hours. After incubation, the inhibition ring formed around the well was confirmed to confirm the presence or absence of antibacterial sulfur, and the results are shown in FIG. 2 below.

As shown in FIG. 2, the Lactobacillus plantarum V135 strain showed high antibacterial activity against Escherichia coli O157, Salmonella typhimurium and Bacillus cereus , and the dead cells of the Lactobacillus plantarum V135 strain also against the three strains. It was confirmed that antibacterial activity was high.

2. Antioxidant activity of Lactobacillus platarum V135 dead cells

The antibacterial activity against dead cells of the novel Lactobacillus plantarum V135 strain was confirmed using a DPPH radical scavenging method. The dead cells of the Lactobacillus plantarum V135 strain were dissolved in sterile water so as to have a concentration of 0.5, 1, 5, and 10% and used in the experiment. The DPPH solution was dissolved in DMSO to prepare a concentration of 0.4 mM. The DPPH radical scavenging ability was measured by mixing 50 μl of sample and 50 μl of DPPH solution, reacting at room temperature for 10 minutes, and then measuring absorbance at 517 nm using a spectrophotometer. As a control, the absorbance value obtained after the reaction by adding 50 μl of sterile water instead of the sample was used. As a positive control, ascorbic acid, a well-known antioxidant, was used to compare the antioxidant activity of dead cells of Lactobacillus plantarum V135 strain. The DPPH radical scavenging activity (%) was calculated by the following [Formula 2], and the results are shown in FIG. 3 below.

[Formula 2]

DPPH radical scavenging activity (%) = (B-A)/B × 100

(A: absorbance of sample, B: absorbance of control)

As a result of measuring the DPPH radical scavenging activity (%), the Lactobacillus platarum V135 dead cells were 14.5, 27.6, 89.2, 94.3, and 94.5% at 0.5, 1, 5, 10, and 20%, respectively, as shown in FIG. 3 . It was confirmed to exhibit the DPPH radical scavenging ability (antioxidant activity). Ascorbic acid used as a positive control showed DPPH radical scavenging activity of 2.14 and 38.4% at 0.001 and 0.01%, respectively. Therefore, it was confirmed that the Lactobacillus plantarum V135 dead cells had an excellent DPPH scavenging ability and had antioxidant activity.

These results show that the dead cells of Lactobacillus plantarum V135 showed high antibacterial activity and antioxidant effect, while the pharmaceuticals of dead cells supplemented the safety of the material, external environment (acid resistance, bile resistance) and heat stability, which are the disadvantages of the strain. It is a result showing the applicability as a red composition and food additive composition.

3. Alcoholic hepatitis improvement effect of Lactobacillus plantarum V135 dead cells

< Experimental group setting and sample administration >

After acclimatizing 5-week-old male C57BL/6 mice supplied by Orient Bio (Seongnam, Korea) in a rodent cage for one week, the general condition was observed during the adaptation period, and only healthy individuals were used for the experiment. In the breeding environment, the temperature was 22±3℃ and the humidity was 50±5%, the light/dark cycle was maintained at 12 hours, and food and water were freely supplied and adapted to the basic diet for 1 week. Only mice determined to be healthy during the acclimatization period were grouped using a randomized method.

Group separation was as shown in Table 4 below, a normal control group (NC), an alcoholic hepatitis induction test control group (EtOH), a positive control group (ursodeoxycholic acid 50 mg/kg/day for 10 days, po) (PC), Lactobacillus planta Room V135 strain low concentration (4×10 ⁹ cfu/g) 125 mg/kg/day administration group (LP-L), Lactobacillus plantarum V135 strain high concentration group (4×10 ⁹ cfu/g) 250 mg/kg/day administration group (LP-H), Lactobacillus plantarum V135 dead cells low concentration (4×10 ⁹ cfu/g) 125 mg/kg/day administration group (HLP-L) and Lactobacillus plantarum V135 dead cells high concentration (4×10 ⁹ cfu/g) was separated as in the 250 mg/kg/day administration group (HLP-H), and a total of 7 groups were composed of 8 animals per group and the experiment was carried out.

