WO2011158998A1 - Strain of bacillus amyloliquefaciens having a high vitamin k2 producing ability - Google Patents

Abstract

The present invention relates to a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens KCTC11712BP) having an outstanding menaquinone synthesising ability, and to a method for making cheonggukjang by using the same. More specifically, provided are conditions for making cheonggukjang optimised to contain a large amount of vitamin K₂, by using a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens KCTC11712BP). The cheonggukjang according to the present invention contains a larger amount of menaquinone than conventional cheonggukjang and can be used to advantage as a functional food able to help prevent osteoporosis.

Description

Bacillus amiriquifaciens strains with high vitamin 생성 2 production

The present invention relates to a Bacillus amyloliquefaciens BY-1 strain (KCTC11712BP) excellent in the ability to synthesize menaquinone (menaquinone), and a method of preparing cheongguk using the same.

With the recent increase in the elderly population, osteoporosis has become a serious threat to human health problems. Osteoporosis is defined as a systemic disease in which bone strength is weakened and fractures occur easily.

Osteoporosis is asymptomatic in itself, but due to low bone mass and the deterioration of bone tissue structure, osteoporosis increases the risk of fractures of the femur, spine and wrist, resulting in high morbidity and mortality. In the United States, as of 1995, US $ 13.8 billion is spent annually for the treatment of diseases related to osteoporosis, and socioeconomic costs related to osteoporosis are increasing. Osteoporosis progresses slowly with age in men, whereas bone density decreases rapidly in menopause, with a relatively high prevalence among menopausal women. In Korea, 50% of Korean women over 50 years have osteopenia and 20-30% have osteoporosis, and according to 2007 statistics, the prevalence of osteoporosis in Korean women aged 60-69 is 24.9%.

Therefore, the magnitude of osteoporotic diseases such as fractures is also expected to increase significantly. The most problematic factor among osteoporosis is the amount of calcium in the bone microstructure, which is suggested to consume less than 75% of the recommended intake, which is 700 ~ 800mg suggested by the Korean nutrition standard. . As a preventive treatment for osteoporosis, vitamine D and vitamine K are taken to help supplement hormones and the absorption of calcium ions into bone microstructure. Recent studies have shown that vitamine K has a function of bone metabolism. Vitamin K is required for the synthesis of proteins that coagulate blood. There are two types of vitamins: K ₁ (phylloquinone) and K ₂ (menaquinone).

Vitamin K ₁ is contained in plants, and vitamin K ₂ is synthesized by bacteria. Analog of vitamin K ₂ are divided into various Bacillius subtilis in particular the production of menaquinone-7 (MK-7) occupies 90 to 96% of the total amount of vitamine K _2.

Therefore, there is an urgent need for the development of strains and health functional foods excellent in the synthesis of vitamin K ₂ .

The present inventors have recognized the necessity of the above, by separating the bacteria having excellent menaquinone synthesizing ability, and clarifying the culture conditions, the present invention tried to produce a functional cheonggukjang to help the absorption of calcium by producing a high meaquinone-containing Cheonggukjang, Bacillus amyloliquefaciens BY-1 strain having excellent quinone synthesis ability was isolated, and the present invention was completed by establishing a method of preparing high-containing menaquinone Chungkukjang.

Therefore, it is an object of the present invention to provide a strain having excellent menaquinone synthesis ability.

It is another object of the present invention to provide a composition for fermenting Cheonggukjang containing the strain.

Still another object of the present invention is to provide a method for preparing Chungkookjang containing a large amount of vitamin K ₂ using the strain.

It is also an object of the present invention to provide a functional food that can help to prevent osteoporosis, including Cheonggukjang prepared with optimized fermentation conditions.

The present invention provides a Bacillus amyloliquefaciens BY-1 strain (KCTC11712BP) excellent in the ability to synthesize menaquinone.

The strain was identified by gyr B gene sequencing, and was identified as Bacillus amiriquifaciens BY-1 (KCTC11712BP). In another aspect, the present invention provides a composition for fermenting Cheonggukjang comprising Bacillus amiriquifaciens BY-1 (KCTC11712BP), a culture of the strain or a culture of the strain.

In another aspect, the present invention provides a method for producing a cheongukjang characterized in that the fermentation of soybean crushed, soybean or black soybeans using the composition for fermentation.

The manufacturing method, the step of increasing the soybean, black beans or soybean crushed; Inoculating the soybean, black soybean or soybean crushed composition for fermentation of Cheonggukjang containing Bacillus amiriquifaciens BY-1 strain; And fermenting the inoculated soybean, black bean or soybean crushed product.

The fermentation of the step of fermentation is preferably carried out at 41 to 45 ℃, more preferably at 43 ℃.

Glycerol (glycerol) may be added in the step of fermentation, preferably, may be added in 8 to 12% by weight, more preferably, may be added in 10% by weight.

The present invention also provides a cheongukjang prepared by the above method.

In another aspect, the present invention provides a food for the prevention or treatment of osteoporosis comprising Cheonggukjang prepared by the above method.

The new strain Bacillus amiriquifaciens BY-1 of the present invention has a higher menaquinone synthesis capacity than the known Bacillus subtils, and the cheonggukjang prepared using the optimized fermentation conditions of the present invention is a conventional fermented food. Compared with higher K ₂ vitamins, it can be useful as a functional food that can help prevent osteoporosis.

Figure 1 shows the gyrase B gene sequence number between BY-1 and other strains. (Sp: AB010081, Bacillus sp .., cer: CP000227, Bacillus cereus Q1., Ant: AB190227, Bacillus anthracis ., Thu: CP000485, Bacillus thuringiensis str., Sub-sub: NZ_ABQK01000001, Bacillus subtilis sub sp. Subtilis str . Thr-kon: Bacillus thuringiensis serovar konkukian str ., Lich, NC_006270, Bacillus licheniformis ATCC 14580., Pum, NC_009848, Bacillus pumilus SAFR-032., Halo, NC_002570, Bacillus halodurans C-125., Amyl, CP000560fa, Bacillens amylolique FZB42., Clau, AP006627, Bacillus clausii KSM-K16., Wei, NC_010184, Bacillus weihenstephanensis KBAB4.)

Figure 2 shows the HPLC chromatogram results of 0.5 ppm menaquinone.

Figure 3 shows the standard curve of the menaquinone (menaquinone (MK-4, 7)) obtained by HPLC.

4 is a schematic view showing the production method of the cheongukjang of the present invention.

Figure 5 shows the HPLC results of MK-7 produced in BY1 incubated for 3 days.

Figure 6 shows the HPLC results of MK-7 produced in BY1 stationary culture for 3 days after shaking culture at 40 ℃ 4 hours.

Figure 7 shows the HPLC results of MK-7 produced in BY1 stationary culture for 3 days after shaking culture for 6 hours at 40 ℃.

Figure 8 shows the HPLC results of MK-7 produced by B. amyloliquefaciens strain BY-1 in soybean extract when 0.2% sodium chloride was added during stationary culture.

Figure 9 shows the HPLC results of MK-7 produced by B. amyloliquefaciens strain BY-1 in soybean extract when 0.4% sodium chloride was added during stationary culture.

Figure 10 shows the HPLC results of MK-7 produced by B. amyloliquefaciens strain BY-1 in soybean extract when 0.6% sodium chloride was added during stationary culture.

Figure 11 shows the result of fermentation at 40 ℃ to determine the effect of fermentation temperature on the production of MK-4,7 in Cheonggukjang by inoculating the strain.

Figure 12 shows the result of fermentation at 43 ℃.

Figure 13 shows the result of fermentation at 46 ℃.

Figure 14 shows the effect of the carbon source on the production of menaquinone.

Figure 15 shows the effect of HPLC on the production of menaquinone upon addition of 5% glycerol.

Figure 16 shows the effect of HPLC on the production of menaquinone upon addition of 10% glycerol.

FIG. 17 shows the effect of HPLC on the production of menaquinone upon addition of 15% glycerol.

FIG. 18 shows the HPLC results of menaquinone produced in Cheonggukjang prepared using strain BY-1.

19 shows HPLC results of menaquinone produced in commercially sold Cheonggukjang.

Figure 20 shows the HPLC results of the menaquinone produced in Cheonggukjang prepared using Bacillus subtilis strain KCTC13241.

The present invention relates to Bacillus amyloliquefaciens BY-1 strain (KCTC11712BP) excellent in menaquinone synthesis ability.

The strain was isolated from conventionally prepared soy sauce, miso, and characterized by having the gyrB gene of SEQ ID NO: 1, which is basophils.

BY-1 strain of the present invention received a high score in the sensory evaluation, it was confirmed that the menaquinone synthesis ability is superior to the known strain, the amount is 7.568 ± 0.140mg / L. Conventionally, Bacillus subtilis was mainly used to produce metaquinone . However, Bacillus amyloliquefaciens was isolated to develop strains with excellent secretion capacity of menaquinone.

The BY-1 strain of the present invention was assigned to the Korean Collection for Type Cultures (KCTC) on June 11, 2010, in accordance with the provisions of the Budapest Treaty on the International Approval of Microbial Deposits in Patent Procedures. Deposited as.

The present invention also relates to a composition for fermenting Cheonggukjang comprising the Bacillus amiriquifaciens BY-1 (KCTC11712BP), a culture of the strain or a culture solution of the strain.

In addition, using the composition to provide a method for producing a soybean crushed soybean crushed, soybean or black soybeans, and provides conditions optimized to increase the metaquinone content of the soybeans.

Cheonggukjang production method of the present invention comprises the steps of increasing the soybean, black beans or soybean crushed; Inoculating the soybean, black bean or soybean crushed with Bacillus amiriquifaciens BY-1; And fermenting the inoculated soybean, black bean or soybean crushed product.

In one embodiment of the present invention, the difference in metaquinone synthesis ability according to the shaking culture time and sodium chloride concentration of the starter as a previous step for the fermentation of the Cheonggukjang. As a result, when the BY-1 strain was shaken for about 4 hours before stationary culture at 40 ° C. for 3 days, the amount of metaquinone synthesis was large, and the content of metaquinone was highest when the sodium chloride concentration was 0.4%.

Therefore, in the present invention, it is preferable that the seed culture is first cultured in 50 mL of TSB medium at 40 ° C. and 120 rpm for 4-6 hours, and second culture at 40 ° C. and 120 rpm in 20 mL medium containing 10% soybean extract for 4 hours. Do.

The method may include soaking soybean, black soybean or soybean crushed product in a salt solution of 0.2 to 0.6%, preferably 0.4% for 10 hours before the steaming step. In addition, the cooking step is preferably carried out for 30 minutes at 121 ℃.

After the inoculation of BY-1 bacteria of the present invention, the fermentation temperature is preferably carried out at 41 to 45 ℃, more preferably 43 ℃ at the time of this culture. Cultures at 43 ° C. may contain the highest amount of metaquinone.

In addition, when the glycerol is added in the fermentation step, the amount of metaquinone may be increased, and the amount may be added in an amount of 8 to 12% by weight, preferably 10% by weight.

The total content of MK-4, 7 contained in Cheonggukjang fermented at 43 ° C for 2 days under conditions without additives using Bacillus amyloliquefaciens BY-1 was fermented using the product produced in Pulmuone or B. subtilis KCTC13241 strain. It was more than twice the amount of Cheonggukjang and the content was 7.54 mg / kg.

