KR101839372B1 - Bacillus subtilis SCGB 634 strain having acid-resistance activity, bile acid-resistance activity, antioxidant activity and antimicrobial activity and not producing biogenic amine and secreting protease, cellulase and amylase and uses thereof - Google Patents

Abstract

The present invention relates to a bacillus subtilis SCGB 634 strain separated from fermented foods and having activities of acid resistance, bile acid resistance, and antioxidation and antibacterial activities and secreting protease, cellulase, and amylase enzymes without producing a biogenic amine, and a purpose thereof. According to the bacillus subtilis SCGB 634 strain, an antibacterial or probiotic composition against harmful microorganisms containing the strain or a culture medium thereof as an effective component can be useful for food, food additives, feed additives, or medicines. More specifically, since the bacillus subtilis SCGB 634 strain of the present invention is able to suppress the proliferation of harmful microorganisms without secreting biogenic amines during a fermentation procedure of fermented foods, the strain is able to bring an effect of producing nontoxic and safe fermented foods, and thus, the strain is able to be industrially useful.

Description

Bacillus subtilis SCGB 634 strain which has acid resistance, biliary properties, antioxidative activity and antimicrobial activity and which secretes protease, cellulase and amylase enzyme without producing biogenic amine, and its use {Bacillus subtilis SCGB 634 strain having acid-resistance activity, antioxidant activity and antimicrobial activity and not producing biogenic amine and secreting protease, cellulase and amylase and uses thereof,

The present invention relates to a method for producing Bacillus subtilis (Bacillus subtilis), which is isolated from a sweetpoty and has antimicrobial activity against acid, bacteriostatic, antioxidant activity and harmful microorganisms and which produces proteases, cellulases and amylase enzymes Bacillus subtilis ) SCGB 634 strain and uses thereof.

Biogenic amines are nitrogen compounds that are produced mainly by chemical action such as decarboxylation of amino acid and amino group transfer, and are caused by microorganism or biochemical activity in protein containing food.

Representative biogenic amines include putrescine, histamine, tyramine, cadaverine, spermidine, and serotonin. Since biogenic amines act directly or indirectly as neurotransmitters in the body and also affect cardiovascular diseases such as blood pressure control and blood flow, various pharmacological phenomena may occur due to ingestion of biogenic amine-containing foods. Among these, ingestion of histamine and tyramine beyond the degradation limit of the human body causes symptoms such as rash, local skin inflammation, allergy, vomiting, nausea and diarrhea. In addition, blood vessel contraction and heart rate activity are increased by biorenic amines to cause elevation of blood pressure, dilation of pupil and eyelid tissue to promote secretion of tears, excessive salivation of saliva, increase of respiration and increase of blood sugar, And the like.

On the other hand, one of the main microorganisms involved in the fermentation of the intestine is the genus Bacillus . B. mycoides , B. thuringiensis , B. anthracis , or B. cereus belonging to the Bacillus subgroup 1 as a bacterium are harmful bacteria . In particular, Bacillus cereus can produce proteins that cause diarrhea or enteritis, such as haemolysin BL (HBL), non-hemolytic enterotoxin (NHE) or cytokinin K (cytK). It also produces certhrax, which causes respiratory diseases similar to anthrax in the form of peptides that cause vomiting and cereulide produced by the proliferation of food-contaminated Bacillus cereus.

Therefore, it is necessary to maintain the taste and flavor of traditional soy sauce, while securing the safety of food hygiene through control of fermentation microorganisms. For this purpose, it is essential to select the fermentation strains capable of inhibiting the growth of harmful strains during fermentation and producing biogenic amines at the same time.

Korean Patent No. 1516588 discloses " a strain of Bacillus sp. Having a biogenic amine-decomposing activity and its use ", but, as in the present invention, 'antibacterial activity against acid resistance, biliary properties, antioxidant activity and harmful microorganisms Bacillus subtilis SCGB 634, which secretes proteases, cellulases and amylase enzymes, and its use, without producing biogenic amines, has never been disclosed.

