Bacillus subtilis SRCM118942 strain producing γ-PGA in mucilage and having antimicrobial activity against pathogenic microorganism for solid Cheongkukjang fermentation and uses thereof

Abstract

The present invention relates to a Bacillus subtilis SRCM118942 strain with accession number KCCM13449P, which has the ability to secrete β-glucosidase and β-galactosidase, has antibacterial activity against harmful microorganisms, secretes amylase, protease, cellulase, and lipase enzymes, and does not produce toxin genes, and a method for producing Cheonggukjang using said strain.

Description

Technology Field

[0001] The present invention relates to a Bacillus subtilis having the ability to secrete β-glucosidase and β-galactosidase, having antimicrobial activity against harmful microorganisms, secreting amylase, protease, cellulase, and lipase enzymes, and not producing toxin genes ( Bacillus subtilis This relates to the SRCM118942 strain and its uses. Background Technology

[0002] Cheonggukjang is a representative traditional Korean fermented food produced through natural fermentation using soybeans, while natto is made using small-grained soybeans and natto bacteria ( Bacillus natto It is a representative Japanese fermented soybean food made by fermenting using Bacillus. Cheonggukjang and natto are similar in that they use soybeans as ingredients and are fermented with Bacillus, but Cheonggukjang is mainly used for soups and stews, while natto is used as a raw side dish.

[0003] Excellent native Bacillus strains have been developed, and Bacillus subtilis and fermentation methods capable of fermenting Cheonggukjang so that it can be eaten raw like Natto have been developed and are being utilized in the industry. In order to use native Bacillus subtilis in Natto production, a large amount of mucilage is produced in the form of threads, and the characteristic odor of Cheonggukjang must be absent so that it can be consumed raw.

[0004] In Korea, the market for Cheonggukjang is declining, while the markets for Natto and raw Cheonggukjang are growing. To address this, it is necessary to develop native Bacillus strains that can reduce the characteristic odor of Cheonggukjang while retaining the quality characteristics of Natto. Through this, the industrialization of high-quality raw Cheonggukjang will also be possible.

[0005] γ-PGA (poly-gamma-glutamic acid) is an anionic high-molecular-weight amino acid produced by fermenting plants (soybeans), and it is attracting attention as a novel functional material for its ability to promote calcium absorption in the intestines. Reported benefits of γ-PGA include enhancement of immune function, inhibition of intracellular bacterial and viral infections, cancer cell death, cell wall protection, and antioxidant effects.

[0006] γ-PGA is a naturally occurring biopolymer made up of repeating units of l-glutamic acid, d-glutamic acid, or both. Because some bacteria are capable of active biosynthesis of γ-PGA from renewable biomass, γ-PGA is considered a promising bio-based chemical and is already widely used in the food, medical, and wastewater industries due to its biodegradability and non-toxicity.

[0007] Korean Registered Patent No. 2219687 discloses a method for producing raw Cheonggukjang using a complex starter strain for Cheonggukjang fermentation, and Korean Registered Patent No. 0630272 discloses instant raw Cheonggukjang using a strain with excellent thrombolytic activity, but these are different from the Bacillus subtilis SRCM118942 strain for raw Cheonggukjang fermentation and the use thereof according to the present invention. The problem to be solved

[0008] The present invention was derived from the above requirements, and in the present invention, Bacillus subtilis (which has the ability to secrete β-glucosidase and β-galactosidase from traditional fermented sauces, has antibacterial activity against harmful microorganisms, secretes amylase, protease, cellulase, and lipase enzymes, and does not produce toxin genes) Bacillus subtilis ) The SRCM118942 strain was isolated.

[0009] The present invention was completed by using the above strain as a starter strain for the production of raw natto, and confirming that it has high content of γ-PGA (Poly-gamma-Glutamic Acid), free amino acids, and amino nitrogen, as well as excellent mucilage production and palatability. means of solving the problem

[0010] To solve the above problem, the present invention relates to a Bacillus subtilis with accession number KCCM13449P, which has the ability to secrete β-glucosidase and β-galactosidase, has antimicrobial activity against harmful microorganisms such as Bacillus cereus and Staphylococcus aureus, secretes amylase, protease, cellulase, and lipase enzymes, and does not produce toxin genes. Bacillus subtilis ) Provides the SRCM118942 strain.

[0011] In addition, the present invention relates to the above Bacillus subtilis ( Bacillus subtilis ) Provides a starter composition for making Cheonggukjang containing the SRCM118942 strain, the culture solution thereof, the concentrate of the culture solution, or the dried product thereof as an active ingredient.

