KR20240038852A - Novel complex strains and use thereof - Google Patents

Abstract

The present invention relates to the use for preventing, improving or treating intestinal disease or malnutrition containing three types of complex strains and amino acids as active ingredients. According to the present invention, a composition containing the three types of complex strains and amino acids is provided. Treatment can alleviate intestinal leakage and improve body weight, small intestine villi length, small intestine depth, and the number of goblet cells and proliferating cells in small intestine villi, so the complex strain composition of the present invention can be used to prevent intestinal disease or malnutrition, It can be useful for improvement or treatment.

Description

Novel complex strains and use thereof}

The present invention relates to the use of novel complex strains to prevent, improve or treat intestinal diseases.

Malnutrition occurs when there is an imbalance between the energy consumed or absorbed and the energy expended. Malnutrition is a major health problem worldwide, accounting for one-fifth of deaths among children under five years of age. Malnutrition in infancy leads to stunted growth and reduced cognitive development in childhood, along with significant changes in the composition of the gut microbiota.

This malnutrition can be caused by chronic inflammatory diseases of the intestines. Environmental enteropathy, also known as tropical enteropathy or environmental intestinal dysfunction, is a chronic intestinal inflammatory disorder that commonly occurs in children living in resource-poor environments, such as developing countries. The causes of environmental enteropathy are diverse. In general, it can develop from exposure to contaminated food and water in areas with poor sanitation, and exposure to microorganisms or changes in the microbial community can be the main cause of the development of environmental enteropathy. It suggests that there is. Environmental enteropathy, widely recognized as a major cause of childhood malnutrition, affects the function of the small intestine, causing chronic intestinal inflammation, villous blunting and increased intestinal permeability, and ultimately reduces nutrient absorption, adversely affecting growth and development. It's crazy.

Additionally, in order to prevent external bacteria or viruses from entering the intestines, intestinal mucosal cells maintain constant and dense gaps between cells. However, if this is damaged, the gaps between cells widen and intestinal permeability increases. do. In other words, when the intestinal bacterial balance is disrupted and inflammation occurs in the intestinal mucosa, these connective tissues become weak, allowing external harmful substances to enter the body, or conversely, substances from the body leak, which is called 'leaky gut syndrome'. If leaky gut syndrome worsens, it can develop into Crohn's disease, which often invades the small intestine, the area where nutrients are absorbed, which can lead to nutritional deficiencies.

Likewise, if inflammation in the intestines causes changes or imbalances in the intestinal microorganisms, this can lead to nutritional deficiencies, which can eventually lead to other diseases such as malnutrition, anemia, slowed growth, developmental delay, and brain development disorders. Therefore, it is difficult to improve the symptoms by simply providing nutrients for the treatment, and it is necessary to treat the intestinal disease or improve the symptoms by fundamentally improving the intestinal tissue and intestinal environment through the treatment of the intestinal disease.

Brown et al. Diet and specific microbial exposure trigger features of environmental enteropathy in a novel murine model, Nat Commun, 2015

The purpose of the present invention is to provide a use for the prevention, improvement or treatment of enteric disease or malnutrition using a novel complex strain.

Specifically, the purpose of the present invention is to provide a use for the prevention, improvement or treatment of intestinal disease or malnutrition of a composition containing three types of complex strains and amino acids as active ingredients.

The present inventors found that a composition containing three types of complex strains and amino acids as active ingredients alleviates intestinal leakage in a mouse model of environmental enteropathy, increases body weight, normalizes small intestine villus length and small intestine villi depth, and reduces goblet of small intestine villi. By confirming that the number of cells and proliferating cells was normalized, it was confirmed that the composition could be usefully used in the prevention or treatment of intestinal disease or malnutrition, and the present invention was completed.

Therefore, in one aspect of the present invention, a pharmaceutical composition for the treatment or prevention of enteric disease or malnutrition is provided, comprising Bacillus subtilis, Bacillus coagulans, Clostridium butyricum, and amino acids as active ingredients.

In another aspect of the present invention, a food composition for preventing or improving intestinal disease or malnutrition is provided, comprising Bacillus subtilis, Bacillus coagulans, Clostridium butyricum, and amino acids as active ingredients. .

A composition containing the complex strain according to the present invention can be usefully used to prevent, improve, or treat intestinal disease or malnutrition.

