KR102008673B1 - Lactobacillus fermentum C-7A and Use thereof - Google Patents
Lactobacillus fermentum C-7A and Use thereof Download PDFInfo
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- KR102008673B1 KR102008673B1 KR1020170167574A KR20170167574A KR102008673B1 KR 102008673 B1 KR102008673 B1 KR 102008673B1 KR 1020170167574 A KR1020170167574 A KR 1020170167574A KR 20170167574 A KR20170167574 A KR 20170167574A KR 102008673 B1 KR102008673 B1 KR 102008673B1
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- Prior art keywords
- glutamic acid
- strain
- strains
- lactobacillus
- present
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Abstract
본 발명은 글루탐산을 생산하는 신규 미생물 및 그로부터 글루탐산을 생산하는 방법에 관한 것으로, 보다 상세하게는 전통발효식품인 청국장으로부터 분리된 락토바실러스 퍼멘텀 C-7A(KCCM12118P) 및 상기 균주를 배양하여 글루탐산을 생산하는 방법에 관한 것이다.
본 발명의 균주는 천연 조미료 등의 식품 분야에서 인체, 환경 친화적으로 응용 가능한 글루탐산을 생산하는 능력을 가진다.The present invention relates to a novel microorganism producing glutamic acid and a method for producing glutamic acid therefrom, and more specifically, to culture glutamic acid by culturing the Lactobacillus fermentum C-7A (KCCM12118P) and the strain isolated from Cheonggukjang, a traditional fermented food. It is about how to produce.
The strain of the present invention has the ability to produce glutamic acid, which is applicable to humans and environmentally friendly in the food field, such as natural seasonings.
Description
본 발명은 글루탐산 생산능을 가지는 락토바실러스 퍼멘텀 C-7A 균주에 관한 것이다.The present invention relates to a Lactobacillus fermentum C-7A strain having a glutamic acid production capacity.
글루탐산은 미생물 발효에 의해 생산되는 대표적인 아미노산으로 의약품, 식품, 동물사료 등에 널리 이용되는 아미노산이다.Glutamic acid is a typical amino acid produced by microbial fermentation and is widely used in medicine, food, animal feed, and the like.
글루탐산의 발효경로를 간단하게 살펴보면, 포도당은 주로 해당경로(EMP)를 거치게 되나 일부는 6탄당 인산경로(HMP)를 거쳐서 2분자의 피루브산(pyruvic acid)으로 대사된다. 그 중 1분자는 CO₂를 고정하여 옥살로아세트산(oxaloacetic acid)으로 되고 다른 1분자는 피루브산으로부터 아세틸코에이(acetyl CoA)와 결합하여 구연산(citric acid)으로 된다. 다시 옥살로아세트산과 구연산은 시트르산 회로(TCA cycle)로 들어가 알파-케토글루타산(α-ketoglutaric acid)이 된다. 여기서, 알파-케토글루타산으로부터 호박산(succinic acid)으로 산화되는 산화대사 경로가 결여되어 있고 또 이소시트레이트 디히드로겐아제(isocitrate dehydrogenase)와 글루타메이트 디히드로겐아제(glutamate dehydrogenase)가 밀접하게 관여하기 때문에 알파-케토글루탄산의 환원적 아미노산화 반응이 능률적으로 진행되어 L-글루탐산이 생성된다.A brief review of the fermentation route of glutamic acid shows that glucose is mainly routed through glycolysis (EMP), but part of it is metabolized to two molecules of pyruvic acid via hexasaccharide phosphate (HMP). One molecule is fixed to CO2 to oxaloacetic acid, and the other molecule is combined with acetyl CoA from pyruvic acid to be citric acid. Oxaloacetic acid and citric acid go back into the citric acid cycle (TCA cycle) to become alpha-ketoglutaric acid. There is a lack of metabolic pathways that oxidize from alpha-ketoglutaric acid to succinic acid, and isocitrate dehydrogenase and glutamate dehydrogenase are closely involved. Because of this, the reductive amino acid reaction of alpha-ketoglutan acid proceeds efficiently, producing L-glutamic acid.
L-글루탐산을 생산하는 통상적인 방법으로는 주로 브레비박테리움(Brevibacterium) 이나 코리네박테리움 속 및 그 변이주를 포함한 코리네형(cornyeform) 박테리아를 이용해 발효를 통해 생산하며(Amino Acid Fermentation, Gakkai Shuppan Center: 195-215, 1986) 그 외에도 대장균(Escherichia coli), 고초균(Bacillus), 방선균(Streptomyces), 페니실리움(Penicillum) 속 및 크렙시엘라(Klebsiella), 어위니아(Erwinia), 판토에아(Pantoea) 속 등의 미생물을 이용하는 방법 등이 있다(미국 특허 제3,220,929호, 제 6,682,912 호).Conventional methods for producing L-glutamic acid are produced primarily by fermentation using corneform bacteria, including Brevibacterium or Corynebacterium genus and its strains (Amino Acid Fermentation, Gakkai Shuppan). Center: 195-215, 1986) Escherichia coli, Bacillus, Streptomyces, Penicillum genus and Klebsiella, Erwinia, Pantoea (Pantoea), and the like using a microorganism (US Pat. No. 3,220,929, 6,682,912).
본 발명자들은 글루탐산을 생산하는 새로운 균주를 개발하고자 예의 노력한 결과, 한국의 전통발효식품으로부터 분리한 신규 락토바실러스 퍼멘텀 C-7A 균주가 글루탐산을 생산하는 것을 확인하고, 본 발명을 완성하게 되었다.As a result of our intensive efforts to develop a new strain that produces glutamic acid, the present inventors have confirmed that the novel Lactobacillus permanent C-7A strain isolated from Korean traditional fermented food produces glutamic acid, thereby completing the present invention.
본 발명자들은 감칠맛의 주성분인 글루탐산을 생산하는 신규 균주를 확보하여 환경 친화적으로 응용 가능한 천연조미료의 재료로서 글루탐산의 생산성을 향상시키고자 노력하였다. 그 결과, 한국 전통발효식품으로부터 글루탐산 생합성 능력이 우수한 균주를 새로이 분리 동정하였으며, 분리한 균주의 형태학적, 배양학적, 생리학적 특성을 조사하여 글루탐산의 생산을 향상시키기 위한 배양조건을 도출함으로써, 본 발명을 완성하였다.The present inventors endeavored to improve the productivity of glutamic acid as a material of natural seasonings that can be applied environmentally friendly by securing a new strain producing glutamic acid as the main component of the umami. As a result, new strains with excellent glutamic acid biosynthesis ability were isolated and identified from Korean traditional fermented foods, and the cultivation conditions for improving the production of glutamic acid were derived by investigating the morphological, culture, and physiological characteristics of the isolated strains. The invention has been completed.
따라서, 본 발명의 목적은 락토바실러스 퍼멘텀(Lactobacillus fermentum) C-7A 균주(KCCM12118P)를 제공하는 데 있다.Therefore, an object of the present invention is to provide a Lactobacillus fermentum C-7A strain (KCCM12118P).
본 발명의 다른 목적은 상술한 본 발명의 균주를 배양하는 단계를 포함하는 글루탐산의 생산 방법을 제공하는 데 있다.Another object of the present invention to provide a method for producing glutamic acid comprising the step of culturing the strain of the present invention described above.
본 발명의 또 다른 목적은 본 발명의 균주에서 생산되는 글루탐산을 제공하는 데 있다.Still another object of the present invention is to provide glutamic acid produced by the strain of the present invention.
상기 목적을 달성하기 위하여, 본 발명은 신규 미생물 락토바실러스 퍼멘텀 C-7A 균주(KCCM12118P)를 제공한다.In order to achieve the above object, the present invention provides a novel microbial Lactobacillus pertumtum C-7A strain (KCCM12118P).
본 발명은 또한 상술한 균주를 배양하는 단계를 포함하는 글루탐산의 제조방법을 제공한다.The present invention also provides a method for producing glutamic acid comprising culturing the above-mentioned strain.
본 발명은 또한, 락토바실러스 퍼멘텀 C-7A 균주(KCCM12118P)가 생산한 글루탐산을 제공한다.The present invention also provides glutamic acid produced by the Lactobacillus permanent C-7A strain (KCCM12118P).
본 발명은 또한, 락토바실러스 퍼멘텀 C-7A 균주(KCCM12118P) 또는 이의 배양물을 함유하는 식품 첨가용 조성물을 제공한다.The present invention also provides a composition for food addition containing Lactobacillus permanum C-7A strain (KCCM12118P) or a culture thereof.
본 발명은 글루탐산 생성능이 우수한 락토바실러스 퍼멘텀 C-7 균주 (KCCM12118P) 및 상기 균주를 이용한 글루탐산의 생산 방법을 제공한다.The present invention provides a Lactobacillus fermentum C-7 strain (KCCM12118P) having excellent glutamic acid producing ability and a method for producing glutamic acid using the strain.
청국장에서 분리한 본 발명의 균주는 우수한 글루탐산 생산능을 가져, 천연 조미료 등의 식품 분야에서 인체, 환경 친화적으로 응용 가능한 글루탐산의 생산성을 향상시켰다.The strain of the present invention isolated from Cheonggukjang has excellent glutamic acid production capability, thereby improving the productivity of glutamic acid, which is applicable to human body and environment-friendly in the food field such as natural seasoning.
특히, 본 발명의 균주는 기본 생산 배지에서 글루탐산을 생산했을 때보다 본 발명의 배지에서 특정 조건으로 배양하였을 때 글루탐산 생산량이 현저하게 증가하였다.In particular, the strain of the present invention significantly increased the amount of glutamic acid produced when cultured under specific conditions in the medium of the present invention than when glutamic acid was produced in the basal production medium.
도 1은 전통발효식품으로부터 분리된 유산균주 중 중복되는 균주를 배제하기 위한 RAPD-PCR 결과를 나타낸 것이다. (A) B-4, D1-2, D1-19, D1-21, D2-9, (B) C-7, C-9, C-20, D3-5, D3-11, D3-22, (C) N1-1, N1-3, N1-8, N1-12, N1-15, N2-1, N2-19, (D) N3-1, N3-2, N3-3, N3-9, N3-21, N3-22, (E) KC1-3, KC1-14, KC1-22, KC2-3, KC2-14, M-1, M-3, G-1, (F) KC3-6, KC3-10, KC3-17, KC3-19, KC4-5, KC4-7, KC4-8, KC4-12, (H) KC5-1, KC5-11, KC5-12, KC5-17(B는 비지, C는 청국장, D는 된장, N은 누룩, KC는 김치, M은 막장, G는 고추장을 의미한다).
도 2는 RAPD-PCR로 분리된 균주 44종 중 Glutamate dehydrogenase 활성이 우수한 상위 10 종의 균주를 나타낸 것이다.
도 3은 GDH 활성을 측정하여 1차 스크리닝한 상위 7개 분리주를 대상으로 glutaminase 활성을 측정한 결과를 나타낸 것이다.
도 4는 글루탐산 우량 생산 균주 3종의 선발과정을 나타낸 것이다.
