KR102479410B1 - Method of preparation for fermented red ginseng having increased content of ginsenoside Compound K and fermented red ginseng using the same - Google Patents
Method of preparation for fermented red ginseng having increased content of ginsenoside Compound K and fermented red ginseng using the same Download PDFInfo
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- KR102479410B1 KR102479410B1 KR1020200137473A KR20200137473A KR102479410B1 KR 102479410 B1 KR102479410 B1 KR 102479410B1 KR 1020200137473 A KR1020200137473 A KR 1020200137473A KR 20200137473 A KR20200137473 A KR 20200137473A KR 102479410 B1 KR102479410 B1 KR 102479410B1
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- KR
- South Korea
- Prior art keywords
- red ginseng
- strain
- fermented red
- ginsenoside
- strains
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Abstract
본 발명은 사포닌에 대해 생물전환능을 갖는 락토바실러스 펜토서스 KK18 균주를 이용하여 진세노사이드 화합물 K의 함량이 증가된 발효 홍삼을 제조하는 방법 및 이에 따른 발효 홍삼에 관한 것이다.
본 발명의 락토바실러스 펜토서스(Lactobacillus pentosus) 균주는 식품학적으로 안전하고, 홍삼에 함유된 사포닌을 홍삼에는 미량으로만 존재하는 진세노사이드 화합물 K 및 F2로 생물전환시키는 능력이 뛰어나다. 특히, 진세노사이드 화합물 K의 함량이 현저히 증가한 발효 홍삼을 제조할 수 있게 한다.
또한, 본 발명의 발효 홍삼의 제조방법에 따르면, 진세노사이드 화합물 K 및 F2의 함량이 현저히 증가된 발효 홍삼을 얻을 수 있다. 본 발명의 발효 홍삼은 체내 흡수가 용이한 화합물 K를 다량 함유하고 있으므로, 건강기능식품 또는 식품 소재로 매우 유용하다.The present invention relates to a method for preparing fermented red ginseng with an increased content of ginsenoside compound K using the Lactobacillus pentosus KK18 strain having bioconversion ability to saponin, and the fermented red ginseng according to the method.
The Lactobacillus pentosus strain of the present invention is food safe, and has an excellent ability to bioconvert saponin contained in red ginseng to ginsenoside compounds K and F2, which are present only in trace amounts in red ginseng. In particular, it is possible to manufacture fermented red ginseng in which the content of ginsenoside compound K is significantly increased.
In addition, according to the method for producing fermented red ginseng of the present invention, fermented red ginseng with significantly increased contents of ginsenoside compounds K and F2 can be obtained. Since the fermented red ginseng of the present invention contains a large amount of compound K that is easily absorbed by the body, it is very useful as a health functional food or food material.
Description
본 발명은 사포닌에 대해 생물전환능을 갖는 락토바실러스 펜토서스 KK18 균주를 이용하여 진세노사이드 화합물 K의 함량이 증가된 발효 홍삼을 제조하는 방법 및 이에 따른 발효 홍삼에 관한 것이다. The present invention relates to a method for preparing fermented red ginseng with an increased content of ginsenoside compound K using the Lactobacillus pentosus KK18 strain having bioconversion ability to saponin, and the fermented red ginseng according to the method.
인삼은 두릅나무과 인삼속 식물의 뿌리를 말한다. 재배산지에 따라 고려인삼(한반도), 미국삼(미국 및 캐나다), 전칠삼(중국), 죽절삼(일본) 등의 명칭으로 불리우며, 인삼의 원형을 유지하는 1차 가공방법에 따라 수삼, 홍삼, 백삼, 당삼 및 봉밀삼 등으로 구분된다.Ginseng refers to the root of a plant in the Araliaceae family. Depending on the place of cultivation, it is called by names such as Korean ginseng (Korean Peninsula), American ginseng (USA and Canada), Jeonchil ginseng (China), and Jukjeol ginseng (Japan). It is classified into white ginseng, tang ginseng and bongmil ginseng.
고려인삼(Panax ginseng C.A. Meyer)은 과거부터 우리나라를 비롯하여 중국, 일본 등에서 주로 건강증진을 위한 목적으로 널리 사용하여 왔다. 인삼의 유효성분들은 인삼의 종류, 뿌리 부위, 수확 년생, 수확 시기, 제조방법에 따라 현저한 차이가 있는 것으로 알려져 있는데, 인삼의 연근별로는 일반적으로 저년근에는 당 함량이 많고 고년근에서는 사포닌 함량이 많은 것으로 알려져 있다. 또한, 수삼을 그대로 건조시킨 백삼과 증기로 쪄서 건조시킨 홍삼 간에는 조사포닌 함량이나 진세노사이드의 종류에서 차이를 보이는 것으로 알려져 있다.Korean ginseng (Panax ginseng C.A. Meyer) has been widely used in Korea, China, and Japan since the past for the purpose of improving health. It is known that the active ingredients of ginseng differ significantly depending on the type of ginseng, the root part, the year of harvest, the harvest time, and the manufacturing method. By lotus root of ginseng, in general, younger roots have a higher sugar content and older ones have a high saponin content. It is known. In addition, it is known that white ginseng, in which fresh ginseng is dried as it is, and red ginseng, which is steamed and dried, show differences in the content of irradiated ginseng or the types of ginsenosides.
건강기능식품에서 말하는 '홍삼'은 인삼을 원재료로 사용하여, 말리지 않은 수삼을 증기 또는 기타 방법으로 쪄서 익혀 말린 것이다. 'Red ginseng' referred to in health functional food uses ginseng as a raw material, and undried fresh ginseng is steamed, cooked, and dried by steam or other methods.
인삼 및 홍삼의 효능 성분으로 알려진 것은 사포닌(saponin)이다. 인삼 및 홍삼의 사포닌은 배당체의 일종으로 다른 식물에서 발견되는 사포닌과는 다른 특이한 화학구조로 되어 있으며, 그 효능도 크게 차이가 난다. 인삼 및 홍삼의 사포닌은 타 식물계 사포닌과 구분하기 위해 인삼(ginseng) 배당체(glycoside)라는 의미로 '진세노사이드(Ginsenoside)'라고 부른다.Saponin is known as an effective component of ginseng and red ginseng. Saponin of ginseng and red ginseng is a type of glycoside and has a unique chemical structure different from saponin found in other plants, and its efficacy is also greatly different. Saponins of ginseng and red ginseng are called 'ginsenosides' in the sense of ginseng glycosides to distinguish them from saponins of other plant origin.
인삼 및 홍삼의 유효성분인 진세노사이드는 트리터페노이드(triterpenoid)의 담마렌(dammarane) 골격에 글루코오스(glucose), 아라비노오스(arabinose), 자일로오스(xylose) 및 람노오스(rhamnose) 등의 당이 하나 또는 여러 개가 결합된 배당체(glycoside)로 현재 약 100 여종 이상 분리되었으며(J Ginseng Res 41 (2017), 435-443), 고분자 진세노사이드인 진세노사이드 Rb1, Rb2, Rc, Rd, Re 및 Rg1을 포함한 6 종 진세노사이드가 총 진세노사이드의 90%를 차지하고 있다.Ginsenoside, an active ingredient of ginseng and red ginseng, contains glucose, arabinose, xylose and rhamnose in the dammarane skeleton of triterpenoids. Ginsenosides Rb1, Rb2, Rc, Rd, which are high-molecular ginsenosides, are currently isolated over 100 species as glycosides in which one or several sugars are linked (J Ginseng Res 41 (2017), 435-443). Six ginsenosides, including , Re and Rg1, account for 90% of the total ginsenosides.
고분자 진세노사이드가 가수분해 되어 생성된 소량의 저분자 진세노사이드 중 하나인 화합물 K(C-K: compound K, 20-O-β-D-glucopyranosyl 20(S)-protopanasadiol)는 기본 골격인 아글리콘(aglycon)의 C-20 번에 하나의 글루코오스가 결합되어 있는 PPD계 저분자 진세노사이드의 하나로 진세노사이드 Rb1, Rb2, Rc가 장내 미생물에 의해 전환된 전환산물이다(Akoa et al., 1998, Akao et al., 1998, Hasegawa et al.,1997; Bae et al., 2000).Compound K (C-K: compound K, 20-O-β-D-glucopyranosyl 20(S)-protopanasadiol), which is one of a small amount of low-molecular-weight ginsenosides produced by hydrolysis of high-molecular ginsenosides, has an aglycone ( It is one of the PPD-based low-molecular-weight ginsenosides in which one glucose is linked to C-20 of the aglycon), and is a conversion product of ginsenosides Rb1, Rb2, and Rc by intestinal microorganisms (Akoa et al., 1998, Akao et al., 1998, Hasegawa et al., 1997; Bae et al., 2000).
상기 화합물 K의 화학 구조는 하기 화학식 1에 나타내었다.The chemical structure of the compound K is shown in
[화학식 1][Formula 1]
Akao 등(1998)의 연구에 의하면 진세노사이드 Rb1을 랫(rat)에 투여했을 때 플라즈마에서 화합물 K가 진세노사이드 Rb1 투여 후 24시간 동안 검출되었으며, 위에서는 거의 대사 되지 않았음이 보고되었고, Karikura 등(1991)이 랫(rat)의 위에서 진세노사이드 Rb2의 대사산물을 조사한 바에 의하면 과산화물의 생성을 제외하고 진세노사이드 Rb2의 대사는 거의 이루어지지 않는 것을 볼 때, 진세노사이드는 위 등의 소화기관에서는 거의 대사가 이루어지지 않고 장내 미생물에 의해서 대사되는 것으로 판단된다. 또한, 김동현 교수의 연구에 따르면 한국인 98명의 장내 미생물을 이용하여 진세노사이드 Rb1의 전환능력을 확인한 결과 20%는 진세노사이드 대사 능력이 없거나 현저히 낮고, 약 60% 정도만이 진세노사이드 전환 가능한 미생물을 가지고 있어 많은 사람들이 진세노사이드를 효율적으로 대사하지 못하는 것으로 보고되었다. 이러한 보고는 개인별 장내 미생물 구성의 차이 및 장내 미생물의 효소활성의 차이로 인삼이나 홍삼을 복용한다 하더라도 개인에 따라 효능의 차이가 있으며 이 차이를 줄이고자 발효 홍삼 섭취를 권장한다.According to a study by Akao et al. (1998), when ginsenoside Rb1 was administered to rats, compound K was detected in plasma 24 hours after administration of ginsenoside Rb1, and it was reported that it was hardly metabolized in the stomach. According to Karikura et al. (1991) examining the metabolites of ginsenoside Rb2 in the stomach of rats, ginsenoside Rb2 is hardly metabolized except for the production of peroxide. Almost no metabolism occurs in the digestive system of , and it is judged that it is metabolized by intestinal microorganisms. In addition, according to Professor Kim Dong-hyun's study, the conversion ability of ginsenoside Rb1 was confirmed using the intestinal microorganisms of 98 Koreans, and as a result, 20% had no or significantly low ginsenoside metabolism ability, and only about 60% of the microorganisms could convert ginsenoside. It has been reported that many people do not metabolize ginsenosides efficiently. These reports show that even if ginseng or red ginseng is taken, there is a difference in efficacy depending on the individual due to differences in the composition of intestinal microorganisms and enzyme activity of intestinal microorganisms, and to reduce this difference, intake of fermented red ginseng is recommended.
종래에는 전통적인 홍삼제조의 단위공정인 증자, 건조, 정형 등의 방법만을 사용하여 제품을 제조하였으나, 최근 홍삼의 발효방법에 따라 효능이 달라짐이 발견되면서 새로운 제조방법이 나타났으며, 주로 열처리, 산도처리, 효소처리 및 미생물을 이용하는 방법들이 다양하게 연구되고 있다.Conventionally, products were manufactured using only methods such as steaming, drying, and forming, which are the unit processes of traditional red ginseng manufacturing. Treatment, enzymatic treatment, and methods using microorganisms are being studied in various ways.
그 중에서도 화합물 K의 제조방법으로 장내 미생물인 비피도박테리움(Bifidobacterium), 곰팡이 또는 각종 미생물에 의한 생산이 공지되어있으나, 상기 방법은 혐기성 조건에서의 배양 및 오랜 배양시간이 요구되며 생성 수율이 극히 낮을 뿐 아니라 부산물이 생성되어 생산적인 면에서 수율이 낮고 식용의 어려움이 있어, 유산균을 이용한 발효를 통해 특정 저분자 진세노사이드인 화합물 K를 다량 함유하는 발효홍삼의 제조방법에 대한 연구 및 개발이 요구되어 진다.Among them, as a method for producing compound K, production by intestinal microorganisms such as Bifidobacterium , fungi, or various microorganisms is known, but the method requires incubation under anaerobic conditions and a long incubation time, and the production yield is extremely low. Research and development of a method for manufacturing fermented red ginseng containing a large amount of compound K, a specific low-molecular-weight ginsenoside, through fermentation using lactic acid bacteria is required. it becomes
본 발명자들은 화합물 K의 함량이 증대된 발효 홍삼을 개발하고자 연구하는 과정에서, 진세노사이드 생물전환능이 우수한 유산균을 선별하였고, 상기 선별된 균주를 이용하여 발효시킨 발효 홍삼에서 화합물 K의 함량이 크게 증가한 것을 확인함으로써 본 발명을 완성하였다.In the course of research to develop fermented red ginseng with increased compound K content, the present inventors selected lactic acid bacteria with excellent ginsenoside bioconversion ability, and fermented red ginseng fermented using the selected strain had a high compound K content. The present invention was completed by confirming the increase.
