KR102616020B1 - Composition for improving intestinal health, intestinal function or intestinal immunity, preventing or improving intestinal inflammation comprising mother's milk oligosaccharide mixture - Google Patents

Abstract

The present invention provides 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 mixture of breast milk oligosaccharides containing galactooligosaccharide and 2'-fucosyllactose as an active ingredient. It relates to pharmaceutical compositions for treatment or prevention, food compositions for infants and young children, or breast milk supplement compositions. The composition containing the human milk oligosaccharide mixture containing galactooligosaccharide and 2'-fucosyllactose of the present invention as an active ingredient improves intestinal health by increasing the content of short-chain fatty acids in the intestine and suppressing the secretion of the intestinal inflammatory cytokine TNF-α. 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 immunity, preventing or improving intestinal inflammation, comprising a breast milk oligosaccharide mixture as an active ingredient {Composition for improving intestinal health, intestinal function or intestinal immunity, preventing or improving intestinal inflammation comprising mother's milk oligosaccharide mixture}

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

Recently, after it was discovered that intestinal microorganisms play a critical role in preventing infectious diseases, regulating immune processes, and absorbing nutrients in the intestines, research on intestinal microorganisms is increasing explosively around the world.

In particular, intestinal microorganisms play an important role in maintaining normal physiological functions of humans (hosts) in the body, and changes in the distribution of intestinal microorganisms can cause metabolic syndromes such as obesity, diabetes, and hyperlipidemia, as well as immune-related diseases such as enteritis and atopy. As it has been revealed that intestinal microorganisms are closely related to induction, research results are continuously being published showing that intestinal microorganisms play a very important role in regulating body metabolism.

These intestinal microorganisms are known to change due to various factors such as aging, inflammatory response, antibiotics, probiotics, diet, eating patterns, and stress. Among them, food intake and eating habits have the greatest impact on changes in intestinal microorganisms. This has been reported in many papers.

Probiotics such as lactic acid bacteria are the most consumed to balance intestinal microorganisms. It is known that consuming these beneficial bacteria has a positive effect on the human body by improving the balance of intestinal microorganisms, but it is known that it is difficult to actually reach the large intestine.

Accordingly, prebiotics refer to non-digestible food ingredients that help the growth and proliferation of microorganisms already existing in the intestines, and it has been reported that when consumed, they can more effectively improve changes in intestinal microorganisms.

Meanwhile, about 200 to 300 types of human milk oligosaccharides (HMOs) exist in human breast milk at a high concentration of 10 to 20 g/L, and among the human milk oligosaccharides, fucosyl oligosaccharides and 2'-foucault room lactose are the highest. It is known to exist in large amounts, and 2'fucosyllactose is known to have a prebiotic effect and prevent infection by preventing pathogens and the intestinal toxins they secrete from sticking to the intestinal epithelium, and is helpful for human health. It is known to play a role in contributing to the formation of intestinal microflora.

Methods for producing 2'-Foucault room lactose include chemical synthesis, synthesis using enzyme reactions, and production using microbial fermentation. However, 2'-Foucault room lactose alone does not have a significant effect on the growth of beneficial intestinal bacteria. , due to its high price, there were limits to its application to food in high enough amounts or ratios to demonstrate functionality.

Meanwhile, galactooligosaccharides, especially 4'-galactosyllactose, are also known as oligosaccharides with a great effect on the growth of beneficial intestinal bacteria as one of the breast milk oligosaccharides, but when 2'-fucosyllactose and galactooligosaccharides are combined in a specific ratio, short-chain fatty acids in the intestine Until now, nothing was known about the effect of increasing the production of or suppressing the secretion of intestinal inflammatory cytokines.

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

The object of the present invention is to provide a food composition for improving intestinal health, intestinal function or intestinal immunity, a food composition for preventing or improving intestinal inflammation, and a food composition containing a mixture of human milk oligosaccharides containing galactooligosaccharide and 2'-fucosyllactose as an active ingredient. The object is to provide a pharmaceutical composition for treating or preventing intestinal diseases, a food composition for infants or 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 mixture of breast milk oligosaccharides containing galactooligosaccharides and 2'-fucosyllactose as an active ingredient.

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

In addition, the present invention provides a pharmaceutical composition for the treatment or prevention of inflammatory bowel disease comprising a mixture of breast milk oligosaccharides containing galactooligosaccharides and 2'-fucosyllactose as an active ingredient.

The galactooligosaccharide and 2'-foucault room lactose 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 based on the total galactooligosaccharides.

The breast milk oligosaccharide mixture may additionally include sialyllactose.

The sialyllactose may contain 1-10 parts by weight based on 100 parts by weight of the total 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 intestinal bacteria, or may inhibit the proliferation or growth of harmful intestinal bacteria.

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 children comprising a composition for improving intestinal health, intestinal function, or intestinal immunity, or a food composition for preventing or improving intestinal inflammation.

The food composition for infants and young children may be infant formula, infant formula, growth formula, or baby food.

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

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 the human milk oligosaccharide mixture containing galactooligosaccharide and 2'-fucosyllactose of the present invention as an active ingredient. Pharmaceutical compositions for treatment or prevention, food compositions for infants, or breast milk supplement compositions can promote the growth of beneficial intestinal bacteria, inhibit the growth of harmful bacteria, increase the content of intestinal short-chain fatty acids, and improve intestinal flora. Additionally, using the composition of the present invention, intestinal health, intestinal function, or intestinal immunity can be improved, and bowel function can be improved.

