KR101956760B1 - Bacillus subtilis SRCM102754 strain having antimicrobial activity, antioxidant activity, extracellular enzyme secretion activity, phenol production activity and not producing harmful metabolite and harmful enzyme and uses thereof - Google Patents

Abstract

The present invention relates to a Bacillus subtilis SRCM102754 strain, which has antimicrobial activities, antioxidant activities, ability to secret extracellular enzymes and produce phenol, and which does not produce harmful substances and harmful enzymes; and uses thereof. It was confirmed that the Bacillus subtilis SRCM102754 strain of the present invention has antimicrobial activities against harmful microorganisms, antioxidant activities, the ability to secret the extracellular enzymes and produce phenol, and does not produce harmful substances and harmful enzymes. Therefore, the Bacillus subtilis SRCM102754 strain of the present invention is considered to be useful in a variety of related industries such as antibiotic microbial preparations and starter strains for producing sauces.

Description

Bacillus subtilis SRCM102754 strain which has antimicrobial activity, antioxidative activity, extracellular enzyme secretion ability and phenol production ability and does not produce harmful substances and harmful enzymes and its use {Bacillus subtilis SRCM102754 strain having antimicrobial activity, antioxidant activity, extracellular enzyme secretion activity , phenol production activity and not producing harmful metabolites and harmful enzymes and uses thereof]

The present invention relates to a strain of Bacillus subtilis SRCM102754 which has antimicrobial activity, antioxidative activity, extracellular enzyme releasing ability and phenol producing ability, and which does not produce harmful substances and harmful enzymes, and uses thereof.

Chungkukjang boiled soybeans, put the rice straw in place, and wrapped in a warm night, the surface turns grayish white and sticky sticky. It is mixed with salt, garlic, and red pepper powder, and it is used as the material of the stew by putting it in the mortar and putting it in the pot. Unlike our traditional soy sauce, miso, and kochujang, Because it is a fermented food, it has traditionally been harvested from soybeans, and has been easily manufactured at home from late autumn to early spring. Chungkukjang has a higher content of soybean protein and lipid than soybean paste, has higher digestion and absorption rate, and is an important source of calcium and vitamins A and B, a sweetening effect of Chungkukjang, prevention of constipation of fiber, effect of in vitro excretion of carcinogens and cholesterol, ) Has been known to have effects such as a seaweed effect due to alcohol absorption, saponin's blood vessel depression, blood circulation promotion and lactic acid decomposition effect, brain aging and dementia of lecithin, prevention of hypertension and arteriosclerosis.

Soybean is a representative fermented food in Korea and uses soybeans as its main material. Soybean is a food material of high protein, but it contains saponin, isoflavone, and ipriflavone as physiologically active substances and has effects such as anticancer action and prevention of osteoporosis. And soybean products including soybean paste are recognized as health foods. It is known that such effective ingredients in soybeans are transferred to soy sauce or soybean paste during fermentation of meju. The manufacturing methods of doenjang are largely classified into the conventional method and the modern method. The traditional method of making miso is somewhat different depending on the region, but basically, the raw soybean is boiled, molded into a brick or spherical shape, dried for 2 to 3 days, and if cracked, it is hung with straw to maintain proper temperature and humidity do. In this way, the various microorganisms around it are naturally mixed and washed, washed meju cleanly, and fermented for 3 ~ 6 months in February ~ March, and the liquid phase is dipped into soy sauce and the rest is made into solid soybean paste.

Kochujang is a traditional seasoned food with a history of about 200 years, and it is an important fermented food indispensable with miso and soy sauce. In recent years, it has become possible to produce on an industrial scale in an improved manner in accordance with changes in dietary patterns, and this trend is gradually expanding along with consumers' desire for convenience. Kochujang usually produces a mixture of raw materials such as rice, wheat flour, barley, sorghum and red bean paste, which is prepared by mixing fermented powder, malt powder, and red pepper powder with salt, A method of producing an improved type of kochujang prepared by using koji or an enzyme agent in flour is known. Kochujang is rich in nutrients such as protein, fat, vitamins A and C. The sweetness of hydrolysis of carbohydrates and the richness of amino acids, hot spices of pepper and saltiness of salt from soybean protein are well harmonized, It is used as a seasoning for raw vegetables, stew, stewed vegetables, and roasted vegetables.

