KR102381973B1 - Composition for improving intestinal health comprising red ginseng residue - Google Patents

Abstract

The present invention relates to a composition for improving intestinal health comprising red ginseng extract as an active ingredient, wherein the composition of the present invention containing red ginseng extract promotes the growth of beneficial intestinal bacteria, inhibits the growth of harmful bacteria, and increases the content of short-chain fatty acids in the intestine It can improve the intestinal flora and reduce the concentration of ammonia nitrogen and skatole. In addition, using the composition of the present invention, it is possible to improve intestinal health or intestinal function, it is possible to improve the digestive function and defecation function. On the other hand, the red ginseng gourd of the present invention has no side effects to the human body and can be effectively used in food or feed compositions.

Description

Composition for improving intestinal health comprising red ginseng residue

The present invention relates to a composition for improving intestinal health comprising red ginseng gourd, and more particularly, while promoting the growth of Bifidobacterium bacteria, while inhibiting the proliferation of Clostridium bacteria, and together with intestinal flora (increasing the proportion of Firmicutes and Bacteroidetes ), increasing the intestinal short chain fatty acid (SCFA) concentration and decreasing the concentration of ammonia nitrogen and/or skatole, thereby improving the intestinal environment. It relates to a composition for improving intestinal health comprising red ginseng extract or an indigestible substance in red ginseng extract as an active ingredient.

Enterobacteriaceae constantly proliferates and excretes by using the ingested food or mucus secreted from the digestive tract as nutrients while maintaining a symbiotic or antagonistic relationship between beneficial and harmful bacteria. If unhealthy eating habits continue during this process, the composition of intestinal bacteria changes, causing abnormalities in the intestinal immune status and adversely affecting health such as intestinal diseases. The incidence of inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, continues to increase worldwide, and these intestinal diseases are also on the rise in Korea. Harmful substances such as putrefactive metabolites (ammonia, hydrogen sulfide, amine, phenol, indole, etc.), bacterial toxins, carcinogens (nitroso compounds, epoxies, etc.) and secondary bile acids produced by harmful bacteria in the intestine directly interfere with the intestinal tract. , some are absorbed and cause long-term disorders in various organs, causing so-called lifestyle-related diseases.

Representative functional ingredients related to intestinal function include dietary fiber and oligosaccharides, which are non-starch polysaccharides, and starchy polysaccharides include indigestible maltodextrin and resistant starch (RS). These substances are called indigestible substances (sugars) or functional dietary fibers. Dietary fiber is a representative non-digestible polysaccharide that is not digested by the digestive enzymes of the human body. It is classified into water-soluble and insoluble according to its solubility in water. The former includes guar gum, indigestible maltodextrin, water-soluble pectin, Inulin and oligosaccharides are included. The latter include cellulose, hemicellulose, lignin, insoluble pectin, and chitosan. In addition, there are four forms of indigestible starch (RS). RS1 refers to starch that is physically indigestible, and RS2 refers to starch that does not gel well like raw potato starch or high-amylose starch (HAS). RS3 mainly refers to aged starch, and RS4 refers to food modified starch. As such, the type and range of indigestible materials are very wide. As the intestinal function for indigestible substances known so far, the effect of improving defecation activity by shortening the residence time of digestive substances in the colon and increasing the amount of bowel movement, and the effect of prebiotics that increase the beneficial bacteria in the intestine are known. Short-chain fatty acids, a component of the final fermentation in the intestine, are attracting attention as a major physiological function factor related to the promotion of intestinal health. In addition to promoting intestinal health, indigestible substances are also recognized as multifunctional health food materials with various physiological activities such as blood cholesterol reduction, blood sugar control, intestinal immunity enhancement and diet effect.

Most of the dietary fibers currently used in Korea are starch-derived low-molecular-weight water-soluble dietary fibers (polydextrose, polydextrose), which are mainly used in beverage manufacturing. It is used in baking, etc.

In Europe, inulin, a water-soluble dietary fiber, is purified from chicory roots and mass-produced. Currently, a fat replacement technology using inulin-type fructan is attracting attention, and when inulin is mixed well, cream-like properties are formed, and the formed cream properties are used for fat replacement. In South America, eggplant-type inulin is extracted from the agave root and used as an alternative sweetener for sugar, such as syrup. In Japan, about 10 types of dietary fiber and oligosaccharides have been commercialized. Commercialized products are mass-produced by some microbial enzyme processes, but most of the products are environmentally friendly products developed for the purpose of recycling by-products or wastes from the main product production process.

Red ginseng mainly contains various kinds of saponin substances called ginsenosides like raw ginseng, and in addition, polyacetylene-based compounds, nitrogen-containing components, trace elements, organic acids and amino acids are contained. As a general ingredient, carbohydrates account for 60-70% of the total. In the case of manufacturing red ginseng products, most of them go through extraction and concentration processes to make a concentrate, and then manufacture them in the form of liquid, powder, or beverage according to the type of each product. Ginseng processing companies extract active ingredients from the original form of red ginseng using hot water or alcohol (ethanol) to produce a concentrate. However, at present, most companies are experiencing difficulties in processing or utilizing the remaining red ginseng gourd after extracting the concentrate. In addition, despite the fact that not only active ingredients but also dietary fiber and acidic polysaccharides remain in red ginseng leaves, recycling of red ginseng leaves is very insufficient due to technical limitations.

In addition, indigestible substances have a very wide range of types and physiological characteristics depending on the type. However, there is an urgent need for systematic research on the physiological functions of each indigestible substance to create demand.

Accordingly, the present inventors conducted a study on the intestinal fermentation characteristics of red ginseng gourd and the balance of intestinal bacteria in an immersion anaerobic culture ( in vitro ) test using pig feces. In addition, it promotes the proliferation of beneficial bacteria (especially Bifidobacterium ), suppresses the relative ratio of harmful bacteria (especially Clostridium ) related to ammonia production, increases the concentration of intestinal short-chain fatty acids (acetic acid and propionic acid), The present invention was completed by discovering that it is possible to improve intestinal health or intestinal function by reducing the concentration of toll.

Accordingly, the technical problem to be solved in the present invention is to provide a composition for improving intestinal health.

In order to solve the above technical problem, the present invention provides a food composition for improving intestinal health comprising red ginseng extract as an active ingredient.

According to one embodiment of the present invention, the composition comprising red ginseng extract as an active ingredient improves intestinal flora (increasing the ratio of Firmicutes and Bacteroidetes ) It provides a food composition for improving intestinal health.