[0012] In addition, the present invention relates to steamed soybeans and the above Bacillus subtilis ( Bacillus subtilis The present invention provides a method for manufacturing Cheonggukjang characterized by including the step of inoculating with the SRCM118942 strain and fermenting.

[0013] In addition, the present invention relates to a Bacillus cereus containing the strain, the culture solution thereof, the concentrate of the culture solution, or the dried product thereof as an active ingredient ( Bacillus cereus ) or Staphilococcus aureus( Staphylococcus aureus Provides an antimicrobial composition for ). Effects of the invention

[0014] Bacillus subtilis of the present invention ( Bacillus subtilis) The SRCM118942 strain has the ability to secrete β-glucosidase and β-galactosidase, and Bacillus cereus ( Bacillus cereus ) and Staphilococcus aureus( Staphylococcus aureus It has antimicrobial activity against harmful microorganisms and secretes amylase, protease, cellulase, and lipase enzymes, and does not produce toxin genes for cereulide, hemolytic enterotoxin, enterotoxin T, enterotoxin FM, non-hemolytic enterotoxin, and cytotoxin K. Furthermore, when used in the production of Cheonggukjang, it can be produced in the form of natto or raw Cheonggukjang that is easy to consume raw, with increased content of amino nitrogen, free amino acids, and γ-PGA (poly-gamma-glutamic acid), abundant production of mucilage and threads, and a weak characteristic Cheonggukjang odor, making it very useful in related industrial fields. Brief explanation of the drawing

[0015] Fig. 1 shows Bacillus subtilis ( Bacillus subtilis This is a photograph showing the degree of string formation in raw natto fermented with the SRCM118942 strain. Figure 2 compares the appearance and packaged photos of raw natto according to the type of strain. Specific details for implementing the invention

[0016] To achieve the objective of the present invention, the present invention relates to a Bacillus cereus having the ability to secrete β-glucosidase and β-galactosidase ( Bacillus cereus ) and Staphilococcus aureus( Staphylococcus aureusBacillus subtilis (accession number KCCM13449P), which has antimicrobial activity against harmful microorganisms, secretes amylase, protease, cellulase, and lipase enzymes, and does not produce toxin genes ( Bacillus subtilis ) Provides the SRCM118942 strain.

[0017] In the present invention, strains were isolated from traditional fermented sauces, and among them, Bacillus subtilis (which exhibits enzyme secretion ability, antibacterial activity against harmful microorganisms, does not produce toxin genes, and shows high amino nitrogen, free amino acid, and γ-PGA (poly-gamma-glutamic acid) content during Cheonggukjang fermentation) Bacillus subtilis A strain was isolated and identified, and named Bacillus subtilis SRCM118942, which was deposited with the Korean Culture Collection of Microorganisms (KCCM) on December 22, 2023 (Deposit number: KCCM13449P).

[0018] In the Bacillus subtilis SRCM118942 strain of the present invention, the toxin gene may be, but is not limited to, the toxin genes of cereulide, hemolytic enterotoxin, enterotoxin T, enterotoxin FM, non-hemolytic enterotoxin, and cytotoxin K.

[0019] The above strain has the functional characteristics described above and, when fermenting Cheonggukjang, exhibits an amino nitrogen content of 110 mg% or more, a free amino acid content of 17,000 mg / kg or more, and a γ-PGA (poly-gamma-glutamic acid) content of 3 mg / g or more, preferably 110~120 mg% amino nitrogen, 17,000~19,000 mg / kg free amino acid, and 3~5 mg / g γ-PGA (poly-gamma-glutamic acid), while reducing the characteristic off-flavor of Cheonggukjang and increasing the amount of mucilage produced, thereby enhancing palatability, making it a suitable strain for producing raw Cheonggukjang (Natto).

[0020] The present invention also provides a starter composition for producing Cheonggukjang containing the strain, the culture solution thereof, the concentrate of the culture solution, or the dried product thereof as an active ingredient.

[0021] In the present invention, a starter for the production of Cheonggukjang refers to a preparation or composition containing microorganisms involved in fermentation for the production of Cheonggukjang. It is used to provide microorganisms capable of growing in the fermented Cheonggukjang or microorganisms capable of growing as dominant species by being added during the production of Cheonggukjang. When Cheonggukjang is produced using the above-mentioned starter for Cheonggukjang fermentation, the microorganisms contained in the starter can achieve the purpose of consistently controlling the quality of the Cheonggukjang, or for specific purposes, such as preventing or reducing off-flavors in the Cheonggukjang, or enhancing the savory or sweet taste in the Cheonggukjang.

[0022] The method for culturing the strain of the present invention can be carried out according to methods commonly used in the art and is not limited to any specific method.

