KR20230104518A - Composition for improving intestinal health, intestinal function or intestinal immunity comprising mother's milk oligosaccharide mixture - Google Patents

Abstract

The present invention relates to a food composition for improving intestinal health, intestinal function or intestinal immunity, a food composition for preventing or improving intestinal inflammation, and inflammatory bowel disease, comprising a human milk oligosaccharide mixture containing galactooligosaccharide and 2'-fucosyllactose as an active ingredient. It relates to a pharmaceutical composition for treatment or prevention, a food composition for infants and young children, or a breast milk supplement composition. The composition comprising the human milk oligosaccharide mixture including galactooligosaccharide and 2'-fucosyllactose of the present invention as an active ingredient increases the content of short-chain fatty acids in the intestine and inhibits the secretion of TNF-α, an inflammatory cytokine in the intestine, thereby improving intestinal health, It can improve intestinal function or intestinal immunity, improve intestinal flora, improve bowel function, improve intestinal inflammation, and further treat or improve inflammatory bowel disease.

Description

Composition for improving intestinal health, intestinal function or intestinal immunity comprising mother's milk oligosaccharide mixture as an active ingredient

The present invention relates to a composition for improving intestinal health, intestinal function or intestinal immunity comprising a human milk oligosaccharide mixture as an active ingredient.

Recently, the fact that intestinal microbes play a decisive role in preventing infectious diseases, regulating the immune process, and nutrient absorption in the intestine has been newly identified.

In particular, intestinal microbes play an important role in maintaining normal physiological functions of humans (hosts) in the body, and changes in the distribution of these intestinal microbes can lead to immune-related diseases such as enteritis and atopy, as well as metabolic syndromes such as obesity, diabetes, and hyperlipidemia. As the fact that it has a close relationship with induction is revealed, research results are continuously being published that the intestinal microbes play a very important role in regulating the body's metabolism.

It is known that these intestinal microbes are changed by various factors such as aging, inflammatory response, antibiotics, probiotics, diet, dietary pattern, and stress. It has been reported in a number of papers.

Probiotics such as lactic acid bacteria are the most consumed for the balance of intestinal microorganisms. It is known that these beneficial bacteria improve the intestinal microbial balance and have a good effect on the human body, but it is known that it is difficult to actually reach the large intestine.

Accordingly, it has been reported that prebiotics refer to non-digestible food components that help the growth and proliferation of microorganisms already present in the intestine, and can improve the quality of intestinal microorganisms more effectively when ingested.

On the other hand, human milk contains about 200 to 300 human milk oligosaccharides (HMOs) at a high concentration of 10 to 20 g/L, and among human milk oligosaccharides, fucosyl oligosaccharides, especially 2'-fucosyllactose, are high. It is known that 2' fucosyllactose has a prebiotics effect, an effect of preventing infection by preventing pathogens and intestinal toxins secreted by them from adhering to the intestinal epithelium, and is useful for human health. It is known to play a role in contributing to the formation of the intestinal microflora.

Methods that can produce 2'-fucosyllactose include chemical synthesis, synthesis using enzymatic reaction, and production using microbial fermentation. However, due to its high price, there were limitations in applying it to food in a content or ratio high enough to demonstrate functionality.

On the other hand, galacto-oligosaccharide, especially 4'-galactosyllactose, is also known as one of the human milk oligosaccharides and has a great effect on the growth of beneficial bacteria in the intestine. Up to now, nothing has been known about the effect of increasing the production or inhibiting the secretion of intestinal inflammatory cytokines.

Korean Patent No. 2268092 Korean Patent No. 2335067 Korean Patent No. 1477693

An object of the present invention is a food composition for improving intestinal health, intestinal function or intestinal immunity, a food composition for preventing or improving intestinal inflammation, comprising a human milk oligosaccharide mixture containing galactooligosaccharide and 2'-fucosyllactose as an active ingredient, To provide a pharmaceutical composition for treating or preventing intestinal disorders, a food composition for infants and young children, or a breast milk supplement composition.

The present invention provides a composition for improving intestinal health, intestinal function or intestinal immunity, comprising a human milk oligosaccharide mixture containing galactooligosaccharide and 2'-fucosyllactose as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving intestinal inflammation comprising a human milk oligosaccharide mixture containing galactooligosaccharide and 2'-fucosyllactose as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for treating or preventing inflammatory bowel disease comprising a mixture of human milk oligosaccharides including galactooligosaccharide and 2'-fucosyllactose as an active ingredient.

The galacto-oligosaccharide and 2'-fucosyllactose may have a weight ratio of 60:40 to 85:15.

The galactooligosaccharide may include 10-20% by weight of 4'-galactosyllactose and 0.05-0.5% by weight of 6'-galactosyllactose out of the total galactooligosaccharide.

The human milk oligosaccharide mixture may further include sialyllactose.

The sialyllactose may be included in an amount of 1 to 10 parts by weight based on 100 parts by weight of the sum of the galactooligosaccharide and 2'-fucosyllactose.

The composition can increase the content of short chain fatty acids in the intestine.

The short-chain fatty acid may be any one or more selected from acetic acid, butyric acid, and propionic acid.

The composition may promote the proliferation or growth of beneficial bacteria in the intestine or inhibit the proliferation or growth of harmful bacteria in the intestine.

The composition may improve bowel function.

The composition may inhibit the secretion of TNF-α.

In addition, the present invention provides a food composition for infants and young children comprising the composition for improving intestinal health, intestinal function or intestinal immunity, or the food composition for preventing or improving intestinal inflammation.

The food composition for infants and young children may be formula milk, formula for infants, formula for growth or weaning formula for infants and young children.

In addition, the present invention provides a breast milk supplement composition comprising the composition for improving intestinal health, intestinal function or intestinal immunity, or the food composition for preventing or improving intestinal inflammation.

Food composition for improving intestinal health, intestinal function or intestinal immunity, food composition for preventing or improving intestinal inflammation, inflammatory bowel disease comprising the human milk oligosaccharide mixture containing galactooligosaccharide and 2'-fucosyllactose of the present invention as an active ingredient A pharmaceutical composition for treatment or prevention, a food composition for infants, or a breast milk supplement composition can promote the growth of beneficial bacteria in the intestine, inhibit the growth of harmful bacteria, increase the content of short-chain fatty acids in the intestine, and improve the intestinal flora. In addition, using the composition of the present invention, intestinal health, intestinal function or intestinal immunity can be improved, and bowel function can be improved.

1 is a graph showing the amount of acetic acid produced when treating a human milk oligosaccharide mixture containing galactooligosaccharide and 2'-fucosyllactose at different mixing ratios in the human fecal system in Experimental Example 1.
Figure 2 is a graph showing the amount of propionic acid produced when treating human milk oligosaccharide mixtures containing galactooligosaccharides and 2'-fucosyllactose at different mixing ratios in the human fecal system in Experimental Example 1.
Figure 3 is a graph showing the amount of butyric acid produced when treating human milk oligosaccharide mixtures containing galactooligosaccharide and 2'-fucosyllactose at different mixing ratios in the human fecal system in Experimental Example 1.
Figure 4 is a graph comparing the degree of inhibition of TNF-α secretion in order to confirm the anti-inflammatory effect in human-derived intestinal epithelial cells (caco-2) in which inflammation was induced using lipopolysaccharide (LPS) in Experimental Example 2 am.
Figure 5 compares the expression level of COX-2 mRNA, an inflammatory factor, to confirm the anti-inflammatory effect in human-derived intestinal epithelial cells (caco-2) in which inflammation was induced using lipopolysaccharide (LPS) in Experimental Example 2. it is a graph
Figure 6 is a graph comparing the expression level of iNOS mRNA, an inflammatory factor, to confirm the anti-inflammatory effect in human-derived intestinal epithelial cells (caco-2) in which inflammation was induced using lipopolysaccharide (LPS) in Experimental Example 2 am.
7 is a graph comparing the nitric oxide generating ability in mouse-derived macrophages (RAW 264.7) in which inflammation was induced using lipopolysaccharide (LPS) in Experimental Example 2.
8 is a photograph for confirming the intestinal length after sacrificing the inflammatory bowel disease animal model mouse in which intestinal epithelial cell damage was induced by DSS administration in Experimental Example 3.
9 is a photograph of a western blot band for confirming the expression of inflammatory factors in intestinal tissue in Experimental Example 3.