Figure 1 is a graph showing the change in body weight of a mouse over time.
Figure 2 is a graph showing the change in body weight of mice in each group after one month.
Figure 3 is a graph showing the amount of FITC leakage into the plasma of mice for each group.
Figure 4 is a graph showing changes in the length of small intestine villi in mice for each group.
Figure 5 is a graph showing the change in small intestine depth of mice for each group.
Figure 6 is a graph showing the change in the number of goblet cells in mice for each group.
Figure 7 is a graph showing the change in the number of proliferating cells in mice for each group.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides a pharmaceutical composition for the treatment or prevention of enteric disease or malnutrition, comprising Bacillus subtilis, Bacillus coagulans, Clostridium butyricum, and amino acids as active ingredients.

The present inventors screened the individual efficacy of each strain in various aspects for various strains isolated from infant feces, kimchi, and cheese, and also evaluated the efficacy in various aspects by using various strains in various combinations. As a result, the present inventors found that when a combination of three strains of Bacillus subtilis , Bacillus coagulans , and Clostridium butyricum was used together, intestinal leakage was alleviated and body weight was improved. An unexpected synergistic effect was confirmed, and it was also confirmed that the actual therapeutic efficacy for intestinal diseases or malnutrition was significant from a pharmacological aspect.

In one embodiment of the present invention, the Bacillus subtilis may be Bacillus subtilis IDCC 1101 (Accession number: KCTC-15062BP), and the Bacillus coagulans may be Bacillus Coagulans IDCC 1201 (Accession number: ATCC BAA- 3143), and the Clostridium butyricuum may be Clostridium butyricuum IDCC 1301 (Accession Number: KCTC-11658BP).

In one embodiment of the present invention, the composition includes Lactobacillus johnsonii , Lactobacillus plantarum , Bifidobacterium animalis subsp. lactis , and Lactobacillus rhamnosus. ( Lactobacillus rhamnosus ), and Bifidobacterium breve ( Bifidobacterium breve ) It may further include at least one type of lactic acid bacteria selected from the group consisting of. For example, the Lactobacillus johnsonii may be Lactobacillus johnsonii IDCC 9203 (accession number: KCTC-10923BP), and the Lactobacillus plantarum may be Lactobacillus plantarum IDCC 3501 (accession number: KCTC-13586BP). In addition, the Bifidobacterium animalis lactis may be Bifidobacterium animalis lactis IDCC 4301 (accession number: KCTC-13587BP), and the Lactobacillus rhamnosus may be Lactobacillus rhamnosus IDCC 3201 (accession number: KCTC-13587BP). KCTC-10833BP), and the Bifidobacterium breve may be Bifidobacterium breve IDCC 4401 (Accession number: KCTC-13169BP). Among the strains listed above, a composition containing Lactobacillus johnsonii IDCC9203 strain, Lactobacillus plantarum IDCC3501 strain, and Bifidobacterium animalis subsp. Lactis IDCC4301 strain as active ingredients is effective in preventing or treating inflammatory bowel disease. Confirmed. Therefore, using the above strains together with the composition of the present invention may be more desirable for improving the intestinal environment. In addition, it was confirmed that Lactobacillus rhamnosus IDCC 3201 strain has protein degrading ability and is effective in improving protein digestion and amino acid absorption. Therefore, using the above strain together with the composition of the present invention may be more desirable for improving the intestinal environment.

The Bacillus subtilis, Bacillus coagulans, Clostridium butyricum, Lactobacillus johnsonii, Lactobacillus plantarum, Bifidobacterium animalis lactis, Lactobacillus rhamnosus, and Bifidobacterium breve are It may exist as live or dead cells, and may also exist in dried or freeze-dried form. The types of strains suitable for inclusion in various compositions and methods of formulation are well known to those skilled in the art. For example, the strains listed above are cultures obtained by culturing in a known liquid medium or solid medium, a concentrate of the culture, a dried product of the culture, or a fermented product obtained by culturing the strain with additional components. Alternatively, it may be formulated in the form of an extract obtained by extracting the strain with an organic solvent, a lysate (or lysate) obtained by dissolving the cell membrane of the strain, or being crushed or homogenized, but is not limited thereto.

In the present invention, the composition may essentially contain one or more amino acids. For example, the amino acid may be BCAA (branched amino acid). In an embodiment of the present invention, when the lactic acid bacteria of the present invention and BCAA are administered together, the metabolic activity of the bacteria can be activated. In addition, it was confirmed that a further improvement effect could be obtained in the body weight of the administered subject and various indicators related to intestinal disease or malnutrition.

The amino acid may be included in an amount of 1 to 50 parts by weight based on 100 parts by weight of the total composition. For example, the amino acid may be included in 1 to 40 parts by weight, 1 to 30 parts by weight, 10 to 40 parts by weight, 10 to 30 parts by weight, 20 to 40 parts by weight, or 20 to 30 parts by weight, based on 100 parts by weight of the total composition. . Preferably, the amino acid may be selected from the group consisting of valine, leucine, isoleucine, and glutamine.