도 5a는 분리 균주 3종의 형태학적 특성을 나타낸 것이다. (A) KC2-14, (B) KC3-17, and (C) C-7A.
도 5b는 분리 균주 3종의 카탈라제 테스트 결과 C-7A 균주가 음성으로 나타나 혐기성 미생물인 것을 확인한 것이다.
도 5c는 API 50 CHL kit를 사용하여 분리 균주 3종의 당 이용 패턴을 나타낸 것이다.
도 6은 HPLC-PDA를 통해 분리 균주 3종의 글루탐산 생성능을 비교한 것으로, (A)는 세포 내(intra-cellular), (B)는 세포 외(extra-cellular), (C)는 총 글루탐산 생산량을 나타낸 것이다.
도 7은 본 발명의 락토바실러스 퍼멘텀 C-7A 균주의 계통도를 나타낸 것이다.
도 8은 락토바실러스 퍼멘텀 C-7A 균주를 온도, pH 및 시간을 달리하여 배양하였을 때 균주의 증식능을 나타낸 것이다.
도 9는 염농도에 따른 락토바실러스 퍼멘텀 C-7A 균주의 증식능을 나타낸 것이다.
도 10은 질소원, CaCl2 및 비오틴 농도에 따른 락토바실러스 퍼멘텀 C-7A 균주의 증식능을 나타낸 것이다.
도 11은 락토바실러스 퍼멘텀 C-7A 균주의 글루탐산 생성능을 HPLC 결과로부터 확인한 것이다.
도 12는 질소원을 다르게 설정하였을 때 락토바실러스 퍼멘텀 C-7A 균주의 세포 내(A), 세포 외(B) 및 총 글루탐산 생산량(C)을 나타낸 것이다.
도 13은 비오틴과 CaCl2를 각각 농도를 달리하여 첨가하였을 때 락토바실러스 퍼멘텀 C-7A 균주의 세포 내(A), 세포 외(B) 및 총 글루탐산 생산량(C)을 나타낸 것이다.
도 14는 발효조 수준에서 락토바실러스 퍼멘텀 C-7A 균주의 대수 증식기를 확인한 것이다.
도 15는 야생 균주(A)와 UV 조사 후 선발된 돌연변이 균주(B)를 나타낸 것이다.Figure 1 shows the results of RAPD-PCR to exclude the overlapping strains of lactic acid strains isolated from traditional fermented foods. (A) B-4, D1-2, D1-19, D1-21, D2-9, (B) C-7, C-9, C-20, D3-5, D3-11, D3-22, (C) N1-1, N1-3, N1-8, N1-12, N1-15, N2-1, N2-19, (D) N3-1, N3-2, N3-3, N3-9, N3-21, N3-22, (E) KC1-3, KC1-14, KC1-22, KC2-3, KC2-14, M-1, M-3, G-1, (F) KC3-6, KC3-10, KC3-17, KC3-19, KC4-5, KC4-7, KC4-8, KC4-12, (H) KC5-1, KC5-11, KC5-12, KC5-17 (B is busy , C stands for Cheonggukjang, D stands for Doenjang, N stands for Yeast, KC stands for Kimchi, M stands for Makjang, and G stands for Gochujang.
Figure 2 shows the top 10 strains excellent in Glutamate dehydrogenase activity among 44 strains isolated by RAPD-PCR.
Figure 3 shows the results of measuring glutaminase activity in the
Figure 4 shows the selection process of three excellent glutamic acid production strains.
Figure 5a shows the morphological characteristics of three isolates. (A) KC2-14, (B) KC3-17, and (C) C-7A.
Figure 5b is a catalase test of three isolated strains C-7A strain is confirmed that the anaerobic microorganisms appear negative.
Figure 5c shows the sugar utilization pattern of the three isolated strains using the
6 is a comparison of the glutamic acid generating ability of the three isolated strains by HPLC-PDA, (A) is intra-cellular, (B) extra-cellular, (C) is total glutamic acid The output is shown.
Figure 7 shows the schematic diagram of the Lactobacillus Permanent C-7A strain of the present invention.
Figure 8 shows the growth ability of the strain Lactobacillus pertumtum C-7A strain when cultured at different temperatures, pH and time.
Figure 9 shows the proliferative capacity of Lactobacillus permanent C-7A strain according to the salt concentration.
Figure 10 shows the proliferative capacity of Lactobacillus fermentum C-7A strain according to the nitrogen source, CaCl 2 and biotin concentration.
Figure 11 confirms the glutamic acid producing ability of the Lactobacillus fermentum C-7A strain from the HPLC results.
Figure 12 shows the intracellular (A), extracellular (B) and total glutamic acid production (C) of the Lactobacillus permanent C-7A strain when the nitrogen source was set differently.
Figure 13 shows the intracellular (A), extracellular (B) and total glutamic acid production (C) of the Lactobacillus permanent C-7A strain when biotin and CaCl 2 were added at different concentrations.
Figure 14 shows the logarithmic growth phase of the Lactobacillus fermentum C-7A strain at the fermentor level.
Figure 15 shows the wild strain (A) and mutant strains (B) selected after UV irradiation.
본 발명의 일 양태에 따르면, 본 발명은 락토바실러스 퍼멘텀(Lactobacillus fermentum) C-7A 균주(KCCM12118P)에 관한 것이다.According to one aspect of the present invention, the present invention relates to Lactobacillus fermentum C-7A strain (KCCM12118P).
본 명세서에서, "글루탐산(glutamic acid)"는 L-글루탐산, DL-글루탐산, L-글루탐산 나트륨염, DL-글루탐산 나트륨염, L-글루탐산 암모늄염, DL-글루탐산 암모늄염을 모두 포함하며, 바람직하게는 L-글루탐산이다.
본 발명의 C-7A 균주는 본 명세서의 발명의 설명 및 도면에서 C-7, C-7A, C7A, C7-A 등으로 기재될 수 있으나, 상기 기재들은 모두 C-7A 균주를 의미하는 것이다.As used herein, "glutamic acid" includes all of L-glutamic acid, DL-glutamic acid, L-glutamic acid sodium salt, DL-glutamic acid sodium salt, L-glutamic acid ammonium salt, DL-glutamic acid ammonium salt, preferably L -Glutamic acid.
The C-7A strain of the present invention may be described as C-7, C-7A, C7A, C7-A, etc. in the description and drawings of the present specification, but all of the above descriptions refer to the C-7A strain.
본 발명에서는 글루탐산을 생산하는 미생물을 얻기 위하여, 대한민국 각지에서 생산, 시판되고 있는 전통발효식품을 수집하여 글루탐산을 생산하는 균주를 탐색분리하였다. 그 결과, 한국의 전통 콩발효식품인 청국장으로부터 글루탐산 생성능이 높은 락토바실러스 균주, '락토바실러스 퍼멘텀 C-7A'를 새로이 분리하였으며 MALDI-TOF와 16S rRNA 염기서열 분석을 통해 동정하였고, 분리된 글루탐산 생산 균주의 형태학적, 배양학적, 생리학적 특성을 조사하였으며 다양한 탄소원과 질소원 등을 이용하여 글루탐산의 생산과 세포 성장에 미치는 영향을 조사하여 분리 균주의 글루탐산 생합성에 필수적인 요인들을 선정한 후 배지 조성 최적화 실험을 수행하였다.In the present invention, in order to obtain a microorganism to produce glutamic acid, by collecting the traditional fermented foods produced and marketed in various parts of the Republic of Korea to search for strains to produce glutamic acid. As a result, the Lactobacillus strain, Lactobacillus Fermentum C-7A, which has high glutamic acid production ability, was newly isolated from Chungkookjang, a traditional Korean fermented soybean, and identified through MALDI-TOF and 16S rRNA sequencing. The morphological, culture, and physiological characteristics of the produced strains were investigated, and the effects of glutamic acid production and cell growth on the production of glutamic acid using various carbon and nitrogen sources were selected. Was performed.
본 발명에 따른 락토바실러스 퍼멘텀 C-7A 균주의 분리 및 동정 과정은 다음과 같다.The isolation and identification of the Lactobacillus permanum C-7A strain according to the present invention is as follows.
미생물의 분리Isolation of Microorganisms
본 발명의 신규 락토바실러스 속 균주는 국내 전통발효식품 15 품목으로부터 유산균 338주를 분리하였고 carbohydrate fermentation test와 RAPD-PCR 시험을 수행하여 총 44개의 분리주를 분리하였다. 상기 분리주들의 glutamate dehydrogenase(GDH) 활성을 각각 측정하여 상대적으로 우수한 활성을 가진 7주를 1차 선별하고 이어서 glutaminase 활성을 비교하여 분리주 KC2-14, KC3-17, C-7A를 2차 선별하였다. 분리주 KC2-14, KC3-17, C-7A의 glutamic acid 생성능을 비교하기 위하여 glutamate determination assay를 실시한 결과 청국장 유래 야생주 C-7A에서 세포 내 glutamic acid의 축적량이 가장 높게 측정되었으며 이로부터 글루탐산 생성능이 가장 우수한 C-7A 균주를 최종 선별하였다.The strain of the genus Lactobacillus of the present invention isolated 338 strains of lactic acid bacteria from 15 traditional fermented foods in Korea, and a total of 44 isolates were isolated by performing a carbohydrate fermentation test and a RAPD-PCR test. Glutamate dehydrogenase (GDH) activity of the isolates was measured first, and seven strains with relatively good activity were first selected, followed by comparison of glutaminase activity, and isolates KC2-14, KC3-17, and C-7A were selected secondarily. In order to compare the glutamic acid production of KC2-14, KC3-17, and C-7A isolates, glutamate determination assay was performed. The best C-7A strain was finally screened.
분리된 미생물의 특성 및 동정Characterization and Identification of Isolated Microorganisms
분리 균주는 간균으로 그람 양성 균주로 나타났으며 주사현미경을 통해 분리 균주를 관찰한 결과는 도 5에 나타난 바와 같다. The isolated strain was a Gram-positive strain as bacilli and the results of observing the isolated strain through a scanning microscope are shown in FIG. 5.
또한, 분리 균주의 생리적 특성에 대한 일차적인 동정으로 API 50 CHL kit와 MALDI-TOF 결과, Lactobacillus fermentum DSM 20391로 동정되었다.In addition, the primary identification of the physiological characteristics of the isolated strain was identified as Lactobacillus fermentum DSM 20391 as a result of
그리고, 정확한 동정을 위해 분리 균주의 16S rRNA 염기서열 분석 결과, Lactobacillus fermentum CECT 562(T)(similarity 99.85%)로 동정할 수 있었으며, Lactobacillus fermentum C-7A로 명명하였고, 이를 한국미생물보존센터(Korean Culture Center of Microorganisms)에 2017년 9월 26일자로 기탁하였다(KCCM12118P).In addition, as a result of 16S rRNA sequencing analysis of the isolated strain, Lactobacillus fermentum CECT 562 (T) (similarity 99.85%) was identified as Lactobacillus fermentum C-7A, which was named Korea Microorganism Conservation Center. Culture Center of Microorganisms) was deposited on September 26, 2017 (KCCM12118P).