The present invention relates to a composition for improving intestinal health, intestinal function or intestinal immunity, comprising a human milk oligosaccharide mixture including galactooligosaccharide and 2'-fucosyllactose as an active ingredient.

In addition, the present invention relates to a food composition for preventing or improving intestinal inflammation comprising a human milk oligosaccharide mixture including galactooligosaccharide and 2'-fucosyllactose as an active ingredient.

In addition, the present invention relates to a pharmaceutical composition for treating or preventing inflammatory bowel disease comprising a mixture of human milk oligosaccharide containing galactooligosaccharide and 2'-fucosyllactose as an active ingredient.

The inflammatory bowel disease may be a disease selected from the group consisting of Crohn's disease, ulcerative colitis, intestinal Behçet's disease, infectious enteritis, ischemic bowel disease, radiation enteritis, and leaky gut syndrome.

The leaky gut syndrome (LGS) refers to the intestinal mucosa in which the intestinal mucosal cells maintain a constant intercellular gap, and when any stimulus or damage is applied during the digestion and absorption process, the intestinal mucosa in which a polymer material can shuttle back and forth through the gap between the cells It indicates a phenomenon in which permeability is increased, and refers collectively to symptoms caused by a phenomenon in which polymeric substances in the blood leak into the lumen of the intestinal tract or polymeric substances in the lumen enter the blood directly due to a failure of the barrier function properly. The above symptoms appear in various clinical conditions such as aging, allergy, multiple trauma, rheumatoid arthritis, inflammatory bowel disease, chronic fatigue syndrome, and hypersensitivity syndrome. In addition, pathogens, antigens, decaying substances, etc. are introduced into the intestinal mucosa due to increased permeability of the intestinal mucosa or damage to the intestinal mucosa, causing an inflammatory reaction, and endotoxins are introduced into the bloodstream, resulting in bacterial translocation and intestinal endotoxemia. etc., resulting in various inflammatory and immune responses. Such leaky gut syndrome complains of various, extensive, and ambiguous symptoms without specific symptoms. Specifically, it is unique in that it has symptoms commonly complained of in various diseases.

The improvement of intestinal immunity or enhancement of intestinal immunity refers to a function of promoting an immune response to an antigen in a non-specific manner during the initial activation of immune cells present in the intestinal tract or a function of enhancing the immunity of the intestinal tract by increasing the activity of cells of the immune system. . Diseases caused by reduced intestinal immune function may be any one or more selected from the group consisting of infectious diseases caused by pathogenic microorganisms and cancer. The composition for improving or enhancing intestinal immunity has an application effect of improving or preventing seasonal flu in humans, improving or preventing bacterial diarrhea, improving or preventing bacterial infections, and enhancing post-wound treatment effects.

The galactooligosaccharide and 2'-fucosyllactose may be in a weight ratio of 60:40 to 85:15, preferably 65:35 to 80:20, and more preferably 70:30 to 75:25. When the ratio of galactooligosaccharide is less than the lower limit or the ratio of 2'-fucosyllactose exceeds the upper limit, the effect of inhibiting the secretion of the inflammatory cytokine TNF-α is not sufficient, and the ratio of galactooligosaccharide exceeds the upper limit or 2' - When the ratio of fucosyllactose is less than the above lower limit, the effect of increasing the production of short-chain fatty acids in the intestine, particularly propionic acid and butyric acid, is not sufficient.

The galactooligosaccharide contains 10-20% by weight of 4'-galactosyllactose, preferably 12-18% by weight and 0.05-0.5% by weight of 6'-galactosyllactose, preferably 0.1-20% by weight of the total galactooligosaccharide. 0.3% by weight.

The human milk oligosaccharide mixture may further include sialyllactose. Sialyllactose may be 3'-sialyllactose, 6'-sialyllactose, or a mixture thereof, and when sialyllactose is further included, the human milk oligosaccharide mixture inhibits the production of short-chain fatty acids in the intestine or the secretion of inflammatory cytokines. effect is increased.

The sialyllactose may be included in an amount of 1 to 10 parts by weight based on 100 parts by weight of the sum of the galactooligosaccharide and 2'-fucosyllactose.

The composition may increase the content of short chain fatty acids in the intestine, and the short chain fatty acids may be any one or more selected from acetic acid, butyric acid and propionic acid, and preferably increases the production of butyric acid.

The composition increases the content of short-chain fatty acids in the intestine, can promote the proliferation or growth of beneficial bacteria in the intestine that increases the content of short-chain fatty acids in the intestine, and may inhibit the proliferation or growth of harmful bacteria in the intestine.

The composition may improve bowel function by increasing the content of short-chain fatty acids in the intestine and promoting the proliferation and growth of beneficial bacteria in the intestine.

The composition may inhibit the secretion of intestinal inflammatory cytokines, particularly TNF-α, by increasing the content of short-chain fatty acids in the intestine and promoting the proliferation and growth of beneficial bacteria in the intestine.

In addition, the present invention provides a food composition for infants and young children comprising the composition for improving intestinal health or improving intestinal function or the food composition for preventing or improving intestinal inflammation.

The food composition for infants and young children may be formula milk, formula for infants, formula for growth or weaning formula for infants and young children.

In addition, the present invention provides a breast milk supplement composition comprising the composition for improving intestinal health or improving intestinal function or the food composition for preventing or improving intestinal inflammation.

The composition of the present invention is a food composition, and various foods containing the human milk oligosaccharide mixture of the present invention as an active ingredient, for example, beverages, gum, tea, vitamin complexes, powders, granules, tablets, capsules, food compositions for infants and young children, breast milk It can be used in supplement form. The food composition for infants and young children may be formula milk, formula for infants, formula for growth or weaning formula for infants and young children.

The food composition according to the present invention may be formulated in the same way as the pharmaceutical composition described later and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, chewing gum, ice cream, vitamin complexes, and health supplements. etc.

The food composition of the present invention may include not only the human milk oligosaccharide mixture as an active ingredient, but also ingredients commonly added during food preparation, such as proteins, carbohydrates, fats, nutrients, seasonings and flavors. . Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides such as conventional sugars such as dextrins and cyclodextrins and sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents, natural flavoring agents [thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is made into drinks and beverages, in addition to the human milk oligosaccharide mixture of the present invention, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be further included. .

The present invention provides a health functional food comprising a food composition for improving, suppressing, or preventing inflammatory bowel disease or leaky gut syndrome containing the human milk oligosaccharide mixture as an active ingredient. Health functional food refers to food produced by adding human milk oligosaccharide mixture to food materials such as beverages, teas, spices, chewing gum, confectionery, etc., or by encapsulation, powdering, suspension, etc. However, unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time using food as a raw material. The health functional food of the present invention obtained in this way is very useful because it can be consumed on a daily basis. The addition amount of the human milk oligosaccharide mixture in such a health functional food cannot be determined uniformly depending on the type of target health functional food, but it can be added within a range that does not impair the original taste of the food. It ranges from 0.01 to 50% by weight, preferably from 0.1 to 20% by weight. In addition, in the case of health functional foods in the form of pills, granules, tablets or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of pills, tablets, capsules or beverages.