According to another embodiment of the present invention, there is provided a food composition for improving intestinal health, characterized in that the composition comprising red ginseng extract as an active ingredient proliferates beneficial intestinal bacteria, particularly Bifidobacterum . to provide.

Here, the proliferation is beneficial intestinal bacteria, particularly Bifidobacterum , the number of bacteria 1.2 to 30 times, preferably 1.5 to 20 times, more preferably 2 to 20 times, even more preferably 2 to 10 times. say to increase

According to another embodiment of the present invention, the present invention provides a food composition for improving intestinal health, comprising red ginseng extract as an active ingredient, which suppresses intestinal ammonia production to reduce blood ammonia concentration.

The present invention also provides a feed composition for improving intestinal health comprising red ginseng gourd as an active ingredient.

The improvement of the intestinal flora means promoting the growth of beneficial bacteria in the intestine and suppressing the growth of bacteria producing harmful substances in the intestine, thereby improving the balance of beneficial bacteria and harmful bacteria in the intestine. In particular, it means to increase the proportion of Firmicutes and Bacteroidetes in the intestinal flora.

Probiotics ( probiotics ) refer to microorganisms that have a good effect on health in the body. It is an effective ingredient.

According to a specific embodiment of the present invention, xylo-oligosaccharide, a representative material, is used as prebiotics serving as food for probiotics (probiotics), and intestinal lactic acid bacteria (especially, Bifidobacterium bacteria) of red ginseng gourd proliferate and harm The potential of red ginseng extract as a prebiotic was confirmed by confirming that there was an inhibitory effect on the relative ratio of substance-producing bacteria (especially, Clostridium genus bacteria).

In one embodiment of the present invention, the red ginseng foil may be red ginseng foil or a red ginseng foil indigestible material.

The red ginseng paste is a residue remaining after extracting red ginseng with a solvent such as water or alcohol, and even if red ginseng is repeatedly extracted several times, all of the active ingredients inside cannot be extracted, and only components soluble in water or alcohol are extracted depending on the extraction solvent. It is a natural substance in which about 20 to 40% of active ingredients such as undissolved saponin, polyacetylene, and protein remain.

The red ginseng meal indigestible material refers to a material that is not hydrolyzed when red ginseng foil enters the body and is hydrolyzed by the digestive enzymes pancretin and alpha-amiroglycosidase, and is produced in the body. .

According to a specific embodiment of the present invention, as a result of performing anaerobic immersion culture of red ginseng gourd using pig feces similar to the composition of human intestinal flora, it can be confirmed that the pH is significantly lower than that of the negative control, cellulose-added group. .

In a state of low intestinal pH, harmful bacteria are sensitive to acid and growth and growth are reduced, but beneficial bacteria actively ferment substrates and have resistance to acids, so growth is maintained. Therefore, in a low pH environment, beneficial bacteria play a role in inhibiting the invasion of harmful bacteria in the intestinal tract by taking an advantage in adhesion competition with harmful bacteria such as pathogens.

According to a specific embodiment of the present invention, as a result of performing anaerobic immersion culture of red ginseng gourd using pig feces similar to the composition of human intestinal flora, the total short-chain fatty acid concentration was significantly higher overall than that of the negative control, cellulose-added group. can check that

The short-chain fatty acid refers to a fatty acid containing 3 or less carbon atoms, and may be acetic acid, propionic acid, or butyric acid.

Fermentable dietary fiber or indigestible starch (RS) flowing into the colon is metabolized into short-chain fatty acids by intestinal bacteria. The produced short-chain fatty acids act as important anions in the colon, stimulating water and sodium absorption, regulating intestinal motility, and reducing the risk of colon cancer. In addition, each short-chain fatty acid has different physiological functions. For example, acetic acid is a short-chain fatty acid produced the most in the large intestine, and is absorbed in the colon epithelial cells together with propionic acid and is used systemically in various ways. In particular, acetic acid moves to the liver and peripheral organs and becomes a substrate for gluconeogenesis and lipogenesis, and propionic acid is known to inhibit the metabolism of major lipid substrates on the contrary. On the other hand, butyric acid is the most important energy source for colon cells.

According to a specific embodiment of the present invention, it can be confirmed that red ginseng leaves have a great effect on propionic acid production. In general, the molecular weight ratio of acetic acid: propionic acid: butyric acid in human feces is 60: 20: 20, and it can be seen that propionic acid increased and butyric acid decreased when red ginseng powder was added. The difference in the molecular weight ratio of short-chain fatty acids and each short-chain fatty acids produced in the red ginseng meal is that non-fermentable dietary fibers such as cellulose are contained in large amounts in red ginseng meal and that xylo-oligosaccharide and other enterobacteriaceae prefer fermentation in red ginseng meal. This is because there is a possibility that the high molecular weight polysaccharide is congested.

According to a specific embodiment of the present invention, anaerobic immersion culture was performed using pig feces similar to the composition of the human intestinal flora of red ginseng gourd to analyze intestinal bacterial growth and microbiome. As a result, Bifidobacterium ( Bifidobacterium ) It can be seen that the proliferation of bacteria and the relative ratio of bacteria of the genus Bifidobacterium among all bacteria increases.

According to another embodiment of the present invention, the composition comprising the red ginseng foil is 1.2 to 30 times, preferably 1.5 to 20 times, more preferably 2 to 20 times, Bifidobacterum bacteria, Even more preferably, it is characterized in that it proliferates 2 to 10 times.

In the present invention, the beneficial intestinal bacteria may refer to microorganisms that live in the intestine and exert beneficial effects on the human body. For example, beneficial intestinal bacteria may include probiotics ( Bifidobacterium , Lactobacillus ). For example, the beneficial intestinal bacteria are not limited thereto, and Lactococcus ( Lactococcus ), Streptococcus ( Streptococcus ), Akkermansia ( Akkermansia ), Faecalibacterium ( Faecalibacterium ), or Enterococcus ( Enterococcus ) may include.

The probiotics refer to probiotics that are beneficial to the human body, and representative probiotics include lactic acid bacteria. However, not all lactic acid bacteria refer to probiotics. On the other hand, prebiotics are food-derived indigestible ingredients that help the growth of probiotics. Representative prebiotics include low molecular weight oligosaccharides (fructooligosaccharide, galactooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, chitin oligosaccharide, etc.) and high molecular weight dietary fiber (digestible dextrin, inulin, guar gum, etc.). there is. In these prebiotics, the preference of beneficial bacteria appears differently depending on the type of constituent material, the type of intermolecular bond, and the length of the chain. For example, Ito et al. administered inulin-type fructans (DP4, DP8, DP16, DP23, average degree of polymerization) with different polymerization degrees to rats. As a result, Lactobacillus bacteria grew on short-chain fructooligosaccharides (DP4, DP8). was increased, and in the case of Bifidobacterium , it is reported that it was generally increased in the group administered with high molecular weight inulin (DP8, DP16, DP23) (Ito et al ., J. Agri. Food Chem., doi: 10.1021/jf200859z).

