KR20240121187A - Novel strains having anti- Helicobacter pylori activity, and the use thereof - Google Patents

Abstract

The present invention relates to a novel strain having anti-Helicobacter pylori activity and its use.

Description

{Novel strains having anti- Helicobacter pylori activity, and the use thereof}

The present invention relates to a novel strain having anti-Helicobacter pylori activity and its use.

Helicobacter pylori infection is widespread throughout the world and is one of the most common chronic infections in humans. Helicobacter pylori, which infects 50% of the world's population, was first isolated and identified from human gastric mucosa by Warren and Marshall in 1983 and has been known as an important factor in the development of gastritis, gastric ulcer, duodenal ulcer, and gastric cancer.

The World Health Organization (WHO) has designated Helicobacter pylori as a group 1 carcinogen for gastric cancer, and Helicobacter pylori causes various gastric diseases in most people, persisting throughout their lives unless eradicated artificially. Helicobacter pylori infection varies depending on age, race, and education level, and is also related to socioeconomic conditions during the growth period. In particular, in developing countries, Helicobacter pylori infection mainly occurs during childhood, but in developed countries, while childhood infection is low, infection continues steadily into adulthood, with an annual rate of 0.3% to 0.5%. Helicobacter pylori infection is a chronic infection that persists throughout most people unless eradication is attempted, but its infection route and prevention have not yet been clearly identified. The age-specific characteristics of Helicobacter pylori infection in Korea were low at an average of 17.2% in children under 15 years of age, and 66.9% in adults over 16 years of age, indicating a transition from the infection pattern of developing countries to the infection pattern of developed countries.

The route of infection of Helicobacter pylori has not been fully elucidated, but current research results have revealed that, firstly, it is transmitted from person to person, and secondly, infection mainly occurs within the family during childhood. Factors that can affect Helicobacter pylori infection have been analyzed, such as gender, body mass index, blood type, education level, economic level, diabetes, fatty liver, peptic ulcer, smoking and drinking history, etc. Helicobacter pylori produces urease to break down urea into carbon dioxide and ammonia, and the ammonia produced during this process neutralizes gastric acid, allowing it to survive in the stomach where strong acid acts. It is known that when infected in the stomach, it attaches to the mucus layer or mucosa to form colonies and directly causes inflammation in the stomach, causing various digestive diseases.

One strategy for treating diseases caused by Helicobacter pylori infection as described above is eradication therapy to eliminate Helicobacter pylori. This is a method of administering triple therapy using 'proton pump inhibitor (PPI)-amoxicillin-clarithromycin' for 1 to 2 weeks, which is the most widely used treatment method to date. However, even the triple therapy as described above does not exceed 80% in eradication rate, and although sequential therapy, simultaneous therapy, and modified sequential therapy are being utilized, the occurrence of fundamental antibiotic resistance cannot be ruled out, and it is difficult to completely eliminate Helicobacter pylori existing in the mucosal layer of the gastric epithelium. Accordingly, in the treatment of diseases caused by Helicobacter pylori infection, in addition to eradication therapy, research and development of new treatments that can restore damage to the gastric mucosa and even the mucus-producing function are needed, as well as new technologies that can suppress the growth of Helicobacter pylori and inhibit attachment to the gastric mucosa in the early stage of infection.

One object of the present invention is to provide a novel strain.

Another object of the present invention is to provide a composition comprising the novel strain.

Another object of the present invention is to provide a use of a composition comprising the novel strain.

To achieve the above object, one aspect of the present invention provides at least one strain selected from the group consisting of Lactiplantibacillus plantarum DS1073 deposited under Accession No. KCTC 15280BP, Lactiplantibacillus plantarum DS1902 deposited under Accession No. KCTC 15281BP, Lactiplantibacillus pentosus DS1989 deposited under Accession No. KCTC 15282BP, and Bifidobacterium longum DS1089 deposited under Accession No. KCTC 15283BP.

In addition, in order to achieve the above purpose, another aspect of the present invention provides a composition comprising at least one selected from the group consisting of the strain, a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution.

In addition, in order to achieve the above purpose, another aspect of the present invention provides a pharmaceutical composition, a health functional food composition, a food composition or a feed/feed additive composition for preventing, improving or treating Helicobacter pylori infection, comprising at least one selected from the group consisting of the strain, a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution and an extract of the culture solution.

The four novel strains of the present invention not only exhibit activity in inhibiting the growth and survival of Helicobacter pylori, but also have the effect of inhibiting the expression or activity of the amiE gene, which is a pathogenic factor of Helicobacter pylori. In addition, they have activity in inhibiting attachment of Helicobacter pylori to the stomach, particularly to the stomach apex, so that these four novel strains have the advantage of being useful as effective ingredients for preventing, improving, or treating diseases caused by infection with Helicobacter pylori.

However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

Figure 1 shows the results of an experiment confirming the growth inhibitory effect of four novel strains of the present invention on Helicobacter pylori P1 strain.
Figure 2 shows the results of an experiment confirming the survival inhibitory effect of four novel strains of the present invention on Helicobacter pylori P1 strain.
Figure 3 shows the results of an experiment confirming the inhibitory effect of four novel strains of the present invention on the expression of the amiE gene in Helicobacter pylori P1 strain.
Figure 4 shows the results of an experiment confirming the inhibitory effect of four novel strains of the present invention on the attachment of Helicobacter pylori P1 strain to the gastric vestibule.
Figure 5 shows the results of an experiment confirming the effect of the novel strain of the present invention on the inhibition of colonization of Helicobacter pylori P1 strain in the stomach.

Hereinafter, the present invention will be described in detail.

1. New strain

One aspect of the present invention provides a novel Lactiplantibacillus plantarum strain, a novel Lactiplantibacillus pentosus strain or a novel Bifidobacterium longum strain.

The Lactibacillus plantarum strain of the present invention may include 16S rRNA having a base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and in particular, may be Lactibacillus plantarum DS1073 strain deposited with the Korean Collection for Type Culture (KCTC) of the Korea Research Institute of Bioscience and Biotechnology on January 10, 2023 under the accession number KCTC 15280BP or Lactibacillus plantarum DS1902 strain deposited under the accession number KCTC 15281BP.

In addition, the Lactibacillus pentosus strain of the present invention may include 16S rRNA having a base sequence of sequence number 3, and in particular, may be the Lactibacillus pentosus DS1989 strain deposited with the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology under the accession number KCTC 15282BP on January 10, 2023.

In addition, the Bifidobacterium longum strain of the present invention may include 16S rRNA having a base sequence of sequence number 4, and in particular, may be the Bifidobacterium longum DS1089 strain deposited with the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology under the accession number KCTC 15283BP on January 10, 2023.

Probiotics are live microorganisms that provide health benefits, and play an important role in suppressing harmful bacteria and increasing beneficial bacteria in the intestinal flora. To be recognized as probiotics, they must be able to survive stomach acid and bile acid, have a beneficial effect in the intestines, be nontoxic, and be nonpathogenic.

