KR20040000639A - Manufacturing method of extract from germinated grain foods with enhanced levels of gamma-aminobutyric acid and immuno-regulator containing of gamma-aminobutyric acid - Google Patents

Manufacturing method of extract from germinated grain foods with enhanced levels of gamma-aminobutyric acid and immuno-regulator containing of gamma-aminobutyric acid Download PDF

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KR20040000639A
KR20040000639A KR1020020035147A KR20020035147A KR20040000639A KR 20040000639 A KR20040000639 A KR 20040000639A KR 1020020035147 A KR1020020035147 A KR 1020020035147A KR 20020035147 A KR20020035147 A KR 20020035147A KR 20040000639 A KR20040000639 A KR 20040000639A
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gamma
aminobutyric acid
germinated
gaba
extract
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KR100478491B1 (en
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오석흥
석태환
심규광
최원규
오찬호
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오석흥
석태환
심규광
최원규
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B60—VEHICLES IN GENERAL
    • B60P—VEHICLES ADAPTED FOR LOAD TRANSPORTATION OR TO TRANSPORT, TO CARRY, OR TO COMPRISE SPECIAL LOADS OR OBJECTS
    • B60P7/00—Securing or covering of load on vehicles
    • B60P7/02—Covering of load
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B60—VEHICLES IN GENERAL
    • B60P—VEHICLES ADAPTED FOR LOAD TRANSPORTATION OR TO TRANSPORT, TO CARRY, OR TO COMPRISE SPECIAL LOADS OR OBJECTS
    • B60P7/00—Securing or covering of load on vehicles
    • B60P7/06—Securing of load
    • B60P7/08—Securing to the vehicle floor or sides
    • B60P7/0884—Securing to the vehicle floor or sides by increasing the friction between the load and the surface
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B60—VEHICLES IN GENERAL
    • B60P—VEHICLES ADAPTED FOR LOAD TRANSPORTATION OR TO TRANSPORT, TO CARRY, OR TO COMPRISE SPECIAL LOADS OR OBJECTS
    • B60P7/00—Securing or covering of load on vehicles
    • B60P7/06—Securing of load
    • B60P7/16—Protecting against shocks
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B60—VEHICLES IN GENERAL
    • B60Y—INDEXING SCHEME RELATING TO ASPECTS CROSS-CUTTING VEHICLE TECHNOLOGY
    • B60Y2200/00—Type of vehicle
    • B60Y2200/10—Road Vehicles
    • B60Y2200/14—Trucks; Load vehicles, Busses
    • B60Y2200/145—Haulage vehicles, trailing trucks

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  • Engineering & Computer Science (AREA)
  • Transportation (AREA)
  • Mechanical Engineering (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

PURPOSE: Provided are a manufacturing method of an extract from germinated grain foods with enhanced levels of gamma-aminobutyric acid(GABA) and an immuno-regulator containing of gamma-aminobutyric acid as an active ingredient. The gamma-aminobutyric acid(GABA) has excellent immune activity, and is thus used as an immuno-regulating agent. CONSTITUTION: A manufacturing method of an extract from germinated grain foods with enhanced levels of gamma-aminobutyric acid(GABA) comprises the steps of: soaking grain and germinating it at 24-26 deg.C for 2-4 days; pulverizing the germinated grain, and dissolving and sterilizing the powder; filtering the dissolved powder twice and freeze-drying it; and dissolving the dried powder in a solvent to give an extract.

Description

감마-아미노부티르산 함량이 증가된 발아곡류 추출액의 제조방법 및 동 발아곡류 추출액 중 감마-아미노부티르산을 유효성분으로 함유한 면역제제 {Manufacturing method of extract from germinated grain foods with enhanced levels of gamma-aminobutyric acid and immuno-regulator containing of gamma-aminobutyric acid}Manufacturing method of extract from germinated grain foods with enhanced levels of gamma-aminobutyric acid and germinated grain extracts with increased gamma-aminobutyric acid content immuno-regulator containing of gamma-aminobutyric acid}

본 발명은 감마-아미노부티르산(gamma-aminobutyric acid, GABA) 함량이 증가된 발아곡류 추출액의 제조방법 및 동 발아곡류 추출액 중 감마-아미노부티르산을 유효성분으로 함유한 면역제제에 관한 것이다.The present invention relates to a method for preparing a germinated grain extract with increased gamma-aminobutyric acid (GABA) content and an immunologic agent containing gamma-aminobutyric acid as an active ingredient in the germinated grain extract.

감마-아미노부티르산(GABA)는 억제성 신경전달물질로서 흥분억제효과, 항경련작용, 정신집중과 기억력강화, 혈압강하 등의 체내 기능을 갖는다. 이와같은GABA의 기능이 알려지면서 의약품으로서의 GABA 뿐만 아니라 최근에는 기능성 식품소재로서의 GABA에 대한 관심이 고조되고 있다. 또한 알콜중독자들의 혈중 GABA 농도는 정상인과 비교시 유의적으로 낮은 값을 보인다. 따라서 체내 GABA의 농도를 높여줄 수 있는 식품을 개발하므로써 이들 질환의 예방 및 치료에 도움을 줄 수 있다.Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter and has body functions such as excitatory effect, anticonvulsant action, mental concentration and memory, and blood pressure lowering. As the function of GABA becomes known, interest in GABA as a functional food material as well as GABA as a medicine has recently increased. In addition, the blood GABA levels of alcoholics were significantly lower than those of normal subjects. Therefore, by developing foods that can increase the concentration of GABA in the body can help prevent and treat these diseases.