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 a method for producing a biodegradable microorganism having protease activity, cellulase activity, antioxidant activity, Bacillus subtilis SCGB 634, which secretes amylase enzyme, was isolated.

The Bacillus subtilis SCGB 634 strain of the present invention has antimicrobial activity against acid, bacteriostatic, antioxidant activity and harmful microorganisms, and is characterized by secreting protease, cellulase and amylase enzyme without producing biorenic amines The present inventors have completed the present invention by confirming that they are more effective than conventional strains for starter strains and probiotics for producing good quality strains.

In order to solve the above-mentioned problems, the present invention provides a bacterium which has acid resistance, biliary properties, antioxidative activity and antimicrobial activity against harmful microorganisms, and which does not produce a biogenic amine, Provides the Southernis SCGB 634 strain (KCCM 11904P).

In addition, the present invention provides a starter composition for the production of intestines containing the Bacillus subtilis SCGB 634 strain or a culture thereof as an active ingredient.

In addition, the present invention provides a method for controlling harmful microbes in a bacterium, comprising the step of treating the Bacillus subtilis SCGB 634 strain or a culture thereof with soy sauce.

The present invention also provides an antimicrobial composition for a harmful microorganism containing Bacillus subtilis SCGB 634 or a culture thereof as an active ingredient.

Also, the present invention provides a probiotic composition comprising the Bacillus subtilis SCGB 634 strain or a culture thereof as an active ingredient.

The Bacillus subtilis SCGB 634 strain of the present invention has antimicrobial activity against acid, bacteriostatic, antioxidant activity and harmful microorganisms, and it is possible to produce protease, cellulase and amylase enzyme without producing biogenic amine Therefore, the antibacterial and probiotic composition for harmful microorganisms containing the strain or the culture liquid thereof as an active ingredient can be very usefully used as a food, a food additive, a feed additive or a medicine. In particular, since the Bacillus subtilis SCGB 634 strain of the present invention can inhibit the growth of harmful microorganisms without secreting the biogenic amine during the fermentation process of the intestinal flour, the strain can be used to produce safe, So that it can be used industrially.

FIG. 1 shows the results of confirming whether or not the strains used in the present invention produce biogenic amines. SCGB 634 represents Bacillus subtilis SCGB 634. -, biogenic amine non-generation; +, Biogenic amine production; x, no reaction
Fig. 2 shows the phylogenetic tree based on the 16S rRNA nucleotide sequence of the SCGB 634 strain isolated in the present invention.
Fig. 3 shows the 16S rRNA nucleotide sequence (SEQ ID NO: 1) of the SCGB 634 strain isolated in the present invention.

In order to achieve the object of the present invention, the present invention provides a method for producing a biodegradable microorganism having protease, cellulase, and amylase enzyme, which has an acid resistance, biliary properties, antioxidative activity and antimicrobial activity against harmful microorganisms, Bacillus subtilis SCGB 634, which secretes < / RTI >

In the present invention, a strain was isolated from a soybean paste of Kochujang, and a strain of Bacillus subtilis , which has excellent antimicrobial activity against harmful microorganisms Bacillus cereus and Staphylococcus aureus , The strain was identified as Bacillus subtilis SCGB 634 and deposited on September 23, 2016 at the Korean Microorganism Conservation Center (KCCM) (Accession No .: KCCM11904P).

In a strain according to an embodiment of the present invention, the biogenic amine may be histamine or tyramine, but is not limited thereto.

By "antimicrobial" is meant the ability to reduce, prevent, inhibit, or eliminate microbial growth or survival at any concentration.

In a strain according to an embodiment of the present invention, the harmful microorganism may be, but is not limited to, Bacillus cereus or Staphylococcus aureus .

In addition, the present invention provides a starter composition for the production of intestines containing the Bacillus subtilis SCGB 634 strain or a culture thereof as an active 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 enhancing the taste or sweet taste which is obtained in the purpose or the soy sauce. In the present invention, the use of Bacillus subtilis SCGB 634 or a culture thereof as a starter strain does not include a biogenic amine, has an antimicrobial activity against a harmful microorganism, and further has an antioxidative activity and an antioxidative activity including a protease, a cellulase and an amylase Soy sauce can be produced.