Figure 1 is a graph showing the amount of acetic acid produced when a human fecal system was treated with a mixture of human milk oligosaccharides containing galactooligosaccharides and 2'-fucosyllactose at different mixing ratios in Experimental Example 1.
Figure 2 is a graph showing the amount of propionic acid produced when a human fecal system was treated with a mixture of human milk oligosaccharides containing galactooligosaccharides and 2'-fucosyllactose at different mixing ratios in Experimental Example 1.
Figure 3 is a graph showing the amount of butyric acid produced when a human fecal system was treated with a mixture of human milk oligosaccharides containing galactooligosaccharides and 2'-fucosyllactose at different mixing ratios in Experimental Example 1.
Figure 4 is a graph comparing the degree of inhibition of secretion of TNF-α 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. This is one 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.
Figure 7 is a graph comparing the nitric oxide production ability in mouse-derived macrophages (RAW 264.7) in which inflammation was induced using lipopolysaccharide (LPS) in Experimental Example 2.
Figure 8 is a photograph to check the intestinal length after sacrificing an inflammatory bowel disease animal model mouse in which intestinal epithelial cell damage was induced by DSS administration in Experimental Example 3.
Figure 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 mixture of breast milk oligosaccharides containing galactooligosaccharides 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 mixture of breast milk oligosaccharides containing galactooligosaccharides and 2'-fucosyllactose as an active ingredient.

Additionally, the present invention relates to a pharmaceutical composition for the treatment or prevention of inflammatory bowel disease comprising a mixture of human milk oligosaccharides containing galactooligosaccharides 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 Behcet's disease, infectious enteritis, ischemic bowel disease, radiation enteritis, and leaky gut syndrome.

The leaky gut syndrome (LGS) refers to the intestinal mucosa, which maintains a certain gap between cells and allows polymer substances to travel back and forth through the gap between cells when any stimulus or damage occurs during the digestion and absorption process. It refers to the phenomenon of increased permeability, which collectively refers to the symptoms caused by polymer substances in the blood leaking into the intestinal lumen or polymer substances in the lumen entering the blood directly due to the barrier not functioning properly. The above symptoms appear in various clinical conditions such as aging, allergies, polytrauma, rheumatoid arthritis, inflammatory bowel disease, chronic fatigue syndrome, and hypersensitivity syndrome. In addition, due to increased permeability of the intestinal mucosa or damage to the intestinal mucosa, pathogens, antigens, and putrefactive substances enter the intestinal mucosa, causing an inflammatory reaction, and endotoxin flows into the bloodstream, causing bacterial translocation and intestinal endotoxemia. This causes various inflammatory and immune reactions to occur. This leaky gut syndrome causes various, widespread, and ambiguous symptoms without any specific symptoms. Specifically, it is unique in that it has symptoms that are common to various diseases.

The improvement of intestinal immunity or enhancement of intestinal immunity refers to the function of promoting an immune response to an antigen non-specifically during the initial activation of immune cells present in the intestinal tract or the function of strengthening the immunity of the intestinal tract by increasing the activity of cells of the immune system. . Diseases caused by decreased 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 the application effects of improving or preventing seasonal influenza in humans, improving or preventing bacterial diarrhea, improving or preventing bacterial infection, and enhancing the treatment effect after wounds.

The galactooligosaccharide and 2'-fucosyllactose may have a weight ratio of 60:40 to 85:15, preferably 65:35 to 80:20, and more preferably 70:30 to 75:25. If the ratio of galactooligosaccharides is less than the above lower limit or the ratio of 2'-fucosyllactose exceeds the above upper limit, the effect of suppressing the secretion of inflammatory cytokine TNF-α is not sufficient, and the ratio of galactooligosaccharides exceeds the above upper limit or 2' -If the proportion of fucosyllactose is less than the above lower limit, the effect of increasing the production of short-chain fatty acids in the intestine, especially 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% by weight, based on the total galactooligosaccharides. It may contain 0.3% by weight.

The breast milk oligosaccharide mixture may additionally include sialyllactose. Sialyllactose may be 3'-sialyllactose, 6'-sialyllactose, or a mixture thereof, and when sialyllactose is additionally included, the effect of the breast milk oligosaccharide mixture on the production of short-chain fatty acids in the intestine or the inhibition of secretion of inflammatory cytokines The effect increases.

The sialyllactose may contain 1-10 parts by weight based on 100 parts by weight of the total of the galactooligosaccharide and 2'-fucosyllactose.

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

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

The composition may inhibit the secretion of intestinal inflammatory cytokines, especially 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 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 infant formula, infant formula, growth formula, or baby food.

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

The food composition according to the present invention can be formulated in the same manner 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, gum, ice cream, vitamin complexes, and health supplements. etc.

The food composition of the present invention may include a mixture of human milk oligosaccharides as an active ingredient, as well as ingredients commonly added during food production, including, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. . Examples of the above-mentioned carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a flavoring agent, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is manufactured as a drink or beverage, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be additionally included in addition to the human milk oligosaccharide mixture of the present invention. .

