KR20220097322A - Increased propionic acid using kestose - Google Patents

Abstract

The present invention relates to a composition comprising kestose as an active ingredient, and an agent for increasing the production and/or content of short chain fatty acid (SCFA) in the intestine, use for promoting SCFA producing bacteria, use of or preventing, treating or ameliorating inflammatory diseases such as atopic dermatitis or intestinal inflammation, and use for regulating intestinal microbiome of the composition. The composition can be a food, pharmaceutical or health functional food composition.

Description

Propionic acid enhancement using kestose {Increased propionic acid using kestose}

The present invention provides a composition comprising kestose as an active ingredient, and an agent for enhancing the production and / or content of short chain fatty acid (SCFA) in the intestine of the composition, the use of promoting bacteria producing short chain fatty acids, inflammatory diseases, for example It relates to a use for preventing, treating or ameliorating skin inflammation or intestinal inflammation, and to a use for regulating the intestinal microbiome.

Recently, probiotics are attracting attention as health supplements according to research results showing that probiotics are effective in relieving atopic symptoms and strengthening immunity due to their efficacy in proliferating beneficial intestinal bacteria and inhibiting harmful bacteria.

However, there are reports that probiotics should be taken with caution due to concerns about side effects in some patients with serosal damage and immunocompromised patients.

Recently, in addition to the term probiotics, research on prebiotics that can maximize their function by activating intestinal probiotics as meaning nutrients that feed probiotics are being actively conducted.

Most prebiotics are in the form of oligosaccharides or polysaccharides. In the past, inulin, lactulose, and lactitol were mainly used as food for beneficial bacteria such as bifidobacteria, and recently, AX (arabinoxylan), etc., is being used more diversely. .

An example of the present invention relates to a use for regulating the intestinal microbiome comprising kestose as an active ingredient, and more particularly, to the use of promoting/promoting the growth of beneficial microorganisms in the intestine and/or inhibiting/reducing the growth of harmful microorganisms. .

A further example of the present invention relates to the use of a composition comprising kestose as an active ingredient to promote growth/promotion of one or more microorganisms selected from the group consisting of intestinal microorganisms, for example, microorganisms of the genus Lactobacillus and microorganisms of the genus Alistipes.

A further example of the present invention relates to the use of a composition comprising kestose as an active ingredient to inhibit/reduce the growth of intestinal microorganisms, for example, microorganisms of the genus Prevotella.

A further example of the present invention relates to the use of promoting/promoting the growth of intestinal microorganisms that produce intestinal short-chain fatty acids, for example, propionic acid, including kestose as an active ingredient.

A further example of the present invention relates to a composition for improving the production and / or content of intestinal short-chain fatty acids, for example, propionic acid, comprising kestose as an active ingredient. The intestinal short-chain fatty acids, for example, the production and / or increase in the content of propionic acid may be achieved by the intestinal microorganisms.

A further example of the present invention relates to an inflammatory disease containing kestose as an active ingredient, for example, skin inflammation (inflammatory skin disease) and / or intestinal inflammation (inflammatory bowel disease) It relates to the prevention, treatment or use of improvement.

An example of the present invention relates to a use for regulating the intestinal microbiome containing kestose as an active ingredient, and more particularly, promoting the growth of beneficial microorganisms in the intestine, inhibiting the growth of harmful microorganisms in the intestine, and/or short-chain fatty acids in the intestine, such as For example, it relates to the use of promoting the growth of intestinal microorganisms that produce propionic acid.

Another embodiment of the present invention relates to a composition for enhancing the production and/or content of intestinal short-chain fatty acid (SCFA), for example, propionic acid, comprising kestose as an active ingredient.

A further example of the present invention relates to an inflammatory disease including kestose as an active ingredient, for example, to the prevention, treatment or improvement of skin inflammation and/or intestinal inflammation.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a composition, use, or method for improving intestinal microbiome imbalance as a use for regulating the intestinal microbiome containing kestose as an active ingredient, and more particularly, inhibiting the growth of harmful microorganisms in the intestine, promoting the growth of beneficial microorganisms in the intestine, and propionic acid It relates to a composition for use in regulating the intestinal microbiome of one or more selected from the group consisting of promoting the growth of producing bacteria. One embodiment of the present invention relates to a method for regulating the intestinal microbiome or improving intestinal microbial imbalance, comprising administering to a subject in need thereof, kestose or a composition comprising kestose.

According to the present invention, by orally providing 1-kestose, an active ingredient, to a subject, for example, a mammal including a human as a pharmaceutical or food or drink, it is possible to improve the intestinal microbial imbalance by controlling the intestinal microbial community of the ingester. . Specifically, by oral ingestion by a mammal of the composition for improving intestinal microbial imbalance and the proliferating agent of beneficial intestinal microbes of the present invention, inhibition of the growth of intestinal harmful microbes, growth of intestinal beneficial microbes, and growth of propionic acid producing bacteria One or more types of intestinal microbiome selected from the group consisting of enhancement may be modulated.

An example of the present invention relates to the use of a composition comprising kestose as an active ingredient to promote growth of one or more microorganisms selected from the group consisting of microorganisms of the genus Alistipes and microorganisms of the genus Alistipes, for example, use of increasing the content or use of beneficial intestinal microorganisms will be.

An example of the present invention relates to the use of a composition containing kestose as an active ingredient to inhibit the growth of harmful microorganisms, for example, microorganisms of the genus Prevotella or to reduce the content thereof.

An example of the present invention relates to the use of a composition comprising kestose as an active ingredient to promote growth of propionic acid-producing bacteria. The composition can increase the content of propionic acid in the intestine.

In one example of the present invention, the composition may be a pharmaceutical composition or a food composition. The composition may be a prebiotic composition, and may additionally be used as synbiotics including probiotics. Probiotics may refer to microorganisms that have a good effect on health in the body. Prebiotics may refer to ingredients that are used by microorganisms including beneficial bacteria to promote the growth or activity of microorganisms, thereby resolving imbalances in the intestinal microbiome and exhibiting a good effect on the health of the host. The probiotic strain or probiotic microorganism may be a living cell or a form of a non-viable cell.

The microorganism applicable to the present invention as the probiotic microorganism includes one or more probiotics selected from the group consisting of microorganisms of the genus Bifidobacterium, microorganisms of the genus Streptococcus, microorganisms of the genus Saccharomyces, microorganisms of the genus bacillus, microorganisms of the genus Alistipe, and microorganisms of the genus Lactobacillus can do.

The active ingredient in the present invention promotes the growth of one or more microorganisms from the group consisting of microorganisms of the genus Lactobacillus and microorganisms of the genus Alistipes in the intestine for a subject in need of improvement of the intestinal microbiome, thereby proliferating bacteria that have a beneficial effect in the intestine and, as a result, it can bring about an improvement effect on the intestinal microbiome.

The composition according to the present invention contains kestose, and the microorganisms of the genus Lactobacillus and the genus Alisipes increase and the microorganisms of the genus Prevotella decrease, which can increase the SCFA production. Therefore, according to the present invention, it is possible to simply and effectively increase the microorganisms of the genus Lactobacillus and/or the microorganisms of the genus Alistipe in the body without worrying about side effects or safety. In addition, according to the present invention, kestose has the advantage of improving the intestinal environment by increasing the microorganisms of the genus Lactobacillus and/or the microorganisms of the genus Alistipe in the body of the subject. In addition, Kestose can achieve intestinal microbial balance by improving the intestinal environment by reducing the microorganisms of the genus Prevotella in the body of humans or animals.

Microorganisms of the genus Alistipe are classified as Gram-negative, rod-shaped, anaerobic and non-spore-forming microorganisms, Alistipes finegoldii, Alistipes putredinis, Alistipes onderdonkii, Alistipes shahii, Alistipes indistinctus, Alistipes senegalensis, Alistipes iptimonensis, Alistipes ihumipes obtimonensis, Alistipes ihumipes obtimonensis, Alistipes ihumipes , Alistipes megaguti and Alistipes provencensis.

Lactobacillus genus microorganisms, L. mucosae, L. salivarius, L.acidophilus, L. plantarum, L.pentosus), L.arizonensis, L. rhamnosus, L.brevis, L.caseii, L.paracasei, L.bulgaricus, L.delbruckii, L.arabinosus, L.caucasicus, L.leishmanni, L.musicus, L.themophilus, L.fermentum and L.helveticus, and the like.

As used herein, the terms "microbiota", "microflora" and "microbiome" refer to a living microbial community that normally inhabits an organ or part of the body, in particular the gastrointestinal tract of a subject, such as an animal or human.

The growth enhancement or inhibition of the microorganisms may be expressed by the content of the intestinal microorganisms, the content of metabolites of the intestinal microorganisms, and / or the relative abundance (%, relative abundance (%)) of the intestinal microorganisms. As used herein, "relative abundance" of a bacterial species means the relative abundance of a particular bacterial species to other bacterial species except for the corresponding bacterium in the human gastrointestinal microflora.