본 발명의 해결하고자 하는 과제는 사포닌에 대해 생물전환능을 갖는 락토바실러스 펜토서스(Lactobacillus pentosus) KK18 균주(수탁번호: KCCM12762P)를 이용하여 진세노사이드 화합물 K의 함량이 증가된 발효 홍삼 제조방법을 제공하는 것이다.The problem to be solved by the present invention is a method for producing fermented red ginseng with increased content of ginsenoside compound K using Lactobacillus pentosus KK18 strain (accession number: KCCM12762P) having bioconversion ability to saponin. is to provide
본 발명의 다른 과제는 상기 방법에 따라 제조되어 발효 전에 비해 진세노사이드 화합물 K의 함량이 증가된 발효 홍삼을 제공하는 것이다.Another object of the present invention is to provide fermented red ginseng prepared according to the above method and having an increased content of ginsenoside compound K compared to before fermentation.
본 발명의 또 다른 과제는 상기 발효 홍삼을 포함하는 식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition containing the fermented red ginseng.
상기한 목적을 달성하기 위하여 본 발명은 본 발명은 (1) 홍삼을 분쇄하여 100 내지 300 메쉬 크기의 홍삼 분말을 제조하는 단계; (2) 정제수에 락토바실러스 펜토서스(Lactobacillus pentosus) KK18 균주(수탁번호: KCCM12762P) 균주를 접종하고 균질화하여 균주 현탁액을 제조하는 단계; 및 (3) 상기 홍삼 분말 100 중량부에 대하여 상기 균주 현탁액 1 내지 10 중량부를 혼합한 후 발효시키는 단계;를 포함하는 진세노사이드 화합물 K의 함량이 증가된 발효 홍삼의 제조방법을 제공한다.In order to achieve the above object, the present invention provides (1) preparing red ginseng powder having a size of 100 to 300 mesh by pulverizing red ginseng; (2) preparing a strain suspension by inoculating a Lactobacillus pentosus strain KK18 (accession number: KCCM12762P) strain into purified water and homogenizing the strain; and (3) mixing 1 to 10 parts by weight of the strain suspension with respect to 100 parts by weight of the red ginseng powder and then fermenting.
본 발명의 일 실시예에 의하면, 상기 (1) 단계의 홍삼 분말은 분쇄 공간의 온도를 50 ℃ 이하로 유지하는 저온 분쇄, 또는 -3 내지 -10℃에서 냉동 분쇄를 수행하여 분쇄된 것일 수 있다.According to one embodiment of the present invention, the red ginseng powder in step (1) may be pulverized by low-temperature pulverization maintaining the temperature of the pulverization space at 50 ° C or less, or frozen pulverization at -3 to -10 ° C. .
본 발명의 일 실시예에 의하면, 상기 (2) 단계의 정제수는 유기산을 이용하여 pH 4.8 내지 6.4로 조정한 것일 수 있다.According to one embodiment of the present invention, the purified water in step (2) may be adjusted to pH 4.8 to 6.4 using an organic acid.
본 발명의 일 실시예에 의하면, 상기 (2) 단계의 균주는 정제수에 대하여 0.5 내지 5 중량%로 접종할 수 있다.According to one embodiment of the present invention, the strain in step (2) may be inoculated at 0.5 to 5% by weight with respect to purified water.
본 발명의 일 실시예에 의하면, 상기 (3) 단계의 발효는 32 내지 42 ℃에서 60 내지 120 시간 동안 수행될 수 있다.According to one embodiment of the present invention, the fermentation in step (3) may be performed at 32 to 42 ° C. for 60 to 120 hours.
또한, 본 발명은 상기 방법에 따라 제조되어, 발효 전에 비해 진세노사이드 화합물 K의 함량이 증가된 발효 홍삼을 제공한다.In addition, the present invention provides fermented red ginseng prepared according to the above method and having an increased content of ginsenoside compound K compared to before fermentation.
본 발명의 일 실시예에 의하면, 상기 발효 홍삼은 진세노사이드 화합물 K의 함량이 3.0 mg/g 이상일 수 있다. According to one embodiment of the present invention, the content of ginsenoside compound K in the fermented red ginseng may be 3.0 mg/g or more.
본 발명의 일 실시예에 의하면, 상기 발효 홍삼은 진세노사이드 F2의 함량이 3.5 mg/g 이상일 수 있다.According to one embodiment of the present invention, the content of ginsenoside F2 in the fermented red ginseng may be 3.5 mg/g or more.
또한, 본 발명은 상기 발효 홍삼을 포함하는 식품 조성물을 제공한다.In addition, the present invention provides a food composition containing the fermented red ginseng.
본 발명의 락토바실러스 펜토서스(Lactobacillus pentosus) 균주는 식품학적으로 안전하고, 홍삼에 함유된 사포닌을 홍삼에는 미량으로만 존재하는 진세노사이드 화합물 K 및 F2로 생물전환시키는 능력이 뛰어나다. 특히, 진세노사이드 화합물 K의 함량이 현저히 증가한 발효 홍삼을 제조할 수 있게 한다.The Lactobacillus pentosus strain of the present invention is food safe, and has an excellent ability to bioconvert saponin contained in red ginseng to ginsenoside compounds K and F2, which are present only in trace amounts in red ginseng. In particular, it is possible to manufacture fermented red ginseng in which the content of ginsenoside compound K is significantly increased.
또한, 본 발명의 발효 홍삼의 제조방법에 따르면, 진세노사이드 화합물 K 및 F2의 함량이 현저히 증가된 발효 홍삼을 얻을 수 있다. 본 발명의 발효 홍삼은 체내 흡수가 용이한 화합물 K를 다량 함유하고 있으므로, 건강기능식품 또는 식품 소재로 매우 유용하다. In addition, according to the method for producing fermented red ginseng of the present invention, fermented red ginseng with significantly increased contents of ginsenoside compounds K and F2 can be obtained. Since the fermented red ginseng of the present invention contains a large amount of compound K that is easily absorbed by the body, it is very useful as a health functional food or food material.
도 1은 β-글루코시다아제 활성을 갖는 균주의 선발을 위해 에스쿨린(esculin)이 함유된 MRS 한천 배지에 균주를 접종하고 배양한 후의 색깔 변화를 관찰한 사진이다.
도 2는 홍삼 추출물에 효소 처리를 하고, β-글루코시다아제 활성이 있는 것으로 확인된 13 종의 균주를 각각 접종한 후 발효시킨 홍삼 발효 추출물을 TLC 분석한 결과이다.
도 3a는 홍삼 추출물에 G69, CK46 및 KK18 균주를 각각 접종한 후 발효시킨 홍삼 발효 추출물을 HPLC 분석한 결과이고, 도 3b는 홍삼 추출물에 G69, CK46 및 KK18 균주를 각각 접종한 후 발효시킨 홍삼 발효 추출물을 TLC 분석한 결과 및 발효기간에 따른 Rg3 함량을 나타내는 그래프이다.
도 4a는 홍삼 추출물에 효소 처리를 한 다음, G69, CK46 및 KK18 균주를 각각 접종한 후 발효시킨 홍삼 발효 추출물을 HPLC 분석한 결과이고, 도 4b는 홍삼 추출물에 효소 처리를 한 다음, G69, CK46 및 KK18 균주를 각각 접종한 후 발효시킨 홍삼 발효 추출물을 TLC 분석한 결과 및 발효기간에 따른 화합물 K의 함량을 나타내는 그래프이다.
도 5는 KK18, G69 및 CK46 균주의 DPPH 라디칼 소거능, ABTS 라디칼 소거능, 및 FRAP 환원력을 나타내는 그래프이다.
도 6은 각 균주의 효소 활성을 확인하고자 KK18, G69 및 CK46 균주와 반응시킨 API ZYM 키트의 색상 변화를 나타내는 사진이다.
도 7은 각 균주의 항생제 내성을 확인하고자 항생제 처리에 의한 KK18, G69 및 CK46 균주의 생육 저해환을 나타내는 사진이다.
도 8은 최종 선별된 본 발명의 락토바실러스 펜토서스 KK18 균주의 유전체 분석 결과서이다.
도 9는 본 발명의 일 실시예에 따라 제조된 발효 홍삼의 진세노사이드 함량을 HPLC를 이용하여 분석한 결과를 나타내는 크로마토그램이다. 1 is a photograph showing color changes after inoculating and culturing strains on MRS agar medium containing esculin for selection of strains having β-glucosidase activity.
Figure 2 is the result of TLC analysis of the fermented red ginseng extract fermented after enzymatic treatment of the red ginseng extract, inoculation of 13 strains confirmed to have β-glucosidase activity, respectively.
Figure 3a is the result of HPLC analysis of the fermented red ginseng extract fermented after inoculating the red ginseng extract with G69, CK46 and KK18 strains, respectively, and Figure 3b is the red ginseng fermentation fermented after inoculating the red ginseng extract with G69, CK46 and KK18 strains, respectively. It is a graph showing the result of TLC analysis of the extract and the Rg3 content according to the fermentation period.
Figure 4a is the result of HPLC analysis of the fermented red ginseng extract fermented after enzymatic treatment of red ginseng extract and then inoculation of G69, CK46 and KK18 strains, respectively. and KK18 strains, respectively, and a graph showing the result of TLC analysis of the fermented red ginseng extract fermented after inoculation and the content of Compound K according to the fermentation period.
5 is a graph showing DPPH radical scavenging ability, ABTS radical scavenging ability, and FRAP reducing power of KK18, G69 and CK46 strains.
Figure 6 is a photograph showing the color change of the API ZYM kit reacted with KK18, G69 and CK46 strains to confirm the enzyme activity of each strain.
Figure 7 is a photograph showing the growth inhibition rings of KK18, G69 and CK46 strains by antibiotic treatment to confirm the antibiotic resistance of each strain.
8 is a genome analysis result of the finally selected Lactobacillus pentosus KK18 strain of the present invention.
9 is a chromatogram showing the results of analyzing the ginsenoside content of fermented red ginseng prepared according to an embodiment of the present invention using HPLC.
이하, 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 일 구현예에 따르면, 본 발명은 사포닌에 대해 생물전환능을 갖는 락토바실러스 펜토서스(Lactobacillus pentosus) KK18 균주(수탁번호: KCCM12762P)를 제공한다. According to one embodiment of the present invention, the present invention provides a Lactobacillus pentosus strain KK18 (accession number: KCCM12762P) having bioconversion ability to saponin.
본 발명의 락토바실러스 펜토서스(Lactobacillus pentosus) KK18 균주(수탁번호: KCCM12762P)는 김치 유래의 락토바실러스 펜토서스 신규 균주이다. 비록 본 발명에서의 락토바실러스 펜토서스 KK18 균주를 김치로부터 분리, 동정하기는 하였으나, 입수 경로가 이에 한정되는 것은 아니다.The Lactobacillus pentosus KK18 strain (Accession Number: KCCM12762P) of the present invention is a novel Lactobacillus pentosus strain derived from kimchi. Although the Lactobacillus pentosus KK18 strain in the present invention was isolated and identified from kimchi, the acquisition route is not limited thereto.
하기 실시예를 통해 김치로부터 분리한 KK18 균주는 미생물 동정 및 분류를 위한 16S rRNA 염기서열 분석 결과, 서열번호 1의 염기서열을 갖는 것으로 나타났다. 상기 염기서열은 NCBI GenBank(http://www.ncbi.nin.gov)에서 제공하는 블라스트 서치 프로그램(blast search program)을 통해 상동성(homology)을 확인해 본 결과, 락토바실러스 펜토서스 124-2 균주와 99% 동일함을 확인할 수 있었다.As a result of 16S rRNA sequencing analysis for microbial identification and classification, the KK18 strain isolated from kimchi through the following examples was found to have the nucleotide sequence of SEQ ID NO: 1. As a result of confirming homology through the blast search program provided by NCBI GenBank (http://www.ncbi.nin.gov), the nucleotide sequence was Lactobacillus pentosus 124-2 strain. was found to be 99% identical.
따라서, 서열번호 1의 16S rRNA 염기서열을 갖는 본 발명의 미생물을 락토바실러스 펜토서스(Lactobacillus pentosus) KK18 균주로 명명하였으며, 한국종균협회(KCCM)에 2020년 7월 8일자로 기탁하였다(수탁번호 KCCM12762P).Therefore, the microorganism of the present invention having the 16S rRNA nucleotide sequence of SEQ ID NO: 1 was named Lactobacillus pentosus KK18 strain, and was deposited with the Korea Breeder Association (KCCM) on July 8, 2020 (accession number KCCM12762P).
