KR101149045B1 - Novel Pediococcus pentosaceus K-643 and extracts from ginseng-fermented products using Pediococcus pentosaceus K-643 for improving glucose homeostasis and manufacturing method thereof - Google Patents
Novel Pediococcus pentosaceus K-643 and extracts from ginseng-fermented products using Pediococcus pentosaceus K-643 for improving glucose homeostasis and manufacturing method thereof Download PDFInfo
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- KR101149045B1 KR101149045B1 KR1020090109950A KR20090109950A KR101149045B1 KR 101149045 B1 KR101149045 B1 KR 101149045B1 KR 1020090109950 A KR1020090109950 A KR 1020090109950A KR 20090109950 A KR20090109950 A KR 20090109950A KR 101149045 B1 KR101149045 B1 KR 101149045B1
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- ginseng
- extract
- fermentation
- fermented
- pediococcus pentosaceus
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Abstract
본 발명은 신규한 페디오코커스 펜토사시우스 케이-643 균주(Pediococcus pentosaceus K-643)(KFCC11464P)(이하 K-643로 약칭함), 상기 균주를 이용한 인삼 발효 추출물 및 이의 제조방법에 관한 것으로서 보다 상세하게는 인삼을 발효시킬 수 있는 신규한 K-643 및 상기 균주로 인삼을 발효시켜 포도당 항상성 개선에 적용할 수 있는 인삼 발효 추출물 및 이의 제조방법에 관한 것이다.The present invention relates to a novel Pediococcus pentosaceus K-643 strain (Pediococcus pentosaceus K-643) (KFCC11464P) (hereinafter abbreviated as K-643), a ginseng fermentation extract using the strain and a method for preparing the same. In detail, the present invention relates to a ginseng fermented extract which can be applied to the improvement of glucose homeostasis by fermenting ginseng with the novel K-643 capable of fermenting ginseng and the strain and a method for preparing the same.
Description
본 발명은 신규한 페디오코커스 펜토사시우스 케이-643 균주(Pediococcus pentosaceus K-643)(KFCC11464P)(이하 K-643로 약칭함), 상기 균주를 이용한 인삼 발효 추출물 및 이의 제조방법에 관한 것으로서 보다 상세하게는 인삼을 발효시킬 수 있는 신규한 K-643 및 상기 균주로 인삼을 발효시켜 포도당 항상성 개선에 적용할 수 있는 인삼 발효 추출물 및 이의 제조방법에 관한 것이다.The present invention relates to a novel Pediococcus pentosaceus K-643 strain (Pediococcus pentosaceus K-643) (KFCC11464P) (hereinafter abbreviated as K-643), a ginseng fermentation extract using the strain and a method for preparing the same. In detail, the present invention relates to a ginseng fermented extract which can be applied to the improvement of glucose homeostasis by fermenting ginseng with the novel K-643 capable of fermenting ginseng and the strain and a method for preparing the same.
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 efficacy (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 진세노사이드는 인삼 건조량의 약 5％를 차지하며, 트리터페노이드(triterpenoid) 계열의 다마란(dammarane) 골격에 포도당(glucose), 아라비노오스(arabinose), 자일로오스(xylose), 람노오스(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 glucose, arabinose, xylose, 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, meaning saponin in ginseng, accounts for about 5% of ginseng dry weight, and contains glucose, arabinose, and xyl on the triterpenoid dammarane skeleton. It is a neutral glycoside that is combined 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 are bound to -OH at the R1, R2 and R3 positions of 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-K로 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 are converted into 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 존재하는데 공복시에는 간, 근육 및 지방에 저장된 포도당이 다시 혈액으로 방출되고 식사를 하면 약 2～3시간 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, exists as blood sugar in the body. When fasting, glucose stored in the liver, muscles, and fats is released into the blood again. After eating, a large amount of glucose absorbed from the gastrointestinal tract for about 2 to 3 hours 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 maintained at 80-120 mg / dL, which is called glucose homeostasis (abbreviated as 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 신규한 페디오코커스 펜토사시우스 케이-643 균주(Pediococcus pentosaceus K-643)(KFCC11464P)(이하 K-643으로 약칭함)를 인체 분변으로부터 선별한 후 균주를 분자 유전학적 측면에서 동정하는 16S rRNA partial sequencing을 통해 균주의 종명, 속명을 확인하여, 한국미생물보존센터에 기탁하였다. Accordingly, the present inventors have identified a novel novel Pediococcus pentosaceus K-643 strain (KFCC11464P) (hereinafter abbreviated as K-643), which is a lactic acid bacterium that complexes ginsenosides. After screening from the strain, the strain was identified by the 16S rRNA partial sequencing in terms of molecular genetics, and the species name and genus were identified and deposited in Korea Microbial Conservation Center.
