KR20230083423A - Human Milk Oligosaccharides for Relieved Intestinal Inflammation and Regulated Gut Microbiome, and Use thereof - Google Patents

Abstract

The present invention is a type of human milk oligosaccharide that is naturally present in breast milk, which helps the proliferation of beneficial bacteria among many commercially available human milk oligosaccharides, and as a result of animal testing, exhibits functionality as a prebiotic by contributing to beneficial changes in the intestinal microbiome, and is expected to contribute to the intestinal health of not only infants and young children who consume formula milk instead of breast milk, but also middle-aged to elderly people with chronic intestinal inflammation.

Description

Human Milk Oligosaccharides for Relieved Intestinal Inflammation and Regulated Gut Microbiome, and Use thereof}

In the present invention, alpha-L-fucopyranosyl-(1→3)-beta-D-galactopyranosyl-(1→4)-di-glucose (α-L-Fucopyranosyl-(1→3)-β- D-galactopyranosyl-(1→4)-D-glucose, hereafter referred to as 2'-FL) and O-N-acetyl-alpha-neuraminyl-(2→6)-O-beta-di-galactopyranosyl- (1→4)-D-glucose monosodium salt (O-(N-acetyl-α-neuraminyl)-(2→6)-O-β-D-galactopyranosyl-(1→4)-D-glucose monosodium salt , Hereinafter, a prebiotics composition comprising 6'-SL) is provided.

Human Milk Oligosaccharides (HMOs) are major physiologically active components contained in breast milk, and are the third most abundant (5 to 15 g/L) component after lactose and lipid. Although there are differences in the components of human milk oligosaccharides according to acquired factors such as genetic factors and nutritional status of nursing mothers, in general, 2'-Fucosyllactose (2'-FL) is the most abundant and 3' -FL, LNT, 6'-SL, and other 5 monosaccharides (glucose, galactose, fucose, acetylglucosamine, acetylneuraminic acid) are combined, and about 200 or more types of human milk oligosaccharides exist.

They are not decomposed by digestive enzymes in the human body, reach the intestine, and then are decomposed into monosaccharides by beneficial bacteria and used as food for their growth. On the other hand, harmful bacteria are not able to use them, so a prebiotic effect in which proliferation is inhibited appears. In addition, since it has a structure similar to the receptor of the intestinal mucosa, it serves to prevent harmful bacteria from penetrating into the intestine by easily binding and exporting harmful bacteria.

Overseas, studies on the functionality of human milk oligosaccharides have already been actively conducted, and new functionalities for human milk oligosaccharides are being revealed one after another. Until now, it has been found that human milk oligosaccharide leads to positive changes in the intestinal microflora through the promotion of beneficial bacteria growth in the intestine and inhibition of harmful bacteria growth, and has the effect of improving immunity.

As the term 'breast milk oligosaccharides' suggests, HMOs are oligosaccharide components unique to human milk that are not found in cow's milk, which is one of the reasons why it is difficult for milk to completely replace breast milk. For this reason, global formula milk manufacturers have been releasing products containing HMOs such as 2'-FL, but in Korea, only powder formula containing general oligosaccharides such as GOS and FOS is still sold. Therefore, infants and young children who consume formula milk are likely to be deprived of the health benefits derived from HMOs.

Although the intestine of newborns is sterile at birth, it is known that the proportion of Bifidobacterium increases in the intestines of breastfed infants and the proliferation of harmful bacteria such as E.coli and C.difficile is suppressed. Thanks to this, the probability of developing necrotizing colitis is reduced, and the key mechanism that makes this possible is the proliferation of beneficial bacteria of the genus Bifidobacterium, especially B. infantis strains, by human milk oligosaccharides.

As such, the need for functional research on a composition in which substances (components) that help propagate beneficial bacteria are selected from among various HMOs present in breast milk and mixed in an optimal ratio is emerging.

Korean Registered Patent No. 10-2029263 Korean Patent Publication No. 10-2019-0060331 Korean Patent Publication No. 10-2021-0057748

Mark A. Underwood, et al., Pediatr Res., 77(0):229-235, 2015 Matsuki T. et al., Nat Commun 7, 11939, 2016 Kunz, C., & Rudloff, S., International Dairy Journal, 16(11):1341-1346, 2006 Barile, D., & Rastall, R. A., Current opinion in biotechnology, 24(2):214-219, 2013 Bode, L. Nutrition reviews, 67(suppl_2):S183-S191, 2009 Wang, C. et al., Molecular nutrition & food research, 1900262, 2019 Wang, Y. et al., Nutrients, 12(5):1284, 2020 Haas, Kelly Nicole, and Jeffrey L Blanchard, International journal of systematic and evolutionary microbiology, 67(2): 402-410, 2017 Moschen AR et al., Cell Host Microbe, 13;19(4):455-69, 2016 Xu, Y. et al., Front Microbiol. 11:219, 2020 Chambers, ES. et al., Curr Nutr Rep., 7(4):198-206, 2018

The present invention is intended to provide a prebiotics composition containing human milk-derived oligocarides as a prebiotics composition that promotes the growth of beneficial bacteria in the intestine and relieves inflammation.

In order to achieve the above object, the following solutions are provided.

In the oligosaccharide complex of the present invention, alpha-L-fucopyranosyl-(1→3)-beta-di-galactopyranosyl-(1→4)-di-glucose (α-L-Fucopyranosyl-(1→ 3) -β-D-galactopyranosyl-(1→4)-D-glucose, 2'-FL); and O-N-acetyl-alpha-neuraminyl-(2→6)-O-beta-di-galactopyranosyl-(1→4)-di-glucose monosodium salt (O-(N-acetyl- α-neuraminyl)-(2→6)-O-β-D-galactopyranosyl-(1→4)-D-glucose monosodium salt, 6′-SL); Provides an oligosaccharide complex comprising a.

In one aspect of the present invention, the 2'-FL and 6'-SL are each independently milk-derived oligosaccharides, and the oligosaccharide complex provides an oligosaccharide complex having an intestinal inflammation relief or microbiome regulating effect.

In one aspect of the present invention, the 6'-SL and 2'-FL are included in a weight ratio of 1: 3 to 7, providing an oligosaccharide complex.

Another aspect of the present invention is a prebiotics composition, wherein the composition is alpha-L-fucopyranosyl-(1→3)-beta-di-galactopyranosyl-(1→4)-di-glucose (α -L-Fucopyranosyl-(1→3)-β-D-galactopyranosyl-(1→4)-D-glucose, 2′-FL); and O-N-acetyl-alpha-neuraminyl-(2→6)-O-beta-di-galactopyranosyl-(1→4)-di-glucose monosodium salt (O-(N-acetyl- α-neuraminyl) -(2→6)-O-β-D-galactopyranosyl-(1→4)-D-glucose monosodium salt, 6'-SL); SL provides a prebiotics composition, each independently being an oligosaccharide derived from human milk.

In another aspect of the present invention, the 6'-SL and 2'-FL are included in a weight ratio of 1: 3 to 7, providing a prebiotics composition.

In another aspect of the present invention, the composition provides a prebiotics composition administered to a subject at 0.10 mg/g/day to 5.00 mg/g/day.

In another aspect of the present invention, the prebiotics composition provides a prebiotics composition for relieving intestinal inflammation or regulating the intestinal microbiome.

Another aspect of the present invention is a pharmaceutical composition for preventing, reducing or treating inflammation in the intestine, wherein the composition is alpha-L-fucopyranosyl-(1→3)-beta-D-galactopyranosyl-(1→ 4)-di-glucose (α-L-Fucopyranosyl-(1→3)-β-D-galactopyranosyl-(1→4)-D-glucose, 2′-FL); and O-N-acetyl-alpha-neuraminyl-(2→6)-O-beta-di-galactopyranosyl-(1→4)-di-glucose monosodium salt (O-(N-acetyl- α-neuraminyl)-(2→6)-O-β-D-galactopyranosyl-(1→4)-D-glucose monosodium salt, 6′-SL); Including, wherein the 2'-FL and 6'-SL are each independently milk-derived oligosaccharides, and the 6'-SL and 2'-FL are included in a weight ratio of 1: 3 to 7, preventing inflammation in the intestine, A pharmaceutical composition for alleviation or treatment is provided.

