KR20170015688A - - SCML458 Lactobacillus brevis SCML458 strain from traditional soy sauce having antimicrobial activity against pathogenic microorganism of soy sauce antibiotic resistance antioxidant activity -glucosidase and enzyme secretion activity and not producing biogenic amine and uses thereof - Google Patents

Abstract

The present invention relates to a traditional fermented paste-derived Lactobacillus brevis SCML458 strain (KACC81008BP) which has an excellent antibacterial activity, antibiotic resistance, -glucosidase activity, antioxidant activity, and an extracellular enzyme secreting function with respect to harmful microorganisms and does not generate biogenic amine; to an antibacterial microbial formulation with respect to fermented paste harmful microorganisms, wherein the antibacterial microbial formulation comprises the Lactobacillus brevis SCML458 strain or a culture medium thereof as an active ingredient; to food including the Lactobacillus brevis SCML458 strain; and to a method for controlling fermented paste harmful microorganisms, wherein the method comprises a step of treating the Lactobacillus brevis SCML458 strain or the culture medium thereof in fermented paste.

Description

Lactobacillus brevis SCML458 strain derived from a traditional herb that is excellent in antimicrobial activity, antibiotic resistance, antioxidative activity, β-glucosidase and extracellular enzyme secretory activity against harmful microorganisms and does not produce a biogenic amine, and its use {Lactobacillus brevis SCML458 antibiotic resistance, antioxidant activity, β-glucosidase and enzyme secretion activity, and not producing biogenic amines and uses thereof.

The present invention relates to a lactobacillus brevis SCML458 strain derived from a traditional herb that is excellent in antimicrobial activity, antibiotic resistance, antioxidative activity, β-glucosidase and extracellular enzyme secretory activity against harmful microorganisms and does not produce a biogenic amine, .

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 Laid-Open Publication No. 2015-0000372 discloses' gamma aminobutyric acid-producing lactobacillus brevis and its use '. However, as in the present invention,' anti-microbial activity against bacteria, antibiotic resistance, antioxidative activity, β - Lactobacillus brevis SCML458 strains derived from traditional herbs that are excellent in glucosidase and extracellular enzyme secretion ability and do not produce biogenic amines, and their uses' have never been disclosed.

The present invention has been made in view of the above-mentioned needs, and an object of the present invention is to provide an antimicrobial activity against Bacillus cereus which is a harmful microorganism, 10 antibiotic resistance, antioxidative activity,? -Glucosidase and protease and cellulase New Lactobacillus brevis ( Lactobacillus), which is derived from a traditional herb that has excellent secretory ability and does not produce a biogenic amine brevis ) The SCML458 strain (KACC81008BP) 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-mentioned problems, the present invention provides a method for producing lactic acid, which is excellent in antimicrobial activity, antibiotic resistance, antioxidative activity, β-glucosidase and extracellular enzyme secretory activity against harmful microorganisms, Lactobacillus brevis ) SCML458 strain (KACC81008BP).

In addition, the present invention provides a method for producing Lactobacillus brevis SCML458 strain or a culture solution thereof as an active ingredient.

In addition, the present invention provides a method for producing Lactobacillus brevis SCML458. ≪ / RTI >

In addition, the present invention provides a method for producing Lactobacillus brevis Which comprises treating a strain of SCML458 or a culture thereof with a soybean curd.

In the present invention, Lactobacillus brevis , The SCML458 strain inhibits the growth of harmful microorganisms that occur during the fermentation process of the soybean, secretes the extracellular enzymes of? -Glucosidase, protease and cellulase, has excellent antibiotic resistance and antioxidant activity, and produces biogenic amines . The Lactobacillus brevis SCML458 strains can be used safely for the production of safe and non - toxic foods.

Figure 1 shows the 16S rRNA base sequence of SCML458 isolate according to the present invention.
FIG. 2 shows the results of SCML458 isolating extracellular enzyme activity assay according to the present invention. (From the left, protease, cellulase)
Fig. 3 shows the result of examining the antioxidative activity against the selected strains.
FIG. 4 shows the results of confirming the growth curve of the SCML458 isolate according to the present invention. Lactobacillus brevis, which was inoculated on MRS medium and cultured at 30 rpm at 150 rpm, Cell dry cell mass and absorbance of SCML458 strain are 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 an antimicrobial agent which is excellent in antimicrobial activity, antibiotic resistance, antioxidative activity,? -Glucosidase and extracellular enzyme secretory activity against harmful microorganisms, Lactobacillus brevis ) SCML458 strain (KACC81008BP).

