KR20160141267A - Bacillus amyloliquefacience having antibacterial activity and uses thereof - Google Patents

Abstract

Bacillus amyloliquefacience RD 7-7 strain (Accession No. KACC 92071P) of the present invention generates a lot of mucilage, when inoculated while fermenting fast-fermented bean paste, has high protease and amylase activity and high content of amino type nitrogen, has excellent fermentation characteristic due to low off-flavor components generated during a fermentation process, has antibacterial activity with respect to various microorganism, and inhibits growth of Bacillu cereus in co-cultivation with Bacillu cereus causing food poisoning, thereby being used for manufacturing a high quality and safe fermented food.

Description

Bacillus amyloliquefacience strains exhibiting antibacterial activity and uses thereof Bacillus amyloliquefacience having antibacterial activity and uses thereof [

The present invention relates to a Bacillus amyloliquefaciens strain exhibiting antibacterial activity and uses thereof.

Soybean has been widely used as a traditional fermented food raw material and a plant-derived functional material in Korea. Soybeans contain many functional ingredients such as isoflavone, saponin, phytic acid, oligo-saccharides, and the like. The enzymes derived from microorganisms during fermentation decompose the fibrous components of soybeans, sugars and proteins in the cells to improve the digestibility, and the digestibility of amides, peptones, polyglutamates, polypeptides and the like It is known that physiologically active substances are generated and blood pressure lowering, thrombolytic ability, anticancer effect and antioxidant are increased.

In soybean fermented foods, the most important factor determining nutrition and quality is the type of microorganisms involved, and various physiologically active substances are produced depending on the strain and fermentation conditions, and thus the functionality is different. When fermented soybean fermented food, Chungkukjang or soybean paste meju, Bacillus existed in rice straw. Bacillus sp. Derived from rice straw produces various enzymes (protease, amylase, cellulase, etc.) and produces various metabolites having physiological activity. The secreted protease has the properties of casein degradation and gelatin degradation, and plays a role in producing a unique taste and flavor during fermentation. It is known that the odor of soybean fermented food is determined by the microorganism growth conditions such as alkylpyrazine, ammonia compound and sulfur compounds produced from Bacillus sp. Bacillus, as well as moisture, salinity and temperature. Studies have been reported on the production of soybean fermentation products using fermentation strains in which the composition of ingredients is changed or the degree of generation of unpleasant compounds is low in order to reduce unpleasant odors. The viscous substance of soybean fermented product is produced by Levan fructan and polyglutamate synthesized by using Bacillus bacteria as a substrate of sugar and proteins of soybean.

The Bacillus strains have excellent productivity of hydrolytic enzymes, and many of them have been developed as industrial enzyme producing bacteria. The production of β-galactosidase from B. licheniformis derived from Chungkukjang and the production of Bacillus subtilis Bacillus amyloliquefaciens and Bacillus subtilis, which produce the proteolytic enzyme, thrombolytic enzyme, and B. pumilus, which is an excellent immunity enhancer. . In addition, studies have also been reported on improving the quality of soybean fermentation by using a strain as a starter. Bacillus bacteria used as a starter are spore-forming bacteria that grow in soil or water, and most of them have the property of decomposing organic matter. Safety is widely accepted.

Bacillus cereus (Bacillus cereus) is a gram-positive bacillus, arranged in a chain, with flagella and mobility. It grows at 7 ~ 49 ℃ under aerobic condition with strong resistance to heat by forming spores and proliferates well under oxygen conditions. The optimum temperature is 28 ~ 35 ℃. It is distinguished from other Bacillus species by β-hemolytic form and produces lecithinase (lecithinase), which shows milky white color around colonies in egg yolk reaction.

Because Bacillus cereus is a kind of soil bacteria, it is widely distributed in the natural world such as soil, dust, sewage, and it is contaminated with plants, causing corruption and food poisoning. The rate of detection is high but the frequency of food poisoning is relatively low. However, it has been attracting attention for a long time due to its high incidence in the UK and Europe. Recently, it has been detected and regulated in various foods in Korea. To prevent food poisoning, food must be killed (spores), germination should be suppressed, and proliferation should be prevented. The thermal resistance of Bacillus cereus spores is reported to be 1.2 to 7.5 minutes at 100 ° C.

Bacillus cereus is known to cause two types of food poisoning. The first is vomiting type food poisoning caused by intake of cereulide produced by the growth of Bacillus cereus in food and the other is the vomiting type food poisoning by ingesting the growth cell or spore of Bacillus cereus bacteria, It is a diarrheal food poisoning which is caused by the production of enterotoxin.

Bacillus amyloliquefacience is a spore-forming aerobic bacterium that is known to have excellent protein and starch hydrolytic activity and produces a natural antibacterial protein, Barnase (ribonuclease). It is also a microorganism that can be used as a food raw material.

The present inventors have been studying to develop a strain having excellent Bacillus cereus inhibiting activity and fermentation characteristics, and when the Bacillus amyloliquefaciens strain RD7-7 isolated from rice doenjang is inoculated at the time of fermentation with Chongkukjang, , Protease, amylase activity and amino nitrogen content, low fermentation odor components, excellent fermentation characteristics, excellent antimicrobial activity against various microorganisms, induce food poisoning Which inhibits the growth of Bacillus cereus during co-culture with Bacillus cereus.

It is an object of the present invention to provide a strain of Bacillus amyloliquefaciens RD7-7 exhibiting antibacterial activity and its use.

In order to achieve the above object, the present invention provides a strain of Bacillus amyloliquefaciens RD7-7 exhibiting antibacterial activity entrusted with the deposit number KACC 92071P.

Further, the present invention relates to a fermentation starter (hereinafter, referred to as " starch ") containing at least one selected from the group consisting of Bacillus amyloliquefaciens RD7-7 strain, a culture of the strain, a concentrate of the culture and a dried product of the culture starter.

The present invention also provides a fermented food product produced using the Bacillus amyloliquefaciens RD7-7 strain of the present invention or the fermentation starter of the present invention.

The present invention also provides a fermented food manufacturing method comprising the step of inoculating the Bacillus amyloliquefaciens strain RD7-7 of the present invention or the fermentation starter of the present invention.

The present invention also provides a pharmaceutical composition for antimicrobial use containing, as an active ingredient, a Bacillus amyloliquefaciens strain RD7-7 strain exhibiting an antimicrobial activity entrusted with the deposit number KACC 92071P.

The present invention also provides an antimicrobial health functional food containing, as an active ingredient, Bacillus amyloliquefaciens strain RD7-7 exhibiting an antimicrobial activity entrusted with the deposit number KACC 92071P.

In addition, the present invention provides an antimicrobial feed additive containing, as an active ingredient, Bacillus amyloliquefaciens strain RD7-7 exhibiting the antimicrobial activity entrusted with the deposit number KACC 92071P.