본 발명의 락토바실러스 펜토서스 KK18 균주는 β-글루코시다아제 활성이 매우 높고, 내산성 및 내담즙성을 가지며, 항산화 활성을 나타내고, 항생제 내성을 가지므로 산업적으로 매우 유용하다.The Lactobacillus pentosus KK18 strain of the present invention has very high β-glucosidase activity, acid resistance and bile resistance, antioxidant activity, and antibiotic resistance, so it is industrially very useful.
본 발명의 락토바실러스 펜토서스 KK18 균주는 홍삼 사포닌을 특정 진세노사이드인 화합물 K 및 F2로 전환시켜 특정 진세노사이드의 함량이 증가된 발효 홍삼을 제조할 수 있게 하므로, 건강 기능성 식품 소재의 제조에 매우 유용하다.The Lactobacillus pentosus KK18 strain of the present invention converts red ginseng saponin into specific ginsenosides, Compounds K and F2, so that fermented red ginseng with increased content of specific ginsenosides can be produced. Very useful.
본 발명의 다른 구현예에 따르면, 본 발명은 (1) 홍삼을 분쇄하여 100 내지 300 메쉬 크기의 홍삼 분말을 제조하는 단계; (2) 정제수에 락토바실러스 펜토서스(Lactobacillus pentosus) KK18 균주(수탁번호: KCCM12762P) 균주를 접종하고 균질화하여 균주 현탁액을 제조하는 단계; 및 (3) 상기 홍삼 분말 100 중량부에 대하여 상기 균주 현탁액 1 내지 10 중량부를 혼합한 후 발효시키는 단계;를 포함하는 진세노사이드 화합물 K의 함량이 증가된 발효 홍삼의 제조방법을 제공한다.According to another embodiment of the present invention, the present invention comprises (1) preparing red ginseng powder having a size of 100 to 300 mesh by grinding red ginseng; (2) preparing a strain suspension by inoculating a Lactobacillus pentosus strain KK18 (accession number: KCCM12762P) strain into purified water and homogenizing the strain; and (3) mixing 1 to 10 parts by weight of the strain suspension with respect to 100 parts by weight of the red ginseng powder and then fermenting.
먼저, 상기 (1) 단계에서는 홍삼을 분쇄하여 100 내지 300 메쉬 크기의 홍삼 분말을 제조한다.First, in step (1), red ginseng is pulverized to prepare red ginseng powder having a mesh size of 100 to 300.
본 발명에 의하면, 상기 홍삼은 홍삼 추출물, 홍삼 농축액, 홍삼 엑기스 또는 홍삼 분말일 수 있으나, 진세노사이드 화합물 K의 증가 면에서 홍삼 분말인 것이 바람직하다. 홍삼 분말의 형태로 발효하는 것이 미생물에 의해 생성되는 효소의 반응표면 면적이 확대되어 효율적으로 유효성분을 증가시킬 수 있기 때문이다. 일반적으로 유산균은 그 크기가 약 50 ㎛이므로, 상기 100 내지 300 메쉬 범위가 유산균이 최적으로 작용할 수 있는 범위이다. According to the present invention, the red ginseng may be red ginseng extract, red ginseng concentrate, red ginseng extract or red ginseng powder, but is preferably red ginseng powder in view of the increase in ginsenoside compound K. This is because fermenting in the form of red ginseng powder can increase the active ingredient efficiently by expanding the reaction surface area of enzymes produced by microorganisms. In general, since lactic acid bacteria have a size of about 50 μm, the 100 to 300 mesh range is the range in which lactic acid bacteria can function optimally.
상기 홍삼 분말은 저온 분쇄 또는 냉동 분쇄하여 100 내지 300 메쉬, 바람직하게는 150 내지 250 메쉬, 더욱 바람직하게는 150 내지 200 메쉬의 입도로 분쇄된 것일 수 있다. The red ginseng powder may be pulverized to a particle size of 100 to 300 mesh, preferably 150 to 250 mesh, and more preferably 150 to 200 mesh by cold pulverization or freeze pulverization.
홍삼은 그 조직이 단단하여 분말화하는 것이 매우 어렵다. 일반적으로 삼 분쇄업소에서 사용하는 사이런스커터로는 홍삼을 100 메쉬 이하로 분쇄하는 것은 기계에 무리가 갈뿐만 아니라 분쇄 과정에서 다량의 쇳가루가 발생되는 문제가 있었다. 이에 본 발명에서는 홍삼을 조분쇄한 후 미세 분말화하는 공정을 수행하여 100 내지 300 메쉬, 바람직하게는 150 내지 250 메쉬, 더욱 바람직하게는 150 내지 200 메쉬 크기의 미세분말로 제조하였다. 이때의 홍삼의 입도가 150 내지 200 메쉬인 경우 약 9.9 내지 14 ㎛가 된다. 시중에 유통되는 일반적인 홍삼 분말은 50 메쉬 크기의 체를 통과하지 못하는 분말의 양이 약 80%이상으로, 이때의 홍삼 분말의 직경은 0.14 mm이다. 상기 일반 홍삼 분말과 본 발명의 홍삼 분말의 표면적을 비교하면 입자의 크기가 1/10 이하이므로 표면적은 약 1000배 이상 차이가 난다. Red ginseng has a hard structure, so it is very difficult to powder it. In general, crushing red ginseng to less than 100 mesh with a silent cutter used in ginseng crushing businesses not only puts strain on the machine, but also causes a large amount of iron powder to be generated during the crushing process. Therefore, in the present invention, red ginseng is coarsely ground and then subjected to a process of finely powdering to prepare a fine powder having a size of 100 to 300 mesh, preferably 150 to 250 mesh, and more preferably 150 to 200 mesh. At this time, when the particle size of red ginseng is 150 to 200 mesh, it is about 9.9 to 14 μm. In general red ginseng powder distributed on the market, the amount of powder that does not pass through a 50 mesh sieve is about 80% or more, and the diameter of red ginseng powder at this time is 0.14 mm. Comparing the surface area of the general red ginseng powder and the red ginseng powder of the present invention, the size of the particles is less than 1/10, so the surface area is about 1000 times or more different.
이와 같이 100 메쉬 이하의 홍삼 미세 분말을 이용하면, 종래의 방법과 같이 반응성을 높이기 위하여 홍삼을 추출하여 추출물 또는 농축액으로 제조하는 공정, 또는 효소를 처리하는 공정을 수행하지 않고, 진세노사이드의 화합물 K로의 전환 반응을 진행시킬 수 있어, 제조 공정을 더욱 단순화하고 생산 단가를 낮출 수 있어 경제적이다. In this way, when red ginseng fine powder of 100 mesh or less is used, the process of extracting red ginseng to increase reactivity and preparing a process of extract or concentrate or the process of treating enzymes is not performed, as in the conventional method, to compound ginsenosides. It is economical because the conversion reaction to K can proceed, further simplifying the manufacturing process and lowering the production cost.
또한, 상기 홍삼은 저온, 예를 들면 분쇄 공간의 온도를 50 ℃ 이하로 유지하여 투입되는 재료를 100 내지 300 메쉬의 입도로 분쇄할 수 있는 저온 분쇄기 또는 냉각기를 부착한 분쇄기를 이용하는 것이 좋다. 본 발명에서 저온 분쇄기 또는 냉각기를 부착한 분쇄기를 이용하여 홍삼을 분쇄하는 이유는 일반 분쇄기를 사용해서 Up(Ultrafine powder)를 생산할 경우, 분쇄 중 물리적인 마찰에 의해 분쇄 공간의 온도가 약 100 ℃까지 상승하므로 열에 약한 홍삼의 생리활성 물질들이 파괴되거나 홍삼 분쇄물이 슬러리 상태로 변할 수 있다. 따라서, 냉각 시스템을 사용하여 분쇄기의 외부를 -18 내지 -20℃로 냉각 시켜서 분쇄 공간의 온도를 50℃ 이하, 바람직하게는 -3 내지 -10℃에서 분쇄함으로서 홍삼의 유효성분의 손실을 최소화하고 슬러리 상태로 변하는 것을 방지하는 것이 바람직하다.In addition, it is preferable to use a low-temperature grinder or a grinder equipped with a cooler that can grind the input material to a particle size of 100 to 300 mesh by maintaining the temperature of the red ginseng at a low temperature, for example, 50 ° C. or less. In the present invention, the reason why red ginseng is pulverized using a low-temperature pulverizer or a pulverizer equipped with a cooler is that when Up (Ultrafine powder) is produced using a general pulverizer, the temperature of the pulverization space rises to about 100 ℃ due to physical friction during pulverization. As it rises, the physiologically active substances of red ginseng, which are weak against heat, may be destroyed or the pulverized red ginseng product may change to a slurry state. Therefore, the outside of the grinder is cooled to -18 to -20 ° C using a cooling system, and the temperature of the grinding space is 50 ° C or less, preferably -3 to -10 ° C. The loss of active ingredients of red ginseng is minimized and It is desirable to prevent turning into a slurry state.
일 구체예에서, 상기 홍삼 분말은 (a) 홍삼을 상온에서 1 내지 50 메쉬로 조분쇄하는 단계; (b) 상기 조분쇄한 홍삼을 분쇄 공간이 -3 내지 -10℃로 유지되는 분쇄기로 분쇄하여 100 내지 300 메쉬로 분쇄하는 단계;를 포함하는 방법으로 제조될 수 있다.In one embodiment, the red ginseng powder is prepared by: (a) coarsely pulverizing red ginseng to a size of 1 to 50 mesh at room temperature; (b) pulverizing the coarsely pulverized red ginseng with a grinder in which the grinding space is maintained at -3 to -10 ° C to pulverize the coarsely pulverized red ginseng into 100 to 300 mesh;
다음으로, 상기 (2) 단계에서는 정제수에 락토바실러스 펜토서스(Lactobacillus pentosus) KK18 균주(수탁번호: KCCM12762P) 균주를 접종하고 균질화하여 균주 현탁액을 제조한다. Next, in step (2), purified water is inoculated with Lactobacillus pentosus strain KK18 (accession number: KCCM12762P) strain and homogenized to prepare a strain suspension.
본 발명에서, 상기 정제수는 유기산을 이용하여 pH 4.8 내지 6.4, 바람직하게는 pH 5.2 내지 6.0, 더욱 바람직하게는 pH 5.4 내지 5.8로 조정하는 것이 본 발명의 락토바실러스 펜토서스 KK18 균주 발효시 진세노사이드 화합물 K의 전환율을 증가시키므로 바람직하다. In the present invention, the pH of the purified water is adjusted to pH 4.8 to 6.4, preferably pH 5.2 to 6.0, and more preferably pH 5.4 to 5.8 using an organic acid. It is preferred because it increases the conversion of compound K.
상기 락토바실러스 펜토서스 KK18 균주의 발효를 위한 균주 현탁액의 pH가 상기 범위를 벗어나는 경우에는 진세노사이드 생물전환율, 특히 화합물 K의 전환율이 기대치를 달성하지 못하게 된다.When the pH of the strain suspension for fermentation of the Lactobacillus pentosus KK18 strain is out of the above range, the ginsenoside bioconversion rate, in particular the conversion rate of compound K, does not achieve the expected value.
상기 락토바실러스 펜토서스 KK18 균주는 상기 정제수에 대하여 0.5 내지 5중량%, 바람직하게는 1.0 내지 3.0 중량%, 더욱 바람직하게는 1.5 내지 2.5 중량%로 접종할 수 있다.The Lactobacillus pentosus KK18 strain may be inoculated at 0.5 to 5% by weight, preferably 1.0 to 3.0% by weight, more preferably 1.5 to 2.5% by weight, based on the purified water.
상기 균주 접종량이 상기 하한치 미만인 경우에는 발효 속도가 지연될 수 있고, 상기 상한치를 초과하는 경우에는 이상 발효가 진행될 수 있고 경제적 효용성이 떨어진다. If the inoculated amount of the strain is less than the lower limit, the fermentation rate may be delayed, and if it exceeds the upper limit, abnormal fermentation may proceed and economic efficiency is reduced.
다음으로, 상기 (3) 단계에서는 상기 홍삼 분말 100 중량부에 대하여 상기 균주 현탁액 1 내지 10 중량부, 바람하게는 2 내지 8 중량부, 더욱 바람직하게는 4 내지 6 중량부를 혼합한 후 고체발효시킨다.Next, in the step (3), 1 to 10 parts by weight, preferably 2 to 8 parts by weight, more preferably 4 to 6 parts by weight of the strain suspension is mixed with respect to 100 parts by weight of the red ginseng powder, followed by solid fermentation. .