The Lactobacillus brevis (Lactobacillus brevis ) The SCML458 strain (KACC81008BP) was isolated from a conventional herbarium, and has antagonistic activity against the harmful microorganism Bacillus cereus , has a β-glucosidase activity, excellent protease and cellular secretion ability, and excellent antioxidant activity And does not produce a biogenic amine. The Lactobacillus brevis (Lactobacillus brevis ) The SCML458 strain was deposited at the National Institute of Agricultural Science and Technology, Center for Agricultural Genetic Resources on Jul. 28, 2015 (Accession No .: KACC 81008BP).

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

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

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.

In addition, the present invention is the Lactobacillus brevis (Lactobacillus brevis ) SCML458 strain or a culture solution thereof as an active ingredient.

In the microorganism preparation according to an embodiment of the present invention, the harmful microorganism may be Bacillus cereus , but is not limited thereto.

The microorganism preparation has antimicrobial activity, antioxidative activity, antibiotic resistance activity and Lactobacillus brevis which does not produce biogenic amine brevis ) SCML458 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 present invention also provides Lactobacillus brevis (Lactobacillus of the present invention brevis ) And culturing the SCML458 strain. The present invention also provides a method for producing an antibacterial microorganism preparation against a harmful microorganism. The Lactobacillus brevis SCML458 strain of the present invention can be cultured by a conventional method known in the art.

In addition, the present invention is the Lactobacillus brevis (Lactobacillus brevis ) SCML458. ≪ / RTI >

The food is Lactobacillus brevis (Lactobacillus of the present invention brevis ) And may be a fermented food using the SCML458 strain as a seed, and more particularly, but not limited to, a soup. 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 present invention also provides Lactobacillus brevis (Lactobacillus of the present invention brevis ) Which comprises treating a strain of SCML458 or a culture thereof with a soybean curd.

The control of the harmful microorganism of the present invention is carried out by the lactobacillus brevis By treating the SCML458 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 proteases and cellulases among the extracellular enzymes that released the isolated microorganisms from the cells, and a solid sorting medium containing components of the substrate to specifically react with each enzyme Were used. The protease secretion ability was determined by using skim milk as a substrate and preparing a skim milk agar medium supplemented with 2% of skim milk (Difco ^™ ) with 1.5% of agar (Vermelho et al ., 1996, Mem. Inst. Oswaldo (CMC, JUNSEI CHEMICAL CO., LTD.) Was used as a substrate and the cells were cultured in CMC agar medium containing 1% of carboxymethylcellulose (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 Cellular secretion ability was investigated with the diameter of the transparent ring.

Bacillus To Sereus  Antimicrobial activity measurement

The antimicrobial activity of the strains selected for food harmful microorganisms was examined. Food-harmful microorganisms are known as Bacillus cereus ( Bacillus cereus) cereus, KCTC 3624), 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.

β- Glucosidase (β- glucosidase ) Active lactic acid bacteria

To determine the activity of β-glucosidase in the strains screened previously, RS (1% beef extract, 1% protease peptone No. 3, yeast extract 0.5%, dextrose 2%, polysorbate 80 0.1 (0.1% of esculin, 0.05% of ferric ammonium citrate, 1.5% of agar) was added to an aqueous solution containing 0.2% of ammonium citrate, 0.5% of sodium acetate, 0.01% of magnesium sulfate, 0.005% of manganese sulfate and 0.2% of dipotassium phosphate) After sterilization, the cells were sterilized and solidified to prepare a solid medium. The selected strains were inoculated and cultured at 37 ° C for 2 days. Then, the color change around the colonies was examined to select a brown or black zone.

Antioxidant activity measurement

Measurement of antioxidant activity for the selected strains is the method of DPPH (2,2-diphenyl-1- picryl-hydrazyl, Sigma-aldrich) (Lee et al ., 2009 Food Sic. Biotechnol, 18, 959-964). 200 μl of the culture supernatant was added to 1 ml of 100 μM DPPH ethanol solution. The reaction product was allowed to react at room temperature for 30 minutes, and the absorbance was measured with a UV / VIS spectrophotometer (SPECORD 200, Analytic jena) at 528 nm. And the degree of antioxidant activity was measured. As a control, the same experiment was carried out using a test tube containing a culture medium.

