KR101734363B1 - Bacillus subtilis SCM688 strain from traditional soy sauce having antimicrobial activity against pathogenic microorganism of soy sauce and enzyme secretion activity, and not producing biogenic amine and uses thereof - Google Patents

Abstract

The present invention relates to a bacterium belonging to the genus Bacillus ( Bacillus subtilis), which is excellent in antimicrobial activity against a harmful microorganism and an extracellular enzyme-releasing ability and does not produce a biogenic amine, subtilis ) The SCM688 strain (KACC81006BP), the Bacillus subtilis A microorganism preparation for antimicrobial use against a harmful microorganism of a genus including SCM688 strain or a culture thereof as an active ingredient, A food containing SCM688 strain, a food containing the Bacillus subtilis SCM688 strain or a culture thereof to a soy sauce, and a method for controlling the soy sauce harmful microorganism.

Description

The present invention relates to a Bacillus subtilis SCM688 strain derived from a traditional herb that has an antimicrobial activity against a harmful microorganism and an ability to secrete an extracellular enzyme and does not produce a biogenic amine, and a use thereof Bacillus subtilis SCM688 strain from a traditional soy sauce having antimicrobial activity against pathogenic microorganisms of soy sauce and enzyme secretion activity, and not producing biogenic amines and uses thereof.

The present invention relates to a Bacillus subtilis SCM688 strain derived from a traditional herb that has an antimicrobial activity against a harmful microorganism and an ability to secrete an extracellular enzyme and does not produce a biogenic amine. More particularly, of the antimicrobial activity and extracellular enzyme secretion is not excellent and to produce a biogenic amine (biogenic amine) traditional soy sauce-derived Bacillus subtilis standing (Bacillus subtilis ) The SCM688 strain (KACC81006BP), the Bacillus subtilis A microorganism preparation for antimicrobial use against a harmful microorganism of a genus including SCM688 strain or a culture thereof as an active ingredient, A food containing SCM688 strain, a food containing the Bacillus subtilis SCM688 strain or a culture thereof to a soy sauce, and a method for controlling the soy sauce harmful microorganism.

The fermentation process of traditional soy sauce is mainly composed of a complex metabolic process of Bacillus, Aspergillus, lactic acid bacteria and yeast. The production of soybean paste is a process of hydrolyzing soybean with high protein content into bacillus and aspergillus which have high protease activity, and fermentation is carried out by appropriate propagation temperature and interaction of various microorganisms. Therefore, biogenic amines It is a suitable condition to be produced.

In fact, the distribution of biogenic amines in domestic fermented foods in 2006 showed that the content of putrescine in traditional doenjang was 99.6 ~ 1453.7 (mean 462.6) mg / kg, histamine 260.1 ~ 952 (mean 569.4) mg / kg, (Average 669.5) mg / kg, which was the highest among the 34 kinds of foods that Koreans mainly eat, followed by anchovy salted fish, modern soy sauce, conventional soy sauce, and modern miso. Considering that 4 of the 7 foods with the highest bioigenic amine content are from Korea, and that the use of soybean paste is higher than that of salted fish due to the nature of the diet, Koreans can consume biogenic amines even to the detriment of health through eating .

Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens), which has amino acid dicarboxylase activity and can produce biogenic amines, amyloliquefaciens), Bacillus subtilis books (Bacillus subtilis and Bacillus licheniformis were identified in Chungkookjang. The histamine decarboxylase gene was deleted, but two species of Bacillus licheniformis containing the tyrosine decarboxylase gene Strains were isolated from Cheonggukjang, which has low content of biogenic amines.

Biogenic amines have been degraded by monoamine oxidase and polyamine oxidase and so far these enzyme activities have been known to be caused by micrococcus, Natrinema, Brevibacterium, Lactobacillus, Staphylococcus, Arthrobacter, Klebsiella, Pseudomonas, Serratia, Salmonella (Naila et < RTI ID = 0.0 > al., 2010 J. Food Sci. 75, 139-150). However, the ability of biosynthetic amine decomposition in Bacillus, which plays a key role in the fermentation of Korean traditional rice cultivars, was not known except for Bacillus licheniformis. In addition, Bacillus licheniformis is not allowed to be used as a food additive in Korea Food and Drug Administration (KFDA) so it can not be applied to actual products. Therefore, we investigated the biogenic amine degradation activity of Bacillus subtilis strains which can be tolerated in the KFDA as GRAS (generally recognized as safe) strains. In the future, traditional pastes should maintain the flavor of traditional soy sauce, but also be able to secure food safety through reduction of biogenic amines. In consideration of this point, the present invention relates to a method for isolating a toxin gene-containing strain, which is capable of decomposing a biogenic amine, which is a harmful substance, mainly Bacillus subtilis and Bacillus amyloliquefaciens, The goal was to separate.