상기 홍삼 분말에 대한 균주 현탁액의 혼합량이 상기 하한치 미만인 경우에는 발효가 제대로 진행되지 않을 수 있으며, 상기 상한치를 초과하는 경우에는 수분 함량이 과다하여 이상 발효가 진행될 수 있다. If the mixing amount of the strain suspension with respect to the red ginseng powder is less than the lower limit, fermentation may not proceed properly, and if it exceeds the upper limit, abnormal fermentation may proceed due to excessive moisture content.
본 발명에서, 상기 발효는 32 내지 42 ℃, 바람직하게는 35 내지 40 ℃에서 60 내지 120 시간, 바람직하게는 80 내지 110 시간, 더욱 바람직하게는 90 내지 105 시간 동안 수행될 수 있다.In the present invention, the fermentation may be carried out at 32 to 42 ° C, preferably 35 to 40 ° C for 60 to 120 hours, preferably 80 to 110 hours, more preferably 90 to 105 hours.
상기 발효 온도가 상기 하한치 미만인 경우에는 발효 기간이 길어져 잡균 오염을 초래할 수 있고, 상기 상한치를 초과하는 경우에는 균주의 생육이 정지될 수 있다. 또한, 상기 발효 시간이 상기 하한치 미만인 경우에는 발효가 충분하지 않아 진세노사이드 전환율이 저조할 수 있으며, 상기 상한치를 초과하는 경우에는 발효에 의해 생성된 진세노사이드가 분해되기 시작하여 오히려 함량이 감소할 수 있다.If the fermentation temperature is less than the lower limit, the fermentation period may be prolonged, resulting in bacterial contamination, and if the fermentation temperature exceeds the upper limit, the growth of the strain may be stopped. In addition, when the fermentation time is less than the lower limit, fermentation is not sufficient and the ginsenoside conversion rate may be low, and when the fermentation time exceeds the upper limit, the ginsenoside produced by fermentation starts to decompose, and the content is rather reduced can do.
또한, 본 발명의 다른 구현예에 따르면, 본 발명은 상기 방법에 따라 제조되어, 발효 전에 비해 진세노사이드 화합물 K의 함량이 증가된 발효 홍삼을 제공한다.In addition, according to another embodiment of the present invention, the present invention provides fermented red ginseng prepared according to the above method and having an increased content of ginsenoside compound K compared to before fermentation.
상기 "진세노사이드 화합물 K의 함량" 및 "발효 홍삼의 제조방법"에 대한 내용은 전술한 바와 같다.The contents of the "content of ginsenoside compound K" and "method for producing fermented red ginseng" are as described above.
상기 발효 홍삼은 진세노사이드 화합물 K를 3.0 mg/g 이상, 바람직하게는 3.0 내지 5.0 mg/g, 더욱 바람직하게는 3.0 내지 4.0 mg/g로 포함할 수 있다. The fermented red ginseng may contain ginsenoside compound K at 3.0 mg/g or more, preferably at 3.0 to 5.0 mg/g, and more preferably at 3.0 to 4.0 mg/g.
또한, 상기 발효 홍삼은 진세노사이드 F2를 3.5 mg/g 이상, 바람직하게는 3.5 내지 5.5 mg/g, 더욱 바람직하게는 3.5 내지 5.0 mg/g로 포함할 수 있다. In addition, the fermented red ginseng may contain ginsenoside F2 at 3.5 mg/g or more, preferably at 3.5 to 5.5 mg/g, and more preferably at 3.5 to 5.0 mg/g.
본 발명의 발효 홍삼은 홍삼을 원료로 하여 제조되고, 홍삼에는 미량으로만 존재하는 진세노사이드 화합물 K 및 F2의 함량이 각각 3.0 mg/g 이상이므로, 건강기능식품 또는 식품 소재로 매우 유용하다.The fermented red ginseng of the present invention is prepared using red ginseng as a raw material, and since the content of ginsenoside compounds K and F2, which exist only in trace amounts in red ginseng, is 3.0 mg/g or more, respectively, it is very useful as a health functional food or food material.
또한, 본 발명의 또 다른 구현예에 따르면, 본 발명은 상기 발효 홍삼을 유효성분으로 포함하는 식품 조성물을 제공한다. In addition, according to another embodiment of the present invention, the present invention provides a food composition comprising the fermented red ginseng as an active ingredient.
상기 식품 조성물은 진세노사이드 화합물 K 및 F2의 함량이 각각 3.0 mg/g 이상인 발효 홍삼을 유효성분으로 포함한다. The food composition includes fermented red ginseng having a content of ginsenoside compounds K and F2 of 3.0 mg/g or more, respectively, as an active ingredient.
본 발명의 식품 조성물은 기능성 식품(functional food), 영양 보조제(nutritional supplement), 건강 식품(health food), 식품 첨가제(food additives) 및 사료 등의 모든 형태를 포함하며, 인간 또는 가축을 비롯한 동물을 취식대상으로 한다.The food composition of the present invention includes all types of functional food, nutritional supplements, health food, food additives, and feed, and can be used for humans or animals, including livestock. target for eating.
상기 유형의 식품 조성물은 당업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다. 일반 식품으로는 이에 한정되지 않지만 음료(알콜성 음료 포함), 과실 및 그의 가공식품(예: 과일통조림, 병조림, 잼, 마아말레이드 등), 어류, 육류 및 그 가공식품(예: 햄, 소시지 콘비이프 등), 빵류 및 면류(예: 우동, 메밀국수, 라면, 스파게이트, 마카로니 등), 과즙, 각종 드링크, 쿠키, 엿, 유제품(예: 버터, 치이즈 등), 식용식물 유지, 마아가린, 식물성 단백질, 레토르트 식품, 냉동식품, 각종 조미료(예: 된장, 간장, 소스 등) 등에 상기 발효 홍삼을 첨가하여 제조할 수 있다. 또한, 영양보조제로는 이에 한정되지 않지만 캡슐, 타블렛, 환 등에 상기 발효 홍삼을 첨가하여 제조할 수 있다. 또한, 건강기능식품으로는 이에 한정되지 않지만 예를 들면, 상기 발효 홍삼을 차, 쥬스 및 드링크의 형태로 제조하여 음용(건강음료)할 수 있도록 액상화, 과립화, 캡슐화 및 분말화하여 섭취할 수 있다. 또한, 상기 발효 홍삼을 식품 첨가제의 형태로 사용하기 위해서는 분말로 제조하거나, 추출한 후 농축액 형태로 제조하여 사용할 수 있다. 또한, 상기 발효 홍삼과 학습 및 기억력 개선, 및 인지기능(coginitive) 증진 효과가 있다고 알려진 공지의 활성 성분과 함께 혼합하여 조성물의 형태로 제조할 수 있다.Food compositions of this type can be prepared in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruits and their processed foods (e.g. canned fruits, bottled products, jams, marmalades, etc.), fish, meat and their processed foods (e.g. ham, sausages) Corned beef, etc.), breads and noodles (e.g. udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine It can be prepared by adding the fermented red ginseng to vegetable protein, retort food, frozen food, various seasonings (eg, soybean paste, soy sauce, sauce, etc.). In addition, nutritional supplements are not limited thereto, but may be prepared by adding the fermented red ginseng to capsules, tablets, pills, etc. In addition, the health functional food is not limited thereto, but for example, the fermented red ginseng is prepared in the form of tea, juice, and drink, and can be consumed by liquefying, granulating, encapsulating, and powdering so that it can be consumed (health drink). there is. In addition, in order to use the fermented red ginseng in the form of a food additive, it can be prepared in the form of a powder or extracted and then used in the form of a concentrate. In addition, it can be prepared in the form of a composition by mixing the fermented red ginseng with a known active ingredient known to have learning and memory improvement and cognitive enhancement effects.
본 발명의 식품 조성물이 건강음료 조성물로 이용되는 경우, 상기 건강음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드; 말토스, 슈크로스와 같은 디사카라이드; 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드; 자일리톨, 소르비톨, 에리트리톨 등의 당알콜일 수 있다. 감미제는 타우마틴, 스테비아 추출물과 같은 천연 감미제; 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 mL 당 일반적으로 약 0.01 내지 0.04 g, 바람직하게는 약 0.02 내지 0.03 g 이다.When the food composition of the present invention is used as a health beverage composition, the health beverage composition may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The aforementioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrins and cyclodextrins; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as thaumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the composition of the present invention.
상기 식품 조성물은 전체 식품 조성물 총 중량에 대하여 발효 홍삼이 0.01 내지 100 중량%, 바람직하게는 0.1 내지 50 중량%로 포함된 것이 바람직하다. The food composition preferably contains fermented red ginseng in an amount of 0.01 to 100% by weight, preferably 0.1 to 50% by weight, based on the total weight of the entire food composition.
상기 외에 본 발명의 건강식품은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산, 펙트산의 염, 알긴산, 알긴산의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올 또는 탄산화제 등을 함유할 수 있다. 이러한 성분은 독립적으로 또는 혼합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물 100 중량부당 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the health food of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, It may contain glycerin, alcohol or a carbonating agent and the like. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected from the range of 0.01 to 0.1 part by weight per 100 parts by weight of the composition of the present invention.
이하, 실시예에 의하여 본 발명을 상세히 설명하겠으나, 다음 실시예에 의해 본 발명이 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples, but the present invention is not limited by the following examples.
<실시예> <Example>
실시예 1: 균주의 분리 및 동정Example 1: Isolation and identification of strains
1-1: 균주의 분리1-1: Separation of strains
각 지역으로부터 입수한 여러 종류의 지역별 김치 및 젓갈 수집하여 균주 분리 김치(젓갈) 시료로 사용하였다. 상기 입수한 지역별 김치 및 젓갈을 하기 표 1에 나타내었다.Several types of kimchi and salted fish obtained from each region were collected and used as strain-separated kimchi (pickled seafood) samples. The obtained kimchi and salted seafood by region are shown in Table 1 below.
상기 김치 또는 젓갈 시료를 채취하여, MRS broth 배지 90 mL에 상기 김치 또는 젓갈 시료 10 g을 혼합하고, 3분 동안 균질화한 후 37 ℃에서 24 시간 동안 배양하였다. 상기 배양액을 MRS 아가 배지에 접종한 후, 유리막대로 도말하였다. 이후, 플레이트를 37 ℃의 항온 배양기에서 24 시간 동안 배양하였다. 생성된 각각의 콜로니를 MRS 아가 플레이트에 획선 접종하였고, 37 ℃에서 24 시간 동안 배양하여 유산균 콜로니들을 분리하였다. 이와 같이 분리한 균주 및 선문대학교 실험실에서 보유 중인 김치 유래 균주를 포함하여 약 1,200 종의 균주를 분리하였다.The kimchi or salted fish sample was collected, mixed with 10 g of the kimchi or salted fish sample in 90 mL of MRS broth medium, homogenized for 3 minutes, and then cultured at 37 ° C. for 24 hours. After inoculating the culture medium into MRS agar medium, it was smeared with a glass rod. Thereafter, the plate was incubated for 24 hours in a 37 °C incubator. Each of the resulting colonies was inoculated on an MRS agar plate with a stroke line, and cultured at 37 ° C. for 24 hours to isolate lactic acid bacteria colonies. About 1,200 strains were isolated, including strains isolated in this way and strains derived from kimchi possessed by the Sunmoon University laboratory.
1-2: β-글루코시다아제 활성 균주 선별1-2: Selection of β-glucosidase active strains
β-글루코시다아제 활성을 갖는 균주의 선발을 위해 MRS 한천(deMan, Rogosa and Sharpe agar) 중의 0.3g/L의 에스쿨린 및 0.2g/L의 구연산철암모늄(ferric ammonium citrate)을 포함하는 에스쿨린-MRS 한천(Esculin-MRS agar) 배지가 사용되었다. 상기 에스쿨린(esculin)이 함유된 MRS 한천 배지에 균주를 접종하고 37 ℃에서 48시간 동안 배양하면서 배지 내에서의 색깔 변화를 관찰하였다(도 1). 에스쿨린은 β-글루코시다아제에 의하여 글루코스(glucose)와 에스쿨린으로 분리되며, 에스쿨린은 구연산철암모늄과 반응하여 콜로니 주위에 흑색 반점(black complex)를 형성하게 된다. 따라서, 에스쿨린 한천 배지에서 배양된 콜로니 주위에 흑색 반점을 형성하는 균주를 β-글루코시다아제 활성을 가지는 균주로 판단하여 선별하였다. 그 결과, 상기 분리한 약 1,200 종의 김치 유래 균주 중에서 흑색 반점을 생성한 136 종의 균주를 확보하였고, 그 중에서 흑색 반점이 가장 진한 29 종의 균주를 선별하였다.Esculin containing 0.3 g/L of esculin and 0.2 g/L of ferric ammonium citrate in MRS agar (deMan, Rogosa and Sharpe agar) for selection of strains having β-glucosidase activity -MRS agar (Esculin-MRS agar) medium was used. The strain was inoculated into the MRS agar medium containing the esculin and cultured at 37 ° C. for 48 hours, while observing color changes in the medium (FIG. 1). Esculin is separated into glucose and esculin by β-glucosidase, and esculin reacts with iron ammonium citrate to form black complexes around colonies. Therefore, strains that form black spots around the colonies cultured on the esculin agar medium were judged as strains having β-glucosidase activity and selected. As a result, among the 1,200 strains derived from kimchi, 136 strains that produced black spots were secured, and among them, 29 strains with the darkest black spots were selected.