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 controlling harmful microbes in a soybean comprising the step of treating the above-mentioned Bacillus subtilis SCGB 634 or a culture thereof with soy sauce. The control of the harmful microorganisms mentioned above means that the strain of Bacillus subtilis SCGB 634 of the present invention is treated to the intestines to delay or inhibit the growth of harmful microorganisms.

The method for culturing the strain of the present invention can be carried out according to a method commonly used in the art.

The present invention also provides an antimicrobial composition for a harmful microorganism containing the strain or a culture solution thereof as an active ingredient.

In the composition for antimicrobial, according to one embodiment of the invention, the microorganisms are Bacillus cereus (Bacillus cereus , or Staphylococcus aureus , but are not limited thereto.

In the antimicrobial 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 medicament, and the food includes dairy products (milk, soy milk, processed milk), fermented milk , Ice cream, soups, beverages, alcoholic beverages, and vitamins, and may be selected from the group consisting of, but not limited to, beverages, beverages, yogurts), drinks, meats, sausages, breads, chocolates, candies, snacks, confections, pizzas, It is not limited.

When the Bacillus subtilis SCGB 634 strain of the present invention is used as a food additive, the strain may be used as it is or may be used together with other food or food ingredients, and may be suitably 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 (prevention, health or therapeutic treatment). Generally, the strain of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, in the production of food or beverage. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range .

There is no particular limitation on the kind of the food. Preferably the food may be in the form of a Bacillus subtilis SCGB 634 strain and may also be used as an additive to other foods. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

The food of the present invention may contain a functional food. In the functional food of the present invention, in addition to the above-mentioned active ingredients, various auxiliary ingredients may be added as necessary. In the case of the functional food of the present invention, vitamins such as vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, folic acid, vitamin C, vitamin D3 and vitamin E, Iron, magnesium, potassium, zinc, etc., or lactic acid bacteria.

In addition, among the functional foods of the present invention, health drinks may contain various flavors or natural carbohydrates as additional components such as ordinary beverages. Examples of the flavoring agent include natural sweetening agents such as tau martin and stevia extract, and synthetic sweetening agents such as saccharine and aspartame. Examples of natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.

The feed additive of the present invention is added to the base feed at a certain ratio. The basic diet may be composed of corn, soybean meal, whey, fish meal, molasses, salt, vitamin premix, and mineral premix. The vitamin premix can be composed of vitamin A, vitamin D, vitamin E, riboflavin and niacin, and the mineral premix can be composed of manganese, iron, zinc, calcium, copper, cobalt and selenium.

The present invention also provides a pharmaceutical product comprising the strain of the present invention or a culture solution thereof as an active ingredient. Since the above medicines contain an antimicrobial substance, diseases caused by harmful bacteria can be prevented or treated. The strain may preferably be Bacillus subtilis SCGB 634 strain, but is not limited thereto.

The present invention also provides a probiotic composition comprising the Bacillus subtilis SCGB 634 strain of the present invention or a culture thereof as an active ingredient.

Since the Bacillus subtilis SCGB 634 of the present invention exhibits an excellent effect on acid resistance and biliary cholesterol, it satisfies all the conditions required as a probiotic agent, and thus can be effectively used as a probiotic composition have.

