KR102169878B1 - Fermented glutinous rice product and food or pharmaceutical composition comprising the same - Google Patents

Abstract

The present invention relates to a glutinous rice fermentation product and a pharmaceutical and food composition containing the same, wherein the glutinous rice fermentation product has low toxicity and can proliferate or activate beneficial bacteria in the intestine, thereby being able to alleviate intestinal bacterial flora. Therefore, the glutinous rice fermentation product can prevent or alleviate inflammatory intestinal disease.

Description

Fermented glutinous rice and a pharmaceutical or food composition containing the same TECHNICAL FIELD [FERMENTED GLUTINOUS RICE PRODUCT AND FOOD OR PHARMACEUTICAL COMPOSITION COMPRISING THE SAME}

The present invention relates to a fermented glutinous rice and a pharmaceutical or food composition containing the same.

Inflammatory bowel disease (IBD) is largely classified into ulcerative colitis (UC) and Crohn's disease (CRD). The exact cause is currently unknown, but both UC and CRD are known to be caused by a complex interaction between genetic and environmental factors and intestinal immune factors.

Among them, UC has abdominal pain, weight loss, and hemogeneic diarrhea as the main symptoms. Until now, the exact cause of UC is not well known, but a remarkable increase in IL-6 content in the intestinal mucosa of UC patients has been observed, and an increase in TNF-α content in blood, colon tissue and feces has also been frequently observed. It is also known that at least inflammatory cytokines are involved in the initiation of UC.

Currently, sulfasalazine, 5-aminosalicylates, steroids, thiopurines, and biological agents are known as UC treatments. Among them, sulfasalazine, which can inhibit NF-κB activity, is most commonly used. However, it has been known that sulfasalazine may cause excessive oxidative stress when used in excessive amounts for a long period of time, resulting in blood disorders, liver toxicity, hypospermia, and male infertility. As such, UC treatments currently being used not only have serious side effects, but are expensive, and thus place a huge economic burden on individuals, families and society. Therefore, there is an urgent need to develop alternative therapies derived from natural products having relatively low toxicity and relatively inexpensive for the treatment of UC.

An object of the present invention is to provide a glutinous rice fermentation product having low toxicity and excellent anti-inflammatory and antioxidant activity against inflammatory bowel disease.

In addition, another object of the present invention is to provide a pharmaceutical composition and food composition capable of preventing or improving inflammatory bowel disease by containing the above-described fermented glutinous rice.

In order to achieve the above object, the present invention is a step of mixing glutinous rice with water and gelatinizing to obtain a glutinous rice hydrate; Obtaining a first fermented glutinous rice by treating the glutinous rice with saccharification enzyme and yeast; Yeast treatment of the first fermented glutinous rice with Saccharomyces sp. yeast to obtain a second fermented glutinous rice; And it provides a fermented glutinous rice prepared by a method comprising; and fermenting the second fermented glutinous rice with Weissella sp. lactic acid bacteria to obtain a third fermented glutinous rice.

In addition, the present invention provides a pharmaceutical composition for preventing or improving inflammatory bowel disease containing the above-described fermented glutinous rice as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving inflammatory bowel disease containing the above-described fermented glutinous rice as an active ingredient.

Since the glutinous rice fermentation product according to the present invention has low toxicity and can proliferate or activate beneficial bacteria in the intestine, the intestinal flora can be improved, and thus, can prevent or improve inflammatory bowel disease.

1 is a graph showing the change in the content of IL-6 in serum for the fermented extracts of Example 1 and Comparative Examples 1-3.
2 is a graph showing changes in IL-6 content by concentration of fermented glutinous rice of Example 1.
3 is a graph showing changes in body weight of experimental groups 1-3 and control groups 1-3.
4 is a photograph showing the degree of improvement of bloody diarrhea in experimental groups 1-3 and controls 1-3.
5 is a photograph showing the separated cecum-rectal portion of the experimental groups 1-3 and the control groups 1-3, and the size of the scale bar is 18.5 mm.
6 is a graph showing the weight of the separated cecum-rectal portion of the experimental groups 1-3 and the control groups 1-3.
7 is a graph showing the length of the separated cecum-rectal portion of experimental groups 1-3 and controls 1-3.
8 is a graph showing the MOP activity in the separated colon tissues of experimental groups 1-3 and control groups 1-3.
9 is an optical micrograph showing the histological profile of the separated colon tissues of experimental groups 1 to 3 and control groups 1 to 3, and the size of the scale bar is 100 μm.
10 is an optical micrograph showing an inflammation-inducing substance and an immune cell marker located in the separated colonic mucosa of experimental groups 1 to 3 and control groups 1 to 3, and the size of the scale bar is 100 μm.
11 is a graph analyzing the glutinous rice fermented product obtained in Example 1.

Hereinafter, the present invention will be described.

In the present invention, "inflammatory bowel disease" refers to a disease that causes inflammation in the intestine, and specifically, may be a chronic recurrent inflammatory disease caused by an abnormal regulation of the immune system in the small intestine and large intestine, and ulcerative colitis, Crohn's disease, etc. Include.

<Fermented glutinous rice and its manufacturing method>

The glutinous rice fermentation product according to the present invention is a glutinous rice fermentation product obtained by fermenting glutinous rice or a product resulting from the fermentation product, specifically, the glutinous rice fermentation product itself, or the glutinous rice fermentation product by solvent extraction, hot water extraction, cold needle extraction, reflux cooling extraction or Contains concentrates extracted by methods such as ultrasonic extraction. The glutinous rice fermented product may be provided in the form of powder, tablet, or soft, and there is no limitation on the form of the provision.

However, the glutinous rice fermented product of the present invention is produced by fermenting glutinous rice through a specific three-stage fermentation process, and according to an example, the steps of mixing glutinous rice with water and gelatinizing to obtain glutinous rice hydrate; Obtaining a first fermented glutinous rice by treating the glutinous rice with saccharification enzyme and yeast; Yeast treatment of the first fermented glutinous rice with Saccharomyces sp. yeast to obtain a second fermented glutinous rice; And it is prepared by a method comprising the step of fermenting the second fermented glutinous rice with Weissella sp. lactic acid bacteria to obtain a third fermented glutinous rice. If necessary, the above-described manufacturing method comprises the steps of filtering and concentrating the third fermented glutinous rice to obtain a fermented glutinous rice concentrate; Mixing the glutinous rice fermentation concentrate with dextrin; And sterilizing the mixture and then drying the mixture. Since the glutinous rice fermented product of the present invention prepared by such a method can proliferate beneficial bacteria in the intestines such as bifidobacterial, the intestinal flora is improved, thereby preventing or improving inflammatory bowel disease.

Hereinafter, a method of preparing the fermented glutinous rice of the present invention will be described by dividing each process.

(a) step of gelatinization of glutinous rice

Glutinous rice is mixed with water and gelatinized to obtain glutinous rice (hereinafter referred to as'S100 step').

According to an example, step S100 may be gelatinized by immersing glutinous rice in water (eg, distilled water) and then applying heat at about 90 to 125° C. for about 10 to 90 minutes. Through this step, the starch particles of glutinous rice are gelatinized into a gel state, so that the micelle particles of glutinous rice are formed widely, which facilitates the action of enzymes, yeast, and lactic acid bacteria, which will facilitate the subsequent fermentation. I can.

Unlike spicy rice, glutinous rice is rice composed mostly of amylopectin, and contains about 5% by weight or less of amylose. Fermented water obtained by fermenting glutinous rice has superior anti-inflammatory efficacy compared to fermented spicy rice obtained by fermenting spicy rice, and thus can significantly reduce the expression of IL-6 protein in serum. These glutinous rice can be crushed and used in the form of powder to promote gelatinization. In addition, when glutinous rice is immersed in water for about 10 to 60 minutes, the gelatinization action can be promoted because the glutinous rice contains adequate moisture.

The content of water may be about 250 to 350 parts by weight based on 100 parts by weight of glutinous rice, but is not limited thereto.

The glutinous rice gelatin obtained through the above-described gelatinization process is a solution containing a translucent colloidal substance in which glutinous rice swells and viscosity is increased.

(b) saccharification enzyme and yeast treatment step

The glutinous rice curd obtained in the step S100 is treated with saccharification enzyme and yeast to obtain a first fermented glutinous rice (hereinafter referred to as'step S200').

Step S200 is a simultaneous saccharification and fermentation process (simultaneous saccharification and fermentation, SSF) in which saccharification of glutinous rice starch by saccharification enzyme and fermentation by yeast are simultaneously performed by adding saccharification enzyme and yeast to the gelatinized glutinous rice. to be.

According to an example, step S200 may be performed by adding a saccharifying enzyme and yeast to the glutinous rice cake obtained in step S100, and then reacting it at a temperature of about 50 to 60° C. for about 90 to 150 minutes.

Non-limiting examples of glycosylation enzymes that can be used in this step include alpha-amylase, beta-amylase, glucoamylase, pullulanase, and the like, The available yeast can be used without any restrictions as long as it is a general yeast sold in the market. By using the saccharification enzyme and yeast together, not only the saccharification rate is improved compared to the case of using only the saccharification enzyme treatment, but the fermentation produced is easier to treat the yeast, so the present invention saccharifies the starch of glutinous rice and at the same time It can be fermented.

