Strain derived from traditional fermented food and having excellent enzyme productivity, and method for preparing fermented cereal enzyme food by using same

Abstract

Disclosed are a novel strain of Bacillus amyloliquefaciens, a method of producing fermented grains using the strain, fermented grains produced using the strain, and a composition for thrombolysis; digestion improvement; prophylaxis, amelioration or treatment of bowel inflammation, serous membrane weakening or intestinal injury; or antioxidation, comprising the fermented grains.

Claims

1. A bacterial composition, comprising: a strain of Bacillus amyloliquefaciens BA245 deposited under accession number KCTC12905BP; wherein the bacterial composition is a powdered and freeze-dried composition.

2. Fermented grains produced with a strain of Bacillus amyloliquefaciens BA245 deposited under accession number KCTC 12905BP.

3. A method of producing fermented grains according to claim 2, comprising: (a) inoculating a grain with the strain of Bacillus amyloliquefaciens BA245 deposited under accession number KCTC 12905BP; and (b) culturing the strain to obtain fermented grains.

4. The method of producing the fermented grains according to claim 3, wherein, in step (a), the grain comprises at least one grain selected from the group consisting of wheat, wheat germ, wheat bran, white rice, brown rice, germinated brown rice, barley, oats, red rice, sticky black rice, sticky rice, rice bran, soybeans, black soybean, black beans, quinoa, lentils, and adlay.

5. The method of producing the fermented grains according to claim 3, wherein, in step (a), the grain comprises wheat germ and wheat bran.

6. The method of producing the fermented grains according to claim 5, wherein the wheat germ and the wheat bran are present in an amount of 40 parts by weight to 100 parts by weight relative to 100 parts by weight of the grain in step (a).

7. The method of producing the fermented grains according to claim 5, wherein, in step (a), the grain further comprises at least one grain selected from the group consisting of oats, lentils, brown rice, sticky barley, and quinoa.

8. The method of producing the fermented grains according to claim 3, wherein, in step (a), the grain has a moisture content of 30% (v/w) to 70% (v/w).

9. The method of producing the fermented grains according to claim 3, further comprising: treating the grain with moisture prior to step (a).

10. The method of producing the fermented grains according to claim 3, further comprising: performing heat treatment of the grain prior to step (a).

11. The method of producing the fermented grains according to claim 3, further comprising: performing heat treatment after treating the grain with moisture prior to step (a).

12. The method of producing the fermented grains according to claim 3, wherein culturing in step (b) is solid culturing.

13. The method of producing the fermented grains according to claim 3, wherein culturing in step (b) is performed at a temperature of 20° C. to 50° C.

14. The fermented grains according to claim 2, wherein the fermented grains are produced by a method producing fermented grains, comprising: (a) inoculating a grain with the strain of Bacillus amyloliquefaciens BA245 deposited under accession number KCTC 12905BP; and (b) culturing the strain to obtain the fermented grains.

15. A food composition comprising a strain of Bacillus amyloliquefaciens BA245 deposited under accession number KCTC12905BP or fermented grains comprising the strain of Bacillus amyloliquefaciens BA245 deposited under accession number KCTC 12905BP.

16. The food composition according to claim 15, wherein the food composition is an enzyme food.

17. A composition for thrombolysis comprising: a strain of Bacillus amyloliquefaciens BA245 deposited under accession number KCTC12905BP or fermented grains comprising the strain of Bacillus amyloliquefaciens BA245 deposited under accession number KCTC 12905BP.

18. The composition for thrombolysis according to claim 17, wherein the composition for thrombolysis is a composition for prophylaxis, amelioration or treatment of myocardial infarction, venous thrombosis, stroke, cerebral infarction, cerebral thrombosis, or cerebral embolism.

19. A composition for digestion improvement, comprising: a strain of Bacillus amyloliquefaciens BA245 deposited under accession number KCTC12905BP or fermented grains comprising the strain of Bacillus amyloliquefaciens BA245 deposited under accession number KCTC 12905BP.

20. A composition for prophylaxis, amelioration or treatment of bowel inflammation, serous membrane weakening or intestinal injury, comprising: a strain of Bacillus amyloliquefaciens BA245 deposited under accession number KCTC12905BP or fermented grains comprising the strain of Bacillus amyloliquefaciens BA245 deposited under accession number KCTC 12905BP.

21. A composition for antioxidation comprising: a strain of Bacillus amyloliquefaciens BA245 deposited under accession number KCTC12905BP or fermented grains comprising the strain of Bacillus amyloliquefaciens BA245 deposited under accession number KCTC 12905BP.

22. A method for prophylaxis, amelioration or treatment of myocardial infarction, venous thrombosis, stroke, cerebral infarction, cerebral thrombosis, or cerebral embolism, comprising administration of the fermented grains according to claim 2 to a subject in need thereof.

23. A method for improving digestive function of a subject, comprising administration of the fermented grains according to claim 2 to a subject in need thereof.

24. A method for prophylaxis, amelioration or treatment of bowel inflammation, serous membrane weakening or intestinal injury, comprising administration of the fermented grains according to claim 2 to a subject in need thereof.

25. A method for reducing active oxygen in a subject, comprising administration of the fermented grains according to claim 2 to a subject in need thereof.