Accordingly, the strains of the present invention may have acid resistance. The acid resistance refers to a property of the strain to survive well in an acidic environment, and may refer to a property of surviving even after 1 hour or more, for example, 1.5 hours or more, 2 hours or more, 2.5 hours or more, and especially 3 hours or more, in an acidic environment of pH 5 or lower, for example, pH 4 or lower, pH 3.5 or lower, and particularly, an environment of pH 3 or lower. In a specific embodiment of the present invention, it was confirmed that the novel strains of the present invention still survived well even when cultured for 3 hours under conditions of pH 3.0, thereby having excellent acid resistance.

In addition, the strains of the present invention may be cholestatic. The cholestatic property refers to a property of the strain to survive well in a biliary environment where bile distributed from the gallbladder exists, and may refer to a property of surviving in a biliary environment where bile such as oxgall and bile salts exists at a concentration of 5% or less, for example, 4.5% or less, 4% or less, 3.5% or less, and especially 3% or less, for 6 hours or more, for example, 10 hours or more, 12 hours or more, 16 hours or more, 20 hours or more, 22 hours or more, and especially 24 hours or more. In a specific embodiment of the present invention, it was confirmed that the novel strains of the present invention still survived well even when cultured for 24 hours under conditions containing 3% (w/v) of bile salt, and thus had excellent cholestatic property.

Meanwhile, the strains of the present invention may have physiological characteristics that enable them to be utilized as probiotics. In particular, the strains may exhibit antibacterial activity against Helicobacter pylori.

Specifically, the strains may have an effect of inhibiting the growth of Helicobacter pylori. The growth of Helicobacter pylori means that the number of Helicobacter pylori cells increases, and inhibition of the growth of Helicobacter pylori may mean that the rate at which the number of Helicobacter pylori cells increases is slowed down, or that the number of Helicobacter pylori cells no longer increases. In a specific embodiment of the present invention, when a culture solution of the novel strain of the present invention was treated to a medium for culturing Helicobacter pylori P1 strain, it was confirmed that the growth rate of the Helicobacter pylori strain was slowed down by about 30 to 70% compared to when it was not treated (see Fig. 1).

In addition, the strains may have an effect of inhibiting the survival of Helicobacter pylori. The survival may mean that Helicobacter pylori exists in an undead state, and the inhibition of the survival may mean that Helicobacter pylori is killed. In a specific embodiment of the present invention, when a culture solution of the novel strain of the present invention was treated to a medium for culturing Helicobacter pylori P1 strain, it was confirmed that about 30 to 40% of Helicobacter pylori strains were killed compared to when the medium was not treated (see Fig. 2).

In addition, the strains may have an effect of inhibiting the expression or activity of a metabolic activity-related gene of Helicobacter pylori. For example, the metabolic activity-related gene may be an amiE gene, and may inhibit the transcription of the amiE gene itself, or may inhibit the activity of a protein expressed from the amiE gene. In a specific embodiment of the present invention, when a culture solution of the novel strain of the present invention was treated to a medium for culturing a Helicobacter pylori P1 strain, it was confirmed that the amount of mRNA of the amiE gene transcribed in the Helicobacter pylori P1 strain was reduced by about 30 to 70% compared to when the medium was not treated (see FIG. 3).

Meanwhile, the strains may have an effect of inhibiting the attachment of Helicobacter pylori to the inner wall of the stomach. In particular, the stomach may be the gastric antrum. If Helicobacter pylori cannot attach to the inner wall of the stomach, Helicobacter pylori cannot approach the inner wall of the stomach in the first place, and thus damage to the stomach by Helicobacter pylori may not occur in the first place. In addition, if the attachment of Helicobacter pylori that has already been attached to the inner wall of the stomach to the inner wall of the stomach is inhibited, it can no longer attach to the inner wall of the stomach, and thus damage to the stomach by Helicobacter pylori may not progress any further. In a specific embodiment of the present invention, three-dimensional hAGOs (human antrum gastric organoids) that mimic the 'antrum' of the stomach at the organ level were manufactured from human pluripotent stem cells (hPSCs), and then a two-dimensional culture platform for evaluating adhesion was constructed using the same. When the two-dimensional culture platform was infected with a Helicobacter pylori strain and the novel strain culture of the present invention was treated, it was confirmed that the level of Helicobacter pylori attached to the hAGOs of the two-dimensional culture platform was reduced to 50% or less compared to when it was not treated (see FIG. 4).

In addition, the strains may have an effect of reducing the colonization of Helicobacter pylori. In a specific embodiment of the present invention, when a culture of the Lactiplantibacillus plantarum DS1073 strain of the present invention was treated to a mouse model infected with Helicobacter pylori, it was confirmed that the colonization of Helicobacter pylori was significantly reduced compared to when it was not treated (see Fig. 5).

Meanwhile, the strains of the present invention may utilize glucose, mannitol, lactose, sucrose, maltose, salicin, xylose, arabinose, esculin, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose, trehalose, etc. as carbon sources in terms of sugar utilization.

In a specific embodiment of the present invention, the culture solution of the strains of the present invention was inoculated into a tube containing glucose (GLU), mannitol (MAN), lactose (LAC), sucrose (SUC), maltose (MAL), salicin (SAL), xylose (XYL), arabinose (ARA), gelatin (GEL), esculin (ESC), glycerol (GLY), cellobiose (CEL), mannose (MNE), melezitose (MLZ), raffinose (RAF), sorbitol (SOR), rhamnose (RHA), or trehalose (TRE), and then the sugar utilization ability of the strains was measured. As a result, it was confirmed that the strains of the present invention can metabolize glucose, mannitol, lactose, sucrose, maltose, salicin, xylose, arabinose, esculin, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose and trehalose as carbon sources, but cannot metabolize gelatin.

In addition, the strains of the present invention may not produce toxic substances, and the toxic substances may be, for example, indole or urea.

Furthermore, the strains of the present invention may not produce β-glucuronidase. The β-glucuronidase refers to an enzyme that promotes hydrolysis, and β-glucuronidase is an enzyme that hydrolyzes various alcohols, phenols, amines, etc. into compounds (glucuronides) containing glucuronic acid, and exists in many living things such as bacteria, fungi, plants, and animals. For example, it is known that glucuronic acid is contained in human sweat and secreted, and that it is involved in the production of substances that cause glandular odor through the metabolism of bacteria that live on the skin, and in particular, it has been reported to be related to colon cancer.

In addition, the strains of the present invention may not exhibit hemolysis. The hemolysis refers to the property of the strain to destroy red blood cells, and in order for the strain to be safely used as a probiotic, it must not exhibit hemolysis.

In a specific embodiment of the present invention, the strains of the present invention were analyzed for their ability to produce indole, urea, and β-glucuronidase. As a result, it was found that the strains of the present invention had no ability to produce toxic substances such as indole and urea, and could not produce β-glucuronidase, which is associated with colon cancer, confirming that the strains of the present invention are safe as probiotics.