The content of the saccharifying enzyme and the yeast is not particularly limited, as an example, the content of the saccharifying enzyme may be about 0.5 to 5 parts by weight based on 100 parts by weight of the glutinous rice hydrate, and the content of the yeast is 100 parts by weight of the glutinous rice hydrate. It may be about 1 to 20 parts by weight based on parts. If the contents of the saccharifying enzyme and the yeast are in the above-described range, optimum reaction conditions can be obtained, thereby reducing production costs, and high-quality glutinous rice fermentation products can be obtained.

The first fermented glutinous rice obtained in this step contains about 50 to 55 mg/g of galato oligosaccharides (eg, stachyose, raffinose, etc.).

(c) Yeast treatment step

The first fermented glutinous rice obtained in step S200 is yeast treated with Saccharomyces sp. yeast to obtain a second fermented glutinous rice (hereinafter referred to as'step S300'). Since the second fermented glutinous rice obtained through this step contains more galacto-oligosaccharide than the first fermented glutinous rice obtained in step S200, the expression of IL-6 protein in the serum can be further reduced.

According to an example, step S300 is inoculated with Saccharomyces sp. yeast in the first fermented glutinous rice obtained in step S200, and then incubated at a temperature of about 25 to 35° C. for about 20 to 36 hours. Secondary fermentation is possible.

A saccharide used in this step My process in (Saccharomyces sp.) Examples of the yeasts in the My process three Levy Jia as Saccharomyces (Saccharomyces cerevisiae), MRS yuba room as Saccharomyces (Saccharomyces uqvarum), Saccharomyces ellipsis Soy Amadeus (Saccharomyces ellipsoideus), MRS with saccharide car's Bergen sheath (Saccharomycescarlsbergensis), MRS sake as Saccharomyces (Saccharomyces sake), to Saccharomyces Correa Taunus (Saccharomyces coreanus), MRS with a Mrs Lee poly urticae (Saccharomyceslipolytica) saccharide saccharide bowl radio (Saccharomyces boulardii ) And Saccharomyces pastorianus , and these may be used alone or in combination of two. According to one example, the yeast of the genus Saccharomyces sp. is Saccharomyces cerevisiae .

The content of the yeast Saccharomyces sp. is not particularly limited, and may be, for example, about 0.5 to 10.0 parts by weight based on 100 parts by weight of the first fermented glutinous rice. If the content of the yeast is within the above-described range, the optimum fermentation rate can be obtained, and the production of by-products can be reduced, thereby obtaining a high-quality glutinous rice fermentation product.

The second fermented glutinous rice obtained in this step contains about 55 to 60 mg/g of galato oligosaccharides (eg, stachyose, raffinose, etc.).

(d) fermentation stage of lactic acid bacteria

The second fermented glutinous rice obtained in step S300 is fermented with Weissella sp. lactic acid bacteria to obtain a third fermented glutinous rice (hereinafter referred to as'step S400'). Since the third fermented glutinous rice obtained through this step contains more galacto-oligosaccharide than the fermented second glutinous rice, the expression of IL-6 protein in the serum can be further reduced.

According to an example, step S400 is fermented by inoculating the second glutinous rice fermented product obtained in step S300 with Weissella sp. lactic acid bacteria and then incubating for about 20 to 36 hours at a temperature of about 25 to 35 °C. I can.

Wei Cellar in used in the step (Weissella sp.) As a lactic acid bacteria derived from lactic acid bacteria sauerkraut, e.g. Wei Cellar cibaria (Weissella cibaria), Wei Salah Collector N-Sys (Weissellakoreensi), Wei Cellar do children (Weissellahanii), Wei Cellar There is a child, such as Kimchi (Weissellakimchii), Wei Cellar Solid (Weissellasoli), four-way Cellar konpu (Weissellaconfusa), it not limited to this. These may be used alone or in combination of two or more.

The content of the Weissella sp. lactic acid bacteria is not particularly limited, and may be, for example, about 0.5 to 10.0 parts by weight based on 100 parts by weight of the second fermented glutinous rice. If the content of the lactic acid bacteria is within the above-described range, an optimum fermentation rate can be obtained, and production of by-products can be reduced, thereby obtaining a high-quality glutinous rice fermentation product.

The third fermented glutinous rice obtained in this step contains about 73 mg/g or more, specifically about 73 to 95 mg/g of galato oligosaccharide (eg, stachyose, raffinose, etc.).

(e) If necessary, the third fermented glutinous rice fermented in step S400 may be filtered and concentrated. At this time, filtration and concentration may be performed according to a conventional method known in the art. According to an example, after filtering the third fermented glutinous rice, it can be concentrated at about 55 ~ 65 ℃.

In addition, the glutinous rice fermentation concentrate may be sterilized and dried. At this time, as the drying method, a method commonly known in the art may be used such as vacuum drying, spray drying, freeze drying, and the like. The powdered glutinous rice fermentation product can be used as a food composition or a pharmaceutical composition.

In addition, the glutinous rice fermentation concentrate may be mixed with dextrin before being sterilized and dried. Dextrin has low water absorption and is excellent in dispersibility, solubility and drying properties. Therefore, by mixing the glutinous rice fermentation concentrate with dextrin, it is possible to improve the powdering characteristics of the final product in the drying process to obtain a powder product of excellent quality. In this case, the ratio (mixing ratio) of the glutinous rice fermentation concentrate and dextrin is not particularly limited, and may be, for example, a weight ratio of 60:40 to 95:5.

Since the glutinous rice fermented product of the present invention prepared by the above-described method can proliferate or activate beneficial bacteria in the intestine such as bifidobacterial, the intestinal flora is improved, thereby preventing or improving inflammatory bowel disease. . In particular, the glutinous rice fermentation product of the present invention not only reduces NF-κB expression when administered orally to an animal model in which inflammatory bowel disease is induced, but also minimizes or inhibits the occurrence of ulcerative colitis (UC) through antioxidant and probiotic effects. I can.

<Pharmaceutical composition containing fermented glutinous rice>

The present invention provides a pharmaceutical composition for preventing or improving inflammatory bowel disease containing the above-described fermented glutinous rice as an active ingredient. The glutinous rice fermentation product can prevent or improve inflammatory bowel disease by proliferating or activating beneficial bacteria in the intestine.

The pharmaceutical composition of the present invention may include oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols according to a conventional method; External preparations; Suppositories; It may be formulated and used in the form of an injection or the like.

At this time, the pharmaceutical composition of the present invention may further include carriers, excipients, and diluents known in the art, in addition to the above-described fermented glutinous rice.

Carriers, excipients, and diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and when administered parenterally, it is possible to select an injection method for external use of the skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection, It is not limited to this.

The dosage of the pharmaceutical composition according to the present invention should be a pharmaceutically effective amount. "Pharmaceutically effective amount" means an amount sufficient to prevent or treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the method of formulation, the condition and weight of the patient, the severity of the disease, the drug It may be appropriately selected according to factors such as form, route of administration and duration, and is not particularly limited. Such administration may be administered once a day or may be divided several times. The above dosage does not in any way limit the scope of the present invention.

<Food composition containing fermented glutinous rice>

The present invention provides a food composition for preventing or improving inflammatory bowel disease containing the above-described fermented glutinous rice as an active ingredient. When the glutinous rice fermented product is included in food and consumed, it can effectively prevent or improve inflammatory bowel disease.

The food composition of the present invention may be added to the glutinous rice fermented product as it is, or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method.

Non-limiting examples of the foods include drinks, meat, sausages, bread, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, alcohol There are beverages, vitamin complexes, etc., and include all health functional foods in the usual sense.

At this time, the content of the fermented glutinous rice is not particularly limited, and may be appropriately adjusted within a range known in the art. At this time, the content of fermented glutinous rice is adjusted in consideration of safety as well as health and hygiene.

According to one example, the food composition of the present invention may be a health functional beverage composition. In this case, in addition to containing the glutinous rice fermented product as an active ingredient, it may further include various flavoring agents or additives such as natural carbohydrates, such as a normal beverage.

Examples of the natural carbohydrates include conventional sugars such as monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), polysaccharides (eg, dextrin, cyclodextrin, etc.) , Sugar alcohols such as xylitol, sorbitol, and erythritol.

In addition, as flavoring agents, there are natural flavoring agents (eg, taumatin, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.).

On the other hand, the food of the present invention, in addition to the above-described components, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its Salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like may further be contained. In addition, the food composition of the present invention may further contain pulp for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The content of these additives is not so important, and may range from about 0 to about 20 parts by weight based on 100 parts by weight of the fermented glutinous rice of the present invention.

Hereinafter, the present invention will be described in detail through examples, but the following examples are only illustrative of the present invention, and the present invention is not limited by the following examples.

