KR20110053112A - Novel enterococcus faecalis k-60 and extracts from ginseng-fermented products using enterococcus faecalis k-60 for improving glucose homeostasis and manufacturing method thereof - Google Patents
Novel enterococcus faecalis k-60 and extracts from ginseng-fermented products using enterococcus faecalis k-60 for improving glucose homeostasis and manufacturing method thereof Download PDFInfo
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- KR20110053112A KR20110053112A KR1020090109938A KR20090109938A KR20110053112A KR 20110053112 A KR20110053112 A KR 20110053112A KR 1020090109938 A KR1020090109938 A KR 1020090109938A KR 20090109938 A KR20090109938 A KR 20090109938A KR 20110053112 A KR20110053112 A KR 20110053112A
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- ginseng
- extract
- fermentation
- fermented
- enterococcus faecalis
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- C—CHEMISTRY; METALLURGY
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- C—CHEMISTRY; METALLURGY
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Abstract
Description
본 발명은 신규한 엔테로코커스 패칼리스 케이-60 균주(Enterococcus faecalis K-60)(KFCC11460P)(이하 K-60으로 약칭한다) 및 상기 균주를 이용한 포도당 항상성 개선용 인삼 발효 추출물 및 그의 제조방법에 관한 것으로서 보다 상세하게는 인삼을 발효시킬 수 있는 신규한 K-60 및 상기 균주로 인삼을 발효시켜 포도당(당) 항상성 개선에 적용할 수 있는 인삼 발효 추출물 및 이의 제조방법에 관한 것이다.The present invention relates to a novel Enterococcus faecalis K-60 strain (Enterococcus faecalis K-60) (KFCC11460P) (hereinafter abbreviated as K-60) and a ginseng fermentation extract for improving glucose homeostasis using the strain and a method for preparing the same. In more detail, the present invention relates to a ginseng fermented extract and a method for preparing the same, which can be applied to improve glucose (sugar) homeostasis by fermenting ginseng with a novel K-60 capable of fermenting ginseng and the strain.
인삼은 식물 분류학상 오가과(Araliaceae) 인삼속(Panax)에 속하는 다년생 숙근초로서 지구상에 약 11종이 알려져 있으며, 대표적인 종으로 고려인삼 (Panax ginseng C.A. Meyer)은 아시아 극동 지역 (한국, 북만주, 러시아 일부)에 자생한 다.Panax ginseng is a perennial root of the genus Araliaceae in the genus Panax , and about 11 species are known on the earth.Panax ginseng CA Meyer is a representative species of Panax ginseng (Panax ginseng CA Meyer). Grows wild).
인삼은 제1형 및 제2형 당뇨병 모델 동물에서 당항상성 개선 효과가 있다는 보고가 있다(Kimura et al., J. Pharmacobio-Dyn. 1981, 4, 410-417; Kimura et al., J. Pharmacobio-Dyn. 1981, 4, 402-409; Yokozawa et al., Chem. Pharm. Bull., 1985, 33, 869-872; Kimura et al., Phytotherpy Res. 1999, 13, 484-488 Kimura et al., Phytotherpy Res. 1999, 13, 484-488; Kimura et al., J. Pharmacobio-Dyn. 1981, 4, 907-915; Chung et al., Arch. Pharmacal. Res. 2001, 24, 214-218; Dey et al., Phytomedicine 2003, 10, 600-605). 또한, 인삼은 인체 실험에서도 당항상성 개선 효과가 있다는 보고가 있다(Sievenpiper et al., Diabetes 2003, 52(Suppl.1), A387; Vuksan et al., Diabetes 2003, 52(Suppl. 1), A137).Ginseng has been reported to have anti-diabetic effects in type 1 and type 2 diabetic animals (Kimura et al., J. Pharmacobio-Dyn. 1981, 4, 410-417; Kimura et al., J. Pharmacobio Dyn. 1981, 4, 402-409; Yokozawa et al., Chem. Pharm. Bull., 1985, 33, 869-872; Kimura et al., Phytotherpy Res. 1999, 13, 484-488 Kimura et al. , Phytotherpy Res. 1999, 13, 484-488; Kimura et al., J. Pharmacobio-Dyn. 1981, 4, 907-915; Chung et al., Arch. Pharmacal.Res. 2001, 24, 214-218; Dey et al., Phytomedicine 2003, 10, 600-605). In addition, ginseng has been reported to improve the sugar always in human experiments (Sievenpiper et al., Diabetes 2003, 52 (Suppl. 1), A387; Vuksan et al., Diabetes 2003, 52 (Suppl. 1), A137 ).
인삼의 성분으로는 사포닌(saponin), 페놀성 성분, 폴리아세칠렌 성분, 알칼로이드 성분, 다당체 등이 알려져 있다. 특히 사포닌 성분은 인삼 특이 성분으로 진세노사이드 (ginsenosides)라 칭하며 30종 이상이 알려져 있으며, 인삼 약효에 있어서 가장 중요한 역할을 하는 것으로 알려져 있다(박종대, 1997. 고려인삼의 화학성분에 관한 고찰. "고려인삼 연구 20년사" 고려인삼학회 p. 69-112). As the components of ginseng, saponin, phenolic component, polyacexylene component, alkaloid component, polysaccharide and the like are known. In particular, saponin is a ginseng-specific ingredient called ginsenosides, and more than 30 kinds are known, and it is known to play the most important role in ginseng medicinal effects (Park Jong-dae, 1997. Consideration on the chemical composition of Korean ginseng. " 20 Years of Korean Ginseng Research "Korean Ginseng Society p. 69-112).
인삼에 있는 사포닌을 의미하는 진세노사이드(Ginsenoside)는 인삼 건조량의 약 5％를 차지하며, 트리터페노이드(triterpenoid) 계열의 다마란(dammarane) 골격에 포도당(glucose), 아라비노즈(arabinose), 자일로즈(xylose), 람노즈(rhamnose) 등이 결합되어 있는 중성 배당체이다. 비당부분에 붙어있는 -OH의 수에 따라 2개인 (3, 12번 위치) 경우 프로토파낙사디올 (protopanaxadiol; PPD) 계열 사포닌, 3개인 (3, 6, 12번 위치) 경우 프로토파낙사트리올 (protopanaxatriol; PPT) 계열 사포닌이라 한다. PPD과 PPT의 R1, R2, R3 위치의 -OH에 글루코오스(glucose), 아라비노오스(arabinose), 자일로오스(xylose), 람노오스(rhamnose) 등이 결합된다. 30종 이상의 진세노사이드가 인삼으로부터 분리 보고되었으나 실제 인삼을 추출하여 분석할 때 상당한 양이 검출되는 것은 Rb1, Rb2, Rc, Rd, Re, Rg1의 6종이 전체 사포닌의 90％ 이상을 차지하고 있으며 나머지 사포닌 성분들은 그 함량이 낮다.Ginsenoside, which means saponin in ginseng, accounts for about 5% of ginseng dry weight. Glucose, arabinose, and triterpenoid-based dammarane skeletons It is a neutral glycoside with xylose and rhamnose. Protopanaxadiol (PPD) family saponins for two (3, 12) depending on the number of -OH attached to the non-sugar moiety, Protopananatriol for three (3, 6, 12) (protopanaxatriol; PPT) family is called saponin. Glucose, arabinose, xylose, rhamnose, and the like are combined with -OH at the positions R1, R2 and R3 of the PPD and PPT. More than 30 species of ginsenosides have been reported from ginseng, but a significant amount of ginseng was detected when extracting and analyzing ginseng. Six species of Rb1, Rb2, Rc, Rd, Re, and Rg1 accounted for more than 90% of the total saponin. Saponin components are low in content.
진세노사이드는 인체내에서 그대로 흡수되는 것이 아니라 인체 장내 Bacteriodes, Fusobactrium, Eubacterium, Provetella 종 등의 미생물에 의하여 분해되어 그 대사체가 흡수된다(Chi et al., 2005). 진세노사이드 Rb1의 경우, 장내 미생물에 의해 진세노사이드 Rd, 컴파운드 케이(compound K, 이하 C-K라로 약칭함)와 프로토파낙사디올로 순차적으로 전환된다. PPD계 진세노사이드인 Rb1, Rb2, Rc, Rd는 장내에서 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol(C-K)로 대사되어 흡수된다(Akao et al., 1998). PPT계 진세노사이드인 진세노사이드 Re, Rf, Rg1은 장내에서 Rh1, F1, protopanaxatriol로 대사된다(Hasegawa 2004; Tawab et al., 2003). Rb1, Rb2, Rc, Rd 등은 장내 미생물에 의하여 20(R)-/20(S)-ginsenoside Rg3로 전환되며(Cheng et al., Phytochemistry 2008), Rg3는 장내 미생물에 의하여 Rh2로 전환된다(Su et al., J Mol Cat B: Enzymatic 2006).Ginsenosides are not absorbed in the human body, but are degraded by microorganisms such as Bacteriodes, Fusobactrium, Eubacterium, and Provetella species in the human intestine to absorb their metabolites (Chi et al., 2005). In the case of ginsenoside Rb1, it is sequentially converted to ginsenoside Rd, compound K (hereinafter abbreviated as C-K) and protopanaxadiol by intestinal microorganisms. PPD ginsenosides Rb1, Rb2, Rc, Rd are metabolized and absorbed by 20-O-β-D-glucopyranosyl-20 (S) -protopanaxadiol (C-K) in the intestine (Akao et al., 1998). Ginsenosides Re, Rf, and Rg1, which are PPT-based ginsenosides, are metabolized to Rh1, F1 and protopanaxatriol in the intestine (Hasegawa 2004; Tawab et al., 2003). Rb1, Rb2, Rc, Rd, etc. are converted to 20 (R)-/ 20 (S) -ginsenoside Rg3 by enteric microorganisms (Cheng et al., Phytochemistry 2008), and Rg3 is converted to Rh2 by enteric microorganisms ( Su et al., J Mol Cat B: Enzymatic 2006).
진세노사이드를 분해하는 장내 미생물은 사람의 체질, 식습관에 따라 그 존재의 유무와 보유하고 있는 정도가 다르며, 대사 산물로 전환하는 전환율에 차이가 있어서 인삼을 섭취한 후 저마다 효능의 차이가 나타날 수 있다. 장내 미생물 균총이 다르기 때문에 진세노사이드의 전환율과 생체 이용율은 다를 수 밖에 없다(J. Pharm. Pharmacol., 50, pp.1155-1160, 1998). 따라서 장내 미생물의 차이로 인한 효능과 흡수율의 차이를 없애기 위해, 진세노사이드를 대사하는 미생물을 발굴하는 것이 필요하다.Intestinal microorganisms that break down ginsenosides differ in their presence and retention according to human constitution and dietary habits, and there is a difference in the conversion rate to metabolites. have. Due to different intestinal microflora, 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 between the intestinal microorganisms, it is necessary to find a microorganism that metabolizes ginsenosides.
한편 인삼의 효능 발현에 진세노사이드가 기인하는 것은 사실이지만 단일 진세노사이드에 의한 것이라기 보다는 여러 개의 진세노사이드에 의한 복합적인 작용이라는 견해가 우세하다. 따라서 진세노사이드를 대사시키는 미생물도 단일 진세노사이드를 전환시키는 미생물보다는 진세노사이드를 복합적으로 전환시키는 미생물이 더 유용할 수 있다.On the other hand, ginsenosides are due to the ginseng side effect, but the view that the ginsenoside is a complex action rather than a single ginsenoside is predominant. Therefore, microorganisms that metabolize ginsenosides may be more useful than microorganisms that convert ginsenosides in combination.