26. A bacterial composition comprising: a pre-cultured strain of Bacillus amyloliquefaciens BA245 deposited under accession number KCTC12905BP in a number of the bacteria of 1×105 CFU/g to 1×1010 CFU/g; and a culture medium for the bacteria.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows selection results on alpha-amylase plate media (FIG. 1A) and protease plate media (FIG. 1B) in order to select strains which highly produce starch degradation enzymes and protein degradation enzymes at the same time.

(2) FIG. 2 shows a phylogenic tree illustrating a phylogenic relationship based on 16S rRNA gene sequence.

(3) FIG. 3 shows a phylogenic tree illustrating a phylogenic relationship based on gyrase A gene sequence suitable for phylogeny of strains in genus bacillus.

(4) FIG. 4 shows chromatography results for the content of low molecular weight peptides of BA245 fermented grains compared with that of raw materials and BA474 fermented grains in which BA474 is a homologous strain.

(5) FIG. 5 shows fibrin plate analysis results illustrating thrombolysis activity.

(6) FIG. 6 shows a bar graph illustrating fibrinolytic activity.

(7) FIG. 7 shows intestinal permeability evaluation results depending upon the content of BA245 fermented grains.

(8) FIG. 8 shows degree of expression of claudin (FIG. 8a) and occludin (FIG. 8b) which are tight junction proteins of intestinal cells, in which the degree of expression represents the degree of serous membrane reinforcement.

(9) FIG. 9 includes photographs illustrating histological observation of recovery of intestine epidermal injury by ingesting BA245 fermented grains.

(10) FIG. 10 shows test results for antioxidant activity (radical scavenging ability) of the grain raw materials, BA245 fermented grains and enzyme food, respectively.

MODE FOR INVENTION

(11) Hereinafter, the present disclosure will be described in more detail with reference to the following examples. It should be understood that these examples are provided for illustration only and are not to be construed in any way as limiting the present disclosure.

Example 1: Selection of Strain Capable of Highly Producing Starch and Protein Degradation Enzymes

(12) In order to isolate strains having high starch and protein degradation enzyme production ability, the present inventors isolated about 3,000 species of microorganisms from various kinds of Korean traditional fermented food (kimchi, jang, yeast, Korean traditional liquor, salted sea foods, and the like), and tried to screen strains possessing high expression degree of starch and protein degradation enzymes mainly from about 1308 species of bacillus suitable for food.

(13) Selection of strains having high starch and protein degradation enzyme production ability was performed by comparing size of transparent circles formed by decomposition of substrates (raw materials) in a YM agar medium (3.0 g of yeast extract, 3.0 g of malt extract, 5.0 g of peptone, 10.0 g of dextrose, 20.0 g of agar) containing 1% (w/v) of soluble starch (Difco, USA) or 2% (w/v) of skim milk (Difco, USA).

(14) Specifically, TSB media (17.0 g of enzymatic digest of casein, 3.0 g of enzymatic digest of soybean meal, 5.0 g of NaCl, 2.5 g of dipotassium phosphate, 2.5 g of dextrose, final pH: 7.3±0.2 at 25° C.) were inoculated with each strain, cultured at 37° C. at 200 rpm for 12 hours, followed by spotting the culture solution by 1.0 μl to the YM agar medium containing a soluble starch and skim milk. After culturing the agar medium at 37° C. for 16 hours, the diameter of transparent circles formed on the medium was measured.

(15) As a result, strain BA245 which exhibited 5.85 mm diameter transparent circles on 1% (w/v) soluble starch medium and 6.14 mm diameter transparent circles on 2% (w/v) skim milk (Difco, USA) medium and possessed excellent starch and protein degradation enzyme activity was selected as a strain for the grain fermentation (FIGS. 1a and 1b).

Example 2: Identification of Strain BA245

(16) 2-1. 16S rRNA Gene Sequencing of Strain BA245

(17) In order to identify the strain BA245 selected in Example 1, 16S RNA gene sequencing was requested to Korean Culture of Microorganisms (KCCM) affiliated with Korean Federation of Culture Collections (KFCC), thereby obtaining a 1420 bp nucleotide sequence (SEQ ID NO: 1) including a 50 to 900 bp nucleotide sequence required for the identification. In addition, the sequence was registered in the database of NCBI GenBank under accession number KR535604.

(18) 2-2. Analysis of Phylogenic Tree According to 16S rRNA Gene Sequencing of Strain BA245

(19) In order to analyze phylogenic taxon of strain BA245, 16S rRNA gene sequences of reference strains of various species which are close to strain BA245 in genus Bacillus were investigated using sequence data registered with GenBank, and these gene sequences were aligned using Bioedit (Hall, 1999) and Clustal X (Thompson et al., 1997). Evolution procedures of strains were pursued using the Kimura two-parameter model (Kimura, 1983), and phylogenic taxon of strains was determined using neighbor-joining and maximum parsimony method of Phylogenic tree (FIG. 2).

(20) 2-3. Phylogenic Tree Analysis of Gyrase a (Gyr A) Gene Sequence of Strain BA245

(21) Identification of Strain BA245 was Performed Using Phylogenic Analysis Based on Gyrase a Gene Sequence.