이와 같은 관심은 GABA 고함유 식품과 식물 탐색 및 이들의 기능성 연구에 대한 동기를 부여하였고 이에 대한 몇몇 연구결과도 발표된 바 있다. 예를 들면, Chang 등은 제주도 다원에서 생산된 녹차 생엽을 채취 시기별로 혐기적으로 처리하여 GABA 및 기타 주요성분의 함량변화를 측정한 결과 GABA의 함량이 증진되는 것을 확인하였다. GABA 함유 현미 배아 추출물의 생리활성 기능으로는 혈압강하 작용, 신장기능의 활성화 작용, 간 기능 개선 작용, 비만 방지 작용, 알코올 대사 촉진 작용, 냄새제거 효과 등이 있는 것으로 보고되어 있다. 또한 현미 배아 추출물은 GABA나 비타민 B군, 미네랄류 등의 영양보조식품으로서 활용이 가능하고, 더 나아가 현미 발아 과정에서 다량으로 축적시킨 GABA를 식품에의 이용이 가능하다.This interest motivated the search for GABA-rich foods and plants and their functional studies, and several studies have been published. For example, Chang et al., Anaerobic treatment of green tea leaves produced in Dawon, Jeju Island by measuring the change in the content of GABA and other major components confirmed that the GABA content is improved. Physiologically active functions of GABA-containing brown rice embryo extract have been reported to have a blood pressure lowering effect, renal function activation, liver function improving effect, anti-obesity effect, alcohol metabolism promoting effect, and odor removing effect. In addition, the brown rice germ extract can be used as a nutritional supplement such as GABA, vitamin B group, minerals, etc. Furthermore, GABA accumulated in a large amount in the germination process of brown rice can be used in food.

현미는 싹이 날 때 각종 비타민, 칼슘, 무기질, 아미노산, 효소, 아라비노키실란, GABA 등이 증가하는 것으로 알려져 있다. 이 중에서 아라비노키실란의 함량이 증가된 곡류의 발아식품 및 그 제조방법에 있어 아라비노키실란은 열에 약하여 아라비노키실란의 효과를 나타내기 위해서는 생식이 권장되고 있다. 하지만 GABA는 비단백태 아미노산의 일종으로 230℃ 정도의 가열에 의한 누룽지를 제조할 때에도 변성의 우려가 없어 GABA 함량이 증진된 발아곡류 및 그 추출물을 이용하여 음료및 식품제조에 있어서 유용하다.Brown rice is known to increase vitamins, calcium, minerals, amino acids, enzymes, arabinoxysilane, GABA, etc. when sprouting. Among them, in the germinated food of grains with increased content of arabinoxysilane and its preparation method, arabinokisilane is vulnerable to heat, and reproduction is recommended in order to show the effect of arabinoxysilane. However, GABA is a kind of non-protein amino acid, so there is no fear of denaturation when manufacturing nurungji by heating at about 230 ° C. Therefore, GABA is useful in the production of beverages and foods using germinated grains and its extracts with enhanced GABA content.

최근에는 GABA 생성에 관련된 유전자 도입에 의한 식물체내 GABA 생성 조절에 관하여 담배 식물체내 글루타메이트 데카르복실라제(glutamate decarboxylase, GAD)나 칼모둘린(calmodulin) 유전자의 도입으로 GABA의 함량을 증진시킬 수 있음이 확인된 바 있다. 그러나 유전자 변형 농산물이 많은 논란을 불러일으키고 있는 시점이기 때문에 대안을 모색하던 중 어떤 유용물질과 식물생리현상을 적절히 이용하여 식물체내 GABA 생성체계를 활성화시키면 GABA의 함량을 획기적으로 증진시킬 수 있었다. 예를들면, 벼를 산소결핍 상태에서 싹을 틔울 경우 단백질 및 아미노산 대사, GABA 생성대사에 칼슘과 칼모듈린이 관련되어 있음도 확인되었다. 따라서 향후 이들 인자에 대한 연구를 계속하면 GABA의 함량이 더욱 증진된 현미를 생산 할 수 있을 것으로 예상된다.Recently, the introduction of glutamate decarboxylase (GAD) or calmodulin genes in tobacco plants can enhance GABA content in the regulation of GABA production in plants by the introduction of genes related to GABA production. It has been confirmed. However, since genetically modified agricultural products are causing a lot of controversy, activating the GABA production system in plants using appropriate useful materials and physiological phenomena during the search for alternatives could dramatically increase the content of GABA. For example, it was also found that calcium and calmodulin are involved in protein and amino acid metabolism and GABA production metabolism when rice sprouts under oxygen deprivation. Therefore, further study of these factors is expected to produce brown rice with an enhanced GABA content.

식물에서 GABA의 합성은 기계적인 자극, 온도, 산소결핍, 수분 스트레스와 같은 여러 외부 환경적 요인에 의해 유도되는 것으로 보고되고 있어, 식물체가 환경적 스트레스에 대항하기 위한 수단의 하나로 GABA 생성체계를 가동시키고 있다고도 생각된다. 이외에도 식물체내에서 GABA는 글루타메이트(glutamate)에서 숙시네이트(succinate)에 이르는 GABA shunt를 통해 TCA 회로에서 산화를 위한 탄소골격의 제공 및 식물이 해충의 공격 중에 있을 때 합성되어 내충성을 보이게 하는 것 등이 제안되었다.The synthesis of GABA in plants has been reported to be induced by several external environmental factors such as mechanical stimulation, temperature, oxygen deficiency, and water stress, so that the plant can operate the GABA production system as a means of combating environmental stress. I think you can. In addition, GABA is synthesized in plants through the GABA shunt, from glutamate to succinate, to provide a carbon skeleton for oxidation in the TCA cycle, and to synthesize and show insect resistance when plants are under attack by pests. Proposed.

본 발명은 곡류를 발아시켜 GABA의 함량이 증가된 발아곡류 추출액을 얻고자한다. 특히 곡류를 발아시 곡류의 침지액으로서 글루탐산 수용액 또는 키토산 수용액과 글루탐산 수용액의 혼합용액을 사용하여 GABA의 함량이 증가된 발아곡류 추출액의 제조방법 제공을 목적으로 한다.The present invention is to germinate grains to obtain a germinated grain extract with increased content of GABA. In particular, it is an object of the present invention to provide a method for producing a germinated grain extract with increased GABA content using a glutamic acid solution or a mixed solution of an aqueous chitosan solution and a glutamic acid solution as a grain dipping solution.