인체가 항상 필요로 하는 포도당은 몸 속에서 혈당으로 존재하는 데 공복시에는 간, 근육 및 지방에 저장된 포도당이 다시 혈액으로 방출되고 식사를 하면 약 2∼3 시간 동안 위장관에서 흡수된 다량의 포도당이 혈액을 거쳐 간, 근육 및 지방에 저장된다. 이러한 체내 에너지 대사가 자동으로 조절되는 것은 바로 췌장 베타세포에서 분비되는 인슐린의 양을 조절함으로써 이루어지게 된다(이현철 등. 당뇨병 백과, 가림출판사, 2007년, p.34). Glucose, which is always needed by the human body, is present in the body as blood sugar. On an empty stomach, glucose stored in the liver, muscles and fats is released into the blood and when eaten, a large amount of glucose absorbed from the gastrointestinal tract for about 2 to 3 hours is consumed. Stored in the liver, muscles and fat. This automatic regulation of energy metabolism in the body is achieved by controlling the amount of insulin secreted from pancreatic beta cells (Lee Hyun-Chul et al., Encyclopedia of Diabetes, 2007, p. 34).
체내에서 혈당(blood glucose)은 80∼120mg/dL으로 항상 일정하게 유지되는데, 이 현상을 포도당 항상성(glucose homeostasis; 이하 당항상성이라 약칭함)이라고 한다. 인체가 당항상성을 유지하는 과정에는 췌장의 베타(β)세포와 알파(α)세포에서 각각 분비되는 인슐린(insulin)과 글루카곤(glucagon)이 관여한다. 식사 직후에는 혈당이 상승하고 이때 분비된 인슐린은 간, 근육, 지방세포 등의 말초조직에 포도당을 에너지원으로 사용할 수 있게 공급하고, 또한 간 및 근육에 포도당을 글리코겐 (glycogen)으로 저장시켜 혈당을 정상화시킨다. 공복에는 혈당이 저하되고 이때 글루카곤이 분비되어 간에서 글리코겐이 포도당으로 분해되어 혈당을 정상화시킨다 (Miyazaki Y, Glass L, Triplitt C, Wajcberg E, Mandarino LJ, DeFronzo RA.2002. Am J Physiol Endocrinol Metab 283, E1135-43). 공복 기간이 길어지면 간의 글리코겐이 고갈되고, 아미노산 등의 당신생합성(gluconeogenesis) 전구체가 간에서 포도당으로 전환되어 혈당을 정상으로 유지시킨다.Blood glucose in the body is always kept constant at 80-120 mg / dL. This phenomenon is called glucose homeostasis. In the process of maintaining the human body glycemic insulin (insulin) and glucagon (glucagon) secreted from the beta (β) cells and alpha (α) cells of the pancreas, respectively. Immediately after a meal, blood sugar rises, and insulin secreted at this time supplies glucose to the peripheral tissues such as liver, muscle, and fat cells to be used as an energy source, and glucose is stored in the liver and muscle as glycogen (glycogen). Normalize Fasting lowers blood sugar and glucagon is secreted at this time to break down glycogen into glucose in the liver to normalize blood sugar (Miyazaki Y, Glass L, Triplitt C, Wajcberg E, Mandarino LJ, DeFronzo RA.2002. Am J Physiol Endocrinol Metab 283 , E1135-43). When the fasting period is longer, glycogen is depleted in the liver, and gluconeogenesis precursors such as amino acids are converted to glucose in the liver to keep blood sugar normal.
당항상성을 유지하는 기능이 저하되면 공복 혈당(fasting glucose)이나 식후 혈당(postprandial glucose)이 조금씩 오르거나 오른 상태가 되어 있는 내당능장애(耐糖能障碍; glucose intolerance)의 단계로 넘어간다. 내당능장애는 사람에 따라서 수개월간 지속되거나 수년에서 10년 이상 지속되기도 한다. 내당능장애 상태가 모두 당뇨병 단계로 진행되지는 않는다. 그러나 내당능장애 상태에서 당항상성을 유지하는 기능 저하가 계속되면 제2형 당뇨병이 발병한다.When the function of maintaining the glycemic state is reduced, fasting glucose or postprandial glucose rises or rises to a state of glucose intolerance (耐 糖 能 障碍; glucose intolerance) is a step up. Impaired glucose tolerance lasts for months or even for years to 10 years, depending on the person. Not all glucose tolerance states progress to the diabetic stage. However, type 2 diabetes develops if the hypoglycemic function continues to be impaired in glucose tolerance.
제2형 당뇨병은 혈당이 정상 이상으로 높아진 상태를 나타내는 것으로 간, 근육, 지방세포 등의 말초조직에서 인슐린 작용의 저하와 췌장 베타세포에서의 인슐린 분비의 균형이 깨어졌을 때 나타나는 질병이다. 말초조직에서의 인슐린 작용의 감소는 대부분의 조직 세포에서 포도당 이용이 저하하고, 특히 간에서는 혈당이 높음에도 불구하고 글리코겐의 합성은 감소하고 당신생합성을 통한 포도당의 합성은 증가시켜 혈당을 더 상승시키는 역할을 한다. 결과적으로 인슐린 분비가 충분히 증가하여 인슐린 저항성을 보상할 수 있으면 당뇨병으로 진전되지 않지만, 인슐린 분비가 충분치 못할 때 당뇨병으로 진전된다. 즉 정상 혈당을 유지하기 위해서는 인슐린 저항성과 인슐린 분비능이 조화를 이루어야 하는데, 제2형 당뇨병은 그 조화가 깨어졌을 때 유발되는 질병이다.Type 2 diabetes is a condition in which blood sugar levels are higher than normal, and a disease occurs when the balance of insulin secretion and the secretion of insulin from pancreatic beta cells is broken in peripheral tissues such as liver, muscle, and fat cells. Reduction of insulin action in peripheral tissues lowers glucose utilization in most tissue cells, and in particular, despite high blood sugar levels in the liver, decreases glycogen synthesis and increases glucose synthesis through biosynthesis, resulting in higher blood sugar levels. Play a role. As a result, if insulin secretion is sufficiently increased to compensate for insulin resistance, it does not progress to diabetes, but when insulin secretion is insufficient, it progresses to diabetes. In other words, in order to maintain normal blood sugar, insulin resistance and insulin secretory ability must be harmonized. Type 2 diabetes is a disease caused when the harmony is broken.
췌장의 베타세포(pancreatic β-cells)는 체내의 포도당 항상성을 조절하는데 있어 중추적인 기능을 담당하고 있을 뿐 아니라, 우리 몸의 에너지 대사를 조절하는 가장 중요한 호르몬인 인슐린을 합성 및 저장하며, 필요에 따라 분비하는 역할을 담당하고 있다. 그러나 인간 수명의 연장, 생활 수준의 향상, 불균형적인 영양분의 과잉 공급, 도시화로 인한 운동량의 감소, 유전적 요인, 환경적 원인 등은 췌장 베타세포의 기능인 인슐린 분비에 이상을 유도하며 심지어 사멸(death)까지 유도한다. 췌장베타세포 기능 이상 및 사멸은 당뇨병의 병인중의 하나이다.Pancreatic β-cells not only play a central role in regulating glucose homeostasis in the body, but also synthesize and store insulin, the most important hormone that regulates our body's energy metabolism. According to the role of secretion. However, prolonging human life, improving living standards, disproportionate supply of nutrients, reducing exercise due to urbanization, genetic factors, and environmental causes can lead to abnormalities in insulin secretion, a function of pancreatic beta cells, and even death. To). Pancreatic beta cell dysfunction and death is one of the etiologies of diabetes.
위에서 살펴본 바와 같이 당뇨병은 어느 한 조직(기관)의 이상에 의하여 발병하는 것이 아니라, 체내의 당항상성 유지에 관여하는 조직간 네트워크의 균형이 파괴됨으로써 발병한다. 따라서 당항상성 개선을 위하여는 당항상성 유지에 관여하는 여러 조직에 작용하는 소재를 섭취하는 것이 이상적이다. 또한 당뇨병 환자 치료를 위하여도 작용기전이 서로 보완적인 약제를 병합하는 것이 좋다. As described above, diabetes is not caused by an abnormality in any one tissue (organ), but is caused by a breakdown of the balance between the tissues involved in the maintenance of glucose in the body. Therefore, in order to improve sugar always, it is ideal to take a material that acts on various tissues involved in maintaining sugar always. In addition, for the treatment of diabetes patients, it is good to combine drugs complementary to each other.
국제당뇨병연맹(International Diabetes Federation, IDF)은 2003년 미국의 당뇨병 유병률(prevalence)은 8.0％, 우리나라는 6.4％라고 보고하였다(International Diabetes Federation, IDF. Diabetes Atlas, 2nd edition, 2003). 그러나 우리나라 국민영양조사(2001, 2005)의 20세 이상 유병률을 적용하 면, 우리나라의 유병률도 8.0％에 육박하며, 현재의 증가속도가 유지된다면 2030년이면 OECD 회원국 중에서 가장 높은 수준에 도달할 수도 있다(당뇨병 기초통계연구 Task Force Team 보고서. Diabetes in Korea 2007. 대한당뇨병학회 2007년).The International Diabetes Federation (IDF) reported in 2003 that the prevalence of diabetes in the United States was 8.0% and 6.4% in Korea (International Diabetes Federation, IDF. Diabetes Atlas, 2nd edition, 2003). However, if the prevalence rate of 20 years or older of Korea's National Nutrition Survey (2001, 2005) is applied, the prevalence rate of Korea is close to 8.0%, and if the current rate of growth is maintained, it may reach the highest level among OECD countries in 2030. (Task Force Team report for Diabetes Basic Statistics Research. Diabetes in Korea 2007. The Korean Diabetes Association 2007).
당뇨병을 예방하거나 치료하기 위하여는 당뇨병의 발병 원인인 균형 잃은 당항상성 조절 기전을 개선시키는 방법이 효과적이다. 이 방법에 약물 치료가 있을 수 있는데, 당뇨병이 이미 진전된 경우에는 약물 치료가 불가피하지만 당뇨병을 예방하기 위한 목적으로는 약물에 대한 부작용을 감안할 때 바람직한 방법이 아니다. 당뇨 예방을 위하여는 약물보다 효과는 약하지만 부작용없는 건강기능식품이 효과적이다. 그러나 현재 국내에 시판되는 혈당강하용 건강기능식품은 탄수화물 소화 효소 저해 등 식후 혈당의 일시적 저하만을 목표로 하기 때문에 그 제품의 섭취에 의한 혈당강하효과는 일시적이며 근본적인 당뇨병의 병인 해소와는 거리가 있다. 또한 당항상성을 유지하는 상호보완적인 여러 기작을 동시에 만족시키는 복합 기능의 건강기능식품이 단일 기작에 근거한 단일 성분인 의약품에 비하여 당뇨 예방 및 치료에 가장 이상적이지만 이러한 소재는 아직 개발되지 못하고 있는 실정이다.In order to prevent or treat diabetes, it is effective to improve the unbalanced glycemic control mechanism that causes diabetes. There may be drug treatment in this method, which is inevitable if diabetes is already advanced, but it is not a desirable method in view of the side effects of the drug for the purpose of preventing diabetes. To prevent diabetes, health functional foods are less effective than drugs, but have no side effects. However, the current hypoglycemic functional foods in Korea target only a temporary decrease in post-prandial blood sugar, such as inhibition of carbohydrate digestive enzymes, so the hypoglycemic effect of ingesting the product is temporary and far from eliminating the underlying etiology of diabetes. . In addition, multi-functional dietary supplements that simultaneously satisfy several complementary mechanisms, which are always ideal, are ideal for the prevention and treatment of diabetes compared to single-component drugs based on a single mechanism, but these materials have not yet been developed. .
