KR100710497B1 - Method for producing ?-aminobutyric acid GABA using Monascus sp. - Google Patents
Method for producing ?-aminobutyric acid GABA using Monascus sp. Download PDFInfo
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- KR100710497B1 KR100710497B1 KR1020050041739A KR20050041739A KR100710497B1 KR 100710497 B1 KR100710497 B1 KR 100710497B1 KR 1020050041739 A KR1020050041739 A KR 1020050041739A KR 20050041739 A KR20050041739 A KR 20050041739A KR 100710497 B1 KR100710497 B1 KR 100710497B1
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- South Korea
- Prior art keywords
- gaba
- buckwheat
- red yeast
- culture
- germinated
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Abstract
본 발명은 홍국균을 이용한 GABA 생산방법에 관한 것으로, 더욱 구체적으로 홍국균을 껍질을 벗기지 않은 발아된 메밀을 기질로 함유하는 배지에서 배양하는 것을 특징으로 하는 GABA (γ-aminobutyric acid)의 생산방법에 관한 것이다.The present invention relates to a method for producing GABA using hongguk bacteria, and more particularly to a method for producing GABA (γ-aminobutyric acid), characterized in that cultured in a medium containing germinated buckwheat as a substrate not peeled hongguk bacteria will be.
본 발명에서는, 신경안정, 혈압강하 효과를 가지는 γ-aminobutyric acid (GABA) 고생산하는 홍국균을 선발하였으며, 이 선발된 홍국균을 이용하여 다양한 곡류를 기질로 이용한 홍국들을 제조하고 그중에서 GABA 생산성이 탁월한 발아된 통메밀을 기질로 선택하여 새로운 고기능성 홍국을 개발하였으며, 또한 발아된 통메밀 홍국의 GABA 생산성을 더욱 향상시키기 위하여 고체배양 최적화 조건을 개발하였다.In the present invention, γ-aminobutyric acid (GABA) high production of red yeast bacteria having a neurostable, blood pressure-lowering effect was selected, using the selected red yeast bacteria to prepare red yeast rice using a variety of grains as a substrate, among them excellent GABA productivity A new high-functional red yeast rice was developed by selecting the germinated whole buckwheat as a substrate, and a solid culture optimization condition was developed to further improve the GABA productivity of the germinated whole buckwheat red yeast rice.
Description
도 1은 GABA standard (a) 와 extracted sample (b)의 HPLC 분석 결과이다.1 shows the results of HPLC analysis of GABA standard (a) and extracted sample (b).
도 2 및 3은 각 균부별 고체배지에서 GABA의 농도(mg/kg)를 나타내는 그래프이다.2 and 3 are graphs showing the concentration (mg / kg) of GABA in each culture medium.
도 4는 다양한 곡류의 GABA 생산능에 대한 효과를 나타낸 그래프이다.Figure 4 is a graph showing the effect on the GABA production capacity of various cereals.
도 5는 Monascus purpureus ATCC 6405에 의해 발효된 다양한 곡류 홍국을 촬영한 사진들이다.Figure 5 is a photograph of the various grain red yeast fermented by Monascus purpureus ATCC 6405.
도 6은 Monascus purpureus ATCC 6405에 의한 다양한 탄소원의 GABA 생산능에 대한 효과를 나타난 그래프이다.Figure 6 is a graph showing the effect on the GABA production capacity of various carbon sources by Monascus purpureus ATCC 6405.
도 7은 Fructose의 농도에 따른 GABA 생산능을 나타낸 그래프이다.7 is a graph showing the GABA production capacity according to the concentration of fructose.
도 8은 Monascus purpureus ATCC 6405에 의한 다양한 질소원의 GABA 생산능에 대한 효과를 나타난 그래프이다.8 is a graph showing the effect on the GABA production capacity of various nitrogen sources by Monascus purpureus ATCC 6405.
도 9는 MSG의 농도에 따른 GABA 생산능을 나타낸 그래프이다.9 is a graph showing the GABA production capacity according to the concentration of MSG.
도 10은 Monascus purpureus ATCC 6405에 의한 다양한 유기화합물의 GABA 생산능에 대한 효과를 나타난 그래프이다.10 is a graph showing the effect on the GABA production capacity of various organic compounds by Monascus purpureus ATCC 6405.
도 11은 Glycerol의 농도에 따른 GABA 생산능을 나타낸 그래프이다.11 is a graph showing the GABA production capacity according to the concentration of glycerin.
도 12는 Monascus purpureus ATCC 6405에 의한 다양한 미네랄의 GABA 생산능에 대한 효과를 나타난 그래프이다.12 is a graph showing the effect on the GABA production capacity of various minerals by Monascus purpureus ATCC 6405.
도 13은 NaH2PO4의 농도에 따른 GABA 생산능을 나타낸 그래프이다.Figure 13 is a graph showing the GABA production capacity according to the concentration of NaH2PO4.
도 14는 Monascus purpureus ATCC 6405에 의한 다양한 pH 조건의 GABA 생산능에 대한 효과를 나타난 그래프이다.14 is a graph showing the effect on the GABA production capacity of various pH conditions by Monascus purpureus ATCC 6405.
도 15는 Monascus purpureus ATCC 6405에 의한 다양한 접종량의 GABA 생산능에 대한 효과를 나타난 그래프이다.15 is a graph showing the effect on the GABA production capacity of various inoculum by Monascus purpureus ATCC 6405.
도 16은 Monascus purpureus ATCC 6405에 의한 다양한 온도 조건의 GABA 생산능에 대한 효과를 나타난 그래프이다.Figure 16 is a graph showing the effect on GABA production capacity of various temperature conditions by Monascus purpureus ATCC 6405.
도 17은 GABA production의 Main effects plot이다.17 is a main effects plot of GABA production.
도 18은 GABA production의 Interaction plot이다.18 is an Interaction plot of GABA production.
도 19는 GABA production의 Three-dimensional response surface plots이다.19 are three-dimensional response surface plots of GABA production.
도 20은 GABA production의 Contour plot이다.20 is a contour plot of GABA production.
도 21은 Desirability의 Three-dimensional response surface plots이다.21 is a three-dimensional response surface plots of desirability.
도 22는 Estimated response surface의 Contour plot이다.22 is a contour plot of the estimated response surface.
본 발명은 홍국균을 이용한 GABA 생산방법에 관한 것으로, 더욱 구체적으로 홍국균을 껍질을 벗기지 않은 발아된 메밀을 기질로 함유하는 배지에서 배양하는 것을 특징으로 하는 GABA (γ-aminobutyric acid)의 생산방법에 관한 것이다.The present invention relates to a method for producing GABA using hongguk bacteria, and more particularly to a method for producing GABA (γ-aminobutyric acid), characterized in that cultured in a medium containing germinated buckwheat as a substrate not peeled hongguk bacteria will be.
홍국 (紅麴)은 붉은 누룩이라는 뜻으로 쌀을 홍국균(Monascus sp.)으로 고체발효시킨 것으로 중국 한(漢)나라황제 유방이 처음 황실 음식으로 채택한 이래 조선 중기 우리나라에 유입되어 한방에서 어혈 해소제로 사용되고 있다. 특히 혈압을 떨어뜨리는데 효과가 높아 이미 홍국을 활용한 여러 가지 기능성 제품들이 선보이고 있다.Hongguk (붉은) means red yeast, which is a fermentation of rice with Monascus sp., And it was introduced into Korea in the middle of Joseon Dynasty since the Chinese emperor Weifang was first adopted as imperial food. It is used. In particular, it is effective in reducing blood pressure, and several functional products using red yeast are already being introduced.
