WO2017213327A1 - 폴리아민 고생산 신균주 바실러스 서브틸리스 ee5 균주 - Google Patents

폴리아민 고생산 신균주 바실러스 서브틸리스 ee5 균주 Download PDF

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WO2017213327A1
WO2017213327A1 PCT/KR2017/001755 KR2017001755W WO2017213327A1 WO 2017213327 A1 WO2017213327 A1 WO 2017213327A1 KR 2017001755 W KR2017001755 W KR 2017001755W WO 2017213327 A1 WO2017213327 A1 WO 2017213327A1
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strain
polyamine
natto
bacillus subtilis
polyamines
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PCT/KR2017/001755
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French (fr)
Korean (ko)
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이해익
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강원대학교 산학협력단
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Priority to JP2018504928A priority Critical patent/JP6454052B2/ja
Publication of WO2017213327A1 publication Critical patent/WO2017213327A1/ko

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/70—Germinated pulse products, e.g. from soy bean sprouts
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus

Definitions

  • the present invention relates to a strain of the genus Bacillus that produces polyamine-containing natto, and more particularly, to a Bacillus subtilis EE5 strain that produces polyamine-containing natto.
  • Polyamines are known to exhibit cell proliferation, anti-allergic, anti-aging and the like as one of the physiologically active substances present in large amounts in cells. In particular, in recent years, it is known to exhibit an inhibitory effect on inflammation, and an effect of suppressing the progression of diseases accompanying aging is expected.
  • a polyamine is extracted from a plant using a salt solution in the same manner as the prior art document 1, and used for health food, cosmetics, food or medicine A description of the method is disclosed.
  • Patent Document 1 Republic of Korea Patent Registration No. 10-1373224 (2014.03.05.)
  • Patent Document 2 Republic of Korea Patent Publication No. 10-2013-0054504 (2013.05.27.)
  • Patent Document 3 Republic of Korea Patent Registration No. 10-1495602 (2015.02.16.)
  • Patent Document 4 Republic of Korea Patent Publication No. 10-2011-0018898 (2011.02.24.)
  • the present inventors have studied the development of foods with improved polyamine content, and found that certain strains among the strains related to fermentation of natto have a significantly higher polyamine biosynthesis.
  • Bacillus subtilis EE5 strain (Accession No. KCTC13019BP) having a high production of polyamine is provided.
  • the strain is characterized by having a 16s RNA of SEQ ID NO: 1, it is possible to produce polyamines, in particular spermidine with high efficiency.
  • a culture fermented using the strain In one embodiment, the culture may be natto.
  • a food composition comprising the culture.
  • a method for producing a polyamine comprising the step of fermenting using the strain.
  • the fermentation may be fermentation of natto, and the polyamine may be spermidine.
  • the natto fermented with the novel strain Bacillus subtilis EE5 appears to contain about 5 times or more polyamines as compared to general natto, and it is possible to supply the required amount of polyamine 1 as one case of natto (50 g). Turned out.
  • Bacillus subtilis EE5 strain of the present invention is usefully applied as a means for economically and efficiently providing high-quality polyamine-containing functional natto with high palatability by being applied to an existing natto manufacturing process in a field requiring a high polyamine-containing functional food. Can be used.
  • FIG. 2 is a chromatogram (A) of HPLC identifying dansyl derivatives of polyamines to identify polyamines produced by the Bacillus subtilis EE5 strain in natto, and MS spectra (B) of corresponding peaks.
  • the present invention provides a Bacillus subtilis EE5 strain (Accession No. KCTC13019BP) having the ability to produce high polyamines.
  • the strain can be obtained by separating the bacteria that form heat-resistant spores from rice straw and hay, and through the mutagenesis process to select a strain that produces a polyamine of high content in natto.
  • the strains selected produce high polyamines, in particular spermidine, and can produce about 5.8 times higher polyamines than in the case of normal strains in fermentation of natto.
  • the strain is characterized by having the 16s RNA of SEQ ID NO: 1.
  • the culture may be a culture of legumes, preferably natto.
  • a food composition comprising the culture.
  • a method for producing a polyamine comprising the step of fermenting using the strain. Fermentation in the production method may be fermentation of natto, the polyamine may be spermidine.
  • the isolated strain was inoculated in a medium in which 5 ml of 5% soy flour was suspended and incubated at 37 ° C. for 24 hours. The culture was centrifuged to determine the amount of polyamine from the supernatant.
  • cyclohexane ethylacetate (3: 2 v / v) was used as a developing solvent, and 1 to 3 ⁇ l of single-treated samples and spurs were used. After loading with 1-3 ⁇ l of midine standard, it was photographed under ultraviolet light and the intensity of spot was measured with a densitometer to determine the amount of spermidine from the standard curve. .
  • natto 3 g of cultured natto was added 30 ml of 3.5% perchloric acid, and the suspension homogeneously pulverized with polytron was shaken at 4 ° C for 1 hour, centrifuged, and the supernatant was dansylation to give TLC or Measured using HPLC.
  • HPLC liquid chromatography
  • polyamines separated by liquid chromatography were analyzed by mass spectrometry (MS / MS: Thermo Scientific TSQ Quantum Ultra, Thermo Scientific, USA) to identify polyamines contained in natto, and the mass spectrometry is shown in Table 2 below. It was performed under the same conditions.
  • Fragment Ion 1 305.00 (304.999-305.001)
  • Beans were soaked at room temperature for 12 hours and then boiled at 121 ° C. for 40 minutes. Inoculated 10 3 spores per gram before cooling the boiled soybeans and put 50 g in a foamed styrene container for natto production and covered with a vinyl membrane, and incubated at 37 °C for 20 hours. After incubating the cultured natto for 12 hours, polyamine content was measured and sensory evaluation was performed.
  • Bacillus subtilis After first selecting about 100 kinds of bacteria as high polyamine-producing bacteria by the above process, the bacteria classified into Bacillus subtilis according to the taxonomic analysis of the strains of the genus Bacillus (Bergey's Manual of Systematic Bacteriology, 2002). Eight species were selected second. The eight strains selected were as listed in Table 4 below.
  • Natto was prepared using each of the eight strains, and finally, the most suitable JR-018 strain was selected for the production of natto according to the high productivity of polyamines, the generation of viscous substances, the fragrance, and the ease of spore formation.
  • the JR-018 strain was treated with nitrosoguanidine (NTG, 100 ⁇ g / ml) at 99.9% mortality to induce mutations, followed by Spizizen containing 5 mM dicyclohexylamine.
  • NVG nitrosoguanidine
  • the amount of polyamine was measured by smearing 10 5 animals per sheet in a minimum medium and incubating for 5 days at 37 ° C. in 5% soybean medium.
  • the most elevated strain was named Bacillus subtilis EE5, and the Korea Biotechnology Research Institute (KCTC) 2016.05.03.
  • the deposit was dated (Accession No .: KCTC13019BP), and natto was prepared to evaluate the amount of polyamine produced and the functionality of the prepared natto.
  • the polyamine content of natto fermented using normal strain, JR-018 strain and Bacillus subtilis EE5 is shown in Table 5 below.
  • the natto fermented using Bacillus subtilis EE5 shows a high polyamine content of about 5.8 times compared to the case of using the general strain, and about 1.9 times higher than the case of using the JR-018 strain. It was.
  • FIG. 1 After inoculating 10 3 natto spores per 1 g of boiled soybeans, fermented at 37 ° C. for 20 hours, refrigerated at 4 ° C. for 12 hours, and extracting a polyamine contained in natto, analyzed by TLC, is shown in FIG. 1. It was. As shown in Figure 1, the natto fermented by the Bacillus subtilis EE5 strain significantly increased the amount of polyamine compared to the general natto.
  • the isolated polyamine high production strain formed a milky white colony on LB agar plate medium, and the colony showed an appearance with irregular milky edges and a large milky white spot at the center.
  • the growth was good in the range of 25 °C to 40 °C, the cell growth was not confirmed above 50 °C.
  • Microscopic observation of the cell growth using LB liquid medium showed that it was a gram-positive, short rod-like appearance similar to the Bacillus subtilis, a comparative strain, the strain was Gram-positive, and the cell size was approximately 0.6-0.7 ⁇ It was 1.5-1.7 micrometers (FIG. 3).
  • the biochemical properties of strains according to the present invention were investigated using the method of analyzing the taxonomic characteristics of the strains of genus Bacillus (Bergey's Manual of Systematic Bacteriology, 2002) and the API50CHB kit (BioMerieux, France).
  • the strain of the present invention is a Gram-positive cylinder, spores are formed in the center of the cell, have a reducing power of nitrate, and do not produce indole. It was also found to degrade casein and starch, produce catalase, and grow only in aerobic conditions.
  • API test results Detailed morphological and biochemical properties (API test results) of the strains according to the present invention are shown in Table 6 below, and biochemical properties (API test results) are shown in Table 7 below.
  • RNA sequencing results of the Bacillus subtilis EE5 strain were analyzed by SEQ ID NO: 1 below.
  • Bacillus subtilis EE5 strain is applied to the existing natto manufacturing process in the field requiring a high polyamine functional food as it is usefully used as a means to economically and efficiently provide a highly functional polyamine high content natto. It was expected.
  • Bacillus subtilis EE5 strain of the present invention can be usefully used in the field requiring a high polyamine functional food.