이에 본 발명자들은 진세노사이드를 복합적으로 전환시키는 유산균인 신규한 엔테로코커스 패칼리스 케이-60 균주(Enterococcus faecalis K-60)(KFCC11460P)(이하 K-60으로 약칭함)를 인체 분변으로부터 선별한 후 균주를 분자 유전학적 측면에서 동정하는 16S rRNA partial sequencing을 통해 균주의 종명, 속명을 확인하여, 한국미생물보존센터에 기탁하였다. Therefore, the present inventors selected a novel Enterococcus faecalis K-60 strain (KFCC11460P) (hereinafter abbreviated as K-60), a lactic acid bacterium, which complexly converts ginsenosides. 16S rRNA partial sequencing, which identifies the strain in terms of molecular genetics, confirmed the species name and genus name and deposited it in the Korea Microbiological Conservation Center.
상기 K-60를 이용하여 인삼에서 생체 이용률이 낮은 진세노사이드 Rg1, Rb1 등을 진세노사이드 Rh1, Rg3, Rh2, C-K 등으로 생성 및/또는 전환을 시킬 수 있다.Using K-60, ginsenosides Rg1 and Rb1 having low bioavailability in ginseng may be generated and / or converted into ginsenosides Rh1, Rg3, Rh2, and C-K.
따라서 본 발명의 목적은 인삼에서 생체 이용률이 낮은 진세노사이드 Rg1, Rb1, Rg1, Re 등을 진세노사이드 Rh1, Rg3, Rh2, C-K 등으로 생성 및/또는 전환 시킬 수 있는 유산균 K-60의 제공과 상기 균주를 이용한 인삼 발효물의 제조방법, 상기 방법에 의해 제조한 인삼 발효물, 상기 인삼 발효물을 포함하는 약학적 제제, 건강기능식품을 제공하고자 한다.Accordingly, an object of the present invention is to provide a lactic acid bacterium K-60 capable of generating and / or converting ginsenosides Rg1, Rb1, Rg1, Re and the like to ginsenosides Rh1, Rg3, Rh2, CK and the like having low bioavailability in ginseng. And to prepare a ginseng fermented product using the strain, ginseng fermented product prepared by the method, to provide a pharmaceutical preparation, health functional food comprising the ginseng fermented product.
또한 본 발명자들은 상기와 같이 인삼의 당항상성 개선 작용이 보고된 바 있으나 그 당항상성 개선 활성이 높지 않았기 때문에, 그 활성을 강화시킬 필요가 있다고 판단하였다. 본 발명자들은 인삼을 K-60로 발효시킴으로써 당항상성 개선 활성이 증가한다는 것을 발견하고, 본 발명을 완성하게 되었다. 따라서, 본 발명의 또다른 목적은 인삼을 K-60로 발효시킨 후 추출하여 당항상성 개선용 인삼 발효 추출물을 제공하는데 있다. In addition, the inventors of the present invention have been reported to improve the glycemic resistance of ginseng as described above, but the glycemic resistance improvement activity was not high, it was determined that it is necessary to enhance the activity. The present inventors have found that the anti-sugar improvement activity is increased by fermenting ginseng with K-60, thereby completing the present invention. Accordingly, another object of the present invention is to provide a ginseng fermentation extract for improving sugar resistance by extracting after fermenting ginseng with K-60.
상기와 같은 목적을 달성하기 위해, 본 발명은 K-60을 제공하며, 또한 K-60로 발효시켜 제조된 당항상성 개선용 인삼 발효 추출물을 제공한다. In order to achieve the above object, the present invention provides K-60, and also provides a ginseng fermentation extract for improving sugar resistance prepared by fermentation with K-60.
또한 본 발명은 (1)인삼을 K-60로 발효시켜 발효물을 얻는 단계; 및 In addition, the present invention (1) the step of fermenting ginseng with K-60 to obtain a fermentation product; And
(2)상기의 발효물을 용매로 추출한 후 여과하는 단계를 포함하도록 하여 당항상성 개선용 인삼 발효 추출물의 제조방법을 제공한다. (2) it provides a method of producing a ginseng fermentation extract for improving sugar sugar by including the step of filtering the fermented product after extraction with a solvent.
상기의 인삼 발효 추출물의 제조방법은 (2)단계 후 여과액을 단독 또는 혼합한 후 감압농축 및 동결건조하여 추출물을 얻는 단계를 추가로 더 포함할 수 있다.The method of preparing the ginseng fermented extract may further include a step of obtaining the extract by concentration or freeze drying under reduced pressure after mixing or mixing the filtrate after step (2) alone.
본 발명은 인삼을 발효시킬 수 있는 신규한 K-60을 제공할 수 있다.The present invention can provide a novel K-60 capable of fermenting ginseng.
본 발명은 상기의 신규한 K-60을 이용하여 인삼에서 생체 이용률이 낮은 진세노사이드 Rg1, Rb1 등을 진세노사이드 Rh1, Rg3, Rh2, C-K 등으로 전환시킬 수 있는 인삼 발효 추출물의 제조방법을 제공할 수 있다.The present invention provides a method for preparing a ginseng fermented extract which can convert ginsenosides Rg1, Rb1, etc., which have low bioavailability in ginseng, to ginsenosides Rh1, Rg3, Rh2, CK, etc. using the novel K-60. Can provide.
본 발명에 의한 당항상성 개선용 인삼 발효 추출물은 동물의 내당능을 개선하고, 췌장 소도에서의 인슐린 분비를 증가시키며, 췌장 베타세포의 사멸을 억제하여, 인삼 자체가 나타내는 효과보다 더욱 증가된 당항상성 개선 효과를 나타낸다.The ginseng fermentation extract for improving glycemic resistance according to the present invention improves glucose tolerance in animals, increases insulin secretion in pancreatic islets, inhibits the death of pancreatic beta cells, and improves glycemic resistance more than the effect of ginseng itself. Effect.
본 발명은 신규한 엔테로코커스 패칼리스 케이-60(Enterococcus faecalis K-60)(KFCC11460P) 균주(이하 K-60으로 약칭한다)를 나타낸다.The present invention represents a novel Enterococcus faecalis K-60 (KFCC11460P) strain (hereinafter abbreviated as K-60).
본 발명은 인삼을 발효시킬 수 있는 신규한 K-60를 나타낸다.The present invention represents a novel K-60 capable of fermenting ginseng.
본 발명은 인삼을 K-60로 발효시켜 제조된 당항상성 개선용 인삼 발효 추출물을 제공한다. The present invention provides a ginseng fermentation extract for improving sugar resistance prepared by fermenting ginseng with K-60.
본 발명은 (1)인삼을 K-60로 발효시켜 발효물을 얻는 단계; (2)상기의 발효물을 용매로 추출한 후 여과하는 단계를 포함하는 당항상성 개선용 인삼 발효 추출물의 제조방법을 제공한다. The present invention comprises the steps of (1) fermenting ginseng with K-60 to obtain a fermented product; (2) it provides a method for producing a ginseng fermentation extract for improving sugar resistance comprising the step of extracting the fermented product as a solvent and then filtering.
본 발명의 신규한 K-60는 인체의 분변을 받아 BL 혈액배지에 계수 희석법을 이용하여 단일 콜로니(colony)를 분리한 후 유산균에 해당하는 특징 및 성상을 보이는 단일 콜로니를 재차 취하여 MRS broth의 액체배지에 접종한 후 배양하여 균주를 얻었다. 이 균주로부터 DNA를 추출하여 16S rRNA를 실시하고 BLAST 프로그램을 사용하여 균주의 상동성을 검색한바, 상기 균주가 엔테로코커스 패칼리스의 스트레인 CTSPL1의 16S rRNA gene과 100％ 상동성을 가짐을 알게되어 엔테로코커스 패칼리스 케이-60 균주(Enterococcus faecalis K-60)로 명명하고 이를 미생물보존센터에 2009년 9월 17일 기탁하여 KFCC11460P의 균주번호를 부여 받았다.The novel K-60 of the present invention receives a fecal from the human body and separates a single colony (colon) by counting dilution method in BL blood medium, and then re-takes a single colony showing characteristics and properties corresponding to lactic acid bacteria and liquid of MRS broth. After inoculating the medium and cultured to obtain a strain. DNA was extracted from the strain, 16S rRNA was performed, and the homology of the strain was searched using the BLAST program. As a result, the strain was found to have 100% homology with the 16S rRNA gene of strain CTSPL1 of Enterococcus faecalis. The strain was named as coccus faecalis K-60 strain (Enterococcus faecalis K-60) and deposited on September 17, 2009 to the microbial preservation center was given a strain number of KFCC11460P.
본 발명의 신규한 K-60는 고농도의 인삼 배지에서도 생장이 가능한 한편, 극성이 높은 진세노사이드인 Rg1, Rb1을 극성이 낮은 진세노사이드인 Rh1, Rg3, C-K 및 Rh2로 대사시킬 수 있다. 본 발명에서는 단순히 Rh1, Rg3, C-K 및 Rh2의 함량 증가만을 목표로 인삼을 발효시킨 것은 아니며, K-60에 의한 발효에 의해 당항상성 이 증진되도록 K-60 발효과정 모니터링의 지표 성분들로서 Rh1, Rg3, C-K 및 Rh2를 설정하였다.The novel K-60 of the present invention can grow in high concentration ginseng medium, while metabolizing the highly polar ginsenosides Rg1, Rb1 into the less polar ginsenosides Rh1, Rg3, C-K and Rh2. In the present invention, the ginseng was not fermented simply to increase the contents of Rh1, Rg3, CK, and Rh2, and Rh1, Rg3 as indicator components of K-60 fermentation process monitoring to enhance sugar resistance by fermentation by K-60. , CK and Rh2 were set.
본 발명의 후술하는 실시예에서는 인삼의 일종인 수삼을 건조하여 백삼을 제조한 후 주근(main roots; MR)과 미삼(hairy roots; HR)으로 분리하여 발효시켰다. 그러나 주근과 미삼으로 분리하지 않고 백삼 자체를 발효시키는 방법도 본 발명의 범위에 포함될 수 있다. 또한 백삼을 증숙하여 홍삼 및 선삼으로 가공한 후 발효시키는 방법도 본 발명의 범위에 들어갈 수 있다. 본 발명에서 인삼은 소정의 크기로 절단한 고상의 인삼, 인삼을 분말화하고 이를 정제수에 현탁시킨 인삼 현탁액, 인삼을 정제수 및/또는 유기용매로 추출하여 얻은 인삼 추출물 중에서 선택된 어느 하나의 형태로 사용할 수 있다. 이때 유기용매는 에탄올 또는 농도 50∼95％의 주정을 사용할 수 있다.In the following examples of the present invention, dried ginseng, which is a kind of ginseng, was prepared to produce white ginseng, and then fermented by separating into main roots (MR) and hairy roots (HR). However, the method of fermenting the white ginseng itself without separating into the main root and the ginseng may also be included in the scope of the present invention. In addition, the method of steaming white ginseng and processing them into red ginseng and ginseng may also fall within the scope of the present invention. In the present invention, ginseng is a solid ginseng cut to a predetermined size, ginseng powdered and suspended in purified water, ginseng suspension, ginseng extracted in purified water and / or an organic solvent to be used in any one selected form of ginseng extract Can be. At this time, ethanol or alcohol having a concentration of 50 to 95% may be used as the organic solvent.