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

Biogenic  Amine ( Biogenic amine ) Confirm creation

For the biosynthetic amine assay, the isolates were inoculated into the MRS broth and incubated at 37 ° C in a suspension incubator (VS-1203P3V, Vision Scientific Co. Ltd., Daejeon, Korea) at 150 rpm for 24 hours 1 ml of the preculture was inoculated into 9 ml of MRS liquid medium containing 0.1% histidine and tyrosine as a 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.

API ZYM Using Lactobacillus Brevis ( Lactobacillus brevis )of  Enzyme activity investigation

The last selected Lactobacillus brevis To investigate the enzymatic activity of SCML458, the activity of alkaline phophatase and 18 other enzymes were examined using API ZYM kit (Biomerieux). Lactobacillus brevis SCML458 was cultured in MRS solid medium, Recovered and suspended in 0.85% NaCl solution (adjusted to Mcfarland turbidity 5-6). After suspending the suspension in each cupule of the ZYM kit and incubating at 37 ° C for 4 hours, the degree of enzyme activity was examined by observing the color change.

Investigation of antibiotics and susceptibility

Antibiotic susceptibility tests were performed by the diffusion method of the National Committee for Clinical Laboratory Standards (NCCLS). Amikacin (30 ㎍, AN), amoxicillin / clavulanic acid (20 ㎍ / 10 ㎍, AMC), and ampicillin (Becton Dickinson Co., Ciprofloxacin (5 ㎍, CIP), colistin (10 ㎍, CL), doxycycline (30 ㎍, D), cephalothin ), Erythromycin (15 ㎍, E), gentamicin (10 ㎍, GM), lincomycin (2 ㎍, L), kanamycin (30 ㎍, K), neomycin 30 μg, N), norfloxacin (10 μg, NOR), streptomycin (10 μg, S), tetracycline (30 μg, TE), trimethoprim / / sulfamethoxazole, 1.25 ㎍ / 23.75 ㎍, SXT). For the susceptibility test, the final selection strain was inoculated on Mueller Hinton Broth (MErck, Germany), incubated at 37 ° C for 5 hours, diluted with 0.5 MacFarland (BioMerieux) broth, plated on Muller-Hinton agar After drying for 30 minutes, 16 antibiotic disks were placed on the plate and incubated at 37 ° C for 1 day. The size of the inhibitory ring for each antibiotic was measured and the susceptibility and tolerance were determined by the CLSI guideline.

Cell growth investigation

In order to investigate the extent of growth according to the culture time, 5% of the strains selected in the MRS medium were inoculated and cultured in a 37 ° C suspension incubator at 150 rpm for 72 hours. The amount of dried cells and the absorbance were measured. The culture was recovered at intervals of 4 hours. The amount of dried cells was measured by centrifuging the culture solution (10 ml) at 13,000 rpm for 30 minutes, washing it three times with sterilized distilled water, drying it at 80 ° C until it reached constant weight, and then measuring its weight. 1 ml of the culture was recovered, centrifuged at 13,000 rpm for 30 minutes, washed three times with sterile 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. Then, 583 kinds of microorganisms were isolated according to the morphological characteristics such as the shape and color of the colon. The strains that had been screened were inoculated on MRS 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 and the like by various enzymes of microorganisms such as amylase, cellulase and lipase. (fatty acid), and thus it is known that a secondary product having various functions as well as flavor is 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) is different to ensure the variety of microbial resources from traditional sauces such as the β- glucosidase activity to remove the superior strains from soybean Microorganisms having functional properties are actively being separated.

As a result, the extracellular enzyme activities of 583 isolates selected above were confirmed to produce two enzymes of protease and cellulase in 29 isolates (FIG. 2).

Example  3. Isolation of isolate Bacillus To Sereus  Antimicrobial activity against

The antimicrobial activity of Bacillus cereus was investigated in 29 isolates selected by enzyme activity assay. The antimicrobial activity of the two kinds of Bacillus cereus was measured and found to have antimicrobial activity against the harmful microorganisms in most cases. As a result, SCML26, SCML125, SCML229, SCML310, SCML337, SCML383, SCML419, SCML458, SCML500 9 had excellent antimicrobial activity against Bacillus cereus. In particular, SCML458 among the isolates showed the most excellent activity, and antimicrobial effect on Bacillus cereus in traditional fermentation and food manufacturing will be effective for foodborne pathogens.

