KR20090083505A - A method for preparing fermented ginseng of ginseng extract using lactobacillus fermentum bljh101 - Google Patents
A method for preparing fermented ginseng of ginseng extract using lactobacillus fermentum bljh101 Download PDFInfo
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- KR20090083505A KR20090083505A KR1020080009331A KR20080009331A KR20090083505A KR 20090083505 A KR20090083505 A KR 20090083505A KR 1020080009331 A KR1020080009331 A KR 1020080009331A KR 20080009331 A KR20080009331 A KR 20080009331A KR 20090083505 A KR20090083505 A KR 20090083505A
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- South Korea
- Prior art keywords
- ginseng
- lactobacillus fermentum
- ginsenoside
- culture
- extract
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Abstract
Description
본 발명은 미생물의 생장을 억제시키는 인삼추출물에서 특이적으로 성장 가능한 락토바실러스 퍼멘텀 BLJH101을 이용한 인삼추출물의 발효물을 제조하는 방법 및 이를 함유하는 조성물에 관한 것으로, 더욱 구체적으로 한국 미생물 보존센터에 기탁번호 KCCM10912P 로 기탁된 락토바실러스 퍼멘텀(Lactobacillus fermentum) BLJH 101(KCCM10912P) 및 인삼 추출액에서 락토바실러스 퍼멘텀(Lactobacillus fermentum)을 배양하는 단계와 상기 배양액을 수득하는 단계를 포함하는 고함량의 진세노사이드 Rg3, 컴파운드 K, 및 진세노사이드 F1을 함유하는 인삼 발효 배양액 제조 방법에 관한 것이다.The present invention relates to a method for preparing a fermentation product of ginseng extract using Lactobacillus pertumtum BLJH101 that can be specifically grown in ginseng extracts that inhibit the growth of microorganisms, and to a composition containing the same. Lactobacillus fermentum (Lactobacillus fermentum) deposited with the accession number KCCM10912P BLJH 101 (KCCM10912P) and ginsenosides comprising the step of culturing Lactobacillus fermentum (Lactobacillus fermentum) in ginseng extract and obtaining the culture medium A method for preparing a ginseng fermentation broth containing side Rg3, compound K, and ginsenoside F1.
인삼(panax ginseng C. A. Mayer)은 오갈피나무과(Aralaiaceae) 인삼속에 속하는 다년생 초본류로 과거 수천 년 전부터 우리나라를 비롯하여 중국, 일본 등에 서 주로 건강증진을 위한 목적으로 널리 사용하여 왔다. 최근에는 수삼을 증기로 수차례 쪄서 가공하여 인체에 유용한 미량의 진세노사이드를 함유한 홍삼의 효능이 뛰어나다고 알려져 있고, 그 사용량 또한 증가하고 있다. 인삼을 물과 에탄올로 추출한 인삼추출물의 주요 효능을 나타내는 사포닌(Saponin)은 30종류 이상의 진세노사이드(ginsenoside)로 구성된다. 진세노사이드는 홍삼추출물의 주요성분으로 면역력강화, 항염증작용, 항알러지작용, 항암효과, 발기부전에 대한 효과, 혈압강하작용, 항콜레스테롤작용, 항혈전작용, 성인병 및 노화에 대한 예방 및 치료효과가 있음이 보고 된다.Ginseng (panax ginseng C. A. Mayer) is a perennial herb belonging to the genus Aralaiaceae, and has been widely used for health promotion in Korea, China, and Japan for thousands of years. Recently, red ginseng containing a small amount of ginsenoside, which is useful for the human body, is processed by steaming several times of steamed ginseng, and its amount of use is also increasing. Saponin, which shows the main efficacy of ginseng extract extracted from ginseng with water and ethanol, is composed of more than 30 kinds of ginsenosides. Ginsenoside is the main ingredient of red ginseng extract, which prevents and cure immunity, anti-inflammatory, anti-allergic, anti-cancer, erectile dysfunction, hypotensive, anti-cholesterol, antithrombotic, adult disease and aging It is reported to be effective.
인삼의 사포닌은 트리터페노이드(triterpenoid)계열의 다마란(dammarane)골격에 글루코즈(glucose), 아라비노즈(arabinose), 자일로즈(xylose) 그리고 람노즈(rhamnose)등이 결합되어 있는 중성배당체이다. 인삼을 물과 주정으로 추출할 때 인삼 사포닌의 진세노사이드 중에서 20-O-β-D-글루코피라노실-20(S)-프로토파낙사디올(20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol; 이하, Rb1), 20-O-[α-L-아라비노피라노실(1→6)-β-D-글루코피라노실]-20(S)-프로토파낙사디올(20-O-[α-L-arabinopyranosyl(1→6)-β-D-glucopyranosyl]-20(S)-protopanaxadiol; 이하, Rb2), 20-O-[α-L-아라비노푸라노실(1→6)-β-D-글루코피라노실]-20(S)-프로토파낙사디올(20-O-[α-L-arabinofuranosyl(1→6)-β-D-glucopyranosyl]-20(S)-protopanaxadiol; 이하, Rc), 20(S)-프로토파낙사트리올(20(S)-protopanaxatriol; 이하, Rg1)등이 다량 존재한다. 이러한 사포닌을 체내에 섭취할 때, 장내에 있는 미생물에 의해 분해되어 체내로 흡수된다(Panta Med. 62, pp453-457, 1996). 진세 노사이드 Rb1의 경우, 장내 미생물에 의해 진세노사이드 Rd, 컴파운드(compound) 케이(K)와 프로토파낙사디올로 순차적으로 전환된다. 인삼사포닌을 분해하는 장내 미생물은 사람의 체질에 따라 그리고 식습관에 따라 그 존재의 유무와 보유하고 있는 정도가 다르며, 대사 산물로 전환하는데 차이가 있어서 인삼을 복용 후 저마다 효능의 차이가 나타날 수 있다. 장내 미생물 균총이 사람마다 다르기 때문에 진세노사이드의 전환율과 생체이용율은 다를 수밖에 없다 (J. Pharm. Pharmacol., 50, pp1155-1160, 1998). 따라서 장내미생물의 차이로 인한 효능과 흡수율의 차이를 없애기 위해, 미생물을 이용한 인삼추출물의 유산균 발효물을 이용하는 것은 개인차를 극복하고 체내 흡수율을 증대시키는 효과가 있다.Ginseng saponins are neutral glycosides that combine glucose, arabinose, xylose, and rhamnose with a triterpenoid-based dammarane skeleton. When ginseng is extracted with water and spirits, 20-O-β-D-glucopyranosyl-20 (S) -protopanaxadiol (20-O-β-D-glucopyranosyl-20) in ginsenosides of ginseng saponins S) -protopanaxadiol; hereinafter Rb1), 20-O- [α-L-arabinofyranosyl (1 → 6) -β-D-glucopyranosyl] -20 (S) -protopanaxadiol (20- O- [α-L-arabinopyranosyl (1 → 6) -β-D-glucopyranosyl] -20 (S) -protopanaxadiol; hereinafter Rb2), 20-O- [α-L-arabinofuranosyl (1 → 6) ) -β-D-glucopyranosyl] -20 (S) -protopanaxadiol (20-O- [α-L-arabinofuranosyl (1 → 6) -β-D-glucopyranosyl] -20 (S) -protopanaxadiol Rc) and 20 (S) -protopanaxatriol (20 (S) -protopanaxatriol; hereinafter Rg1) are present in large amounts. When these saponins are ingested in the body, they are broken down and absorbed by the microorganisms in the intestine (Panta Med. 62, pp453-457, 1996). In the case of ginsenoside Rb1, it is sequentially converted to ginsenoside Rd, compound K (K) and protopanaxadiol by intestinal microorganisms. The intestinal microorganisms that break down ginseng saponins vary depending on the constitution and dietary habits of humans, and their presence and degree of retention, and there is a difference in the conversion to metabolites, which may result in different effects after taking ginseng. Since the intestinal microflora varies from person to person, ginsenoside conversion and bioavailability are inevitably different (J. Pharm. Pharmacol., 50, pp 1155-1160, 1998). Therefore, in order to eliminate the difference in efficacy and absorption rate due to the difference in intestinal microorganisms, the use of lactic acid bacteria fermentation of ginseng extract using microorganisms has the effect of overcoming individual differences and increasing the absorption rate in the body.
이와 같이 인삼추출물의 우수한 기능성과 더불어 생체 이용률을 높이기 위해서 다양한 시도들이 행해지고 있다. As such, various attempts have been made to increase the bioavailability with excellent functionality of ginseng extract.