Meanwhile, in the present invention, intestinal harmful bacteria may refer to microorganisms that live in the intestine, produce harmful substances, and have harmful effects on the human body, such as enteritis. For example, the intestinal harmful bacteria is not limited to Clostridium bacteria, but may include Fusobacterium or Porphyromonas .

According to one embodiment of the present invention, the composition of the present invention can promote the proliferation or growth of Bifidobacterium , and can inhibit the proliferation or growth of Clostridium .

According to a specific embodiment of the present invention, through meta-analysis by next-generation sequencing of anaerobic immersion culture medium using pig feces similar to the composition of human intestinal flora with red ginseng extract indigestible material, a significant difference in the number of enterobacterial species was found. appeared, and the principal coordinate analysis (beta-diversity) also confirmed a large difference between the addition groups.

According to a specific embodiment of the present invention, as a result of performing anaerobic immersion culture of red ginseng gourd using pig feces similar to the composition of human intestinal flora, ammonia nitrogen production concentration was significantly inhibited compared to the cellulose-treated group, which is a negative control. can check that

In general, the fermentation of amino acids decomposed from food and proteins derived from the host produces a wide variety of metabolites. For example, branched-chain fatty acids (BCFAs) such as isonyric acid, isovaleric acid and 2-methyl butyric acid in feces are indicators of branched-chain amino acid fermentation, and their feces My concentration increases with a high-protein meal. In addition, depending on the environment of the large intestine, intermediate metabolites such as lactic acid, succinic acid, and alpha-keto acid may accumulate in the large intestine. Protein fermentation products include those having potential toxicity, representative examples of which include ammonia (NH ₃ ), N -nitroso compounds, amines, and p -cresol. In particular, ammonia production in the large intestine is produced when amino acids derived from food are decomposed by bacteria or urea is decomposed by bacteria urease. Ammonia in the large intestine is produced more at high pH than at low pH. Ammonia in the living body is formed by deamination to amino acids during protein metabolism and is converted to urea in the urea cycle of the liver. When the concentration of ammonia in the blood rises, it acts as a neurotoxic substance and can become a major cause of minimal hepatic encephalopathy. Intestinal ammonia-producing bacteria include Clostridium spp., Peptostreptococcus , Fusobacterium , Actinomyces , Bacteroides , Megasphaera , and Propionibacterium (Richardson et al ., BMC Microbiol., doi: org/10.1186/1471-2180-13-6), Lactobacilli form very little ammonia (Vince & Burridge, J Med Microbiol, doi: 10.1099/00222615-13-2-177).

According to a specific embodiment of the present invention, as a result of performing anaerobic immersion culture of red ginseng powder using pig feces similar to the composition of human intestinal flora, it was found that the concentration of Skatole production was statistically significantly lower than that of the cellulose-added group. can be checked

The skatole is an indole derivative produced by decomposition of tryptophan by microorganisms in the digestive tract, and has a structure (3-methylindole) in which a methyl group is attached to the indole, and causes pig boar or fecal odor.

Therefore, the composition comprising red ginseng gourd according to the present invention as an active ingredient can be usefully used as a food or feed composition as a composition for improving intestinal health, improving intestinal flora, and enhancing the efficacy of probiotics.

Red ginseng gourd included as an active ingredient in the composition of the present invention may be included in an amount of 0.001 to 99% by weight, preferably 0.01 to 80% by weight, more preferably 0.01 to 50% by weight based on the total weight of the total composition. . When the content of the red ginseng gourd is less than 0.001% by weight, there is a problem in that the effect of improving intestinal health is weak.

As used herein, the term “improvement” refers to any action in which a disease or symptom of a disease is improved in an individual.

As used herein, the term "subject" refers to mammals such as chimpanzees, monkeys, pigs, dogs, cats, rats, and mice, including humans, having diseases whose symptoms can be improved by administering the composition of the present invention. .

As used herein, the term "indigestible material" refers to carbohydrates and nitrogen-containing components greater than or equal to disaccharides that are not decomposed by digestive enzymes of mammals, and refers to dietary fiber, oligosaccharides, indigestible starch, indigestible proteins and indigestible peptides.

When the composition of the present invention is used as a food composition, the red ginseng paste may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be suitably determined according to the purpose of use (prevention, health or therapeutic treatment), and since the composition of the present invention is environmentally friendly and there is no problem in terms of stability, there is no great limitation on the mixing amount.

Since the food composition of the present invention can be ingested on a daily basis, the effect of improving colon function can be expected, so it can be usefully used as a health functional food for the purpose of health promotion.

As used herein, the term "health functional food" is the same term as food for special health use (FoSHU), and is a food with high medical and medical effects processed to efficiently exhibit bioregulatory functions in addition to nutrition supply. means Here, the term "function" refers to obtaining a useful effect for health purposes, such as regulating nutrients or physiological action with respect to the structure and function of the human body. The health functional food of the present invention can be manufactured by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, the formulation of the health functional food may also be manufactured without limitation as long as it is a formulation recognized as a food. The health functional food of the present invention can be manufactured in various forms, and unlike general drugs, it has the advantage that there are no side effects that may occur when taking the drug for a long period of time by using food as a raw material, and it is excellent in portability. The health functional food of the present invention can be ingested as a supplement to enhance the effect of improving intestinal function.

The health functional food means a food having an active health maintenance or promotion effect compared to general food, and health supplement food means a food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and health supplement are used interchangeably.

Specifically, the health functional food is a food prepared by adding the composition of the present invention to food materials such as beverages, teas, spices, gum, and confectionery, or encapsulating, powdering, suspension, etc. It means to bring an effect, but unlike general drugs, it has the advantage that there are no side effects that may occur when taking the drug for a long time using food as a raw material.

The health functional food may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used.

In addition, the health functional food may include additional ingredients that are commonly used in food to improve odor, taste, vision, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu); and amino acids such as lysine, tryptophan, cysteine, and valine.