또한 GABA의 함량이 증가된 발아곡류 추출액 중에서 GABA의 특성을 조사한 바 GABA 함량이 증가된 발아곡류 추출액을 복용한 쥐의 면역력 증가 효과가 있음을 알게되어 본 발명은 GABA를 유효성분으로 함유하는 면역제제의 제공을 또다른 목적으로 한다.In addition, as a result of investigating the properties of GABA in the germinated grain extract with increased GABA content, it was found that there was an effect of increasing immunity of rats taking germinated grain extract with increased GABA content. The present invention provides an immunologic agent containing GABA as an active ingredient. The provision of is for another purpose.

도 1은 각각의 발아구 현미 추출액의 면역능에 미치는 효과를 나타낸 그래프이다.1 is a graph showing the effect on the immune capacity of each germinated brown rice extract.

본 발명의 감마-아미노부티르산 함량이 증가된 발아곡류 추출액의 제조방법 은 곡류를 침지액에 침지시켜 24∼26℃에서 2∼4일 동안 1∼5mm의 싹이 나도록 발아시키는 단계와, 발아한 곡류를 분말화하고 수용액에 용해한 후 살균하는 단계와, 살균 후 찌꺼기를 제거하고 여과한 후 건조하는 단계와, 건조 후 건조물을 증류수에 용해하여 추출하는 단계를 포함하는 것을 특징으로 한다.Method for producing a germinated grain extract with increased gamma-aminobutyric acid content of the present invention is the step of immersing the grains in immersion liquid to germinate 1-5mm for 2 to 4 days at 24 to 26 ℃, and germinated grains Powdered and dissolved in an aqueous solution and then sterilized, sterilized after removing the residue, filtered and dried, and drying and drying the dried material in distilled water, characterized in that it comprises a step of extracting.

본 발명에서 곡류는 현미, 보리, 밀, 수수, 콩 중에서 선택된 어느 하나를 사용할 수 있으며, 보다 바람직하게는 우리몸에 유용한 영양성분이 많으며, 쉽게 구할 수 있는 현미를 이용하는 것이 좋다.In the present invention, the grains may use any one selected from brown rice, barley, wheat, sorghum and soybeans, and more preferably, there are many useful nutrients in our bodies, and it is preferable to use brown rice which is easily available.

발명에서 곡류를 침지한 침지액은 5∼10 mM 글루탐산 수용액 또는 5∼10 mM 글루탐산 수용액과 50∼100 ppm 키토산 수용액이 1:9∼9:1의 비율로 혼합된 혼합용액을 사용할 수 있는데 이러한 침지액은 곡류의 발아를 촉진하기 위해서 12시간 마다 침지액을 새로이 교환하여 곡류를 침지시키는 것이 좋다.In the present invention, the dipping solution in which cereals are immersed may use a mixed solution of 5-10 mM glutamic acid solution or 5-10 mM glutamic acid solution and 50-100 ppm chitosan solution in a ratio of 1: 9-9: 1. In order to promote the germination of the grains, it is recommended that the grains be immersed by renewing the dipping solution every 12 hours.

침지액을 5∼10 mM 글루탐산 수용액을 사용하는 경우 글루탐산 수용액의 농도가 5 mM 미만일 경우에는 GABA 생성에 미치는 영향이 크지 않고, 10 mM 초과하는 경우에는 오히려 발아가 지연될 수 있어 본 발명에서 곡류를 발아시키기 위해 사용하는 침지액을 글루탐산 수용액으로 하는 경우 5∼10 mM 글루탐산 수용액을 사용하는 것이 좋다.When 5 to 10 mM glutamic acid solution is used as the immersion liquid, the concentration of the aqueous glutamic acid solution is less than 5 mM, the effect on GABA production is not large, and when the concentration is greater than 10 mM, germination may be delayed. When the immersion liquid used for germination is made into an aqueous glutamic acid solution, it is preferable to use an aqueous 5-10 mM glutamic acid solution.

또한 곡류의 침지액으로 상기의 글루탐산 수용액 이외에도 5∼10 mM 글루탐산 수용액과 50∼100 ppm 키토산 수용액이 1:9∼9:1의 비율로 혼합된 혼합용액을 사용할 수 있는데, 혼합용액에서 키토산 수용액이 50 ppm 미만이면 GABA 생성 증진 및 항균 효과가 미미하고, 100 ppm 초과하는 경우 항균 효과는 좋지만 GABA 생성 증진이 글루탐산 단독처리에 비하여 감소될 수 있어 키토산 수용액의 농도는 50∼100 ppm 인 것을 사용하는 것이 좋다.In addition to the glutamic acid solution, a mixed solution containing 5 to 10 mM glutamic acid solution and 50 to 100 ppm chitosan solution in a ratio of 1: 9 to 9: 1 may be used as the grain dipping solution. If it is less than 50 ppm, the GABA production and antimicrobial effect is insignificant. If it exceeds 100 ppm, the antibacterial effect is good, but the GABA production enhancement may be reduced compared to glutamic acid treatment alone. good.

발아한 곡류를 마쇄하여 분말화하고 수용액에 용해한 후 120℃ 및 1.5atm에서 고압살균한 다음 거즈로 1차 여과하여 찌꺼기를 제거하고 재차 여과지로 2차 여과하여 여과액을 동결건조 후 건조물을 증류수 또는 살균수로 용해시켜 감마-아미노부티르산 함량이 증가된 발아곡류 추출액을 얻을 수 있다.The germinated grains were ground and powdered, dissolved in an aqueous solution, autoclaved at 120 ° C. and 1.5 atm, filtered first with gauze to remove debris, and filtered again with a filter paper. The filtrate was lyophilized, and the dried product was dried or dried. Dissolved in sterile water to obtain a germinated grain extract with increased gamma-aminobutyric acid content.

한편 본 발명은 상기의 방법으로 제조한 감마-아미노부티르산 함량이 증가된 발아곡류 추출액 중 감마-아미노부티르산을 유효성분으로 함유한 면역제제를 제공할 수 있다.On the other hand, the present invention can provide an immunological agent containing gamma-aminobutyric acid as an active ingredient in the germinated grain extract with increased gamma-aminobutyric acid content prepared by the above method.