분리된 미생물의 글루탐산 생성능Glutamic Acid Generating Ability of Isolated Microorganisms
유산균 표준 균주 대비 L. fermentum C-7A의 글루탐산 생산량은 HPLC 분석을 통하여 비교한 결과 세포 내, 세포 외에서 C-7A의 글루탐산 생산능이 가장 우수한 것을 확인하였다.Glutamic acid production of L. fermentum C-7A compared to the standard strain of lactic acid bacteria was confirmed by the HPLC analysis, the best glutamic acid production of C-7A in the cell and extracellular.
본 발명의 신규한 균주는 락토바실러스 속 균주이다.The novel strain of the present invention is the genus Lactobacillus.
본 발명의 일 구현예에 따르면, 상기 균주는 청국장에서 분리된 것을 특징으로 한다.According to one embodiment of the present invention, the strain is characterized in that isolated from the Cheonggukjang.
본 발명의 다른 구현예에 따르면 상기 균주는 서열번호 1의 16S rRNA 염기서열을 갖는다.According to another embodiment of the present invention the strain has a 16S rRNA nucleotide sequence of SEQ ID NO: 1.
본 발명의 다른 양태에 따르면, 본 발명은 상술한 본 발명의 락토바실러스 퍼멘텀 C-7A 균주를 배양하는 단계를 포함하는 글루탐산의 생산 방법에 관한 것이다.According to another aspect of the present invention, the present invention relates to a method for producing glutamic acid comprising the step of culturing the Lactobacillus fermentum C-7A strain of the present invention described above.
본 발명의 방법은 상술한 본 발명의 균주를 이용하여 글루탐산을 생산하는 것이기 때문에, 이 둘 사이에 공통된 내용은 반복 기재에 따른 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다.Since the method of the present invention is to produce glutamic acid using the above-described strain of the present invention, the contents common between the two are omitted in order to avoid excessive complexity of the specification according to the repetitive description.
본 발명자들은 분리 미생물을 동정한 후 다양한 조건과 첨가제를 이용하여 글루탐산 생산과 균주 생장에 미치는 영향을 조사하여 배양 조건 및 배지 조성의 최적화 실험을 수행하였다.After identifying the isolated microorganisms, the inventors investigated the effects on the production of glutamic acid and strain growth using various conditions and additives, and performed optimization experiments on culture conditions and medium composition.
본 발명의 바람직한 구현예에 따르면, 정치 배양 혹은 호기적으로 35~40℃, pH 5~8에서 12~24시간 동안 실시하고, 보다 바람직하게는 호기적으로 35~40℃, pH 5~7에서 15~24시간 동안 실시하며, 가장 바람직하게는 호기적으로 40℃, pH 6.0±0.3에서 24시간 동안 실시한다.According to a preferred embodiment of the present invention, it is carried out for 12 to 24 hours at 35 ~ 40 ℃,
본 발명의 신규 분리한 균주는 통상적인 바실러스 또는 락토바실러스 속 균의 배양방법을 통해 배양할 수 있다. 배지로는 천연배지 또는 합성배지를 사용할 수 있다. 배지의 탄소원으로는 예를 들어, 글루코오스, 수크로오스, 덱스트린, 글리세롤, 녹말 등이 사용될 수 있고, 질소원으로는 펩톤, 육류 추출물, 효모 추출물, 건조된 효모, 대두, 암모늄염, 나이트레이트 및 기타 유기 또는 무기 질소 함유 화합물이 사용될 수 있으나, 이러한 성분에 한정되는 것은 아니다. 배지에 포함되는 무기염으로는 마그네슘. 망간, 칼슘. 철, 칼륨 등이 사용될 수 있으나, 이들에 한정되는 것은 아니다. 상기 탄소원, 질소원 및 무기염의 성분 이외에 아미노산, 비타민, 핵산 및 그와 관련된 화합물들이 배지에 첨가될 수 있다.The novel isolated strain of the present invention can be cultured through a conventional method for culturing Bacillus or Lactobacillus genus. The medium may be natural or synthetic medium. As the carbon source of the medium, for example, glucose, sucrose, dextrin, glycerol, starch and the like can be used, and as the nitrogen source, peptone, meat extract, yeast extract, dried yeast, soybean, ammonium salt, nitrate and other organic or inorganic Nitrogen containing compounds may be used, but are not limited to these components. Inorganic salts contained in the medium include magnesium. Manganese, calcium. Iron, potassium and the like can be used, but are not limited thereto. In addition to the components of the carbon source, nitrogen source and inorganic salts, amino acids, vitamins, nucleic acids and related compounds may be added to the medium.
본 발명의 균주 L. fermentum C-7A는 modified MRS 배지 중에서 ammonium citrate, ammonium nitrate, biotin이 첨가될 때 균체 증식에 영향을 받았으며, 이중 ammonium citrate가 가장 우수하였다.The strain L. fermentum C-7A of the present invention was affected by cell growth when ammonium citrate, ammonium nitrate, and biotin were added in modified MRS medium, and ammonium citrate was the best.
HPLC를 수행하여 글루탐산의 수율 증가량을 계산한 결과, 접종 전 MRS 액체배지의 L-글루탐산 함량은 410.06±0.39mg/L이며 본 발명의 균주 C-7A를 동일 배지에 접종하여 온도 37℃에서 24시간 동안 배양 후 측정된 총 글루탐산의 함량은 510.97±0.21로서 배양 전 대비 약 24.6%의 수율 증대가 확인되었다.As a result of calculating the yield increase of glutamic acid by HPLC, the L-glutamic acid content of the MRS liquid medium before inoculation was 410.06 ± 0.39mg / L, and the strain C-7A of the present invention was inoculated in the same medium for 24 hours at 37 ° C. The total glutamic acid content measured after incubation was 510.97 ± 0.21, and the yield increase of about 24.6% was confirmed.
첨가제가 L. fermentum C-7A의 글루탐산 생성량에 미치는 영향을 비교하기 위하여 L. fermentum C-7A를 48시간 배양 후 HPLC를 이용하여 총(세포내/외) 글루탐산을 각각 측정한 결과 CaCl2의 농도가 높아질수록 글루탐산 축적량이 증가하였으며, CaCl2 0.02%에서 첨가 전 대비 약 17.1% 수율 증대가 확인되었다.Additives for L. fermentum 7A-C the L. fermentum C-7A in order to compare the effects of glutamic acid production amount of after 48 hours total with the HPLC (cells and / or other) As a result of measuring the concentration of glutamic acid, respectively CaCl 2 The higher the amount of glutamic acid accumulated, the higher the yield of CaCl 2 0.02% compared to before the addition of about 17.1%.
Glutamic acid 생산능이 높은 돌연변이주를 얻고자 야생주 C-7A를 UV조사를 이용하여 돌연변이를 유도하였고 4개의 변이주를 얻었으나, 이들의 총 glutamic acid 생성능은 야생주보다 열성인 것을 확인하였다.Mutant strain C-7A was induced by UV irradiation to obtain mutant strains with high glutathic acid production ability, and four mutant strains were obtained, but their total glutamic acid producing ability was more recessive than wild strain.
본 발명의 다른 양태에 따르면, 본 발명은 락토바실러스 퍼멘텀 C-7A 균주(KCCM12118P)로부터 생산된 글루탐산에 관한 것이다.According to another aspect of the present invention, the present invention relates to glutamic acid produced from Lactobacillus permanent C-7A strain (KCCM12118P).
본 발명의 또 다른 양태에 따르면, 본 발명은 락토바실러스 퍼멘텀 C-7A 균주(KCCM12118P) 또는 이의 배양물을 포함하는 식품 첨가용 조성물에 관한 것이다.According to another aspect of the present invention, the present invention relates to a composition for adding food comprising the Lactobacillus fermentum C-7A strain (KCCM12118P) or a culture thereof.
상기 균주의 배양물은 균체를 포함한 배양 원액일 수 있으며, 또한 배양 상등액을 제거하거나 농축한 균체일 수 있다. 상기 배양물의 조성은 통상의 바실러스나 락토바실러스 균주 배양에 필요한 성분뿐 아니라, 바실러스나 락토바실러스 균주의 생장에 상승적으로 작용하는 성분을 추가적으로 포함할 수 있으며, 이에 따른 조성은 당 업계의 통상의 기술을 가진 자에 의하여 용이하게 선택될 수 있다.The culture of the strain may be a culture stock solution including the cells, and may also be a cultured cell from which the culture supernatant is removed or concentrated. The culture composition may further include components necessary for culturing Bacillus or Lactobacillus strains, as well as components that act synergistically to the growth of Bacillus or Lactobacillus strains, the composition according to the conventional techniques in the art It can be easily selected by those who have it.
또한, 균주의 상태는 액상상태 또는 건조상태일 수 있으며, 건조방법은 통풍건조, 자연건조, 분무건조 및 동결건조가 가능하지만, 이에 제한되는 것은 아니다.In addition, the strain may be in a liquid state or a dry state, and the drying method may be, but is not limited to, ventilation drying, natural drying, spray drying, and freeze drying.
다른 양태로서 본 발명은 락토바실러스 퍼멘텀 균주, 이의 배양물, 이의 농축물, 또는 이의 건조물을 포함하는 조성물에 관한 것이다. 상기 락토바실러스 퍼멘텀 균주는 구체적으로 락토바실러스 퍼멘텀 KCCM12118P일 수 있으나, 이에 한정되는 것은 아니다.In another aspect, the present invention relates to a composition comprising a Lactobacillus permanent strain, a culture thereof, a concentrate thereof, or a dried product thereof. The Lactobacillus fermentum strain may specifically be Lactobacillus fermentum KCCM12118P, but is not limited thereto.
상기 조성물은 글루탐산을 함유하는 식품 조성물일 수 있으며, 식품 조성물의 일 양태로는 조미료 식품일 수 있다.The composition may be a food composition containing glutamic acid, and one aspect of the food composition may be a seasoning food.
이하, 본 발명을 실시예를 통해 상세히 설명한다. 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail through examples. The following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.