The composition according to one aspect of the present invention, milk powder and beverages prepared including the composition are helpful in improving the intestinal health of consumers.

The composition according to one aspect of the present invention, the milk powder and beverages prepared therefrom, are prebiotics, regulating the microbiome in the intestines of consumers; It helps to increase beneficial bacteria and suppress harmful bacteria.

The composition according to one aspect of the present invention, the milk powder and beverage prepared by including the same, are helpful in alleviating the inflammatory markers in the intestines of consumers.

The effects of the present invention are not limited to the effects mentioned above. It should be understood that the effects of the present invention include all effects that can be inferred from the following description.

Figure 1 is the result of measuring the growth of four strains according to the composition of each ratio (5: 1, 1: 1, 1: 5) of 2'-FL and 6'-SL.
Figure 2 is the result of measuring the expression level of the tight junction protein ZO-1 gene after treating the human milk oligosaccharide in enterocytes.
Figure 3 is the result of measuring the expression level of the tight junction protein Claudin gene after treating the human milk oligosaccharide in enterocytes.
Figure 4 is the result of measuring the expression level of the inflammatory Cytokine CD-14 gene after processing the human milk oligosaccharide in enterocytes.
Figure 5 is the result of measuring the amount of short-chain fatty acids in feces at 2 weeks after administration of human milk oligosaccharides to rats inducing inflammation.
6 is a microscopic observation result of H&E-stained colonic mucosa after administration of human milk oligosaccharide to mice inducing inflammation.
7 is a result of measuring the expression level of the ZO-1 gene, a close junction protein of the large intestine, after administration of human milk oligosaccharides to mice inducing inflammation.
8 is a result of measuring the expression level of the Occludin gene, a close junction protein of the small intestine, after administration of human milk oligosaccharides to mice inducing inflammation.
Figure 9 is a result of measuring the expression level of the inflammatory Cytokine IL-1b gene in the small intestine after administering human milk oligosaccharide to the mouse inducing inflammation.
Figure 10 is the result of measuring the expression level of the inflammatory Cytokine IL-6 gene in the small intestine after administering human milk oligosaccharide to mice inducing inflammation.
11 is a result of measuring the expression level of the inflammatory Cytokine COX-2 gene in the small intestine after administration of human milk oligosaccharides to mice inducing inflammation.
Figure 12 is the result of measuring the length of the intestine after sacrificing after administration of human milk oligosaccharide at each concentration to the rat inducing inflammation.
Figure 13 is the result of measuring the weight of the cecum after sacrificing after administration of human milk oligosaccharide by concentration to the rats inducing inflammation.
14 is a result of measuring the concentration of Glucose in the serum after administration of human oligosaccharide by concentration to rats inducing inflammation.
15 is a result of measuring triglyceride concentration in serum after administration of human milk oligosaccharide by concentration to rats inducing inflammation.
16 is a microscope observation result of H&E-stained colonic mucosa after administration of human milk oligosaccharide at various concentrations to mice inducing inflammation.
17 is a microscopic observation result of small intestine mucosa stained with H&E after administration of human milk oligosaccharide at various concentrations to mice inducing inflammation.
18 is a result of measuring the expression level of the ZO-1 gene, a close junction protein in the large intestine, after administration of human milk oligosaccharides at different concentrations to mice inducing inflammation.
19 is a result of measuring the expression level of Claudin4 gene, a close junction protein in the large intestine, after administration of human milk oligosaccharide at different concentrations to mice inducing inflammation.
Figure 20 is the result of measuring the expression level of the inflammatory Cytokine IL-8 gene in the large intestine after administration of human milk oligosaccharide by concentration to mice inducing inflammation.
Figure 21 is the result of measuring the expression level of the inflammatory Cytokine TNF-alpha gene in the large intestine after administration of human milk oligosaccharide by concentration to mice inducing inflammation.
Figure 22 is the result of measuring the expression level of the anti-inflammatory factor eNOS gene in the large intestine after administration of human milk oligosaccharide by concentration to mice inducing inflammation.
Figure 23 is the result of measuring the expression level of the inflammatory Cytokine IL-6 gene in the small intestine after administration of human milk oligosaccharide by concentration to mice inducing inflammation.
Figure 24 is the result of measuring the expression level of the inflammatory Cytokine IL-8 gene in the small intestine after administration of human milk oligosaccharide by concentration to mice inducing inflammation.
Figure 25 is the result of measuring the expression level of the anti-inflammatory Cytokine IL-10 gene in the small intestine after administration of human milk oligosaccharide by concentration to mice inducing inflammation.
26 is a result of measuring the amount of short-chain fatty acids in feces after administering human milk oligosaccharide by concentration to rats inducing inflammation.
27 is a diagram showing the NGS analysis process in order.
28 is a result of phylum level obtained by performing intestinal flora analysis of feces after administration of human milk oligosaccharide by concentration to rats inducing inflammation.
Figure 29 is the result of family level obtained by performing intestinal flora analysis of feces after administration of human milk oligosaccharide by concentration to rats inducing inflammation.
30 to 31 to 32 to 33 are measurement results for each family obtained by administering human milk oligosaccharide by concentration to rats inducing inflammation and then performing intestinal flora analysis of feces.
Figure 34 is the result of the genus level obtained by performing the intestinal flora analysis of feces after administration of human milk oligosaccharide by concentration to the rat inducing inflammation.
35 to 36 to 37 to 38 are measurement results for each genus obtained by administering human milk oligosaccharide at different concentrations to rats inducing inflammation and then performing intestinal flora analysis of feces.
39 is a result of confirming diversity (Inverse Simpson) by performing intestinal flora analysis of feces after administration of human milk oligosaccharides at different concentrations to mice inducing inflammation.

Unless otherwise specified, all numbers, values and/or expressions expressing components, reaction conditions, or amounts of components used herein are intended to indicate that such numbers, among other things, are inherently different from the measurement taken to obtain such values. Since these are approximations that reflect uncertainty, they should be understood as being qualified by the term "about" in all cases. Also, when numerical ranges are disclosed herein, such ranges are contiguous and include all values from the minimum value of such range to the maximum value inclusive, unless otherwise indicated. Furthermore, where such ranges refer to integers, all integers from the minimum value to the maximum value inclusive are included unless otherwise indicated.

In this specification, where ranges are stated for a variable, it will be understood that the variable includes all values within the stated range inclusive of the stated endpoints of the range. For example, a range of "5 to 10" includes values of 5, 6, 7, 8, 9, and 10, as well as any subrange of 6 to 10, 7 to 10, 6 to 9, 7 to 9, and the like. inclusive, as well as any value between integers that fall within the scope of the stated range, such as 5.5, 6.5, 7.5, 5.5 to 8.5 and 6.5 to 9, and the like. Also, for example, the range of "10% to 30%" includes values such as 10%, 11%, 12%, 13%, etc., and all integers up to and including 30%, as well as values from 10% to 15%, 12% to 12%, etc. It will be understood to include any sub-range, such as 18%, 20% to 30%, and the like, as well as any value between reasonable integers within the scope of the stated range, such as 10.5%, 15.5%, 25.5%, and the like.

Hereinafter, the present invention will be described in detail.

The present invention aims to provide a prebiotics composition containing human milk-derived oligocarides as a prebiotics composition that promotes the growth of beneficial bacteria in the intestine and relieves inflammation. To achieve the above object, the following solutions are provided. do. In the present invention, alpha-L-fucopyranosyl-(1→3)-beta-D-galactopyranosyl-(1→4)-di-glucose (α-L-Fucopyranosyl-(1→3)-β- D-galactopyranosyl-(1→4)-D-glucose, hereafter 2'-FL) and O-N-acetyl-alpha-neuraminyl-(2→6)-O-beta-μ-galactopyranosyl- (1→4)-D-glucose monosodium salt (O-(N-acetyl-α-neuraminyl)-(2→6)-O-β-D-galactopyranosyl-(1→4)-D-glucose, monosodium A prebiotics composition containing a salt, hereinafter 6'-SL) is provided.

