KR102092528B1 - Method for producing soy sauce using Bacillus subtilis SRCM103716 and Aspergillus oryzae strain - Google Patents

Abstract

The present invention relates to a method for preparing sesame dregs-fermented soy sauce and sesame dregs-fermented soy sauce prepared by the same. The method for preparing sesame dregs-fermented soy sauce comprises the following steps: (1) adding water to ground sesame dregs, cooling the sesame dregs, and inoculating the cooled sesame dregs with Bacillus subtilis SRCM103716 and fermenting the sesame dregs to obtain sesame dregs Bacillus subtilis-fermented substance having increased total polyphenol and flavonoid contents and enhanced antioxidant activity and β-glucosidase activity; (2) adding water to ground sesame dregs, cooling the sesame dregs, and inoculating the cooled sesame dregs with Aspergillusoryzae strain and fermenting the sesame dregs to obtain a sesame dregs-fermented substance; (3) mixing the sesame dregs Bacillus subtilis-fermented substance prepared in the step (1) with the sesame dregs-fermented substance prepared in the step (2) to prepare a sesame dregs mixture-fermented substance having an increased lignan content and enhanced antioxidant activity; and (4) adding salt water to the sesame dregs mixture-fermented substance prepared in the step (3), aging the mixture, and filtering the mixture. According to the present invention, it is possible to provide high-functional soy sauce with excellent preferences.

Description

Method for producing soy sauce using Bacillus subtilis SRCM103716 and Aspergillus oryzae strain

The present invention (1) by inoculating the strain of Bacillus subtilis SRCM103716 after fermentation by increasing water by adding water to the crushed sesame seeds and fermenting them to inoculate the total polyphenol and total flavonoid content and antioxidant activity and preparing β-glucosidase (β-glucosidase) activity-enhanced sesame seed archaea fermentation; (2) After adding water to the crushed sesame seeds, increase the amount, and then cool the cooled sesame seeds with Aspergillus oryzae ) producing a sesame leaf fermentation product by inoculating the strain and then fermenting it; (3) preparing a mixed fermented sesame seeds with enhanced lignan content and antioxidant activity by mixing the fermented sesame seeds produced in step (1) and the fermented sesame seeds prepared in step (2); And (4) adding a brine to the sesame-bak mixed fermentation product prepared in step (3), followed by aging and filtering, thereby producing a method of producing sesame-bak fermented soy sauce and prepared by the above method. It is about fermented soy sauce in sesame seeds.

Soy sauce is an important food that is indispensable in the diet of Korean people, and is a traditional fermented food made through fermentation and aging process as one of the main ingredients of soybeans. It is a processed food that is excellent in storage at the same time as a seasoning.

Soy sauce is a very characteristic food, and it depends on the type of fermentation microorganism involved in it and the difference in manufacturing technology. Although these sauces have been manufactured and used in each household, the consumption and consumption of the sauces have been greatly improved as the trend of purchasing them in the market rather than those manufactured at home has changed due to economic growth and changes in dietary habits.

Sesamol and sesaminol are low-molecular vegetable compounds, and are known to inhibit antioxidant activity and cholesterol, atherosclerosis, and hyperglycemia. It is also known as an active ingredient with a structure similar to that of the female hormone estrogen, which binds to the estrogen receptor in the body to relieve female menopausal symptoms and prevent chronic degenerative diseases such as cancer and cancer by activating estrogen. In particular, sesaminol glycosides, which are lignan glycosides in sesame, are known to defend against oxidative disorders such as biomembranes and show synergistic action of α-tocopherol, thereby exhibiting anti-aging activity in the body. On the other hand, sesaminol glycosides are consumed as food rather than antioxidant activity in the sesame seed seed itself, which has a phenolic hydroxyl group that exhibits antioxidant activity and is associated with sugar, and thus aglycone (aglycon) under the action of β-glucosidase of intestinal bacteria ) There is a research report that it is hydrolyzed in the form to help absorb in the intestine and prevent oxidation in the body. Sesamol is known as a representative component of roasted sesame oil, and it is known that the antioxidant activity of sesame oil's lignan component greatly contributes to the oxidative stability of sesame oil. It exists in the form of sesaminol glucoside. Despite these advantages, studies using sesame seeds are still insufficient, and the utilization of food materials is also low.

