KR102189342B1 - Bacillus subtilis SRCM103716 strain having excellent β-glucosidase and antioxidant activity and uses thereof - Google Patents

Abstract

The present invention relates to a Bacillus subtilis SRCM103716 strain (KFCC11832P), a microorganism agent for fermenting sesame oil meal including the strain or a fermented liquid thereof, a method of manufacturing a mixed fermented sesame oil meal material with the increased lignan content and antioxidant activity using the strain, and a mixed fermented sesame oil meal material with the increased lignan content and antioxidant activity manufactured thereby. According to the present invention, the Bacillus subtilis SRCM103716 strain provides excellent β-glucosidase activity, provides antibacterial activity and β-hemolytic activity for harmful microorganisms, does not generate harmful substances, harmful enzymes, and biogenic amine, and increases a total polyphenol content, a total flavonoid content, and antioxidation activity when sesame oil meal is fermented.

Description

Bacillus subtilis SRCM103716 strain having excellent β-glucosidase and antioxidant activity and uses thereof {Bacillus subtilis SRCM103716 strain having excellent β-glucosidase and antioxidant activity and uses thereof}

The present invention has excellent β-glucosidase activity, has antibacterial activity against harmful microorganisms and β-hemolytic activity, does not produce harmful substances, harmful enzymes and biogenic amines, fermenting sesame seeds Bacillus subtilis SRCM103716 strain (KFCC11832P), characterized in that it increases the total polyphenol and total flavonoid content and antioxidant activity, a microbial preparation for fermentation of sesame seeds containing the strain or a culture solution thereof as an active ingredient, It relates to a method for preparing a sesame seed mixture fermented product with improved lignan content and antioxidant activity using the strain, and to a sesame seed mixed fermented product with improved lignan content and antioxidant activity prepared by the above method.

Sesame is a one-year herb belonging to the sesame family (Pedaliaceae. Sesamum), and is a worldwide paid crop grown from tropical to cold and temperate zones. When looking at the nutritional components of sesame seeds, it contains 51% lipid, 20% protein, and 15% sugar. Compared to rice and wheat, it is a food rich in calcium, phosphorus, zinc, iron, vitamins B1, B2, and unsaturated fatty acids. In addition, sesame seeds contain 0.1-1.0% sesamin and 0.3-0.7% sesamin, which have antioxidant effects.

In addition, after the sesame seeds are roasted at an appropriate temperature and milked by a compression method, the milking foil, which is an essential by-product, contains a large amount of ingredients effective for the human body. The main active ingredients present in sesame seeds include lignan components such as sesamin, sesaminol, sesamolinol, and pinoresinol. They mainly exist in the form of water-soluble glycosides bound to glucose, and their content is about 1%. The lignan glycosides have a lipid peroxidation inhibitory effect, and it has been reported that the lipid peroxidation inhibitory effect increases with the added concentration.

In addition, the lignan components of sesame seeds are known to prevent oxidation of low density lipoprotein (LDL). In particular, among the lignan components, sesaminol glucosides have been reported to have strong antioxidant power and antiarteriosclerosis.

Korean Patent Registration No.1786938 discloses Bacillus subtilis SY07 strain isolated from traditional paste and its use, and Korean Patent Publication No. 2015-0101789 discloses Bacillus subtilis BK418 strain having complex enzyme-producing ability and antibacterial activity. And the use thereof, but are different from the Bacillus subtilis SRCM103716 strain having excellent β-glucosidase activity and antioxidant activity of the present invention and use thereof.

The present invention was conceived by the above requirements, and in the present invention, Bacillus subtilis ( Bacillus subtilis) from traditional foods is intended to select Bacillus subtilis, which is excellent in total polyphenol and total flavonoid content and antioxidant activity, and has enzyme secretion ability. ) SRCM103716 strain was isolated, and the strain increased total polyphenol and total flavonoid content and antioxidant activity during sesame seed fermentation, while protease, amylase, cellulase, and β-glucosidase ( β-glucosidase), antimicrobial activity against harmful microorganisms, and β-hemolysis activity, and it was confirmed that no harmful substances, harmful enzymes, and biogenic amines were produced. Therefore, by confirming that the Bacillus subtilis SRCM103716 strain isolated in the present invention can be used importantly for the production of high-quality sesame seed mixed fermented products, the present invention was completed.

In order to solve the above problems, the present invention has excellent β-glucosidase activity, has antibacterial activity against harmful microorganisms and β-hemolytic activity, and generates harmful substances, harmful enzymes and biogenic amines. Without, it provides a Bacillus subtilis ( Bacillus subtilis ) SRCM103716 strain (KFCC11832P), characterized in that it increases the total polyphenol and total flavonoid content and antioxidant activity during sesame seed fermentation.

In addition, the present invention provides a microbial preparation for fermentation of sesame seeds containing Bacillus subtilis SRCM103716 strain or a culture solution thereof as an active ingredient.

In addition, the present invention is (1) inoculated with Bacillus subtilis SRCM103716 strain in the cooled sesame seed after adding water to the pulverized sesame meal and fermented to increase the total polyphenol and total flavonoid content and antioxidant. Preparing a fermented product of sesame gourd Bacillus bacillus with improved activity and β-glucosidase activity; (2) adding water to the pulverized sesame meal to increase the steam, and then inoculating the cooled sesame seed with Aspergillus oryzae strain and fermenting to prepare a sesame seed fermented product; And (3) mixing the fermented sesame seed meal prepared in step (1) with the fermented sesame seed meal prepared in step (2), and enhancing antioxidant activity. It provides a method for producing fermented sesame seed mixture.