현재까지 발효 홍삼을 제조하는 방법으로는 김치유산균을 이용한 발효인삼 또는 발효 홍삼의 제조방법(한국특허공개 10-2006-0001834)에서 김치 유산균을 사용하여 인삼 및 홍삼을 발효시킨 후 유기산을 처리하여 발효인삼 및 발효 홍삼을 제조하는 방법을 사용하였다. 이는 인삼 및 홍삼에 김치유산균을 발효 후에 유기산을 추가로 처리하여 산 분해 공정이 추가된 점과 유기산 처리 후에 추출액의 pH 하락에 의한 신맛이 발생하는 문제점이 있다. 기능성 및 관능성이 우수한 인삼발효액의 제조방법(한국공개특허 10-2003-0041630)에서 퍼핑 처리한 인삼을 아스퍼질러스 속 균을 이용하여 발효하여 유효성분의 함량을 증가시켰다. 유효성분은 증가 되었지만 체내에 이용률을 높이는 성분으로의 전환되지는 않았다. 인삼 사포닌 대사에 관여해 생리활성도를 증가시키는 장내세균제제(한국공개특허 10-2003-0029165)에서는 인삼 사포닌 대사에 관여하여 생리활성도를 증가시키는 효과를 나타내는 장내 세균제제에 관한 것으로 위산에 안정하고 컴파운드 K와 프로토파낙사디올의 전환율이 높은 장내 세균제제를 제시하였다. 세균제제를 인삼 혹은 추출물과 동시에 섭취하였을 경우 체내에서의 전환율에 대한 근거는 미약하다. Up to now, fermented red ginseng is fermented by fermenting ginseng or red ginseng using Kimchi lactic acid bacteria or fermented ginseng and red ginseng using kimchi lactic acid bacteria in the method of producing fermented red ginseng (Korean Patent Publication No. 10-2006-0001834). The method of preparing ginseng and fermented red ginseng was used. This is a kimchi lactic acid bacteria in the ginseng and red ginseng after the fermentation of the organic acid is added to the acid decomposition process and there is a problem that the acidity caused by the pH drop of the extract after the organic acid treatment. The ginseng puffed in the method of producing ginseng fermentation solution with excellent functionality and functionality (Korean Patent Publication No. 10-2003-0041630) was fermented using Aspergillus bacteria to increase the content of the active ingredient. The active ingredient was increased, but not converted into ingredients that increase utilization in the body. Intestinal bacterial preparations that increase physiological activity by participating in ginseng saponin metabolism (Korea Patent Publication No. 10-2003-0029165) relates to intestinal bacterial preparations that have an effect of increasing physiological activity by participating in saponin metabolism of ginseng. Intestinal bacterial preparations with high conversion of K and protoparnaxadiol were suggested. If bacterial products are taken simultaneously with ginseng or extract, the evidence for conversion in the body is weak.
이에 본 발명자들은 홍삼에 특이적으로 생육하는 유산균을 한국 정통 발효 식품인 김치에서 선별하였으며, 균주의 생화학적 패턴을 통해 균주를 동정하는 API kit, 균주를 분자 유전학적 측면에서 동정하는 16S rDNA partial sequencing을 통해 인삼 추출물에 특이적인 성장을 보이는 균주의 종, 속명을 확인하였다. 상기 균주 배양을 통해서 생체 이용률이 낮은 진세노사이드 Rg1, Rb1, Rg, Re등을 진세노사이드 Rg3, Rh2, 컴파운드 K, 진세노사이드 F1등으로 획기적인 전환을 시켰고, 동시에 유산균의 기능이 강화된 항균효능을 접목시킨 본 발명을 완성하게 되었다. Accordingly, the present inventors selected lactic acid bacteria specifically grown in red ginseng from Kimchi, a traditional Korean fermented food, and a 16S rDNA partial sequencing that identifies an API kit and strains in terms of molecular genetics. Through the species of the strain showing a specific growth in ginseng extract was identified. Ginsenosides Rg1, Rb1, Rg, Re, etc. with low bioavailability through the strain culture was transformed into ginsenosides Rg3, Rh2, compound K, ginsenoside F1, and at the same time, the antibacterial function of lactic acid bacteria enhanced The present invention combining the efficacy has been completed.
이에, 본 발명자들은 상기 종래기술들의 문제점들을 극복하기 위하여 예의 연구 노력한 결과, 락토바실러스 퍼멘텀(Lactobacillus fermentum) BLJH 101(KCCM10912P)을 이용하여 인삼 추출물을 발효시키는 경우, 고함량의 진세노사이드 Rg3, 컴파운드 K, 진세노사이드 Rh2, 프로토파낙트리올 및 진세노사이드 F1을 함유하고, 병원균에 대한 항균능이 증대된 인삼 발효 배양액을 제조 할 수 있음을 확인하고, 본 발명을 완성하게 되었다.Accordingly, the present inventors have made diligent research efforts to overcome the problems of the prior art, when fermenting ginseng extract using Lactobacillus fermentum BLJH 101 (KCCM10912P), high content of ginsenoside Rg3, It was confirmed that the ginseng fermentation broth containing compound K, ginsenoside Rh2, protopanactriol and ginsenoside F1, and having increased antibacterial activity against pathogens, was completed.
따라서, 본 발명의 주된 목적은 미생물의 생장을 억제시키는 인삼 추출물에서 특이적으로 생육하는 유산균인 락토바실러스 퍼멘텀 BLJH101을 제공하는 데 있다.Therefore, the main object of the present invention is to provide a lactobacillus fermentum BLJH101 which is a lactic acid bacteria specifically grown in ginseng extracts that inhibit the growth of microorganisms.
본 발명의 다른 목적은 상기 락토바실러스 퍼멘텀 BLJH101을 이용하여 미생물 배양시 필요한 다른 첨가물이 없는 순수한 인삼 추출액에서 락토바실러스 퍼멘텀(Lactobacillus fermentum)을 배양하는 단계와 상기 배양액을 수득하는 단계에 의해 제조된 고 함량의 진세노사이드 Rg3, 컴파운드 K, 및 진세노사이드 F1을 함유하는 인삼 발효 배양액 및 그 제조 방법을 제공하는 데 있다.Another object of the present invention is to prepare the Lactobacillus fermentum (Lactobacillus fermentum) in a pure ginseng extract without the other additives required for microbial culture using the Lactobacillus Permentum BLJH101 and the culture medium prepared by the step of obtaining To provide a ginseng fermentation broth containing a high content of ginsenoside Rg3, Compound K, and ginsenoside F1 and a method for producing the same.
본 발명의 한 양태에 따르면, 본 발명은 한국 미생물 보존센터에 기탁번호 KCCM10912P로 기탁된 락토바실러스 퍼멘텀(Lactobacillus fermentum) BLJH 101을 제공한다. 본 발명의 상기 락토바실러스 퍼멘텀(Lactobacillus fermentum) BLJH 101(KCCM10912P)은 인삼 추출액에서 특이적으로 생장하는 특성을 갖는 균주이다. 인삼 추출액은 본래 그 자체로 미생물의 성장을 억제하는 성분을 갖기 때문에 본 발명의 락토바실러스 퍼멘텀 BLJH 101과 같이 인삼 추출액에서 특이적으로 생장하여 대사과정을 통해 인삼 추출액의 성분을 변환할 수 있는 균주는 인삼 추출물의 이용에 있어서 중요한 의미를 갖는다. 상기 락토바실러스 퍼멘텀은 API 분석시 Lactobacillus fermentum과 86.4%의 유사성을 갖는다(도 3a). 본 발명의 상기 락토바실러스 퍼멘텀 BLJH 101은 인삼 추출물에서 배양할 때 타 균주와 비교하여 현저히 높은 생균수를 보이는 장점을 갖는다(실시예 1-2, 도 1a 및 도 1b). 이는 균주 성장에 있어서 본 발명의 락토바실러스 퍼멘텀 BLJH 101의 인삼 추출물 이용도가 타균주에 비해 월등히 높다는 것을 의미한다.According to one aspect of the present invention, the present invention provides Lactobacillus fermentum BLJH 101 deposited in the Korean microbial conservation center with the accession number KCCM10912P. The Lactobacillus fermentum BLJH 101 (KCCM10912P) of the present invention is a strain having specific growth characteristics in ginseng extract. Since ginseng extract itself has a component that inhibits the growth of microorganisms in itself, it is a strain capable of specifically growing in ginseng extract, such as Lactobacillus fermentum BLJH 101 of the present invention, and converting the components of the ginseng extract through metabolic processes. Has an important meaning in the use of ginseng extract. The Lactobacillus fermentum had a similarity of 86.4% with Lactobacillus fermentum in the API analysis (FIG. 3A). The Lactobacillus
본 발명의 한 양태에 따르면, 본 발명은 인삼 추출액에서 락토바실러스 퍼멘텀(Lactobacillus fermentum)을 배양하는 단계 및 상기 락토바실러스 퍼멘텀에 의해 발효된 인삼 배양액을 수득하는 단계를 포함하는 고함량의 진세노사이드 Rg3, 컴파운드 K, 진세노사이드 Rh2, 프로토파낙트리올 및 진세노사이드 F1을 함유하는 인삼 발효 배양액의 제조 방법을 제공한다. 본 발명에서 고함량이란 락토바실러스 퍼멘텀 BLJH 101을 배양하기 전의 인삼 추출물보다 진세노사이드 Rg3, 컴파운드 K, 진세노사이드 Rh2, 프로토파낙트리올 및 진세노사이드의 함량이 증대된 것을 의미한다. According to an aspect of the present invention, the present invention comprises the step of culturing Lactobacillus fermentum (Lactobacillus fermentum) in ginseng extract and obtaining a high content of ginsenosides comprising the step of obtaining a ginseng culture fermented by the Lactobacillus fermentum Provided is a method for preparing a ginseng fermentation broth containing Side Rg3, Compound K, Ginsenoside Rh2, Protopanactriol, and Ginsenoside F1. High content in the present invention means that the content of ginsenoside Rg3, compound K, ginsenoside Rh2, protopanactriol and ginsenosides is increased than the ginseng extract before culturing Lactobacillus fermentum BLJH 101.