이하 본 발명을 다음의 비교예, 실시예 및 시험예에 의하여 설명하고자 한다. 그러나 이들은 본 발명의 일예로서 이들이 본 발명의 권리범위를 한정하는 것은 아니다.Hereinafter, the present invention will be described by the following comparative examples, examples and test examples. However, these are examples of the present invention and they do not limit the scope of the present invention.

<비교예 1>Comparative Example 1

현미를 분말화하고 물에 용해한 후 120℃ 및 1.5atm에서 고압살균한 다음 거즈로 1차 여과하여 찌꺼기를 제거하고 재차 여과지로 2차 여과하여 여과액을 동결건조 후 증류수로 용해하여 발아하지 않은 현미(N구) 추출액을 얻었다.The brown rice is powdered and dissolved in water, sterilized by autoclaving at 120 ℃ and 1.5atm, filtered first with gauze to remove debris, and filtered secondly with filter paper. The filtrate is lyophilized and dissolved with distilled water to not germinate brown rice. (N sphere) An extract was obtained.

<비교예 2>Comparative Example 2

현미를 물에 침지시켜 24∼26℃에서 2∼4일 동안 1∼5mm의 싹이 나도록 발아시켰다. 현미를 물에 침지시 12시간 마다 물을 교환하였다. 발아한 현미를 분말화하고 수용액에 용해한 후 120℃ 및 1.5atm에서 고압살균한 다음 거즈로 1차 여과하여 찌꺼기를 제거하고 재차 여과지로 2차 여과하여 여과액을 동결건조 후 증류수로 용해하여 물 발아구(W구) 현미 추출액을 얻었다.The brown rice was immersed in water and germinated to shoot 1-5 mm at 24-26 ° C. for 2-4 days. The water was changed every 12 hours when the brown rice was immersed in water. The germinated brown rice is powdered, dissolved in an aqueous solution, autoclaved at 120 ° C and 1.5 atm, filtered first with gauze to remove debris, and filtered again with a filter paper. The filtrate is lyophilized and dissolved with distilled water to germinate water. Gu (W) brown rice extract was obtained.

<비교예 3>Comparative Example 3

현미를 50 ppm 키토산 수용액에 침지시켜 24∼26℃에서 2∼4일 동안 1∼5mm의 싹이 나도록 발아시켰다. 현미를 50 ppm 키토산 수용액에 침지시 12시간 마다 50 ppm 키토산 수용액을 교환하였다.The brown rice was immersed in 50 ppm aqueous chitosan solution and germinated to shoot 1-5 mm at 24-26 ° C. for 2-4 days. When the brown rice was immersed in 50 ppm chitosan solution, the 50 ppm chitosan solution was exchanged every 12 hours.

발아한 현미를 분말화하고 수용액에 용해한 후 120℃ 및 1.5atm에서 고압살균한 다음 거즈로 1차 여과하여 찌꺼기를 제거하고 재차 여과지로 2차 여과하여 여과액을 동결건조 후 증류수로 용해하여 50 ppm 키토산 발아구(C구) 현미 추출액을 얻었다.The germinated brown rice is powdered, dissolved in an aqueous solution, autoclaved at 120 ° C and 1.5 atm, filtered first with gauze to remove debris, and filtered again with a filter paper. The filtrate is lyophilized, dissolved in distilled water, and 50 ppm. Chitosan germinated (C sphere) brown rice extract was obtained.

상기에서 키토산 수용액은 게껍질로부터 얻은 키틴을 조제하여 탈아세틸화 정도가 90% 이상이고 평균분자량이 20,000인 키토산을 공지의 방법으로 제조한 것이다.In the chitosan aqueous solution, chitin obtained from crab shell is prepared, and chitosan having a deacetylation degree of 90% or more and an average molecular weight of 20,000 is prepared by a known method.

<실시예 1><Example 1>

현미를 5 mM 글루탐산 수용액에 침지시켜 24∼26℃에서 2∼4일 동안 1∼5mm의 싹이 나도록 발아시켰다. 현미를 5 mM 글루탐산 수용액에 침지시 12시간 마다 5 mM 글루탐산 수용액을 교환하였다.The brown rice was immersed in an aqueous 5 mM glutamic acid solution and germinated to shoot 1-5 mm at 2 to 4 days at 24 to 26 ° C. When the brown rice was immersed in 5 mM glutamic acid aqueous solution, 5 mM glutamic acid aqueous solution was exchanged every 12 hours.

발아한 현미를 분말화하고 수용액에 용해한 후 120℃ 및 1.5atm에서 고압살균한 다음 거즈로 1차 여과하여 찌꺼기를 제거하고 재차 여과지로 2차 여과하여 여과액을 동결건조 후 증류수로 용해하여 5 mM 글루탐산 수용액 발아구(G구) 현미 추출액을 얻었다.The germinated brown rice is powdered, dissolved in an aqueous solution, autoclaved at 120 ° C and 1.5 atm, filtered first with gauze to remove debris, and filtered again with a filter paper. The filtrate is lyophilized and dissolved in distilled water to give 5 mM. Glutamic acid aqueous solution germinated (G sphere) brown rice extract was obtained.

<실시예 2><Example 2>

현미를 100 ppm 키토산 수용액과 10 mM 글루탐산 수용액이 1:1의 비율로 혼합된 용액(50 ppm 키토산 수용액/5 mM 글루탐산 수용액)에 침지시켜 24∼26℃에서 2∼4일 동안 1∼5mm의 싹이 나도록 발아시켰다. 현미를 상기 혼합용액에 침지시 12시간 마다 혼합용매를 교환하였다.Brown rice is immersed in a solution in which a 100 ppm aqueous chitosan solution and a 10 mM glutamic acid solution are mixed at a ratio of 1: 1 (50 ppm aqueous chitosan solution / 5 mM glutamic acid solution), and shoots of 1 to 5 mm for 2 to 4 days at 24 to 26 ° C. This germinated to sprout. When the brown rice was immersed in the mixed solution, the mixed solvent was changed every 12 hours.