In addition, the present invention provides a sesame seed mixed fermented product with improved lignan content and antioxidant activity prepared by the above method.

In the present invention, Bacillus subtilis SRCM103716 strain isolated from traditional food significantly increases total polyphenol and total flavonoid content and antioxidant activity during sesame seed fermentation, and β-glucosidase activity It was confirmed that this was excellent, had antimicrobial activity against harmful microorganisms and β-hemolytic activity, and did not produce harmful substances, harmful enzymes and biogenic amines. It is believed that the Bacillus subtilis SRCM103716 strain of the present invention can be used to prepare a high-quality fermented product when producing a sesame seed fermented product, and thus can be usefully used in the food industry.

1 is a phylogenetic tree of Bacillus subtilis SRCM103716 of the present invention.
Figure 2 is a schematic diagram of the manufacturing process of sesame seed Bacillus fermented product.
3 is a graph comparing the number of viable cells of fermented sesame seeds before and after fermentation with Bacillus bacillus.
4 is a graph comparing DPPH radical scavenging ability of fermented sesame seeds before and after fermentation with Bacillus bacillus.
5 is a graph comparing the total polyphenol content of fermented sesame seeds before and after fermentation with Bacillus bacillus.
6 is a graph comparing the total flavonoid content of fermented sesame seeds before and after fermentation with Bacillus bacillus.
7 is a graph comparing the DPPH radical scavenging ability of a fermented sesame seed mixture according to the mixing ratio.
Figure 8 is a schematic diagram of the manufacturing process of soy sauce using a sesame seed fermentation mixture.

In order to achieve the object of the present invention, the present invention has excellent β-glucosidase activity, antibacterial activity against harmful microorganisms and β-hemolytic activity, and harmful substances, harmful enzymes and biogenic amines. It provides a Bacillus subtilis ( Bacillus subtilis ) SRCM103716 strain (KFCC11832P), characterized in that it increases the total polyphenol and total flavonoid content and antioxidant activity during fermentation of sesame seeds without generating.

In the strain according to an embodiment of the present invention, the harmful microorganisms are Bacillus cereus , Staphylococcus aureus , and Listeria monocytogenes , and the harmful substances are phenylalanine. (phenylalanine), and the harmful enzymes are urease and β-glucuronidase, and the biogenic amine may be tyramine and histamine, but is not limited thereto.

In addition, in the strain according to an embodiment of the present invention, the strain may additionally have the ability to secrete protease, amylase, and cellulase, but is not limited thereto.

In addition, the present invention provides a microbial preparation for fermentation of sesame seeds containing Bacillus subtilis SRCM103716 strain (KFCC11832P) or a culture solution thereof as an active ingredient.

The microbial preparation for fermentation of sesame seeds according to the present invention may be prepared as a solution, powder, suspension, dispersion, emulsion, oily dispersion, paste, dust, scattering material or granules, but is not limited thereto.

The present invention also,

(1) After adding water to the crushed sesame seed meal, water was added, and then Bacillus subtilis SRCM103716 strain was inoculated in the cooled sesame seed meal, and fermented to obtain total polyphenol and total flavonoid content, antioxidant activity, and β-glucose. Preparing a fermented product of sesame gourd Bacillus bacillus with enhanced sidase (β-glucosidase) activity;

(2) adding water to the pulverized sesame meal to increase the steam, and then inoculating the cooled sesame seed with Aspergillus oryzae strain and fermenting to prepare a sesame seed fermented product; And

(3) The lignan content and antioxidant activity are enhanced, comprising the step of mixing the fermented sesame seed meal prepared in step (1) and the fermented sesame seed meal prepared in step (2). It provides a method of manufacturing a sesame seed mixed fermented product.

The method for producing a fermented sesame seed mixture of the present invention,

(1) Water is added 1.2 to 1.8 times to the crushed sesame seed, and then increased at 110 to 130°C for 10 to 20 minutes, and then Bacillus subtilis SRCM103716 strain was inoculated into the cooled sesame seed meal. Fermenting at 40° C. for 20 to 28 hours to prepare a fermented sesame gourd Bacillus fermented product with enhanced total polyphenol and total flavonoid content, antioxidant activity, and β-glucosidase activity;

(2) After adding water 0.4 to 0.6 times to the crushed sesame seed, add water at 110 to 130°C for 10 to 20 minutes, and inoculate the cooled sesame seed with Aspergillus oryzae strain. Fermenting at 32° C. for 68 to 76 hours to prepare a sesame seed fermented product; And

(3) mixing the fermented sesame seed meal prepared in step (1) and the fermented sesame seed meal prepared in step (2) in a weight ratio of 4 to 6: 94 to 96,

More specifically

(1) After adding water 1.5 times to the pulverized sesame seed, it was increased for 15 minutes at 121°C, and then Bacillus subtilis SRCM103716 strain was inoculated into the cooled sesame seed and fermented at 37°C for 24 hours. Preparing a fermented product of sesame gourd Bacillus with enhanced total polyphenol and total flavonoid content, antioxidant activity and β-glucosidase activity;

(2) After adding water 0.5 times to the pulverized sesame meal and increasing it at 121°C for 15 minutes, inoculating the cooled sesame seed with Aspergillus oryzae strain and fermenting it at 30°C for 72 hours. Preparing a fermented sesame seed meal; And

(3) The fermented sesame seed meal prepared in step (1) and the fermented sesame seed meal prepared in step (2) may be mixed in a weight ratio of 5:95.