The Bacillus amyloliquefaciens strain RD7-7 of the present invention inhibits the growth of Bacillus cereus when co-cultured with Bacillus cereus causing food poisoning, has excellent antimicrobial activity against various microorganisms, , It produces a lot of viscous substances, has high protease, amylase activity and amino nitrogen content, and low odor components generated during the fermentation process. Therefore, it has excellent fermentation characteristics and quality, Can be usefully used.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a scanning electron microscope (TEM) image of a strain of Bacillus amyloliquefaciens RD7-7. FIG.
2 is a diagram showing a standard curve of a real-time PCR.
FIG. 3 is a graph showing the results of reverse transcription polymerase chain reaction (PCR) of the groEL and nheA genes of Bacillus cereus in a co-culture solution of Bacillus cereus and Bacillus amyloliquefaciens strain RD7-7.
FIG. 4 is a graph showing the results of quantitative real-time polymerase chain reaction of the groEL and nheA genes of Bacillus cereus in a co-culture solution of Bacillus cereus and Bacillus amyloliquefaciens strain RD7-7.
FIG. 5 is a diagram showing western blotting of the expression level of AtpB protein of Bacillus cereus in a co-culture solution of Bacillus cereus and Bacillus amyloliquefaciens RD7-7.
6 shows the fermentation state of Chungkookjang inoculated with Bacillus cereus and Bacillus amyloliquefaciens strain RD7-7 at different ratios.
Fig. 7 is a diagram showing western blotting of the expression level of AtpB protein of Bacillus cereus in fermented Chungkukjang co-inoculated with Bacillus cereus and Bacillus amyloliquefaciens RD7-7.
8 is a diagram showing the result of measurement of the number of microorganisms in a sample extract of soybean fermented product inoculated with various Bacillus bacteria:
Control group: experimental group not inoculated with Bacillus;
HJ18-4: an experimental group inoculated with Bacillus subtilis HJ18-4;
RD7-7: an experimental group inoculated with Bacillus amyloliquefaciens RD7-7;
HJ5-2: an experimental group inoculated with Bacillus amyloliquefaciens HJ5-2; And
HJ39-5: Experimental group inoculated with Bacillus amyloliquefaciens HJ39-5.
FIG. 9 is a graph showing the measurement results of protease and amylase activity in a sample extract of soybean fermented product inoculated with various Bacillus sp.
Control group: experimental group not inoculated with Bacillus;
HJ18-4: an experimental group inoculated with Bacillus subtilis HJ18-4;
RD7-7: an experimental group inoculated with Bacillus amyloliquefaciens RD7-7;
HJ5-2: an experimental group inoculated with Bacillus amyloliquefaciens HJ5-2; And
HJ39-5: Experimental group inoculated with Bacillus amyloliquefaciens HJ39-5.
10 is a graph showing the results of measurement of reducing sugar content in a sample extract of soybean fermented product inoculated with various Bacillus bacteria:
Control group: experimental group not inoculated with Bacillus;
HJ18-4: an experimental group inoculated with Bacillus subtilis HJ18-4;
RD7-7: an experimental group inoculated with Bacillus amyloliquefaciens RD7-7;
HJ5-2: an experimental group inoculated with Bacillus amyloliquefaciens HJ5-2; And
HJ39-5: Experimental group inoculated with Bacillus amyloliquefaciens HJ39-5.
FIG. 10 is a graph showing the results of measurement of amino-nitrogen content in a sample extract of soybean fermented product inoculated with various Bacillus species:
Control group: experimental group not inoculated with Bacillus;
HJ18-4: an experimental group inoculated with Bacillus subtilis HJ18-4;
RD7-7: an experimental group inoculated with Bacillus amyloliquefaciens RD7-7;
HJ5-2: an experimental group inoculated with Bacillus amyloliquefaciens HJ5-2; And
HJ39-5: Experimental group inoculated with Bacillus amyloliquefaciens HJ39-5.

Hereinafter, the present invention will be described in detail.

The present invention provides a strain of Bacillus amyloliquefacience RD7-7 exhibiting antimicrobial activity entrusted with the deposit number KACC 92071P.

Preferably, the strain is isolated from rice doenjang, but is not limited thereto.

The antimicrobial activity is for any one selected from the group consisting of Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Salmonella enteric, and Bacillus cereus. And more preferably Bacillus cereus according to a specific embodiment of the present invention.

The Bacillus amyloliquefaciens strain RD7-7 of the present invention is excellent in antimicrobial activity against various microorganisms (see Table 1), and when co-cultured with Bacillus cereus that causes food poisoning, the strain GROEL and nheA of Bacillus cereus (See FIGS. 3 to 5) and produce a lot of viscous substance during fermentation of chongkukjang (see FIG. 6), and a protease, an amylase activity and an amino nitrogen content (See Figs. 9 to 11), and since the off-flavor components generated during the fermentation process are low (Table 9), they can be used for manufacturing fermented foods having excellent safety and quality.

Further, the present invention relates to a fermentation starter (hereinafter, referred to as " starch ") containing at least one selected from the group consisting of Bacillus amyloliquefaciens RD7-7 strain, a culture of the strain, a concentrate of the culture and a dried product of the culture starter.

The fermentation starter means a preparation or composition comprising any one selected from the group consisting of a microorganism including lactic acid bacteria or bacteria involved in fermentation, a culture thereof, a concentrate of the culture and a dried product of the culture, Starter is used to provide a microorganism capable of growing in a fermented food or a microorganism capable of growing as a dominant species when added to a fermented food or the like during its production. When the fermented food is produced using the starter for fermentation, the quality of the fermented food can be controlled by the microorganisms contained in the starter for fermentation, and the speed or step of the fermentation can be controlled. Food can be produced. Further, the fermentation starter may further contain an additive usually used.

The Bacillus amyloliquefaciens strain RD7-7 of the present invention inhibits the growth of Bacillus cereus when co-cultured with Bacillus cereus causing food poisoning, has excellent antimicrobial activity against various microorganisms, , It is possible to produce a lot of viscous substance and to have a high content of protease, amylase activity and amino nitrogen and low odor components generated during the fermentation process, so that it has excellent fermentation characteristics and quality and is useful as a starter for fermentation Lt; / RTI >

The present invention also provides a fermented food product produced using the Bacillus amyloliquefaciens RD7-7 strain or the fermentation starter of the present invention.

The fermented food refers to a food prepared by adding one or more microorganisms such as lactic acid bacteria or enzymes and using the fermentation action of the microorganism and more specifically to a food prepared by adding a fermented food seed to a food substrate, it means. The fermented foods include all non-sterilized open fermented foods such as liquor, bread, kimchi, salted fish, miso, soy sauce, cheese, butter, yogurt and the like.

