KR20130085602A - Fermentation products of probiotic mixed cultures and functional health foods, medicinal composition including the same - Google Patents

Abstract

PURPOSE: Functional health food and a medicinal composition are provided to use a fermentation product of mixed strains using three different types of strains with excellent enzyme activity and functionality. CONSTITUTION: A fermentation product is fermented by Bacillus subtilis-SKm (KFCC 11520P), Lactococcus lactis, and Aspergillus oryzae. Lactococcus lactis is Lactococcus lactis-GAm (KFCC 11510P). 0.1-1 parts by weight of Aspergillus oryzae is added to 100 parts by weight of fermentation object material. 10^4-10^8 cfu/g of Bacillus subtilis-SKm and 10^4-10^8 cfu/g of Lactococcus lactis are used for 100 parts by weight of fermentation object material. The fermentation product is selected from a group consisting of fermented soybeans, fermented soybean blocks, fermented soybean paste, soy sauce, and red pepper paste.

Description

FIELD OF THE INVENTION The present invention relates to a fermentation product of a probiotic mixed strain and a health functional food and a medicinal composition containing the same,

The present invention relates to a fermented product having improved quality of a probiotic mixed strain and a health functional food and a pharmaceutical composition containing the fermented product.

 Conventionally, the conventional method of making meju is mostly used. However, in the case of using different kinds of water, salt or the like used in the manufacture of meju and miso or adding other ingredients (medicinal herb, etc.) in order to improve the quality and the sensibility of meju Mostly. In addition, there was also an invention to improve the fermentation speed and quality by an enzyme produced by a microorganism by inoculating a microorganism into Meju, but most microorganisms used in the invention were a single microorganism such as Aspergillus sp. Fungi were often applied.

Korean Patent Laid-Open No. 2011-126447 (published on November 23, 2011) discloses that Rhizopus, Aspergillus usami and Aspergillus oryzae are cultured and mixed A method for preparing meju using a culture of a fungus strain, natto bacterium and lactic acid bacterium is described.

Korean Patent Publication No. 2011-126447 (published on November 23, 2011)

It is an object of the present invention to provide a fermented product having excellent quality and functionality, which is fermented by the strain, by mixing three strains having excellent enzyme activity and functionality, and a health functional food and a pharmaceutical composition containing the fermented product.

1. Fermentations fermented with Bacillus subtilis-SKM (KFCC 11520P), Lactococcus lactis and Aspergillus oryzae.

2. The fermentation product according to item 1 above, wherein the lactococcus lactis is Lactococcus lactis-GAm (KFCC 11510P).

3. In the first place, Aspergillus duck material 0.1-1 parts by weight based on 100 parts by weight of the fermented substance, Bacillus subtilis -SKm is 10 ⁴ -10 ⁸ cfu / g and Lactococcus lactis 10 ⁴ - 10 ⁸ cfu / g.

4. The fermentation product according to 1 above, wherein the fermented product is selected from the group consisting of meju, soybean meal, soybean paste, soy sauce, kochujang, and chonggukjang.

5. A health functional food comprising the fermented product of any one of the above 1 to 4.

6. The health functional food having at least one efficacy selected from the group consisting of hyperlipemia, obesity, cancer, inflammatory diseases of the large intestine and small intestine, and improvement of functional bowel disease.

7. Above 5, dairy products, confectionery products, seasonings, beverages and drinks, snacks, candies, ice cream and frozen desserts, breakfast cereals, nutrition bars, snack bars, chocolate products, processed foods, cereals products and pasta, soups and sauces And dressings, confectionery products, oil and fat products, dairy drinks and milk drinks, soy dairy-like products, frozen foods, cooked foods and alternative foods, meat products, cheese, yogurt, bread And bread, yeast products, cakes, cookies, and crackers.

8. A pharmaceutical composition for the prophylaxis or treatment of hyperlipemia, obesity, inflammatory diseases of the large intestine and small intestine, functional bowel disease, and cancer comprising the fermented product of any one of the above 1 to 4.

9. The pharmaceutical composition according to item 8, wherein the inflammatory disease of the large intestine and the small intestine is selected from the group consisting of acute enteritis, Crohn's disease, ulcerative colitis, and ischemic colitis.