The composition of the present invention may be prepared according to a conventional method for producing a prod- uct composition, and may be in the form of a lyophilized or encapsulated culture or a suspension or a dry powder. In addition, one or two or more pharmaceutically acceptable conventional carriers or one or two or more additives may be selected as the active ingredient of the above-mentioned Bacillus subtilis SCGB 634 strain or a culture thereof, . ≪ / RTI >

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

Strain isolation and culture conditions

Kochujang samples, which were prepared by conventional methods, were collected from Sunchang County, Jeollabuk-do and used for the experiment. 1 g of collected kochujang was added to 9 mL of sterile physiological saline solution (0.85% NaCl) and diluted by decidual method. The sample was plated on LB (1% tryptone, 0.5% yeast extract and 0.5% sodium chloride) Time. To isolate pure Bacillus subtilis strains, a single colony was isolated and cultured in LB liquid medium at 30 ° C for 24 hours. The selected strains were mixed in LB liquid medium containing 20% glycerol and stored at 4 ° C. After storage, activation of the cells was activated by incubation in LB liquid medium at 30 ° C for 24 hours. The activity of the enzyme was measured by centrifugation at 12,000 rpm at 4 ° C for 30 minutes, and the supernatant was recovered and sterilized by 0.45 μm membrane filter (Sartorius, Frankfurt, Germany).

Biogenic  Amine Whether to generate  Confirmation (qualitative analysis)

Qualitative analysis of whether biogenic amines were produced was carried out as follows. One platinum of a pure Bacillus strain colony was plated on a biogenic amine production medium. The medium for confirming the generation of the biogenic amine was prepared by adding 1.5% agar, 0.25% glycerol, 0.006% bromocresol purple to LB medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, , 0.1% histidine or tyrosine, and cultured at 37 DEG C for 24 hours to confirm the color development. As a control, the same test was carried out on a medium not containing only amino acid in the medium, and then used as a control.

Biogenic  Amine Whether to generate  Confirmation (quantitative analysis)

Quantitative analysis of tyramine and histamine was analyzed by HPLC. For the analysis, tyramine, histamine, tyrosine, histidine dansyl chloride and 1,7-diaminoheptane were purchased from Sigma Aldrich, and proline, ether and hydrochloric acid were purchased from Samchon Chemical. Acetonitrile and water were purchased from JT Baker. The standard solution is dissolved in 0.1 N hydrochloric acid to give about 1 g / L And then diluted with two standard solutions at a concentration of 0.1 to 100 mg / L to prepare a calibration curve. In order to confirm the generation of the biogenic amine analysis, the selected strain was inoculated into LB liquid medium containing 200 ppm of tyrosine and histidine, which are precursors of histamine, and cultured at 30 ° C for 24 hours. Respectively. 0.5 mL of the culture solution and standard solution were added to each test tube, and then 0.25 mL of 1,7-diaminoheptane (0.1 g / L) was added. 0.25 ml of saturated sodium carbonate solution and 0.4 ml of 1% dansyl chloride acetone solution were added and mixed. The mixture was then plugged and derivatized at 45 ° C for 1 hour. After the derivatization, add 0.25 mL of 10% proline solution to remove excess dynechyl chloride, add 2.5 mL of ether to the test tube and shake for 3 minutes. Take the supernatant, evaporate it completely in a nitrogen concentrator, add 0.5 mL of acetonitrile And filtered through a 0.45 [mu] l filter and analyzed using HPLC. The analysis conditions are shown in the table below.

HPLC instrumental analysis conditions for biogenic amine, alcohol, sugar analysis HPLC analysis conditions for biogenic amine analysis Instrument Agilent 1200 series
(Agilent Technologies, Santa Clara, CA, USA) Column CapcellPak C18 Column Detector DAD detector (254 μm) Mobile phase A: 0.1% formic acid in H 2 O B: 0.1% formic acid in ACN Gradient condition A: B = 45:55, 0 to 10 min A: B = 35:65, 10-15 min A: B = 20: 80, 15 - 20 min A: B = 10: 90, 20 - 30 min A: B = 10: 90, 40 min over Flow rate 1.0 mL / min Temperature 40 ℃ Injection volume 20 μL

Extracellular  Activity measurement of enzyme (qualitative analysis)