[0023] The present invention also,

[0024] (1) A step of draining the water from the soaked soybeans, steaming them, and cooling them;

[0025] (2) Bacillus subtilis (in the cooled soybeans of step (1) above) Bacillus subtilis ) Step of inoculating with the SRCM118942 strain; and

[0026] (3) A method for manufacturing Cheonggukjang is provided, characterized by including the step of fermenting soybeans inoculated with the strain of step (2) above.

[0027] In the method for manufacturing Cheonggukjang according to the present invention, step (1) can preferably be performed by draining the water from soybeans soaked at 3 to 5°C for 10 to 14 hours, steaming at 100 to 120°C for 5 to 15 minutes, and cooling to 45 to 50°C, and more preferably by draining the water from soybeans soaked at 4°C for 12 hours, steaming at 110°C for 10 minutes, and cooling to 45 to 50°C. Although the soybeans soaked as in step (1) can be saturated to a state suitable for steaming, if soaked for less than the above time, the soybeans do not swell sufficiently and steaming is not done well, and if soaked for more than the above time, the water-soluble nutrients of the soybeans are lost as they leach into the water during the soaking process. In addition, by steaming the soaked soybeans at a high temperature as described above, it was possible to prevent contamination by mold or other bacteria and prepare soybeans suitable for making Cheonggukjang without a beany smell. Furthermore, by cooling to the temperature as described above and then inoculating with bacteria to ferment, it was possible to ferment into Cheonggukjang that not only has excellent flavor and improved palatability but also possesses a viscosity suitable for raw Cheonggukjang and exhibits excellent viscous formation.

[0028] In addition, in the method for manufacturing Cheonggukjang of the present invention, the inoculation of step (2) is preferably Bacillus subtilis (relative to the total weight of soaked soybeans before steaming) Bacillus subtilis ) By inoculating the SRCM118942 strain at 0.5~1.5% (v / w), the bacterial concentration was 10 4~6It can be made to be CFU / g, and more preferably, Bacillus subtilis (relative to the total weight of soaked soybeans before steaming) Bacillus subtilis ) When the SRCM118942 strain was inoculated at 1% (v / w), the bacterial concentration was 10 5 It can be made so that CFU / g is used. If the strain is inoculated in excess of the above range, there is a problem that the Cheonggukjang changes into a porridge-like form due to over-fermentation, and if it is inoculated in excess of the above range, there is a problem that fermentation does not occur properly.

[0029] In addition, in the method for manufacturing Cheonggukjang according to the present invention, the fermentation in step (3) can preferably be carried out at 42 to 48°C for 25 to 35 hours, and more preferably at 45°C for 30 hours. Producing Cheonggukjang by fermenting under the above conditions maximizes the activity of the nutritional components and live bacteria of Cheonggukjang, while suppressing the production of substances that cause unpleasant odors during the fermentation process, resulting in excellent palatability. Furthermore, a large amount of mucilage is produced, allowing for the production of raw Cheonggukjang in the form of natto. However, if fermentation is carried out at a temperature and time below the above, there is a problem that the savory taste of Cheonggukjang is reduced, and if fermentation is carried out at a temperature and time above, there is a problem that harmful bacteria proliferate excessively and palatability decreases.

[0030] The method for manufacturing Cheonggukjang according to the present invention, more specifically, is

[0031] (1) A step of draining water from soybeans soaked at 3~5℃ for 10~14 hours, steaming at 100~120℃ for 5~15 minutes, and cooling to 45~50℃;

[0032] (2) Bacillus subtilis (in the cooled soybeans of step (1) above) Bacillus subtilis ) Step of inoculating with the SRCM118942 strain; and

[0033] (3) The method may include a step of fermenting soybeans inoculated with the strain of step (2) at 42 to 48°C for 25 to 35 hours, and

[0034] More specifically,

[0035] (1) A step of draining water from soybeans soaked at 4℃ for 12 hours, steaming at 110℃ for 10 minutes, and cooling to 45~50℃;

[0036] (2) Bacillus subtilis (in the cooled soybeans of step (1) above) Bacillus subtilis ) Step of inoculating with the SRCM118942 strain; and

[0037] (3) The step of fermenting soybeans inoculated with the strain of step (2) above at 45°C for 30 hours may be included.

[0038] The present invention also provides Cheonggukjang prepared by the above method.

[0039] The present invention also comprises a Bacillus cereus containing the strain, the culture solution thereof, the concentrate of the culture solution, or the dried product thereof as an active ingredient ( Bacillus cereus ) or Staphilococcus aureus( Staphylococcus aureus Provides an antimicrobial composition for ).