In the present invention, the composition may further include minerals. The mineral may be included in an amount of 0.1 to 5 parts by weight based on 100 parts by weight of the total composition. For example, the mineral may be included in an amount of 0.1 to 3 parts by weight, 0.1 to 2.5 parts by weight, 1 to 3 parts by weight, or 1 to 2.5 parts by weight based on 100 parts by weight of the total composition. In the present invention, the mineral may be, for example, magnesium or zinc.

In the present invention, the composition may further include vitamins. The vitamin may be included in an amount of 0.01 to 1 part by weight based on 100 parts by weight of the total composition. For example, the vitamin may be included in an amount of 0.01 to 0.5 parts by weight, 0.01 to 0.25 parts by weight, 0.1 to 0.5 parts by weight, and 0.1 to 0.25 parts by weight, based on 100 parts by weight of the total composition. In the present invention, the vitamins may be, for example, vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, folic acid, vitamin C, vitamin D3, and vitamin E.

In the present invention, the composition may further include prebiotics. The prebiotics may be included in an amount of 0.1 to 30 parts by weight based on 100 parts by weight of the total composition. For example, the prebiotics are used in an amount of 0.1 to 3 parts by weight, 1 to 3 parts by weight, 1 to 5 parts by weight, 1 to 30 parts by weight, 1 to 25 parts by weight, 10 to 30 parts by weight, based on 100 parts by weight of the total composition. It may be included in 20 to 30 parts by weight, or 20 to 25 parts by weight. In the present invention, the prebiotics may be, for example, oligosaccharides such as fructooligosaccharides and galactooligosaccharides, starch, sugar alcohol, and dietary fiber.

In the present invention, "intestinal disease" may be caused by an imbalance in the intestinal flora, affecting intestinal function, causing chronic intestinal inflammation, villous blunting and increased intestinal permeability, and ultimately reducing nutrient absorption, which is negative for growth and development. It may refer to any intestinal dysfunction that affects it. The intestinal disease may be impaired intestinal absorption function or decreased intestinal absorption function. For example, the intestinal disease may be environmental enteropathy, leaky gut syndrome, malabsorption syndrome, or disorder of chronic intestinal inflammation, but is not limited thereto.

In the present invention, the onset of the intestinal disease may result in decreased absorption of nutrients, resulting in nutritional deficiency. If nutritional deficiency persists, it can cause fatigue and weakness, as well as decreased immunity and infection. Children suffering from severe nutritional deficiency cannot grow normally, their behavioral development is significantly delayed, and mild intellectual disability may occur. Ultimately, the above intestinal diseases can cause malnutrition, anemia, slowed growth, and brain development disorders.

In the present invention, “malnutrition” refers to an abnormal state of the body that occurs due to lack of nutrients, including malnutrition that occurs due to lack of food supply and nutritional deficiency due to acute or chronic disease in people who receive adequate nutrition. It may refer to any condition that has occurred.

In an embodiment of the present invention, a composition containing three complex strains (Bacillus subtilis IDCC 1101, Bacillus coagulans IDCC 1201, Clostridium butyricum IDCC 1301) and amino acids as active ingredients was used in an animal model of environmental enteropathy. The degree of leakage of orally administered FITC particles and the final weight after one month were measured. As a result, it was confirmed that the intestinal leakage phenomenon was alleviated in the experimental group administered the composition compared to the control group. In addition, the composition was administered to a mouse model of environmental enteropathy, and it was confirmed that the small intestine villus length, small intestine villi depth, and the number of goblet cells and proliferating cells in the small intestine villi of the mouse were improved, and accordingly, the composition of the present invention was found to improve intestinal disease or It was confirmed that malnutrition can be improved.

Accordingly, the present invention relates to the use of Bacillus subtilis, Bacillus coagulans, Clostridium butyricum, and amino acids for the production of pharmaceutical compositions for the prevention or treatment of intestinal diseases or malnutrition; and administering Bacillus subtilis, Bacillus coagulans, Clostridium butyricum, and amino acids to a subject in need thereof.

In the above uses and treatment methods, the description of strains and diseases can be applied as described above.

As used in the present invention, the term "prevention" refers to all actions that suppress or delay intestinal disease by administering the pharmaceutical composition according to the present invention, and the term "treatment" refers to all actions that inhibit or delay intestinal disease by administering the pharmaceutical composition according to the present invention. It refers to all actions that improve or benefit the symptoms of intestinal disease.

