KR20140087518A - New lactic acid bacteria having GABA-producing activity and composition comprising a cultured broth of the strain - Google Patents
New lactic acid bacteria having GABA-producing activity and composition comprising a cultured broth of the strain Download PDFInfo
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Abstract
Description
본 발명은 GABA 생산 활성을 갖는 신규 유산균 균주 및 이의 발효 배양물을 포함하는 조성물에 관한 것으로, 보다 상세하게는 고 GABA 생산 활성을 갖는 락토바실러스 플란타룸 DSR GB150(KCCM11343P) 균주, 상기 균주를 발효시켜 수득한발효 배양물 및 상기 배양물을 함유하는 조성물에 관한 것이다.The present invention relates to a novel lactic acid bacterial strain having a GABA production activity and a composition comprising the fermentation culture, and more particularly to a lactobacillus plantarum DSR GB150 (KCCM11343P) strain having high GABA production activity, And a culture containing the fermentation culture.
감마-아미노부티르산 (γ-aminobutyric acid; GABA)은 비단백질 구성 아미노산으로서 사람에 있어서는 신경계 및 혈액에 함유되어 있고, 이의 대부분은 뇌의 골수에 존재하여 아세틸 콜린이라 불리우는 신경전달 물질은 증가시키고, 뇌 기능을 촉진시키는 등의 생리작용을 한다.Gamma-aminobutyric acid (GABA) is a nonprotein constituent amino acid that is contained in the nervous system and blood in humans, and most of it is present in the bone marrow of the brain, thereby increasing neurotransmitters called acetylcholine, And physiological functions such as promoting the function.
GABA는 신경전달물질, 뇌기능 촉진 뿐만 아니라, 다른 여러 생리활성을 가지고 있는데 오래 전부터 혈압저하작용, 이뇨작용 등을 가진다는 것이 알려져 있으며, 특히 뇌졸중 치료제로서 의약품에도 등록되어 있다.GABA has not only a neurotransmitter, brain function, but also various other physiological activities. It has been known for a long time that it has blood pressure lowering action, diuretic effect and the like, and it is also registered as a medicine for stroke.
GABA 함유 차, 쌀 및 콩 발효 식품, 김치 등에서 분리한 유산균들이 GABA를 고농도로 생산한다는 연구 보고가 있는데 실제 일본에서는 김치에서 분리한 젖산균을 이용하여 GABA를 고농도 생산함으로써 식품에 실제 응용하고 있다. There is a report that lactic acid bacteria isolated from GABA-containing tea, rice and soybean fermented food, kimchi produce high concentration of GABA. In fact, in Japan, lactic acid bacteria isolated from kimchi are used to produce high concentration of GABA.
또한, 일본 소주에서부터 락토바실러스 브레비스 (Lactobacillus brevi)를 배양하여 직접 GABA를 만드는 방법까지 보고되고 있다. 이런 고혈압 예방, 뇌기능 촉진 등의 효과는 계속 건강에 대한 관심이 높아지고 있는 시점에서 GABA의 산업적 응용을 더욱 가속화할 것으로 예측된다In addition, Japanese Soju from Lactobacillus brevis (Lactobacillus brevi ) to produce GABA directly. These effects of preventing hypertension and promoting brain function are expected to accelerate the industrial application of GABA at a time when the interest in health continues to increase
식품중에 GABA의 함유량을 증가시키는 방법으로서는 시판되고 있는 가바론차 (gabaron ,tea)와 같이, 찻잎을 혐기적 조건하에서 처리하는 것에 의해 GABA의 함량을 증가시키는 방법이나, 쌀의 배아 등을 물에 침지하는 것에 의해 GABA 및 유리 아미노산을 현저히 증가시믐 방법이 개발되어 있다.As a method for increasing the content of GABA in food, there is a method of increasing the content of GABA by treating tea leaves under anaerobic conditions, such as gabaron (tea), which is commercially available, or a method of immersing the embryo of rice in water , A significant increase in GABA and free amino acids has been developed.
미생물 발효법을 이용하여 GABA를 풍부하게 한 식품도 개발되어 있으며, 예를 들면, 2종의 유산균을 접종하여 고농도의 GABA를 얻는 방법, 유제품을 프로테아제로 처리하여 GABA의 출발원료인 글루타민산을 생산하고, 유산발효시키는 방법, 김치로부터 단리된 유산균을 이용하는 방법, 붕어초밥으로부터 단리된 유산균을 사용하는 방법, 글루타민산 디카르복실라아제를 갖는 유산균을 사용하여 GABA가 부가된 대두(콩)를 제조하는 방법 등이 보고되고 있다.Foods enriched with GABA using microbial fermentation methods have been developed. For example, a method of obtaining high concentration of GABA by inoculating two kinds of lactic acid bacteria, a method of producing glutamic acid as a starting material of GABA by treating dairy products with a protease, A method of fermenting lactic acid, a method of using lactic acid bacteria isolated from kimchi, a method of using lactic acid bacteria isolated from cruciferous sushi, a method of producing GABA-added soybean (soybean) using lactic acid bacteria having glutamic acid decarboxylase Is being reported.