In addition, the health functional food contains preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butyl Hydroxytoluene (BHT), etc.), coloring agents (tar pigments, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclmate, saccharin, sodium, etc.), fragrances (vanillin, lactones, etc.), swelling agents (alum, D-potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (foaming agents), film agents, gum base agent, foam inhibitor, solvents, improving agents, etc. It may contain food additives. The additive may be selected according to the type of food and used in an appropriate amount.

As an example of the health functional food of the present invention, it may be used as a health drink composition, and in this case, it may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional drink. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; sugar alcohols such as xylitol, sorbitol, and erythritol. Sweeteners include natural sweeteners such as taumatine, stevia extract; A synthetic sweetener such as saccharin or aspartame may be used. The ratio of the natural carbohydrate may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 mL of the health beverage composition of the present invention.

In addition to the above, the health drink composition includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid, pectic acid salts, alginic acid, alginic acid salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol, a carbonation agent, or the like. In addition, it may contain the pulp for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. These components may be used independently or in combination. Although the ratio of these additives is not very important, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

As another aspect, there is provided a feed composition for improving intestinal health comprising red ginseng gourd.

As used herein, the term "feed" refers to any natural or artificial diet, one-meal meal, etc. or a component of the one-meal meal that is suitable for or for livestock and companion animals to eat, ingest, and digest. The feed including the active ingredient can be prepared in various types of feed known in the art, and specifically, it may include a concentrated feed, a roughage and/or a special feed.

The composition of the present invention included in the feed composition of the present invention may vary depending on the purpose and conditions of use of the feed, for example, 0.01 to 100% by weight, more specifically 5 to 20% by weight based on the total weight of the livestock feed composition It may be included in weight %.

When the composition comprising red ginseng extract of the present invention as an active ingredient is used in combination with other probiotics, a synergistic effect with probiotics can be obtained.

On the other hand, the red ginseng paste of the present invention is a harmless substance without cytotoxicity. Therefore, the red ginseng gourd of the present invention can be safely used even when used for a long period of time, and in particular, it can be safely used in the food or feed composition as described above.

As such, the composition containing red ginseng extract of the present invention as an active ingredient promotes the growth of beneficial intestinal bacteria, inhibits the growth of harmful bacteria, increases the content of short-chain fatty acids in the intestine, improves intestinal flora, Reduce the concentration of skatole. In addition, by using the composition of the present invention, it is possible to improve intestinal health or intestinal function. On the other hand, the composition comprising red ginseng powder of the present invention has no side effects on the human body and can be effectively used in food or feed compositions.

1 is a graph showing the change in pH during the incubation period of the indigestible material of red ginseng gourd.
2 is a graph showing the change in the concentration of short-chain fatty acids during the incubation period of the indigestible material of red ginseng.
3 is a graph showing the change in the molecular weight ratio of short-chain fatty acids during the culture period of the indigestible material of red ginseng foil.
Figure 4 shows the trend of the total number of general anaerobes and E. coli during the culture period of the indigestible material of red ginseng.
Figure 5 shows the trend of the number of lactic acid bacteria during the culture period of the indigestible material of red ginseng.
6 is an analysis of the diversity of enterobacteriaceae.
7 shows the cluster distribution.
8 shows the relative distribution ratio of enterobacteriaceae between samples in a phylum unit.
9 shows the relative composition ratio of enterobacteriaceae at the genus level.
10 shows the relative proportions of intestinal flora in genus affiliation.
Figure 11 shows the change in ammonia nitrogen production during the culture period of the indigestible material of red ginseng.
12 shows the change in skatole production during the incubation period of the red ginseng meal indigestible material.

Hereinafter, examples and the like will be described in detail to help the understanding of the present invention. However, the embodiments according to the present invention may be modified in various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Example 1> Indigestible material derived from red ginseng gourd Produce

In 100 mM maleic acid buffer (pH 6.0), 300 U/100 mL of alpha-amiroglucosidase (Sigma-Aldrich, St. Louis, USA) and pancreatin (Sigma-Aldrich) were added at a ratio of 0.75 g/100 mL. It was dissolved to prepare a digestive enzyme solution. After pulverizing red ginseng powder with a general grinder, 20 g of powdered red ginseng powder was placed in an Erlenmeyer flask, and 100 mL of the digestive enzyme solution was added. Then, after culturing for 16 hours in a 37° C. water bath, 99% ethanol was added and allowed to stand for 3 hours. To remove the hydrolyzed protein and starch, the solution in the Erlenmeyer flask was dispensed into a centrifuge tube, and centrifugation (3,000 rpm) was performed to remove the supernatant. Then, an appropriate amount of 99% ethanol was added to the precipitate, followed by centrifugation to wash the precipitate. Finally, after adding an appropriate amount of acetone to wash the residue, the precipitate in the tube was collected and naturally dried overnight in the draft. After that, the weight of the dried precipitate was measured, and finally, the yield of indigestible substances from red ginseng foil was calculated. The indigestible material of red ginseng powder was pulverized using a mixer.

<Test Example 1> Analysis of general components of red ginseng powder indigestible material

The protein content in the red ginseng powder was measured according to the Kjeldahl nitrogen quantification method (AOAC 920.87 nitrogen-protein conversion factor 6.25). The total lipid content in the red ginseng powder was measured according to the soxhlet extraction method (AOAC 920.85). The ash content in the red ginseng powder was measured at 550° C. according to the direct sintering method (AOAC 923.03). The water content in the red ginseng powder was measured according to the heat-drying method (dried at 110°C for 2 hours, AOAC 925.10) through loss on drying. The dietary fiber content in red ginseng meal was measured by Prosky's method (AOAC 991.43). The total amount of soluble dietary fiber and insoluble dietary fiber was calculated as total dietary fiber. The indigestible starch (RS) content in red ginseng meal was measured using a commercially available RS assay kit (Megazyme, Wieklow, Ireland, AOAC 2002.02). Indigestible starch and digestible starch were analyzed through this kit analysis, and the total amount of starch was calculated using the sum of the two.

Table 1 shows the results of component analysis of red ginseng extract and red ginseng extract indigestible substances.