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 breast milk oligosaccharide mixture as an active ingredient. Health functional foods are foods manufactured by adding a mixture of breast milk oligosaccharides to food ingredients such as beverages, teas, spices, gum, and confectionery, or by encapsulating, powdering, or suspending them, and bringing about specific health effects when consumed. However, unlike regular drugs, it has the advantage of not having any side effects that may occur when taking the drug for a long time since it is made from food. 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 amount of breast milk oligosaccharide mixture added in such health functional foods cannot be uniformly specified as it depends on the type of health functional food being targeted. However, it can be added within the range that does not damage the original taste of the food, and is usually the same for the target food. It is in the range of 0.01 to 50% by weight, preferably 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 a pill, tablet, capsule, or beverage.

In another example, the breast milk oligosaccharide mixture as described above can be used in various ways as a pharmaceutical composition for preventing or treating inflammatory bowel disease or leaky gut syndrome. In addition to the active ingredient, the human milk oligosaccharide mixture, it can be manufactured using pharmaceutically suitable and physiologically acceptable auxiliaries, which include excipients, disintegrants, sweeteners, binders, coating agents, bulking agents, lubricants, lubricants, or flavors. You can use my back.

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

The pharmaceutical composition may be in the form of 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, etc. Additionally, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacance or sodium oleate, sodium stearate, magnesium stearate, sodium Includes benzoate, sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, etc.

Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these ingredients can be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

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

The pharmaceutical composition of the present invention can be administered orally. The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and is usually A skilled doctor can easily determine and prescribe an effective dosage for desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10 g/kg.

The pharmaceutical composition of the present invention can be prepared in unit dose form by formulating using a pharmaceutically acceptable carrier and/or excipient, or can be prepared by placing it in a multi-dose container. At this time, 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 dispersant or stabilizer.

Additionally, 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, preferably a human, that is the subject of treatment, observation or experiment.

As used herein, the term “effective amount” means the amount of an active ingredient or pharmaceutical composition that is believed by a researcher, veterinarian, physician, or other clinician to induce a biological or medical response in a tissue system, animal, or human, which means that Includes amounts that induce symptom relief. 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 dosage to be administered can be easily determined by a person skilled in the art, including 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 the antigen, and the effectiveness contained in the composition. Depending on various factors, including the content of the ingredients and other ingredients, the type of dosage form, and the patient's age, middle and general health, gender and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs. It can be adjusted. In the prevention, treatment or improvement method of the present invention, for adults, it is preferable to administer the breast milk oligosaccharide mixture once or several times a day at a dose of 0.001 g/kg to 10 g/kg.

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

[Production Example 1]

To determine the amount of short-chain fatty acid production 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'-Foucault room lactose, galactooligosaccharide and 2'-Foucault room lactose were mixed. It was mixed as shown in Table 1 below. The galactooligosaccharide is a powder containing 75% by weight of galactooligosaccharide, 20% by weight of lactose, glucose, and galactose, and 5% by weight of moisture. Of the total galactooligosaccharides, 4'-galactosyl lactose is 13.5% by weight, 6'-gal. Lactosyllactose contains 0.15% by weight, and the 2'-Foucault room lactose was used as a powder containing 95% by weight of 2'-Foucault room lactose and 5% by weight of moisture. The mixing weight ratio in Table 1 is the mixing ratio of the solid content of galactooligosaccharide and 2'-fucosyllactose, respectively.

division Galactooligosaccharide 2'-Foucault room lactose G.L. 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 intestinal short-chain fatty acid production in the human fecal system]

A fecal slurry was prepared by mixing 5 g of mixed feces, 1 g each, from five infants with an average age of 3 months, and mixing them with 50 mL of pH 7.7 PBS buffer. The human milk oligosaccharide mixture in Table 1 was mixed with the fecal slurry at a concentration of 1 mg/mL, and 1 mL of the human milk oligosaccharide mixture-treated fecal slurry 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) inoculated into 9 mL, mixed, and incubated at 37°C for 48 hours under anaerobic conditions. cultured for some time.

The culture was centrifuged at 12,100 After mixing for 10 minutes, centrifugation at 1,932 -BDMCS) was added to each vial, vortexed each vial, and incubated in a drying oven at 70°C for 45 minutes. 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 in Table 1 is denoted 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 are They are shown in Figures 1, 2, and 3, respectively.

In Figure 1, acetic acid 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 the 2'-fucosyllactose-only group (FL), and among the breast milk oligosaccharide mixtures, galactooligosaccharides and 2'-foucaultyl lactose were used at a ratio of 0.7:0.3 to 0.7:0.3. The significant increase was greater in the GF1, GF2, and GF3 groups mixed at 0.88:0.12.

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

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-only group (GL) and the 2'-fucosyllactose-only group (FL), as well as the GF1, GF2, GF4, and GF5 groups mixed with galactooligosaccharide and 2'-fucosyllactose in different ratios. In the GF3 group, which mixed lactooligosaccharide and 2'-fucosyllactose at a weight ratio of 0.7:0.3, the amount of butyric acid produced was significantly increased by about two times.

As a result of confirming the effect of intestinal short-chain fatty acid production in the human fecal system, galactooligosaccharide and 2'-fucosyllactose were 0.7% higher than the fecal group, galactooligosaccharide only group (GL), and 2'-fucosyllactose only group (FL). In the GF3 group mixed at a weight ratio of :0.3, the production of acetic acid, propionic acid, and butyric acid significantly increased.

In particular, the GF3 group is used for industrial purposes in that it has a significantly higher intestinal short-chain fatty acid production effect compared to the 2'-fucosyllactose only group (FL), even though the content of expensive 2'-fucosyllactose is lowered. It is highly likely.