Korean Patent No. 1516588 discloses a strain of Bacillus sp. Having a biogenic amine-decomposing activity and its use. However, as in the present invention, there is an antimicrobial activity and an extracellular enzyme secretory activity against harmful microorganisms , A strain of Bacillus subtilis SCM688 derived from a traditional herb that does not produce a biogenic amine and its use 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 an antimicrobial activity against harmful microorganisms such as Bacillus cereus and Staphylococcus aureus, and to provide a protease, amylase, cellulase and lipase the new Bacillus of excellence, and does not produce biogenic amines derived from traditional sauces standing subtilis (Bacillus subtilis ) The strain SCM688 (KACC81006BP) was isolated. The inventors of the present invention have completed the present invention by confirming that the strain isolated in the present invention can be effectively used as a microorganism which can effectively control the harmful microorganisms occurring in the fermentation process of the traditional microorganisms.

In order to solve the above problems, the present invention other antibacterial activity on microorganisms and cells, enzyme secretion is excellent, biogenic amines (biogenic amine) that does not produce a traditional sauces standing Bacillus subtilis (Bacillus derived subtilis ) SCM688 strain (KACC81006BP).

In addition, the present invention relates to the above- SCM688 strain or a culture solution thereof as an active ingredient.

In addition, the present invention relates to the above- SCM688. ≪ / RTI >

In addition, the present invention relates to the above- The method comprising the step of treating the strain SCM688 or a culture thereof with soybean curd.

In the present invention, Bacillus subtilis ( Bacillus subtilis) subtilis ) It was confirmed that the SCM688 strain inhibited the proliferation of harmful microorganisms occurring during the fermentation process of the soybean, secreted extracellular enzymes of protease, amylase, cellulase and lipase, and did not produce biogenic amine. The Bacillus subtilis SCM688 strains can be used safely in food industry because they can produce safe and safe food without toxicity.

Figure 1 shows the 16S rRNA nucleotide sequence of the SCM688 isolate according to the present invention.
FIG. 2 shows the results of qualitative analysis of biogenic amines against 5 strains among the selected strains. -, do not produce biogenic amines; +, Biogenic amine production
FIG. 3 shows the results of assaying extracellular enzymatic activity of SCM688 isolate according to the present invention. (From the left, cellulase, protease, amylase, lipase)
FIG. 4 shows the result of confirming the growth curve of SCM688 separation according to the present invention. Bacillus subtilis inoculated on TSB medium and cultured at 30 rpm at 150 rpm Cell dry cell mass and absorbance of SCM688 strain were shown by incubation time. The experiment was repeated 3 times and the data were averaged (n = 3) and expressed as mean ± standard deviation.

In order to accomplish the object of the present invention, the present invention provides a method for producing a microorganism which is excellent in antimicrobial activity against an harmful microorganism and an extracellular enzyme-releasing ability, and which is produced from a Bacillus subtilis ( Bacillus subtilis) subtilis ) SCM688 strain (KACC81006BP).

The Bacillus subtilis subtilis ) The SCM688 strain (KACC81006BP) was isolated from a traditional herb, has antagonistic ability against harmful microorganisms, has excellent extracellular enzyme secretion ability, and does not produce a biogenic amine. The Bacillus subtilis subtilis ) The strain SCM688 was deposited on Jul. 28, 2015 (Accession No .: KACC 81006BP) at the National Institute of Agricultural Science and Technology.

In the strain in accordance with an embodiment of the invention, the microorganisms are Bacillus cereus (Bacillus cereus , or Staphylococcus aureus , but are not limited thereto.

In a strain according to an embodiment of the present invention, the extracellular enzyme may be, but not limited to, protease, amylase, cellulase or lipase.

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

The biogenic amine of the present invention is formed from an amino acid by the action of an amino acid decarboxylase of a microorganism. When a biogenic amine is ingested from a food in excess of the decomposition limit of the human body, the biogenic amine may cause rash, local skin inflammation, Vomiting, nausea and diarrhea.

The present invention also relates to the use of the above Bacillus subtilis subtilis ) SCM688 strain or a culture solution thereof as an active ingredient.