Sample name carbohydrate protein moisture ash lipid Dietary Fiber starch RS etc red ginseng gourd 41.7 4.11 27.9 11.7 7.97 4.90 1.76 red ginseng gourd
indigestible substances 61.8 7.29 17.9 10.6 6.98 0.22

On a dry basis, red ginseng gourd contained 41.7% dietary fiber and 4.11% indigestible starch (RS). As a result of hydrolysis of dried red ginseng powder with amyloglycosidase and pancreatin, the yield of unhydrolyzed red ginseng powder was 67.4% on a dry matter basis. In this experiment, these unhydrolyzed products were defined as indigestible substances. Most of the unhydrolysates were carbohydrates (64.3%), followed by proteins (17.9%). As a result of calculating the residual amount of each indigestible substance using the yield (67.4%), the dietary fiber content in the enzyme-treated red ginseng meal was 61.8% (41.7 g/0.674 = 61.8 g). For indigestible starch, the yield was 6.1% (4.11 g/0.674 = 6.10 g) and the actual measurement was 7.3%. One peculiar thing discovered in this experiment is that most of the proteins were not degraded even when the red ginseng gourd was enzymatically treated. There was no significant difference even when comparing the calculated protein content of 17.4% (11.7 g/0.674 = 17.4 g) in the enzyme-treated red ginseng gourd with 17.9% of the actual measured value in the enzyme-treated red ginseng gourd. From this, the protein in red ginseng meal is also considered to be indigestible.

<Test Example 2> Measurement of intestinal environment improvement effect of indigestible substances of red ginseng foil

1) Anaerobic fermentation using Jarfermentor (immersion anaerobic incubator)

A jarfermentor is an ecosystem device that models the intestinal environment so that the inside of the fermenter is anaerobically stirred while culturing the intestinal flora or single microorganisms, and the dynamics of the intestinal bacteria can be grasped. The main body has a microorganism inoculation port, a temperature sensitive device, a pH electrode, a thermometer, an alkali inlet, a sample collecting port, a medium inlet port, and a gas outlet port. In addition, by supplying carbon dioxide into the fermenter, the intestinal environment, anaerobic state, is maintained, and by injecting an alkaline solution into the abnormal intestinal environment caused by acid fermentation, it is possible to culture the intestinal microbiota in an environment suitable for the intestinal pH conditions.

In this experiment, the effect on the improvement of the intestinal environment such as fermentation characteristics of indigestible substances of red ginseng gourd was investigated using a self-fermenter. The pH in the initial culture tank was set to 5.6, and when the pH is tilted toward acid due to acid fermentation, a sterile sodium hydroxide solution (2N NaOH) is automatically injected into the culture tank to prevent it from falling below pH 5.5, which is an unphysiological situation did The culture tank temperature was maintained at 37 ° C., and the culture solution was homogeneously stirred at a speed of 160 rpm, and 0.4 L of carbon dioxide (CO ₂ ) gas was injected per minute to maintain an anaerobic state. As seen from the composition of the experimental group shown in Table 2 below, 44 mL of fecal excrement corresponding to 2% of the total culture solution (220 mL), a carbon source (sample) corresponding to 3%, and a protein source corresponding to 0.83% (nutrient broth) After addition, culture was carried out in anaerobic conditions for 48 hours.

Incubator (220 mL total) carbon source
(3.0% level) cellulose xylo-oligosaccharide Polysaccharides derived from red ginseng gourd protein source
(0.83% level) Nutrient broth Nutrient broth Protein derived from red ginseng gourd bacterium
(2.0% level) Pig feces

2) Preparation of fecal excrement suspension

For immersion anaerobic culture, fresh pig excrement was used as a source of intestinal bacteria. In this experiment, feces were directly collected from the anus of three pigs from pigs (about 3-5 months old) exempted from antimicrobial administration within at least one month. The collected feces were immediately placed in a double zipper bag maintained anaerobically with AnaeroPack (AnaeroPack, a deoxidizer, Mitsubishi Gas Chem., Tokyo, Japan) and stored at 4°C until analysis. Fecal collection was performed 5 times in total. After weighing 2.3 g each of the feces of 3 animals collected each time, they were mixed in a transparent plastic bag. Thereafter, sterile physiological saline (0.85%) was added 10 times for dilution, and the solid was removed to prepare a filtrate. This filtrate was used as a fecal fecal suspension. 44 mL of fecal suspension was added to 2% (v/v) of the volume of the culture mixture (220 mL) in the culture tank (Able & Biott, Tokyo, Japan). For stabilization of the intestinal flora, only the fecal fecal suspension was cultured for the first 12 hours.

3) Add Medium

As a medium for bacterial culture, Nutrient broth (Difco, Sparks, MD, USA) was used. Nutrient broth is a medium composed of beef extract and peptone in a ratio of 3 to 5. The protein source in the culture medium was added to the culture tank so that the total amount was 0.83% (1.83 g). Nutrient broth medium was dissolved in distilled water (1.83 g/50 mL) in advance and then sterilized at 121°C for 15 minutes. 50 mL of the sufficiently cooled medium was injected into the culture tank under anaerobic conditions.

4) Addition of carbon source

As the carbon source, cellulose, xylooligosaccharide, and indigestible powder of red ginseng powder were used. So that the amount of carbohydrate added in the culture tank was 3% of the total, 6.60 g of sterilized cellulose and xylo-oligosaccharide were mixed with 126 mL of sterile physiological saline, and then injected into each culture tank. In addition, 10.26 g (126 mL) of sterilized red ginseng powder indigestible was injected. The amount of red ginseng indigestible material added was determined by analyzing the concentration of carbohydrates and protein in the red ginseng foil indigestible material.

5) pH analysis

The pH in the culture medium was observed and recorded in real time for 48 hours of incubation. Cultures were collected in sterile tubes every 0, 6, 12, 24, and 48 hours of culture. The culture solution collected in the 1.5 mL EPEN tube was stored at -80°C until analysis for next-generation sequencing (for NGS analysis), and the culture solution collected in the 2.0 mL EPEN tube was immediately used for bacterial testing after collection were stored in a -30°C freezer until each experimental analysis.

1 is a graph showing the change in pH during the incubation period of the indigestible material of red ginseng gourd. As shown here, the cellulose-added port increased from pH 5.6 at the beginning of the culture to pH 6.5 at the end of the culture. On the other hand, the pH of the group added with xylo-oligosaccharide and red ginseng powder indigestible material hardly changed during the entire incubation period, and both groups were statistically significantly lower than the pH of the group added with cellulose (p<0.05).