The present invention also provides a sesame seed mixed fermented product with improved lignan content and antioxidant activity prepared by the above method.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Manufacturing example One. Sesame seeds Fermentation mixture

(1) Add 1.5 times (v/w) water to the pulverized sesame meal, increase the water at 121℃ for 15 minutes, and then add Bacillus subtilis SRCM103716 strain to the sesame leaf cooled to 60℃. After inoculation to a concentration of 3-4 log CFU/mL, fermentation was performed at 37° C. for 24 hours to prepare a fermented product of Sesame Bacteria Bacillus.

(2) 0.5 times (v/w) of water was added to the crushed sesame meal, and then increased at 121°C for 15 minutes, and then 0.3% ( Aspergillus oryzae ) strain was added to the sesame seeds cooled to 60°C. v/w) After inoculation, fermentation was performed at 30° C. for 72 hours to prepare a sesame seed fermented product.

(3) The fermented sesame seed meal prepared in step (1) and the fermented sesame seed meal prepared in step (2) were mixed at a weight ratio of 5:95.

Example 1. Characteristics of Bacillus bacteria

The experiment was conducted by selecting 15 species of Bacillus Bacillus having β-glucosidase activity out of 200 species of Bacillus previously owned by Jinheungwon isolated from soy sauce, red pepper paste, soybean paste, and salted fish. Table 1 shows a list of experimental strains.

1) Analysis of extracellular secretion enzyme activity (Amylase, Protease, Cellulase)

According to the agar diffusion method, Bacillus strains were inoculated into 5 ml of LB broth and cultured in a shaking incubator at 30°C and 150 rpm for 24 hours, and the culture solution was centrifuged at 13,000 rpm and then sterilized using a 0.45 μm filter. It was used as a test solution.

Agar diffusion method was used to verify the activity of amylase, protease, and cellulase among the enzymes of cells released outside the cells of the Bacillus strain, and a substrate that reacts specifically with each enzyme A selection medium containing the ingredients of was prepared and used. In order to measure amylase activity, a medium containing 2% agar containing 1% soluble starch was used by selecting soluble starch (starch soluble, Junsei) as a substrate, and 2% skim milk (BD, Difco) was used to measure the protease activity. ) Was added to prepare and use 1.5% agar medium. In order to verify the cellulase activity, CMC (carboxylmethyl cellulose, Junsei) was selected as a substrate to prepare a 1.5% agar medium to which 1% CMC was added. After forming an 8 mm hole in the medium containing each substrate, 100 μl of the strain test solution was dispensed into each well, and the size of the clear zone formed by incubating at 37°C for 24 hours was measured. Enzymatic activity of the strain was measured, Lugol staining was used to measure amylase activity, and congo red staining was used to measure cellulase activity.

List of 15 experimental strains No. Sympathy strain Separation Enzyme activity (unit: cm) amylase protease cellulase β-glucosidase One Bacillus subtilis BCH3509 Soy sauce 1.4 2.1 2.0 + ^* 2 Bacillus subtilis BCH3520 Soy sauce 1.6 2.3 2.3 + 3 Bacillus subtilis BCH3558 Kochujang 1.6 1.2 2.1 + 4 Bacillus subtilis BCH3572 Kochujang 2.2 2.0 1.8 + 5 Bacillus subtilis BCH3623 Miso 1.7 1.9 2.2 + 6 Bacillus subtilis BCH3637 Miso 1.7 2.1 2.1 + 7 Bacillus subtilis BCH3678 Miso 2.0 2.2 2.3 + 8 Bacillus subtilis BCH3700 Salted fish 1.9 2.3 2.4 + 9 Bacillus subtilis BCH3704 Salted fish 1.9 2.1 2.4 + 10 Bacillus subtilis BCH3711 Salted fish 1.8 2.2 2.4 + 11 Bacillus subtilis SRCM103716 Salted fish 1.9 2.0 2.5 + 12 Bacillus subtilis BCH3723 Cheonggukjang 1.7 2.2 2.2 + 13 Bacillus subtilis BCH3751 Cheonggukjang 1.9 2.2 2.3 + 14 Bacillus subtilis BCH3755 Cheonggukjang 1.9 2.1 2.4 + 15 Bacillus subtilis BCH3765 Cheonggukjang 2.3 2.4 1.7 +

^* +; positive, -; negative

2) Measurement of antibacterial activity against food harmful microorganisms

According to the agar diffusion method, Bacillus strain was inoculated into 5 ml of LB broth and cultured in a shaking incubator at 30°C and 150 rpm for 24 hours, and the culture solution was centrifuged at 13,000 rpm and then sterilized using a 0.45 μm filter as a test solution. Was used as.