실시예 1: 글루탐산 생산균의 분리 및 선발Example 1 Isolation and Selection of Glutamic Acid Producing Bacteria
1-1. 전통발효식품으로부터 유산균주의 분리1-1. Isolation of Lactic Acid Bacteria from Traditional Fermented Foods
전통발효식품으로부터 유산균 신균주를 분리하기 위하여 비지 1종, 된장 3종, 김치 5종, 청국장 1종, 막장 1종, 고추장 1종, 누룩 3종 총 15품목의 시료를 수집하여 검체 10 g(ml)에 9배 희석액을 가하여 100 ml가 되게 하고 균질화하였다(10-1용액). 시험용액(10-1용액) 1 ml에 희석액을 가하여 10 ml가 되게 하고 10-2 희석용액을 만든 후 10-5 희석용액까지 동일하게 조작하였다. 0.02%의 sodium azide 첨가 BCP-MRS 고체 배지에 각 희석 시험용액 0.1 ml씩을 2매 이상에 접종하여 멸균 초자봉으로 도말하였다. 시료가 접종된 페트리 디쉬는 37℃에서 48±3시간 배양하였다. 배양 후 생성된 황색의 집락을 각각 백금이를 이용하여 무균적으로 순수분리하여 MRS broth 5 ml에 접종하고 37℃에서 24±3시간 배양하였다. 배양균체를 glycerol의 농도가 최종 35%가 되도록 균일하게 혼합한 다음 -70℃에서 보존하였다. 동결보존된 분리주는 이후 각 시험을 실시하기 이전에 MRS broth에 2% 를 접종하고 35~37℃에서 24±3시간 계대배양하여 사용하였다.In order to separate lactic acid bacteria mycobacteria from traditional fermented foods, a total of 15 samples of 1 type of bean curd, 3 kinds of doenjang, 5 kinds of kimchi, 1 type of Cheonggukjang, 1 type of makjang, 1 type of red pepper paste, and 3 types of yeast were collected and 10 g ( ml) to 9 ml dilution to make 100 ml and homogenized (10-1 solution). Diluted solution was added to 1 ml of test solution (10-1 solution) to make 10 ml, and 10-2 dilutions were prepared. 0.1 ml of each diluted test solution was inoculated into 0.02% sodium azide-added BCP-MRS solid medium and plated with sterile chopping sticks. Petri dishes inoculated with the samples were incubated at 37 ° C. for 48 ± 3 hours. After colonization, the yellow colonies were aseptically separated from each other using platinum, and then inoculated in 5 ml of MRS broth and incubated at 37 ° C. for 24 ± 3 hours. The cultured cells were uniformly mixed so that the final concentration of glycerol was 35% and then stored at -70 ° C. Cryopreserved isolates were then inoculated with 2% MRS broth before each test and passaged at 35 ± 37 ° C for 24 ± 3 hours.
1-2. 당 이용성 패턴으로부터 젖산 발효 균주의 분리1-2. Isolation of Lactic Acid Fermentation Strains from Sugar Availability Patterns
1-1에서 분리된 유산균주들을 MRS 배지에 접종한 후 37℃에서 24±3시간 배양된 MRS broth 배양액 0.5 ml를 8,000 ×g로 5분간 원심분리하여 배지와 균체를 분리하고 균체는 0.85% NaCl 0.5 ml에 재현탁하였다. 이 용액 0.02 ml를 glucose, lactose, xylose, sucrose, non-carbohydrate purple broth에 각각 접종하고 37℃에서 24±3시간 배양 후 황색으로 변한 시험주는 젖산발효가 일어난 것으로 판정하였다.After inoculating the lactic acid strains isolated in 1-1 to MRS medium, 0.5 ml of MRS broth culture medium incubated at 37 ° C. for 24 ± 3 hours at 8,000 × g for 5 minutes to separate the medium and the cells, and the cells were 0.85% NaCl. Resuspend in 0.5 ml. 0.02 ml of this solution was inoculated into glucose, lactose, xylose, sucrose, and non-carbohydrate purple broth, respectively, and the test strain turned yellow after incubation at 37 ° C for 24 ± 3 hours.
1-3. RAPD-PCR에 의해 중복 균주 배제1-3. Exclusion of duplicate strains by RAPD-PCR
RAPD-PCR(Random Amplified Polymorphic DNA Polymerase Chain Reaction)은 primer P1254(5'-CCGCAGCCAA-3')를 사용하여 설정하였으며(Torriani et al., 1999), Thermalcycler (Model PTC-100 TM, MJ Research Inc. Watertown, MA, USA)를 사용하였다. PCR mixture는 master mix 5X 4 μl, genomic DNA 2 μl, 증류수 13 μl, 10 pmol/μl 농도의 primer(Bioneer, Deajeon, Korea)는 각 1 μl로 total volume은 20 μl로 하였다. 반응 조건은 pre-denaturation은 94℃/4 min, denaturation 94℃/30 sec, annealing 50℃/1 min, extension 72℃/2 min로 30 cycle 간 반응시킨 후. PCR product를 2% agarose gel을 이용하여 확인하였다. 각 밴드의 위치가 동일하게 나타나는 것은 동일 균주로 판단하고 이후 실험에서 배제하였다.Random Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR) was established using primer P1254 (5'-CCGCAGCCAA-3 ') (Torriani et al. , 1999) and Thermalcycler (Model PTC-100 ™, MJ Research Inc. Watertown, Mass., USA). PCR mixture was composed of 4 μl of
1-4. 글루탐산 생성능이 우수한 유산균주의 선발1-4. Selection of Lactic Acid Bacteria with Excellent Glutamic Acid Generating Capacity
1-3에서 분리된 44종의 유산균을 배양 후 10분 원심분리한 다음 PBS 1.0 ml로 균체를 2회 세척하여 동일 완충액에 재현탁 시켰다. Sonicator (VCX130 Digital ultrasonic processor grade C, Vibra cell, Sonics & Materials Inc., CT, USA)로 30% amplitude에서 1분 30초간(3 sec on and 10 sec off, 30 times) 균체를 파쇄한 다음 8,000 ×g 5분 원심분리하여 상등액(cell-free extract)을 시험용액으로 사용하고 총 단백질 농도는 Bradford법(1976)으로 측정하였다.44 lactic acid bacteria isolated at 1-3 were incubated for 10 minutes after incubation, and the cells were washed twice with 1.0 ml of PBS and resuspended in the same buffer. Crush the cells with Sonicator (VCX130 Digital ultrasonic processor grade C, Vibra cell, Sonics & Materials Inc., CT, USA) at 30% amplitude for 1 minute and 30 seconds (3 sec on and 10 sec off, 30 times), then 8,000 × g Centrifuged for 5 minutes to use a cell-free extract as a test solution and the total protein concentration was measured by Bradford method (1976).
Glutamate dehydrogenase 활성은 다음과 같이 측정하였다. 반응용액은 40 μl의 50 mM potassium phosphate-50 mM triethylamine buffer (pH 8.0), 1% Triton X-100, 20 μl의 100 mM glutamic acid, 20 μl의 2 mM iodonitrotetrazolium, 20 μl의 diaphorase 1.76 U/ml, 20 μl NADP+ 13.8 mM 혹은 NAD+ 17.33 mM, 180 μl의 증류수로 제조하고, 37℃에서 1시간 동안 반응시키면서 NAD(P)H의 농도를 492 nm 흡광도로 측정하였다. Glutamic acid를 추가하지 않은 동일 조성의 반응용액을 대조군으로 하고 GDH 활성단위는 0.01/min의 흡광도를 올리는데 필요한 효소의 양으로 정하였다(Catherine et al. 2002).Glutamate dehydrogenase activity was measured as follows. The reaction solution was 40 μl of 50 mM potassium phosphate-50 mM triethylamine buffer (pH 8.0), 1% Triton X-100, 20 μl of 100 mM glutamic acid, 20 μl of 2 mM iodonitrotetrazolium, 20 μl of diaphorase 1.76 U / ml , 20 μl NADP + 13.8 mM or NAD + 17.33 mM, 180 μl of distilled water, and reacted for 1 hour at 37 ℃ was measured the concentration of NAD (P) H 492 nm absorbance. The reaction solution of the same composition without adding glutamic acid was used as a control, and the GDH active unit was determined as the amount of enzyme required to increase the absorbance of 0.01 / min (Catherine et al. 2002).
Glutaminase 활성은 다음과 같이 측정하였다. 신선하게 제조한 0.04 M L-glutamine 용액 0.1 ml, 0.1 M phosphate buffer (pH 8.0) 0.1 ml에 미리 파쇄해 놓은 cell-free extract 0.1 ml를 가한 후 37℃에서 30분간 반응을 유지한 뒤, 1.5 M trichloroacetic acid 0.1 ml을 가하여 반응을 중지하였다. Blank test(공시험)는 동일 mixture에 trichloroacetic acid 처리를 한 이후 cell-free extract를 가한 것으로 제조하였다. 반응용액 0.1 ml에 증류수 3.7 ml, Nessler's reagent 0.2 ml을 가한 뒤 spectrophotometer를 이용하여 450 nm에서 OD값을 측정하여 glutaminase 활성을 측정하고 활성단위는 37℃에서 1 mM의 암모니아를 생산하는데 소요되는 효소의 양으로 정하였다(Renu et al., 2003).Glutaminase activity was measured as follows. 0.1 ml of freshly prepared 0.04 M L-glutamine solution and 0.1 ml of 0.1 M phosphate buffer (pH 8.0) were added, and 0.1 ml of pre-crushed cell-free extract was added. 0.1 ml of trichloroacetic acid was added to stop the reaction. Blank test was prepared by adding trichloroacetic acid to the same mixture and then adding cell-free extract. To 0.1 ml of the reaction solution, 3.7 ml of distilled water and 0.2 ml of Nessler's reagent were added, and the OD value was measured at 450 nm by using a spectrophotometer to measure glutaminase activity. The active unit of enzyme was used to produce 1 mM ammonia at 37 ° C. Amount was determined (Renu et al. , 2003).
실시예 2. 선발된 글루탐산 생산균의 특성 및 동정Example 2. Characterization and Identification of Selected Glutamic Acid Producing Bacteria
2-1. 분리 균주의 생화학적 동정2-1. Biochemical Identification of Isolated Strains
그람 염색 결과는 광학현미경을 통해 확인하였으며 보라색으로 염색된 것은 그람 양성(positive), 붉게 염색된 것은 음성(negative)으로 판정하였다. 또한, 현미경 검경을 통하여 미생물의 형태적 특징에 따라서 구균, 단간균, 간균으로 구분하였다.Gram staining results were confirmed by optical microscopy, and purple stained was determined to be gram positive and red stained to be negative. In addition, microscopic examination was divided into microorganisms according to the morphological characteristics of microorganisms, bacilli, bacilli.
Catalase test를 위하여 slide glass에 분리균주를 도말한 후 3% H2O2 용액을 떨어뜨려 기포의 생성 유무에 따라 양성(positive)과 음성(negative)으로 판정하였고 대조군으로 그람 음성 E. coli ATCC 25922를 사용하였다.For the catalase test, after spreading the isolated strain on the slide glass, 3% H 2 O 2 solution was dropped to determine positive and negative depending on the presence or absence of bubbles. Gram negative
당 이용성을 확인하기 위하여 API 50 CHL kit(bioMerieux, France)를 사용하였으며 노란색으로 변한 것은 양성(positive, +), 그렇지 않은 것은 음성(negative, -) 판정하였다.