<Example 1>-Preparation of fermented glutinous rice

Glutinous rice was immersed in three times the amount of water (about 30° C.) for 60 minutes to soak, and then 4 kg of the mixture was gelatinized at a temperature of about 121° C. for about 1 hour to obtain glutinous rice. Thereafter, 20 ml of alpha-amylase and 5 g of malt were added to 4 kg of glutinous rice, and the first fermented glutinous rice was obtained by simultaneous saccharification fermentation at a temperature of about 55° C. for about 2 hours. Subsequently, 100 g of yeast Saccharomyces cerevisiae (ATCC 9804; ATCC, Manassas, VA, USA) was inoculated to 4 kg of the first fermented glutinous rice and fermented for about 24 hours at a temperature of about 30°C. Thus, a second fermented glutinous rice was obtained. Thereafter, 200 g of lactic acid bacteria Weissella cibaria (KACC 11845; KACC, Suwon, Korea) was inoculated into 4 kg of the second fermented glutinous rice and fermented for about 24 hours at a temperature of about 30 °C to ferment the third glutinous rice. Got water. Thereafter, the third fermented glutinous rice was filtered, concentrated at a temperature of 60° C., and then dextrin was mixed thereto. Then, the mixture (glutinous rice concentrate: dextrin = 90:10 weight ratio) was sterilized at a temperature of about 80° C. for about 1 hour and spray-dried to obtain a glutinous rice fermented product (galacto-oligosaccharide content: about 73.02 mg/g). Was prepared.

The results of analyzing the final glutinous rice fermented product obtained above are shown in Table 1 and FIG. 11 below.

oligosaccharide Concentration of fermented glutinous rice (μg/mL) Final volume (mL) Sample collection amount (g) Dilution factor Results (mg/g) Stachyose 419.5390 50 0.8323 One 25.2036 Raffinose 795.9573 50 0.8323 One 7.8167 Total amount of galactooligosaccharide 73.0203

<Comparative Example 1>-Preparation of fermented glutinous rice

Glutinous rice flour and water were mixed in a weight ratio of 1:3, and then 4 kg of this mixture was gelatinized at a temperature of about 121° C. for about 1 hour to obtain glutinous rice flour. Thereafter, 20 ml of alpha-amylase and 5 g parts by weight of malt were added to 4 kg of glutinous rice, followed by simultaneous saccharification fermentation at about 55° C. for about 2 hours to obtain a glutinous rice fermentation product. Thereafter, the glutinous rice fermented product was filtered, concentrated at a temperature of 60° C., and then dextrin was mixed thereto. Subsequently, the mixture (glutinous rice concentrate: dextrin = 90:10 weight ratio) was sterilized at a temperature of about 80° C. for about 1 hour and spray-dried to obtain a glutinous rice fermented product (galacto-oligosaccharide content: about 52.07 mg/g). Was prepared.

<Comparative Example 2>-Preparation of fermented glutinous rice

Glutinous rice flour and water were mixed in a weight ratio of 1:3, and then 4 kg of this mixture was gelatinized at a temperature of about 121° C. for about 1 hour to obtain glutinous rice flour. Thereafter, 20 ml of alpha-amylase and 5 g of malt were added to 4 kg of glutinous rice, followed by fermentation at a temperature of about 55° C. for about 2 hours to obtain a first fermented glutinous rice. Subsequently, 100 g of yeast Saccharomyces cerevisiae (ATCC 9804; ATCC, Manassas, VA, USA) was inoculated to 4 kg of the first fermented glutinous rice and fermented for about 24 hours at a temperature of about 30°C. Thus, a second fermented glutinous rice was obtained. Thereafter, the second fermented glutinous rice was filtered, concentrated at a temperature of 60° C., and then dextrin was mixed thereto. Then, the mixture (glutinous rice concentrate: dextrin = 90:10 weight ratio) was sterilized at a temperature of about 80° C. for about 1 hour and spray-dried to obtain a glutinous rice fermented product (galacto-oligosaccharide content: about 59.59 mg/g). Was prepared.

<Comparative Example 3>-Preparation of fermented rice extract

A fermented rice extract was prepared in the same manner as in Example 1, except that rice powder was used instead of the glutinous rice powder used in Example 1.

<Experimental Example 1>-Inhibitory efficacy of fermented glutinous rice

In order to confirm the inhibitory effect of inflammatory bowel disease of the fermented extracts prepared in Example 1 and Comparative Examples 1 to 3, respectively, changes in IL-6 content in serum were measured as follows. The measurement results are shown in FIG. 1.

After stabilizing colon cells by culturing in a 12-well plate, 1 μg/ml of LPS (Lipopolysaccharide) was treated, and the glutinous rice fermented products of Examples 1 and 1 to 2 and the rice fermented products of Comparative Example 3 were prepared. Each was treated at 200 μg/ml. The cells treated with each fermentation product were cultured in a CO ₂ incubator for 24 hours, and then the supernatant of each cell medium was taken, and then stored in a -20° C. freezer, and analyzed. At this time, as a control, colon cells not treated with LPS (hereinafter,'Con group'), and colon cells treated with only LPS (LPS group) were used.

The amount of IL-6 in the medium ejected from the cells was detected with a commercially available ELISA kit and a microplate reader. Each value was expressed as the mean ± standard deviation of cells in 4 wells, and the unit was pg/mg protein. The ELISA kit used in this assay was the Human IL-6 ELISA kit from eBioscience.

As can be seen from Figure 1, the glutinous rice fermented product of Example 1 had a lower IL-6 content in serum than that of the glutinous rice fermented product of Comparative Examples 1-2. In addition, the glutinous rice fermented product of Example 1 had about two times lower IL-6 content in serum than the rice fermented extract of Comparative Example 3 using rice flour.

<Experimental Example 2>-Inhibitory efficacy of glutinous rice fermented product by concentration

In order to confirm the inhibitory effect of inflammatory bowel disease according to the concentration of the glutinous rice fermented product prepared in Example 1, the change of IL-6 content in the serum was measured as follows. The measurement results are shown in FIG. 2.

After stabilizing colon cells by culturing them in a 12-well plate, LPS (Lipopolysaccharide) was treated with 1 μg/ml, and the glutinous rice fermented product of Example 1 was concentration-dependently 0, 12, 5, 25, 50, 100 , 200 μg/ml were each treated. After the cells treated with the glutinous rice fermentation by concentration were cultured in a CO ₂ incubator for 24 hours, the supernatant of each cell medium was taken, and then stored in a -20°C freezer and analyzed. At this time, as a control, colon cells not treated with LPS (hereinafter,'Con group') were used. The amount of IL-6 in the medium ejected from the cells was detected with a commercially available ELISA kit and a microplate reader. Each value was expressed as the mean ± standard deviation of cells in 4 wells, and the unit was pg/mg protein. The ELISA kit used in this assay was the Human IL-6 ELISA kit from eBioscience.

As can be seen from Figure 2, as the concentration of the glutinous rice fermented product prepared in Example 1 increased, the IL-6 content in the serum was lowered.

<Experimental Example 3>-Effect of preventing or improving inflammatory bowel disease of fermented glutinous rice

In order to confirm the effect of preventing or improving inflammatory bowel disease of the fermented glutinous rice prepared in Example 1, each of the intestinal DSS-induced enteritis mouse models were evaluated as follows.

VAF/SPF C57BL/6N male mice [6 weeks old, body weight (average 20.53±0.77 g; 18.77-22.1 g)] to the glutinous rice fermentation product of Example 1 (FRe 300, 200, 100 mg/kg) once daily 7 Daily forced oral administration, body weight change, Disease Activity Index (DAI) change, cecum-rectal weight change, colon-rectal length change, microbiological change in colon contents, change in serum Ig content, change in MPO activity in colon tissue, Changes in serum cytokine content, changes in cytokine content in colon tissue, changes in lipid peroxidation and antioxidant defense systems in colon tissue, changes in mRNA expression in colon tissue, histopathological changes in colon, and immunohistochemical changes were observed, respectively. Here, FRe represents the glutinous rice fermented product of Example 1.

At this time, 8 male mice were prepared per group, a total of 6 groups as follows.

I) Experimental group 1 (FRe300 group): After dissolving DSS (Dextran sodium sulfate) (Sigma-Aldrich, No. 42867) at a concentration of 3.5% (35 mg/ml) in normal tap water, it was male with negative water for 7 days. After inducing UC by supplying it to mice, 300 mg/kg of FRe was forcibly orally administered once daily to male mice for 7 days.

Ii) Experimental group 2 (FRe200 group): DSS (Dextran sodium sulfate) (Sigma-Aldrich) was dissolved in normal tap water at a concentration of 3.5% (35 mg/ml), and then supplied to male mice in negative water for 7 days. After UC was induced, 200 mg/kg of FRe was forcibly orally administered once daily to male mice for 7 days.

Iii) Experimental group 3 (FRe100 group): After dissolving DSS (Dextran sodium sulfate) (Sigma-Aldrich) in general tap water at a concentration of 3.5% (35 mg/ml), it was supplied to male mice in negative water for 7 days. After inducing UC, 100 mg/kg of FRe was forcibly orally administered once daily to male mice for 7 days.

Iv) Control 1: General tap water was supplied to male mice for 7 days, and then 100 mg/kg of sterile distilled water was forcibly orally administered once daily to male mice for 7 days.

V) Control 2: DSS (Dextran sodium sulfate) (Sigma-Aldrich) was dissolved in normal tap water at a concentration of 3.5% (35 mg/ml), and then it was supplied to male mice for 7 days in negative water to induce UC. Then, 100 mg/kg of sterile distilled water was forcibly orally administered once daily to male mice for 7 days.