In addition, in a specific embodiment of the present invention, the strains of the present invention were cultured in a blood medium and analyzed for α, β and γ hemolysis, and as a result, the strains of the present invention did not exhibit hemolysis, confirming that they are safe as probiotics.

In addition, the strains of the present invention may have intestinal adhesion. The intestinal adhesion refers to a property of the strain to adhere to intestinal cells and not be detached. If the intestinal adhesion is excellent, the time for which the strain can remain in the intestines increases, and the effect of improving intestinal flora, bowel movement, and promoting intestinal health as a probiotic can be maintained for a long time. Specifically, the intestinal adhesion refers to a condition in which, after the strain reaches the intestines, it adheres to intestinal cells and remains in the intestinal environment for 12 hours or more, for example, 15 hours or more, 18 hours or more, 21 hours or more, and especially 24 hours or more, at least 0.5%, for example, 1% or more, 1.5% or more, 2% or more, 2.5% or more, and especially 3% or more of the initial number of viable cells are attached to the intestinal cells.

In a specific embodiment of the present invention, the strains of the present invention were treated to Caco-2 cells and cultured using glucose as a carbon source, and after washing with a PBS solution, the intestinal adhesion rate (%) of the strains was confirmed. As a result, the intestinal adhesion rate of the strains was measured to be about 3.35% to 12.63%, confirming that they have excellent intestinal adhesion ability.

In addition, the strains may not exhibit resistance to antibiotics, or may have low resistance to the antibiotics. The resistance may be extrinsic resistance, and the extrinsic resistance refers to resistance that can be induced by the introduction of a resistance gene to an antibiotic into another external bacterium through a mobile means such as a plasmid or transposon. Therefore, the strains of the present invention that do not exhibit extrinsic resistance to antibiotics do not horizontally transfer the resistance gene to harmful bacteria existing in the intestines, so there is no concern that harmful bacteria in the intestines will acquire extrinsic resistance and cause resistance problems to antibiotics.

The antibiotics to which the strains of the present invention do not exhibit resistance are not particularly limited, as long as they are known in the technical field to which the present invention belongs, and may be, for example, penicillin antibiotics, tetracyclines antibiotics, macrolide antibiotics, sulfonamide antibiotics, amphenicol antibiotics, aminoglycoside antibiotics, etc., and specifically, vancomycin, erythromycin, tetracycline, etc.

In a specific embodiment of the present invention, antibiotic resistance was tested for the strains using MTS ^TM (MIC Test Strip) (Liofilchem). As a result, the Lactibacillus plantarum DS1073 strain was confirmed to have lower resistance to ampicillin, clindamycin, tetracycline, and chloramphenicol than that described in the EFSA guide (European Food Safety Authority antibiotic safety criteria). In addition, the Lactibacillus plantarum DS1902 strain was confirmed to have lower resistance to ampicillin and clindamycin than that described in the EFSA guide, and to have the same antibiotic resistance as the reference value for erythromycin. Meanwhile, the Lactibacillus pentosus DS1989 strain was confirmed to have lower resistance to ampicillin, clindamycin, and tetracycline than that described in the EFSA guide, and to have the same antibiotic resistance as the reference value for erythromycin and chloramphenicol. In particular, in Europe, it has been said for a long time that lactic acid bacteria cannot be commercialized if they can horizontally transfer antibiotic resistance genes to other bacteria, and in Korea, the Ministry of Food and Drug Safety recently inserted a clause on antibiotic resistance transfer into the standards for judging food ingredients of microorganisms, so the strains of the present invention have the advantage of being recognized as safe as probiotics and thus are advantageous for commercialization.

2. Composition comprising the novel strain of the present invention

Another aspect of the present invention provides a composition comprising at least one selected from the group consisting of the novel strain of the present invention described above, a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution as an effective ingredient.

The above culture medium is obtained by culturing the novel strain of the present invention, and may be the culture medium itself containing cells of the strain, or a culture supernatant obtained by removing cells therefrom, and may also be a filtrate, concentrate or dried product thereof. The culture medium from which the cells have been removed contains components produced and secreted by the novel strain of the present invention, such as metabolites, and thus may have a probiotic effect with excellent antibacterial activity against Helicobacter pylori.

The above concentrate increases the solid concentration of the culture solution, and may be a concentrate of a culture solution containing cells of the novel strain of the present invention, or a concentrate of a culture supernatant from which cells of the novel strain of the present invention have been removed. The concentrate may be concentrated by vacuum concentration, plate concentration, thin film concentration, etc., but is not limited thereto, and may be performed at a temperature of 40°C to 60°C using, for example, a known concentrator. The content of the culture solution included in the composition of the present invention may be appropriately adjusted depending on the concentration of the concentrate.

The above dried product includes, but is not limited to, a product dried by a method such as freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, spray drying, foam drying, high-frequency drying, or infrared drying.

The above extract means an extract obtained from the culture solution or a concentrate thereof, and may include an extract, a diluted or concentrated extract, a dried product obtained by drying the extract, or a adjusted or purified product thereof, or a fraction obtained by fractionating the same.

In addition, the composition is suitable for ingestion together with the novel strain of the present invention and may additionally contain other known ingredients or lactic acid bacteria having antibacterial activity against Helicobacter pylori when ingested.

The composition containing the novel strain of the present invention can be manufactured in the form of a unit dose or by being placed in a multi-dose container by formulating it using a carrier, excipient and/or additive according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and can be manufactured. At this time, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablets, capsules, gels (e.g., hydrogels) or lyophilizer, and may additionally include a dispersant, stabilizer or cryoprotectant as the additives.

Specifically, in the case of the lyophilizer, it includes using the strain in the form of a powder by lyophilizing it together with a cryoprotectant, and the cryoprotectant may be skimmed milk powder, maltodextrin, dextrin, trehalose, maltose, lactose, mannitol, cyclodextrin, glycerol and/or honey. In addition, it includes using it by mixing it with a preservation carrier, adsorbing it, drying it and solidifying it, and the preservation carrier may be diatomaceous earth, activated carbon and/or defatted steel.

A composition including the novel strain of the present invention can be manufactured through a step of mixing at least one selected from the group consisting of a strain, a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution with any one of the carriers, excipients, or additives.

The description of the strain, carrier, excipient, and additive is as described above. When using a cryoprotectant as the additive, the novel strain of the present invention and the cryoprotectant may be mixed, the mixture may be frozen at -45 degrees Celsius to -30 degrees Celsius, dried at 30 degrees Celsius to 40 degrees Celsius, ground in a mixer, and manufactured in the form of a freeze-dried powder. Specifically, the freezing process may be a vacuum freezing process under temperature conditions of -45 degrees Celsius to -30 degrees Celsius and pressure conditions of 5 to 50 mTorr for 65 to 75 hours.

