KR101156161B1 - Bacillus subtilis hj18-4 oligotrophic strain isolated from memil soksungjang and method for making fermentation food by using same - Google Patents

Abstract

The present invention relates to a low-nutritional fermentation strain derived from a fast-acting field using buckwheat, producing fermentation enzymes, having antimicrobial activity against food poisoning bacteria and E. coli, and having a thrombolytic ability, and a method for preparing fermented food using the same. Low-nutritional strains isolated from fast-growing intestines, with fermentative enzyme-producing ability; food poisoning bacteria and E. coli Strain The accession number for having antibacterial activity and fibrinolytic functions claim KACC 91541P favor Bacillus subtilis (Bacillus subtilis) strain HJ18-4 cellulase (cellulase), amylase (amylase), Protease (protease), lipase (lipase) and chair ILA Kinase (enzyme production and Candida (Candida, such as xylanase)), Listeria monocytogenes (Listeria) related to the fermentation Salmonella (Salmonella), Staphylococcus (Staphilococcus), Bacillus cereus (Bacillus cereus ) Such as Food poisoning bacteria and E. coli Strain Since it has antimicrobial activity and thrombolytic ability, it is expected to be able to produce fermented enzymes using this, as well as to produce fermented foods with improved quality.

Description

BACILLUS SUBTILIS HJ18-4 OLIGOTROPHIC STRAIN ISOLATED FROM MEMIL SOKSUNGJANG AND METHOD FOR MAKING FERMENTATION FOOD BY USING SAME}

The present invention relates to a low-nutritional fermentation strain derived from fast-acting field using buckwheat, producing fermentation enzymes, having antimicrobial activity against food poisoning bacteria and Escherichia coli, and having a thrombolytic ability, and a method for preparing fermented food using the same.

Fermentation technology using its own spawn, such as European yogurt and Japanese natto, is not only spreading to the whole world, but also earning a lot of foreign currency by intellectualizing it. Therefore, in Korea, a systematic study on the development and preservation management of excellent traditional fermented microorganisms for quality improvement, quality uniformity, and extension of preservation centering on traditional fermented foods is required. In addition, domestic industries are also required to pay foreign royalties to pay royalties, so considering the long-term industrial development, it is necessary to secure our excellent strains.

Microbes account for 60% of the world's biomass, but only about 1% is known to be isolated or cultured. Recently, the necessity of technology development to utilize the infinite function of these cultured microorganisms has emerged.

Buckwheat fasteners are fast-paced well-being cages containing bioactive ingredients, unlike normal doenjang, which takes five months from buckwheat to maturation. By adding brine to short-term fermented soybeans prepared by short-term fermentation using steamed soybean and buckwheat flour, mixed with brine and then fermented into jars, it has excellent physiological activities such as antithrombotic effect and antioxidant activity. It is a safety well-being attribute book that has an inhibitory effect on (see Korean Patent Application No. 10-2009-134375).

Therefore, the inventors of the present invention, while continuing to research and identify new strains having antibacterial activity against food poisoning bacteria and E. coli and thrombolytic activity from buckwheat fasteners, Bacillus subtilis (a new strain isolated from buckwheat fasteners) Bacillus subtilis) strain cellulase (cellulase related to the fermentation as a low nutritional strain), amylase (amylase), protease (protease), lipase (lipase) and chair ILA kinase (enzyme production and Candida (Candida, such as xylanase)), Stephen Lee Ria (Listeria), Salmonella (Salmonella), Staphylococcus (Staphilococcus), Bacillus cereus (Bacillus cereus) Such as Food poisoning bacteria and E. coli Strain The present invention has been completed by discovering that it has antimicrobial activity and thrombolytic ability.

An object of the present invention is to provide an enzyme related to fermentation derived from fast-acting field using buckwheat, and to provide a low-nutrition fermentation strain having antibacterial activity against food poisoning bacteria and Escherichia coli and having thrombolytic ability.

Another object of the present invention is to provide a method for producing a fermented food using the low nutritional fermentation strain.