The fermented food may be Chungkookjang. In the case of Chungkukjang prepared by adding the Bacillus amyloliquefaciens RD7-7 strain or a starter for fermentation, the fermented food may be Bacillus amyloliquefaciens RD7-7 strain, The growth of Cereus is inhibited, the activity of protease, amylase enzyme and amino nitrogen is high, so the safety, taste and flavor of Chonggukjang is improved.

The Bacillus amyloliquefaciens RD7-7 strain or fermentation starter can be applied to any food field produced by fermenting food raw materials including soybean.

The fermented food may be prepared by adding the Bacillus amyloliquefaciens RD7-7 strain or a starter for fermentation to a food and aging the mixture. The amount of the strain used is appropriately adjusted according to the type of the fermented food and the kind of the bacterium good.

The Bacillus amyloliquefaciens strain RD7-7 of the present invention inhibits the growth of Bacillus cereus when co-cultured with Bacillus cereus causing food poisoning, has excellent antimicrobial activity against various microorganisms, , It is possible to produce a lot of viscous substance, have a high content of protease, amylase activity and amino nitrogen, and a low odor component generated during the fermentation process, which is excellent in fermentation characteristics and quality and can be usefully used in the production of safe fermented food have.

The present invention also provides a fermented food manufacturing method comprising the step of inoculating a strain of Bacillus amyloliquefaciens RD7-7 or a starter for fermentation of the present invention.

The fermented food manufacturing method may be a method of producing chungkukjang. In the case of the method for producing chungkugojang including the step of inoculating the Bacillus amyloliquefaciens RD7-7 strain or fermentation starter, the soybean is immersed for 18 hours at 20 ° C And thereafter increasing the temperature at 100 DEG C for 2 hours; Cooling the thus-grown beans to 40 ° C or lower, inoculating and mixing the Bacillus amyloliquefaciens strain RD7-7 or fermentation starter-selecting strain into soybeans; And fermentation for 36 hours in an incubator with a humidity of 70% and a temperature of 37 ° C.

The Bacillus amyloliquefaciens strain RD7-7 of the present invention inhibits the growth of Bacillus cereus when co-cultured with Bacillus cereus causing food poisoning, has excellent antimicrobial activity against various microorganisms, , The protein content, the amylase activity, and the amino nitrogen content are high and the odor components generated during the fermentation process are low, so that the fermentation characteristics and quality are excellent and they are usefully used in a safe fermented food manufacturing method .

The present invention also provides a pharmaceutical composition for antimicrobial use comprising a Bacillus amyloliquefaciens RD7-7 strain exhibiting an antimicrobial activity entrusted with the deposit number KACC 92071P.

The Bacillus amyloliquefaciens strain RD7-7 is preferably isolated from rice doenjang, but is not limited thereto.

The antimicrobial activity may be any one or more selected from the group consisting of Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Salmonella enteric and Bacillus cereus , And more preferably Bacillus cereus according to a specific embodiment of the present invention.

The composition of the present invention contains 0.1 to 99.9% by weight of the active ingredient containing the Bacillus amyloliquefaciens strain RD7-7 of the present invention, based on the total weight of the composition, and contains a pharmaceutically acceptable carrier, excipient or diluent can do.

The compositions of the present invention may be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.

Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of suppositories include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The composition of the present invention may be administered orally or parenterally, and it is preferable to select the intraperitoneal, rectal, rectal, intravenous, intramuscular, subcutaneous, intrauterine or intracerebral injection methods for parenteral administration, It is used for external skin.

The dosage of the composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease, and the daily dose is Bacillus amyloliquefaciens Is 0.01 to 1000 mg, preferably 30 to 500 mg / kg, more preferably 50 to 300 mg / kg, based on the amount of the active ingredient including the strain RD7-7, and is administered 1 to 6 times a day .

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

In addition, the present invention provides an antibacterial health functional food comprising Bacillus amyloliquefacience RD7-7 strain exhibiting antimicrobial activity entrusted with the deposit number KACC 92071P.

The health functional food of the present invention may contain various flavors or natural carbohydrates as an additional ingredient. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau Martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate may be selected from the range of 0.01 to 0.04 part by weight, specifically about 0.02 to 0.03 part by weight per 100 parts by weight of the health functional food of the present invention.

In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, protective colloid thickener, pH adjuster, stabilizer, preservative, glycerin, A carbonating agent used in a carbonated beverage, and the like. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health food of the present invention.

In addition, the present invention provides an antimicrobial feed additive comprising Bacillus amyloliquefaciens strain RD7-7 exhibiting antibacterial activity entrusted with the deposit number KACC 92071P.

Since the feed additive has antimicrobial activity, it is possible to prevent bacterial diseases by continuously feeding the feed additives to poultry, domestic animals, and the like, and it is possible to improve already existing bacterial diseases. Feeds can be classified into various categories according to nutritional value, main ingredient, distribution, moisture content, processing type, etc. Feeds can be used as feed, concentrate feed, supplementary feed, protein feed, starch feed, fat feed or fiber feed .

The feed additive may contain an additional acceptable carrier. For example, the feed additive may be added as it is or a known carrier, stabilizer or the like may be added. If necessary, various nutrients such as vitamins, amino acids, and minerals, antioxidants, antibiotics, antibacterial agents, and other additives may be added, and the form thereof may be a proper state such as powder, granule, pellet, . The feed additive of the present invention can be supplied singly or in a feed mixture.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following Examples and Experimental Examples.

Example 1 Identification of Various Antimicrobial Spectrum of Fermented Microorganisms Derived from Soybean Fermented Foods

In order to confirm the antimicrobial spectrum of fermenting microorganisms derived from soybean fermented foods, Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Salmonella enteric and Bacillus cereus ) Was subjected to an agar spot assay.

<1-1> Identification of Bacillus amyloliquefaciens RD7-7 strain

The isolates of Bacillus amyloliquefaciens RD7-7 were analyzed by 16 r DNA sequencing. For 16S rDNA gene sequencing, the chromosomal DNA of the strain was extracted according to the manufacturer's instructions using a DNA separation kit (GenEX genomic Sx DNA kit, GeneAll Biotechnology Co., Seoul, Korea) (SEQ ID NO: 13) and 1492R (5'-GGATACCTTGTTACGACTT-3 ', SEQ ID NO: 14) primers were used to amplify the 16S rDNA gene fragment of the isolated strain by polymerase chain reaction (PCR) The nucleotide sequence was analyzed (Genocell, Daejeon, Korea). The results were compared with the nucleotide sequences of the NCBI GenBank database (http://ncbi.nlm.nih.gov) to confirm phylogenetic relationships.

<1-2> Agaspot assay

Nutrient agar containing 0.5% of food poisoning bacteria (OD: 0.4) was dispensed into petri dishes and allowed to stand at room temperature. 10 μl of the selected strain (OD: 0.4) (-, not shown; +, 0.30 mm; ++, 0.31-0.5 mm; and ++) in the clear zone produced after incubation at 37 ° C for 24-48 hours. +, 0.51-0.80 mm).