Comparison of Enzyme Activity and Antimicrobial Activity of Selected Strains Test Items Selected strains SCML
26 SCML
125 SCML
229 SCML
310 SCML
337 SCML
383 SCML
419 SCML
458 SCML
500 Enzyme Activity (mm) ^a Protease 1.4 1.4 1.4 1.3 1.5 1.4 1.4 1.4 1.3 Cellulase 0.8 0.8 1.0 0.8 0.8 1.0 0.8 1.2 1.2 Antimicrobial activity (mm) KCTC 3624 ^b 1.8 1.7 1.7 1.5 1.6 1.6 1.8 1.5 1.6 KCCM 40935 ^c 1.4 1.3 1.6 1.6 1.5 1.4 1.3 1.8 1.4

^a Clear zone size (diameter)

^bKCTC 3624: Bacillus cereus (Bacillus cereus),^eKCCM40935: Bacillus cereus (Bacillus cereus)

Example  4. Antioxidant activity measurement

Based on the previous experiments, the antioxidative activity of isolates with antimicrobial activity was measured by measuring the free radical scavenging effect by DPPH. DPPH is one of the indicators of antioxidant activity against phenolic substances as a representative substrate for measuring antioxidant activity. It inhibits lipid oxidation in foods by donating electrons to free radicals, inhibits aging by reactive oxygen species in the body, And prevent aging (Lee et < RTI ID = 0.0 > al ., 1997, Korean J. Food Sci. Technol., 29, 432-436).

Therefore, SCML26, SCML458 and SCML500 showed more than 15% DPPH activity and SCML458 had the highest antioxidative activity of 24.25% SCML458 was finally selected as the most excellent strain through measurement of activity and antioxidant activity (Fig. 3).

Comparison of antioxidant activity against selected strains Test Items Selected strains SCML
26 SCML
125 SCML
229 SCML
310 SCML
337 SCML
383 SCML
419 SCML
458 SCML
500 Antioxidant activity (%) 16.04
± 0.12 13.83 + 0.04 14.89 ± 0.15 10.87 ± 0.31 5.79 ± 0.24 14.00 0.32 6.13 + 0.02 24.25 + - 0.81 15.02 + - 0.22

Example  5. 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 acid and measuring the content of histidine and tyrosine produced by the strain. Biogenic amines were detected in some isolates of SCML26, SCML125, SCML229, SCML337 and SCML500, but the detection values were not quantified. Histamine in food was found to be weak in 840 mg, severe in 4080 mg, (Maijala and Eerola, 1993; Meat Sci, 35, 387-395). It has been reported that all of the strains selected above are not harmful Respectively.

In particular, no histidine or tyrosine was detected in the isolates of SCML310, SCML383, SCML419 and SCML458.

Quantitative comparison of biogenic amines to selected strains Test Items Selected strains SCML
26 SCML
125 SCML
229 SCML
310 SCML
337 SCML
383 SCML
419 SCML
458 SCML
500 Biogenic amine
(mg / L) Histamine NQ ^a N.Q N.D. N.D. N.D. N.D. N.D. N.D. N.D. Tyramine ND ^b N.D. N.Q N.D. N.Q N.D. N.D. N.D. N.Q

^a ND: Not detected

^b NQ:

Example  6. β- Glucosidase (β- glucosidase ) Active measurement

Foods fermented with soybeans have an aglycone-type glycoside mixed with sugar components, and a representative substance is isoflavone. Isoflavones are called phytoestrogens and are called daidzein and genistein as active substances in the body. Isoflavones present in the form of glycosides are converted into β-glucosidase, an enzyme produced by gastric acid and intestinal microorganisms, that is, non-glycosylated forms of daidzein and genistein, which are absorbed in the intestines. Recently, it has been confirmed that genistein has a strong anticancer effect. In particular, fermented foods using microorganisms that produce such enzymes are expected to become more and more functional foods. Therefore, the results of β-glucosidase activities of selected strains showed that all of them except SCML458, which is a selective strain, were found to be inactive, which was superior to the results of the previous enzyme activity, antimicrobial activity and antioxidant activity Were selected as the final selection strains.

Test Items Selected strains SCML
26 SCML
125 SCML
229 SCML
310 SCML
337 SCML
383 SCML
419 SCML
458 SCML
500 ? -glucosidase activity
(Degree of color development) - - - - - - - +++ -

Active: + Ha, ++ Middle, +++ Phase

Example  7. Identification of the strain

The 16S rRNA gene sequence analysis of SCML458 finally screened revealed BLAST as Lactobacillus brevis , and finally Lactobacillus brevis SCML458. SCML458 was deposited at the Korean Agricultural Culture Collection (KACC) with the Lactobacillus brevis KACC 81008BP.

