KR20230076270A - Composition for anti-inflammatory and anti-Helicobacter pylori containing fermented product of Ulmus Davidiana - Google Patents

Abstract

The present invention relates to an anti-inflammatory and anti-Helicobacter composition containing a fermented product of Ulmus davidiana bark. The Ulmus davidiana bark extract fermented with Bacillus licheniformis FLa3 strain (accession number KACC 81191BP) of the present invention inhibits the production of NO, TNF-α, and IL-8, increases the inhibition function of gastric mucosa adhesion, and is confirmed to have excellent antioxidant activity, so it can be usefully used as a health functional food for gastritis, gastric ulcer, duodenal ulcer, and gastric cancer.

Description

Composition for anti-inflammatory and anti-Helicobacter pylori containing fermented product of Ulmus Davidiana}

The present invention relates to a functional fermented material having anti-inflammatory and anti-Helicobacter pylori effects by fermenting radishes with Bacillus licheniformis FLa3 strain and a method for producing the same.

Helicobacter pylori is a gram-negative bacterium, which was first isolated and identified from human gastric mucosal tissue by Warren and Marshall in 1983. At first, it was named Campylobacter pyloridis, but it was named 'Helicobacter pylori' by citing the shape in the body and the name of the pylorus, which mainly lives in the stomach.

Helicobacter pylori is also found in mucous membranes such as the esophagus or duodenum, but most of them colonize and proliferate in contact with epithelial cells in the inside of the stomach, especially in the gastric mucous layer with a thickness of about 0.5 mm. Helicobacter pylori is known to be associated with the development of gastritis, gastric ulcer, peptic ulcer, duodenal ulcer and gastric cancer. Once infected with Helicobacter pylori, the infection persists for decades and is rarely eliminated spontaneously, so Helicobacter pylori is the main cause of chronic gastritis.

Currently, representative treatment methods for Helicobacter pylori rely on antibiotics such as metronidazole and amoxicillin, and repeated use of these drugs has been reported to cause increased antibiotic resistance and various side effects. Currently, efforts are being made to find extracts and active ingredients capable of inhibiting Helicobacter pylori using various natural materials.

Until recently, patents and reports related to Yugeunpi have been published on a composition for preventing and treating obesity containing Yugeunpi extract as an active ingredient (Publication No. 10-2013-0060806), and a composition for external application for skin containing citron, Yugeunpi and seaweed extracts (public disclosure). No. 10-2013-0015565), an anti-aging composition containing low- and high-molecular-weight hyaluronic acid and a polysaccharide extract isolated from Radix root bark (Publication No. 10-2009-0098083), containing Radix root bark extract, and having skin protection ability from ultraviolet rays and cosmetic compositions (Publication No. 10-2017-0015711).

Most of the studies on the physiological activity of Yugeunpi so far are mostly physiological activity assays such as inhibition of inflammation, anti-obesity, and skin moisturizing based on antioxidant activity. Research on physiological activity assays, extraction conditions and fermentation processes for maximum production of active ingredients is lacking.

Therefore, this technology shows that the fermented extract of Yugeunpi, which has not been previously studied, has the ability to inhibit adhesion to the gastric mucosa for anti-Helicobacter and the inflammatory substances induced by Helicobacter NO, TNF-a. By inhibiting the production of IL-8, it is effective for gastritis, gastric ulcer, gastric duodenal ulcer and gastric cancer, etc., in the field of utilization of health foods, pharmaceutical compositions and beverages, etc., as well as in the production and utilization of functional fermented products using useful microorganisms. It is a field related to

(001) Korean Registered Patent KR 10-1164876 (002) Korean registered patent KR 10-1789506

The present invention provides a composition for preventing or improving gastrointestinal diseases, including a fermented product of fermented radishes root with Bacillus licheniformis FLa3 (Accession No. KACC 81191BP).

The present invention provides a food composition for preventing or improving gastrointestinal diseases, including a fermented product of fermented radish root bark fermented with Bacillus licheniformis FLa3.

The present invention provides an antibacterial composition against Helicobacter pylori, including fermented radish root bark fermented with Bacillus licheniformis FLa3.

The present invention provides a composition for preventing or improving gastrointestinal diseases, including a fermented product of fermented radishes root with Bacillus licheniformis FLa3 (Accession No. KACC 81191BP).

In the present invention, "prevention" means any action that suppresses or delays the onset of a disease by administration of a composition according to an embodiment, and "improvement" refers to a state in which a disease is treated by administration of a composition according to an embodiment and It can mean any action that at least reduces the relevant parameter, eg, the severity of the symptom. The disease may mean a gastrointestinal disease.

Bacillus licheniformis FLa3 strain (KACC 81191BP) of the present invention may have celluase and pectinase activities.