The regulation of the intestinal microflora of the composition according to the present invention can be achieved by increasing the relative abundance (%, relative abundance (%)) of microorganisms of the genus Lactobacillus and/or Alistipe in the intestine, and the relative abundance (%, relative abundance) of microorganisms of the genus Prevotella in the intestine (%)).

The composition according to the present invention has an increase in the relative abundance (%) of microorganisms of the genus Lactobacillus in feces or intestines, or microorganisms of the genus Alistipe in feces or intestines by 5% or more, or 7% or more, for example 5 % to 100%, which may be 105% or more or 107% or more, for example, 105% to 200%, based on 100% of the level before ingestion of the active ingredient to the subject.

Specifically, the composition according to the invention has an increase in the relative abundance (%) of microorganisms of the genus Lactobacillus in feces or in the intestine by 50 to 70%, 50 to 65%, or 50 to 60%, and/or feces A composition comprising kestose as an active ingredient, which achieves an increase in the relative abundance (%) of microorganisms of the genus Alistipe in the intestine or intestine by 5 to 25%, 5 to 20%, or 5 to 15% can The relative abundance (%) of microorganisms of the genus Lactobacillus in the feces or intestines is 150 to 170% based on 100% of the level before ingestion of the active ingredient to the target, and microorganisms of the genus Alistipe in the feces or intestines The relative abundance (%) of the active ingredient may be 105 to 125% based on 100% of the level before ingestion to the subject.

The composition according to the present invention is effective in reducing the decrease in the relative abundance (%) of microorganisms of the genus Prevotella in feces by 0.5% to 2.5%, 0.5 to 2%, or 0.5 to 1.5% It may be a composition as a component. The relative abundance (%, relative abundance (%)) of the microorganisms of the genus Prevotella in the feces or intestines may be 97.5 to 99.5% based on 100% of the level before ingestion of the active ingredient to the subject.

Another embodiment of the present invention is a composition for increasing the content of short-chain fatty acids in the intestine, for example, propionic acid containing kestose as an active ingredient, or a composition for growth or increase of propionic acid-producing bacteria in the intestine containing kestose as an active ingredient it's about

In addition, the present invention relates to a method for increasing the content of pyropionic acid in the intestine, comprising administering a composition containing kestose to a subject in need thereof, or a method for proliferating intestinal pyropionic acid-producing bacteria will be.

The composition may be a food composition, a health functional food, or a pharmaceutical composition.

The increase in the content of propionic acid in the feces or in the intestine may be due to the proliferation of propionic acid-producing microorganisms in the intestine. The proliferation of the microorganisms may be expressed as an increase in the content of intestinal microorganisms, an increase in the content of propionic acid, and/or an increase in the relative abundance (%, relative abundance (%)).

The intestinal propionic acid-producing microorganism may include microorganisms known in the art, for example, Lactobacillus spp. And Alisipes spp. may include one or more microorganisms from the group consisting of.

Specifically, in the experimental group (Advanced Fos: powder and syrup-medium concentration/high concentration) administration group according to the present invention, all short-chain fatty acids producing bacteria Lactobacillus spp. with Alisipes spp. was significantly increased, and Prevotella spp. It was confirmed that the significantly decreased (see Test Example 4). Prevotella spp. is associated with autoimmune diseases, insulin resistance, diabetes, and intestinal inflammation, and is known to play a role in reducing the production of short-chain fatty acids.

In one embodiment of the present invention, the propionic acid content in the feces is 100±30mg/L, 150±30mg/L, 200±30mg/L, 250±30mg/L or 300±30mg/L or more Kestose-containing composition provides

In one example of the present invention, the propionic acid content in the feces is 100 to 700%, 100 to 500%, 100 to 300%, 130 to 500%, 130 to 700%, 150 to 500%, 150 to 700 based on the level before ingestion. %, 150 to 300%, or 130 to 300%.

In one embodiment of the present invention, the weight ratio of butyric acid: propionic acid in feces or intestines is 4:1 to 1:3, 3:1 to 1:3, 3:1 to 1:2, 2:1 to 1:2 , More preferably, it provides a kestose-containing composition, characterized in that 1:1 to 1:2. By increasing the ratio of propionic acid to fecal or intestinal butyric acid in the corresponding ratio, it is possible to maintain the intestinal pH in an acidic environment, and to induce the maximization of the effect of improving inflammation by calming immune cells.

A further embodiment of the present invention relates to a composition for preventing, ameliorating or treating intestinal diseases and/or inflammatory diseases containing kestose. In addition, the disease may be a disease caused by an immune hypersensitivity reaction, for example, an inflammatory skin disease and/or inflammatory bowel disease caused by an immune hypersensitivity reaction. The composition may be a food composition, a health functional food, or a pharmaceutical composition.

The composition disclosed herein not only improves bowel function and intestinal function by improving the intestinal flora by promoting the growth or growth of beneficial intestinal bacteria and inhibiting the growth or growth of harmful bacteria in the intestine, but also acts as a prebiotic when combined with probiotics There is a synergistic effect that enhances the efficacy of probiotics. In an exemplary embodiment, the composition may be, for example, a composition for preventing, improving, or treating constipation, diarrhea, or intestinal-related diseases.

In addition, it is known that dysbiosis is highly associated with inflammatory diseases, such as inflammatory bowel disease (IBD) and inflammatory skin diseases. Accordingly, the composition comprising kestose according to the present invention can provide a composition for preventing, improving or treating inflammatory diseases.

Various possibilities, such as genetic factors and environmental factors, have been discussed as to the pathogenesis of inflammatory bowel disease, but in recent years, the possibility of abnormal intestinal immunity (digestive tract mucosal immunity) is increasing. Recent studies suggest the possibility that an abnormal immune response to a certain species of flora may become a chronic inflammatory response. The mechanism of the onset of inflammation and allergy in the intestinal tract due to abnormal intestinal immunity is supported by the analysis of the function and differentiation of the patient's T cells and the production pattern of cytokines in the lesion site and serum. Moreover, it became clear that this abnormality in mucosal immunity causes chronic inflammation in the intestinal tract in the analysis of various recently developed animal models of inflammatory bowel disease (Gastroenterology, Vol.109, p.1344-1367, 1995).

As used herein, the terms “subject”, “subject”, “host”, and “patient” may be used interchangeably herein, and are preferably any animal subject, including humans, laboratory animals, livestock, and domestic pets. refers to humans. The subject may host a variety of microorganisms. A subject may be diagnosed or suspected of being at high risk for a disease, and may have a microbiome condition (ie, a microbial imbalance) that contributes to the disease.

The inflammatory disease according to the present invention may be an inflammatory skin disease or inflammatory bowel disease caused by an immune hypersensitivity reaction, and the inflammatory skin disease includes atopic dermatitis, contact dermatitis, allergic contact dermatitis, etc., and the inflammatory bowel disease includes Crohn's disease (CD) and ulcerative colitis (UC), and UC is known as a chronic inflammatory disease that mainly targets the colon.

The inflammatory disease may be an inflammatory skin disease or inflammatory bowel disease caused by an immune hypersensitivity reaction, and the experimental group (Advanced Fos: powder and syrup-medium concentration and high concentration) contributes to restoring the immune system damaged in the immune response, and It was shown that the atopic symptoms were improved by inhibiting the activity (see Test Example 8).

The inflammatory skin disease, for example, atopic dermatitis is caused by an imbalance of the intestinal microbiome and Th1/Th2, typically due to a decrease in Th1 IFN-r involved in IgE expression and an increase in Th2 IL-13. The symptoms of AD are known to worsen. In addition, in histological analysis, IL-5, IL-13, TARC, and Eotaxin, which are associated with eosinophils, induce an increase in the number of eosinophils, influx into the skin, and degranulation, leading to inflammation and tissue damage.

Specifically, in the present invention, the Advanced Fos group is Lactobacillus spp. and Alisipes spp. increased and Prevotella spp. decreased, which was verified by the increase in SCFA secretion, and this result confirmed that it had an effect on the decrease in the increased Th2 expression in patients with atopic dermatitis. In addition, it is to improve AD symptoms by upregulating the expression of Th1-related factors and down-regulating the expression of Th2-related factors in order to balance Th1/Th2 in the intestine and skin as well as blood.

The present invention confirmed the improvement efficacy of atopic dermatitis (AD) in each formulation (powder, syrup) and concentration of fructooligosaccharide containing kestose.

Specifically, in an animal model inducing atopic dermatitis, when the composition according to the present invention was administered, the serum IgE content decreased, the secretion of IL-13 involved in IgE production decreased, and the number of eosinophils increased and The secretion of related TARC decreased, the secretion of Eotaxin involved in skin influx and degranulation of eosinophils decreased, and the secretion of IL-4 and IL-5, which caused Th1/Th2 imbalance, decreased. Therefore, the administration of the experimental group (Advanced Fos: powder and syrup-medium concentration/high concentration) improved the atopic symptoms by controlling the secretion of cytokines and suppressing the immune response of Th2 to restore the balance of Th1/Th2.