2-2. MALDI-TOF를 이용한 동정2-2. Identification using MALDI-TOF
Bruker Microflex LT(Bruker Daltonics, Germany) metrix assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF MS)를 이용하여 미생물을 동정하였다. MRS agar 배지에서 48시간 동안 배양된 colony를 target steel plate에 옮긴 후 1 μl의 α-Cyano-4-hydroxycinnamic acid (CHCA)와 50% (v/v) actonitrile, 2.5% (v/v) trifluoroacetic acid가 포함된 matrix solution으로 덮어준 후 건조하였다. 준비된 plate를 Microflex LT 기기의 MALDI Biotyper software version 3.0(Bruker Daltonics)로 분석하였다.Microorganisms were identified using Bruker Microflex LT (Bruker Daltonics, Germany) metrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Colonies cultured in MRS agar medium for 48 hours were transferred to a target steel plate, followed by 1 μl of α-Cyano-4-hydroxycinnamic acid (CHCA) and 50% (v / v) actonitrile, 2.5% (v / v) trifluoroacetic acid. Covered with a matrix solution containing and dried. The prepared plate was analyzed by MALDI Biotyper software version 3.0 (Bruker Daltonics) of the Microflex LT instrument.
2-3. 분리 균주의 유전학적 동정2-3. Genetic Identification of Isolated Strains
분리균주의 genomic DNA를 추출하여 Universal primer 27F와 1492R (Weisburg et al., 1991)를 이용한 16S rRNA gene PCR을 진행하였다. 기기는 Thermalcycler(Model PTC-100 TM, MJ Research Inc. Watertown, MA, USA)를 사용하고 PCR mixture는 master mix 5X 4 μl, genomic DNA 2 μl, 증류수 13 μl, 10 pmol/μl 농도의 primer는 각 1 μl로 total volume은 20 μl로 하였다. 반응 조건은 pre-denaturation은 95℃/5 min, denaturation 95℃/30 sec, annealing 52℃/30 sec, extension 72℃/2 min, final extension 72℃/5 min로 30 cycle 간 반응시킨 후. PCR amplicon을 2 % agarose gel을 이용하여 확인하였다.Genomic DNA of the isolate was extracted and subjected to 16S rRNA gene PCR using Universal primer 27F and 1492R (Weisburg et al. , 1991). The instrument uses a thermalcycler (Model PTC-100TM, MJ Research Inc. Watertown, MA, USA), and the PCR mixture contains 4 μl of
16S rRNA gene PCR 생성물을 수탁업체(Cosmogenetech, Seoul, Korea)에 sequencing을 의뢰하여 DNA Analyzer(model 3730xl, Applied Biosystems, Waltham, MA, USA)로 염기서열을 분석하고 분석된 염기서열을 바탕으로 NCBI(National Center for Biotechnology Information)의 BLAST(Basic Local Alignment Search Tood) 검색과 Ezbiocloud의 데이터베이스(Kim et al., 2012)를 통해 표준 균주와 유사성을 확인하고 MEGA6 프로그램의 neighbor-joining method를 적용하여 phylogenetic tree를 작성하였다.Sequencing the 16S rRNA gene PCR product to a consignment company (Cosmogenetech, Seoul, Korea) and analyzed the sequencing with DNA Analyzer (model 3730xl, Applied Biosystems, Waltham, MA, USA) and based on the analyzed sequencing NCBI ( The Byl (Basic Local Alignment Search Tood) search of the National Center for Biotechnology Information and the database of Ezbiocloud (Kim et al. , 2012) confirm similarity with standard strains and apply the neighboring-joining method of MEGA6 program to generate phylogenetic tree. Created.
실시예 3. 글루탐산 생산능 검토를 통해 최종 균주의 선발Example 3 Selection of Final Strains through Glutamic Acid Production Capacity Review
글루탐산 생산능을 검토하기 위해 glutamate assay kit (Sigma-Aldrich, USA)를 이용하여 동일 배양 조건에서 공시 균주들의 실제 글루탐산의 양을 비교하고 이를 통해 최종 공시 균주를 선발하였다. Glutamate assay를 시행하는 기본 원리는 GDH의 보충대사반응을 이용한 것으로, Glutamate assay를 시행하기 위하여 먼저 107~108 cells/ml의 균체를 2회 세척 후 파쇄하여 상등액을 spin filter(10 kDa MWCO)를 사용하여 10,000 ×g에서 10분간 원심분리하여 시료를 얻었다. Glutamate assay buffer 90 μl, glutamate developer 8 μl, glutamate enzyme mix 2 μl를 제조하여 100 μl의 반응용액(reaction mix)을 제조한 다음 각 well에 넣고 기포가 생기지 않게 pipetting을 통해 조심스럽게 혼합하여 빛을 피해 37℃에서 30분간 반응시킨 뒤 450 nm의 파장에서 흡광도를 측정하였으며 측정결과 흡광도가 가장 높게 나타난 균주를 최종 균주로 정하였다.In order to examine glutamic acid production ability, the glutamate assay kit (Sigma-Aldrich, USA) was used to compare the actual glutamic acid content of the disclosed strains under the same culture conditions, and the final disclosed strains were selected. The basic principle of the Glutamate assay is the supplementary metabolic reaction of GDH.In order to perform the Glutamate assay, the supernatant is spin-filtered (10 kDa MWCO) after washing twice with 10 7 ~ 10 8 cells / ml. Samples were obtained by centrifugation at 10,000 x g for 10 minutes. Prepare 90 μl of Glutamate assay buffer, 8 μl of glutamate developer, and 2 μl of glutamate enzyme mix to prepare 100 μl of reaction mixture, and put them into each well and carefully mix them through pipetting to avoid bubbles to avoid light. After reacting at 37 ° C. for 30 minutes, absorbance was measured at a wavelength of 450 nm, and the strain showing the highest absorbance was determined as the final strain.
최종 공시균주의 유효성을 예비 확인하고자 기존 GDH 활성이 있는 것으로 알려진 표준균주 L. fermentum ATCC 9338와 L. casei ATCC 393를 선정하고 최종 균주와 상기 표준균주를 각각 107~108 정도의 균체만을 MRS 액체배지에 접종하고 온도 37.5℃로 고정하여 48시간 동안 배양하였다. 배양액을 경시적으로 sampling하고 세포내(intra-cellular) 및 세포외(extra-cellular) L-글루탐산 함량을 HPLC를 이용해 분석하였다.In order to preliminarily confirm the validity of the final test strain, the standard strains L. fermentum
실시예 4. 글루탐산의 최적 생산조건Example 4 Optimal Production Conditions for Glutamic Acid
pH를 각각 4, 5, 6, 7, 8±0.3로 맞추어 둔 MRS broth 10 ml에 동량의 균을 접종하여 각 온도별로 3시간마다 spectrophotometer(Thermo Scientific Multiskanⓡ FC Microplate Photometer, USA)를 이용하여 540 nm에서의 흡광도를 측정하였다. 염도의 경우 pH6±0.3의 MRS broth에 37.5℃ incubator에서 3시간마다 동일기기를 사용하여 측정한 것으로 앞의 최적 pH 및 온도 실험과 독립적으로 시행되었다.Inoculate an equal amount of bacteria in 10 ml of MRS broth with pHs of 4, 5, 6, 7, 8 ± 0.3 and 540 using a spectrophotometer (Thermo Scientific Multiskan® FC Microplate Photometer, USA) every 3 hours for each temperature. Absorbance at nm was measured. Salinity was measured every 3 hours in 37.5 ℃ incubator in MRS broth of
Ammonium citrate를 제외한 나머지 시약은 동일하게 혼합하여 제조한 modified-MRS broth에 ammonium citrate를 0부터 최대 0.4%까지 0.1% 간격으로 첨가하였다. Except for Ammonium citrate, ammonium citrate was added to the modified-MRS broth prepared in the same mixture from 0 to 0.4% at 0.1% intervals.
Ammonium nitrate도 동일 배지에 농도가 0부터 0.5% 간격으로 최대 2.0%까지 되도록 하였다(Jeon et al., 2009; Zareian et al., 2012). Ammonium nitrate was also concentrated in the same medium from 0 to 0.5% up to 2.0% (Jeon et al ., 2009; Zareian et al. , 2012).
CaCl2와 biotin은 MRS broth (Difco, Detroit, MI, USA)에 직접 첨가하였으며 그 농도는 CaCl2는 0.005% 간격으로 최대 0.020%까지 조정하였고 biotin은 3 μg/l, 15 μg/l, 30 μg/l까지 조정하였다(Meng et al., 2016; Sato et al., 2008).CaCl 2 and biotin were added directly to MRS broth (Difco, Detroit, MI, USA), the concentration was adjusted to up to 0.020% with CaCl 2 at 0.005% intervals, and biotin was 3 μg / l, 15 μg / l, 30 μg / l (Meng et al ., 2016; Sato et al. , 2008).
각 배지에 동량의 균을 접종한 뒤 37.5℃ incubator에서 배양하였고 최초 접종된 시간으로부터 3시간마다 540nm에서의 흡광도를 측정하여 발효능을 간접 측정하였다.The same amount of bacteria was inoculated into each medium, followed by incubation in an incubator at 37.5 ° C., and the absorbance at 540 nm was measured every three hours from the time of initial inoculation.
Ortho-phthalaldehyde(OPA)-유도체화 방식을 적용하여 다음과 같이 amino acid를 정량 분석하였다(Winspear et al., 1983). HPLC-DAD를 수행하기 위하여 Mobile phase A는 25 mM NaH2PO4를 초순수증류수로 제조하고 pH 6.8±0.05로 설정하였으며 Mobaile phase B는 acetonitrile:methanol: water(45:45:10, v:v:v)를 혼합하였다. Mobile phase는 모두 HPLC grade로 제조되었으며 감압여과방식을 이용하여 45 μm의 cellulose membrane으로 여과하고 ultra-sonicator (JAC-1505, 40kHz, Jinwoo-ALEX, Korea)를 통해 1시간의 bubble decay 과정을 거친 다음 실험에 사용되었음(Bartolomeo et al., 2006).Using ortho-phthalaldehyde (OPA) -derivatization method, amino acid was quantitatively analyzed as follows (Winspear et al. , 1983). To perform HPLC-DAD, Mobile phase A was prepared with 25 mM NaH 2 PO 4 ultrapure distilled water and set to pH 6.8 ± 0.05. Mobaile phase B was prepared with acetonitrile: methanol: water (45:45:10, v: v: v) was mixed. All mobile phases were manufactured with HPLC grade, filtered through 45 μm cellulose membrane using reduced pressure filtration, and then bubble decay for 1 hour through ultra-sonicator (JAC-1505, 40kHz, Jinwoo-ALEX, Korea). Used in experiments (Bartolomeo et al ., 2006).