10. The pharmaceutical composition according to the above 8, wherein the functional bowel disease is selected from the group consisting of diarrhea, constipation, enteric diverticulum and irritable bowel syndrome.

11. (S1) growing soybeans; (S2) inoculating aspergillus oryzae and Bacillus subtilis-SKm to the expanded soybeans to firstly ferment; And (S3) a step of secondary inoculation with lacto-coccus lactis after the first fermentation to obtain a fermented product.

12. The aspergillus oryzae according to claim 11, wherein 0.1 to 1 part by weight of the aspergillus oryzae is contained in 100 parts by weight of the soybean, bacillus subtilis-SKM is 10 ⁴ -10 ⁸ cfu / g, and lacto-coccus lactis is 10 ⁴ -10 ⁸ cfu / g. < / RTI >

13. The process of claim 11, wherein said primary fermentation step is performed at 20-45 < 0 > C for 24-72 hours.

14. The method of claim 11, wherein said secondary fermentation step is performed at 20-45 < 0 > C for 12-36 hours.

The fermented product of the probiotic mixed strain of the present invention exhibits a higher microorganism distribution than the fermented product of commercial Meju and other strains and is well fermented and has excellent enzyme activity of 30 to 50% . Antioxidant effect was increased and cancer cell growth inhibition effect was more than 20% higher than that of commercial Meju and other strains.

It is also effective for reducing hyperlipidemia and obesity in blood. It can be used for prevention of obesity, increase in intestinal lactic acid bacterium, decrease in harmful substances, high-quality fermented product having functional bowel disease and inflammatory diseases of bowel and small intestine, And a pharmaceutical composition.

Fig. 1 shows the pH of a soybean meal fermented with a probiotic mixed strain.
FIG. 2 shows amino acid and ammonia nitrogen values of the soybean meal fermented with the probiotic mixed strain.
Fig. 3 shows the distribution of microorganisms in the soybean meju fermented with the probiotic mixed strain.
Fig. 4 shows the enzymatic activity of the soybean meju fermented with the probiotic mixed strain.
FIG. 5 shows changes in the moisture content and the number of lactic acid bacteria in the feces of rats fed orally with the soybean fermented with the probiotic mixed strain.
FIG. 6 shows changes in the content of harmful enzymes in the feces of rats fed orally with the soybean fermented with the probiotic mixed strain.
FIG. 7 shows changes in body weight of rats fed with orally administered soybean meju fermented with a probiotic mixed strain.
8 shows the content of lipid components in the blood of rats fed orally with the soybean fermented with the probiotic mixed strain.
Figure 9 shows the length of the colon according to the Meju treatment in colitis-induced rats.
Fig. 10 shows changes in colon tissues according to Meju treatment in colitis-induced rats.
Fig. 11 shows histological evaluation of colonic tissues according to Meju treatment in colitis-induced rats.
Fig. 12 shows changes in inflammatory cytokines according to Meju treatment in colitis-induced rats.
FIG. 13 shows changes in pH and salinity during the fermentation period of doenjang prepared with a soybean fermented with a probiotic mixed strain.
FIG. 14 shows changes in amino acid and ammonia nitrogen contents during fermentation of soybean paste prepared with soybean meal fermented with probiotic mixed strain.
FIG. 15 shows changes in enzyme activity during the fermentation period of doenjang prepared with soybean meal fermented with a probiotic mixed strain.
16 shows the appearance of doenjang prepared with soybean fermented with a probiotic mixed strain.
Fig. 17 shows the DPPH free radical scavenging activity of doenjang prepared from the fermented soybean meal with the probiotic mixed strain.
FIG. 18 shows the inhibitory effect of soybean fermented soybean fermented with probiotic meju on growth of HT-29 colon cancer cells.

The present invention relates to a fermented product of a probiotic mixed strain and a health functional food and a pharmaceutical composition containing the fermented product.

Hereinafter, the present invention will be described in more detail.

The mixed strain according to the present invention is characterized by comprising Aspergillus oryzae, Lactococcus lactis, Bacillus subtilis-SKm KFCC 11520P. The three strains have excellent enzyme activity and functionality. The three strains can be obtained by various methods, preferably from a traditional meju.