Korean Registered Patent No. 1714417 discloses a method for producing soy sauce using a tomato fermentation broth, and Korean Registered Patent No. 1399276 discloses a method for producing short-term mature soy sauce, but Bacillus subtilis SRCM103716 and Aspergillus of the present invention. It is different from the method for producing soy sauce using the duck strain.

The present invention was devised by the above-mentioned demands, and in the present invention, it is excellent in total polyphenol and total flavonoid content and antioxidant activity, isolates archaea bacteria having enzyme secretion ability, and uses the separated strain to produce high-quality sesame seeds. It is to provide a method of manufacturing fermented soy sauce.

In order to solve the above problems, the present invention is (1) by increasing water by adding water to the crushed sesame seeds, and then inoculating Bacillus subtilis SRCM103716 strain on the cooled sesame seeds to ferment and inoculate the total polyphenols. Preparing a sesame seed archaea fermentation product having enhanced total flavonoid content, antioxidant activity, and β-glucosidase activity; (2) After adding water to the crushed sesame seeds, increase the amount, and then cool the cooled sesame seeds with Aspergillus oryzae ) producing a sesame leaf fermentation product by inoculating the strain and then fermenting it; (3) preparing a mixed fermented sesame seeds with enhanced lignan content and antioxidant activity by mixing the fermented sesame seeds produced in step (1) and the fermented sesame seeds prepared in step (2); And (4) provides a method for producing sesame seeds fermented soy sauce, characterized in that it comprises a step of aging after aging by adding brine to the mixed fermented sesame seeds prepared in step (3).

In addition, the present invention provides a fermented soy sauce sesame seeds prepared by the above method.

The soy sauce of the present invention can provide a high-functional soy sauce having excellent quality, enhanced free amino acid content and aroma components, and excellent palatability because it uses the mixed fermented sesame seeds with enhanced lignan content and antioxidant activity as the main ingredients. .

1 is a phylogenetic tree of the Bacillus subtilis SRCM103716 of the present invention.
Figure 2 is a schematic diagram showing the manufacturing process of sesame seed archaea fermentation.
Figure 3 is a graph comparing the number of live bacteria before and after fermentation with sesame leaf fermentation as an archaebacteria.
Figure 4 is a graph comparing the DPPH radical scavenging ability of sesame seed fermentation before and after fermentation with archaea.
Figure 5 is a graph comparing the total polyphenol content of sesame leaf fermentation before and after fermentation with archaea.
Figure 6 is a graph comparing the total flavonoid content of sesame leaf fermentation before and after fermentation with archaea.
7 is a graph comparing the DPPH radical scavenging ability of the sesame seed fermentation mixture according to the mixing ratio.
8 is a schematic diagram of a process for producing soy sauce using a fermented mixture of sesame seeds.

In order to achieve the object of the present invention, the present invention

(1) After adding water to the crushed sesame seeds and increasing the amount, the cooled sesame seeds were inoculated with Bacillus subtilis SRCM103716 strain and fermented to give total polyphenol and total flavonoid content, antioxidant activity, and β-gluco Preparing a fermented sesame-pseudomonas aeruginosa fermentation with enhanced cydase (β-glucosidase) activity;

(2) After adding water to the crushed sesame seeds, increase the amount, and then cool the cooled sesame seeds with Aspergillus oryzae ) producing a sesame leaf fermentation product by inoculating the strain and then fermenting it;

(3) preparing a mixed fermented sesame seeds with enhanced lignan content and antioxidant activity by mixing the fermented sesame seeds produced in step (1) and the fermented sesame seeds prepared in step (2); And

(4) provides a method for producing a sesame seed fermented soy sauce, characterized in that it is prepared by adding brine to the fermented sesame meal prepared in the step (3), followed by filtration.