As a result, as shown in Table 1, the antimicrobial activity of the fermenting microorganisms was confirmed, and Bacillus amyloliquefaciens RD7-7 strains excellent in antimicrobial activity against Bacillus cereus were selected. (Fig. 1).

name
Bell Antimicrobial activity R1 ¹ R2 ² R3 ³ R4 ⁴ R11 ⁵ RD5-4 Bacillus amyloliquefaciens subsp. plantarum FZB42 (T) + + + + - RD7-5 Bacillus tequilensis NRRL B-41771 (T) +++ ++ + ++ - RD7-7 Bacillus amyloliquefaciens subsp. amyloliquefaciens DSM 7 (T) ++ ++ + ++ +++ RD12-3 Bacillus amyloliquefaciens subsp. amyloliquefaciens DSM 7 (T) - - - - - RD13-6 Bacillus subtilis subsp. inaquosorum BGSC 3A28 (T) - - - - - RD13-9 Bacillus methylotrophicus CBMB205 (T) - - - - - HJ11-1 Bacillus subtilis strain ZT-4-5 + - + + - HJ11-3 Bacillus subtilis strain BRZ6 ++ - + + +

^1. Listeria monocytogenes (steria monocytogenes), ² of E. coli (Escherichia coli), ³ Staphylococcus aureus (Staphylococcus aureus), ⁴ Salmonella Entebbe rigs (Salmonella enteric) and ⁵ Bacillus cereus (Bacillus cereus)

Experimental Example 1 Confirmation of Bacillus amyloliquefaciens RD7-7 Growth Inhibitory Effect on Bacillus cereus (KACC10004)

<1-1> Confirmation of growth inhibition of Bacillus cereus strain by measuring the number of bacteria

In order to confirm the inhibitory effect of Bacillus amyloliquefaciens RD7-7 on the growth of Bacillus cereus, two bacteria were co-cultured and confirmed by measuring the number of bacteria.

Concretely, 0.5% of Bacillus cereus and 0.125%, 0.25%, 0.5% and 1% of Bacillus amyloliquefaciens RD7-7 were mixedly inoculated in 5 ml of LB medium (Luria-Bertani broth) and cultured at 37 ° C for 24 hours , And then 10-fold dilution was performed in sterile saline with decimal dilution. Then, the diluted solution was dispensed into CHROMagar, a selective medium for Bacillus cereus, and cultured at 37 DEG C for 24 hours. Then, the number of Bacillus cereus strains was measured.

As a result, as shown in Table 2, the experimental group co-cultivated with Bacillus cereus and Bacillus amyloliquefaciens RD7-7 inhibited the growth of Bacillus cereus depending on the concentration of Bacillus amyloliquefaciens RD7-7 And the experimental group co-cultured with the control strain Bacillus amyloliquefaciens R48 KACC 15866 did not inhibit the growth of Bacillus cereus (Table 2).

Sample cfu / ml Survival rate (%) The control (Bacillus cereus KACC10004) 2.65 x 10 ⁸ Bacillus amyloliquefaciens
RD 7-7 0.13% 2.71 × 10 ⁷ 88.24 0.25% 1.36 x 10 ⁷ 84.67 0.50% 9.35 × 10 ⁵ 70.88 One% 4.20 x 10 ³ 43.01 Bacillus amyloliquefaciens
KACC 15866 0.13% 3.40 x 10 ⁸ 101.28 0.25% 2.70 x 10 ⁸ 100.10 0.50% 3.05 x 10 ⁸ 100.72 One% 3.00 x 10 ⁸ 100.64

<1-2> Confirmation of growth inhibition of Bacillus cereus by real-time polymerase chain reaction

Real time polymerase chain reaction (PCR) was performed to confirm the growth inhibition effect of Bacillus cereus in the culture solution in which the Bacillus cereus and Bacillus amyloliquefaciens strain RD7-7 in the experimental example <1-1> were co-cultured. Respectively.

Concretely, the culture medium in which 0.5% of Bacillus cereus and 0.125%, 0.25%, 0.5% and 1% of Bacillus amyloliquefaciens RD7-7 were co-cultured was diluted stepwise to 10, 100, 500 and 1000 times to prepare QIAamp DNA mini (QIAGEN, USA) according to the manufacturer's protocol. Then, real-time PCR was performed using the Bacillus cereus real time PCR kit under the conditions shown in the following Table 3 with the groEL gene as a target, wherein the standard curve was diluted with 10, 100, 500 and 1000 times The number of strains on one medium and the value of the threshhold cycle (C (t)) of real time PCR were used.

PCR step Temperature time Number of cycles Uracil-N-glycosylase 50 ℃ 2 minutes One preheat 95 ℃ 10 minutes One Denaturation 95 ℃ 30 seconds 40 Annealing 55 ° C 50 seconds

As a result, a standard curve was derived as shown in Fig. 2 (Fig. 2). As shown in Table 4, it was confirmed that the growth of Bacillus cereus was inhibited by the Bacillus amyloliquefaciens strain RD7-7, &Lt; 1-1 > opinion.

Sample C (t) y = -3.9756x + 49.693
log cfu / ml The control (Bacillus cereus KACC10004) 18.46 7.86 Bacillus amyloliquefaciens
RD7-7 0.125% 19.08 7.70 0.25% 21.29 7.14 0.5% 23.41 6.61 One% 25.07 6.19 Bacillus amyloliquefaciens
KACC 15866 0.125% 16.85 8.26 0.25% 15.99 8.48 0.5% 16.47 8.36 One% 16.64 8.31

<1-3> Confirmation of inhibition of Bacillus cereus growth by confirming the transcription amount of toxin-related gene

In order to confirm the inhibitory effect of growth of Bacillus cereus, the amount of transfer of the toxin related genes of Bacillus cereus, groEL and nheA gene was confirmed.