When a cryoprotectant is further included in the composition of the present invention, damage or death of the strain can be suppressed during the freeze-drying process of the composition, and the composition in the freeze-dried form has advantageous advantages during the storage, distribution, and preservation processes. In addition, the composition in the freeze-dried form can be ingested in the form of a powder, and in this case, the strain in the composition can exhibit its activity while growing or metabolizing in the body.

3. Use of the novel strain of the present invention or a composition containing the same

Another aspect of the present invention provides a composition comprising the novel strain of the present invention described above for the prevention, improvement or treatment of Helicobacter pylori infection.

The above Helicobacter pylori infection refers to all diseases that can be caused by Helicobacter pylori, such as gastritis, gastric ulcer, duodenal ulcer, nonulcer dyspepsia syndrome, gastric MALT lymphoma, gastric hyperplastic polyp, gastric cancer, gastrointestinal cancer, pancreatitis, inflammatory bowel disease, or functional digestive disorders such as upper abdominal fullness, upper abdominal pain, belching, abdominal distension, early fullness, nausea, vomiting, reflux, heartburn, and decreased appetite.

The above prevention may mean any action that blocks, suppresses or delays symptoms caused by infection with Helicobacter pylori. In addition, the above improvement or treatment may mean any action that improves or benefits symptoms caused by infection with Helicobacter pylori.

In particular, the composition may be a medicine, a food, or a feed. If the composition is a medicine, it may be a pharmaceutical composition for preventing or treating Helicobacter pylori infection, if the composition is a food, it may be a health functional food composition for preventing or improving Helicobacter pylori infection or a general food composition, and if the composition is a feed, it may be a feed composition or a feed additive composition for preventing or improving Helicobacter pylori infection.

In one embodiment of the present invention, when the composition is a pharmaceutical composition for preventing or treating Helicobacter pylori infection, the pharmaceutical composition may be prepared in a unit dosage form or may be prepared by introducing the composition into a multi-dose container by formulating the composition using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains. At this time, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablets, capsules or gel (e.g., hydrogel), and may additionally include a dispersant or a stabilizer.

In addition, the strain contained in the pharmaceutical composition may be delivered in a pharmaceutically acceptable carrier such as a colloidal suspension, powder, saline solution, lipids, liposomes, microspheres, or nano-spheres. They may form a complex with or be associated with a carrier, and may be delivered in vivo using a carrier system known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances, or fatty acids.

In addition, pharmaceutically acceptable carriers may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, which are commonly used in formulations. In addition, lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, etc. may be further included in addition to the above ingredients. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition according to the present invention can be administered orally or parenterally during clinical administration, and can be used in the form of a general pharmaceutical preparation. That is, the pharmaceutical composition of the present invention can be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing a herbal extract or a fermented herbal product with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include withepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerol, and gelatin.

The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiotherapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention and treatment of Helicobacter pylori infection.

The concentration of the effective ingredient included in the composition of the present invention can be determined in consideration of the purpose of treatment, the condition of the patient, the required period, etc., and is not limited to a specific range of concentrations. The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the 'pharmaceutically effective amount' means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dosage level can be determined according to the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, the concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent, or can be administered in combination with a therapeutic agent for a disease caused by another pollutant or a therapeutic agent for improving skin aging, and can be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects by considering all of the above factors, and this can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption rate of the active ingredient in the body, inactivation rate, excretion rate, type of disease, and concomitantly administered drugs, and may increase or decrease depending on the route of administration, severity of Helicobacter pylori infection, sex, weight, age, etc.

In another embodiment of the present invention, when the composition is a health functional food composition or a general food composition for preventing or improving Helicobacter pylori infection, the health functional food composition or the food composition can inhibit the growth, survival, or expression/activity of a pathogenic factor of Helicobacter pylori, or inhibit the ability of Helicobacter pylori to adhere to the stomach.

When the health functional food composition of the present invention is used as a food additive, the health functional food composition can be added to food as it is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The amount of the effective ingredient can be used appropriately depending on its intended use (prevention or improvement). Generally, when manufacturing food or beverage, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw material. However, in case of long-term intake for health purposes, the amount can be less than the above range, and since there is no problem in terms of safety, the effective ingredient can be used in an amount greater than the above range.

There are no special restrictions on the types of the above health functional foods or foods. Examples of the above health functional foods or foods include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea drinks, alcoholic beverages, vitamin complexes, dairy products, fermented milk, etc., and include all health functional foods or foods in the conventional sense.

In particular, the above health functional food is a food with high medical and healthcare effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition, and can obtain useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects. The above health functional food can be manufactured by a method commonly used in the technical field of the present invention, and can be manufactured by adding raw materials and ingredients commonly added in the industry. In addition, the formulation of the above health functional food can be manufactured in various forms without limitation as long as it is a formulation recognized as a health functional food, and unlike general drugs, it has the advantage of having no side effects that may occur when taking drugs for a long period of time since it uses food as a raw material, and has the advantage of excellent portability.

The health functional food of the present invention includes ingredients that are usually added during food manufacturing, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when manufactured as a drink, it may include natural carbohydrates or flavoring agents as additional ingredients in addition to the effective ingredients. The natural carbohydrates are preferably monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, etc.), or sugar alcohols (e.g., xylitol, sorbitol, erythritol, etc.). The flavoring agent may be a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) or a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).

In addition to the above health functional food composition, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. may be further contained. The ratio of these added components is not particularly important, but is generally selected in the range of 0.01 to 0.1 parts by weight with respect to 100 parts by weight of the health functional food composition of the present invention.

In addition, in another embodiment of the present invention, when the composition is a feed composition or feed additive composition for preventing or improving Helicobacter pylori infection, the feed composition or feed additive composition may be added as a feed additive composition for the purpose of preventing or improving Helicobacter pylori infection. The feed additive of the present invention corresponds to an auxiliary feed under the Feed Management Act.

The term "feed" in the present invention may mean any natural or artificial diet, meal, etc., or a component of said meal, which is suitable for an animal to eat, ingest, or digest. The type of said feed is not particularly limited, and feed commonly used in the relevant technical field may be used. Non-limiting examples of said feed include plant-based feed such as grains, roots, food processing by-products, algae, fibers, pharmaceutical by-products, fats, starches, meal, or grain by-products; animal-based feed such as proteins, inorganic substances, fats, minerals, fats, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

Meanwhile, the present invention provides a method for preventing or treating Helicobacter pylori infection, comprising a step of administering the pharmaceutical composition to a subject.

The subject may be a human or an animal other than a human, and may not be infected with Helicobacter pylori or may be infected with Helicobacter pylori. If the subject is not infected with Helicobacter pylori, Helicobacter pylori infection can be prevented by administering a pharmaceutically effective amount of the pharmaceutical composition to the subject, and if the subject is infected with Helicobacter pylori, Helicobacter pylori infection can be treated by administering a pharmaceutically effective amount of the pharmaceutical composition to the subject.