Ⅵ) Control 3: DSS (Dextran sodium sulfate) (Sigma-Aldrich) was dissolved in normal tap water at a concentration of 3.5% (35 mg/ml), and then it was supplied to male mice in negative water for 7 days to induce UC. Thereafter, 100 mg/kg of Sulfasalazine (Sigma-Aldrich) was forcibly orally administered once daily to male mice for 7 days.

(1) weight change

The weight of each mouse was measured once a day using an automatic electronic balance (XB320M, Precisa Instrument, Dietikon, Switzerland), and the results are shown in FIG. 3. Mouse weight of a dual DDS supply before the mouse weight of the day (W _0), mice weight of the administration of the first test substance work (W _1), and the final sacrifice day (administered 7 days after the test substance has passed 24 hours) (W ₂ ) Was measured, and it is shown in Table 2 below. At this time, in order to reduce individual differences, the weight gain (ΔW) during the 7-day test period was calculated according to Equation 1 below. At this time, in order to minimize individual differences, all experimental animals were fasted for 18 hours before the day of administration of the first test substance and the day of final sacrifice, and drinking water was not restricted. In Table 1 below, each value is expressed as the weight mean ± standard deviation (SD) of 8 mice.

[Equation 1]

(In the above formula,

W ₁ is the weight of the mouse on the day of administration of the first test substance,

W ₂ is the body weight of the mouse after 24 hours of administration of the test substance on the 7th day).

Mouse weight ΔW (g) W ₀ (g) W ₁ (g) W ₂ (g) Control 1 20.54±0.76 17.25±0.66 17.93±0.66 0.67±0.35 Control 2 20.50±1.21 17.24±1.00 13.86±0.97 ^a -3.38±1.09 ^a Control 3 20.50±0.41 17.21±0.34 15.95±0.82 ^ab -1.26±0.80 ^ab Experimental group 1 20.56±1.00 17.18±0.84 16.06±0.64 ^ab -1.11±0.92 ^ab Experimental group 2 20.51±0.73 17.19±0.67 15.55±0.83 ^ab -1.64±0.94 ^ab Experimental group 3 20.55±0.51 17.26±0.54 15.24±0.32 ^ab -2.03±0.55 ^{ab a} p<0.01: compared with control 1 according to the LSD test
^b p<0.01: compared with control 2 according to the LSD test

As a result of the measurement, weight loss was observed in control group 2, which caused ulcerative colitis. On the other hand, in control 3 (administered sulfasalazine) and experimental group 1 (administered FRe 300 mg/kg) from 4 days after administration, in experimental group 2 (administered FRe 200 mg/kg) and in experimental group 3 (administered FRe 100 mg/kg), administration 5 And after 6 days, the weight was significantly increased compared to the control group 2. In addition, the amount of weight gain during the 7-day administration period was also significantly increased compared to the control group 3 as well as the experimental groups 1 to 3 compared to the control group 2 (see Tables 2 and 3).

In addition, the amount of weight gain during the administration period (7 days) changed by -600.00% in the case of control 1, compared to the control 1, whereas in the case of experimental groups 1 to 3, about 68.04%, about 52.48% and about 52.48%, respectively, compared to the control 2 It showed a change of about 41.00%. This was similar to the control group 3, in which the weight gain during the administration period (7 days) was changed by about 63.59% compared to the control group 2.

(2) Changes in disease activity index (DAI)

Disease (colitis) activity index (Disease Activity Index, DAI) was previously established [Kim et al., 2013b; Han et al., 2015] in consideration of stool consistency, weight loss, and bleeding, and scored from 0 to 4 (see Table 3), and the results are shown in Tables 4 and 4 . Each parameter in Table 3 was recorded on the basis of the final sacrifice date (24 hours after administration of the last 7th test substance). In Table 4 below, each value is expressed as a score mean±standard deviation of 8 mice.

Parameter Criteria Score Weight loss(%) <1 0 1-5 One 6-10 2 11-15 3 >15 4 stool consistency Well-formed pellets-Diarrhoea 0-4 Rectal bleeding Negative-Gross bleeding 0-4 DAI (Weight loss score + stool hardness score + rectal bleeding score)/3 0-4

Score (Max=4) DAI [(A+B+C)/3] Weight loss [A] Stool[B] Bleeding[C] Control 1 0.00±0.00 0.25±0.46 0.00±0.00 0.08±0.15 Control 2 3.63±0.53 ^a 4.00±0.00 ^a 3.75±0.46 ^a 3.79±0.31 ^a Control 3 1.88±1.13 ^ab 2.75±0.89 ^ab 2.13±0.35 ^ab 2.25±0.71 ^ab Experimental group 1 1.88±0.99 ^ab 2.88±0.99 ^ab 2.13±0.64 ^ab 2.29±0.86 ^ab Experimental group 2 2.25±1.28 ^ab 3.00±0.93 ^ac 2.50±0.76 ^ac 2.58±0.94 ^ab Experimental group 3 2.75±0.71 ^ac 3.50±0.53 ^ac 2.88±0.64 ^ac 3.04±0.53 ^{ab a} p<0.01: compared with control 1 according to MW test
^b p<0.01 and ^c p<0.01: compared with control 2 according to MW test

In control group 2, hemophilic diarrhea was observed with significant weight loss, and DAI was significantly increased compared to control group 1. On the other hand, in the experimental groups 1-3 and the control group 3, DAI significantly decreased compared to the control group 2, respectively. In particular, the DAI of experimental groups 1 to 3 were 2.29±0.86, 2.58±0.94, and 3.04±0.53, respectively, and it was confirmed that there was an inhibitory effect on the increase of DAI depending on the dose of the fermented glutinous rice. In addition, the experimental group 1 suppressed the increase in DAI similar to the control group 3 (see Table 4 and Figure 4).

Control 2 showed about 4450% change in DAI compared to control 1, whereas control 3 and experimental groups 1 to 3 showed changes of -40.66%, -39.56%, -31.87% and -19.78%, respectively, compared to control 2, respectively. Done.

(3) Changes in cecum-rectal weight and colon-rectal length

24 hours after administration of the last 7th test substance, using a rodent inhalation anesthesia device (Surgivet, Waukesha, WI, USA) and a rodent ventilator (Model 687, Harvard Apparatus, Cambridge, UK), N ₂ O and O _In a mixture of ₂ (N ₂ O=70 vol%, O ₂ =28.5 vol%), the cecum-rectal part was separated under anesthesia with 2 to 3% isoflurane (Hana Pharm. Co., Hwasung, Korea). Figure 5 is a picture showing a separate cecum-rectal portion of each mouse.

The weight of the separated cecum-rectal part was measured at g level (absolute wet-weights) with an automatic electronic balance, and in order to reduce the difference in individual weight, the weight and absolute weight of the final sacrifice date were used according to Equation 2 below. Relative weight (% body weight) was calculated. The measurement and calculation results are shown in FIG. 6. In FIG. 6, ^a p<0.01 and ^b p<0.05 are compared with control 1 according to the LSD test, and ^c p<0.01 is compared with control 2 according to the LSD test.

Using an electronic digital caliper (CD-15CPX, Mytutoyo, Tokyo, Japan), the length of the separated colon-rectal portion was measured at the cm level, and the results are shown in FIG. 7.

[Equation 2]

(In the above formula,

W _Re is the relative weight of the cecum-rectal part,

W _ab is the absolute weight of the cecum-rectal part,

W ₂ is the weight of the mouse 24 hours after administration of the last 7th test substance).

1) In the control group 2, the absolute weight and the relative weight of the cecum-rectal portion were significantly increased compared to the control group 1. On the other hand, in the control group 3 and the experimental groups 1 to 3, the absolute weight and the relative weight of the cecum-rectum were significantly decreased compared to the control group 2, respectively. In particular, the absolute and relative weights of the cecum-rectal portion of the experimental groups 1 to 3 were about 2.2 to 2.6 g and about 0.4 g, respectively. It was confirmed that there is an effect of suppressing an increase in the absolute weight and relative weight of the cecum-rectal part according to the dosage of the glutinous rice fermented product. In addition, Experimental Group 1 had an effect of suppressing an increase in the absolute weight and relative weight of the cecum-rectal portion, similar to that of the control group 3 (see FIGS. 5 and 6).

In addition, the control group 2 showed a change of about 39.41% in the absolute weight of the cecum-rectal portion compared to the control group 1. On the other hand, experimental groups 1 to 3 showed a change of about -26.62%, about -22.13%, and about -18.54% in the absolute weight of the cecum-rectal part, respectively, compared to the control group 2. In particular, the absolute weight change of the cecum-rectal portion of the experimental group 1 was similar to the absolute weight change of the cecum-rectal portion of the control group 3, which showed a change of about -27.91% compared to the control group 2.

In addition, control 2 showed a change in the relative weight of the cecum-rectal part of about 81.10% compared to the control 1, whereas in the experimental groups 1 to 3, the relative weight of the cecum-rectal part was about -36.87% and about -36.87% compared to the control 2, respectively. It showed a change of -30.78% and about -26.22%. In particular, the change in the relative weight of the cecum-rectal portion of the experimental group 1 was similar to that of the cecum-rectal portion of the control group 3, which showed a change of -37.56% compared to the control group 2.