GABA (γ-aminobutyric acid)는 동, 식물의 자연계에 널리 분포하는 비단백질 구성 아미노산의 일종으로, 동물에서는 뇌나 척수에 존재하는 억제계의 신경전달물질로서 작용하기 때문에, 신경 억제기능, 정신 안정기능, 혈압상승 억제작용, 뇌의 대사촉진작용, 동맥경화의 예방, 숙취해소, 피부노화방지 등의 효과를 나타내고, 식물에서는 해충의 공격이나 종자의 발아시 또는 기계적 자극, 온도, 수분, 빛, 산소 등의 여러 외부 환경적 요인에 의해 그 함량이 급격히 증진되므로, 식물세포의 활성화를 통한 성장과 자기보호기능에 중요한 역할을 하게 된다. 따라서, 식물체에서의 GABA는 외부환경을 적절히 조절하여 줌으로써, 그 함량을 크게 증진시킬 수 있는 물질인 것으로 알려져 있다. 최근에, GABA는 기능성 식품의 성분으로 그 활용성이 증가되고 있다.GABA (γ-aminobutyric acid) is a type of non-protein constituent amino acid widely distributed in the natural world of animals and plants. In animals, it acts as a neurotransmitter of inhibitory system in the brain or spinal cord. It has the effect of inhibiting blood pressure rise, promoting the metabolism of the brain, preventing arteriosclerosis, relieving hangovers, and preventing skin aging.In plants, insects attack or germination of seeds or mechanical stimulation, temperature, moisture, light, oxygen Because of its rapid increase in content due to various external environmental factors such as, it plays an important role in the growth and self-protection function through the activation of plant cells. Therefore, GABA in plants is known to be a substance that can greatly enhance its content by appropriately controlling the external environment. Recently, GABA has been increasing in utility as a component of functional foods.
메밀(Fagopyrum esculentum Moench; 일반적으로는 'buckwheat'이라고 부름)은 마디풀과에 속하는 일년생 초본식물로, 분류학상 곡류와 구별되지만 낱알의 성분조성이 곡류와 비슷하여 보통 잡곡으로 취급된다. 원산지는 시베리아로 현재는 동남아 각지에서 많이 재배하고 있으며, 우리나라에서는 삼국시대 이전부터 재배되 어 왔다고 하는데 구체적인 문헌에 메밀이 처음으로 나타난 것은 1417년에 간행된 우리나라 최고의 의서인 향약구급방(鄕藥救急方)이다. 메밀은 과거에 식량이 부족할 때 구황(救荒)작물로 이용되어 왔으나, 최근들어 뛰어난 영양성분과 약리활성 그리고 독특한 맛으로 건강기능성 별미식으로 인기가 높다.Buckwheat (Fagopyrum esculentum Moench, commonly called 'buckwheat') is an annual herb that belongs to the genus Madipulaceae and is distinguished from cereal grains, but the composition of the grain is similar to grains and is usually treated as cereals. It is native to Siberia and is now cultivated in many parts of Southeast Asia, and it has been cultivated in Korea before the Three Kingdoms era. )to be. Buckwheat has been used as a yellow crop when food was scarce in the past, but recently it is popular as a health functional specialty food because of its excellent nutrition, pharmacological activity and unique taste.
메밀에는 단백질 13%, 지방 2.5%, 전분 67% 정도 함유되어 있으며, 식이섬유 함유량이 높다. 특히, 메밀에 함유된 단백질은 균형있는 아미노산 조성을 가지고 있으며 필수 아미노산인 트립토판, 트레오닌, 라이신 등의 비율도 높을 뿐 아니라, 토코페롤, 페놀산, 플라보노이드(flavonoid)와 같은 항산화 물질을 다량 함유하고 있다. 플라보노이드는 담황색 또는 노란색의 색소화합물로서, 기본구조인 플라본(flavone)과 플라보놀(flavonol), 플라바논(flavanone), 플라바놀(flavanol) 및 이소플라본(isoflavone) 등의 유도체들이 있다. 특히, 메밀에는 플라보놀의 하나이며 비타민 P로 알려진, 루틴(rutin)이 많이 함유되어 있다 (참조: 성환길, 식품은 약이다, 한국메디칼인덱스, 1988; 및 Marshall, H.G. 및 Pomeranz, Y., Buckwheat: Description, Breeding, Production and Utilization, Advances in Cereal Science and Technology, Am. Ass. of Cereal Chem. Volume V, p.157, 1982).Buckwheat contains 13% protein, 2.5% fat, and 67% starch, and is high in fiber. In particular, the protein contained in buckwheat has a balanced amino acid composition and high ratios of essential amino acids tryptophan, threonine, and lysine, as well as high amounts of antioxidants such as tocopherol, phenolic acid, and flavonoids. Flavonoids are pale yellow or yellow pigment compounds, and include basic structures such as flavone, flavonol, flavanone, flavanone, flavanol, and isoflavone. In particular, buckwheat is one of flavonols and contains a lot of rutin, also known as vitamin P. (Sung Hil-gil, food is a drug, Korean Medical Index, 1988; and Marshall, HG and Pomeranz, Y., Buckwheat: Description, Breeding, Production and Utilization, Advances in Cereal Science and Technology, Am.Ass. Of Cereal Chem.Volume V, p.157, 1982).
홍국균의 발효에 관한 종래기술로는, 홍국균으로 발효시킨 메밀에 관한 특허출원(출원번호: 10-2002-0079448), 모나스쿠스속 균류에 의해 발효된 곡물을 포함하는 다류의 제조방법에 관한 특허출원(출원번호: 10-2000-0024564) 및 발아현미 또는 발아현미 함유곡류를 배지로 한 홍국의 제조방법 및 이를 이용한 식품의 제조방법에 관한 특허출원(출원번호:10-2001-0056427)이 있다. 그러나, GABA 고함유 홍국을 개발하기 위하여 다양한 곡류들을 적용한 특허출원이나 논문들은 아직 보고되지 않았으며, 홍국을 통한 GABA 고생산 배양법은 아직 국내외에 보고되지 않았다.As a prior art related to the fermentation of red yeast bacteria, a patent application for buckwheat fermented with red yeast bacteria (application number: 10-2002-0079448), a patent application for a method for producing a variety of teas including grains fermented by the genus Monascus (Application No .: 10-2000-0024564) and a patent application (Application No.:10-2001-0056427) relating to a manufacturing method of red yeast rice using germinated brown rice or germinated brown rice containing cereals as a medium, and a food manufacturing method using the same. However, no patent applications or papers applying various grains to develop GABA-rich red yeast soup have been reported, and GABA high production culture method through red yeast rice has not been reported at home and abroad.