The present invention also relates to Cheonggukjang prepared by the above method. Cheonggukjang fermented without the use of additives MK-4,7 is very high content of the concentration of 7.54 mg / kg.

In another aspect, the present invention provides a functional food that can help prevent osteoporosis, including Cheonggukjang prepared by the above method.

The osteoporosis refers to a systemic disease in which bone strength is weakened and fractures easily occur.

The food may be a health functional food.

The health functional food means a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients having useful functions for the human body. Functionality refers to obtaining useful effects on health use such as nutrient control or physiological action on the structure and function of the human body.

It is a food prepared by adding Cheonggukjang with high content of MK-4,7 as an active ingredient to food ingredients or manufactured in various forms of formulations, unlike general medicines, which may occur when taking medicines for a long time. There are no side effects and the like, and excellent in portability, the health functional food of the present invention can be taken as a supplement for the prevention or treatment of osteoporosis. The health functional food may be prepared by including food supplements acceptable food additives in addition to Cheonggukjang.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are provided to illustrate the present invention more easily, and the content of the present invention is not limited by the examples.

Preparation of Reagents and Instruments

The standard for vitamin K analysis was menaquinone-4,7 (Sigma-Aldrich Co., Louis Mo, USA) and the analytical solvent, Merk Cemical Co., Used. Pretreatment of the sample was performed using a centrifuge (Supra 21K, Hanil Co, Korea) and shaker (VS8480SFN, Vision Co. Korea) and spectrophotometer (UV-1650PC, Shimazu, Japan) for absorbance measurement. All used strain Difco (USA) medium.

Strain selection

In order to detect and isolate Bacillus sp. Strains and other strains that can be used for the production of Cheonggukjang from conventionally prepared soy sauce and soybean paste, 1 g of each sample was added to 9 mL of sterile water and diluted with LB agar (Difco Co., USA). , Pseudomonas isolation agar (Difco Co., USA), Lactobacillus was plated on MRS agar (Difco Co., USA), potato dextrose agar, actinomycetes isolation agar medium (Difco Co., USA). Strains were cultured for 1 to 3 days under aerobic conditions at 37 ° C. to isolate morphological differences.

Preparation of Soy Hydrothermal Leachate

After washing 2 kg of soybeans, they were immersed in tap water for 10 hours and then dehydrated. 20 L of distilled water was added to the dehydrated soybeans, and the mixture was steamed at 121 ° C. for 60 minutes to recover 18.5 L. The collected extract was packaged 100 mL each and used while storing in a refrigerator.

Viable cell count

Total aerobic bacteria were serially diluted 10-fold with physiological saline (0.8% NaCl) according to the APHA standard method, and then incubated at 40 ° C for 24 hours using plate count agar (Difco, Co., USA). After incubation, the colony was counted, and the colony forming unit per 1 mL of sample was expressed as CFU. Absorbance (O.D) was measured by diluting the culture broth with distilled water 1: 1 (v: v) in the same way as measuring the number of viable cells, and measured the optical density at 660 nm using a spectrophotometer.

Cheonggukjang Manufacturing

Raw soybean was purchased from 2008 Korean white baektae and soaked for 12 hours at room temperature and then cooled to 40 ℃ after 30 minutes steaming at 30 ℃. In order to confirm the fermentation capacity of the isolated strains, each strain was cooled in 10% soybean extract and inoculated at 5% (v / w) in the soybean extract. Humidity was used for 48 hours after incubation in a constant temperature and humidity chamber (VS-9111H-800, Vision Scientific Co. Korea).

Biochemical Analysis by API Kit

Among the isolates, the strains of the genus Bacillus were analyzed using API 20E and 50CHB system kit (Biomereux Co., France). Investigation of sugar fermentation capacity using the API kit was carried out according to the kit manual and transferred to the kit strip and incubated for 24 hours at 43 ℃ ATB identification computer system (bio Merieux Co., France) Investigate by typing. 96-99% excellent, 93-95% very good, 82-92% good, 85-88% acceptable, Low discrimination, unidentified strains requiring further differentiation were considered.

Made by Primer

primer (primer) of the gyrB gene is a conserved domain of the gyrB gene of the Bacillus sokdeul (conserved domain) for comparison to Bacillus amino Lowry quinolyl Pacific Enschede (Bacillus amyloliquefaciens) FZB42 (CP000560) of numbering 127 ~ 142 bp and 1495 ~ 1511 bp A forward primer: 5′-CCCAAGCTTAACTGCACTGGGAAATYGTHGAYAAYAG-3 ′ GyrB135 and a reverse primer: 5′-CGGAATTCGGATCCACRTCGGCRTTCATRAT-3 ′ GyrB1510 were prepared. Each primer inserted two restriction enzymes respectively for cloning (GyrB135: Hin dIII Pst I, GyrB1510: Eco RI Bam HI).

Genomic DNA Extraction

Genomic DNA extraction is described by Smith et al. It tested according to the method of. That is, inoculated with 5 mL of nutrient broth medium, pre-cultured at 37 ° C. for 12-16 hours, and then inoculated again with 50 mL of NB and incubated for 12-16 hours. . Cells were obtained by centrifugation at 4 ° C., 13,000 rpm for 10 minutes. 5 μL (4 μg / mL) of lysozyme was added to 567 μL of TE buffer, and the resulting cells were reacted at room temperature for 5 minutes. 30 μL of 10% SDS, 3 μL of proteinase K (20 mg / mL) were added, homogenized and incubated at 37 ° C. for 1 hour. 100 μL of 5 M NaCl was added, homogenized, and 80 μL of CTAB / NaCl solution was added thereto, followed by vortexing and reaction at 65 ° C. for 10 minutes. An equal volume of chloroform: isoamylalcohol (24: 1) was added and centrifuged for 5 minutes to transfer the supernatant to a new tube. Equal amounts of phenol: chloroform: isoamylalcohol (25: 24: 1) were added and centrifuged for 10 minutes to transfer the supernatant to a new tube. 0.65 times volume of isopropanol was added to the obtained supernatant, and then slowly inverted to form genomic DNA. When the white genomic DNA strands were visible to the naked eye, it was removed by a glass rod and 1 mL of 70% ethanol was added. Then, the supernatant was discarded and dried by centrifugation at 4 ° C. and 13,000 rpm for 10 minutes. Suspended in sterile distilled water added with RNase and reacted for 30 minutes at 37 ℃ to remove RNA and stored at -20 ℃.

Polymerase chain reaction and cloning of reaction products

Polymerase chain reaction (PCR) reactions to amplify the gyrB gene were performed by known methods. Genomic DNA extracted from the strain was used as template DNA, and PCR amplification was performed using My Genie 96 Thermal block (Bioneer Co., Daejon, Korea). PCR reaction solution was prepared by adding 3 μL of primer (10 pmol / μL) prepared in PCR pre-mixer (Bioneer Co., Daejon, Korea), 3 μL of template DNA (10 ng Genomic DNA), 44 μL of sterile distilled water. After adding and mixing and applying 1 unit of pfu polymerase, it was performed by adding mineral oil. PCR reaction conditions were 1 cycle of denaturation at 94 ° C for 4 minutes, denaturation at 94 ° C for 1 minute, and annealing at 40 ° C for 1 minute, followed by elongation at 72 ° C for 2 minutes and 30 seconds. The reaction was 30 cycles. And amplified through final extension (final extension) for 10 minutes at 72 ℃. The amplified gyrB gene was confirmed by electrophoresis on a 1% agarose gel.

gyrB  Gene sequencing and analysis

For sequencing of the gyrB gene, sequencing was requested from the Center for Genetic Analysis (Cosmo Genetech Co., Seoul., Korea). The determined nucleotide sequence was analyzed by sequence homology using BLAST search. The homologous gyrB gene sequence found in the data base was analyzed for similarity and phylogenetic tree using the ClustalW multiple sequence alignment program developed by Huggins of EMBL.

Sensory evaluation

The same amount of distilled water was mixed with 100 g of each Cheonggukjang prepared using 21 isolated strains, and the sample was liquid. Then, a sample having a unique aroma was selected for 20 undergraduate and graduate students. The test items were evaluated by a 5-point rating method (1: very bad, 2: slightly bad, 3: moderate, 4: relatively good, 5: very good).

Free amino acid analysis

Amino acid analysis was performed using Pico-Tag system (Water Co., Milford, MA, USA) according to Heinrikson and Meredith's method. 2 g of the sample was taken, 10 mL of 0.1% formic acid was added, homogenized and centrifuged (6,000 rpm, 15 min). Take 3 mL of the supernatant, filter it with a 0.45 μm syringe filter (Millipore Co., MA, USA), and use the AccQ® Fluor ^TM reagent kit (Waters Co., Milford, MA, USA) to prepare fluorescent derivatives. Total amino acids were analyzed. During HPLC analysis, the sample was injected with 10 μL and passed through the AccQTag column (3.9 × 150 mm, Waters, USA) at 37 ° C in gradient mode at a flow rate of 1 mL / min, and a fluorescence detector (λ _ex : 250). nm, λ _em : 395 nm). Amino acid standards were used as amino acid standards (Sigma-Aldrich Co., St. Louis, Missouri, USA).

Vitamin K ₂  analysis

Vitamin K ₂ analysis was determined according to the method of Sato et al . After extracting with 2-propanol: n-hexane (2-propanol: n-hexan e (v / v, 1: 2)) mixed solvent in 4g of Chungkukjang, 2 mL of the nucleic acid layer was taken and concentrated with nitrogen gas. 2 mL of methanol was added to the concentrate, filtered through a 0.45 μm syringe filter (syringe filters (Millipore, Billerica, Mass., USA)), followed by HPLC (model 1200 series, Agilent Technologies, Inc., Wilmington, DE, USA) and fluorescence. It was measured using a fluorescence detector. The column used was an octadecyl-silyl (ODS) -silica-gel-packed column (L-column, 250 mm × 4.6 mm, Chemical Inspection and Testing Institute, Tokyo, Japan). The mobile phase was 100% methanol, the flow rate flowed at a flow rate of 0.8 mL / min, the injection volume was 5 μl and the detector was a fluorescence detector (λ _ex : 320 nm). , λ _em : 430 nm)).

Example 1: Isolation and Selection of Strains

Cheonggukjang was prepared after first screening 50 strains from raw materials for the purpose of separating the excellent strains to enhance the fermentation degree of Cheonggukjang. Viscous substance production ability and sensory test were performed on the prepared Cheonggukjang to select the most preferred strain 3 colony (colony) (Table 1).

BY-01's color remained soybean yellow, and it was slightly tasted immediately after incubation. The most viscous substance was produced, but the viscosity was slightly weak compared to BY-03.

BY-02 retained the yellow color of the beans and showed a slight taste immediately after incubation, but after a period of time the aftertaste was not good. Viscosity was similar to BY-01.

In the case of BY-03, the color was closer to dark brown as time went on, and immediately after incubation, strong bitter taste appeared, and the aroma preference was high. Overall sensory evaluation showed BY-01> BY-02> BY-03.

Table 1 Sensory evaluation results

Strain	evaluation
	color	incense	flavor	Overall acceptance
BY-01	+++++	++++	+++++	+++++
BY-02	+++++	++++	++++	++++
BY-03	+	++++	+++	+++

Although the morphological and physiological characteristics of the isolated strains vary with each strain, most colony shows the same characteristics as Bacillus genus. The morphological and physiological differences were largely different from those of the isolated strains. Biochemical reaction results by API 50CHB kitB. subtilis / amyloliuefaciensWow A similarity rate of 99.8% was identified, and it was determined to be a highly flame resistant microorganism from the results of growth at 7% salinity.