Various aspects of the present invention are described below.

In the oligosaccharide complex of the present invention, alpha-L-fucopyranosyl-(1→3)-beta-di-galactopyranosyl-(1→4)-di-glucose (α-L-Fucopyranosyl-(1→ 3) -β-D-galactopyranosyl-(1→4)-D-glucose, 2'-FL); and O-N-acetyl-alpha-neuraminyl-(2→6)-O-beta-di-galactopyranosyl-(1→4)-di-glucose monosodium salt (O-(N-acetyl- α-neuraminyl)-(2→6)-O-β-D-galactopyranosyl-(1→4)-D-glucose monosodium salt, 6′-SL); Provides an oligosaccharide complex comprising a.

In one aspect of the present invention, the 2'-FL and 6'-SL are each independently milk-derived oligosaccharides, and the oligosaccharide complex provides an oligosaccharide complex having an intestinal inflammation relief or microbiome regulating effect.

In one embodiment, the oligosaccharide complex relates to a method for treatment or prophylactic treatment of a disorder related to a disturbed composition or function of the intestinal microbiome, the disorder being metabolic disorder and intestinal barrier dysfunction. (intestinal barrier dysfunction), the method comprising orally administering a prebiotic composition to a subject.

In one aspect of the present invention, the 6'-SL and 2'-FL are included in a weight ratio of 1: 3 to 7, providing an oligosaccharide complex.

In one embodiment, the oligosaccharide complex may be provided in the form of a composition, and the oligosaccharide complex may be included in an amount of 0.1 to 100% based on the total weight of the composition. In the above aspect, the composition may be a health food composition or a pharmaceutical composition. The dosage form of the health food composition according to one aspect of the present invention is not particularly limited, but may be formulated into, for example, tablets, granules, powders, liquids such as drinks, caramels, gels, bars, and the like. Food compositions include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective It may include a colloidal thickener, a pH adjusting agent, a stabilizer, a preservative, glycerin, alcohol, a carbonating agent used in carbonated beverages, and the like. The food composition of each formulation can be selected and blended without difficulty by a person skilled in the art according to the formulation or use purpose other than the active ingredient, and synergistic effect when applied simultaneously with other raw materials, especially probiotics-containing compositions. can happen In the pharmaceutical composition of one aspect of the present invention, the pharmaceutical composition may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, soft or hard capsules, etc. These solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose in one or more compounds. Alternatively, it is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. In terms of the above, the composition may be administered parenterally or orally as desired, and may be divided into 1 to several times to be administered at 0.01 to 2000 mg/kg/day. The dosage for a specific patient may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of the disease, and the like. The pharmaceutical composition according to one aspect of the present invention, according to a conventional method, including oral formulations such as powders, granules, tablets, soft or hard capsules, suspensions, emulsions, syrups, aerosols, injections and sterile injection solutions, etc. It can be formulated and used in any form suitable for pharmaceutical preparations.

Another aspect of the present invention is a prebiotics composition, wherein the composition is alpha-L-fucopyranosyl-(1→3)-beta-di-galactopyranosyl-(1→4)-di-glucose (α -L-Fucopyranosyl-(1→3)-β-D-galactopyranosyl-(1→4)-D-glucose, 2′-FL); and O-N-acetyl-alpha-neuraminyl-(2→6)-O-beta-di-galactopyranosyl-(1→4)-di-glucose monosodium salt (O-(N-acetyl- α-neuraminyl) -(2→6)-O-β-D-galactopyranosyl-(1→4)-D-glucose monosodium salt, 6'-SL); SL provides a prebiotics composition, each independently being an oligosaccharide derived from human milk.

In another aspect of the present invention, the 6'-SL and 2'-FL are included in a weight ratio of 1: 3 to 7, providing a prebiotics composition. When the above numerical range is satisfied, the effect of improving intestinal health, relieving inflammation in the intestine, and/or regulating microbiome is excellent.

In another aspect of the present invention, the composition provides a prebiotics composition administered to a subject at 0.10 mg/g/day to 5.00 mg/g/day. When the above numerical range is satisfied, the effect of improving intestinal health, relieving inflammation in the intestine, and/or regulating microbiome is excellent.

In another aspect of the present invention, the prebiotics composition provides a prebiotics composition for relieving intestinal inflammation or regulating the intestinal microbiome.

In one embodiment, the composition can promote the proliferation or growth of beneficial bacteria in the intestine, or inhibit the proliferation or growth of harmful bacteria in the intestine. In the present specification, intestinal beneficial bacteria may collectively refer to microorganisms that inhabit the intestines and exert beneficial effects on the human body. For example, intestinal beneficial bacteria may include probiotics. For example, the intestinal beneficial bacteria are not limited, but Bifidobacterium , Lactobacillus , Lactococcus , Streptococcus , Akkermansia , and Faecalibacterium , or Enterococcus . On the other hand, in the present specification, intestinal harmful bacteria may be a general term for microorganisms that live in the intestine and exert harmful effects on the human body, such as enteritis. For example, the intestinal harmful bacteria may include, but are not limited to, Escherichia coli, Fusobacterium , Clostridium , or Porphyromonas .

Another aspect of the present invention is a pharmaceutical composition for preventing, reducing or treating inflammation in the intestine, wherein the composition is alpha-L-fucopyranosyl-(1→3)-beta-D-galactopyranosyl-(1→ 4)-di-glucose (α-L-Fucopyranosyl-(1→3)-β-D-galactopyranosyl-(1→4)-D-glucose, 2′-FL); and O-N-acetyl-alpha-neuraminyl-(2→6)-O-beta-di-galactopyranosyl-(1→4)-di-glucose monosodium salt (O-(N-acetyl- α-neuraminyl)-(2→6)-O-β-D-galactopyranosyl-(1→4)-D-glucose monosodium salt, 6′-SL); Including, wherein the 2'-FL and 6'-SL are each independently milk-derived oligosaccharides, and the 6'-SL and 2'-FL are included in a weight ratio of 1: 3 to 7, preventing inflammation in the intestine, A pharmaceutical composition for alleviation or treatment is provided. When the above numerical range is satisfied, the effect of improving intestinal health, relieving inflammation in the intestine, and/or regulating microbiome is excellent.

Hereinafter, the present invention will be described in more detail through specific manufacturing examples. The following preparation examples are merely examples to aid understanding of the present invention, and the scope of the present invention is not limited thereto.

Experimental Example 1. HMOs bacterial growth test

1) Subculture

606, 9595, 5273, and 5310 stock-made bacteria (600 μl of bacteria, 360 μl of glycerol) were dispensed into each MRS Broth medium (10 ml) and cultured in a 37° C. incubator for 18 hours. After 18 hours, 100 μl of the cultured medium was added to 10 ml of fresh MRS Broth medium and passaged (second passage). After 18 hours, the activity of the bacteria was made suitable for the experiment by proceeding to the third passage in the same manner.

In order to confirm that beneficial bacteria can use 2'-FL and 6'-SL as carbon sources, the composition of MRS medium containing Glucose as a carbon source is modified (carbon source is removed), and the composition of m (modified) MRS medium is shown in Table 1 below. shown in

ingredient name g/L Peptone 10 Beef Extract 10 Yeast Extract 5 Tri-ammoniumcitrate 2 Sodium acetate 3 Magnesium sulphate heptahydrate 0.1 Manganese sulphate monohydrate 0.038 Dipotassium phosphate 2 Tween 80 1ml

After preparing 350 ml of mMRS at the same ratio as above, autoclave was performed (121 degrees, 15 minutes). After subdividing 48ml per bottle into 7 previously sterilized 250ml glass bottles, dissolving sugar (total 960mg) in 2ml D.W., filtering using a 0.22μ sterilization filter, subdividing 12ml into 50ml conical tubes, and Glucose as shown in Table 2 below. and HMOs. Among various HMOs, 2'-FL, which is present in the highest proportion in breast milk, and sialyllactose, which are contained in colostrum and are known to have effects such as improving brain development and cognition, inhibiting bacterial infection and autoimmunity, and activating intestinal immunity, are the most It was intended to reveal the effect of each of the 6'-SLs present in a large ratio and the composition in which they were mixed in various ratios on the growth of the strain.