본 발명의 인삼 발효 배양액 제조 방법에서, 상기 락토바실러스 퍼멘텀(Lactobacillus fermentum) BLJH 101은 서열번호 1의 16s RNA를 갖는 락토바실러스 퍼멘텀(Lactobacillus fermentum) BLJH 101도 포함하나, 바람직하게는 한국 미생물 보존센터에 기탁번호 KCCM10912P로 기탁된 락토바실러스 퍼멘텀(Lactobacillus fermentum) BLJH 101인 것이 좋다.In the method for preparing the ginseng fermentation broth of the present invention, the Lactobacillus fermentum BLJH 101 also includes the Lactobacillus fermentum BLJH 101 having 16s RNA of SEQ ID NO: 1, but preferably preserves Korean microorganisms. Lactobacillus fermentum BLJH 101 deposited in the center with the accession number KCCM10912P is preferred.
본 발명의 인삼 발효 배양액 제조 방법에서, 상기 인삼 추출액은 어떠한 인삼도 될 수 있으나, 바람직하게는 재배삼, 장뇌삼, 산삼, 수삼, 백삼, 홍삼, 흑삼 및 태극삼으로 이루어진 군에서 선택된 어느 하나의 추출액인 것이 좋고, 더욱 바람직하게는 홍삼 추출액인 것이 좋다. 이는 홍삼을 사용하는 경우 수삼에 비해 수분함량이 현저히 낮아 변질의 우려가 없고, 수삼을 증기로 찌고 말리는 과정에서 유효성분이 더욱 농축되기 때문이다. 본 발명의 실시예에서는 홍삼을 이용하여 락토바실러스 퍼멘텀 BLJH 101을 분리 동정하였다.In the method of preparing ginseng fermentation broth of the present invention, the ginseng extract may be any ginseng, but preferably any one extract selected from the group consisting of cultivated ginseng, camphor ginseng, wild ginseng, ginseng, white ginseng, red ginseng, black ginseng, and taegeuk ginseng. It is good that it is more preferably a red ginseng extract. This is because when the red ginseng is used, the moisture content is significantly lower than that of the ginseng, so there is no fear of deterioration, and the active ingredient is more concentrated in steaming and drying the ginseng. In the embodiment of the present invention, the red ginseng was isolated and identified Lactobacillus permanent BLJH 101.
본 발명의 인삼 발효 배양액 제조 방법에서, 상기 배양액은 병원균인 Escherichia coli , Pseudomonas aeruginosa , 또는 Staphylococcus aureus 에 대하여 항균능이 증대되고, 항산화능이 증대된 것을 특징으로 한다. 본 발명의 실시예 5 및 실시예 6에서는 본 발명의 인삼 발효 배양액이 발효하지 않은 인삼 추출액과 비교하여 항균능과 항산화능이 증가하였음을 증명하였다.In the ginseng fermentation broth production method of the present invention, the culture medium is Escherichia coli , Pseudomonas pathogens aeruginosa , or Staphylococcus aureus It is characterized in that the antimicrobial activity is increased, and the antioxidant activity is increased. In Example 5 and Example 6 of the present invention, the ginseng fermentation broth of the present invention was proved to increase the antimicrobial activity and antioxidant activity compared to the non-fermented ginseng extract.
본 발명의 한 양태에 따르면, 본 발명은 인삼 추출액에서 락토바실러스 퍼멘텀(Lactobacillus fermentum) BLJH 101을 배양하는 단계 및 상기 배양액을 수득하는 단계를 포함하는 방법에 따라 제조된 고함량의 진세노사이드 Rg3, 컴파운드 K, 진세노사이드 Rh2, 프로토파낙트리올 및 진세노사이드 F1을 함유하는 인삼 발효 배양액을 제공한다.According to an aspect of the present invention, the present invention provides a high content of ginsenoside Rg3 prepared according to a method comprising culturing Lactobacillus fermentum BLJH 101 in a ginseng extract and obtaining the culture solution. A ginseng fermentation broth containing Compound K, ginsenoside Rh2, protopanactriol and ginsenoside F1 is provided.
본 발명의 한 양태에 따르면, 본 발명은 인삼 추출액에서 락토바실러스 퍼멘텀(Lactobacillus fermentum)을 배양하는 단계, 상기 락토바실러스 퍼멘텀에 의해 발효된 인삼 배양액을 수득하는 단계, 상기 수득한 인삼 배양액을 멸균하는 단계, 및 상기 멸균한 인삼 배양액을 농축하는 단계를 포함하는 유산균 사균 함유 인삼 배양 농축액의 제조 방법 및 이에 따라 제조된 유산균 사균 함유 인삼 배양 농축액 을 제공한다.According to an aspect of the present invention, the present invention comprises the steps of culturing the Lactobacillus fermentum (Lactobacillus fermentum) in the ginseng extract, obtaining a ginseng culture fermented by the Lactobacillus fermentum, the obtained ginseng culture sterilized It provides a method for producing a lactic acid bacteria bacteria-containing ginseng culture concentrate comprising the step of, and concentrating the sterilized ginseng culture solution and thus prepared ginseng culture concentrate containing the lactic acid bacteria bacilli.
본 발명의 한 양태에 따르면, 본 발명은 인삼 추출액에서 락토바실러스 퍼멘텀(Lactobacillus fermentum)을 배양하는 단계, 상기 락토바실러스 퍼멘텀에 의해 발효된 인삼 배양액을 수득하는 단계, 및 상기 수득한 인삼 발효 배양액을 건조하는 단계를 포함하는 인삼 배양 분말의 제조 방법 및 이에 따라 제조된 인삼 배양 분말을 제공한다.According to an aspect of the present invention, the present invention comprises the steps of culturing Lactobacillus fermentum (Lactobacillus fermentum) in the ginseng extract, obtaining a ginseng culture fermented by the Lactobacillus fermentum, and the obtained ginseng fermentation broth It provides a method for producing a ginseng culture powder comprising the step of drying and the ginseng culture powder prepared accordingly.
본 발명의 한 양태에 따르면, 본 발명은 인삼 추출액에서 락토바실러스 퍼멘텀(Lactobacillus fermentum) BLJH 101을 배양하는 단계 및 상기 배양액을 수득하는 단계를 포함하는 방법에 따라 제조된 고함량의 진세노사이드 Rg3, 컴파운드 K, 진세노사이드 Rh2, 프로토파낙트리올 및 진세노사이드 F1을 함유하는 인삼 발효 배양액을 동결건조하여 1.0 × 107 CFU/g 이상의 락토바실러스 퍼멘텀(Lactobacillus fermentum) BLJH 101을 포함하는 인삼 배양액 생균제 제조 방법 및 이에 따라 제조된 인삼 배양액 생균제를 제공한다.본 발명의 상기 인삼 배양액 생균제는 바람직하게는 1.0 × 107 CFU/g 이상의 락토바실러스 퍼멘텀(Lactobacillus fermentum) BLJH 101을 포함하는 것이 좋다. According to an aspect of the present invention, the present invention provides a high content of ginsenoside Rg3 prepared according to a method comprising culturing Lactobacillus fermentum BLJH 101 in a ginseng extract and obtaining the culture solution. Ginseng containing Lactobacillus fermentum BLJH 101 with 1.0 × 10 7 CFU / g or more of lyophilized ginseng fermentation broth containing Compound K, ginsenoside Rh2, protopanactriol and ginsenoside F1 Provided is a method for producing a culture medium probiotic and a ginseng culture probiotic prepared according to the present invention. The ginseng culture probiotic of the present invention is preferably Lactobacillus at least 1.0 × 10 7 CFU / g. fermentum ) BLJH 101 may be included.
본 발명의 한 양태에 따르면, 본 발명은 본 발명의 락토바실러스 퍼멘텀(Lactobacillus fermentum) BLJH 101을 인삼 추출물에서 배양하여 제조한 인삼 발효 배양액, 유산균 사균 함유 인삼 배양 농축액, 또는 인삼 배양액 생균제를 유효성분으로 함유하는 항균용 조성물을 제공한다.According to an aspect of the present invention, the present invention is a ginseng fermentation broth prepared by culturing the Lactobacillus fermentum BLJH 101 of the present invention in ginseng extract, ginseng culture concentrate containing lactic acid bacteria, or ginseng culture probiotic active ingredient It provides an antimicrobial composition containing.
상기 목적을 달성하기 위하여, 본 발명에서는 한국 정통식품인 김치에서 홍삼 근에 전이된 유산균을 분리하고, 특이적으로 인삼추출물만을 이용하여 성장하는 유산균을 분리 동정하였다. 또한 본 발명의 분리 동정된 균주 락토바실러스 퍼멘텀(Lactobacillus fermentum) BLJH 101을 인삼추출물로 단독 배양함으로써 유산균의 대사산물인 진세노사이드 Rg3, Rh2, 컴파운드 K, 및 진세노사이드 F1등을 포함하고, 인삼 추출물의 항균능이 증대된 발효액, 발효분말, 발효 농축액, 또는 발효 생균제와 이를 함유하는 조성물을 제공한다.In order to achieve the above object, in the present invention, lactic acid bacteria transferred to red ginseng root from Kimchi, which is a traditional Korean food, was isolated and specifically identified as lactic acid bacteria grown using only ginseng extract. In addition, the isolated and identified strain Lactobacillus fermentum BLJH 101 of the present invention by culturing alone with ginseng extract includes ginsenosides Rg3, Rh2, compound K, and ginsenoside F1, which are metabolites of lactic acid bacteria, Provided is a fermentation broth, a fermentation powder, a fermentation concentrate, or a fermentation probiotic and a composition containing the same, which have an increased antibacterial activity.