TABLE 2 Sugar Fermentation of Strain BY-1 Isolated from Soy Sauce (using api 20E Kit)

No.	substrates	Reactions / Enzyme	Result
One	ONPG *	Beta-galactosidase	-
2	Arginine	Arginine dihydrolase	-
3	Lysine	Lysine decarboxylase	-
4	Ornithine	Ornithine decarboxylase	+
5	Sodium citrate	Citrate utilization	+
6	Sodium thiosulfate	H2S production	-
7	Urea	Urease	-
8	Tryptophane	Ttryptophane deaminase	-
9	tryptophane	Indole production	-
10	Creatine sodium pyruvate	Acetoin production	-
11	Kohn's gelatin	Gelatinase	+
12	Glucose	fermentation	+
13	Mannitol	fermentation	-
14	Inositol	fermentation	+
15	Sorbitol	fermentation	+
16	Rhamnose	fermentation	-
17	Sucrose	fermentation	-
18	Melibiose	fermentation	+
19	Amygdalin	fermentation	-
20	Arabinose	fermentation	+

ONPG ^* : Ortho-nitro-phl-β-D galactopyranodide (ONPG) isopropylthiogalacto-pyranosoide (IPTG)

+: Positive;-： negative

TABLE 3 Sugar fermentation of strain BY-1 isolated from soy sauce (using api 50CHE. Kit)

Carbohydrate	Result 1)	Carbohydrate	Result
Blank	-	Esculin ferric citrate	+
Glycerol	+	Salicin	+
Erythritol	-	D-Celiobiose	+
D-Arabinose	-	D-Maltose	+
L-Arabinose	-	D-Lactose	+
D-Ribose	+	D-Melibiose	-
D-Xylose	+	D-Saccharose (sucrose)	+
L-Xylose	-	D-Trehalose	+
D-Adonitol	-	Inulin	-
Methyl- -D-xylopyranoside	-	D-Melezitose	-
D-Galactose	-	D-Raffinose	+
D-Glucose	+	Amidon (starch)	+
D-Fructose	+	Glycogen	+
D-Mannose	+	Xylitol	-
L-Sorbose	-	Geniobiose	+
L-Rhamnose	-	D-Turanose	-
Dulcitol	-	D-Lyxose	-
Inositol	+	D-Tagatose	-
D-Mannitol	+	D-Fucose	-
D-Sorbitol	-	L-Fucose	-
Methyl-aD-Mannopyranoside	-	D-Arabitol	-
Methyl-aD-Glucopyranoside	+	L-Arabitol	-
N-Acetylglcosamine	-	Potassium gluconate	-
Amygdalin	+	Potassium 2-ketogluconate	-
Arbutin	+	Potassium 5-ketogluconate	-

¹⁾ Symbol denotes positive (+) and negative (-) in carbohydrate utilization patterns of API 50CHB kit.

Table 4 Characterization of BY-1 Strains

Sodium chloride Con.	BY-01
2% NaCl	++
5% NaCl	++
7% NaCl	++
10% NaCl	+

+: positive,-: negative, NG: No growth.

Example 2: gyrB Isolation strain identification according to gene sequence analysis

The nucleotide sequence of the gyrB gene was analyzed for classification and identification of searched bacteria. Analysis of the determined base sequence 1335 bp by ClustalW is as follows.