Korean Patent No. 1811241 discloses that Bacillus subtilis SCGB 180, which has acid resistance, biliary properties, antioxidative activity, lipase inhibitory activity and antimicrobial activity, and which secretes protease and amylase enzyme without producing biogenic amine And Korean Patent No. 1816124 discloses 'Bacillus subtilis SCDB 257 strain which has an ability to produce glutamate and an antimicrobial activity against harmful microorganisms and does not produce a biogenic amine, and its use' However, as in the present invention, the Bacillus subtilis SRCM102754 strain which has an antimicrobial activity, an antioxidative activity, an extracellular enzyme secretory ability and a phenol-producing ability and does not produce harmful substances and harmful enzymes and its use has been disclosed Not at all.

SUMMARY OF THE INVENTION The present invention has been made in view of the above needs, and it is an object of the present invention to provide an antimicrobial activity, an antioxidative activity, a protease, a cellulase and an amylase secretion activity and a phenol production ability of Bacillus cereus, Staphylococcus aureus and Enterococcus faecalis Bacillus subtilis which does not produce urease and 棺 -glucuronidase which is a harmful enzyme and indole and phenylpyruvic acid which are harmful substances, The strain SRCM102754 (KCCM12198P) was isolated.

The Bacillus subtilis Since the SRCM102754 strain has all of the above characteristics, it can be used as an antimicrobial agent against starter strains for producing high quality soybean products and against harmful microorganisms such as Bacillus cereus, Staphylococcus aureus and Enterococcus faecalis By confirming, the present invention has been completed.

In order to solve the above-mentioned problems, the present invention relates to a method for producing a microorganism having Bacillus subtilis , which has antimicrobial activity, antioxidative activity, extracellular enzyme secretory ability and phenol production ability against harmful microorganisms and which does not generate toxic substances and harmful enzymes, SRCM102754 strain (KCCM12198P).

In addition, the present invention provides a starter composition for the production of soy sauce, comprising the strain, a culture solution thereof, a concentrate of the culture solution or a dried product of the culture solution as an effective ingredient.

The present invention also provides an antimicrobial composition for a harmful microorganism containing the strain, a culture solution thereof, a concentrate of the culture solution or a dried product of the culture solution as an effective ingredient.

The Bacillus subtilis of the present invention, The SRCM102754 strain (KCCM12198P) has antimicrobial activity, antioxidant activity, extracellular enzyme secretion ability and phenol production ability against harmful microorganisms, and it is confirmed that it does not produce harmful substances and harmful enzymes. Thus, the Bacillus subtilis The SRCM102754 strain is considered to be a useful strain that can be used in a variety of industries including starter strains and antibiotic microbial preparations for the production of soy sauce.

FIG. 1 shows a flow diagram of the SRCM102754 strain isolated in the present invention.
Fig. 2 shows DPPH scavenging activity (%) of SRCM102754 strain isolated in the present invention.
Figure 3 shows the total phenol content of the SRCM102754 strain isolated in the present invention.

In order to accomplish the object of the present invention, the present invention provides a method for producing a microorganism having Bacillus subtilis , which has antimicrobial activity, antioxidative activity, extracellular enzyme secretory ability and phenol production ability against harmful microorganisms and which does not produce harmful substances and harmful enzymes, SRCM102754 strain (KCCM12198P).

In the present invention, a strain was isolated from a conventional soybean. Among them, antimicrobial activity, antioxidative activity, protease, cellulase and amylase releasing ability and phenol producing ability against Bacillus cereus, Staphylococcus aureus and Enterococcus faecalis were observed, The substance indole and phenylpyruvic acid, the β-glucuronidase which is a harmful enzyme, and the strain Bacillus subtilis which did not produce urease were identified, It was deposited on Jan. 11, 2018 (Accession No .: KCCM12198P) to the Korean Microorganism Conservation Center (KCCM) identified as SRCM102754.