In another example, a pharmaceutical composition for preventing or treating inflammatory bowel disease or leaky gut syndrome comprising the above-described human milk oligosaccharide mixture as an active ingredient may be variously used. In addition to the human milk oligosaccharide mixture as an active ingredient, it can be prepared using pharmaceutically suitable and physiologically acceptable adjuvants, such as excipients, disintegrants, sweeteners, binders, coating agents, expanding agents, lubricants, lubricants, or flavoring agents. You can use my back.

The pharmaceutical composition may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration.

Formulations of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, or injectable solutions. 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, and the like. In addition, 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, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

In compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

Furthermore, using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field, it can be preferably formulated according to each disease or component.

The pharmaceutical composition of the present invention can be administered orally. The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate and reaction sensitivity, usually This allows the skilled physician to readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.001-10 g/kg.

The pharmaceutical composition of the present invention may be prepared in a unit dose form by formulation using a pharmaceutically acceptable carrier and/or excipient, or prepared by putting it into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.

In addition, the present invention provides a method for improving, preventing or treating inflammatory bowel disease or leaky gut syndrome comprising administering an effective amount of a human milk oligosaccharide mixture to a mammal.

As used herein, the term "mammal" refers to a mammal that is a subject of treatment, observation or experimentation, and preferably refers to a human.

As used herein, the term "effective amount" means an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, which is considered by a researcher, veterinarian, physician or other clinician, which is Includes an amount that induces relief of symptoms. The effective amount and frequency of administration of the active ingredient of the present invention may vary depending on the desired effect. Therefore, the optimal dose to be administered can be easily determined by a person skilled in the art, and the degree of symptoms of inflammatory bowel disease, the degree of intestinal permeability for leaky gut syndrome, the degree of immune cell activation, the degree of immune response to antigens, and the effective amount contained in the composition Depending on various factors, including the content of ingredients and other ingredients, the type of dosage form, and the patient's age, middle, general health condition, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs can be regulated. In the prevention, treatment or improvement method of the present invention, in the case of adults, it is preferable to administer the human milk oligosaccharide mixture at a dose of 0.001 g/kg to 10 g/kg when administered once to several times a day.

Hereinafter, the present invention will be described in more detail through examples, but the present invention is not limited thereto.

[Production Example 1]

Galactooligosaccharide and 2'-fucosyllactose were tested to determine the amount of short-chain fatty acids produced in the intestine and the anti-inflammatory effect in human-derived intestinal epithelial cells and mouse-derived macrophages according to the mixing ratio of galactooligosaccharide and 2'-fucosyllactose. They were mixed as shown in Table 1 below. The galactooligosaccharide is a powder comprising 75% by weight of galactooligosaccharide, 20% by weight of lactose, glucose and galactose, and 5% by weight of water, and 13.5% by weight of 4'-galactosyllactose and 6'-gal Lactosyllactose contains 0.15% by weight, and the 2'-fucosyllactose was used as a powder containing 95% by weight of 2'-fucosyllactose and 5% by weight of moisture. The mixing weight ratios in Table 1 are the mixing ratios of the solids of galacto-oligosaccharide and 2'-fucosyllactose, respectively.

division Galactooligosaccharides 2'-fucosyllactose GL 100 0 GF1 88 12 GF2 83 17 GF3 70 30 GF4 44 56 GF5 21 79 FL 0 100

[Experimental Example 1: Confirmation of short-chain fatty acid production in the intestine in human feces system]

Fecal slurry was prepared by mixing 5 g of mixed feces obtained by mixing 1 g each of feces obtained from 5 infants of an average age of 3 months with 50 mL of pH 7.7 PBS buffer. The fecal slurry was mixed with the human milk oligosaccharide mixture of Table 1 at a concentration of 1 mg/mL, and 1 mL of the fecal slurry treated with the human milk oligosaccharide mixture was mixed with BHI broth medium (Brain Heart Infusion, composition (g/L); Brain Heart Infusion 37 g, Hemin solution (1%) 0.5 mL, L-Cysteine.HCL 0.5 g, Resazurin (0.1%) 0.5 mL, Distilled water 1 L) 9 mL and then mixed, 37 ℃, 48 ℃ under anaerobic conditions time incubated.

The culture solution was centrifuged at 12,100 x g and 4 ° C for 20 minutes, and 900 μL of the centrifuged supernatant was transferred to a 2 mL EP tube, and then 400 mM acetic acid was added as an internal standard, 0.5 mL hexane was added, and at room temperature After mixing for 10 minutes, 180 μL of the hexane layer obtained by repeating the process of collecting the hexane layer by centrifugation at 1,932 x g and 4 ° C for 10 minutes was added twice to an insert vial, and then the derivative reagent MTBSTFA (+1% t -BDMCS) was added to each vial by 6 μL, each vial was vortexed, and after incubation at 70 ° C. for 45 minutes in a drying oven, three types of short-chain fatty acids, namely acetic acid, propionic acid and butyric acid, were analyzed by GC-MS.

The negative control group in which only the fecal slurry was cultured without treatment with the human milk oligosaccharide mixture of Table 1 was indicated as "fecal", and the acetic acid, propionic acid and butyric acid contents (mM) of the fecal slurry treated with the human milk oligosaccharide alone and the mixture in Table 1 1, 2 and 3, respectively.

In FIG. 1, acetic acid was significantly increased in the GF1, GF2, GF3, and GF5 groups compared to the negative control group (fecal). That is, there was no significant increase in acetic acid production in the galactooligosaccharide-only group (GL) and 2'-fucosyllactose-only group (FL), and among the human milk oligosaccharide mixtures, galactooligosaccharide and 2'fucosyllactose were administered at a concentration of 0.7:0.3 to 0.7:0.3. A significant increase was greater in the GF1, GF2, and GF3 groups mixed at 0.88:0.12.

In FIG. 2, propionic acid was significantly increased in the GF3, GF4, GF5, and GL groups compared to the negative control group (fecal). In particular, the concentration of butyric acid in the fecal group was 4.84±0.02 mM, whereas the concentration of butyric acid in the GF3 group was 5.68±0.27 mM, showing the highest increase in butyric acid production.

In Figure 3, butyric acid was significantly increased in all human milk oligosaccharide alone and mixed groups compared to the negative control group (fecal). In particular, compared to the galactooligosaccharide alone group (GL) and 2'-fucosyllactose alone group (FL) as well as the GF1, GF2, GF4, and GF5 groups mixed with galactooligosaccharide and 2'-fucosyllactose at different ratios, In the GF3 group in which lacto-oligosaccharide and 2'-fucosyllactose were mixed at a weight ratio of 0.7:0.3, the production of butyric acid was significantly increased by about two times.

As a result of confirming the effect of short-chain fatty acid production in the intestine in the human fecal system, galactooligosaccharide and 2'-fucosyllactose were reduced to 0.7 compared to the fecal group, the galactooligosaccharide alone group (GL), and the 2'-fucosyllactose alone group (FL). : The GF3 group mixed at a weight ratio of 0.3 significantly increased the production of acetic acid, propionic acid and butyric acid.

In particular, the GF3 group is used for industrial purposes in that it shows a significantly higher intestinal short-chain fatty acid production effect than the 2'-fucosyllactose alone group (FL), even though the content of 2'-fucosyllactose, which is expensive, is lowered. Most likely.

[Experimental Example 2: Confirmation of Inhibitory Effect of Inflammation in Intestinal Epithelial Cells and Macrophages]

1. Confirmation of the secretion inhibitory effect of TNF-α, an inflammatory cytokine in intestinal epithelial cells

Inflammation was induced in human-derived intestinal epithelial cells (caco-2) with 1 μg/mL of lipopolysaccharide (LPS, Sigma-Aldrich), and the human milk oligosaccharide mixture of Preparation Example 1 was treated with 1 mg/mL of TNF. -α concentration was measured according to the measurement method in the kit using an ELISA kit purchased from Goma Biotex and shown in FIG.