In order to measure the activity of protein, fiber and starch hydrolyzate among extracellular enzymes, the solid plate medium containing substrate components to be specifically reacted with each enzyme was prepared. Each culture supernatant of the separated microorganisms was sterilized with a 0.45 μm membrane filter (Sartorius, Frankfurt, Germany), and 100 μl of each microorganism was inoculated into 8 mm of prepared wells. The secretion of protease was determined by inoculating a culture supernatant of the isolate with 1.5% agar (Junsei Chemical Co. Ltd., Tokyo, Japan) in 2% skim milk (Difco ^TM , MI, USA) The cellulase activity was determined by inoculating 1.5% agar in 1% CMC (carboxylmethyl cellulose, Junsei Chemical Co. Ltd., Tokyo, Japan) Stained with Congolad (Sigma-aldrich, MO, USA), washed with 1 M NaCl and observed for the size of the low support. The activity of starch hydrolyzate (amylase) was measured by using a starch as a substrate and adding the culture supernatant of each selected strain to a starch medium containing 1% soluble starch (Junsei chemical Co. Ltd., Tokyo, Japan) After sterilization in the same manner as described above and dispensing into 100 μl of each well, the cells were reacted at 25 ° C for 24 hours, stained with Lugol solution (Sigma-aldrich, MO, USA) Respectively.

Identification of antimicrobial activity against harmful pathogenic microorganisms related to food poisoning

The antimicrobial activity against Bacillus cereus and Staphylococcus aureus , which causes food poisoning by decaying or contamination of food, was measured. Bacillus cereus KCTC3624 was purchased from KCTC (Korea Research Institute of Bioscience and Biotechnology), Bacillus cereus KCCM 40935 and Staphylococcus aureus KCCM 11593 were purchased from Korea Microorganism Conservation Center (KCCM) Respectively. The culture medium (3.0 × 10 ⁵ ) of each indicator strain was added to soft medium containing 0.8% agar (NB) soft medium to prepare a plate culture medium. The culture supernatant of the selected strain 100 μl of the culture supernatant was dispensed into the wells, and the culture supernatant was allowed to diffuse in the test plate medium. The culture was incubated for 18 hours at the optimum growth temperature of the indicated strain, and the size of the inhibitory circle was measured.

Antioxidant activity

DPPH (2,2-diphenyl-1-picryl-hydrazyl) method was used to measure the electron donating ability of the selected strains. Add 200 μL of culture supernatant to 1 mL of 100 μM DPPH ethanol solution. After incubation for 30 minutes at room temperature, the absorbance was measured at 528 nm with UV / VIS spectrophotometer (SPECORD 200, Analytic jeca) The degree of antioxidant activity was measured according to the formula.

Antioxidant activity (%) = [1- (Abs _{528 nm of sample} ) / (Abs _{528 nm of control} )] 100

Identification of the final selection strains

For identification of the final selection strains, the cells were recovered from agar plate medium and their DNA was extracted using ZR Fungal / Bacterial DNA MiniPrep kit. The 16S rRNA gene was amplified using two known universal primers. The nucleotide sequences of the two universal primers are as follows. First, the forward primer is (27F) 5'-AGAGTTTGATCMTGGCTCAG-3 '(SEQ ID NO: 2), and the reverse primer (1492R) 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO: 3). The conditions of each reaction for PCR are as follows. DNA denaturation was performed at 95 ° C for 1 minute, annealing at 60 ° C for 1 minute, and DNA strand synthesis at 72 ° C for 1 minute or 2 minutes. The PCR product was reacted with 1% agarose gel 5 μl, X-gal 30 mg / ml). After confirming the bands, the amplified PCR products were identified by Cosmojin Tech Co., Ltd.

Of the final selection strains Bacillus Cereus ( Bacillus cereus ) Safety analysis through toxin gene analysis

In order to confirm the safety of the final selection strains in food, PCR method was used to confirm the presence of 6 diarrhea toxin and vomit - type toxin genes produced by Bacillus cereus. PCR (Veriti 96-well Thermal Cycler, Life technologies) was investigated using Koza Biotech's PowerChek ^™ Bacillus cereus Toxin 60 plex Detection kit. Six toxin genes including CER, hblC, bceT, entFM, nheA and cytK The presence of genes was investigated. The PCR reaction conditions were 95 ° C for 10 minutes, 35 cycles (95 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds), and 72 Post-extension for 10 min. The amplified PCR product was electrophoresed on a 1.0% agarose gel containing 0.5 μg / mL SYBR green in 0.5% TAE buffer, and the band was confirmed on a UV transilluminator (Gel documentation, Bio-Rad) .