In the strain according to an embodiment of the present invention, the extracellular enzymes are protease, cellulase and amylase, the harmful microorganisms are Bacillus cereus, Staphylococcus aureus and Enterococcus faecalis, and harmful substances include indole and phenyl Pyruvic acid, and the noxious enzyme may be? -Glucuronidase and urease, but is not limited thereto.

In addition, the present invention provides a starter composition for the production of soy sauce, comprising the strain, a culture solution thereof, a concentrate of the culture solution or a dried product of the culture solution as an effective ingredient.

In the present invention, a starter for the production of an intestinal product means a preparation or a composition containing microorganisms involved in fermentation for the production of intestines. And is used to provide a microorganism capable of growing in a fermented soybean or a microorganism capable of growing as a dominant species by adding at the time of producing a soybean milk. When the starter for the long-term fermentation is used, the microorganism contained in the starter for long-term fermentation may be used to control the quality of the starter constantly or to control the quality of the starter for a specific purpose, It is possible to achieve the purpose of lowering the taste or sweetness of the object or the soup.

The method for culturing the strain of the present invention can be carried out according to a method commonly used in the art, and is not limited to a specific method.

When the strain obtained in the step of culturing the strain of the present invention or the culture of the strain or the concentrated liquid of the culture medium of the strain is used as an additive, the culture of the strain or the strain or the concentrated liquid of the culture of the strain is directly added Other additives may be used together, and may be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the purpose of use thereof.

In addition, the present invention provides a method for producing soy sauce using the strain or a culture thereof as a starter strain.

The present invention also provides an antimicrobial composition for a harmful microorganism containing the strain, a culture solution thereof, a concentrate of the culture solution or a dried product of the culture solution as an effective ingredient.

The antimicrobial composition of the present invention may have the same meaning as an antibiotic, which is a generic term for an antimicrobial agent, and may have the same meaning as an antifungal agent, a bactericide, an antiseptic, a preservative or a bactericidal agent, and preferably Bacillus cereus , Staphylococcus ( Staphylococcus aureus) but are not limited to, substances capable of inhibiting or inhibiting the development and function of pathogenic microorganisms, particularly Gram-positive pathogenic microorganisms, including aureus and Enterococcus faecalis .

The antimicrobial composition of the present invention comprises Bacillus subtilis , Proteins, lipids, vitamins and minerals in addition to the SRCM102754 strain (KCCM12198P) strain, the culture solution thereof, the concentrate of the culture solution or the dried product of the culture solution. The saccharide may be selected from the group consisting of honey, dextrin, sucrose, palatinose, glucose, fructose, starch syrup, sugar alcohol and the like. The saccharide, protein, lipid, vitamins or minerals, , Sorbitol, xylitol and maltitol. The protein may be milk-derived proteins such as casein, whey protein, soybean protein, animal-derived enzymes such as trypsin and pepsin of these proteins, and neutrase The lipid may be a first saturated fatty acid, a sunflower oil containing a polyunsaturated fatty acid, a rapeseed oil, an olive oil, a safflower oil, a corn oil, Derived fat such as soybean oil, palm oil and palm oil, middle-chain fatty acid, EPA, DHA, soybean-derived phospholipid, milk-derived phospholipid, The RAL fluids may be potassium phosphate, potassium carbonate, potassium chloride, sodium chloride, calcium lactate, calcium gluconate, calcium pantothenate, casein calcium, magnesium chloride, ferrous sulfate, sodium bicarbonate, but are not particularly limited by each example .

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Isolation and culture

In order to isolate Bacillus strains, 1 g of the maltose sample was mixed well with 9 mL of sterile physiological saline, and 10 μl of each step was diluted. Then, 100 μl of the dilution was applied to Luria-Bertani agar (LB agar, BD Difco, Sparks, MD, USA) The culture was incubated at 30 ° C for 18 hours. The strain was firstly selected using the difference in the shape of the colonies formed, and then the strain was isolated by pure culture. The isolated microorganisms were suspended in a 10% skim milk (BD Difco, Sparks, MD, USA) solution and stored at -80 ° C for use in the next study.