In order to measure the food harmful activity, the indicator strains were sold and used in KCCM (Korean Culture Center of Microorganisms) and KCTC (Korean Collection for Type Cultures). Each indicator strain was cultivated in an appropriate medium, cultivated so that the OD (optical density) was 0.4 to 0.6 at 600 nm, and then mixed with 0.8% agar medium to 1% to prepare a medium, followed by agar diffusion method. According to this, after forming an 8 mm hole in the medium containing each indicator strain, 100 µl of the strain test solution was dispensed into each well, cultured at 30-37°C for 24 hours, and then formed around the hole. The size of the inhibitory ring was measured to measure the antibacterial activity of the selected strain.

As a result, SRCM103716 strain was confirmed antimicrobial activity against Bacillus cereus , Staphylococcus aureus , and Listeria monocytogenes .

Comparison of antimicrobial activity of 15 experimental strains No. Sympathy strain Antibacterial activity (unit: cm) B. cereus KCCM40935 S. aureus KCCM11335 L. monocytogenes KCCM43155 E. faecalis KCCM11814 One Bacillus subtilis BCH3509 1.5 2 1.5 ND 2 Bacillus subtilis BCH3520 1.9 One 2.1 1.6 3 Bacillus subtilis BCH3558 One ND 1.6 ND 4 Bacillus subtilis BCH3572 1.2 1.1 2 1.1 5 Bacillus subtilis BCH3623 One 1.2 2 1.3 6 Bacillus subtilis BCH3637 1.5 0.8 1.8 ND 7 Bacillus subtilis BCH3678 ND ND 1.5 ND 8 Bacillus subtilis BCH3700 ND 1.3 ND ND 9 Bacillus subtilis BCH3704 1.3 One 1.5 ND 10 Bacillus subtilis BCH3711 1.5 1.3 ND 1.1 11 Bacillus subtilis SRCM103716 1.4 1.4 1.5 ND 12 Bacillus subtilis BCH3723 ND ND ND ND 13 Bacillus subtilis BCH3751 ND ND 1.7 1.2 14 Bacillus subtilis BCH3755 ND One 1.3 1.2 15 Bacillus subtilis BCH3765 1.1 ND ND ND

3) Analysis of hemolytic activity (Hemolysis type) of selected strains

To analyze the hemolytic activity of Bacillus strains, Blood agar (Kisanbio, Korea) was purchased and the selected strain was streaked on each blood agar medium, and then cultured at 30°C for 24 hours. If there is no change in the agar around the colony Hemolysis was evaluated as β-hemolysis, when red blood cells were completely destroyed and a clear zone was formed around the colony, and α-hemolysis was determined when only some red blood cells were destroyed and green around the colony.

As a result, it was confirmed that the SRCM103716 strain had β-hemolytic activity, and did not produce harmful metabolites of phenylalanine, urease, and β-glucuronidase.

Comparison of hemolytic activity of 15 experimental strains No. Sympathy strain Hemolytic Harmful metabolites phenyl alanine urease β-glucuronidase (TBX) One Bacillus subtilis BCH3509 β ND ND ND 2 Bacillus subtilis BCH3520 β ND ND ND 3 Bacillus subtilis BCH3558 β ND ND ND 4 Bacillus subtilis BCH3572 β ND ND ND 5 Bacillus subtilis BCH3623 β ND ND ND 6 Bacillus subtilis BCH3637 β ND ND ND 7 Bacillus subtilis BCH3678 γ ND ND ND 8 Bacillus subtilis BCH3700 β ND ND ND 9 Bacillus subtilis BCH3704 β ND ND ND 10 Bacillus subtilis BCH3711 β ND ND ND 11 Bacillus subtilis SRCM103716 β ND ND ND 12 Bacillus subtilis BCH3723 α ND ND ND 13 Bacillus subtilis BCH3751 α ND ND ND 14 Bacillus subtilis BCH3755 α ND ND ND 15 Bacillus subtilis BCH3765 α ND ND ND

4) Confirmation of production of biogenic amine

As a result of checking the presence or absence of biogenic amine production, it was confirmed that all strains except for the BCH3520 strain did not produce tyramine and histamine.

Whether or not biogenic amines of 15 experimental strains are produced No. Sympathy strain Biogenic amines ctrl Tyramine Histamine One Bacillus subtilis BCH3509 ND ND ND 2 Bacillus subtilis BCH3520 + + + 3 Bacillus subtilis BCH3558 ND ND ND 4 Bacillus subtilis BCH3572 ND ND ND 5 Bacillus subtilis BCH3623 ND ND ND 6 Bacillus subtilis BCH3637 ND ND ND 7 Bacillus subtilis BCH3678 ND ND ND 8 Bacillus subtilis BCH3700 ND ND ND 9 Bacillus subtilis BCH3704 ND ND ND 10 Bacillus subtilis BCH3711 ND ND ND 11 Bacillus subtilis SRCM103716 ND ND ND 12 Bacillus subtilis BCH3723 ND ND ND 13 Bacillus subtilis BCH3751 ND ND ND 14 Bacillus subtilis BCH3755 ND ND ND 15 Bacillus subtilis BCH3765 ND ND ND

ND: not detected

5) β-glucosidase activity

A β-glucosidase activity assay kit (Sigma) was used to measure the β-glucosidase activity of 15 strains of the experiment. After pre-culture of 15 strains of the experiment, the main culture solution was inoculated with 1% (v/v) in LB broth (Difco) and cultured with shaking at 37°C for 24 hours. After collecting the supernatant of the main culture solution, the cells were dissolved in 50 mM phosphate buffer (pH 7.0), and then used as a sample. 220 µl distilled water, 20 µl distilled water, and 200 µl calibrator were added to 1 well, 200 µl of assay buffer and 8 µl of β-NPG substrate were added to each well of the sample, and then 20 µl of the sample was added. Thereafter, the initial absorbance was measured at 405 nm, and then left at 37°C for 20 minutes, and the final absorbance was measured at 405 nm. Thereafter, the β-glucosidase activity value was confirmed using the following calculation formula. The β-glucosidase activity values for 15 experimental strains are shown in Table 5.