In the method of preparing the fermented soy sauce of the present invention, the Bacillus subtilis SRCM103716 strain has excellent β-glucosidase activity, and Bacillus cereus , Staphylococcus Aureus ( Staphylococcus aureus ) and Listeria monocytogenes ( Listeria monocytogenes ) have antimicrobial and β-hemolytic activity against harmful microorganisms, harmful substances of phenylalanine, urease and β-glucuro Total poly during fermentation of sesame seeds, having the ability to secrete proteases, amylases and cellulases without producing harmful enzymes of β-glucuronidase and biogenic amines of tyramine and histamine A strain with a high increase rate of phenol and total flavonoid content and antioxidant activity was selected, and deposited with the Korea Microbial Conservation Center (KCCM) on July 10, 2019. Endowed with panoramic number KFCC11832P.

In addition, in the method of manufacturing the fermented soy sauce of sesame seeds of the present invention, the sesame leaves Bacillus fermentation product of step (1) is preferably 1.2 to 1.8 times water is added to the crushed sesame leaves to 10 to 20 at 110 to 130 ° After inoculating the cooled sesame seeds for a minute and then inoculating Bacillus subtilis SRCM103716 strain, fermentation can be performed at 34-40 ° C. for 20-28 hours, more preferably crushed sesame seeds. It can be prepared by inoculating Bacillus subtilis SRCM103716 strain with cooled sesame seeds after increasing the water for 1.5 minutes by adding water 1.5 times at 121 ° C. and fermenting at 37 ° C. for 24 hours. The sesame seed archaea fermentation product prepared under the above conditions has an advantage of total polyphenol and total flavonoid content and excellent antioxidant activity and β-glucosidase activity.

In addition, in the method for producing fermented soy sauce of sesame seeds of the present invention, the fermented sesame seeds of step (2) is preferably 0.4 to 0.6 times water is added to the crushed sesame seeds for 10 to 20 minutes at 110 to 130 ° C. It can be prepared by inoculating strains of Aspergillus oryzae on cooled sesame seeds after increasing the amount, and fermenting at 28-32 ° C for 68-76 hours, more preferably water in crushed sesame seeds. Was added 0.5 times and increased for 15 minutes at 121 ℃, and then cooled to Aspergillus or Aspergillus oryzae ) can be prepared by inoculating the strain and fermenting at 30 ° C for 72 hours.

In addition, in the method of preparing the fermented sesame soy sauce of the present invention, the mixing of step (3) is preferably a sesame gourd bacillus fermentation product and a sesame leaf fermentation product can be mixed in a weight ratio of 4-6: 94-96, , More preferably, the fermentation of sesame seeds and Bacillus spp. May be mixed at a weight ratio of 5:95. The production of soy sauce using the fermented product mixed under the above conditions increased the fragrance component, and the high amino acid content was high, and it could be prepared as a mixed fermented sesame seeds suitable for soy sauce production.

In addition, in the method of preparing the fermented soy sauce of sesame seeds of the present invention, step (4) preferably adds 23 to 25% (w / v) saline 2 to 3 times (v / w) brine to the mixed fermented sesame seeds By aging at 30 ~ 35 ℃ for 3 ~ 4 weeks, it can be filtered, and more preferably, 30 ~ 35 by adding 24% (w / v) brine 2.5 times (v / w) to sesame leaf mixed fermentation product After aging for 3-4 weeks at ℃, it can be filtered. The production of soy sauce by aging under the above-described conditions was high in total nitrogen content and enhanced umami and savory taste, so that it could be prepared with excellent quality soy sauce.

The method for producing the fermented soy sauce of sesame seeds of the present invention, more specifically

(1) Water is added to the crushed sesame seeds 1.2 ~ 1.8 times, and the water is increased at 110 ~ 130 ℃ for 10 ~ 20 minutes. After cooling, the cooled sesame seeds are inoculated with Bacillus subtilis SRCM103716 strain, and then 34 ~ Fermentation at 40 ° C. for 20-28 hours to produce a sesame-pseudomonas aeruginosa fermentation product with enhanced total polyphenol and total flavonoid content, antioxidant activity, and β-glucosidase activity;

(2) Water is added to the crushed sesame seeds 0.4 ~ 0.6 times, and the water is increased at 110 ~ 130 ℃ for 10 ~ 20 minutes, and then cooled to the cooled sesame seeds. Aspergillus oryzae ) producing a sesame leaf fermentation product by inoculating the strain and fermenting at 28-32 ° C for 68-76 hours;