Of the starting strain Extracellular  enzyme Secretory function  Measure

The agar well diffusion method was used to verify the activity of amylase, cellulase, and protease among the enzymes of the cells that released the isolated microorganism from the cells, and a solid selection including the components of the substrate to specifically react with each enzyme Medium was used. To measure the protease activity, skim milk was selected as a substrate and skim milk agar was prepared by adding 1.5% agar (Duchefa Biochemio, harrlem, Netherland) to 2% skim milk (BD Difco) . The activity of cellulase was determined by adding 1.5% agar (Duchefa Biochemio), 0.01% trypan blue (Sigma) to 1% CMC (Junsei chemical Co., -aldrich, St. Louis, Mo., USA) was added to prepare CMC agar. Starch hydrolyzate (amylase) was prepared by using soluble starch as a substrate and starch agar added with soluble starch (Junsei chemical) and 2% agar (Duchefa).

Measurement of antimicrobial activity against food harmful microorganisms

The antimicrobial activity of strains selected for harmful microorganisms was investigated in food. Bacillus cereus 0.8% soft LB agar medium was prepared using KCTC3624, Bacillus cereus KCCM 40935, Staphylococcus aureus KCCM 11593 and Enterococcus faecalis KCCM 11814 as indicator strains. To investigate the antimicrobial activity of the selected strains, strains were inoculated in LB broth (BD Difco) medium and cultured at 30 ° C. for 24 hours. The culture broth was centrifuged at 14,000 rpm for 10 minutes, and the supernatant was taken. A 0.45 μm syringe filter (Sartorius) and used as a supernatant sample. In accordance with the agar well diffusion method, 100 μL of the supernatant of the selected strain was dispensed into 6-mm wells and cultured at 30 ° C. for 24 hours. The size of the inhibitory circle was measured to determine antagonistic ability against the harmful microorganism of the selected strain Respectively.

Antioxidant Activity Analysis DPPH  free radical scavenging activity)

The antioxidative activity against the selective strains was analyzed by Kedare & Singh 's method. 20 μL of the culture supernatant was mixed with 180 μL of 0.1 mM 2,2-diphenyl-1-picrylhydrazide (DPPH, Sigma-aldrich) and reacted for 30 minutes. Then, the mixture was reacted with UV / VIS spectrophotometer (SPECOR200, analyticjena , jena, Germany), and the antioxidant activity was measured by substituting the following equation. The negative control was used for the culture medium and the measured values were measured after the same treatment as compared with the control group.

Antioxidant activity (%) = [1- (Abs _{517 nm of sample} ) / (Abs _{517 nm of control} )] 100

API ZYM  KIT, CHB50  kit Bacillus  Enzyme activity and sugar availability of strains

To investigate the enzyme activities of the strains, 18 enzymatic activities such as alkaline phosphatase were examined using API ZYM kit (bioMeriux Co., France). Also, the glycosylation activity of the isolated strains was confirmed using the API CHB50 kit.

Toxic gene analysis

Genomic DNA was extracted using the QIAamp DNA mini QIAcube kit (Qiagen, Hilden, Germany) for analysis of the Bacillus cereus toxic gene of the isolate. The extracted genomes were used to detect the presence of six toxic genes, hblC (hemolytic enterotoxin), cytK ( cytotoxin K), nheA ( nonhemolytic enterotoxin), CER (emetic toxin), bceT (enterotoxin T), and entFM DNA was used as a template and the gene was amplified by PCR using Bacillus cereus toxin 6-plex detection kit (KOGENEBIOTECH, Seoull, Korea), and the presence of the gene was confirmed on 1% agarose gel.