When the composition for preventing, improving, or treating gastrointestinal diseases according to one embodiment includes a fermented product fermented with Bacillus licheniformis FLa3 (Accession No. KACC 81191BP), Bacillus licheniformis ) Other types of Bacillus licheniformis other than FLa3 ( Bacillus licheniformis ) Compared to compositions containing FLa3 bacteria, (i) prevention, improvement, or treatment of gastrointestinal diseases and / or (ii) anti-Helicobacter pylori efficacy may be superior.

In one example, that the composition has excellent anti-Helicobacter pylori efficacy means that it has excellent antibacterial activity against Helicobacter pylori strains or excellent activity to prevent apoptosis of cells (eg, gastric mucosal cells) infected by Helicobacter pylori. can mean

The "fermented product" means a product of enzymatic or metabolic decomposition of organic substances using microorganisms. In this application, "fermentation" may mean any activity or process including enzymatic or metabolic degradation of organic matter using microorganisms, but not a decay reaction.

The fermentation product may be a whole culture of the Bacillus licheniformis FLa3 strain, a dilution thereof, a concentrate, a dried product, a lyophilized product, a lysate, and/or a fraction thereof, and the concentrate is the culture It can be obtained by centrifugation or evaporation, and the dried product can be obtained by drying the culture using a dryer or the like, and the lyophilisate can be obtained by freeze-drying the culture using a freeze dryer or the like. The lysate may be obtained by physically or ultrasonically treating the strain or culture, and the fraction may be obtained by applying a method such as centrifugation or chromatography to the culture or lysate.

In one example, Bacillus licheniformis ( Bacillus licheniformis ) FLa3 (Accession No. KACC 81191BP) fermentation product fermented root bark is optimal fermentation conditions when the inoculation amount of 36 hours 37 ℃, pH 6, 10% (v / v) may be, but is not limited thereto.

In one example, the gastrointestinal disease may be caused by Helicobacter pylori infection.

In one example, the gastrointestinal disease may be at least one selected from the group consisting of gastritis, gastric ulcer, duodenal ulcer, peptic ulcer, and gastric cancer.

The gastritis refers to a state in which the inner wall of the stomach is inflamed, and the gastric ulcer may occur when a balance between a defense factor that protects the mucous membrane and an attack factor that causes mucosal damage is broken in the stomach. The attack factors that can be regarded as the cause of gastric ulcer include gastric acid, various digestive enzymes, bile, ingested drugs, alcohol, Helicobacter pylori infection, non-steroidal anti-inflammatory drugs, smoking, and the like.

The present invention provides a food composition for preventing or improving gastrointestinal diseases, including a fermented product of fermented radish root bark fermented with Bacillus licheniformis FLa3.

The above foods include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, health functions There are (sex) food and health food, and includes all food in the usual sense.

The health functional food (functional food) is the same term as food for special health use (FoSHU), and is a medicine processed to efficiently display bioregulatory functions in addition to nutritional supply, and has high medical effect. means food. Here, "function (sex)" means to obtain useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological functions. The food of the present application can be manufactured by a method commonly used in the art, and can be prepared by adding raw materials and ingredients commonly added in the art during the manufacture. In addition, the formulation of the food may also be prepared without limitation as long as the formulation is recognized as a food. The composition for food of the present application can be prepared in various types of dosage forms, and unlike general drugs, it has the advantage of using food as a raw material and has no side effects that may occur when taking drugs for a long time, and has excellent portability, Of the foods can be consumed as supplements to enhance the effect of preventing or improving gastric ulcer.

The health food (health food) means a food having an active health maintenance or promotion effect compared to general food, health supplement food (health supplement food) means a food for the purpose of health supplement. In some cases, the terms health functional food, health food, and health supplement food may be used interchangeably.

Specifically, the health functional food is a food prepared by adding the composition according to the example to food materials such as beverages, teas, spices, gum, confectionery, etc., or encapsulated, powdered, suspended, etc., when ingested. It means to bring about a specific effect, but unlike general drugs, it has the advantage of not having side effects that can occur when taking drugs for a long time by using food as a raw material.

Since the food composition according to one embodiment can be consumed on a daily basis, a high effect on the prevention or improvement of gastrointestinal diseases can be expected, so it can be used very usefully.

The food composition may further include a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier commonly used in the art may be used.

In addition, the food composition may include additional ingredients that are commonly used in food compositions and can improve smell, taste, and vision. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chrome (Cr) may be included. In addition, amino acids such as lysine, tryptophan, cysteine, and valine may be included.

In addition, the food composition may include preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder, high bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butyl hydroxy Loxytoluene (BHT), etc.), coloring agents (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite, etc.), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclemate, saccharin) , sodium, etc.), flavoring (vanillin, lactones, etc.), expanding agent (alum, D-potassium hydrogen stannate, etc.), strengthening agent, emulsifier, thickener (thickener), coating agent, gum base agent, foam inhibitor, solvent, improver, etc. food May contain food additives. The additive may be selected according to the type of food and used in an appropriate amount.