(22) In order to analyze gyrase A gene sequence, a forward primer (SEQ ID NO: 2: 5′-CAG TCA GGA AAT GCG TAC GTC CTT-3′) and a reverse primer (SEQ ID NO: 3: 5′-CAA GGT AAT GCT CCA GGC ATT GCT-3′) were constructed, and then Microgen Inc. performed sequence analysis of gyrase A. Gyrase A gene sequences of reference strains of various species close to strain BA245 in genus Bacillus were investigated using sequence data registered with GenBank, and these gene sequences were aligned using Bioedit (Hall, 1999) and Clustal X (Thompson et al., 1997). Development procedures of strains were chased using the Kimura two-parameter model (Kimura, 1983) and phylogenic taxon of strains was determined using neighbor-joining and maximum parsimony method of phylogenic tree (Kimura et al., 2004) (FIG. 3).

(23) 2-4. Analysis of Biochemical Properties of BA245 Using API Kit

(24) Biochemical properties of Bacillus amyloliquefaciens BA245 were examined using API 50CH (bioMerieux Co., France) used in identification of Bacillus. API kit was used in accordance with manufacturer's guidelines, and results thereof are summarized in Table 1 below.

(25) A TSB liquid medium (17.0 g if enzymatic digest of casein, 3.0 g of enzymatic digest of soybean meal, 5.0 g of NaCl, 2.5 g of dipotassium phosphate, 2.5 g of dextrose, final pH: 7.3±0.2 at 25° C.) was inoculated with Bacillus amyloliquefaciens BA245, cultured at 37° C., dispensed to Eppendorf tubes, and then centrifuged at 10,000 rpm for 5 minutes to collect cells, which were washed once with a sterilized saline solution (0.85%). The cells were suspended in sterilized distilled water, and an ampoule of API 50CH medium aseptically broken was inoculated with the suspension, followed by pipetting homogenously, which in turn was dispensed in an amount of 150 μl to microtubes of each test strip. After cells were dispensed, the cells were cultured in an incubator at 37° C. for 24 to 48 hours and color change was investigated using API kit.

(26) TABLE-US-00001 TABLE 1 Property Result Property Result Glycerol + Salicin + Erythritol − Cellobiose + D-arabinose − Maltose + L-arabinose + Lactose + Ribose + Melibiose (+) D-xylose + Sucrose + L-xylose − Trehalose + Adonitol − Inulin − Methyl-B-D- − Melezitose − xylopyranside Galactose (+) Raffinose + Glucose + Starch + Fructose + Glycogen + Mannose + Xylitol − Sorbose − Gentiobiose + Rhamnose − D-turanose (+) Dulcitol − D-lyxose − Inositol + D-tagatose − Mannitol + D-fucose − Sorbitol + L-fucose − Methyl-α,D- − D-arabitol − Mannopyranside Methyl-α,D- + L-arabitol − glucoside N-acetyl- + Gluconate − glucosamine Amygdalin + 2-keto- − gluconate Arbutin + 5-keto- (+) gluconate Esculin +

(27) 2-5. Results of Identification

(28) Considering the overall results of biochemical properties and phylogenic relationship analysis, the selected strain was designated as Bacillus amyloliquefaciens BA245 and was deposited at the Korean Collection for Type Cultures (KCTC) on Sep. 23, 2015 under accession number KCTC12905BP under the provisions of the Budapest Treaty.

Example 3: Production of Fermented Grains Using Strain BA245

(29) Fermented grains was produced using Bacillus amyloliquefaciens BA245 (hereinafter referred to as ‘strain BA245’) selected in Example 2 as follows.

(30) First, moisture was added to 300 g of grains [wheat bran; wheat germ; oats; brown rice; quinoa; lentils; sticky barley; wheat germ and wheat bran grain mixture (60 wt % of wheat germ and 40 wt % of wheat bran); and an entire grain mixture (40 wt % of wheat germ, 30 wt % of wheat bran, 10 wt % of oats, 5 wt % of lentils, 5 wt % of brown rice, 5 wt % of sticky barley and 5 wt % of quinoa, hereinafter the entire the grain mixture is referred to as ‘the grain raw material’)], followed by steaming the grains at 121° C. for 30 minutes and then cooling to 40° C. or less. Next, the strain BA245 was streaked on a TSB agar medium (17.0 g of enzymatic digest of casein, 3.0 g of enzymatic digest of soybean meal, 5.0 g of NaCl, 2.5 g of dipotassium phosphate, 2.5 g of dextrose, 15.0 g of agar, final pH: 7.3±0.2 at 25° C.), followed by culturing at 37° C. for 12 hours, thereby activating the strain. The activated strain was suspended in 9 ml of 0.8% sterilized NaCl solution, the strain was diluted to about 0.2 in A.sub.660 nm using a loop and the resulting suspension was used as seed culture. 40 ml of a TSB medium (17.0 g of enzymatic digest of casein, 3.0 g of enzymatic digest of soybean meal, 5.0 g of NaCl, 2.5 g of dipotassium phosphate, 2.5 g of dextrose, final pH: 7.3±0.2 at 25° C.) was inoculated with the seed culture suspension such that the concentration was 1%, followed by shake culturing at 37° C. and 180 rpm.