이하, 본 발명의 균주를 분리하여 인삼 발효 배양액, 발효분말, 발효 농축액, 또는 발효 생균제와 이를 함유하는 조성물을 제조하는 방법을 상세하게 설명한다.Hereinafter, the method for preparing a ginseng fermentation broth, fermentation powder, fermentation concentrate, or fermentation probiotic and a composition containing the same by separating the strain of the present invention will be described in detail.
한국 정통 발효 식품인 배추김치에 멸균한 홍삼을 넣고, 한 달 동안 숙성시킨 후, 김치와 숙성된 홍삼을 믹서로 분쇄하고, 멸균된 생리 식염수로 희석한 액을 체로 걸러 얻은 용액을 한천배지에 37℃, 2일 배양하여, 균체 주변이 노란 콜로니를 57개 확보하였다. 확보한 콜로니를 MRS 한천배지에 스트리킹하여, 37℃에서 3일 배양한 후 간균 형태의 콜로니 10종을 선별하였다. 각각의 콜로니를 5% 인삼 추출액에 접종한 후 1.0 × 108 CFU/mL 이상의 생균력을 보이는 특이적인 균주 1종을 선별하였고, 이를 BLJH 101이라 명명하였다(도 2). 선발된 인삼 추출물 이용 균주를 MRS 한천 배지에 접종하여 37℃, 2일 배양 후, 단일 콜로니를 얻었다. 상기 균주에 대하여 생화학적 패턴으로 균체를 동정하는 방법인 API 분석을 수행하였다(도 3a 및 도 3b). 그 결과 본 발명의 BLJH 101는 락토바실러스 퍼멘텀(Lactobacillus fermentum)과 86.4%의 유사성을 나타내었다(도 3a). 또한 본 발명의 BLJH 101는 라이보오스, 자일로오스, 갈락토오스, 프록토오스, 만노오스, 말토오스, 멜리비오스, 및 글루코네이트를 분해하여 산을 생성하였다(도 3b). Sterilized red ginseng was added to cabbage kimchi, a Korean traditional fermented food, and aged for one month. The kimchi and matured red ginseng were pulverized with a mixer, and the solution diluted with sterile saline solution was sieved. After culturing at 2 ° C. for 2 days, 57 colonies were obtained around the cells. Secured colonies were streaked on MRS agar medium, and then cultured at 37 ° C. for 3 days, and 10 colonies of bacilli were selected. After inoculating each colony into 5% ginseng extract, one specific strain showing viability of 1.0 × 10 8 CFU / mL or more was selected and named BLJH 101 (FIG. 2). The selected ginseng extract using the strain was inoculated in MRS agar medium and cultured at 37 ° C. for 2 days to obtain a single colony. The strain was subjected to API analysis, which is a method of identifying cells in biochemical patterns (FIGS. 3A and 3B). As a result,
또한 분자 유전학적 균주 동정 방법인 16S rDNA partial sequecing을 수행하여 균주를 종 수준에서 동정하였다(도 4). 그 결과, 락토바실러스 퍼멘텀(Lactobacillus fermentum)CECT 562 AJ575812과 가장 유사하므로 Lactobacillus fermentum BLJH 101로 명명하였다. 상기 균주는 2007년 12월 23일부로 대한민국 특허균주 기탁기관인 한국 미생물 보존센터에 기탁하여 수탁번호 KCCM10912P를 부여받았다. In addition, strains were identified at the species level by performing 16S rDNA partial sequecing, a molecular genetic strain identification method (FIG. 4). As a result, Lactobacillus fermentum (Lactobacillus fermentum) CECT 562 AJ575812 because it is the most similar to the name
본 균주의 특성을 다른 유산균 균주와 비교하기 위해, 한국 미생물 보존센터에서 분양받은 공시 균주들과 MRS 배지 및 인삼 추출물(15%)에 각각 접종하여 배양하였다. 그리고 각각의 균주들의 MRS 배지 대비 인삼 추출물에서의 생장을 측정 하였다. In order to compare the characteristics of this strain with other lactic acid bacteria strains, the strains inoculated in MRS medium and ginseng extract (15%), which were distributed in Korea microbial conservation center were incubated. And the growth in ginseng extract compared to MRS medium of each strain was measured.
인삼 추출물은 그 자체로 미생물의 생장을 억제하는 항균능을 갖는다. 본 발명에서도 인삼추출물(30%)을 증류수(70%)에 혼합하여 실험한 경우 본 발명의 Lactobacillus fermentum BLJH 101을 제외한 다른 실험 균들은 하루가 경과하여 모두 사멸하였다. 인삼 추출물(10%)을 이용한 단독배양 시 Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus 등의 병원균은 물론, 같은 유산균 속인 Lactobacillus sakei, Lactobacillus heveticus, Lactobacillus sporogenesis, Pediococcus pentosaceus 등도 모두 성장이 저해되었다. 또한 인삼추출물(5%)을 증류수(95%)에 혼합하여 실험한 경우에도 역시 하루가 지나서 균들이 사멸하는 결과를 나타내었는데, 이는 상기 혼합물 내의 영양소가 결핍되어 나타나는 결과라고 판단된다. Ginseng extract itself has an antimicrobial activity that inhibits the growth of microorganisms. In the present invention, when the experiment was mixed with ginseng extract (30%) in distilled water (70%), all other test bacteria except
따라서 본 발명자들은 인삼추출물(15%)을 증류수(85%)에 혼합하여 제조한 혼합액에 본 발명의 균주인 Lactobacillus fermentum BLJH 101과 다른 균주들을 접종하여 생장을 관찰하였다. 관찰결과 본 발명의 Lactobacillus fermentum BLJH 101이 독보적으로 인삼 추출물을 이용하여 생장함을 확인하였다(도 1a 및 도 1b). 인삼 추출물 배지에서 본 발명의 균주인 Lactobacillus fermentum BLJH 101의 생균수가 많다는 것은 균주 성장에 있어서 타 균주에 비해 인삼 추출물 이용도가 월등히 높다는 것을 의미하며, Lactobacillus fermentum BLJH 101이 생체 이용률이 낮은 인삼 추출물의 진세노사이드 Rg1, Rb1, Rg, Re등을 인체에 흡수가 높은 진세노사이드 Rg3, 컴파운드 K, 진세노사이드 Rh2, 프로토파낙트리올, 진세노사이드 F1 등으로 효율적으로 전환할 수 있음을 나타내는 증거이다. Therefore, the present inventors inoculated the
본 발명의 인삼추출물의 유산균 배양액을 제조하는 방법을 설명한다.It describes a method for producing a lactic acid bacteria culture medium of the ginseng extract of the present invention.
인삼을 그 중량의 약 3 내지 10 배, 바람직하게는 약 5 내지 7 배에 달하는 부피의 물, 에탄올, 또는 이들의 혼합용매, 바람직하게는 물 및 에탄올 혼합용매로 에탄올을 기준으로 40 내지 80 v/v%, 바람직하게는 약 60 내지 70 v/v% 비율이 되도록 약 1 내지 10시간, 바람직하게는 약 2 내지 5시간 동안 1 내지 10회, 바람직하게는 4 내지 6회 반복하여, 열수추출, 초음파 추출, 환류냉각추출 등의 추출방법, 바람직하게는 환류냉각 추출방법을 이용하여 추출한 추출액을 감압 여과하여 얻은 추출물을 약 40 내지 80℃, 바람직하게는 약 50 내지 60℃에서 감압 농축하여 추출물을 수득하는 제 1 단계; 상기 제 1 단계의 인삼추출물을 고형분 함량이 약 10 내지 60 w/v%, 바람직하게는 약 10 내지 40 w/v%가 되도록 정제수를 첨가하여 희석하여 인삼추출액을 얻는 제 2 단계; 인삼추출액을 멸균한 후, Lactobacillus fermentum BLJH 101을 접종하여 내압 0.3 내지 1.0 bar, 바람직하게는 0.5 내지 0.7 bar, 회전속도를 50 내지 300 rpm, 바람직하게는 100 내지 200 rpm, 통기량을 0.1 내지 1.0 vvm, 바람직하게는 0.3 ~ 0.5 vvm을 유지하면서, 1 내지 4일간, 바람직하게는 2 내지 3일간 배양하는 3단계를 통하여 인삼추출물의 유산균 배양물을 제조할 수 있다.Ginseng is about 3 to 10 times its weight, preferably about 5 to 7 times the volume of water, ethanol, or a mixed solvent thereof, preferably water and ethanol mixed solvent, 40 to 80 v based on ethanol Hot water extraction is repeated 1 to 10 times, preferably 4 to 6 times for about 1 to 10 hours, preferably about 2 to 5 hours, so that the ratio is / v%, preferably about 60 to 70 v / v%. , Extracts obtained by ultrasonic extraction, reflux cooling extraction, and the like, and preferably, extracts obtained by filtration under reduced pressure using a reflux cooling extraction method are concentrated under reduced pressure at about 40 to 80 ° C, preferably about 50 to 60 ° C. A first step of obtaining; A second step of diluting the ginseng extract of the first step by adding purified water so that the solid content is about 10 to 60 w / v%, preferably about 10 to 40 w / v%; After sterilizing the ginseng extract, inoculated with
상기 방법을 통해 얻은 인삼 추출액 Lactobacillus fermentum BLJH 101 발효 분말을 적정량 메탄올에 녹여, 발효를 실시하지 않은 인삼 추출액과 비교하여, 분석한 결과 진세노사이드 Rb1은 3.8배 감소하고, 진세노사이드 Rg1은 검출이 되지 않았다. Rg3(Rg3-S, Rg3-R 합한 것)의 함량은 배양 후 약 4배가 증가하였고, 컴파운드 K의 경우도 약 4배가 증가하였으며, 진세노사이드 F1의 경우도 약 1.5배 증가하였다. 배양 후, 진세노사이드 Rh2는 0.22% 생성되었으며, 프로토파낙트리올도 1.22% 생성되었다. 이로써, 인삼 추출액의 Lactobacillus fermentum BLJH 101 발효 를 통하여, 인체 내 흡수가 용이한 진세노사이드 Rg3, 컴파운드 K, 진세노사이드 F1, 진세노사이드 Rh2, 및 프로토파낙트리올의 성분이 증가되었음을 알 수 있었다(실시예 5의 표 3).Ginseng extract Lactobacillus obtained through the above method fermentum The
상기 발효액 분말의 항균 활성 측정을 위해 MIC(Minimum Inhibitory Concentration)테스트를 하여, 병원균 Escherichia coli, Pseudomonas aeruginosa, 및 Staphylococcus aureus에 대한 최소 저해 농도를 설정하였다. 그 결과 실시예의 분말에 있어서 Escherichia coli.에 대한 항균활성은 3.3배, Pseudomonas aeruginosa에 대한 항균활성은 1.6배, Staphylococcus aureus에 대한 항균활성은 증가하였다(도 6).In order to determine the antimicrobial activity of the fermentation broth powder was tested by Minimum Inhibitory Concentration (MIC), and the minimum inhibitory concentrations for the pathogen Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were set. As a result, the antimicrobial activity against Escherichia coli. Was 3.3 times, the antimicrobial activity against Pseudomonas aeruginosa was 1.6 times, and the antimicrobial activity against Staphylococcus aureus was increased in the powder of Example (Fig. 6).