In order to evaluate the efficacy of the mixed strain according to the present invention as a fermentation strain, the yield and quality of the bean meju were compared with those of the bean meju fermented with commercial meju and mixed. However, soybean meal is only an example of fermented product, and can be applied to miso, soy sauce, hot pepper paste, and chongkukjang.

Hereinafter, one embodiment of a method for preparing a fermented soybean meal according to the present invention will be described in more detail.

First, the beans are added (S1).

The soybean can be carried out by washing the soybeans, immersing in water for 5-24 hours, then adding 100-150 ° C for 20-40 minutes and cooling to 30-60 ° C. One of the factors that has the greatest influence on the degree of soybean growth is the time to immerse in water. The soaking time of the beans may represent the degree of growth most suitable to be carried out for 5-24 hours, preferably 10-15 hours.

After the beans are grown, Aspergillus oryzae and Bacillus subtilis-SKm are inoculated and primary fermented (S2).

In the present invention, the Aspergillus oryzae and Bacillus subtilis-SKm are inoculated first because the fermentation rate is slower than that of Lactococcus lactis-GAm. The amount of the inoculum of the strain is not particularly limited. Preferably, the aspergillus oryzae is inoculated in an amount of 0.1-1 parts by weight with respect to 100 parts by weight of the soybean which is a substance to be fermented, and Bacillus subtilis-SKM with 10 ⁴ -10 ⁸ cfu / g . Under the above conditions, optimal growth conditions are exhibited and fermentation can be most effectively achieved. Depending on the strain, the fermentation time and temperature may be appropriately selected, but preferably fermentation can be carried out at 20-45 ° C for 24-72 hours.

Lactococcus lactis may be further inoculated after the primary fermentation and secondary fermentation may be performed to produce a soybean meal (S3).

In the present invention, Lactococcus lactis is characterized in that it is inoculated in the second fermentation step because it can inhibit the growth of two strains of different fermentation products with faster fermentation rates than the other two strains. The amount of the strain to be inoculated is not particularly limited, but may preferably be inoculated at 10 ⁴ -10 ⁸ cfu / g. The best fermentation effect can be obtained in the inoculation amount. Depending on the strain, the fermentation time and temperature may be appropriately selected, but preferably fermentation can be performed at 20-45 ° C for 12-36 hours. Under the above conditions, the optimum growth conditions are exhibited and fermentation can be performed more favorably.

Lactococcus lactis according to the present invention is preferably Lactococcus lactis-GAm KFCC 11510P.

The fermented product of the present invention is effective for the improvement of inflammatory diseases and functional intestinal diseases of hyperlipidemia, obesity, cancer, large intestine and small intestine and can be used as a health functional food containing the same. It may be formulated into capsules, tablets, powders, liquid suspensions, pills, granules and the like, but is not limited thereto.

The health functional food of the present invention can be used as a food product for dairy, bakery, seasoning, beverage and drink, snack, candy, ice cream and frozen dessert, breakfast cereal, nutrition bar, snack bar chocolate product, And dressings, confectionery products, oil and fat products, dairy drinks and milk drinks, soy dairy-like products, frozen foods, cooked foods and alternative foods, meat products, cheese, yogurt, bread And a bread, a yeast product, a cake and a cookie, and a cracker.

The health functional food in the form of tablets can be prepared as it is, or it can be prepared by putting an excipient, a binder, a disintegrant or other additives into the granules in an appropriate manner and then compressing them by putting in a lubricant or the like, It may be prepared by direct compression molding of an even mixture of excipients, binders, disintegrants, or other suitable additives, or by mixing the pre-formed granules with the health-functional food as it is or by adding appropriate additives and then by compression molding, A binder or another suitable additive is added to wet the powder evenly mixed with a solvent, the wet powder is molded into a mold at a low pressure, and dried by a suitable method. In addition, a mating agent or the like may be added to the health-functional food in the form of tablets, if necessary.

The hard capsule of the capsule-type health functional food is usually prepared by mixing the capsules with an appropriate excipient such as a health functional food or a health functional food, or by granulating the mixture into a granular form by a suitable method or by granulating the granular product into a granular form suitable for granulation The soft capsules are usually capsules filled with health-functional food or an appropriate excipient for health-functional food, which are then encapsulated in capsules containing an appropriate capsule base, such as gelatin, or glycerin or sorbitol, , And if necessary, a coloring agent preservative or the like may be added to the capsule base.