5) Measurement of short-chain fatty acid concentration

The concentrations of short-chain fatty acids, acetic acid, propionic acid, and butyric acid, in the culture medium were measured using high-performance liquid chromatography (LC-10AD, Shimadzu, Kyoto, Japan). To prepare a sample for short-chain fatty acid, the culture medium was centrifuged (16,000 rpm, 4° C., 15 minutes) to take the supernatant, and 0.5N HClO ₄ was added to remove the protein in the supernatant and left at room temperature for 5 minutes. Centrifugation (16,000 rpm, 4° C., 10 minutes) was performed again, and the filtered sample was donated to HPLC using a disposable filter for HPLC (0.45 μm, Advantec, Tokyo, Japan). HPLC setting conditions are RSpak KC-811 (8.0 mm × 300 mm, Shodex, Tokyo, Japan) post column with 2 mM perchloric acid as the mobile phase, and pass through the column at 47°C so that it becomes 1.0 mL per minute, and then in a mixer at a flow rate of 0.5 mL/ Each SCFA was detected at a wavelength of 430 nm using a UV-detector (Shimadzu) by reacting with a min of ST3-R (Shodex, Tokyo, Japan) solution. Identification of various short-chain fatty acids was qualitative by comparison of retention time (RT) of samples obtained from Chromat Pack (C-R6A, Shimadzu) and standard solutions, and quantitative analysis was performed by comparing peak areas. In addition, the sum of the three types of SCFA concentration was calculated as the total SCFA concentration.

2 is a graph showing the change in the concentration of short-chain fatty acids during the incubation period of the indigestible material of red ginseng. As shown here, the concentration of acetic acid was significantly increased in the group added with xylo-oligosaccharide and red ginseng powder indigestible material compared to the group added with cellulose at 6 hours of culture ( p <0.05). From the 12th hour of culture, the group added with xylo-oligosaccharide significantly increased (p<0.05) than those added with cellulose and red ginseng foil indigestible substances. The group added with red ginseng powder indigestible material increased significantly ( p <0.05) than the group added with cellulose after 24 hours of incubation. Overall, the acetic acid fermentation took precedence over the group added with xylo-oligosaccharide compared to the group added with the indigestible substance of red ginseng. The propionic acid concentration was significantly increased in the xylo-oligosaccharide-added group than in the cellulose-added group from 6 hours after the initiation of culture ( p <0.05). The group added with red ginseng powder indigestible material increased significantly compared to the group with cellulose after 12 hours of culture ( p <0.05), but there was no statistically significant difference with the group with xylo-oligosaccharide added at 48 hours. The concentration of butyric acid was significantly increased in the group added with xylo-oligosaccharide compared to the group added with cellulose and red ginseng foil indigestible substances from 6 hours of incubation ( p <0.05). In the case of red ginseng foil, the concentration was similar to that of the cellulose-added group. During the entire incubation period, the total concentration of short-chain fatty acids in the indigestible substance added to red ginseng meal was lower than that of xylo-oligosaccharide. This difference is thought to be due to the high content of cellulose and hemicellulose, which are non-fermentable dietary fibers, in red ginseng gourd.

3 is a graph showing the change in the molecular weight ratio of short-chain fatty acids during the culture period of the indigestible material of red ginseng foil. As shown here, the molecular weight ratio of short-chain fatty acids in the culture tank for each culture time showed different patterns for each sample. It was confirmed that the indigestible material of red ginseng extract had little effect on butyric acid production, similar to cellulose. In general, the acetic acid: propionic acid: butyric acid molecular weight ratio (molar proportion) in human feces is 60: 20: 20. In this experiment, it was found to be 54:28:17 in the initial culture of the pig feces used. 3, in the case of the cellulose-added group, acetic acid increased and propionic acid decreased, and in the case of the xylo-oligosaccharide-added group, acetic acid decreased and butyric acid increased. On the other hand, propionic acid increased and butyric acid decreased in the case of red ginseng meal with indigestible substances added. The difference in the molecular weight ratio of short-chain fatty acids and each short-chain fatty acid produced by the addition of indigestible substances in red ginseng powder is that non-fermentable dietary fibers such as cellulose are contained in large amounts in red ginseng meal and that xylo-oligosaccharide and other enterobacteriaceae are present in red ginseng meal. It is judged that this is because there is a possibility that the preferred fermentable polymer polysaccharide is crowded.

6) Measurement of the number of various bacteria in the culture medium

Enterobacteriaceae was visually measured for the number of colonies after culturing by a plate plating method using a selective medium. BL agar medium (Eiken, Tokyo, Japan) for general anaerobic culture, EMB agar medium (Eiken) for E. coli culture, MRS agar medium (Oxoid, UK) for lactic acid bacteria, Rogosa agar medium (Kanto Chemical, Tokyo, Japan) for Lactobacillus culture ), and TOS propionic acid agar medium (Yakult Pharmaceutocal Industry, Tokyo, Japan) was used for culturing Bifidobacterium . The culture solution collected for each time period was diluted 10-fold with sterile physiological saline (up to 10 ^-7 ), and then 100 μL was smeared on the agar medium and cultured. It was cultured in anaerobic conditions according to the GasPak method at 37°C in the incubator. Colonies were counted after 24 hours for coliform bacteria, 48 hours for general anaerobes, total lactic acid bacteria and Lactobacillus , and 72 hours for Bifidobacterium . The number of colonies formed in each medium (Colony Forming Unit) was expressed as CFU and finally calculated as a log (log ₁₀ ).

Figure 4 shows the trend of the total number of general anaerobes and E. coli during the culture period of the indigestible material of red ginseng. As shown here, the number of general anaerobic bacteria increased significantly in the group in which xylo-oligosaccharide and red ginseng indigestible substances were added compared to the group in which cellulose was added after 12 hours of incubation ( p <0.05). At 24 hours of incubation, the total number of E. coli was significantly decreased in the group with xylo-oligosaccharide added compared to the group with cellulose, and at 48 hours of culture, growth was significantly reduced in the group with xylo-oligosaccharide added compared to the group with cellulose and red ginseng powder indigestible material. decreased ( p <0.05). During the entire incubation period, the number of coliform bacteria in the group added with red ginseng foil indigestible material was almost the same as that of the group added with cellulose.