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PCT/KR2017/001755 2016-06-08 2017-02-17 폴리아민 고생산 신균주 바실러스 서브틸리스 ee5 균주 WO2017213327A1 (ko)

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JP7376023B2 (ja) * 2018-11-14 2023-11-08 池田食研株式会社 老化抑制剤
KR102288725B1 (ko) 2019-02-14 2021-08-11 강원대학교산학협력단 대두 배아를 이용한 폴리아민 고함유 발효물의 제조 방법
KR20240010582A (ko) * 2022-07-13 2024-01-24 이해익 폴리아민 고생산 바실러스 코아귤란스 균주

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KR101485970B1 (ko) * 2011-11-11 2015-01-27 안동대학교 산학협력단 신규한 락토바실러스 속 аｍｌ 2-1 균주, 이를 이용한 폴리아민과 감마 아미노 부티르산의 제조방법 및 폴리아민과 감마 아미노 부티르산의 용도
KR101495602B1 (ko) * 2013-10-16 2015-02-26 한국식품연구원 청국장 유래 바실러스속 미생물 및 이를 이용하여 s-아데노실-l-메티오닌을 대량 생산하는 방법

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JP5754009B1 (ja) * 2014-04-24 2015-07-22 茨城県 糸引性低下納豆菌株及び該納豆菌株による納豆の製造方法と納豆

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KR101495602B1 (ko) * 2013-10-16 2015-02-26 한국식품연구원 청국장 유래 바실러스속 미생물 및 이를 이용하여 s-아데노실-l-메티오닌을 대량 생산하는 방법

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Title
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