Hemolytic and Toxic metabolites and  Hazardous enzyme production analysis

Blood agar medium was prepared by adding 5% sheep blood (MBcell, SeouL, Korea) in order to confirm the hemolytic form of the selected strains. The strain was streaked and cultured at 37 ° C for 24 hours, The hemolytic form was confirmed in the form of the ring of the. It was also confirmed whether or not the harmful metabolites of indole, phenylpyruvic acid and urease and β-glucuronidase were produced. The strain was inoculated into TSB (BD, Difco) liquid culture medium and incubated at 37 ° C for 24 hours with shaking. An indoligent dropper (BBL, BD Difco) Confirmation of indole formation. To confirm the production of phenylpyruvic acid, strain was inoculated in a phenylalanine agar (fluka, Buchs, switzerland) medium, cultured at 37 ° C for 24 hours and 48 hours, and 3-4 drops of 10% MBcell was added to the strain The presence of phenylpyruvic acid was confirmed by the color change of the surroundings. To confirm the production of urease, a urea rapid test kit (MB cell) was inoculated with a starting strain and vaseline oil (MBcell) was added to the surface and incubated at 37 ° C for 4-24 hours in an oxygen-free state The urease production was confirmed by color change observation. To confirm β-glucuronidase activity, a selection strain was inoculated into tryptone-V-X glucuronide (TBX, Oxoid) agar medium and cultured at 37 ° C. for 24 hours. The change was observed to confirm the activity of [beta] -glucuronidase.

Analysis of Total Phenol Content in Culture Media

The selected strains were cultured overnight at 30 ° C in an LB agar medium and incubated overnight at 30 ° C and 150 rpm. Single colonies were inoculated into 5 ml of TSB broth (OXOID). 2 ml of the culture was taken in a 2 ml tube and then centrifuged at 13,000 rpm for 15 minutes. The supernatant was taken in a new 2 ml tube and gallic acid monohydrate (Sigma-Aldrich) was used as a standard solution. The total phenolic content of the TSB broth was determined using the total phenolic content of the functional food platform. 10 μl of the standard solution and the sample were put into a 96-well plate (SPL), and then 90 μl of distilled water and 10 μl of Folin ciocalteu reagent (Sigma-Aldrich) were added and incubated at room temperature for 5 minutes. Then 100 μl of 7% (w / v) sodium carbonate solution (Sigma-Aldrich) was added to each well and incubated for 20 minutes at room temperature. Absorbance was measured at 750 nm using a microplate reader (TECAN). The total phenol content was calculated from the calibration curve of gallic acid and the absorbance value was set to 0 by measuring the blank (TSB broth). The linear equation of the calibration curve is as follows.

y = 0.002x + 0.044, R ² = 1

y is the absorbance, x is the total phenol content (· ml ^-1 (ppm)),

Identification of selected strains and preparation of phylogenetic trees

The strains were inoculated into TSB broth and cultured at 37 ° C for 24 hours. The cells were recovered by centrifugation, and the ZR Fungal / Bacterial DNA Miniprep kit (Zymo Research Corp., CA., USA). The extracted DNA was synthesized with universal primer 785F (5'-GGATTAGATACCCTGGTA-3 ', SEQ ID NO: 2) and 907R (5'-CCGTCAATTCMTTTRAGTTT-3', SEQ ID NO: 3) to amplify the 16S rRNA gene fragment. Was purified using a QIAquick PCR Purification Kit (QIAGEN), and then subjected to nucleotide sequence analysis by Macrogen Co., Ltd. The nucleotide BLAST of the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) was used to compare the homology with the nucleotide sequence of GeneBank. The nucleotide sequences of the standard strains were obtained through the ExTaxone-e server ( http://www.eztaxon.org ), and the MEGA 7.0 program was used. Neighbor-joining algorithm was used for the tree diagram, and bootstrapping was performed over 1,000 iterations to confirm the robustness of the tree.

Example  1. Selection of strain

The doenjang samples prepared by the conventional method were diluted stepwise and cultivated in a LB agar (BD Difco) medium. The strain was firstly selected for its morphological characteristics, and the strain was secondly selected using a medium for selection of Bacillus cereus . Four isolates of Bacillus were selected by extracellular enzyme activity, antimicrobial activity and antioxidant activity.