김치 내에는 여러 종류의 유산균이 생육하고 있고 이 유산균 중에는 항균 물질에 관한 보고는 있지만, 항세균 효과에 집중되어 있으며 김치 유래 유산균 중 GABA 생산 활성을 지니는 유산균에 관한 보고는 전무한 상태이다. There are several kinds of lactic acid bacteria in kimchi. Although there are reports of antimicrobial substances in these lactic acid bacteria, they are concentrated on the antibacterial effect, and there is no report on lactic acid bacteria having GABA production activity among kimchi - derived lactic acid bacteria.
따라서, 지금까지 연구된 GABA 생성능의 한계를 해결하기 위하여, GABA 고 생성능을 갖는 새로운 미생물의 탐색 및 상기 미생물이 생산하는 물질에 대한 연구가 절실히 요구되고 있다.Therefore, in order to solve the limitation of the GABA production ability so far, it is urgently required to search for a new microorganism having a high GABA production ability and to study the substance produced by the microorganism.
본 발명자들은 상기와 같은 종래기술의 문제점을 해결하고자 지속적으로 연구를 수행하던 중, 유용 미생물을 다량 함유하고 있는 김치로부터 분리한 유산균이 고 GABA 생산 활성을 지님을 알아내고, 본 발명을 완성하였다.The inventors of the present invention have found that lactic acid bacteria isolated from kimchi containing a large amount of useful microorganisms have high GABA production activity while studying to overcome the problems of the prior art as described above, and completed the present invention.
따라서, 본 발명의 목적은 고 GABA 생산 활성을 갖는 신규한 유산균을 제공하는 것이다.Therefore, an object of the present invention is to provide a novel lactic acid bacteria having high GABA production activity.
본 발명의 다른 목적은 상기 유산균을 발효시켜 얻은발효 배양물을 제공하는 것이다.Another object of the present invention is to provide a fermentation culture obtained by fermenting the above-mentioned lactic acid bacteria.
본 발명의 또 다른 목적은 상기에서 얻은배양물을 함유하는 조성물을 제공하는 것이다.It is still another object of the present invention to provide a composition containing the culture obtained above.
본 발명은 상기와 같은 목적을 달성하기 위하여, GABA(γ-aminobutyric acid) 생산 활성을 갖는 락토바실러스 플란타룸 DSR GB150 (KCCM11343P) 균주를 제공한다.In order to achieve the above object, the present invention provides Lactobacillus plantarum DSR GB150 (KCCM11343P) strain having GABA (gamma -aminobutyric acid) production activity.
본 발명은 또한, 상기 락토바실러스 플란타룸 DSR GB150 (KCCM11343P) 균주를 발효시켜 얻은 발효 배양물을 제공한다.The present invention also provides a fermentation culture obtained by fermenting the Lactobacillus plantarum DSR GB150 (KCCM11343P) strain.
본 발명은 또한, 상기 발효 배양물을 함유하는 조성물을 제공한다.The present invention also provides a composition containing said fermentation culture.
본 발명에 의한 신규 유산균인 락토바실러스 플란타룸 DSR GB150 (KCCM11343P) 균주는 GABA 생산 활성이 우수하기 때문에, 이를 발효시켜 얻은 발효 배양물을 이용하여 절임류 식품용 조미액 등과 같은 식품첨가용 조성물, 생균제 조성물, 정장제, 사료 조성물, 화장료 조성물 등에 유용하게 사용될 수 있다. Since the lactobacillus plantarum DSR GB150 (KCCM11343P) strain of the novel lactic acid bacterium according to the present invention has excellent GABA production activity, the fermentation culture obtained by fermenting the fermented culture can be used as a food additive composition such as a seasoning for pickled foods, , A dressing agent, a feed composition, a cosmetic composition and the like.
도 1a 내지 도 1d는 본 발명의 락토바실러스 플란타룸 DSR GB150 (KCCM11343P) 균주와 공지의 락토바실러스 플란타룸 (GenBanK Accession No. HF562939)의 16S rRNA의 상동성을 분석한 결과를 나타낸 것이다.Figures 1A-1D are photographs showing the Lactobacillus plantarum DSR GB150 (KCCM11343P) strain of the present invention and the known The results of analysis of the homology of 16S rRNA of Lactobacillus plantarum (GenBank accession No. HF562939) are shown.
본 발명의 일태양은 GABA(γ-aminobutyric acid) 생산 활성을 갖는 락토바실러스 플란타룸 DSR GB150 (KCCM11343P) 균주에 관한 것이다..One aspect of the present invention relates to a Lactobacillus plantarum DSR GB150 (KCCM11343P) strain having GABA (gamma -aminobutyric acid) production activity.
본 발명에 의한 상기 균주는 서열번호 1의 16S rRNA 염기서열을 갖는데, 락토바실러스 플란타룸 (GenBanK Accession No. HF562939)의 16S rRNA와 99%의 상동성을 갖는다..The strain according to the present invention has the 16S rRNA base sequence of SEQ ID NO: 1 and has 99% homology with 16S rRNA of Lactobacillus flutarium (GenBank Accession No. HF562939).