Acid resistance  And My bile  analysis

To investigate the effect of pH and bile salt tolerance, the strain was cultured in LB liquid medium at 37 ℃ for 18 hours. For acid resistance analysis, the culture broth was adjusted to pH 2.0 and 7.0 with HCl and NaOH for 18 hours at 37 ° C in LB liquid medium, followed by incubation at 37 ° C for 3 hours with shaking. The culture was serially diluted with 0.85% sterile physiological saline, and 100 쨉 l was plated on LB solid medium and cultured at 37 째 C for 18 hours to measure viable cell count. The survival rate was checked by using the number of viable cells before the pH control and the degree of acid resistance was confirmed. Bacilli were added to the culture medium at 37 ° C for 18 hours at 0, 0.3, 0.6% in LB liquid medium, and incubated at 37 ° C for 3 hours with shaking. The culture was continuously diluted with 0.85% sterile physiological saline, And cultured at 37 DEG C for 18 hours to measure viable cell count. The survival rate of the control group was checked by the number of viable cells before the addition of bile.

Example  1. Isolation of microorganisms from traditional kochujang

In order to select bacillus with various enzyme activity, antimicrobial activity and antioxidant activity, 104 kinds of kochujang prepared by traditional methods in Sunchang - gun, North Jeolla Province were collected and used in the experiment. A total of 786 isolates were obtained from kochujang, and the isolates were stored at -20 ℃ for subsequent study.

Example  2. Biogenic  Amine Whether to generate  Confirmation (qualitative analysis)

Genetic analysis of biogenic amines was carried out to isolate isolates. As a result, 322 isolates from 786 isolated isolates did not produce biogenic amines, and 322 isolates were studied.

Example  3. Biogenic  Amine Whether to generate  Confirmation (quantitative analysis)

Studies have reported that bioenic amines can cause symptoms such as nausea, vomiting, hypertension, and headache in the presence of biogenic amines in food. The distribution of biogenic amines in kochujang, which is the main food of Koreans, showed that histamine was 56.1 mg / kg (average 569.4 mg / kg) and tiramycin was 284.4-1430 mg / kg (mean 669.5 mg / kg) The average of food was high. Therefore, we investigated the production of biogenic amines from strains isolated from the genus, and conducted a survey aimed at isolating strains that can be used for food using the isolated strains.

Therefore, quantitative analysis of isolates selected on the basis of the above-mentioned qualitative analysis confirmed the same results as in qualitative analysis in most isolates. In some isolates, tyramine or histamine However, the following experiment was carried out without showing the numerical value of quantification.

Analysis of Biogenic Amine Production of Selected Strains Strain No. Histamine Tyramine SCGB 634 ND ND SCGB 635 ND ND SCGB 636 ND ND SCGB 637 ND ND SCGB 638 ND 19.24 ppm SCGB 639 ND ND

ND: Not detected (not detected)

Example  4. Extracellular  Enzyme activity measurement

Enzyme activity was measured in protease, cellulase, and amylase for the assay of various organic compounds. Each of the skim milk, carboxymethyl cellulose (CMC), and starch media was sterilized, and the culture broth after each medium was completely solidified was cultured using a well diffusion method. The cleavage of skim milk, CMC and starch was investigated at a clear zone diameter after activating at 25 ℃ for 12 hours.

As shown in Table 3 below, the SCGB 634 strain was found to have enzyme activity in protein, starch, and fibrinolytic enzyme according to the qualitative analysis method. Especially, Respectively.