(31) To the steamed grains in which the moisture content was adjusted to 36%, 30 ml of a culture solution (6.0×10.sup.7 cfu/ml) was added and mixed sufficiently, followed by solid fermentation by stationary culturing at 37° C. for 24 hours under constant humidity conditions, thereby producing fermented grains.

(32) Meanwhile, as a control group of strain BA245, Bacillus amyloliquefaciens BA474 (hereinafter referred to as ‘strain’ BA474) which belongs to the same species as strain BA245 was used, and fermented grains were produced using an entire the grain mixture as a raw material in the same manner as in Example 3 except that strain BA474 was used instead of strain BA245.

Example 4: Analysis of Starch Degradation Enzyme Activity

(33) In order to analyze starch degradation enzyme activity of the fermented grains, the following experiment was performed.

(34) Fermented grains fermented using strain BA245 and strain BA474 of Example 3 were dried in a dryer at 60° C. until the moisture content reached 10% or less, followed by crushing to obtain specimens for measurement of starch degradation enzyme activity (hereinafter referred to as ‘BA245 fermented grains’ and ‘BA474 fermented grains’ depending upon the sort of grain used in fermentation of the grain-fermented substances, wherein if an entire grain mixture (40 wt % of wheat germ, 30 wt % of wheat bran, 10 wt % of oats, 5 wt % of lentils, 5 wt % of brown rice, 5 wt % of sticky barley and 5 wt % of quinoa) was used as a raw material, the specimen was referred to as ‘BA245 fermented grains’ without designating raw materials, and if a grain mixture composed of grain components or a grain mixture composed of wheat germ and wheat bran was used as a raw material, the specimen was referred to as ‘BA245 fermented grains’ with raw materials designated in parentheses. Starch degradation enzyme activity was measured in accordance with the enzyme food alpha-amylase test method described in “Standards and specifications of foods”.

(35) Specifically, 5.0 g of each specimen was precisely weighed, dissolved in water to produce 100 ml of a solution, followed by filtering the solution, which in turn was used as a liquid specimen, while two 20 ml tubes were prepared, one for test and the other for blank test. To the test tube, 5 ml of 1% soluble starch solution, 13 ml of Mcilvaine buffer (pH 6.0) and 1 ml of 0.1% calcium chloride solution were added and warmed to 37° C., and 1 ml of liquid specimen was further added thereto, and left at 37° C. for 30 minutes, thereby preparing a reaction solution for test. Separately, in the tube for blank test, 1 ml of a liquid specimen, which was heated at 100° C. for 30 minutes to deactivate the specimen, was treated in the same manner as in the test tube, thereby preparing a reaction solution for blank test. To 0.2 ml of each reaction solution for test and blank test, 10 ml of an iodine reagent solution was added to prepare a test solution, which in turn was subjected to absorbance measurement with a liquid layer of 1 cm at a wavelength of 660 nm using water as a comparison liquid. The absorbance of the test solution should be within 0.030 of that of the blank test. If the absorbance was undetectable due to excessive color development, the absorbance was measured using a diluted liquid specimen by applying times of dilution. The content of starch was calculated using a standard curve obtained from colorimetric reaction of an iodine (I.sub.2) solution, in which 1 unit of alpha-amylase was defined as an amount of enzyme to be used to digest 10 mg of starch for 30 minutes.

(36) TABLE-US-00002 TABLE 2 Sample Alpha-amylase (U/g) BA245 fermented grains 2762 BA245 fermented grains 2300 (grain mixture of wheat germ and wheat bran) BA474 fermented grains 169

(37) As a result, it was confirmed from Table 2 that starch degradation enzyme activity of the BA245 fermented grains and BA245 fermented grains (grain mixture of wheat germ and wheat bran) was much better than the BA474 fermented grains as a control group.

Example 5: Analysis of Protein Degradation Enzyme Activity

(38) In order to analyze protein degradation enzyme activity of the BA254 fermented grains, the following experiment was performed.

(39) Specimens obtained in the same manner as in Example 3 was subjected to an enzyme food protease test method prescribed in .sup.┌Standards and Specification of Foods.sub.┘.

(40) Specifically, 5.0 g of each specimen was precisely weighed, dissolved in water to produce 100 ml of a solution, followed by filtering the solution, which in turn was used as a liquid specimen. To a test tube, 1 ml of 0.6% casein solution was added and warmed in a thermostat water bath at 37° C., and 1 ml of liquid specimen was precisely weighed and added thereto, shaken sufficiently and left in a thermostat water bath at 37° C. for exactly 10 minutes. To the resulting solution, 2 ml of 0.4 M acetic trichloroacetic acid solution was added, left again at 37° C. for 25 minutes, and then filtered. 1 ml of the resulting filtered solution was precisely placed in a test tube, and 5 ml of 0.4 M sodium carbonate solution and 1 ml of Folin's reagent (prepared by diluting a stock solution three times) were added thereto, followed by shaking sufficiently. The resulting solution was left at 37° C. for 20 minutes and the resulting colored solution was used as a test solution. Separately, 1 ml of a liquid specimen was precisely weighed and placed in a test tube, and left at 37° C. for 10 minutes, and then 2 ml of 0.4 M trichloroacetic acid was added and mixed therewith. Then, 1 ml of 0.6% casein solution was added to the mixture, which in turn was left at 37° C. for 25 minutes, followed by the same treatment as in the test solution. The resulting solution was used as a solution for blank test. The absorbance was measured at a wavelength of 660 nm with a liquid layer of 1 cm using water as a comparison liquid. The absorbance of the test solution should be within 0.030 of that of the blank test. If the absorbance was undetectable due to excessive color development, the absorbance was measured using a diluted liquid specimen by applying times of dilution. A standard curve was constructed using tyrosine and protease activity was analyzed in comparison with detected tyrosine content, in which 1 unit of protease was defined as a microgram of tyrosine produced for one minute.