In the microorganism preparation according to the embodiment of the present invention, the microorganisms are Bacillus cereus (Bacillus cereus , or Staphylococcus aureus , but are not limited thereto.

The microorganism preparation has antimicrobial activity against harmful microorganisms isolated from the genus and bacillus subtilis ( Bacillus subtilis, subtilis ) SCM688 strain as an active ingredient. The microbial formulation according to the present invention may be prepared in the form of a liquid, and may be added in the form of a powdered powder or may be granulated by formulating it into a powder. However, the formulation is not particularly limited.

The invention also standing Bacillus subtilis (Bacillus of the present invention subtilis ) And culturing the SCM688 strain. The present invention also provides a method for producing an antimicrobial microorganism preparation against a harmful microorganism. The Bacillus subtilis SCM688 strain of the present invention can be cultured by a conventional method known in the art.

The present invention also relates to the use of the above Bacillus subtilis subtilis ) SCM688. ≪ / RTI >

The food may be selected from the group consisting of Bacillus subtilis subtilis ) SCM688 strain as a seed culture, and more particularly, may be a soup, but is not limited thereto. In the food according to one embodiment of the present invention, the soup is selected from the group consisting of meju, Korean miso, miso, seasoned miso, kochujang, seasoned kochujang, chunjang, chungkukjang, mixed soup, Korean soy sauce, brewed soy sauce and mixed soy sauce But it is not limited thereto.

The invention also standing Bacillus subtilis (Bacillus of the present invention subtilis ) The method comprising the step of treating the strain SCM688 or a culture thereof with soybean curd.

The control of the harmful microbes of the present invention can be carried out using the Bacillus subtilis By treating the SCM688 strain with the intestinal flora, it means delaying or inhibiting the growth of the harmful microorganism.

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

A total of about 200 samples of traditional meju, doenjang, and kochujang samples were collected from 11 surface units of Sunchang County, Jeollabuk-do, and 1 g of the collected samples were suspended in 9 ml of sterilized 0.85% NaCl solution. 100 희 of dilution was plated on Luria- bertani agar (LB, Difco ^TM ) medium and cultured at 30 캜 for 24 hours. In order to isolate the pure strains, the strains were firstly selected using the morphological difference of the cultured microorganisms, and then the strains were separated by pure culture. The isolated microorganisms were mixed with LB liquid medium containing 20% glycerol for use in the next study and stored at -80 ° C.

Selective strain Extracellular  enzyme Secretory function  Measure

The agar well diffusion method was used to verify the activity of protease and cellulase among extracellular enzymes that released the isolated microorganism out of the cells, and a solid sorting medium containing the components to be specifically reacted with each enzyme Amylase secretion activity was determined by using a starch agar medium containing 1% soluble starch (JUNSEI CHEMICAL Co., Ltd., Tokyo, Japan) using starch as a substrate, measuring the lipase hydrolysis activity using Spirit Blue Agar Lipase plate (Difco ^TM ) was prepared and used. The protease secretion ability was obtained by preparing a skim milk culture medium in which skim milk was selected as a substrate and 1.5% agar was added to 2% of Difco ^™ (Vermelho et al ., 1996, Mem. Inst. Oswaldo Cruz, 91, 755-760), and the cell-releasing ability was determined by using carboxylmethyl cellulose (CMC, JUNSEI chemical Co. Ltd.) as a substrate and culturing in CMC agar medium containing 1% (Teather and Wood, 1982, Appl. Environ. Microbiol, 43, 777-780). The culture supernatant of each strain was sterilized with a 0.45-μm membrane filter (Sartorius), and 100 μl of each supernatant was dispensed into a well of a diameter of 8 mm and reacted at 25 ° C. for 24 hours. Then, the protease and The cellulase secretion ability was examined with the diameter of the transparent ring.

Antimicrobial activity measurement

The antimicrobial activity of the strains selected for food harmful microorganisms was examined. Food and harmful microorganisms are Bacillus cereus (Bacillus cereus , KCTC 3624), Staphylococcus ( Staphylococcus aureus) aureus subsp. aureus, KCCM 11593, KCCM 41331) , Bacillus cereus (Bacillus cereus , and KCCM 40935). The antimicrobial activity of harmful microorganisms was investigated in accordance with an agar diffusion method using 0.8% soft agar plates containing harmful microorganisms (Park et al ., 2002, Kor. J. Life science, 12, 200-207) 100 [mu] l of the culture supernatant of the isolated strain isolated with membrane filter (Sartorius) was dispensed into wells of each antimicrobial activity measuring medium, and cultured in a 30 [deg.] C incubator for 24 hours to measure the antimicrobial activity according to the size of the inhibitory loop.