본 발명자들은 세계 최초로 껍질을 벗기지 않은 발아된 통메밀을 기질로 이용한 홍국을 이용한 GABA의 고생산 배양기술을 개발하였으며, 신경안정, 혈압강하효능을 가지는 GABA를 고함유 홍국의 개발을 위하여 수집된 36 종의 홍국균으로부터 GABA 고생산성을 가지는 균주를 새롭게 선발하였으며, 이 선발된 홍국균를 다양한 기질(곡류)에 배양시킨 후 HPLC를 이용하여 GABA 생산성을 정량적으로 분석한 결과 발아된 통메밀을 GABA 고생산을 위한 최적의 기질로 선발하였으며, 새로운 발아된 통메밀을 기질로 이용한 홍국의 GABA 고생산을 위한 고체배양 기술을 개발하였다.The inventors of the present invention developed the world's first high-production culture technology of GABA using red ginseng, which is a non-peeled whole wheat buckwheat as a substrate, and collected for the development of red ginseng containing GABA having neurostable and blood pressure lowering efficacy. A new strain of high yielding GABA was selected from hongguk bacteria, and after culturing the selected hongguk bacteria on various substrates (grains), quantitative analysis of GABA productivity using HPLC showed that germinated whole buckwheat was grown for high production of GABA. The optimum substrate was selected, and a new solid culture technology was developed for the high production of GABA in red yeast rice using the new germinated whole buckwheat as a substrate.
따라서, 본 발명의 주된 목적은 홍국균의 고체배양기술을 이용한 고레벨의 GABA 생산방법을 제공하는 데 있다.Therefore, the main object of the present invention is to provide a high level GABA production method using the solid culture technology of hongguk bacteria.
본 발명의 다른 목적은 상기 방법으로 생산된 GABA를 함유하는 발아 메밀 홍국을 제공하는데 있다.Another object of the present invention to provide a germinated buckwheat red yeast rice containing GABA produced by the above method.
본 발명의 목적을 달성하기 위하여, 본 발명은 홍국균을 껍질을 벗기지 않은 발아된 메밀을 기질로 함유하는 배지에서 배양하는 것을 특징으로 하는 GABA (γ-aminobutyric acid)의 생산방법을 제공한다.In order to achieve the object of the present invention, the present invention provides a method for producing GABA (γ-aminobutyric acid), characterized in that the culturing in a medium containing germinated buckwheat as a substrate not peeled.
본 발명에 있어서, 바람직하게는 상기 홍국균은 모나스쿠스 퍼퍼루스(Monascus purpureus) (ATCC 6405), 모나스쿠스 퍼퍼루스(Monascus purpureus) (IFO 32228) 또는 모나스쿠스 퍼퍼루스(Monascus purpureus) (ATCC 16358)인 것을 특징으로 한다.In the present invention, preferably, the erythrocyte is Monascus purpureus (ATCC 6405), Monascus purpureus (IFO 32228) or Monascus purpureus (ATCC 16358). It is characterized by.
본 발명에 있어서, 바람직하게는 상기 배지는 탄소원으로서 5% 내지 15% 농도의 푸럭토스(Fructose) 또는 글루코스(Glucose)를 함유하는 것을 특징으로 한다.In the present invention, the medium is preferably characterized as containing 5% to 15% of fructose (Fructose) or glucose (Glucose) as a carbon source.
본 발명에 있어서, 바람직하게는 상기 배지는 질소원으로서 1% 내지 4% 농도의 모노소디움 글루타메이트(MSG), 알라닌(Alanine) 또는 (Glutamate)를 함유하는 것을 특징으로 한다.In the present invention, preferably, the medium contains monosodium glutamate (MSG), alanine or (Glutamate) at a concentration of 1% to 4% as a nitrogen source.
본 발명에 있어서, 바람직하게는 상기 배지는 유기화합물로서 0.5% 내지 1.5% 농도의 글리세롤(Glyerol) 또는 에탄올(Ethanol)를 함유하는 것을 특징으로 한다.In the present invention, preferably, the medium is characterized in that it contains glycerol (Glyerol) or ethanol (Ethanol) at a concentration of 0.5% to 1.5% as an organic compound.
본 발명에 있어서, 바람직하게는 상기 배지는 미네랄로서 0.1% 내지 0.5% 농도의 인산나트륨(NaH2PO4) 또는 질산나트륨(NaNO3)을 함유하는 것을 특징으로 한다.In the present invention, preferably, the medium contains sodium phosphate (NaH 2 PO 4) or sodium nitrate (NaNO 3) at a concentration of 0.1% to 0.5% as a mineral.
본 발명에 있어서, 바람직하게는 상기 배양 pH는 pH 3 내지 6인 것을 특징으로 하며, 상기 배양 온도는 26 내지 30℃인 것을 특징으로 한다.In the present invention, preferably the culture pH is characterized in that
본 발명에 있어서, 바람직하게는 상기 배지의 탄소원은 5-10% 푸럭토스(Fructose), 질소원은 1-3% 모노소디움 글루타메이트(MSG), 유기화합물은 0.5-1% 글리세롤(glycerol), 미네랄은 0.1-0.3% 소디움 포스페이트(NaH2PO4), 배양 pH는 3-4, 배양 온도는 26-28℃인 것을 특징으로 한다.In the present invention, preferably, the carbon source of the medium is 5-10% Fructose, the nitrogen source is 1-3% monosodium glutamate (MSG), the organic compound is 0.5-1% glycerol, minerals are 0.1-0.3% sodium phosphate (NaH 2 PO 4), the culture pH is characterized in that 3-4, the culture temperature is 26-28 ℃.
본 발명의 다른 목적을 달성하기 위해, 본 발명은 상기 본 발명의 방법으로 생산된 GABA (γ-aminobutyric acid)를 함유하는 발아 메밀 홍국을 제공한다.In order to achieve another object of the present invention, the present invention provides a germinated buckwheat red yeast rice containing GABA (γ-aminobutyric acid) produced by the method of the present invention.
본 발명의 발아 메일 홍국은 혈압강하성분물질인 GABA를 고함유할뿐 아니라, 메밀의 함유된 유용대사산물을 부가적으로 함유하고 있다. 특히, 껍질을 벗기지 않은 발아된 통메밀에는 껍질에 다량 존재하는 항산화, 혈압강하 성분으로 잘 알려진 rutin과 quercetin이 대부분 껍질에 존재하며 게다가 발아시킬 경우는 더욱 향상 시킬 수 있다. 본 발명에서는 홍국의 GABA와 일반메밀보다 껍질을 벗기지 않은 발아메밀 속에 존재하는 다량으로 존재하는 rutin과 quercetin 같은 유용생리활성 물질들이 함께 함유된다. The germinated mail red yeast of the present invention not only contains GABA, a blood pressure lowering substance, but also additionally contains useful metabolites of buckwheat. In particular, unshelled germinated whole buckwheat contains rutin and quercetin, which are well known as antioxidants and blood pressure lowering ingredients, which are present in the skin in large quantities, and can be further improved when germinated. In the present invention, useful bioactive substances such as rutin and quercetin, which are present in large amounts present in germinated buckwheat without peeling than GABA and buckwheat of red yeast rice, are contained together.