The health functional food of the ring form is usually prepared by mixing an excipient, a binder and a disintegrant in an ordinary healthy functional food, shaping it into a spherical form by an appropriate method, and then adding the gelatin to starch or other suitable skin care agent, It may also be an objectionable substance.

The health functional food of the granular formulation is usually prepared by mixing the health functional food as it is or the excipient, binder, disintegrant etc. into the health functional food, and then granulating it by a proper method and evenly grinding the particles as much as possible. , A mating agent, and the like.

The health functional food of the beverage formulation may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. Functional materials such as antler extract, fructooligosaccharide which helps defecation activity and absorption of calcium, acacia honey, complex gold extract which is a natural preservative, and gellan gum which is a precipitation improvement enhancer may be added, but not limited thereto, Suitable components may be suitably used.

The pharmaceutical composition of the present invention has medicinal use for the prevention or treatment of hyperlipemia, obesity, cancer, inflammatory diseases of the large intestine and small intestine, and functional intestinal diseases. Inflammatory diseases include acute enteritis, Crohn's disease, ulcerative colitis and ischemic colitis. Functional bowel diseases include diarrhea, constipation, enteric diverticulitis and irritable bowel syndrome.

In the case of pharmaceutical compositions containing the fermented product of the present invention, pharmaceutically acceptable and physiologically acceptable adjuvants can be used. The adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, A lubricant, a lubricant or a flavoring agent can be used.

The pharmaceutical composition of the present invention may be formulated into a pharmaceutical composition containing one or more pharmaceutically acceptable carriers for administration. The pharmaceutical preparation form can be a granule, a powder, a tablet, a coating, a capsule, a suppository, a liquid, a syrup, a juice, a suspension, an emulsion or the like.

For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include but are not limited to natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, corn sweeteners, acacia, trackercance or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, but are not limited to, starch, methylcellulose, agarose, bentonite, xanthan gum and the like.

Acceptable pharmaceutical carriers for pharmaceutical compositions that are formulated into liquid solutions include those suitable for sterilization and in vivo such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol And one or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders and lubricants can be additionally added and formulated into pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

Example  1. Recipe of bean meju

Soybeans were purchased and used in 2011 from Yeonye-myeon, Goesan-gun, Chungbuk Province. Aspergillus oryzae were supplied and used in Chungmu fermentation. Bacillus subtilis-SKm was inoculated on nutrient medium (Difco Co., Sparks, MI, USA) and cultured at 40 ° C for 24-48 hours, Kochus lactis-GAm (KFCC 11510P) was cultured at 37 ° C for 24-48 hours using MRS medium (Difco Co.) and adjusted to 10 ⁶ cfu / g.

The soybean was subjected to regular washing and washing and immersed in 1.5 times of water for 12 hours. The soybean was then heated at 121 DEG C for 30 minutes and cooled to 50 DEG C to obtain 0.2 part by weight of Aspergillus oryzae and 100 parts by weight of Bacillus subtilis- ⁶ cfu / g was inoculated and primary fermentation was carried out at 30 DEG C for 48 hours. After the first fermentation, 10 ⁶ cfu / g of Lactococcus lactis-GAm was inoculated and secondary fermentation was carried out at 37 ° C for 24 hours to prepare a bean meze.

In the following experimental examples, CG-m is commercial bean meju, Am is soybean fermented with a single strain of Aspergillus oryzae, AB-m is Aspergillus oryzae and Bacillus subtilis- AL-m is a soybean fermented with Aspergillus oryzae and Lactococcus lactis, and a soybean fermented with ABL-m is Aspergillus oryzae, Bacillus subtilis-SKm and Lactococcus lactis. .

The subjects were divided into two groups: normal diet group with ND, control group with high cholesterol diet + saline group, CG-m group with high cholesterol diet + commercial soybean meal group, Am with high cholesterol diet + Am group, AB-m with high cholesterol diet + AB -m oral administration group, AL-m high cholesterol diet + AL-m oral administration group and ABL-m high cholesterol diet + ABL-m oral administration group.