Biogenic  Amine ( Biogenic amine ) Confirm creation

The primary selection was carried out through the qualitative experiment to determine whether the biogenic amines were produced from the isolated microorganisms. Qualitative experiments to confirm the generation of biogenic amines were conducted by Chang and Chang (Chang et al ., 2012, Int. J. Food Microbiol, 150, 269-274). First, the solid medium for the detection of the biogenic amines was dissolved in 0.25% glycerol (JUNSEI Chemical Co. Ltd., Tokyo, Japan), 0.006% Bromocresol Purple (BCP, Sigma-aldrich, MO, USA), 0.1% histidine and tyrosine (aldrich, MO, USA) was prepared and used. The cells were washed three times with sterilized distilled water and 10 μl each was dispensed on an 8 mm ADVANTEC paper disk (Toyo Roshi Kaisha Ltd., Tokyo, Japan). After incubation at 37 ° C. for 24 hours, the cells were washed with normal LB medium The generation of the biogenic amine was confirmed. In addition, the biosynthetic amines were inoculated into the LB broth and inoculated at 30 ° C in a suspension incubator (VS-1203P3V, Vision Scientific Co. Ltd., Daejeon, Korea) at 150 rpm for 24 hours After pre-culturing, 1 ml of the preculture was inoculated in 9 ml of LB liquid medium containing 0.1% histidine and tyrosine precursor and incubated at 37 ° C in a suspension culture incubator at 150 rpm for 24 hours. The culture broth was centrifuged at 13,000 rpm for 30 minutes and the supernatant was used as a pretreatment sample for biogenic amine analysis. 0.5 ml of each of the pretreatment sample solution and the standard solution was taken and then 0.25 μl 1,7-diaminoheptane (Sigma-aldrich, MO, USA) and 0.25 ml saturated Na ₂ CO ₃ (Sigma-Aldrich, MO, USA), 1% acetone (Sigma-aldrich, MO, USA) and 0.4 ml of dansyl chloride Lt; / RTI > To the derivatized sample, 0.25 ml of 10% proline (Sigma-aldrich, MO, USA) was added and the excess dyne chloride was removed. 2.5 ml of ethyl ether (Samchun, Seoul, Korea) was added and shaken for 3 minutes. The separated supernatant was evaporated and the residue was filtered through a 0.45 μm syringe filter (Sartorius, Frankfurt, Germany) using 0.5 ml acetonitrile (Mallinckrodt JT Baker, Phillipsberg, NJ, USA) . The instrumental analysis conditions for biogenic amine analysis (Jeong et al ., 2014, J. Korean Soc. Food Sci. Nutr, 43, 550-556) are as follows.

HPLC analysis conditions for biogenic amine detection Instrument Agilent 1200 series
(Agilent Technologies, Santa Clara, CA, USA) Column CapcellPak C18 Column Detector DAD detector (254 nm) Mobile phase A: 0.1% formic acid in H 2 O B: 0.1% formic acid in Acetonitrile 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 쨉 l

Identification of the strain

16S rRNA gene analysis was performed to identify the final selection strains. For identification of the strains, the cells were inoculated into Nutrient broth (NB, Difco ^™ ), cultured at 30 ° C. for 24 hours, centrifuged to recover the cells, and then treated with ZR Fungal / Bacterial DNA Miniprepkit (Zymo Research Corp.) DNA was extracted. The 16S rRNA gene was synthesized with universal primers, 27F and 1492R, and the gene fragment was amplified by PCR (Baek et The amplified PCR products were purified with QIAquick PCR Purification kit (QIAGEN) and analyzed with the aid of Cosmogentech Co. to analyze the nucleotide sequences. Biochemical properties of the fermentation broth were investigated using API 50CH kit (bioMerieux) based on Bergey's manual of systematic bacteriology (Leadbetter, 1974, Bergey's Manual of Determinative Bacteriology, 8th ed.