2) In the control group 2, the length of the colon-rectal portion was significantly decreased compared to the control group 1. On the other hand, in the case of the control group 3 and the experimental groups 1 to 3, the length of the colon-rectal portion was significantly increased compared to the control group 2. In particular, in the case of experimental groups 1 to 3, there was an effect of suppressing the decrease in the length of the colon-rectal portion according to the dose of the glutinous rice fermented product. In particular, Experimental Group 1 had an effect of inhibiting length reduction of the colon-rectal portion similar to that of the control group 3 (see FIGS. 5 and 7 ).

In addition, in the case of the control group 2, the length of the colon-rectal portion showed a change of about -42.52% compared to the control 1, whereas the length of the colon-rectal portion of the experimental groups 1 to 3 was about 17.11% and about 17.11% compared to the control group 2, respectively. It showed a change of 16.32% and about 13.68%. In particular, in the case of experimental group 1, the change in the length of the colon-rectal portion was similar to that of the control group 3, which showed a change of about 16.05% compared to the control group 2.

(4) microbiological changes in colon contents

After 24 hours of administration of the last 7th test substance, colon contents were aseptically collected from all mice after cecal-rectal separation, and then diluted 10-fold and cultured in 3-fold. Chromocult agar and Bile Esculin Azide Agar were used to increase the number of aerobic bacteria ( Escherichia coli, Coliforms, and Enterococci ). The plate was incubated in aerobiosis at about 37° C. for about 24 to 48 hours. Brain heart Infusion agar, Mann Rogosa Sharpe agar (MRS), modified MRS agar, 0.3% (w/v) sodium propionate, 0.2% (w/v) lithium chloride, 0.05% (w/v) cysteine hydrochloride, and 5% (v/v) of defibrinated sheep blood was used to increase anaerobic bacteria ( Lactobacillus spp. and Bifidoacterium spp. ). The plate was routinely incubated for about 48-72 hours at about 37° C. using anaerojars (candle jars) and Anaerogen sachets. After counting the number of colonies, the results are shown in Table 5. All agar and chemicals used in this assay were purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). In Table 5 below, the unit of each colon content is CFU×log ₁₀ /g.

E.coli Enterococci Anaerobes Lactobacilli Bifidobacteria Control 1 5.11±0.27 6.08±0.14 10.90±0.39 8.24±0.31 8.25±0.21 Control 2 6.51±0.26 6.81±0.18 12.29±0.29 7.13±0.17 6.75±0.30 Control 3 6.40±0.27 6.64±0.22 12.19±0.27 7.27±0.45 6.88±0.30 Experimental group 1 5.69±0.33 6.28±0.18 11.43±0.17 8.05±0.19 7.89±0.22 Experimental group 2 6.00±0.16 6.40±0.23 11.68±0.14 7.93±0.15 7.60±0.39 Experimental group 3 6.15±0.12 6.54±0.14 11.90±0.13 7.81±0.17 7.40±0.31

As can be seen from Table 5, in the case of control 1, the number of E. coli, Enterococci and Anaerobes in the colon contents increased, and the number of Lactobacilli and Bifidobacteria decreased compared to the control 1. On the other hand, in the case of experimental groups 1 to 3, the number of E. coli, Enterococci, and Anaerobes in the colon contents significantly decreased, and the number of Lactobacilli and Bifidobacteria significantly increased compared to the control group 2. The change in the number of microorganisms in these experimental groups 1 to 3 was dependent on the dose of glutinous rice fermentation. On the other hand, in the case of the control group 3, compared to the control group 2, the number of microorganisms in the colon contents was not significantly changed.

Specifically, with respect to the number of E. coli in the contents of the colon, the control group 2 showed a change of about 27.38% compared to the control group 1, whereas the control group 3 and experimental groups 1 to 3 showed a change of about -1.73% compared to the control group 2, respectively. It showed changes of about -12.67%, about -7.87% and -5.57%.

In addition, with respect to the number of Enterococci in the contents of the colon, control 2 showed a change of about 12.14% compared to control 1, whereas control 3 and experimental groups 1 to 3 showed about -2.57% and about -7.89% compared to control 2, respectively. , About -6.06% and -4.04%.

In addition, with respect to the number of Anaerobes in the colon contents, control 2 showed a change of about 12.73% compared to control 1, whereas control 3 and experimental groups 1 to 3 showed a change of about -0.81% and about -7.02% compared to control 2, respectively. , About -4.98% and about -3.15%.

In addition, with respect to the number of Lactobacilli in the colon contents, control 2 showed a change of about -13.51% compared to control 1, whereas control 3 and experimental groups 1 to 3 showed a change of about 2.09%, about 12.98%, and about 2, respectively, compared to control 2 It showed a change of 11.23% and about 9.58%.

In addition, with respect to the number of Bifidobacteria in the colon contents, control 2 showed a change of about -18.18% compared to control 1, whereas control 3 and experimental groups 1 to 3 showed a change of about 1.85%, about 16.85%, about 1.85%, about 16.85%, about control 2 compared to control 1, respectively. It showed a change of 12.59% and about 9.63%.

(5) Changes in Ig (Immunoglobulin) content in serum

24 hours after administration of the last 7th test substance, about 1 ml of venous blood was collected from the vena cava under inhalation anesthesia. All blood samples were centrifuged for about 10 minutes at about 12,500 ppm under low temperature (about 4° C.) using clotting active serum tubes. Thereafter, after storing in a cryogenic cooler (MDF-1156, Sanyo, Tokyo, Japan) at -150 ℃, using a microplate reader (Sunrise, Tecan, Mgennedorf, Switzerland) at 450 nm of the manufacturer's According to the guidelines, the concentrations (contents) of Ig A, Ig G, and Ig M in serum were measured with enzyme-linked immunosorbent assay (ELISA) quantitative kits, respectively, and the results are shown in Table 6. The ELISA kit used in this assay was Mouse Ig A ELISA kit (MBS720755; MyBioSource San Diego, CA, USA), Mouse Ig G ELISA kit (MBS702698; MyBioSource San Diego, CA, USA), and Mouse Ig M ELISA kit (MBS703424 ; MyBioSource San Diego, CA, USA). In Table 6 below, each value represents the mean ± standard deviation of the Ig concentration of 8 mice.

Ig A (㎍/mL) Ig G (㎍/mL) Ig M (㎍/mL) Control 1 67.64±6.48 588.08±103.79 142.48±5.36 Control 2 102.99±14.86 306.75±38.45 165.78±3.92 Control 3 82.23±2.91 434.94±39.18 150.11±4.66 Experimental group 1 76.63±5.15 477.45±46.71 142.47±4.01 Experimental group 2 81.58±6.44 447.11±44.29 149.97±4.50 Experimental group 3 86.64±3.64 402.76±22.03 157.53±3.22

As a result of the measurement, in the case of the control group 2, the content of Ig A and Ig M that appeared during infection was significantly increased compared to that of the control group 1, and the content of Ig G, which showed a protective function against infection, was significantly decreased. On the other hand, in the case of experimental groups 1 to 3, the content of Ig A and Ig M in serum was significantly decreased compared to the control group 2, while the content of Ig G in serum was significantly increased. In particular, the experimental group 1 had a lower content of Ig A and Ig M in the serum than the control group 3, and the content of Ig G in the serum was higher. In addition, according to the serum Ig content of experimental groups 1 to 3, as the dose of glutinous rice fermented product increased, the serum contents of Ig A and Ig M decreased, while the serum contents of Ig G increased.

In addition, with respect to the content of Ig A in serum, control 2 showed a change of about 52.27% compared to control 1, whereas control 3 and experimental groups 1 to 3 showed about -20.16% and about -25.59%, respectively, compared to control 2 , About -20.79% and about -15.88%.

In addition, with respect to the content of Ig G in serum, control 2 showed a change of about -47.84% compared to control 1, whereas control 3 and experimental groups 1 to 3 showed a change of about 41.79%, about 55.65%, compared to control 2, respectively. It showed a change of about 45.76% and about 31.30%.

In addition, with respect to the content of Ig M in serum, control 2 showed a change of about 16.35% compared to control 1, whereas control 3 and experimental groups 1 to 3 showed about -9.45%, about -14.06%, respectively, compared to control 2, It showed a change of about -9.53% and about -4.98%.

(6) Changes in MPO (Myeloperoxidase) activity in colon tissue

Using a bead beater (TacoTMPre, GeneResearch Biotechnology Corp., Taichung, Taiwan) and an ultrasonic cell disruptor (KS-750, Madell Technology Corp., Ontario, CA, USA) of 10 volumes A colon tissue sample (about 0.2 g) was homogenized in an ice-cold potassium phosphate buffer (50 mM K ₂ HPO ₄ , pH 6.0; Sigma-Aldrich, St. Louise, MO, USA). After centrifuging the homogenate at about 12,000 × g at about 4° C. for about 10 minutes, the supernatant was separated from the pellet and removed. Thereafter, the pellet was stored at a cryogenic cooling of about -150 °C, and then 50 mM of K ₂ HPO ₄ containing about 0.5% (v/v) HETAB (hexa-decyl-trimethyl-ammonium bromide) and about 10 mM Of EDTA (Sigma-Aldrich, St. Louise, MO, USA) was re-homogenized to an equivalent volume. MPO activity was evaluated by measuring the H ₂ O ₂ -dependent oxidation of o-dianizidine·2HCl, and the results are shown in FIG. 8. Here, one unit (U) of the enzyme activity is present per 1 g of tissue weight causing a change in absorbance of 1.0/min at 460 nm and 37°C using a UV/Vis spectrometer (OPTIZEN POP, Mecasys, Daejeon, Korea). It was defined as the MPO content.