The composition according to one example may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, the food composition of the present application may be added in an amount of 50 parts by weight or less, specifically 20 parts by weight or less, based on the food or beverage when preparing food or beverage. However, when ingested for a long period of time for health and hygiene purposes, the amount below the above range may be included, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

An example of the food composition may be used as a health beverage composition, and in this case, as in conventional beverages, various flavoring agents or natural carbohydrates may be included as additional ingredients. The aforementioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrins and cyclodextrins; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as thaumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame may be used. The ratio of the natural carbohydrates may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 mL of the health drink composition of the present application.

In addition to the above, the health beverage composition includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol or a carbonating agent, and the like. In addition, it may contain fruit flesh for the manufacture of natural fruit juice, fruit juice beverages, or vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present application.

The food composition according to one embodiment may include various weight % if it can exhibit a preventive or ameliorative effect of gastrointestinal diseases, but, for example, the composition according to one embodiment is 0.00001 to 100% by weight relative to the total weight of the food composition, 0.01 to 80% by weight, or 10 to 50% by weight, but is not limited thereto.

The present invention provides an antibacterial composition against Helicobacter pylori, including fermented radish root bark fermented with Bacillus licheniformis FLa3.

The present invention Bacillus licheniformis ( Bacillus licheniformis ) It was confirmed that the fermented root bark extract fermented with the FLa3 strain suppressed the production of NO, TNF-α, and IL-8, enhanced the gastric mucosal adhesion inhibitory function, and had excellent antioxidant activity. It will be useful as a health functional food for gastritis, gastric ulcer, duodenal ulcer and stomach.

Figure 1 is a result of comparing and analyzing celluase and pectinase activities of strains isolated from salted fish.
Figure 2 is a result of phylogenetic analysis of the genetic information of the Bacillus licheniformis FLa3 strain isolated from salted fish.
Figure 3 is the result of evaluating the cytotoxicity and anti-inflammatory activity in order to select the optimal extraction solvent of Yugeunpi.
Figure 4 is the result of evaluating the cytotoxicity and anti-inflammatory activity to select the optimal extraction time of yugeunpi.
Figure 5 is the result of analyzing the optimal fermentation conditions of yugeunpi using Bacillus licheniformis strain.
Figure 6 is the result of analyzing the cytotoxicity according to the inoculated amount of extract and Helicobacter before and after fermentation of Yugeunpi for gastric cancer (AGS) cells.
Figure 7 is the result of analyzing the anti-Helicobacter effect of the extract before and after fermentation of Yugeunpi.
Figure 8 shows the analysis conditions of catechin, epicatechi, kaempferol and quercetin in the extract before and after fermentation of Yugeunpi.

Hereinafter, examples will be described in detail to aid understanding of the present application. However, the following examples are merely illustrative of the contents of the present application, but the scope of the present application is not limited to the following examples. The embodiments of the present application are provided to more completely explain the present application to those skilled in the art.

Example 1. Bacillus licheniformis ( Bacillus licheniformis ) Isolation and identification of FLa3 strain

In order to isolate microorganisms from Bandaengi salted seafood (traditional fermented food), salted seafood was diluted by a stepwise dilution method. After spreading the diluted salted fish broth on LB solid medium, it was cultured aerobically at 37° C. for 24 hours to isolate microorganisms. An experiment was conducted to select microorganisms having cellulase and pectinase activities of the isolated microorganisms. The agar medium used was cellulase clearing medium and pectinase screening medium. The previously cultured microbial suspension was inoculated into punched wells with a sterilized 8 mm cork borer and incubated at 37°C for 2 days. After culturing, strains producing cellulase and pectinase showing clear zones were selected by congo red staining. In order to select strains suitable for fermentation of yu geun pi, 1 g of yu geun pi powder was added to 27 mL of distilled water and sterilized at 121 ^° C for 15 minutes and used as a medium. After sufficiently cooling the medium, 3 mL of bacterial suspension (OD _{600 nm} = 1.0) was inoculated in PBS. Fermentation was carried out at 120 rpm and 37 ° C for 2 days, and the supernatant was used as an enzyme solution. The cellulase and pectinase activities of the primary selected strains were evaluated using the 3,5-dinitrosalicylic acid (DNS) method. Cellulase activity was measured using a 1% CMC solution (0.1 M citrate buffer, pH 5.5) as a substrate, and pectinase activity was measured using a 1% pectin solution (0.1 M citrate buffer, pH 5.5) as a substrate. 0.4 mL of substrate and 0.4 mL of enzyme solution were mixed and reacted at 50 ^° C for 30 minutes to hydrolyze the substrate, and 0.6 mL of DNS reagent was added and heated at 100 ^° C for 15 minutes. After cooling the mixture, the absorbance was measured at 540 nm. In order to measure the content of reducing sugar contained in the enzyme solution, the mixture (blank) was measured by adding a buffer instead of the substrate. Reducing sugar was calculated using a calibration curve of glucose and galacturonic acid, and relative enzyme activity was shown by comparing the concentrations of the produced reducing sugar.