본 발명의 당항상성 개선용 인삼 발효 추출물의 제조의 일예로서 (1)인삼 현탁액을 K-60로 발효시켜 발효물을 얻는 단계, (2)상기의 발효물을 에탄올, 주정 또는 부탄올의 용매로 추출한 후 여과하는 단계를 포함하는 당항상성 개선용 인삼 발효 추출물의 제조방법을 제공할 수 있다.As an example of the preparation of ginseng fermentation extract for improving sugar resistance of the present invention, (1) fermenting a ginseng suspension with K-60 to obtain a fermentation product, (2) extracting the fermentation product with a solvent of ethanol, alcohol or butanol It can provide a method for producing a ginseng fermentation extract for improving sugar resistance, including the step of filtering after.
본 발명에 따른 인삼 발효 추출물은 상기 인삼 분말을 사용한 배지 혹은 인삼 추출물을 사용한 액체 배지에 K-60를 접종하여 발효시켜 얻은 인삼 발효물을 에탄올, 주정 또는 부탄올의 용매로 추출한 후 얻어진 추출물을 여과하여 얻을 수 있다.The ginseng fermentation extract according to the present invention is filtered by extracting the ginseng fermentation product obtained by inoculating fermentation by inoculating K-60 in the medium using the ginseng powder or the liquid medium using the ginseng extract with a solvent of ethanol, alcohol or butanol You can get it.
본 발명의 당항상성 개선용 추출물은 기존의 인삼 발효물에 비하여 진세노사 이드 Rh1(프로토파나사트리올; protopanaxatriol)과 Rh2(프로토파나사다이올; protopanaxadiol)를 동시에 많이 함유한다는 특징이 있으며, 이는 기존의 인삼 발효물 특허에서 진일보한 것이다.Glycosides Rh1 (protopanaxatriol) and Rh2 (protopanasadiol; protopanaxadiol) at the same time, compared to the conventional ginseng fermentation extract of the present invention is characterized in that it contains a lot This is a further step in the ginseng fermentation patent.
본 발명의 당항상성 개선용 인삼 발효 추출물은 포도당의 경구 투여에 의한 혈당치 상승을 낮춘다. 즉 내당능 (glucose tolerance)을 개선한다. Ginseng fermentation extract for improving sugar glycation of the present invention lowers the blood sugar level rise by oral administration of glucose. That is, it improves glucose tolerance.
본 발명의 당항상성 개선용 추출조성물은 흰쥐에서 분리한 췌장 소도(pancreatic islets)에서 인슐린의 분비를 촉진시킨다.The extract composition for improving sugar resistance of the present invention promotes insulin secretion in pancreatic islets isolated from rats.
본 발명의 당항상성 개선용 추출조성물은 췌장 베타 세포에서 사이토카인 혹은 유리 지방산으로 유도된 세포사멸을 억제하여, 인삼만을 원료로 하였을 때 보다 훨씬 증가된 효과를 나타낸다.The extract composition for improving sugar resistance of the present invention inhibits apoptosis induced by cytokines or free fatty acids in pancreatic beta cells, and shows a much increased effect than when only ginseng was used as a raw material.
또한, 본 발명의 인삼 발효 추출물은 당항상성을 개선함으로써 베타세포 이상, 내당능장애 (glucose intolerance), 공복혈당장애, 당뇨병 전단계 (prediabetes), 당뇨병을 예방 및/또는 치료할 수 있다.In addition, the ginseng fermented extract of the present invention can prevent and / or treat beta cell abnormality, glucose intolerance, fasting glucose disorders, prediabetes, and diabetes by improving glycemic condition.
본 발명은 상기의 신규한 K-60을 이용한 인삼 발효 추출물의 제조방법을 나타낸다.The present invention shows a method for producing a ginseng fermented extract using the novel K-60.
본 발명은 상기의 신규한 K-60를 이용하여 인삼에서 생체 이용률이 낮은 진세노사이드 Rg1, Rb1, Rg1, Re 등을 진세노사이드 Rh1, Rg3, Rh2, C-K 등으로 전환시킬 수 있는 인삼 발효 추출물의 제조방법을 나타낸다.The present invention ginseng fermentation extract that can convert ginsenosides Rg1, Rb1, Rg1, Re, etc., which have low bioavailability in ginseng to ginsenosides Rh1, Rg3, Rh2, CK, etc. using the novel K-60 The manufacturing method of this is shown.
본 발명은 상기의 신규한 K-60를 이용하여 당항상성을 유지하거나 개선시킬 수 있는 인삼 발효 추출물의 제조방법을 나타낸다.The present invention shows a method for producing a ginseng fermented extract that can maintain or improve sugar compatibility using the novel K-60.
본 발명은 (1)인삼 현탁액을 K-60로 발효시켜 발효물을 얻는 단계; 및 (2)상기의 발효물을 용매로 추출한 후 여과하는 단계를 포함하는 인삼 발효 추출물의 제조방법을 나타낸다. The present invention comprises the steps of (1) fermenting the ginseng suspension with K-60 to obtain a fermentation product; And (2) extracting the fermented product as a solvent and then filtering the ginseng fermented extract.
본 발명은 (1)백삼주근 분말, 백삼미삼 분말, 인삼 분말, 홍삼 분말 또는 선삼 분말을 정제수에 첨가하여 얻은 인삼분말 현탁액을 K-60로 35∼37℃에서 3∼10일간 동안 발효시켜 발효물을 얻는 단계; 및 (2)상기의 발효물을 60℃∼80℃에서 2∼4시간 동안 에탄올, 주정 또는 부탄올의 용매로 추출한 후 여과하는 단계를 포함한다.The present invention (1) white ginseng root powder, white ginseng powder, ginseng powder, red ginseng powder or ginseng powder is added to purified water to ferment the ginseng powder suspension obtained by fermentation at 35-37 ° C. for 3-10 days with K-60. Obtaining; And (2) extracting the fermented product from 60 ° C. to 80 ° C. for 2 to 4 hours with a solvent of ethanol, alcohol or butanol, and then filtering the fermented product.
상기의 용매중 에탄올은 50∼99％ 농도의 에탄올을 사용할 수 있다.Ethanol in the solvent may be used at a concentration of 50 to 99%.
본 발명의 신규한 K-60를 이용한 인삼 발효 추출물의 제조방법에 대해 조사한바, 본 발명의 목적을 달성하기 위해서는 상기에서 언급한 조건에 의해 신규한 K-60를 이용한 인삼 발효 추출물의 제조방법을 제공하는 것이 바람직하다.Investigation on the preparation method of the ginseng fermentation extract using the novel K-60 of the present invention, in order to achieve the object of the present invention, a method for producing a ginseng fermentation extract using the novel K-60 under the above-mentioned conditions It is desirable to provide.
본 발명은 상기에서 언급한 신규한 K-60를 이용한 인삼 발효 추출물을 포함한다.The present invention includes the ginseng fermented extract using the novel K-60 mentioned above.
본 발명은 상기에서 언급한 신규한 K-60를 이용하여 제조한 인삼 발효 추출물을 포함하는 약학제 제제를 나타낸다.The present invention represents a pharmaceutical formulation comprising a ginseng fermented extract prepared using the novel K-60 mentioned above.
본 발명은 상기에서 언급한 신규한 K-60를 이용하여 제조한 인삼 발효 추출 물을 포함하는 당항상성 개선용 약학제 제제를 나타낸다.The present invention represents a pharmaceutical formulation for improving sugar resistance including ginseng fermentation extract prepared using the novel K-60 mentioned above.
본 발명은 상기에서 언급한 신규한 K-60를 이용하여 제조한 인삼 발효 추출물을 포함하는 건강기능식품을 나타낸다.The present invention represents a health functional food comprising a ginseng fermented extract prepared using the novel K-60 mentioned above.
본 발명은 상기에서 언급한 신규한 K-60를 이용하여 제조한 인삼 발효 추출물을 포함하는 당항상성 개선용 건강기능식품을 나타낸다.The present invention represents a health functional food for improving sugar resistance, including a ginseng fermented extract prepared using the novel K-60 mentioned above.
이하 본 발명의 내용을 실시예 및 시험예를 통하여 구체적으로 설명한다. 그러나, 이들은 본 발명을 보다 상세하게 설명하기 위한 것으로 본 발명의 권리범위가 이들에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples. However, these are intended to explain the present invention in more detail, and the scope of the present invention is not limited thereto.
<실시예 1> 엔테로코커스 패칼리스 케이-60 균주(Enterococcus faecalis K-60)(KFCC11460P)의 분리Example 1 Isolation of Enterococcus faecalis K-60 (KFCC11460P)
사람의 분변을 받아 BL혈액배지에 계수 희석법을 이용하여 단일 콜로니를 분리하였다. 그 중 유산균에 해당하는 특징 및 성상을 보이는 단일 콜로니를 취하여 MRS broth의 액체배지에 접종한 후, 37℃ 항온기에서 24 시간 동안 배양한 다음 상기 배양액에서 균주를 분리하였다. Human feces were isolated to separate single colonies by counting dilution in BL blood medium. Among them, a single colony showing characteristics and properties corresponding to lactic acid bacteria was taken and inoculated into a liquid medium of MRS broth, and then cultured in a 37 ° C incubator for 24 hours, and then strains were separated from the culture solution.
상기 균주로부터 DNA를 추출하여 16S rRNA를 제작하여 BLAST 프로그램을 사용하여 상동성을 검색하였다.DNA was extracted from the strain, 16S rRNA was produced, and homology was searched using the BLAST program.
DNA 정제 키트(purification kit)를 이용하여 상기 균주로부터 DNA를 추출하고 이를 16S rRNA 분자의 양끝에 존재하는 보존성이 높은 염기서열 영역을 표적으 로 삼아 PCR primer[27f (5'-agagtttgatcctggctcag-3')와 1492r(5'-ggctaccttgttacgactt-3']를 제작하여 PCR 하여 전 영역 대비 약 67％에 해당하는 크기의 PCR product를 얻을 수 있었다.DNA primer was extracted from the strain using a DNA purification kit, and the PCR primer [27f (5'-agagtttgatcctggctcag-3 ') was used to target a highly conserved sequence region at both ends of the 16S rRNA molecule. And 1492r (5'-ggctaccttgttacgactt-3 '] were prepared and PCR to obtain a PCR product of about 67% of the total area.
PCR product 의 검출은 일반적인 아가로스 겔(agarose gel) 전기영동을 사용하였다. 이로부터 목적 밴드를 잘라내어 용출한 후 새로 제작한 시퀀싱용 프라이머(sequencing primer) [r4L(5'-ACGGGCGGTGTGTACAAG-3')와 f2L(5'-CCAGCAGCCGCGGTAATAG- 3']를 이용하여 시퀀싱(sequencing) 하였고 온라인상에서 서열 데이터를 분석하는 프로그램인 BLAST 프로그램을 사용하여 상동성을 검색하였다.PCR product detection was performed using agarose gel electrophoresis. The target band was cut out and eluted, and then sequencing was performed using newly prepared sequencing primers [r4L (5'-ACGGGCGGTGTGTACAAG-3 ') and f2L (5'-CCAGCAGCCGCGGTAATAG-3']. Homology was searched using the BLAST program, a program that analyzes sequence data on the bed.
균주의 생성물(product)을 블라스트 조사(blast search)한 결과 Enterococcus faecalis strain CTSPL1의 16S rRNA gene과 100％ 동일함(identities)을 갖는 것을 알 수 있어 상기 균주를 엔테로코커스 패칼리스 케이-60 균주(Enterococcus faecalis K-60)로 명명하고 이를 미생물보존센터에 2009년 9월 17일 KFCC11460P 로 기탁하였다.A blast search of the product of the strain revealed that the bacterium had 100% identity with the 16S rRNA gene of Enterococcus faecalis strain CTSPL1, and the strain was Enterococcus faecalis K-60 strain (Enterococcus). faecalis K-60) and it was deposited with KFCC11460P on September 17, 2009 at the Center for Microbiological Conservation.