[0040] In an antibacterial composition according to one embodiment of the present invention, the composition may preferably be in the form of a food, a food additive, a feed additive, or a pharmaceutical, and the food may be selected from the group consisting of dairy products (milk, soy milk, processed milk), fermented milk (liquid yogurt, solid yogurt), drinks, meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, chewing gum, ice cream, soup, beverages, alcoholic beverages, and vitamin complexes, but is not limited thereto.

[0042] The present invention will be described in detail below with reference to manufacturing examples and embodiments. However, the following manufacturing examples and embodiments are merely illustrative of the present invention, and the content of the present invention is not limited to the following manufacturing examples and embodiments.

[0044] Preparation Example 1. Raw Cheonggukjang

[0045] For the production of raw cheonggukjang, selected soybeans were washed, soaked in water at refrigerator temperature (4℃) for 12 hours, and then naturally dehydrated at room temperature for 1 hour to prepare the soaked soybeans. Next, they were steamed in an NK steamer at 110℃ for 10 minutes and cooled to 40–45℃; subsequently, they were placed in a fermentation tray and Bacillus subtilis ( Bacillus subtilis ) The SRCM118942 strain was inoculated. The above strain was inoculated at 0.1% (w / w) of the weight of soaked soybeans before steaming, and the total bacterial concentration was 10 5 The concentration was adjusted to CFU / g. Subsequently, it was fermented in an incubator at a temperature of 45°C for 30 hours.

[0047] A. Characteristics of the strain

[0048] 1. Antimicrobial activity

[0049] The antimicrobial activity of the isolated strains was evaluated using the well diffusion assay. The isolated strains were inoculated into LB (Luria-Bertani) liquid medium and incubated at 37°C for 24 hours. After centrifugation at 13,000 rpm for 30 minutes, the supernatant was collected. The collected supernatant was filtered through a 0.2 µm syringe filter (Sartorius, Frankfurt, Germany), and 100 µl of the filtered supernatant was inoculated into 8 mm wells prepared according to the well diffusion assay using 0.8% soft agar plates. The samples were then incubated at 30°C for 24 hours. Subsequently, the antimicrobial activity was evaluated by checking for the formation of a zone of inhibition around the wells.

[0051] 2. Enzyme Activity Analysis

[0052] ① Amylase activity analysis

[0053] A medium containing 10.0 g / L of soluble starch and 20.0 g / L of agar was prepared and sterilized (autoclave, 121°C, 15 min). Wells were then prepared using an 8 mm cork borer, and 100 µl of the supernatant of the strain was inoculated. After culturing for 1 day, Lugol's iodine solution (20.0 g / L KI, 2.0 g / L I2) was added. After reacting for 1 minute, the solution was removed, and the presence or absence of a clear zone was checked to evaluate amylase activity.

[0055] ② Protease activity analysis

[0056] The protease activity of the isolated strain was evaluated by mixing 2.0% skim milk sterilized at 110°C for 10 minutes and 1.5% agar sterilized at 121°C for 15 minutes after cooling for a certain period. After mixing, 25 mL was dispensed per plate to prepare a solid medium. Wells were made using an 8 mm cork borer, and 100 µl of the strain supernatant was dispensed into each well. After incubating at 30°C for 24 hours, protease activity was evaluated based on the presence or absence of a clear zone formed around the wells.

[0058] ③ Cellulase activity analysis

[0059] A medium containing 10.0 g / L of carboxymethyl cellulose and 15.0 g / L of agar was prepared and sterilized (autoclave, 121°C, 15 min). Wells were then made using an 8 mm cork borer, and 100 µl of the supernatant of the strain was inoculated. After culturing for 1 day, the cells were stained with 0.1% Congo Red solution for about 15 minutes, and then washed with 1M NaCl for 15 minutes to check for the formation of a clear zone, thereby evaluating cellulase activity.

[0061] ④ Lipase activity analysis

[0062] After preparing and sterilizing (autoclave, 121°C, 15 min) a medium containing 10.0 mL / L Tween 80 and 15.0 g / L agar, wells were made using an 8 mm cork borer and 100 µL of the supernatant of the strain was inoculated. After culturing for 3 days, lipase activity was evaluated by checking for the formation of precipitates.

[0064] ⑤ β-glucosidase activity analysis

[0065] After preparing and sterilizing (autoclave, 121°C, 15 min) bile-esculin solid medium (M340, HIMEDIA), wells were made using an 8 mm cork bore, and 100 µl of the supernatant of the strain was inoculated. After culturing for 1 day, β-glucosidase activity was evaluated by checking for the formation of a black zone.