experimental group test substance Dosage number of animals NC DW - 8 EtOH EtOH - 8 PC EtOH + ursodeoxycholic acid 50 mg/kg/day 8 LP-L EtOH + L. plantarum V135 strain 125 mg/kg/day 8 LP-H EtOH + L. plantarum V135 strain 250 mg/kg/day 8 HLP-L EtOH + L. plantarum V135 dead cells 125 mg/kg/day 8 HLP-H EtOH + L. plantarum V135 dead cells 250 mg/kg/day 8

The NC and EtOH groups were orally administered with DW once a day for a total of 10 days, and for the PC, LP-L, LP-H, HLP-L, and HLP-H groups, each test substance was orally administered once a day for a total of 10 days. After administration, 10 ml/kg of 60% alcohol was orally administered at intervals of 12 hours from 60 hours (2.5 days) before the last test substance administration to induce alcoholic hepatitis. Mice were sacrificed 4 hours after the last alcohol administration, and cardiac blood and liver tissues were extracted.

< Observation of pathological changes in liver tissue caused by alcohol of Lactobacillus plantarum V135 dead cells >

After fixing the extracted liver tissue in 10% formalin, a paraffin block was prepared, and a slide was prepared by making a section with a thickness of 5㎛. After staining with H&E (Hematoxylin & Eosin) solution, it was observed using a light microscope, and inflammatory cell infiltration, adipocyte infiltration, and sinusoidal vasodilation were scored. The results of observing the pathological changes in liver tissue caused by the Lactobacillus plantarum V135 strain and dead cells in mice induced by alcohol administration are shown in FIG. 4 below.

As shown in FIG. 4, in the alcoholic hepatitis induction test control group (EtOH), infiltration of inflammatory cells, fat glomerular infiltration and sinus vasodilation were observed in the liver tissue, Lactobacillus plantarum V135 strain (LP), the It was confirmed that the dead cells (HLP) of the strain significantly reduced the alcohol-induced clinical findings in a concentration-dependent manner. In particular, the Lactobacillus plantarum V135 dead cells (HLP) showed a tendency to further reduce the alcohol-induced clinical findings compared to the Lactobacillus plantarum V135 strain (LP).

< Observation of AST and ALT concentration in serum by alcohol of Lactobacillus plantarum V135 dead cells >

Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzyme activities were measured using a commercial assay kit. The results of observation of changes in AST and ALT, hepatocyte damage indexes, by the Lactobacillus plantarum V135 strain and dead cells in mice induced by alcohol administration are shown in FIG. 5 below.

As shown in FIG. 5, the Lactobacillus plantarum V135 strain (LP), the dead cell (HLP) of the strain showed a tendency to decrease the concentration of serum ALT and AST increased by alcohol in a concentration-dependent manner. became In particular, the Lactobacillus plantarum V135 dead cell high concentration group (HLP-H) showed a significant decrease in serum ALT and AST compared to the alcoholic hepatitis induction test control group (EtOH), and showed a superior effect than the positive control group (PC).

< Observation of inflammatory cytokine gene expression in liver tissue by alcohol of Lactobacillus plantarum V135 dead cells >

Total RNA was extracted from liver tissue using TRIzol solution, and absorbance was measured at 260 nm and 280 nm to confirm RNA concentration and purity. cDNA was synthesized from 100 ng of total RNA using the Prime Script RT solution kit. Real-time PCR was performed in a thermal cycler using SYBR Premix Ex Taq, and the reaction conditions were pre-denaturation at 95°C for 5 minutes, followed by 15 seconds at 95°C and 1 minute at 60°C, a total of 40 times. Using GAPDH as a control gene, the relative expression levels of inflammatory cytokines IL-1β and IL-6 were calculated, and the primer sequences used were interleukin-1β (IL-1β) and interleukin-6 (IL-6). The results of observing the change in the expression of inflammatory cytokines in the liver tissue by the Lactobacillus plantarum V135 strain and dead cells in mice induced by alcohol administration are shown in FIG. 6 below.