상기 선별한 29 종의 균주들을 대상으로 하여 다시 β-글루코시다아제 활성 시험을 진행하였으며, 그 결과를 하기 표 2에 나타내었다.The β-glucosidase activity test was conducted again for the 29 strains selected above, and the results are shown in Table 2 below.
상기 표 2를 살펴보면, CK45, KK18, G17, G33, G69 및 G88의 6종의 균주가 강한 흑색 반점을 나타내는 것을 확인할 수 있다.Referring to Table 2, it can be seen that six strains of CK45, KK18, G17, G33, G69 and G88 exhibit strong black spots.
1-3: β-글루코시다아제 효소 활성 측정1-3: Measurement of β-glucosidase enzyme activity
상기 에스쿨린 MRS 한천 배지에서 흑색 반점을 형성하는 29 종의 균주 중에서 상대적으로 강한 흑색 반점을 형성한 균주 6종(CK45, KK18, G17, G33, G69 및 G88), 및 그 다음으로 강한 흑색 반점을 형성한 균주 7종(G16, G42, G64, NJ22, CK31, CK46, SY40)을 대상으로 β-글루코시다아제 활성을 측정하였다.Among the 29 strains forming black spots on the Esculin MRS agar medium, 6 strains (CK45, KK18, G17, G33, G69 and G88) that formed relatively strong black spots, and the next strong black spots β-glucosidase activity was measured for 7 strains (G16, G42, G64, NJ22, CK31, CK46, SY40).
β-글루코시다아제 활성 측정은 1% 카르복시메칠셀루로스(carboxymethyl cellulose, CMC)가 첨가된 시판용 MRS 배지에 12 시간 전배양한 균주를 접종하여 37℃에서 24 시간 배양 후, 배양액을 4℃에서 3000 rpm으로 15 분간 원심분리하여 세포를 제거하고, 상층액 0.3 ml를 취하여 0.7 ml의 5 mM ρ-nitrophenyl-β-D-glucopynanoside(ρ-NPG)용액과 혼합한 후 37℃에서 10 분간 반응시켰다. 반응액은 1 ml의 0.5 M Na2CO3 용액을 첨가하여 반응을 중지시키고, 생성된 p-nitrophenol를 450 nm에서 흡광도를 측정한 후 ρ-NP의 검량곡선을 이용하여 농도를 환산하여 하기 표 3에 나타내었다.β-glucosidase activity was measured by inoculating a strain pre-cultured for 12 hours in a commercially available MRS medium supplemented with 1% carboxymethyl cellulose (CMC), incubating at 37 ° C for 24 hours, and then incubating the culture medium at 4 ° C for 3000 °C. Cells were removed by centrifugation at rpm for 15 minutes, and 0.3 ml of the supernatant was mixed with 0.7 ml of 5 mM ρ-nitrophenyl-β-D-glucopynanoside (ρ-NPG) solution and reacted at 37° C. for 10 minutes. The reaction solution was stopped by adding 1 ml of 0.5 M Na 2 CO 3 solution, the absorbance of the produced p-nitrophenol was measured at 450 nm, and the concentration was converted using the calibration curve of ρ-NP, and the following table 3.
상기 표 3을 살펴보면, 선발된 13종의 균주는 28.05 ~ 33.53 mU/mL 범위의 높은 β-글루코시다아제 활성을 나타내는 것을 알 수 있다.Looking at Table 3, it can be seen that the selected 13 strains exhibit high β-glucosidase activity in the range of 28.05 to 33.53 mU/mL.
상기 선별된 13종의 균주를 대상으로 16s rRNA 유전자 염기서열 분석을 수행하였다. 상기 균주들을 염기서열 분석을 통해 동정한 결과, 모두 락토바실러스 펜토서스 또는 락토바실러스 플란타럼에 속하는 것으로 확인되었다. 상기 균주들을 동정한 결과를 하기 표 4에 정리하여 나타내었다. 16s rRNA gene sequencing was performed on the 13 selected strains. As a result of identifying the above strains through sequencing, it was confirmed that all of them belong to Lactobacillus pentosus or Lactobacillus plantarum. The results of identifying the strains are summarized in Table 4 below.
1-4: 진세노사이드 생물전환능 우수 균주 선별1-4: Selection of strains with excellent ginsenoside bioconversion ability
상기 β-글루코시다아제 활성이 있는 것으로 확인된 13 종의 균주들 중에서 진세노사이드 생물전환능이 우수한 균주를 선별하고자 하였다.Among the 13 strains confirmed to have the β-glucosidase activity, strains having excellent ginsenoside bioconversion ability were selected.
이를 위하여, 홍삼 추출물(고형분 5%)에 효소 처리를 한 다음, 상기 균주를 1%(w/w) 첨가하고 37 ℃에서 진탕 배양한 후, TLC 분석을 수행하여 F2 및 화합물 K(CK) 밴드가 선명한 균주, 즉 Rb1 감소에 의한 진세노사이드 생물전환능이 우수한 균주를 선별하고자 하였다. To this end, enzyme treatment was performed on red ginseng extract (5% solid content), 1% (w/w) of the above strain was added, shaken culture was performed at 37 ° C, and TLC analysis was performed to perform F2 and compound K (CK) bands. It was intended to select strains with clear strains, that is, strains with excellent ginsenoside bioconversion ability by reducing Rb1.
이때, 홍삼 추출물은 70%(w/w) 발효주정에 홍삼 분말을 10 중량%로 첨가하고 75 ℃에서 5 시간 동안 추출하여 15 브릭스의 추출액을 얻은 후, 회전농축기를 이용하여 50 ℃의 항온수조에서 30 브릭스로 농축하여 얻은 것이다.At this time, the red ginseng extract was obtained by adding 10% by weight of red ginseng powder to 70% (w/w) fermented alcohol and extracting at 75 ° C for 5 hours to obtain an extract of 15 brix, and then using a rotary concentrator to heat a constant temperature water bath at 50 ° C. was obtained by concentration to 30 Brix.
또한, 상기 효소처리는 상기 홍삼 추출물(고형분 5%)에 Rapidase C80max 5%(w/w) 및 Pyr-flo 5%(w/w)를 첨가하고 24시간 동안 반응시킨 후, Ultimase MFC 5%(w/w)를 추가하고 72 시간 동안 반응시키는 것이다. In addition, the enzyme treatment was performed by adding
상기와 같이 홍삼 추출물을 효소 처리한 후 균주를 접종하여 발효시킨 홍삼 발효 추출물을 이용하여 TLC 분석한 결과를 도 2에 나타내었다.2 shows the results of TLC analysis using the fermented red ginseng extract fermented by inoculating the strain after enzymatically treating the red ginseng extract as described above.
도 2를 살펴보면, G69, CK46 및 KK18 균주에서 F2 및 화합물 K(CK) 밴드가 선명하게 나타났으며, KK18 균주에서 상기 F2 및 화합물 K(CK) 밴드가 가장 진하게 나타났다.Referring to FIG. 2, the F2 and compound K (CK) bands appeared clearly in the G69, CK46, and KK18 strains, and the F2 and compound K (CK) bands were the most intense in the KK18 strain.
이에 따라, 상기 TLC 분석을 통해 F2 및 화합물 K(CK) 밴드가 선명하게 나타난 G69, CK46 및 KK18 균주를 진세노사이드 생물전환능 우수 균주로 선별하였고, 상기 밴드가 가장 진하게 나타난 KK18 균주를 우선적으로 선별하였다.Accordingly, the G69, CK46, and KK18 strains, in which the F2 and compound K (CK) bands clearly appeared through the TLC analysis, were selected as excellent strains for ginsenoside bioconversion ability, and the KK18 strain, in which the bands were the most intense, was preferentially selected.
1-5: 선별된 균주의 생물전환능 확인1-5: Confirmation of bioconversion ability of selected strains
상기 선별된 KK18 균주, 및 G69, CK46 균주의 Rb1 감소에 의한 생물전환능을 확인하고자, 홍삼 추출물(고형분 5%)에 상기 균주를 각각 5%(w/w) 접종하여 37 ℃에서 진탕 배양한 후 TLC 분석 및 HPLC 분석을 수행하였다(도 3a 및 도 3b).In order to confirm the bioconversion ability of the selected KK18 strain, G69 and CK46 strains by reducing Rb1, 5% (w / w) of each of the above strains was inoculated into red ginseng extract (5% solid content) and cultured with shaking at 37 ° C. After that, TLC analysis and HPLC analysis were performed (FIGS. 3a and 3b).
도 3a는 홍삼 추출물에 G69, CK46 및 KK18 균주를 각각 접종한 후 발효시킨 홍삼 발효 추출물을 HPLC 분석한 결과이고, 도 3b는 홍삼 추출물에 G69, CK46 및 KK18 균주를 각각 접종한 후 발효시킨 홍삼 발효 추출물을 TLC 분석한 결과 및 발효기간에 따른 Rg3 함량을 나타내는 그래프이다.Figure 3a is the result of HPLC analysis of the fermented red ginseng extract fermented after inoculating the red ginseng extract with G69, CK46 and KK18 strains, respectively, and Figure 3b is the red ginseng fermentation fermented after inoculating the red ginseng extract with G69, CK46 and KK18 strains, respectively. It is a graph showing the result of TLC analysis of the extract and the Rg3 content according to the fermentation period.
상기 도 3을 살펴보면, 홍삼 추출물의 발효에 의해 Rb1이 감소하고 Rg3 함량이 증가한 것을 확인할 수 있다. 이때, 상기 발효에 의해 F2 및 화합물 K는 생성되지 않았다.Referring to FIG. 3, it can be seen that Rb1 decreased and Rg3 content increased due to fermentation of the red ginseng extract. At this time, F2 and compound K were not produced by the fermentation.
또한, 홍삼 추출물(고형분 5%)에 효소 처리를 한 다음, 상기 균주를 각각 1%(w/w) 접종하여 37 ℃에서 진탕 배양한 후 TLC 분석 및 HPLC 분석을 수행하였다(도 4a 및 도 4b). 상기 효소 처리는 상기 홍삼 추출물(고형분 5%)에 Rapidase C80max 5%(w/w) 및 Pyr-flo 5%(w/w)를 첨가하고 24시간 동안 반응시킨 후, Ultimase MFC 5%(w/w)를 추가하고 72 시간 동안 반응시키는 것이다. In addition, after enzymatic treatment of red ginseng extract (5% solid content), each of the above strains was inoculated at 1% (w/w), shaken and cultured at 37 ° C, and then TLC analysis and HPLC analysis were performed (FIGS. 4a and 4b). ). The enzyme treatment was performed by adding
도 4a는 홍삼 추출물에 효소 처리를 한 다음, G69, CK46 및 KK18 균주를 각각 접종한 후 발효시킨 홍삼 발효 추출물을 HPLC 분석한 결과이고, 도 4b는 홍삼 추출물에 효소 처리를 한 다음, G69, CK46 및 KK18 균주를 각각 접종한 후 발효시킨 홍삼 발효 추출물을 TLC 분석한 결과 및 발효기간에 따른 화합물 K의 함량을 나타내는 그래프이다.Figure 4a is the result of HPLC analysis of the fermented red ginseng extract fermented after enzymatic treatment of red ginseng extract and then inoculation of G69, CK46 and KK18 strains, respectively. and KK18 strains, respectively, and a graph showing the result of TLC analysis of the fermented red ginseng extract fermented after inoculation and the content of Compound K according to the fermentation period.
상기 도 4를 살펴보면, 발효 전후의 발효물을 이용하여 TLC를 실시한 결과 세 균주 모두 발효 2일 차에 F2 밴드가 증가하였으며, 특히 KK18 균주는 발효 2일차 및 3일차에 화합물 K(CK) 밴드가 확연히 증가하는 것을 확인할 수 있다.Referring to FIG. 4, as a result of TLC using the fermented product before and after fermentation, the F2 band increased on the 2nd day of fermentation in all three strains, and in particular, the KK18 strain had a compound K (CK) band on the 2nd and 3rd day of fermentation. A marked increase can be seen.