As can be seen in Figure 8, in the case of the experimental groups 1 to 3, the MPO activity was lower than that of the control group 2. This indicated that the experimental groups 1 to 3 had an effect of inhibiting MPO activity compared to the control group 2. In addition, according to the MPO activity levels of the experimental groups 1 to 3, the MPO activity was suppressed as the dose of the glutinous rice fermented product increased. In addition, experimental group 2 had almost the same MPO activity as control group 3, and experimental group 3 had lower MPO activity than control group 3.

In addition, control group 2 showed a 1520.00% change in colonic tissue MPO activity compared to control group 1, whereas control group 3 and experimental groups 1 to 3 had about -45.03% and about -67.25 of MPO activity in colon tissue compared to control 2, respectively. %, about -45.97% and about -27.81%.

(7) Changes in cytokine content in serum and colon tissue

Colon samples were digested in 5 volumes of ice-cold radioimmunoprecipitation assay (RIPA) buffer, incubated on ice for 30 minutes, and then centrifuged twice at 20,000 × g for 15 minutes at about 4°C. The resulting supernatant and serum were stored in a cryogenic cooler at -150°C and used for analysis. The levels of TNF-α, IL-6, and IL-8 in serum and colon were detected with a commercially available ELISA kit and microplate reader at 450 nm, and the results are shown in Tables 7 and 8. Done. In Table 7, each value was expressed as the mean ± standard deviation of 8 mice, and its unit was pg/mg protein.

ELISA kits used in this assay include Mouse TNF-α ELISA kit (MBS825075; MyBioSource San Diego, CA, USA), Mouse IL-8 ELISA kit (DElA1355; Creative Diagnostics, Shirley, NY, USA), and Mouse IL-6 ELISA kit (MBS2508516; MyBioSource San Diego, CA, USA).

Serum levels TNF-α IL-6 IL-8 Control 1 93.00±18.78 55.50±18.91 29.88±12.53 Control 2 304.00±71.00 218.88±24.89 111.63±10.16 Control 3 179.25±27.70 115.13±23.94 67.88±12.70 Experimental group 1 124.63±29.95 84.50±15.68 50.50±13.07 Experimental group 2 180.75±28.74 116.25±32.68 67.13±11.99 Experimental group 3 213.25±16.18 155.00±36.77 81.38±11.04

Colon tissue levels TNF-α IL-6 IL-8 Control 1 162.00±29.62 239.13±31.03 159.38±18.48 Control 2 419.50±70.46 542.63±67.38 478.13±52.86 Control 3 259.38±46.34 361.13±46.15 307.13±51.93 Experimental group 1 201.25±21.74 293.13±24.57 233.88±44.49 Experimental group 2 259.88±32.32 357.13±40.10 311.38±58.28 Experimental group 3 314.00±20.28 397.50±42.14 357.88±36.00

1) As can be seen in Table 7, control 2 significantly increased the content of TNF-α, IL-6 and IL-8 in serum compared to control 1 (p<0.01). On the other hand, experimental groups 1 to 3 significantly decreased the content of TNF-α, IL-6, and IL-8 in serum compared to the control group 2 (p<0.01). In particular, experimental group 1 showed a decrease in the content of TNF-α, IL-6, and IL-8 in serum compared to control 3. In addition, according to the content of TNF-α, IL-6, and IL-8 in the serum of experimental groups 1 to 3, as the dose of glutinous rice fermentation increases, the increase in the content of TNF-α, IL-6 and IL-8 in the serum is suppressed. The effect was improved.

In addition, with respect to the content of TNF-α in serum, control 2 showed a change of about 226.88% compared to control 1, whereas control 3 and experimental groups 1 to 3 showed a change of about -41.04% and about -59.00% compared to control 2, respectively. , About -40.54% and about -29.85%.

In addition, with respect to the IL-6 content in serum, control 2 showed a change of about 294.37% compared to control 1, whereas control 3 and experimental groups 1 to 3 showed a change of about -47.40% and about -61.39% compared to control 2, respectively. , About -46.89% and about -29.18%.

In addition, with respect to the serum IL-8 content, control 2 showed a change of about 273.64% compared to control 1, but control 3 and experimental groups 1 to 3 showed a change of about -39.19% and about -54.76%, respectively, compared to control 2 , About -39.87% and -27.10%.

2) As can be seen from Table 8, the content of TNF-α, IL-6, and IL-8 in colon tissue was significantly increased in the control group 2 compared to the control 1 (p<0.01). On the other hand, experimental groups 1 to 3 significantly decreased the content of TNF-α, IL-6, and IL-8 in colon tissue compared to control group 2 (p<0.01). In particular, experimental group 1 showed a decrease in the content of TNF-α, IL-6, and IL-8 in colon tissue compared to the control group 3. In addition, according to the content of TNF-α, IL-6, and IL-8 in the colon tissue of experimental groups 1 to 3, the increase in the amount of TNF-α, IL-6 and IL-8 in the serum was suppressed as the dose of glutinous rice fermentation increased. The effect was improved.

In addition, with respect to the content of TNF-α in the colon tissue, control 2 showed a change of about 158.95% compared to control 1, whereas control 3 and experimental groups 1 to 3 showed -38.17, -52.03, -38.05 compared to control 2, respectively And -25.15%.

The IL-6 content in the colon tissue showed a change of about 126.92% in control 2 compared to control 1, but in control 3 and experimental groups 1 to 3, respectively, about -33.45%, about -45.98%, and about -34.19 compared to control 2, respectively. % And about -26.74%.

The IL-8 content in the colon tissue showed a change of about 200.00% in control 2 compared to control 1, but in control 3 and experimental groups 1 to 3, respectively, compared to control 2, about -35.76%, about -51.08%, and about -34.88 % And about -25.15%.

(10) Changes in lipid peroxidation and antioxidant defense system in colon tissue

After weighing the separated colon tissue, it was homogenized in ice-cooled 0.01 M Tris-HCl (pH 7.4), and then stored in a cryogenic cooler at about -150 °C, as described in Kavutcu et al [1996], 12,000 × Centrifuged at g for 15 minutes.

The concentration of lipid peroxidation in the colon was measured by calculating MDA (malondialdehyde) using thiobarbituric acid at an absorbance of 525 nm, and its unit was nM MDA/mg protein [Jamall and Smith, 1985]. The total protein content was measured by a conventional method [Lowry et al., 1951] using bovine serum albumin (Invitrogen, Carlsbad, CA, USA) as an internal standard. The measurement results are shown in Table 9 below. In Table 9 below, each value is expressed as the mean ± standard deviation of 8 mice.

Concentration of lipid peroxidation
(nM MDA/mg protein) GSH content
(μM/g tissue) CAT active
(U/mg protein) SOD activity
(U/mg protein) Control 1 6.76±1.22 2.12±0.33 69.51±12.45 34.46±4.54 Control 2 26.37±4.42 0.95±0.10 37.94±10.27 15.98±4.19 Control 3 14.89±3.75 1.40±0.20 53.71±7.19 24.47±3.52 Experimental group 1 10.29±2.00 1.76±0.17 61.85±6.10 29.52±1.99 Experimental group 2 15.33±1.72 1.43±0.23 54.10±6.27 24.11±3.62 Experimental group 3 18.04±2.81 1.29±0.18 49.98±4.67 22.72±3.72 * MDA: Malondialdehyde
* GSH: Glutathione
* CAT: Catalase
* SOD: Superoxide dismutase

As can be seen from Table 9, the control group 2 significantly increased the lipid peroxidation concentration and MDA content in the colon tissue compared to the control group 1 (p<0.01), and the GSH content, SOD activity, and CAT activity in the colon tissue were significantly increased. It decreased significantly (p<0.01). On the other hand, in all experimental groups 1 to 3, lipid peroxidation concentration and MDA content in colon tissue were significantly decreased (p<0.01) compared to control 2, and GSH content, SOD activity and CAT activity in colon tissue were significantly ( p<0.01) increased. In particular, experimental group 1 had lower lipid peroxidation concentration and MDA content in colon tissue, and higher GSH content, SOD activity, and CAT activity in colon tissue compared to control group 3. In addition, according to the lipid peroxidation concentration, MDA content, GSH content, SOD activity, and CAT activity levels in colon tissues of experimental groups 1 to 3, as the dose of glutinous rice fermentation increased, the lipid peroxidation concentration and MDA content in the colon tissue were suppressed. Not only the effect was improved, but also the effect of inhibiting the decrease in GSH content, SOD activity and CAT activity in colon tissue was improved.

In addition, with respect to the concentration of lipid peroxidation in the colon tissue, the control group 2 showed a change of about 289.87% compared to the control group 1, but the control group 3 and the experimental groups 1 to 3 showed a change of about -43.53% and about -60.98% compared to the control group 2, respectively. , About -41.88% and -31.57%.