(41) TABLE-US-00003 TABLE 3 Sample Protease (U/g) BA245 fermented grains 3868 BA245 fermented grains 3481 (grain mixture of wheat germ and wheat bran) BA474 fermented grains 106

(42) As a result, it was confirmed from Table 3 that protein degradation enzyme activity of the BA245 fermented grains was much better than the BA474 fermented grains as a control group.

(43) It was also confirmed from the results of Examples 3 and 4 that BA245 was useful for preparation of an enzyme food comprising the fermented grains having a high fermentation titer value.

Example 6: Analysis of Low Molecular Weight Protein Content

(44) Since it was confirmed in Example 4 that fermented grains having good protein degradation enzyme activity were produced in grain fermentation using BA245, the content of proteins in the BA245 fermented grains was analyzed according to the molecular weight in order to identify whether proteins in the grain were present in hydrolyzed peptides or low molecular weight proteins.

(45) 0.1 g of the specimen of Example 3 was precisely weighed and subjected to extraction using 5 ml of 8 M urea. After centrifuging the resulting mass at 8,000 rpm for 10 minutes, the supernatant was taken and filtered with a 0.22 μm syringe filter, and finally used as a sample for analysis. The injection volume was 25 μl and a mixture of 50 mM NaPO.sub.4 (pH 7.2) and 150 mM NaCl was used as a mobile phase. The flow rate was 0.5 ml/min and detection was performed at a UV wavelength of 214 nm.

(46) As a result, it could be seen that the BA245 fermented grains had a larger peak area for low molecular weight proteins than the grain raw material and the BA474 fermented grains, thereby showing a shift of protein density to a central band side (FIG. 4). In chromatography graphs of FIG. 4, the left bands refer to high molecular weight proteins, the central bands refer to low molecular weight proteins, and the right bands refer to a solvent. Numerical data of chromatography of FIG. 4 are shown in Table 4.

(47) TABLE-US-00004 TABLE 4 MW (kDa) 30 or 5 or Sample more 10 to 30 5 to 10 less Total (%) Grain raw material 58.05 17.31 8.07 16.57 100 BA245 fermented grains 10.34 10.06 15.01 64.59 100 BA474 fermented grains 36.15 13.70 11.75 38.40 100

(48) From the results, it could be seen that the high molecular weight proteins in the grain raw material were hydrolyzed by fermentation using BA245 into the low molecular weight proteins and that the degree of hydrolysis by BA245 was much better than the degree of hydrolysis by BA474.

Example 7: Nutrient Components Analysis

(49) In order to confirm that the content of carbohydrates is reduced, the content of dietary fiber is increased, the content of crude proteins and essential free amino acids is increased to increase nutrient components and nutrient absorption rate in grain fermentation using BA245, a grain raw material, a BA245 fermented grains, a BA474 fermented grains and three enzyme foods fermented from commercially available grains [Secret of Fermented Enzyme produced by Daesang Wellife Co., Ltd., (hereinafter referred to as “DAESANG”); Seed fermented enzyme produced by LG Life Health (hereinafter referred to as “LG”); Fullvita Naemome Gain Enzyme Digest produced by ORGA Food (hereinafter referred to as “PULMUONE”) were prepared and offered to the Korea Health Supplement Institute to conduct nutrient component analysis.

(50) TABLE-US-00005 TABLE 5 Item BA245 BA245 BA245 Fermented BA245 BA245 BA245 Fermented Fermented Grains Fermented Fermented Fermented Grains Grains (grain mixture of Grains Grains Grains (Brown (Sticky wheat germ and (oat) (Quinoa) (Lentil) rice) barley) wheat bran) Carbohydrate (% w/w) 70.12 72.49 61.67 79.57 76.05 58.55 Crude protein (% w/w) 12.27 13.77 29.10 9.02 13.44 23.84 Saccharide (mg/g) 44.78 18.99 ND 100.52 102.10 ND Essential Threonine 1.24 9.78 8.63 2.80 2.50 4.67 Free Lysine 4.11 12.43 70.93 7.40 24.37 78.85 Amino Isoleucine 1.05 7.77 9.87 5.56 7.74 16.95 Acid Leucine 2.96 12.76 9.48 10.24 13.76 61.21 (mg/100 g) Methionine 1.75 2.97 1.58 10.14 6.23 10.64 Phenylalanine 12.20 12.52 68.76 27.96 63.37 109.41 Tryptophan 5.98 6.03 5.78 5.63 18.15 89.63 Valine 7.17 11.37 27.15 26.95 54.03 77.85 Total 36.46 75.63 202.18 96.68 190.15 449.21 ND: Non-detectable