In addition, in an animal model inducing atopic dermatitis, when the composition according to the present invention was administered, the secretion amount of TNF-α was relatively increased, the secretion amount of Th1-related IFN-γ was relatively increased, and the secretion amount of Th1-related IL-12 was relatively increased. This relatively increased, administration of the experimental group (Advanced Fos: powder and syrup-medium concentration/high concentration) improved atopic symptoms by regulating the secretion of Th1-related cytokines to restore the balance of Th1/Th2 (see FIG. 17) .

In an animal model inducing atopic dermatitis, when the composition according to the present invention is administered, a decrease in the secretion of Treg-related cytokine, for example, IL-1β, a decrease in the secretion of IL-10 that causes an imbalance of Th1/Th2, by suppressing the excessive Treg immune response, suppressing the inflammatory response, and improving the atopic symptoms by restoring the Th1/Th2 balance.

In addition, as a result of administering the composition according to the present invention to an animal model inducing atopic dermatitis and evaluating the itching score and severity score for the effect of improving the severity of atopic dermatitis, it was confirmed that dermatitis was alleviated in the experimental group (see FIG. 15 ).

Kestose according to the present invention may be used alone or as a sugar composition comprising the same, and may be used in liquid or powder form, and the powder may be amorphous or crystalline. The Kestose may be used by purchasing a commercially available product, or may be prepared using a predetermined raw material.

Kestose usable in the present invention may be used in liquid or powder form, and may be included in the composition according to the present invention in various amounts.

The kestose may be used as a single component or may be used as a mixed composition including other saccharides together.

In the composition containing the kestose, the kestose in liquid or powder form is 50 (w/w) % or more, 60 (w/w) % or more, 70 (w/w) % or more, based on the total solids content of sugars; It may be provided as a composition having a content of 80 (w/w) % or more, or 85 (w/w) % or more, such as fructooligosaccharide. In addition, kestose in crystalline form may be a composition having a content of 90% or more, 95% or more, or 98% or more based on total solids.

The composition containing kestose may be fructooligosaccharide (FOS). For example, it contains a high content of 1-kestose (1-kesotse, GF2), nystose (Nystose, GF3) and 1-F-Fructosyl nystose (1-F-Fructosyl nystose, GF4) It may further include one or more saccharides selected from the group consisting of. FOS consists of a linear chain with 1 to 9 fructose residues linked to sucrose molecules by β2→1 bonds. As for the kestose-containing fructooligosaccharide, the higher the 1-kestose content, the more preferably 1-kestose is the main component. For example, it may contain a high content of 1-kestose (1-kesotse, GF2) in an amount of 50% by weight or more based on the sugar solid content.

There are products that contain a small amount of Kestose (1-kestose) in a small amount of about 20 to 35 (w/w)% based on the total solids in FOS distributed in the market, but in the present invention, Kestose, an active ingredient that exhibits an effective effect, By containing tosses (1-kestose) in an amount of 50% (w/w) or more based on the total solids of sugar, it is possible to effectively control the daily intake (dose) of kestose, and through this, it is possible to effectively control the The balance of body metabolism can be induced through the regulation of microbiome and microbial metabolites. When a sugar composition having a low kestose content, such as fructooligosaccharide, is ingested, the intake of fructooligosaccharide is increased in order to ingest an effective amount of kestose according to the present invention, and thus the digestive organs (diarrhea, bloating) . Therefore, by ingesting a sugar composition having a high kestose content, such as fructooligosaccharide, undesirable problems caused by excessive intake of fructooligosaccharide can be lowered, and furthermore, an effective amount of kestose intake according to the present invention can be easily achieved. Efficacy can be further increased.

Kestose or a sugar composition containing kestose usable in the present invention is not particularly limited, and may be prepared using an enzyme having kestose conversion activity or a microorganism producing the enzyme, usually using sugar as a substrate. The prepared kestose-containing product may be used as it is, or it may be used as a sugar composition in which the kestose content is increased by performing extraction and purification processes.

The enzyme having the kestose conversion activity is an enzyme having an activity to convert fructooligosaccharide containing kestose from a substrate containing sugar, for example, Aspergillus niger (Aspergillus niger) strain, Pichia fly Derived from at least one selected from the group consisting of Pichia farinose strain, Yarrowia lipolytica, Millerozyma farinose, and Aspergillus oryzae strain It may be an enzyme.

The dosage form of the drug, quasi-drug, or supplement containing kestose is not particularly limited, and a dosage form suitable for the administration method can be appropriately selected. For example, in the case of oral administration, solid or liquid formulations such as powders, tablets, dragees, capsules, granules, dry syrups, liquids, syrups, drops, and drinks can be used.

The total daily intake of Kestose as an active ingredient in the Kestose composition for an adult 60 kg is 2 to 30 g, 2 to 27 g, 2 to 26 g, 2 to 25 g, 2 to 24 g, 2 to 20 g , 2 to 15 g, 2 to 10 g, or 2 to 8 g. Such an intake amount is not limited to once a day, but can be ingested by dividing it into a plurality of times. It can be set to an appropriate amount in consideration of the side effects of the intestinal function, intestinal function improvement, calcium absorption enhancement function and “feel bloated” according to the intake of the kestose or fructooligosaccharide containing it.

In addition, the kestose composition contains kestose, nystose, and fructofuranosylnistose in an amount of 400 mg/g or more, 500 mg/g or more, 800 mg/g or more, or 900 mg/g or more. It may be a composition that In the kestose composition, the content weight ratio of kestose to the total weight of nystose and fructofuranosylnistose is 1:1 to 10:1, 1:1 to 5:1, 1:1 to 3: 1, or 1:1 to 2:1 composition.

The subject or individual administering or ingesting the composition containing kestose as an active ingredient according to the present invention is an animal including a human, for example, includes a human, a mouse, a rat, a monkey, and the like.

The composition containing kestose as an active ingredient according to the present invention can be ingested before, after, or simultaneously with a meal, and the intake conditions are not particularly limited. The composition containing the kestose as an active ingredient may be ingested for a period effective to improve inflammatory diseases, improve intestinal function, or achieve changes in the intestinal microbial community or environment, for example, 4 weeks or more, 8 weeks or more, or It may be a method of intake for 15 weeks or more, for example, 4 to 20 weeks. An effective effect can be derived only by ingesting the composition according to the present invention once, but it can be ingested twice or more.

The formulation unit for ingestion of the composition containing kestose as an active ingredient is, for example, to contain 1, 2, 3 or 4 times or 1/2, 1/3 or 1/4 times the individual intake amount. can be manufactured. The individual intake preferably contains an amount administered once a day of the active ingredient, which is usually formulated in an amount corresponding to all, 1/2, 1/3 or 1/4 times the daily dose and divided may be administered.

The composition comprising kestose according to the present invention may be a food composition or a pharmaceutical composition.

A suitable dosage of the composition according to the present invention is a formulation method, an administration method, the patient's age, weight, sex, pathological condition, food, administration time, administration route,. It can be prescribed in various ways depending on factors such as excretion rate and response sensitivity.

The composition containing kestose as an active ingredient according to the present invention may be a food, a food additive, a beverage, a beverage additive, a health food, or a functional food. In the present invention, "health functional food" means a food manufactured (including processing) using raw materials or ingredients useful for the human body in accordance with the Health Functional Food Act. It refers to obtaining useful effects for health purposes such as maintaining and improving health by maintaining the normal function of the body or activating physiological functions. The kestose can be added to general foods, or it can be prepared by encapsulation, powdering, suspension, or the like. When consumed, it has specific health effects, and unlike general drugs, it has the advantage of not having side effects that may occur when taking the drug for a long time because it is made from food.

When the kestose of the present invention is used as a food additive, the kestose can be added as it is, used with other foods or food ingredients, or used appropriately according to other conventional methods. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of its use (prophylactic, health or therapeutic treatment).

The kestose or the composition containing kestose may be administered as it is or in the form of food or medicine by oral ingestion to a subject, for example, a human or an animal.

Kestose (1-kestose) can be added to and used in the normal manufacturing process of various foods, food additives, and animal feed. The sweetness of 1-kestose is 30, and its quality of taste), physical properties, and processability are close to that of sucrose. Treated like sugar, it can be used in various foods, beverages, food additives, medicines, or feed.

Specific embodiments of the composition according to the present invention include, for example, beverages, dairy products, granules provided for food, paste, seasoning, retort food, baby food, fermented food, preserved food, processed aquatic products, processed meat products, grains Processed foods such as processed products, food additives, health foods, animal feeds, etc. are mentioned.

The present invention has the advantage of improving intestinal microbiome imbalance by simply and effectively increasing beneficial intestinal microbes in humans or animals and reducing harmful microbes without worrying about side effects or safety.