As a result, four cellulose-degrading strains and pectin-degrading strains were inoculated, respectively, to proceed with fermentation of radishes. In order to select the optimal fermentation strain, the cellulase and pectinase activities of the fermentation broth inoculated with various strains were evaluated. Among the four strains, the FLa3 strain showed the highest cellulase and pectinase activities, and it was confirmed that the extraction yield was improved the most (FIG. 1).

In order to identify the FLa3 strain, the 16S rRNA nucleotide sequence of the strain was analyzed. The analyzed gene sequence was analyzed for homology with previously known microorganisms using NCBI's BLAST program, and a phylogenetic tree was created using MEGA7 program. As a result of the analysis, it was confirmed that the FLa3 strain showed 99% homology with Bacillus licheniformis and was a new strain that did not exist previously.

Therefore, the FLa3 strain with excellent cellulase and pectinase activity was finally named Bacillus licheniformis FLa3 and deposited at the National Institute of Agricultural Sciences Microorganism Bank (KACC) on November 15, 2021 (Accession No. KACC 81191BP), and is shown in SEQ ID NO: 1 (Fig. 2).

Example 2. Exploration of Optimal Extraction Conditions for Yugeunpi

Radiant root bark was dried and powdered in a dry oven at 50 ° C and used in the experiment. In order to select the optimal extraction solvent conditions, 200 mL of various solvents (distilled water, EtOH 30, 60, 95%) were added to 10 g of powder raw material and extracted for 5 hours. In order to select the optimal extraction time condition, 200 mL of the optimal solvent was added to 10 g of powder raw material, Extraction was performed for various times (1, 3, 5, 7, 9). The extract was lyophilized using a lyophilizer, and the dried powder was dissolved in an extraction solvent and used in the experiment. The extraction yield was calculated by measuring the weight of the lyophilized solid.

The anti-inflammatory effect was evaluated to select the optimal extraction conditions of radishes. The cytotoxicity range of each extract prepared according to various extraction conditions was confirmed. Using RAW264.7 cells, the cytotoxicity of the Eugenia extract was evaluated through the MTT assay. RAW264.7 cells were inoculated into a 96 well plate at a density of 1×10 ⁵ cells/mL and cultured for 24 hours. The culture medium was removed and dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) solution at concentrations of 62.5, 125, 250, 500 and 1,000 μg/mL of the radicle root. ) diluted in medium. Cells were treated with the diluted medium and cultured for 24 hours at 5% CO ₂ and 37°C. After incubation, 50 μL of MTT solution (5 mg/mL) was added to each well and incubated for 4 hours. Thereafter, the supernatant was removed, and 150 μL/well of DMSO was added to dissolve MTT formazan, and absorbance was measured at 540 nm using a microplate reader. Cytotoxicity was calculated using the formula:

Cytotoxicity (%) = (absorbance _sample / absorbance _control ) × 100

The optimal extraction conditions were selected by comparing the anti-inflammatory effects of the extracts prepared under various conditions. The concentration of NO produced in the inflammatory response induced by LPS was measured through griess reagent analysis. RAW264.7 cells were inoculated into a 24 well plate at a density of 1×10 ⁵ cells/mL and cultured for 24 hours. The culture medium was removed and each sample was diluted to 250 μg/mL in DMEM medium containing 10% FBS and 1% P/S solution. The diluted medium was treated with cells and cultured for 2 hours at 37°C and 5% CO ₂ conditions. Inflammation was then induced for 24 hours by adding a medium containing 1 μg/mL of LPS. After inducing inflammation, 100 μL of griess reagent was mixed with 100 μL of cell culture supernatant, reacted for 10 minutes, and absorbance was measured at 540 nm using a microplate reader. The nitrite concentration was calculated using the calibration curve of sodium nitrite, and NO production was calculated using the following formula.

NO production (%) = (concentration _sample / concentration _control ) × 100

As a result, the extract in all solvent conditions did not show cytotoxicity up to 250 μg/mL. As for the anti-inflammatory activity, the highest anti-inflammatory activity was shown by reducing NO production by 66.5% in the extract extracted with EtOH 95% compared to the LPS-treated group (100%), and EtOH 95% was selected as the optimal extraction solvent (FIG. 3 ).

In addition, the cytotoxicity and anti-inflammatory effects of the extracts extracted for 1, 3, 5, 7, and 9 hours were evaluated in order to select the optimal extraction time of radishes. No cytotoxicity was observed up to 250 μg/mL of the extract. Compared to the LPC treatment group (100%), the extract reduced NO production by 64.8-66.5%, but the difference according to the extraction time was not statistically significant, so the extraction time was selected as 1 hour (FIG. 4).