In order to achieve the above object, in the present invention, as a low-nutritional strain isolated from buckwheat fasteners, it has the ability to produce fermentation enzymes, food poisoning bacteria and E. coli ( E. coli ) Strain The accession number for having antibacterial activity and fibrinolytic functions claim KACC 91541P favor Bacillus subtilis (Bacillus subtilis ) provides the HJ18-4 strain.

In order to achieve the above object, the present invention provides a method for producing a fermented food using the Bacillus subtilis strain HJ18-4 strain.

Bacillus subtilis HJ18-4, a new strain isolated from buckwheat tract field according to the present invention, is a cellulase, amylase, protease, lipase and xylase related to fermentation. such as the enzyme and Candida (Candida), Listeria monocytogenes (Listeria), Salmonella (Salmonella), Staphylococcus (Staphilococcus), Bacillus cereus (Bacillus cereus ) Such as Food poisoning bacteria and E. coli Strain Since it has antimicrobial activity and thrombolytic ability, it is expected to be able to produce fermented enzyme using this, as well as to produce fermented food with improved quality.

FIG. 1 is Escherichia coli (E. coli), fatigue star nose Syracuse aureus (Staphylococcus aureus) and Bacillus cereus (Bacillus cereus ) shows the results of cultivation of buckwheat fasteners in selective medium.
Figure 2 is a low-nutrition bacterium Bacillus subtilis separated by buckwheat fasteners according to the present invention The morphological observations of the HJ18-4 strain and the Bacillus subtilis KACC 10114 strain, which is a standard strain, are shown.
Figure 3 is a low nutritional Bacillus subtilis separated into buckwheat fastener field according to the present invention This is a result of comparing colony morphology in low nutrient production medium of HJ18-4 strain and Bacillus subtilis KACC 10114 strain which is a standard strain.
Figure 4 is a low nutritional Bacillus subtilis separated by buckwheat fastener field according to the present invention This is a result of comparing the protease, cellulose, amylase and lipase enzyme activities of the HJ18-4 strain and the standard strain Bacillus subtilis KACC 10114 strain.
Figure 5 is a low nutritional Bacillus subtilis separated by buckwheat fastener field according to the present invention The antimicrobial activity of HJ18-4 strain and Bacillus subtilis KACC 10114 strain, which is a standard strain, was compared.
Figure 6 is a low nutritional Bacillus subtilis separated by buckwheat fastener field according to the present invention It was systematically classified by 16s rDNA sequencing of the HJ18-4 strain.
Figure 7 is a low nutritional Bacillus subtilis separated by buckwheat fastener field according to the present invention It shows the thrombolytic ability of the HJ18-4 strain.
Figure 8 is a low nutritional Bacillus subtilis separated by buckwheat fastener field according to the present invention HJ18-4 strain hemolysis is shown.

The technical terms and scientific terms used in the present invention can be construed as meaning ordinary meanings understood by those of ordinary skill in the art without departing from the scope of the present invention.

Food poisoning bacteria and Escherichia coli E. coli as low-nutritive strains isolated from the buckwheat extract field of the present invention Strain Have antimicrobial activity and thrombolytic ability. The strain isolated from the present invention was isolated from the buckwheat field, and as a result of morphological characteristics, biochemical properties, and sequencing of 16s rDNA, it was confirmed that the strain had 99% homology with Bacillus subtilis. It was named Bacillus subtilis HJ18-4, and deposited on March 29, 2010, with the accession number KACC 91541P to the National Institute of Agricultural Science. Isolation of this strain is not possible with the usual high nutrient medium, but after isolation in low nutrient medium, its proliferation can be cultured by the normal high nutrient medium culture method.

Bacillus subtilis low nutritional strain HJ18-4 of the present invention has the following morphological and molecular biological characteristics.

Morphological features of the Bacillus subtilis low nutritional strain HJ18-4 of the present invention are shown in FIG. 2 by optical microscopy, and as shown in FIG. 3, typical Bacillus forms such as Bacillus subtilis KACC 10114. Representative Gram-positive bacterium and low-nutritional bacteria that produce sugar are the optimum growth temperature.

In addition, the biochemical experiments of Bacillus subtilis HJ18-4 of the present invention were very similar to the Bacillus subtilis KACC 10114 strain, a standard strain as shown in FIG.