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.
실시예Example 1. One. 락토바실러스Lactobacillus 퍼멘텀Permanent BLJH101BLJH101 의 분리 동정Separation Identification of
1-1. 1-1. 락토바실러스Lactobacillus 퍼멘텀Permanent BLJH101BLJH101 의 분리Separation of
한국 정통 발효 식품인 배추김치에 멸균한 홍삼을 넣고, 한 달 동안 숙성시킨 후, 김치와 숙성된 홍삼을 믹서로 분쇄하고, 멸균된 생리 식염수로 희석한 액을 체로 걸러 얻은 용액을 BCP plate count 한천배지(100그램 당 펩톤 0.5그램 중, 이 스트 발효추출물 0.25그램 중, 포도당 0.1그램 중, 폴리솔베이트 80 0.1그램 중, 엘 시스테인 0.01그램 중, 브로모크레졸 퍼플 0.005그램 중, 한천 1.5그램 중)에 37℃, 2일 배양하여, 균체 주변이 노란 콜로니를 57개 확보하였다. Sterilized red ginseng was added to cabbage kimchi, a Korean traditional fermented food, and aged for one month. The kimchi and matured red ginseng were pulverized with a mixer, and the solution obtained by sieving the diluted solution with sterile saline was filtered through a BCP plate count agar. Medium (in 0.5 grams of peptone per 100 grams, in 0.25 grams of yeast fermentation extract, in 0.1 grams of glucose, in 0.1 grams of polysorbate 80, in 0.01 grams of L cysteine, in 0.005 grams of bromocresol purple, in 1.5 grams of agar) The cells were cultured at 37 ° C. for 2 days to secure 57 yellow colonies around the cells.
확보된 콜로니를 MRS(100그램 당 포도당 2그램 중, 트윈 80 0.1그램 중, 암모늄 시트레이트 0.2그램 중, 초산염 0.5그램 중, 가수황산마그네슘 0.01그램 중, 무수황산망간 0.005그램 중, 2염기 인산칼륨 0.2그램 중, 비프 추출물 1그램 중, 효모 추출물 0.5그램 중, 단백질효소처리 펩톤 1그램 중) 한천배지에 스트리킹하여, 37℃에서 3일 배양한 후, 검경하여 간균 형태의 콜로니 10종을 선별하였다. 각각의 콜로니를 5% 인삼 추출액에 접종한 후 37℃에서, 1 일간 배양하고, 1.0 × 108 CFU/mL 이상의 생균력을 보이는 특이적인 균주 1종을 선별하였다. 이를 BLJH 101이라 명명하였다. The obtained colonies were MRS (in 2 grams of glucose per 100 grams, in 0.1 grams of Tween 80, in 0.2 grams of ammonium citrate, in 0.5 grams of acetate, in 0.01 grams of magnesium sulfate, in 0.005 grams of anhydrous manganese sulfate, in potassium dibasic phosphate. Among 0.2 grams, 1 gram of beef extract, 0.5 grams of yeast extract, 1 gram of proteinase-treated peptone) were streaked on agar medium, incubated at 37 ° C. for 3 days, and then examined and selected 10 kinds of colony in bacillus form. . Each colony was inoculated in 5% ginseng extract, and then cultured at 37 ° C. for 1 day, and one specific strain showing a viability of 1.0 × 10 8 CFU / mL or more was selected. It was named
선발된 인삼 추출물 이용 균주를 MRS 한천 배지에 접종하여 37℃, 2일 배양 후, 단일 콜로니를 얻었다. 상기 균주에 대하여 생화학적 패턴으로 균체를 동정하는 방법인 API 분석을 수행하였다. Lactobacillus fermentum과 86.4% 유사성이 있음을 확인하였다. 또한 분자 유전학적 균주 동정 방법인 16S rDNA partial sequecing을 수행하여 균주를 종 수준에서 동정하였다. 16S rDNA partial sequencing으로 염기서열을 결정하였고, National Center for Biotechnological Information (http://www.ncbi.nlm.nih.gov/blast)의 자료를 검색한 결과(도 5), 락토바실러스 퍼멘텀(Lactobacillus fermentum)CECT 562 AJ575812와 99% 분자적 동 일성을 보였으므로, 이를 Lactobacillus fermentum BLJH 101로 명명하였다. 본 균주의 16S rRNA의 염기서열은 도4에 나타내었고, 2007년 12월 23일부로 대한민국 특허균주 기탁기관인 한국 미생물 보존센터에 기탁하여 수탁번호 KCCM10912P를 부여받았다. The selected ginseng extract using the strain was inoculated in MRS agar medium and cultured at 37 ° C. for 2 days to obtain a single colony. The strain was subjected to API analysis, which is a method for identifying cells in biochemical patterns. It was confirmed that there is 86.4% similarity with Lactobacillus fermentum. Also, 16S rDNA partial sequecing was performed to identify species at the species level. The nucleotide sequence was determined by 16S rDNA partial sequencing, and the result of searching the data of the National Center for Biotechnological Information (http://www.ncbi.nlm.nih.gov/blast) (FIG. 5), Lactobacillus latent (Lactobacillus) fermentum) CECT 562 AJ575812 showed 99% molecular identity, so it was named
1-2. 1-2. 락토바실러스Lactobacillus 퍼멘텀Permanent BLJH101BLJH101 의 인삼추출액에서 성장From ginseng extract
본 균주의 특성을 다른 유산균 균주와 비교하기 위해, 한국 미생물 보존센터에서 분양받은 공시균주 Lactobacillus reuteri KCCM 40717, Lactobacillus sporogenesis ATCC 8038, Pediococcus pentosaceus ATCC 8081, Lactobacillus plantarum KCCM 12116, Lactobacillus sakei KCCM 40264, Lactobacillus acidophilus KCCM 40265, Lactobacillus heveticus ATCC 13866, 및 Lactobacillus casei KCCM 12452와 MRS 대비 인삼 추출물 발효물의 생균수를 측정하는 실험을 하였다. To compare the characteristics of this strain with other lactic acid bacteria strains, Lactobacillus reuteri KCCM 40717, Lactobacillus sporogenesis ATCC 8038, Pediococcus pentosaceus ATCC 8081, Lactobacillus plantarum KCCM 12116, Lactobacillus sakei KCCM 40264, Lactobacillus reuteri 40265, Lactobacillus heveticus ATCC 13866, and Lactobacillus casei KCCM 12452 and MRS were measured to determine the viable cell count of the fermented ginseng extract fermentation.