In addition, according to the present invention, there is an advantage of improving the intestinal environment by increasing propionic acid or propionic acid-producing strains of human or animal intestinal short-chain fatty acids simply and effectively without worrying about side effects or safety.

1 is an experimental protocol for analyzing the effect of kestose on the intestinal microflora using an animal model according to an example of the present invention.
2 is a result of measuring the change in body weight of an experimental animal model according to an example of the present invention.
3 is a graph showing the species diversity of intestinal microbes through alpha-diversity analysis for gut microbes in an experimental animal model according to an example of the present invention.
4 is a graph showing the difference between groups of intestinal microorganisms through beta-diversity analysis on the intestinal microorganisms in an experimental animal model according to an example of the present invention.
5 shows the results of phylum profiling of the intestinal microbial community using fecal samples from animal models collected at the 8th, 12th, and 16th weeks of the study period according to an example of the present invention.
6 shows the results of phylum profiling of the intestinal microbial community using fecal samples of animal models collected at the 16th week of the study period according to an example of the present invention.
7 shows the results of top30 genus profiling through heatmap analysis of the intestinal microbial community using fecal samples of animal models collected at 8, 12, and 16 weeks of the study period according to an example of the present invention.
8 shows the results of top30 genus profiling through heatmap analysis of the intestinal microbial community using fecal samples of animal models collected at the 16th week of the study period according to an example of the present invention.
9 shows a significant increase in Lactobacillus spp. by administration of the experimental group according to an example of the present invention.
10 shows a significant increase in Alistipe spp. by administration of the experimental group according to an example of the present invention.
11 shows a significant decrease in Prevetella spp. by administration of an experimental group according to an example of the present invention.
12 is a result of measuring the change in butyric acid content according to the administration of the experimental group using the fecal sample of the animal model collected at the 16th week of the study period according to an example of the present invention.
13 is a result of measuring the change in acetic acid content according to the administration of the experimental group using the fecal sample of the animal model collected at the 16th week of the study period according to an example of the present invention.
14 is a result of measuring the change in propionic acid content according to the administration of the experimental group using the fecal sample of the animal model collected at the 16th week of the study period according to an example of the present invention.
15 is an evaluation result of improving the severity of atopic dermatitis through a total score (itching and severity) according to an example of the present invention.
16 is an analysis result of serum IgE secretion of experimental animals according to an example of the present invention.
17 is an analysis result of the secretion amount of serum TNF-α, IFN-γ, and IL-12 of an experimental animal according to an embodiment of the present invention.
18 is an analysis result of the secretion amount of serum L-4, IL-5, IL-13, TARC, and Eotaxin of experimental animals according to an embodiment of the present invention.
19 is an analysis result of the secretion amount of serum IL-1β and IL-10 of an experimental animal according to an example of the present invention.
20 is a diagram showing the CD86 expression level of splenocytes analyzed by flow cytometry according to an embodiment of the present invention.
21 is a diagram showing the CD274 expression level of splenocytes analyzed by flow cytometry according to an embodiment of the present invention.
22 is a result of measuring the number of mast cells (mast cell count) in the skin tissue of an experimental animal according to an example of the present invention.
23 is a result of measuring the number of eosinophils (eosinophil count) in the skin tissue of an experimental animal according to an example of the present invention.
24 is a result of measuring the number of mast cells (mast cell count) in the intestinal tissue of an experimental animal according to an example of the present invention.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not intended to be limited to the following examples.

Preparation Example 1: Animal model construction

The overall schedule of animal model construction and dietary administration experiments in this experiment is shown in FIG. 1 .

Specifically, in order to construct an animal model with atopic dermatitis, five-week-old female balb/c mice were separated from each group by 6 mice and were acclimatized for 1 week. For all groups except for the negative control group (Neg), the OVA/Al(OH)3 mixture was prepared by dissolving OVA and Alum (Sigma Aldrich) in PBS. Administered intraperitoneally (once in 2 weeks, a total of 4 doses were administered for 8 weeks). Also, hair removal on the back of Balb/c mice was continued for 8 weeks, and OVA/Al(OH)3 was continuously contacted with the skin of the back. AD was induced, so the atopic dermatitis induced experimental animal was used as an experimental group animal model in subsequent experiments.

As a negative control group, 5 week-old female balb/c mice were separated from each group by 6 mice and were acclimatized for 1 week. Thereafter, similar to (1) construction of the atopic animal model, but without inducing atopy for 8 weeks using the OVA/Al(OH)3 mixture, PBS at the same dose as the experimental group was orally administered during the subsequent administration period.

As a positive control group, five-week-old female balb/c mice were separated from each group by 6 and were acclimatized for 1 week. Thereafter, atopy was induced through OVA/Al(OH)3 for 8 weeks similar to the construction of the atopic animal model, and PBS at the same dose as the experimental group was orally administered during the administration period.

Fecal samples were collected at 8, 12, and 16 weeks during the entire study period from the experimental animals to be used as the experimental group, negative control group, and positive control group, and were used for microbiome and SCFA analysis later. In addition, at the end of the experiment, cecal fecal samples were collected and used for microbiota profiling and metabolite analysis.

Comparative Examples 1 and 2: maltose diet experiment

The maltose diet animal model is an experimental group animal model in which atopy was induced through OVA/Al(OH) ₃ for 8 weeks in Example 1 and then proceeded with the same diet. ) was dissolved in 200ul of PBS at the standard concentration for one mouse and orally administered at the same concentration.

Specifically, Comparative Example 1-1 was applied at a maltose-medium concentration of 8 g/day ratio, and in Comparative Example 1-2, Maltose-high concentration was applied at a ratio of 16 g/day.

Comparative Examples 3 and 4: Low Kestose Containing FOS Powder Diet Experiment

The experimental group administered with FOS induced atopy through OVA/Al(OH) ₃ for 8 weeks, and then dissolved FOS in 200ul of PBS based on one mouse relative to the kestose content (33%) during the administration period for oral administration. It was prepared as a sample.

The low-content Kestose-containing FOS powder had a fructooligosaccharide content of 95% based on total solids, and the kestose content included in the fructooligosaccharide was 33% by weight, and thus the dietary powder had a kestose content of 31.35% by weight based on the total solids. will contain

The experiment was carried out by dividing the solution obtained by dissolving each sample in PBS into two doses, administered twice a day, and for adults (60 kg), Comparative Example 3 was 8 g/day medium concentration group and Comparative Example 4 was 16 g. Each of the /day high-dose groups was applied and administered for 8 weeks.

The high- and medium-concentration diets were divided into diets of the same composition according to the daily dose, and the medium concentration group was applied at a dose of 8 g/day and the high concentration group at a dose of 16 g/day.

group group description formulation negative control negative PBS positive control Positive PBS Comparative Example 1 Maltose-Medium concentration applied at 8 g/day rate powder Comparative Example 2 Maltose - applied at a high concentration of 16 g/day powder Comparative Example 3 Low-content Kestose FOS-Medium concentration 8g/day ratio applied powder Comparative Example 4 Low-content Kestose FOS-High concentration 16g/day ratio applied powder Example 1 Advanced Fos-Medium concentration 8g/day rate applied powder Example 2 Advanced Fos-High concentration 16g/day rate applied powder Example 3 Advanced Fos-Medium concentration 8g/day rate applied syrup Example 4 Advanced Fos-High concentration 16g/day rate applied syrup

Examples 1 and 2: High Kestose Containing FOS Powder Diet Experiment

The experimental group administered with FOS powder containing high content of kestose induced atopy through OVA/Al(OH)3 for 8 weeks. It was dissolved to prepare a sample for oral administration.

The high-content Kestose-containing FOS powder used in this Example had a fructooligosaccharide content of 91.2% based on the total solids, and the kestose content included in the fructooligosaccharides was 85.5% by weight, so the dietary powder was 77.98 based on the total solids. It contains kestose in weight percent.

Each sample was administered by dissolving it in PBS, and the experiment was performed by dividing it into two doses, administered twice a day, and based on an adult (60 kg), Example 1 is a dose of 8 g of the medium concentration group and Example 2 was administered for 8 weeks each at a dose of 16 g in the high concentration group.

A diet was fed to the animal model in substantially the same manner as in Comparative Example 3, except that the FOS powder containing high kestose prepared in the present invention was fed instead of the FOS powder of Comparative Example 3.

Examples 3 and 4: High Kestose Containing FOS Syrup Diet Experiment

The experimental group administered with FOS syrup containing high kestose induced atopy through OVA/Al(OH)3 for 8 weeks. (85.6%), dissolved in 200ul PBS at a concentration of 1 mouse relative to the solid content of FOS syrup (75.3%), and prepared as a sample for oral administration.

The FOS syrup containing high kestose used in this Example had a fructooligosaccharide content of 91.2% based on total solids, and the kestose content contained in the fructooligosaccharides was 89.9% by weight, so the dietary powder was 81.998 based on total solids. It contains kestose in weight percent.