또한, 발효 시간에 따른 진세노사이드 F2 및 화합물 K 함량을 HPLC로 정량한 결과, KK18 균주는 발효 3일차까지 화합물 K의 함량을 12.5% 증가시키는 것으로 나타났다. 따라서 KK18 균주는 진세노사이드 F2에서 화합물 K로의 생물전환 활성을 갖는 균주로 확인되었다. 또한, KK18 균주를 접종한 후 발효 5일차에는 화합물 K의 함량이 감소하는 것으로 나타나, KK18 균주를 첨가한 후 3일 동안 배양하는 조건이 효소전환 및 유산균 발효 병행에 의한 화합물 K의 생성에 최적인 것으로 나타났다.In addition, as a result of quantifying the contents of ginsenoside F2 and compound K according to the fermentation time by HPLC, it was found that the KK18 strain increased the compound K content by 12.5% until the 3rd day of fermentation. Therefore, strain KK18 was identified as a strain having bioconversion activity from ginsenoside F2 to compound K. In addition, the content of compound K was found to decrease on the 5th day of fermentation after inoculation of strain KK18, and the conditions for culturing for 3 days after adding strain KK18 are optimal for the production of compound K by simultaneous enzyme conversion and lactic acid fermentation. appeared to be
한편, G69 균주 및 CK46 균주를 접종한 발효물에서는 F2 함량이 증가하는 것으로 나타났으나 화합물 K의 함량은 거의 증가하지 않았다. 따라서, 상기 두 균주는 진세노사이드 F2를 화합물 K로 생물전환하는 활성은 갖지 않았거나 미미한 것으로 보인다.On the other hand, in the fermented product inoculated with the G69 strain and the CK46 strain, the F2 content was found to increase, but the compound K content hardly increased. Thus, the two strains appear to have no or negligible activity to bioconvert ginsenoside F2 to compound K.
1-6: 내산성 및 내담즙성1-6: acid resistance and bile resistance
유산균이 프로바이오틱스 활성을 갖기 위해서는 위액과 담즙에 대한 내성을 갖고 생존할 수 있어야 장에 도달하여 생리활성을 발휘할 수 있다. 따라서, 상기 선별된 균주들의 내산성 및 내담즙성을 확인하고자 하였다.In order for lactic acid bacteria to have probiotic activity, they must be able to survive with resistance to gastric juice and bile so that they can reach the intestine and exert their physiological activity. Therefore, it was attempted to confirm the acid resistance and bile resistance of the selected strains.
상기 위산 및 담즙산에 대한 내성 효과를 알아보기 위하여 선발 균주를 LB 액체배지에서 30℃에서 18시간 배양 후, 이를 실험의 초기 접종 배양액으로 사용하였다. In order to examine the effect of resistance to gastric acid and bile acid, the selected strain was cultured in LB liquid medium at 30 ° C. for 18 hours, and then used as an initial inoculation culture medium for the experiment.
내산성 분석은 각각 pH 2.5로 조절한 LB 액체배지에 초기 접종 배양액을 5%씩 접종하여 30℃에서 30분 진탕배양한 후, 생균수를 측정하여 내산성 정도를 확인하였다. 그리고, 내담즙성 분석은 담즙(oxgall)을 0.3% 첨가한 LB 액체배지에 초기 접종 배양액을 5%씩 접종하여 30℃에서 6시간 진탕배양한 후 생균수를 측정하여 내담즙성을 확인하였다. 각 실험의 대조군(control)는 pH를 조절하지 않은 배지, 담즙을 첨가하지 않은 배지에서 반응한 생균수로 정의하였고, 각 균주의 내산성 및 내담즙성을 대조군에 대한 % 비율로서 하기 표 5에 나타내었다. Acid resistance analysis was inoculated with 5% of the initial inoculation culture medium in LB liquid medium adjusted to pH 2.5, incubated with shaking at 30 ° C. for 30 minutes, and then the degree of acid resistance was confirmed by measuring the number of viable cells. And, in the analysis of bile resistance, 5% of the initial inoculation culture medium was inoculated into LB liquid medium containing 0.3% of bile (oxgall), incubated with shaking at 30 ° C. for 6 hours, and then the number of viable cells was measured to confirm bile resistance. The control group of each experiment was defined as the number of viable cells that reacted in a medium without adjusting the pH and a medium without the addition of bile, and the acid resistance and bile resistance of each strain are shown in Table 5 below as a % ratio with respect to the control group. was
G69 Lactobacillus plantarum
G69
CK46 Lactobacillus plantarum
CK46
KK18 Lactobacillus pentosus
KK18
상기 표 5를 살펴보면, 본 발명에서 선별된 균주인 KK18 균주, 및 G69 및 CK46 균주는 각각 위산과 담즙에서 생존률이 80% 이상인 것으로 나타나, 위산과 담즙에 대한 내성을 갖고 있는 것으로 확인되었다. 따라서, 상기 균주들은 프로바이오틱스로서 균주 섭취 시 안정적으로 장내까지 많은 생균이 도달할 것으로 판단된다. Referring to Table 5, the strains KK18 and G69 and CK46 strains selected in the present invention showed a survival rate of 80% or more in gastric acid and bile, respectively, and it was confirmed that they had resistance to gastric acid and bile. Therefore, the strains are considered to be probiotics, and many live bacteria will stably reach the intestine when ingested.
1-7: 항산화 활성1-7: antioxidant activity
상기 선별된 KK18 균주, 및 G69 및 CK46 균주의 항산화 활성을 확인하고자 하였다. 이를 위하여, DPPH 라디칼 소거능, ABTS 라디칼 소거능, 및 FRAP 환원력을 하기와 같이 측정하였다(도 5).Antioxidant activities of the selected KK18 strain, and the G69 and CK46 strains were examined. To this end, DPPH radical scavenging ability, ABTS radical scavenging ability, and FRAP reducing power were measured as follows (FIG. 5).
<DPPH 라디칼 소거능>< DPPH radical scavenging activity >
DPPH 라디칼 소거능은 선별된 KK18 균주를 LB 액체배지에서 30℃에서 18시간 배양한 후 원심분리하여 얻은 상층액을 분석시료로 사용하였다. 분석시료 0.2 ml와 DPPH 용액 0.8 ml를 혼합하고 암실에서 30분간 반응시키고 그 반응물을 517 nm에서 흡광도를 측정하였다. DPPH 라디칼 소거능은 음성 대조구의 경우 각 조성물 대신 증류수로 대체하여 상기 방법과 동일하게 수행하여 흡광도 차이를 아래와 같은 식에 대입하여 백분율(%)로 산출하였다.For DPPH radical scavenging activity, the supernatant obtained by centrifugation after culturing the selected KK18 strain in LB liquid medium at 30 ° C. for 18 hours was used as an analysis sample. 0.2 ml of the analysis sample and 0.8 ml of the DPPH solution were mixed and reacted in a dark room for 30 minutes, and the absorbance of the reactant was measured at 517 nm. DPPH radical scavenging ability was calculated as a percentage (%) by substituting the difference in absorbance into the following formula by replacing each composition with distilled water in the case of the negative control group and performing the same method as above.
라디칼 소거능(%) = [1-(음성대조구 흡광도 ㆇ 실험구 흡광도)] × 100Radical scavenging ability (%) = [1-(absorbance of negative control group ㆇ absorbance of experimental group)] × 100
<ABTS 라디칼 소거능>< ABTS radical scavenging activity >
ABTS 라디칼 소거능은 청색의 라디칼이 점차 무색으로 변색하는 즉, 항산화 활성에 따라 점점 퇴색하는 원리를 이용하여 측정하는 방법이다. 이 분석은 탈색반응이 1분 내에 빠르게 종료되므로 짧은 시간에 측정이 가능한 장점이 있다. ABTS 라디칼 소거능은 우선 K2S2O8 시약과 메탄올을 2:1로 섞어 16시간 암실에서 ABTS 양이온 라디칼을 형성시키고 이를 다시 메탄올로 희석하여 최종 농도가 732 nm에서 흡광도 값이 0.8±0.2가 되도록 조절한 ABTS 시약을 사용하였다. ABTS radical scavenging ability is a method for measuring using the principle that blue radicals gradually change color to colorless, that is, gradually fade according to antioxidant activity. This assay has the advantage of being able to measure in a short time because the decolorization reaction is quickly completed within 1 minute. ABTS radical scavenging ability is firstly mixed with K2S2O8 reagent and methanol at a ratio of 2:1 to form ABTS cation radicals in the dark for 16 hours, and then diluted with methanol again to adjust the final concentration to an absorbance value of 0.8±0.2 at 732 nm. used
구체적으로는 ABTS 시약 0.9 ml에 각각의 분석시료 0.1 ml를 첨가하여 1분간 반응시키고 신속히 732 nm에서 흡광도를 측정하였다. 음성 대조구는 시료 대신 증류수를 사용하여 동일한 방법으로 수행하여 흡광도의 차이를 상기 식에 의해 백분율(%)로 산출하였다. Specifically, 0.1 ml of each analysis sample was added to 0.9 ml of ABTS reagent, reacted for 1 minute, and absorbance was quickly measured at 732 nm. The negative control was performed in the same manner using distilled water instead of the sample, and the difference in absorbance was calculated as a percentage (%) by the above formula.
<FRAP 환원력>< FRAP reducing power >
FRAP(ferric reducing antioxidant power) 환원력은 산화환원 반응 중 환원에 해당되는 것으로 Fe3+이온을 Fe2+이온으로 환원시킬 때 흡광도 수치를 측정하여 이를 항산화 활성의 지표로써 나타낸 것이다.FRAP (ferric reducing antioxidant power) reduction power corresponds to reduction in the redox reaction, and when Fe 3+ ion is reduced to Fe 2+ ion, the absorbance value is measured and expressed as an index of antioxidant activity.
구체적으로는 30 mM 아세테이트 버퍼(30 mM, pH 3.6), TPTZ 시약(10 mM, 6 N 염산에 녹인)과 FeCl3 용액(20 mM, 증류수에 녹인)을 100:10:10(v/v/v)의 비율로 먼저 혼합하여 FRAP 시약을 조제하였다. 상기 FRAP 시약을 37℃의 수욕 상에서 15분간 예비반응시켜 두고 각각의 분석시료를 시험관에 0.05 ml씩 첨가하고 여기에 미리 반응해두었던 FRAP 시약을 0.85 ml씩 첨가하여 상기와 같은 방법으로 37℃에서 15분 반응시켰다. 반응 후 일부분을 593 nm에서 흡광도를 측정하였다.Specifically, a 100: 10 :10 (v/v/ The FRAP reagent was prepared by first mixing in the ratio of v). The FRAP reagent was pre-reacted in a water bath at 37 ° C. for 15 minutes, each analysis sample was added to the test tube at 0.05 ml each, and 0.85 ml of the previously reacted FRAP reagent was added thereto. minutes reacted. After the reaction, the absorbance of a portion was measured at 593 nm.
도 5는 상기 선별된 KK18 균주, 및 G69 및 CK46 균주 배양액의 DPPH 라디칼 소거능, ABTS 라디칼 소거능, 및 FRAP 환원력을 나타내는 그래프이다. 5 is a graph showing the DPPH radical scavenging ability, ABTS radical scavenging ability, and FRAP reducing power of the culture solutions of the selected KK18 strain and G69 and CK46 strains.
도 5를 살펴보면, G69 균주가 가장 높은 DPPH 라디칼 소거능(43%) 및 ABTS 라디칼 소거능(30%)을 나타내는 것을 확인할 수 있다. KK18 균주는 상대적으로 높은 FRAP 환원력을 나타냈으며, DPPH 라디칼 소거능 및 ABTS 소거능이 각각 30% 및 25%로 양호한 항산화 활성이 있는 것으로 확인되었다.Referring to FIG. 5, it can be seen that the G69 strain exhibits the highest DPPH radical scavenging activity (43%) and ABTS radical scavenging activity (30%). The KK18 strain showed relatively high FRAP reducing power, and DPPH radical scavenging ability and ABTS scavenging ability were 30% and 25%, respectively, and it was confirmed to have good antioxidant activity.