Example 3. Exploration of optimal fermentation conditions for Yugeun bark

Bacillus licheniformis FLa3 with high cellulase and pectinase activity was inoculated into LB broth and pre-cultured at 37°C for 24 hours. The cultured strain was harvested and suspended in PBS to prepare an inoculum of OD 1.0. In order to select the optimal fermentation conditions for radish root bark, various temperatures (25℃, 30℃, 37℃, 45℃), pH (5, 6, 7, 8, 9) and inoculation amount (5%, 10%, 15%) were used. compared. After mixing 1 g of Yugeunpi powder and 27 mL of distilled water, it was sterilized at 121 ° C. for 15 minutes. Thereafter, the strain suspension was inoculated and fermentation was performed under various conditions. In addition, the extraction yield of the fermented product was evaluated to confirm the relationship between the enzyme activity and the extraction yield. The optimal fermentation time was selected by comparing the extraction yield, pH, and Log (CFU/mL of FLa3) for 72 hours.

As a result, microbial fermentation is influenced by factors such as temperature, pH and inoculation amount. These factors show a high correlation between microbial growth and enzyme activity. Fermentation was carried out at 25, 30, 37, and 45 ° C. to select temperature conditions, which are one of the factors that play an important role in enzyme activity. The cellulase and pectinase activities of Bacillus licheniformis FLa3 according to the fermentation temperature were selected as the optimal fermentation temperature condition because the extraction yield was higher than that of other fermentation temperatures with a yield increase of about 37% compared to the control group at 37 ° C (Fig. 5A). Initial pH is one of the factors affecting fermentation efficiency, and the activities of pectinase and cellulase produced by Bacillus licheniformis FLa3 were evaluated under various pH conditions (5, 6, 7, 8, 9). When the initial pH was adjusted to 6, the highest active cellulase and pectinase activities appeared, and the initial pH was selected as 6 (FIG. 5B). Enzyme activity and extraction yield were compared according to the inoculation amount of microorganisms, and the highest enzymatic activity was obtained when the inoculation amount was 10% (v/v) (FIG. 5C). High microbial densities increase competition between substrates, nutrients and bacteria. Changes according to the fermentation period were evaluated under the condition of maximizing the activity of cellulase and pectinase to increase the extraction yield. Although the initial number of strains was 10.9 log CFU/mL, it tended to decrease continuously as fermentation progressed, and the change in pH showed similar results to the number of viable cells. As fermentation proceeded, the pH decreased from 6.1 to 5.5. The extraction yield increased from 3.60% to 4.91% in 36 hours. The long fermentation time was yielded due to unnecessary bioconversion, so the optimal fermentation time was selected as 36 hours (Fig. 5D).

Example 4. Cytotoxicity assay for AGS cells

The cytotoxicity of the extract before and after fermenting Yugeunpi on AGS (above) cells was evaluated by MTT assay. AGS cells were inoculated into a 96 well plate at a density of 1×10 ⁵ cells/mL and cultured for 24 hours. The culture medium was removed and samples were diluted to concentrations of 62.5, 125, 250, 500, 1,000 and 2,000 μg/mL in RPMI 1640 medium containing 10% FBS and 1% P/S solution. Cells were treated with the diluted medium and cultured at 5% CO ₂ and 37°C for 24 hours. After incubation, 50 μL of MTT solution (5 mg/mL) was added to each well and reacted for 4 hours. Thereafter, the supernatant was removed, and 150 μL/well of DMSO was added to dissolve MTT formazan, and absorbance was measured at 540 nm using a microplate reader. Cytotoxicity (%) was calculated using the formula:

Cytotoxicity (%) = (absorbance _sample / absorbance _control ) × 100

The cytotoxicity of Helicobacter pylori infection on AGS cells was evaluated by trypan blue exclusion test. The cells were inoculated into a 24 well plate at a density of 1×10 ⁵ cells/mL and cultured for 24 hours. The culture medium was removed and the Helicobacter pylori suspension was inoculated into RPMI 1640 medium containing 0.1% yeast extract. Cells were treated with the inoculated medium (MOI; 25, 50, 100, 200, 400) and cultured for 6 hours at 37°C and 5% CO ₂ conditions. Cells were separated from the cell culture dish using TrypLE express enzyme and mixed with the same amount of cell suspension 0.4% trypan blue. The number of viable cells was measured using a TC20 automated cell counter. Cytotoxicity (%) was calculated using the formula:

Cytotoxicity (%) = (number of cells _{Helicobacter treated group} / number of cells _{Helicobacter untreated group} ) × 100

As a result, it is the result of analyzing whether the treatment of the extract before and after fermentation of Yugeunpi shows cytotoxicity in AGS cells (Fig. 6A). Extracts of 6.25, 125, 250, 500, 1,000 and 2,000 μg/mL were treated and cytotoxicity was measured through MTT assay. As a result, toxicity was not observed at concentrations within 500 μg/mL, so the treatment concentration of each extract was determined in subsequent experiments. 125, 250 and 500 μg/mL. The result is that Helicobacter infection is cytotoxic to AGS cells. After treating AGS cells with Helicobacter suspensions at MOI (multiplicity of infection) 25, 50, 100, 200, and 400, cytotoxicity was measured by trypan blue analysis. 200 was selected (Fig. 6B).