Each sample was administered by dissolving it in PBS, and the experiment was performed by dividing it into two doses, administered twice a day, and based on an adult (60 kg), Example 3 is a medium concentration group having a dose of 8 g and Examples 4 was administered for 8 weeks as a high-dose group having a high dose of 16 g.

A diet was fed to the animal model in substantially the same manner as in Comparative Example 3, except that the FOS syrup containing high kestose prepared in the present invention was fed instead of the FOS powder of Comparative Example 3.

Test Example 1: Confirmation of mouse weight gain and dietary intake

During the administration period, the weight change of the mice according to the administration of each experimental group was recorded, and the mouse weight was measured at the same time period before oral administration every week.

In order to check whether obesity was caused by fructooligosaccharide intake, the dietary intake of mice according to the administration of each experimental group was checked during the administration period, and the dietary intake was confirmed at the same time every week.

2 is a result of the change in body weight of the experimental animal model according to the administration of the experimental group, and it was shown that the amount of weight gain decreased due to atopy in the positive control group and the maltose administration group according to Comparative Examples 1 and 2. In the case of the experimental group according to Examples 1 to 4, since the amount of weight gain was similar to that of the negative control group, the amount of weight gain was recovered according to the improvement effect of Advanced FOS according to Examples 1 to 4 on atopy.

Test Example 2: Species Diversity Analysis of the Intestinal Microbiome

(1) Analysis of microbial community in fecal samples

For the samples of Comparative Examples 1 to 4 and Examples 1 to 4, for the experimental animals of Preparation Example 1, fecal samples were collected at 8, 12, and 16 weeks during the entire study period. DNA was extracted from the collected 8, 12, 16, fecal samples using a DNA soil kit.

180 DNAs were sequenced through Illumina Miseq platform, and a total of 13,117,845 16S rRNA sequence read counts were obtained, and an average of 85,737 reads were read per sample, and all groups obtained more than 2500 reads. The 16S rRNA sequences were analyzed using the qiime2 pipeline, and sequences without or unclear at the 5' or 3' end were removed. Trimmed sequences less than 250 bp were removed, and chimera sequences were removed using the chimera of the qiime2 pipeline. This allows the identification of each sequencing read in the sample to view the microbial community structure.

Shannon index, Simpson index and InvSimpson index were calculated for alpha-diversity of the gut microbiome. For the analysis of species community diversity (beta-diversity), cluster analysis was performed through Adonis analysis using Bray-Curtis distance metrics. The graph was visualized through R software (version 4.0.3).

(2) Analysis of species diversity and group differences in the gut microbiome

In order to analyze the species diversity of the intestinal microbiome and differences between groups, for the experimental animals of Comparative Examples 1 to 4 and Examples 1 to 4, fecal samples obtained at the 16th week of the study period were removed from low-quality sequences and averaged sequencing. was confirmed to be 85,737. In order to examine the similarity between the positive control and the negative control, which are atopic-induced mice, the alpha-diversity of the intestinal microbiota was confirmed. Alpha diversity results showed that there was no difference between the groups in Shannon, Simpson, and InvSimpson diversity indices (FIG. 3).

However, in the microbial beta-diversity, it was confirmed that each group was divided, and in particular, although the positive control group and the maltose group were on the right, it could be seen that the experimental group changed similarly to the negative control group (Fig. 4). ).

Test Example 3: Confirmation of intestinal microbiome profiling

For the experimental animals of Comparative Examples 1 to 4 and Examples 1 to 4, fecal samples were collected at 8, 12, and 16 weeks during the entire study period. DNA was extracted from the collected 8-week, 12-week and 16-week fecal samples using a DNA soil kit. Using the DNA sample, profiling analysis of the intestinal microbiome between the control group and the experimental group for 8 weeks, 12 weeks, and 16 weeks was performed.

The microbiome can be designated as a taxonomy that organizes the lineages and subordinations of living things based on specific criteria. The samples at week 8, week 12, and week 16 by group were confirmed at the Phylum level. It was confirmed that Bacteroidetes and Firmicutes were predominant in all groups overall at the Phylum level of the 8-week, 12-week, and 16-week samples ( FIG. 5 ).

In addition, when compared with the positive control group at the Phylum level of the 16-week sample, Firmicutes in the Advanced Fos powder high concentration administration group of Example 2 and the Advanced Fos syrup medium concentration and high concentration administration group of Example 4 increased, and Bacteroidetes decreased.

More specifically, compared with the positive control group (1.83% ± 0.05), the relative abundance ratio of Firmicutes: Bacteroidetes was the high concentration of FOS powder containing high kestose of Example 2 group (2.72% ± 0.15). , p = 0.0048) and the FOS syrup medium dose group containing high kestose of Example 3 and the FOS syrup high concentration group containing high kestose contents according to Example 4 (medium concentration; 2.42% ± 0.15, p = 0.0245, It was confirmed that it was significantly increased at high concentration; 3.34% ± 0.15, p < 0.0001).

There was no significant difference between the positive control group and the maltose group of Comparative Examples 1 and 2 (medium concentration of Comparative Example 1; 2.11% ± 0.07, p = 0.2184, high concentration of Comparative Example 2; 1.58% ± 0.10, p = 0.26) ( Fig. 6).

In summary, at the Phylum level, it was confirmed that Bacteroidetes and Firmicutes were dominant, and at week 16, unlike the Maltose group of the positive control group and Comparative Examples 1 and 2, in the experimental group administration group of Examples 1 to 4, Firmicutes was relatively higher than Bacteroidetes. was confirmed to increase. It was confirmed that the ratio of Bacteroidetes did not decrease in the medium concentration group of Fos powder containing high content of kestose of Example 1, but it was confirmed that it was associated with a significant increase such as Alisipes through genus profiling.

Through heatmap analysis at the genus level through Top30, the intestinal microflora of the 16-week samples for each group were identified. At the Genus level, Lactobacillus, Alisipes, Prevotella, Muribaculum, and Bacteroides were confirmed as dominant species in the intestine. More specifically, Lactobacillus (Lactobacillus), Muribaculum ( Muribaculum ), Alistipes ( Alistipes ), Bacteroides ( Bacteroides ) and Prevotella ( Prevotella ) Four genera (Genus) was dominant. Lactobacillus confirmed high occupancy in the normal control group and the Advanced Fos syrup high concentration group of Example 4 (63.70% ± 2.31, p = 0.0022). On the other hand, it was visually confirmed that the Advanced Fos powder medium concentration administration group of Example 2 increased significantly than the Advanced Fos syrup medium concentration group of Example 3 and the Advanced Fos syrup high concentration administration group of Example 4 in Alisipes (Fig. 7, and Fig. 8).

Test Example 4: Analysis of SCFA-producing bacteria

(1) Increase of SCFA-producing bacteria by administration of test group

For the experimental animals of Comparative Examples 1 to 4 and Examples 1 to 4, when compared with the positive control group and the maltose group in the fecal sample obtained at the 16th week of the study period, the experimental group according to Examples 1 to 4 (Advanced Fos : Lactobacillus spp, a short-chain fatty acids producing bacteria in both powder and syrup-medium/high concentration) administration groups. with Alisipes spp. was confirmed to be significantly increased.

(2) Lactobacillus spp.

Lactobacillus spp. It was confirmed that it was significantly increased in the Advanced Fos powder high concentration according to Examples 1 and 2 and the Advanced Fos syrup medium/high concentration administration group according to Examples 3 and 4. Lactobacillus spp. showed a gradually increasing pattern over 8 weeks, 12 weeks, and 16 weeks.

In particular, at 16 weeks, when compared to the positive control group (39.61% ± 4.61), the high concentration of Fos powder according to Example 2 (63.70% ± 2.31, p = 0.0022) and the intermediate concentration of Advanced Fos syrup according to Examples 3 and 4 / It was confirmed that the high concentration group (medium concentration; 58.91% ± 3.28, p = 0.0043, high concentration; 66.31% ± 4.35, p = 0.0022) significantly increased. There was no significant difference between the positive control group and the maltose group according to Comparative Examples 1 and 2 (medium concentration; 40.71% ± 5.48, p = 0.8571, high concentration; 35.85% ± 0.61, p = 0.0905). Lactobacillus spp. (family Lactobacillaceae, phylum Firmicutes) are short-chain fatty acid producing bacteria, known as beneficial strains for the intestine.