상기 K-643을 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-643, 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-643의 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-643 capable of producing and / or converting ginsenosides Rg1, Rb1, Rg1, Re and the like to ginsenosides Rh1, Rg3, Rh2, CK and the like with 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-643로 발효시킴으로써 당항상성 개선 활성이 증가한다는 것을 발견하고, 본 발명을 완성하게 되었다. 따라서, 본 발명의 또다른 목적은 인삼을 K-643로 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-643, thereby completing the present invention. Therefore, another object of the present invention is to provide a ginseng fermentation extract for improving sugar resistance by fermenting ginseng with K-643.
상기와 같은 목적을 달성하기 위해, 본 발명은 K-643을 제공하며, 또한 K-643로 발효시켜 제조된 당항상성 개선용 인삼 발효 추출물을 제공한다. In order to achieve the above object, the present invention provides K-643, and also provides a ginseng fermentation extract for improving sugar resistance prepared by fermentation with K-643.
또한 본 발명은 (1)인삼을 K-643로 발효시켜 발효물을 얻는 단계; 및 In addition, the present invention (1) the step of fermenting ginseng K-643 to obtain a fermented 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 페디오코커스 펜토사시우스 케이-643 균주(Pediococcus pentosaceus K-643)(KFCC11464P)를 제공할 수 있다.The present invention can provide a novel Pediococcus pentosaceus K-643 strain (KFCC11464P) capable of fermenting ginseng.
본 발명은 상기의 신규한 K-643을 이용하여 인삼에 함유되어 있으며 생체 이용률이 낮은 진세노사이드 Rg1, Rb1, Rg1, Re 등을 진세노사이드 Rh1, Rg3, Rh2, C-K 등으로 전환시킬 수 있는 인삼 발효 추출물의 제조방법을 제공할 수 있다.The present invention is capable of converting ginsenosides Rg1, Rb1, Rg1, Re and the like contained in ginseng using the novel K-643 to ginsenosides Rh1, Rg3, Rh2, CK and the like. It can provide a method for producing ginseng fermented extract.
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 페디오코커스 펜토사시우스 케이-643 균주(Pediococcus pentosaceus K-643)(KFCC11464P)(이하 K-643로 약칭한다)를 나타낸다.The present invention represents a novel Pediococcus pentosaceus K-643 strain (Pediococcus pentosaceus K-643) (KFCC11464P) (hereinafter abbreviated as K-643).
본 발명은 인삼을 발효시킬 수 있는 신규한 K-643을 나타낸다.The present invention represents a novel K-643 capable of fermenting ginseng.
본 발명은 인삼을 K-643로 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-643.
본 발명은 (1)인삼을 K-643로 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-643 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-643은 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 페디오코커스 펜토사시우스의 스트레인 CTSPL1의 16S rRNA gene과 100％ 상동성을 가짐을 알게되어 페디오코커스 펜토사시우스 케이-643(Pediococcus pentosaceus K-643)로 명명하고 이를 미생물보존센터에 2009년 9월 17일 기탁하여 KFCC11464P의 미생물 기탁번호를 부여 받았다.The novel K-643 of the present invention receives a fecal from the human body and isolates 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. 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. The strain was found to have 100% homology with 16S rRNA gene of strain CTSPL1 of Pediococcus pentosassius. It was named Pediococcus pentosaceus K-643 and deposited it on the microorganism preservation center on September 17, 2009 and was given the microbial deposit number of KFCC11464P.