In addition, with respect to the GSH content in the colon tissue, the control group 2 showed a change of about -55.20% compared to the control group 1, but the control group 3 and the experimental groups 1 to 3 were about 47.89%, about 85.88%, about 47.89%, about 85.88% compared to the control group 2, respectively. It showed a change of 51.19% and 36.28%.

In addition, with regard to CAT activity in colon tissue, control 2 showed a change of about -45.42% compared to control 1, but control 3 and experimental groups 1 to 3 showed a change of about 41.54%, about 63.01%, and about, respectively, compared to control 2 It showed a change of 42.57% and about 31.74%.

In addition, with regard to SOD activity in colon tissue, control 2 showed a change of about -53.62% compared to control 1, but control 3 and experimental groups 1 to 3 showed about 53.14%, about 84.73%, and about respectively compared to control 2 It showed a change of 50.91% and about 42.18%.

(11) Changes in mRNA expression in colon tissue

NF-κB (signal molecules essential for promotion of inflammation), cytokines (TNF-α, IL-6, IL-8), TJ proteins (ZO-1, ZO-2), Claudin-1, Occludin, and Expression of mucin (Mucin-1 and Mucin-2 mRNA) was measured through real-time RT-PCR analysis according to a previously established method [Han et al., 2015]. Briefly, RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) from hippocampal tissue obtained from mice immediately after completion of the passive avoidance test. The concentration and quality of the RNA were measured by the CFX96TM Real-Time System (Bio-Rad, Hercules, CA, USA). To prevent contamination of DNA, samples were treated with recombinant DNase I (DNA-free; Ambion, Austin, TX, USA). RNA was reverse transcribed using the reagent High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Analysis was performed using the ABI Step One Plus Sequence Detection System (Applied Biosystems, Foster City, CA, USA), and their expression levels were calculated in proportion to the vehicle control group. 10 minutes at 94° C. under thermal conditions; And 39 cycles of 15 seconds at 94°C, 20 seconds at 57°C and 30 seconds at 72°C were applied. The data were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression using a comparative threshold cycle method [Schmittgen and Livak, 2008]. The sequences of PCR oligonucleotide primers (Bioneer, Daejeon, Korea) used in this analysis are shown in Table 10. For quantitative analysis, intact control crystal tissue was used as a control, and NF-κB, TNF-α, IL-6, IL-8, ZO-1, ZO-2, Claudin-1, Occludin, Mucin-1 And the relative expression of Mucin-2 was calculated using the 2- ^ΔΔCt method [Livak and Schmittgen, 2001]. The results are shown in Tables 11 and 12 below.

Control 1 Control 2 Control 3 NF-κB 1.06±0.11 6.84±1.06 4.55±0.83 TNF-α 1.01±0.08 4.78±0.78 2.76±0.49 IL-6 0.98±0.07 3.11±0.57 1.86±0.28 IL-8 1.02±0.07 2.95±0.20 1.74±0.20 ZO-1 1.07±0.12 0.48±0.08 0.67±0.09 ZO-2 1.04±0.09 0.25±0.06 0.43±0.12 Claudin-1 1.00±0.09 0.43±0.07 0.60±0.07 Occludin 1.00±0.08 0.50±0.09 0.69±0.08 Mucin-1 1.03±0.09 0.33±0.08 0.53±0.10 Mucin-2 1.01±0.07 0.47±0.09 0.72±0.09

Experimental group 1 Experimental group 2 Experimental group 3 NF-κB 3.10±0.88 4.38±0.52 5.07±0.81 TNF-α 1.98±0.40 2.41±0.47 3.15±0.46 IL-6 1.44±0.29 1.86±0.20 2.14±0.24 IL-8 1.30±0.23 1.69±0.26 1.97±0.17 ZO-1 0.85±0.07 0.64±0.11 0.60±0.07 ZO-2 0.67±0.09 0.41±0.08 0.33±0.04 Claudin-1 0.82±0.11 0.64±0.10 0.55±0.08 Occludin 0.87±0.06 0.70±0.07 0.64±0.05 Mucin-1 0.69±0012 0.57±0.07 0.45±0.08 Mucin-2 0.92±0.07 0.76±0.12 0.64±0.12

As can be seen in Tables 11 and 12, in control 2 compared to control 1, TNF-α, a cytokine regulated through NF-κB, along with an increase in NF-κB, a signaling system such as inflammatory response control and immune system regulation, In addition to the increased expression of IL-6 and IL-8 mRNA, the expression of ZO-1, ZO-2, Claudin-1, Occludin, Mucin-1 and Mucin-2 mRNA was decreased. On the other hand, in the case of experimental groups 1 to 3, the expression of NF-κB, TNF-α, IL-6 and IL-8 mRNA decreased compared to control 2, and ZO-1, ZO-2, Claudin-1, Occludin, and Mucin The expression of -1 and Mucin-2 mRNA was increased compared to the control group 2. In particular, experimental group 1 decreased the expression of NF-κB, TNF-α, IL-6 and IL-8 mRNA compared to control 3, and ZO-1, ZO-2, Claudin-1, Occludin, Mucin-1 and Mucin The expression of -2 mRNA was increased. In addition, according to experimental groups 1 to 3, as the dose of glutinous rice fermentation increased, the expression of NF-κB, TNF-α, IL-6 and IL-8 mRNA decreased, while ZO-1, ZO-2, Claudin-1 , Occludin, Mucin-1 and Mucin-2 mRNA expressions were increased.

In addition, with respect to the expression of NF-κB mRNA in colon tissue, control 2 showed a change of about 543.31% compared to control 1, whereas control 3 and experimental groups 1 to 3 showed a change of about -33.50% and about- It showed changes of 54.64%, about -35.87% and -25.80%.

In addition, with respect to the expression of TNF-α mRNA in the colon tissue, control 2 showed a change of about 374.78% compared to control 1, but control 3 and experimental groups 1 to 3 showed about -48.51% and about- It showed a change of 58.56%, about -49.56% and about -34.07%.

In addition, with respect to the expression of IL-6 mRNA in the colon tissue, it showed a change of about 216.92% compared to the control 1, but the control group 3 and the experimental groups 1-3 were about -40.22%, about -53.83%, respectively compared to the control group 2, It showed a change of about -40.43% and about -31.27%.

In addition, with respect to the expression of IL-8 mRNA in the colon tissue, it showed a change of about 190.04% compared to the control 1, but the control group 3 and the experimental groups 1 to 3 were about -41.01%, about -55.98%, respectively, compared to the control group 2, It showed a change of about -42.58% and about -33.29%.

In addition, with respect to the expression of ZO-1 mRNA in the colon tissue, it showed a change of about -55.56% compared to the control 1, but the control group 3 and the experimental groups 1 to 3 were about 41.58%, about 79.21%, and about the control group 2, respectively. It showed a change of 33.95% and about 25.26%.

In addition, with respect to the expression of ZO-2 mRNA in the colon tissue, it showed a change of about -75.57% compared to the control 1, but the control group 3 and the experimental groups 1 to 3 were about 69.46%, about 162.07%, respectively compared to the control group 2, It showed a change of 61.58% and about 30.54%.

In addition, in relation to the expression of Claudin-1 mRNA in the colon tissue, it showed a change of about -57.39% compared to the control 1, but the control group 3 and the experimental groups 1 to 3 showed a change of about 41.76%, about 93.82% and about 93.82% compared to the control 2, respectively. It showed a change of 50.00% and about 28.24%.

In addition, with respect to the expression of Occludin mRNA in the colon tissue, it showed a change of about -50.19% compared to the control 1, but the control group 3 and the experimental groups 1 to 3 were about 37.84%, about 74.19%, and about 41.10% compared to the control 2, respectively. And about 28.82%.

In addition, with respect to the expression of Mucin-1 mRNA in the colon tissue, it showed a change of about -68.08% compared to the control 1, but the control group 3 and the experimental groups 1 to 3 were about 61.98%, about 109.89% and about 109.89% compared to the control group 2, respectively. It showed a change of 73.38% and about 36.88%.

In addition, with respect to the expression of Mucin-2 mRNA in the colon tissue, it showed a change of about -53.58% compared to the control 1, but the control group 3 and the experimental groups 1 to 3, respectively, about 52.93%, about 96.54%, about 52.93%, about 96.54%, about the control group 2, respectively. It showed a change of 61.97% and about 35.11%.