[0067] ⑥ β-galactosidase activity analysis

[0068] After preparing and sterilizing (autoclave, 121°C, 15 min) a medium containing 0.15 g / L X-gal and 15.0 g / L agar, wells were made using an 8 mm cork borer and 100 µl of the supernatant of the strain was inoculated. After incubation for 1 day, β-galactosidase activity was evaluated by checking for the formation of a blue zone.

[0070] 3. Bacillus cereus through PCR analysis ( Bacillus cereus ) Toxin gene search

[0071] To confirm the possession of Bacillus cereus toxin genes, the presence of six genes—cereulide (CER), hemolytic enterotoxin (hblC), enterotoxin T (bceT), enterotoxin FM (entFM), non-hemolytic enterotoxin (nheA), and cytotoxin K (CytK)—was checked using the PowerChek™ Bacillus cereus toxin 6-plex detection kit (P0171C, Kogenbiotech Co. Ltd., Seoul, Korea). 10 µl of PCR premix, 5 µl of primer mix, and 5 µl of colony suspension were added sequentially to a 250 µl PCR tube, and PCR was performed at 95°C for 10 min → (95°C, 30 sec → 60°C, 30 sec → 72°C, 30 sec) × 35 cycles → 72°C for 10 min. The PCR products were loaded onto a 1.5% agarose gel prepared with 0.5 × buffer and subjected to electrophoresis, and the bands were visualized using a Chemidoc system (c600, Azure Biosystems, Inc., Dublin, CA, USA). The expected PCR product sizes for each toxin gene were CER (114 bp), hblC (195 bp), bceT (303 bp), ent FM (360–400 bp), nheA (475 bp), and cytK (585 bp).

[0073] 4. γ-PGA Analysis

[0074] 5 g of Cheonggukjang was placed in a test tube, 5 mL of an equal amount of distilled water was added, and homogenized using a homogenizer for 3 minutes. Then, it was washed with 2 mL of distilled water and centrifuged at 15,000 × g and 4°C for 10 minutes. The supernatant was recovered, and cold ethanol was added in a volume four times that of the supernatant and mixed. After standing overnight at 4°C, the supernatant was removed by centrifuging at 15,000 × g and 4°C for 10 minutes. A small amount of distilled water was added to the precipitate to dissolve and recover it. A certain amount of the recovered precipitate was taken, and the dry constant weight was determined to calculate the γ-PGA production yield.

[0075] γ-PGA production capacity = γ-PGA dry constant weight (mg) / Cheonggukjang sample weight (mg)

[0077] B. Preparation of raw cheonggukjang

[0078] 1. Preliminary experiment comparing Natto bacteria and isolated strains

[0079] Strains isolated from commercially available natto ( Bacillus natto Raw Cheonggukjang was prepared by fermenting soybeans using 72 strains isolated through identification by the Foundation for the Promotion of Fermented Microorganism Industry. The specific method for preparing raw Cheonggukjang is as follows. The seed was prepared by inoculating a single colony of the test bacteria with 5 mL of TSB (tryptic soy broth) into a test tube and incubating it at 37°C for 24 hours. Domestic soybeans (white soybeans) were purchased, selected, soaked in distilled water at a refrigerator temperature (4°C) for 12 hours, and then naturally dehydrated at room temperature for 1 hour to prepare soaked soybeans. 15 g of the soaked soybeans were placed in an Erlenmeyer flask and steamed at 121°C for 30 minutes, then cooled to 40–45°C at room temperature. 200 µl of TSB (tryptic soy broth) and 100 µl of seed were inoculated into the cooled soybeans, and the total bacterial concentration was 10 5 The concentration was adjusted to CFU / g. Subsequently, raw natto was produced by fermenting in an incubator at 37°C and 80% humidity for 24 hours.

[0081] 2. Experiment on the production of raw natto using the Bacillus subtilis SRCM118942 strain

[0082] The finally selected natto characteristic Bacillus held by the Fermentation Microorganism Industry Promotion Foundation ( B. subtilis SRCM118942), Bacillus currently used as a Cheonggukjang strain ( B. subtilis SRCM102059) and Bacillus used by other companies ( B. subtilis Fresh Cheonggukjang was prepared using ). For the preparation of fresh Cheonggukjang, selected soybeans were washed, soaked in water at a refrigerator temperature (4℃) for 12 hours, and naturally dehydrated at room temperature for 1 hour to prepare soaked soybeans. Next, they were steamed in an NK steamer at 110℃ for 10 minutes, and then placed on a fermentation plate and inoculated with Bacillus subtilis. Bacillus subtilis was inoculated at 0.1% of the weight of the soaked soybeans before steaming (10 5 Fermented at 45℃ for 30 hours to a concentration of CFU / g.