본 발명에 의한 상기 균주는 300 mg/㎖ 내지 450 mg/㎖, 바람직하게는 350 mg/㎖ 내지 400 mg/㎖,의 GABA 생산 활성을 갖는 것을 특징으로 한다.The strain according to the present invention has a GABA production activity of 300 mg / ml to 450 mg / ml, preferably 350 mg / ml to 400 mg / ml.
본 발명에 의한 상기 균주는 김치에서 분리한 유산균일 수 있으나, 분리원은 이에 한정되는 것은 아니다. The strain according to the present invention may be lactic acid bacteria isolated from kimchi, but the source of the isolate is not limited thereto.
본 발명의 다른 태양은 상기 락토바실러스 플란타룸 DSR GB150 (KCCM11343P) 균주를 발효시켜 얻은 발효 배양물에 관한 것이다.Another aspect of the invention relates to a fermentation culture obtained by fermenting the Lactobacillus plantarum DSR GB150 (KCCM11343P) strain.
본 발명에서, 상기에서 "발효물"이라 함은 상기 균주를 발효시켜 얻은 발효액 자체이거나, 상기 발효액을 고ㆍ액분리하여 배지 성분을 제거한 발효액이거나, 배지성분 및 상기 균주를 제거한 발효액의 상등액을 의미한다. In the present invention, the term "fermented product" refers to a fermentation broth obtained by fermenting the strain, or a fermentation broth in which the broth component is removed by subjecting the fermentation broth to solid-liquid separation or a supernatant of the broth and the fermentation broth from which the strain has been removed do.
본 발명의 또 다른 태양은 상기 락토바실러스 플란타룸 DSR GB150 (KCCM11343P) 균주를 발효시켜 얻은 발효 배양물을 함유하는 조성물에 관한 것이다.Another aspect of the present invention relates to a composition containing a fermentation culture obtained by fermenting the Lactobacillus plantarum DSR GB150 (KCCM11343P) strain.
본 발명에 의한 상기 조성물은 생균제(probiotics) 조성물일 수 있다. The composition according to the present invention may be a probiotics composition.
본 발명에서, 상기 "생균제"라 함은 유해 미생물에 대한 저항성 증진을 통한 인간의 내질병성을 향상시커나, 가축의 생산성 증진을 위해 가축에 급여하기 위한 살아있는 유산균을 의미한다.In the present invention, the term " probiotic agent "means a live lactic acid bacterium for improving the disease resistance in humans through the enhancement of resistance to harmful microorganisms, or for feeding livestock to improve the productivity of livestock.
본 발명에 의한 상기 조성물은 식품첨가용 조성물일 수 있는데, 상기 식품첨가용 조성물은 쌈무, 단무지, 피클류 등과 같은 절임류 식품용 조미액일 수 있다.The composition according to the present invention may be a food additive composition, and the food additive composition may be a seasoning liquid for pickled foods such as bamboo shoots, radishes, pickles, and the like.
본 발명에 의한 상기 조성물은 정장용 약학 조성물일 수 있다. The composition according to the present invention may be a pharmaceutical composition for dressing.
본 발명에서, 상기 "정장용" 이라 함은 장내 해로운 세균은 죽이고, 유익한 세균은 더 좋게 해주는 등의 작용을 통하여, 장을 깨끗하게 해서 장기능을 좋게 해주기 위하여 사용되는 것을 의미한다.In the present invention, the term "dressing" means to be used for cleansing the intestines and improving the intestinal function by, for example, killing intestinal bacteria and beneficial bacteria.
상기 약학 조성물은 정제, 환제, 캡슐제 등 통상의 제형으로 제조할 수 있다.The pharmaceutical composition may be prepared in conventional formulations such as tablets, pills, capsules, and the like.
본 발명에서, 상기 조성물은 사료 조성물일 수 있다. In the present invention, the composition may be a feed composition.
상기 사료 조성물은 유해 미생물에 대한 저항성 증진을 통한 가축의 생산성 증진을 위해 가축에 급여하는데, 통상의 사료에 본 발명의 상기 발효 배양물을 첨가하여 제조할 수 있다. The feed composition is fed to the livestock to improve the productivity of the livestock through the enhancement of resistance to harmful microorganisms. The feed composition can be prepared by adding the fermentation culture of the present invention to a common feed.
본 발명에 의한 상기 조성물은 화장료 조성물일 수 있다. The composition according to the present invention may be a cosmetic composition.
상기 화장료 조성물은 로션, 크림, 젤 등의 제형이 바람직하나, 이에 한정되는 것은 아니다.
The cosmetic composition is preferably a formulation such as lotion, cream, gel and the like, but is not limited thereto.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 실시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
<실시예 1> 김치 유산균의 분리 및 동정Example 1 Isolation and Identification of Kimchi Lactic Acid Bacteria
통상적인 김치 제조방법으로 제조된 김치에서 유산균을 분리하였다. Lactic acid bacteria were isolated from kimchi prepared by conventional kimchi manufacturing method.