Cell growth investigation

In order to investigate the extent of growth according to the incubation time, 5% of strains selected in LB medium were inoculated and cultured at 30 ° C in a suspension incubator at 150 rpm for 72 hours. Then, the amount of dried cells and the absorbance were measured. The culture was recovered at intervals of 4 hours. The amount of dry cell mass was measured by centrifuging 10 ml of the culture solution at 13,000 rpm for 30 minutes, washing it three times with sterilized distilled water, drying at 80 ° C until it reached constant weight, and then measuring its weight. 1 ml of the culture broth was collected and centrifuged at 13,000 rpm for 30 minutes, washed three times with sterilized distilled water, resuspended in 1 ml of sterilized distilled water, and analyzed by UV / VIS spectrophotometer (SPECORD 200, Analytic jena) Absorbance was measured at 600 nm.

Example  1. Isolation of strain

In order to isolate native fermenting microorganisms with various functionalities and physiological activities, approximately 200 samples of Meju, Doenjang and Kochujang prepared by traditional methods were collected from Sunchang - gun, North Jeolla Province. The collected samples were suspended in sterilized distilled water and cultured in a bacterial culture medium using a stepwise dilution method. 612 kinds of microorganisms were isolated according to morphological characteristics such as the shape and color of the colon. The isolates were subjected to qualitative tests using a solid medium for the detection of biogenic amines for the primary screening of non-producing strains of biogenic amines, and it was confirmed that 203 isolates did not produce biogenic amines (Fig. 2). The strains that had completed the first screening were inoculated on LB medium and cultured for 2 days before being used in the next study.

Example  2. Extracellular  enzyme Secretory function  Measure

Bacillus subtilis , B. licheniformis , B. citreas , etc. are known to be representative strains involved in fermentation of the fermented soybean, Free sugars, isoflavones, aglycons, fatty acids (such as amylases, amylases, cellulases, and lipases) by various enzymes of microorganisms such as yeast, fatty acid, etc., and it is known that secondary products having various functions as well as flavor are produced. In particular, proteases have been shown to degrade proteins during fermentation, thereby affecting the content of free amino acids, a unique taste component (Kim et al ., 2005, Kor, J. Life Science, 15, 749-754) Ra et al ., 2004, J. Korean Soc. Food Sci. Nutr, 33, 439-442) has been actively separating microorganisms having various functions in order to secure various microorganism resources from traditional herbs such as isolating strains having excellent activity of? -Glucosidase activity from doenjang. Thus, the extracellular enzyme activities of 203 isolates from the previously selected isolates showed that protease, amylase, cellulase and lipase were produced in 86 isolates. Respectively.

In particular, SCM688 was excellent in proteolytic activity and amylase and cellulase activity (FIG. 3).

Example  3. Antimicrobial activity of isolates

The antimicrobial activity against pathogenic microorganisms was investigated in 86 isolates. The antimicrobial activity of all four pathogenic microorganisms was determined to be antimicrobial activity against at least one harmful microorganism. As a result, 9 kinds of antimicrobial activities such as SCM57, SCM103, SCM119, SCM148, SCM193, SCM200, SCM501, SCM688 and SCM699 It has antimicrobial activity against harmful pathogenic microorganisms of various food and has excellent inhibitory effect. In particular, strains of SCM688 has showed the best activity against pathogenic bacteria of the four species, Bacillus cereus (B. cereus) KCTC 3624 and Staphylococcus aureus (Staphylococcus aureus subsp. aureus ) KCCM 11593 and KCCM 41331 showed high antibacterial activity.

Comparison of Enzyme Activity and Antimicrobial Activity of Selected Strains Test Items Selected strains SCM
57 SCM
103 SCM
119 SCM
148 SCM
193 SCM
200 SCM
501 SCM
688 SCM
699 Enzyme Activity (mm) ^a Protease 1.2 w - - 1.0 1.0 1.3 2.1 - Amylase - 1.1 1.1 1.0 - - 1.2 1.4 w Cellulase w 1.3 - 1.2 - 1.0 - 1.4 1.5 Lipase - w - - - - - w w Antimicrobial activity (mm) KCTC 3624 ^b w - 1.0 - - 1.2 w 1.4 1.3 KCCM 11593 ^c - w - - 1.0 - w 1.2 - KCCM 41331 ^d 1.1 - 1.0 1.1 - w w 1.3 - KCCM 40935 ^e - - - w 1.3 - 1.0 w1.4 -

^a Clear zone size (diameter)