발아한 현미를 분말화하고 수용액에 용해한 후 120℃ 및 1.5atm에서 고압살균한 다음 거즈로 1차 여과하여 찌꺼기를 제거하고 재차 여과지로 2차 여과하여 여과액을 동결건조 후 건조물을 증류수로 용해하여 50 ppm 키토산 수용액/5 mM 글루탐산 수용액 발아구(CG구) 현미 추출액을 얻었다.The germinated brown rice is powdered, dissolved in an aqueous solution, autoclaved at 120 ° C and 1.5 atm, filtered first with gauze to remove debris, and filtered again with a filter paper. The filtrate is lyophilized and the dried product is dissolved in distilled water. A 50 ppm chitosan aqueous solution / 5 mM glutamic acid aqueous solution germinated (CG sphere) brown rice extract was obtained.

<시험예 1><Test Example 1>

상기 비교예 및 실시예에서 추출한 각각의 발아구 현미 추출액(mL)에 따른 총단백질 함량(mg)을 측정하여 그 결과를 아래의 표 1에 나타내었다.The total protein content (mg) according to the germinated brown rice extract (mL) extracted in Comparative Examples and Examples was measured, and the results are shown in Table 1 below.

각각의 발아구 현미 추출액에 따른 총 단백질 함량은 감마-글로블린을 표준 단백질로 사용하고 공지의 브래드포드법(Bradford, M. M. (1976),Anal. Biochem72, 248-254)을 이용하여 측정하였다.The total protein content of each germinated brown rice extract was measured using gamma-globulin as a standard protein and using a known Bradford method (Bradford, MM (1976), Anal. Biochem 72, 248-254).

표 1. 비교예 및 실시예 각각의 발아구에 따른 총 단백질 함량(mg/mL)Table 1. Total protein content (mg / mL) according to the germination of each of the Comparative Examples and Examples

항목Item 비교예 1(N구)Comparative example 1 (N ball) 비교예 2(W구)Comparative example 2 (W sphere) 비교예 3(C구)Comparative Example 3 (C Ward) 실시예 1(G구)Example 1 (G sphere) 실시예 2(CG구)Example 2 (CG sphere) 총단백질 함량Total protein content 3.6±0.43.6 ± 0.4 2.4±0.32.4 ± 0.3 1.5±0.11.5 ± 0.1 1.8±0.11.8 ± 0.1 1.1±0.11.1 ± 0.1

상기 표 1의 결과를 보면 비교예 1의 N구 현미 추출액은 다른 발아구에 비하여 유의적으로 높은 값이었으며, 특히 실시예 2의 CG구 현미 추출액에 비하여 총 단백질 함량이 약 3.3배 높았다.In the results of Table 1, the N-sphere brown rice extract of Comparative Example 1 was significantly higher than the other germinated bulbs, and in particular, the total protein content was about 3.3 times higher than the CG-brown rice extract of Example 2.

<시험예 2><Test Example 2>

상기 비교예 및 실시예 각각의 발아구 현미 추출액 중에서 GABA를 포함하는 수용액 층은 각 현미 추출물에 메탄올:클로로포름:물이 12:5:3의 비율로 혼합된 혼합용매를 1:1의 비율로 가한 후 혼합하여 원심분리(12,000 × g, 15분, 4℃)를 통하여 얻은 후 냉동건조 하였다. 이어 200㎕의 증류수로 용해한 후 0.45 ㎛ 폴리비닐리덴플루오라이드(Poly Vinylidene Fluoride, PVDF) 필터(Millipore)로 여과하여 아미노산자동분석기(AccQ·Tag Amino Acid Analysis System, Waters, USA)로 각 구별 현미 추출액 mL당 총 유리아미노산 함량(nmole) 및 GABA 함량을 분석하였고, 표준 GABA(Sigma, USA)의 분석결과와 비교하여 GABA의 함량(nmole)을 산출하여 그 결과를 아래의 표 2에 정리하여 나타내었다.In the germinated brown rice extract of each of the Comparative Examples and Examples, the aqueous solution layer containing GABA was added to each brown rice extract in a ratio of 1: 1: methanol: chloroform: water mixed at a ratio of 12: 5: 3. After mixing was obtained through centrifugation (12,000 × g, 15 minutes, 4 ℃) and lyophilized. After dissolving with 200 μl of distilled water, it was filtered with 0.45 μm Poly Vinylidene Fluoride (PVDF) filter (Millipore) and extracted with brown rice extract using an automatic amino acid analyzer (AccQ, Tag Amino Acid Analysis System, Waters, USA) The total free amino acid content (nmole) and GABA content per mL were analyzed, and the content of GABA (nmole) was calculated in comparison with the standard GABA (Sigma, USA) analysis results and the results are summarized in Table 2 below. .

아미노산자동분석기 사용시 아미노산의 형광 유도체화를 위해 6-아미노퀴놀릴-N-하이드록시숙신이미딜 카보네이트(6-aminoquinolyl-N-hydroxysyccinimidyl carbonate)(AccQ·Fluor Reagent)를 사용하였으며 이들 유도체의 분리를 위해 3.9×150mm AccQ·TagTM(Nova-PakTMC18, Waters, USA) 컬럼을 사용하였다. 컬럼(column)으로부터 유도체를 용출시키기 위해서는 아세테이트-포스페이트 완충액(acetate-phosphate buffer)(AccQ·Tag Eluent A)와 60% 아세토니트릴(acetonitrile)을 98:2의 비율로 1mL/min의 유속으로 흘려주었다.6-aminoquinolyl-N-hydroxysyccinimidyl carbonate (AccQ Fluor Reagent) was used for fluorescence derivatization of amino acids in the amino acid autoanalyzer. A 3.9 × 150 mm AccQ · Tag ™ (Nova-Pak ™ C 18 , Waters, USA) column was used. To elute the derivative from the column, acetate-phosphate buffer (AccQ-Tag Eluent A) and 60% acetonitrile were flowed at a flow rate of 1 mL / min at a ratio of 98: 2. .