BY-1. -------- TGCCCTGGCCGGTTATTGTACAGATATTAATATCGAGATTGAAAAAGATAAC 52

Amyl_ ATTGACGAAGCCCTGGCCGGTTATTGTACAGATATTAACATCGAGATTGAAAAAGATAAC 60

Sub-sub_ ATTGACGAAGCCCTCGCCGGTTATTGTACGGATATCAATATCCAAATCGAAAAAGACAAC 60

Lich_ ATCGATGAAGCATTGGCCGGTTACTGCACTGAAATCAATGTCGCAATTGAAAAAGACAAC 60

Pum_ ATTGATGAGGCTTTAGCTGGATATTGCACAGATATTACCGTGCAAATTGAAAAGGATAAT 60

Ant_ ATCGATGAAGCACTTGCAGGGTACTGTGACGAAATTAACGTTAGTATCGAAGAAGATAAT 60

Thu_ ATCGATGAAGCACTTGCAGGGTACTGTGACGAAATTAACGTTAGTATCGAAGAAGATAAT 60

Thr-kon_ ATCGATGAAGCACTTGCAGGGTACTGTGACGAAATTAACGTTAGTATCGAAGAAGATAAT 60

cer._ ATTGATGAAGCACTTGCAGGTTACTGTGATGAAATTAACGTTAGTATCGAAGAAGATAAT 60

Wei_ ATTGATGAAGCTTTAGCAGGATATTGTGATGAAATTAATGTCAGTATTGAAGAAGATAAT 60

Sp_ ATTGACGAAGCGATGGCTGGCTTTTGTGATGAGATCGGGGTTGTAATCGAAGAGGATAAT 60

Halo_ ATTGACGAAGCGATGGCTGGCTTTTGTGATGAGATCGGGGTTGTAATCGAAGAGGATAAT 60

Clau_ ATTGACGAAGCGATGGCTGGTTTCTGTGATGAAATTAAAGTAACGATCGAGGAAGGAAAC 60

                     ** * ** ** * ** ** ** * ** ** * * **

BY-1. AGCATTACCGTTAAGGACAACGGGCGCGGAATTCCGGTCGGTATCCAGGAGAAGATGGGC 112

Amyl_ AGCATTACCGTTAAGGACAACGGGCGCGGAATTCCGGTCGGTATCCAGGAGAAGATGGGC 120

Sub-sub_ AGTATCACGGTTGTAGATAATGGCCGCGGTATTCCAGTCGGTATTCATGAAAAAATGGGC 120

Lich_ AGCATCACAGTAAAAGACAACGGACGGGGTATCCCGGTCGGTATTCATGAGAAGATGGGT 120

Pum_ AGCATTACAGTGAAAGATAATGGTCGCGGAATTCCTGTTGGGATTCATGAGAAAATGGGA 120

Ant_ AGTATTCGTGTAACAGATAATGGACGTGGTATTCCAGTTGGTATACAAGAAAAAATGGGA 120

Thu_ AGTATTCGTGTAACAGATAATGGACGTGGTATTCCAGTTGGTATACAAGAAAAAATGGGA 120

Thr-kon_ AGTATTCGTGTAACAGATAATGGACGTGGTATCCCAGTTGGTATACAAGAAAAAATGGGA 120

cer._ AGTATTCGTGTAACGGATAATGGACGTGGTATTCCAGTTGGTATACAAGAAAAAATGGGA 120

Wei_ AGTATTGTTGTAACAGATGATGGTCGTGGTATCCCTGTTGGTATACAAGAAAAAATGGGA 120

Sp_ AGCATTACAGTTACCGACAATGGTCGAGGCATTCCTGTTGGGATTCATGAAAAGATGGGG 120

Halo_ AGCATTACAGTTACCGACAATGGTCGAGGCATTCCTGTTGGGATTCATGAAAAGATGGGG 120

Clau_ GCCCTTACGGTTGAGGATAATGGCCGTGGCATTCCTGTCGGCATCCAAGAAAAAATGGGC 120

             * ** ** * ** ** ** ** ** ** ** ** ** ** ** *****

BY-1. CGCCCTGCGGTTGAAGTCATCATGACCGTTCTCCACGCCGGCGGTAAATTTGACGGAAGC 172

Amyl_ CGCCCTGCGGTTGAAGTTATCATGACCGTTCTCCACGCCGGCGGTAAATTTGACGGAAGC 180

Sub-sub_ CGTCCTGCGGTAGAAGTCATTATGACGGTGCTTCATGCCGGAGGAAAATTTGACGGAAGC 180

Lich_ CGTCCCGCTGTGGAAGTCATCATGACTGTCCTGCACGCCGGAGGAAAGTTTGACGGAAGC 180

Pum_ CGTCCTGCTGTAGAGGTTATTATGACTGTTCTTCACGCTGGCGGTAAATTTGACGGCAGT 180

Ant_ CGTCCTGCTGTAGAAGTTATTATGACCGTTCTTCATGCTGGTGGTAAGTTTGGCGGCGGC 180

Thu_ CGTCCTGCTGTAGAAGTTATTATGACCGTTCTTCATGCTGGTGGTAAGTTTGGCGGCGGG 180

Thr-kon_ CGTCCTGCTGTAGAAGTTATTATGACCGTTCTTCATGCTGGTGGTAAGTTTGGCGGCGGC 180

cer._ CGTCCTGCTGTAGAGGTTATTATGACCGTTCTTCATGCTGGTGGTAAGTTTGGCGGCGGC 180

Wei_ CGTCCTGCTGTAGAAGTTATTATGACGGTACTCCATGCCGGTGGTAAATTTGGCGGTGGT 180

Sp_ CGTCCGGCTGTCGAGGTCATTATGACCGTTCTCCATGCAGGAGGGAAGTTCGGCGGCGGT 180

Halo_ CGTCCGGCTGTCGAGGTCATTATGACCGTTCTCCATGCAGGAGGGAAGTTCGGCGGCGGT 180

Clau_ CGTCCGGCGGTCGAGGTCATTATGACTACCCTTCATGCCGGAGGGAAATTTGGCGGTGGC 180

            ** ** ** ** ** ** ** ***** ** ** ** ** ** ** ** * *** *

BY-1. GGATATAAAGTATCCGGCGGTCTTCACGGTGTAGGGGCGTCCGTCGTAAACGCCTTGTCG 232

Amyl_ GGATATAAAGTATCCGGCGGTCTTCACGGTGTAGGGGCGTCCGTCGTAAACGCCTTGTCG 240

Sub-sub_ GGCTATAAAGTATCCGGAGGATTACACGGTGTAGGTGCGTCGGTCGTAAACGCACTATCA 240

Lich_ GGATATAAAGTTTCGGGCGGTTTGCACGGCGTCGGTGCTTCTGTTGTTAACGCCCTTTCA 240

Pum_ GGTTATAAAGTATCTGGCGGTCTGCATGGCGTAGGGGCATCTGTTGTTAATGCGTTATCT 240

Ant_ GGTTATAAAGTTTCTGGTGGTTTGCATGGTGTTGGGGCATCTGTAGTAAATGCTCTATCA 240

Thu_ GGTTATAAAGTTTCTGGTGGTTTGCATGGTGTTGGGGCATCTGTAGTAAATGCTCTATCA 240

Thr-kon_ GGTTATAAAGTTTCTGGTGGTTTGCATGGTGTTGGGGCATCTGTAGTAAATGCTCTATCA 240

cer._ GGTTATAAAGTTTCTGGTGGTTTGCATGGTGTTGGGGCATCTGTAGTAAATGCCCTATCA 240

Wei_ GGTTATAAAGTTTCTGGTGGTTTGCATGGTGTAGGTGCATCTGTTGTAAATGCTTTATCA 240

Sp_ GGTTATAAAGTATCTGGTGGTTTGCACGGTGTAGGTGCATCCGTCGTAAATGCTTTATCG 240

Halo_ GGTTATAAAGTATCTGGTGGTTTGCACGGTGTAGGTGCATCCGTCGTAAATGCTTTATCG 240

Clau_ GGCTATAAAGTATCAGGCGGCCTTCACGGAGTTGGTGCTTCCGTCGTCAACGCGCTGTCG 240

            ** ******** ** ** ** * ** ** ** ** ** ** ** ** ** ** * **

BY-1. ACCACTCTTGACGTTACGGTTCATCGTGACGGAAAAATCCACTATCAGGCGTACGAGCGC 292

Amyl_ ACCACTCTTGACGTTACGGTTCATCGTGACGGAAAAATCCACTATCAGGCGTACGAGCGC 300

Sub-sub_ ACAGAGCTTGATGTGACGGTTCACCGTGACGGTAAAATTCACCGCCAAACCTATAAACGC 300

Lich_ ACCGAGCTGGATGTAACGGTTTACAGAGATGGAAAAATCCATTATCAGGAATTTGAACGT 300

Pum_ ACGACCTTAGACGTGACCGTATATCGTGATGGGAAAATTCATTATCAACAATTCAAACGC 300

Ant_ ACAGAACTAGAGGTATTTGTACATCGTGAAGGTAAAATCCATTATCAAAAATACGAAAGA 300

Thu_ ACAGAACTAGAGGTATTTGTACATCGTGAAGGTAAAATCCATTATCAAAAATACGAAAGA 300

Thr-kon_ ACAGAACTAGAGGTATTTGTACATCGTGAAGGTAAAATCCATTATCAAAAATACGAAAGA 300

cer._ ACAGAACTAGAGGTATTTGTACATCGTGAAGGTAAAATCCATTATCAAAAATACGAAAGA 300

Wei_ ACAGAATTAGAGGTATTTGTACATCGAGATGGAAAAATACACTATCAAAAATATGAACGC 300

Sp_ ACGATGCTGGAAGTAGAAGTTCACCGTGAAGGGAAAGTCCACTATCAAAAATTTCATCGC 300

Halo_ ACGATGCTGGAAGTAGAAGTTCACCGTGAAGGGAAAGTCCACTATCAAAAATTTCATCGC 300

Clau_ ACACACTTAACGGTCAATGTCCATTTAGACGGGAAAATCCATAGCCAATCTTATAGCCGT 300

            ** * ** ** * ** ** *** * ** ** * *

BY-1. GGTGTGCCTGTGGCCGATCTTGAAGTGATCGGTGATACTGATAAGACCGGAACGATTACG 352

Amyl_ GGTGTACCTGTGGCTGATCTTGAAGTGATCGGTGATACTGATAAGACCGGAACGATTACG 360

Sub-sub_ GGAGTTCCGGTTACAGACCTTGAAATCATTGGCGAAACGGATCATACAGGAACGACGACA 360

Lich_ GGCGTTCCGAAAGCTGATTTGAAAGTCATTGGAGATACGGAAGTGACGGGAACGACCACA 360

Pum_ GGTGTTCCAGTTGGAGATTTAGAGATCATTGGTGAAACAGATGTTACAGGGACAACCACT 360

Ant_ GGTATTCCGGTTGCGGATTTAAAAGTCATTGGTGATACAGATCAAACGGGAACGATAACT 360

Thu_ GGTATTCCGGTTGCGGATTTAAAAGTCATTGGTGATACAGATCAAACGGGAACGATAACT 360

Thr-kon_ GGTATTCCGGTTGCGGATTTAAAAGTCATTGGTGACACAGATCAAACGGGAACGATAACT 360

cer._ GGTATTCCGGTTGCGGATTTAAAAGTCATTGGTGACACAGATCAAACAGGAACGATAACT 360

Wei_ GGGGTCCCGGCTGCGGATTTAAAAGTAATAGGTGAAACTGATCGTACAGGAACGATCACA 360

Sp_ GGTGTACCTGCTGCTGATTTAGAAGTCATTGGCACGACCGATCGAACAGGCACGAAAATC 360

Halo_ GGTGTACCTGCTGCTGATTTAGAAGTCATTGGCACGACCGATCGAACAGGCACGAAAATC 360

Clau_ GGTGTTCCCGATGCTGATTTGACCGTCATAGGTGAAACCGATAAAACCGGCACGACTATT 360

            ** * ** ** * * ** ** ** ** ** ** ** * *

BY-1. CACTTCGTTCCGGATCCGGAAATCTTCAAAGAAACAACTGTATACGACTATGATCTGCTT 412

Amyl_ CACTTCGTTCCGGATCCGGAAATCTTCAAAGAAACAATCGTATACGACTATGATCTGCTT 420

Sub-sub_ CATTTTGTCCCGGACCCTGAAATTTTCTCAGAAACAACCGAGTATGATTACGATCTGCTT 420

Lich_ CACTTCAAGCCTGATCCGGAAATATTCACGGAAACGACTGAATACGACTATGATACGCTC 420

Pum_ CATTTTGTGCCAGATCCAGAAATTTTTACTGAAACCATTGAATTTGATTACGACACACTT 420

Ant_ CGATTTAAACCAGATCCAGAAATTTTTCAGGAAACAACAGTATACGAATTTGATACACTA 420

Thu_ CGATTTAAACCAGATCCAGAAATTTTTCAGGAAACAACAGTATACGAATTTGATACACTA 420

Thr-kon_ CGATTTAAACCAGATCCAGAAATTTTTCAGGAAACAACAGTATACGAATTTGATACACTA 420

cer._ CGATTTAAACCAGATCCAGAAATTTTTCAGGAAACAACAGTATACGAATTTGATACGCTC 420

Wei_ CGCTTTAAGCCAGATTCAGAAATTTTTACAGAGACGACAGAATACGAATTTGATACATTA 420

Sp_ CACTTTAAACCAGACGGTGACATTTTTACGGAAACAACGGTTTTCGAATATGATACTCTC 420

Halo_ CACTTTAAACCAGACGGTGACATTTTTACGGAAACAACGGTTTTCGAATATGATACTCTC 420

Clau_ ACGTTCCAGCCAGACCCAGAAATTTTCCGGGAGACGGTTGAATTTGATTATGAAACACTT 420

               ** ** ** ** ** ** ** ** * * ** * ** *

BY-1. TCAAACCGTGTCCGGGAATTGGCCTTCCTGACAAAAGGCGTAAACATCACGATTGAAGAC 472

Amyl_ TCAAACCGTGTCCGGGAATTGGCCTTCCTGACAAAAGGCGTAAACATCACGATTGAAGAC 480

Sub-sub_ GCCAACCGCGTGCGTGAATTAGCCTTTTTAACAAAGGGCGTAAACATCACGATTGAAGAT 480