(3) The sesame leaf fermentation product produced in the step (1) and the sesame leaf fermentation product prepared in the step (2) are mixed at a weight ratio of 4 to 6:94 to 96 to improve the lignan content and antioxidant activity. Preparing a sesame-bak mixed fermentation product; And

(4) 23 ~ 25% (w / v) brine is added 2-3 times (v / w) to the mixed fermented sesame leaf prepared in the step (3), aged at 30 ~ 35 ℃ for 3-4 weeks After filtering it may include a step,

More specifically

(1) Water was added 1.5 times to the crushed sesame seeds and increased at 121 ° C for 15 minutes, followed by inoculation of Bacillus subtilis SRCM103716 strain on the cooled sesame leaves and fermentation at 37 ° C for 24 hours. Preparing total sesame seed archaea fermentation with enhanced polyphenol and total flavonoid content, antioxidant activity, and β-glucosidase activity;

(2) increase the water to ground Sesame at 121 ℃ to 0.5 times the singer for 15 minutes and then cooled to Aspergillus Sesame's duck material (Aspergillus oryzae ) preparing a sesame leaf fermentation product by inoculating the strain and fermenting at 30 ° C. for 72 hours;

(3) The sesame leaf fermentation product produced in step (1) and the sesame leaf fermentation product prepared in step (2) are mixed at a weight ratio of 5:95 to improve the lignan content and antioxidant activity. Preparing water; And

(4) 24% (w / v) brine is added 2.5 times (v / w) to the mixed fermentation product prepared in step (3), aged at 30-35 ° C. for 3-4 weeks, and filtered. It may include steps.

The present invention also provides a fermented soy sauce sesame seeds prepared by the above method.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, the content of the present invention is not limited to the following examples.

Manufacturing example  One. Sesame seeds  Fermentation mixture

(1) Water was added 1.5 times (v / w) to the crushed sesame seeds and increased for 15 minutes at 121 ° C, and then Bacillus subtilis SRCM103716 strain was first added to the sesame seeds cooled to 60 ° C. After inoculation to be 3 to 4 log CFU / mL, fermentation was performed at 37 ° C. for 24 hours to prepare a sesame seed archaea fermentation product.

(2) 0.5 times (v / w) water was added to the crushed sesame seeds, and the mixture was increased at 121 ° C for 15 minutes, and then cooled to 60 ° C in the sesame leaves. Aspergillus oryzae ) was inoculated with 0.3% (v / w) strain, and fermented at 30 ° C for 72 hours to prepare a sesame leaf fermentation product.

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

Manufacturing example  2. Sesame seeds  Fermented soy sauce

To the fermentation mixture prepared in Preparation Example 1, 24% (w / v) brine was added 2.5 times (v / w), aged at 30-35 ° C. for 3-4 weeks, filtered and separated from the fermentation broth. The separated fermentation broth was sterilized at 100 ° C. for 3 minutes, and then filtered with filter paper (Advantec No. 2, Toyo Roshi Kaisha Ltd., Japan).

Example  1. Characteristics by archaea

Among the 200 Bacillus species that were previously held by the Promotion Agency isolated from soy sauce, red pepper paste, miso, and salted fish, 15 Bacillus subtilis possessing β-glucosidase activity were selected and tested. Table 1 shows the list of experimental strains.

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

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

Among the enzymes of cells released into the cells of the Bacillus strain, amylase, protease, and cellulase activities were used to verify the agar diffusion method, and substrates that specifically react with each enzyme The starting medium containing the components of was prepared and used. In order to measure amylase activity, a medium containing 2% agar containing 1% of soluble starch was used by selecting soluble starch (starch soluble, Junsei) as a substrate, and 2% skim milk (BD, Difco) to measure protease activity ) Was added and used to prepare 1.5% agar medium. To verify the cellulase activity, CMC (carboxylmethyl cellulose, Junsei) was selected as a substrate to prepare 1.5% agar medium with 1% CMC added. After forming 8 mm holes 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. The enzyme activity of the strain was measured, Lugol staining method was used to measure amylase activity, and congo red staining was used to measure cellulase activity.