(51) TABLE-US-00006 TABLE 6 Item The grain Material (Entire BA245 BA474 The grain Fermented Fermented Mixture) Grains Grains DAESANG LG PULMUONE Carbohydrate (% w/w) 62.16 57.21 61.71 64.62 69.05 71.36 Dietary fiber (% w/w) 23.37 27.32 23.52 9.67 7.75 12.74 Crude protein (% w/w) 18.00 23.97 18.38 18.68 21.02 14.16 Saccharide (mg/g) 32.87 ND 43.34 116.70 164.91 155.16 Essential Threonine 16.78 57.81 20.91 7.00 3.89 15.47 Free Lysine 9.18 141.06 25.06 18.11 6.21 35.96 Amino Isoleucine 3.40 96.67 13.23 4.35 3.57 8.75 Acid Leucine 4.16 192.30 60.73 39.43 5.06 24.89 (mg/100 g) Methionine 1.31 45.44 9.48 6.92 ND 4.11 Phenylalanine 4.54 206.15 56.39 35.75 4.98 15.45 Tryptophan 29.44 78.09 33.01 9.79 ND 5.78 Valine 6.79 211.79 65.30 7.88 5.64 14.40 Total 75.60 1029.31 284.11 129.23 29.35 124.81 ND: Non-detectable

(52) As a result, in Tables 5 and 6, it could be seen that the fermented grains produced using BA245 possessed enhanced nutrient components as compared with the raw materials in view of the reduced content of carbohydrates and saccharides, the increased content of dietary fibers, crude proteins and essential free amino acids, and the like, and that the BA245 fermented grains and the BA245 fermented grains (grain mixture of wheat germ and wheat bran) were superior to the BA474 fermented grains and other enzyme foods in view of the nutrient components.

Example 8: Analysis of Fibrinolytic Enzyme Activity

(53) In order to determine whether the BA245 fermented grains have thrombolysis activity, fibrinolytic enzyme activity of the grain raw material and the BA245 fermented grains was measured. As control groups, plasmin (1 U/ml) and fermented soybean foods called ‘Chungkukjang’ (produced by Taekwang Co., Ltd.), Natto (produced by Pulmuone Co., Ltd., Azuma Co., Ltd., and Takano Co., Ltd.) and commercially available food (DAESANG; HI-SAENG produced by Hi-mo Natural Health Division), which are reported to have thrombolysis activity, were used.

(54) After 1 g of a sample was mixed with 9 ml of a sterilized saline solution, the resulting solution was extracted using a stirrer (Wiseshaker, Daihan) at 4° C. for 30 minutes while mixing, followed by centrifugation (8,000×g, 4° C., 30 minutes) and filtering, and the resulting supernatant was used as an analysis sample.

8-1. Fibrin Plate Assay

(55) To 10 ml of 2.4% completely dissolved fibrin solution (pH 7.0), 10 ml of 1.5% agarose solution was added and mixed, directly poured into a petri dish, and left at room temperature for 2 to 3 days until gel could be completely hardened, thereby obtaining a fibrin plate. The prepared fibrin plate was punched such that openings having a diameter of 4.5 mm were consistently formed, and to the openings, 20 μl of an analysis sample was added dropwise, and cultured at 37° C. for 24 hours. In order to clearly observe the size of transparent circles formed, 0.11M of trichloroacetic trichloroaceticacid was poured onto the fibrin gel.

(56) As a result, the BA245 fermented grains exhibited thrombolytic activity of 220% higher than plasmin, 177% higher than Chungkukjang, 122% higher than Natto (PULMUNONE), 117% higher than Natto (TAKANO) and 131% higher than Natto (AZUMA), 119% higher than enzyme food (DAESANG), and 142% higher than enzyme food (HI-SAENG) (Table 7 and FIG. 5).

(57) TABLE-US-00007 TABLE 7 Sample Dissolve zone (mm) Control group (Plasmin)  8.6 Grain raw material — BA245 fermented grains 19.0 Enzyme food (DAESANG) 15.9 Enzyme food (HI-SAENG) 13.4 Natto (PULMUONE) 15.6 Natto (TAKANO) 16.3 Natto (AZUMA) 14.5 Chungkukjang powder 10.7 (TAEKWANG)

8-2. Analysis of Fibrinolytic Activity

(58) To 0.1 ml of the sample prepared in Example 8, 0.3 ml of 0.1M Tris-HCl buffer (pH 7.8) containing 10 mM calcium chloride was added, and warmed in a thermostat water bath at 30° C. for 5 minutes. To this solution, 0.3 ml of 1.2% fibrin solution (pH 7.8) was added, mixed, and left at 30° C. for 10 minutes, followed by adding 0.6 ml of 0.11 M trichloroacetic acid to the mixture in order to inhibit enzyme reaction. The resulting solution was used as a test solution. Separately, 0.3 ml of 0.1M Tris-HCl buffer (pH 7.8) was added to 0.1 ml of the sample and left in a thermostat water bath at 30° C. for 10 minutes, followed by adding 0.6 ml of 0.11 M trichloroacetic acid thereto. To the resulting solution, 0.3 ml of 1.2% fibrin solution (pH 7.8) was added, and the resulting solution was used as a blank test solution. Each of the test solution and the blank test solution was centrifuged at 12,000 rpm for 5 minutes, and a supernatant was collected and subjected to absorbance measurement at 275 nm for quantitative comparison of fibrinolytic enzyme activity.