β-glucosidase activity (Unit/L) = (final absorbance-initial absorbance × 250 units/L)/(calibrator absorbance-water absorbance)

Results of measuring β-glucosidase activity of experimental strains No. Strain β-glucosidase (unit/L) One BCH3509 4.91±0.97 2 BCH3520 6.03±0.62 3 BCH3558 1.43±0.79 4 BCH3572 2.42±0.97 5 BCH3623 2.67±1.85 6 BCH3637 1.43±0.26 7 BCH3678 2.55±1.32 8 BCH3700 1.06±0.26 9 BCH3704 1.49±0.70 10 BCH3711 9.63±1.67 11 SRCM103716 12.6±2.89 12 BCH3723 0.99±0.35 13 BCH3751 2.42±3.42 14 BCH3755 3.42±1.85 15 BCH3765 3.85±0.53

β-glucosidase activity was highest in BCH3711 and SRCM103716 at 9.63 unit/L and 12.6 unit/L, respectively.

Example 2. According to the type of Bacillus Sesame seeds Fermented Quality characteristics

Fermentation was carried out in the same manner as in FIG. 2 for fermentation of sesame seed meal.

1) Number of fermented products of 15 kinds of Bacillus bacteria in sesame seed meal

The results of the number of viable cells before and after fermentation are shown in FIG. 3. As a result, 8 log CFU/mL or more was measured in all fermented samples. All 15 species were confirmed to grow well on sesame seed material.

2) β-glucosidase activity

Β-glucosidase activity was measured for the sesame seed fermented product according to the strain. As a sample, the supernatant was collected, dissolved in 50 mM phosphate buffer (pH 7.0), and used as a sample. Table 6 shows the results measured in the same manner as before using the β-glucosidase activity assay kit (Sigma). As a result, Bacillus subtilis In sesame seeds fermented with SRCM103716, β-glucosidase activity was the highest at 16.88 unit/L.

Results of measuring β-glucosidase activity of sesame seeds fermented product according to strains No. sample β-glucosidase (unit/L) One BCH3509 9.96±0.95 2 BCH3520 9.66±0.59 3 BCH3558 10.59±0.64 4 BCH3572 7.97±0.87 5 BCH3623 8.85±0.35 6 BCH3637 9.26±0.21 7 BCH3678 9.32±0.15 8 BCH3700 8.26±0.68 9 BCH3704 8.70±0.49 10 BCH3711 11.72±0.65 11 SRCM103716 16.88±0.26 12 BCH3723 9.35±0.54 13 BCH3751 10.42±0.68 14 BCH3755 7.42±0.61 15 BCH3765 8.85±0.84

3) DPPH radical scavenging ability measurement

DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging ability was measured by modifying the method of Blois (1958) to measure the reducing power of each sample as an effect on electron donation capabilities (EDA). That is, 150 µl of a 0.15 mM DPPH solution (dissolved in 99% MeOH) was added to 50 µl of a sample, and then allowed to stand at room temperature for 30 minutes, and then the change in absorbance at 517 nm was measured using a spectrophotometer. The radical scavenging ability of each sample was expressed as a percentage of the difference in absorbance between the addition and no addition of the sample by the following equation.

Electron donating ability (%) = (1-absorbance of sample addition/absorbance of untreated group) × 100

The DPPH radical analysis results of the sesame seed material before and after fermentation are shown in FIG. 4. Before fermentation, it had an activity value of 43.24~45.34%, but after fermentation, all activity increased. Among them, fermented sesame seeds using BCH3711 and SRCM103716 increased the highest at 63.82% and 66.45%, respectively.

4) Total polyphenol content

The total phenol content was measured after appropriately diluting the sample extracted according to the Folin-Denis variant for each concentration. After mixing and stirring 12.5 µl of Folin-Ciocalteu reagent to 200 µl of each concentration sample, the mixture was allowed to stand at room temperature for 120 minutes to react. The absorbance value of the reaction solution was analyzed at 750 nm using a UV-Vis spectrophotometer (DU 800, Beckman coulter, Fullerton, CA, USA). The total phenol content was calculated from the standard calibration curve obtained by analyzing gallic acid by the same method as the analytical sample, and the total polyphenol content was expressed as mg gallic acid equivalent in 1 L.

The results of measuring the polyphenol content before and after fermentation of the sesame seed material are shown in FIG. 5. After fermentation, it was confirmed that the polyphenol content increased in all strains except for the control, and among them, the three species, BCH3637, BCH3678, and SRCM103716, increased the highest at 2490.2, 2559.4, and 2803.3 mg/L, respectively.