본 발명은 Monascus 균주의 이차대사산물로 잘 알려진 GABA의 생산성 최적화를 위하여 GABA 고생산 균주를 선발하였으며 GABA의 생산성에 적합한 기질을 새롭게 선별하고 고체배양에 필요한 영양적인 조건과 외부의 환경적인 조건의 최적화를 통하여 새로운 고기능성 홍국의 고체배양 최적화 기술을 개발하고자 수행하였다. 이를 위하여 수집된 36 종의 Monascus 균주들을 고체배양 한 후 TLC와 HPLC를 이용하여 GABA의 함량을 분석하였으며 분석결과 GABA 생산성(1104 mg/kg)이 가장 우수한 Monascus purpureus ATCC 6405 균주를 선발하게 되었다. 그리고 이 균주를 10 여종의 다양한 곡류에 배양하여 껍질을 벗기지 않은 발아된 메밀이 GABA를 증대시키는 최적의 기질임을 확인하게 되었다. GABA 생산성 향상을 위한 영양학적, 환경학적 최적화 요소를 선별하기 위하여 고전적인 A factor at a time method를 이용 하였다. 분석한 결과 GABA에 영향을 미치는 중요한 요인으로 MSG, pH, 온도가 선발되었으며 이들 3 요소를 이용하여 GABA 생산성을 증진시킬 수 있는 고체배양최적조건을 결정하기 위한 방법으로 반응표면분석법(response surface methodology, RSM)을 이용하였으며 실험계획법으로 중심합성계획법을 사용하였다. 그 결과 MSG 1%, 26℃, pH 3.28일 때 GABA의 최적조건임을 알 수 있었으며, 이 조건에서 GABA는 3820 mg/kg으로 최적화 되지 않은 조건과 비교하여 GABA 생산성은 4 배 정도 증진 시킬 수 있었다.In order to optimize the productivity of GABA, which is well known as a secondary metabolite of Monascus strains, the present invention was selected for the high production of GABA strains and newly selected substrates suitable for the productivity of GABA and optimization of nutritional conditions and external environmental conditions for solid culture. The purpose of this study was to develop a solid culture optimization technology for new high functional red yeast rice. To this end, the 36 Monascus strains collected were solid cultured and analyzed for GABA content using TLC and HPLC. As a result, GAAS productivity (1104 mg / kg) was selected as the best Monascus purpureus ATCC 6405 strain. The strains were then cultured in more than 10 different grains to confirm that unshelled germinated buckwheat is the optimal substrate for increasing GABA. The classical A factor at a time method was used to select the nutritional and environmental optimization factors for improving GABA productivity. As a result of analysis, MSG, pH, and temperature were selected as important factors affecting GABA, and response surface methodology was used as a method to determine the optimal conditions for solid culture that can improve GABA productivity using these three factors. RSM) was used and the central synthesis planning method was used as the experimental design method. As a result, it was found that the optimum condition of GABA at 1% MSG, 26 ℃, and pH 3.28. In this condition, GABA productivity could be increased by 4 times compared to the condition that is not optimized to 3820 mg / kg.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.
(실시예 1) GABA 고생산능을 가진 균주의 선별Example 1 Selection of Strains with High GABA Production Capacity
GABA 고생산능을 가진 홍국 균주의 선별을 위하여 표 1과 같이 수집된 36 종 Monascus 균주를 시험에 사용하였다. ATCC는 미국의 American Type Culture Collection (URL: www.atcc.org)에서 구입하였으며, IFO는 일본의 Institute for Fermentation, Osaka (URL: www.ifo.or.jp)에서 구입하였다.For the screening of red yeast strains with high GABA production capacity, 36 Monascus strains collected as shown in Table 1 were used for the test. ATCC was purchased from the American Type Culture Collection (URL: www.atcc.org) in the United States, and IFO was purchased from the Institute for Fermentation, Osaka (URL: www.ifo.or.jp) in Japan.
[표 1]TABLE 1
1) 종배양 (seed culture)Seed culture
PDA(Potato dextrose agar) 사면배지에 Monascus 균주를 접종하여 28℃, 7일 동안 배양된 plate를 0.05% Tween-80 멸균수용액으로 세척하고, 얻어진 포자를 희석액에 현탁하여 stock (1.5x107spores/ml)을 얻었다.Monascus strain was inoculated onto PDA (Potato dextrose agar) slope medium, and the plates incubated for 7 days at 28 ° C. were washed with 0.05% Tween-80 sterile aqueous solution, and the resulting spores were suspended in diluent to obtain stock (1.5 × 107 spores / ml). Got it.
포자현탁액 2.5 ml을 50 ml Mizutani 배지(Glucose 5.0%, Bacto peptone 2.0%, KH2PO4 0.8%, CH3COOK 0.2%, NaCl 0.1%, MgSO4·7H2O 0.05%)(pH 5.0)에, 30℃, 150 rpm, 2 일 동안 배양하였다. 2.5 ml of spore suspension was added to 50 ml Mizutani medium (Glucose 5.0%, Bacto peptone 2.0%, KH2PO4 0.8%, CH3COOK 0.2%, NaCl 0.1%, MgSO4.7H2O 0.05%) (pH 5.0), 30 ° C, 150 rpm, 2 Incubated for days.
2) 고체배양방법2) Solid culture method
쌀 20g 과 증류수 14ml을 121℃에서 30 min동안 증기로 찐 후 50℃ 건조기에 2 시간~3 시간 정도 넣은 후 수분을 적당히 제거하여 Clean bench에서 2 ml의 종배양액을 접종한 후 10 일 동안 배양하였다. 고체배양 시 3일 동안은 30℃에서 4~10일째까지는 28℃의 낮은 온도에서 배양하였다.20 g of rice and 14 ml of distilled water were steamed at 121 ° C. for 30 min, and then placed in a 50 ° C. dryer for 2 hours to 3 hours, and then water was adequately inoculated, inoculated with 2 ml of the seed culture solution on a clean bench, and incubated for 10 days. . The solid culture was incubated at a low temperature of 28 ℃ for 3 days at 30
3) GABA의 분석방법 3) GABA analysis method
(1) 홍국샘풀에서 추출 (1) Extracted from Hongguk Sam
1 g 홍국 샘플을 분말화한후 70 % Ethanol 9 ml을 혼합하여 150 rpm (30℃)으로 12 시간 동안 추출하였다. After powdering 1 g red yeast sample, 9 ml of 70% Ethanol was mixed and extracted at 150 rpm (30 ° C.) for 12 hours.
(2) 홍국추출물의 유도체화(2) Derivatization of Red Rice Extract
20 ㎕의 홍국추출물에 AccQ.Fluor Borate buffer 100 ㎕를 첨가한 후 AccQ.Fluor reagent (1 mg/ml) 20 ㎕를 넣어 잘 혼합하여 항온조에서 55℃, 10 min동안 유도체화 시켰다.100 μl of AccQ.Fluor Borate buffer was added to 20 μl of red yeast extract, and 20 μl of AccQ.Fluor reagent (1 mg / ml) was mixed well and derivatized in a thermostat at 55 ° C. for 10 min.