In the present invention, “subject” refers to a subject in need of prevention or treatment of a disease, and more specifically, any mammal in need of prevention or treatment of an enteric disease, such as humans and primates, as well as It includes livestock such as cows, pigs, sheep, horses, dogs, and cats without limitation, but is preferably a human.

The pharmaceutical composition according to the present invention can be administered in a conventional manner through routes such as oral, intravenous, intraarterial, intraperitoneal, intramuscular, or intrasternal.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not significantly irritate living organisms and does not inhibit the biological activity and properties of the administered ingredient. Additionally, preferably, the composition of the present invention may further include fillers, excipients, disintegrants, binders, and lubricants. Additionally, the compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal.

In the present invention, “administration” means introducing the composition of the present invention into a patient by any appropriate method, and the route of administration of the composition of the present invention is oral or parenteral, as long as it can reach the target tissue. It can be. It may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically, intranasally, intrapulmonaryly, or rectally, but is not limited thereto.

In the case of a formulation for oral administration, the composition of the present invention can be used as an orally dispersible film, powder, granule, tablet, pill, sugar-coated tablet, capsule, solution, gel, syrup, slurry, suspension, etc., methods known in the art. It can be formulated using . For example, oral preparations can be prepared by combining the active ingredient with solid excipients, grinding them, adding suitable auxiliaries, and processing them into a granule mixture to obtain tablets or dragees. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, starches including corn starch, wheat starch, rice starch and potato starch, cellulose, Fillers such as celluloses including methyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethyl-cellulose, gelatin, polyvinylpyrrolidone, etc. may be included. Additionally, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives.

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.

The composition of the present invention is not limited thereto, but may be formulated so that the three strains (Bacillus subtilis, Bacillus coagulans, Clostridium butyricum) are present in one container in the form of a mixture, or Each of the three strains is provided packaged in a separate container, but can be formulated to be administered simultaneously or sequentially when used.

In the present invention, “effective amount” means the amount necessary to delay or completely stop the onset or progression of a specific disease to be treated, and the effective amount of the three strains included in the pharmaceutical composition of the present invention is effective for intestinal diseases or nutritional disorders. It refers to the amount required to achieve the effect of preventing or treating ataxia. Therefore, the effective amount depends on the type of disease, the severity of the disease, the type and content of other ingredients contained in the composition, and the patient's age, weight, general health, gender and diet, administration time, administration route, treatment period, and simultaneous use. It can be adjusted depending on various factors, including the drug used. It is obvious to those skilled in the art that the appropriate total daily usage amount can be determined by the treating physician within the scope of sound medical judgment.

For the purposes of the present invention, a specific therapeutically effective amount for a particular patient refers to the type and degree of response to be achieved, the specific composition, including whether other agents are used as the case may be, the patient's age, weight, general health, and gender. It is desirable to apply it differently depending on various factors including diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used together or simultaneously with the specific composition, and similar factors well known in the medical field.

Another aspect of the present invention provides a food composition for preventing or improving intestinal disease or malnutrition, comprising Bacillus subtilis, Bacillus coagulans, Clostridium butyricum, and amino acids as active ingredients.

In the food composition, the description of strains and diseases can be applied as described above.

"Food" of the present invention means to include raw materials and all processed substances consumed for maintaining health and growth and development, and includes functional foods, health functional foods, and medical food, etc.

As an example, the food may be a functional food. In the present invention, “functional food” refers to food with physiological function or activity, and refers to food containing biologically active ingredients at a level that can provide specific health effects. In this aspect, the present invention provides a functional food composition for preventing or improving intestinal disease or malnutrition, comprising Bacillus subtilis, Bacillus coagulans, Clostridium butyricum, and amino acids as active ingredients.

As an example, the food may be a health functional food. In the present invention, "health functional food" is a food manufactured using specific nutrients or raw materials or ingredients with useful functions for the human body, that is, functional raw materials, and helps human health by maintaining normal functions of the human body or activating physiological functions. It means food that can be given. In this aspect, the present invention provides a health functional food composition for preventing or improving intestinal disease or malnutrition, comprising Bacillus subtilis, Bacillus coagulans, Clostridium butyricum, and amino acids as active ingredients.

As an example, the food may be medical food. In the present invention, “medical food” refers to food manufactured for people who need special dietary management due to disease or health problems, or manufactured for treatment. In this aspect, the present invention provides a medical food composition for preventing or improving intestinal disease or malnutrition, comprising Bacillus subtilis, Bacillus coagulans, Clostridium butyricum, and amino acids as active ingredients.

In one embodiment of the present invention, the Bacillus subtilis may be Bacillus subtilis IDCC 1101, the Bacillus coagulans may be Bacillus coagulans IDCC 1201, and the Clostridium butyricum may be Clostridium butyricum. It may be liquid IDCC 1301.