The formulation, administration method, dosage, and concentration of the active ingredient contained in the pharmaceutical composition are as described above.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[Example 1]

Establishment of four new strains

[1-1] Isolation and identification of strains of the present invention

Four strains (Representative 28, Representative 295, Representative 299, and Representative 150) were isolated from the feces of a 21-month-old baby and kimchi samples. For the four strains isolated as described above, the base sequences of 16S rRNA of the four strains were analyzed by PCR using the universal primers 27F (5'-AGAGTTTGATCMTGGCTCA-3': SEQ ID NO: 5) and 1492R (5'-TACGGYTACCTTGTTACGACTT-3': SEQ ID NO: 6).

As a result, the 16S rRNA base sequences of the above four strains were as shown in Table 1 below, as shown in Sequence Numbers 1 to 4, respectively. And, as a result of analysis based on the base sequence of the above 16S rRNA, it was confirmed that 28 representative strains showed 99.86% homology with the previously reported Lactiplantibacillus plantarum standard strain, 295 representative strains showed 99.60% homology with the previously reported Lactiplantibacillus plantarum standard strain, 299 representative strains showed 99.93% homology with the previously reported Lactiplantibacillus pentosus standard strain, and 150 representative strains showed 99.85% homology with the previously reported Bifidobacterium longum standard strain. At this time, sequence comparison with each of the above standard strains was performed on the EZ BioCloud server (https://eabiocloud.net/identify).

Accordingly, each of the four strains above was named as DS1073 for the representative 28 strain, DS1902 for the representative 295 strain, DS1989 for the representative 299 strain, and DS1089 for the representative 150 strain, as shown in Table 1 below. Thereafter, each strain was deposited at the Korean Collection for Type Culture (KCTC) of the Korea Research Institute of Bioscience and Biotechnology on January 10, 2023, and was assigned a deposit number as shown in Table 1 below.

Strain designation 16S rRNA Accession number Representative 28 Lactiplantibacillus plantarum DS1073 Sequence number 1 KCTC 15280BP Representative 295 Lactiplantibacillus plantarum DS1902 Sequence number 2 KCTC 15281BP Representative 299 Lactiplantibacillus pentosus DS1989 Sequence number 3 KCTC 15282BP Representative 150 Bifidobacterium longum DS1089 Sequence number 4 KCTC 15283BP

Each of the four new strains described above was inoculated onto the MRS (Man, Rogosa, and Sharpe) medium and cultured under anaerobic conditions at 37°C for 24 to 36 hours to reach the stationary phase, and then the culture was centrifuged at 3,000xg for 10 minutes. The precipitated pellet was obtained, stored at 65°C for 30 minutes for low-temperature sterilization, passed through a 0.22 μm filter, and stored at -80°C for use in the experiment.

[1-2] Whole genome analysis of the strain of the present invention

The whole genomes of the novel strains of the present invention separated as described above were analyzed. Specifically, complete whole genome sequencing (WGS) and draft genome analysis were performed, and the identity and functionality of the strains, such as genome homology analysis with a standard strain, phylogenomic characteristic analysis, and specific gene and metabolic pathway analysis, were investigated using the genome analysis results. In addition, in order to investigate the stability at the genome level, the whole genome was secured, the amino acid sequence of the coding sequence (CDS) was extracted, and then the homology of toxic genes was searched from the predicted protein encoding genes in the genome to confirm whether the strains had toxic genes. The genome homology analysis was searched using the Antibiotic Resistance Genes DB and the Comprehensive Antibiootic Resistance DB. In addition, in order to search for functional genes of the strains of the present invention, errors in the sequencing results were corrected through manual curation of the assembled genome, the structure prediction of the genes and functional gene tagging were performed, and a genome map was created. Specifically, standard strains and functional comparison strains were selected from GeneBank DB, CDSs were extracted from genome sequences, and then orthologous gene clustering of predicted protein sequences was performed using All to All BLAST and Markov Cluster Algorithm (MCL). Thereafter, the genetic characteristics of the strains of the present invention were analyzed by comparing and analyzing the genes of the strains of the present invention with the strains of the comparison target strains.

As a result, as described in Table 2, among the four novel strains of the present invention, the Bifidobacterium longum DS1089 strain possesses the antibiotic resistance gene erm(X), but the other three strains (DS1073, DS1902, and DS1989) were confirmed to not possess resistance and virulence genes, indicating that they can be utilized as probiotics.

Strain name Separation circle Whole genome (Mb) Resistance and virulence genes Lactiplantibacillus plantarum DS1073 Feces 3.25 doesn't exist Lactiplantibacillus plantarum DS1902 kimchi 3.22 doesn't exist Lactiplantibacillus pentosus DS1989 kimchi 3.82 doesn't exist Bifidobacterium longum DS1089 Feces 2.41 erm(X)

[Example 2]

Characterization of four new strains

In order to confirm whether the four new strains isolated and identified in Example 1 above have characteristics that can be utilized as probiotics, experiments were conducted on the overall metabolic activity including sugar utilization, acid resistance, bile resistance, intestinal adhesion, and antibiotic resistance in MRS medium.

[2-1] Confirmation of overall metabolic activity

In order to confirm the sugar utilization, toxic substance productivity, gelatin decomposition ability, enzyme activity including β-glucuronidase productivity, and hemolysis of the strains of the present invention, MRS agar medium was spread and prepared. The sugar utilization, toxic substance productivity, and gelatin decomposition ability analyses were conducted by suspending the strain colonies in API 20A Medium to a turbidity of 3 McFarland or higher and inoculating them into each well using the API 20A kit (Bio-Merieux, France) according to the provided instructions. In addition, since the β-glucuronidase has been reported to be associated with colon cancer, the enzyme activity including the productivity of β-glucuronidase was analyzed using the API ZYM kit. Next, the hemolysis evaluation confirmed α, β, and γ hemolysis on a sheep blood agar plate.

The types of sugars confirmed in this experiment are glucose (GLU), mannitol (MAN), lactose (LAC), sucrose (SUC), maltose (MAL), salicin (SAL), xylose (XYL), arabinose (ARA), gelatin (GEL), esculin (ESC), glycerol (GLY), cellobiose (CEL), mannose (MNE), melezitose (MLZ), raffinose (RAF), sorbitol (SOR), rhamnose (RHA), and trehalose (TRE). The types of toxic substances confirmed in this experiment are indole and urea.

The prepared fungal solution was inoculated into each sugar tube and cultured for 24 hours. Then, one drop of BCP reagent was added to all sugar tubes (except URE, GEL, and ESC) and the reaction results were read according to the reading table. For each reaction, + was indicated if positive and - was indicated if negative, and the results are shown in Table 3.