Example 5. Anti-Helicobacter Efficacy Evaluation of Yugeunpi Extract

The adhesion inhibitory ability of the extract before and after fermentation of Yugeunpi against Helicobacter was evaluated by measuring the urease activity. AGS cells were inoculated into a 6 well plate at a density of 1×10 ⁵ cells/mL and cultured for 24 hours. Cells were treated with Helicobacter pylori suspension (MOI 200) and various concentrations (125, 250, 500 μg/mL) of the extract diluted in RPMI medium containing 0.1% yeast extract and incubated for 6 hours at 37°C and 5% CO ₂ . did Non-attached Helicobacter strains were removed by washing twice with PBS, and the raw amount of attached Helicobacter strains was evaluated using urease reagent. 2 mL of Urease reagent (20% urea, 0.012% phenol red-containing PBS, pH 6.5) was added to each well and incubated at 37°C for 1 hour. The absorbance was measured at 562 nm using a microplate reader, and the adhesion capacity (%) was calculated using the following formula.

Attachment activity (%) = (absorbance _sample / absorbance _Helicobacter ) × 100

The effect of the extract before and after fermenting Yugeunpi on the inflammatory response induced by Helicobacter was evaluated by measuring the production of NO, TNF-α and IL-8. AGS cells were seeded in a 24 well plate at a density of 1×10 ⁵ cells/mL and cultured for 24 hours. Helicobacter pylori suspension (MOI 200) and extracts of various concentrations (125, 250, 500 μg/mL) were treated with diluted cells in RPMI medium containing 0.1% yeast extract and cultured at 37°C and 5% CO ₂ for 6 hours. did To evaluate NO production, 100 μL of griess reagent was mixed with 100 μL of cell culture supernatant, reacted at room temperature for 10 minutes, and absorbance was measured at 540 nm using a microplate reader. The nitrite concentration was calculated using a calibration curve of sodium nitrite, and NO production was calculated using the following formula.

NO production (%) = (concentration _sample / concentration _control ) × 100

In order to measure the production of pro-inflammatory cytokines, TNF-α and IL-8, the cell culture supernatant was measured using an ELISA kit according to the manufacturer's protocol.

As a result, as a result of analyzing the urease activity to measure the Helicobacter adhesion inhibitory ability of the Yugeunpi extract, the efficacy was increased by about 15% compared to before fermentation with 50% adhesion inhibitory ability at 500 μg / mL of the fermented extract (Fig. 7A). In order to suppress the production of NO induced by Helicobacter pylori, as a result of treating the extract before and after fermentation of yugeunpi, it was found that the production of NO was further suppressed by fermentation at 500 μg/mL to 69% before fermentation and 57% after fermentation. (Fig. 7B). It was found that the production of pro-inflammatory cytokines produced by Helicobacter was reduced by treating the extract before and after fermentation of yugeunpi, and in the case of TNF-α at 500 μg/mL, 78% of the extract before fermentation compared to the Helicobacter-treated group (100%), fermentation 74% of the extract was produced (FIG. 7C), and in the case of IL-8, compared to the Helicobacter pylori-treated group (100%), 75% of the pre-fermentation extract and 65% of the fermented extract were produced (FIG. 7D). In conclusion, it was found that the anti-inflammatory effect was enhanced through fermentation.

Example 6. Extraction yield, total polyphenol content and antioxidant activity of Yugeunpi extract

The fermented product prepared under each condition was lyophilized to remove moisture, and extracted for 1 hour by adding 20 mL of 95% ethanol. After concentrating the extract using a vacuum rotary evaporator (Eyela N-1100, Japan), it was freeze-dried using a freeze dryer. The extraction yield was calculated by weight of lyophilized solid.

The Folin-Cilcalteu method was used to evaluate the content of total polyphenols known to have antioxidant activity. 0.1 mL of methanol, 0.5 mL of distilled water, and 0.1 mL of sample were mixed. Then, the mixture was treated with 50 μL of 1 N Folin-Ciocalteu's phenol reagent and reacted for 5 minutes. Then, 0.1 mL of 5% Na ₂ CO ₃ was added and reacted for 1 hour. The absorbance was measured at 725 nm using a microplate reader, and the total polyphenol content was calculated through a gallic acid calibration curve.