[0083] Quality analysis samples were collected at intervals of 0, 6, 20, and 30 hours during the production of Cheonggukjang, and analysis was conducted by the Fermented Microorganism Industry Promotion Agency and commissioned to some accredited testing and inspection institutions. The analyzed parameters included the number of Bacillus bacteria, E. coli, coliforms, Bacillus cereus, amino nitrogen content (AN), pH, acidity, free amino acids, free sugars, and γ-PGA.

[0085] Example 1. Evaluation of antimicrobial activity against foodborne pathogenic microorganisms

[0086] Bacillus subtilis Bacillus subtilis The results of the antimicrobial activity of the SRCM118942 strain culture against six types of harmful food bacteria are shown in Table 1 below, and it was found that the proliferation of Bacillus subtilis around the wells inhibited the proliferation of harmful food bacteria ( Bacillus subtilis ) The SRCM118942 strain is Bacillus cereus ( Bacillus cereus ) and Staphilococcus aureus( Staphylococcus aureus It was found to have antibacterial activity against ).

[0087] Antimicrobial activity of Bacillus subtilis SRCM118942 strain Harmful bacteria in food Antibacterial activity (mm) Bacillus cereus Bacillus cereus ) KCTC1661 1.41 Bacillus cereus Bacillus cereus ) KCCM11204 1.36 Escherikia Colai ( Escherichia coli ) KCCM11234 1.00 Staphilococcus aureus Staphylococcus aureus ) KCCM11593 1.27 Salmonella typhimurium ( Salmonella typhimurium ) KCCM1926 1.00 Listeria monocytogenes Listeria monocytogenes ) KCCM43155 1.00

[0089] Example 2. Enzyme Activity Analysis

[0090] (1) Amylase, protease, cellulase and lipase activity

[0091] During the fermentation process of traditional soy sauce, various extracellular enzymes derived from microorganisms, such as amylase, protease, cellulase, and lipase, are secreted, and these enzymes produce secondary metabolites that have various functionalities as well as flavor. Proteases decompose proteins during fermentation, affecting the content of free amino acids, which are the components of the characteristic savory taste. Cellulose is decomposed and utilized by cellulase secreted by fungi and bacteria, and it is known that isoflavone glycosides are converted into aglycones by endo-1,4-β-D-glucanase, exo-1,4-β-D-glucanase, and β-glucosidase, which are components of cellulase, thereby exhibiting functionalities such as antioxidant properties.

[0092] Bacillus subtilis Bacillus subtilis As a result of investigating the production performance of amylase, protease, cellulase, and lipase using the SRCM118942 strain via well diffusion, it was confirmed that the strain produced all four enzymes and also exhibited excellent activity (Table 2).

[0093] Amylase, protease, cellulase, and lipase activity of Bacillus subtilis SRCM118942 strain Types of enzymes Enzyme activity (mm) amylase 2.67 protease 18.82 cellulase 19.35 lipase 9.77

[0095] (2) β-glucosidase activity

[0096] Fermented foods made from soybeans contain glycosides in the form of aglycones mixed with sugar components; a representative substance is isoflavone, which is referred to as a phytoestrogen. The active substances in the body are daidzein and genistein. Isoflavones existing in glycoside form are converted into non-glycolytic forms, such as daidzein and genistein, by gastric acid and β-glucosidase, an enzyme produced by intestinal microorganisms, and are absorbed in the intestines. Recent reports indicate that genistein has been confirmed to possess strong anticancer effects, and it is predicted that fermented foods utilizing microorganisms that produce such enzymes will gain even more attention as functional foods.

[0097] The selected strain in the present invention, Bacillus subtilis SRCM118942, was found to have high β-glucosidase activity.

[0098] β-glucosidase activity of Bacillus subtilis SRCM118942 strain Types of enzymes Enzyme activity (mm) β-glucosidase 12.72

[0100] (3) β-galactosidase activity

[0101] β-galactosidase is an enzyme that helps alleviate lactose intolerance caused by lactose in milk, which affects 80% of Koreans. Also known as lactase, it has long been known as a typical inducible enzyme. In particular, for individuals with a deficiency in lactase activity, consuming milk results in the lactose not being absorbed but accumulating in the lumen of the small intestine, causing an increase in osmotic pressure. This leads to excess water, and fermentation by microorganisms in the cecum generates carbon dioxide, lowers the pH in the large intestine, and irritates the intestines, causing discomfort. Furthermore, it induces temporary diarrhea or chronic inflammation of the large intestine, ultimately leading to chronic diarrhea.