(59) As a result, it could be seen that the BA245 fermented grains exhibited the highest fibrinolytic activity and much better fibrinolytic activity than Natto (Table 8 and FIG. 6).

(60) TABLE-US-00008 TABLE 8 Sample Fibrinolytic activity (FU/g) The grain raw material 19.86 BA245 fermented grains 136.17 Enzyme food (DAESANG) 53.90 Enzyme food (HI-SAENG) 25.53 Natto (PULMUONE) 68.06 Natto (TAKANO) 52.48 Natto (AZUMA) 36.88 Chungkukjang powder 22.7 (TAEKWANG)

8-3. Fibrinolytic Activity Analysis of Strain BA245

(61) In order to analyze fibrinolytic activity of the strain, the strain BA245 was compared with three strains isolated from commercially available Natto.

(62) As a result, as shown in Table 9, it could be seen that the strain BA245 exhibited much better thrombolytic enzyme activity than the strains isolated from commercially available Natto.

(63) TABLE-US-00009 TABLE 9 Sample Fibrinolytic activity (FU/g) Strain BA245 1617 Strain isolated from Natto (PULMUONE) 1209 Strain isolated from Natto (TAKANO) 1072 Strain isolated from Natto (AZUMA) 1098

Example 9: Evaluation of Solid Meal Gastric Emptying Improvement by Feeding BA245 Fermented Grains

(64) 8-week old male SD (Sprague-Dawley) rats were allotted to experiment groups each consisting of ten rats, wherein all animals were acclimated to experimental diet and circumstance for one week. After acclimation, rats were fasted for 20 hours while freely drinking water in order to measure gastric emptying ability.

(65) Experimental Example 1 was performed to evaluate gastric emptying per same hours under the condition that the same solid meal was fed. Specifically, on the day of experiment, 3 g of an experimental diet prepared by suspending 50% of barium sulfate, 0.6% of agarose, feed (AIN-93, Dyets) in water was orally administered to SD rats. Experiment was performed for a total of three experimental groups, wherein 15% of feed was added to a control group, while the diet formulated with 5% of the grain raw materials+10% of feed or formulated with 5% of BA245 fermented grains+10% of feed was fed to the experimental groups such that the fed amount could be the same. 40 minutes after administration, the stomach was excised and gastric emptying (%) was evaluated by measuring the weight of food remaining in the stomach.

(66) Experimental Example 2 is a model in which an actual dose method in the human body is reflected, and improvement in gastric emptying when the same amount of feed was added and the BA245 fermented grains was further added was evaluated, in which the human equivalent dose corresponds to six times based on rat standard. 15% of feed was fed to the control group, and 600 mg/kg of the grain raw material and 600 mg/kg of the BA245 fermented grains were further fed to experimental groups. 40 minutes after administration, the stomach was excised and gastric emptying (%) was evaluated by measuring the weight of the diet remained in the stomach.

(67) As a result, it could be seen that Experimental Example 1 exhibited significant increase in gastric emptying (p<0.05) as compared with the case where BA245 fermented grains or the grain raw material was administered (Table 10), and that Experimental Example 2 exhibited significant increase in gastric emptying (p<0.05) as compared with the case the grain raw material was fed (Table 11).

(68) Gastric emptying is known to clinically decline in functional indigestion and it can be evaluated that digestion capability is enhanced when the BA245 fermented grains is digested together with food.

(69) TABLE-US-00010 TABLE 10 Experimental group Gastric emptying (%, ±SD) Feed (15%) 23 ± 20 Feed (10%) + Grain raw material (5%)  9 ± 11 Feed (10%) + BA245 fermented grains (5%) 37 ± 21

(70) TABLE-US-00011 TABLE 11 Experimental group Gastric emptying (%, ±SD) Feed 23 ± 20 Feed + Grain raw material (600 mg/kg)  9 ± 11 Feed + BA245 fermented grains (600 mg/kg) 37 ± 21

Example 10: Evaluation of Intestinal Permeation Improvement Through BA245 Fermented Grains Ingestion in an Acute Enteritis Animal Model

(71) An acute enteritis animal model was constructed by administering alcohol to rats, and the resulting rats were fed BA245 fermented grains with different intake concentrations to measure the degree of recovery in stomach function depending upon ingestion amount.

(72) Animals used in experiment were 8-week old SD rats weighing 200 to 250 g, which were grown under 12 hours on/12 hours off light in a polycarbonate cage. After one week of acclimation, rats were treated in accordance with the following experiment design together with free diet.