Example  2. Identification of the strain

Based on the results of analysis of 16S rRNA gene sequence (SEQ ID NO: 1) of SRCM102754, BLAST was found to be Bacillus subtilis , and the homology with the standard strain was compared and analyzed on Eztaxon server. The parameters and option values used for the sequence alignment analysis were the Gonnet series as the protein weight matrix and the gap opening 15, the gap extension 6.66, the delay transition A gap transition is set to 0.5, a gap penalty is set to 5, a K-tuple size is set to 2, a top diagonals is set to 4, a window size is set to 0.5, Lt; / RTI >

As a result, SRCM102754 (1692 bp, SEQ ID NO: 1) showed 99% homology with Bacillus subtilisin, aquosome KCTC13429 (AMXN01000021). The inference of evolutionary distances to create a phylogenetic tree using nucleotide sequences was modeled using a maximum composite Likehood method. Finally, the selected strain was designated as Bacillus subtilis SRCM102754, and SRCM102754 was deposited at Korean Center for Microorganisms (KCCM) as deposit number KCCM12198P.

Example  3. Selection of the strain Extracellular  enzyme Secretory function  analysis

Amylase derived from microorganisms has been used continuously as a key enzyme for processing starch in the food industry for a long time in processing or improving the properties of foodstuffs (Natasa B et al., 2011, Biochem Eng J, Vol.53; pp.203-209). It is also known to be involved in the sweetness component of sugar. Proteases have been shown to degrade proteins during fermentation and thus to affect the content of free amino acids, a unique taste component (Kim et al., 2005, Kor. J. Life Science, 15, 749-754) It plays a role of decomposing cellulose which is a main component of the cell wall and improving intestinal availability. The cellulase components secreted by fungi and bacteria include endo-1, 4-β-D-glucanase, exo-1,4-β-glucanase and β-glucosidase, - It has been known that isoflavone glycosides are converted to aglycon by glucosidase and exhibit functions such as antioxidation (Ra, KS et al., 2004. J. Kor. Soc. Food Sci. Nutr. 33: 439-442 ).

The activity of four strains found in doenjang was compared and analyzed. The activity of SRCM102754 was higher than that of other strains of amylase, cellulase and protease (Table 1).

Determination of the extracellular enzyme secretion ability of the selected strain No. Strains No. Extracellular enzyme activity Amylase Protease Cellulase One SRCM102754 +++ ++++ +++ 2 SRCM102756 + ++++ +++ 3 SRCM102747 +++ ++++ ++ 4 SRCM102750 ++ ++++ -

+: <1 cm, ++:> 1 cm, +++:> 1.5 cm, ++++:> 2 cm, +++++:> 2.5 cm

SRCM102756: Bacillus subtilis

SRCM102747: Bacillus velezensis

SRCM102750: Bacillus subtilis

Example  4. Antimicrobial activity analysis of the selected strain against food harmful microorganisms

Bacillus cereus KCTC3624, Bacillus cereus KCCM40935, Staphylococcus aureus KCCM11593, and Enterococcus faecalis KCCM11814. As a result, the SRCM102754 strain had antimicrobial activity against various food harmful pathogenic microorganisms and also showed excellent inhibitory effect (Table 2) ).

Measurement of antimicrobial activity against food harmful microorganisms No Strains No. Antimicrobial activity B. cereus
KCTC 3624 B. cereus
KCCM 40935 S aureus
KCCM 11593 E. faecalis
KCCM 11814 One SRCM102754 ++ ++ ++ ++++ 2 SRCM102756 ++ ++ - ++++ 3 SRCM102747 ++ ++ ++ ++++ 4 SRCM102750 + + - +++

+: <1 cm, ++:> 1 cm, +++:> 1.5 cm, ++++:> 2 cm, +++++:> 2.5 cm

Example  5. Analysis of toxin gene of selected strain

Extracting the genomic DNA of the 4 strains Bacillus cereus The most known hemolytic that produce toxins (hemolytic enterotoxin, hblC), Saito toxin K (Cytotoxin K, CytK), cost septic enterotoxin (non-hemolytic enterotoxin, nheA) , emetic toxin (emetic toxin, CER), enterotoxin T (enterotoxin T, bceT) gene and enterotoxin FM (enterotoxin FM, entFM) results confirm the existence of selected strains selected strain via the PCR for the 6 genes (Table 3).