동결 보존한 Lactobacillus fermentum BLJH 101와 상기 생물 보존센터에서 분양받은 공시균주들을 40mL의 MRS배지에 2mL 접종한 후, 37℃에서 2일간 정치배양을 하였다. 정치배양한 40mL의 MRS 배지를 15%의 인삼 추출액 40mL에 접종하여, 37℃에서 2일간 정치배양 한다. 상기 배양액을 생균수 확인 법을 통하여 생균수를 확인하였고, 인삼 추출액 40mL의 생균수를 정치배양한 40mL의 MRS 배지의 생균수로 나누어 측정한 결과를 아래 에 나타내었다. The cryopreserved
[표 2]. MRS 배지 대비 인삼 추출 배지 생균력 확인TABLE 2 Confirmation of ginseng extract media viability compared to MRS media
MRS 배지와 비교하여 생균수를 확인한 결과, 분리 균주인 락토바실러스 퍼멘텀 BLJH101의 생균수는 MRS 배지 대비 69.9%로 타 균주에 비해 현저히 높은 생균수를 보였다. MRS 배지 대비 인삼 추출물 배지 생균수가 많다는 것은 균주 성장에 있어서 락토바실러스 퍼멘텀 BLJH 101이 타 균주에 비해 인삼 추출물 이용도가 월등히 높다는 것을 의미하는 증거이다.As a result of confirming the viable cell number in comparison with the MRS medium, the viable cell number of the isolated strain Lactobacillus permanent BLJH101 was 69.9% compared to the MRS medium, which was significantly higher than other strains. The large number of ginseng extract medium viable cells compared to MRS medium is evidence that Lactobacillus
실시예Example 2. 2. 락토바실러스Lactobacillus 퍼멘텀Permanent BLJH101BLJH101 의 인삼 추출 배양 농축액 제조Preparation of Ginseng Extract Culture Concentrate
인삼 8.5kg에 5배 분량의 이온수를 넣고, 85℃에서 8시간 씩 3회 일반 물 추출 방법으로 30L 반응조(B.E. MARUBISH SH(TOKYO), MODEL MSJ-V3)를 이용하여 추출하였다. 추출한 액을 농축하여 고형분 60%의 인삼 추출물 액기스를 만들었다. 60% 홍삼 농축액 5.0 kg을 발효조에 넣고 4배 분량의 이온수를 첨가하였다. 상기액을 121℃에서 30분간 가압 가온처리 한 후, 37℃까지 온도를 낮추었다. MRS배지에서 37℃, 2일 정치 배양한 락토바실러스 퍼멘텀 BLJH101 200mL을 접종한 후, 0.5bar, 37℃, 100rpm의 발효조 조건에서 2일간 발효하였다. 상기 발효액을 121℃에서 30분간 가압 가온 처리 한 후, 농축하여 4.5kg의 60% 인삼 추출 발효 농축액을 얻었다.Five times the amount of ionized water was added to 8.5 kg of ginseng, and extracted using a 30L reactor (B.E. The extracted solution was concentrated to make a 60% solids ginseng extract extract. 5.0 kg of 60% red ginseng concentrate was added to the fermenter and 4 times the amount of ionized water was added. After the solution was pressurized and warmed at 121 ° C. for 30 minutes, the temperature was lowered to 37 ° C. After inoculating 200 mL of Lactobacillus pertumtum BLJH101 cultured at 37 ° C. for 2 days in a MRS medium, it was fermented at 0.5 bar, 37 ° C., and 100 rpm for 2 days. The fermentation broth was pressurized and warmed at 121 ° C. for 30 minutes, and then concentrated to obtain 4.5 kg of 60% ginseng extract fermentation concentrate.
실시예Example 3. 3. 락토바실러스Lactobacillus 퍼멘텀Permanent BLJH101BLJH101 의 인삼 추출 배양액 추출물 분말의 제조Of Ginseng Extract Culture Extract Extract Powder
인삼 8.5kg에 5배 분량의 이온수를 넣고, 85℃에서 8시간 씩 3회 일반 물 추출 방법으로 30L 반응조(B.E. MARUBISH SH(TOKYO), MODEL MSJ-V3)를 이용하여 추출하였다. 추출한 액을 농축하여 고형분 60%의 인삼 추출물 액기스를 만들었다. 60% 홍삼 농축액 5.0 kg을 발효조에 넣고 4배 분량의 이온수를 첨가하였다. 상기액을 121℃에서 30분간 가압 가온처리 한 후, 37℃까지 온도를 낮추었다. MRS배지에서 37℃, 2일 정치 배양한 락토바실러스 퍼멘텀 BLJH101 200mL을 접종한 후, 0.5bar, 37℃, 100rpm의 발효조 조건에서 2일간 발효하였다. 상기 발효액을 121℃에서 30분간 가압 가온 처리 한 후, 분무건조(스프레이 드라이어, 아인시스템)하여 1.5kg의 인삼 추출 배양액 추출물 분말을 얻었다.Five times the amount of ionized water was added to 8.5 kg of ginseng, and extracted using a 30L reactor (B.E. The extracted solution was concentrated to make a 60% solids ginseng extract extract. 5.0 kg of 60% red ginseng concentrate was added to the fermenter and 4 times the amount of ionized water was added. After the solution was pressurized and warmed at 121 ° C. for 30 minutes, the temperature was lowered to 37 ° C. After inoculating 200 mL of Lactobacillus pertumtum BLJH101 cultured at 37 ° C. for 2 days in a MRS medium, it was fermented at 0.5 bar, 37 ° C., and 100 rpm for 2 days. The fermentation broth was pressurized and warmed at 121 ° C. for 30 minutes, followed by spray drying (spray dryer, ein system) to obtain 1.5 kg of ginseng extract culture extract powder.
실시예Example 4. 4. 락토바실러스Lactobacillus 퍼멘텀Permanent BLJH101BLJH101 의 인삼 추출 배양액 생균 분말 제조Probiotic Powder Preparation of Ginseng Extract
인삼 8.5kg에 5배 분량의 이온수를 넣고, 85℃에서 8시간 씩 3회 일반 물 추출 방법으로 30L 반응조(B.E. MARUBISH SH(TOKYO), MODEL MSJ-V3)를 이용하여 추출하였다. 추출한 액을 농축하여 고형분 60%의 인삼 추출물 액기스를 만들었다. 60% 홍삼 농축액 5.0 kg을 발효조에 넣고 4배 분량의 이온수를 첨가하였다. 상기액을 121℃에서 30분간 가압 가온처리 한 후, 37℃까지 온도를 낮추었다. MRS배지에서 37℃, 2일 정치 배양한 락토바실러스 퍼멘텀 BLJH101 200mL을 접종한 후, 0.5bar, 37℃, 100rpm의 발효조 조건에서 2일간 발효하였다. 상기 발효액을 -75℃에서 동결한 후, 동결 건조기(ilshin Freeze Dryer, FD-5510. 10L)로 건조하여, 믹서기로 분쇄하여 2.7kg의 생균 건조분말을 얻었다. Five times the amount of ionized water was added to 8.5 kg of ginseng, and extracted using a 30L reactor (B.E. The extracted solution was concentrated to make a 60% solids ginseng extract extract. 5.0 kg of 60% red ginseng concentrate was added to the fermenter and 4 times the amount of ionized water was added. After the solution was pressurized and warmed at 121 ° C. for 30 minutes, the temperature was lowered to 37 ° C. After inoculating 200 mL of Lactobacillus pertumtum BLJH101 cultured at 37 ° C. for 2 days in a MRS medium, it was fermented at 0.5 bar, 37 ° C., and 100 rpm for 2 days. After freezing the fermentation broth at -75 ° C, it was dried with a freeze dryer (ilshin Freeze Dryer, FD-5510. 10L), ground in a blender to obtain a dry powder of 2.7kg.
실시예Example 5. 5. 락토바실러스Lactobacillus 퍼멘텀Permanent BLJH101BLJH101 의 of 인삼추출물발효물의Ginseng Extract Fermented 사포닌 함량 측정 Saponin Content Determination
5-1. 5-1. 비교예Comparative example (발효하지 않은 인삼 추출물의 제조)(Preparation of Unfermented Ginseng Extract)
인삼 8.5kg에 5배 분량의 이온수를 넣고, 85℃에서 8시간 씩 3회 추출한다. 추출한 액을 농축하여 고형분 60%의 인삼 추출물 액기스 5.0kg를 만든 후, 30 brix로 희석한 후, 분무건조 하여, 인삼 추출 분말 2.8kg을 얻었다.Add 5 times the amount of ionized water to 8.5kg ginseng, and extract three times at 85
5-2. 5-2. HPLCHPLC 를 이용한 인삼추출물발효액의 사포닌 함량 측정Saponin Content of Ginseng Extract Fermentation Solution
인삼추출물 발효물의 진세노사이드의 함량을 측정하기 위해서 하기와 같이 실험을 수행하였다. 실시예 3의 인삼추출물발효분말 1g과 실시예 5-1의 발효하지 않은 인삼 추출 분말을 정확히 둥근 플라스크에 각각 정량하여 넣고, 50% (v/v) 메탄올 70ml를 가한 후 수욕조에서 가열하면서 1시간 동안 환류추출한 후에 원심 분리하여 상등액을 취했다. 또한 잔사에 50% (v/v) 메탄올 50ml를 넣고 반복 추출한 후, 원심 분리하여 얻은 상등액을 합한 후 60℃ 아래 감압 농축하였다. 완충액 20ml를 넣어 녹인 후 25ml 용량 플라스크에 옮기고, 소량의 완충액으로 세척하여 25ml로 채운 다음 활성화시킨 C18 카트리지(Sep-Pak Plus, WAT020515, Waters, USA)에 5ml를 통과시켜 물과 30%(v/v) 메탄올로 세척한 후 메탄올 20ml로 용리한 다음 60℃ 아래에서 감압농축한 후 메탄올 2ml로 용해하여 분석 시 사용하였다. In order to measure the content of ginsenoside of ginseng extract fermentation, the experiment was performed as follows. 1 g of ginseng extract fermented powder of Example 3 and the non-fermented ginseng extract powder of Example 5-1 were respectively quantified into exactly round flasks, and 70 ml of 50% (v / v) methanol was added thereto, followed by heating in a water bath. After refluxing for an hour, the supernatant was taken by centrifugation. Further, 50 ml of 50% (v / v) methanol was added to the residue, followed by repeated extraction. The supernatants obtained by centrifugation were combined and concentrated under reduced pressure at 60 ° C. Dissolve 20 ml of buffer, transfer to a 25 ml volumetric flask, wash with a small amount of buffer, fill with 25 ml, and pass 5 ml through an activated C18 cartridge (Sep-Pak Plus, WAT020515, Waters, USA). v) After washing with methanol eluted with 20ml of methanol and concentrated under reduced pressure at 60 ℃ and dissolved in 2ml of methanol was used for analysis.