Lich_ GCCACTCGTGTCCGCGAACTCGCTTTCTTGACAAAAGGCGTCAAAATCACGATCGAAGAC 480

Pum_ GCTAACCGTGTACGTGAATTAGCTTTCTTAACAAAAGGTGTAAACATCATTATTGAAGAT 480

Ant_ GCAACTCGTATGCGTGAATTAGCATTTTTAAATCGTAATATTAAACTGACGATTGAAGAT 480

Thu_ GCAACTCGTATGCGTGAATTAGCATTTTTAAATCGTAATATTAAACTGACGATTGAAGAT 480

Thr-kon_ GCAACTCGTATGCGTGAATTAGCATTTTTAAATCGTAATATTAAATTGACGATTGAAGAT 480

cer._ GCAACTCGTATGCGTGAATTAGCATTTTTAAATCGTAATATTAAATTAACAATTGAAGAT 480

Wei_ GCGACTCGTATGCGTGAGTTGGCGTTTTTAAATCGTAATATTAAATTAACAATTGAAGAT 480

Sp_ GCATCACGGTTGCGTGAATTGGCCTTTTTAAATAAAGGATTGCGCATTAAGATTACCGAT 480

Halo_ GCATCACGGTTGCGTGAATTGGCCTTTTTAAATAAAGGATTGCGCATTAAGATTACCGAT 480

Clau_ GCTGCCCGCATTCGCGAGTTGGCGTTTTTAAACAAAGGGCTGACGATCAAAATTGAGGAC 480

             * ** * ** ** * ** ** * * * * * ** **

BY-1. AAACGTGAAGGACAAGAACGGAAAAACGAGTACCACTACGAAGGCGGAATCAAAAGCTAT 532

Amyl_ AAACGTGAAGGACAAGAACGGAAAAACGAGTACCACTACGAAGGCGGAATCAAAAGCTAT 540

Sub-sub_ AAACGTGAAGGACAAGAGCGCAAAAATGAATACCATTACGAAGGCGGAATTAAAAGTTAT 540

Lich_ AAGCGAGAAGGAAAAGAACGCAAGAATGAATACTGCTATGAAGGCGGTATTAAAAGCTAT 540

Pum_ TTACGCGAAGGTAAAGAACGACGAAATGAATACTGCTACGAAGGCGGTATTAAGAGCTAT 540

Ant_ AAACGTGAA --- CATAAGCAAAAAAAAGAATTCCATTATGAAGGTGGAATTAAATCATAT 537

Thu_ AAACGTGAA --- CATAAGCAAAAAAAAGAATTCCATTATGAAGGTGGAATTAAATCATAT 537

Thr-kon_ AAACGTGAA --- CATAAGCAAAAGAAAGAATTCCATTATGAAGGTGGAATTAAATCATAT 537

cer._ AAACGTGAA --- CATAAGCAAAAGAAAGAATTCCATTATGAAGGTGGAATTAAATCATAC 537

Wei_ AAGCGTGAA --- CATAAGCAAAAGAAAGAATTCCATTATGAAGGTGGAATTAAATCATAT 537

Sp_ ATGCGT --- GAGGAAGAAAAGTCGGACGAATTTCACTATGAGGGCGGGATCGCTTCATTT 537

Halo_ ATGCGT --- GAGGAAGAAAAGTCGGACGAATTTCACTATGAGGGCGGGATCGCTTCATTT 537

Clau_ AAACGAACAGAGGACGGCAAAAAGGCCACCTACCATTACGAGGGTGGAATTTCGTCGTTT 540

               ** * * ** ** ** ** ** *

BY-1. GTTGAGTACTTAAACCGTTCCAAAGAAGTCGTTCATGAAGAGCCGATTTATATCGAAGGC 592

Amyl_ GTTGAGTACTTAAACCGTTCCAAAGAAGTCGTTCATGAAGAGCCGATTTATATCGAAGGC 600

Sub-sub_ GTAGAGTATTTAAACCGCTCTAAAGAGGTTGTCCATGAAGAGCCGATTTACATTGAAGGC 600

Lich_ GTTGAACACTTGAACCGTTCGCGGGAAGTTATTCATGAAGAGCCGGTCTATATTGAAGGA 600

Pum_ GTAGAACATTTAAATCGCTCAAAAGAAGTCGTTCATGAAGAACCTGTTTACATCGAAGGT 600

Ant_ GTTGAGCATTTAAACCGCTCAAAACAACCAATCCATGAAGAGCCTGTATATGTAGAAGGA 597

Thu_ GTTGAGCATTTAAACCGCTCAAAACAACCAATCCATGAAGAGCCTGTATATGTAGAAGGA 597

Thr-kon_ GTTGAGCATTTAAACCGTTCGAAACAACCAATCCATGAAGAGCCTGTATATGTAGAAGGA 597

cer._ GTCGAGCATTTAAATCGCTCAAAACAACCAATCCATGAAGAACCTGTATATGTAGAAGGA 597

Wei_ GTTGAACATTTAAATCGTTCAAAACAACCGATTCATGAAGAACCTGTATATGTTGACGGT 597

Sp_ GTGGAGCATTTAAACCGCACGCGTGAGACTTTGCACGAAACGCCGATCCATATCGAAGGA 597

Halo_ GTGGAGCATTTAAACCGCACGCGTGAGACTTTGCACGAAACGCCGATCCATATCGAAGGA 597

Clau_ GTCAAACACCTCAACCGTTCAAAAGAAACGCTCCATGAAGAGCCGATTTATGTGGAAAGC 600

            ** * * * ** ** * * * ** *** ** * * * ** *

BY-1. GAGAAAGACGGCATAACGGTTGAAGTTGCATTGCAATACAACGACAGCTATACAAGCAAT 652

Amyl_ GAGAAAGACGGCATAACGGTTGAAGTTGCGTTGCAATACAACGACAGCTATACAAGCAAC 660

Sub-sub_ GAAAAGGACGGCATTACGGTTGAAGTGGCTTTGCAATACAATGACAGCTACACAAGCAAC 660

Lich_ TCCAAAGACGGCATTACAGTCGAGGTGGCTCTTCAATACAATGACAGCTATACAAGCAAC 660

Pum_ GAAAGAGACGGAATTACGGTTGAAGTGGCATTACAATATAACGATTCCTATACAAGCAAC 660

Ant_ TCAAAAGATGGTATTCAAGTTGAAGTTTCCTTACAGTATAACGAAGGATATACAAATAAT 657

Thu_ TCAAAAGATGGTATTCAAGTTGAAGTTTCCTTACAGTATAACGAAGGATATACAAATAAT 657

Thr-kon_ TCAAAAGATGGTATTCAAGTTGAGGTTTCTTTACAGTATAACGAAGGATATACAAATAAT 657

cer._ TCAAAAGATGGTATTCAAGTTGAGGTTTCCTTACAGTATAACGAAGGATATACAAATAAT 657

Wei_ TCAAAAGATGGGATTCAGGTTGAGGTTGCTCTTCAATATAACGAAGGCTATACAAATCAT 657

Sp_ GAGAAGCAAGGCGTTTACGTAGAGATCGCCGTCCAATATAACGATGGATTCACGAGCAAT 657

Halo_ GAGAAGCAAGGCGTTTACGTAGAGATCGCCGTCCAATATAACGATGGATTCACGAGCAAT 657

Clau_ GAAAAAGACGGCATTTCTGTTGAAGTAGCCGTCCAATACAATGACGGATTTACGAGCAGT 660

               * * ** * ** ** * * * ** ** ** ** * ** *

BY-1. ATTTATTCTTTCACAAATAATATCAACACATACGAAGGCGGGACGCACGAAGCCGGATTT 712

Amyl_ ATTTATTCTTTCACAAATAACATCAACACATACGAAGGCGGGACGCACGAAGCCGGATTT 720

Sub-sub_ ATTTACTCGTTTACAAACAACATTAACACGTACGAAGGCGGTACCCATGAAGCTGGCTTC 720

Lich_ ATTTATTCATTTGCTAACAACATTCATACGTATGAAGGCGGAACCCATGAAGCCGGCTTT 720

Pum_ ATCTATTCCTTCGCCAACAATATCAACACGTATGAAGGCGGAACACACGAAGCAGGCTTT 720

Ant_ ATTTACTCATTTACGAACAACATTCACACGTATGAAGGTGGAACACATGAAGTAGGGTTT 717

Thu_ ATTTACTCATTTACGAACAACATTCACACGTATGAAGGTGGAACACATGAAGTAGGGTTT 717

Thr-kon_ ATTTACTCATTTACGAATAACATTCATACGTATGAAGGTGGAACACATGAAGTAGGGTTT 717

cer._ ATTTACTCATTTACGAATAACATTCATACGTATGAAGGTGGAACACATGAAGTAGGGTTT 717

Wei_ ATTTATTCATTTACAAATAATATTCATACGTATGAAGGCGGTACACATGAGGTAGGTTTT 717

Sp_ ATTTACTCGTTTGCTAATAATATCAACACCCATGAAGGGGGAACGCACGAGTCAGGGTTT 717

Halo_ ATTTACTCGTTTGCTAATAATATCAACACCCATGAAGGGGGAACGCACGAGTCAGGGTTT 717

Clau_ ATTTACTCCTTCGCGAATAACATTAATACCCATGAAGGGGGCACGCATGAATCAGGCTTT 720

            ** ** ** ** * ** ** ** * ** * ***** ** ** ** ** ** **

BY-1. AAAACCGGTCTGACCCGTGTCATAAACGACTATGCGAGAAGAAAAGGGATTTTCAAAGAA 772

Amyl_ AAAACCGGTCTGACCCGTGTCATAAACGACTATGCAAGAAGAAAAGGGATTTTCAAAGAA 780

Sub-sub_ AAAACGGGCCTGACTCGTGTTATCAACGATTACGCCAGAAAAAAAGGGCTTATTAAAGAA 780

Lich_ AAGACCGGTTTGACGAGGGTCATCAATGATTACGCGAGAAGAAACGGCGTATTCAAAGAA 780

Pum_ AAAACAGGTCTGACGCGTGTGATCAATGATTATGCTCGTAAAAATGGCGTATTCAAAGAT 780

Ant_ AAAACAGCTTTAACTCGTGTGATTAACGATTATGGGCGTAAAAATAGTATTCTAAAAGAT 777

Thu_ AAAACAGCTTTAACTCGTGTGATTAACGATTATGGGCGTAAAAATAGTATTCTAAAAGAT 777

Thr-kon_ AAAACAGCTTTAACTCGTGTGATTAACGATTATGGACGTAAAAATAGTATTCTAAAAGAT 777

cer._ AAAACAGCTTTAACTCGTGTGATTAACGATTATGGACGTAAAAATAGTATTTTAAAAGAT 777

Wei_ AAGACTGCTCTAACACGTGTAATTAACGATTATGGTCGTAAAAATAATATTTTAAAAGAT 777

Sp_ AAAACAGGTCTTACTCGTGTCATTAACGATTATGCTCGCAAGCATAATTTGTTCAAAGAA 777

Halo_ AAAACAGGTCTTACTCGTGTCATTAACGATTATGCTCGCAAGCATAATTTGTTCAAAGAA 777

Clau_ AAAACAGGCTTGACGCGCGTTATTAATGATTACGCCCGCAAAAATGGGTTGTTTAAAGAA 780

            ** ** * * ** * ** ** ** ** ** * * * * * * *****

BY-1. AATGATCCGAATTTAAGCGGGGATGATGTGAGAGAAGGGCTGACTGCCATTATTTCAATT 832

Amyl_ AATGATCCGAATTTAAGCGGGGATGATGTGAGAGAAGGGCTGACTGCCATTATTTCAATT 840

Sub-sub_ AATGATCCAAACCTAAGCGGAGATGACGTAAGGGAAGGGCTGACAGCGATTATTTCAATC 840

Lich_ AGCGATCCGAACTTAAGCGGAGAAGACGTCCGGGAAGGTTTGACAGCGATTATTTCAATC 840

Pum_ GGAGACTCGAATTTGAGCGGTGAAGATGTGCGAGAAGGCTTAACAGCCATTATCTCTATC 840

Ant_ GCAGACAGTAATTTAACTGGTGAGGACGTTCGTGAAGGTTTAACTGCAATTGTATCAATT 837

Thu_ GCAGACAGTAATTTAACTGGTGAGGACGTTCGTGAAGGTTTAACTGCAATTGTATCAATT 837

Thr-kon_ GCAGACAGTAATTTAACTGGTGAGGACGTTCGTGAAGGTTTAACTGCAATTGTATCAATT 837

cer._ GCAGACAGTAACTTAACGGGTGAGGATGTTCGTGAAGGTTTAACTGCAATTGTATCAATT 837

Wei_ GCGGATAGTAATTTAACTGGTGAAGATGTTCGTGAAGGCTTAACAGCAATCGTGTCAATT 837

Sp_ AGCGATCCGAACTTAACGGGGGAAGATGTACGTGAAGGATTAACCGCCATTATCTCGGTA 837

Halo_ AGCGATCCGAACTTAACGGGGGAAGATGTACGTGAAGGATTAACCGCCATTATCTCGGTA 837

Clau_ AACGATCCAAACTTAAGTGGCGAGGATGTCCGTGAAGGGCTGACAGCGATCATTTCCGTC 840

              ** ** * * ** ** ** ** * ***** * ** ** ** * ** *

BY-1. AAGCACCCTGATCCGCAATTCGAAGGGCAGACGAAAACCAAGCTCGGCAACTCTGAAGCG 892

Amyl_ AAGCACCCTGATCCGCAATTCGAAGGGCAGACGAAAACGAAGCTCGGCAACTCCGAAGCG 900

Sub-sub_ AAACACCCTGATCCGCAGTTTGAGGGCCAAACAAAAACAAAGCTGGGCAACTCAGAAGCA 900

Lich_ AAGCACCCGGATCCTCAATTTGAAGGGCAGACGAAAACAAAGCTCGGCAACTCAGAAGCG 900

Pum_ AAACATCCAGATCCTCAATTCGAAGGGCAAACGAAGACAAAGCTTGGGAACTCAGAAGCA 900

Ant_ AAACATCCAAATCCACAATTTGAAGGACAAACGAAGACGAAACTTGGGAATAGTGAAGCG 897