The normal control group (Con) is a group not treated with LPS and human milk oligosaccharide mixture, the negative control group (LPS) is a group treated only with LPS, and the remaining experimental group of human milk oligosaccharide mixture of Preparation Example 1 is treated with LPS and human milk oligosaccharide mixture at the same time. The OD value at 450 nm, that is, the TNF-α content (pg/mL) was measured.

In the negative control group (LPS) treated only with LPS, there was a significant increase compared to the normal control group (Con), and in the GF4 group, the amount of TNF-α secretion increased significantly compared to the negative control group (LPS), and in the GL, GF4 and GF5 groups There was no significant difference from the negative control group (LPS), and there was a significant decrease compared to the negative control group (LPS) in the GF2, GF3 and FL groups.

The amount of inflammatory cytokine TNF-α secretion was not observed in the galactooligosaccharide-only group (GL) and was found in the 2'-fucosyllactose-only group (FL), but galactooligosaccharide and 2'-fucosyllactose were mixed at 0.3:0.7 to 0.3:0.7 When mixed at a weight ratio of 0.83:0.17, activity similar to that of the 2'-fucosyllactose alone group (FL) was exhibited.

2. Confirmation of inhibitory effect on inflammatory factor expression in intestinal epithelial cells

Human-derived intestinal epithelial cells (Caco-2) were co-treated with 1 mg/mL of the human milk oligosaccharide mixture of Preparation Example 1 and 1 μg/mL of LPS, respectively, for 24 hours and then cultured, followed by mRNA expression of the inflammatory factors COX-2 and iNOS. level was confirmed.

The mRNA expression level is confirmed by dissolving the cell membrane using trizol and then extracting the mRNA. Afterwards, the amount of extracted mRNA was quantified in the same amount using nanodrop, and cDNA was synthesized using the LeGene premium express first-strand cDNA synthesis system, and the cDNA was mixed with HiPi real-time PCR 2x master mix SYBR green mix to make a PCR reaction solution. After confirming the expression levels of the inflammatory factors using an iQ5 thermal cycler, they are shown in FIGS. 5 and 6 , respectively.

The mRNA expression level of COX-2 significantly increased in the negative control group (LPS) treated only with LPS compared to the normal control group (Con), and the galacto-oligosaccharide alone group (GL) showed no significant difference from the negative control group (LPS). , The 2'-fucosyllactose alone group (FL) and the GF2, GF3, GF4 and GF5 groups were significantly reduced compared to the negative control group (LPS).

The mRNA expression level of iNOS increased significantly in the negative control group (LPS) treated only with LPS compared to the normal control group (Con), and the galacto-oligosaccharide alone group (GL) showed no significant difference from the negative control group (LPS), but its expression The amount was greatly increased, and the 2'-fucosyllactose alone group (FL) and the GF2 and GF3 groups were significantly decreased compared to the negative control group (LPS).

The activity of suppressing the mRNA expression levels of COX-2 and iNOS, which are inflammatory factors, was not shown in the galactooligosaccharide alone group (GL) and was shown in the 2'-fucosyllactose alone group (FL), but galactooligosaccharide and 2' - When fucosyllactose was mixed in a weight ratio of 0.21:0.79 to 0.83:0.17, preferably 0.44:0.56 to 0.7:0.3, it exhibited similar or superior activity to that of the 2'-fucosyllactose alone group (FL).

3. Confirmation of the inhibitory effect on nitric oxide production in macrophages

In a mouse-derived macrophage (RAW 264.7) model in which inflammation was induced using lipopolysaccharide (LPS), the nitric oxide (NO) production ability of the human milk oligosaccharide mixture of Preparation Example 1 was confirmed to confirm anti-inflammatory efficacy. .

In the preparation, each of the human milk oligosaccharide mixtures in 1 was simultaneously treated with 100 ng/mL LPS at a concentration of 0.25 mg/mL for 12 hours, then 50 μL of the supernatant was taken, 50 μL of 1% Sulfanilamide/5% phosphoric acid solution was added, and 0.1% After adding 50 μL of NEAD, the absorbance was measured at 540 nm to measure the nitric oxide concentration in the supernatant, which is shown in FIG. 7 .

The normal control group (Con) is a group not treated with LPS and human milk oligosaccharide mixture, the negative control group is a group treated only with LPS, and the other experimental group of human milk oligosaccharide mixture of Preparation Example 1 is 540 when LPS and human milk oligosaccharide mixture are simultaneously treated. OD values in nm, i.e., nitric oxide content (μM) were measured.

In the negative control group (LPS) treated only with LPS, there was a significant increase compared to the normal control group (Con), and in the GL and FL groups, the concentration of nitric oxide was significantly increased compared to the negative control group (LPS), but GF1, GF2, and GF3 and in the GF4 group, nitric oxide production was suppressed.

The anti-inflammatory effect through inhibition of nitric oxide production was not shown in the galactooligosaccharide-only group (GL) and 2'-fucosyllactose-only group (FL), and galactooligosaccharide and 2'-fucosyllactose were administered at 0.44:0.56 to 0.88 : When mixed at a weight ratio of 0.12, it suppressed the production of nitric oxide compared to the negative control group (LPS) and showed an excellent anti-inflammatory effect.

[Experimental Example 3: Confirmation of Intestinal Inflammation Improvement Effect in Inflammatory Bowel Disease Animal Model]

1. Confirmation of intestinal permeability inhibition effect, intestinal inflammation and intestinal length

In Experimental Examples 1 and 2, the GF2 and GF3 groups with excellent short-chain fatty acid production and anti-inflammatory effects were selected, and galactooligosaccharide alone group (GL) and 2'-fucosyllactose alone group (FL) were selected as positive control groups. Selected and compared the effect of improving intestinal inflammation in inflammatory bowel disease animal models.

When dextran sulfate sodium (DSS) is administered to mice, it induces damage to intestinal epithelial cells, induces intestinal inflammation, and induces intestinal leakage. Male C57BL/6 mice (6 weeks old) weighing 250-300 g were supplied with sufficient food and water and controlled for 12 hours of daytime and nighttime (daytime from 8:00 am) with appropriate artificial lighting. And a constant temperature (20 ~ 24 ℃) and humidity (45 ~ 65%) were maintained. The sleep cycle was maintained for a week to adapt to the changed environment, and abnormal behavior was checked.

After undergoing an adaptation period for 2 weeks as described above, enteritis was induced by administering 2% DSS in negative water for 5 days. For the experimental animals, only healthy animals with constant weight change were selected, and a normal control group (CON) administered with physiological saline by a random batch method, a negative control group (DSS) administered with DSS, and GF2 or GF3 of Preparation Example 1 after administration of DSS The administration group and the positive control group were divided into GL or FL administration groups. Among the above groups, the groups administered with the human milk oligosaccharide mixture of Preparation Example 1 dissolved each of the human milk oligosaccharide mixtures in 0.1M PBS (pH 7.4) at a concentration of 1 g/kg and orally administered 200 μL each once a day. Feed and drinking water were supplied autonomously during the 4-week administration period (supplied until the evening before sacrifice), feed was stopped 12 hours before sacrifice, and FITC-dextran was orally administered at 200 μg/g 4 hours before sacrifice. At the time of sacrifice, anesthesia was performed using CO ₂ , and blood was then collected from the abdominal vena cava.

In order to confirm the intestinal permeability of FITC orally administered 4 hours before sacrifice, the collected blood was centrifuged at 15 ° C and 2000 rpm for 15 minutes, and transferred to a 96 black well to measure the concentration of FITC in serum obtained by excitation 490 nm, Fluorescence was measured at emission 520 nm and shown in Table 2 below.