(3) HPLC를이용한 GABA의 분석 조건(3) Analysis conditions of GABA using HPLC
HPLC 분석은 Varian Prostar 210 (USA) 기기를 이용하며, 분석용 컬럼으로는 Xterra MS C18 5 ㎛ column (reverse phase, 4.5×150 ㎜, Waters)을 사용한다. 용매조건으로 아래에 설명된 gradient mobile phase를 사용하였으며, 사용 용매는 Acetonitrile - 0.025 M sodium Acetate (pH 5.03), 유속은 1 ml/min 샘플 injection volume은 20 ㎕의 조건에서 실시한다. 검출은 248 nm 에서 photodiode array detector (Varian Protstar 320)를 사용하여 분석하였다. - Gradient conditions: 초기 100% A: 17 min, 93% A: 21 min, 90% A: 32 min, 66% A (all liner). 분석시간은 (injection-to-injection) 45 분이다.HPLC analysis was performed using a Varian Prostar 210 (USA) instrument, and an
표준 GABA 용액은 GABA를 1,000 mg/l distilled water 농도로 준비하여 working solutions을 위해 100 또는 1,000 mg/l 로 희석하였다. 200, 400, 600, 800, 1000 ppm 농도로 GABA 표준 곡선을 직선으로 얻었다. HPLC는 체류시간(retention times) 28.858 min에서 GABA를 효과적으로 분리하였으며, 홍국추출물에서도 같은 체류시간에 GABA를 발견할 수 있었다. 도 1은 GABA standard (a) 와 extracted sample (b)의 HPLC 분석 결과이다.Standard GABA solutions were prepared with 1,000 mg / l distilled water concentration and diluted to 100 or 1,000 mg / l for working solutions. GABA standard curves were obtained linearly at concentrations of 200, 400, 600, 800 and 1000 ppm. HPLC effectively separated GABA at retention times 28.858 min and found GABA at the same retention time in red yeast extract. 1 shows the results of HPLC analysis of GABA standard (a) and extracted sample (b).
상기 표 1의 36종의 균주을 GABA 생산능에 대해 스크리닝하였다. 증기로 찐 쌀에서서의 7일째 배양물에 대해 GABA 생산능을 비교 평가하였다. 그 결과, 시험한 36 종의 균주들 가운데에서 Monascus purpureus ATCC 6405, Monascus purpureus IFO 32228 및 Monascus purpureus ATCC 16358의 3종이 820 to 1104 mg/kg의 GABA를 생산하여 우수한 것으로 나타났으며, 그중에서 Monascus purpureus ATCC 6405가 1104 ppm으로 GABA 생산능이 가장 우수한 균주로 선정되었다. 도 2 및 3은 각 균부별 고체배지에서 GABA의 농도(mg/kg)를 나타내는 그래프이다. 36 strains of Table 1 were screened for GABA production capacity. The GABA production capacity was evaluated for culture on
(실시예 2) GABA 고생산을 위한 최적의 기질 선택 Example 2 Optimal Substrate Selection for High GABA Production
실시예 1에서 GABA 생산능이 우수한 균주로 선정된 Monascus purpureus ATCC 6405의 GABA 생산성을 더욱 향상시키기 위하여 본 실시예에서는 기존의 홍국의 기질로써 사용되었던 곡류(쌀, 보리)들과 최적의 신규한 기질 선택을 위하여 다양한 곡류(기장, 차조, 율무, 수수, 메밀, 발아메밀, 발아현미, 현미)들을 대상으로 검토하였다. 종배양은 실시예 1의 실험방법과 동일하고, 고체배양방법은 쌀외에 보리, 수수, 차조, 기장, 현미, 발아현미, 율무, 메밀 및 발아메밀 각각을 대상으로 한 것을 제외하고 실시예 1의 실험방법이 동일하였으며, GABA 분석방법은 실시예 1과 동일하였다.In order to further improve the GABA productivity of Monascus purpureus ATCC 6405, which was selected as a strain having excellent GABA production in Example 1, in this example, grains (rice and barley) that were used as substrates of conventional red yeast rice and optimal new substrate selection were selected. For this purpose, various cereals (millet, green tea, barley, sorghum, buckwheat, germinated buckwheat, germinated brown rice, brown rice) were examined. Seed culture was the same as the experimental method of Example 1, the solid culture method of Example 1 except that each of the barley, sorghum, tea, millet, brown rice, germinated brown rice, yulmu, buckwheat and germinated buckwheat in addition to rice Experimental method was the same, GABA analysis method was the same as in Example 1.
분석 결과, 시험한10 종의 다양한 곡류를 이용한 홍국들 가운데에서 발아된 메밀 홍국의 GABA 함량이 1495 ppm을 생산하여 GABA 생산능이 가장 우수한 균주로 선정되었다. 도 4는 다양한 곡류의 GABA 생산능에 대한 효과를 나타낸 그래프이다. 도 5는 Monascus purpureus ATCC 6405에 의해 발효된 다양한 곡류 홍국을 촬영한 사진들이다.As a result, GABA content of germinated buckwheat red yeast rice produced 1495 ppm among the 10 different cereals tested, and was selected as the best GABA producing strain. Figure 4 is a graph showing the effect on the GABA production capacity of various cereals. Figure 5 is a photograph of the various grain red yeast fermented by Monascus purpureus ATCC 6405.
GABA 생산능이 우수한 Monascus purpureus ATCC 6405와 발아메밀을 기질로써 이용하여 생산력은 향상시켰지만 국외적으로(대만) 보고된 최적화한 홍국의 GABA 함유량(2000ppm~6000ppm)에는 미치지 못하였기 때문에 새롭게 발아된 메밀을 이용한 홍국의 영양학적, 환경학적 관점에서 최적화된 GABA 고 생산성 홍국의 고체배양기술의 개발을 시도하였다.Monascus purpureus ATCC 6405 and germinated buckwheat, which have excellent GABA production capacity, were used as substrates to improve productivity, but did not meet the GABA content (2000ppm ~ 6000ppm) of optimized red yeast rice reported overseas (Taiwan). Attempts were made to develop GABA high productivity red rice culture technology optimized from the nutritional and environmental point of view.