Figure 5 shows the trend of the number of lactic acid bacteria during the culture period of the indigestible material of red ginseng. As shown here, the total number of lactic acid bacteria (including lactobacilli and lactobacilli) increased significantly in the group added with red ginseng powder indigestible material compared to the group added with cellulose and xylo-oligosaccharide ( p <0.05) at 6 and 12 hours of incubation. . The total number of lactic acid bacteria in the group added with red ginseng powder indigestible material continued to decrease from 12 hours to 48 hours after incubation, and at 24 hours of culture, the group with xylo-oligosaccharide and cellulose added showed a statistically higher number of viable bacteria ( p < 0.05), and at 48 hours of incubation, the number of bacteria was similar to that of the cellulose-added group. The group added with xylo-oligosaccharide showed the maximum number of growth until the 24th hour of culture and decreased at the 48th hour, but it was still significantly higher than that of the group with cellulose ( p <0.05). During the entire culture period, the number of Lactobacillus bacteria in the group added with xylo-oligosaccharide and red ginseng powder indigestible material was significantly higher than that of the group with cellulose ( p <0.05). The number of Lactobacillus bacteria in the group added with red ginseng powder indigestible substance increased significantly from 6 hours to 12 hours of incubation compared to the group with xylo-oligosaccharide ( p <0.05), but showed similar values between the two groups starting from 24 hours of incubation, but at 48 hours It was reversed and significantly decreased compared to the xylo-oligosaccharide-added group ( p <0.05). However, it was significantly higher than the number of viable cells in the cellulose-added group ( p <0.05). The number of Bifidobacterium bacteria increased significantly in the group in which the indigestible substance was added and in the group in which xylo-oligosaccharide was added, compared to the group in which cellulose was added, throughout the entire incubation period except for the 24 hour culture ( p <0.05). Red ginseng gourd was more friendly to the growth of Bifidobacterium than xylooligosaccharide.

Since red ginseng gourd contains various indigestible sugars, it is presumed that the low molecular weight indigestible sugars in red ginseng gourd were used as a nutrient source for Lactobacillus bacteria from the beginning of culture to 24 hours, and then almost digested after 24 hours. On the other hand, the fact that the growth of Bifidobacterium by red ginseng gourd showed a significantly higher number of viable bacteria than in the xylo-oligosaccharide-added group over the entire culture period, it is inferred that the indigestible high-molecular polysaccharide in red ginseng meal is the preferred nutrient source for Bifidobacterium compared to oligosaccharide. In terms of growth, it suggests the possibility of being a more useful prebiotic than xylooligosaccharide.

7) Next-generation sequencing (NGS) analysis

Microbial DNA was extracted from the culture medium of each experimental group at 48 hours of in vitro culture using QIAamp ^® DNA Stool Mini Kit (QIAGEN ^® ). The extracted culture medium Microbial DNA was diluted to a concentration of 5ng/μl using sterile distilled water. Thereafter, for sequencing of the intestinal microbiota, the amplicon was prepared according to the Illumina 16S Metagenomic sequencing library protocols. PCR amplification was performed using PCR forward primer 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3' (SEQ ID NO: 1) and reverse primer 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3' (SEQ ID NO: 2) After denaturing the DNA at 95 ° C for 3 minutes, 95 ° C- A total of 25 cycles were repeated in cycles of 30 seconds, 55° C.-30 seconds, and 72° C.-30 seconds. Finally, the V3-4 variable region of V1-9 of the bacterial 16S rDNA was amplified at 72° C. for 5 minutes and stored at 4° C. (Klindworth et al ., Nucleic Acids Res., doi: org/10.1093/nar/han) 808). PCR amplified using V3-4 primer was mixed in the same amount and the amplicon (about 200 bp) was confirmed by electrophoresis, and then purified using AMPure XP beads (Beckman Coulter, High Wycombe, UK), and then After denaturing DNA for 3 minutes at 95°C for index PCR to add multiplex indexes and Illumina sequencing adapter, 8 cycles at 95°C-30 seconds, 55°C-30 seconds, 72°C-30 seconds, and finally 72°C for 5 minutes did. After obtaining the DNA concentration in the final product, normalization and pooling, the base sequence was analyzed using the MiSeq ^TM platform (Illumina inc., San Diego, CA, USA). In order to know the type of intestinal flora in the sample and its ratio, the nucleotide sequences were grouped based on the similarity (97%) between the nucleotide sequences obtained from the sample, and the microbial diversity in the sample was evaluated by using this as a unit. The operational taxonomic unit (hereinafter referred to as OTU) and the composition of the intestinal flora were analyzed by QIIME (Quantitative Insights Into Microbial Ecology), and Calypso software was used for data visualization.

The culture medium at 48 hours, in which the concentration difference of short-chain fatty acids was clearly observed between each treatment group, was collected and meta-analysis of the intestinal flora was performed by the next-generation sequencing method.

6 is an analysis of the diversity of enterobacteriaceae. Multivariate analysis of sequencing data by meta 16S analysis was applied, and diversity analysis was performed by principal coordinate analysis (PCoA) from the types and number of OTUs that emerged. As a result, the number of intestinal bacterial species in each sample showed a statistically significant difference through alpha-diversity analysis (Kruskal-Wallis test, p <0.05, FIG. 6(a)). In addition, it was observed that the distribution (plot) distance between the cellulose, xylo-oligosaccharide, and red ginseng powder indigestible substances was far apart from each other through principal coordinate analysis. These results mean that the three types of samples have different effects on the formation of enterobacteriaceae (type and number of OTUs) (beta-diversity, FIG. 6(b)).

In addition, the 16S rRNA gene sequencing data was compared with the existing database to identify enterobacteriaceae at the phylum level. As a result, a total of 8 phyla were found, and the relative distribution ratios of the top 4 phyla were Actinobacterium (approximately 3%, 3%, 6%, cellulose, xylooligosaccharide, and red ginseng gourd), Bacteroidetes (approximately 20%, 3%, 38%, cellulose, xylo-oligosaccharide and red ginseng extract net), Firmicutes (approx. 73%, 90%, 54%, cellulose, xylo-oligosaccharide and red ginseng extract net) and Proteobacterium (approximately 2.2%, 0.3%, 0.8%, cellulose) , xylo-oligosaccharide and red ginseng gourd) accounted for more than 97% of the total. In addition, cluster analysis was performed according to the relative composition ratio of the intestinal flora observed in each of the 15 samples.

7 shows the cluster distribution. Each culture medium was composed of the same unit to form 3 clusters for each sample. Such cluster formation was close to cellulose and red ginseng, but far from xylo-oligosaccharide. Because red ginseng gourd contains cellulose, it is judged that a pattern similar to cellulose appeared rather than xylo-oligosaccharide.

8 shows the relative distribution ratio of enterobacteriaceae between samples in a phylum unit. As can be seen here, the relative ratio of phyla between each sample was different (Kruskal-Wallis test, p <0.05). For example, Bacteroidetes , which is reported to be universally increased in the large intestine by dietary fiber intake, and Firmicutes , which is reported to be decreased, significantly increased and decreased in the red ginseng extract treated group compared to other treatments, respectively. Also, the relative distribution ratio of each sample was significantly different in Actinobacterium and proteobacterium phyla.