[0102] Therefore, among the selected strains, a strain with excellent activity against β-galactosidase was selected, and measurements were conducted to alleviate lactose intolerance using the strain. Activity was measured by reacting the strains in the same manner using β-galactosidase derived from Aspergillus oryzae, and then constructing standard curves of enzyme activity and absorbance, expressed in units per mL of strain culture solution. As a result, the SRCM118942 strain was found to have β-galactosidase activity.

[0103] β-galactosidase activity of Bacillus subtilis SRCM118942 strain Types of enzymes Enzyme activity β-galactosidase 0.12

[0105] Example 3. Toxin gene analysis

[0106] Bacillus subtilis Bacillus subtilis ) Bacillus cereus of strain SRCM118942 ( Bacillus cereu Analysis of toxin genes known to be produced by s) revealed the absence of genes for cereulide (CER), hemolytic enterotoxin (hblC), enterotoxin T (bceT), enterotoxin FM (entFM), non-hemolytic enterotoxin (nheA), and cytotoxin K (CytK). Therefore, it was confirmed that the S2-3 strain possesses safety against food poisoning and can be utilized in various application fields such as food and food additives.

[0107] Confirmation of possession of Bacillus cereus toxin gene gene Agreement rate (%) CER ND 1) hblC ND bceT ND entFM ND nheA ND CytK ND

[0108] 1) Not detected

[0110] Example 4. Characteristics of soybean fermentation using natto bacteria and isolated strains

[0111] A sensory evaluation was conducted in four replicates using a 10-point scale on raw cheonggukjang prepared using commercially available natto bacteria and 72 types of Bacillus strains isolated through identification by the Korea Fermentation Microorganism Industry Promotion Agency. As a result, raw cheonggukjang fermented with the SRCM118942 strain exhibited fermentation characteristics most similar to those prepared with commercially available natto strains, such as mucilage and thread formation, clumping, and stickiness, while having a weak characteristic cheonggukjang aroma, Bacillus subtilis ( Bacillus subtilis ) The SRCM118942 strain was selected as the starter strain for the final production of raw natto (Table 6).

[0112] Soybean Fermentation Characteristics strain name Commercially available natto bacteria SRCM118942 strain Degree of fermentation (degree of surface mucus formation) 7 8 Degree of thread generation 8~9 8 Degree of clumping 2 2~2.5 degree of stickiness 7 6~7 Cheonggukjang scent 2~2.5 0.5

[0114] Bacillus subtilis selected through Table 6 above ( Bacillus subtilis ) SRCM118942 strain culture solution (10 8 Fresh Cheonggukjang was prepared by inoculating 0.1% (v / w) of (cfu / mL or higher) and fermenting at 37°C for 24 hours, after which a sensory evaluation was qualitatively expressed as relative values ​​of strong, medium, weak, and high, low. As a result, sufficient mucilage was produced to produce natto, and the Cheonggukjang odor was very weak, making it suitable for the production of natto and fresh Cheonggukjang (Table 7, Fig. 1).

[0115] Evaluation results of raw cheonggukjang strain name SRCM118942 strain Surface viscosity plenty Scent intensity approximately Flavor intensity approximately degree of elasticity of viscous material river Amount of viscous material plenty

[0117] Example 5. Quality characteristics of Cheonggukjang according to strain and fermentation time

[0118] The results of the quality characteristics of Cheonggukjang according to strain and fermentation time are shown in Table 8. There were no significant differences in pH range and acidity content among all samples, and acidity content tended to increase as fermentation progressed. Amino nitrogen (AN) content increased as fermentation progressed, with the highest content of 115.26 mg% observed in SRCM118942 fermented for 30 hours. Escherichia coli and coliforms were not detected in any samples, and the number of viable Bacillus cells increased as fermentation progressed, confirming cell growth. Bacillus cereus was also not detected in any samples.

[0119] Quality characteristics of Cheonggukjang according to strain and fermentation time Sample name pH Acidity (%) AN(mg%) Microbiological test (CFU / g) Fermentation time Sample name E. coli coliform bacteria Bacillus viable count Bacillus cereus 0 hours Cheonggukjang bacteria used by the company 6.15 0.73 66.36 Non-detectable Non-detectable 1.5×10 6 Non-detectable SRCM102059 6.45 0.55 54.46 Non-detectable Non-detectable 7.0×10 5 Non-detectable SRCM118942 5.84 0.88 60.51 Non-detectable Non-detectable 3.7×10 5 Non-detectable 6 hours Cheonggukjang bacteria used by the company 6.76 0.37 53.57 Non-detectable Non-detectable 3.0×10 6 Non-detectable SRCM102059 6.27 0.65 52.22 Non-detectable Non-detectable 3.0×10 5 Non-detectable SRCM118942 6.04 0.69 62.86 Non-detectable Non-detectable 2.3×10 5 Non-detectable 20 hours Cheonggukjang bacteria used by the company 6.32 1.26 92.40 Non-detectable Non-detectable 1.7×10 8 Non-detectable SRCM102059 6.73 0.73 72.98 Non-detectable Non-detectable 1.5×10 8 Non-detectable SRCM118942 6.10 1.15 74.90 Non-detectable Non-detectable 1.4×10 8 Non-detectable 30 hours Cheonggukjang bacteria used by the company 6.58 0.95 109.34 Non-detectable Non-detectable 2.1×10 8 Non-detectable SRCM102059 6.73 0.69 87.31 Non-detectable Non-detectable 1.8×10 8 Non-detectable SRCM118942 6.35 1.11 115.26 Non-detectable Non-detectable 2.3×10 8 Non-detectable