Bacillus cereus toxin gene analysis result of SRCM102754 No. Strains No. PowerChek ^™ Bacillus cereus Toxin 6-plex Detection Kit CytK nheA entFM bceT hblC CER One SRCM102754 ND ND ND ND ND ND 2 SRCM102756 ND ND ND ND ND ND 3 SRCM102747 ND ND ND ND ND ND 4 SRCM102750 ND ND ND ND ND ND

ND: Not detected

Example  6. Antioxidant activity analysis of selected strains

Antioxidants inhibit lipid oxidation in foods that donate electrons to free radicals to prevent rancidity and discoloration of fatty foods. Therefore, antioxidative activities of SRCM102754, SRCM102747, SRCM102750 and SRCM102756 were 20.29%, 9.93%, 7.64% and 11.54%, respectively, as measured by the DPPH method (FIG. 2).

Example  7. Analysis of Total Phenol Content of Selected Cultures

Among the many physiologically active substances contained in plant foods, phenol is the most abundant and is known to have a high sulfation activity (Park et al., 2007, Kor. J. FOOD CULTURE, 22 (3), 353- 358). Total phenol analysis of the culture supernatant showed that the SRCM102754 strain contained high phenol at 63.50 ppm, SRCM102747 at 52.78 ppm, SRCM102750 at 35.55 ppm and SRCM102756 at 25.25 ppm (FIG. 3).

Example  8. Selection of the strain Toxic metabolites  And harmful enzyme production

The SRCM1002754 strain was tested for hemolysin by dissolving red blood cells or hemolysing to discolor blood. As a result, the selected strain was gamma-hemolytic (gamma-hemolysin) for haemolysis by hemolytic streptococci or pneumococcal streptococci -hemolysis). In addition, it is known that the selective strain is a tumor-associated substance produced by decomposing tryptophan by tryptophanase, indole and phenylfibric acid, urease, which is an enzyme involved in carcinogenesis, and? -Glucuronidase (? glucuronidase). As a result, it was confirmed that the selected strain did not produce a carcinogen-related substance as shown in Table 4.

Bacillus subtilis SRCM102754 Hemolysis γ-hemolysis Glucosidase &lt; RTI ID = 0.0 &gt; ND Glucuronidase &lt; RTI ID = 0.0 &gt; ND Urease ND Indole ND Phenyl pyruvic acid ND

Example  9. Enzyme activity analysis of selected strains

As a result of the enzyme activity test, the strain SRCM102754 was found to have esterase activity (Table 5).

Bacillus subtilis SRCM102754 Control - alkaline phosphatase - esterase (C4) + esterase lipase (C8) - lipase (C14) - leucine arylamidase - valine arylamidase - cystine arylamidase - trypsin - alpha-chymotrypsin - asit phosphatase - naphthol-AS-BI-phosphohydrolase - alpha -galactosidase - β-galactosidase - β-glucuronidase - α-glucosidase - β-glucosidase - N-acetyl-β-glucosaminidase - alpha-nabbisudase - alpha-fucosidase -

Example  10. Glucose availability analysis of selected strains

The sugar availability of the SRCM102754 strain was analyzed using the API 50 CHB kit (Table 6). Glycerol, L-arabinose, D-ribose, D-xylose, D-glucose, D- fructose, D-mannose, Inositol, Mannitol, Sorbitol, Methyl-α-D-glucoside, N-acetyl-D D-glucosamine, Amygdalin, Arbutin, Esculin, Salicin, D-cellobiose, D-maltose, starch, Glycogen, Gentiobiose, D-turanose, and D-puerose, which are known to those skilled in the art. (D-fucose).

Glycosylation of the selected SRCM102754 strain Test Reaction Test Reaction Contorl - Esculin + Glycerol + Salicin ++ Erythritol - D-cellobiose ++ D-arabinose - D-maltose ++ L-arabinose ++ D-lactose - D-ribose + D-melibiose - D-xylose + Sucrose ++ L-Xylose - Trehalose + D-adonitol - Inulin + Methyl-D-xylopyranoside - D-melezitose - D-galactose - D-raffinose - D-glucose ++ Starch + D-fructose ++ Glycogen + D-mannose ++ Xylitol - L-sorbose - Gentiobiose + Rhamnose - D-turanose + Dulcitol - D-lyxose - Inositol + D-tagatose - Mannitol ++ D-fucose + Sorbitol ++ L-fucose - Methyl-alpha-D-glucopyranose - D-arabitol - Methyl-α-D-glucoside + L-arabitol - N-Acetyl-D-glucosamine + Glycogen - Amygdalin + 2-Ketogluconate - Arbutin + 5-Ketogluconate -