김치시료를 0.85 % 식염수로 10 배 희석하였고, 0.1 ㎖씩 MRS 아가 배지(MRS 아가, 디프코(Difco.); 박토 펩톤 10 g, 소고기추출물 10 g, 효모추출물 5 g, 글루코스 20 g, 트윈 801 g, 구연산 2 g, 제2 인산칼륨 2 g, 초산나트륨 5 g, 황산망간 0.1 g, 황산마그네슘 0.05 g, 아가 15 g/증류수 1 ℓ)플레이트에 접종한 후, 유리막대로 도말하였다. Kimchi samples were diluted 10 times with 0.85% saline, and 0.1 ml each of MRS agar medium (MRS agar, Difco .; 10 g of bovine peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, g, citric acid 2 g, potassium dibasic 2 g, sodium acetate 5 g, manganese sulfate 0.1 g, magnesium sulfate 0.05 g, agar 15 g / distilled water 1 l).
이후, 플레이트를 37 ℃의 항온 배양기에서 1일 동안 배양하였다. 생성된 각각의 콜로니를 MRS 아가 플레이트에 획선 접종하였고, 37 ℃에서 1일 동안 배양하여 유산균 콜로니를 분리하였다.
Plates were then incubated for 1 day in a 37 < 0 > C incubator. Each of the resulting colonies was inoculated on MRS agar plates and cultured at 37 ° C for 1 day to isolate lactic acid bacteria colonies.
<실시예 2> GABA 생성 균주의 분리Example 2: Isolation of GABA producing strains
상기 실시예 1에 의해 김치에서 분리한 유산균 17종을 1% L-글루타민산이 첨가된 MRS 배지에서 48시간 동안 배양한 후 상등액만 취하여 TLC(thin layer cgromatography)로 1차 탐색하였다.Seventeen kinds of lactic acid bacteria isolated from kimchi according to Example 1 were cultured in MRS medium supplemented with 1% L-glutamic acid for 48 hours, followed by primary screening by TLC (thin layer cgromatography).
상기 MRS 배지는 MRS 브로스, 디프코(Difco.); 박토펩톤 10 g, 소고기추출물 10 g, 효모추출물 5 g, 글루코스 20 g, 트윈80 1 g, 구연산 2 g, 제2 인산칼륨 2 g, 초산나트륨 5 g, 황산망간 0.1 g, 황산마그네슘 0.05 g, 아가 15 g/증류수 1 ℓ로 구성되어 있다.The MRS medium was MRS broth, Difco .; 10 g of bovine peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 80 g of tween, 2 g of citric acid, 2 g of dibasic potassium phosphate, 5 g of sodium acetate, 0.1 g of manganese sulfate, 15 g of agar / 1 l of distilled water.
TLC는 silica gel 60 (Merk 사)를 사용하였고, 전개 용매는 부탄올, 아세트산, 및 물을 각각 4:1:1 비율로 섞어서 사용하였다. 2% 닌히드린 시약(Sigma 사)를 상용하여 발색하였고, GABA 표준 시약과 비교하여 Rf 값이 동일한 시료를 선별하여 GABA 생성균주를 분리하였다. Silica gel 60 (Merk) was used for the TLC, and the developing solvent was a mixture of butanol, acetic acid, and water at a ratio of 4: 1: 1. 2% ninhydrin reagent (Sigma) was used for color development. GABA-producing strains were isolated by selecting samples having the same Rf value as those of GABA standard reagents.
그 결과, 후보 균주 6종을 선별하였다. 선별된 유산균의 배양액은 0.45um 실린지 필터로 여과하여 AccQ-Tag 방법으로 유도체화한 후, 선형 구배 조건하에서 분석하였다. 이때, HPLC 칼럼으로는 Nova-PakTM C18, 4Um 컬럼을 사용하였다. HPLC 분석 결과, 락토바실러스 플란타룸 DSR GB150 (KCCM11343P) 균주가 가장 많은 GABA를 생성하는 것으로 나타났다 (표 1 참조).As a result, six candidate strains were selected. The cultures of the selected lactic acid bacteria were filtered with a 0.45um syringe filter, derivatized with AccQ-Tag method, and analyzed under linear gradient conditions. At this time, as the HPLC column, Nova-Pak 占 C18, 4 占 퐉 column was used. HPLC analysis showed that Lactobacillus plantarum DSR GB150 (KCCM11343P) strain produced the most GABA (see Table 1).
(mg/ml)GABA content
(mg / ml)
<실시예 3> 선별된 유산균의 형태학적 및 생화학적 특성 조사 Example 3 Morphological and Biochemical Characterization of Selected Lactic Acid Bacteria
a. 형태학적 특성a. Morphological characteristics
현미경을 이용하여, 상기 동정한 균주의 형태학적 특성을 관찰하였다. 분리된 유산균은 그람 양성균이며, 막대 형태를 갖고 있음을 확인할 수 있었다. 집락은 불투명의 연한 노란색으로 집락의 표면은 매끄러웠다(표 2).