실시예 3의 인삼추출발효분말과 비교예 5-1의 발효하지 않은 인삼 추출분말의 유효성분인 진세노사이드의 함량을 각각 측정하기 위해 고성능액체크로마토그래피(High Performance Liquid Chromatography; HPLC; 717 plus autosampler, 1525 binary pump and 2487 dual wavelength UV detector, Waters, US)를 사용하였고, 컬럼은 캅-셀팍 C18(Cap-Cell Pak C18, 250 × 4.6 mm, 5 ㎛, Shiseido, Japan)을 사용하였다. 검출 스펙트럼은 UV 203 nm를 사용하여 측정하였다. 이동상은 아세토니트릴(CH3CN)과 물을 농도구배를 두어 사용하였고, 유속은 1.6 ㎖/min으로 하였다. 표준품으로는 진세노사이드 Rb1, Rg1, Rg3, F1, 컴파운드 K를 1 mg/㎖의 농도로 메탄올에 녹인 후 스톡(stock) 용액으로 사용하여, 1, 0.1, 0.01 ㎎/㎖의 검액을 만들어 검량선용 표준용액으로 사용하였고, 검량선을 작성하였다. 실시예 3의 인삼추출발효분말과 비교예 5-1의 발효하지 않은 인삼 추출분말의 진세노사이드 함량 변화를 측정하였고, 그 결과를 표 3 및 도 7에 나타내었다.In order to measure the contents of ginsenoside which is the active ingredient of the ginseng extract fermented powder of Example 3 and the non-fermented ginseng extract powder of Comparative Example 5-1, respectively, High Performance Liquid Chromatography (HPLC) 717 plus autosampler , 1525 binary pump and 2487 dual wavelength UV detector, Waters, US) and Cap-Cell Pak C18 (Cap-Cell Pak C18, 250 × 4.6 mm, 5 μm, Shiseido, Japan) were used. Detection spectra were measured using UV 203 nm. The mobile phase used acetonitrile (CH3CN) and water at a concentration gradient, and the flow rate was 1.6 ml / min. As a standard product, ginsenosides Rb1, Rg1, Rg3, F1, and compound K were dissolved in methanol at a concentration of 1 mg / ml, and then used as a stock solution to prepare a test solution of 1, 0.1, 0.01 mg / ml. A standard solution was used and a calibration curve was prepared. The ginsenoside content change of the ginseng extract fermented powder of Example 3 and the non-fermented ginseng extract powder of Comparative Example 5-1 was measured, and the results are shown in Table 3 and FIG. 7.
[표 3]. 인삼추출물 배양분말의 진세노사이드 성분 함량 변화 TABLE 3 Changes of Ginsenoside Components in Ginseng Extract Culture Powder
실시예 3의 진세노사이드 성분 함량을 분석한 결과 진세노사이드 Rb1은 15배 감소하고, 진세노사이드 Rg1은 검출이 되지 않았다. Rg3(Rg3-S, Rg3-R 합한 것)의 함량은 배양 후 약 4배가 증가하였고, 컴파운드 K의 경우도 약 4배가 증가하였으며, 진세노사이드 F1의 경우도 약 1.5배 증가하였다. 배양 후, 진세노사이드 Rh2는 0.2% 생성되었으며, 프로토파낙트리올(PPT)도 1.2% 생성되었다. 상기 결과를 통해 인삼 추출액을 본 발명의 신 균주 락토바실러스 퍼멘텀 BLJH101으로 발효시킨 경우, 생체 이용율이 낮은 진세노사이드 Rg1, Rb1, Rg, Re 등을 생체 이용률이 놓은 진세노사이드 Rg3, Rh2, 컴파운드 K, 진세노사이드 F1등으로 획기적인 전환을 시킴을 확인할 수 있었다.As a result of analyzing the ginsenoside component content of Example 3, ginsenoside Rb1 was reduced by 15 times, and ginsenoside Rg1 was not detected. The content of Rg3 (Rg3-S and Rg3-R combined) increased about 4 times after incubation, about 4 times for compound K, and about 1.5 times for ginsenoside F1. After incubation, ginsenoside Rh2 was produced 0.2% and protopanactriol (PPT) 1.2%. When the ginseng extract was fermented with the new strain Lactobacillus latent BLJH101 of the present invention, ginsenosides Rg1, Rb1, Rg, Re, etc. having low bioavailability, ginsenosides Rg3, Rh2, compound K, ginsenoside F1 was found to be a significant conversion.
실시예Example 6. 6. 락토바실러스Lactobacillus 퍼멘텀Permanent BLJH101BLJH101 의 of 인삼추출물발효물의Ginseng Extract Fermented 항균능Antimicrobial activity 측정 Measure
실시예 3의 인삼추출물발효분말과 발효하지 않은 비교예 5-1에서 제조한 시료를 TSA (100그램 당 판크레아틱 효소 처리 카제인 1.5그램 중, 효소 처리 소이빈 밀 0.5그램 중, 소금 0.5그램 중, 한천 1.5그램 중) 20mL배지에 각각 10%, 7%, 5%, 3%, 1%, 0% 첨가한 후, 121℃에서 30분간 멸균하여 60℃가 되었을 때 페트리 디쉬에 부었다. 상기 인삼추출물발효분말과 인삼추출물분말이 섞인 TSA 배지에, 37℃ TS(100그램 당 판크레아틱 효소 처리 카제인 1.5그램 중, 효소 처리 소이빈 밀 0.5그램 중, 소금 0.5그램 중)배지에서 1일 배양한 병원균 Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus을 105 희석하여 20㎕ 점적한 후, 37℃에서 1일간 배양하였다. 상기 한천 배지에서 균 성장을 확인할 수 없는 농도 MIC(Minimum Inhibitory Concentration)를 확인한다. 비교예의 인삼 추출물의 Escherichia coli에 대한 MIC는 10%, Pseudomonas aeruginosa는 5%, Staphylococcus aureus는 5% 이며, 실시 예의 인삼추출발효분말의 Escherichia coli에 대한 MIC는 3%, Pseudomonas aeruginosa는 3%, Staphylococcus aureus는 3%를 보였다. 각 병원균의 MIC 값에 역수를 취한 값을 항균 활성이라 하고, 값을 비교하였을 때, 실시 예의 분말에 있어서 Escherichia coli.에 대한 항균활성은 3.3배, Pseudomonas aeruginosa에 대해서는 1.6배, Staphylococcus aureus에 대해서는 1.6배 증가하였다(도 6).The ginseng extract fermented powder of Example 3 and the sample prepared in Comparative Example 5-1, which were not fermented, were prepared in TSA (1.5 grams of pancreatic enzyme-treated casein per 100 grams, 0.5 grams of enzyme-treated soybean wheat, and 0.5 grams of salt). , 1.5 g of agar) 10%, 7%, 5%, 3%, 1%, and 0% were added to 20mL medium, respectively, and sterilized at 121 ° C. for 30 minutes and poured into Petri dishes at 60 ° C. On the TSA medium in which the ginseng extract fermented powder and the ginseng extract powder were mixed, one day in a medium at 37 ° C TS (1.5 grams of pancreatic enzyme-treated casein per 100 grams, 0.5 grams of enzyme-treated soybean wheat, and 0.5 grams of salt) The cultured pathogens Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus were diluted 10 5 and dipped in 20 µl, and then cultured at 37 ° C for 1 day. Confirm the concentration MIC (Minimum Inhibitory Concentration) can not confirm the growth of the bacteria in the agar medium. The ginseng extract of Comparative Example was 10% MIC for Escherichia coli, 5% for Pseudomonas aeruginosa, and 5% for Staphylococcus aureus. aureus showed 3%. The inverse of the MIC value of each pathogen is called antimicrobial activity. When the values are compared, the antimicrobial activity against Escherichia coli. Is 3.3 times, 1.6 times for Pseudomonas aeruginosa, 1.6 times for Staphylococcus aureus. It increased fold (Figure 6).
실시예Example 7. 7. DPPHDPPH 에 의한 항산화 활성 측정 Antioxidant Activity by
실시예 3 및 비교예 5-1에서 제조한 시료를 적정한 농도(400㎍/mL)로 희석한 후, 시료 1ml에 0.2mM DPPH(1,1-diphenyl-2-picrylhydrazyl, Sigma Chemical Co., USA) 용액(99.9% 에탄올) 0.5ml를 가했다. Vortex mixer로 10초간 진탕하고, 10,000rpm으로 3분 동안 원심 분리시킨 후, 상등액에 DPPH 용액(0.5mL)을 첨가한 10분 후에 분광광도계(Jasco, Germany)를 이용하여 525nm에서 흡광도를 측정하였다. 흡광도를 측정할 때 시료에 의한 흡광도의 차이는 DPPH용액을 제외한 시료의 흡광도를 측정하여 보정해주고, DPPH 저해율(%)을 50% 감소시키는 IC50을 구하였다. DPPH에 의한 항산화 활성 측정 원리는 DPPH를 산화제로 사용하여 산화를 개시시킨 후, 실험하고자 하는 샘플에 의한 자유 라디칼의 소거를 측정하는 것이다. 따라서 DPPH 저해율이 높다는 것은 항산화 활성도가 높다는 것을 의미한다.After diluting the samples prepared in Example 3 and Comparative Example 5-1 to the appropriate concentration (400 μg / mL), 0.2 ml DPPH (1,1-diphenyl-2-picrylhydrazyl, Sigma Chemical Co., USA) in 1 ml of the sample ) 0.5 ml of a solution (99.9% ethanol) was added. After 10 seconds of shaking with a Vortex mixer, centrifugation at 10,000 rpm for 3 minutes, the absorbance was measured at 525 nm using a spectrophotometer (Jasco, Germany) 10 minutes after adding DPPH solution (0.5 mL) to the supernatant. When measuring the absorbance, the difference in absorbance by the sample was corrected by measuring the absorbance of the sample except for the DPPH solution, and the IC 50 was obtained to reduce the DPPH inhibition rate (%) by 50%. The principle of measuring antioxidant activity by DPPH is to measure the scavenging of free radicals by the sample to be tested after initiating oxidation using DPPH as an oxidant. Therefore, high DPPH inhibition rate means high antioxidant activity.