Thu_ AAACATCCAAATCCACAATTTGAAGGACAAACGAAGACGAAACTTGGGAATAGTGAAGCG 897

Thr-kon_ AAACATCCAAATCCACAATTTGAAGGACAAACGAAGACGAAACTTGGGAATAGTGAAGCG 897

cer._ AAACATCCAAACCCACAATTTGAAGGACAAACGAAGACGAAACTTGGGAATAGTGAAGCA 897

Wei_ AAGCATCCAAATCCACAATTTGAAGGACAAACAAAGACGAAACTTGGAAATAGTGAAGCG 897

Sp_ AAAATTCCTGATCCTCAGTTTGAAGGGCAAACGAAAACGAAGCTAGGAAATAGCGAAGCA 897

Halo_ AAAATTCCTGATCCTCAGTTTGAAGGGCAAACGAAAACGAAGCTAGGAAATAGCGAAGCA 897

Clau_ AAAATTCCTGAGCCCCAGTTTGAAGGGCAAACGAAAACAAAGCTAGGAAACAGTGAAGCA 900

            ** ** * ** ** ** ** ** ** ** ** ** ** ** ** ** *****

BY-1. AGAACGATCACTGACACGCTGTTTTCTTCTGCGCTGGAAACATTCCTTCTTGAAAATCCG 952

Amyl_ AGAACGATCACTGATACGCTGTTTTCTTCTGCGCTGGAAACATTCCTTCTTGAAAATCCG 960

Sub-sub_ CGGACGATCACCGATACGTTATTTTCTACGGCGATGGAAACATTTATGCTGGAAAATCCA 960

Lich_ CGTACGATAACAGATGCGCTATTTTCAGAAGCGCTTGAAAAGTTTCTGCTAGAAAACCCG 960

Pum_ AGAACCATTACCGACTCCCTTTTCTCTGAAGCACTTGAGAAATTCCTCTTAGAGAACCCT 960

Ant_ AGAACGATTACAGAGTCTGTGTTTTCAGAGGCATTTGAAAAGTTCTTACTAGAAAACCCG 957

Thu_ AGAACGATTACAGAGTCTGTGTTTTCAGAGGCATTTGAAAAGTTCTTACTAGAAAACCCG 957

Thr-kon_ AGAACAATTACAGAGTCTGTGTTTTCAGAGGCATTTGAAAAGTTCTTACTAGAAAACCCG 957

cer._ AGAACGATTACAGAGTCTGTGTTTTCAGAGGCATTTGAAAAGTTCTTACTAGAAAACCCG 957

Wei_ AGAACGATTACAGAGTCTGTATTCTCAGAGGCGTTTGAAAAGTTCTTACTAGAAAATCCC 957

Sp_ CGTACGATCACTGATTCCCTTTTCACAGAGCATTTTTCCCGGTTTCTGATCGAACATCCG 957

Halo_ CGTACGATCACTGATTCCCTTTTCACAGAGCATTTTTCCCGGTTTCTGATCGAACATCCG 957

Clau_ CGCACAATTACTGACTCTTTATTCAGCGAAAGTTTTGCAAGGTTCCTGTCTGAGAATCCA 960

             * ** ** ** ** * * ** * ** * ** * **

BY-1. GACTCAGCCCGCAAAATCGTTGAAAAAGGTTTAATGGCCGCAAGAGCGCGGATGGCAGCG 1012

Amyl_ GACTCAGCCCGCAAAATCGTTGAAAAAGGTTTAATGGCCGCAAGAGCGCGGATGGCAGCG 1020

Sub-sub_ GATGCAGCCAAAAAAATTGTCGATAAAGGTTTAATGGCGGCAAGAGCAAGAATGGCTGCG 1020

Lich_ GATTCAGCGAAAAAAATCGTTGAAAAAGGGGTTATGGCCGCCAGAGCACGGATGGCTGCA 1020

Pum_ GATGCGGCAAAGAAAATTGTGGAGAAAGGCGTGATGGCAGCTCGTGCAAGAATGGCTGCC 1020

Ant_ AACGTTGCACGAAAAATCGTAGAAAAAGGTACGATGGCAGCGCGTGCACGTGTTGCAGCG 1017

Thu_ AACGTTGCACGAAAAATCGTAGAAAAAGGTACGATGGCAGCGCGTGCACGTGTTGCAGCG 1017

Thr-kon_ AACGTTGCACGAAAAATCGTAGAAAAAGGTACGATGGCAGCGCGTGCACGTGTTGCAGCG 1017

cer._ AATGTTGCACGAAAAATCGTAGAAAAAGGTACGATGGCAGCACGTGCACGTGTTGCAGCG 1017

Wei_ AATGTTGCACGTAAAATTATAGATAAAGGGACTATGGCAGCACGTGCACGTGTAGCGGCT 1017

Sp_ CAAGTGGCACGTAAGCTAGTTGACAAAGGACTCATGGCTTCAAGGGCTAGGGAAGCAGCG 1017

Halo_ CAAGTGGCACGTAAGCTAGTTGACAAAGGACTCATGGCTTCAAGGGCTAGGGAAGCAGCG 1017

Clau_ GGAGTTGCCCGCAAGATTGTCGATAAAGGGTTGATGGCTTCCCGTGCCCGTGAGGCTGCT 1020

                  ** ** * * ** ***** ***** * * ** * ** **

BY-1. AAAAAAGCGCGGGAATTGACCCGGCGCAAAAGTGCGCTTGAGATTTCCAATCTGCCGGGC 1072

Amyl_ AAAAAAGCGCGGGAATTGACCCGCCGCAAAAGTGCGCTTGAGATTTCCAATCTGCCGGGC 1080

Sub-sub_ AAAAAAGCGCGTGAACTAACACGCCGTAAGAGTGCTTTGGAAATTTCAAACCTGCCCGGT 1080

Lich_ AAGAAAGCACGCGAATTGACGCGCAGAAAAAGCGCCCTTGAAGTGTCCAATCTGCCGGGG 1080

Pum_ AAAAAGGCACGTGAGCTGACAAGACGTAAAAGCGCACTGGAAGTCTCCAGCTTACCGGGG 1080

Ant_ AAAAAAGCACGTGAATTGACACGTCGTAAGAGCGCGTTAGAAGTTTCAAGTTTACCTGGT 1077

Thu_ AAAAAAGCACGTGAATTGACACGTCGTAAGAGCGCGTTAGAAGTTTCGAGTTTACCTGGT 1077

Thr-kon_ AAAAAAGCACGTGAATTGACACGTCGTAAGAGCGCGTTAGAAGTTTCAAGTTTACCTGGT 1077

cer._ AAAAAAGCACGTGAATTGACACGCCGCAAGAGCGCATTAGAAGTTTCAAGTTTACCTGGT 1077

Wei_ AAAAAGGCTCGTGAACTAACGCGCCGAAAGAGTGCTCTAGAAGTTTCAAGTTTACCAGGG 1077

Sp_ AAAAAAGCACGTGAGCTTACGCGGAGAAAGAGCGCTCTTGAGGTAAGCTCTCTCCCAGGA 1077

Halo_ AAAAAAGCACGTGAGCTTACGCGGAGAAAGAGCGCTCTTGAGGTAAGCTCTCTCCCAGGA 1077

Clau_ AAAAAGGCCCGTGAGTTGACACGGCGGAAAAGCGCCTTAGAAGTAAGCTCGCTTCCTGGA 1080

            ** ** ** ** ** * ** * * ** ** ** * ** * * ** **

BY-1. AAACTGGCGGACTGTTCTTCTAAAGATCCGAGCATTTCCGAGCTGTATATCGTAGAGGGT 1132

Amyl_ AAACTGGCGGACTGTTCTTCTAAAGATCCGAGCATTTCCGAGCTGTATATCGTAGAGGGT 1140

Sub-sub_ AAGTTAGCGGACTGCTCTTCAAAAGATCCGAGCATCTCCGAGTTATATATCGTAGAGGGT 1140

Lich_ AAACTTGCTGACTGTTCTTCTAAAGACCCGACGATTTCCGAACTTTACATCGTTGAGGGT 1140

Pum_ AAACTAGCGGACTGTTCTTCTAAAGACCCATCCATCTCTGAACTCTATATCGTAGAGGGA 1140

Ant_ AAATTAGCAGATTGCTCTTCAAAAGATCCAGCAATTAGTGAAATTTACATTGTAGAGGGT 1137

Thu_ AAATTAGCAGATTGCTCTTCAAAAGATCCAGCAATTAGTGAAATTTACATTGTAGAGGGT 1137

Thr-kon_ AAATTAGCAGACTGCTCTTCAAAGGATCCAGCAATTAGTGAAATTTACATTGTAGAGGGT 1137

cer._ AAATTAGCAGATTGCTCTTCGAAAGATCCAGCAATTAGTGAAATTTACATTGTAGAGGGT 1137

Wei_ AAGCTAGCTGATTGTTCTTCTAAAGATCCAGCAATTAGCGAAATTTACATCGTGGAGGGT 1137

Sp_ AAACTAGCAGACTGTTCGTCACGCGACGCTTCGATTAGTGAGATTTACATTGTGGAGGGG 1137

Halo_ AAACTAGCAGACTGTTCGTCACGCGACGCTTCGATTAGTGAGATTTACATTGTGGAGGGG 1137

Clau_ AAGCTAACCGACTGTACATCAAAAGATGCGTCGATTAGCGAATTGTTTATCGTTGAGGGC 1140

            ** * * ** ** * ** ** * ** ** * * ** ** *****

BY-1. GACTCTGCGGGCGGATCAGCGAAACAGGGACGGGACCGCCATTTCCAAGCCATTCTGCCG 1192

Amyl_ GACTCTGCGGGCGGATCAGCGAAACAGGGACGGGACCGTCATTTCCAAGCCATTCTGCCG 1200

Sub-sub_ GACTCTGCCGGAGGATCTGCTAAACAAGGACGCGACAGACATTTCCAAGCCATTTTGCCG 1200

Lich_ GACTCTGCGGGCGGATCGGCAAAACAGGGCCGCGACCGTCATTTCCAAGCAATTTTGCCT 1200

Pum_ GATTCAGCGGGCGGATCTGCTAAGCAAGGTCGTGATCGTCACTTCCAAGCCATTTTGCCG 1200

Ant_ GACTCTGCCGGTGGATCAGCAAAGCAAGGGCGTGATCGTCACTTCCAAGCGATTTTACCA 1197

Thu_ GACTCTGCCGGTGGATCAGCAAAGCAAGGGCGTGATCGTCACTTCCAAGCGATTTTACCA 1197

Thr-kon_ GACTCTGCCGGTGGATCAGCAAAGCAAGGGCGTGATCGTCACTTCCAAGCGATTTTACCA 1197

cer._ GACTCTGCTGGTGGATCAGCAAAACAAGGGCGTGATCGTCACTTCCAAGCGATTTTACCG 1197

Wei_ GACTCTGCGGGCGGATCTGCAAAACAAGGACGCGATCGTCATTTTCAAGCGATTTTACCA 1197

Sp_ GACTCTGCTGGCGGATCGGCCAAACAAGGCCGTGATCGGCATTTCCAAGCGATTCTCCCA 1197

Halo_ GACTCTGCTGGCGGATCGGCCAAACAAGGCCGTGATCGGCATTTCCAAGCGATTCTCCCA 1197

Clau_ GATTCTGCCGGCGGTTCGGCAAAAGGGGGCCGAGATCCCCATTTCCAAGCGATTCTTCCT 1200

            ** ** ** ** ** ** ** ** ** ** ** ** ** ***** *** * **

BY-1. CTGCGCGGTAAGATTCTGAACGTTGAGAAAGCCAGACTTGATAAGATTCTCTCAAACAAT 1252

Amyl_ CTGCGCGGTAAGATTCTGAACGTTGAGAAAGCCAGACTTGATAAAATTCTCTCAAACAAT 1260

Sub-sub_ CTTAGAGGTAAAATCCTAAACGTTGAAAAGGCCAGACTGGATAAAATCCTTTCTAACAAC 1260

Lich_ TTGAGAGGGAAAATTTTGAACGTCGAAAAAGCCCGCCTGGACAAAATATTGTCCAACAAT 1260

Pum_ TTAAGAGGGAAGATCCTAAACGTTGAAAAAGCGCGACTAGATAAAATTCTATCGAACAAC 1260

Ant_ CTGAAAGGTAAAATTATTAACGTTGAAAAGGCAAGATTAGATAAAATCTTATCTAACGAT 1257

Thu_ CTGAAAGGTAAAATTATTAACGTTGAAAAGGCAAGATTAGATAAAATCTTATCTAACGAT 1257

Thr-kon_ CTGAAAGGTAAAATTATTAACGTTGAAAAGGCAAGATTAGATAAAATCTTATCTAACGAT 1257

cer._ CTAAAAGGTAAAATTATTAACGTTGAAAAAGCAAGATTGGATAAAATTTTATCTAACGAT 1257

Wei_ CTGAAGGGTAAAATTATTAACGTTGAAAAGGCACGTTTAGATAAGATTTTATCAAATGAT 1257

Sp_ TTGCGCGGGAAAATCTTAAATGTAGAGAAGGCTCGATTAGATAAAATATTAGCGAACAAT 1257

Halo_ TTGCGCGGGAAAATCTTAAATGTAGAGAAGGCTCGATTAGATAAAATATTAGCGAACAAT 1257

Clau_ TTGCGAGGGAAAATCCTTAACGTAGAAAAAGCCCGTCTGGACAAAATTTTAGCAAACAAT 1260

             * ** ** ** * ** ** ** ** ** * * ** ** ** * * ** *

BY-1. GAGGTCAGATCAATGATCACGGCCCTCGGAACAGGAATCGGCGAAGATTTTAATCT-GAA 1311

Amyl_ GAGGTCAGATCAATGATCACGGCCCTCGGAACAGGAATCGGAGAAGATTTTAATCTTGAA 1320

Sub-sub_ GAAGTTCGCTCTATGATCACAGCGCTCGGCACAGGTATCGGAGAAGACTTCAACCTTGAG 1320

Lich_ GAGGTTCGTTCTATGATCACCGCCCTTGGCACCGGAATCGGGGAAGATTTCAACCTTGAA 1320

Pum_ GAGGTTCGTTCAATGATTACAGCATTAGGAACTGGAATCGGAGAAGACTTCAATTTAGAG 1320

Ant_ GAAGTGCGTACAATTATTACTGCAATTGGTACGAACATTGGCGGAGATTTTGATATTGAG 1317

Thu_ GAAGTGCGTACAATTATTACTGCAATTGGTACGAACATTGGCGGAGATTTTGATATTGAG 1317