In one embodiment of the present invention, the composition includes at least one selected from the group consisting of Lactobacillus johnsonii, Lactobacillus plantarum, Bifidobacterium animalis lactis, Lactobacillus rhamnosus, and Bifidobacterium breve. It may include more than one species.

In the present invention, the composition may essentially contain one or more amino acids. Preferably, the amino acid may be selected from the group consisting of valine, leucine, isoleucine, and glutamine.

In one embodiment of the present invention, the composition may further include minerals. Preferably, the mineral may be magnesium or zinc.

In one embodiment of the present invention, the intestinal disease refers to reduced small intestine absorptive function or small intestine absorptive dysfunction, and may specifically be environmental enteropathy, leaky gut syndrome, malabsorption syndrome, or chronic intestinal inflammatory disorder, but is limited thereto. That is not the case.

As used herein, the term "improvement" means any action that at least reduces the severity of a parameter, such as a symptom, associated with the alleviation or treatment of a condition.

The food composition according to the present invention can be prepared in the form of a composition by mixing with known active ingredients known to be effective in treating intestinal diseases or malnutrition, and can be used as a functional food, nutritional supplement, or health food. Includes all forms of food and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, the food composition may include health functional foods or seasonings, beverages, bars, etc. Additionally, the food composition containing the strain as an active ingredient may include beverages such as fermented milk, and the food composition containing the strain as an active ingredient may be granulated, encapsulated, or powdered for consumption.

The food composition of the present invention can be prepared using food-chemically suitable and physiologically acceptable auxiliaries in addition to the above active ingredients, and the auxiliaries include excipients, disintegrants, sweeteners, binders, coating agents, leavening agents, lubricants, and lubricants. Agents or flavoring agents can be used.

For administration, the food composition may be preferably formulated as a pharmaceutical composition by including one or more foodologically acceptable carriers in addition to the active ingredients described above.

For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, etc. Additionally, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacance or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate. Includes sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc. When formulated as a liquid solution, it includes suspensions, oral solutions, emulsions, syrups, etc., and includes, but is not limited to, diluents such as water and liquid paraffin, humectants, sweeteners, fragrances, and preservatives.

The food composition according to the present invention can be added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, vitamin complexes, health supplements, etc.

The food composition of the present invention may include ingredients commonly added during food production, and includes, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the above-mentioned carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a flavoring agent, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. Additionally, it may contain vitamins such as vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, folic acid, vitamin C, vitamin D3, and vitamin E. For example, when the food composition of the present invention is manufactured as a drink or beverage, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be additionally included.

In the composition of the present invention, the effective amount of colony forming units (CFU) of each strain may be determined by a person skilled in the art according to the purpose (disease prevention, health or therapeutic treatment) or need, and may also be determined depending on the final formulation. can be decided.

For example, when producing food or drugs, live or dead cells of each of the three strains of the present invention are used at 1 x 10 ⁶ to 1 x 10 ¹² bacteria (bacterial count or CFU) per 1 g (or mL) of the total composition. It can be added, preferably in the amount of 1 x 10 ⁷ to 1 x 10 ¹⁰ , but is not limited thereto.

In addition, the daily intake of the composition containing the above-mentioned live or dead cells is 1 x 10 ⁶ to 1 x 10 ¹² CFU/kg, preferably 1 x 10 ⁷ to 1 x 10 ¹⁰ based on the total number of cells. It may be CFU/kg, and intake may be taken once a day or divided into several times, but is not limited thereto.

The composition of the present invention contains 1 to 10 parts by weight of three complex strains of Bacillus subtilis , Bacillus coagulans , and Clostridium butyricum , based on 100 parts by weight of the total composition. It may include For example, based on 100 parts by weight of the total composition, the total content of the three complex strains may be 1 to 5 parts by weight, 1 to 3 parts by weight, or 2 to 3 parts by weight.

The composition of the present invention may further include additional lactic acid bacteria in addition to the above three complex strains. For example, the additional lactic acid bacteria include Lactobacillus johnsonii, Lactobacillus plantarum , Bifidobacterium animalis subsp. lactis , and Lactobacillus rhamnosus . ), and Bifidobacterium breve . The additional lactic acid bacteria may be included in an amount of 0.1 to 5.0 parts by weight based on 100 parts by weight of the total composition. For example, the additional lactic acid bacteria may be included in an amount of 0.1 to 3.0 parts by weight, 0.1 to 1.0 parts by weight, or 0.1 to 0.5 parts by weight based on 100 parts by weight of the total composition.