In addition, by measuring MPO activity in the colon tissue of the mouse, intestinal inflammation was quantified and shown in Table 2 below. For MPO activity, take 25 mg of colon tissue, add 8.7 g/L dibasic potassium phosphate to HTAB buffer (6.8 g/L monobasic potassium phosphate, adjust the pH to 6.0, and then add 5 g of hexadecyltrimethylammonium bromide (HTAB) to 1 L of potassium phosphate). Reaction solution prepared by dissolving in buffer (50 mM, pH 6.0)) to 25 mg / mL, homogenize the colon tissue, and centrifuge at 4 ° C, 13400 xg, 6 min and other conditions to collect the supernatant. The supernatant was dispensed in 3 repetitions of 7 μL in a 96 well plate, and 200 μL of O-dinisidine solution was mixed and the absorbance was measured at 450 nm.

In addition, the intestinal length photograph of each experimental group is shown in FIG. 8, and the intestinal weight/length ratio is shown in Table 2 together.

division FITC concentration (μg/mL) Activate MPO (opponent unit) Sheet weight/length ratio (mg/cm) Con 24.8 ^a ±0.53 1.45 ^a ±0.34 ^133.4b ±17.3 DSS ^50.6b ±1.99 1.99 ^b ±0.57 165.6 ^a ±26.5 GL ^36.1c ±0.83 1.70 ^c ±0.44 149.7 ^a ±26.7 FL ^35.5c ±1.33 1.75 ^c ±0.36 154.4 ^a ±34.8 GF2 31.7 ^ac ±1.24 1.46 ^ac ±0.57 ^125.7b ±24.7 GF3 30.6 ^ac ±0.93 1.48 ^ac ±0.47 ^133.7b ±13.9

In Table 2, the concentration of FITC in plasma was significantly increased by more than 2 times compared to the normal control group (con) in the negative control group (DSS) due to intestinal leakage due to DSS administration. In addition, the galactooligosaccharide alone group (GL), the 2'-fucosyllactose alone group (FL), and the GF2 and GF3 groups, which had excellent anti-inflammatory effects by mixing galactooligosaccharide and 2'-fucosyllactose, were negative control groups ( DSS), the FITC concentration was significantly reduced, and in particular, in the GF2 and GF3 groups, it was significantly reduced to a level with no significant difference from the normal control group (Con).

Meanwhile, in Table 2, MPO activity was significantly increased in the negative control group (DSS) compared to the normal control group (con) due to intestinal leakage due to DSS administration. In addition, in the galactooligosaccharide alone group (GL), the 2'-fucosyllactose alone group (FL), and the GF2 and GF3 groups mixed with galactooligosaccharide and 2'-fucosyllactose, MPO activity was higher than that of the negative control group (DSS). was significantly reduced, especially in the GF2 and GF3 groups, to a level that was not significantly different from that of the normal control group (Con).

Meanwhile, in FIG. 8, due to DSS administration, the intestinal length of the negative control group (DSS) was significantly reduced compared to the normal control group (Con), and the galacto-oligosaccharide alone group (GL) and 2'-fucosyllactose alone group (FL) were negative. Compared to the control group (DSS), the intestinal length increased to some extent, and in the GF2 and GF3 groups in which galacto-oligosaccharide and 2'-fucosyllactose were mixed, the intestinal length significantly increased compared to the GL and FL groups.

Meanwhile, in Table 2, the intestinal weight/length ratio was significantly increased in the negative control group (DSS) compared to the normal control group (con) due to intestinal leakage due to DSS administration. In addition, there was no significant difference from the negative control group (DSS) in the intestinal weight/length ratio in the galactooligosaccharide-only group (GL) and 2'-fucosyllactose-only group (FL), but galacto-oligosaccharide and 2'-fucose In the GF2 and GF3 groups mixed with room lactose, compared to the negative control group (DSS), the intestinal weight/length ratio was significantly reduced to a level that was not significantly different from that of the normal control group (Con).

2. Check the amount of short-chain fatty acids produced in feces

After collecting 10 mg of caecal feces from each experimental group, 350 μL of extraction solution containing internal standard (Acetic acid-d4), hydrochloric acid, and ether at a ratio of 2:1:4 was dispensed, homogenized, and prepared. 80 μL of the supernatant obtained by centrifugation at °C, 1000 g, 10 min was placed in an insert vial, and 16 μL of the derivative reagent - MTBSTFA (+1% t-BDMCS) was added to each vial. The vial to which the supernatant and the derivative reagent was added was reacted at 80 ° C. for 20 minutes and then incubated at room temperature for 48 hours. The concentrations of acetic acid, propionic acid and butyric acid were analyzed by GC-MS and are shown in Table 3.

division Acetic acid (mM) Propionic acid (mM) Butyric acid (mM) Con 1556 ^a ±73.9 ^34.7b ±6.48 ^0.45ab ±0.03 DSS 1340 ^b ±175.0 22.5 ^a ±3.32 0.40 ^bc ±0.06 GL 1146 ^c ±272.1 23.8 ^a ±5.68 ^0.35c ±0.01 FL 1178 ^bc ±289.2 ^23.7a ±6.46 ^0.37c ±0.02 GF2 1628 ^a ±342.6 ^45.8b ±17.5 ^0.40ab ±0.05 GF3 1790 ^a ±249.3 45.7 ^b ±15.9 0.48 ^a ±0.00

Acetic acid was 1556 mM in the normal control group (con), but significantly decreased to 1340 mM in the negative control group (DSS), and in the galactooligosaccharide alone group (GL) and 2'-fucosyllactose alone group (FL), the negative control group (DSS) showed a tendency equal to or rather decreasing. However, in the GF2 and GF3 groups, which had excellent anti-inflammatory effects by mixing galacto-oligosaccharide and 2'-fucosyllactose, the concentration of acetic acid increased significantly compared to the negative control group (DSS), and the concentration was higher than that of the normal control group (con). showed up

Propionic acid was 34.7 mM in the normal control group (con), but significantly decreased to 22.5 mM in the negative control group (DSS), and in the galactooligosaccharide alone group (GL) and 2'-fucosyllactose alone group (FL), the negative control (DSS) did not increase the propionic acid content. However, in the GF2 and GF3 groups, the concentration of propionic acid increased significantly compared to the negative control group (DSS), and showed a higher concentration than that of the normal control group (con).

Butyric acid was higher in the normal control group (con) than in the negative control group (DSS), but did not show a significant difference. However, in the galacto-oligosaccharide alone group (GL) and 2'-fucosyllactose alone group (FL), the concentration was significantly lower than that of the normal control group (con), and in the GF2 and GF3 groups, the concentration was significantly lower than that of the normal control group (con). there was no significant difference However, the GF3 group showed a significantly higher concentration of butyric acid than the negative control group (DSS).

Therefore, through the analysis of short-chain fatty acid content in the feces of inflammatory bowel disease animal models showing leaky gut, the galacto-oligosaccharide-only group (GL) and the 2'-fucosyllactose-only group (FL) showed significant short-chain compared to the negative control group (DSS). Although it was difficult to confirm the effect of increasing fatty acid production, the GF2 and GF3 groups, which had excellent anti-inflammatory effects by mixing galacto-oligosaccharide and 2'-fucosyllactose, showed a significant increase in short-chain fatty acid production compared to the negative control group (DSS). It was confirmed that the production of short-chain fatty acids was equal to or rather higher than that of the normal control group (con).