9 shows the relative composition ratio of enterobacteriaceae at the genus level. By comparing the 16S rRNA gene sequencing data with the existing database, the relative composition ratio of enterobacteriaceae was observed at the genus level. In particular, as a result of comparing homology according to the relative composition ratio of the intestinal flora at the genus level, the composition ratio of the enterobacteriaceae was different for each sample as the 58 genera enteric bacteria were expressed in different colors. In this experiment, 41 genera of enterobacteriaceae could be classified, but 17 genera could not be classified.

As a result of comparing the difference according to the relative composition ratio of the intestinal flora at the genus level, the relative distribution of the intestinal bacteria was found to be similar to the difference at the phylum level.

10 shows the relative proportions of intestinal flora in genus affiliation. In particular, as a result of analyzing the differences between each sample addition group by selecting characteristic bacteria from the genus unit, the group administered with the indigestible substance of red ginseng extract showed higher levels of bacteria of the genus Prevotella of the phylum Bacteroidetes and the bacteria of the genus Bifidobacterium of the phylum Actinoacteria compared to the other two administration groups. The relative ratio was significantly increased compared to other administration groups. Interestingly, the results of the Bifidobacterium genus in the meta-analysis were similar to the results of the Bifidobacterium colonies observed in the selective medium. However, the relative ratio of bacteria of the genus Lactobacillus of the phylum Firmicutes was significantly higher only in the xylooligosaccharide treatment group. On the other hand, the bacteria of the genus Prevotella are mainly found in the oral cavity and intestines, rumen or soil of humans, and are known to exist in large numbers in the intestines of people living in Africa or Southeast Asia. It has been reported that the bacteria of the genus Prevotella predominate in those who consume carbohydrates, especially fiber (Wu et al., Science, doi:10.1126/science.1208344). In particular, the Prevotella copri species is It has a high ability to break down dietary fiber and is known to produce succinic acid or acetic acid as a major metabolite. Also in this experiment, the relative distribution ratio of Prevotella copri was significantly higher in the group administered with red ginseng meal indigestible than the other two samples.

8) Ammonia nitrogen measurement

Ammonia nitrogen concentration in the culture was measured with an ammonia analysis kit (Wako, Osaka, Japan). For analysis, the culture medium was centrifuged (16,000 rpm, 4°C, 15 minutes) to take the supernatant, and the supernatant was appropriately diluted with 0.1 M phosphate buffer (pH5.5). Finally, the absorbance was measured at a wavelength of 620 nm using a microplate reader (Multiscan FC, ThermoFisher). Ammonia nitrogen concentration was calculated using a calibration curve prepared from a standard solution.

Figure 11 shows the change in ammonia nitrogen production during the culture period of the indigestible material of red ginseng. As shown here, the ammonia nitrogen concentration in the cellulose-added group continued to rise from the initiation of the culture to the end of the culture. At 6 hours after the initiation of culture, the ammonia nitrogen concentration in the group with the indigestible substance added to red ginseng foil was significantly (p<0.05) suppressed than the ammonia nitrogen concentration in the group with cellulose and xylo-oligosaccharide added. The ammonia nitrogen concentration in the xylo-oligosaccharide-added group peaked at the 6th hour of incubation, and then decreased significantly (p<0.05) compared to the cellulose-added group as well as the red ginseng meal indigestible group.

In this experiment, the suppression of ammonia nitrogen production in the fermenter with indigestible substances added to the fermenter is highly likely due to reduced ammonia production at low pH due to organic acid production.

9) Skatole measurement

The previously reported analysis method (Walstra et al ., Livest. Prod. Sci., doi: org/10.1016/S0301-6226(99)00054-8) was measured with small modifications. Specifically, as a standard, 10 mg of skatole (3-methyl indole) was dissolved in a small amount of 99% ethanol and dissolved in 100 mL of 0.1 M Tris-HCl buffer (pH7.5) (final 100 μg/mL). As a coloring solution, 0.5 g of p -dimethyl aminobenzenealdehyde was dissolved in 50 mL of alcohol containing sulfuric acid (20 mL of sulfuric acid + 30 mL of 95% ethanol). The sample for analysis was used by adding 20 µL of 2 M Tris-HCl buffer (pH7.5) to 380 µL of the culture supernatant. A standard solution or a sample solution was added to each well of a 96-well plate, a coloring solution was added, mixed well, and the reaction was allowed to stand at room temperature for 20 minutes. Then, the absorbance was measured at a wavelength of 570 nm using a microplate reader (Multiscan FC).

12 shows the change in skatole production during the incubation period of the red ginseng meal indigestible material. In this experiment, the addition of xylooligosaccharide inhibited the formation of skatole the most. In particular, it peaked at 6 hours of incubation time and decreased statistically significantly (p<0.05) compared to the cellulose-added group after 12 hours. In contrast, the group added with red ginseng powder indigestible material peaked at 12 hours of incubation time and showed a mild decrease thereafter. 0.05) showed a low production concentration.

Claims (6)

A food composition for improving intestinal health comprising, as an active ingredient, an unhydrolyzed product of red ginseng gourd remaining after hydrolyzing red ginseng gourd with alpha-amiroglucosidase and pancreatin digestive enzymes, wherein the unhydrolyzed product of red ginseng gourd is insoluble A food composition for improving intestinal health, characterized in that it contains indigestible protein in addition to dietary fiber and indigestible starch. According to claim 1,
The composition is Firmicutes ( F irmicutes ) and Bacteroidetes ( Bacteroidetes ) Food composition for improving intestinal health, characterized in that it improves the ratio of the phylum in the intestinal flora. According to claim 1,
The composition is Bifidobacterium ( Bifidobacterum ) A food composition for improving intestinal health, characterized in that it proliferates. According to claim 1,
Food composition for improving intestinal health, characterized in that the composition suppresses intestinal ammonia production to lower blood ammonia concentration. A feed composition for improving intestinal health comprising, as an active ingredient, an unhydrolyzed product of red ginseng gourd remaining after hydrolyzing red ginseng gourd with alpha-amiroglucosidase and pancreatin digestive enzymes, wherein the unhydrolyzed product of red ginseng gourd is insoluble A feed composition for improving intestinal health, comprising: indigestible protein in addition to dietary fiber and indigestible starch. The method of claim 5, wherein the composition improves the ratio of Firmicutes and Bacteroidetes in the intestinal flora, and Bifidobacterum to proliferate and inhibit intestinal ammonia production. A feed composition for improving intestinal health, characterized in that it lowers the blood ammonia concentration.

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