컬럼 내의 농도구배는 0분에서 25분까지 50% B, 25분부터 26분까지 100% B, 26분부터 33분까지 100% B, 33분에서 34분까지 0% B, 그리고 34분에서 40분까지 100% B로 유지하였음. Mobile phase의 유속은 1.0 ml/min으로 고정하였다. 유도체화는 autosampler (Agilent, USA)의 injection program을 Fig. 7과 같이 설정하였다. Intra-cellular glutamic acid를 측정하기 위한 전처리는 assay를 위한 preperation과 동일하게 시행하나 sonication 처리 전 완충액을 25 mM NaH2PO4 (pH 6.8)를 사용하였으며 균 파쇄 후 상등액만을 취하여 10 kDa MWCO spin filter를 이용해 10,000 ×g에서 10분간 원심분리하여 시료를 얻었다(Manza et al., 2005). Extra-cellular glutamic acid의 경우를 내려 MWCO filter 바로 시료를 얻고, 농도가 높으므로 여과된 eluent A로 10배 희석하여 분석에 사용하였다.The gradient in the column is 50% B from 0 to 25 minutes, 100% B from 25 to 26 minutes, 100% B from 26 to 33 minutes, 0% B from 33 to 34 minutes, and 40 to 34 minutes. Maintained at 100% B by minute. The flow rate of the mobile phase was fixed at 1.0 ml / min. Derivatization is illustrated in the injection program of the autosampler (Agilent, USA). It was set as 7. The pretreatment for measuring intra-cellular glutamic acid was performed in the same manner as the preperation for the assay, but 25 mM NaH 2 PO 4 (pH 6.8) was used as the buffer before sonication treatment. Samples were obtained by centrifugation at 10,000 x g for 10 minutes (Manza et al. , 2005). Extra-cellular glutamic acid was lowered to obtain a sample directly from the MWCO filter, and the concentration was high, so that 10-fold dilution with filtered eluent A was used for analysis.
전처리를 한 시료는 auto sampler(Agilent, USA) 전용의 vial에 150 μl를 기포가 생기지 않게 옮겨 담고 컬럼은 Symmetry C18이 사용되었고, 분석용 기기는 Agilent 1260 infinity HPLC(Agilent, USA)가 사용되었으며, 검출기는 DAD(Agilent, USA)가 사용되었다. Glutamate 함량을 구하기 위한 표준물질은 0.5 μmole/ml amino acid standard(Sigma-Aldrich, USA)가 사용되었으며 시료의 정량 계산식은 아래와 같다.The pretreated sample was transferred to a vial dedicated to the auto sampler (Agilent, USA) without bubbles. The column was Symmetry C18, and the analytical instrument was Agilent 1260 infinity HPLC (Agilent, USA). The detector was DAD (Agilent, USA). 0.5 μmole / ml amino acid standard (Sigma-Aldrich, USA) was used as a standard to determine the glutamate content.
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최적발효조건 확립에서 실시한 각각의 농도로 설정된 ammonium citrate, ammonium nitrate, CaCl2와 biotin을 첨가한 modified-MRS 액체 배지에 동량의 균주를 접종하여 37.5℃에서 48시간 배양하고, 채취한 sample을 HPLC를 이용해 세포내(intra-cellular) 및 세포외(extra-cellular) 글루탐산의 양을 측정하였다.Inoculate the same amount of strain in ammonium citrate, ammonium nitrate, modified-MRS liquid medium containing CaCl 2 and biotin, and incubate at 37.5 ℃ for 48 hours. The amount of intra-cellular and extra-cellular glutamic acid was measured.
실시예 5. 소형발효조(5L jar fermentor)의 적용Example 5 Application of 5 L jar fermentor
최종 분리 균주의 실제 산업적 이용 가능성을 검토하기 위하여 소형발효조 (5L jar fermentor) 수준에서 MRS 배지의 발효능을 비교하였다. MRS medium을 이용하여 분리주를 5L jar fermentor(LiFlus GX PF1G2SL05, Biotron)을 사용하여 대량배양하였다. Fermentor의 woking volume은 각각 3L로 배지를 제조하여 배양하였다. 유산균은 통성혐기성 미생물로서 산소는 통기하지 않았으며, 200 rpm으로 교반하였다. 배양 중에는 5N NaOH 용액을 이용하여 pH를 6.0이 유지되도록 조절하였으며, 배양온도는 37℃로 고정하였다.In order to examine the actual industrial applicability of the final isolated strain, the fermentation capacity of MRS medium was compared at the level of 5 L jar fermentor. MRS medium was incubated in 5L jar fermentor (LiFlus GX PF1G2SL05, Biotron) in bulk. The woking volume of the fermentor was cultured by preparing a medium in 3L each. The lactic acid bacteria were aerobic anaerobic microorganisms and did not vent with oxygen and were stirred at 200 rpm. During the incubation, the pH was adjusted to 6.0 using 5N NaOH solution, and the incubation temperature was fixed at 37 ° C.
비교예. Ultraviolet(UV)을 이용한 돌연변이 유도 시험 수행Comparative example. Performing Mutation Induction Tests Using Ultraviolet (UV)
본 발명의 균주보다 높은 글루탐산 생산능을 가진 돌연변이주를 유도하기 위해서 먼저 107~108 CFU/ml의 cell suspention 10 ml를 culture dish에 담고, Sample과의 거리가 30cm가 되도록 15W UV lamp (VL-215.LC, VILBER Lourmat, France)를 위치시킨 다음 조심스럽게 magnetic stirrer를 작동시키면서 0, 30, 45, 60, 75초간 254 nm 파장의 UV를 조사하였다. 각각의 조사 시간대에서 pH 6±0.3의 MRS 고체 배지에 conventional plate counting method를 실시하고 37.5℃에서 48시간 동안 배양한 뒤, 치사율(lethality)과 생존율(survival rate)을 계산하였다(Kadam et al.,2006). Ma et al. (2010)에 의하면 통상적으로 적절한 돌연변이 발생 민감도가 가장 높을 때는 생존율이 0.001~10%일 때를 뜻하며 UV-induced lethality는 다음과 같이 계산할 수 있다.In order to induce a mutant strain having a higher glutamic acid production capacity than the strain of the present invention, first put 10 ml of cell suspention of 10 7 ~ 10 8 CFU / ml in a culture dish, and then use a 15W UV lamp (VL -215.LC, VILBER Lourmat, France) and then carefully irradiated with UV at 254 nm wavelength for 0, 30, 45, 60 and 75 seconds with magnetic stirrer. At each irradiation time point, the conventional plate counting method was performed on MRS solid medium at
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colony를 선별하여 MRS 액체배지에 재접종하고 37.5℃에서 24시간 동안 배양한 뒤, 배양균체를 glycerol의 농도가 최종 35%가 되도록 균일하게 혼합한 다음 -70℃에서 보존하였다. 동결된 돌연변이주는 이후 각 실험에 사용되기 전 배지의 2%가 되도록 접종하고 37.5℃에서 24시간 이상 계대 배양한 후 사용하였다.Colonies were screened, re-inoculated in MRS broth and incubated for 24 hours at 37.5 ° C. The cultured cells were then uniformly mixed to achieve a final glycerol concentration of 35% and then stored at -70 ° C. Frozen mutants were then inoculated to 2% of the medium before being used in each experiment and passaged at 37.5 ° C. for at least 24 hours before use.
MRS 액체배지에서 37.5℃, 48시간 배양된 돌연변이주의 세포 내(intra-cellular) 및 세포 외(extra-cellular) 글루탐산 생산량을 측정하기 위하여 이전 실험에 적용한 방법과 동일하게 HPLC를 이용하여 각각 실험을 수행하였다.In order to measure intra-cellular and extra-cellular glutamic acid production in mutant strains cultured at 37.5 ° C. for 48 hours in MRS liquid medium, each experiment was carried out using HPLC as in the previous method. It was.
실험결과 및 고찰Experimental Results and Discussion
1. 글루탐산 생산균의 분리 및 선발1. Isolation and Selection of Glutamic Acid Producing Bacteria
1-1. 유산균의 분리1-1. Isolation of Lactobacillus
재래 비지, 된장, 청국장, 누룩, 막장, 고추장, 김치와 같은 15개의 국내 전통발효식품 시료로부터 총 유산균 338주를 분리하였다.A total of 338 strains of lactic acid bacteria were isolated from 15 Korean traditional fermented food samples, such as Korean traditional bean curd, Doenjang, Cheonggukjang, Nuruk, Makjang, Gochujang, and Kimchi.
1-2. Carbohydrate fermentation test (CFT)1-2. Carbohydrate fermentation test (CFT)
한 시료로부터 각기 다른 패턴의 당 이용성을 보이는 균주를 분리한 결과, 비지 4개 균주, 된장1에서 4개 균주, 된장 2에서 4개 균주, 된장3에서 5균주, 청국장에서 5균주, 누룩1에서 7균주, 누룩2에서 5균주, 누룩3에서 10균주, 막장에서 4균주, 고추장에서 4균주, 김치1에서 4균주, 김치2에서 4균주, 김치3에서 6균주, 김치4에서 5균주, 김치5에서 7균주로서 총합 78균주이며 이외의 균주들은 이후 실험에서 배제되었다.As a result of isolates of sugar-soluble strains of different patterns from one sample, four strains of bean curd, four strains of
1-3. RAPD-PCR1-3. RAPD-PCR
각 밴드의 위치가 동일하게 나타나는 균주들을 배제한 결과, 비지 1균주, 된장1에서 3균주, 된장 2에서 1균주, 된장3에서 3균주, 청국장에서 3균주, 누룩1에서 5균주, 누룩2에서 2균주, 누룩3에서 6균주, 막장에서 2균주, 고추장에서 1균주, 김치1에서 3균주, 김치2에서 2균주, 김치3에서 4균주, 김치4에서 4균주, 김치5에서 4균주로서 총합 44균주이며 이외의 균주들은 이후 실험에서 배제되었다(표 1 및 도 1).As a result of excluding strains that show the same position of each band, 1 strain of bean curd, 1 to 3 strains of Doenjang, 1 to 1 strain of
1-4. Glutamate dehydrogenase 활성 측정1-4. Measurement of Glutamate dehydrogenase Activity
1-3에서 분리한 총 44종의 분리주에 대한 GDH 활성을 측정한 결과 김치에서 분리한 균주 KC3-17에서 가장 높은 활성이 나타났으며 청국장 분리 균주 C7-A, 김치 분리 균주 KC2-14, KC5-17, 된장 분리 균주 D3-11, D3-16, D3-5 순으로 활성이 감소하였다(도 2). 그 이외의 균주들에서는 매우 낮은 활성으로 인하여 이후의 실험에서 제외하였다.As a result of measuring the GDH activity of 44 isolates isolated from 1-3, the highest activity was observed in the strain KC3-17 isolated from Kimchi, and the isolates of Chonggukjang isolate C7-A and KC2-14 and KC5 were isolated. -17, soybean isolate strains D3-11, D3-16, D3-5 in order of decreasing activity (Fig. 2). Other strains were excluded from subsequent experiments due to their very low activity.
1-5. Glutaminase 활성 측정1-5. Measurement of Glutaminase Activity
1-4에서 GDH 활성을 측정하여 1차 스크리닝한 상위 7개 분리주 (KC3-17, C7-A, KC2-14, KC5-17, D3-11, D3-16, D3-5)를 대상으로 glutaminase 활성 측정 실험을 진행한 결과 KC2-14, D3-5, C-7A, D3-16의 순으로 높았으며, KC5-17가 효소활성이 가장 약했다(도 3 및 표 2).Glutaminase in the top 7 isolates (KC3-17, C7-A, KC2-14, KC5-17, D3-11, D3-16, D3-5) screened by measuring GDH activity in 1-4 As a result of the activity measurement experiment, KC2-14, D3-5, C-7A, and D3-16 were the highest, and KC5-17 had the weakest enzyme activity (FIG. 3 and Table 2).