DS1073 DS1902 DS1989 D-glucose + + + D-mannitol + + + D-lactose + + + sucrose + + + D-maltose + + + salicin + + + D-xylose + + + L-arabinose + + + gelatin - - - esculin + + + glycerol + + + D-cellobiose + + + D-mannose + + + D-melezitose + + + D-raffinose + + + D-sorbitol + + + D-rhamnose + + + D-trehalose + + + Indol production - - - Urea production - - - Gelatin resolution - - - β-glucuronidase - - - Hemolysis - - - Alkaline phosphatase + + + Esterase (C4) - - + Esterase Lipase (C8) - - + Lipase (C14) - - - Leucine arylamidase + + + Valine arylamidase + + + Crystine arylamidase + + + Trypsin - - - α-chymotrypsin - - - Acid phospatase + + + Naphtol-AS-BI-phosphohydrolase + + + α-galactosidase + + + β-galactosidase + + + α-glucosidase + + + β-glucosidase + + + N-acetyl-β-glucosaminidase + + + α-mannosidase - - - α-fucosidase - - -

As a result, as described in Table 3 above, it was confirmed that the novel strains of the present invention metabolize glucose, mannitol, lactose, sucrose, maltose, salicin, xylose, arabinose, esculin, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose and trehalose. In addition, it was confirmed that they did not produce toxic substances such as indole and urea, did not produce β-glucuronidase, and did not have the ability to decompose arabinose, gelatin, glycerol and rhamnose. In addition, it was observed that the strains of the present invention did not exhibit hemolysis.

[2-2] Acid resistance check

The three new strains isolated and identified in the above Example 1 were inoculated into MRS medium and pre-cultured at 37°C, and the concentration of each strain was adjusted to OD ₆₀₀ = 1. Then, each strain was inoculated at a concentration of 1% (v/v) into 10 mL of MRS medium adjusted to pH 3.0, and the survival rate was confirmed after culturing for 3 hours.

Strain Total number of colonies forming units (CFU) at 0h Total bacterial count (CFU) at 3h Survival rate (%) Lactiplantibacillus plantarum DS1073 4.0 x 10 ⁷ CFU 3.0 x 10 ⁷ CFU 75.0 Lactiplantibacillus plantarum DS1902 2.9 x 10 ⁷ CFU 1.7 x 10 ⁷ CFU 58.0 Lactiplantibacillus pentosus DS1989 4.9 x 10 ⁷ CFU 6.9 x 10 ⁶ CFU 139.4 Bifidobacterium longum DS1089 - - -

As a result, as described in Table 4 above, it was confirmed that all three novel strains of the present invention are basically resistant to an acidic environment.

[2-3] Confirmation of biliary tract

Each strain cultured in the above Example [2-2] was inoculated into 10 mL of MRS medium containing 3% (w/v) of bile salt (Oxoid ^TM ) at a concentration of 10 ⁵ CFU/mL, and the survival rate was confirmed after culturing for 12 hours.

Strain Total number of colonies forming units (CFU) at 0h Total bacterial count (CFU) at 12h Survival rate (%) Lactiplantibacillus plantarum DS1073 3.4 x 10 ⁶ CFU 4.3 x 10 ⁵ CFU 12.0 Lactiplantibacillus plantarum DS1902 3.9 x 10 ⁶ CFU N.C >200 Lactiplantibacillus pentosus DS1989 1.4 x 10 ⁶ CFU N.C >200 Bifidobacterium longum DS1089 - - -

As a result, as described in Table 5 above, it was confirmed that all three novel strains of the present invention have resistance to an environment containing bile.

[2-4] Antibiotic resistance confirmation

In order to confirm the antibiotic resistance of the strains of the present invention, each strain was spread on MRS agar medium and prepared. An antibiotic resistance test was performed according to the manufacturer's instructions using MTS ^TM (MIC Test Strip) (Liofilchem) for antibiotics commonly used in the EFSA (European Food Safety Authority) guide.

The antibiotics used in this experiment were ampicillin, clindamycin, erythromycin, tetracycline, and chloramphenicol, and the concentration at the point where the antibiotic strip and the inhibition zone met was set as the minimum inhibitory concentration. After each of the strains of the present invention was smeared on the prepared medium, the antibiotic strip was placed, and the medium was cultured at 37°C for 48 hours, and the minimum inhibitory concentration (MIC) at which growth was inhibited was measured, and the results are shown in Table 6.

control Ampicillin
(Amp) Erythromycin
(Ery) Clindamycin
(Cln) Tetracycline
(Tet) Chloramphenicol
(Chr) EFSA guide
(μg/ml) 2 1 2 32 8 DS1073
(μg/ml) Growth 0.94 1.5 0.75 12 6 DS1902
(μg/ml) Growth 0.19 1 1 64 12 DS1989
(μg/ml) Growth 0.12 1 1 16 8

As a result, as described in Table 6 above, all three novel strains of the present invention were confirmed to have antibiotic resistance lower than the reference value of the EFSA guide for ampicillin and clindamycin. Meanwhile, in the case of the DS1073 strain, the antibiotic resistance was lower than the reference value for tetracycline and chloramphenicol, in the case of the DS1902 strain, the antibiotic resistance was the same as the reference value for erythromycin, and in the case of the DS1989 strain, the antibiotic resistance was lower than the reference value for tetracycline, and the antibiotic resistance was the same as the reference value for erythromycin and chloramphenicol. Therefore, since the strains of the present invention do not horizontally transfer resistance genes to harmful bacteria existing in the intestine, there is no concern that harmful bacteria in the intestine will acquire external resistance and cause resistance problems to antibiotics, and thus, they have an advantage in that they are also advantageous for commercialization as probiotics.

[2-5] Checking the attachment ability

In order to evaluate the intestinal adhesion ability of the strains of the present invention, Caco-2 cell line was used. Specifically, Caco-2 cell line was cultured at 37℃ in a 5% CO ₂ incubator (PHC, MCO-230AIC, Indonesia) using MEM medium containing 10% FBS and 1% penicillin-streptomycin, dispensed into a 24-well plate at a concentration of 1x10 ⁵ cells/well, and cultured for 14 days. Then, the cultured cells were suspended in MEM medium to adjust the final concentration to 1x10 ⁸ CFU/ml, inoculated into each well at 1ml, and cultured for 2 hours. At this time, glucose was used as the carbon source for each strain. Thereafter, the cultured strains were washed five times with PBS (Phosphate Buffered Saline) solution, treated with 0.5% trypsin-EDTA to detach non-attached Caco-2 cells from the plate, diluted with PBS and plated on MRS plate medium, and then the number of viable cells after 24 hours was measured to evaluate the intestinal adhesion ability of the strains of the present invention. At this time, Lacticazei Bacillus rhamnosus GG strain (KCTC 5033) and Bifidobacterium longum R0175 strain (Hyperbiotics, PRO-Bifido) were used as controls.

As a result, as described in Table 7 below, all three novel strains of the present invention were confirmed to have excellent intestinal adhesion ability. Specifically, the DS1073 strain had an intestinal adhesion ability of about 3.35%, the DS1989 strain had an intestinal adhesion ability of about 5.02%, and the DS1902 strain was particularly confirmed to have a significantly high intestinal adhesion ability of 12.63%.