(3) Alisipes spp

Aliipes spp. was not significantly different at 8 weeks and 12 weeks, but increased significantly at 16 weeks. It was confirmed that the Advanced Fos powder medium concentration group according to Example 1 was significantly increased compared to the Advanced Fos syrup medium concentration/high concentration administration group according to Examples 3 and 4 in Alisipes spp. In particular, at 16 weeks, when compared to the positive control group (3.47% ± 0.99), Advanced Fos powder medium/high concentration according to Examples 1 and 2 (medium concentration; 14.73% ± 1.43, p = 0.0043, high concentration; 14.38% ± 1.97) , p = 0.0043) and the Advanced Fos syrup medium/high concentration administration group according to Examples 3 and 4 (medium concentration; 8.74% ± 0.40, p = 0.0095, high concentration; 6.59% ± 0.25, p = 0.0152) significantly increased. confirmed to be. There was no significant difference between the positive control group and the maltose group according to Comparative Examples 1 and 2 (medium concentration of Comparative Example 1; 4.22% ± 1.51, p = 0.4429, high concentration of Comparative Example 2; 1.76% ± 0.43, p = 0.6095) . Aliipes spp. (family Rikenellaceae, phylum Bacteroidetes) are short-chain fatty acid producing bacteria, known as beneficial strains for the gut.

(4) Prevotella spp.

Compared with the positive control group and the maltose group of Comparative Examples 1 and 2, Advanced Fos (powder and syrup-medium and high concentration) administration according to Examples 1 to 4, Prevotella spp. It was confirmed that significantly decreased. In particular, at 16 weeks, when compared to the positive control group (5.37% ± 1.40), Advanced Fos powder medium concentration / high concentration according to Examples 1 and 2 (medium concentration of Example 1; 1.56% ± 0.37, p = 0.0043, Example 2 high concentration; 0.90% ± 0.35, p = 0.0043) and the Advanced Fos syrup medium/high concentration administration group according to Examples 3 and 4 (Example 3 medium concentration; 1.58% ± 0.36, p = 0.0087, Example 4) High concentration of ; 1.69% ± 0.33, p = 0.0043) was confirmed to be significantly reduced. There was no significant difference between the positive control group and the maltose group (medium concentration; 2.80% ± 0.16, p = 0.1077, high concentration; 4.31% ± 0.47, p = 0.8571). Prevotella spp. is associated with autoimmune diseases, insulin resistance, diabetes, and intestinal inflammation, and is known to play a role in reducing the production of short-chain fatty acids.

Test Example 5: Intestinal metabolite (SCFA) analysis

(1) Measurement of butyrate content in feces

A stool sample (100mg) was homogenized in NaOH containing 5ug/ml caproic acid (Sigma-Aldrich) and centrifuged (12,000g, 4℃) for 20 minutes. The supernatant was transferred to a Tube (Corning, USA) and stored at -80°C until HPLC analysis. SCFA was analyzed using an Aminex 87H column (300×10 mm; Bio-Rad, USA), and butyric acid was detected using an RI-detector (ERC, RefractoMax 520, Japan).

The low-content kestose-containing Fos (powder) according to Comparative Examples 3 and 4, the high-kestose-containing Advanced Fos (powder) according to Examples 1 and 2, and the high-kestose-containing Advanced Fos syrup according to Examples 3 and 4 were prepared. In the administered experimental group, the butyrate content in the feces was high at 16 weeks, and the level was similar to that of the negative control group (Neg).

12 is a graph comparing the content of butyric acid in mouse fecal samples according to the administration of the experimental group. As a result, at 16 weeks, the content of butyric acid in the positive control group and the group administered with maltose (medium/high concentration) of Comparative Examples 1 and 2 is 32.2±4.9mg/L, 45.5±3.9mg/L, and 29.5±3.5mg/L, respectively, while the experimental group according to Examples 1 to 4 (Advanced Fos: powder and syrup-medium concentration/high concentration) was 172.8± A significant increase was observed at 5.8 mg/L, 232.6±43.2 mg/L, 188.1±27.4 mg/L, or 221.9±23.9 mg/L. Butyric acid is important for maintaining intrinsic immunity and intestinal health and is involved in inhibiting inflammation, so the ingestion of the experimental group (Advanced Fos: powder and syrup-medium concentration/high concentration) according to Examples 1 to 4 improved the immune response and intestinal health to normal levels. Atopic disease was improved by recovering to

(2) Measurement of acetic acid (Acetate) content in feces

The content of acetic acid in the stool sample was measured in the same manner as in Test Example 5-(1).

13 is a graph comparing the acetic acid content in mouse fecal samples according to the administration of the experimental group. As a result of the comparison, the content of acetic acid in the positive control group and the group administered with maltose (medium concentration/high concentration) at week 16 was 296.1±40.4, respectively. mg/L, 304.9±46.3 mg/L, 277.1±20.8 mg/L, whereas the experimental group according to Examples 1 to 4 (Advanced Fos: powder and syrup-medium concentration/high concentration) was 471.1±38.0 mg/L, respectively, Significant increases were observed at 561.2±75.4 mg/L, 561.0±29.7 mg/L, and 653.4±103.7 mg/L.

(3) Measurement of propionic acid content in feces

The content of propionic acid in the stool sample was measured in the same manner as in Test Example 5-(1).

14 is a graph comparing the propionic acid content in the mouse fecal sample according to the administration of the experimental group, and as a result, the content of propionic acid in the positive control group and the group administered with maltose (medium concentration/high concentration) at week 16 was 28.1±2.7 mg, respectively; /L, 96.1±17.2mg/L, 42.8±6.2mg/L, whereas the experimental groups according to Examples 1 to 4 (Advanced Fos: powder and syrup-medium concentration/high concentration) were 112.1±19.2mg/L, 158.2, respectively Significant increases were observed as ±35.6mg/L, 143.2±12.7mg/L, and 211.5±19.6mg/L.

Test Example 6: Evaluation of improvement in severity of atopic dermatitis

The number of scratching of the mouse for 15 minutes was measured at the time when atopy induction with OVA/Al(OH)3 was completed (8 weeks) and when the administration of the experimental group was completed (16 weeks). In addition, the severity of dermatitis was quantified by applying a severity score of 0 (none), 1 (mild), 2 (moderate), and 3 (severe) based on the symptoms of dryness, erythema, and keratin. The final score was the number of scratches and the severity score. were summed.

15 is As a graph comparing the effect of improving the severity of atopic dermatitis by administration of the experimental group by numerical values such as itching score and severity score, the Advanced according to Examples 1 to 4 compared to Fos (powder) containing low content of kestose according to Comparative Examples 3 and 4 It was confirmed that dermatitis was alleviated in the experimental group to which Fos (powder and syrup) was administered, and in particular, it was confirmed that the improvement effect was excellent in the experimental group to which Advanced Fos according to Examples 1 to 4 was administered.

Test Example 7: Serological analysis of experimental animals

Blood was obtained from mice sacrificed on the day of the end of administration of the experimental group. The blood was stored at 4° C. for 1 hour and centrifuged at 5,000×g for 1 hour to separate the serum.

Serum IgE and Cytokine (TNF-α, IFN-γ, IL-12, IL-4, IL-5, IL-13, TARC, Eotaxin, IL-1β, IL-10) analysis was performed with an ELISA kit (R&D systems, USA). ), and absorbance was measured at 450 nm using a microplate reader (Tecan, Mδnnedorf, Switzerland).

16 is a graph analyzing the amount of serum IgE secretion by AD induction and administration of the experimental group. As a result of comparing the secretion amount of IgE that aggravates the symptoms of AD by stimulating mast cells, the positive control group and maltose (medium concentration/high concentration) were administered IgE secretion in the group was 16.7±1.8pg/ml, 16.4±1.1pg/ml, and 13.13±1.3pg/ml, respectively, whereas Fos (powder) according to Comparative Examples 3 and 4 was at medium/high concentrations, respectively. 10.9±1.1pg/ml, 10.7±0.64pg/ml levels were shown, and Advanced Fos (powder) according to Examples 1 and 2 was 10.6±1.1pg/ml and 9.9±1.3pg/ml at medium/high concentrations, respectively. , Advanced Fos (syrup) according to Examples 3 and 4 was 9.2±1.6pg/ml and 8.3±0.6pg/ml at medium/high concentrations, respectively, in the experimental group in which Fos (powder) and Advanced Fos (powder and syrup) were administered It was confirmed that the secretion of IgE was relatively decreased.

17 shows the results of analyzing the Th1-related cytokine secretion by AD induction and administration to the experimental group. In the graph comparing the secretion of TNF-α, the TNF-α secretion of the positive control group and the group administered with maltose (medium/high concentration) were 23.8±2.6pg/ml, 23.9±2.22pg/ml, and 23.1±1.3pg/ml, respectively. While the level of ml was shown, Fos (powder) according to Comparative Examples 3 and 4 exhibited 29.8±1.2 pg/ml and 31.7±0.9 pg/ml at medium/high concentrations, respectively.

In addition, Advanced Fos (powder) according to Examples 1 and 2 is 32.4±1.3pg/ml and 35.8±2.4pg/ml, respectively, at medium concentration/high concentration, Advanced Fos (syrup) according to Examples 3 and 4 is medium concentration TNF in the experimental group administered with Fos (powder) according to Comparative Examples 3 and 4 and Advanced Fos (powder and syrup) according to Examples 1 to 4 at 32.3±1.1pg/ml and 34.9±2.8pg/ml, respectively, at high concentrations It was confirmed that the secretion amount of -α was relatively increased.