한편 K-60의 16s DNA 여기서열을 염기서열목록 5에 나타내었다.Meanwhile, the 16s DNA sequence of K-60 is shown in SEQ ID NO: 5.
<실시예 2> K-60의 균학적 성질Example 2 Mycological Properties of K-60
K-60의 균학적 성질은 미생물의 분류학적 동정은 Hammes 등[The prokaryotes, 1563-1578, 2nd Edition, Springer-Verlag Co. (1992)]의 방법에 따라 수행하였으며, 그 결과는 표 1에서 보는 바와 같다. 이 균주는 그람 양성, 구균 이며, 산소유무와 상관없이 잘 생장하였고, 카탈라아제는 양성이었다.The bacteriological properties of K-60 are classified by Hammes et al., The prokaryotes, 1563-1578, 2nd Edition, Springer-Verlag Co. (1992)], and the results are shown in Table 1. The strain was Gram positive, cocci, well grown with or without oxygen, and catalase positive.
표 1. K-60의 생리적 및 생화학적 특성Table 1. Physiological and biochemical properties of K-60
<제조예> 인삼 분말의 제조Preparation Example Preparation of Ginseng Powder
인삼은 안성산 5년근을 건조하여 백삼 주근, 백삼 미근(미삼)으로 분리하여 분말을 만들었다. Ginseng was dried 5 years old root of Anseong and separated into white ginseng root and white ginseng root (misam) to make powder.
인삼은 안성산 5년근을 건조한 다음 96∼100℃에서 3∼5시간 동안 쪄서 건조한 후 홍삼 분말로 만들었다. Ginseng was dried 5 years old Anseong and then steamed for 3 to 5 hours at 96 ~ 100 ℃ to make red ginseng powder.
인삼은 안성산 5년근을 건조한 다음 110∼130℃에서 2∼3시간 쪄서 건조한 후 선삼 분말로 만들었다.Ginseng was dried 5 years old Anseong and then steamed for 2 to 3 hours at 110 ~ 130 ℃ to make ginseng powder.
<인삼 증류수 추출물 분말의 제조><Preparation of Ginseng Distilled Water Extract Powder>
상기의 <인삼 분말의 제조>에서 얻은 백삼 분말, 홍삼 분말 및 선삼 분말을 각각 인삼 분말 1kg당 증류수 10리터를 가하여 60℃∼100℃에서 0.5∼10시간 추출하여 동결건조하여 분말로 제조하였다.White ginseng powder, red ginseng powder and ginseng powder obtained in the above <Production of ginseng powder> were added with 10 liters of distilled water per 1 kg of ginseng powder, respectively, extracted at 60 ° C. to 100 ° C. for 0.5 to 10 hours, and lyophilized to prepare a powder.
<인삼 에탄올 추출물 분말의 제조><Production of Ginseng Ethanol Extract Powder>
상기의 <인삼 분말의 제조>에서 얻은 백삼, 홍삼 및 선삼의 분말을 각각 인삼 1kg당 에탄올 10리터를 가하여 60℃∼80℃에서 0.5∼10시간 추출하고 감압건조후 동결건조하여 분말로 제조하였다.White ginseng, red ginseng, and ginseng powder obtained in the above <Production of ginseng powder> were added with 10 liters of ethanol per 1 kg of ginseng, respectively, extracted at 60 ° C. to 80 ° C. for 0.5 to 10 hours, dried under reduced pressure, and lyophilized to prepare a powder.
<실시예 3-1> 백삼 주근 발효물의 제조Example 3-1 Preparation of White Ginseng Root Ferment
상기 제조예에서 얻은 백삼 주근 분말 20g에 증류수 2,000㎖를 가하고 멸균한 다음 K-60 2g을 넣고 37℃에서 7일간 발효시켜 발효물을 얻었다.2,000 g of distilled water was added to 20 g of white ginseng fructus powder obtained in the above preparation, sterilized, and 2 g of K-60 was added thereto, followed by fermentation at 37 ° C. for 7 days to obtain a fermented product.
상기의 발효물을 에탄올 2,000㎖로 70℃에서 3시간 동안 추출하고 여과하여 백삼 주근 발효 추출물을 제조하였다. The fermented product was extracted with 2,000 ml of ethanol at 70 ° C. for 3 hours and filtered to prepare a white ginseng root fermentation extract.
<실시예 3-2> 백삼 주근 발효물의 제조Example 3-2 Preparation of White Ginseng Root Ferment
상기 제조예에서 얻은 백삼 주근 분말 20g에 증류수 2,000㎖를 가하고 멸균한 다음 K-60 2g을 넣고 37℃에서 7일간 발효시켜 발효물을 얻었다.2,000 g of distilled water was added to 20 g of white ginseng fructus powder obtained in the above preparation, sterilized, and 2 g of K-60 was added thereto, followed by fermentation at 37 ° C. for 7 days to obtain a fermented product.
상기의 발효물을 부탄올 2,000㎖로 70℃에서 3시간 동안 추출하고 여과하여 백삼 주근 발효 추출물을 제조하였다. The fermented product was extracted with 2,000 ml of butanol at 70 ° C. for 3 hours and filtered to prepare a white ginseng root fermentation extract.
<실시예 4-1> 백삼 미근(미삼) 발효물의 제조Example 4-1 Preparation of White Ginseng Roots
상기 제조예에서 얻은 백삼 미근(미삼) 분말 20g에 증류수 2,000㎖를 가하고 멸균한 다음 K-60 2g을 넣고 37℃에서 7일간 발효시켜 발효물을 얻었다.2,000 g of distilled water was added to 20 g of white ginseng root (misam) powder obtained in the above preparation, and sterilized. Then, 2 g of K-60 was added thereto, followed by fermentation at 37 ° C. for 7 days to obtain a fermented product.
상기의 발효물을 에탄올 2,000㎖로 70℃에서 3시간 동안 추출하고 여과하여 백삼 미근(미삼) 발효 추출물을 제조하였다. The fermented product was extracted with 2,000 ml of ethanol at 70 ° C. for 3 hours and filtered to prepare a white ginseng root (misam) fermented extract.
<실시예 4-2> 백삼 미근(미삼) 발효물의 제조Example 4-2 Preparation of White Ginseng Roots
상기 제조예에서 얻은 백삼 미근(미삼) 분말 20g에 증류수 2,000㎖를 가하고 멸균한 다음 K-60 2g을 넣고 37℃에서 7일간 발효시켜 발효물을 얻었다.2,000 g of distilled water was added to 20 g of white ginseng root (misam) powder obtained in the above preparation, and sterilized. Then, 2 g of K-60 was added thereto, followed by fermentation at 37 ° C. for 7 days to obtain a fermented product.
상기의 발효물을 부탄올 2,000㎖로 70℃에서 3시간 동안 추출하고 여과하여 백삼 미근(미삼) 발효 추출물을 제조하였다. The fermented product was extracted with 2,000 mL of butanol at 70 ° C. for 3 hours and filtered to prepare a white ginseng root (misam) fermented extract.
<실시예 5-1> 홍삼 발효물의 제조Example 5-1 Preparation of Red Ginseng Fermented Products
상기 제조예에서 얻은 홍삼분말 20g에 증류수 2,000㎖를 가하고 멸균한 다음 K-60 2g을 넣고 37℃에서 7일간 발효시켜 발효물을 얻었다.2,000 g of distilled water was added to 20 g of the red ginseng powder obtained in Preparation Example, sterilized, and 2 g of K-60 was added thereto, followed by fermentation at 37 ° C. for 7 days to obtain a fermented product.
상기의 발효물을 에탄올 2,000㎖로 70℃에서 3시간 동안 추출하고 여과하여 홍삼 발효 추출물을 제조하였다. The fermented product was extracted with 2,000 ml of ethanol at 70 ° C. for 3 hours and filtered to prepare a red ginseng fermented extract.
<실시예 5-2> 홍삼 발효물의 제조Example 5-2 Preparation of Red Ginseng Fermented Products
상기 제조예에서 얻은 홍삼분말 20g에 증류수 2,000㎖를 가하고 멸균한 다음 K-60 2g을 넣고 37℃에서 7일간 발효시켜 발효물을 얻었다.2,000 g of distilled water was added to 20 g of the red ginseng powder obtained in Preparation Example, sterilized, and 2 g of K-60 was added thereto, followed by fermentation at 37 ° C. for 7 days to obtain a fermented product.
상기의 발효물을 부탄올 2,000㎖로 70℃에서 3시간 동안 추출하고 여과하여 홍삼 발효 추출물을 제조하였다. The fermented product was extracted with 2,000 ml of butanol at 70 ° C. for 3 hours and filtered to prepare a red ginseng fermented extract.
<실시예 6-1> 선삼 발효물 제조<Example 6-1> Preparation of fermented ginseng
상기 제조예에서 얻은 선삼분말 20g에 증류수 2,000㎖를 가하고 멸균한 다음 K-60 2g을 넣고 37℃에서 7일간 발효시켜 발효물을 얻었다.2,000 g of distilled water was added to 20 g of the ginseng powder obtained in Preparation Example, sterilized, and 2 g of K-60 was added thereto, followed by fermentation at 37 ° C. for 7 days to obtain a fermented product.
상기의 발효물을 에탄올 2,000㎖로 70℃에서 3시간 동안 추출하고 여과하여 홍삼 발효 추출물을 제조하였다. The fermented product was extracted with 2,000 ml of ethanol at 70 ° C. for 3 hours and filtered to prepare a red ginseng fermented extract.
<실시예 6-2> 선삼 발효물 제조<Example 6-2> Preparation of fermented ginseng
상기 제조예에서 얻은 선삼분말 20g에 증류수 2,000㎖를 가하고 멸균한 다음 K-60 2g을 넣고 37℃에서 7일간 발효시켜 발효물을 얻었다.2,000 g of distilled water was added to 20 g of the ginseng powder obtained in Preparation Example, sterilized, and 2 g of K-60 was added thereto, followed by fermentation at 37 ° C. for 7 days to obtain a fermented product.
상기의 발효물을 부탄올 2,000㎖로 70℃에서 3시간 동안 추출하고 여과하여 홍삼 발효 추출물을 제조하였다. The fermented product was extracted with 2,000 ml of butanol at 70 ° C. for 3 hours and filtered to prepare a red ginseng fermented extract.
<비교예 1> 백삼 주근 추출물의 제조Comparative Example 1 Preparation of White Ginseng Extract
백삼 주근 분말 20g에 증류수 2,000㎖를 가하여 멸균한 후 에탄올 2,000㎖로 70℃에서 3시간 추출하고 여과하여 백삼 주근 추출물을 제조하였다.2,000 g of distilled water was added to 20 g of white ginseng root powder and sterilized, followed by extraction with 2,000 ml of ethanol at 70 ° C. for 3 hours, followed by filtration to prepare white ginseng root extract.
<비교예 2> 백삼 미삼 추출물의 제조Comparative Example 2 Preparation of White Ginseng Misam Extract
백삼 미삼 분말 20g에 증류수 2,000㎖를 가하여 멸균한 후 에탄올 2,000㎖로 70℃에서 3시간 추출하고 여과하여 백삼 미삼 추출물을 제조하였다.2,000 g of distilled water was added to 20 g of white ginseng rice ginseng powder and sterilized, followed by extraction with 2,000 ml of ethanol at 70 ° C. for 3 hours, followed by filtration.
<비교예 3> 홍삼 추출물의 제조Comparative Example 3 Preparation of Red Ginseng Extract
홍삼분말 20g에 증류수 2,000㎖를 가하여 멸균한 후 에탄올 2,000㎖로 70℃에서 3시간 추출하고 여과하여 홍삼 추출물을 제조하였다.2000 g of red ginseng powder was added to sterilize 2,000 ml of distilled water, and then extracted with 2,000 ml of ethanol at 70 ° C. for 3 hours and filtered to prepare a red ginseng extract.