Thr-kon_ GAAGTGCGTACAATTATTACTGCAATTGGTACGAACATTGGCGGAGATTTTGATATTGAG 1317

cer._ GAAGTGCGTACAATTATTACTGCAATTGGTACGAACATTGGCGGCGATTTTGATATCGAG 1317

Wei_ GAAGTTCGTACAATTATTACAGCAATCGGTACAAATATTGGTGGGGATTTTGCTATTGAA 1317

Sp_ GAAATTCGCGCGATCATTACCGCCCTTGGAACAGGAATTGGTGATGATTTCGACATTGAA 1317

Halo_ GAAATTCGCGCGATCATTACCGCCCTTGGAACAGGAATTGGTGATGATTTCGACATTGAA 1317

Clau_ GAAATTCGGATGATCATCACGGCAATTGGCACGGGAATTGGCGATGAATTTGATATTTCA 1320

            ** * * ** ** ** ** * ** ** ** ** * ** ** *

BY-1. AAAGCGCGT-ATCATAAAGTGTACC 1335

Amyl_ AAAGCGCGTTATCATAAA ------- 1338

Sub-sub_ AAAGCCCGTTACCACAAA ------- 1338

Lich_ AAAGCCCGCTACCACAAA ------- 1338

Pum_ AAGGCTCGCTATCACAAA ------- 1338

Ant_ AAAGCTCGTTATCATAAA ------- 1335

Thu_ AAAGCTCGTTATCATAAA ------- 1335

Thr-kon_ AAAGCTCGTTATCATAAA ------- 1335

cer._ AAAGCTCGTTATCATAAA ------- 1335

Wei_ AAAGCTCGCTATCATAAA ------- 1335

Sp_ AAGGCTCGTTACCATAAA ------- 1335

Halo_ AAGGCTCGTTACCATAAA ------- 1335

Clau_ AAAGCACGCTACCATAAA ------- 1338

            ** ** ** * ** ***

Each strain of the sequence was as follows.

Sp: AB010081, Bacillus sp .., cer: CP000227, Bacillus cereus Q1., Ant: AB190227, Bacillus anthracis ., Thu: CP000485, Bacillus thuringiensis str., Sub-sub: NZ_ABQK01000001, Bacillus subtilis sub sp. subtilis str ., Thr-kon: Bacillus thuringiensis serovar konkukian str ., 8: Lich, NC_006270, B acillus licheniformis ATCC 14580., 9: Pum, NC_009848, Bacillus pumilus SAFR-032., 10: Halo, NC_002570, Bacillus halodurans C 125: Amyl, CP000560, Bacillus amyloliquefaciens FZB42., 12: Clau, AP006627, Bacillus clausii KSM-K16., 13: Wei, NC_010184, Bacillus weihenstephanensis KBAB4.

BY-1 strain (KCTC11712BP) showed 98% homology with Bacillus amyloliquefaciens FZB42. In addition, in the relationship between closely related (Fig. 1), Bacillus amino Lowry quinolyl Pacific Enschede (Bacillus amyloliquefaciens) as shown in the phylogenetic tree BY-1 strains and Bacillus amyloliquefaciens strain will be able to be classified into the same species. According to Drancourt, more than 99% homology is the same at the species level, and more than 97% homology is at the genus level. The results showed 98% homology with B. amyloliquefaciens, and physiological biochemical identification was also confirmed as B. amyloliquefaciens .

Table 5 GyrB sequence similarity matrix between BY-1 and other strains

	One	2	3	4	5	6	7	8	9	10	11	12	13
One	100
2	67	100
3	69	71	100
4	69	70	96	100
5	69	70	96	99	100
6	79	67	70	71	71	100
7	69	70	96	98	98	70	100
8	78	69	70	71	71	76	71	100
9	74	69	73	73	73	75	73	75	100
10	67	100	71	70	70	67	70	69	69	100
11	98	68	70	70	70	80	70	78	75	68	100
12	69	71	69	68	68	70	68	69	68	71	69	100
13	68	70	87	86	86	70	87	71	73	70	69	68	100

1: BY-1., 2: Sp, AB010081, Bacillus sp .., 3: cer, Bacillus cereus Q1, CP000227, 4: Ant, AB190227, Bacillus anthracis ., 5: Thu, CP000485, Bacillus thuringiensis str., 6 : Sub-sub, NZ_ABQK01000001, Bacillus subtilis sub sp. subtilis str ., 7: Thr-kon, NC_005957, Bacillus thuringiensis serovar konkukian str ., 8: Lich, NC_006270, B acillus licheniformis ATCC 14580., 9: Pum, NC_009848, Bacillus pumilus SAFR-032., 10: Halo, NC_002570 , Bacillus halodurans C-125., 11: Amyl, CP000560, Bacillus amyloliquefaciens FZB42., 12: Clau, AP006627, Bacillus clausii KSM-K16., 13: Wei, NC_010184, Bacillus weihenstephanensis KBAB4.

Example 3: Vitamin K ₂  analysis

 Menaquinone-4, 7, which is a standard substance, was dissolved in methanol at a concentration of 1.0 ppm, and then mixed (v / v 1: 1) and 5 μL injection was shown in FIGS. 2 and 3. MK-4 peaked around 6 minutes and MK-7 peaked around 20 minutes, and the correlation coefficients of concentration were 0.9948 and 0.994.

Example 4: Menaquinone (Vitamin K ₂ ) -7 Isolation of microorganisms with high production capacity

Three kinds of Bacillus sp. B. subtilis KCTC1324, a strain of soybean and soybean fermented food, was obtained from the Korea Institute of Biotechnology and Biotechnology Center (Daejeon, Korea) and used as a control to measure the production of MK-7. After culturing for 5 days at 40 ° C. in soybean extract extracted with 10-fold water, one of the best strains for producing menaquinone (vitamin K ₂ ) -7 was selected. Bacillus amyloliquefaciens BY-1 was 7.568 mg / L of MK. Produced -7.

Therefore, the gene was analyzed by gyrase method and identified as Bacillus amyloliquefaciens . Rowland and Taber (15) and Rowland et al. Has been extensively studied on the mechanism of MK production using Bacillus subtilis strains, and Sato et al , B. subtilis strains, Tsukamoto et al and Tani et al. al reported Flavobacterium strain, and recently, Morisita et al reported the results using lactic acid bacteria strain. However, Bacillus amyloliquefaciens was isolated and developed for the first time.

Table 6 Comparison of the production capacity of menaquinone-7 at the optimum temperature of each strain

Bacillus strain	OD 660 1)	MK-7 (mg / L)
BY-1 2)	2.889 ± 0.042 4) a5)	7.568 ± 0.140 a
BY-2 2)	2.813 ± 0.014 a	7.417 ± 0.048 a
BY-3 2)	2.782 ± 0.056 a	5.283 ± 0.003 b
KCTC1324 3)	2.418 ± 0.062 c	5.324 ± 0.149 b

¹⁾ OD ₆₆₀ (absorbance at ₆₆₀ nm) is measured after incubation. Each sample is analyzed as supernatant. ²⁾ incubate at 40 ° C.

³⁾ Incubate at 37 ° C. ⁴⁾ Mean ± SD (n = 3) ⁵⁾ There was a significant difference at p <0.01 by Duncan's multiple range test.

Example 5 Effect of Shaking Culture Time and Sodium Chloride Concentration on Menaquinone-7 Production

As a preliminary step for the fermentation of Cheonggukjang, the total bacterial counts, spores, and MK-7 contents according to the shaking culture time of the starter are shown in Table 8.

TABLE 7 <I> B. In soy extract. amyloliquefaciens </ i> Effect of Shake Time on Melaquinone-7 Production by Strain BY-1

Shaking time (h)	OD 660 1)	Total viable cells (10 7 / mL)	Spore (10 2 / mL)	MK-7 (mg / L)
0	0.54 ± 0.006 2) e3)	2.77 ± 0.95 d	0.00 ± 0.00 c	3.44 ± 0.39 b
4	2.50 ± 0.021 d	94.00 ± 25.94 b	0.33 ± 0.58 c	4.22 ± 0.06 a
6	3.51 ± 0.083 c	185.67 ± 23.75 a	7.00 ± 1.00 c	3.77 ± 0.24 ab
8	4.00 ± 0.140 a	55.33 ± 5.86 bc	10.33 ± 2.62 c	3.66 ± 0.31 b
10	3.98 ± 0.031 a	16.00 ± 4.58 cd	104.00 ± 65.48 b	3.70 ± 0.26 ab
12	3.71 ± 0.110 b	30.00 ± 16.64 cd	758.00 ± 98.80 a	3.25 ± 0.29 b

¹⁾ OD ₆₆₀ (absorbance at ₆₆₀ nm) is measured after incubation. Each sample is analyzed as supernatant.

²⁾ Mean ± SD (n = 3) ³ There is a significant difference at p <0.01 by Duncan's multiple range test.

After inoculating 1 mL (42.75 × 10 ⁷ CFU) of the entire culture of Bacillus amyloliquefaciens BY-1 in 20 mL of soybean extract while shaking at 120 rpm at 40 ° C., incubated for 4, 6, 7, 8, 10, 12 hours, The highest content of 4.22 ± 0.06 mg / L of menaquinone-7 was shown in the cultured cultures after 4 hours shaking culture in the cultured cultured cells at 40 ° C for 3 days. The number of viable cells was 185.67 × 10 ⁷ CFU / mL when stationary cultured for 3 days after 6-hour shaking culture, and 700 CFU / mL spores were generated when spores were produced. After shaking for 6 hours or more, the number of spores was significantly increased while the contents of menaquinone-7 were significantly decreased in each culture group.