As a result of comparing the secretion of Th1-related IFN-γ, the IFN-γ secretion of the positive control group and the group administered with maltose (medium/high concentration) were 12.7±0.9pg/ml, 13.1±0.3pg/ml, and 13.7±0.3, respectively. While it showed a level of pg/ml, Fos (powder) according to Comparative Examples 3 and 4 was 16.4±0.6pg/ml and 17.3±0.7pg/ml, respectively, at medium/high concentrations, Advanced Fos according to Examples 2-3 (Powder) is 21.1±1.4pg/ml and 23.3±0.9pg/ml at medium/high concentration, respectively, Advanced Fos (syrup) according to Examples 3 and 4 is 17.3±0.5pg/ml at medium/high concentration, respectively, It was confirmed that the secretion of IFN-γ was relatively increased in the experimental group administered with Fos (powder) according to Comparative Examples 3 and 4 and Advanced Fos (powder and syrup) according to Examples 1 to 4 at 18.3±1.3 pg/ml. .

As a result of comparing the secretion of Th1-related IL-12, the IL-12 secretion of the positive control group and the group administered with maltose (medium/high concentration) were 103.3±5.5pg/ml, 101.5±4.6pg/ml, and 106.3±9.6, respectively. While the level of pg/ml was shown, Fos (powder) according to Comparative Examples 3 and 4 was 115.3±5.6pg/ml, 122.5±1.6pg/ml, and Advanced Fos according to Examples 1 and 2 at medium/high concentrations, respectively. (Powder) is 128.5±2.9pg/ml at medium/high concentration, respectively, 129.2±2.7pg/ml according to Examples 3 and 4, Advanced Fos (syrup) is 126.5±3.5pg/ml at medium/high concentration, respectively, It was confirmed that the secretion amount of IL-12 was relatively increased in the experimental group administered with Fos (powder) according to Comparative Examples 3 and 4 and Advanced Fos (powder and syrup) according to Examples 1 to 4 at 125.8±3.0 pg/ml. .

Therefore, administration of the experimental group (Advanced Fos: powder and syrup-medium concentration/high concentration) according to Examples 1 to 4 improved the atopic symptoms by regulating the secretion of Th1-related cytokines to restore the balance of Th1/Th2.

18 is a comparison result of Th2-related cytokine secretion by AD induction and administration to the experimental group. Specifically, as a result of comparing the secretion amount of IL-4, which causes Th1/Th2 imbalance by inhibiting Th1 cell differentiation and promoting Th2 cell differentiation, the positive control group and the maltose (medium/high concentration) group administered IL- 4 The secretion amount was 46.9±1.0pg/ml, 43.5±1.6pg/ml, and 42.1±2.2pg/ml, respectively, whereas Fos (powder) according to Comparative Examples 3 and 4 was 26.5± 1.7pg/ml, 23.1±3.5pg/ml, Advanced Fos (powder) according to Examples 1 and 2 were 12.8±0.9pg/ml, 9.3±0.6pg/ml, Examples 3 and 4 at medium/high concentrations, respectively Advanced Fos (syrup) according to Fos (powder) according to Comparative Examples 3 and 4 and Advanced Fos ( It was confirmed that the secretion amount of IL-4 was relatively decreased in the experimental group administered with powder and syrup).

As a result of comparing the secretion of IL-5 that causes Th1/Th2 imbalance with IL-4, the IL-5 secretion of the positive control group and the group administered with maltose (medium/high concentration) was 136.9±6.8 pg/ml, respectively. , 130.9±8.3pg/ml, 136.1±14.7pg/ml, whereas Fos (powder) was 122.5±10.9pg/ml, 79.8±20.3pg/ml, Examples 1 and 2 at medium/high concentrations, respectively The Advanced Fos (powder) according to In the experimental group administered with Fos (powder) according to Comparative Examples 3 and 4 and Advanced Fos (powder and syrup) according to Examples 1 to 4 at pg/ml, 68.9±12.7 pg/ml, the secretion amount of IL-5 was relatively It was confirmed that it decreased.

As a result of comparing the secretion of IL-13 involved in IgE production, the IL-13 secretion of the positive control group and the group administered with maltose (medium/high concentration) were 324.8±31.4pg/ml, 316.6±58.4pg/ml, respectively, respectively. While it showed a level of 316.6±32.6pg/ml, Fos (powder) according to Comparative Examples 3 and 4 was 225.6±5.7pg/ml, 168.7±7.1pg/ml, Examples 1 and 2 at medium/high concentrations, respectively. Advanced Fos (powder) according to each of 143.0±11.3pg/ml and 128.4±10.6pg/ml at medium/high concentration, Advanced Fos (syrup) according to Examples 3 and 4 was 178.4±14.5pg at medium/high concentration, respectively In the experimental group administered with Fos (powder) according to Comparative Examples 3 and 4 and Advanced Fos (powder and syrup) according to Examples 1 to 4 at /ml, 138.8±6.9 pg/ml, the secretion amount of IL-13 was relatively decreased. confirmed that.

As a result of comparing the TARC secretion associated with the increase in the number of eosinophils, the TARC secretion in the positive control group and the maltose (medium/high concentration) group were 27.8±1.95 pg/ml and 26.6±1.1 pg/ml, respectively. ml, while showing a level of 22.8±1.2pg/ml, Fos (powder) according to Comparative Examples 3 and 4 was 22.4±1.5pg/ml, 21.2±2.0pg/ml, Example 1 and Advanced Fos (powder) according to 2 is 18.0±0.9pg/ml and 17.7±1.8pg/ml at medium/high concentration, respectively, Advanced Fos (syrup) according to Examples 3 and 4 is 19.7± at medium/high concentration, respectively In the experimental group administered with Fos (powder) according to Comparative Examples 3 and 4 and Advanced Fos (powder and syrup) according to Examples 1 to 4 at 1.1pg/ml and 19.1±0.4pg/ml, the TARC secretion was relatively decreased. confirmed that.

As a result of comparing the secretion of Eotaxin involved in skin inflow and degranulation of eosinophils, the secretion of Eotaxin in the positive control group and the group administered with maltose (medium/high concentration) were 144.7±3.7pg/ml and 141.9±3.5pg, respectively. /ml, the level of 137.0±5.8pg/ml was shown, while Fos (powder) according to Comparative Examples 3 and 4 was 125.3±6.1pg/ml, 122.3±1.2pg/ml, Example 1 at medium/high concentrations, respectively And Advanced Fos (powder) according to 2 is 117.9±5.7pg/ml and 113.6±1.5pg/ml at medium/high concentration, respectively, Advanced Fos (syrup) according to Examples 3 and 4 is 123.0 at medium/high concentration, respectively In the experimental group administered with Fos (powder) according to Comparative Examples 3 and 4 and Advanced Fos (powder and syrup) according to Examples 1 to 4 at ±2.5pg/ml, 118.6±7.4pg/ml, the secretion amount of Eotaxin was relatively It was confirmed that it decreased.

Administration of the experimental group (Advanced Fos: powder and syrup-medium concentration / high concentration) according to Examples 1 to 4 suppresses the immune response of Th2 by controlling the secretion of cytokines, thereby improving the atopic symptoms by restoring the balance of Th1/Th2 did

19 is a graph comparing and analyzing Treg-related cytokine secretion by AD induction and administration of the experimental group. First, as a result of comparing the secretion amount of IL-1β, which induces systemic inflammation due to overexpression, the positive control group and maltose (medium concentration / high concentration) were While the IL-1β secretion of the administered group was 50.7±2.3pg/ml, 49.8±1.4pg/ml, and 45.1±3.2pg/ml, respectively, Fos (powder) according to Comparative Examples 3 and 4 had a medium concentration / At high concentrations, 34.2±4.2pg/ml, 21.1±3.0pg/ml, Advanced Fos (powder) according to Examples 1 and 2, respectively, 14.0±3.8pg/ml, 11.1±2.6pg/ml at medium/high concentrations , Advanced Fos (syrup) according to Examples 3 and 4 was 7.3±2.3pg/ml and 10.3±3.6pg/ml at medium/high concentration, respectively, Fos (powder) and Examples 1 to 4 according to Comparative Examples 3 and 4 In the experimental group administered with Advanced Fos (powder and syrup) according to 4, it was confirmed that the secretion amount of IL-1β was relatively decreased.

In the case of IL-10 secretion that causes Th1/Th2 imbalance, the IL-10 secretion of the positive control group and the group administered with maltose (medium/high concentration) was 173.7±20.8pg/ml and 171.8±20.7pg/ml, respectively. , 170.4±18.3pg/ml, while Fos (powder) according to Comparative Examples 3 and 4 was 126.2±25.9pg/ml and 90.9±8.7pg/ml, Examples 1 and 2 at medium/high concentrations, respectively The Advanced Fos (powder) according to In the experimental group administered with Fos (powder) according to Comparative Examples 3 and 4 and Advanced Fos (powder and syrup) according to Examples 1 to 4 at pg/ml, 38.1±10.4 pg/ml, the secretion amount of IL-10 was relatively It was confirmed that it decreased.