Bacteria Group Treatment(/12ml) 1. Lactobacillus acidophilus
606
2. Lacticaseibacillus rhamnosus
ATCC 9595
3. Lactiplantibacillus plantarum
5273
4. Lactiplantibacillus plantarum
5310 Positive Control Glucose 240mg 2'-FL 2'-FL 240mg 6'-SL 6'-SL 240mg 2'-FL : 6'-SL = 1:5 2'-FL 200mg, 6'-SL 40mg 2'-FL : 6'-SL = 1:1 2'-FL 120mg, 6'-SL 120mg 2'-FL : 6'-SL = 1:1 2'-FL 40mg, 6'-SL 200mg

After centrifugation of the 4 strains that had undergone the third passage in MRS Broth medium, the supernatant was removed and washed 2-3 times with PBS (Phosphate-Buffered Saline). After that, the bacterial pellet was dispersed again in PBS, and 1% of the bacteria was dispensed in each medium according to _OD (Optical Density) = 0.3.

_OD and pH were measured at 0h, 24h, and 48h, and the number of bacteria grown at 0h and 48h was confirmed by the plate medium method. The number of viable cells at 48 h of the four strains is shown in FIG. 1 . Overall, it was confirmed that the growth of the strain at a ratio of 2'-FL: 6'-SL = 5: 1 was excellent.

Experimental Example 2. Induction of inflammation and experimental design in cell models

Cells used in the experiment were HT-29 (KCLB), cultured at 37°C, 5% CO ₂ , RPMI 1640 medium, and human milk oligosaccharides were dissolved in the medium and treated for 48 hours. Then, an inflammatory response was induced with 1 ug/ml Lipopolysaccharide for 16 hours. n=6 per group, treatment groups are shown in Table 3 below. At this time, the ratio of 2'-FL and 6'-SL was determined and applied as 5:1 according to the experimental results of Experimental Example 1.

group group name process One Normal - 2 LPS 1 μg/ml LPS 3 FL+LPS 1ug/ml LPS + 2mg/ml FL 4 SL+LPS 1ug/ml LPS + 0.4mg/ml SL 5 FL+SL+LPS 1ug/ml LPS + 2mg/ml FL + 0.4mg/ml SL 6 FL 2 mg/ml FL 7 SL 0.4 mg/ml S L 8 FL+SL 2 mg/ml FL + 0.4 mg/ml SL

Experimental Example 3. Tight junction protein and inflammation-related gene expression level evaluation test in cells

Analysis factors for each analysis item are shown in Table 4 below.

analysis items analysis factor Inflammation and pain transmission biomarkers IL-1β, CD14, IL-8, iNOS cell bonding ZO-1, claudin, occludin

To extract the mRNA of the prepared cells, 1ml of TRIZOL reagent was added to the plate containing the cells, scrapped, pipetted, transferred to a tube, disassembled, and allowed to stand at room temperature for 5 minutes. After that, 200 μl of Chloroform was added per 1 ml of TRIZOL reagent, stirred at high speed for 15 seconds with a Vortex Mixer, and allowed to stand at room temperature for 2 to 3 minutes. After centrifugation at 12,000 rpm and 4℃ for 15 minutes, the supernatant was separated into a new micro tube, and 500 μl of 100% Isopropanol was added thereto and allowed to stand at room temperature for 10 minutes. Centrifugation was performed again at 12,000 rpm and 4°C for 15 minutes to remove the supernatant except for the transparent RNA pellet. 200 μl of 75% ethanol diluted with DEPC was dispensed into the pellet, washed 2 to 3 times, vortexed for 5 seconds, and centrifuged at 7,500 rpm and 4℃ for 5 minutes. After removing the supernatant, the remaining pellet and liquid were dried using air on a clean bench. Then, 50 μl of the DEPC solution was added and the purity and concentration were measured with a nano drop to confirm whether the mRNA was accurately extracted. After confirming the measured concentration, DEPC was added to each other sample based on the sample having the lowest concentration to adjust the concentration, and then stored at -80 ° C.

For cDNA synthesis, a high capacity cDNA reverse transcription kit (Part#4368813, Applied biosystems), distilled water, PCR tube, and PCR equipment were prepared before the experiment, and 10 μl of the PCR mix prepared as shown in Table 5 was added per 10 μl of RNA sample. did

Component Volume (μL) Total RNA 10.0 10 X RT buffer 2.0 25 X dNTP Mix (100 mM) 0.8 10 X RT random primers 2.0 Multiscribe reverse transcriptase 1.0 Nuclease-free H20 4.2 Total Volume per RXN 10.0

PCR was performed by preparing a PCR program for 20 μl of the prepared sample as shown in Table 6 below.

Step 1 Step 2 Step 3 Step 4 Temp (℃) 25 37 85 4 Time (min) 10 120 5 hold

The PCR-performed sample was stored at 4°C because cDNA was more stable at a lower temperature than RNA.

As a third step, qRT-PCR was performed by preparing Power SYBR green PCR master mix (Cat No.436759, Applied biosystem), cDNA sample, and PCR plate (flat). The mixing ratio of reagents is shown in Table 7 below.

Component for duplex PCR setup Volume (μl) for 20 μl rxn cDNA template 4 Power SYBR green PCR master mix (2X) 7.5 Primer mix 0.75 H ₂ O 2.75 Total 15

Then, qRT-PCR was performed by entering a program as shown in Table 8 below.

Step AmpliTaq gold Enzyme activation PCR Hold Cycles (40 cycles) Denature Anneal / Extend Time 10min 15 seconds 60 seconds Temp (℃) 95 95 60

In fact, the ratios of primers and mixes used in the execution of CSP qRT-PCR are shown in Table 9 below.

Per 15 μl rxn 23S rRNA CSP SYBR green mix 7.5 μl 90 μl 90 μl Primer 0.75 μl 9 μl 9 μl DDW 2.75 μl 33 μl 33 μl cDNA template 4 μl 48 μl 48 μl

Finally, qRT-PCR was performed to confirm the difference in gene expression level of each treatment group compared to the housekeeping gene. The oligonucleotide primers used in the cell experiment are shown in Table 10 below, and the measurement results are shown in Figs. 2 to 4 was

As can be seen in Figure 2, the gene expression level of the tight junction protein ZO-1 was decreased in the LPS treatment group, and when treated with LPS and 2'-FL, LPS and 2'-FL + 6'-SL (5: 1) composition showed signs of recovery. In particular, the synergistic effect of the composition was confirmed as the expression level of tight junction proteins increased more in the 2'-FL + 6'-SL (5: 1) treated group than in the 2'-FL and 6'-SL alone treated group. .

The expression level of claudin, a tight junction protein in FIG. 3, decreased only in the LPS-treated group and increased in all experimental groups treated with HMOs. Through this, it was found that HMOs can help intestinal health by preventing the decrease in gene expression of tight junction proteins caused by LPS.

The expression level of the inflammation-related gene CD-14 shown in FIG. 4 increased in the LPS-treated group and decreased in the LPS + HMOs-treated group, confirming the effect of HMOs on improving intestinal inflammation.