The normal group was fed with PBS without induction of colitis. Control was induced by DSS and colitis induced by PBS. CG-m was induced by DSS and CG-m group. ABL-m was DSS. Colitis And CG-m.

Experimental Example  One. Probiotic  Soybean fermented with mixed strain Meju pH  Measure

The samples were diluted with distilled water and measured at room temperature with a pH meter (TP-93, Toko Chemical Laboratories, Tokyo, Japan). The acidity was determined by titration with 0.1 N NaOH until the pH reached 8.4 by taking 20 ml of a 20-fold diluted sample in accordance with the Association of Official Analytical Chemists (AOAC) standard test method and expressed as the amount of 0.1 N NaOH required.

The pH was similar to that of AB-m, Al-m and ABL-m 7.1, and was lower than that of CG-m and Am to 6.9 (see Fig.

Experimental Example 2. Amino acid content and ammonia nitrogen content of soybean fermented with probiotic mixed strain

 Amino acid was determined by the pomolite titration method by diluting the sample 10 times. That is, 20 ml of a neutral formalin solution was added to 20 ml of the sample solution, and then titrated with 0.1 N NaOH until the pH reached 8.4, and the amount of amino acid nitrogen was calculated by measuring the number of ml of 0.1 N NaOH solution consumed. Ammonia was measured by the indene phenol method by AM 505-K (Asan, Pharmaceutical, Hwasung, Korea) after diluting the sample 40 times.

Amino nitrogen was more than 10% higher in ABL-m than in CG-m or AL-m. Ammonia nitrogen showed similar values for all meju (see FIG. 2).

Experimental Example 3. Measurement of microbial distribution of soybean fermented with soybean meal with probiotic mixed strain

The samples were diluted step by step and plated on a plate count agar medium (Difco Co.) at a rate of 100 μl. The cells were cultured at 30 ° C for 48 hours and counted. Yeast and mold counts were counted by counting colonies that were cultured for 48 hours at 30 ° C using a potato agar medium (Difco Co.).

The total aerobic microorganisms 9-10 log cfu / g, the lactic acid bacteria 7-10 log cfu / g, the yeast and mold 7-8 log cfu / g, and the ABL-m showed a significantly higher microbial distribution ).

Experimental Example 4. Measurement of Enzyme Activity of Soybean Meal Fermented with Probiotic Mixture

The protease activity was determined by measuring the tyrosine produced using 0.6% casein as a substrate by the polyene method, and the activity of the enzyme was expressed as the number of 쨉 g of tyrosine produced at 30 째 C for 1 minute. The activity of alpha-amylase was determined by coloring the reaction solution with iodine solution using 1% soluble starch as a substrate and measuring the absorbance at 700 nm. The activity of the enzyme was expressed as the number of soluble starches dissolved at 40 ° C. The lipase activity was measured by titrating the fatty acid produced with olive oil as a substrate with 0.05N NaOH.

The enzymatic activity of meju prepared by using the combined strain was higher than that of meju with a single strain as a starter. Especially, the enzyme activity of ABL-m was 30 ~ 50% superior to that of CG-m or AL-m (See Fig. 4).

Experimental Example 5. Measurement of moisture content, lactic acid bacterial count and noxious enzyme content in feces of rats fed orally with soybean fermented with probiotic mixed strain

Fecal samples were collected at weekly intervals and stored at -80 ° C.

Moisture content was analyzed using a dryer (105 ° C, 24 h) and calculated as follows.

Fecal Moisture Content (%) = (Wwet - Wdry) / Wwet x 100

Wwet: weight of feces before drying, Wdry: weight of water after drying

The number of lactic acid bacteria in the feces was counted by counting the number of colonies after the feces were diluted stepwise and dispensed into MRS medium (Difco Co.) and incubated at 37 ° C for 48 hours.