[0121] Example 6. Quality characteristics of Cheonggukjang according to strain type

[0122] The results of the quality characteristics of finished Cheonggukjang products fermented for 30 hours by strain are shown in Table 9, and a photograph of the Cheonggukjang is shown in Figure 2. Total free amino acids were highest in the SRCM118942 sample at 18,007.7 mg / kg, and γ-PGA was also highest at 3.82 mg / g. Three types of free sugars were detected; fructose content was highest in SRCM102059 at 9,375.6 mg / kg, and glucose was highest in SRCM118942 at 10,329.6 mg / kg. Maltose was highest in the Cheonggukjang strain used by another company at 1,909.4 mg / kg. Based on the results of this experiment, the SRCM118942 strain, which has high content of amino nitrogen, free amino acids, and γ-PGA while being rich in the flavor and mucilage of Cheonggukjang, is considered the most suitable for the production of Cheonggukjang.

[0123] Quality characteristics of Cheonggukjang according to strain type Sample name Total free amino acids (mg / kg) γ-PGA(mg / g) Free sugars (mg / kg) fruit sugar glucose Malt sugar natto bacteria used by other companies 16,033.0 2.42 1,817.6 8,402.6 1,909.4 SRCM102059 13,772.8 1.42 9,375.6 7,519.6 1,177.7 SRCM118942 18,007.7 3.82 5,640.0 10,329.6 1,281.1

[0125] Depository Name: Korean Culture Collection of Microorganisms (Overseas) Trustee Number: KCCM13449P Date of Deposit: 2023-12-22

Claims

Claim 1 It has the ability to secrete β-glucosidase and β-galactosidase, and Bacillus cereus ( Bacillus cereus ) and Staphilococcus aureus( Staphylococcus aureus Bacillus subtilis (accession number KCCM13449P), which has antimicrobial activity against harmful microorganisms, secretes amylase, protease, cellulase, and lipase enzymes, and does not produce toxin genes for cereulide, hemolytic enterotoxin, enterotoxin T, enterotoxin FM, non-hemolytic enterotoxin, and cytotoxin K ( Bacillus subtilis ) SRCM118942 strain. Claim 2 Bacillus subtilis according to claim 1, characterized in that the strain exhibits an amino nitrogen content of 110–120 mg%, a free amino acid content of 17,000–19,000 mg / kg, and a γ-PGA (poly-gamma-glutamic acid) content of 3–5 mg / g during Cheonggukjang fermentation. Bacillus subtilis ) SRCM118942 strain. Claim 3 delete Claim 4 A starter composition for producing Cheonggukjang containing the strain of claim 1, a culture solution thereof, a concentrate of said culture solution, or a dried product thereof as an active ingredient. Claim 5 (1) A step of draining the water from the soaked soybeans, steaming them, and cooling them; (2) A step of adding the Bacillus subtilis of claim 1 to the cooled soybeans from step (1) ( Bacillus subtilis A method for manufacturing Cheonggukjang characterized by comprising the steps of: (3) inoculating a SRCM118942 strain; and (3) fermenting soybeans inoculated with the strain of step (2). Claim 6 In claim 5, (1) a step of draining the water from soybeans soaked at 3~5℃ for 10~14 hours, steaming at 100~120℃ for 5~15 minutes, and cooling to 45~50℃; (2) the cooled soybeans of claim 1 ( Bacillus subtilis A method for producing Cheonggukjang characterized by comprising the steps of: (3) inoculating the soybeans inoculated with the strain of SRCM118942; and (3) fermenting the soybeans inoculated with the strain of step (2) at 42 to 48°C for 25 to 35 hours. Claim 7 Bacillus cereus containing the strain of claim 1, its culture solution, a concentrate of the culture solution, or a dried product thereof as an active ingredient ( Bacillus cereus ) or Staphilococcus aureus( Staphylococcus aureus Antimicrobial composition for )

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