Gene Sequence of primers Annealing temperature GAPDH F: 5'-ATGACCACAGTCCATGCCATC-3'R: 5'-CCTGCTTCACCACCTTCTTG-3' 65.2 Zo-1 F: 5'-CAACATACAGTGACGCTTCACA-3'
R: 5'-CACTATTGACGTTTCCCCACTC-3' 55 Claudin F: 5'-AGAGAGCCTGACCAAAATTCGT-3'
R: 5'-TCTGGCTATTTTAGTTGCCACAG-3' 64 Ocln F: 5'-ACAAGCGGTTTTATCCAGAGTC-3'R: 5'-GTCATCCACAGGCGAAGTTAAT-3' 55.6 IL-1b F: 5'-ATGCCACCTTTTGACAGTGATG-3' R: 5'-TGTCGTTGCTTGGTTCTCCT-3' 61.2 IL-6 F: 5'-GGTACATCCTCGACGGCATCT-3' R: 5'-GTGCCTCTTTGCTGCTTTCAC-3' 68.3 IL-8 F: 5'-GTTGTGAGGACATGTGGAAGCACT-3' R: 5'-CACAGCTGGCAATGACAAGACTGG-3' 66.4 CD14 F: 5'-ACGCCAGAACCTTGTGAGC-3' R: 5'-GCATGGATCTCCACCTCTACTG-3' 62 TLR4 F: 5'-GACGTGGAACTGGCAGAAGAGG-3'R: 5'-TGACGGCAGAGAGGAGGGAC-3' 67 iNOS F: 5'-ACCCAAGGTCTACGTTCAGG-3' R: 5'-CGCACATCTCCGCAAATGTA-3' 62

Experimental Example 4. Method for Inducing Inflammation in Animals and Experimental Design (1st)

group group name diet One Normal Normal feed + PBS 2 LPS Normal feed + PBS 3 LPS+HMOs General feed + FL 0.25mg/g + SL 0.05mg/g 4 HMOs General feed + FL 0.25mg/g + SL 0.05mg/g

Experimental animals (rats) were C57BL/6N (male, n=20, 3 weeks old, 15-20 g), and were raised in an environment of 23°C and 12 h light-dark cycles. Human milk oligosaccharides were mixed with 2'-FL and 6'-SL at a ratio of 5:1, diluted in PBS, and administered orally separately from feed. n = 5 per group, and experiments were conducted by dividing groups as shown in Table 11 according to the concentration of human milk oligosaccharide. The rats received human milk oligosaccharide for 2 weeks after a 1-week adaptation period.

A total of 4 experimental groups were used, and inflammation was induced by intraperitoneal injection of 1 μg/g/day of LPS (lipopolysaccharide, Sigma Chemical Co.) for 5 days before sacrifice to verify the inflammation mitigating effect of human milk oligosaccharide, which is the purpose of this experiment. The control group was injected with PBS instead of LPS in the same way.

Experimental Example 5. Measurement of SCFAs in body weight, food intake, and fecal samples

1) Measurement of body weight and food intake

Body weight and food intake were measured once a week to confirm the change in body weight before and after administration of human milk oligosaccharides and before and after the induction of LPS inflammation and the degree of effect of human oligosaccharides and LPS.

2) Measurement of SCFAs (Short-chain Fatty Acids) in feces samples

Fecal samples were collected every week when bedding was changed, and the collected fecal samples were stored in a -80°C Deep freezer until analysis.

GC-MS was used to measure SCFAs. First, a 50 mg fecal sample was homogenized with 500 μl of internal standard (100 μM crotonic acid), and then extracted with 250 μl HCL and 1 ml of a solvent such as ether. This solution was mixed well using a Vortex mixer, centrifuged at 1,000 Хg for 10 minutes, and the supernatant was transferred to a new glass vial. After mixing 400 μl of the extract supernatant and 80 μl of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA), sealing the lid, heating in a water bath at 80 ° C. for 20 minutes, and leaving it at room temperature for 48 hours. The stationary sample was measured using an HP-5MS column (0.25mm x 30m x 0.25㎛) on GC-MSD 6890/5973N (Agilent Technologies). Helium (99.9999% purity) was used as the carrier gas, which was delivered at a flow rate of 1.2ml/min. The head pressure was set to 97 kpa with a split 20:1, and the temperature of the transfer line was set to 260 °C and 250 °C. The temperature program used for analysis was 60°C for 3 minutes, 60~120°C (5°C per minute), and 120~300°C (20°C/minute). Each sample was injected by 1 μl, and analysis time was about 30 minutes per sample. The concentration of fatty acids was measured compared to the peak area of the standard solution (Volatile Free Acid Mix, Supelco, USA), and the results are shown in FIG. 5.

Short-chain fatty acids in feces are an indicator that reflects the flora of feces. Before the start of the experiment, there was no difference between groups in short-chain fatty acids, but after 2 weeks of experiment, the amount of acetate, propionate, and butyrate in the HMOs-fed group increased significantly. I was able to confirm that Total SCFA was the sum of the three SCFAs and showed a significant difference between the groups treated with LPS and HMOs.

Experimental Example 6. H&E staining of colon tissue

H&E staining was used to evaluate the occurrence of inflammation in the large intestine and small intestine, and the structures of goblet cells and gut epithelial cells were analyzed under a microscope.

In case of inflammation, the size and number of inflammatory cells were measured by taking pictures of stained tissues of the small and large intestine.

As a result of examining the morphological difference, as shown in FIG. 6, in the group treated with human milk oligosaccharide, the intestinal inflammatory parts seen in the LPS group were not seen, and the thickness of the muscle layer of the large intestine, which is the outer wall of the large intestine, also showed a significant difference and was recovered.

Experimental Example 7. Tight junction protein and inflammation-related gene expression level evaluation

To extract mRNA from the prepared tissue (small intestine, large intestine, caecum), 400 μl of TRIZOL reagent was added to the tube containing the tissue, disassembled using a homogenizer (Tissue tearor, Biospec products inc.), and allowed to stand at room temperature for 2 minutes. . After that, after putting 80 μl of Chloroform, it was stirred at high speed for 15 seconds with a Vortex Mixer and allowed to stand for 2 to 3 minutes at room temperature again. After centrifugation at 12,000 rpm and 4℃ for 15 minutes, the supernatant was separated into a new micro tube, and 240 μl of 100% Isopropanol was added thereto and allowed to stand at room temperature for 10 minutes.

Centrifugation was performed again at 12,000 rpm and 4°C for 15 minutes to remove the supernatant except for the transparent RNA pellet. 200 μl of 75% ethanol diluted with DEPC was dispensed into the pellet, washed 2 to 3 times, vortexed for 5 seconds, and centrifuged at 7,500 rpm and 4℃ for 5 minutes. After removing the supernatant, the remaining pellet and liquid were dried using air on a clean bench. Then, 50 μl of the DEPC solution was added and the purity and concentration were measured with a nano drop to confirm whether the mRNA was accurately extracted. After confirming the measured concentration, DEPC was added to each other sample based on the sample having the lowest concentration to adjust the concentration, and then stored at -80 ° C.

For cDNA synthesis, a high capacity cDNA reverse transcription kit (Part#4368813, Applied biosystems), distilled water, PCR tube, and PCR equipment were prepared before the experiment, and 10 μl of the PCR mix prepared as shown in Table 12 below was added per 10 μl of RNA sample. did

Component Volume (μl) Total RNA 10.0 10 X RT buffer 2.0 25 X dNTP Mix (100 mM) 0.8 10 X RT random primers 2.0 Multiscribe reverse transcriptase 1.0 Nuclease-free H20 4.2 Total Volume per RXN 10.0

PCR was performed by creating a PCR program for 20 μl of the prepared sample as shown in Table 13 below.

Step 1 Step 2 Step 3 Step 4 Temp (℃) 25 37 85 4 Time (min) 10 120 5 hold

The PCR-performed sample was stored at 4°C because cDNA was more stable at a lower temperature than RNA.

As a third step, qRT-PCR was performed by preparing Power SYBR green PCR master mix (Cat No.436759, Applied biosystem), cDNA sample, and PCR plate (flat). The mixing ratio of reagents is shown in Table 14 below.

Component for duplex PCR setup Volume (μl) for 20μl rxn cDNA template 2 Power SYBR green
PCR master mix (2X) 10 Home-made gene
expression assay (2X) One H ₂ O 7 Total 20

Then, qRT-PCR was performed by entering a program as shown in Table 15 below.

Step AmpliTaq gold Enzyme activation PCR Hold Cycles (40 cycles) Denature Anneal / Extend Time 10min 15 seconds 60 seconds Temp (℃) 95 95 60

In fact, the ratios of primers and mixes used in the execution of CSP qRT-PCR are shown in Table 16 below.