3. Check the expression level of inflammatory factors in intestinal tissue

The expression of NF-κB pathway related factors INF-α, iNOS, COX-2, and p-NF-κB/NF-κB was confirmed through Western blotting using the intestinal tissue of each experimental group, and shown in FIG. 9 and Table 4. . To this end, protein was extracted from intestinal tissue cut into pieces of about 2 mm using RIPA buffer, a protein extraction buffer, and after protein quantification, separated by 10% SDS-PAGE, PVDF membrane transfer, and Tris-buffered saline Tween 20 (TBST) After blocking with 5% skim milk dissolved in , TNF-α, iNOS, COX-2, and p-NF-κB/NF-κB were confirmed. Expression levels of TNF-α, iNOS, and COX-2 were expressed as relative expression folds for GAPDH.

division TNF-α (multiple) iNOS (multiple) COX-2 (multiple) p-NF-κB/NF-κB
(Drainage) Con 1.00 ^bc ±0.23 1.00 ^a ±0.49 1.00 ^a ±0.58 1.00 ^a ±0.21 DSS 1.42 ^a ±0.10 1.98 ^b ±0.06 2.22 ^b ±0.11 3.29 ^b ±0.47 GL 1.37 ^ac ±0.28 ^1.46ab ±0.22 ^1.64ab ±0.74 ^2.14ab ±0.55 FL 1.43 ^a ±0.35 1.80 ^b ±0.32 2.31 ^b ±0.30 ^2.16ab ±0.65 GF2 0.95 ^bc ±0.10 0.99 ^a ±0.41 ^1.01a ±0.69 1.61 ^a ±0.85 GF3 0.72 ^b ±0.09 1.09 ^a ±0.14 0.97 ^a ±0.19 1.47 ^a ±0.66

Expressions of TNF-α, iNOS, COX-2, and p-NF-κB/NF-κB inflammatory factors were significantly increased in the negative control group (LPS) compared to the normal control group (Con).

The galacto-oligosaccharide alone group (GL) and 2'-fucosyllactose alone group (FL) were compared to the negative control group (DSS) in TNF-α, iNOS, COX-2 and p-NF-κB/NF-κB all inflammatory factors. No significant difference was shown. However, the GL group showed slightly lower expression of inflammatory factors than the FL group. On the other hand, in the GF2 and GF3 groups in which galacto-oligosaccharide and 2'-fucosyllactose were mixed, the expression of all inflammatory factors was significantly suppressed compared to the negative control group (DSS), and showed the same level as the normal control group (Con).

[Production Example 2]

In order to confirm the synergistic effect of increased short-chain fatty acid production and anti-inflammatory effect when sialyllactose was added to the GF2 and GF3 groups, which had excellent anti-inflammatory effects in the human milk oligosaccharide mixture of Preparation Example 1, the human milk oligosaccharide mixture was prepared in the ratio shown in Table 5 below. was manufactured. As the sialyllactose, a mixture of 3'-sialyllactose and 6'-sialyllactose in a weight ratio of 1:1 was used.

division Galactooligosaccharides 2'-fucosyllactose Sialyllactose GL 100 0 0 FL 0 100 0 SL1 and SL2 0 0 100 GF2 83 17 0 GF3 70 30 0 GF2S 78.9 16.1 5 GF3S 66.5 28.5 5

[Experimental Example 4: Confirmation of Short-Chain Fatty Acid Production in Human Fecal System of Sialyllactose-Containing Human Milk Oligosaccharide Mixture]

All human milk oligosaccharides or mixtures thereof except for the SL1 group were mixed at 1 mg/mL and tested in the same manner as in Experimental Example 1, and the results are shown in Table 6. The SL1 group was administered low-concentration sialyllactose, with a difference only in that it was mixed at 50 μg/mL, 1/10 of SL2. The dose of SL1 was the same as that of sialyllactose in the GF2S and GF3S groups.

The negative control group in which only the fecal slurry was cultured without treatment with the human milk oligosaccharide mixture of Table 4 was indicated as "fecal", and the acetic acid, propionic acid and butyric acid contents (mM) of the fecal slurry treated with the human milk oligosaccharide mixture of Table 4 were respectively shown in Table 6 shown in

division Acetic acid (mM) Propionic acid (mM) Butyric acid (mM) Fecal ^71.68a ±4.19 4.84 ^a ±0.02 2.50 ^a ±0.13 GL ^78.86ab ±1.27 5.07 ^b ±0.04 3.87 ^b ±0.03 FL ^78.54ab ±2.92 ^5.46ab ±0.29 ^3.8bc ±0.58 SL1 ^78.52ab ±1.85 ^4.95ab ±0.12 3.96 ^b ±0.17 SL2 85.48 ^bc ±2.24 5.22 ^b ±0.05 ^4.62c ±0.12 GF2 ^82.93b ±2.06 ^5.17ab ±0.17 4.70 ^c ±0.16 GF3 81.36 ^b ±0.71 5.66 ^b ±0.18 ^4.82c ±0.41 GF2S ^88.24c ±1.82 ^5.85b ±0.35 5.35 ^cd ±0.71 GF3S ^90.12c ±2.21 5.72 ^b ±0.24 5.42 ^d ±0.25

In Table 6, acetic acid was significantly increased in the SL2, GF2, GF3, GF2S and GF3S groups compared to the negative control group (fecal). That is, there was no significant increase in acetic acid production in the galacto-oligosaccharide alone group (GL), the 2'-fucosyllactose alone group (FL), and the low sialyllactose concentration group (SL1).

Acetic acid production was higher in the sialyllactose high concentration group (SL2), in which sialyllactose was increased 20 times and administered at a high concentration, and in the GF2 and GF3 groups in which galactooligosaccharide and 2'fucosyllactose were mixed in the human milk oligosaccharide mixture, compared to the negative control group (fecal). increased significantly.

The GF2S and GF3S groups, in which low concentrations of sialyllactose were added to the GF2 and GF3 groups, respectively, showed a significant increase in acetic acid production compared to the GF2 and GF3 groups, and higher production than the high-concentration sialyllactose group (SL2). was

In Table 6, propionic acid was significantly increased in the GL, SL2, GF3, GF2S and GF3S groups compared to the negative control group (fecal). There was no significant increase in acetic acid production in the 2'-fucosyllactose alone group (FL) and the sialyllactose low concentration group (SL1).

Sialyllactose high concentration group (SL2) in which sialyllactose was increased 20 times and administered at high concentration, GF3 group in which galactooligosaccharide and 2'fucosyllactose were mixed in human milk oligosaccharide mixture, and sialyllactose in GF2 and GF3 groups, respectively The GF2S and GF3S groups added with a low concentration showed a significant increase in acetic acid production compared to the negative control group (fecal).

In Table 6, butyric acid was significantly increased in all human milk oligosaccharide alone and mixed groups compared to the negative control group (fecal). In particular, sialyllactose high concentration group (SL2), GF2 and GF3 groups mixed with galactooligosaccharide and 2'-fucosyllactose, and GF2S and GF3S mixed with galactooligosaccharide, 2'-fucosyllactose and sialyllactose The group significantly increased the concentration of butyric acid production compared to the galacto-oligosaccharide alone group (GL) and the low-sialyllactose group (SL1).

In particular, the GF2S and GF3S groups lower the content of expensive 2'-fucosyllactose in industrial use, and even though sialyllactose is administered at a low concentration, the 2'-fucosyllactose alone group (FL) or high concentration of sialyllactose Compared to the group (SL2), it has a high possibility of utilization in that it shows a significantly higher effect on short-chain fatty acids production in the intestine.

[Production Example 3]

Among the human milk oligosaccharide mixtures of Preparation Example 1, the GF2 and GF3 groups, which had excellent anti-inflammatory effects, were each mixed with 100 parts by weight of conventional milk powder containing no human milk oligosaccharides at a weight ratio of 0.5. Conventional milk powder containing no human milk oligosaccharides was represented as the IF1 group, formula mixed with GF2 as the IF2 group, and formula mixed with GF3 as the IF3 group. The negative control group in which only LPS was administered without adding the formula was denoted as LPS, and the normal control group in which both the formula and LPS were not administered was denoted as Con.