1-8: API ZYM 키트를 이용한 효소 활성 확인1-8: Confirmation of enzyme activity using API ZYM kit
선별 균주의 효소활성을 확인하기 위하여 총 19종의 각종 효소의 기질 이용성을 기초로 제작된 API ZYM 키트(BioMerieux, Marcy-I'Etoile, France)를 사용하였다. 선별 유산균을 MRS 아가 배지에서 획선 도말하여 37℃에서 48시간 동안 배양한 콜로니를 2mL 현탁 배지에 재부유하고, McFarland(BioMerieux)로 탁도 5~6으로 조정하여 각 튜브에 60 ㎕씩 분주하고 37 ℃에서 4시간 배양한 후 ZYM A 및 ZYM B 시약을 각 튜브에 한 방울씩 떨어뜨리고 5분간 실온에서 반응시켜 색 변화로 각각의 기질 효소에 대한 활성 여부를 판독하였다. 색의 변화 정도에 따라 0~5까지의 값으로 표시하였으며, 0은 음성반응, 5 (= 40 nmol)는 최대 강도의 반응이고 4~1은 각각 30, 20, 10 및 5 nmol의 중간 값을 나타내며 2 이상일 경우 양성으로 판정하였다. 또한, 색의 변화 정도가 2~3일 경우 "+", 3~4일 경우 "++", 4~5일 경우 "+++"로 표시하였다. 일반적으로, 유산균이 프로바이오틱스로 사용되기 위해서는 이들이 생산하는 효소 또한 매우 중요한 부분을 차지하고 있어 최종 선별한 KK18 균주의 효소 생산 여부를 확인하기 위해 API ZYM 키트를 이용하여 조사하였다. 그 결과, 에스테라제 리파아제(Esterase lipase), 리파아제(Lipase), 루신 아릴아미다아제(leucin arylamidase), 발린 아릴아미다아제(valine arymidase), 산성인산가수분해효소(Acid phosphatase), 나프톨-AS-포스포히드롤라아제(Naphol-AS-phosphohydrolase), β-갈락토시다아제(β-galactosidase), β-글루코시다아제(β-glucosidase) 및 N-아세틸-β-글루코사미니다아제(β-glucosaminidase) 활성이 있음을 확인하였다(표 6 및 도 6 참조).In order to confirm the enzymatic activity of the selected strain, an API ZYM kit (BioMerieux, Marcy-I'Etoile, France), which was designed based on substrate availability of a total of 19 various enzymes, was used. The selected lactic acid bacteria were smeared on MRS agar medium and cultured at 37 ° C for 48 hours. Colonies were resuspended in 2 mL suspension medium, adjusted to turbidity 5-6 with McFarland (BioMerieux), dispensed 60 μl into each tube, and incubated at 37 ° C. After incubation for 4 hours, ZYM A and ZYM B reagents were added dropwise to each tube and reacted at room temperature for 5 minutes, and the activity of each substrate enzyme was read by color change. The degree of color change was expressed as a value from 0 to 5, where 0 is a negative reaction, 5 (= 40 nmol) is the maximum intensity reaction, and 4 to 1 is the median value of 30, 20, 10 and 5 nmol, respectively. Indicated, if it was 2 or more, it was judged positive. In addition, when the degree of color change was 2 to 3, "+", 3 to 4, "++", and 4 to 5 were marked with "+++". In general, in order for lactic acid bacteria to be used as probiotics, the enzymes they produce also occupy a very important part, so the API ZYM kit was investigated to confirm the enzyme production of the finally selected KK18 strain. As a result, esterase lipase, lipase, leucin arylamidase, valine arymidase, acid phosphatase, naphthol-AS -Phosphohydrolase (Naphol-AS-phosphohydrolase), β-galactosidase (β-galactosidase), β-glucosidase (β-glucosidase) and N-acetyl-β-glucosaminidase (β- glucosaminidase) activity was confirmed (see Table 6 and FIG. 6).
phosphohydrolaseNaphthol-AS-
phosphohydrolase
β-glucosaminidaseN-acetyl-
β-glucosaminidase
상기 표 6을 살펴보면, G69, CK46 및 KK18 균주 모두 β-글루코시다아제 활성을 나타내었으며, KK18 균주가 G69 및 CK46 균주에 비해 높은 β-글루코시다아제 활성을 나타내었다.Referring to Table 6, strains G69, CK46 and KK18 all showed β-glucosidase activity, and strain KK18 showed higher β-glucosidase activity than strains G69 and CK46.
또한, 상기 G69, CK46 및 KK18 균주 모두 발암 효소인 β-글루쿠로니다아제에 대한 활성을 나타내지 않아 발암에 대한 안전성을 확인하였다.In addition, all of the strains G69, CK46 and KK18 showed no activity against the carcinogenic enzyme β-glucuronidase, confirming their safety against carcinogenesis.
1-9: 항생제 감수성 분석1-9: Antibiotic susceptibility analysis
유산균의 경우 식품과 의약품의 소재로 많이 이용되고 있으며, 이로 인해 항생제 내성을 확인하는 것은 매우 중요한 요소이므로 상기 선별된 G69, CK46 및 KK18 균주를 대상으로 항생제 감수성 분석을 하였다. In the case of lactic acid bacteria, they are widely used as a material for food and medicine, and therefore, it is a very important factor to confirm antibiotic resistance. Therefore, antibiotic susceptibility analysis was performed on the selected strains G69, CK46, and KK18.
항생제 감수성 시험은 NCCLS(National Committee for Clinical Laboratory Standards)의 디스크 확산법(disk diffusion method)으로 실시하였다. 사용한 항생제 디스크(BBL, Sensi-disc, Becton Dickinson Co., NJ, USA)는 암피실린(ampicillin), 젠타마이신(gentamicin), 카나마이신(kanamycin), 스트렙토마이신(Streptomycin), 테트라사이클린(Tetracycline), 시프로플록사신(Ciprofloxacin), 클로람페니콜(Chloramphenicol) 및 독시사이클린(Doxycycline)로 8종을 사용하였다. 선별 균주는 MRS 액체배지에서 배양한 후, 1.0×105 CFU/mL가 되도록 뮬러 힌트 아가(Merck, Darmstadf, Germany)에 도말한 다음, 8종의 항생제 디스크를 놓고 37℃에서 24시간 방치하였으며, 각 항생제에 대한 억제환의 크기를 측정하고 NCCLS(National Committee for Clinical Laboratory Standards) 가이드라인에 따라 감수성과 내성을 판정하였다(도 7 참조). 데이터 값은 3회 반복 측정하여 평균±표준편차로 나타내었다.The antibiotic susceptibility test was performed by the disk diffusion method of NCCLS (National Committee for Clinical Laboratory Standards). The used antibiotic disc (BBL, Sensi-disc, Becton Dickinson Co., NJ, USA) contains ampicillin, gentamicin, kanamycin, streptomycin, tetracycline, ciprofloxacin ( Ciprofloxacin), chloramphenicol and doxycycline were used. Selected strains were cultured in MRS liquid medium, smeared on Mueller Hint Agar (Merck, Darmstadf, Germany) to a concentration of 1.0 × 10 5 CFU / mL, and then placed on 8 antibiotic disks and left at 37 ° C. for 24 hours, The size of the ring of inhibition for each antibiotic was measured, and susceptibility and resistance were determined according to the National Committee for Clinical Laboratory Standards (NCCLS) guidelines (see FIG. 7). Data values were measured three times and expressed as mean ± standard deviation.
KK18 L. pentosus
KK18
GG L. rhamnosus
GG
* S; Sensitive, R; Resistance*S; Sensitive, R; Resistance
상기 표 7을 살펴보면, G69, CK46 및 KK18 균주 모두 상업균주인 락토바실러스 람노서스(Lactobacillus rhamnosus) GG 균주와 동일한 내성을 나타내는 것으로 확인되었다. 즉, 암피실린, 테트라사이클린, 클로람페니콜, 독시사이클린에 의해 저해되었으나, 젠타마이신, 카나마이신, 스트렙토마이신 및 시프로플록사신에 대해서는 내성을 나타내었다. 내성을 나타내는 계열의 항생제에 대해서는 락토바실러스 속 균주가 고유 내성을 갖고 있는 것으로 알려져 있으므로, 상기 3종의 균주는 산업적으로 사용이 가능할 것으로 판단된다.Referring to Table 7, it was confirmed that all strains G69, CK46 and KK18 exhibit the same resistance as the commercial strain, Lactobacillus rhamnosus GG strain. That is, it was inhibited by ampicillin, tetracycline, chloramphenicol, and doxycycline, but showed resistance to gentamicin, kanamycin, streptomycin, and ciprofloxacin. Since strains of the genus Lactobacillus are known to have inherent resistance to antibiotics of a resistant class, it is considered that the three strains can be used industrially.
1-10: 선별한 균주의 분자생물학적 동정1-10: Molecular biological identification of selected strains
상기 실시예 1-1 내지 1-9를 통해, β-글루코시다아제 활성이 높고, 진세노사이드 생물전환능이 우수하며, 특히 진세노사이드 F2를 화합물 K로의 생물전환 활성을 가지고 있고, 내산성 및 내담즙성이 우수하며, 항산화 활성 및 항생제 감수성이 있는 균주로서 락토바실러스 펜토서스 KK18 균주를 최종 선별하였다.Through Examples 1-1 to 1-9, β-glucosidase activity is high, ginsenoside bioconversion ability is excellent, and in particular, ginsenoside F2 has bioconversion activity to compound K, acid resistance and resistance The Lactobacillus pentosus KK18 strain was finally selected as a strain having excellent choleretic properties, antioxidant activity and antibiotic sensitivity.
상기 최종 선별한 락토바실러스 펜토서스 KK18 균주의 분자생물학적 특징을 조사하기 위해 균주 배양액으로부터 염색체 DNA를 분리하였고, 상기 염색체 DNA를 이용하여 16s rRNA gene의 전체 염기서열 분석을 수행하였다. 상기 동정한 균주는 락토바실러스 펜토서스(Lactobacillus pentosus)에 속하는 균주인 것으로 조사되었고, 상기 동정한 균주의 염기서열(16s rRNA 서열)을 서열번호 1에 나타내었다. In order to investigate the molecular biological characteristics of the finally selected Lactobacillus pentosus KK18 strain, chromosomal DNA was isolated from the strain culture, and whole sequencing of the 16s rRNA gene was performed using the chromosomal DNA. The identified strain was found to be a strain belonging to Lactobacillus pentosus , and the nucleotide sequence (16s rRNA sequence) of the identified strain is shown in SEQ ID NO: 1.
상기 균주를 GenBank에 등록된 다른 균주들과 염기서열을 비교한 결과 표준균주인 락토바실러스 펜토서스 124-2 표준균주와 99%의 상동성을 나타내었으며, 서열번호 1과 100% 상동성을 갖는 균주는 기존에 존재하지 않는 것을 확인하였다(도 8 참조). 따라서 본 발명자들은 상기 락토바실러스 펜토서스 KK18 균주를 2020년 7월 8일자로 한국종균협회(KCCM)에 기탁하여 수탁번호 KCCM12762P를 부여받았다. As a result of comparing the base sequence of this strain with other strains registered in GenBank, the standard strain It showed 99% homology with the Lactobacillus pentosus 124-2 standard strain, and it was confirmed that a strain having 100% homology with SEQ ID NO: 1 did not exist (see FIG. 8). Therefore, the present inventors deposited the Lactobacillus pentosus KK18 strain on July 8, 2020 with the Korea Breeding Association (KCCM) and received accession number KCCM12762P.
실시예 2: 발효 홍삼의 제조Example 2: Preparation of fermented red ginseng
(1) 홍삼 분말 제조(1) Manufacture of red ginseng powder
홍삼은 한국인삼공사의 제품을 구입하여 사용하였다. 홍삼은 1차로 가정용 분쇄기(한일전기 후드프로세서)를 이용하여 10 mesh 크기로 조분쇄를 한 후 냉동고에 24시간 이상 보관하여 영하 5 ℃가 되도록 한 후 냉각기를 부착한 분쇄기(silant cutter, USA)를 이용하여 150 mesh로 분쇄하여 본 발명에 사용하였다. Red ginseng was purchased and used by Korea Ginseng Corporation. Red ginseng is first coarsely pulverized into a size of 10 mesh using a household grinder (Hanil Electric hood processor), stored in a freezer for more than 24 hours to reach
(2) 균주 현탁액 제조(2) Preparation of strain suspension
정제수에 유기산을 첨가하여 pH를 5.6으로 조정하였다. 상기 pH 5.6의 정제수에 전배양한 락토바실러스 펜토서스 KK18 균주 배양액(1×108 cfu/mL)을 2.0 중량%로 접종한 후 균질화하여 균주 현탁액을 제조하였다. An organic acid was added to the purified water to adjust the pH to 5.6. A strain suspension was prepared by inoculating 2.0% by weight of the Lactobacillus pentosus KK18 strain culture medium (1×10 8 cfu/mL) pre-cultured in purified water of pH 5.6 and then homogenizing.
(3) 발효(3) Fermentation
상기 (1)에서 제조한 홍삼 분말 100 중량부에 상기 (2)에서 제조한 균주 현탁액 5 중량부를 분무한 후 37 ℃에서 96 시간 동안 발효시켰다. 상기 발효가 완료된 발효물을 90 ℃에서 5 시간 동안 열풍 건조하여 발효 홍삼을 제조하였다.5 parts by weight of the strain suspension prepared in (2) was sprayed on 100 parts by weight of the red ginseng powder prepared in (1) above, and then fermented at 37 ° C. for 96 hours. Fermented red ginseng was prepared by hot air drying the fermented product at 90 ° C. for 5 hours.
비교예 1: pH 3.5에서 발효Comparative Example 1: Fermentation at pH 3.5
실시예 2와 동일한 방법으로 실시하되, (2) 단계의 균주 현탁액 제조시 정제수의 pH를 pH 5.6 대신 pH 3.5로 조정하여 발효 홍삼을 제조하였다.Fermented red ginseng was prepared in the same manner as in Example 2, but by adjusting the pH of the purified water to pH 3.5 instead of pH 5.6 when preparing the strain suspension in step (2).