Strain name Adhesion (%) Standard Deviation (SD) Lactiplantibacillus plantarum DS1073 3.35** 0.627 Lactiplantibacillus plantarum DS1902 12.63** 3.752 Lactiplantibacillus pentosus DS1989 5.02** 3.515 Lacticazybacillus rhamnosus GG 3.09** 0.759 Bifidobacterium longum R0175 0.27* 0.662

(*p<0.5; **p<0.05; ***p<0.001)

[Example 3]

Confirmation of antibacterial effects of new strains against Helicobacter pylori

The antibacterial effect against Helicobacter pylori was confirmed for the four new strains isolated and identified in Example 1 above.

[3-1] Confirmation of growth inhibition effect of Helicobacter pylori P1 strain

Helicobacter pylori strain _P1 was cultured in Brucella medium containing 10% FBS (Gibco), 1 ug/mL nystatin (Kisanbio), 5 ug/mL trimethoprim (Kisanbio), and 10 ug/mL vancomycin (Kisanbio) under microaerobic culture conditions at ₃₇ °C with 5-10% O 2 and 5-10% CO 2 and grown until OD ₆₀₀ reached 0.18-0.2 (before exponential-mid log phase). Then, the cultures of the four novel strains isolated and identified in Example 1 were treated twice every 24 hours at a concentration of 3.3% and cultured for 48 hours again. And the OD ₆₀₀ value of the culture solution was measured to confirm the degree of growth inhibition of the Helicobacter pylori strain.

As a result, as illustrated in Fig. 1, it was confirmed that the growth rate of the Helicobacter pylori strain was reduced to about 30 to 70% when the four novel strains of the present invention were treated, compared to when the Helicobacter pylori strain was treated only with a lactic acid bacteria basic medium (MRS medium).

From the above results, it can be seen that all four novel strains of the present invention significantly inhibit the growth of Helicobacter pylori strains.

[3-2] Confirmation of the effect of lowering the survival rate of Helicobacter pylori P1 strain

In addition, as in the above Example [3-1], the Helicobacter pylori P1 strain grown until the OD ₆₀₀ reached 0.18-0.2 was dispensed into 96-deep well plates at 300 uL each, and then the culture solutions of the four new lactic acid bacteria established in the above Example 1 were treated twice every 24 hours at a concentration of 3.3%, and then cultured for another 48 hours. After 48 hours, 100 uL of the culture solution was transferred to a 96-well plate, and the CCK-8 (WST-8) assay was performed to measure the absorbance of formazan at 450 nm to confirm the survival rate of the Helicobacter pylori strain.

As a result, as shown in Fig. 2, it was confirmed that the survival rate of the Helicobacter pylori strains was reduced to about 60 to 70% when the four novel strains of the present invention were treated compared to when the Helicobacter pylori strains were cultured in MRS medium.

From the above results, it can be seen that all four novel strains of the present invention significantly reduce the survival rate of Helicobacter pylori strains.

[3-3] Genes associated with metabolic activity of Helicobacter pylori P1 strain amiE Confirmation of the effect of suppressing the expression of

In addition, it was confirmed whether the expression of amiE , a metabolic activity-related gene of Helicobacter pylori strains, was inhibited by the four novel strains of the present invention. Specifically, total bacterial RNA of Helicobacter pylori was isolated from a culture of Helicobacter pylori strains cultured by treating the novel strains of the present invention as in the above Example [3-1] using NucleoSpin RNA Plus (Macherey-Nagel) according to the manufacturer's instructions. Then, cDNA was synthesized and amplified using an RT-qPCR kit (NanoHelix Co. Ltd.), and then real-time PCR was performed using the primer pairs described in Table 8 ^{below using a LightCycler ® 96} System (Roche), thereby measuring the expression level of the amiE gene. SYBR Green technology was used to quantify the expression level of the amiE gene as described above, and the expression level of the amiE gene was normalized based on 16s rRNA. All experiments were repeated three times, and the CT value of each target gene was calculated using the software provided by the manufacturer.

Target gene direction Base sequence (5'->3') Sequence number amiE Forward GGTTTGCCTGGGTTGGAT 7 amiE Reverse GATTTTGCGGTATTTTTG 8 16S rRNA Forward CTGGTAGTCCACGCCGTAAA 9 16S rRNA Reverse CGAATTAAACCACATGCTCCAC 10 GAPDH Forward GAAGGTGAAGGTCGGAGTC 11 GAPDH Reverse GAAGATGGTGATGGGATTTC 12 Hp16S rRNA Forward TCGGAATCACTGGGCGTAA 13 Hp16S rRNA Reverse TTCTATGGTTAAGCCATAGGATTTCAC 14

As a result, as shown in FIG. 3, it was confirmed that the expression level of the amiE gene in the Helicobacter pylori strain was reduced to about 40 to 60% when the culture solution of the four novel strains of the present invention was treated compared to when the MRS medium was treated in the Helicobacter pylori strain. From the results as described above, it can be seen that all four novel strains of the present invention significantly inhibit the expression of the amiE gene in the Helicobacter pylori strain.

[Example 4]

Confirmation of the effect of reducing the adhesion of Helicobacter pylori using hAGOs (human antral gastric organoids)

[4-1] Production of 3D hAGOs

Human pluripotent stem cells (hPSCs) were cultured using a previously described method (Yoon et al. , Molecular carcinogenesis, 55 (4): 387-396 (2016), Son et al. , Proteomics, 15 (13): 2220-2229 (2015)), and three-dimensional human antral gastric organoids (hAGOs) were produced using a previously described method (McCracken et al. , Nature, 516 (7531): 400-404 (2014)).

Specifically, hPSCs were cultured on 5% Matrigel-coated dishes, grown to 90% confluency, and then cultured in endoderm differentiation medium [RPMI 1640 (Gibco), 100 ng/mL Activin A (R&D Systems), 50 ng/mL BMP4 (R&D systems), and 0%, 0.2%, and 2% purified fetal bovine serum (dFBS, Gibco)] for 3 days. Then, they were cultured for 3 days in hindgut differentiation medium [DMEM/F12, 2 μM CHIR99021 (TOCRIS), 500 ng/mL FGF4 (R&D Systems), 100 ng/mL Noggin (R&D systems), 2% dFBS], and 1 μM retinoic acid was added on the third day of culture, and cultured for 1 more day to differentiate into 3D posterior-foregut spheroids. The 3D posterior-vestibular spheroids differentiated as described above were cultured by inserting them into Matrigel (BD Biosciences) and cultured for 3 days in the vestibular organoid differentiation medium [advanced DMRM/F12 (Gibco), 2 mM L-glutamine (Gibco), 1% Penicillin-Streptomycin (Gibco), and 15 mM HEPES buffer (Gibco), 1X N2 (Invitrogen), 1X B27 (Invitrogen), 100 ng/ml EGF (R&D Systems), 100 ng/ml Noggin, 1 uM Retinoic acid], and then transferred to the vestibular organoid differentiation medium [advanced DMRM/F12, 2 mM L-glutamine, 1% Penicillin-Streptomycin, and 15 mM HEPES buffer, 1X N2, 1X B27, Human vestibular organoids were produced by culturing in [100 ng/mL of EGF] for a total of 30 days.