Specifically, mRNA was extracted using the RNeasy plus mini kit (QIAGEN) in the co-culture solution of Experimental Example <1-1>, and cDNA was synthesized from the mRNA using cDNA synthesis kit (amfiRivert Platinum cDNA synthesis Master Mix, GenDEPOT) Were synthesized. Then, using the synthesized cDNA, the expression level of the gene was confirmed by reverse transcription PCR (RT-PCR), and the expression of CFX96 Touch ™ Real-Time (Bio-Rad) using iQ ™ SYBR® Green Supermix Signals were quantified and compared quantitatively in the PCR Detection System. PCR conditions and primers were as shown in Tables 5 and 6 below.

step Temperature time Number of cycles Initial denaturation and enzyme activity 95 ℃ 3 minutes One denaturalization 95 ℃ 15 seconds 33 (RT-PCR)
40 (qPCR) Combination 55 ° C 30 seconds kidney 72 ℃ 30 seconds (qPCR) Melting curve 72 ℃ 30 seconds One

SEQ ID NO: primer The column (5'-3 ') Usage One groEL -L 5'-TGCAACTGTATTAGCACAAGCT-3 ' RT-PCR, qPCR 2 groEL -R 5'-TTACCAACGCGCTCCATTGCTT-3 ' RT-PCR, qPCR 3 nheA -L 5'-GTTAGGATCACAATCACCGC-3 ' RT-PCR, qPCR 4 nheA -R 5'-ACGAATGTAATTTGAGTCGC-3 ' RT-PCR, qPCR 5 nheC- L 5'-TGGATTCCAAGATGTAACG-3 ' RT-PCR, qPCR 6 nheC- R 5'-ATTACGACTTCTGCTTGTGC-3 ' RT-PCR, qPCR 7 entFM -L 5'-AAAGAAATTAATGGACAAACTCAAACTCA-3 ' RT-PCR, qPCR 8 entFM -R 5'-GTATGTAGCTGGGCCTGTACGT-3 ' RT-PCR, qPCR 9 ces -L 5'-GGTGACACATTATCATATAAGGTG-3 ' RT-PCR, qPCR 10 ces -R 5'-ATTCAACATAATATTATACGCCGT-3 ' RT-PCR, qPCR 11 16s-rRNA-L 5'-AGAGTTTGATCCTGGCTCAG-3 ' RT-PCR, qPCR 12 16s-rRNA-R 5'-GGCTACCTTGTTACGACTT-3 ' RT-PCR, qPCR

As a result, as shown in FIG. 3 and FIG. 4, the Bacillus amyloliquefaciens strain RD7-7 decreased the transcription amount of the groEL and nheA genes of Bacillus cereus strain in a concentration-dependent manner (FIGS. 3 and 4) It was confirmed that the growth of the mouse was inhibited.

<1-4> Confirmation of inhibition of Bacillus cereus growth by confirming the expression level of AtpB protein

Western blotting was performed to confirm the expression inhibition of AtpB protein in order to confirm the inhibitory effect of growth of Bacillus cereus.

Specifically, SDS sample buffer was added to the cell pellet obtained by centrifuging the co-culture solution at 10,000 × g for 5 minutes, denatured at 100 ° C. for 10 minutes, and electrophoresed on a 12% SDS polyacrylamide gel After migration, the gel was transferred to a nitrocellulose membrane. (Tris-buffered saline with 0.1% TBS-T) and incubated for 1 hour with anti-AtpB primary antibody (Affinity purified rabbit anti-AtpB, Agrisera, Vannas, Sweden) Tween-20) solution at 10 min intervals. The membranes were then placed in a solution of horseradish peroxidase (HRP) -conjugated goat IgG secondary antibody (Bio-Rad, USA) and reacted for 1 hour. Then, the membrane was washed three times at 10-minute intervals with TBS-T solution, and developed on a x-ray film by using a chemiluminescence blotting substrate (POD) (Roche, Mannheim, Germany) .

As a result, as shown in FIG. 5, it was confirmed that the strain of Bacillus amyloliquefaciens RD7-7 decreased the expression of AtpB protein of Bacillus cereus in a concentration-dependent manner (FIG. 5).

<Experimental Example 2> Fermentation characteristics of Bacillus amyloliquefaciens RD 7-7 in soybean fermented product (Chungkukjang)

In order to confirm the fermentation characteristics of Bacillus amyloliquefaciens RD7-7 and the inhibitory effect of Bacillus cereus on the fermented soybean, the following experiment was conducted.

&Lt; 2-1 > Production of soybean fermentation product

Specifically, in order to prepare soybean fermented product, the soybean was immersed at 20 ° C for 18 hours and then at 100 ° C for 2 hours. The soybeans were cooled to below 40 ° C, and then cultured in 1 kg of soybeans in a culture medium (OD: 0.5, 10 ⁸ CFU / ml) obtained by culturing the selected strains in LB medium (Difco, ), And the mixture was placed in a styrofoam box (21 × 27 × 1.8 cm), and the control group was spontaneously fermented without inoculating the strain. Bacillus amyloliquefaciens RD7-7 strain and Bacillus cereus were inoculated at different ratios, and the fermentation conditions were fermented for 36 hours in an incubator at a humidity of 70% and a temperature of 37 ° C to prepare soybean fermented products.

<2-2> Fermentation characteristics of Bacillus amyloliquefaciens RD7-7 strain in soybean fermentation and inhibition effect on Bacillus cereus growth

As a result, as shown in FIG. 6, it was confirmed that Sample 1, which was fermented by inoculation with Bacillus amyloliquefaciens RD7-7, produced much more impurities than the control group not inoculated with bacillus amyloliquefaciens RD7-7, It was found that the fermentation characteristics of Cyanthus RD7-7 strain were excellent. In addition, Sample 2, which had been fermented by inoculation with Bacillus cereus, did not show any impurities and showed a pleasant smell, indicating that the fermentation did not proceed normally. In particular, in Samples 3 and 4 which were fermented by mixing Bacillus amyloliquefaciens RD7-7 and Bacillus cereus at 9: 1 or 5: 5, the effect of Bacillus amyloliquefaciens RD7-7 . (Fig. 6).

<Experimental Example 3> Confirmation of growth inhibitory effect of Bacillus cereus in soybean fermented product

In order to confirm the inhibitory effect of Bacillus amyloliquefaciens strain RD7-7 on the growth of Bacillus cereus in the fermented soybean, the protein was isolated from the soybean fermentation cells collected after the pretreatment according to the method of Experimental Example <1-4> And analyzed for the expression level of AtpB protein.

As a result, as shown in FIG. 7, when the RD7-7 strain and the Bacillus cereus were co-inoculated, the expression level of the AtpB protein was decreased depending on the concentration of the RD7-7 strain, indicating that the growth of Bacillus cereus was inhibited (Fig. 7).

<Experimental Example 4> Determination of fermentation characteristics of Bacillus amyloliquefaciens RD 7-7

In order to confirm the fermentation characteristics of Bacillus amyloliquefaciens RD 7-7, the following experiment was conducted.

<4-1> Preparation of soybean fermented product and preparation of sample extract

The soybean fermented product was prepared according to the method of Experimental Example < 2-1 >, and the inoculation strain was Bacillus amyloliquefaciens RD7-7, Bacillus subtilis HJ18-4, Bacillus amyloliquefaciens HJ5-2, and Bacillus amyloliquefaciens HJ39-5 were used.

The extract solution was prepared by homogenizing 100 g of distilled water with 20 g of soybean fermented product. The supernatant was used as a sample solution by centrifugation (8,000 × g, 10 min).