1-6. 글루탐산 생성능이 우수한 균주 선발1-6. Selection of strains with excellent glutamic acid production ability
15품목의 국내 전통발효식품 시료로부터 분리한 유산균 338주에서 서로 다른개별 특성을 갖는 균주를 확보하기 위하여 carbohydrate fermentation test를 수행함으로서 78균주가 얻어졌고, RAPD-PCR을 통해 1차 스크리닝을 한 결과, 총 44균주를 확보하였다. 이들 44분리주에 대한 2차 스크리닝 방법으로 효소활성실험을 수행하여 최종 3종의 최우량 분리주 KC3-17, C7-A, KC2-14를 확보하였다(도 4).In 338 strains of lactic acid bacteria isolated from 15 Korean traditional fermented food samples, 78 strains were obtained by performing carbohydrate fermentation test to obtain strains with different individual characteristics, and as a result of primary screening through RAPD-PCR, A total of 44 strains were obtained. Enzyme activity experiments were carried out by the secondary screening method for these 44 isolates to secure the final three best isolates KC3-17, C7-A, KC2-14 (Fig. 4).
2. 선발된 글루탐산 생산균의 특성 및 동정2. Characterization and Identification of Selected Glutamic Acid Producing Bacteria
2-1. 분리 균주의 생화학적 동정2-1. Biochemical Identification of Isolated Strains
분리균주 KC3-17, C7-A, KC2-14 모두 그람 양성으로 나타났으며 각각 단간균, 간균, 구균으로 나타났고(도 5a) Catalase test 결과는 KC3-17, C-7A, KC2-14는 모두 음성으로 나타났다(도 5b). API 50 CHL kit (bioMerieux, France)를 사용한 당 이용 패턴은 도 5c와 같으며 당 이용의 양성과 음성 판정은 하기 표 3에 나타내었다.The isolates KC3-17, C7-A, and KC2-14 were all Gram-positive and were shown to be monobacterium, bacillus, and cocci (Fig. 5a). Catalase test results were KC3-17, C-7A, and KC2-14. All were negative (FIG. 5B). The sugar use pattern using the
2-2. MALDI-TOF를 이용한 동정2-2. Identification using MALDI-TOF
Bruker microflex LT(Bruker Daltonics, Germany) 기기를 이용하여 MALDI-TOF를 수행하였으며, 동정된 결과를 표 4에 나타냈다. 제조사의 설명에 따르면 score의 경우는 속의 value 2.300~3.000 동정이 확실하고 종의 동정까지 높은 가능성을 나타낸다고 하였으며, 2.000~2.299의 score value는 속의 동정은 확실하나, 종의 동정에서는 가능성을 나타낸다고 하였다. 선발 균주 KC2-14, KC3-17, C-7A는 각각 Pediococcus acidilactici 146 RLT, Lactobacillus plantarum DSM 15312, Lactobacillus fermentum DSM 20391으로 동정되었다.MALDI-TOF was performed using a Bruker microflex LT (Bruker Daltonics, Germany) instrument, and the identified results are shown in Table 4. According to the manufacturer's explanation, the scores of the genus value 2.300 ~ 3.000 were clearly identified and showed high possibility of species identification, while the score values of 2.000 ~ 2.299 indicated the genus identification, but the species identification indicated the possibility. The selected strains KC2-14, KC3-17 and C-7A were identified as Pediococcus acidilactici 146 RLT, Lactobacillus plantarum DSM 15312, and Lactobacillus fermentum DSM 20391, respectively.
2-3. 글루탐산 생성능으로부터 최종 균주 선발2-3. Selection of final strain from glutamic acid production ability
Glutamaic acid determination kit (Sigma-Aldrich, USA)를 이용하여 동일 조건에서 선발 균주들의 실제 글루탐산 생산능을 비교한 결과 C-7A의 세포내(intra-cellular) 글루탐산은 0.082±0.007 nmol/μL이었으며 글루탐산의 농도는 12.07±0.121 ng/μl로서 생산량이 가장 높은 것을 확인하였다. 나머지 KC2-14와 KC3-17 균주는 총 글루탐산의 양이 매우 낮아서 제외하였으며 이에 따라 C-7A를 최종 공시균주로 선정하였다.The intracellular cellular glutamic acid of C-7A was 0.082 ± 0.007 nmol / μL using the glutamatic acid determination kit (Sigma-Aldrich, USA). The concentration was 12.07 ± 0.121 ng / μl confirmed that the highest yield. The remaining KC2-14 and KC3-17 strains were excluded because the total amount of glutamic acid was very low. Therefore, C-7A was selected as the final test strain.
젖산을 생성하는 표준 균주 대비 선발 균주의 글루탐산 생산능을 HPLC를 이용하여 검토한 결과, 선발 균주 L. fermentum C-7A의 intra-cellular glutamic acid는 24시간까지 빠르게 축적되었으나 그 이후 점차 감소되었다. 표준균주 L. fermentum ATCC 9338과 L. casei ATCC 393도 유사한 경향을 보이는 듯 했으나 거의 축적되지 않았다. Extra-cellular glutamic acid의 생산량은 모든 균주에서 24시간 이후 시간이 지날수록 점차 감소되었으며 L. fermentum C-7A에서만 96시간에서 다소 값이 증가하는 것을 보였다. 총 glutamic acid는 L. fermentum C-7A가 가장 높았으며 L. fermentum ATCC 9338, L. casei ATCC 393 순으로 감소되는 것을 확인하였다(도 6).As a result of examining the glutamic acid production ability of the selected strain compared to the standard strain producing lactic acid using HPLC, the intra-cellular glutamic acid of the selected strain L. fermentum C-7A accumulated rapidly up to 24 hours, but gradually decreased thereafter. The standard strains L. fermentum
2-4. 최종 선발 균주에 대한 유전학적 동정2-4. Genetic Identification of Final Selected Strains
16S rRNA gene PCR 생성물로 염기서열을 분석한 결과, 최종 선발 균주 C-7A의 분석된 염기서열은 총 1319 bp로서 Lactobacillus fermentum CECT 562(T)와 99.85 %로 가장 유사성이 높은 것을 확인하였으며 Lactobacillus fermentum DSM 20391으로 동정된 MALDI-TOF 결과와 차이를 보였다. 이에, 최종 선발 균주인 C-7A를 "락토바실러스 퍼멘텀 C-7A"로 명명하였으며, 한국미생물보존센터(Korean Culture Center of Microorganisms)에 2017년 9월 26일자로 기탁하여 수탁번호 KCCM12118P를 부여 받았다.As a result of sequencing with 16S rRNA gene PCR product, the analyzed sequencing of the final selected strain C-7A was 1319 bp, which showed the highest similarity with Lactobacillus fermentum CECT 562 (T), 99.85%, and Lactobacillus fermentum DSM The MALDI-TOF result was identified as 20391. Therefore, the final selection strain C-7A was named "Lactobacillus fermentation C-7A", and was deposited with the Korean Culture Center of Microorganisms on September 26, 2017 and was given accession number KCCM12118P. .
본 균주와 유전학적으로 비슷한 균들 간의 계통발생학적 위치를 MEGA6 프로그램의 neighbor-joining method를 적용하여 phylogenetic tree를 작성하였다(도 7).The phylogenetic tree was constructed by applying the neighbor-joining method of the MEGA6 program to the phylogenetic location among the strains genetically similar to this strain (FIG. 7).
3. 글루탐산의 최적 생산조건3. Optimal Production Conditions for Glutamic Acid
3-1. 온도와 산도의 영향3-1. Effect of temperature and acidity
온도 40℃에서 배양하였을 pH 6±0.3 때 가장 빠르게 대수 증식기로 진입하는 것을 확인하였다(도 8).When incubated at a temperature of 40 ℃ it was confirmed that the fastest entering the logarithmic growth when the
3-2. 염도의 영향3-2. Effect of Salinity
NaCl의 농도는 2%까지는 증식에 지장이 없었으나 3% 이상에서는 증식이 저해되는 현상을 확인하였다(도 9).It was confirmed that the concentration of NaCl did not interfere with proliferation until 2%, but proliferation was inhibited at 3% or more (FIG. 9).
3-3. 질소원 및 기타 중요인자의 농도별 영향3-3. Concentration Effects of Nitrogen Sources and Other Important Factors
Ammonium citrate를 첨가한 군이 ammonium nitrate를 첨가한 군보다 전반적으로 증식능이 높았다. Ammonium nitrate 첨가 군에서는 오히려 암모니아를 전혀 첨가하지 않았을 때 가장 증식이 활발한 것으로 측정되었다. Ammonium citrate 첨가 군에서는 첨가량이 많아질수록 농도의존적(concentration-dependent)으로 증식이 촉진되었다. 한편, 암모니아의 양은 0.2%로 동일하게 고정된 MRS 액체 배지에 CaCl2의 농도를 증가할수록 미세하게 성장률이 향상되는 것을 보였으나 biotin의 경우에는 첨가량과 성장률에 유의적 차이를 보이지 않았다(도 10).Ammonium citrate added group showed higher proliferative capacity than ammonium nitrate group. In the Ammonium nitrate addition group, the most proliferation was observed when no ammonia was added. In the ammonium citrate group, the concentration increased depending on the concentration. On the other hand, the amount of ammonia was 0.2% in the same fixed MRS liquid medium showed a slight increase in the growth rate as the concentration of CaCl 2 increased, but in the case of biotin did not show a significant difference in the addition amount and growth rate (Fig. 10) .
3-4. 글루탐산 생산 증대 효과 확인3-4. Identify the effect of increasing glutamic acid production
글루탐산의 생산 증대 효과를 확인하기 위하여 HPLC를 수행하여 amino acid를 측정한 결과 24시간 동안 배양된 intra-cellular amino acid는 glutamic acid와 isoleucine, lysine이 다른 amino acid보다 높게 측정되었다. 0.5μmole/ml amino acid standard와 비교하여 각 peak의 순서와 시간대를 파악하고 계산식을 통해 비교 정량하였다(도 11).In order to confirm the effect of increasing the production of glutamic acid, the measurement of amino acid by HPLC was performed. The intra-cellular amino acid incubated for 24 hours was higher in glutamic acid, isoleucine and lysine than other amino acids. The order and time period of each peak were compared with 0.5 μmole / ml amino acid standard and compared and quantified through a calculation formula (FIG. 11).