(실시예 3) 일회일요인법(A factor at a time method)에 의한 GABA 고생산 고체배양기술개발Example 3 GABA High Production Solid Culture Technology Development by A Factor at a Time Method
1) 영양요인 최적화1) Optimizing Nutrition Factors
고전적인 최적화 방법인 “A factor at a time method”를 이용하여 고체배양 시 영양학적, 환경학적으로 중요한 최적화 요소들과 그들의 최적농도를 결정하여 최적화하였다. 고체 배양 시 영양물질 중에서 GABA 생산성 향상의 최적요인을 분석하기 위하여 8 종 탄소원(glucose, fructose, xylose, galactose, mannitol, lactose, sucrose, maltose), 13 종 질소원(monosodium glutamic acid, yeast extract, beef extract, soytone, peptone, valine, orinitine, methionine, lysine, leucine, glycine, glutamate, alanine, control), 9 종 미네랄(NaNO3, FeSO4, ZnSO4, Na2HPO4, NaHPO4, KH2PO4, K2HPO4, MgSO4), 6 종 유기화합물(Olive oil, soybean oil, oleic acid, ethanol, polyethylene glycol 2000, glycerol)을 배양 시 각각 따로 첨가하여 배양하였다.Classical optimization method “A factor at a time method” was used to determine and optimize nutritionally and environmentally important optimization factors and their optimal concentration in solid culture. Eight kinds of carbon sources (glucose, fructose, xylose, galactose, mannitol, lactose, sucrose, maltose), 13 kinds of nitrogen sources (monosodium glutamic acid, yeast extract, beef extract) , soytone, peptone, valine, orinitine, methionine, lysine, leucine, glycine, glutamate, alanine, control Olive oil, soybean oil, oleic acid, ethanol,
고체배양방법을 위해, 250ml 삼각플라스크에 발아메밀 20g 과 20ml 증류수 (다양한 탄소원, 질소원, 미네랄, 유기원 첨가)을 121℃에서 30 min동안 증기로 찐 후 50℃ 건조기에 2 시간~3 시간 정도 넣은 후 수분을 적당히 제거하여 clean bench에서 2 ml의 종배양액을 접종한 후 10 일 동안 배양하였다. 고체배양 시 3일 동안은 30℃에서 4~10일째까지는 28℃의 낮은 온도에서 배양하였다.For the solid culture method, 20 g of germinated buckwheat and 20 ml of distilled water (added various carbon sources, nitrogen sources, minerals, and organic sources) were steamed at 121 ° C. for 30 min in a 250 ml Erlenmeyer flask, and then placed in a 50 ° C. dryer for 2 to 3 hours. After the water was appropriately removed and inoculated with 2 ml of the culture medium in a clean bench and incubated for 10 days. The solid culture was incubated at a low temperature of 28 ℃ for 3 days at 30
분석결과, 탄소원으로서는 푸럭토스(Fructose) 또는 글루코스(Glucose)가 좋은 탄소원으로 나타났으며, 이중에서 Fructose이 GABA 생합성에 가장 좋은 탄소원으로 선정되었으며, 최적 첨가 농도는 10%였다. 도 6은 Monascus purpureus ATCC 6405에 의한 다양한 탄소원의 GABA 생산능에 대한 효과를 나타난 그래프이다. 도 7은 Fructose의 농도에 따른 GABA 생산능을 나타낸 그래프이다.As a result, Fructose or Glucose were found to be the best carbon sources. Among them, Fructose was selected as the best carbon source for GABA biosynthesis, and the optimal concentration was 10%. Figure 6 is a graph showing the effect on the GABA production capacity of various carbon sources by Monascus purpureus ATCC 6405. 7 is a graph showing the GABA production capacity according to the concentration of fructose.
질소원으로서는 모노소디움 글루타메이트(MSG), 알라닌(Alanine) 또는 (Glutamate)가 좋은 것으로 나타났으며, 이중에서 MSG는 GABA 생합성에 가장 좋은 질소원으로 선정되었으며, 최적 첨가 농도는 3%였다. 도 8은 Monascus purpureus ATCC 6405에 의한 다양한 질소원의 GABA 생산능에 대한 효과를 나타난 그래프이다. 도 9는 MSG의 농도에 따른 GABA 생산능을 나타낸 그래프이다.As a nitrogen source, monosodium glutamate (MSG), alanine (Glantamate) or (Glutamate) was found to be good, among which MSG was selected as the best nitrogen source for GABA biosynthesis, and the optimum concentration was 3%. 8 is a graph showing the effect on the GABA production capacity of various nitrogen sources by Monascus purpureus ATCC 6405. 9 is a graph showing the GABA production capacity according to the concentration of MSG.
유기화합물로서는 글리세롤(Glyerol) 또는 에탄올(Ethanol)이 좋은 것으로 나타났으며, 이중에서 Glycerol은 GABA 생산성 향상에 가장 좋은 유기화합물 선정되었으며, 최적 첨가 농도는 1%였다. 도 10은 Monascus purpureus ATCC 6405에 의한 다양한 유기화합물의 GABA 생산능에 대한 효과를 나타난 그래프이다. 도 11은 Glycerol의 농도에 따른 GABA 생산능을 나타낸 그래프이다.Glycerol or ethanol were found to be good as organic compounds. Among them, Glycerol was selected as the best organic compound to improve GABA productivity, and the optimum concentration was 1%. 10 is a graph showing the effect on the GABA production capacity of various organic compounds by Monascus purpureus ATCC 6405. 11 is a graph showing the GABA production capacity according to the concentration of glycerin.
미네랄로서는 인산나트륨(NaH2PO4) 또는 질산나트륨(NaNO3)이 좋은 것으로 나타났으며, 이중에서 NaH2PO4은 GABA 생산성 향상에 가장 좋은 유기화합물 선정되었으며, 최적 첨가 농도는 0.25%였다. 도 12는 Monascus purpureus ATCC 6405에 의한 다양한 미네랄의 GABA 생산능에 대한 효과를 나타난 그래프이다. 도 13은 NaH2PO4의 농도에 따른 GABA 생산능을 나타낸 그래프이다.Sodium phosphate (NaH 2 PO 4) or sodium nitrate (NaNO 3) was good as minerals. Among them, NaH 2
2) 환경요인 최적화2) Environmental Factor Optimization
고체 배양 시 환경요인 중에서 GABA 생산성 향상의 최적요인을 분석하기 위하여 각기 다른 온도 (26℃, 28℃, 30℃, 32℃, 34℃, 36℃), pH (2,3,4,5,6,7), 접종량(5%, 10%, 15%, 20%, 25%)을 적용하여 배양 후 분석하였다.Different temperature (26 ℃, 28 ℃, 30 ℃, 32 ℃, 34 ℃, 36 ℃), pH (2,3,4,5,6) to analyze the optimal factors for improving GABA productivity among environmental factors in solid culture , 7), inoculation amount (5%, 10%, 15%, 20%, 25%) was applied after incubation.
고체배양방법을 위해, 250ml 삼각플라스크에 발아메밀 20g 과 20ml 증류수 (다양한 pH 조절)을 121℃에서 30 min동안 증기로 찐 후 50℃ 건조기에 2 시간~3 시간 정도 넣은 후 수분을 적당히 제거하여 clean bench에서 2 ml의 종배양액을 접종한 후 10 일 동안 배양한다. 고체배양 시 3일 동안은 30℃에서 4~10일째까지는 다양한 온도에서 배양하였다.For the solid culture method, 20 g of germinated buckwheat and 20 ml of distilled water (various pH control) were steamed at 121 ° C. for 30 min in a 250 ml Erlenmeyer flask, and then placed in a 50 ° C. dryer for 2 hours to 3 hours, and then the water was properly removed.
분석 결과, 배양 pH는 3 내지 6이고, 배양 온도는 26 내지 30℃인 것이 좋은 것으로 나타났다. 이중에서도, 고체배양 시 pH 5.0 일때 GABA 생산성이 가장 높았으며, 고체배양 시 온도는 28℃ 일때 GABA 생산성이 가장 높았으며, 고체배양 시 접종량 10% 일때 GABA 생산성이 가장 높았다. 도 14는 Monascus purpureus ATCC 6405에 의한 다양한 pH 조건의 GABA 생산능에 대한 효과를 나타난 그래프이다. 도 15는 Monascus purpureus ATCC 6405에 의한 다양한 접종량의 GABA 생산능에 대한 효과를 나타난 그래프이다. 도 16은 Monascus purpureus ATCC 6405에 의한 다양한 온도 조건의 GABA 생산능에 대한 효과를 나타난 그래프이다.As a result, it was found that the culture pH was 3 to 6 and the culture temperature was 26 to 30 ° C. Among them, GABA productivity was the highest at pH 5.0 in solid culture, GABA productivity was highest at 28 ° C in solid culture, and GABA productivity was highest at 10% inoculum in solid culture. 14 is a graph showing the effect on the GABA production capacity of various pH conditions by Monascus purpureus ATCC 6405. 15 is a graph showing the effect on the GABA production capacity of various inoculum by Monascus purpureus ATCC 6405. Figure 16 is a graph showing the effect on GABA production capacity of various temperature conditions by Monascus purpureus ATCC 6405.