(73) Group 1: Control group

(74) Group 2: Ethanol

(75) Group 3: Ethanol+50 mg/kg/day of BA245 fermented grains

(76) Group 4: Ethanol+100 mg/kg/day of BA245 fermented grains

(77) Group 5: Ethanol+150 mg/kg/day of BA245 fermented grains

(78) Group 6: Ethanol+100 mg/kg/day of grain raw material

(79) Alcohol was orally administered to Groups 2 to 6 for four weeks to induce irritable bowel syndrome, thereby causing reduction in intestinal function. An initial dose was 2 g/kg/day. The amount of alcohol to be orally administered was increased weekly by 1 g/kg/day, and by the time of the fourth and final week, 5 g/kg/day was orally administered. After four weeks, intestinal permeation ability was measured.

(80) Specifically, intestinal permeation ability was determined by sacrificing rats, isolating the ileum, and soaking the isolated ileum in Krebs-Henseleit bicarbonate buffer (KHBB: bicarbonate buffer), followed by suturing one end of the ileum, and injecting 100 μl of FITC-dextran into the lumen. The other end of the intestine was sutured to form 8 cm of a gut sac. The gut sac was washed with KHBB, followed by putting in 2 mL of KHBB and incubating at 37° C. for 20 minutes. FD-4 of FTTC-dextran passed from the lumen to incubation buffer was measured at 530 nm using a spectrophotometer. Permeability of FD-4 is represented in μg at 1 cm per minute.

(81) As a result, it could be seen that there was significant reduction in intestinal permeability due to alcohol administration by feeding the BA245 fermented grains, and particularly, it could be seen that the group administered 100 mg or more per kg of body weight exhibited recovery close to the normal group (FIG. 7).

Example 11: Analysis of Serous Membrane Enhancement by BA245 Fermented Grains Intake Through an Acute Enteritis Animal Model

(82) If serous fluid comes to leak due to serous membrane weakening, serous inflammation can be induced, and in order to quantitatively measure recovery of intestinal permeability increased due to alcohol, analysis of tight junction proteins of intestinal cells participating in intestinal permeability was performed using the animal model designed in Example 10. Representative main tight junction proteins participating in cellular bonding of serous membranes are ZO-1, claudin, and occludin, wherein the expression degree of genes corresponding to each protein was measured through RT-PCR analysis, and the bonding degree between intestinal cells was identified, thereby confirming effects of fermented grains intake. In RNA extraction, TRIzol was utilized and concentration thereof was measured using a NanoDrop spectrophotometer.

(83) As a result, it could be seen that expression of claudin and occludin was increased in proportion to BA245 fermented grains administration and feeding concentration, thereby enhancing the serous membrane (p<0.05) (FIGS. 8A and 8B).

Example 12: Evaluation of Structural Damage Amelioration by BA245 Fermented Grains Intake Through Intestinal Histological Analysis

(84) Intestinal histological analysis was performed using an animal model designed in Example 10. Intestinal tissues sampled from each group were fixed in a 10% formalin solution, followed by dehydration using ethanol, and then fixed in paraffin. A 4 m thick section was dyed with hematozylin-Eosin and the dyed fragments were observed through a microscope.

(85) It could be confirmed that crypt and villus structures of intestinal walls were broke down when intestinal injury was developed by alcohol, and it could be observed that the BA245 fermented grains intake group, particularly the group to which 100 mg or more of BA245 fermented grains per kg of body weight was fed, exhibited recovery close to normal tissues (FIG. 9).

Example 13: Measurement of Antioxidant Activity of BA245 Fermented Grains

(86) Antioxidant activity is known to control various kinds of disease caused by oxidative stress (lipid and oxide accumulation prevention, enzyme inactivation, cell aging, arteriosclerosis, diabetes, stroke, cancer, DNA synthesis decline, adult diseases and aging, and the like). A grain raw material, a BA245 fermented grains, and two kinds of commercially available enzyme food (DAESANG and HI-SAENG) were compared.

(87) Specifically, samples and 70% ethanol were mixed in a ratio of 1:9, followed by extracting while stirring at 30° C. for 3 hours using a stirrer (Wiseshaker, Daihan). The resulting mixture was centrifuged at 8,000×g at 4° C. for 10 minutes, followed by collecting the supernatant, which was subjected to identical extraction, and the finally collected supernatant was filtered. The filtered supernatant was dried using a freeze dryer until solvents could be completely removed, and the resulting mass was utilized as an analysis sample.

(88) 10 mg of the sample was completely dissolved in 1 ml of 70% ethanol to prepare a test solution. 38.5 mg of ABTS and 6.6 mg of potassium persulfate were dissolved in 5 ml of distilled water, followed by stirring, and then were left under dark conditions for 12 to 16 hours to generate radicals. Thereafter, an ABTS solution was prepared so as to have an absorbance at 734 nm of 0.7±0.02.

(89) To 10 μl of the test solution, 990 μl of the ABTS solution was added, mixed, and left in a dark room for 10 minutes, followed by measuring absorbance at 734 nm. All experiments were repeated three times or more, and then mean values and standard deviation values thereof were calculated.

(90) As a result, it could be seen that free radical scavenging ability of the BA245 fermented grains was 43.7%, which was much higher than those of the grain raw material and commercially available enzyme foods (FIG. 10). Through this experiment, it was also confirmed that antioxidant activity could be increased by fermentation using BA245 and was much higher than those of conventional two enzyme foods.