본 발명의 신규한 K-643은 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, 인삼을 K-643을 이용하여 발효시 당항상성이 증진되도록 K-643 발효과정 모니터링의 지표 성분들로서 Rh1, Rg3, C-K 및 Rh2를 설정하였다. The novel K-643 of the present invention is capable of growing in high concentration ginseng medium, while metabolizing the highly polar ginsenosides Rg1 and Rb1 into the less polar ginsenosides Rh1, Rg3, C-K and Rh2. In the present invention, the ginseng is not fermented with the aim of merely increasing the contents of Rh1, Rg3, CK and Rh2, and Rh1 as an indicator component of K-643 fermentation process monitoring so that sugar always improves during fermentation using K-643. , Rg3, CK and Rh2 were set.
본 발명에서는 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 페디오코커스 펜토사시우스 케이-643 균주로 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 present invention, the ginseng was dried to prepare 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, steamed white ginseng and processed into red ginseng and ginseng and then fermented with Pediococcus pentosassius K-643 strain 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, the organic solvent may be ethanol or alcohol of 50 to 95% concentration.
본 발명의 당항상성 개선용 인삼 발효 추출물의 제조의 일예로서 (1)인삼 현탁액을 K-643로 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-643 to obtain a fermentation product, and (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-643을 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-643 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 추출조성물은 기존의 인삼 발효물에 비하여 진세노사이드 Rh1(프로토파나사트리올; protopanaxatriol)과 Rh2(프로토파나사다이올; protopanaxadiol)를 동시에 많이 함유한다는 특징이 있으며, 이는 기존의 인삼 발효물 특허에서 진일보한 것이다.Extract composition for improving sugar compatibility of the present invention is characterized in that it contains more ginsenoside Rh1 (protopanaxatriol) and Rh2 (protopanaxadiol) than conventional ginseng fermentation, which This is a step further from the existing ginseng fermentation patent.
본 발명의 당항상성 개선용 추출조성물은 흰쥐의 당부하검사에서 당부하 후 2시간 동안의 혈당 상승량을 측정하였던 바, 인삼을 발효시킨 시험군이 인삼을 발효시키지 않은 시험군 보다 낮추는 효과가 있었다.The extract composition for improving glycemic efficacy of the present invention was measured in the blood glucose test for 2 hours after the glucose load in the glucose load test of the rat, the test group fermented ginseng was lower than the test group not fermented ginseng.
혈당치가 상승하면 췌장 소도의 베타세포로부터 인슐린이 분비되어 혈당이 제거된다. 그러나 여러 병인에 의하여 혈당치가 높은데도 불구하고 적절한 인슐린이 분비되지 못하면 당항상성의 균형이 깨진다. 본 발명의 당항상성 개선용 추출조성물은 흰쥐에서 분리한 췌장 소도(pancreatic islets)에서 인슐린의 분비를 촉진시킨다.When blood glucose levels rise, insulin is secreted from the beta cells of pancreatic islets to remove blood sugar. However, even if the blood sugar is high due to various etiologies, proper insulin is not secreted, the balance of sugar always breaks. 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 추출조성물은 당항상성을 개선함으로써 베타세포 이상, 내당능장애(glucose intolerance), 공복혈당장애, 당뇨병 전단계(prediabetes), 당뇨병을 예방 및 치료할 수 있다.In addition, the extract composition of the present invention can prevent and treat beta-cell abnormality, glucose intolerance, fasting glucose disorders, prediabetes, and diabetes by improving glycemic condition.
본 발명은 상기의 신규한 K-643을 이용한 인삼 발효 추출물의 제조방법을 나타낸다.The present invention shows a method for producing a ginseng fermented extract using the novel K-643.