List of 15 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 1.9 2.3 2.4 +  9 Bacillus subtilis BCH3704 Salted 1.9 2.1 2.4 + 10 Bacillus subtilis BCH3711 Salted 1.8 2.2 2.4 + 11 Bacillus subtilis SRCM103716 Salted 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, the Bacillus strain was inoculated into 5 ml of LB broth and cultured in a shaking culture incubator at 30 ° C and 150 rpm for 24 hours. It was used as.

In order to measure food harmful activity, indicator strains were pre-distributed from KCCM (Korean Culture Center of Microorganisms) and KCTC (Korean Collection for Type Cultures). Each indicator strain was cultivated in an appropriate medium, and OD (optical density) was incubated to be 0.4 to 0.6 at 600 nm, followed by mixing with 1% in 0.8% agar medium in the medium to prepare a medium. After forming a hole of 8 mm in the medium containing each indicator strain, 100 μl of the strain test solution was dispensed into each well and cultured at 30 to 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, the SRCM103716 strain confirmed antibacterial activity against Bacillus cereus , Staphylococcus aureus , and Listeria monocytogenes .

Comparison of 15 antibacterial activities 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 hemolysis type of selected strains

In order to analyze the hemolytic activity of Bacillus strains, blood agar (Kisanbio, Korea) was purchased, and the selected strains were streaked on each blood agar medium and cultured at 30 ° C. for 24 hours. Hemolysis, when red blood cells were completely destroyed and a clear zone was formed around the colony, β-hemolysis was observed, and when only some red blood cells were destroyed and observed in green around the colonies, it was determined as α-hemolysis.

As a result, it was confirmed that the SRCM103716 strain has β-hemolytic activity, and does 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) Check if biogenic amine is generated

As a result of confirming the presence or absence of biogenic amine production, it was confirmed that tyramine and histamine were not produced in all strains except the BCH3520 strain.

Presence of 15 biogenic amines in experimental strains 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

The β-glucosidase activity assay kit (Sigma) was used to measure the β-glucosidase activity of 15 types of experimental strains. After pre-culturing 15 kinds of experimental strains, the main culture solution was inoculated with 1% (v / v) in LB broth (Difco) and shake cultured at 37 ° C for 24 hours. After collecting the supernatant of the main culture solution, the cells were released in 50 mM phosphate buffer (pH 7.0) and used as a sample. 220 µl distilled water to 1 well, 20 µl distilled water, 200 µl calibrator was added to 1 well, and 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, then left at 37 ° C for 20 minutes, and then the final absorbance was measured at 405 nm. Thereafter, the value of β-glucosidase activity was confirmed using the following formula. The β-glucosidase activity values for 15 test strains are shown in Table 2.

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

Measurement results of 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, 9.63 unit / L and 12.6 unit / L, respectively.

Example  2. According to the type of archaea Sesame seeds Fermentation  Quality characteristics

The fermentation was performed in the same manner as in FIG. 2 for the fermentation of Sesame seeds.

1) The number of live bacteria of 15 types of archaea bacteria from sesame seeds

The results of the viable cell count before and after fermentation are shown in FIG. 3. As a result, 8 log CFU / mL or more was measured in the whole fermented sample. All 15 species were confirmed to grow well in sesame seeds.

2) β-glucosidase activity

Β-glucosidase activity was measured for the fermented sesame seeds according to the strain. The supernatant was collected and released 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.

Measurement results of β-glucosidase activity of sesame seeds according to strain 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 modified by the method of Blois (1958) to measure the reducing power of each sample as an effect on electron donation ability (EDA). That is, after adding 150 µl of 0.15 mM DPPH solution (dissolved in 99% MeOH) to 50 µl of the sample and standing at room temperature for 30 minutes, 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 sample addition and non-addition zones by the following equation.