[Experimental Example 2: Confirmation of inflammation inhibition effect in intestinal epithelial cells and macrophages]

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

Inflammation was induced in human-derived intestinal epithelial cells (caco-2) with 1 μg/mL lipopolysaccharide (LPS, Sigma-Aldrich), and after treatment with 1 mg/mL of the human milk oligosaccharide mixture of Preparation Example 1, TNF -α concentration was measured using an ELISA kit purchased from Goma Biotex according to the measurement method in the kit, and is shown in Figure 4.

The normal control group (Con) was a group not treated with the LPS and human milk oligosaccharide mixture, the negative control group (LPS) was a group treated with only LPS, and the remaining human milk oligosaccharide mixture experimental group of Preparation Example 1 was simultaneously treated with the LPS and human milk oligosaccharide mixture. 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, the amount of TNF-α secretion was significantly increased compared to the normal control group (Con), and in the GF4 group, the amount of TNF-α secretion was significantly increased 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 it significantly decreased compared to the negative control group (LPS) in the GF2, GF3, and FL groups.

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

2. Confirmation of the effect of suppressing the expression of inflammatory factors in intestinal epithelial cells

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

To confirm the mRNA expression level, the cell membrane was dissolved using trizol and the mRNA was extracted. Afterwards, the amount of extracted mRNA was quantified in equal amounts using nanodrop, and then cDNA was synthesized using the LeGene premium express first-strand cDNA synthesis system. The cDNA was mixed with HiPi real-time PCR 2x master mix SYBR green mix to create a PCR reaction solution. Then, the expression levels of the inflammatory factors were confirmed using the iQ5 thermal cycler and are shown in Figures 5 and 6, respectively.

The mRNA expression level of COX-2 was significantly increased in the LPS-only negative control group (LPS) compared to the normal control group (Con), and there was no significant difference in the galactooligosaccharide-only group (GL) from the negative control group (LPS). , the 2'-fucosyllactose only group (FL) and the GF2, GF3, GF4, and GF5 groups decreased significantly compared to the negative control group (LPS).

The mRNA expression level of iNOS was significantly increased in the LPS-only negative control group (LPS) compared to the normal control group (Con), and there was no significant difference in the expression of the galactooligosaccharide-only group (GL) compared to the negative control group (LPS). The amount greatly increased, and the 2'-fucosyllactose only group (FL) and the GF2 and GF3 groups significantly decreased compared to the negative control group (LPS).

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

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

Anti-inflammatory efficacy was confirmed by confirming the nitric oxide (NO) producing ability of the breast milk oligosaccharide mixture of Preparation Example 1 in a mouse-derived macrophage (RAW 264.7) model in which inflammation was induced using lipopolysaccharide (LPS). .

In preparation, each of the human milk oligosaccharide mixtures in step 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 and 50 μL of 1% Sulfanilamide/5% phosphoric acid solution was added, followed by 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 Figure 7.

The normal control group (Con) is a group not treated with the LPS and human milk oligosaccharide mixture, the negative control group is a group treated with only LPS, and the remaining human milk oligosaccharide mixture experimental group of Preparation Example 1 is 540% when treated simultaneously with the LPS and human milk oligosaccharide mixture. The OD value in nm, that is, the nitric oxide content (μM), 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 GL and FL groups, the nitric oxide concentration 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 observed in the galactooligosaccharide-only group (GL) and the 2'-foucault room lactose-only group (FL), and the ratio of galactooligosaccharide and 2'-foucault room lactose was 0.44:0.56 to 0.88. : When mixed at a weight ratio of 0.12, it showed an excellent anti-inflammatory effect by suppressing nitric oxide production compared to the negative control (LPS).

[Experimental Example 3: Confirmation of intestinal inflammation improvement effect in inflammatory bowel disease animal model]

1. Inhibitory effect on intestinal permeability, confirmation of intestinal inflammation and intestinal length

In Experimental Examples 1 and 2, the GF2 and GF3 groups, which had excellent short-chain fatty acid production effects and anti-inflammatory effects, were selected, and the galactooligosaccharide only group (GL) and the 2'-fucosyllactose only group (FL) were used as positive controls. The effect of improving intestinal inflammation was compared in an inflammatory bowel disease animal model.

When Dextran Sulfate Sodium (DSS) is administered to mice, it induces damage to intestinal epithelial cells, leading to intestinal inflammation and intestinal leakage. Male C57BL/6 mice (6 weeks old) weighing 250 to 300 g were supplied with sufficient food and water and maintained for 12 hours day and night with appropriate artificial lighting (daytime from 8:00 am). And constant temperature (20-24℃) and humidity (45-65%) were maintained. We monitored them for a week to help them adapt to the changed environment, maintained their sleep cycle, and checked for abnormal behavior.

After an adaptation period of 2 weeks as described above, enteritis was induced by administering 2% DSS in the drinking water for 5 days. The experimental animals were selected only from healthy animals with constant body weight changes, and were randomly selected to form a normal control group (CON) administered saline solution, a negative control group (DSS) administered DSS, and GF2 or GF3 of Preparation Example 1 after administration of DSS. The group was divided into GL or FL administration group as the positive control group. Among the above groups, the human milk oligosaccharide mixture administration group of Preparation Example 1 dissolved each human milk oligosaccharide mixture in 0.1M PBS (pH 7.4) at a concentration of 1 g/kg and orally administered 200 μL each once a day. During the 4-week administration period, feed and water were supplied autonomously (supplied until the evening before sacrifice), feed was stopped 12 hours before sacrifice, and 200 μg/g FITC-dextran was administered orally 4 hours before sacrifice. At the time of sacrifice, the animal was anesthetized using _CO2 , and blood was then collected from the abdominal vena cava.