비교예 2: pH 7.0에서 발효Comparative Example 2: Fermentation at pH 7.0
실시예 2와 동일한 방법으로 실시하되, (2) 단계의 균주 현탁액 제조시 정제수의 pH를 pH 5.6 대신 pH 7.0으로 조정하여 발효 홍삼을 제조하였다.Fermented red ginseng was prepared in the same manner as in Example 2, but by adjusting the pH of the purified water to pH 7.0 instead of pH 5.6 when preparing the strain suspension in step (2).
비교예 3: 홍삼 추출물 이용Comparative Example 3: Use of red ginseng extract
실시예 2와 동일한 방법으로 실시하되, (3) 단계에서 홍삼 분말 대신 홍삼 추출물을 이용하여 발효 홍삼을 제조하였다.It was carried out in the same manner as in Example 2, but in step (3), fermented red ginseng was prepared using red ginseng extract instead of red ginseng powder.
이때, 홍삼 추출물은 70%(w/w) 발효주정에 (1) 단계에서 얻은 홍삼 분말을 10 중량%로 첨가한 후 75 ℃에서 5 시간 동안 추출하여 15 브릭스의 추출액을 얻은 후, 회전농축기를 이용하여 50 ℃의 항온수조에서 30 브릭스로 농축하여 얻은 것이다. At this time, the red ginseng extract was obtained by adding 10% by weight of the red ginseng powder obtained in step (1) to 70% (w / w) fermented alcohol, followed by extraction at 75 ° C. for 5 hours to obtain an extract of 15 brix, and then a rotary condenser It was obtained by concentrating to 30 Brix in a constant temperature water bath at 50 ° C.
비교예 4: 효소처리 홍삼 추출물 이용Comparative Example 4: Use of enzyme-treated red ginseng extract
비교예 3과 동일한 방법으로 실시하되, 홍삼 추출물 대신 효소처리 홍삼 추출물을 이용하여 발효 홍삼을 제조하였다.Fermented red ginseng was prepared in the same manner as in Comparative Example 3, but using the enzyme-treated red ginseng extract instead of the red ginseng extract.
이때, 효소처리 홍삼 추출물은 상기 홍삼 추출물에 Rapidase C80max 5%(w/w) 및 Pyr-flo 5%(w/w)를 첨가하고 24시간 동안 반응시킨 후, Ultimase MFC 5%(w/w)를 추가하고 72 시간 동안 반응시켜 얻은 것이다. At this time, for the enzyme-treated red ginseng extract,
비교예 5: 락토바실러스 플란타럼 G69 균주로 발효Comparative Example 5: Fermentation with Lactobacillus plantarum G69 strain
실시예 2와 동일한 방법으로 실시하되, (2) 단계의 균주 현탁액 제조시 균주를 락토바실러스 펜토서스 KK18 균주 대신 락토바실러스 플란타럼 G69 균주를 접종하여 발효 홍삼을 제조하였다. It was carried out in the same manner as in Example 2, but when preparing the strain suspension in step (2), the strain was inoculated with the Lactobacillus plantarum G69 strain instead of the Lactobacillus pentosus KK18 strain to prepare fermented red ginseng.
비교예 6: 락토바실러스 플란타럼 CK46 균주로 발효Comparative Example 6: Fermentation with Lactobacillus plantarum CK46 strain
실시예 2와 동일한 방법으로 실시하되, (2) 단계의 균주 현탁액 제조시 균주를 락토바실러스 펜토서스 KK18 균주 대신 락토바실러스 플란타럼 CK46 균주를 접종하여 발효 홍삼을 제조하였다. Fermented red ginseng was prepared by inoculating the strain Lactobacillus plantarum CK46 strain instead of the Lactobacillus pentosus KK18 strain as the strain when preparing the strain suspension in step (2), except in the same manner as in Example 2.
<시험예> < Test Example>
시험예: 진세노사이드 함량Test Example: Ginsenoside Content
상기 실시예 및 비교예에서 제조한 발효 홍삼에 함유된 진세노사이드의 함량을 분석하기 위해 HPLC를 이용하였다.HPLC was used to analyze the content of ginsenosides contained in the fermented red ginseng prepared in the above Examples and Comparative Examples.
구체적으로, 진세노사이드의 함량을 측정하기 위하여, 진세노사이드 표준품 5종(Rb1, Rb2, Rg2, F2 및 화합물 K)을 각 5mg씩 정밀하게 측정한 후, 메탄올 1 ml에 용해시켰다. 그 후, 10배 희석한 다음, 다시 50배 및 100배로 희석하여 3개 농도의 표준액을 제조하였다. 상기 제조된 표준액을 진세노사이드의 함량을 측정하는 기준으로 설정하였다.Specifically, in order to measure the content of ginsenoside, 5 mg of each of 5 standard ginsenosides (Rb1, Rb2, Rg2, F2 and compound K) was precisely measured and then dissolved in 1 ml of methanol. Thereafter, the sample was diluted 10 times, and then further diluted 50 times and 100 times to prepare standard solutions of three concentrations. The prepared standard solution was set as a standard for measuring the content of ginsenoside.
한편, 상기 실시예 및 비교예에 따라 제조된 발효 홍삼 10 g을 70% 메탄올 100 ml에 용해시킨 후, 80 ℃ 항온 수조에서 환류법으로 3회 추출하고 감압농축하였다. 그 후, 증류수를 가한 후, 디에칠에테르를 가하여 지용성 성분을 제거하고, 수포화 n-부탄올로 3회 추출한 뒤, 감압농축하고 HPLC를 이용하여 진세노사이드 함량을 분석하였다(도 9). 도 9는 실시예에 따라 제조된 발효 홍삼의 진세노사이드 함량을 HPLC를 이용하여 분석한 결과를 나타내는 크로마토그램이다. On the other hand, 10 g of fermented red ginseng prepared according to the above Examples and Comparative Examples was dissolved in 100 ml of 70% methanol, extracted three times by the reflux method in a constant temperature water bath at 80 ° C, and concentrated under reduced pressure. Then, after adding distilled water, diethyl ether was added to remove fat-soluble components, extracted three times with saturated n-butanol, concentrated under reduced pressure, and analyzed for ginsenoside content using HPLC (FIG. 9). 9 is a chromatogram showing the results of analyzing the ginsenoside content of fermented red ginseng prepared according to Example using HPLC.
상기 표 8을 살펴보면, 실시예에 따른 발효 홍삼이 비교예에 비해 진세노사이드 화합물 K 및 F2의 함량이 현저히 증가된 것을 확인할 수 있다.Looking at Table 8, it can be seen that the content of ginsenoside compounds K and F2 in the fermented red ginseng according to the Example was significantly increased compared to the Comparative Example.
이러한 결과들을 통해, 본 발명의 균주 및 제조방법을 이용하는 경우 발효 홍삼의 특정 진세노사이드 함량을 효과적으로 증가시킬 수 있음을 확인하였다. Through these results, it was confirmed that the specific ginsenoside content of fermented red ginseng can be effectively increased when using the strain and manufacturing method of the present invention.
비록 본 발명이 상기에 언급된 바람직한 실시예로서 설명되었으나, 발명의 요지와 범위로부터 벗어남이 없이 다양한 수정이나 변형을 하는 것이 가능하다. 또한, 첨부된 특허청구범위는 본 발명의 요지에 속하는 이러한 수정이나 변형을 포함한다.Although the present invention has been described in terms of the preferred embodiments mentioned above, various modifications and variations are possible without departing from the spirit and scope of the invention. The appended claims also cover such modifications and variations as fall within the subject matter of this invention.
<110> Sunmoon University Industry-Academic Collaboration Foundation <120> Method of preparation for fermented red ginseng having increased content of ginsenoside Compound K and fermented red ginseng using the same <130> HPC-9613 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 720 <212> DNA <213> Lactobacillus pentosus <400> 1 gacgaacgct ggcggcgtgc ctaatacatg caagtcgaac gaactctggt attgattggt 60 gcttgcatca tgatttacat ttgagtgagt ggcgaactgg tgagtaacac gtgggaaacc 120 tgcccagaag cgggggataa cacctggaaa cagatgctaa taccgcataa caacttggac 180 cgcatggtcc gagtttgaaa gatggcttcg gctatcactt ttggatggtc ccgcggcgta 240 ttagctagat ggtggggtaa cggctcacca tggcaatgat acgtagccga cctgagaggg 300 taatcggcca cattgggact gagacacggc ccaaactcct acgggaggca gcagtaggga 360 atcttccaca atggacgaaa gtctgatgga gcaacgccgc gtgagtgaag aagggtttcg 420 gctcgtaaaa ctctgttgtt aaagaagaac atatctgaga gtaactgttc aggtattgac 480 ggtatttaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg 540 gcaagcgttg tccggattta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat 600 gtgaaagcct tcggctcaac cgaagaagtg catcggaaac tgggaaactt gagtgcagaa 660 gaggacagtg gaactccatg tgtagcggtg aaatgcgtag atatatggaa gaacaccagt 720 720 <110> Sunmoon University Industry-Academic Collaboration Foundation <120> Method of preparation for fermented red ginseng having increased content of ginsenoside Compound K and fermented red ginseng using the same <130> HPC-9613 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 720 <212> DNA <213> Lactobacillus pentosus <400> 1 gacgaacgct ggcggcgtgc ctaatacatg caagtcgaac gaactctggt attgattggt 60 gcttgcatca tgatttacat ttgagtgagt ggcgaactgg tgagtaacac gtgggaaacc 120 tgccccagaag cgggggataa cacctggaaa cagatgctaa taccgcataa caacttggac 180 cgcatggtcc gagtttgaaa gatggcttcg gctatcactt ttggatggtc ccgcggcgta 240 ttagctagat ggtggggtaa cggctcacca tggcaatgat acgtagccga cctgagaggg 300 taatcggcca cattgggact gagacacggc ccaaactcct acgggaggca gcagtaggga 360 atcttccaca atggacgaaa gtctgatgga gcaacgccgc gtgagtgaag aagggtttcg 420 gctcgtaaaa ctctgttgtt aaagaagaac atatctgaga gtaactgttc aggtattgac 480 ggtatttaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg 540 gcaagcgttg tccggattta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat 600 gtgaaagcct tcggctcaac cgaagaagtg catcggaaac tgggaaactt gagtgcagaa 660 gaggacagtg gaactccatg tgtagcggtg aaatgcgtag atatatggaa gaacaccagt 720 720
Claims (9)
(2) 유기산을 이용하여 pH 5.4 내지 5.8로 조정한 정제수에 서열번호 1의 염기서열로 표시되는 락토바실러스 펜토서스(Lactobacillus pentosus) KK18 균주(수탁번호: KCCM12762P)를 접종하고 균질화하여 균주 현탁액을 제조하는 단계; 및
(3) 상기 홍삼 분말 100 중량부에 대하여 상기 균주 현탁액 1 내지 10 중량부를 혼합한 후 고체발효시키는 단계;를 포함하는 진세노사이드 화합물 K의 함량이 증가된 발효 홍삼의 제조방법.(1) preparing red ginseng powder having a size of 100 to 300 mesh by grinding red ginseng;
(2) Lactobacillus pentosus KK18 strain (accession number: KCCM12762P) represented by the nucleotide sequence of SEQ ID NO: 1 is inoculated into purified water adjusted to pH 5.4 to 5.8 using an organic acid and homogenized to prepare a strain suspension doing; and
(3) mixing 1 to 10 parts by weight of the strain suspension with respect to 100 parts by weight of the red ginseng powder, followed by solid fermentation; manufacturing method of fermented red ginseng with increased content of ginsenoside compound K, including.
(1) 단계의 홍삼 분말은 분쇄 공간의 온도를 50 ℃ 이하로 유지하는 저온 분쇄, 또는 -3 내지 -10℃에서 냉동 분쇄를 수행하여 분쇄된 것임을 특징으로 하는 진세노사이드 화합물 K의 함량이 증가된 발효 홍삼의 제조방법.According to claim 1,
The red ginseng powder in step (1) has an increased content of ginsenoside compound K, characterized in that it is pulverized by low-temperature pulverization maintaining the temperature of the pulverization space at 50 ° C or less, or frozen pulverization at -3 to -10 ° C. Method for producing fermented red ginseng.
(2) 단계의 균주는 정제수에 대하여 0.5 내지 5 중량%로 접종하는 것을 특징으로 하는 것을 특징으로 하는 진세노사이드 화합물 K의 함량이 증가된 발효 홍삼의 제조방법.According to claim 1,
The method for producing fermented red ginseng with increased content of ginsenoside compound K, characterized in that the strain in step (2) is inoculated at 0.5 to 5% by weight with respect to purified water.
(3) 단계의 발효는 32 내지 42 ℃에서 60 내지 120 시간 동안 수행되는 것을 특징으로 하는 진세노사이드 화합물 K의 함량이 증가된 발효 홍삼의 제조방법.According to claim 1,
The method of producing fermented red ginseng with increased content of ginsenoside compound K, characterized in that the fermentation in step (3) is performed at 32 to 42 ° C. for 60 to 120 hours.
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