Electron donating ability (%) = (1-absorbance of sample addition / absorption of no treatment) × 100

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

4) Total polyphenol content

The total phenol content was measured after appropriately diluting samples extracted according to the Folin-Denis method by concentration. After mixing and stirring 12.5 µl of Folin-Ciocalteu reagent in 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 in the same way as the analysis sample, and the total phenol content of the sample extract was calculated, 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 seeds are shown in FIG. 5. After fermentation, it was confirmed that the polyphenol content increased in all strains except the control group. Among them, 3 types of BCH3637, BCH3678, and SRCM103716 were the highest, respectively, 2490.2, 2559.4, and 2803.3 mg / L.

5) Total flavonoid content

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

The results of measuring the content of flavonoids before and after fermentation of sesame seeds are shown in FIG. 6. After fermentation, it was confirmed that the flavonoid content was increased in all strains except the control group. Among these, 3 types of BCH3637, BCH3678, and SRCM103716 were found to be the highest at 1423.27, 1688.67, and 1553.08 mg / L, respectively.

5. Sesame leaf archaea fermentation strain selection

Thus, as a strain for fermentation of sesame seeds, it was selected as Bacillus subtilis SRCM103716, 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 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 assigned a deposit number KFCC11832P.

Example  3. Sesame seeds Fermentation  Mixing ratio setting

Sesame leaf Bacillus subtilis SRCM103716 Fermentation and Sesame leaf Aspergillus oryzae ) Samples were prepared as shown in Table 7 to set the mixing ratio of the fermentation product. By confirming the DPPH radical scavenging ability, it was decided to set the fermented soy sauce production section at the rate with the highest antioxidant activity.

Section for setting the mixing ratio of sesame seeds sample Sesame seeds 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 Erasing ability  result

Results of DPPH radical scavenging ability according to the mixing ratio of sesame seeds are shown in FIG. 7. DPPH radical activity at 84.12% was highest in Aspergillus oridae ( A. oryzae ) 95%, B. subtilis SRCM103716 5%, followed by Aspergillus ori . oryzae ) from 100% to 77.28%. In order to Sesame fermented soy sauce production Aspergillus duck material (A. oryzae) 95%, Bacillus subtilis (B. subtilis) it was confirmed that the best SRCM103716 5%.

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

Finally, it was decided to mix 95% of the fermented sesame seed Aspergillus orijae with 5% of the Bacillus subtilis SRCM103716 fermentation for use in the production of soy sauce. Accordingly, DPPH radical scavenging activity and lignan components of the non-fermented sesame seeds and the fermented mixture of sesame seeds were measured.

(2-1) Sesame seeds  Of the fermentation mixture DPPH Radical  Scavenging activity result

The results of DPPH radical scavenging activity of the fermentation mixture of sesame seeds and non-fermentation using Bacillus subtilis SRCM103716 strain and Aspergillus oryzae are shown in Table 8. As a result of the measurement, it was confirmed that at 100-fold dilution, the fermented mixture of non-fermented sesame and fermented sesame seeds was 28.4% and 73.3%, respectively, and the antioxidant activity increased after fermentation.

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

(2-2) Sesame seeds  Of the fermentation mixture Lignan  Ingredient analysis results

The lignan component of the fermentation was extracted and analyzed, and the method is as follows. 50 mL methanol was added to 2.5 g of the fermentation product sample, followed by extraction using a sonicator (JAC4020) for 1 hour. After extraction, solids were removed by centrifugation at 3,000 rpm for 10 minutes using a centrifuge (Hanil MF80). After filtering with a 0.45 μm syringe filter, the analysis conditions are the same as in Table 9.

Lignan component analysis conditions Analysis conditions HPLC Agilent 1200 HPLC Detection Multi-wavelength 290 nm Column Capcell PAK MGII C18 Column
(4.6 × 150mm ID, 3) Column temp. 35 ℃ Flow rate 0.5 mL / min Injection 15 μl Elution 0.1% Formic acid aq. (A) / 10% acetonitrile aq. (B) as the gradient (5% B → 85% B) Extraction solvent 80% aqeous methanol Data acquisition Agilent Chemstation Markers Sesamol (1), Sesaminol (2),
Sesamin (3), Sesamolin (4)

Table 10 shows the results of analysis of the lignan content of the fermented mixture of sesame seeds and non-fermentation 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, and sesaminol diglucoside and sesaminol as the change in each lignan composition. ) Was found to increase by 1340.44% and 2071.43%, respectively. This was confirmed that the sesaminol glycoside bond is decomposed by fermentation metabolism as well as the overall lignan content increase, and the polymer glycoside bond decreases while the content of the low molecular sesaminol glycoside increases.