5) Total flavonoid content

The total flavonoid content was measured after appropriately diluting the sample extracted according to the Davis variant for each concentration. To 0.5 mL of each concentration sample, 0.1 mL of 10% aluminum nitrate, 0.1 mL of 1M potassium acetate, and 4.3 mL of ethanol were sequentially added and mixed, and then allowed to stand at room temperature for 40 minutes to react, thereby increasing the absorbance at 450 nm. Measured. As a standard material, the total flavonoid content of the sample extract was calculated from a standard calibration curve obtained by analyzing gallic acid in the same manner as the sample, and the total flavonoid content was expressed as mg gallic acid equivalent in 1 L.

The results of measuring the flavonoid content before and after fermentation of the sesame seed material are shown in FIG. 6. After fermentation, it was confirmed that the content of flavonoids increased in all strains except for the control, and among them, the three species BCH3637, BCH3678, and SRCM103716 were found to increase the highest at 1423.27, 1688.67 and 1553.08 mg/L, respectively.

5. Selection of sesame seeds Bacillus fermentation strains

Therefore, Bacillus subtilis SRCM103716 was selected as a strain for fermentation of sesame seed material, which has excellent β-glucosidase activity and DPPH radical scavenging activity, and has a high increase rate of total polyphenol content and flavonoid content after fermentation ( The 16S rRNA sequence is SEQ ID NO: 1), and the schematic diagram of the selected strain is as shown in FIG. 1, and was deposited with the Korea Microbial Conservation Center (KCCM) on July 10, 2019, and was given the accession number KFCC11832P.

Example 3. Sesame seeds Fermented Mixing ratio setting

Sesame Seed Bacillus subtilis SRCM103716 Fermented Product and Sesame Seed Aspergillus Duck ( Aspergillus oryzae ) Samples were prepared as shown in Table 7 in order to set the mixing ratio of the fermented product. By confirming the DPPH radical scavenging ability, it was decided to set the sesame meal fermented soy sauce production section with the ratio having the highest antioxidant activity.

Section for setting the mixing ratio of fermented sesame seeds sample Sesame seed fermentation mixing ratio (weight ratio) A. oryzae B. subtilis SRCM103716 A100 100 0 A95B5 95 5 A75B25 75 25 A50B50 50 50 B100 0 0

One) Sesame seeds Fermentation According to the mixing ratio DPPH Radical Scavenging ability result

The DPPH radical scavenging ability results according to the mixing ratio of fermented sesame seeds are shown in FIG. 7. Aspergillus duck material ( A. oryzae ) 95%, Bacillus subtilis ( B. subtilis ) SRCM103716 DPPH radical activity at a mixing ratio of 5% was measured the highest as 84.12%, followed by Aspergillus duck material ( A. oryzae ) It was measured from 100% to 77.28%. Therefore, it was confirmed that 95% of Aspergillus ducks ( A. oryzae ) and 5% of Bacillus subtilis ( B. subtilis ) SRCM103716 are most suitable for the production of sesame seed fermented soy sauce.

2) Sesame seeds Non-fermented products Sesame seeds Fermentation mixture DPPH Radical Scavenging activity and League I measure the ingredients

Finally, 95% of sesame seed Aspergillus duckweed fermented product and 5% of Bacillus subtilis SRCM103716 fermented product were mixed and decided to be used in the production of soy sauce. Accordingly, DPPH radical scavenging activity and lignan components of the mixture of non-fermented sesame seeds and sesame seeds were measured.

(2-1) Sesame seeds Fermentation mixture DPPH Radical Scavenging activity result

Table 8 shows the results of DPPH radical scavenging activity of the fermented mixture and non-fermented sesame seeds using Bacillus subtilis SRCM103716 strain and Aspergillus oryzae . As a result of the measurement, it was confirmed that at 100 times dilution, the non-fermented sesame seed and the sesame seed fermented mixture increased about 160% after fermentation to 28.4% and 73.3% respectively.

DPPH radical scavenging activity result of fermented sesame seed mixture sample DPPH activity (%) of Sesame bark extracts Ascorbic acid 100㎍/mL 50㎍/mL 20㎍/mL 10㎍/mL Non-fermented sesame seeds 92.84 28.4±0.9 18.1±3.0 7.6±3.2 3.1±0.6 Sesame seed fermentation mixture 92.84 73.3±19.6 51.3±14.4 20.3±4.5 9.7±1.2 Increase rate (%) - 158.1 183.4 167.1 212.9

(2-2) Sesame seeds Fermentation mixture Lignan Component analysis result

Table 9 shows the results of the analysis of the lignan content of the fermented mixture and non-fermented sesame seeds using Bacillus subtilis SRCM103716 strain and Aspergillus oryzae . As a result of the analysis, the total lignan content increased by 57.94%, and the content of sesaminol triglucoside decreased by 18.93% after fermentation due to changes in each lignan composition, and sesaminol diglucoside and sesaminol ) Was found to increase by 1340.44% and 2071.43%, respectively. It was confirmed that the sesaminol glycoside bond was decomposed by fermentation metabolism as well as the increase of the overall lignan content, so that the polymeric glycoside bond decreased, while the content of the low molecular sesaminol glycoside increased.