The advantages and features of the present invention and methods for achieving them will become clear with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below and will be implemented in various different forms. The present embodiments are merely intended to ensure that the disclosure of the present invention is complete and that common knowledge in the technical field to which the present invention pertains is not limited. It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims.

[Example]

Experiment method

1) Preparation of 3 types of complex strain (Forbaby) preparation

The three complex strain preparations of the present invention used in the experiment were prepared by mixing three live bacterial strains: Bacillus subtilis IDCC 1101, Bacillus coagulans IDCC 1201, and Clostridium butyricum IDCC 1301. In addition, Lactobacillus johnsonii IDCC 9203, Lactobacillus plantarum IDCC 3501, Bifidobacterium animalis lactis IDCC 4301, Lactobacillus rhamnosus IDCC 3201, and Bifidobacterium as additional lactic acid bacteria to the above three complex strains. Breve IDCC 4401 strains were mixed. The specific ingredients and contents of the preparation are shown in Table 1.

ingredient Unit (mg) glucose 371.256 xylitol 600 chocolate powder 600 chocolate flavor powder 45 Galactooligosaccharide 60 Chicory Extract Powder 60 vegetable cream powder 147 silicon dioxide 45 milk flavor powder 30 Whole milk powder 93 zinc oxide 11.2 Vitamin B12 Blend Powder 2.6 Vitamin B6 2 dry yeast powder 2 Enzyme Mixture Formulation 2 Vitamin C One alpha differential One folic acid 0.44 thiamine 0.5 3 complex strains 66 leucine 600 isoleucine 50 Valine 50 glutamine 100 Magnesium chloride 50 Additional lactic acid bacteria 10 total amount 3000

2) Preparation of control (placebo) preparation

In order to confirm the effectiveness of the above three complex strain preparations, A preparation containing the three complex strains, no additional lactic acid bacteria, and no amino acids was prepared. The specific ingredients and their contents of the preparation are shown in Table 2.

ingredient Unit (mg) glucose 236.259 xylitol 150 chocolate powder 600 chocolate flavor powder 45 Galactooligosaccharide 60 Chicory Extract Powder 60 vegetable cream powder 135 silicon dioxide 45 milk flavor powder 30 Whole milk powder 75 zinc oxide 11.199 Vitamin B12 Blend Powder 2.601 Vitamin B6 2.001 dry yeast powder 2.001 Enzyme Mixture Formulation 2.001 Vitamin C 0.999 alpha differential 0.999 folic acid 0.441 thiamine 0.501 maltodextrin 1541.01 total amount 3000

3) Environmental enteropathy (EE) model in mice

The mouse EE model was described by Brown, E., Wlodarska, M., Willing, B. et al . Diet and specific microbial exposure trigger features of environmental enteropathy in a novel murine model. It was produced with reference to the contents of Nat Commun 6, 7806 (2015) (https://doi.org/10.1038/ncomms8806).

Weaned 3-week-old young mice (C57BL/6 black mice) were divided into 4 to 5 mice per group, and were divided into normal control group (normal), untreated group (EE), control group (EE+placebo), and experimental group (EE). +Forbaby) was prepared. Excluding the normal control group, each group was fed nutritionally deficient feed for 4 weeks to induce nutritional deficiency, and in the 3rd week of feeding, infectious bacteria were orally administered 3 times to induce infection to prepare an environmental enteropathy model. did. At the 4th week of observation, mice were sacrificed to confirm the effect.

Specifically, the normal control group was fed normal feed (28% protein, 22% fat) for 4 weeks, and the untreated group, control group, and experimental group were each fed nutritionally deficient feed (9% protein, 13% fat) for 4 weeks. did. Among them, the control group was fed with the control preparation of 2) (placebo preparation), and the experimental group was fed with the three types of complex strain preparation of 1) (Forbaby preparation). Specifically, 3000 mg of each of the three complex strain preparations or the control preparation was mixed with 50 ml of drinking water every 24 hours and provided to each cage (based on 5 mice).

Meanwhile, a total of 7 types of infectious bacteria isolated from humans were used, specifically Bacteroides vulgatus 3/1/40A, Bacteroides fragilis 3/1/12, Bacteroides ovatus 3/8/47, and Bacteroides dorei 5/1/36. (D4), Parabacteroides distasonis 2/1/33B, Escherichia coli 3/2/53, and Escherichia coli 4/1/47 were used. Each bacteria was cultured under anaerobic conditions in FAA (fastidious anaerobe) medium. One day after inoculation, equal amounts of each bacteria were mixed in PBS to make ¹⁰⁹ cells/ml, and 100μl was injected into each mouse's stomach through gavage.