<비교예 4> 선삼 추출물의 제조Comparative Example 4 Preparation of Ginseng Extract
선삼분말 20g에 증류수 2,000㎖를 가하여 멸균한 후 에탄올 2,000㎖로 70℃에서 3시간 추출하고 여과하여 선삼 추출물을 제조하였다.2,000 g of distilled water was added to 20 g of ginseng powder and sterilized, and extracted with 2,000 ml of ethanol at 70 ° C. for 3 hours and filtered to prepare ginseng extract.
<시험예 1> 함량분석 실험Test Example 1 Content Analysis Experiment
상기 실시예 3-1, 실시예 4-1과 비교예 1, 비교예 2 에서 얻은 각각의 추출물에 각각의 추출물과 동량의 메탄올(methanol)을 넣고 원심분리하여 상등액을 얻었다.Each extract obtained in Example 3-1, Example 4-1, Comparative Example 1, and Comparative Example 2 was added with the same amount of methanol (methanol) and centrifuged to obtain a supernatant.
상기에서 얻은 상등액 중에서 100 ml을 감압농축하고 각각의 추출물을 0.2g씩 취하여 메탄올 100ml씩 3회 추출하였다. 이 추출물을 감압 농축시킨 후 물 100ml를 넣어 현탁시키고, 에테르 100ml씩으로 3회 추출하고 나머지 물분획을 다시 100ml씩으로 3회 추출하고 감압 농축시켰다. 이어서 MeOH 1ml에 용해시켜 HPLC로 분석하여 표 1과 같은 사포닌 함량 분석결과를 얻었다.100 ml of the supernatant obtained above was concentrated under reduced pressure, 0.2 g of each extract was extracted, and 100 ml of methanol were extracted three times. The extract was concentrated under reduced pressure, suspended in 100 ml of water, extracted three times with 100 ml of ether, and the remaining water fraction was extracted three times with 100 ml each and concentrated under reduced pressure. Subsequently, it was dissolved in 1 ml of MeOH and analyzed by HPLC to obtain a saponin content analysis result as shown in Table 1.
HPLC 분석조건 :영린 에이치피엘씨 시스템: Younglin HPLC system - [Younglin SP930D quaternary pump (Seoul, Korea)와 ELSD (Attech Co., USA) detector]: Column, Develosil ODS-UG-5 Batch #10054 (NOMURA CHEMICAL, Japan); 용매, 증류수-아세토나이트릴 [0-35 min (90:10), 35-30min(5:95)] HPLC analysis conditions: Younglin HPLC system: Younglin HPLC system-[Younglin SP930D quaternary pump (Seoul, Korea) and ELSD (Attech Co., USA) detector]: Column, Develosil ODS-UG-5 Batch # 10054 (NOMURA CHEMICAL, Japan); Solvent, distilled water-acetonitrile [0-35 min (90:10), 35-30 min (5:95)]
상기와 같이 실험한 결과 실시예 3-1 및 실시예 4-1에서 K-60 발효에 의하여 백삼 주근에는 함유되어 있지 않은 Rh1, Rg3, C-K, Rh2가 발효 인삼 주근에는 각각 4.6％, 0.5％, 0.7％, 4.5％ 함유되었으며, 미삼에는 함유되어 있지 않은 Rh1, Rg3, C-K, Rh2가 발효 미삼에는 각각 2.1％, 3.1％, 0.7％, 1.5％ 함유되었다(표 2 참조). 즉 K-60 발효에 의하여 인삼에는 함유되지 않은 성분들이 발효 백삼에 생성되었다.As a result of the experiment as described above, Rh1, Rg3, CK, Rh2, which are not contained in white ginseng root by K-60 fermentation in Examples 3-1 and 4-1, were 4.6%, 0.5%, 0.7%, 4.5%, Rh1, Rg3, CK, Rh2, which were not contained in rice ginseng, contained 2.1%, 3.1%, 0.7%, and 1.5%, respectively, in fermented rice ginseng (see Table 2). In other words, K-60 fermentation produced ingredients not contained in ginseng fermented white ginseng.
즉, 표 2의 결과로부터 K-60를 이용하여 인삼을 발효시킨 발효물에는 단순한 인삼에 함유되지 않은 성분들이 생성되어, 상기 K-60는 인삼에서 진세노사이드 Rh1, Rg3, C-K, Rh2를 생성 및/또는 전환시킬 수 있음을 알 수 있었다.That is, from the results of Table 2, the fermentation product of the ginseng using K-60 produces components not contained in simple ginseng, and the K-60 produces ginsenosides Rh1, Rg3, CK, and Rh2 in ginseng. And / or can be converted.
표 2. K-60에 의한 인삼의 발효가 진세노사이드 함량에 미치는 영향Table 2. Effect of Ginseng Fermentation on Ginsenoside Content by K-60
sample
1)- 측정할 수 없었음 (not detected). 1) -not detected.
<시험예 2> 흰쥐에서의 경구당부하 검사에 미치는 영향 평가<Test Example 2> Evaluation of the effect on oral glucose tolerance test in rats
스프래그 돌리 흰쥐 (Sprague-Dawley rats; 체중 약 400g; 수컷) 12마리를 16시간 절식시킨 후 포도당을 경구 투여(2g/kg 체중)한 다음 이를 4마리씩 3개군으로 나눈 후 대조군에는 아무런 처리를 하지 않고, 실시예 3-2의 백삼 주근을 K-60으로 발효 후 부탄올로 추출한 백삼 주근 발효 추출물을 투여한 군 및 비교예 1의 백삼 주근 추출물을 투여한 군으로 각각 처리하였다. 투여 후 0분, 30분, 60분, 90분, 120분에 꼬리 끝 부분을 절개하여 혈액을 채취하여 아쿠첵 혈당측정기(로슈사 제품)를 이용하여 혈당치를 측정한다. After 12 hours of fasting 12 Sprague-Dawley rats (approximately 400 g; males), oral administration (2 g / kg body weight) of glucose, divided them into three groups of four animals, and no treatment in the control group After fermenting the white ginseng fructus of Example 3-2 with K-60, the white ginseng fructus fermented extract extracted with butanol and the white ginseng fructus extract of Comparative Example 1 were treated. At 0, 30, 60, 90, and 120 minutes after the administration, the tail tip is incised to collect blood and the blood glucose level is measured using an acujun glucose meter (Roche).
그 결과 본 발명의 백삼 주근 K-60 발효물의 부탄올 추출물은 포도당 부하에 의한 90분후의 혈당치가 인삼군은 130mg/dl인데 비하여 116 mg/dl로 11％ 낮추었으며, 혈당증가곡선 아래 면적(Area under the curve; AUC)은 인삼군은 16069 mg/dl·min인데 비하여 14471 mg/dl·min로 10％ 낮추었으며, 이를 표 3에 나타내었다.As a result, the butanol extract of the fermented K-60 fermented white ginseng of the present invention lowered the blood glucose level by 11% to 116 mg / dl compared to 130 mg / dl in the ginseng group after 90 minutes due to glucose load, and the area under the blood glucose increase curve (Area under The curve; AUC) was lowered by 10% to 14471 mg / dl · min compared to 16069 mg / dl · min in the ginseng group, which is shown in Table 3.
표 3. K-60에 의한 백삼 발효물의 부탄올 추출물이 흰쥐의 경구 당부하에 의한 혈당 상승에 미치는 영향Table 3. Effect of Butanol Extract of White Ginseng Ferment by K-60 on Blood Glucose Elevation by Oral Glucose Loading in Rats
6.466.4 ±
6.4
32.0170 ±
32.0
19.5156 ±
19.5
17.2128 ±
17.2
8.7123 ±
8.7
172116464 ±
1721
0.588.8 ±
0.5
13.5160 ±
13.5
23.7143 ±
23.7
11.6130 ±
11.6
15.2120 ±
15.2
152116069 ±
1521
5.082.5 ±
5.0
16.1129 ±
16.1
6.2140 ±
6.2
7.6116 ±
7.6
5.1114 ±
5.1
633.014471 ±
633.0
1)Values are mean± SD (n=4). 1) Values are mean ± SD (n = 4).
2)AUC : Area under the curve (포도당 부하 후 120분 동안의 혈당치 상승 곡선 아래의 면적이며 단위는 mg/dl·min) 2) AUC: Area under the curve (area under the blood glucose level rise curve for 120 minutes after glucose loading, in mg / dl · min)
3)포도당만 투여한 군 3) group administered with glucose only
<시험예 3> 췌장 소도에서의 인슐린 분비에 미치는 영향Test Example 3 Effect on Insulin Secretion in Pancreatic Islets
- 포도당에 반응하는 실험계인 흰쥐 췌장 소도의 일차 배양으로 인슐린 분비 효과를 검증하였다.-Insulin secretion was verified by primary culture of rat pancreatic islets, a laboratory system that responds to glucose.
- 췌장 소도의 분리 및 배양Isolation and Culture of Pancreatic Islets
스프래그 돌리 흰쥐 (Sprague-Dawley rats)로부터 췌장 소도의 분리는 콜라게나제(collagenase)를 총담관에 직접 주입하는 방법을 이용하였다. 떼어낸 췌장은 피콜(ficoll) 경사를 이용하여 소도를 분리함. 소도를 크렙스 링거 이탄화 버퍼(KRB; Krebs-Ringer bicarbonate buffer, pH 7.2)에 담아 37℃, 5％ CO2 배양기에서 1일간 배양 후 사용하였다.Isolation of pancreatic islets from Sprague-Dawley rats was performed by injecting collagenase directly into the bile duct. The detached pancreas is isolated by using a ficoll gradient. Sodium was contained in Krebs-Ringer bicarbonate buffer (KRB, pH 7.2) and used after incubation in 37 ° C., 5% CO 2 incubator for 1 day.
- 인슐린 분비의 검사Examination of insulin secretion
24시간 배양한 췌장 소도를 KRB로 2회 수세 후 각 well 당 일정 크기의 것들로 3개씩 옮긴 다음 포도당 농도에 따른 분비능과 배양 시간에 따른 분비능 그리고 생물전환물에 의한 인슐린 분비능을 측정함. 인슐린 농도의 측정은 효소면역방법 (EIA; enzyme immunoassay) 이용한 키트 사용하였다. Pancreatic islets incubated for 24 hours were washed twice with KRB, and then transferred to three of each well with a predetermined size. The secretion ability according to the glucose concentration, the secretion ability according to the culture time, and the insulin secretion ability by the bioconversion were measured. The insulin concentration was measured using a kit using enzyme immunoassay (EIA).
이상의 결과로부터, 본 발명의 백삼 주근의 K-60 발효물의 에탄올 추출물은 인삼 추출물을 단독으로 사용한 것에 비하여 배지 농도 3.3mM에서의(기저) 인슐린 분비에는 영향주지 않았으나, 배지 농도 16.7mM에서의 인슐린 분비(포도당 자극)는 인삼 추출물을 단독으로 사용한 것에 비하여 증가시켰다. 이는 포도당에 의한 인슐린 분비를 증강시켜주는 효과로서 당항상성 조절이 안 되는 사람들에게는 이상적인 효과이며, 이를 표 4에 나타내었다.From the above results, the ethanol extract of the K-60 fermentation of white ginseng root of the present invention did not affect the insulin secretion at the media concentration of 3.3 mM but the insulin secretion at the media concentration of 16.7 mM compared with the ginseng extract alone. (Glucose Stimulation) was increased compared to the ginseng extract alone. This is an effect that enhances insulin secretion by glucose is an ideal effect for those who do not always control the glucose, it is shown in Table 4.