12) Histopathological and immunohistochemical changes in the colon

a) After 24 lapse of treatment with the last 7th test substance, all mice were sacrificed, and samples obtained from the colon were fixed in 10% neutral buffered formalin (NBF). The proximal part of the colon (1.5 cm away from the cecum) was cross-cut into portions in each mouse. All cross-sectioned colon tissue portions were re-fixed in 10% NBF for 24 hours at least at histopathological observations. Paraffin embedding was performed using an automated tissue processor (Shandon Citadel 2000, Thermo Scientific, Waltham, MA, USA) and an embedding center (Shandon Histostar, Thermo Scientific, Waltham, MA, USA), and 3-4 Five consecutive sections of µm thickness were prepared using an automated microtome (RM2255, Leica Biosystems, Nussloch, Germany). Representative sections were hematoxylin-eosin (HE) for general histopathology or Masson's trichrome (MT) for collagen fibers. Then, the histological profile of the individual cross-cut colon tissue was observed with an optical microscope (Model Eclipse 80i, Nikon, Tokyo, Japan) In order to see the change in more detail, the average total colon thickness (µm), The height and width of the colon villi (μm), and the number of inflammatory cells penetrating onto the colonic mucosa (cells/mm ² ) were measured under HE staining with an automated image analyzer (iSolution FL ver 9.1, IMT i-solution Inc., Vancouver, British Colombia, Canada), and according to a previously established method [Park et al., 2012; Kim et al., 2013b], the percentage of the average collagen fiber occupied area for the colonic mucosa under MT staining (% /Mm2) was calculated using an automatic image analyzer In addition, as shown in Table 13 below, tissue lesions were previously established in a microscopic damage evaluation system [Hamamoto et al., 1999; Kim et al., 2013a]. Here, mucosal damage was expressed as 0-4 points according to the loss of crypt (mucous membrane) and infiltration of inflammatory cells (Max = 4 ). The results are shown in Tables 15-16 and 9 below. When performing this analysis, the histopathologist did not see the group distribution.

Score standard 0 Normal mucosa One 1/3 loss of crypt 2 2/3 loss of crypt 3 Lamina propria covered with a monolayer of epithelial cells with mild inflammatory cell penetration 4 Corrosion and marked inflammatory cell infiltration

b) The immune response to proinflammatory cytokines (TNF-α) with T cell subset markers (CD4 and CD8) in the colonic mucosa, respectively, was evaluated by the avidin-biotin-peroxidase complex (ABC) method and peroxidase substrate, respectively. It was observed by the kit (Vector Labs, Burlingame, CA, USA) (see Table 14). Here, for unstained sections, endogenous peroxidase activity was blocked by incubating in methanol and 0.3% H ₂ O ₂ for 30 minutes, and non-specific binding of immunoglobulins was pretreated with citrate buffer antigen (epitope) retrieval and then in a constant temperature and humidity chamber. Blocked with normal horse serum blocking solution for 1 hour. The primary antisera was treated overnight in an incubator at 4°C, and then incubated with biotinylated universal secondary antibody and ABC reagent for 1 hour in an incubator at room temperature. Finally, the sections were reacted with the peroxidase substrate kit for 3 minutes at room temperature. At each step, all sections were washed 3 times in 0.01 M phosphate buffered saline (PBS). Cells occupying more than 20% of the immune response and density of each antibody against CD4, CD8 and TNF-α were considered positive. Previous reports [Park et al., 2014; According to Kim et al., 2015a], the average number of each immunolabeled cell located in the colonic mucosa was counted as cells/mm 2 by a computer based on an automatic image analyzer. The results are shown in Tables 15 to 16 and Fig. 10 below. When performing this analysis, the histopathologist did not see the group distribution.

Control 1 Control 2 Control 3 Damage scores* 0.25±0.46 3.63±0.52 2.13±0.64 Total diameters(㎛) 2156.36±145.45 2228.98±169.70 2222.16±77.47 Villus height(㎛) 848.91±43.94 202.20±20.36 628.81±107.18 Villus width(㎛) 291.80±27.94 172.10±25.75 240.83±26.14 Collagen fibers(%/㎟) 18.90±7.03 64.05±7.02 43.06±4.37 Cell numbers on the mucosa
(cells/㎟) Inflammatory cells 28.00±10.14 325.25±49.64 212.38±30.98 TNF-α+ cells 8.75±3.99 76.25±12.53 26.75±8.14 CD4+ cells 8.50±3.99 187.25±26.60 86.00±31.96 CD8+ cells 15.00±3.55 247.88±33.82 124.50±26.01

Experimental group 1 Experimental group 2 Experimental group 3 Damage scores* 1.25±0.71 20.. ±0.76 2.38±0.52 Total diameters(㎛) 2168.90±149.66 2214.10±92.83 2201.66±151.11 Villus height(㎛) 688.98±78.47 629.83±121.07 466.01±112.81 Villus width(㎛) 262.20±14.90 238.64±18.01 209.59±16.94 Collagen fibers(%/㎟) 30.61±5.85 42.14±5.38 50.49±10.64 Cell numbers on the mucosa
(cells/㎟) Inflammatory cells 157.25±44.04 220.50±26.40 257.25±16.49 TNF-α+ cells 15.00±4.27 27.00±4.14 42.25±10.50 CD4+ cells 49.00±19.36 90.25±27.14 112.25±27.87 CD8+ cells 61.25±21.64 127.00±29.53 188.63±19.49

c) As can be seen in Tables 15 to 16 and FIG. 9, in Control 2, colon villi atrophy, mucosal erosion, inflammatory cell infiltration and local fibrosis were observed, and accordingly, the microscopic damage score increased. In addition, in the case of the control group 2, the average colon villi height and thickness decreased, and the number of infiltrating inflammatory cells in the mucous membrane and the proportion of collagen fibers also increased.

On the other hand, in experimental groups 1 to 3, the average colon villi height and thickness increased, and the microscopic damage score, the number of infiltrating inflammatory cells in the mucous membrane, and the proportion of collagen fibers decreased.

d) As can be seen in Tables 15-16 and FIG. 10, the number of TNF-α, CD4, and CD8 immune response cells in the colonic mucosa increased in the control group 2, whereas the experimental groups 1-3 were TNF-α and CD4 in the colonic mucosa. , The number of CD8 immune response cells decreased.

Claims (14)

Mixing glutinous rice with water and gelatinizing to obtain glutinous rice hydrate;
Obtaining a first fermented glutinous rice containing galacto-oligosaccharide by adding saccharification enzyme and yeast to the glutinous rice saccharification and fermentation (simultaneous saccharification and fermentation);
Obtaining a second fermented glutinous rice containing more galacto-oligosaccharides than the first fermented glutinous rice by yeast treatment of the first fermented glutinous rice with Saccharomyces sp. And
Fermenting the second fermented glutinous rice with Weissella sp. lactic acid bacteria to obtain a third fermented glutinous rice containing more galacto-oligosaccharides than the second fermented glutinous rice;
Including,
The saccharification enzyme is included in a range of 2 to 5 parts by weight based on 100 parts by weight of glutinous rice,
A glutinous rice fermented product containing galacto-oligosaccharide prepared by a method comprising the yeast in the range of 1 to 20 parts by weight based on 100 parts by weight of glutinous rice. The method of claim 1,
The gelatinization step is performed for 10 to 90 minutes at a temperature of 90 to 125 ℃, glutinous rice fermentation. The method of claim 1,
The saccharification enzyme and yeast treatment step is performed for 90 to 150 minutes at a temperature of 50 to 60 ℃, glutinous rice fermentation. delete The method of claim 1,
The yeast of the genus Saccharomyces sp. is Saccharomyces cerevisiae , Saccharomyces uqvarum , Saccharomyces ellipsoideus , Saccharomyces ellipsoideus Karl's Bergen sheath (Saccharomycescarlsbergensis), saccharose in MRS sake (Saccharomyces sake), saccharose in MRS Correa Taunus (Saccharomyces coreanus), saccharose in Mrs Lee poly urticae (Saccharomyceslipolytica) saccharide in MRS bowl radio (Saccharomyces boulardii) and MRS in Saccharomyces Pas Torianus ( Saccharomyces pastorianus ) is selected from the group consisting of, glutinous rice fermentation. The method of claim 1,
The Saccharomyces sp. The content of yeast is 0.5 to 10 parts by weight based on 100 parts by weight of the first fermented glutinous rice, fermented glutinous rice. The method of claim 1,
The yeast treatment step will be carried out for 20 to 36 hours at a temperature of 25 to 35 ℃, glutinous rice fermentation. The method of claim 1,
The Weissella sp. lactic acid bacteria are Weissella cibaria , Weissella koreensi , Weissella hanii , Weissella kimchii , Weissella kimchii , Weissella Soli ( Weissella soli ), and Weissella confusa ( Weissella confusa ) that is selected from the group consisting of, glutinous rice fermentation. The method of claim 1,
The Weissella sp. The content of lactic acid bacteria is 0.5 to 10 parts by weight based on 100 parts by weight of the second fermented glutinous rice, glutinous rice fermented product. The method of claim 1,
The fermentation step of the lactic acid bacteria will be carried out for 20 to 36 hours at a temperature of 25 to 35 ℃, glutinous rice fermentation. The method of claim 1,
Filtering and concentrating the third fermented glutinous rice to obtain a fermented glutinous rice concentrate;
Mixing the glutinous rice fermentation concentrate with dextrin; And
Sterilizing the mixture and then spray drying
In addition, the glutinous rice fermentation product further comprising. The method of claim 11,
The glutinous rice fermentation concentrate and dextrin are mixed in a weight ratio of 60:40 to 95:5. A pharmaceutical composition for preventing or improving inflammatory bowel disease, comprising the fermented glutinous rice according to any one of claims 1 to 3 and 5 to 12 as an active ingredient. A food composition for preventing or improving inflammatory bowel disease, comprising the fermented glutinous rice according to any one of claims 1 to 3 and 5 to 12 as an active ingredient.

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