Using a microscope, the morphological characteristics of the strain identified above were observed. The isolated lactic acid bacteria were Gram - positive bacteria and had rod shape. The colonies were light yellow opaque and the surface of the colonies was smooth (Table 2).
b. 생화학적 특성b. Biochemical property
API 시스템(API system; La Balme-les-Grottes, France)으로 상기 분리된 균주를 동정하였다. 우선, 멸균한 백금이로 균 콜로니를 각각 취한 후 멸균 증류수 2 ㎖에 부유시켰다. The isolated strain was identified by an API system (La Balme-les-Grottes, France). First, the sterilized platinum-fungus colonies were each taken and suspended in 2 ml of sterilized distilled water.
멸균 증류수 5 ㎖에 API 50CH 키트(BioMerieux, France)에서 제공되는 맥팔란드 표준 용액 No.2 (MccFaland Standard Solution No.2)의 농도로 상기 균액을 부유시켰다. 상기 부유액을 API 50CH 키트의 액체배지에 첨가하여 균질화시킨 후, API 50CH 키트의 50개 각 튜브에 200 ㎕씩 접종하였다. 미네랄 오일로 튜브 위를 엎어준 후, 30 ℃에서 48 시간 동안 배양시켰다. The bacterial solution was suspended in 5 ml of sterilized distilled water at a concentration of MccFaland Standard Solution No. 2 supplied by API 50CH kit (BioMerieux, France). The supernatant was added to the liquid medium of the API 50CH kit and homogenized, and 200 μl of each of the 50 tubes of the API 50CH kit was inoculated. The tube was overlaid with mineral oil and then cultured at 30 DEG C for 48 hours.
상기 배양액을 API 시스템으로 분석하여 49종의 탄수화물 발효패턴을 확인한 후, 이 결과를 ATB 동정 컴퓨터 시스템에 입력하여 동정하였다. 그 결과, DSR GB150 (KCCM11343P)은 하기 표 3(본 발명에 따른 락토바실러스 플란타룸 GB150 (KCCM11343P) 균주의 탄수화물 발효패턴 분석 결과)의 탄수화물 발효패턴을 갖고 있는 것으로 판명되어 락토바실러스 플란타룸으로 동정되었다. The culture solution was analyzed by an API system to identify 49 types of carbohydrate fermentation patterns, and the results were entered into an ATB identification computer system. As a result, DSR GB150 (KCCM11343P) was found to have a carbohydrate fermentation pattern of the following Table 3 (result of analysis of carbohydrate fermentation pattern of Lactobacillus plantarum GB150 (KCCM11343P) strain according to the present invention) It was identified.
상기 동정된 균주를 "락토바실러스 플란타룸 DSR GB150"이라고 명명하고, 2012년 12월20일자로, 한국미생물보존센터에 KCCM11343P로 기탁되었다.
The identified strain was named "Lactobacillus plantarum DSR GB150 ", and deposited on December 20, 2012 as KCCM 11343P at the Korean Society for Microbiological Care.
<실시예 4> 선별된 유산균의 유전학적 특성 조사 Example 4: Investigation of genetic characteristics of selected lactic acid bacteria
본 발명의 유산균 균주의 동정 및 분류를 위한 16S rRNA유전자의 염기서열을 분석하였다. Genomic DNA를 분리하여 16S ribosomal DNA 부분을 증폭하여 증폭된 DNA 단편을 전기영동으로 확인하였다. The base sequence of the 16S rRNA gene for the identification and classification of the lactic acid bacteria strain of the present invention was analyzed. Genomic DNA was isolated and the 16S ribosomal DNA fragment was amplified and the amplified DNA fragment was confirmed by electrophoresis.
DNA 단편을 Qiagen PCR purification kit (Giagen,Hilden,Germany)으로 정제하여 d-Rhodamine dye-labeling dd-NTP를 포함하는 반응액과 혼합하여 시퀀싱 PCR을 한 후 얻어진 DNA를 Ethanol/Sodium acetate 침전법을 이용하여 정제하였다. DNA fragments were purified by Qiagen PCR purification kit (Giagen, Hilden, Germany) and mixed with a reaction solution containing d-Rhodamine dye-labeled dd-NTP and subjected to sequencing PCR. The obtained DNA was subjected to ethanol / sodium acetate precipitation Lt; / RTI >
상기 정제한 DNA를 TSR(Template Suppression Reagent)에 녹여 ABI prism 310 Genetic Analyzer(PE AppliedBiosystems, U.S.A)로 분석하고, 분석된 염기서열은 Genebank(http://www.ncbi.nlm.nih.gov/) Accession No. HF562939를 이용하여 동정하였다. The purified DNA was dissolved in Template Suppression Reagent (TSR) and analyzed with ABI prism 310 Genetic Analyzer (PE Applied Biosystems, USA). The analyzed base sequence was Genebank (http://www.ncbi.nlm.nih.gov/) Accession No. HF562939.