Administration of the experimental group (Advanced Fos: powder and syrup-medium concentration / high concentration) according to Examples 1 to 4 suppresses the inflammatory response by suppressing the excessive Treg immune response and improves atopic symptoms by restoring the Th1/Th2 balance, In addition to achieving better effects due to the high toss content, the intake of fructooligosaccharide decreases while consuming a high kestose content, so It is more preferable because discomfort can be reduced.

Test Example 8: Flow cytometry analysis

(1) Analysis method

A spleen was obtained from a sacrificed mouse, homogenized using a 50-mm Petri dish (Nunclon, Denmark) and ACK buffer (Gibco, USA), and red blood cells were lysed. The homogenized sample was filtered with a cell strainer (SPL, Korea) and washed with 2% RPMI (Hyclone, USA). After centrifugation at 2,000xg, 4℃ for 10 minutes, the supernatant was discarded and resuspended in 2% RPMI. All cells were equally diluted and treated with anti-mouse antibodies, CD86 and CD274 (BD Pharmigen, USA), and reacted at 4°C for 20 minutes. The expression level on the cell surface was confirmed using a flow cytometer (FACSCalibur; Becton Dickinson, USA). Mean fluorescence intensity (MFI) was analyzed using Cell Quest software (version 6.0) of a FACSCalibur instrument.

(2) Confirmation of CD86 expression level by AD induction and administration of experimental group

20 is a graph showing the expression level of CD86 involved in activating T cells and B cells. The expression level of CD86, a costimulatory factor that activates T cells and B cells, was confirmed when atopy was induced from splenocytes isolated from the spleen. As a result, the CD86 expression levels of the positive control group and the group administered with maltose (medium/high concentration) were 25.4±2.2%, 25.8±0.4%, and 23.1±1.2%, respectively, whereas Fos according to Comparative Examples 3 and 4 (Powder) is 21.7±1.9%, 20.9±1.1%, respectively, at medium/high concentration, Advanced Fos (powder) according to Examples 1 and 2 is 16.1±0.4%, 14.0±0.9%, respectively, at medium/high concentration, respectively Advanced Fos (syrup) according to Examples 3 and 4 was 16.9±1.0% and 17.7±0.8%, respectively, at medium/high concentrations, Fos (powder) according to Comparative Examples 3 and 4 and Advanced Fos according to Examples 1 to 4 ( In the experimental group administered with powder and syrup), the expression level of CD86 was relatively decreased.

(3) Confirmation of the expression level of CD274 by AD induction and administration of the experimental group

21 is a graph showing the expression level of CD274 by AD induction and administration to the experimental group. As a result, the expression level of CD274, a negative immune regulatory factor involved in impaired immunomodulatory function, was confirmed from spleen cells isolated from the spleen, positive control group. CD274 expression levels of the group administered with and maltose (medium/high concentration) were 14.1±1.5%, 14.9±0.2%, and 14.8±1.6%, respectively, whereas Fos (powder) according to Comparative Examples 3 and 4 was At medium/high concentrations, 18.8±1.3%, 20.4±0.1%, respectively, Advanced Fos (powder) according to Examples 1 and 2 were 22.1±1.2% and 23.1±0.2%, respectively, at medium/high concentrations, Examples 3 and 4 Advanced Fos (syrup) according to Fos (powder) according to Comparative Examples 3 and 4 and Advanced Fos according to Examples 1 to 4 (powder and syrup) was 20.9±1.0% and 21.0±0.9%, respectively, at medium/high concentrations, respectively. In the experimental group administered with , CD274 expression level was relatively increased.

The experimental group according to Examples 1 to 4 (Advanced Fos: powder and syrup-medium and high concentration) contributed to repairing the immune system damaged in the immune response and suppressed the activity of excessive immune cells to improve atopic symptoms.

Test Example 9: Tissue analysis of animal models

(1) Experimental method

The back skin of the sacrificed mice was fixed with a 10% formalin solution and embedded in paraffin. The skin tissue section was cut to a thickness of 4-5um and stained with Toluidine blue and Congo red dyes. The number of mast cells and eosinophils was measured at 400 magnification using a DM 4000B microscope (Leika).

(2) Measurement of mast cells in dorsal skin

22 is a graph confirming mast cells in the skin tissue (dorsal skin). As a result of measuring the number of mast cells that produce histamine and cause inflammation and itchiness in the skin tissue, a positive control group and maltose ( While the number of mast cells in the group administered at medium/high concentration) was 328±19, 366±13, and 357±31, respectively, Fos (powder) according to Comparative Examples 3 and 4 was At medium/high concentration, 234±1, 226±8, Advanced Fos (powder) according to Examples 1 and 2, respectively, 214±7, 206±11, respectively, Examples 3 and 4 at medium/high concentration Advanced Fos (syrup) according to is 231±9 pieces and 221±18 pieces, respectively, at medium/high concentration, respectively, Fos (powder) according to Comparative Examples 3 and 4 and Advanced Fos (powder and syrup) according to Examples 1 to 4 In the administered experimental group, the number of mast cells was relatively decreased.

(3) Measurement of eosinophils in dorsal skin.

23 is a graph measuring eosinophils in dorsal skin. As a result of confirming the number of eosinophils acting on hypersensitivity and exacerbating atopic symptoms in skin tissues, a positive control group and maltose (medium concentration/high concentration) ), the number of eosinophils in the administered group showed levels of 6.5±2.6, 6±2.5, and 6.3±2.5, respectively, whereas Fos (powder) according to Comparative Examples 3 and 4 was at medium/high concentrations, respectively. 4.3±1.7, 4.6±1.9, Advanced Fos (powder) according to Examples 1 and 2, respectively, 4±1.6, 2.8±1.5, and Advanced Fos (syrup) according to Examples 3 and 4 at medium/high concentrations Eosinophils ( eosinophil) was relatively decreased.

(4) Measurement of mast cells in intestinal tissue (ileum)

24 is a graph of measuring mast cells in intestinal tissue (ileum). As a result of confirming the number of mast cells, mast cells of a positive control group and a group administered with maltose (medium concentration / high concentration) ) The number showed 48±6, 56±12, and 59±11 levels, respectively, whereas Fos (powder) according to Comparative Examples 3 and 4 was 33±7 and 32±7 at medium/high concentrations, respectively. , Advanced Fos (powder) according to Examples 1 and 2 is 23 ± 6 pieces and 24 ± 4 pieces, respectively at medium/high concentration, Advanced Fos (syrup) according to Examples 3 and 4 is 25 at medium/high concentration, respectively In the experimental group administered with Fos (powder) according to Comparative Examples 3 and 4 and Advanced Fos (powder and syrup) according to Examples 1 to 4, the number of mast cells was relatively reduced to ±6 and 19±8. .

Claims (14)

A composition for increasing the content of propionic acid in the intestine containing kestose as an active ingredient. The composition according to claim 1, wherein the content of propionic acid is increased by the proliferation of propionic acid-producing bacteria in the intestine. The composition of claim 2, wherein the intestinal propionic acid-producing bacteria are one or more microorganisms selected from the group consisting of microorganisms of the genus Lactobacillus and microorganisms of the genus Alistipe. According to claim 1, wherein the composition, the propionic acid content in the feces or intestines, the composition characterized in that the increase to 101 to 700% based on the level before ingestion of kestose. The composition according to claim 1, wherein the composition has a propionic acid content in feces or intestines of 100±30 mg/L or more. The composition of claim 1, wherein the weight ratio (w/w) of butyric acid and propionic acid in feces or intestines is 4:1 to 1:3. The composition of claim 1, wherein the kestose is provided as a fructooligosaccharide composition comprising a kestose content of 50 wt% or more based on the solid content. The composition of claim 1, wherein the daily dose of kestose is provided in the range of 2 to 30 g per day for an adult weighing 60 kg. According to claim 1, wherein the composition is a prebiotic (prebiotics) composition. The composition of claim 1, wherein the composition further comprises a probiotic composition comprising one or more microorganisms selected from the group consisting of microorganisms of the genus Bifidobacterium, microorganisms of the genus Streptococcus, microorganisms of the genus Saccharomyces, and microorganisms of the genus bacillus. The composition for increasing the content of propionic acid in the intestine according to claim 1, which is a composition for preventing, improving or treating inflammatory diseases by controlling the intestinal microbial community, containing kestose as an active ingredient. The composition of claim 11, wherein the inflammatory disease is an inflammatory skin disease or inflammatory bowel disease caused by an immune hypersensitivity reaction. The composition of claim 12, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis. The composition of claim 12, wherein the inflammatory skin disease is atopic dermatitis, contact dermatitis, or allergic contact dermatitis.

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