Analysis of Lignan Components in Sesame Seed Fermented Mixture sample Contents (㎍/g) Sesamol Sesaminol
triglucoside Sesaminol
diglucoside Sesaminol Total lignan Non-fermented sesame seeds 30.2±2.1 999.3±56.6 54.9±2.7 3.5±0.5 1087.9±61.9 Sesame seed fermentation mixture 41.4±3.4 810.1±1.9 790.8±120.1 76.0±22.4 1718.2±56.2 Increase rate (%) 37.1 -18.9 1340.4 2071.4 57.9

Example 4. Sesame seeds Quality Characteristics of Soy Sauce Using Fermented Mixture

The manufacturing process of soy sauce using the sesame seed fermentation mixture is shown in FIG.

One) Sesame seeds Analysis of fragrance components of soy sauce

Volatile fragrance components were separated and identified from sesame seed soy sauce, and the analysis conditions are shown in Table 10.

Conditions Gas chromatography TRACE 1300 Series GC Mess selective detector TSQ Series Column DB-WAX (Agilent)
(60m length × 0.25mm ID × 0.50㎛) Column temperature 40℃-3min
4℃/min ~ 150℃-5min
5℃/min ~ 230℃-10min Injector temperature 250℃ Detector temperature 250℃ Carrier gas N ₂ Column pressure 400.00 (kPa) Run time 60 min Mass range 35~550 m/z

Tables 11 to 13 show the results of analysis of fragrance components of sesame seed soy sauce according to the ratio of the sesame seed fermentation mixture using Bacillus subtilis SRCM103716 strain and Aspergillus oryzae . A total of 61 compounds were identified as a result of analyzing the volatile components of sesame seed fermented soy sauce. Among them, 5 aldehydes, 10 aromatic compounds, 10 alcohols, 3 acids, 10 ketones, 4 esters, and 19 other compounds were identified. Characteristically, compared to soy sauce using a single strain, the content of 3-methyl butanal with fruit and malt flavor was 5.28% in soy sauce using complex strains, and 2 with smoking, wood flavor and bacon flavor. -It was confirmed that 2-methoxy phenol increased by 17.01%, and 2,3-butanediol (2,3-Butanediol) having fruit flavor, cream flavor, and butter flavor increased by 1.19%, respectively. In addition, the content of other volatile compounds that do not show aroma showed a tendency to decrease by 13.47% from 34.3% to 20.83%. As a result, Aspergillus in the liver of a complex strain using SRCM103716 strain and Aspergillus oryzae It was confirmed that the fragrant compound was increased compared to the liver of a single strain inoculated with duck material ( Aspergillus oryzae ) alone.

2) Sesame seeds Measurement of free amino acids in soy sauce

For free amino acids, take 2 g of a sample, add 30 mL of tertiary distilled water, stir, dilute to 50 mL, extract for 20 minutes using ultrasound, centrifuge (3000 rpm, 10 minutes), and add 2 mL of 5% TCA to 2 mL of the supernatant. Was then centrifuged (10,000 rpm, 10 minutes), the supernatant was taken, diluted with 0.02N-HCl, passed through a 0.2 μm syringe filter, and analyzed by an amino acid analyzer under the analysis conditions shown in Table 14 below.

Conditions Instument Amino acid analysis (Hitachi L-8900) Detector UV/Vis (440nm-570nm) Column Hitachi 4.6×60mm (speration)
Hitachi 4.6×40mm (Ammonia filtering) Buffer flow rate 0.40 mL/min Ninhydrin flow rate 0.35 mL/min Temperature 50℃ Injection volume 20 μl Mobile phase Buffer set (PH-SET KANTO)

The higher the content of free amino acids produced by enzyme action during fermentation, the better it is evaluated as an excellent food with excellent taste and nutrition. Table 15 shows the results of free amino acids in fermented soy sauce according to the ratio of the fermented sesame seed mixture using Bacillus subtilis SRCM103716 strain and Aspergillus oryzae of this experiment. Aspergillus duck jae 100% fermented soy sauce and Aspergillus duck jae 95%, Bacillus subtilis SRCM103716 The total free amino acid content of 5% fermented soy sauce was measured to be 319.3 mg/L and 333.6 mg/L, respectively, and glutamic acid in the paste was 100% of Aspergillus duck material, and 95% of fermented soy sauce and Aspergillus duck material. + Bacillus subtilis SRCM103716 5% fermented soy sauce was found to be 47.82 mg/L and 70.44 mg/L, respectively. In addition, the content of aspartic acid was measured as 20.99 mg/L and 25.29 mg/L, and the ingredients that closely affect the taste and the unique taste of soy sauce are Aspergillus ducks 95% + Bacillus subtilis. SRCM103716 was higher in 5% fermented soy sauce. All three kinds of free amino acids that exhibit bitter taste, such as isoleucine, leucine, and phenylalanine, resulted in SRCM103716 strain + Aspergillus duckweed ( Aspergillus oryzae ) in the liver of a complex strain utilizing Aspergillus duck material ( Aspergillus oryzae ) appeared lower than the soy sauce of a single strain inoculated only, and it was confirmed that the savory taste, the unique taste of soy sauce increased, and the bitter taste decreased.