<4-2> Measurement of the number of microorganisms

In the sample extract obtained from the soybean fermentation product of Experimental Example <6-1>, the number of microorganisms was measured by the following method.

Specifically, 1 g of each sample extract was diluted to 10 levels by a decanteration method using sterile physiological saline, and then the diluted solution was cultured in a plate count agar (Difco) (37 캜 for 24 hours) The number of lactic acid bacteria was counted by incubating the diluted solution with MRS agar (Difco) in a gas pack (Gas Pack, Oxoid, Hampshire, England) under anaerobic conditions (37 ℃ for 48 hours) CFU / g).

As a result, as shown in Fig. 8, the total number of bacteria was the lowest in the control group of 1.12 x 108 CFU / ml, and the fermented product inoculated with Bacillus amyloliquefaciens HJ5-2 was 3.00 x 109 CFU / ml Respectively. As a result of measuring the number of lactic acid bacteria, the number of bacteria was the highest at 1.40 × 10 8 CFU / ㎖ in the control group not inoculated with the strain, and the soybean fermented inoculated with the strain was 2.50 × 10 2 ~ 7.30 × 10 4 CFU / ㎖, respectively. It is believed that this was dominant by inoculation of Bacillus spp. And inhibited the production of other microbes (Fig. 8).

<4-3> Measurement of enzyme activity

The activity of the proteolytic enzyme was measured by modifying a known method (Korean J Food Sci Technol 30: 1333-13380). Specifically, 0.6% casein substrate solution (0.2 M phosphate buffer, pH 7.0) was added to 1 ml of the sample extract, followed by reaction at 37 ° C for 10 minutes. After the reaction, 5 ml of 0.44 M trichloroacetic acid (TCA) was added to terminate the reaction. Then, the solution was allowed to stand at room temperature for 30 minutes, and 5 ml of a 0.55 M sodium carbonate (Na2CO3) solution and 4 ml of a Folin reagent solution (4 times diluted) were added to 2 ml of the filtrate filtered through a filter paper (No.2, Whatman, Buckinghamshire, UK) 1 ml was added, and the reaction was carried out at room temperature for 30 minutes, and the absorbance was measured at 660 nm. The standard substance, tyrosine, was diluted stepwise and analyzed in the same manner, and 1 unit was calculated by converting the amount of 1 μg of tyrosine liberated for 1 minute using a standard curve.

Alpha-amylase activity was measured by DUN (Dextrinogenic Unit of Nagase, Yoon KS. 1988., Kunkuk University, Seoul, Korea). Specifically, 1 ml of the sample extract was added to 3 ml of 1% soluble starch (0.02 M phosphate buffer, pH 7.0), reacted at 40 ° C for 10 minutes, and 10 ml of 1 M hydrochloric acid was added to stop the reaction. Iodine solution (0.005% I2 + 0.05% KI) (10 ml) was added, and the absorbance at 660 nm was measured. For the blank test, 10 ml of 1 M hydrochloric acid (HCl) was added to stop the reaction. The reaction solution was added to the reaction solution to measure color development. The amount of 0.1 mg of starch was dissolved in 1 ml of the crude enzyme solution for 1 minute. unit.

As a result, as shown in FIG. 9, the protease activity of the soybean fermented product inoculated with the Bacillus amyloliquefaciens RD7-7 strain was the highest at 647.92 unit / ml, and the strain was inoculated The lowest activity of the control group was 318.13 unit / ㎖. It has been reported that the strain having high protein activity has been reported to reduce the odor of chongkukjang and improve the taste, and thus it has been confirmed that the Bacillus amyloliquefaciens strain RD7-7 of the present invention can be usefully used for the production of chungkukjang ).

In addition, as shown in FIG. 9, the activity of alpha-amylase activity was the highest at 33.39 unit / ml in the control group not inoculated with the strain, which is considered to be caused by various microorganisms due to natural fermentation. The soybean fermented inoculated with Bacillus amyloliquefaciens RD7-7 or Bacillus subtilis HJ18-4 showed the highest activity at 20.82 and 18.45 unit / ㎖, respectively. Amylase activity is involved in the sweetness component of sugar and is considered to be an enzyme important in terms of quality. Therefore, it was confirmed that the RD7-7 strain of the present invention can be effectively used in the production of chungkukjang (Fig. 10).

<4-4> Measurement of reducing sugar

The reducing sugar content was measured by the DNS method (dinitrosalicylic acid, Anal Biochem 2: 127-132). Specifically, 1 ml of the sample extraction solution diluted in 3 ml of the DNS reagent was added, and the reaction was carried out at 37 ° C for 5 minutes. After cooling, the absorbance was measured at 550 nm, and the reducing sugar content was calculated from a standard curve using glucose.

As a result, as shown in FIG. 10, the content of reducing sugar was 1.31 ~ 2.35%, and the content of soybean fermented inoculated with Bacillus subtilis HJ18-4 was high (2.35%).

<4-5> Amino nitrogen measurement

Amino nitrogen content was determined by the well-known Formol titration method (Korean J Food Preserv 14: 356-363). Specifically, 2 ~ 3 drops of 0.5% phenolphthalein solution was added to a mixed solution of 5 ml of sample extract, 10 ml of formalin solution and 10 ml of distilled water, and 0.1 N sodium hydroxide (NaOH) was added thereto. Were calculated using the titration amount until the color became red and the appropriate amount of the experiment.

As a result, as shown in Fig. 11, the amino acid nitrogen content was 112.85 ~ 227.69 mg%, and the content of Bacillus amyloliquefaciens RD7-7, which had a high protease activity, was 227.96 mg%. Amino nitrogen is used as a measure of the degree of fermentation of soybean fermented food because it is due to the amino acid produced by the proteolytic enzyme produced by the microorganism during fermentation and thus soybean fermented product inoculated with Bacillus amyloliquefaciens RD7-7 It was confirmed that the fermentation was increased.

<4-6> Amino acid and organic acid measurement

Free amino acid content was analyzed according to the known method (Korean J Food & Nutr 25: 505-512.). Specifically, 3 g of the sample extract was taken, 30 ml of 70% ethanol was added, homogenized for 1 hour, and then centrifuged at 21,000 x g for 15 minutes. Then, the supernatant was repeatedly extracted twice with 25 ml of 70% ethanol, and the combined extracts were concentrated under reduced pressure using a rotary evaporator (EYELA A-3S, Rikakikai Co., Ltd., Tokyo, Japan). The concentrate was dissolved in 20 ml of 0.02 N hydrochloric acid, diluted 10-fold and filtered through a 0.45 ㎛ membrane filter (Merck Millipore, Darmstadt, Germany) and analyzed using an amino acid automatic analyzer (Blochrom 30, Biochrom Ltd., Cambrdge, UK) Respectively.