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

In addition, MPO activity was measured in the colon tissue of the mouse to quantify intestinal inflammation, and is shown in Table 2 below. MPO activity was measured by taking 25 mg of colon tissue and adjusting the pH to 6.0 by adding 8.7 g/L dibasic potassium phosphate to 6.8 g/L monobasic potassium phosphate, then adding 5 g of hexadecyltrimethylammonium bromide (HTAB) to 1 L of potassium phosphate. The reaction solution (prepared by dissolving in buffer (50 mM, pH 6.0)) was adjusted to 25 mg/mL, the colon tissue was homogenized, and the supernatant was collected by centrifugation at 4°C, 13,400 xg, 6 min. The supernatant was dispensed three times, 7 μL each, into a 96 well plate, mixed with 200 μL of O-dinisidine solution, and the absorbance was measured at 450 nm.

Additionally, photographs of the intestine length of each experimental group are shown in Figure 8, and the intestine weight/length ratio is shown in Table 2.

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

In Table 2, the FITC concentration in plasma significantly increased by more than two times in the negative control group (DSS) compared to the normal control group (con) due to intestinal leakage caused by DSS administration. In addition, the galactooligosaccharide-only group (GL), the 2'-fucosyllactose-only group (FL), and the GF2 and GF3 groups, which showed excellent anti-inflammatory effects by mixing galactooligosaccharide and 2'-fucosyllactose, were used as negative controls ( FITC concentration was significantly reduced compared to DSS), and especially 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 caused by DSS administration. In addition, the galactooligosaccharide-only group (GL), the 2'-fucosyllactose-only group (FL), and the GF2 and GF3 groups mixed with galactooligosaccharide and 2'-fucosyllactose showed MPO activity compared to the negative control group (DSS). was significantly reduced, especially in the GF2 and GF3 groups, to a level where there was no significant difference from the normal control group (Con).

Meanwhile, in Figure 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 in the galactooligosaccharide only group (GL) and the 2'-foucault room lactose only group (FL), the intestine length was significantly decreased compared to the normal control group (Con). Intestinal length increased to some extent compared to the control group (DSS), and in the GF2 and GF3 groups containing a mixture of galactooligosaccharide and 2'-fucosyllactose, the intestinal length increased significantly 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 caused by 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 the 2'-Foucault room lactose-only group (FL), but the In the GF2 and GF3 groups mixed with pylactose, the intestinal weight/length ratio was significantly reduced compared to the negative control group (DSS) to a level with no significant difference from the normal control group (Con).

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

After collecting 10 mg of cecal feces from each experimental group, 350 μL of extraction solution containing internal standard (Acetic acid-d4), hydrochloric acid, and ether in a ratio of 2:1:4 was dispensed and homogenized, followed by 4 80 μL of the supernatant obtained by centrifugation at ℃, 1000 g, 10 min was placed in an insert vial, and 16 μL of derivative reagent - phosphorus MTBSTFA (+1% t-BDMCS) was added to each vial. The vial containing the supernatant and the derivative reagent was reacted at 80°C for 20 minutes, then incubated at room temperature for 48 hours, and 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 34.7 ^b ±6.48 0.45 ^ab ±0.03 DSS 1340 ^b ±175.0 22.5 ^a ±3.32 0.40 ^bc ±0.06 G.L. 1146 ^c ±272.1 23.8 ^a ±5.68 ^0.35c ±0.01 FL 1178 ^BC ±289.2 23.7 ^a ±6.46 ^0.37c ±0.02 GF2 1628 ^a ±342.6 45.8 ^b ±17.5 0.40 ^ab ±0.05 GF3 1790 ^a ±249.3 45.7 ^b ±15.9 0.48 ^a ±0.00

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

Propionic acid was 34.7mM in the normal control group (con), but significantly decreased to 22.5mM in the negative control group (DSS), and in the galactooligosaccharide only group (GL) and the 2'-fucosyllactose only group (FL) in the negative control group. It did not increase the propionic acid content compared to (DSS). However, in the GF2 and GF3 groups, the concentration of propionic acid increased significantly compared to the negative control group (DSS), and the concentration was higher 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 there was no significant difference. However, the galactooligosaccharide only group (GL) and the 2'-fucosyllactose only group (FL) showed significantly lower concentrations compared to the normal control group (con), and the GF2 and GF3 groups showed significantly lower concentrations than the normal control group (con). There was no significant difference. However, the GF3 group showed significantly higher butyric acid concentration than the negative control group (DSS).

Therefore, through analysis of the short-chain fatty acid content in the feces of an inflammatory bowel disease animal model showing intestinal leakage, the galactooligosaccharide-only group (GL) and the 2'-fucosyllactose-only group (FL) had significant short-chain fatty acid content 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 galactooligosaccharide and 2'-fucosyllactose, significantly increased 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 higher than that of the normal control group (con).