Superoxide dismutase (SOD)-like activity was measured using the SOD Assay kit-WST according to the manufacturer's protocol. To measure the SOD activity of each extract, extracts were prepared at concentrations of 3.2, 16, 80, 400, 2,000, and 10,000 μg/mL, and the sigmoidal (sigmoid, 4 parameter) equation of SigmaPlot (SigmaPlot 10.0 Windows version, USA) was used. inhibition concentration 50 (IC ₅₀ ) was calculated using

The hydrogen peroxide scavenging activity was measured using the 2,2-azinobis(3-ethylbenzthiazolin)-6-sulfonicacid(ABTS)-peroxidase system. To 0.4 mL of samples of various concentrations (3.2, 16, 80, 400, 2,000, 10,000 μg/mL), 0.1 mL of 10 mM H ₂ O ₂ and 0.5 mL of PBS (pH 5.0) were added and reacted at 37°C for 5 minutes. made it Thereafter, 0.15 mL of 1.25 mM ABTS and 0.15 mL of 1 U/mL peroxidase were added to the mixture and reacted at 37°C for 10 minutes. Then, it was measured at 405 nm using a microplate reader. IC ₅₀ was calculated using the sigmoidal (sigmoid, 4 parameter) equation of SigmaPlot.

As a result, as shown in Table 1 below, the extraction yield, total polyphenol content, SOD-like activity, and hydrogen peroxide scavenging activity of the extract before and after fermentation of Yugeunpi were shown. Through fermentation, the extraction yield was 4.91%, which increased by about 36.4% compared to the extract before fermentation. In addition, the total polyphenol content was increased by 26.2% through fermentation from 125.56 mgGAE/g of the extract before fermentation to 158.46 mgGAE/g of the fermented extract. IC ₅₀ values of SOD-like activity and hydrogen peroxide scavenging activity of the fermented extract were about 164.06 and 4246.20 μg/mL, confirming that the antioxidant activity was enhanced through fermentation.

sample transference number(%) Total polyphenol content
(mg GAE/g) SOD-like activity
(IC ₅₀ , μg/mL) Hydrogen peroxide scavenging activity
(IC ₅₀ , μg/mL) Eugene Bark Extract 3.60±0.03 126.56±0.63 302.00±1.08 4920.40±1.17 Yugeunpi fermented extract 4.91±0.05 158.46±1.46 164.06±1.53 4246.20±1.17 ascorbic acid 103.87±1.25 96.07±1.19

Example 7. HPLC (High performance liquid chromatography)

Catechin and epicatechin of the extract before and after fermentation were analyzed using an Agilent 1220 Infinity II system equipped with a ZORBAX Eclipse Plus C18 column (5 μm, 250 × 4.6 mm, Agilent Technologies, USA) of a Variable Wavelength Detector.

Analysis conditions are shown in FIG. 8 . To evaluate the contents of catechin, epicatechin, kaempferol, and quercetin, 5,000 mg/L extract and 5, 10, 15, 20, and 25 mg/L standard materials prepared with methanol were used. The contents of catechin, epicatechin, kaempferol, and quercetin were calculated using a standard curve.

As a result, as shown in Table 2, the change in the effective content according to fermentation was quantitatively analyzed using an HPLC analyzer. Representatively, the contents of catechin, epicatechin, quercetin, and kaempferol were increased through fermentation. Catechin increased from 1.59 mg/g before fermentation to 4.43 mg/g after fermentation, about 171.7%, and epicatechin increased from 0.88 mg/g before fermentation to 2.15 mg/g after fermentation, about 144.3%. Quercetin was increased by about 27.3% from 0.11 mg/g before fermentation to 0.14 mg/g after fermentation, and kaempferol was increased by about 16.7% from 0.06 mg/g before fermentation to 0.07 mg/g after fermentation.

sample Content (mg/g) Catechin Epicatechin Quercetin Kaempferol Eugene Bark Extract 1.59±0.01 0.88±0.01 0.11±0.00 0.06±0.00 Yugeunpi fermented extract 4.32±0.02 2.15±0.01 0.14±0.00 0.07±0.00

Example 8. Antibacterial activity evaluation of Yugeunpi extract

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extracts before and after fermentation were evaluated against Helicobacter pylori SS1, Salmonella typhimurium KVCC-BA0000693 and Listeria monocytogenes KVCC-BA0000087 strains.

Pathogenic microbial suspensions were inoculated into extracts before and after Yugeunpi at various concentrations. The concentration of the extract was adjusted to 5, 7.5, 10, 25, 37.5, 50, 75, 100, 125, 150, 175 and 200 mg/mL for evaluation. The initial concentration of pathogenic bacteria was adjusted to 1.0×10 ⁸ CFU/mL ( Helicobacter pylori SS1) and 2.0×10 ⁸ CFU/mL ( Salmonella typhimurium KVCC-BA0000693, Listeria monocytogenes KVCC-BA0000087). A 96-well plate was incubated at 37°C for 24 hours (Helicobacter culture under 10% CO ₂ conditions), and the turbidity of the bacterial growth medium was measured at 600 nm. The concentration at which turbidity did not increase was determined as the MIC. Concentrations higher than the MIC were spread on BHI agar or CB agar and incubated at 37° C. for 24 hours to evaluate MBC. MBC was determined as the concentration at which colonies were not observed.