Soy amino acid analysis result (mg/L) Amino acids A. oryzae 100% fermented soy sauce A. oryzae 95%, B. subtilis SRCM103716 5% fermented soy sauce Phosphoserine - - Taurine - - Aspartic Acid 20.99±1.55 25.29±0.31 Threonine 12.27±0.48 11.18±0.36 Serine 5.02±0.24 1.66±0.09 Glutamic Acid 47.82±3.32 70.44±1.27 Sarcosine - - α-Aminoadipic Acid 2.59±0.06 3.44±0.10 Glycine 12.52±0.40 12.96±0.21 Citrulline 8.44±0.56 6.31±0.37 Alanine 17.22±1.46 17.79±0.24 α-Aminobutyric Acid 2.01±0.22 2.02±0.14 Valine 20.88±1.57 19.18±0.52 Cystine - - Methionine 13.96±0.05 11.77±0.20 Tyrosine 18.38±0.19 16.09±0.28 Cystionine - - Isoleucine 15.20±0.87 13.33±0.16 Leucine 26.59±0.66 20.70±0.19 Phenylalanine 19.14±0.85 17.12±0.47 β-aminoisobutyric acid 4.57±1.22 7.11±0.57 γ-Aminoisobutyric Acid 2.62±0.52 1.67±0.47 Ammonia 10.74±2.10 18.37±1.18 Ornithine 25.62±2.77 36.85±1.14 Lysine 6.70±0.41 6.45±0.14 Histidine 6.46±0.35 6.38±0.34 Anserine 2.24±0.82 2.02±0.65 Arginine 10.29±0.02 0.18±0.01 Hydroxyproline - - Proline 6.26±0.11 3.63±0.11 Total 319.26±4.65 333.60±6.60

Korea Microorganism Conservation Center (domestic) KFCC11832P 20190710

<110> Microbial Institute for Fermentation Industyry <120> Bacillus subtilis SRCM103716 strain having excellent beta-glucosidase and antioxidant activity and uses thereof <130> PN19311 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 478 <212> DNA <213> Bacillus subtilis <400> 1 cttctcagga cgaacgctgg cggcgtgcct aatacatgca agtcgagcgg acagatggga 60 gcttgctccc tgatgttagc ggcggacggg tgagtaacac gtgggtaacc tgcctgtaag 120 actgggataa ctccgggaaa ccggggctaa taccggatgg ttgtttgaac cgcatggttc 180 aaacataaaa ggtggcttcg gctaccactt acagatggac ccgcggcgca ttagctagtt 240 ggtgaggtaa cggctcacca aggcaacgat gcgtagccga cctgagaggg tgatcggcca 300 cactgggact gagacacggc ccagactcct acgggaggca gcagtaggga atcttccgca 360 atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg aaggttttcg gatcgtaaag 420 ctctgttgtt agggaagaac aagtaccgtt cgaatagggc ggtaccttga cggtacct 478

Claims (7)

β-glucosidase (β-glucosidase) activity is excellent, Bacillus cereus ( Bacillus cereus ), Staphylococcus aureus ( Staphylococcus aureus ) and Listeria monocytogenes ( Listeria monocytogenes ) antimicrobial activity and β- It has hemolytic activity, does not produce phenylalanine, urease, β-glucuronidase, tyramine and histamine, while fermenting sesame seeds with total polyphenol and total flavonoid content and antioxidant Bacillus subtilis ( Bacillus subtilis ) SRCM103716 strain (KFCC11832P) characterized by increasing the activity. delete The strain according to claim 1, wherein the strain further has the ability to secrete protease, amylase, and cellulase. Claim 1 Bacillus subtilis ( Bacillus subtilis ) SRCM103716 strain or a microorganism preparation for fermentation of sesame seeds containing the culture medium thereof as an active ingredient. (1) After adding water to the pulverized sesame seed meal, water was added and then inoculated with the Bacillus subtilis SRCM103716 strain of Clause 1 in the cooled sesame seed meal, and fermented to obtain total polyphenol and total flavonoid content and antioxidant activity. Preparing a fermented product of sesame gourd Bacillus bacillus with enhanced β-glucosidase activity;
(2) adding water to the pulverized sesame meal to increase the steam, and then inoculating the cooled sesame seed with Aspergillus oryzae strain and fermenting to prepare a sesame seed fermented product; And
(3) The lignan content and antioxidant activity are enhanced, comprising the step of mixing the fermented sesame seed meal prepared in step (1) and the fermented sesame seed meal prepared in step (2). Method for producing fermented sesame seed mixture. The method of claim 5,
(1) Water was added 1.2 to 1.8 times to the pulverized sesame seed, and then increased at 110 to 130°C for 10 to 20 minutes, and then Bacillus subtilis SRCM103716 strain of Paragraph 1 was inoculated into the cooled sesame seed meal. After fermentation at 34-40° C. for 20-28 hours to prepare a sesame gourd Bacillus fermented product with improved total polyphenol and total flavonoid content, antioxidant activity, and β-glucosidase activity;
(2) After adding water 0.4 to 0.6 times to the crushed sesame seed, add water at 110 to 130°C for 10 to 20 minutes, and inoculate the cooled sesame seed with Aspergillus oryzae strain. Fermenting at 32° C. for 68 to 76 hours to prepare a sesame seed fermented product; And
(3) characterized by comprising the step of mixing the fermented sesame seed meal prepared in step (1) and the fermented sesame seed meal prepared in step (2) at a weight ratio of 4 to 6:94 to 96 Method for producing a mixed fermented product of sesame seed meal with improved lignan content and antioxidant activity. delete

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