Bacillus subtilis HJ18-4 strain of the present invention produces enzymes such as cellulase (amylase), amylase, protease (lipase) and xylase (xylanase) enzymes related to fermentation ( see Fig. 1), Candida (Candida), Listeria monocytogenes (Listeria), Salmonella (Salmonella), Staphylococcus (Staphilococcus), Bacillus cereus (Bacillus cereus ) Such as Food poisoning bacteria and E. coli Strain Have antimicrobial activity (see FIGS. 4 to 5), and have thrombolytic ability (see FIGS. 7 and 8).

Therefore, the Bacillus subtilis HJ18-4 strain of the present invention can be used not only to produce fermentation enzymes, but also to improve fermented foods, for example, to produce quality fermented foods, which can be very useful in the field of fermented foods. It is expected to be.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited by these Examples.

Example  1: Strain Separation Using Low Nutrition Medium

To isolate the strain producing enzymes and sugars and antibacterial substances of the present invention, samples of buckwheat growth were collected, 10 g of the sample was placed in a sterile bag and 100 ml of 0.85% physiological saline was added for 10 minutes using BAGMIXER (interscience, France). Samples were prepared by homogenizing for a while.

Low nutrient medium composition, yeast extract, 0.5 g; Proteose peptone no3, 0.5 g; Casamino acids, 0.5 g; 0.5 g of dextrose; 0.5 g of soluble starch; Sodium pyruvate, 0.3 g; Dipotassium phosphate, 0.3 g; Magnesium sulfate, 0.05 g; Distilled water, 1000 mL was prepared by adjusting to pH 7.0 ~ 7.2. Isolate the strains that grow after incubating various low-nutrition bacteria in R2A plate medium with 10 ^-1 , 10 ^-2 , 10 ^-3 and 10 ^-4 times diluted in distilled water in R2A, a low nutrient bacterial separation medium It was.

Example  2: enzyme activity and antimicrobial activity Excellent strain  Selection

Using the strain isolated in Example 1 for the enzyme activity and sugar production and food poisoning bacteria Antimicrobial activity was measured. Among them, the enzyme production of cellulose, amylase, protease, lipase and xylanase is excellent, and of Candida, Listeria, Salmonella, Staphylococcus and Bacillus cereus Bacillus subtilis HJ18-4 strain, which has the ability to inhibit the growth of food poisoning bacteria and E. coli E. coli , was selected.

Example  3: HJ18 Morphological and Biochemical Properties of -4

After examining the morphological and biochemical characteristics of the Bacillus subtilis HJ18-4 strain selected in Example 2, classification was identified through a flexible relationship with other species based on the analysis of 16S rDNA.

Morphological characteristics of the selected Bacillus subtilis HJ18-4 strain are observed in an optical microscope and are shown in FIG. 2. Figure 3 compares the colony morphology of the low nutrient Bacillus subtilis HJ18-4 strain and the standard strain Bacillus subtilis KACC 10114 strain of the present invention separated by buckwheat field in a low nutrient medium (colony morphology) Indicated. As shown here, the Bacillus subtilis HJ18-4 strain of the present invention is a typical Bacillus genus bacillus, such as the standard strain KACC 10114, which is a typical Gram-positive and low-trophic bacterium that produces sugars. Appeared. Figure 6 is a low nutritional Bacillus subtilis separated into buckwheat fastener field according to the present invention The phylogenetic taxonomy by 16s rDNA sequencing of the HJ18-4 strain is shown.

In addition, biochemical experiments were performed using the microbial identification kit API20NE kit, and the biochemical results of the Bacillus subtilis HJ18-4 and the standard strain KACC10114 strain are shown in Table 1 below. As shown here, it can be seen that the Bacillus subtilis HJ18-4 strain of the present invention is very similar to the KACC10114 strain which is a standard strain.

Example  4 : Buckwheat  Antimicrobial effect HJ18 - 4 strains  Comparative Analysis of Enzyme Activity and Antibacterial Activity

The antimicrobial activity of the food poisoning causing bacteria of the buckwheat growth, a specimen of the Bacillus subtilis HJ18-4 strain of the present invention was examined and the results are shown in FIG. 1. Buckwheat growth fermented for two weeks or more was isolated from E. coli , Staphylococcus aureus and Bacillus cereus (Bacillus cereus ) No harmful microorganisms were detected as a result of plating on selective medium.