Extracellular enzyme activity assay (protease, cellulase, amylase) Strain No. Protease Cellulase Amylase SCGB 634 16 17 11 SCGB 635 15 w - SCGB 636 - 11 -

Unit: mm, diameter

w: Weak

Example  5. Antimicrobial activity against harmful pathogenic microorganisms related to food poisoning

The isolates were selected by measuring the antimicrobial activity against pathogenic microorganisms related to food poisoning using well diffusion method. As a result, SCGB 634 showed high activity against three kinds of indicator strains and showed the highest level among the isolated strains. Therefore, it was confirmed that the most effective isolate for inhibition of food poisoning harmful microorganisms was SCGB 634.

The antimicrobial activity of isolate isolated from kochujang No. Strains No. Antibacterial activity (diameter, mm) Bacillus cereus KCTC 3624 Bacillus cereus
KCCM 40935 Staphylococcus aureus
KCCM 11593 One SCGB 634 16 16 12 2 SCGB 635 - - - 3 SCGB 637 10 10 10 4 SCGB 638 10 10 10

-, no response

Example  6. Antioxidant activity

Among the isolated strains isolated from the conventional kochujang of the present invention, the antioxidative effect of the isolate SCGB 634, which is excellent in biogenic amine production and antimicrobial activity, was measured by measuring the free radical scavenging effect by DPPH. The antioxidative effect of DPPH on the selective radical scavenging activity was 14.96 ± 0.41%.

Antioxidative activity of DPPH isolated from kochujang No. Strains No. Identification DPPH (%) One SCGB 634 Bacillus subtilis 14.96 + - 0.41 2 SCGB 635 Unidentified 12.15 ± 0.27 3 SCGB 637 Unidentified 9.87 + - 0.31

Example  7. Final selection Separator SCGB  Identification of 634

SCS 634 was identified as Bacillus subtilis based on the results of the BLAST search. Based on the results, the sequence of the 16S rRNA gene was identified as the standard of Eztaxon serve As a result of analysis of homology with the strain, SCGB 634 was found to be highly similar to Bacillus subtilis DSM 10, and a phylogenetic tree was constructed based on the 16S rRNA nucleotide sequence. Finally, it was named Bacillus subtilis SCGB 634 and deposited with Bacillus subtilis KCCM 11904P in the Korean Culture Center of Microorganisms (KCCM).

Example  8. Bacillus Cereus ( Bacillus cereus ) Safety analysis through toxin gene analysis

SCGB 634, identified as a GRAS strain, was selected as a functional strain to be used as a final food product by evaluation of strain characteristics and functional efficacy of isolates from Kochujang and identification of the strain and identification of strain. Finally, And to ensure safety. Using the PowerChek ^™ Bacillus cereus Toxin 6-plex Detection kit on the Bacillus cereus toxin gene for SCGB 634, no genes were found to produce six toxins, indicating that it is safe for food poisoning, And can be used for various applications such as the above.

Genetic analysis of Bacillus cereus toxin of SCGB 634 Strain No. Identification
(16s rRNA seq ) PowerChek ^TM Bacillus cereus  Toxin 6- plex  Detection Kit CytK nheA ent  FM bceT hblC CER SCGB 634 Bacillus subtilis ND ND ND ND ND ND

ND: Not detected (not detected)

Example  9. Acid resistance  And My bile  Confirm

As a result of confirmation experiment of in vitro probiotics, it has been widely used for acid tolerance and bile test because it has a stable gastric juice and tolerance to gastric juice and bile to reach the intestines. In order to confirm the acidity of the selected strains, the viability of selected strains was compared with the number of viable cells before pH control by adjusting the pH to 7.0 and pH 2.0 after culturing the selected strains. As a result, the SCGB 634 strain It was confirmed that about 1.89% of the strains were able to survive for 3 hours under the condition of pH 2.0. Since bile acid affects the cell membrane of microorganisms and the microorganisms die, the survival rate of the selected strain was checked by adding 0.3, 0.6% bile after the selection culture to identify the biliary properties. The survival rate of SCGB 634 was 0.3% after 3 hours of bile supplementation and 85% of survival rate.