표 2. 각 구별 mL당 총 유리아미노산 함량 및 GABA 함량(nmole)Table 2. Total free amino acid content and GABA content (nmole) per mL of distinction

아미노산 및 GABA 함량Amino Acid and GABA Content 비교예 1(N구)Comparative example 1 (N ball) 비교예 2(W구)Comparative example 2 (W sphere) 비교예 3(C구)Comparative Example 3 (C Ward) 실시예 1(G구)Example 1 (G sphere) 실시예 2(CG구)Example 2 (CG sphere) AspAsp 136(±15)136 (± 15) 40(±5)40 (± 5) 16(±2)16 (± 2) 14(±2)14 (± 2) 14(±3)14 (± 3) SerSer 181(±16)181 (± 16) 55(±6)55 (± 6) 83(±7)83 (± 7) 164(±21)164 (± 21) 169(±19)169 (± 19) GluGlu 195(±13)195 (± 13) 65(±4)65 (± 4) 44(±5)44 (± 5) 38(±5)38 (± 5) 58(±4)58 (± 4) GlyGly 38(±3)38 (± 3) 38(±4)38 (± 4) 21(±3)21 (± 3) 16(±3)16 (± 3) 24(±2)24 (± 2) HisHis 26(±7)26 (± 7) 35(±2)35 (± 2) 34(±2)34 (± 2) 27(±5)27 (± 5) 34(±1)34 (± 1) ArgArg 53(±5)53 (± 5) 70(±3)70 (± 3) 61(±5)61 (± 5) 48(±4)48 (± 4) 72(±4)72 (± 4) ThrThr 19(±2)19 (± 2) 21(±2)21 (± 2) 19(±3)19 (± 3) 15(±2)15 (± 2) 21(±1)21 (± 1) AlaAla 225(±21)225 (± 21) 307(±18)307 (± 18) 267(±15)267 (± 15) 416(±34)416 (± 34) 418(±33)418 (± 33) GABAGABA 274(±24)274 (± 24) 340(±10)340 (± 10) 520(±42)520 (± 42) 820(±21)820 (± 21) 897(±36)897 (± 36) ProPro 53(±7)53 (± 7) 44(±7)44 (± 7) 41(±5)41 (± 5) 27(±3)27 (± 3) 28(±3)28 (± 3) CysCys 2(±1)2 (± 1) 2(±1)2 (± 1) 3(±1)3 (± 1) 2(±1)2 (± 1) 2(±2)2 (± 2) TyrTyr 12(±2)12 (± 2) 24(±2)24 (± 2) 25(±1)25 (± 1) 28(±3)28 (± 3) 30(±2)30 (± 2) MetMet 20(±3)20 (± 3) 59(±4)59 (± 4) 40(±3)40 (± 3) 35(±4)35 (± 4) 51(±6)51 (± 6) ValVal 5(±2)5 (± 2) 17(±3)17 (± 3) 14(±3)14 (± 3) 9(±2)9 (± 2) 16(±3)16 (± 3) LeuLeu 8(±2)8 (± 2) 32(±3)32 (± 3) 18(±1)18 (± 1) 15(±2)15 (± 2) 23(±2)23 (± 2) LysLys 16(±3)16 (± 3) 21(±3)21 (± 3) 17(±3)17 (± 3) 17(±2)17 (± 2) 24(±4)24 (± 4) IleIle 14(±2)14 (± 2) 49(±3)49 (± 3) 40(±4)40 (± 4) 30(±2)30 (± 2) 46(±3)46 (± 3) PhePhe 9(±1)9 (± 1) 25(±2)25 (± 2) 26(±2)26 (± 2) 17(±2)17 (± 2) 29(±1)29 (± 1) TotalTotal 1,286(±129)1,286 (± 129) 1,244(±82)1,244 (± 82) 1,289(±107)1,289 (± 107) 1,738(±118)1,738 (± 118) 1,956(±129)1,956 (± 129)

표 2에서와 같이 발아에 의한 GABA 함량 변화는 매우 뚜렷함을 쉽게 알 수 있었고 키토산/글루탐산 처리구(CG구)의 GABA 함량이 가장 높았다. 또한 GABA 생성효소인 글루탐산 탈탄산효소의 기질이 되는 글루탐산(Glu)은 모든 발아구에서 현저하게 감소되는 경향을 보였다. 기타 아스파틱산(Asp)은 현저하게 감소하였고, 알라닌(Ala)의 함량은 증진되었다. 또한 G구 및 CG구 에서는 다른 발아구에서는 감소되던 Ser의 함량이 감소되지 않고 회복되었다. GABA를 포함하여 각 구별 추출액 mL 당 총 유리아미노산 함량(nmole)에 있어서 N구는 1,286(±129), W구는1,244(±82), C구는 1,289(±107), G구는 1,738(±118), CG구는 1,956(±129)으로 CG구와 G구가 다른 구에 비하여 유의적으로 높은 값을 가짐을 알 수 있었다As shown in Table 2, the GABA content change by germination was very clear and showed the highest GABA content in chitosan / glutamic acid treatment (CG). In addition, glutamic acid (Glu), which is a substrate of glutamate decarboxylase, a GABA synthase, tended to be markedly decreased in all germinations. Other aspartic acid (Asp) was significantly reduced, and the content of alanine (Ala) was enhanced. In addition, in G and CG groups, Ser content, which was decreased in other germinated cells, was recovered without decreasing. Total free amino acid content (nmole) per mL of extract, including GABA, was found to be 1,286 (± 129) for N, 1,244 (± 82) for W, 1,289 (± 107) for C, 1,738 (± 118) for G, The CG group was 1,956 (± 129), and the CG and G groups were significantly higher than the other groups.