[Experimental Example 5: Confirmation of Inhibition of Intestinal Inflammation and Intestinal Barrier Protection Effect of Formulated Milk Containing Human Milk Oligosaccharide Mixture]

1. Check the effect of increasing the TEER (rans epithelial electrical resistance) of the damaged intestinal barrier

A transwell plate is used to create a co-culture model, and Caco-2 is dispensed at a density of 1×10 ⁵ cells/mL on the apical side, which is the upper part of the insert, and cultured in DMEM low glucose media containing 10% FBS. The basolateral side was cultured for 21 days to form a monolayer by dispensing DMEM low glucose medium without FBS without cells to induce differentiation. In order to confirm whether the cultured Caco-2 formed a stable monolayer, trans-epithelial electrical resistance (TEER) values were measured every 3 days for 21 days. THP-1 culture was carried out in RPMI media containing 10% inactivated FBS and 40 nM of phorbol 12-myristate 13-actate (PMA) at a concentration of 1×10 ⁵ cells/mL by dispensing 1 mL each into a 12-well plate for 72 hours 37 ℃, 5% CO2 conditions were incubated. On day 21 of Caco-2 culture, co-culture was initiated by inserting Caco-2-cultured insert wells into a 12-well plate containing differentiated THP-1 macrophages. It was confirmed that the TEER value, which peaked between days 9 and 12, maintained an average of 600 Ωcm on day 21, and the IF1, IF2, and IF3 groups of Preparation Example 3 were cultured on day 21 at a concentration of 1 mg/mL , and DMEM low glucose medium was dispensed into the normal control group (Con) and the negative control group (LPS). THP-1, a human-derived immune cell located on the basolateral side, was treated with 1 μg/mL LPS in all experimental groups except for the normal control group (Con) and cultured for 24 hours.

The change in TEER value of each experimental group compared to the normal control group (Con) is shown in Table 7 below in %.

division TEER (%) Con 100.0 ^a ±9.80 LPS ^94.6b ±3.06 IF1 ^118.9ab ±15.01 IF2 ^128.7a ±17.02 IF3 ^136.5a ±9.15

In the negative control group (LPS), TEER was significantly reduced compared to the normal control group (Con) because the monolayer was collapsed due to inflammation and the TEER value was lowered. Compared to the negative control group (LPS), the existing formula (IF1) showed a tendency to increase TEER, but there was no significant difference. confirmed to increase.

2. Confirmation of inhibitory effect on inflammatory factor expression

In order to determine whether each milk powder product suppresses LPS-induced inflammation, co-cultured Caco-2 is used to confirm the suppression of the expression of inflammatory factors TNF-α, IL-6, iNOS, COX-2, and IL-8 using qPCR. and are shown in Table 8 below. In order to use qPCR, mRNA was extracted after breaking the cell membrane using trizol. Afterwards, the amount of extracted mRNA was quantified in the same amount using nanodrop, and cDNA was synthesized using LeGene premium express first-strand cDNA synthesis system. cDNA was mixed with HiPi real-time PCR 2x master mix SYBR green mix to create a PCR reaction solution, which was used for qPCR. The expression suppression effect was expressed as a multiple of the relative expression level when the expression level of the normal control group (Con) was set to 1.

division TNF-α IL-6 iNOS COX-2 IL-8 Con 1.00 ^a ±0.07 1.00 ^a ±0.07 ^1.00ab ±0.15 1.00 ^ac ±0.14 1.00 ^a ±0.14 LPS 1.52 ^b ±0.13 1.63 ^b ±0.05 1.14 ^a ±0.19 1.77 ^b ±0.19 1.56 ^b ±0.10 IF1 0.83 ^a ±0.04 0.93 ^a ±0.08 0.70 ^bc ±0.01 ^1.13a ±0.26 0.86 ^a ±0.04 IF2 ^0.61c ±0.04 0.70 ^c ±0.01 ^0.56c ±0.03 0.84 ^c ±0.05 0.59 ^c ±0.04 IF3 ^0.56c ±0.01 0.68 ^c ±0.02 ^0.56c ±0.05 0.87 ^ac ±0.09 ^0.57c ±0.04

In the negative control group (LPS), the expression of TNF-α, IL-6, COX-2, and IL-8 inflammatory factors increased significantly compared to the normal control group (Con), and in the case of iNOS, there was no significant difference, but there was a tendency to increase. was

Compared to the negative control group (LPS), the conventional formula group (IF1), which did not contain the human milk oligosaccharide mixture, significantly reduced the expression of all inflammatory factors such as TNF-α, IL-6, iNOS, COX-2, and IL-8, The IF2 and IF3 groups containing the human milk oligosaccharide mixture significantly reduced the expression of inflammatory factors NF-α, IL-6, COX-2 and IL-8 compared to the IF1 group.

3. Confirm intestinal barrier protection effect

In order to confirm the intestinal barrier protective effect, the expression of tight junction factors Claudin-1, Occludin and ZO-1) in the co-cultured Caco-2 was confirmed by qPCR and is shown in Table 9 below. The expression suppression effect was expressed as a multiple of the relative expression level when the expression level of the normal control group (Con) was set to 1.

division Claudin-1 Occludin ZO-1 Con ^1.00ab ±0.01 1.00 ^a ±0.01 1.00 ^a ±0.06 LPS 0.90 ^a ±0.03 0.80 ^b ±0.03 0.88 ^a ±0.09 IF1 1.08 ^bc ±0.08 ^0.66c ±0.03 0.98 ^a ±0.07 IF2 ^1.21c ±0.06 1.04 ^a ±0.04 1.61 ^b ±0.20 IF3 ^1.22c ±0.05 1.08 ^a ±0.03 1.69 ^b ±0.10

The negative control group (LPS) showed a significant increase in Occludin expression compared to the normal control group (Con), and there was no significant difference in Claudin-1 and ZO-1, but showed an increasing tendency.

Compared to the negative control group (LPS), the conventional formula group (IF1) without human milk oligosaccharide mixture significantly increased Claudin-1, significantly decreased Occludin, and had no effect on ZO-1. However, the IF2 and IF3 groups containing the human milk oligosaccharide mixture significantly increased Claudin-1, Occludin, and ZO-1 compared to the negative control group (LPS) as well as the normal control group (Con), confirming that the intestinal barrier protective effect was excellent. did

Statistical treatment of the above experiments was performed by the students t-test ( p <0.05) method, and in the experimental results, a, b, c, d, and e each indicate a significant difference at p value < 0.05.

Examples of formulation of a composition containing the human milk oligosaccharide mixture of the present invention are described below, but the present invention is not intended to be limited thereto, but only specifically described.

Formulation Example 1. Preparation of powder

GF2, GF3 of Preparation Example 1, or GF2S, GF3S of Preparation Example 2 500 mg

Lactose 100 mg

Talc 10 mg

A powder is prepared by mixing the above ingredients and filling them in an airtight bag.

Formulation Example 2. Preparation of tablets

GF2, GF3 of Preparation Example 1, or GF2S, GF3S of Preparation Example 2 300 mg

Corn Starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet manufacturing method.

Formulation Example 3. Preparation of capsule formulation

200 mg of GF2, GF3 of Preparation Example 1, or GF2S and GF3S of Preparation Example 2

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium stearate 0.2 mg

Capsules are prepared by mixing the above ingredients and filling them into gelatin capsules according to a conventional capsule preparation method.

Formulation Example 4. Preparation of liquid formulation

4 g of GF2, GF3 of Preparation Example 1, or GF2S and GF3S of Preparation Example 2

Isomerized sugar 10 g

5 g mannitol

Appropriate amount of purified water

According to the conventional liquid formulation manufacturing method, each component is dissolved in purified water, lemon flavor is added in an appropriate amount, the above components are mixed, and then purified water is added to adjust the total amount to 100g, and then filled into a brown bottle to be sterilized. to prepare a liquid.