Per 15 μl rxn 23S rRNA CSP SYBR green mix 7.5 μl 90 μl 90 μl Primer 0.75 μl 9 μl 9 μl DDW 2.75 μl 33 μl 33 μl cDNA template 4 μl 48 μl 48 μl

Finally, qRT-PCR was performed to confirm the difference in gene expression level of each treatment group compared to the housekeeping gene. 11.

As a result of examining the mRNA expression levels of the tight junction protein ZO-1 in the large intestine (Fig. 7) and the tight junction protein Occludin in the small intestine (Fig. 8), the LPS-treated group decreased, but the HMOs-treated group recovered to a level similar to that of the normal group. appeared to be

The expression levels of the inflammatory cytokines IL-1b, IL-6, and COX-2 in the small intestine shown in FIGS. 9 to 11 were increased in the LPS-treated group, but recovered to levels similar to those of the normal group in the HMOs-treated group.

Gene Sequence of primers Annealing temperature GAPDH F: 5'-ATGACCACAGTCCATGCCATC-3'R: 5'-CCTGCTTCACCACCTTCTTG-3' 65.2 Zo-1 F: 5'-CAACATACAGTGACGCTTCACA-3'
R: 5'-CACTATTGACGTTTCCCCACTC-3' 55 Claudin F: 5'-AGAGAGCCTGACCAAAATTCGT-3'
R: 5'-TCTGGCTATTTTAGTTGCCACAG-3' 64 Ocln F: 5'-ACAAGCGGTTTTATCCAGAGTC-3'R: 5'-GTCATCCACAGGCGAAGTTAAT-3' 55.6 IL-1b F: 5'-ATGCCACCTTTTGACAGTGATG-3' R: 5'-TGTCGTTGCTTGGTTCTCCT-3' 61.2 IL-6 F: 5'-GGTACATCCTCGACGGCATCT-3' R: 5'-GTGCCTCTTTGCTGCTTTCAC-3' 68.3 IL-8 F: 5'-GTTGTGAGGACATGTGGAAGCACT-3'R: 5'-CACAGCTGGCAATGACAAGACTGG-3' 66.4 CD14 F: 5'-ACGCCAGAACCTTGTGAGC-3' R: 5'-GCATGGATCTCCACCTCTACTG-3' 62 TLR4 F: 5'-GACGTGGAACTGGCAGAAGAGG-3'R: 5'-TGACGGCAGAGAGGAGGGAC-3' 67 iNOS F: 5'-ACCCAAGGTCTACGTTCAGG-3' R: 5'-CGCACATCTCCGCAAATGTA-3' 62

Experimental Example 8. Induction of inflammation in animal models and experimental design (secondary)

Experimental animals (rats) were C57BL/6N (male, n=70, 2 weeks old), and were raised in an environment of 23° C. and 12 h light-dark cycles. Human milk oligosaccharides were mixed with 2'-FL and 6'-SL at a ratio of 5:1, diluted in PBS, and administered orally separately from feed. n = 10 per group, and the experiment was conducted by dividing the groups according to the concentration of human milk oligosaccharide as shown in Table 18 below. The rats received human milk oligosaccharide for 2 weeks after a 1-week adaptation period.

Group LPS HMOs Control PBS PBS LPS 1 μg/g (5 days) PBS ×0.5 HMOs 1 μg/g (5 days) 0.15mg/g (2’FL:6’SL=5:1) ×1 HMOs 1 μg/g (5 days) 0.3mg/g (2’FL:6’SL=5:1) ×2 HMOs 1 μg/g (5 days) 0.6mg/g (2’FL:6’SL=5:1) ×5 HMOs 1 μg/g (5 days) 1.5mg/g (2’FL:6’SL=5:1) ×10 HMOs 1 μg/g (5 days) 3.0 mg/g (2’FL:6’SL=5:1)

A total of 7 experimental groups were administered, and inflammation was induced by intraperitoneal injection of 1 μg/g/day of LPS (lipopolysaccharide, Sigma Chemical Co.) for 5 days before sacrifice in order to verify the inflammatory effect of human milk oligosaccharide, which is the purpose of this experiment. The control group was injected with PBS instead of LPS in the same way.

Experimental Example 9. Body weight, food intake, colon length, caecal weight measurement

1) Measurement of body weight and food intake

By measuring daily body weight and food intake, the weight change before and after administration of human milk oligosaccharide and before and after induction of LPS inflammation and the degree of effect of human milk oligosaccharide and LPS were confirmed.

2) Measurement of colon length and cecum weight

After sacrificing the experimental animals, changes were confirmed by measuring the length of the colon and the weight of the caecum between the LPS inflammation induction group and the HMOs treatment group. The results of measuring the length of the large intestine are shown in FIG. 12 and the results of measuring the weight of the cecum are shown in FIG. 13 .

As shown in FIG. 12, the length of the large intestine was decreased by LPS treatment and increased again in the 0.30 HMOs group, but no significant difference was found in the HMOs group with other concentrations.

The weight of the cecum shown in FIG. 13 was decreased by treatment with LPS, and a tendency for the weight to increase as the concentration of HMOs treatment increased, and a significant difference was found at high concentrations of HMOs (0.60 HMOs, 3.0 HMOs).

Experimental Example 10. Measurement of glucose and TG levels in blood samples and evaluation of expression levels of inflammation-related proteins

1) Serum glucose, TG level measurement

Serum was separated from blood collected after sacrifice, and serum glucose (MBL glucose test, MBL Co., Ltd., Gyeonggi-do) and TG level (MBL TG test, MBL Co., Ltd., Gyeonggi-do) were measured enzymatically.

Both serum glucose and TG levels tended to decrease as the treatment concentration of HMOs increased, as shown in FIGS. 14 and 15 .

2) Assessment of inflammation-related proteins

Serum was isolated from blood collected after sacrifice, and inflammatory cytokines (IL-6, TNF-alpha, IL-10) expressed in serum were measured enzymatically using a Cytokine ELISA kit (Labiskoma, Seoul).

Experimental Example 11. H&E staining of large intestine and small intestine tissues

H&E staining was used to evaluate the occurrence of inflammation in the large intestine and small intestine, and the structures of goblet cells and gut epithelial cells were analyzed under a microscope.

In case of inflammation, the size and number of inflammatory cells were measured by taking pictures of stained tissues of the small and large intestine.

As a result of examining the morphological differences in the colon tissue, it was confirmed that Immune infiltrate appeared in the LPS-treated group, and inflammation in the colon was induced through the damaged Villi-crypt structure, as shown in FIG. 16 . In the group treated with HMOs, the inflammatory parts of the intestine seen in the LPS group were not seen, and it was observed that the villi-crypt structural damage was partially restored.

Immune infiltrate was formed in the small intestine tissue shown in FIG. 17 in the LPS-treated group, and this structure was not seen in the HMOs-treated group.

Experimental Example 12. Tight junction protein (tight junction protein) and inflammation-related gene expression level evaluation

To extract mRNA from the prepared tissue (small intestine, large intestine), 400 μl of TRIZOL reagent was added to the tube containing the tissue, digested using a Homogenizer (Tissue tearor, Biospec products inc.), and allowed to stand at room temperature for 2 minutes. After that, after putting 80 μl of Chloroform, it was stirred at high speed for 15 seconds with a Vortex Mixer and allowed to stand for 2 to 3 minutes at room temperature again. After centrifugation at 12,000 rpm and 4℃ for 15 minutes, the supernatant was separated into a new micro tube, and 240 μl of 100% Isopropanol was added thereto and allowed to stand at room temperature for 10 minutes.

Centrifugation was performed again at 12,000 rpm and 4°C for 15 minutes to remove the supernatant except for the transparent RNA pellet. 200 μl of 75% ethanol diluted with DEPC was dispensed into the pellet, washed 2 to 3 times, vortexed for 5 seconds, and centrifuged at 7,500 rpm and 4℃ for 5 minutes. After removing the supernatant, the remaining pellet and liquid were dried using air on a clean bench. Then, 50 μl of the DEPC solution was added and the purity and concentration were measured with a nano drop to confirm whether the mRNA was accurately extracted. After confirming the measured concentration, DEPC was added to each other sample based on the sample having the lowest concentration to adjust the concentration, and then stored at -80 ° C.