<시험예 3><Test Example 3>

MTT법에 의한 비장, 흉선 및 장간막 림프절 세포의 증식반응 측정하기 위해 생쥐의 경추를 탈구시켜 비장, 흉선 및 장간막 림프절을 무균적으로 적출하고 각각의 세포를 세정(1500rpm, 10분)해서 세포 현탁액을 조제한 후, 1× 106cells/well이 되도록 세포수를 조정하고 비장세포 현탁액에는 lipopolysaccharide(Sigma, USA) 5㎍/ml를, 흉선과 장간막 림프절세포 현탁액에는 Concanavalin A(Sigma, USA) 0.5 ㎍/ml를 T-세포 활성화제로 첨가하고 상기 비교예 및 실시예의 N구, W구, C구, G구, CG구의 현미 추출액을 각각 100 ㎍/ml 되게 첨가하여 48시간 동안 37℃의 CO2배양기(5%-CO2, 95%-air) 내에서 배양하였다.To determine the proliferative response of spleen, thymus and mesenteric lymph node cells by MTT method, the cervical spine of mice was dislocated, the spleen, thymus and mesenteric lymph nodes were removed aseptically, and each cell was washed (1500 rpm, 10 minutes). After preparation, the cell number was adjusted to 1 × 10 6 cells / well, lipopolysaccharide (Sigma, USA) 5 μg / ml for splenocyte suspension, and Concanavalin A (Sigma, USA) 0.5 μg / ml for thymic and mesenteric lymph node suspensions. addition of an activator ml T- cell and comparing the examples and the embodiment, obtain N, W old, C-ku, G old, 100 CG ㎍ the sphere rice extract, respectively / ml to be added in a CO 2 incubator at 37 ℃ for 48 h ( 5% -CO 2 , 95% -air).

배양 종료 4시간 전에 덱스트로스(Dextrose)가 가미된 인산완충액인 Dulbecco's phosphate buffered saline-A(DPBS-A, pH 7.4)(Sigma, USA)에 5 mg/ml 농도로 희석된 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide 시약(Sigma, USA) 20㎕를 각 well에 첨가하고, 0.1N HCl에 녹인 10% sodium dodecyl sulfate(SDS, Sigma, USA) 100 ㎕로 용해시켜 18시간 동안 은박지로 빛을 차단하였다. 발색된 각 well의 흡광도를 ELISA 판독기(reader)(Dynatech., MR5000, USA)를 이용해서 570nm에서 측정하고 대조군(Con A)의 흡광도와 비교하여 세포생존율을 백분율로 환산하여 그 결과를 도 1에 나타내었다.3- [4,5 diluted to 5 mg / ml in Dulbecco's phosphate buffered saline-A (DPBS-A, pH 7.4) (Sigma, USA), 4 hours prior to the end of incubation. 20 μl of -Dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide reagent (Sigma, USA) was added to each well and dissolved in 100 μl of 10% sodium dodecyl sulfate (SDS, Sigma, USA) dissolved in 0.1N HCl. The light was blocked with tinfoil for 18 hours. The absorbance of each color well was measured at 570 nm using an ELISA reader (Dynatech., MR5000, USA) and compared with the absorbance of the control group (Con A) to convert the cell viability in percentage and the results are shown in FIG. Indicated.

도 1은 현미 추출액의 투여가 면역능에 미치는 효과를 알아보기 위해 BALB/c 마우스의 림프절에서의 면역세포의 증식반응에 미치는 효과를 관찰한 결과로서 면역세포의 증식반응에 있어서는 특히 G구 및 CG구에서 다른구에 비하여 장간막 림프절세포에서 유의성 있는 증식효과가 관찰되었다.1 is a result of observing the effect on the proliferation of immune cells in lymph nodes of BALB / c mice in order to determine the effect of the administration of brown rice extract on the immune function, especially G and CG spheres in the immune cell proliferation response Significant proliferative effect was observed in mesenteric lymph node cells compared to other cells in.

<시험예 4><Test Example 4>

비장 및 흉선세포의 아집단(Subpopulation) 측정을 위해 생쥐에 7일 동안 N구, W구, C구, G구, CG구의 현미 추출액을 각각 500 mg/kg body weight 되게 경구 투여(p.o)한 다음 생쥐를 경추 탈구시켜 비장 및 흉선을 적출한 후, 비장세포 및 흉선세포 현탁액을 조제하고 1×106cells/well에 PE/FITC conjugated-anti B220 및 Thy1 monoclonal antibody와 PE-anti CD4/FITC-anti CD8 monoclonal antibody (1:40 dilution)로 이중 염색하여 4℃에서 30분간 반응시키고 laser flow cytometer(excitation: 488 nm. emission: 525 nm-FITC, 575 nm-PE)를 이용하여 비장세포(splenocytes) 및 흉선세포(thymocytes) 중의 B 혹은 T세포의 아집단을 측정하여 그 결과를 아래의 표 3에 나타내었다.To determine the subpopulation of spleen and thymus cells, mice were orally administered brown rice extracts of N, W, C, G and CG at 500 mg / kg body weight for 7 days ( po ). After splenic dislocation of mice, spleen and thymus were extracted, and splenocyte and thymic cell suspensions were prepared, and PE / FITC conjugated-anti B220 and Thy1 monoclonal antibody and PE-anti CD4 / FITC-anti in 1 × 10 6 cells / well. Double staining with CD8 monoclonal antibody (1:40 dilution) and reaction at 4 ° C. for 30 minutes and splenocytes using a laser flow cytometer (excitation: 488 nm. Emission: 525 nm-FITC, 575 nm-PE) Subpopulations of B or T cells in thymocytes were measured and the results are shown in Table 3 below.

통계처리에서 실험결과는 평균±표준편차로 표시하였으며, 각 군간의 통계적 유의성 검정은 GraphPad InStat Software(San Diego, CA, USA)를 이용하여 p<0.05 수준에서 투키 테스트(Tukey test)를 통하여 검증하였다.The statistical results were expressed as mean ± standard deviation, and the statistical significance test between each group was verified by Tukey test at p <0.05 level using GraphPad InStat Software (San Diego, CA, USA). .