As a result, as shown in Table 3 below, the antibacterial activity was analyzed by evaluating the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extract before and after fermenting radishes root against intestinal pathogenic microorganisms. The antibacterial activity of Yugeunpi against Helicobacter was confirmed as MIC 150 mg/mL, MBC 175 mg/mL before fermentation, and MIC 100 mg/mL, MBC 125 mg/mL after fermentation, indicating that the antibacterial activity against Helicobacter was enhanced through fermentation. could In the case of salmonal, the MIC was 125 mg/mL and MBC 175 mg/mL before fermentation, and the MIC 100 mg/mL and MBC 125 mg/mL after fermentation were confirmed. Listeria was identified as MIC 100 mg/mL and MBC 150 mg/mL before fermentation and 75 mg/mL and 125 mg/mL after fermentation.

strain sample Minimum inhibitory concentration
(mg/mL) Minimum sterilization concentration
(mg/mL) Helicobacter pylori Eugene Bark Extract 150 175 Yugeunpi fermented extract 100 125 Salmonella typhimurium Eugene Bark Extract 125 175 Yugeunpi fermented extract 100 125 Listeria monocytogenes Eugene Bark Extract 100 150 Yugeunpi fermented extract 75 125

From the above description, those skilled in the art to which this application pertains will be able to understand that this application can be implemented in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present application should be construed as including all changes or modifications derived from the meaning and scope of the following patent claims and their equivalent concepts rather than the detailed description above.

Name of Depositary Institution: Research Institute of Agricultural Biotechnology

Accession number: KACC81191BP

Entrusted date: 20211115

<110> Chonbuk National University Industry-Academic Cooperation Foundation <120> Composition for anti-inflammatory and anti-Helicobacter pylori containing fermented product of Ulmus Davidiana <130> DHP21-483 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1170 <212> DNA <213> Bacillus licheniformis <400> 1 gggggggtgt tttggttttt tggatggcgg gtctatacat gcaagtcgag cggacagatg 60 ggagcttgct ccctgatgtt agcggcggac gggtgagtaa cacgtgggta acctgcctgt 120 aagactggga taactccggg aaaccggggc taataccgga tgcttgtttg aaccgcatgg 180 ttcaaatata aaaggtggct tttagctacc acttacagat ggacccgcgg cgcattagct 240 agttggtgag gtaacggctc accaaggcaa cgatgcgtag ccgacctgag agggtgatcg 300 gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc 360 cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt 420 aaaactctgt tgttagggaa gaacaagtac cgttcgaata gggcggtacc ttgacggtac 480 ctgaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag 540 cgttgtccgg aattattggg cgtaaagcgc gcgcaggcgg tttcttaagt ctgatgtgaa 600 agcccccggc tcaaccgggg agggtcattg gaaactgggg aacttgagtg cagaagagga 660 gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca ccagtggcga 720 aggcgactct ctggtctgta actgacgctg aggcgcgaaa gcgtggggag cgaacaggat 780 tagataccct ggtagtccac gccgtaaacg atgagtgcta agtgttagag ggtttccgcc 840 ctttagtgct gcagcaaacg cattaagcac tccgcctggg gagtacggtc gcaagactga 900 aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc 960 aacgcgaaga accttaccag gtcttgacat cctctgacaa ccctagagat agggcttccc 1020 cttcgggggc agagtgacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg 1080 ggttaagtcc cgcaacgagc gcaacctttg atcttagttg ccagcattca gttggggcac 1140 tctaagggtg actggccggt gacaaacccg 1170

Claims (5)

Bacillus licheniformis ( Bacillus licheniformis ) A composition for preventing or improving gastrointestinal diseases, including fermented yugeunpi fermented with FLa3 (Accession No. KACC 81191BP). The composition according to claim 1, wherein the gastrointestinal disease is caused by Helicobacter pylori infection. The composition according to claim 1, wherein the gastrointestinal disease is at least one selected from the group consisting of gastritis, gastric ulcer, duodenal ulcer, peptic ulcer, and gastric cancer. Bacillus licheniformis ( Bacillus licheniformis ) A food composition for preventing or improving gastrointestinal diseases comprising a fermented product of fermented radishes with FLa3. Bacillus licheniformis ( Bacillus licheniformis ) Antimicrobial composition for Helicobacter pylori ( Helicobacter pylori ) containing fermented radish root bark fermented with FLa3.

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