For cDNA synthesis, high capacity cDNA reverse transcription kit (Part#4368813, Applied biosystems), distilled water, PCR tube, and PCR equipment were prepared before the experiment, and 10 μl of PCR mix prepared as shown in Table 19 below was added per 10 μl of RNA sample. did

Component Volume (μl) Total RNA 10.0 10 X RT buffer 2.0 25 X dNTP Mix (100 mM) 0.8 10 X RT random primers 2.0 Multiscribe reverse transcriptase 1.0 Nuclease-free H20 4.2 Total Volume per RXN 10.0

PCR was performed by preparing a PCR program for 20 μl of the prepared sample as shown in Table 20 below.

Step 1 Step 2 Step 3 Step 4 Temp (℃) 25 37 85 4 Time (min) 10 120 5 hold

The PCR-performed sample was stored at 4°C because cDNA was more stable at a lower temperature than RNA.

As a third step, qRT-PCR was performed by preparing Power SYBR green PCR master mix (Cat No.436759, Applied biosystem), cDNA sample, and PCR plate (flat). The mixing ratio of reagents is shown in Table 21 below.

Component for duplex PCR setup Volume (ul) for 20 ul rxn cDNA template 4 Power SYBR green
PCR master mix (2X) 7.5 Home-made gene
expression assay (2X) 0.75 H ₂ O 2.75 Total 15

Then, qRT-PCR was performed by entering a program as shown in Table 22 below.

Step AmpliTaq gold Enzyme activation PCR Hold Cycles (40 cycles) Denature Anneal / Extend Time 10min 15 seconds 60 seconds Temp (℃) 95 95 60

In fact, the ratio of primers and mixes used in the execution of CSP qRT-PCR is shown in Table 23 below.

Per 15 μl rxn 23S rRNA CSP SYBR green mix 7.5 μl 90 μl 90 μl Primer 0.75 μl 9 μl 9 μl DDW 2.75 μl 33 μl 33 μl cDNA template 4 μl 48 μl 48 μl

Finally, qRT-PCR was performed to confirm the difference in gene expression level of each treatment group compared to the housekeeping gene, and oligonucleotide primers used in animal experiments are shown in Table 24 below.

The mRNA expression levels of the tight junction proteins Claudin4 and ZO-1 in the large intestine are shown in FIGS. 18 to 19. The expression level was lowered in the LPS-treated group, and the mRNA expression level lowered by LPS was recovered in the 0.15mg/g HMOs-treated group, but no concentration-specific trend was found. The expression level of inflammatory Cytokine mRNA was increased in the LPS-treated group as shown in FIGS. 20 to 21 . As the concentration of HMOs treated increased, the expression level of inflammatory cytokines tended to recover to the level of the control group. As shown in FIG. 22 , the mRNA expression level of the anti-inflammatory factor eNOS, which was lowered in the LPS-treated group, was increased again by HMOs. It was confirmed that the expression level of eNOS was higher in the low-concentration HMOs group.

As shown in FIGS. 23 and 24 , the expression level of inflammatory cytokines in the small intestine increased in the LPS-treated group and tended to recover to the control level in the HMOs-treated group at all concentrations. As shown in FIG. 25, the expression level of the anti-inflammatory cytokine IL-10, which was lowered in the LPS-treated group, increased again in the HMOs-treated group at all concentrations.

Gene Sequence of primers Annealing temperature GAPDH F: 5′-AGGTCGGTGTGAACGGATTTG-3′ R: 5′-TGTAGACCATGTAGTTGAGGTCA-3′ 61.3 IL-6 F: 5'-TAGTCCTTCCTACCCCAATTTCC-3'
R: 5'-TTGGTCCTTAGCCACTCCTTC-3' 61.9 IL-10 F: 5'-GCTCTTACTGACTGGCATGAG-3'
R: 5'-CGCAGCTTCTAGGAGCATGTG-3' 61.9 IL-1b F: 5'-GCAACTGTTCCTGAACTCAACT-3'R: 5'-ATCTTTTGGGGTCCGTCAACT-3' 59.9 IL-8 F: 5'-TTTCCACCGGCAATGAAG-3'R: 5'-TAGAGGTCTCCCGAATTGGA-3' 59.7 TNF-alpha F: 5'-CTGAACTTCGGGGTGATCGG-3'R: 5'-GGCTTGTCACTCGAATTTTTGAGA-3' 61.9 TLR4 F: 5'-ATGGCATGGCTTACACCACC-3'R: 5'-GAGGCCAATTTTGTCTCCACA-3' 59.9 iNOS F: 5'-GTTCTCAGCCCAACAATACAAGA-3'R: 5'-GTGGACGGGTCGATGTCAC-3' 61.3 ZO-1 F: 5'-GCCGCTAAGAGCACAGCAA-3' R: 5'-TCCCCACTCTGAAAATGAGGA-3' 59.3 Claudin4 F: 5'-GTCCTGGGAATCTCCTTGGC-3' R: 5'-TCTGTGCCGTGACGATGTTG-3' 61.3 Occludin F: 5'-TTGAAAGTCCACCTCCTTACAGA-3' R: 5'-CCGGATAAAAAGAGTACGCTGG-3' 61.5 Muc2 F: 5'-AGGGCTCGGAACTCCAGAAA-3'R: 5'-CCAGGGAATCGGTAGACATCG-3' 61.9 eNOS F: 5'-TCAGCCATCACAGTGTTCCC-3'R: 5'-ATAGCCCGCATAGCGTATCAG-3' 60 TGF-beta F: 5'-TCTGCATTGCACTTATGCTGA-3'R: 5'-AAAGGGCGATCTAGTGATGGA-3' 58.5

Experimental Example 13. Intestinal flora analysis and comparison of short-chain fatty acids (SCFA) changes in fecal samples

1) Measurement of short chain fatty acids in fecal samples

Fecal samples were collected every week when bedding was changed, and the collected fecal samples were stored in a -80°C Deep freezer until analysis.

GC-MS was used to measure SCFAs. First, a 50 mg fecal sample was homogenized with 500 μl of internal standard (100 μM crotonic acid), and then extracted with 250 μl HCL and 1 ml of a solvent such as ether. This solution was mixed well using a Vortex mixer, centrifuged at 1,000 Хg for 10 minutes, and the supernatant was transferred to a new glass vial. After mixing 400 μl of the extract supernatant and 80 μl of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA), sealing the lid, heating in a water bath at 80 ° C. for 20 minutes, and leaving it at room temperature for 48 hours. The stationary sample was measured using an HP-5MS column (0.25mm x 30m x 0.25㎛) on GC-MSD 6890/5973N (Agilent Technologies). Helium (99.9999% purity) was used as the carrier gas, which was delivered at a flow rate of 1.2ml/min. The head pressure was set to 97 kpa with a split 20:1, the temperature of the moving line was set to 260 °C and the temperature of the inlet to 250 °C. The temperature program used for analysis was 60°C for 3 minutes, 60~120°C (5°C per minute), and 120~300°C (20°C/minute). Each sample was injected by 1 μl, and analysis time was about 30 minutes per sample. The concentration of fatty acids was measured compared to the peak area of the standard solution (Volatile Free Acid Mix, Supelco, USA), and the results are shown in FIG. 26 .

Short-chain fatty acids in feces are an indicator that reflects the flora of the feces. At week 1, the total amount of short-chain fatty acids (Total SCFA) tended to decrease by solid diet, and treatment with LPS recovered Total SCFA at week 2. reduced the degree. Treatment with HMOs seemed to partially suppress the effect of LPS, but no concentration-dependent trend was observed. Acetate was increased by LPS treatment at 2 weeks, and HMOs treatment tended to decrease this increase. In the high-concentration HMOs group, the amount of propionate was found to increase. Butyrate was decreased in the LPS-treated group, but the decrease tended to be alleviated in the HMOs-treated group, and significantly increased in the 2nd week of the 1.5mg/g HMOs-treated group.