Separation of acidity and biliary properties No. Strains No. Survival rate (%) pH 7.0 pH 2.0 Oxgall 0% Oxgall 0.3% Oxgall 0.6% One SCGB 1 100 0 100 46 16 2 SCGB 634 100 1.89 100 85 37 3 SCGB 574 100 1.56 100 9 4

Korea Microorganism Conservation Center (overseas) KCCM11904P 20160923

<110> Microbial Institute for Fermentation Industry <120> Bacillus subtilis SCGB 634 strain having acid-resistance          activity, even acid-resistance activity, antioxidant activity and          antimicrobial activity and not producing biogenic amine and          secreting protease, cellulase and amylase and uses thereof <130> PN16421 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1487 <212> DNA <213> Bacillus subtilis <400> 1 cgtcaggacg aacgctggcg gcgtgcctaa tacatgcaag tcgagcggac agatgggagc 60 ttgctccctg atgttagcgg cggacgggtg agtaacacgt gggtaacctg cctgtaagac 120 tgggataact ccgggaaacc ggggctaata ccggatggtt gtttgaaccg catggttcaa 180 acataaaagg tggcttcggc taccacttac agatggaccc gcggcgcatt agctagttgg 240 tgaggtaacg gctcaccaag gcaacgatgc gtagccgacc tgagagggtg atcggccaca 300 ctgggactga gacacggccc agactcctac gggaggcagc agtagggaat cttccgcaat 360 ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa ggttttcgga tcgtaaagct 420 ctgttgttag ggaagaacaa gtaccgttcg aatagggcgg taccttgacg gtacctaacc 480 agaaagccac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt 540 ccggaattat tgggcgtaaa gggctcgcag gcggtttctt aagtctgatg tgaaagcccc 600 cggctcaacc ggggagggtc attggaaact ggggaacttg agtgcagaag aggagagtgg 660 aattccacgt gtagcggtga aatgcgtaga gatgtggagg aacaccagtg gcgaaggcga 720 ctctctggtc tgtaactgac gctgaggagc gaaagcgtgg ggagcgaaca ggattagata 780 ccctggtagt ccacgccgta aacgatgagt gctaagtgtt agggggtttc cgccccttag 840 tgctgcagct aacgcattaa gcactccgcc tggggagtac ggtcgcaaga ctgaaactca 900 aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960 aagaacctta ccaggtcttg acatcctctg acaatcctag agataggacg tccccttcgg 1020 gggcagagtg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa 1080 gtcccgcaac gagcgcaacc cttgatctta gttgccagca ttcagttggg cactctaagg 1140 tgactgccgg tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat 1200 gacctgggct acacacgtgc tacaatggac agaacaaagg gcagcgaaac cgcgaggtta 1260 agccaatccc acaaatctgt tctcagttcg gatcgcagtc tgcaactcga ctgcgtgaag 1320 ctggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc gggccttgta 1380 cacaccgccc gtcacaccac gagagtttgt aacacccgaa gtcggtgagg taacctttta 1440 ggagccagcc gccgaaggtg ggacagatga ttggggtgaa gtctaca 1487 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 agagtttgat cmtggctcag 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tacggytacc ttgttacgac tt 22

Claims (5)

Antioxidant activity and antimicrobial activity against Bacillus cereus and Staphylococcus aureus in acid-resistant, 0.3-0.6% bacterium , pH 2 to 7, isolated from confectioners, Bacillus subtilis SCGB 634 strain (KCCM 11904P) which secretes proteases, cellulases and amylase enzymes without producing tyramine and histamine. A starter composition for the production of intestines containing the Bacillus subtilis strain SCGB 634 of claim 1 or a culture thereof as an active ingredient. A method for controlling Bacillus cereus or Staphylococcus aureus comprising the step of treating the Bacillus subtilis SCGB 634 strain of claim 1 or a culture thereof with soy sauce. A composition for antibacterial activity against Bacillus cereus or Staphylococcus aureus containing the Bacillus subtilis SCGB 634 strain of claim 1 or a culture thereof as an active ingredient. A probiotic composition comprising the Bacillus subtilis SCGB 634 strain of claim 1 or a culture thereof as an active ingredient.

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