[표 4]. DPPH 활성TABLE 4 DPPH active
실시예 3의 인삼추출물발효분말에 의한 항산화 활성은 비교예 5-1에서 제조한 인삼추출물분말에 대비하여 약 25배 정도 증가된 것을 관찰하였다.The antioxidant activity of the ginseng extract fermented powder of Example 3 was observed to be increased by about 25 times compared to the ginseng extract powder prepared in Comparative Example 5-1.
이상 설명한 바와 같이, 본 발명에 따르면 미생물의 성장을 억제시키는 인삼추출물에 특이적으로 생육하는 유산균인 락토바실러스 퍼멘텀 BL101을 분리 동정할 수 있다. As described above, according to the present invention, it is possible to isolate and identify Lactobacillus fermentum BL101, which is a lactic acid bacterium that specifically grows on ginseng extracts that inhibit the growth of microorganisms.
또한 본 발명에 따르면 인삼추출물에 특이적으로 생육하는 유산균인 락토바실러스 퍼멘텀 BL101을 배양하여, 진세노사이드 Rg1, Rb1 등의 대사산물을 진세노사이드 Rg3, 컴파운드 K, 진세노사이드 F1 등의 성분으로 전환시켜 생체 이용률이 높은 진세노사이드 성분을 함유하고, 인삼 추출물의 항균력을 향상시킨 유산균 발효액, 발효분말, 농축액, 및 발효 생균제를 제조하고, 이를 함유하는 조성물을 제조할 수 있다. In addition, according to the present invention by culturing Lactobacillus Permanent BL101, a lactic acid bacterium that grows specifically in ginseng extract, metabolites such as ginsenoside Rg1, Rb1, ginsenoside Rg3, compound K, ginsenoside F1, etc. The lactic acid bacteria fermentation broth, fermentation powder, concentrate, and fermentation probiotic containing the ginsenoside component having high bioavailability and improving the antibacterial activity of ginseng extract may be prepared, and the composition containing the same may be prepared.
또한 인삼 추출액을 제외한 유산균 배양을 위한 여타 다른 배지 성분이 들어 가지 않기 때문에 공정을 단순화 할 수 있으며, 첨가물에 의한 제품 함량의 손실도 줄일 수 있다.In addition, the process can be simplified because other media components for cultivation of lactic acid bacteria except for ginseng extract can be simplified, and the loss of product content by additives can be reduced.
도 1a는 인삼추출물에 특이적으로 성장하는 유산균을 생균수 측정법으로 확인한 그래프이다(세포 수는 대수스케일로 표시).Figure 1a is a graph confirming the growth of lactic acid bacteria specifically grown in ginseng extract by viable cell count (cell number is shown in logarithmic scale).
도 1b는 인삼추출물 및 MRS 한천배지에서 성장하는 유산균을 각각 생균수 측정법으로 확인한 그래프이다(세포 수는 로그스케일로 표시).Figure 1b is a graph confirming the lactic acid bacteria growing in ginseng extract and MRS agar medium by viable cell count, respectively (cell number is shown in log scale).
도 2는 김치에 절인 인삼에서 분리한 락토바실러스 퍼멘텀 BLJH 101균의 균주 사진이다.Figure 2 is a photograph of the strain of
도 3a는 API 킷트로 동정한 락토바실러스 퍼멘텀 BLJH 101의 분석 결과이다.Figure 3a is an analysis of the
도 3b는 API 킷트로 동정한 락토바실러스 퍼멘텀 BLJH 101의 분석 결과이다.3B is an analysis result of Lactobacillus
도 4은 락토바실러스 퍼멘텀 BLJH101 균주의 16s rDNA 염기 서열이다.4 is the 16s rDNA nucleotide sequence of the Lactobacillus permanent BLJH101 strain.
도 5는 락토바실러스 퍼멘텀 BLJH101 균주의 분자 유전학적 분류의 위치를 나타낸 것이다.Figure 5 shows the location of the molecular genetic classification of the Lactobacillus permanent BLJH101 strain.
도 6은 락토바실러스 퍼멘텀 BLJH101 균주로 발효한 인삼추출발효 분말과 인삼추출물의 항균활성 효능을 비교한 도표이다.Figure 6 is a chart comparing the antibacterial activity of the ginseng extract fermentation powder and ginseng extract fermented with Lactobacillus fermentum BLJH101 strain.
도 7는 락토바실러스 퍼멘텀 BLJH101 균주에 의해 변화된 인삼추출발효 분말의 사포닌 HPLC 패턴 그림이다.7 is a saponin HPLC pattern of the ginseng extract fermented powder changed by the Lactobacillus fermentum BLJH101 strain.
도 8은 미생물 발효에 의한 사포닌 변화를 모식화한 그림이다.8 is a diagram schematically illustrating the change in saponin by microbial fermentation.
<110> BIOLAND Ltd. <120> A method for preparing fermented ginseng of ginseng extract using Lactobacillus fermentum BLJH101 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 687 <212> RNA <213> Lactobacillus fermentum BLJH101 <400> 1 cgugcuaucu gcagucgacg cguuggccca auugauugau ggugcuugca ccugauugau 60 uuuggucgcc aacgaguggc ggacggguga guaacacgua gguaaccugc ccagaagcgg 120 gggacaacau uuggaaacag augcuaauac cgcauaacaa cguuguucgc augaacaacg 180 cuuaaaagau ggcuucucgc uaucacuucu ggauggaccu gcggugcauu agcuuguugg 240 ugggguaacg gccuaccaag gcgaugaugc auagccgagu ugagagacug aucggccaca 300 augggacuga gacacggccc auacuccuac gggaggcagc aguagggaau cuuccacaau 360 gggcgcaagc cugauggagc aacaccgcgu gagugaagaa ggguuucggc ucguaaagcu 420 cuguuguuaa agaagaacac guaugagagu aacuguucau acguugacgg uauuuaacca 480 gaaagucacg gcuaacuacg ugccagcagc cgcgguaaua cguagguggc aagcguuauc 540 cggauuuauu gggcguaaag agagugcagg cgguuuucua agucugaugu gaaagccuuc 600 ggcuuaaccg gagaagugca ucggaaacug gauaacuuga gugcagaaga ggguagugga 660 acuccaugug uagcggugga augcgua 687 <110> BIOLAND Ltd. <120> A method for preparing fermented ginseng of ginseng extract using Lactobacillus fermentum BLJH101 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 687 <212> RNA <213> Lactobacillus fermentum BLJH101 <400> 1 cgugcuaucu gcagucgacg cguuggccca auugauugau ggugcuugca ccugauugau 60 uuuggucgcc aacgaguggc ggacggguga guaacacgua gguaaccugc ccagaagcgg 120 gggacaacau uuggaaacag augcuaauac cgcauaacaa cguuguucgc augaacaacg 180 cuuaaaagau ggcuucucgc uaucacuucu ggauggaccu gcggugcauu agcuuguugg 240 ugggguaacg gccuaccaag gcgaugaugc auagccgagu ugagagacug aucggccaca 300 augggacuga gacacggccc auacuccuac gggaggcagc aguagggaau cuuccacaau 360 gggcgcaagc cugauggagc aacaccgcgu gagugaagaa ggguuucggc ucguaaagcu 420 cuguuguuaa agaagaacac guaugagagu aacuguucau acguugacgg uauuuaacca 480 gaaagucacg gcuaacuacg ugccagcagc cgcgguaaua cguagguggc aagcguuauc 540 cggauuuauu gggcguaaag agagugcagg cgguuuucua agucugaugu gaaagccuuc 600 ggcuuaaccg gagaagugca ucggaaacug gauaacuuga gugcagaaga ggguagugga 660 acuccaugug uagcggugga augcgua 687
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| KR101285681B1 (en) * | 2010-12-17 | 2013-07-11 | 성금수 | Saponin-biotransforming activity and processes for preparing fermented ginseng using the same |
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| KR102180886B1 (en) * | 2019-06-14 | 2020-11-19 | 주식회사 더가든오브내추럴솔루션 | Extract containing roots fermented and whole body of ginseng cultivated in cold wind and cosmetic composition containing the same |
| CN111363774A (en) * | 2020-03-18 | 2020-07-03 | 江苏菌钥生命科技发展有限公司 | Method for fermenting ginseng by lactobacillus fermentum, ginseng fermentation product and application |
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