For analysis of the organic acid content, 3 g of the sample extract and 30 ml of distilled water were placed in a 50 ml flask and sonicated for one hour. After cooling to room temperature, the distilled water was filled up to the marking, filtered through filter paper (No. 2, Whatman), and filtered through a 0.45 μm membrane filter (Merck Millipore). The organic acid standard material was dissolved in the same solvent as the sample and used for preparing a calibration curve. The column was packed with cap cell pak C18-AQ (80A, Shiseido, Tokyo, Japan) and analyzed with a detector The detector was analyzed with UV 210 nm and the mobile phase with 0.1 M ammonium phosphate (NH ₄ H ₂ PO ₄ , pH 2.5) at a flow rate of 1.0 ml / min.

As a result, as shown in Table 7, the amino acid content of glutamic acid was 3.18 ~ 3.39%, aspartic acid was 1.87 ~ 2.07%, leucine was 1.28 ~ 1.48% Lysine was detected in the order of 1.05-1.18% and arginine was detected in the order of 1.03-1.32%. The total amino acid content of soybean fermented product inoculated with Bacillus amyloliquefaciens HJ5-2 and Bacillus amyloliquefaciens HJ39-5 was the highest at 18.38 and 17.07% (Table 7). Aspartic acid, bitter valine, isoleucine, and leucine, which break down the protein by various enzymes derived from Bacillus sp. During soybean fermentation, It is known that free amino acids such as methionine and phenylalanine, sweet alanine, glycine and lysine are produced and combined to give a unique taste and flavor of soybean fermented product (J Agric Food Chem 48: 716-723.).

amino acid(%) Control ¹ RD7-7 ² HJ5-2 ³ HJ18-4 ⁴ HJ39-5 ⁵ Aspartic acid 1.87 1.90 2.07 1.89 1.92 Threonine 0.57 0.57 0.61 0.54 0.57 Serine 0.83 0.82 0.85 0.81 0.84 Glutamic acid 3.18 3.25 3.39 3.38 3.31 Proline 0.90 0.99 1.09 0.91 0.93 Glycine 0.69 0.71 0.75 0.69 0.71 Alanine 0.78 0.76 0.91 0.77 0.78 Cysteine 0.09 0.07 0.08 0.08 0.08 Balin 0.89 0.90 0.99 0.89 0.93 Methionine 0.20 0.19 0.23 0.19 0.22 Isoleucine 0.81 0.83 0.91 0.80 0.83 Leucine 1.38 1.38 1.48 1.28 1.37 Tyrosine 0.60 0.60 0.70 0.54 0.64 Phenylalanine 0.94 0.97 1.07 1.02 0.94 Histidine 0.46 0.46 0.51 0.48 0.46 Lysine 1.08 1.07 1.18 1.05 1.06 Arginine 1.31 1.22 1.32 1.03 1.21 Tryptophan 0.27 0.27 0.27 0.25 0.27 Total amount 16.83 16.94 18.38 16.62 17.07

¹ control: test group not inoculated with Bacillus, ² HJ18-4: test group inoculated with Bacillus subtilis HJ18-4, ³ RD7-7: test group inoculated with Bacillus amyloliquefaciens RD7-7, ⁴ HJ5-2 : Experimental group inoculated with Bacillus amyloliquefaciens HJ5-2, and ⁵ HJ39-5: Bacillus amyloliquefaciens HJ39-5.

As shown in Table 8, the organic acid content of oxalic acid was 36.51 ~ 63.57 ㎎ / 100 g in all experimental groups, and succinic acid was detected in the control and RD7-7 inoculated soybean fermentations at 600.15 ~ 429.49 mg / 100 g was detected (Table 8). In the control group, the content of organic acid was higher than that of the soybean fermented inoculated with the strain, which is due to the production of organic acids by fermentation with lactic acid, which is considered to be related to the higher lactic acid bacteria count in the control group than the inoculated fermented Bacillus strain.

Organic acid (mg / 100 g) Control ¹ RD7-7 ² HJ5-2 ³ HJ18-4 ⁴ HJ39-5 ⁵ Citric acid Non-detection Non-detection Non-detection Non-detection Non-detection Suche mountain 600.15 429.49 Non-detection Non-detection Non-detection Lactic acid Non-detection Non-detection Non-detection Non-detection Non-detection Oxalic acid 63.57 57.05 40.3 65.28 36.51 Fumaric acid Non-detection Non-detection Non-detection Non-detection Non-detection Malian Non-detection Non-detection Non-detection Non-detection Non-detection

¹ control: test group not inoculated with Bacillus, ² HJ18-4: test group inoculated with Bacillus subtilis HJ18-4, ³ RD7-7: test group inoculated with Bacillus amyloliquefaciens RD7-7, ⁴ HJ5-2 : Experimental group inoculated with Bacillus amyloliquefaciens HJ5-2, and ⁵ HJ39-5: Bacillus amyloliquefaciens HJ39-5.

<4-7> Fragrance analysis

Add 10 ml of the extraction solvent (diethyl ether: pentane = 1: 1) to 2 g of the sample extract, shake the extract (20 ° C, 15 hr) and centrifuge at 8,000 rpm for 10 min. Sodium sulfate ) Was added to adsorb moisture. Thereafter, the supernatant was filtered (No. 2, Whatman), and the filtrate was re-filtered using a 0.2 μm syringe filter (Merck Millipore) and used as an analytical sample. The fractions were analyzed by GC / MS (QP2010, Shimadzu, Kyoto, Japan) and the columns were HP-innowax (60 m / ID 0.25 μm, Agilent, Santa Clara, CA, USA). The oven temperature was maintained at 40 ° C for 5 minutes, then increased to 230 ° C at 2 ° C per minute and held for 30 minutes. The injection temperature was 250 ° C, the carrier gas was helium (He), and the column flow rate was 0.74 ml / min. The compound was identified by searching the mass spectrum obtained by GC-MS using a Wiley 275 data base. For quantitative analysis, the content of each compound was determined using 4-methyl-2-pentanol (50 ppm) .

As a result, as shown in Table 9, three esters, six ketones, two aldehydes, four alcohols, two hydrocarbons, pyrazines, Two kinds of acids, four kinds of acids and four kinds of fragrance components were detected. 23 kinds of fragrance components were detected in soybean fermented product inoculated with Bacillus amyloliquefaciens RD7-7 or Bacillus subtilis HJ18-4 and 15 fragrance components were detected in soybean fermented product inoculated with HJ39-5 . Trimethyl pyrazine and tetramethyl-pyrazine were detected in the pyrazines known as volatile fragrance components in the chungkukjang. Tetramethyl-pyrazine, which is known as a stimulant of Chungkookjang, was detected in the RD7-7 and HJ39-5 strains But not in fermented products (Table 9). Bacillus amyloliquefaciens RD7-7 and Bacillus subtilis HJ18-4 were found to be slightly lower in the butyric acid and valeric acid than the other strains. Therefore, it was confirmed that the Bacillus amyloliquefaciens strain RD7-7 of the present invention produced less off-taste during fermentation.