This infection occurred on the 16th, 18th, and 20th days after feeding a nutritionally deficient diet, and the mice were sacrificed and sampled on the 28th day, 8 days after infection.

4) Preparation of paraffin blocks for mouse intestinal sampling and observation of small intestine tissue

In the mouse environmental enteropathy model, mice were dissected at 4 weeks of observation to obtain intestinal samples. After anesthetizing the mouse, the intestines are removed, rolled using the Swiss-rolling technique, and stored in a 4% paraformaldehyde solution for more than 24 hours. Afterwards, a paraffin block was prepared to confirm the length of small intestine villi, the depth of small intestine villi, the number of goblet cells present in the small intestine villi, and the number of proliferating cells in the small intestine villi.

The tissue fixed above was washed in water to remove the fixation reagent. To remove moisture from the tissue, the tissue was placed in an automatic tissue processor and processed for dehydration and transparency (Table 3).

Automatic tissue processing machine One 70% EtOH 4 hours 2 80% EtOH 1 hours 3 90% EtOH 1 hours 4 95% EtOH 1 hours 5 100% EtOH 1 hours 6 100% EtOH 1 hours 7 100% EtOH 1 hours 8 xylene 1 hours 9 xylene 1 hours 10 xylene 1 hours 11 paraffin 1 hours 12 paraplast 1 hours

To allow paraffin, which does not mix with water, to penetrate into the tissue, the tissue was dehydrated using alcohol. As shown in Table 3 above, dehydration was accomplished through gradual treatment from a low-concentration alcohol solution to a high-concentration alcohol solution, followed by treatment with pure 100% alcohol and xylene. Paraffin dissolved in xylene, an organic solvent, was infiltrated into the dehydrated tissue and then treated with liquid, pure paraffin at high temperature (60°C) to completely infiltrate. The tissue in the mold was cooled and the paraffin was made into a solid state, then appropriated. Cut to size and trimmed. Afterwards, it was cut to an appropriate thickness (usually 5μm) using a thin slicer. The cut paraffin sections were floated in a 55°C water bath containing a 10% alcohol solution, and when the paraffin was slightly melted, they were floated onto a slide and dried. The slide was dried at room temperature for a day, and then the paraffin of the tissue was removed.

One xylene 10 minutes 2 xylene 10 minutes 3 100% EtOH 5 minutes 4 100% EtOH 5 minutes 5 95% EtOH 5 minutes 6 70% EtOH 5 minutes 7 50% EtOH 5 minutes 8 ADW 5 minutes 9 ADW 5 minutes 10 3% H ₂ O ₂ 20 minutes 11 ADW 5 minutes 12 PBS 5 minutes

The prepared slides were stained with hematoxylin and eosin. After dehydrating the slide by performing the reverse process of Table 4, the cover slide was covered with a mounting solution and observed using a microscope.

Experimental Example 1: Confirmation of weight improvement effect of 3 types of complex strain preparation

According to the description in experimental method 3), a normal control group and an experimental group consisting of 4 to 5 mice per group were prepared, and each was treated with the three types of complex strain preparation and the control preparation. Food was mixed with drinking water every day and body weight was tracked every two days. The final weight after 1 month was expressed as a ratio based on the initial weight of the 3-week-old mouse.

As a result, it was confirmed that body weight was significantly increased in the group treated with the three types of complex strain preparation compared to the untreated group (EE), and especially in the group treated with the three types of complex strain preparation compared to the control (placebo) preparation. It was confirmed that body weight increased significantly (Figures 1 and 2).

Experimental Example 2: Confirmation of intestinal leakage improvement effect of 3 types of complex strain preparation

An experiment was conducted to confirm the degree of intestinal leakage in an animal model by observing how much orally administered FITC particles leak into the blood depending on the degree of intestinal leakage.

In the same manner as the mouse model described in 3) of the experimental method, a normal control group (normal), an untreated group (EE), a control group (EE+placebo), and an experimental group (EE+Forbaby) consisting of 4 to 5 mice per group were used. Prepared and treated with feed, three types of complex strain preparation, and control preparation, respectively. At 4 weeks of observation, mice were dissected to check for intestinal leakage (FITC) indicators.

To measure intestinal leakage, the fluorescent substance FITC-dextran (Fluorescein isothiocyanate-dextran, sigma #FD4) was used and the same amount of FITC-dextran was orally administered to each mouse. Four hours after administration, blood was collected to obtain a plasma sample, and FITC fluorescence in the sample was measured.