[4-2] Evaluation of Helicobacter pylori adhesion using 2D hAGOs

After the 3D hAGOs produced in the above Example [4-1] were separated from Matrigel, washed with cold PBS, treated with 0.25% trypsin-EDTA (Gibco), and cultured at 37°C for 5 minutes. The hAGOs dissociated into single cells were washed by centrifugation at 1000 rpm, and seeded at ^1x106 cells per well in a 12-well dish coated with 1% Matrigel and cultured with gastric vestibular organoid differentiation medium. After 3 days, the cultured hAGOs were dissociated with 0.25% trypsin-EDTA, and the cell number was counted and prepared.

The Helicobacter pylori P1 strain cultured as in the above Example [3-1] was co-cultured at a concentration of 10 MOI per well, and the culture solutions of the four novel strains of the present invention isolated and identified in the above Example 1 were added at a concentration of 1% and co-cultured for 4 hours.

Then, the cells were washed five times with 1 ml PBS containing 1% DEPC, RNA was extracted with NucleoSpin RNA (MN), and cDNA was synthesized from the extracted RNA using Superscript IV (Invitrogen). qRT-PCR was performed using a 7500 Fast Real-time PCR system (Applied Biosystems, Foster City, CA, USA) to measure the amount of GAPDH gene and 16S rRNA of Helicobacter pylori strains in human gastric vestibular organoids, thereby quantifying the number of Helicobacter pylori strains attached to the two-dimensional hAGOs.

As a result, as shown in Fig. 4, it was confirmed that the adhesion of the Helicobacter pylori strain to hAGOs was reduced to a level of less than 50% when the culture solution of the four novel strains of the present invention was treated compared to when the MRS medium was treated to the Helicobacter pylori strain.

From the above results, it can be seen that all four novel strains of the present invention have the activity of reducing the adhesion of Helicobacter pylori strains to the gastric vestibule.

[Example 5]

Confirmation of the effect of reducing colonization of Helicobacter pylori based on mouse model

[5-1] Production of Helicobacter pylori infection model and treatment of new strain culture solution

Among the four novel strains isolated and identified in Example 1 above, Lactibacillus plantarum DS1073 was utilized to observe the efficacy in reducing Helicobacter pylori colonization in mice.

Six-week-old C57BL/6 male mice were fed a standard diet and maintained at a constant temperature (20-22°C) with a 12-h day/night cycle. MRS medium and 0.5x or 5x lactic acid bacteria culture were orally administered (200 μL each) for 3 weeks. On days 8, 10, and 12, the mice were infected with 1 x 10 ⁹ Helicobacter pylori, and on day 21, the stomachs of the mice were removed and analyzed.

[5-2] Evaluation of Helicobacter pylori colonization in the above tissues

Total RNA of the excised gastric tissue was extracted using Trizol and reverse transcribed using Superscript IV cDNA synthesis kit (Invitrogen). qRT-PCR was performed using 7500 Fast Real-time PCR system (Applied Biosystems). The number of Helicobacter pylori strains attached to the mouse stomach was measured by measuring the amount of b-actin gene and 16S rRNA of Helicobacter pylori strains in the mouse stomach. Primer sequences for the b-actin gene are shown in Table 9 below.

Target gene direction Base sequence (5'->3') Sequence number b-actin Forward AGCCATGTACGTAGCCATCC 15 b-actin Reverse CTCTCAGCTGTGGTGGTGAA 16

As a result, as shown in Fig. 5, it was confirmed that the colonization of Helicobacter pylori was reduced to 90% at 0.5x and to 63% at 5x when the culture solution of Lactibacillus plantarum DS1073 was treated compared to when the MRS medium was treated. From the results as described above, it can be seen that the Lactibacillus plantarum DS1073 strain has the activity of reducing the colonization of the Helicobacter pylori strain as described above.

[Manufacturing example]

Preparation of a crude composition containing novel strains

A crude composition containing four novel strains of the present invention was prepared.

Each of the four novel strains of the present invention was cultured using an optimized enrichment medium and culture conditions. After cell recovery, 5-50 (volume/weight)% of maltodextrin, 5-50 (volume/weight)% of trehalose, or 5-50 (volume/weight)% of cellulose as a cryoprotectant was added to the concentrate, freeze-dried, and then ground to produce lactic acid bacteria powder.

Although the present invention has been described in detail above only with respect to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the technical scope of the present invention, and it is natural that such modifications and variations fall within the scope of the appended claims.

Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC) KCTC15280BP 20230110 Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC) KCTC15281BP 20230110 Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC) KCTC15282BP 20230110 Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC) KCTC15283BP 20230110

Attach electronic file of sequence list

Claims (12)

Lactiplantibacillus plantarum strain DS1073 deposited under accession number KCTC 15280BP.
Lactiplantibacillus plantarum strain DS1902 deposited under accession number KCTC 15281BP.
Lactiplantibacillus pentosus strain DS1989 deposited under accession number KCTC 15282BP.
Bifidobacterium longum strain DS1089 deposited under accession number KCTC 15283BP.
A composition comprising at least one strain selected from the group consisting of Lactiplantibacillus plantarum DS1073 deposited under Accession Number KCTC 15280BP, Lactiplantibacillus plantarum DS1902 deposited under Accession Number KCTC 15281BP, Lactiplantibacillus pentosus DS1989 deposited under Accession Number KCTC 15282BP, and Bifidobacterium longum DS1089 deposited under Accession Number KCTC 15283BP, and at least one selected from the group consisting of a culture solution of the strain, a concentrate of the culture solution, a dried product of the culture solution, and an extract of the culture solution.
In claim 5,
The above composition is a composition that inhibits the growth or survival of Helicobacter pylori.
In claim 5,
The above composition is a composition that inhibits the expression of the amiE gene in Helicobacter pylori.
In claim 5,
The above composition is a composition that reduces the adhesion of Helicobacter pylori to the gastric vestibule.
In claim 5,
The above composition is a composition that reduces the colonization of Helicobacter pylori.
In any one of claims 5 to 9,
The composition above is a pharmaceutical composition for preventing or treating Helicobacter pylori infection.
In claim 10,
A composition wherein the above Helicobacter pylori infection is at least one selected from the group consisting of gastritis, gastric ulcer, duodenal ulcer, nonulcer dyspepsia syndrome, gastric MALT lymphoma, gastric hyperplastic polyp, gastric cancer, gastrointestinal cancer, pancreatitis, inflammatory bowel disease, upper abdominal fullness, upper abdominal pain, belching, abdominal distension, early satiety, nausea, vomiting, reflux, heartburn, and decreased appetite.
In any one of claims 5 to 9,
The composition above is a food composition for preventing or improving Helicobacter pylori infection, or a health functional food composition for preventing or improving Helicobacter pylori infection.