본 발명의 미삼의 K-60 발효물의 에탄올 추출물은 미삼 추출물을 단독으로 사용한 것에 비하여 배지 농도 3.3mM에서의 (기저) 인슐린 분비는 감소하였으나, 배지 농도 16.7mM에서의 인슐린 분비(포도당 자극)는 인삼 추출물을 단독으로 사용한 것에 비하여 증가시켰다. 이는 포도당에 의한 인슐린 분비를 증강시켜주는 효과로서 당항상성 조절이 안 되는 사람들에게는 이상적인 효과이며, 이를 표 5에 나타내었다.The ethanol extract of K-60 fermented ginseng of the present invention decreased the (base) insulin secretion at a medium concentration of 3.3 mM but the insulin secretion (glucose stimulation) at a medium concentration of 16.7 mM compared to that of the ginseng extract alone. The extract was increased compared to the one used alone. This is an effect that enhances insulin secretion by glucose is an ideal effect for those who do not always control the glucose, it is shown in Table 5.
본 발명의 미삼의 K-60 유산균 발효물의 부탄올 추출물은 미삼 추출물을 단독으로 사용한 것에 비하여 배지 농도 3.3mM에서의 (기저) 인슐린 분비는 약간 증가시켰으며, 배지 농도 16.7mM에서의 인슐린 분비 (포도당 자극)는 미삼 추출물을 단독으로 사용한 것에 비하여 크게 증가시켰다. 이는 포도당에 의한 인슐린 분비를 증강시켜주는 효과로서 당항상성 조절이 안 되는 사람들에게는 이상적인 효과이며, 이를 표 6에 나타내었다.Butanol extract of the K-60 lactic acid bacteria fermentation of the ginseng of the present invention slightly increased the (base) insulin secretion at 3.3 mM medium, and the insulin secretion (glucose stimulation) at the medium concentration of 3.3 mM compared to that of the ginseng extract alone. ) Was significantly increased compared to the use of the rice extract alone. This is an effect that enhances insulin secretion by glucose is an ideal effect for those who do not always control the glucose, which is shown in Table 6.
표 4. 백삼 주근 K-60 발효물의 에탄올 추출물의 췌장 소도에서 인슐린 분비효과Table 4. Effect of Insulin Secretion on Pancreatic Islet of Ethanol Extract of White Ginseng Root K-60 Fermentation
Concentration (㎍ / mL)
Misam
K-60 fermented product **
*Values are mean± SEM (n=6).Values are mean ± SEM (n = 6).
*Significantly different from control group at *P<0.05, ***P<0.001.* Significantly different from control group at * P <0.05, *** P <0.001.
**백삼 주근 K-60 발효물의 에탄올 추출물을 나타낸다.** Represents the ethanol extract of white ginseng root K-60 fermentation.
표 5. 미삼의 유산균 K-60 발효물의 에탄올 추출물의 췌장 소도에서의 인슐린 분비 효과Table 5. Effect of Insulin Secretion on Pancreatic Islet of Ethanol Extract of Lactic Acid Bacteria K-60 Fermentation
Concentration (㎍ / mL)
Misam
K-60 fermented product **
*Values are mean± SEM (n=6).Values are mean ± SEM (n = 6).
*Significantly different from control group at **P<0.01, ***P<0.001.* Significantly different from control group at ** P <0.01, *** P <0.001.
**미삼의 K-60 발효물의 에탄올 추출물을 나타낸다.** Represents the ethanol extract of the ginseng K-60 fermentation.
표 6. 미삼의 유산균 K-60 발효물의 부탄올 추출물의 췌장소도에서의 인슐린 분비 효과Table 6. Insulin Secretion Effect of Butanol Extract of Lactic Acid Bacteria K-60 Fermentation
Concentration (㎍ / mL)
Misam
K-60 fermented product **
*Values are mean± SEM (n=6).Values are mean ± SEM (n = 6).
*Significantly different from control group at *P<0.05, **P<0.01, ***P<0.001.* Significantly different from control group at * P <0.05, ** P <0.01, *** P <0.001.
**미삼의 K-60 발효물의 부탄올 추출물을 나타낸다.** Butanol extract of K-60 fermentation of ginseng.
<시험예 4> 췌장 베타세포 사멸에 미치는 영향Test Example 4 Effect on Pancreatic Beta-cell Death
- 세포주 : MIN6N8 세포주 Cell line: MIN6N8 cell line
- 세포사멸 유도 조건-Apoptosis induction condition
- 사이토카인에 의한 유도 : 인터페론-γ(IFN-γ10 ng/mL), 종양괴사인자(TNF)-α (10 ng/mL), 인터루이킨(interleukin; IL)-1β (10 ng/mL)의 혼합물을 48시간 배지에 첨가하였다. 본 발명의 인삼발효 추출물인 백삼 주근의 K-60 발효물의 에탄올 추출물, 백삼 주근의 K-60 발효물의 부탄올 추출물, 미삼의 K-60 발효물의 에탄올 추출물, 또는 미삼의 K-60 발효물의 부탄올 추출물을 사이토카인과 동시에 처리하였다.Induction by cytokines: interferon-γ (IFN-γ 10 ng / mL), tumor necrosis factor (TNF) -α (10 ng / mL), interleukin (IL) -1β (10 ng / mL) Was added to the medium for 48 hours. Ethanol extract of K-60 fermentation of white ginseng root, ginseng fermentation extract of the present invention, butanol extract of K-60 fermentation of white ginseng root, ethanol extract of K-60 fermentation of ginseng, or butanol extract of K-60 fermentation of ginseng Treatment was performed concurrently with cytokines.
- 유리지방산에 의한 유도 : 팔미트산 50mM을 50％ 에탄올에 용해하여 0.5mM이 되도록하고, 48시간 후 배지에 첨가하였고 본 발명의 인삼발효 추출물인 미삼의 K-60 발효물의 에탄올 추출물 또는 미삼의 K-60 발효물의 부탄올 추출물을 팔미트 산과 동시에 처리하였다-Induction by free fatty acid: 50mM of palmitic acid was dissolved in 50% ethanol to 0.5mM, and added to the medium after 48 hours. Butanol extract of K-60 fermentation was treated simultaneously with palmitic acid
- 세포사멸 측정 Apoptosis measurement
사멸된 세포에서 생성되는 세포질성 히스톤단백질 관련 DNA 절편(cytoplasmic histone associated-DNA-fragments 모노 및 올리고 뉴클레오솜)을 항히스톤 항체(anti-histone-antibody) 및 항DNA-POD(anti-DNA-POD)로 접합하여 ABTS로 발색시키는 photometric enzyme-immunoassay 이용하였다.Cytoplasmic histone associated-DNA-fragments mono and oligo nucleosomes produced in killed cells were treated with anti-histone antibodies and anti-DNA-PODs. A photometric enzyme-immunoassay was used to conjugate and develop with ABTS.
그 결과, 본 발명의 백삼 주근의 K-60 발효물의 에탄올 추출물은 사이토카인 유도 베타세포 사멸을 백삼 주근 추출물을 단독으로 사용한 것에 비하여 더 억제하는 효과를 보였다(표 7 참조). As a result, the ethanol extract of K-60 fermentation of white ginseng root of the present invention showed more inhibitory effect on cytokine-induced beta cell death compared to white ginseng root extract alone (see Table 7).
본 발명의 백삼 주근의 K-60 발효물의 부탄올 추출물은 사이토카인 유도 베타세포 사멸을 주근 추출물을 단독으로 사용한 것에 비하여 더 억제하는 효과를 보였다(표 8 참조).Butanol extract of K-60 fermentation of white ginseng root of the present invention showed more inhibitory effect on cytokine-induced beta cell death as compared with the use of the root extract alone (see Table 8).
본 발명의 미삼의 K-60 발효물의 에탄올 추출물은 사이토카인 유도 베타세포 사멸을 백삼 주근 추출물을 단독으로 사용한 것에 비하여 더 억제하는 효과를 보였다(표 9 참조).Ethanol extract of the K-60 fermentation of the ginseng of the present invention showed more inhibitory effect on cytokine-induced beta cell death compared to the white ginseng fructus extract alone (see Table 9).
본 발명의 미삼의 K-60 발효물의 에탄올 추출물은 팔미틴산 유도 베타세포 사멸을 미삼 추출물을 단독으로 사용한 것에 비하여 더 억제하는 효과를 보였다(표 10 참조).Ethanol extract of the K-60 fermentation of the ginseng of the present invention showed more inhibiting effect of palmitic acid-induced beta cell death than that of the ginseng extract alone (see Table 10).
본 발명의 미삼의 K-60 발효물의 부탄올 추출물은 사이토카인 유도 베타세포 사멸을 미삼 추출물을 단독으로 사용한 것에 비하여 더 억제하는 효과를 보였다(표 11 참조).Butanol extract of the K-60 fermentation of the ginseng of the present invention showed a more inhibitory effect on cytokine-induced beta cell death as compared to using the ginseng extract alone (see Table 11).
본 발명의 미삼의 K-60 발효물의 부탄올 추출물은 팔미틴산 유도 베타세포 사멸을 미삼 추출물을 단독으로 사용한 것에 비하여 더 억제하는 효과를 보였다(표 12 참조).Butanol extract of the K-60 fermentation of the ginseng of the present invention showed a more inhibitory effect compared to palmitate-induced beta cell death compared to using the ginseng extract alone (see Table 12).
상기 결과를 종합하면, 본 발명의 백삼 주근의 K-60 발효물의 에탄올 추출물은 흰쥐 췌장소도에서의 포도당 자극에 의한 인슐린 분비를 증가시키며, 사이토카인 유도 베타세포 사멸을 억제하는 효과를 보였다.Taken together, the ethanol extract of the K-60 fermentation of the white ginseng root of the present invention increases the insulin secretion by stimulating glucose in the rat pancreatic islet and inhibited cytokine-induced beta cell death.
백삼 주근의 K-60 발효물의 부탄올 추출물은 포도당 부하에 의한 혈당상승을 억제하였으며, 사이토카인 유도 베타세포 사멸을 억제하는 효과를 보였다.Butanol extract of K-60 fermentation of white ginseng root inhibited glucose rise due to glucose loading and suppressed cytokine-induced beta cell death.
미삼의 K-60 발효물의 에탄올 추출물은 흰쥐 췌장소도에서의 포도당 자극에 의한 인슐린 분비를 증가시키며, 사이토카인 유도 베타세포 사멸을 억제하는 효과를 보였으며, 팔미틴산 유도 베타세포 사멸을 억제하는 효과를 보였다. Ethanol extracts of the K-60 fermentation of M. ginseng increased insulin secretion by glucose stimulation in rat pancreatic islets, inhibited cytokine-induced beta cell death, and inhibited palmitate-induced beta cell death. .
미삼의 K-60 발효물의 부탄올 추출물은 미삼 추출물을 단독으로 사용한 것에 비하여 흰쥐 췌장소도에서의 포도당 자극에 의한 인슐린 분비를 증가시키며, 사이토카인 유도 베타세포 사멸을 억제하는 효과를 보였으며, 팔미틴산 유도 베타세포 사멸을 억제하는 효과를 보였다.Butanol extract of K-60 fermentation of M. ginseng increased insulin secretion by glucose stimulation in pancreatic islet of rats, and inhibited cytokine-induced beta cell death, compared with M. ginseng alone. It showed an effect of inhibiting cell death.