Korea Microorganism Conservation Center (overseas) KCCM12198P 20180111

<110> Microbial Institute for Fermentation Industry <120> Bacillus subtilis SRCM102754 strain having antimicrobial          activity, antioxidant activity, extracellular enzyme secretion          activity, phenol production activity and not producing harmful          metabolites and harmful enzymes and uses thereof <130> PN18020 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1692 <212> DNA <213> Bacillus subtilis SRCM102754 <400> 1 ctctgtcagg acgaacgctg gcggcgtgcc taatacatgc aagtcgagcg gacagatggg 60 agcttgctcc ctgatgttag cggcggacgg gtgagtaaca cgtgggtaac ctgcctgtaa 120 gactgggata actccgggaa accggggcta ataccggatg gttgtttgaa ccgcatggtt 180 caaacataaa aggtggcttc ggctaccact tacagatgga cccgcggcgc attagctagt 240 tggtgaggta acggctcacc aaggcaacga tgcgtagccg acctgagagg gtgatcggcc 300 acactgggac tgagacacgg cccagactcc tacgggaggc agcagtaggg aatcttccgc 360 aatggacgaa agtctgacgg agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa 420 gctctgttgt tagggaagaa caagtaccgt tcgaataggg cggtaccttg acggtaccta 480 accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt 540 tgtccggaat tattgggcgt aaagggctcg caggcggttt cttaagtctg atgtgaaagc 600 ccccggctca accggggagg gtcattggaa actggggaac ttgagtgcag aagaggagag 660 tggaattcca cgtgtagcgg tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg 720 cgactctctg gtctgtaact gacgctgagg agcgaaagcg tggggagcga acaggattag 780 ataccctggt agtccacgcc gtaaacgatg agtgctaagt gttagggggt ttccgcccct 840 tagtgctgca gctaacgcat taagcactcc gcctggggag tacggtcgca agactgaaac 900 tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac 960 gcgaagaacc ttaccaggtc ttgacatcct ctgacaatcc tagagatagg acgtcccctt 1020 cgggggcaga gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt 1080 taagtcccgc aacgagcgca acccttgatc ttagttgcca gcattcagtt gggcactcta 1140 aggtgactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca tcatgcccct 1200 tatgacctgg gctacacacg tgctacaatg gacagaacaa agggcagcga aaccgcgagg 1260 ttaagccaat cccacaaatc tgttctcagt tcggatcgca gtctgcaact cgactgcgtg 1320 aagctggaat cgctagtaat cgcggatcag catgccgcgg tgaatacgtt cccgggcctt 1380 gtacacaccg cccgtcacac cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt 1440 ttaggagcca gccgccgaag gtgggacaga tgattggggt gaagtcgtaa aagggtaacc 1500 aaaaaagccg gctccctgac actggggtcc agaataagct aaagtgtacg ggaagtagtc 1560 ttttatagaa ggtgcgttcg taaatagtag ataacttttg aggttacaca attagtaaga 1620 catttaacta aatggaagaa ggagatttgc gtgtttagga gacatgctat ggttgtatgt 1680 aacaagggtt at 1692 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggattagata ccctggta 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ccgtcaattc mtttragttt 20

Claims (4)

Antioxidant activity, protease, cellulase and amylase releasing ability and phenol producing ability against Bacillus cereus , Staphylococcus aureus and Enterococcus faecalis , Bacillus subtilis , which does not produce phenylpyruvic acid,? -Glucuronidase and urease, SRCM102754 strain (KCCM12198P). delete A starter composition for the production of soy sauce, comprising the strain of claim 1, a culture thereof, a concentrate of the culture solution, or a dried product of the culture solution as an effective ingredient. ( Bacillus cereus , Staphylococcus aureus , or Enterococcus faecalis ) containing the strain of claim 1, a culture thereof, a concentrate of the culture broth, or a dried product of the culture broth as an active ingredient, / RTI &gt;

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