본 발명의 유산균 균주의 16S rRNA 염기서열(서열번호 1)은 도 1에서 보는 바와 같이, 분석 결과 락토바실러스 플란타룸(Lactobacillus plantarum)과 가장 높은 상동성(99 %)을 나타내었다.
As shown in FIG. 1, the 16S rRNA nucleotide sequence (SEQ ID NO: 1) of the lactic acid bacteria of the present invention showed the highest homology (99%) with Lactobacillus plantarum.
<제조예 1> GABA 생성 유산균 발효액을 이용한 절임류 조미액 제조<Preparation Example 1> Production of GABA-producing seasoning liquid using fermentation broth of lactic acid bacteria
정제수 90.945% (단위는 중량%이며, 이하 %로만 표기한다)에 고과당 5%, 효모추출물 0.5%, sodium citrate 1%, sodium acetate 0.5%, MgSO4 0.005%, MnSO4 0.05% 를 제조한 후, GABA 생성 유산균 GB150 (KCCM11343P)을 2% 접종하여 48시간 동안 발효시켰다.5% of fructose, 0.5% of yeast extract, 1% of sodium citrate, 0.5% of sodium acetate, and 0.5% of MgSO 4 were added to purified water 90.945% 0.005%, MnSO 4 0.05%, and then GABA-producing lactic acid bacteria GB150 (KCCM11343P) was inoculated at 2% and fermented for 48 hours.
발효액 91.1%에 효소처리스테비아 0.15%, 식초 5.3%, 구연산 0.625%, 식염 2.75%, 자몽종자 추출물 0.075%를 첨가하여 GABA 함유 쌈무 조미액을 완성하였다.Enzymatic treatment of stevia with 0.15%, vinegar with 5.3%, citric acid with 0.625%, salt with 2.75% and grapefruit seed extract with 0.075% was completed in 91.1% of the fermentation broth.
실시예 2와 같은 방법으로 HPLC를 이용하여 조미액의 GABA 함유량을 시판 중인 쌈무와 비교한 결과를 표 2에 나타내었다.
Table 2 shows the results of comparing the GABA content of the seasoning solution with commercially available samsam using HPLC in the same manner as in Example 2. [
상기 표 4에서 보는 바와 같이, GABA 생성 균주를 첨가한 발효 쌈무에서만 GABA가 52.1mg/ml가 생성되었음을 확인하였다.
As shown in Table 4, it was confirmed that GABA was produced at 52.1 mg / ml only in the fermented samsum supplemented with GABA producing strains.
<제조예 2> GABA 생성 유산균 발효액을 이용한 생균제 조성물의 제조Preparation Example 2 Preparation of a Probiotic Compound Using GABA-Producing Lactic Acid Fermentation Liquid
본 발명의 식물성 유산균 락토바실러스 플란타룸 DSR GB150 (KCCM11343P) 2%를 2.0 % 글루코스, 1.0 % 펩톤, 1.0 % 효모추출물, 0.2 % 제이인산, 0.05 % 황산마그네슘 및 0.05 % 시스테인이 함유된 배지에 접종하여 37 ℃에서 2 일 동안 호기적으로 배양하였다. 2% of the plant lactobacillus Lactobacillus plantarum DSR GB150 (KCCM11343P) of the present invention was inoculated into a medium containing 2.0% glucose, 1.0% peptone, 1.0% yeast extract, 0.2% ferric phosphate, 0.05% magnesium sulfate and 0.05% cysteine And cultured aerobically for 2 days at 37 ° C.
탈지강, 대두박, 옥분 및 당밀이 각각 3:1:1:1의 중량비로 혼합되어 들어 있는 고체 배양기에 상기 각 균주의 배양액을 10 중량 %로 접종한 후, 여기에 물을 45 내지 60 중량%로 첨가하였다. 이를 40 ℃에서 3일 동안 교반시키면서 혐기적으로 고체발효를 수행하여 생균제 조성물을 제조하였으며, 이때 제조된 생균제 조성물은 본 발명의 유산균이 1×109 cfu/g 이상으로 포함되도록 하였다.
The culture broth of each of the above strains was inoculated at 10% by weight in a solid culture medium in which a defatted steel, soybean meal, corn and molasses were mixed at a weight ratio of 3: 1: 1: 1, Lt; / RTI > The fermentation broth composition was prepared by anaerobically fermenting the fermentation broth with stirring at 40 ° C for 3 days. The broth composition prepared herein contained 1 × 10 9 cfu / g or more of the lactic acid bacteria of the present invention.
<제조예 3> GABA 생성 유산균 발효액을 이용한 약학적 조성물의 제조<Preparation Example 3> Preparation of pharmaceutical composition using GABA-producing lactic acid fermentation broth
상기 제조예 2에서 얻어진 생균 조성물의 분말을 각각 통상적인 방법에 따라 정제, 환제 및 캡슐제로 성형하여 정정용 약학조성물로 제조하였으며, 이때 제조된 조성물은 본 발명의 유산균이 1×109 cfu/g 이상으로 포함되도록 하였다.