3. Confirmation of expression level of inflammatory factors in intestinal tissue

Expression of NF-κB pathway-related factors INF-α, iNOS, COX-2, and p-NF-κB/NF-κB was confirmed through Western blotting using intestinal tissues of each experimental group, and is shown in Figure 9 and Table 4. . For this purpose, proteins were extracted from intestinal tissue cut to about 2 mm using RIPA buffer, a protein extraction buffer, and after protein quantification, the proteins were separated using 10% SDS-PAGE, a PVDF membrane was transferred, and Tris-buffered saline Tween 20 (TBST) was used. After blocking with 5% skim milk dissolved in , TNF-α, iNOS, COX-2, and p-NF-κB/NF-κB were confirmed. The expression levels of TNF-α, iNOS, and COX-2 were expressed as relative expression folds to 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 G.L. 1.37 ^AC ±0.28 1.46 ^ab ±0.22 1.64 ^ab ±0.74 2.14 ^ab ±0.55 FL 1.43 ^a ±0.35 1.80 ^b ±0.32 2.31 ^b ±0.30 2.16 ^ab ±0.65 GF2 0.95 ^bc ±0.10 0.99 ^a ±0.41 1.01 ^a ±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

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

The galactooligosaccharide only group (GL) and the 2'-fucosyllactose only group (FL) were compared to the negative control group (DSS) in all inflammatory factors including TNF-α, iNOS, COX-2, and p-NF-κB/NF-κB. There was no significant difference. However, the GL group showed somewhat lower expression of inflammatory factors than the FL group. Meanwhile, in the GF2 and GF3 groups mixed with galactooligosaccharide and 2'-fucosyllactose, 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 increase in short-chain fatty acid production and the synergistic effect of anti-inflammatory effect when sialyllactose was added to the GF2 and GF3 groups, which have excellent anti-inflammatory effects among 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. The sialyllactose used was a mixture of 3'-sialyllactose and 6'-sialyllactose in a 1:1 weight ratio.

division Galactooligosaccharide 2'-Foucault room lactose Sialyllactose G.L. 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 intestinal short-chain fatty acid production in the human fecal system of a mixture of breast milk oligosaccharides containing sialyllactose]

Except for the SL1 group, all human milk oligosaccharides or their mixture groups were mixed at 1 mg/mL and tested in the same manner as Experiment 1, and the results are shown in Table 6. The SL1 group was administered a low concentration of sialyllactose, and the only difference was that it was mixed at 50 ㎍/mL, which is 1/10 of SL2. The administered concentration of SL1 is the same as the concentration of sialyllactose included 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 in Table 4 is denoted as "fecal", and the acetic acid, propionic acid, and butyric acid contents (mM) of the fecal slurry treated with the human milk oligosaccharide mixture in Table 4 are shown in Table 6, respectively. shown in

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

In Table 6, acetic acid 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 galactooligosaccharide only group (GL), the 2'-fucosyllactose only group (FL), and the sialyllactose low concentration group (SL1).

In the sialyllactose high concentration group (SL2), which was administered at a high concentration of sialyllactose 20 times higher, and in the GF2 and GF3 groups, which were mixed galactooligosaccharides and 2'foucault room lactose in the breast milk oligosaccharide mixture, acetic acid production was higher compared to the negative control group (fecal). It increased significantly.

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

In Table 6, propionic acid 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 only group (FL) and the sialyllactose low concentration group (SL1).

The sialyllactose high concentration group (SL2), which was administered a high concentration of sialyllactose 20 times higher, the GF3 group, which was a mixture of galactooligosaccharides and 2'foucault room lactose from a human milk oligosaccharide mixture, and the GF2 and GF3 groups were administered sialyllactose, respectively. In the GF2S and GF3S groups added at low concentrations, acetic acid production significantly increased compared to the negative control group (fecal).

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

In particular, the GF2S and GF3S groups reduced the content of 2'-fucosyllactose, which is expensive for industrial use, and even though sialyllactose was administered at a low concentration, the 2'-fucosyllactose only group (FL) or the high-concentration sialyllactose group It has high potential for use in that it has a significantly higher effect on producing short-chain fatty acids in the intestines compared to the group (SL2).

[Production Example 3]

Among the human milk oligosaccharide mixtures of Preparation Example 1, the GF2 and GF3 groups, which have excellent anti-inflammatory effects, were mixed at a weight ratio of 0.5 with 100 parts by weight of existing formula that does not contain human milk oligosaccharides. Existing formula that did not contain human milk oligosaccharides was represented as the IF1 group, formula mixed with GF2 was represented as the IF2 group, and formula mixed with GF3 was represented as the IF3 group. The negative control group administered only LPS without adding the formula was indicated as LPS, and the normal control group administered neither the formula nor LPS was indicated as Con.

[Experimental Example 5: Confirmation of the effect of infant formula containing breast milk oligosaccharide mixture on inhibiting intestinal inflammation and protecting the intestinal barrier]

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

To create a co-culture model, a transwell plate is used, and Caco-2 is dispensed at a density of 1 × 10 ⁵ cell/mL on the apical side, the upper part of the insert, and cultured in DMEM low glucose media containing 10% FBS, and on the lower part, the apical side. On the basolateral side, DMEM low glucose medium without FBS was dispensed and cultured for 21 days to form a monolayer to induce differentiation. To check whether the cultured Caco-2 formed a stable monolayer, trans-epithelial electrical resistance (TEER) values were measured at 3-day intervals for 21 days. THP-1 culture was performed in RPMI media containing 10% inactivated FBS and ⁴⁰ nM of phorbol 12-myristate 13-actate (PMA) at 1 Cultivated under conditions of ℃ and 5% CO2. On the 21st day of Caco-2 culture, co-culture was started by inserting an insert well cultured with Caco-2 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 Ω㎠ on day 21, and the IF1, IF2, and IF3 groups of Preparation Example 3 were incubated at a concentration of 1 mg/mL on the 21st day of culture. and DMEM low glucose medium was dispensed into the normal control group (Con) and 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 the normal control group (Con) and cultured for 24 hours.