글루탐산의 수율 증가량을 계산한 결과, 접종 전 MRS 액체배지(pH 6±0.3)의 L-글루탐산 함량은 410.06±0.39mg/L이며 분리균주 C-7A를 동일 배지에 접종하여 온도 37℃에서 24시간 동안 배양 후 측정된 총 글루탐산의 함량은 510.97±0.21로서 배양 전 대비 약 24.6%의 수율 증대가 확인되었다. 총 글루탐산의 양은 세포 내(intra-cellular)의 글루탐산과 세포 외(extra-cellular) 글루탐산을 더한 값을 의미한다.As a result of calculating the yield increase of glutamic acid, the L-glutamic acid content of MRS liquid medium (
질소원을 다르게 설정했을 때의 세포 내 글루탐산은 amonium citrate 첨가 군에서 ammonium nitrate 군보다 전반적으로 높게 나타났으며, 그 농도가 낮을수록 글루탐산 축적이 높게 나타났다. 세포 외 글루탐산의 경우 첨가군 간에 큰 차이는 없었다. 총 글루탐산의 양을 보면 질소원을 전혀 첨가하지 않은 군에서 상당한 글루탐산이 축적됨을 나타낸다. 특히 ammonium nitrate는 glutamic acid를 생산하기 위한 질소원으로 적합하지 않은 것이 확인되었다(도 12).Intracellular glutamic acid was higher in the amonium citrate-added group than in the ammonium nitrate group when the nitrogen source was set differently. The lower the concentration, the higher the glutamic acid accumulation. In the case of extracellular glutamic acid, there was no significant difference between the addition groups. The total amount of glutamic acid indicates that significant glutamic acid accumulates in the group without adding any nitrogen source. In particular, it was confirmed that ammonium nitrate is not suitable as a nitrogen source for producing glutamic acid (FIG. 12).
본 발명의 균주의 효율적인 글루탐산 축적을 위하여 biotin과 CaCl2를 각각 농도별로 첨가한 결과 biotin을 첨가한 군에서는 유의적인 효용성을 보이지 않았다. 그러나 CaCl2를 첨가한 군에서 다소 글루탐산의 양이 높게 측정되었다. 특히 0.02%로 가장 높은 농도의 CaCl2를 첨가하였을 때 세포외(extra-cellular) 글루탐산이 가장 많이 측정되었다(도 13).For the efficient glutamic acid accumulation of the strains of the present invention, biotin and CaCl 2 were added at different concentrations. However, the amount of glutamic acid was slightly higher in the group added with CaCl 2 . In particular, extra-cellular glutamic acid was most measured when CaCl 2 was added at the highest concentration of 0.02% (FIG. 13).
4. 소형발효조(5 L jar fermentor)의 적용4. Application of 5 L jar fermentor
5 L 소형발효조에서 3 L volume의 MRS 액체 배지에 L. fermentum C-7A를 접종하고 4시간마다 생균수를 측정한 결과, 기존의 12시간 정도에서 대수 증식기로 도입되었던 플라스크 수준과는 달리 발효조 수준에서는 약 8시간 단축되어 4시간 정도에서 대수 증식기로 도입되는 것을 확인하였다(도 14).Inoculate L. fermentum C-7A into 3 L volume of MRS liquid medium in a 5 L small fermenter and measure the viable cell count every 4 hours. In about 8 hours it was confirmed that the introduction into the logarithmic multiplier in about 4 hours (Fig. 14).
5. 돌연변이 균주의 글루탐산 생성능 확인5. Confirmation of Glutamic Acid Production Capacity of Mutant Strains
수율 증대를 위한 공시균주의 UV-induced mutagenesis를 수행하기 위하여 야생주 C-7A의 UV 조사시간에 따른 치사율을 계산하였다. Ma et al. (2010)에 의하면 돌연변이율이 높을 때는 생존율이 0.001~10%일 때를 뜻하며, 생존율이 낮을수록 돌연변이 발생 확률이 높다. 야생주 C-7A는 15초 조사로 94.3%, 30초는 97.6%, 45초는 99.9%, 60초는 100%의 치사율을 나타내어 45초가 돌연변이주를 유도하기 위한 적절한 조사시간으로 설정되었다.In order to perform UV-induced mutagenesis of the strains to increase the yield, lethality was calculated according to the UV irradiation time of wild strain C-7A. Ma et al. According to (2010), when the mutation rate is high, the survival rate is 0.001 to 10%, and the lower the survival rate, the higher the probability of mutation. Wild strain C-7A showed a mortality of 94.3%, 30 seconds 97.6%, 45 seconds 99.9%, and 60
254nm 파장의 15W UV를 45초간 조사하여 mutagenesis가 일어난 것으로 간주되는 8개의 colony를 선별하였다. 선별된 돌연변이주와 야생주의 탄소원 이용성을 비교 검토한 결과 4주의 돌연변이주를 얻음(도 15).Eight colonies were considered to have occurred mutagenesis by irradiation of 15 W UV at 254 nm for 45 seconds. A comparison of the selected mutants with wild-carbon availability revealed that four mutants were obtained (FIG. 15).
4주의 돌연변이주를 48시간 배양하여 glutamic acid 생성능이 향상되는지 여부를 확인하기 위해 HPLC를 이용하여 야생균주와 비교 분석한 결과 세포내(intra-cellular) 및 세포외(extra-cellular) glutamic acid의 생성량이 야생주의 그것과 비교할 때 측정이 어려울 정도로 농도가 낮았다(표 5).The production of intra-cellular and extra-cellular glutamic acid as a result of comparative analysis with wild strains using HPLC to confirm whether glutamic acid production ability was improved by incubating mutant strains for 4 hours for 48 hours The concentrations were so low that it was difficult to measure compared to that of this wild strain (Table 5).
수탁번호 : KCCM12118PAccession number: KCCM12118P
기탁기관명 : 한국미생물보존센터(국외)Depositary Name: Korea Microorganism Conservation Center (overseas)
수탁일자 : 20170926Deposit Date: 20170926
<110> DAESANG CORPORATION <120> Lactobacillus fermentum C-7A and Use thereof <130> PH17-10916 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1319 <212> DNA <213> Artificial Sequence <220> <223> 16s rRNA sequence of Lactobacillus fermentum C-7A <400> 1 aacgagtggc ggacgggtga gtaacacgta ggtaacctgc ccagaagcgg gggacaacat 60 ttggaaacag atgctaatac cgcataacaa cgttgttcgc atgaacaacg cttaaaagat 120 ggcttctcgc tatcacttct ggatggacct gcggtgcatt agcttgttgg tggggtaacg 180 gcctaccaag gcgatgatgc atagccgagt tgagagactg atcggccaca atgggactga 240 gacacggccc atactcctac gggaggcagc agtagggaat cttccacaat gggcgcaagc 300 ctgatggagc aacaccgcgt gagtgaagat agggtttcgg ctcgtaaagc tctgttgtta 360 aagaagaaca cgtatgagag taactgttca tacgttgacg gtatttaacc agaaagtcac 420 ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttat ccggatttat 480 tgggcgtaaa gagagtgcag gcggttttct aagtctgatg tgaaagcctt cggcttaacc 540 ggagaagtgc atcggaaact ggataacttg agtgcagaag agggtagtgg aactccatgt 600 gtagcggtgg aatgcgtaga tatatggaag aacaccagtg gcgaaggcgg ctacctggtc 660 tgcaactgac gctgagactc gaaagcatgg gtagcgaaca ggattagata ccctggtagt 720 ccatgccgta aacgatgagt gctaggtgtt ggagggtttc cgcccttcag tgccggagct 780 aacgcattaa gcactccgcc tggggagtac gaccgcaagg ttgaaactca aaggaattga 840 cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagctacgcg aagaacctta 900 ccaggtcttg acatcttgcg ccaaccctag agatagggcg tttccttcgg gaacgcaatg 960 acaggtggtg catggtcgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1020 gagcgcaacc cttgttacta gttgccagca ttaagttggg cactctagtg agactgccgg 1080 tgacaaaccg gaggaaggtg gggacgacgt cagatcatca tgccccttat gacctgggct 1140 acacacgtgc tacaatggac ggtacaacga gtcgcgaact cgcgagggca agcaaatctc 1200 ttaaaaccgt tctcagttcg gactgcaggc tgcaactcgc ctgcacgaag ttggaatcgc 1260 tgtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgccc 1319 <110> DAESANG CORPORATION <120> Lactobacillus fermentum C-7A and Use <130> PH17-10916 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1319 <212> DNA <213> Artificial Sequence <220> <223> 16 s rRNA sequence of Lactobacillus fermentum C-7A <400> 1 aacgagtggc ggacgggtga gtaacacgta ggtaacctgc ccagaagcgg gggacaacat 60 ttggaaacag atgctaatac cgcataacaa cgttgttcgc atgaacaacg cttaaaagat 120 ggcttctcgc tatcacttct ggatggacct gcggtgcatt agcttgttgg tggggtaacg 180 gcctaccaag gcgatgatgc atagccgagt tgagagactg atcggccaca atgggactga 240 gacacggccc atactcctac gggaggcagc agtagggaat cttccacaat gggcgcaagc 300 ctgatggagc aacaccgcgt gagtgaagat agggtttcgg ctcgtaaagc tctgttgtta 360 aagaagaaca cgtatgagag taactgttca tacgttgacg gtatttaacc agaaagtcac 420 ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttat ccggatttat 480 tgggcgtaaa gagagtgcag gcggttttct aagtctgatg tgaaagcctt cggcttaacc 540 ggagaagtgc atcggaaact ggataacttg agtgcagaag agggtagtgg aactccatgt 600 gtagcggtgg aatgcgtaga tatatggaag aacaccagtg gcgaaggcgg ctacctggtc 660 tgcaactgac gctgagactc gaaagcatgg gtagcgaaca ggattagata ccctggtagt 720 ccatgccgta aacgatgagt gctaggtgtt ggagggtttc cgcccttcag tgccggagct 780 aacgcattaa gcactccgcc tggggagtac gaccgcaagg ttgaaactca aaggaattga 840 cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagctacgcg aagaacctta 900 ccaggtcttg acatcttgcg ccaaccctag agatagggcg tttccttcgg gaacgcaatg 960 acaggtggtg catggtcgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1020 gagcgcaacc cttgttacta gttgccagca ttaagttggg cactctagtg agactgccgg 1080 tgacaaaccg gaggaaggtg gggacgacgt cagatcatca tgccccttat gacctgggct 1140 acacacgtgc tacaatggac ggtacaacga gtcgcgaact cgcgagggca agcaaatctc 1200 ttaaaaccgt tctcagttcg gactgcaggc tgcaactcgc ctgcacgaag ttggaatcgc 1260 tgtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgccc 1319
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A food additive composition comprising Lactobacillus fermentum C-7A strain (KCCM12118P) or a culture thereof.
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| US3220929A (en) | 1964-02-10 | 1965-11-30 | Kyowa Hakko Kogyo Kk | Microbiological production of amino acid by reductive amination |
| AU746542B2 (en) | 1998-03-18 | 2002-05-02 | Ajinomoto Co., Inc. | L-glutamic acid-producing bacterium and method for producing L-glutamic acid |
| WO2006028298A2 (en) * | 2004-09-10 | 2006-03-16 | Ajinomoto Co., Inc. | L-glutamic acid-producing microorganism and a method for producing l-glutamic acid |
| JP4595506B2 (en) * | 2004-11-25 | 2010-12-08 | 味の素株式会社 | L-amino acid-producing bacterium and method for producing L-amino acid |
| CN101090911B (en) * | 2004-12-28 | 2014-05-07 | 味之素株式会社 | L-glutamic acid-producing microorganism and a method for producing l-glutamic acid |
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