As a result, it was observed that intestinal leakage was promoted and the amount of FITC increased in the untreated group. On the other hand, when the experimental group with malnutrition was treated with the three-type complex strain preparation, the amount of FITC was reduced compared to the untreated group and the control group, and intestinal leakage was observed to be improved (Figure 3).

Experimental Example 3: Confirmation of the effect of improving small intestine function of three types of complex strain preparations

In order to confirm the effect of the three types of complex strain preparation on improving small intestine function, the small intestine villus length, small intestine villi depth, number of goblet cells present in the small intestine villi, and the number of proliferating cells in the small intestine villi were confirmed.

In the same manner as the mouse model described in 3) of the experimental method, a normal control group (normal), an untreated group (EE), a control group (EE+placebo), and an experimental group (EE+Forbaby) consisting of 4 to 5 mice per group were used. Prepared and treated with feed, three types of complex strain preparation, and control preparation, respectively. At the fourth week of observation, mice were dissected and intestinal samples were obtained, and each indicator was confirmed using the paraffin block prepared in experimental method 5).

As a result, it was confirmed that the length of small intestinal villi was significantly increased in the group treated with the lactic acid bacteria preparation compared to the untreated group (EE), and especially in the group treated with the three types of complex strain preparation compared to the control (placebo) preparation. It was confirmed that the length of small intestine villi was significantly increased (Figure 4).

In addition, it was confirmed that the depth of small bowel cavities significantly increased in the group treated with the lactic acid bacteria preparation compared to the untreated group. In particular, the depth of small bowel cavities was significant in the group treated with the three-type complex strain preparation compared to the control preparation. It was confirmed that there was a significant increase (Figure 5).

It was confirmed that the number of goblet cells and the number of proliferating cells present in the small intestinal villi were significantly increased in the group treated with the lactic acid bacteria preparation compared to the untreated group. In particular, compared to the case of treatment with the control preparation, the treatment of the three types of complex strain preparation It was confirmed that the number of cells was significantly increased in one group (Figures 6 and 7).

Biological Resource Center KCTC15062BP 20220829 Biological Resource Center KCTC11658BP 20100305 Biological Resource Center KCTC10923BP 20060315 Biological Resource Center KCTC13586BP 20180717 Biological Resource Center KCTC13587BP 20180717 Biological Resource Center KCTC10833BP 20050801 Biological Resource Center KCTC13169BP 20161202

Claims (12)

Containing Bacillus subtilis, Bacillus coagulans, Clostridium butyricum, and amino acids as active ingredients,
Food composition for preventing or improving intestinal disease or malnutrition. According to paragraph 1,
Further comprising at least one species selected from the group consisting of Lactobacillus johnsonii, Lactobacillus plantarum, Bifidobacterium animalis lactis, Lactobacillus rhamnosus, and Bifidobacterium breve,
Food composition for preventing or improving intestinal disease or malnutrition. According to paragraph 1,
The amino acid is one or more selected from the group consisting of valine, leucine, isoleucine, and glutamine,
Food composition for preventing or improving intestinal disease or malnutrition. According to paragraph 1,
containing more minerals,
Food composition for preventing or improving intestinal disease or malnutrition. According to paragraph 4,
The mineral is magnesium or zinc,
Food composition for preventing or improving intestinal disease or malnutrition. According to paragraph 1,
The intestinal disease is environmental enteropathy, leaky gut syndrome, malabsorption syndrome, or disorder of chronic intestinal inflammation.
Food composition for preventing or improving intestinal disease or malnutrition. Containing Bacillus subtilis, Bacillus coagulans, Clostridium butyricum, and amino acids as active ingredients,
Pharmaceutical composition for the treatment or prevention of intestinal disease or malnutrition. In clause 7,
Further comprising one or more species selected from the group consisting of Lactobacillus johnsonii, Lactobacillus plantarum, Bifidobacterium animalis lactis, Lactobacillus rhamnosus, and Bifidobacterium breve,
Pharmaceutical composition for the treatment or prevention of intestinal disease or malnutrition. In clause 7,
The amino acid is one or more selected from the group consisting of valine, leucine, isoleucine, and glutamine,
Pharmaceutical composition for the treatment or prevention of intestinal disease or malnutrition. In clause 7,
containing more minerals,
Pharmaceutical composition for the treatment or prevention of intestinal disease or malnutrition. In clause 7,
The mineral is magnesium or zinc,
Pharmaceutical composition for the treatment or prevention of intestinal disease or malnutrition. In clause 7,
The intestinal disease is environmental enteropathy, leaky gut syndrome, malabsorption syndrome, or disorder of chronic intestinal inflammation.
Pharmaceutical composition for the treatment or prevention of intestinal disease or malnutrition.