Analysis results of lignan composition of sesame seeds fermentation 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 seeds fermentation mixture 41.4 ± 3.4 810.1 ± 1.9 790.8 ± 120.1 76.0 ± 22.4 1718.2 ± 56.2 Growth 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 fermented mixture of sesame seeds is shown in FIG. 8.

1) Analysis of general ingredients of sesame seeds soy sauce

The pH was measured using a pH meter (pH / mV / TEMP Meter P25, ISTEK, Korea). Salinity was calculated by the following formula (End point: reddish brown) titrated with 0.1N-AgNO ₃ (F = 1.000) by taking 10 ml of the pre-treated sample.

NaCl (%) = (0.1N-AgNO3 consumption (ml) × 0.00585 × D × F × 100) / S

D: dilution factor (250/10)

S: sampling amount (g)

0.00585: amount of NaCl (g) corresponding to the number of ml of 0.1N-AgNO ₃

F: Factor of 0.1N-AgNO ₃

In addition, the total nitrogen content (T.N) was measured by the total nitrogen content by the Micro Kjeldahl Method. 1 ml of soy sauce was placed in a decomposition flask, 7 g of a decomposition catalyst and 12 ml of sulfuric acid were added, followed by heating and decomposition for 50 minutes. After cooling, distillation was performed using a Kjeldahl distillation apparatus, and the ammonia separated at this time was collected in a 4% boric acid solution and titrated with a 0.1N HCl standard solution.

Total nitrogen content (%) = (T-B)

0.1N-AgNO3 consumption (ml) × 0.00585 × D × F × 100) / S

T: Sample 0.1N HCl consumption

B: Blank 0.1N HCl consumption

S: sampling amount

The net solid content was taken 5 ml of soy sauce, mixed well with seaweed, dried for 4 hours in a 105 ° C dryer, then cooled and weighed to obtain the total solid content to obtain the net solid content.

Net solids content (%) = (Weight after drying-Blank container weight × 20)-Salinity (%)

As for the chromaticity (ABS value), 10 ml of the sample was diluted 50-fold, and absorbance was measured at 500 nm using a UV-VIS spectrophotometer (Shimadzu, UV mini-1240).

ABS value = 500 nm ABS value × 50 (dilution multiple)

Table 11 shows the general analysis results of sesame soy sauce using Bacillus subtilis SRCM103716 strain and Aspergillus oryzae . As the aging time elapsed, the pH tended to decrease slightly. This is due to the organic acid produced in the fermentation process. As the aging period increases, it can be seen that the pH of the organic acid increases and the pH decreases slightly. In addition, the total nitrogen content, net extract, and brownness were found to increase with the aging period, and the L value indicating brightness decreased as aging progressed.

2) Sesame seeds  Analysis of aroma components in soy sauce

Volatile fragrance components were separated and identified from sesame seed soy sauce, and the analysis conditions are shown in Table 12.

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

Table 13 to 15 show the results of the analysis of the fragrance components of sesame seeds soy sauce according to the ratio of Bacillus subtilis SRCM103716 strain and Aspergillus oryzae using Aspergillus oryzae . As a result of analyzing the volatile components of the fermented soybean sesame seeds, a total of 61 compounds were identified. Among them, 5 aldehydes, 10 aromatic compounds, 10 alcohols, 3 acids, 10 ketones, 4 esters, and 19 other compounds were identified. Characteristically, the content of 3-methyl butanal with fruity and malt scent in the soy sauce using the complex strain is 5.28%, smoked, wood flavor, and bacon flavor compared to the soy sauce using the single strain. -It was confirmed that 2,3-butanediol (2,3-butanediol) having 2-methoxy phenol (17.01%), fruit flavor, cream flavor, and butter flavor increased by 1.19%. In addition, the content of other non-aromatic volatile compounds showed a tendency to decrease by 13.47% from 34.3% to 20.83%. As a result, aspergillus in the soy sauce of the complex strain using SRCM103716 strain and Aspergillus oryzae It was confirmed that the compound of the aroma component was increased more than the soy sauce of a single strain inoculated with Orijae ( Aspergillus oryzae ) alone.