Table 7 below shows the percentage change in TEER value of each experimental group compared to the normal control group (Con).

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

In the negative control group (LPS), TEER was significantly decreased compared to the normal control group (Con) because the monolayer collapsed due to inflammation and the TEER value was lowered. The existing formula (IF1) showed a tendency to increase TEER compared to the negative control group (LPS), but there was no significant difference, and in the IF2 and IF3 groups mixed with human milk oligosaccharides, TEER was significantly higher than the negative control group (LPS). An increase was confirmed.

2. Confirmation of the effect of suppressing the expression of inflammatory factors

To confirm whether each powdered milk product suppresses LPS-induced inflammation, co-cultured Caco-2 was used to check the effect of suppressing the expression of inflammatory factors TNF-α, IL-6, iNOS, COX-2, and IL-8. This is shown in Table 8 below. To use qPCR, the cell membrane was broken using trizol and mRNA was extracted. Afterwards, the amount of extracted mRNA was quantified in equal amounts using nanodrop, and then cDNA was synthesized using the 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 inhibition 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 1.00 ^ab ±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 1.13 ^a ±0.26 0.86 ^a ±0.04 IF2 ^0.61c ±0.04 ^0.70c ±0.01 ^0.56c ±0.03 ^0.84c ±0.05 0.59 ^c ±0.04 IF3 ^0.56c ±0.01 0.68 ^c ±0.02 0.56 ^c ±0.05 0.87 ^AC ±0.09 0.57 ^c ±0.04

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

The conventional formula group (IF1), which did not contain the human milk oligosaccharide mixture, significantly reduced the expression of all inflammatory factors, including TNF-α, IL-6, iNOS, COX-2, and IL-8, compared to the negative control group (LPS). 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. Confirmation of intestinal barrier protection effect

To confirm the effect of protecting the intestinal barrier, the expression of tight junction factors (Claudin-1, Occludin, and ZO-1) in co-cultured Caco-2 was confirmed by qPCR and is shown in Table 9 below. The expression inhibition 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 Claudine-1 Occludin ZO-1 Con 1.00 ^ab ±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

Occludin expression was significantly increased in the negative control group (LPS) compared to the normal control group (Con), and there was no significant difference in Claudin-1 and ZO-1, but it showed an increasing trend.

The existing formula group (IF1), which did not contain the human milk oligosaccharide mixture, significantly increased Claudin-1, significantly decreased Occludin, and had no effect on ZO-1 compared to the negative control group (LPS). 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 protection effect was excellent. did.

Statistical processing of the above experimental examples was performed using 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.

Below, a formulation example of a composition containing the human milk oligosaccharide mixture of the present invention is described, but the present invention is not intended to be limited, but merely explained in detail.

Formulation Example 1. Preparation of powder

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

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled into an airtight bubble to prepare a powder.

Formulation Example 2. Preparation of tablets

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

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are manufactured by compressing them according to a typical tablet manufacturing method.

Formulation Example 3. Preparation of capsules

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

Crystalline cellulose 3 mg

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 typical capsule manufacturing method.

Formulation Example 4. Preparation of liquid formulation

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

10 g isomerized sugar

5 g mannitol

Proper amount of purified water

According to the usual liquid preparation method, add and dissolve each ingredient in purified water, add an appropriate amount of lemon flavor, mix the above ingredients, add purified water, adjust the total to 100g by adding purified water, and then fill it in a brown bottle and sterilize it. to produce a liquid.

Formulation Example 5. Preparation of granules

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

Vitamin mixture dosage

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 ㎍

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

Folic acid 50 μg

Calcium pantothenate 0.5 mg

Mineral mixture appropriate amount

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Monobasic Potassium Phosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

The composition ratio of the above vitamin and mineral mixture is a mixture of components relatively suitable for granules in a preferred embodiment, but the mixing ratio may be modified arbitrarily. The above components are mixed according to a typical granule manufacturing method, and then the granules are mixed. It can be prepared and used to manufacture 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 oligosaccharides

2 g plum concentrate

1 g taurine

Add purified water to make a total of 900 ml.

After mixing the above ingredients according to a typical health drink manufacturing method, stirring and heating at 85°C for about 1 hour, the resulting solution was filtered, placed in a sterilized 2 L container, sealed, sterilized, stored in the refrigerator, and then refrigerated. Used for manufacturing the functional beverage composition of the invention.

The composition ratio is a preferred embodiment of mixing ingredients relatively suitable for beverages of preference, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as demand class, country of demand, and intended use.