As shown in Figure 6, the expression of IL-1β was significantly decreased compared to the alcoholic hepatitis induction test control group (EtOH) in both the Lactobacillus plantarum V135 strain (LP) and dead cells (HLP) group, but IL-6 was confirmed to show a significant decrease only in the Lactobacillus plantarum V135 dead cells. These results indicate that the Lactobacillus plantarum V135 dead cells inhibit cytokine expression more than equivalent to that of the Lactobacillus plantarum V135 strain (LP).

< Observation of MAPK/AP-1 signaling and inflammatory signal protein expression in liver tissue by alcohol of Lactobacillus plantarum V135 dead cells >

Excessive hepatocyte apoptosis is a prominent pathological feature of alcohol-induced liver disease and plays an important role in hepatocellular apoptosis in liver diseases including acute and chronic hepatitis. Hepatocyte apoptosis leads to several forms of pathological liver damage, including hepatic dysfunction, fibrosis/cirrhosis and tumorigenesis. Thus, the balance between apoptotic activity and anti-apoptotic acitivity plays a pivotal role in liver disease progression and is a target for liver disease treatment. Mitogen-activated protein kinase (MAPK) is involved in cell apoptosis by various external stimuli (alcohol, stress, etc.), extracellular signal-regulated kinas (ERK), c-Jun N-terminal kinase (JNK/SAPK), It is composed of p38 kinase and regulates the expression of various genes by phosphorylation of transcriptional regulators.

After the liver tissue was put into RIPA buffer and homogenized, the supernatant was separated at 14,000 rpm for 15 minutes. The protein concentration of the sample thus obtained was quantified using a protein assay. 50 μg of liver tissue homogenate solution was mixed with 2× loading buffer, boiled at 95° C. for 5 minutes, and then separated by 10% SDS-PAGE. The isolated protein was transferred to PVDF paper and confirmed by Ponceasu S solution staining. After that, it was treated with a 5% non-fat milk powder solution diluted in TBS-T solution for 2 hours or more. Primary antibodies are phophorylated-SAPK/JNK (p-SAPK/JNK), SAPK/JNK, phosphorylated-Erk, Erk, phosphorylated-p38 MAPK, p38 MAPK, c-Jun, c-Fos, phosphorylated-nuclear factor kappa B ( p-NFκB), NFκB, iNOS, and COX-2 were diluted at a ratio of 1:500-1,000 and used. After washing thoroughly with TBS-T solution, the secondary antibody was diluted with goat anti-rabbit IgG(H+L)-horse radish peroxidase conjugate at a ratio of 1:5,000 and reacted. After reacting with ECL solution, the intensity of the band was quantified using a chemiluminescence image system. The expression level of the phosphorylated protein was calculated as a relative value based on the total protein expression level of the protein. The results of observing the protein expression change of the hepatitis-inducing factor by the Lactobacillus plantarum V135 strain and dead cells in mice induced by alcohol administration are shown in FIG. 7 below.

As shown in FIG. 7 , as a result of evaluating the expression of proteins related to apoptosis in liver cells, the administration of Lactobacillus plantarum V135 dead cells (HLP) showed that the expression of hepatocyte apoptosis-related protein factors by alcohol was all concentration-dependent. was found to decrease. Lactobacillus plantarum V135 strain (LP) showed a decrease in protein expression more than equivalent compared to.

< Observation of p65NF-κB protein expression in liver tissue by alcohol of Lactobacillus plantarum V135 dead cells >

Stimulation such as alcohol activates nuclear factors-κB (NF-κB), a transcription factor of the inflammatory response, and as a result, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (C0X-2) are expressed, resulting in excess nitric oxide (NO) and PGE2, causing inflammation. The results of observation of changes in the expression of inflammatory proteins in the liver tissue by the Lactobacillus plantarum V135 strain and dead cells in mice induced by alcohol administration are shown in FIG. 8 below.