From these results, it was shown that the viable cell number was the highest at 4 hours shaking culture time before the spores were produced, and thus the optimum incubation time was 4-5 hr.

In order to investigate the effect of NaCl concentration on the production of menaquinone of Bacillus amyloliquefaciens BY-1 isolated strain, 0.2 ~ 0.8% (w / v) NaCl was added to soy extract culture, and NaCl concentration was 0.4% as shown in Table 9. Menaquinone (MK-4, 7) was the highest content in comparison with the control (Fig. 8, 9, 10).

Table 8 <I> B. In soy extract during 3-day stationary culture. amyloliquefaciens </ i> Effect of Sodium Chloride (NaCl) on Melaquinone-7 Production by Strain BY-1

Sodium chloride concentration (%)	Viable count (10 7 / mL)	MK-7 (mg / kg)
0.0	28.67 ± 7.09 1) b2)	2.79 ± 0.06 b
0.2	29.00 ± 7.55 b	3.01 ± 0.01 ab
0.4	47.00 ± 3.00 a	3.22 ± 0.02 a
0.6	34.67 ± 5.03 ab	3.03 ± 0.19 ab
0.8	40.67 ± 10.69 ab	2.86 ± 0.29 b
1.0	29.67 ± 8.96 b	2.80 ± 0.11 b

¹⁾ Mean ± SD (n = 3)

²⁾ There was a significant difference at p <0.01 by Duncan's multiple range test.

Example 6: Determination of menaquinone, amino type nitrogen and amino acid according to the culture temperature

After inoculation of B. amyloliquefaciens BY-1 strain to soybean extract and fermentation at 40 ° C. for 48 hours, the extract was extracted with methanol and the content of menaquinone (MK-4, 7) in the filtrate was measured. When fermented at 37, 40, 43 and 46 ℃, the content of menaquinone was 5.45 ± 0.28, 6.29 ± 0.72, 8.21 ± 0.77 and 6.31 ± 0.32 mg / kg, respectively, and the optimum incubation temperature for the production of menaquinone was 43 ℃. Judging by

Table 9 Effect of temperature on the production of MK-4 and MK-7 in Chungkookjang

Tem. (℃)	MK-4 (mg / kg)	MK-7 (mg / kg)	Ration (MK4 / MK7)	Total (mg / kg)
37	0.84 ± 0.06 1) a2)	4.61 ± 0.32 c	0.18 ± 0.01 a	5.45 ± 0.28 b
40	0.71 ± 0.07 b	5.58 ± 0.66 bc	0.13 ± 0.01 b	6.29 ± 0.72 b
43	0.75 ± 0.06 ab	7.46 ± 0.72 a	0.10 ± 0.01 bc	8.21 ± 0.77 a
46	0.64 ± 0.02 c	5.67 ± 0.33 b	0.11 ± 0.00 bc	6.31 ± 0.32 b

¹⁾ Mean ± SD (n = 3)

²⁾ There was a significant difference at p <0.01 by Duncan's multiple range test.

The results of analyzing the amino acids are shown in Table 11. The result of the highest amino acid content when incubated at 43 ° C. indicates that the protein resolution is also optimal, which means that the fermentation is not inhibited as the content of menaquinone is increased.

Table 10 Amino Acid Composition of Peptides Isolated from Cheonggukjang

Amino acid		Content (pmole)
		37	40	43	46
Essential free amino acids	Histidine	5.51 b	6.28 b	8.43 a	6.86 ab
	Throneine	2.09 b	3.03 b	5.28 a	5.23 a
	Arginine	0.04 c	0.13 bc	0.54 a	0.35 ab
	Valine	7.63 b	9.02 b	12.20 a	11.63 a
	Methionine	3.64 b	4.03 ab	4.54 a	3.84 ab
	Isoleucine	5.28 b	7.00 b	11.06 a	10.86 a
	Leucine	8.33 c	12.33 b	18.24 a	18.29 a
	Lysine	0.60 c	1.00 c	1.80 b	2.27 a
	Phenylalanine	15.20 b	17.71 b	22.94 ab	19.02 a
Flavor enhancer free amino acids	Asprtic acid	0.00	0.00	0.00	0.00
	Glutaminc acid	1.94	3.39	4.78	3.00
	Serine	0.60	0.94	0.89	0.73
	Glycine	5.77 b1)	10.45 b	19.57 a	19.75 a
	Alanine	5.08 b	7.36 b	15.07 a	14.47 a
Free amino acids and derivatives	Prosphoserine	6.47 b	7.86 b	12.87 a	12.15 a
	Tyrosine	9.91 b	11.43 ab	14.36 a	12.90 ab
Total		78.09	101.96	152.57	139.08

¹⁾ There was a significant difference at p <0.01 by Duncan's multiple range test.

Example 7 Influence of Carbon Sources on the Production of Menaquinone

The carbohydrate utilization by API 50CHB kit was examined and preliminary tests were performed with the addition of strong positive carbohydrates, followed by glucose, glycerol, maltose, mannose, soluble starch ( soluble starch) was thought to increase the production of menaquinone. Therefore, in order to investigate the effect of carbon source on the production of menaquinone by Bacillus amyloliquefaciens BY-1, soybean extract was used as a control medium, and glucose, glycerol, maltose, mannose ( mannose) and soluble starch (2%) were added to prepare a culture solution, and then cultured at 43 ° C. for 2 days. Table 12 shows the results of measuring Mk-4 and MK-7 production. When glucose was added to glycerol, maltose, mannose, and soluble starch, the production of MK-7 was significantly different from that of the control, whereas glucose was added. Was not significantly different from the control. In addition, the addition of 2% carbon source of soluble starch increased the MK-4 production significantly compared to the control. When glycerol was added, Mk-4 and MK-7 were the highest. Cheonggukjang was prepared by adding 2.5, 5, 10, and 15% of glycerol, respectively, and the production of Mk-4 and MK-7 was measured. In this study, 10% of glycerol was measured. The highest value was 12.47 mg / kg when added. The soybean extract contained a large amount of free carbohydrates, while the increased soybeans had relatively low carbohydrate availability, which required high glycerol concentrations. In addition, 2% addition of maltose and mannose significantly increased Mk-4 and MK-7 production, suggesting a good carbon source to replace glycerol.

Table 11 Effect of Carbon Sources on Menaquinone Formation

Carbon source (2%)	MK-4 (mg / kg)	MK-7 (mg / kg)	Ration (MK4 / MK7)	Total (mg / kg)
Control	0.62 ± 0.09 1) b2)	7.35 ± 0.73 c	0.085 ± 0.012	7.97 ± 0.82 d
Glucose	0.69 ± 0.07 b	7.77 ± 0.07 c	0.089 ± 0.009	8.46 ± 0.01 cd
Glycerol	0.76 ± 0.02 b	11.71 ± 0.61 a	0.065 ± 0.002	12.47 ± 0.63 a
Maltose	0.69 ± 0.05 b	9.77 ± 1.10 b	0.070 ± 0.005	10.45 ± 1.04 b
Mannose	0.71 ± 0.09 b	9.41 ± 0.29 b	0.075 ± 0.009	10.12 ± 0.20 b
Starch	0.95 ± 0.11 a	8.67 ± 0.64 bc	0.113 ± 0.001	9.61 ± 0.56 bc

¹⁾ Mean ± SD (n = 3)

²⁾ There was a significant difference at p <0.01 by Duncan's multiple range test.

Table 12 Effect of Glycerol Concentration on Menaquinone Production

Glycerol (%)	MK-4 (ug / g)	MK-7 (ug / g)	Ration (MK4 / MK7)	Total (ug / g)
0	0.75 ± 0.06 1) b2)	7.46 ± 0.72 c	0.10 ± 0.01	8.21 ± 0.77 c
2.5	0.84 ± 0.01 b	8.71 ± 0.67 bc	0.10 ± 0.00	9.55 ± 0.68 bc
5.0	0.78 ± 0.05 b	9.84 ± 0.42 ab	0.08 ± 0.01	10.63 ± 0.37 ab
10.0	1.02 ± 0.08 a	11.13 ± 0.64 a	0.09 ± 0.01	12.15 ± 0.62 a
15.0	0.90 ± 0.07 ab	9.93 ± 0.97 ab	0.09 ± 0.01	10.73 ± 1.04 ab

¹⁾ Mean ± SD (n = 3)

²⁾ There was a significant difference at p <0.01 by Duncan's multiple range test.

Comparative Example 1: Comparison of MK-4,7 Content with Other Cheonggukjang

Isolation of strains with high production of vitamin K ₂ from soybean fermented foods is progressing in Japan and China, but most of them were Bacillus subtilis strains, but no Bacillus amyloliquefaciens strains were reported. The total content of MK-4, 7 contained in Cheonggukjang fermented at 43 ° C for 2 days under conditions without additives using Bacillus amyloliquefaciens BY-1 was fermented using the product produced in Pulmuone or B. subtilis KCTC13241 strain. It was more than twice the amount of Cheonggukjang and the content was 7.54 mg / kg.

Table 13 Menaquinone Content Comparison

Sample	MK-4 (ug / g)	MK-7 (ug / g)	Total (ug / g)
B. amyloliquefaciens BY-1 1)	0.88 ± 0.04 4) a5)	6.66 ± 0.50 a	7.54 ± 0.49 a
Commercial Cheonggukjang 2)	0.00 ± 0.00 b	2.79 ± 0.12 b	2.79 ± 0.12 b
B. subtilis KCTC13241 3)	0.74 ± 0.08 a	2.71 ± 0.15 b	3.45 ± 0.09 b

¹⁾ Incubation at 43 ℃ ²⁾ Pulmuone food Co .. ³⁾ Incubation at 37 ℃ ⁴⁾ Mean ± SD (n = 3)

⁵⁾ There was a significant difference at p <0.01 by Duncan's multiple range test.

The present invention can provide a composition for fermentation of Cheonggukjang containing a large amount of vitamin K ₂ by using a strain having excellent menaquinone synthesis ability, and can also provide a functional food that can help prevent osteoporosis. .

Microbial deposit

Claims (10)

1. Bacillus amyloliquefaciens BY-1 strain (KCTC11712BP) excellent in menaquinone synthesis.
2. The strain of claim 1, wherein the Bacillus amiriquifaciens BY-1 has a gyrB gene of SEQ ID NO: 1.
3. Bacillus amiriquifaciens BY-1 (KCTC11712BP), a composition for fermenting Cheonggukjang comprising a culture of the strain or a culture solution of the strain.
4. Using the composition of claim 3, soybean crushed, soybean or black soybean fermentation method characterized in that the fermentation.
5. The method of claim 4, wherein

   Increasing the soybeans, black beans or soy crushes;

   Inoculating the composition of claim 3 on the soybean, black soybean or soybean shreds; And

   Fermenting the inoculated soybeans, black beans or soybean crushed.
6. The method according to claim 5, wherein the fermentation step is carried out at 41 to 45 ℃.
7. The method according to claim 5, wherein glycerol is added in the fermentation step.
8. 8. A process according to claim 7, wherein the glycerol is added in an amount of 8 to 12% by weight.
9. Cheonggukjang prepared by the method according to any one of claims 4 to 8.
10. Food for preventing or ameliorating osteoporosis comprising the cheongukjang prepared by the method according to any one of claims 4 to 8.

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