Each of the powders of the viable composition obtained in Preparation Example 2 was individually molded into tablets, pills and capsules according to a conventional method to prepare a pharmaceutical composition for correction. The composition prepared herein had a lactic acid bacterium of the present invention having a concentration of 1 × 10 9 cfu / g or more .
<제조예 4> GABA 생성 유산균 발효액을 이용한 사료 조성물의 제조<Preparation Example 4> Preparation of feed composition using GABA-producing lactic acid fermentation broth
본 발명의 락토바실러스 플란타륨 DSR GB150 유산균을 2.0 % 글루코스, 1.0 % 펩톤, 1.0 % 효모추출물, 0.2 % 제이인산, 0.05 % 황산마그네슘 및 0.05 % 시스테인이 함유된 배지에 접종하여 37 ℃에서 2일 동안 호기적으로 배양하였다. 각 균주의 배양액과 부형제 탈지강을 1:1의 중량비로 단순 혼합한 후 40 ℃ 이하에서 건조시켰으며, 건조된 혼합물을 분쇄하여 사료용 조성물을 제조하였다. 이때 제조된 사료용 조성물은 본 발명의 유산균이 1×109 cfu/g 이상으로 포함되도록 하였다.
Lactobacillus plantaium Lactobacillus plantarium of the present invention was inoculated into a medium containing 2.0% glucose, 1.0% peptone, 1.0% yeast extract, 0.2% ferric phosphate, 0.05% magnesium sulfate and 0.05% cysteine at 37 ° C for 2 days Were cultivated aerobically. The culture medium of each strain and the excipient degreasing solution were simply mixed at a weight ratio of 1: 1, followed by drying at 40 ° C or lower, and the dried mixture was pulverized to prepare a feed composition. At this time, the prepared feed composition contained the lactic acid bacteria of the present invention at a concentration of 1 × 10 9 cfu / g or more.
<제조예 5> GABA 생성 유산균 발효액을 이용한 화장료 조성물의 제조PREPARATION EXAMPLE 5 Preparation of Cosmetic Composition Using GABA-Producing Lactic Acid Fermentation Liquid
아래 표 5의 조성물을 순서대로 계량하고 균일하게 혼합한 후, 정제수로 잔량을 채워, 실시예에 따라 제조한 유산균 발효 상등액을 함유하는 유연화장수(스킨)를 제조하였다.
The compositions of Table 5 below were weighed out in order and mixed uniformly. The remaining amount was filled with purified water to prepare a softened longevity (skin) containing the lactic acid fermentation supernatant prepared according to the examples.
본 발명에 의한 신규 유산균인 락토바실러스 플란타룸 DSR GB150 (KCCM11343P) 균주는 GABA 생산 활성이 우수하기 때문에, 이를 발효시켜 얻은 발효 배양물을 이용하여 절임류 식품용 조미액 등과 같은 식품첨가용 조성물, 생균제 조성물, 정장제, 사료 조성물, 화장료 조성물 등에 유용하게 사용될 수 있다. Since the lactobacillus plantarum DSR GB150 (KCCM11343P) strain of the novel lactic acid bacterium according to the present invention has excellent GABA production activity, the fermentation culture obtained by fermenting the fermented culture can be used as a food additive composition such as a seasoning for pickled foods, , A dressing agent, a feed composition, a cosmetic composition and the like.
<110> Daesang FNF Co., Ltd. <120> New lactic acid bacteria having GABA-producing activity and composition comprising a cultured broth of the strain <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1358 <212> DNA <213> Lactobacillus plantarum <220> <221> gene <222> (1)..(1358) <223> 16S rRNA <400> 1 gaactggtga gtaacacgtg ggaaacctgc ccagaagcgg gggataacac ctggaaacag 60 atgctaatac cgcataacaa cttggaccgc atggtccgag cttgaaagat ggcttcggct 120 atcacttttg gatggtcccg cggcgtatta gctagatggt ggggtaacgg ctcaccatgg 180 caatgatacg tagccgacct gagagggtaa tcggccacat tgggactgag acacggccca 240 aactcctacg ggaggcagca gtagggaatc ttccacaatg gacgaaagtc tgatggagca 300 acgccgcgtg agtgaagaag ggtttcggct cgtaaaactc tgttgttaaa gaagaacata 360 tctgagagta actgttcagg tattgacggt atttaaccag aaagccacgg ctaactacgt 420 gccagcagcc gcggtaatac gtaggtggca agcgttgtcc ggatttattg ggcgtaaagc 480 gagcgcaggc ggttttttaa gtctgatgtg aaagccttcg gctcaaccga agaagtgcat 540 cggaaactgg gaaacttgag tgcagaagag gacagtggaa ctccatgtgt agcggtgaaa 600 tgcgtagata tatggaagaa caccagtggc gaaggcggct gtctggtctg taactgacgc 660 tgaggctcga aagtatgggt agcaaacagg attagatacc ctggtagtcc ataccgtaaa 720 cgatgaatgc taagtgttgg agggtttccg cccttcagtg ctgcagctaa cgcattaagc 780 attccgcctg gggagtacgg ccgcaaggct gaaactcaaa ggaattgacg ggggcccgca 840 caagcggtgg agcatgtggt ttaattcgaa gctacgcgaa gaaccttacc aggtcttgac 900 atactatgca aatctaagag attagacgtt cccttcgggg acatggatac