Unit (ppm) Fragrance component RT1 (min) RD7-7 HJ5-2 HJ18-4 HJ39-5 Ester
(esters) 2-propenoic acid, 2-propenyl ester 6.233 0.46 0.45 0.16 Non-detection formic acid, ethyl ester 7.074 1.52 0.60 1.92 1.42 acetic acid, ethyl ester 8.486 78.00 71.82 71.65 76.45 Ketone
(ketones) 2-methyl-3-heptanone 6.445 2.44 2.53 2.08 2.36 2-butanone 8.893 Non-detection 6.11 Non-detection Non-detection 2,3-butanedione 11.905 2.74 2.09 3.04 2.05 acetone 30.852 185.25 93.28 290.90 246.37 2,2-dimethyl-cyclohexanone-4,4-d2 59.011 Non-detection 4.02 Non-detection Non-detection 2-oxazolidinone 59.023 4.27 3.04 Non-detection Aldehyde
(aldehydes) 2-methyl-propanal 8.928 2.29 Non-detection 2.56 2.52 acetaldehyde 11.733 0.76 0.75 0.64 0.63 Alcohol
(alcohols) ethanol 9.875 0.91 1.04 0.32 0.63 2-butanol 14.072 4.57 4.32 4.16 4.41 1,3-butanediol 47.951 58.20 22.05 44.14 69.51 1-butanol 61.597 1.37 Non-detection Non-detection Hydrocarbon
(hydrocarbons) benzene 10.164 89.73 87.62 83.48 90.95 1 H-indene 44.410 0.46 0.75 Non-detection Non-detection Pyrazine
(pyrazines) trimethyl-pyrazine 38.684 0.61 3.43 1.92 Non-detection tetramethyl-pyrazine 43.192 Non-detection 2.68 2.24 Non-detection mountain
(acids) 이소 부추리산 49.061 0.76 8.34 2.56 Non-detection isovaleric acid 55.069 1.68 46.34 2.08 3.94 propanoic acid 60.788 0.91 34.57 4.00 0.95 karbonic acid 63.165 0.76 Non-detection 1.76 Non-detection Other cyclohexane 6.107 17.22 17.29 16.15 17.50 1-phenyl-4-penten-1-yne 65.112 Non-detection Non-detection 6.24 Non-detection 2-methyl-naphthalene 66.999 2.74 2.24 1.60 1.42 3 - [(trimethylsilyl) oxy] furan 70.852 1.07 Non-detection 0.64 Non-detection

<4-8> Statistical Analysis

The significance of the mean values was verified by using the soft-ware package (version 12) of the SPSS system (SPSS Inc., Chicago, IL, USA) And tested by Duncan's multiple range test.

<110> Foundation of Agri. Tech. Commercialization & Transfer <120> Bacillus amyloliquefacience having antibacterial activity and          uses thereof <130> 2015P-04-044 <160> 14 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Bacillus cereus <400> 1 tgcaactgta ttagcacaag ct 22 <210> 2 <211> 22 <212> DNA <213> Bacillus cereus <400> 2 ttaccaacgc gctccattgc tt 22 <210> 3 <211> 20 <212> DNA <213> Bacillus cereus <400> 3 gttaggatca caatcaccgc 20 <210> 4 <211> 20 <212> DNA <213> Bacillus cereus <400> 4 acgaatgtaa tttgagtcgc 20 <210> 5 <211> 19 <212> DNA <213> Bacillus cereus <400> 5 tggattccaa gatgtaacg 19 <210> 6 <211> 20 <212> DNA <213> Bacillus cereus <400> 6 attacgactt ctgcttgtgc 20 <210> 7 <211> 29 <212> DNA <213> Bacillus cereus <400> 7 aaagaaatta atggacaaac tcaaactca 29 <210> 8 <211> 22 <212> DNA <213> Bacillus cereus <400> 8 gtatgtagct gggcctgtac gt 22 <210> 9 <211> 24 <212> DNA <213> Bacillus cereus <400> 9 ggtgacacat tatcatataa ggtg 24 <210> 10 <211> 24 <212> DNA <213> Bacillus cereus <400> 10 attcaacata atattatacg ccgt 24 <210> 11 <211> 20 <212> DNA <213> Bacillus cereus <400> 11 agagtttgat cctggctcag 20 <210> 12 <211> 19 <212> DNA <213> Bacillus cereus <400> 12 ggctaccttg ttacgactt 19 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotide <400> 13 agagtttgat catggctcag 20 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> synthetic nucleotide <400> 14 ggataccttg ttacgactt 19

Claims (13)

Bacillus amyloliquefacience RD7-7 strain exhibiting antimicrobial activity entrusted with accession number KACC 92071P.
The Bacillus amyloliquefaciens strain RD7-7 according to claim 1, wherein the strain is isolated from rice doenjang.
The method according to claim 1, wherein the antimicrobial activity is selected from the group consisting of Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Salmonella enteric, and Bacillus cereus. Wherein the bacterium belongs to any one or more of the bacterium belonging to the genus Bacillus amyloliquefaciens RD7-7.
A starter for fermentation comprising as an active ingredient at least one selected from the group consisting of the Bacillus amyloliquefaciens RD7-7 strain of claim 1, a culture of the strain, a concentrate of the culture and a dried product of the culture, .
A fermented food produced using the Bacillus amyloliquefaciens strain RD7-7 of claim 1 or the fermentation starter of claim 4.
The fermented food according to claim 5, wherein the fermented food is soybeans.
The fermented food according to claim 5, wherein the fermented food is chunggukjang.
A method for producing a fermented food comprising the step of inoculating the strain of Bacillus amyloliquefaciens RD7-7 of claim 1 or the fermentation starter of claim 4.
A pharmaceutical composition for antimicrobial use containing Bacillus amyloliquefacience strain RD7-7, which exhibits antimicrobial activity entrusted with the deposit number KACC 92071P, as an active ingredient.
10. The antimicrobial pharmaceutical composition according to claim 9, wherein the Bacillus amyloliquefaciens RD7-7 strain is isolated from rice doenjang.
10. The method of claim 9, wherein the antimicrobial activity is selected from the group consisting of Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Salmonella enteric and Bacillus cereus. Wherein the antimicrobial agent is one or more of the following strains.
Bacillus amyloliquefacience RD7-7 strain exhibiting antimicrobial activity entrusted with deposit number KACC 92071P as an active ingredient.
An antimicrobial feed additive containing Bacillus amyloliquefacience strain RD7-7 as an active ingredient exhibiting antimicrobial activity entrusted with the deposit number KACC 92071P.

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