상기 결과를 토대로, 본 발명의 당항상성 개선용 조성물은 약제학적 조성물로 사용되는 경우 당항상성 유지을 유지하는 기능을 회복시킬 수 있어서, 공복혈당 장애자, 내당능 장애자, 당뇨병 전단계로 진단받은 자, 당뇨병 환자의 췌장 베타세포 보호제 및 인슐린 저항성 개선제로 사용될 수 있는 효과가 있음을 확인하였다.Based on the above results, the composition for improving glycemic improvement of the present invention can restore the function of maintaining the glycemic maintenance when used as a pharmaceutical composition, impaired fasting glucose, impaired glucose tolerance, diagnosed with prediabetes, diabetic patients It was confirmed that there is an effect that can be used as a pancreatic beta cell protectant and insulin resistance improver.
표 7. 백삼 주근의 K-60 발효물의 에탄올 추출물의 췌장베타세포에서의 사이토카인 유도 세포사멸에 미치는 영향Table 7. Effect of Ethanol Extract of K-60 Fermentation of White Ginseng Root on Cytokine-induced Apoptosis in Pancreatic Beta Cells
*Values are mean± SEM (n=6).Values are mean ± SEM (n = 6).
**백삼 주근의 K-60 발효물의 에탄올 추출물을 나타낸다.** Represents the ethanol extract of K-60 fermentation of white ginseng root.
표 8. 백삼 주근의 유산균 K-60 발효물의 부탄올 추출물의 췌장베타세포에서의 사이토카인 유도 세포사멸에 미치는 영향Table 8. Effect of Butanol Extract of Lactic Acid Bacteria K-60 Fermentation of Panax ginseng CA Meyer on Cytokine-induced Apoptosis in Pancreatic Beta Cells
White ginseng root
K-60 fermented product **
*Values are mean± SEM (n=6).Values are mean ± SEM (n = 6).
**백삼 주근의 유산균 K-60 발효물의 부탄올 추출물** Butanol Extract of Lactic Acid Bacteria K-60 Fermentation of White Ginseng Root
표 9. 미삼의 유산균 K-60 발효물의 에탄올 추출물의 췌장베타세포에서의 사이토카인 유도 세포사멸에 미치는 영향Table 9. Effect of Ethanol Extract of Lactic Acid Bacteria K-60 Fermentation of Panax ginseng on Cytokine-induced Apoptosis in Pancreatic Beta Cells
Misam
K-60 fermented product **
*Values are mean± SEM (n=6).Values are mean ± SEM (n = 6).
**미삼의 K-60 발효물의 에탄올 추출물을 나타낸다.** Represents the ethanol extract of the ginseng K-60 fermentation.
표 10. 미삼의 유산균 K-60 발효물의 에탄올 추출물의 췌장베타세포에서의 팔미틴산 유도 세포사멸에 미치는 영향Table 10. Effect of Ethanol Extract of Lactic Acid Bacteria K-60 Fermentation of Panax ginseng on Palmitate-induced Apoptosis in Pancreatic Beta Cells
Misam
K-60 fermented product **
*Values are mean± SEM (n=6).Values are mean ± SEM (n = 6).
**미삼의 K-60 발효물의 에탄올 추출물을 나타낸다.** Represents the ethanol extract of the ginseng K-60 fermentation.
표 11. 미삼의 유산균 K-60 발효물의 부탄올 추출물의 췌장베타세포에서의 사이토카인 유도 세포사멸에 미치는 영향Table 11. Effect of Butanol Extract of Lactic Acid Bacteria K-60 Fermentation on Cytokine-induced Apoptosis in Pancreatic Beta Cells
Misam
K-60 fermented product **
*Values are mean± SEM (n=6).Values are mean ± SEM (n = 6).
**미삼의 K-60 발효물의 부탄올 추출물을 나타낸다.** Butanol extract of K-60 fermentation of ginseng.
표 12. 미삼의 유산균 K-60 발효물의 부탄올 추출물의 췌장베타세포에서의 팔미틴산 유도 세포사멸에 미치는 영향Table 12. Effect of Butanol Extract of Lactic Acid Bacteria K-60 Fermentation on Pancreatic Beta-induced Apoptosis in Pancreatic Beta Cells
Misam
K-60 fermented product **
*Values are mean± SEM (n=6).Values are mean ± SEM (n = 6).
**미삼의 K-60 발효물의 부탄올 추출물을 나타낸다.** Butanol extract of K-60 fermentation of ginseng.
상술한 바와 같이, 본 발명의 바람직한 실시예를 참조하여 설명하였지만 해당 기술 분야의 숙련된 당업자라면 하기의 특허청구범위에 기재된 본 발명의 사상 및 영역으로부터 벗어나지 않는 범위 내에서 본 발명을 다양하게 수정 및 변경시킬 수 있음을 이해할 수 있을 것이다. As described above, although described with reference to a preferred embodiment of the present invention, those skilled in the art will be variously modified and modified within the scope of the present invention without departing from the spirit and scope of the invention described in the claims below. It will be appreciated that it can be changed.
본 발명의 신규한 페디오코커스 펜토사시우스 케이-60 균주는 인삼 또는 인삼 추출물을 발효시킬 수 있어 인삼 발효 추출물을 얻을 수 있다.The novel Pediococcus pentosassius K-60 strain of the present invention can ferment ginseng or ginseng extract to obtain a ginseng fermented extract.
본 발명의 엔테로코커스 패칼리스 케이-60 균주(Enterococcus faecalis K-60)(KFCC11460P)는 인삼에서 생체 이용률이 낮은 진세노사이드 Rg1, Rb1, Rg1, Re 등을 진세노사이드 Rh1, Rg3, Rh2, C-K 등으로 전환 및/또는 생성시킬 수 있어 본 발명에 의한 인삼 발효 추출물이 함유된 약제학적 제제, 건강기능식품에 적용할 수 있다. Enterococcus faecalis K-60 strain (KFCC11460P) of the present invention is a ginsenoside Rg1, Rb1, Rg1, Re, etc. with low bioavailability in ginseng ginsenosides Rh1, Rg3, Rh2, CK It can be converted to and / or produced, etc. can be applied to pharmaceutical preparations, health functional food containing the ginseng fermented extract according to the present invention.
본 발명의 당항상성 개선용 인삼 발효 추출물은 약제학적 조성물로 사용되는 경우 당항상성을 유지하는 기능을 회복시킬 수 있어서, 공복혈당 장애자, 내당능 장애자, 당뇨병 전단계로 진단받은 자, 당뇨병 환자의 췌장 베타세포 보호제 및 인슐린 저항성 개선제로 사용될 수 있는 효과가 있음을 확인하였다. 또한 인슐린 저항성은 당뇨병 뿐만 아니라 대사성 증후군 환자도 가지는 것으로 일반화되어 있으므로 대사성 증후군 환자의 인슐린 저항성 개선제로도 사용될 수 있을 것이다.The ginseng fermentation extract for improving glycemic efficacy of the present invention can restore the function of maintaining glycemic efficacy when used as a pharmaceutical composition, impaired fasting glucose, impaired glucose tolerance, those diagnosed with prediabetes, pancreatic beta cells of diabetic patients It was confirmed that there is an effect that can be used as a protective agent and an insulin resistance improving agent. In addition, since insulin resistance is generalized to have metabolic syndrome patients as well as diabetes, it may be used as an insulin resistance improving agent of metabolic syndrome patients.
<110> KOREA FOOD RESEARCH INSTITUTE <120> Novel Enterococcus faecalis K-60 and extracts from ginseng-fermented products using Enterococcus faecalis K-60 for improving glucose homeostasis and manufacturing method thereof <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> A primer for sequencing of 27f <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> A primer for sequencing of 1492r <400> 2 ggctaccttg ttacgactt 19 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> A primer for swquencing of r4L <400> 3 acgggcggtg tgtacaag 18 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> A primer for sequencing of f2L <400> 4 ccagcagccg cggtaatag 19 <210> 5 <211> 599 <212> DNA <213> Enterococcus faecalis K-60 <400> 5 cgagcgcagg cggtctttta agtctaatgt gaaagccttc ggctcaaccg aagaagtgca 60 ttggaaactg ggagacttga gtgcagaaga ggacagtgga actccatgtg tagcggtgaa 120 atgcgtagat atatggaaga acaccagtgg cgaaggcggc tgtctggtct gcaactgacg 180 ctgaggctcg aaagcatggg tagcgaacag gattagatac cctggtagtc catgccgtaa 240 acgatgatta ctaagtgttg gagggtttcc gcccttcagt gctgcagcta acgcattaag 300 taatccgcct ggggagtacg accgcaaggt tgaaactcaa aagaattgac gggggcccgc 360 acaagcggtg gagcatgtgg tttaattcga agctacgcga agaaccttac caggtcttga 420 catcttctga cagtctaaga gattagaggt tcccttcggg gacagaatga caggtggtgc 480 atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgccacg agcgcaaccc 540 ttattactag ttgccagcat taatttgggc actctagtga gactgccggt gacaaaccc 599 <110> KOREA FOOD RESEARCH INSTITUTE <120> Novel Enterococcus faecalis K-60 and extracts from ginseng-fermented products using Enterococcus faecalis K-60 for improving glucose homeostasis and manufacturing method <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> A primer for sequencing of 27f <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> A primer for sequencing of 1492r <400> 2 ggctaccttg ttacgactt 19 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> A primer for swquencing of r4L <400> 3 acgggcggtg tgtacaag 18 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> A primer for sequencing of f2L <400> 4 ccagcagccg cggtaatag 19 <210> 5 <211> 599 <212> DNA <213> Enterococcus faecalis K-60 <400> 5 cgagcgcagg cggtctttta agtctaatgt gaaagccttc ggctcaaccg aagaagtgca 60 ttggaaactg ggagacttga gtgcagaaga ggacagtgga actccatgtg tagcggtgaa 120 atgcgtagat atatggaaga acaccagtgg cgaaggcggc tgtctggtct gcaactgacg 180 ctgaggctcg aaagcatggg tagcgaacag gattagatac cctggtagtc catgccgtaa 240 acgatgatta ctaagtgttg gagggtttcc gcccttcagt gctgcagcta acgcattaag 300 taatccgcct ggggagtacg accgcaaggt tgaaactcaa aagaattgac gggggcccgc 360 acaagcggtg gagcatgtgg tttaattcga agctacgcga agaaccttac caggtcttga 420 catcttctga cagtctaaga gattagaggt tcccttcggg gacagaatga caggtggtgc 480 atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgccacg agcgcaaccc 540 ttattactag ttgccagcat taatttgggc actctagtga gactgccggt gacaaaccc 599
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| KR101418748B1 (en) * | 2012-04-30 | 2014-07-11 | 충남대학교산학협력단 | A composition comprising extract of fermented ginseng using Enterococcus faecalis CRNB-A3 for treating or preventing atopic dermatitis |
| WO2019045263A1 (en) * | 2017-08-28 | 2019-03-07 | 주식회사 쎌바이오텍 | Antibacterial lactopad composition |
| WO2020093237A1 (en) * | 2018-11-06 | 2020-05-14 | 中国科学院动物研究所 | Use of enterococcus faecalis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101418748B1 (en) * | 2012-04-30 | 2014-07-11 | 충남대학교산학협력단 | A composition comprising extract of fermented ginseng using Enterococcus faecalis CRNB-A3 for treating or preventing atopic dermatitis |
| WO2019045263A1 (en) * | 2017-08-28 | 2019-03-07 | 주식회사 쎌바이오텍 | Antibacterial lactopad composition |
| WO2020093237A1 (en) * | 2018-11-06 | 2020-05-14 | 中国科学院动物研究所 | Use of enterococcus faecalis |
| CN113164530A (en) * | 2018-11-06 | 2021-07-23 | 中国科学院动物研究所 | Application of enterococcus faecalis |
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