As shown in FIG. 8 , as a result of confirming the protein expression in liver tissue of NF-κB, iNOS and C0X-2, which are inflammation-related factors, the administration of Lactobacillus plantarum V135 dead cells (HLP) was caused by alcohol-induced liver tissue. All of the inflammatory-related protein factor expression was decreased in a concentration-dependent manner, and it was confirmed that the Lactobacillus plantarum V135 strain (LP) showed a decrease in protein expression equal to or greater than that of the strain.

Taken together, these results can explain that Lactobacillus plantarum V135 dead cells can inhibit liver cell damage through alcohol-induced apoptosis and inflammation inhibition of liver cells.

In conclusion, through the experimental results of Examples 1 to 4, Lactobacillus plantarum V135 strain, dead cells of the strain or metabolites of the strain are gross liver damage, pathological changes and inflammatory cytokines for alcoholic hepatitis. There is an advantage that can be used as a pharmaceutical composition for the prevention, improvement or treatment of alcoholic hepatitis or a food additive composition for the prevention or improvement of alcoholic hepatitis because it has the effect of preventing, improving or treating hepatitis by inhibiting the production of caine, and Lactobacillus plus Lactobacillus plantarum V135 (Lactobacillus plantarum V135) KCTC strain kills through heat treatment to improve the disadvantages of live cells (stability, product applicability, etc.) There are advantages to providing

Above, the present invention has been described in detail with reference to examples, but the present invention is not limited to the above examples, and can be modified in various forms, and those of ordinary skill in the art within the technical spirit of the present invention It is clear that many variations are possible by the ruler. In addition, within the scope that does not depart from the technical spirit of the present invention described in the claims, various forms of substitution, modification and change will be possible by those skilled in the art, and it is also said that it falls within the scope of the present invention. something to do.

Claims (6)

Lactobacillus plantarum V135 ( Lactobacillus plantarum V135 ) KCTC18796P strain, a dead cell of the strain or a pharmaceutical composition for the prevention, improvement or treatment of alcoholic hepatitis containing the metabolite of the strain as an active ingredient. The method according to claim 1,
The pharmaceutical composition for the prevention, improvement or treatment of alcoholic hepatitis,
Lactobacillus plantarum V135 (Lactobacillus plantarum V135) KCTC18796P strain, the dead cells of the strain or metabolites of the strain per 1 g of the composition 1 × 10 ⁵ cfu to 1 × 10 ¹² cfu of alcoholic hepatitis, characterized in that it contains A pharmaceutical composition for prevention, improvement or treatment. The method according to claim 1,
The dead cells of the strain,
The process of preparing a culture solution by inoculating Lactobacillus plantarum V135 KCTC18796P strain into an edible medium, heating the culture solution to a temperature of 80°C or higher, and then rapidly cooling to a temperature of 60°C or lower is repeated at least once A pharmaceutical composition for the prevention, improvement or treatment of alcoholic hepatitis, characterized in that the heat treatment is carried out. Lactobacillus plantarum V135 (Lactobacillus plantarum V135) KCTC18796P strain, a dead cell of the strain or a food additive composition for the prevention or improvement of alcoholic hepatitis containing a metabolite of the strain as an active ingredient. 5. The method according to claim 4,
The food additive composition for the prevention or improvement of alcoholic hepatitis,
Lactobacillus plantarum V135 (Lactobacillus plantarum V135) KCTC18796P strain, the dead cells of the strain or metabolites of the strain per 1 g of the composition 1 × 10 ⁵ cfu to 1 × 10 ¹² cfu of alcoholic hepatitis, characterized in that it contains Food additive composition for prevention or improvement. 5. The method according to claim 4,
The dead cells of the strain,
After preparing a culture solution by inoculating Lactobacillus plantarum V135 KCTC18796P strain into an edible medium, heating the culture solution to a temperature of 80°C or higher, and then performing a rapid cooling process to a temperature of 60°C or lower at least once. Food additive composition for the prevention or improvement of alcoholic hepatitis, characterized in that the heat treatment is repeatedly carried out.

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