(실시예 4) 반응표면분석법(Response surface methodology)에 의한 GABA 고생산 고체배양기술개발Example 4 GABA High Production Solid Culture Technology Development by Response Surface Methodology
"A factor at a time method"는 고전적인 최적화 배양방법의 하나로써 하나의 조건에서 하나의 요인이 미치는 영향에 대해서 분석하기 위한 좋은 방법이지만 최적화된 각각의 요인들이 혼합되면 요인들 상호간의 영향에 의해서 최적화된 요인들의 최적 농도에서 배양결과가 반드시 최대의 생산량을 나타내지 못하기 때문에 각 요인들과 농도 상호간에 있을 수 있는 모든 경우의 수에 대한 결과를 통계학적 방법으로 최근 다양하게 응용 되어지고 있는 반응표면분석법을 이 실험에 도입하게 되었다."A factor at a time method" is a classical method of optimizing culture, which is a good way to analyze the effect of a factor under one condition, but when each optimized factor is mixed, Since the results of cultures do not necessarily show the maximum yield at the optimal concentration of the optimized factors, the response surface, which has been recently applied in various ways by the statistical method, for the number of cases in which each factor and concentration can be mutually different. The method was introduced in this experiment.
1) Basal medium 1) Basal medium
A factor at a time method를 통해서 인정된 중요한 요인(MSG, pH)들을 기본배양액으로 첨가하여 주었다. 이 기본 배양액은 고체배양할 때 증류수를 대신하여 첨가하였다. Important factors (MSG, pH) recognized through A factor at a time method were added to the basic culture solution. This base culture was added in place of distilled water when solid culture.
- 탄소원: 5% fructose (1g fructose /발아메밀 20g)-Carbon Source: 5% fructose (1g fructose / 20g buckwheat)
- 미네랄: 0.1% NaH2PO4 (0.02g NaH2PO4 /발아메밀 20g)Minerals: 0.1% NaH 2 PO 4 (0.02
- 유기화합물: 0.5% glycerol (0.1ml glycerol /발아메밀 20g)-Organic compound: 0.5% glycerol (0.1ml glycerol / 20 g of germinated buckwheat)
- 질소원: 1~5%MSG (0.2~ 2g MSG /발아메밀 20g)-Nitrogen source: 1 ~ 5% MSG (0.2 ~ 2g MSG / Germinated buckwheat 20g)
- 초기 pH를 2~6으로 조절하였다(Acetic acid, NaOH 사용).Initial pH was adjusted to 2-6 (Acetic acid, NaOH).
2) 고체배양방법 2) Solid culture method
250 ml 삼각플라스크에 발아메밀 20 g 과 20 ml basal medium을 넣은 후 autoclave를 이용하여 121℃에서 30 min동안 증기로 찐 후 50℃ 건조기에 2 시간~3 시간 정도 넣은 후 수분을 적당히 제거하여 clean bench에서 2 ml의 종배양액을 접종한 후 10 일 동안 배양하였다. 고체배양 시 3일 동안은 30℃에서 4~10일째까지는 RSM에 적용된 온도 맞추어서 각각 배양하였다.After putting 20 g of germinated buckwheat and 20 ml basal medium into a 250 ml Erlenmeyer flask, steam with an autoclave for 30 min at 121 ° C, put it in a 50 ° C dryer for 2 to 3 hours, and then remove the water appropriately to clean the bench. 2 ml of seed culture solution was inoculated and then cultured for 10 days. Solid cultures were incubated for 3 days at 30 ° C. and 4-10 days at the temperature applied to the RSM.
3) 분석 결과3) Analysis result
본 실험에서 처리 조합을 할당하기 위해 Central Composite Design로서 3-factor 5-level response surface experiment를 사용하였다. 본 실험에서, Monascus purpureus ATCC 6405의 응답인 GABA의 양(mg/kg)은 하기 표 2에 기술된 3가지 요인(MSG, pH, Temperature)의 영향하에 있다고 가정하였다. 데이터는 STATGRAPHICS Plus Version 5.1 (USA)를 사용하여 분석하였다. Fitted model의 등식은 다음과 같다.To assign treatment combinations in this experiment, we used a 3-factor 5-level response surface experiment as the Central Composite Design. In this experiment, it was assumed that the amount of GABA (mg / kg), the response of Monascus purpureus ATCC 6405, was under the influence of three factors (MSG, pH, Temperature) described in Table 2 below. Data was analyzed using STATGRAPHICS Plus Version 5.1 (USA). The fitted model's equation is
GABA = -3035.66-2936.16 × MSG-1072.29 × Temperature-2405.81 × pH - 95.5365 × MSG2+116.583 × MSG × Temperature-64.834 × MSG × pH - 39.6737 × Temperature2 + 215.056 × Temperature × pH - 475.57 × pH2 GABA = -3035.66-2936.16 × MSG-1072.29 × Temperature-2405.81 × pH-95.5365 × MSG2 + 116.583 × MSG × Temperature-64.834 × MSG × pH-39.6737 × Temperature 2 + 215.056 × Temperature × pH-475.57 × pH 2
[표 2] Central composite design(CCD)을 위한 변수들과 그 레벨Table 2. Variables and Levels for Central Composite Design (CCD)
[표 3] Central composite design (CCD)의 실험디자인과 결과[Table 3] Experimental Design and Results of Central Composite Design (CCD)
이 모델의 fit은 0.88로 계산된 coefficient of determination R2로 체크되었으며, 이는 응답에서 88.3397%의 variability를 가리킨다. 이 모델의 최대점을 얻기 위한 3가지 변수의 최적수치는 MSG, temperature, pH 각각 1.0 %, 26 ℃, 3.28로 계산되었다. 이 최대점에서 모델은 3820 mg/kg GABA의 최대 응답을 예상하였다.The fit of this model was checked with a coefficient of determination R2 of 0.88, indicating 88.3397% variability in the response. The optimal values of the three variables to obtain the maximum point of this model were calculated as 1.0%, 26 ° C, and 3.28% for MSG, temperature and pH, respectively. At this maximum, the model expected a maximum response of 3820 mg / kg GABA.
따라서, 최적의 고체배양조건은 아래와 같으며, 이때, 3820 mg/kg GABA의 최대 응답을 보이는 것으로 예상되었다.Therefore, the optimum solid culture conditions were as follows, where it was expected to show a maximum response of 3820 mg / kg GABA.
- 탄소원: 5% fructose (1g fructose /발아메밀 20g)-Carbon Source: 5% fructose (1g fructose / 20g buckwheat)
- 미네랄: 0.1% NaH2PO4 (0.02g NaH2PO4 /발아메밀 20g)Minerals: 0.1% NaH 2 PO 4 (0.02
- 유기화합물: 0.5% glycerol (0.1ml glycerol /발아메밀 20g)-Organic compound: 0.5% glycerol (0.1ml glycerol / germinated buckwheat 20g)
- 질소원: 1 %MSG (0.2 g MSG /발아메밀 20g)Nitrogen source: 1% MSG (0.2 g MSG / 20 g germinated buckwheat)
- 초기 pH를 3.28으로 조절하였다(Acetic acid, NaOH 사용).Initial pH was adjusted to 3.28 (Acetic acid, NaOH).