Claims (33)

Contains a mixture of human milk oligosaccharides containing galacto-oligosaccharides and 2'-fucosyllactose as an active ingredient,
The galactooligosaccharide and 2'-foucault room lactose are in a weight ratio of 60:40 to 85:15,
The galactooligosaccharide is a food composition for improving intestinal immunity, characterized in that it contains 10-20% by weight of 4'-galactosyllactose and 0.05-0.5% by weight of 6'-galactosyllactose based on total galactooligosaccharides. delete delete According to paragraph 1,
A food composition for improving intestinal immunity, characterized in that the breast milk oligosaccharide mixture further contains sialyllactose. According to paragraph 4,
A food composition for improving intestinal immunity, characterized in that the sialyllactose contains 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 paragraphs 1, 4, and 5,
The composition is a food composition for improving intestinal immunity, characterized in that it increases the content of short chain fatty acid in the intestine. According to clause 6,
A food composition for improving intestinal immunity, wherein the short-chain fatty acid is at least one selected from acetic acid, butyric acid, and propionic acid. According to any one of paragraphs 1, 4, and 5,
The composition is a food composition for improving intestinal immunity, characterized in that it promotes the proliferation or growth of beneficial intestinal bacteria or inhibits the proliferation or growth of harmful intestinal bacteria. According to any one of paragraphs 1, 4, and 5,
A food composition for improving intestinal immunity, characterized in that the composition improves bowel function. According to any one of paragraphs 1, 4, and 5,
A food composition for improving intestinal immunity, characterized in that the composition inhibits the secretion of TNF-α. Contains a mixture of human milk oligosaccharides containing galacto-oligosaccharides and 2'-fucosyllactose as an active ingredient,
The galactooligosaccharide and 2'-foucault room lactose are in a weight ratio of 60:40 to 85:15,
The galactooligosaccharide is a food composition for preventing or improving intestinal inflammation, characterized in that it contains 10-20% by weight of 4'-galactosyllactose and 0.05-0.5% by weight of 6'-galactosyllactose based on total galacto-oligosaccharides. . delete delete According to clause 11,
A food composition for preventing or improving intestinal inflammation, characterized in that the breast milk oligosaccharide mixture further contains sialyllactose. According to clause 14,
A food composition for preventing or improving intestinal inflammation, characterized in that the sialyllactose contains 1-10 parts by weight based on 100 parts by weight of the total of the galactooligosaccharide and 2'-fucosyllactose. According to any one of claims 11, 14 and 15,
The composition is a food composition for preventing or improving intestinal inflammation, characterized in that it increases the content of short chain fatty acids in the intestine. According to clause 16,
A food composition for preventing or improving intestinal inflammation, wherein the short-chain fatty acid is at least one selected from acetic acid, butyric acid, and propionic acid. According to any one of claims 11, 14 and 15,
The composition is a food composition for preventing or improving intestinal inflammation, characterized in that it promotes the proliferation or growth of beneficial intestinal bacteria or inhibits the proliferation or growth of harmful intestinal bacteria. According to any one of claims 11, 14 and 15,
The composition is a food composition for preventing or improving intestinal inflammation, characterized in that it improves bowel function. According to any one of claims 11, 14 and 15,
The composition is a food composition for preventing or improving intestinal inflammation, characterized in that it inhibits the secretion of TNF-α. Contains a mixture of human milk oligosaccharides containing galacto-oligosaccharides and 2'-fucosyllactose as an active ingredient,
The galactooligosaccharide and 2'-foucault room lactose are in a weight ratio of 60:40 to 85:15,
The galactooligosaccharide is a pharmaceutical for treating or preventing inflammatory bowel disease, characterized in that it contains 10-20% by weight of 4'-galactosyllactose and 0.05-0.5% by weight of 6'-galactosyllactose based on total galactooligosaccharides. Composition. delete delete According to clause 21,
A pharmaceutical composition for treating or preventing inflammatory bowel disease, characterized in that the breast milk oligosaccharide mixture further contains sialyllactose. According to clause 24,
A pharmaceutical composition for treating or preventing inflammatory bowel disease, characterized in that the sialyllactose contains 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 paragraphs 21, 24 and 25,
The composition is a pharmaceutical composition for treating or preventing inflammatory bowel disease, characterized in that it increases the content of short chain fatty acid in the intestine. According to clause 26,
A pharmaceutical composition for treating or preventing inflammatory bowel disease, wherein the short-chain fatty acid is at least one selected from acetic acid, butyric acid, and propionic acid. According to any one of paragraphs 21, 24 and 25,
The composition is a pharmaceutical composition for treating or preventing inflammatory bowel disease, characterized in that it promotes the proliferation or growth of beneficial intestinal bacteria or inhibits the proliferation or growth of harmful intestinal bacteria. According to any one of paragraphs 21, 24 and 25,
The composition is a pharmaceutical composition for treating or preventing inflammatory bowel disease, characterized in that it improves bowel function. According to any one of paragraphs 21, 24 and 25,
The composition is a pharmaceutical composition for treating or preventing inflammatory bowel disease, characterized in that it inhibits the secretion of TNF-α. Infants and children comprising the food composition for improving intestinal immunity according to any one of claims 1, 4, and 5, or the food composition for improving intestinal inflammation according to any one of claims 11, 14 to 20. For food composition. The food composition for infants and young children according to claim 31, wherein the food composition for infants and young children is infant formula, infant formula, growth formula, or baby food. The food composition for improving intestinal immunity according to any one of claims 1, 4, and 5, or breast milk containing the food composition for improving intestinal inflammation according to any one of claims 11, 14 to 20. Supplement composition.