In addition, the enzyme activity and antimicrobial activity of the HJ18-4 strain were examined, and the enzyme activity and the antimicrobial activity were much higher than those of the standard strain KACC10114. The results are shown in FIGS. 4 and 5, and are shown in Table 2 below.

In addition, 16S rDNA was isolated to confirm the nucleotide sequence of HJ18-4. As a result, it was 99% homologous to Bacillus subtilis and analyzed for the soft relationship with other species. Therefore, the selected HJ18-4 strain was Bacillus subtilis ( Bacillus subtilis ) was named HJ18-4 and deposited on March 29, 2010, with the accession number KACC 91541P to the National Institute of Agricultural Science.

Example  5: HJ18 -4 strains Thrombolytic ability

Low nutritional Bacillus subtilis separated by buckwheat fastener according to the present invention The thrombolytic ability of the HJ18-4 strain was measured as follows. Fibrinogen was dissolved to 0.3% in 10 mM phosphate buffer (pH 7.8) containing 0.15 M NaCl, and 5 mL of a 1% agarose solution was added to 5 mL of the completely dissolved fibrinogen solution and mixed well. 0.1 mL of thrombin (100 NIH / mL) was added thereto, mixed, immediately poured into a petri dish, and allowed to solidify by standing at room temperature for 5 to 10 minutes to form a fibrin plate. After spotting 2uL of the group culture solution and reacting at 37 ° C for 18 hours, the dissolution area of the fibrin plate was measured. The results are shown in FIG. 7, and as a result, low nutritional Bacillus subtilis separated by the buckwheat fastener according to the present invention. The HJ18-4 strain showed superior thrombolytic activity compared to the standard strain Bacillus subtilis KACC 10114 strain.

Figure 8 is a low nutritional Bacillus subtilis separated by buckwheat fastener field according to the present invention HJ18-4 strain hemolysis was shown, which is summarized in Table 3 below.

Strain name Bacillus subtilis HJ18-4 Bacillus subtilis D8-3 Whether hemolysis - ++

As shown in Table 3, the low nutritional Bacillus subtilis separated by the buckwheat fastener field according to the present invention The HJ18-4 strain did not show hemolysis, and the bacterium Bacillus subtilis D8-3 showed hemolysis.

Bacillus subtilis HJ18-4, a new strain isolated from buckwheat tract field according to the present invention, is a cellulase, amylase, protease, lipase and xylase related to fermentation. such as the enzyme and Candida (Candida), Listeria monocytogenes (Listeria), Salmonella (Salmonella), Staphylococcus (Staphilococcus), Bacillus cereus (Bacillus cereus ) Such as Food poisoning bacteria and E. coli Strain Since it has antimicrobial activity and thrombolytic ability, it is expected to be able to produce fermented enzyme using this, as well as to produce fermented food with improved quality.

Agricultural Biotechnology Research Institute KACC91541 20100329

Claims (5)

As a low-nutritional strain isolated from buckwheat has grown in the fermentation enzyme-producing ability, sikjungdokgyun and Escherichia coli (E. coli) Strain The accession number for having antibacterial activity and fibrinolytic functions claim KACC 91541P favor Bacillus subtilis (Bacillus subtilis ) HJ18-4 strain.
The method of claim 1,
Bacillus subtilis HJ18-4 strain, characterized in that the fermentation enzyme (cellulase), amylase (amylase), protease (lipase) or xylase (xylanase).
The method of claim 1,
Sikjungdokgyun Candida (Candida), Lee Ste Liao (Listeria), Salmonella (Salmonella), Staphylococcus (Staphilococcus) or Bacillus cereus Bacillus subtilis strain, characterized in that HJ18-4 (Bacillus cereus).
Method for producing a fermented food using Bacillus subtilis strain HJ18-4 strain according to any one of claims 1 to 3.
The method of claim 4, wherein
Fermented food is a manufacturing method characterized in that the enteric.

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