aggtggtgca 960 tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1020 tattatcagt tgccagcatt aagttgggca ctctggtgag actgccggtg acaaaccgga 1080 ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta 1140 caatggatgg tacaacgagt tgcgaactcg cgagagtaag ctaatctctt aaagccattc 1200 tcagttcgga ttgtaggctg caactcgcct acatgaagtc ggaatcgcta gtaatcgcgg 1260 atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatga 1320 gagtttgtaa cacccaaagt cggtggggta acctttta 1358 ≪ 110 > Daesang FNF Co., Ltd. <120> New lactic acid bacteria having GABA-producing activity and composition of a cultured broth of the strain <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 1358 <212> DNA <213> Lactobacillus plantarum <220> <221> gene ≪ 222 > (1) .. 1358 <223> 16S rRNA <400> 1 gaactggtga gtaacacgtg ggaaacctgc ccagaagcgg gggataacac ctggaaacag 60 atgctaatac cgcataacaa cttggaccgc atggtccgag cttgaaagat ggcttcggct 120 atcacttttg gatggtcccg cggcgtatta gctagatggt ggggtaacgg ctcaccatgg 180 caatgatacg tagccgacct gagagggtaa tcggccacat tgggactgag acacggccca 240 aactcctacg ggaggcagca gtagggaatc ttccacaatg gacgaaagtc tgatggagca 300 acgccgcgtg agtgaagaag ggtttcggct cgtaaaactc tgttgttaaa gaagaacata 360 tctgagagta actgttcagg tattgacggt atttaaccag aaagccacgg ctaactacgt 420 gccagcagcc gcggtaatac gtaggtggca agcgttgtcc ggatttattg ggcgtaaagc 480 gagcgcaggc ggttttttaa gtctgatgtg aaagccttcg gctcaaccga agaagtgcat 540 cggaaactgg gaaacttgag tgcagaagag gacagtggaa ctccatgtgt agcggtgaaa 600 tgcgtagata tatggaagaa caccagtggc gaaggcggct gtctggtctg taactgacgc 660 tgaggctcga aagtatgggt agcaaacagg attagatacc ctggtagtcc ataccgtaaa 720 cgatgaatgc taagtgttgg agggtttccg cccttcagtg ctgcagctaa cgcattaagc 780 attccgcctg gggagtacgg ccgcaaggct gaaactcaaa ggaattgacg ggggcccgca 840 caagcggtgg agcatgtggt ttaattcgaa gctacgcgaa gaaccttacc aggtcttgac 900 atactatgca aatctaagag attagacgtt cccttcgggg acatggatac aggtggtgca 960 tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1020 tattatcagt tgccagcatt aagttgggca ctctggtgag actgccggtg acaaaccgga 1080 ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta 1140 caatggatgg tacaacgagt tgcgaactcg cgagagtaag ctaatctctt aaagccattc 1200 tcagttcgga ttgtaggctg caactcgcct acatgaagtc ggaatcgcta gtaatcgcgg 1260 atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatga 1320 gagtttgtaa cacccaaagt cggtggggta acctttta 1358
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Cited By (4)
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KR101961149B1 (en) * | 2018-11-30 | 2019-03-25 | 주식회사 아미코스메틱 | Lactobacillus paraplantarum ami-1101 and use thereof |
KR20210070712A (en) | 2019-12-05 | 2021-06-15 | 주식회사 휴온스내츄럴 | Novel gaba producing lactobacillus brevis hbt strain and its culture, and composition comprising a culture broth of the strain |
KR102483081B1 (en) | 2022-07-25 | 2023-01-02 | 김미진 | Bacteria and bacteria manufacturing method |
KR20230076923A (en) | 2021-11-23 | 2023-06-01 | 강성우 | New strain Lactobacillus brevis KU-024 and method for production of high concentration GABA by fed-batch cultivation of its strain |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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KR101961149B1 (en) * | 2018-11-30 | 2019-03-25 | 주식회사 아미코스메틱 | Lactobacillus paraplantarum ami-1101 and use thereof |
KR20210070712A (en) | 2019-12-05 | 2021-06-15 | 주식회사 휴온스내츄럴 | Novel gaba producing lactobacillus brevis hbt strain and its culture, and composition comprising a culture broth of the strain |
KR20210092173A (en) | 2019-12-05 | 2021-07-23 | 주식회사 휴온스내츄럴 | Novel gaba producing lactobacillus brevis hbt strain and composition comprising a culture broth of the strain |
KR20230076923A (en) | 2021-11-23 | 2023-06-01 | 강성우 | New strain Lactobacillus brevis KU-024 and method for production of high concentration GABA by fed-batch cultivation of its strain |
KR102483081B1 (en) | 2022-07-25 | 2023-01-02 | 김미진 | Bacteria and bacteria manufacturing method |
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