2) Analysis of NGS (Next Generation Sequencing) results

The NGS process proceeded in the order shown in FIG. 27, and Fecal samples for each group were stored at -80 ° C, and NGS analysis was requested to Macrogen. Through NGS, it is possible to see the microbial flora of each group and changes in the flora by week, and it is possible to compare the flora of each group by analyzing the composition and diversity of the intestinal flora up to the Family and Genus levels.

Based on the NGS results, the contents of Phylum, Family, Genus, and Species of the flora were compared by group and parking lot. In addition to the OTUs analysis of the intestinal flora, the distribution of various microorganisms in the intestinal flora was confirmed using Simpson's index and Shannon index as indicators to statistically confirm alpha diversity between groups.

As shown in FIG. 28, at the Phylum Level, Bacteroidetes and Firmicutes were found to have a large composition ratio. The ratio of Firmicutes decreased by LPS treatment at 2 weeks, and Firmicutes increased again in the HMOs treated group. In general, it is known that the F/B (Firmicutes/Bacteroidetes) ratio decreases in inflammatory conditions such as inflammatory bowel disease (IBD).

At the family level, as shown in FIG. 29, Muribaculaceae, Rikenellaceae, Lactobacillaceae and Lachnospiraceae showed a high composition ratio. While Muribaculaceae shown in FIG. 30 decreased at 2 weeks in the LPS-treated group, it tended to increase in the high-concentration HMOs-treated group. On the other hand, the composition ratio of Rikenellaceae in FIG. 31 increased significantly only in the LPS-treated group at 2 weeks. Lachnospiraceae in FIG. 32 decreased at 2 weeks in the LPS-treated group, and showed a tendency to increase in the low-concentration HMOs-treated group. In the case of Akkermansiaceae, as shown in FIG. 33, the ratio tended to increase in the high-concentration HMOs treatment group, and showed a significant difference in the 3.0 HMOs treatment group.

At the Genus level, as shown in FIG. 34, Bacteroides, Muribaculum, Alistipes, Mucispirillum, Lactobacillus, and Kineothrix were found to be the main ones. As shown in FIG. 35 , the ratio of Muribaculum belonging to Muribaculaceae appeared to increase in the HMOs-treated group compared to the LPS-treated group, but no significant difference was observed. In the case of Alistipes in FIG. 36, the ratio was greatly increased by the treatment of LPS for 2 weeks, and this effect was not shown in the HMOs treatment group. Alistipes belongs to Bacteriodetes and is normally present at a low rate, and it has recently been found to be present at a high rate in dysbiosis and various diseases (Moschen et al. 2016). On the other hand, Kineothrix in FIG. 37 was decreased in the LPS-treated group, which tended to increase in the low-concentration HMOs-treated group. Kineothrix is a recently discovered genus that requires additional research, and Kineothrix alysoides is known to produce butyrate by fermenting sugar (Haas et al. 2017). A significant increase in Akkermansia in the 3.0 mg/g HMOs treatment group is shown in FIG. 38 . Akkermansia muciniphila , a species of Akkermansia, inhabits the intestinal mucosal layer and regulates basal metabolism, such as promoting mucin secretion (Xu et al. 2020). A.muciniphila is a short-chain fatty acid (SCFA) producer and has been reported to convert dietary fiber into acetate, propionate, and butyrate, and these metabolites can affect glucose and lipid homeostasis (Chambers et al., 2018). According to a recent study, it was observed that treatment with 2'-FL increased the proportion of SCFA-producing bacteria such as Akkermansia and Ruminococcaceae, and also increased the amount of SCFA (Wang et al. 2020).

As shown in FIG. 39, there was no difference in diversity between groups at week 0 and week 1, and it was confirmed that the diversity of the LPS group at week 2 decreased in the inverse simpson value.

As a result of comprehensively examining the intestinal microflora regulating and anti-inflammatory effects of human milk oligosaccharides using bacterial growth experiments and animal experiments, the growth of lactic acid bacteria was further promoted in a medium with a high content of 2'-FL. Human milk oligosaccharides significantly inhibited the inflammatory response induced by LPS in animal experiments. In the animal experiment, each experimental group was treated with human milk oligosaccharide in which 2'-FL and 6'-SL were mixed at a ratio of 5:1. Glucose and TG levels in serum after sacrifice were improved as the concentration of human milk oligosaccharide increased, and the expression of inflammation-related factors in the large intestine and small intestine also showed a significant inhibitory effect depending on the concentration. In feces, the effect of human milk oligosaccharide on intestinal microflora was confirmed through short-chain fatty acid analysis and NGS analysis.

Through these results, it was confirmed that the protective effect of human milk oligosaccharides against the inflammatory response induced by LPS. The higher the concentration of human milk oligosaccharide, the better the effect tended to be, but even at the minimum concentration of 0.15mg/g, the effect was not significantly different, so there would be no major problem in the effect even if a commercial product was made by selecting this concentration from an economic point of view. It is expected.

Claims (8)

In the oligosaccharide complex,
Alpha-L-Fucopyranosyl-(1→3)-beta-D-galactopyranosyl-(1→4)-D-glucose (α-L-Fucopyranosyl-(1→3)-β-D-galactopyranosyl -(1→4)-D-glucose, 2′-FL); and
O-N-acetyl-alpha-neuraminyl-(2→6)-O-beta-di-galactopyranosyl-(1→4)-di-glucose monosodium salt (O-(N-acetyl-α -neuraminyl)-(2→6)-O-β-D-galactopyranosyl-(1→4)-D-glucose monosodium salt, 6′-SL);
An oligosaccharide complex comprising a.
According to claim 1,
The 2'-FL and 6'-SL are each independently an oligosaccharide derived from human milk,
The oligosaccharide complex has an effect of relieving inflammation or regulating the microbiome in the intestine, the oligosaccharide complex.
According to claim 2,
The 6'-SL and 2'-FL are 1: contained in a weight ratio of 3 to 7, the oligosaccharide complex.
In the prebiotics composition, the composition
Alpha-L-Fucopyranosyl-(1→3)-beta-D-galactopyranosyl-(1→4)-D-glucose (α-L-Fucopyranosyl-(1→3)-β-D-galactopyranosyl -(1→4)-D-glucose, 2′-FL); and
O-N-acetyl-alpha-neuraminyl-(2→6)-O-beta-di-galactopyranosyl-(1→4)-di-glucose monosodium salt (O-(N-acetyl-α -neuraminyl)-(2→6)-O-β-D-galactopyranosyl-(1→4)-D-glucose monosodium salt, 6′-SL);
Including,
The 2'-FL and 6'-SL are each independently an oligosaccharide derived from human milk, a prebiotics composition.
According to claim 4,
The 6'-SL and 2'-FL are 1: contained in a weight ratio of 3 to 7, a prebiotics composition.
According to claim 4,
The composition is administered to the subject at 0.10 mg / g / day to 5.00 mg / g / day, a prebiotics composition.
According to claim 4,
The prebiotics composition is for relieving intestinal inflammation or regulating the intestinal microbiome, a prebiotics composition.
In the pharmaceutical composition for preventing, reducing or treating inflammation in the intestine, the composition
Alpha-L-Fucopyranosyl-(1→3)-beta-D-galactopyranosyl-(1→4)-D-glucose (α-L-Fucopyranosyl-(1→3)-β-D-galactopyranosyl -(1→4)-D-glucose, 2′-FL); and
O-N-acetyl-alpha-neuraminyl-(2→6)-O-beta-di-galactopyranosyl-(1→4)-di-glucose monosodium salt (O-(N-acetyl-α -neuraminyl)-(2→6)-O-β-D-galactopyranosyl-(1→4)-D-glucose monosodium salt, 6′-SL);
Including,
The 2'-FL and 6'-SL are each independently an oligosaccharide derived from human milk,
The 6'-SL and 2'-FL are included in a weight ratio of 1: 3 to 7,
A pharmaceutical composition for preventing, reducing or treating intestinal inflammation.

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