Formulation Example 5. Preparation of granules

GF2, GF3 of Preparation Example 1, or GF2S, GF3S of Preparation Example 2 1,000 mg

Appropriate amount of vitamin mixture

Vitamin A Acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

10 μg of biotin

Nicotinamide 1.7 mg

Folic acid 50 μg

Calcium Pantothenate 0.5 mg

Appropriate amount of mineral mixture

Ferrous sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium Carbonate 25.3 mg

Potassium Phosphate Monobasic 15 mg

Dibasic Calcium Phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium Chloride 24.8 mg

Although the composition ratio of the above vitamin and mineral mixture was prepared by mixing ingredients suitable for granules in a preferred embodiment, the mixing ratio may be arbitrarily modified, and after mixing the above ingredients according to a conventional granule manufacturing method, It can be prepared and used for preparing a health functional food composition according to a conventional method.

Formulation Example 6. Preparation of functional beverage

GF2, GF3 of Preparation Example 1, or GF2S, GF3S of Preparation Example 2 1,000 mg

Citric Acid 1,000 mg

100 g of oligosaccharides

2 g plum concentrate

1 g of taurine

Add purified water to make a total of 900 ml

After mixing the above ingredients according to the normal health drink manufacturing method, stirring and heating at 85 ° C. for about 1 hour, the resulting solution is filtered and collected in a sterilized 2 L container, sealed and sterilized, and then refrigerated. It is used for preparing the functional beverage composition of the present invention.

Although the composition ratio is a mixture of ingredients suitable for a relatively favorite beverage in a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the class of demand, the country of demand, and the purpose of use.

Claims (33)

A composition for improving intestinal health, intestinal function or intestinal immunity, comprising a mixture of human milk oligosaccharides including galacto-oligosaccharide and 2'-fucosyllactose as an active ingredient. According to claim 1,
The composition for improving intestinal health, intestinal function or intestinal immunity, characterized in that the galacto-oligosaccharide and 2'-fucosyllactose have a weight ratio of 60:40 to 85:15. According to claim 2,
The galacto-oligosaccharide comprises 10-20% by weight of 4'-galactosyllactose and 0.05-0.5% by weight of 6'-galactosyllactose out of the total galacto-oligosaccharide. Food composition for improvement. According to claim 1,
The human milk oligosaccharide mixture is a food composition for improving intestinal health, intestinal function or intestinal immunity, characterized in that it further comprises sialyllactose. According to claim 4,
The sialyllactose is a food composition for improving intestinal health, intestinal function or intestinal immunity, characterized in that it comprises 1-10 parts by weight based on 100 parts by weight of the sum of the galactooligosaccharide and 2'-fucosyllactose. According to any one of claims 1 to 5,
The composition is a food composition for improving intestinal health, intestinal function or intestinal immunity, characterized in that it increases the content of short chain fatty acids in the intestine. According to claim 6,
The short-chain fatty acid is a food composition for improving intestinal health, intestinal function or intestinal immunity, characterized in that at least one selected from acetic acid, butyric acid and propionic acid. According to any one of claims 1 to 5,
The composition is a food composition for improving intestinal health, intestinal function or intestinal immunity, characterized in that it promotes the proliferation or growth of beneficial bacteria in the intestine or inhibits the proliferation or growth of harmful bacteria in the intestine. According to any one of claims 1 to 5,
The composition is a food composition for improving intestinal health, intestinal function or intestinal immunity, characterized in that to improve bowel function. According to any one of claims 1 to 5,
The composition is a food composition for improving intestinal health, intestinal function or intestinal immunity, characterized in that for inhibiting the secretion of TNF-α. A food composition for preventing or improving intestinal inflammation comprising a mixture of human milk oligosaccharides containing galacto-oligosaccharide and 2'-fucosyllactose as an active ingredient. According to claim 11,
The galacto-oligosaccharide and 2'-fucosyllactose are a food composition for preventing or improving intestinal inflammation, characterized in that the weight ratio of 60:40 to 85:15. According to claim 12,
The galactooligosaccharide is a food composition for preventing or improving intestinal inflammation, characterized in that it comprises 10-20% by weight of 4'-galactosyllactose and 0.05-0.5% by weight of 6'-galactosyllactose out of the total galacto-oligosaccharide. . According to claim 11,
The human milk oligosaccharide mixture is a food composition for improving intestinal health or improving intestinal function, characterized in that it further comprises sialyllactose. According to claim 14,
The sialyllactose is a food composition for preventing or improving intestinal inflammation, characterized in that it comprises 1-10 parts by weight based on 100 parts by weight of the sum of the galactooligosaccharide and 2'-fucosyllactose. According to any one of claims 11 to 15,
The composition is a food composition for preventing or improving intestinal inflammation, characterized in that to increase the content of short chain fatty acids (short chain fatty acid) in the intestine. According to claim 16,
The short-chain fatty acid is a food composition for preventing or improving intestinal inflammation, characterized in that at least one selected from acetic acid, butyric acid and propionic acid. According to any one of claims 11 to 15,
The composition is a food composition for preventing or improving intestinal inflammation, characterized in that it promotes the proliferation or growth of beneficial bacteria in the intestine or inhibits the proliferation or growth of harmful bacteria in the intestine. According to any one of claims 11 to 15,
The composition is a food composition for preventing or improving intestinal inflammation, characterized in that to improve bowel function. According to any one of claims 11 to 15,
The composition is a food composition for preventing or improving intestinal inflammation, characterized in that for inhibiting the secretion of TNF-α. A pharmaceutical composition for treating or preventing inflammatory bowel disease, comprising a mixture of human milk oligosaccharides including galacto-oligosaccharide and 2'-fucosyllactose as an active ingredient. According to claim 21,
The galacto-oligosaccharide and 2'-fucosyllactose is a pharmaceutical composition for treating or preventing inflammatory bowel disease, characterized in that the weight ratio of 60:40 to 85:15. The method of claim 22,
The galactooligosaccharide comprises 10-20% by weight of 4'-galactosyllactose and 0.05-0.5% by weight of 6'-galactosyllactose out of the total galacto-oligosaccharide. composition. According to claim 21,
The human milk oligosaccharide mixture is a pharmaceutical composition for treating or preventing inflammatory bowel disease, characterized in that it further comprises sialyllactose. According to claim 24,
The pharmaceutical composition for treating or preventing inflammatory bowel disease, characterized in that the sialyllactose comprises 1-10 parts by weight based on 100 parts by weight of the sum of the galactooligosaccharide and 2'-fucosyllactose. The method of any one of claims 21 to 25,
The composition is a pharmaceutical composition for treating or preventing inflammatory bowel disease, characterized in that to increase the content of short chain fatty acids (short chain fatty acid) in the intestine. The method of claim 26,
The short-chain fatty acid is a pharmaceutical composition for treating or preventing inflammatory bowel disease, characterized in that any one or more selected from acetic acid, butyric acid and propionic acid. The method of any one of claims 21 to 25,
The pharmaceutical composition for treating or preventing inflammatory bowel disease, characterized in that the composition promotes the proliferation or growth of beneficial bacteria in the intestine or inhibits the proliferation or growth of harmful bacteria in the intestine. The method of any one of claims 21 to 25,
The composition is a pharmaceutical composition for treating or preventing inflammatory bowel disease, characterized in that to improve bowel function. The method of any one of claims 21 to 25,
The composition is a pharmaceutical composition for treating or preventing inflammatory bowel disease, characterized in that for inhibiting the secretion of TNF-α. Food for infants and young children comprising the food composition for improving intestinal health, intestinal function or intestinal immunity according to any one of claims 1 to 5, or the food composition for improving intestinal inflammation according to any one of claims 11 to 15 composition. The food composition for infants and young children according to claim 31, wherein the food composition for infants and young children is formula milk, formula for infants, formula for growth, or baby food for infants and young children. Breast milk supplement composition comprising the food composition for improving intestinal health, intestinal function or intestinal immunity according to any one of claims 1 to 5, or the food composition for improving intestinal inflammation according to any one of claims 11 to 15 .

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