본 발명은 상기의 신규한 K-643을 이용하여 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 provides a ginseng fermentation extract capable of converting ginsenosides Rg1, Rb1, Rg1, Re and the like into ginsenosides Rh1, Rg3, Rh2, CK, etc. using the novel K-643. The method is shown.
본 발명은 상기의 신규한 K-643을 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-643.
본 발명은 (1)인삼을 K-643로 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 ginseng with K-643 to obtain a fermented 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-643로 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-643. 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, followed by filtration.
상기의 용매 중 에탄올은 50～99％ 농도의 에탄올을 사용할 수 있다.Ethanol in 50-99% concentration can be used for ethanol in said solvent.
본 발명의 신규한 K-643을 이용한 인삼 발효 추출물의 제조방법에 대해 조사한바, 본 발명의 목적을 달성하기 위해서는 상기에서 언급한 조건에 의해 신규한 K-643을 이용한 인삼 발효 추출물의 제조방법을 제공하는 것이 바람직하다.Investigation on the preparation method of the ginseng fermentation extract using the novel K-643 of the present invention, in order to achieve the object of the present invention, a method for preparing the ginseng fermentation extract using the novel K-643 under the above-mentioned conditions It is desirable to provide.
본 발명은 상기에서 언급한 K-643을 이용한 인삼 발효 추출물을 포함한다.The present invention includes the ginseng fermented extract using K-643 mentioned above.
본 발명은 상기에서 언급한 신규한 K-643을 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-643 mentioned above.
본 발명은 상기에서 언급한 신규한 K-643을 이용하여 제조한 인삼 발효 추출물을 포함하는 당항상성 유지 또는 개선용 약학제 제제를 나타낸다.The present invention represents a pharmaceutical formulation for maintaining or improving glycation always including a ginseng fermented extract prepared using the novel K-643 mentioned above.
본 발명은 상기에서 언급한 신규한 K-643을 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-643 mentioned above.
본 발명은 상기에서 언급한 신규한 K-643을 이용하여 제조한 인삼 발효 추출물을 포함하는 당항상성 유지 또는 개선용 건강기능식품을 나타낸다.The present invention represents a health functional food for maintenance or improvement of sugar always including the ginseng fermented extract prepared using the novel K-643 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> ≪ Example 1 >
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 표적으로 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 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. 1492r (5'-ggctaccttgttacgactt-3 '] was prepared and PCR to obtain a PCR product having a size 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 Pediococcus pentosaceus strain CTSPL1의 16S rRNA gene과 100％ 동일함(identities)을 갖는 것을 알 수 있어 상기 균주를 페디오코커스 펜토사시우스 케이-643 균주(Pediococcus pentosaceus K-643)로 명명하고 이를 미생물보존센터에 2009년 11월 17일 KFCC11464P로 기탁하였다.A blast search of the product of the strain revealed that it had 100% identity with the 16S rRNA gene of Pediococcus pentosaceus strain CTSPL1, and the strain was identified as Pediococcus pentosassius K-643 strain. (Pediococcus pentosaceus K-643) and it was deposited with KFCC11464P on November 17, 2009 to the Center for Microbiological Conservation.
한편 K-643의 16s DNA 여기서열을 염기서열목록 5에 나타내었다.Meanwhile, the sequence of 16s DNA of K-643 is shown in SEQ ID NO: 5.
<실시예 2> <Example 2>
상기 실시예 1의 페디오코커스 펜토사시우스 케이-643 균주(Pediococcus pentosaceus K-643)의 균학적 성질과 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. 이들 균주는 그람양성, 구균이며, 산소유무와 상관없이 잘 생장하였고, 카탈라아제는 양성이었다.The bacteriological properties and microbial identification of microorganisms of Pediococcus pentosaceus K-643 strain of Example 1 were identified by Hammes et al. [The prokaryotes, 1563-1578, 2nd Edition, Springer-Verlag Co. . (1992)], and the results are shown in Table 1. These strains were Gram-positive, cocci, well grown with or without oxygen, and catalase was positive.