^b KCTC 3624: Bacillus cereus (Bacillus cereus ), ^c KCCM 11593: Staphylococcus ( Staphylococcus aureus) aureus subsp. aureus ), ^d KCCM41331: Staphylococcus ( Staphylococcus aureus ) aureus subsp. aureus), ^e KCCM40935: Bacillus cereus (Bacillus cereus )

w: weak activity

Example  4. Biogenic  Confirmation of amine formation

Quantitative analysis was carried out by adding the selected strains to a liquid medium containing histidine and tyrosine, measuring the decarbonic acid activity of the amino acids, and measuring histamine and tyramine contents produced by the strain. In some isolates of SCM57, SCM193, and SCM699, biogenic amines were detected, but not detected quantitatively. Histamine in the food was weak in 8-40 mg, severe in 40-80 mg, severe in 100 mg (Maijala and Eerola, 1993, Meat Sci, 35, 387-395). It has been reported that all of the previously selected strains are not harmful Respectively.

Especially, in the isolates of SCM103, SCM119, SCM148, SCM200, SCM501, and SCM688, histamine and tyramine were not detected, and the same qualitative analysis as the result of the above proceeded using the solid medium for detecting biogenic amine was confirmed. Based on the above results, SCM688, which showed excellent results in both extracellular enzyme activity, antimicrobial activity, and production of biogenic amine, was selected as the final strain.

Quantitative comparison of biogenic amines to selected strains Test Items Selected strains SCM
57 SCM
103 SCM
119 SCM
148 SCM
193 SCM
200 SCM
501 SCM
688 SCM
699 Biogenic amine
(mg / L) Histamine NQ ^a N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. Tyramine ND ^b N.D. N.D. N.D. N.Q N.D. N.D. N.D. N.Q

^a ND: Not detected

^b NQ:

Example  5. Identification of the strain

Based on the results of 16S rRNA gene sequencing analysis of SCMB688, BLAST was found to be Bacillus subtilis , Because the genus can survive by generating spores in various environments, it can be classified as a different species even if it exhibits 100% homology with the 16S rRNA sequence (FIG. 1) as a bacterium with a large biodiversity depending on the environment. 50CH kit was used to analyze glucose metabolism. The result of analysis of glucose metabolism was as follows. It was identified as Bacillus subtilis and confirmed to be consistent with 16S rRNA sequencing result. Finally, it was named Bacillus subtilis SCM688. SCM688, the final selection strain, was introduced into the Korean Agricultural Culture Collection (KACC) by Bacillus subtilis KACC 81006BP.

Results of the analysis of the metabolism ability of SCM688 strain by API50 CHB kit Enzyme ^a SCM688 Enzyme SCM688 Control - Esculin + Glycerol + Salicin + Erytyritol - D-cellobiose + D-arabinose - D-maltose + L-arabinose + D-lactose - D-ribose + D-melibiose + D-Xylose - D-saccharose + L-Xylose - D-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 + L-rhamnose - D-turanose - Dulcitol - D-lyxose - Inositol + D-tagatose - D-mannitol + D-fucose - D-sorbitol + L-fucose - Methyl-D-mannopyranoside - D-arabitol - Methyl-D-glucopyranoside + L-arabitol - N-acetyl glycosamine - Potassium gluconate - Amygdalin + Potassium 2-Ketogluconate - Arbutin + Potassium 5-Ketogluconate -

Example  6. Cell growth investigation

In order to confirm the growth curve of SCM688, the cells were inoculated on TSB (Oxoid CM0129) medium and suspension cultured at 30 ° C and 150 rpm. Absorbance and dry cell mass were measured at intervals of 4 hours (FIG. Growth curves showed the logarithmic period from 8 hours to 28 hours. After 28 hours, the proliferation gradually slowed down to 32 hours. The highest cell growth was observed at 28 hours with an absorbance of 1.2143 (OD600) and a dry cell weight of 1.3684 g / L. The cell growth of Bacillus subtilis was in the quiescent period from 24 hours to 28 hours. Based on these results, the optimum incubation time was set to 24 hours from the peak stage to the stationary phase.

Agricultural Biotechnology Research Institute KACC81006BP 20150728

Claims (4)

It has antimicrobial activity against Bacillus cereus and Staphylococcus aureus and secretes protease, amylase, cellulase and lipase, and histamine ( Bacillus subtilis ) SCM688 strain (KACC81006BP) derived from a traditional herb that does not produce histamine and tyramine. delete An antimicrobial microbial preparation for Bacillus cereus or Staphylococcus aureus comprising the Bacillus subtilis SCM688 strain of claim 1 or a culture thereof as an active ingredient. Bacillus subtilis of claim 1 , Mushrooms containing SCM688 strain.

Images