3) Sesame seeds  Measurement of free amino acids in the liver

For free amino acids, take 2 g of sample, add 30 mL of tertiary distilled water, stir, 50 mL, extract for 20 minutes using ultrasonic waves, centrifuge (3000 rpm, 10 min), and 2 mL of 5% TCA in 2 mL of supernatant. After inserting and centrifuging (10,000 rpm, 10 minutes), the supernatant was taken, diluted with 0.02N-HCl, passed through a 0.2 μm syringe filter, and analyzed with an amino acid analyzer under the analysis conditions in Table 16 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 enzymatic action during the fermentation process, the better the taste and nutrition. The free amino acid results of fermented soy sauce according to the ratio of the Bacillus subtilis SRCM103716 strain and the sesame leaf fermentation mixture using Aspergillus oryzae are shown in Table 17. 100% fermented soy sauce and 95% aspergillus raw material, 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 the glutamic acid of the enteric acid was 100% aspergillus ory and 95% of aspergillus ory + Bacillus subtilis SRCM103716 was found to be 47.82 mg / L and 70.44 mg / L in 5% fermented soy sauce, respectively. In addition, the content of aspartic acid (aspartic acid) was measured as 20.99 mg / L, 25.29 mg / L, and the ingredients that closely affect the savory taste and the unique taste of soy sauce were Aspergillus ducks 95% + Bacillus subtilis SRCM103716 was higher in 5% fermented soy sauce. As a result, all three of the free amino acids, isoleucine, leucine, and phenylalanine, which are bitter tastes, result in SRCM103716 strain + Aspergillus orijae ( Aspergillus oryzae ) in the soy sauce of a complex strain using Aspergillus ( Aspergillus) oryzae ) showed lower than the soy sauce of the single strain inoculated only, and it was confirmed that the savory taste, soy sauce-specific taste 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 Microbial Conservation Center KFCC11832P 20190710

<110> CHARM GO EUL CO., LTD. <120> Method for producing soy sauce using Bacillus subtilis SRCM103716          and Aspergillus oryzae strain <130> PN19312 <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 (5)

(1) Water is added to the crushed sesame seeds 1.2 to 1.8 times, and the water is increased at 110 to 130 ° C for 10 to 20 minutes, and the cooled sesame seeds have excellent β-glucosidase activity, and Bacillus Bacillus cereus , Staphylococcus aureus and Listeria monocytogenes have antimicrobial and β-hemolytic activity against harmful microorganisms, harmful substances of phenylalanine, The ability to secrete protease, amylase and cellulase without producing harmful enzymes of urease and β-glucuronidase and biogenic amines of tyramine and histamine Bacillus subtilis (Bacillus subtilis) SRCM103716 strain (Accession No .: KFCC11832P) was inoculated and fermented at 34-40 ° C. for 20-28 hours to total polyphenol and total flavonoid content and β-glucosidase The (β-glucosidase) comprising: an activity of promoting the production Sesame Bacillus subtilis fermentation;
(2) Water is added to the crushed sesame seeds 0.4 ~ 0.6 times, and the water is increased at 110 ~ 130 ℃ for 10 ~ 20 minutes. After cooling, the cooled sesame seeds are inoculated with Aspergillus oryzae strain, and then 28 ~ Preparing fermented sesame seeds by fermenting at 32 ° C. for 68 to 76 hours;
(3) The sesame leaf fermentation product produced in the step (1) and the sesame leaf fermentation product prepared in the step (2) are mixed at a weight ratio of 4 to 6:94 to 96 to improve the lignan content and antioxidant activity. Preparing a sesame-bak mixed fermentation product; And
(4) 23 ~ 25% (w / v) brine is added 2-3 times (v / w) to the mixed fermented sesame leaf prepared in the step (3), aged at 30 ~ 35 ℃ for 3 ~ 4 weeks Method of producing a fermented soy sauce with sesame seeds, characterized in that it comprises a step of filtering after the let. delete delete delete delete

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