- 배양초기는 30℃로 동일하며 4일~10일 동안의 최적배양온도는 26℃ 였다.-The initial culture was the same as 30 ℃ and the optimal culture temperature was 26 ℃ for 4-10 days.
도 17은 GABA production의 Main effects plot이다. 도 18은 GABA production의 Interaction plot이다 (A: MSG, B: Temperature, C: pH). 도 19는 GABA production의 Three-dimensional response surface plots이다. 온도 26℃에서 GABA 생산에 대한 MSG, temperature 및 pH의 combined effect를 나타낸다. 도 20은 GABA production의 Contour plot이다. 온도 26℃에서 GABA 생산에 대한 MSG, temperature 및 pH의 combined effect를 나타낸다. 도 21은 Desirability의 Three-dimensional response surface plots이다. 온도 26℃에서 GABA 생산에 대한 MSG, temperature 및 pH의 combined effect를 나타낸다. 도 22는 Estimated response surface의 Contour plot이다. 온도 26℃에서 GABA 생산에 대한 MSG, temperature 및 pH의 combined effect를 나타낸다. 도 22는 optimum conditions (MSG 1%, 26℃, pH 3.28)에서 GABA content가 3820 mg/kg로 증가할 것으로 예상한다. 17 is a main effects plot of GABA production. 18 is an Interaction plot of GABA production (A: MSG, B: Temperature, C: pH). 19 are three-dimensional response surface plots of GABA production. The combined effect of MSG, temperature and pH on GABA production at a temperature of 26 ° C is shown. 20 is a contour plot of GABA production. The combined effect of MSG, temperature and pH on GABA production at a temperature of 26 ° C is shown. 21 is a three-dimensional response surface plots of desirability. The combined effect of MSG, temperature and pH on GABA production at a temperature of 26 ° C is shown. 22 is a contour plot of the estimated response surface. The combined effect of MSG, temperature and pH on GABA production at a temperature of 26 ° C is shown. 22 anticipates that GABA content will increase to 3820 mg / kg at optimum conditions (
이상 설명한 바와 같이, 신경안정, 혈압강하 효과를 가지는 γ-aminobutyric acid (GABA) 고생산하는 홍국균을 선발하였으며, 이 선발된 홍국균을 이용하여 다양한 곡류를 기질로 이용한 홍국들을 제조하고 그중에서 GABA 생산성이 탁월한 발아된 통메밀을 기질로 선택하여 새로운 고기능성 홍국을 개발하였으며, 또한 발아된 통메밀 홍국의 GABA 생산성을 더욱 향상시키기 위하여 고체배양 최적화 조건을 개발하였다. 특히, “A factor at a time” 방법을 이용하여 GABA의 최적화 배양방법 개발하였으며, 새로운 고체배양방법에 껍질을 벗기지 않은 통메밀을 기질로 이용한 홍국에서는 GABA 농도가 일반적인 쌀을 기질로한 홍국과 비교하여 4 배 이상 높일 수 있었으며, 통메밀을 발아시켜 배양함으로써 메밀의 유용성분인 rutin과 quercetin의 함량을 최대화 시킬 수 있었다. 본 발명에 따르면, 홍국의 발효기질로써 GABA 생산성을 증진시키는 껍질이 벗겨지지 않은 발아된 통메밀을 이용한 고체배양방법 확립함으로써, 새로운 발효기질 사용으로 다기능성 홍국의 개발을 통하여 활용도를 높일 수 있을 것으로 기대되며, 기능성이 강화된 홍국을 다양한 식품의 원재료로 활용할 수 있으며, 이를 통한 새로운 식음료의 개발 또한 가능하며, 국내외 기능성식품 시장에서 홍국의 활용도가 점차 높아지고 있으므로, 다양한 기능성 식품의 원재료로 활용성이 뛰어날 것으로 기대된다.As described above, erythrocytes with high production of γ-aminobutyric acid (GABA), which have neurostable and blood pressure-lowering effects, were selected, and erythrocytes using various grains as substrates were prepared using the selected erythrocytes. A new high functional red yeast rice was developed by selecting excellent germinated whole buckwheat as a substrate, and a solid culture optimization condition was developed to further improve the GABA productivity of germinated whole buckwheat red yeast rice. In particular, we developed an optimized culture method for GABA using the “A factor at a time” method, and compared to the red rice plants with GABA concentrations compared to the general rice plants in red yeast rice using unbuckled whole buckwheat as a substrate for the new solid culture method. The buckwheat was increased by more than 4 times, and by culturing whole buckwheat, the contents of rutin and quercetin, which are useful components of buckwheat, were maximized. According to the present invention, by establishing a solid culture method using unbuckled germinated whole buckwheat to enhance GABA productivity as a fermentation substrate of red yeast rice, it will be possible to increase the utilization through the development of multifunctional red yeast rice using a new fermentation substrate. It is expected that the functionally enhanced red soup can be used as a raw material for various foods, and new foods and beverages can be developed through it. It is expected to excel.
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| KR102254560B1 (en) | 2020-12-08 | 2021-05-21 | (주)안온바이오 | Extract of Liriope platyphylla with increased gaba content and anti-inflammatory composition comprising the same |
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| JP2000060536A (en) | 1998-08-27 | 2000-02-29 | Okayama Prefecture | Production of koji |
| KR20020070241A (en) * | 2002-08-14 | 2002-09-05 | 김일두 | Cultivation method of functional germinated brown rice by using Hongkook |
| KR20030023281A (en) * | 2001-09-13 | 2003-03-19 | 박동기 | Method for the production of monascus inoculation germinated rice brown and it productive method for food |
| US20040047842A1 (en) * | 2002-08-15 | 2004-03-11 | Food Industry Research And Development Institute | Monascus purpureus mutants and their use in preparing fermentation products having blood pressure lowering activity |
| KR20040051783A (en) * | 2002-12-13 | 2004-06-19 | 이성구 | Buckwheat fermented with fungi monascus species |
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| KR20030023281A (en) * | 2001-09-13 | 2003-03-19 | 박동기 | Method for the production of monascus inoculation germinated rice brown and it productive method for food |
| KR20020070241A (en) * | 2002-08-14 | 2002-09-05 | 김일두 | Cultivation method of functional germinated brown rice by using Hongkook |
| US20040047842A1 (en) * | 2002-08-15 | 2004-03-11 | Food Industry Research And Development Institute | Monascus purpureus mutants and their use in preparing fermentation products having blood pressure lowering activity |
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| KR101507470B1 (en) | 2013-08-27 | 2015-04-01 | (주)건우애듀 | Solid phase fermentation method of grains using useful microorganism and functional fermentation composition manufactured by the same |
| KR102254560B1 (en) | 2020-12-08 | 2021-05-21 | (주)안온바이오 | Extract of Liriope platyphylla with increased gaba content and anti-inflammatory composition comprising the same |
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