The tryptophanase activity was determined by adding 0.2 ml of the complete reagent solution (2.75 mg of pyridoxal phosphate, 19.6 mg of disodium edetate dihydrate and 10 mg of bovine serum albumin in 100 ml of 0.05 M phosphate buffer, pH 7.5) 20 mM tryptophan and 0.1 ml of crude enzyme solution (fecal dilution) are added and left at 37 ° C for 1 hour. Thereafter, 2 ml of a color reagent solution (14.7 g of para-dimethylaminobenzaldehyde in 52 ml of H ₂ SO ₄ and 948 ml of 95% ethanol) was added to stop the reaction, and the reaction was then centrifuged at 3,000 rpm for 10 minutes The supernatant was measured for absorbance at 550 nm.

The β-glucuronidase activity was determined by centrifuging the reaction mixture (1.6 ml of 2 mM para-nitrophenyl-β-D-glucuronide, 0.4 ml of crude enzyme solution) at 37 ° C. for 30 minutes, 1 ml of 0.5 N NaOH is added to stop the reaction. This was centrifuged at 3,000 rpm for 10 minutes, and the supernatant was taken and absorbance was measured at 405 nm.

The water content of the feces of Meju - treated group increased with time. In the control group, water content in the feces decreased to 50% or less due to high cholesterol diet, and in the AL-m group and the ABL-m group, the water content in the feces increased and the ABL-m increased to 60 % (See Fig. 5A).

In the feces, the lactic acid bacteria decreased in the early stage of high cholesterol diet, gradually increased over time, and the abundance of the lactic acid bacteria in the ABL-m group was the largest, which was higher than that of ND (see FIG.

The activity of tryptophanase, beta - glucuronidase, which is the major toxic substance inducing enzyme, was remarkably decreased after 4 weeks of meju intake. ND, the control group showed much lower activity than the meju-treated group and ABL-m showed the lowest activity (see FIG. 6).

Experimental Example  6. Probiotic  Measurement of weight change in rats fed orally with soybean fermented with mixed strains

SD rats (male, 5 weeks old, Sam Taco Co.) were used as experimental animals. The temperature of the feeding room was adjusted to 22 ± 2 ℃ and the light / dark cycle was adjusted at intervals of 12 hours. Feed and water were supplied freely during the experiment.

Changes in body weight were observed by oral administration of soybean meal to SD rats for 4 weeks.

Although there was no significant difference according to the experimental group, the weight gain of meju-treated group was smaller than that of the control group, and the weight of ABL-m was the lowest (see Fig. 7).

Experimental Example  7. Probiotic  Measurement of Lipid Content in Blood of Rats Fed Orally Fed Soybean Meju with Mixed Strains

Blood was collected and centrifuged at 3,500 rpm for 10 minutes to isolate plasma.

Serum triglyceride concentrations were analyzed using a commercial kit (AM202K, AM1575K, 077K9806, Asan Pharmaceutical).

The amount of triglyceride in blood was lower in the meju-treated group than in the control group, and the lowest level of ABL-m was found (see Fig. 8).

Experimental Example  8. Probiotic  Colitis of bean meju fermented with mixed strain Inhibition  Measure

The animals used in this experiment were 6-week-old female Balb / c mice (Sumatako, Osan) and weighing about 20-25 g. Animals were fed a sufficient amount of water and feed, and the animals were maintained at a temperature of 22 ± 1 ° C and a humidity of 55 ± 5%, and maintained at a light-dark cycle for 12 weeks.

Colitis was induced by sodium dextran sulfate (DSS) in BALB / C mice and oral effect of meju for 2 weeks was observed to prevent colitis.

For the histopathological analysis of the colon, the residual intestinal fluid was removed with 0.6% NaCl and fixed with 10% formalin for 24 hours. Paraffin tissue samples were prepared and cut into 5 ㎛ sections. The tissues were then stained with hematoxylin and eosin and observed under a microscope.

Serum concentrations of IL-12 and IL-17α were measured by enzyme-linked immunosorbent assay (ELISA MAX Deluxe Sets, Inc.) and quantitative evaluation of inflammatory cytokines. Absorbance was measured at 450 nm using Biolegend.

(P <0.05) (see FIG. 9). In the case of ABL-m treated group, there was a significant decrease in the length of the colon, which is the most typical symptom of ulcerative colitis, .

Colonic tissues were observed with hematoxylin and eosin staining. As a result, the colon tissues of the meju-treated group showed a much reduced lesion and inflammation, and villous and epithelial tissues were restored 10). As a result of this numerical analysis, the symptoms of ulcerative colitis were significantly alleviated in the meju-treated group (P <0.05), and the ABL-m showed the best evaluation (see FIG. 11).