표 3. 각각의 발아구 현미 추출액을 경구투여한 쥐의 비장세포(splenocytes) 및 흉선세포(thymocytes)의 아집단을 측정결과Table 3. Results of measurement of subpopulations of splenocytes and thymocytes of rats administered orally with each germinated brown rice extract

항목Item Splenocytes(%)Splenocytes (%) Thymocytes(%)Thymocytes (%) B cellB cell THT cellTHT cell TH T H TC/TSTC / TS TH T H TC/TS T C / T S ControlControl 43.8±1.743.8 ± 1.7 22.1±2.322.1 ± 2.3 11.4±0.611.4 ± 0.6 3.1±0.33.1 ± 0.3 13.7±1.113.7 ± 1.1 5.8±0.25.8 ± 0.2 N구N ball 45.2±2.945.2 ± 2.9 23.5±2.423.5 ± 2.4 12.7±1.112.7 ± 1.1 3.3±0.53.3 ± 0.5 14.8±1.414.8 ± 1.4 6.3±0.46.3 ± 0.4 W구W sphere 44.1±1.944.1 ± 1.9 23.2±2.223.2 ± 2.2 12.9±0.612.9 ± 0.6 3.5±0.43.5 ± 0.4 14.7±1.214.7 ± 1.2 6.4±0.56.4 ± 0.5 C구C sphere 47.6±2.347.6 ± 2.3 23.4±1.323.4 ± 1.3 2.9±0.32.9 ± 0.3 2.9±0.22.9 ± 0.2 13.9±1.113.9 ± 1.1 5.8±0.35.8 ± 0.3 G구G sphere 57.8±2.8* 57.8 ± 2.8 * 28.5±1.6* 28.5 ± 1.6 * 14.5±0.6*14.5 ± 0.6 * 4.3±0.44.3 ± 0.4 17.5±1.5*17.5 ± 1.5 * 7.1±0.87.1 ± 0.8 CG구CG District 51.2±2.1* 51.2 ± 2.1 * 26.7±1.826.7 ± 1.8 14.1±0.2*14.1 ± 0.2 * 3.1±0.23.1 ± 0.2 16.7±1.0*16.7 ± 1.0 * 6.2±0.56.2 ± 0.5

*: Significantly different from control group(p<0.05)*: Significantly different from control group (p <0.05)

상기 표 3에서처럼 G구 및 CG구 현미 추출액을 투여한 쥐의 비장세포 중 B세포 및 T세포의 아집단을 각각 유의성있게 증가시켰고, T세포 중 특히 CD4+세포인 TH세포가 증가되었다. 흉선세포에서도 G구 및 CG구에서 TH세포가 증가됨을 알 수 있었다.As shown in Table 3, the subpopulations of B cells and T cells were significantly increased in spleen cells of rats administered with G and CG brown rice extracts, and T H cells, particularly CD4 + cells, were increased. In thymus cells, T H cells were increased in G and CG cells.

상기 시험예의 결과로부터 글루탐산 수용액 또는 키토산 수용액과 글루탐산수용액의 혼합용액에 침지시킨 곡류는 감마-아미노부티르산 함량이 증가되었음을 알 수 있으며, 이러한 곡류의 추출액 중 감마-아미노부티르산은 면역에 대한 효과가 높아 면역제제로 이용할 수 있음을 알 수 있다.From the results of the test example, grains immersed in the mixed solution of glutamic acid solution or chitosan solution and glutamic acid solution showed that the gamma-aminobutyric acid content was increased. It can be seen that it can be used as a formulation.

Claims (7)

곡류를 침지액에 침지시켜 24∼26℃에서 2∼4일 동안 발아시키는 단계와,Immersing the grains in dipping solution for 2-4 days at 24-26 ° C., 발아한 곡류를 분말화하고 수용액에 용해한 후 살균하는 단계와,Powdered germinated grains, dissolved in an aqueous solution and then sterilized; 살균 후 1차 여과하여 찌꺼기를 제거하고 2차 여과한 여과액을 동결건조하는 단계와,After sterilization to remove the residue by the first filtration and freeze-drying the second filtered filtrate, 건조 후 건조물을 용매에 용해하여 추출하는 단계를 포함하는 것을 특징으로 하는 감마-아미노부티르산 함량이 증가된 발아곡류 추출액의 제조방법Method for producing a germinated grain extract with increased gamma-aminobutyric acid content, characterized in that it comprises the step of dissolving and drying the dried product in a solvent after drying 제 1항에 있어서, 곡류는 현미, 보리, 밀 수수, 콩 중에서 선택된 어느 하나인 것을 특징으로 하는 감마-아미노부티르산 함량이 증가된 발아곡류 추출액의 제조방법The method of claim 1, wherein the grains are any one selected from brown rice, barley, wheat sorghum, and soybean. 제 1항에 있어서, 침지액을 12시간 마다 교환하여 곡류를 침지시키는 것을 특징으로 하는 감마-아미노부티르산 함량이 증가된 발아곡류 추출액의 제조방법The method of preparing a germinated grain extract with increased gamma-aminobutyric acid content according to claim 1, wherein the dipping solution is soaked every 12 hours. 제 1항에 있어서, 침지액은 5∼10 mM 글루탐산 수용액 또는 5∼10 mM 글루탐산 수용액과 50∼100 ppm 키토산 수용액이 1:9∼9:1의 비율로 혼합된 혼합용액 임을 특징으로 하는 감마-아미노부티르산 함량이 증가된 발아곡류 추출액의 제조방법The method of claim 1, wherein the immersion solution is a mixed solution in which a 5-10 mM glutamic acid solution or a 5-10 mM glutamic acid solution and a 50-100 ppm chitosan aqueous solution are mixed in a ratio of 1: 9-9: 1. Method for preparing germinated grain extract with increased aminobutyric acid content 제 1항에 있어서, 곡류를 1∼5mm의 싹이 나도록 발아시키는 것을 특징으로 하는 감마-아미노부티르산 함량이 증가된 발아곡류 추출액의 제조방법The method of preparing a germinated grain extract with increased gamma-aminobutyric acid content according to claim 1, wherein the grains are germinated to sprout 1 to 5 mm. 제 1항에 있어서, 용매는 증류수 또는 살균수임을 특징으로 하는 감마-아미노부티르산 함량이 증가된 발아곡류 추출액의 제조방법The method of claim 1, wherein the solvent is distilled or sterilized water, wherein the gamma-aminobutyric acid content is increased. 특허청구범위 제1항 내지 제6항의 방법으로 제조한 감마-아미노부티르산 함량이 증가된 발아곡류 추출액 중 감마-아미노부티르산을 유효성분으로 함유한 면역제제An immunologic agent containing gamma-aminobutyric acid as an active ingredient in germinated grain extracts with increased gamma-aminobutyric acid content prepared by the method of claims 1 to 6
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