Example  8. API ZYM Kit  Used Lactobacillus Brevis  Enzyme activity of SCML458

The last selected Lactobacillus brevis In order to confirm the production of the enzyme of SCML458, an API ZYM kit was used. As shown in the table below, SCML458 produced protease enzymes of valine arylamidase and cystine arylamidase, and α and β-glucosidase activities were also confirmed.

Analysis of enzyme activity of SCML458 strain by API ZYM kit Enzyme Result Control - Alkaline phosphatase - Esterase (C4) - Esterase lipase (C8) + Lipase (C14) - Leucine arylamidase - Valine arylamidase + Cystine arylamidase + Trypsin - alpha-chymotrypsin - Acid phosphatase - Naphol-AS-BI-phosphohydrolase + alpha -galactosidase - β-glucuronidase + β-glucosidase + α-glucosidase + β-glucosidase + N-acetyl-β-glucosaminidase + alpha-mannosidase - alpha-fucosidase -

Example  9. Investigation of antibiotic resistance

The antimicrobial susceptibility of SCML458, a selective strain, was found to be inhibited against amoxicillin / clavulanic acid, doxycycline and erythromycin, and amikacin, But are not limited to, cephalothin, ciprofloxacin, colistin, lincomycin, kanamycin, norfloxacin, streptomycin, tetracycline, trimethoprim / It was confirmed that the antibiotics based on trimethoprim / sulfamethoxazole were tolerant to antibiotics.

Investigation of antibiotic resistance and susceptibility of Lactobacillus brevis SCML458 Enzyme Code / Disc potency Result ^a Amikacin AN / 30 [mu] g ND Amoxicillin / clavulanic acid AMC / 20 [mu] g, 10 [mu] g SS Ampicillin AM / 10 [mu] g SS Cephalothin CF / 30 [mu] g ND Ciprofloxacin CIP / 5 [mu] g ND Colistin CL / 10 [mu] g ND Doxycycline D / 30 [mu] g SS Erythromycin E / 15 [mu] g SS Gentamicin GM / 10 [mu] g S Lincomycin L / 2 [mu] g ND Kanamycin K / 30 ㎍ ND Neomuycin N / 30 g S Norfloxacin NOR / 10 [mu] g ND Streptomycin S / 10 [mu] g ND Tetracycline TE / 30 [mu] g ND Trimethoprim / sulfamethoxazole SXT / 1.25 [mu] g, 23.75 [mu] g ND

^a diameter, cm; ND, Note; S, 1.2 to 1.5; SS, 1.6 ~ 2.0

Example  10. Cell growth investigation

To determine the growth curve of SCML458, the cells were inoculated on MRS (Difco) medium and cultured at 37 ° C in a suspension culture incubator at 150 rpm. The absorbance and the amount of dried cells were measured at intervals of 4 hours (FIG. 4). Growth curves showed the logarithmic period from 8 to 32 hours, and after 36 hours, the growth gradually slowed down and entered the stopper from 40 hours. The highest cell growth was observed at 36 hours with an absorbance of 1.8505 (OD ₆₀₀ ) and a dry cell weight of 2.4090 g / L. Based on these results, the optimum culture time was set to 36 hours, which is from the peak stage to the stationary stage.

Agricultural Biotechnology Research Institute KACC81008BP 20150728

Claims (4)

Lactobacillus brevis ( Lactobacillus brevis), which is excellent in antimicrobial activity, antibiotic resistance, antioxidative activity, β-glucosidase and extracellular enzyme secretory activity against harmful microorganisms and does not produce biogenic amine brevis ) SCML458 strain (KACC81008BP). The method according to claim 1, wherein the harmful microorganism is Bacillus cereus , and the antibiotic is selected from the group consisting of amikacin, cephalothin, ciprofloxacin, colistin, lincomycin, kanamycin, norfloxacin, streptomycin, tetracycline, and trimethoprim / sulfamethoxazole. The extracellular enzyme is a protease, And a cellulase, wherein the biogenic amine is histamine and tyramine. Claim 1 or claim 2 Lactobacillus brevis (Lactobacillus brevis ) An antimicrobial microbial preparation for a harmful microorganism which contains SCML458 strain or a culture solution thereof as an active ingredient. Claim 1 or claim 2 Lactobacillus brevis (Lactobacillus brevis ) Mushrooms containing SCML458 strain.

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