IL-12, and IL-17α, which are involved in the pathogenesis of ulcerative colitis, were significantly reduced in the mouse serum, and in the ABL-m group, 30% more (see Fig. 12).

Experimental Example  9. Probiotic  During the fermentation period of soybean paste made from soybean fermented with mixed strain pH  And salinity change measurement

Meju, water and salt were mixed at a ratio of 1: 1.7: 0.46 and fermented at 37 ℃ for 6 weeks.

Soybean paste prepared with commercially available meju and commercially available soybean meal was used as a comparative group. Each commercially available meju was made with Almei Traditional Meju (Allii Food Co., Ltd., Gyeongbuk Goryeong) and commercially available soybean Meju was Allium Meju (Allii Food Co., Ltd., Gyeongbuk Goryeong).

The pH did not change significantly from 6.4 to 7.6 at 0 day and 6.0 to 7.3 after 6 weeks, and the salinity did not show any significant change in the range of 8.7 to 9.2% during the fermentation period (see FIG. 13).

Experimental Example  10. Probiotic  During the fermentation period of soybean paste made from soybean fermented with mixed strain Amino acid  And ammonia nitrogen content change measurement

The experimental method is the same as the method of Experimental Example 2 above.

In case of amino nitrogen, 6 kinds of soybean paste showed increasing tendency during fermentation period of 6 weeks. The amino acid content of soybean paste made with soybean paste was higher than that of commercial soybean paste (CB-d, CG-d). Especially, the amino acid content of ABL-d was high. In addition, the amino acid nitrogen content of soybean paste made with soybean meal was higher than that of CG-d, suggesting that the fermentation rate is faster than soybean paste made with commercially available soybean meal.

Ammonia nitrogen also showed similar tendency to amino nitrogen, and ammonia nitrogen of all doenjang increased during fermentation period. All the doenjang showed values within the normal range, and the ammonia nitrogen content of CG-d was very low (see FIG. 14).

Experimental Example  11. Probiotic  Measurement of enzyme activity during fermentation of soybean paste prepared with soybean fermented with mixed strain

The experimental method is the same as that of Experimental Example 4 above.

Protease activity was more than 5-fold higher in ABL-d than in commercial Meju and more than 20% higher than in AL-d.

Alpha-amylase activity was more than 70% higher in ABL-d than in commercial Meju.

The lipase activity was about 30 to 250% higher in ABL-d than in meju and other strains (see Fig. 15).

Experimental Example  12. Probiotic  Observation of fermentation period of soybean paste prepared with fermented soybean meal with mixed strain

AL-d and ABL-d, which had a large increase in amino nitrogen and high enzymatic activity, showed that soybean was decomposed much in appearance and fermented well. In the case of CG-d, the shape of the soybean was not decomposed. CB-d showed a good fermentation, but the color of the bean was black due to a lot of change of color (see FIG. 16).

Experimental Example  13. Probiotic  The soybean fermented with soybean meal fermented with mixed strains DPPH  freedom Radical Scatters  Measure

The lyophilized sample was ground, and 20 times (w / v) of methanol was added to the sample. The mixture was stirred for 12 hours for 3 times, filtered and concentrated using a rotary vacuum concentrator (EYELA, Tokyo Rikakikai Co., Tokyo, Japan) An extract was obtained. These extracts were diluted to appropriate concentrations in dimethylsulfoxide (DMSO) and used in the experiments.

The DPPH free radical scavenging effect was measured by Blois et al. 100 μl of the sample and 100 μl of a 150 μl DPPH (1,1-diphenyl-2picrylhyrazyl, Sigma Co., Louis, MO, USA) solution were mixed in a 96-well plate and reacted at room temperature for 30 minutes in a state of blocking light. Absorbance was measured using a spectrophotometer (UV / VIS spectrophotometer, Jasco, Tokyo, Japan). At all concentrations of 1 mg / ml and 2 mg / ml, ABL-d showed a significantly higher elimination (see FIG. 17).