표 1. 신규 K-643의 균학적 성질Table 1. Mycological Properties of New K-643
<제조예> 인삼 분말 제조Preparation Example Ginseng Powder Preparation
인삼은 안성산 5년근을 잘 건조하여 백삼 주근과 백삼 미근(미삼)으로 분리한 다음 분쇄하여 백삼주근 분말, 백삼미근 분만을 얻었다. Ginseng was dried 5 years old root in Anseong, separated into white ginseng root and white ginseng root (misam), and then pulverized to obtain white ginseng root powder and white ginseng root.
인삼은 안성산 5년근을 잘 건조하여 98℃에서 4시간 동안 쪄서 홍삼을 만든 다음 이를 건조한 후 분쇄하여 홍삼분말을 얻었다. Ginseng was well dried 5 years old Anseong acid, steamed for 4 hours at 98 ℃ to make red ginseng, then dried and ground to obtain a red ginseng powder.
인삼은 안성산 5년근을 잘 건조하여 120℃에서 2.5시간 쪄서 선삼을 만든 다음 이를 건조한 후 분쇄하여 선삼분말을 얻었다. The ginseng was dried 5 years old Anseong acid well and steamed for 2.5 hours at 120 ℃ to make ginseng, then dried and pulverized to obtain a ginseng 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 페디오코커스 펜토사시우스 케이-643 균주(Pediococcus pentosaceus K-643)(KFCC11464P) 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 and sterilized. Then, 2 g of Pediococcus pentosaceus K-643 strain (KFCC11464P) was added and fermented at 37 ° C. for 7 days. Got.
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 페디오코커스 펜토사시우스 케이-643 균주(Pediococcus pentosaceus K-643)(KFCC11464P) 2g을 넣고 37℃에서 7일간 발효시켜 발효물을 얻었다.2,000 g of distilled water was added to 20 g of white ginseng fructose powder obtained in the above preparation and sterilized. Got.
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 페디오코커스 펜토사시우스 케이-643 균주(Pediococcus pentosaceus K-643)(KFCC11464P) 2g을 넣고 37℃에서 7일간 발효시켜 발효물을 얻었다.Distilled water 2,000ml was added to 20g of the white ginseng root (misam) powder obtained in the above preparation and sterilized. Then, 2g of Pediococcus pentosaceus K-643 strain (KFCC11464P) was added and fermented 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 페디오코커스 펜토사시우스 케이-643 균주(Pediococcus pentosaceus K-643)(KFCC11464P) 2g을 넣고 37℃에서 7일간 발효시켜 발효물을 얻었다.Distilled water 2,000ml was added to 20g of the white ginseng root (misam) powder obtained in the above preparation and sterilized. Then, 2g of Pediococcus pentosaceus K-643 strain (KFCC11464P) was added and fermented 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 페디오코커스 펜토사시우스 케이-643 균주(Pediococcus pentosaceus K-643)(KFCC11464P) 2g을 넣고 37℃에서 7일간 발효시켜 발효물을 얻었다.20 g of red ginseng powder obtained in the preparation example was added 2,000 ml of distilled water and sterilized. Then, 2 g of Pediococcus pentosaceus K-643 strain (KFCC11464P) was added and fermented at 37 ° C. for 7 days. Got it.
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 페디오코커스 펜토사시우스 케이-643 균주(Pediococcus pentosaceus K-643)(KFCC11464P) 2g을 넣고 37℃에서 7일간 발효시켜 발효물을 얻었다.20 g of red ginseng powder obtained in the preparation example was added 2,000 ml of distilled water, and sterilized. Got it.
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 페디오코커스 펜토사시우스 케이-643 균주(Pediococcus pentosaceus K-643)(KFCC11464P) 2g을 넣고 37℃에서 7일간 발효시켜 발효물을 얻었다.To 20 g of the ginseng powder obtained in the preparation example, 2,000 ml of distilled water was added and sterilized. Then, 2 g of Pediococcus pentosaceus K-643 strain (KFCC11464P) was added and fermented at 37 ° C. for 7 days. Got it.