Experimental Example  14. Probiotic  The soybean fermented with soybean meal fermented with mixed strains HT -29 Growth of colon cancer cells Inhibition  Measure

HT-29 human colon cancer cells used in the experiment were used for experiments while being cultured from a Korean cell line bank. Namely, cancer cells were cultured in RPMI 1640 medium (GIBCO, Grand Island, NY, USA) containing 100 units / ml of penicillin-streptomycin and 10% FBS in a 5% CO ₂ incubator at 37 ° C, Each cancer cell was subcultured every 6 to 7 days while re-sowing 2 to 3 times a week. The cultured cancer cells were dispensed into 96-well plates at a rate of 2 × 10 ⁴ cells / ml per well, and 20 μl of a sample prepared at a constant concentration was added and cultured at 37 ° C. in a 5% CO ₂ incubator for 72 hours. After addition of 20 μl of MTT solution prepared at a concentration of 5 mg / ml using PBS, the cells were further cultured for 4 hours under the same incubation conditions. The resulting formazan crystals were dissolved in DMSO, and the resulting solution was analyzed with an enzyme immunoassay (ELISA reader) Absorbance at 540 nm.

ABL-d showed significantly higher inhibition of cancer cell growth at all concentrations of 0.5 mg / ml and 1.0 mg / ml (see FIG. 18).

Claims (14)

Fermentation fermented with Bacillus subtilis-SKm (KFCC 11520P), Lactococcus lactis and Aspergillus duck.
The fermentation product of claim 1, wherein the Lactococcus lactis is Lactococcus lactis-GAm (KFCC 11510P).
The method according to claim 1, Aspergillus duck material is 0.1 to 1 parts by weight based on 100 parts by weight of the fermentation target material, Bacillus subtilis-SKm is 10 ⁴ ~ 10 ⁸ cfu / g and Lactococcus lactis is 10 ⁴ ~ 10 ⁸ Fermented product formed by inoculation at cfu / g.
The fermented product according to claim 1, which is selected from the group consisting of meju, soybean meju, soybean paste, soy sauce, red pepper paste, and cheongukjang.
Health functional food containing the fermented product of any one of Claims 1-4.
The health functional food according to claim 5, wherein the health functional food having at least one effect selected from the group consisting of hyperlipidemia, obesity, cancer, large intestine and small intestinal inflammatory diseases and functional intestinal diseases.
The method according to claim 5, further comprising the steps of: preparing the dairy product, the confectionery, the seasoning, the beverage and the drink, the snack, the candy, the ice cream and the frozen dessert, the morning cereal, the nutrition bar, , Confectionery products, oil and fat products, dairy drink and milk beverage, soy dairy-like product, frozen food, cooked food and substitute food, meat products, cheese, yogurt, bread and bun , Yeast products, cakes, cookies and crackers.
A pharmaceutical composition for the prophylaxis or treatment of at least one disease selected from the group consisting of hyperlipidemia, obesity, inflammatory diseases of the large intestine and small intestine, functional bowel disease and cancer comprising the fermentation product of any one of claims 1 to 4.
The pharmaceutical composition of claim 8, wherein the inflammatory diseases of the large intestine and the small intestine are selected from the group consisting of acute enteritis, Crohn's disease, ulcerative colitis and ischemic colitis.
The pharmaceutical composition of claim 8, wherein the functional bowel disease is selected from the group consisting of diarrhea, constipation, intestinal tract and irritable bowel syndrome.
(S1) increasing the beans;
(S2) first fermentation by inoculating Aspergillus duck with Bacillus subtilis-SKm in the increased soybeans; And
(S3) secondary fermentation by additional inoculation of lactococcus lactis after the first fermentation;
Fermented product manufacturing method comprising a.
The method according to claim 11, Aspergillus duck soybean material based on 100 parts by weight of 0.1-1 parts by weight, Bacillus subtilis -SKm is 10 ⁴ -10 ⁸ cfu / g and Lactococcus lactis is 10 ⁴ -10 ⁸ cfu / Method of producing a fermented product that is made by inoculating g.
The method of claim 11, wherein the first fermentation step is performed at 20-45 ° C. for 24-72 hours.
The method of claim 11, wherein the secondary fermentation step is performed at 20-45 ° C. for 12-36 hours.

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