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 페디오코커스 펜토사시우스 케이-643 균주(Pediococcus pentosaceus K-643)(KFCC11464P) 2g을 넣고 37℃에서 7일간 발효시켜 발효물을 얻었다.To 20 g of the ginseng powder obtained in the preparation example, 2,000 ml of distilled water was added and sterilized. Then, 2 g of Pediococcus pentosaceus K-643 strain (KFCC11464P) was added and fermented at 37 ° C. for 7 days. Got it.
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 여과하였다.2,000 g of distilled water was added to 20 g of white ginseng root powder and sterilized, and extracted with 2,000 ml of ethanol at 70 ° C. for 3 hours and filtered.
<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 여과하였다.2,000 g of distilled water was added to 20 g of white ginseng misam powder and sterilized, and extracted with 2,000 ml of ethanol at 70 ° C. for 3 hours and filtered.
<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 여과하였다.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.
<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 여과하였다.2,000 g of distilled water was added to 20 g of the ginseng powder and sterilized, and extracted with 2,000 ml of ethanol at 70 ° C. for 3 hours and filtered.
<시험예 1> 진세노사이드 함량분석 실험Test Example 1 Ginsenoside Content Analysis Experiment
상기 실시예 3-1, 실시예 4-1과 비교예 1, 비교예 2에서 얻은 각각의 추출물에 각각의 추출물과 동량의 메탄올(methanol)을 넣고 원심분리하여 상등액을 얻었다. 이중 100ml을 감압농축하고 각각의 추출물을 각 0.2g씩 취하여 메탄올 100ml씩 3회 추출하였다. 이 추출물을 감압 농축시킨 후에 이에 물 100ml를 넣어 현탁시키고, 에테르 100ml씩으로 3회 추출하고 나머지 물분획을 다시 100ml씩으로 3회 추출하고 감압 농축시켰다. 이어서 MeOH 1ml에 용해시켜 HPLC로 분석하여 표 2와 같은 사포닌 함량 분석결과를 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. 100 ml were 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 2.
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)]
상기의 분석 결과로부터 실시예 3-1 및 실시예 4-1에서 백삼을 K-643 발효 후 추출한 백삼 주근 발효 추출물은 백삼 주근에는 함유되어 있지 않은 Rh1, Rg3, C-K, Rh2가 발효 백삼 주근에는 각각 3.8％, 2.0％, 1.1％, 11.4％ 함유되었으며, 미삼에는 함유되어 있지 않은 Rh1, Rg3, C-K, Rh2가 발효 미삼에는 각각 4.8％, 1.2％, 1.0％, 11.1％ 함유되었다. 즉, 백삼에는 함유되지 않은 성분들이 K-643 발효에 의하여 발효 백삼 중에 Rh1, Rg3, C-K, Rh2 등이 생성되었다(표 2 참조).From the above analysis results, the white ginseng root fermented extract extracted after fermentation of white ginseng in Example 3-1 and Example 4-1 was fermented to Rh1, Rg3, CK, and Rh2 which were not contained in white ginseng root. 3.8%, 2.0%, 1.1%, and 11.4% were contained, and Rh1, Rg3, CK, and Rh2, which were not contained in rice ginseng, contained 4.8%, 1.2%, 1.0%, and 11.1%, respectively. That is, Rh1, Rg3, C-K, Rh2, etc. were produced in fermented white ginseng by the K-643 fermentation, which was not contained in white ginseng (see Table 2).
표 2. 인삼의 유산균 K-643에 의한 발효가 진세노사이드 함량에 미치는 영향Table 2. Effect of Ginseng Fermentation by Lactic Acid Bacteria K-643 on Ginsenoside Content
sample
1)- 측정할 수 없었음 (not detected). 1) -not detected.
<시험예 2> 간세포에서의 당항상성 관련 기능 평가Experimental Example 2 Evaluation of Functions Related to Glucose Tolerance in Hepatocytes
간에서의 인슐린 저항성은 혈당치가 높은데도 불구하고 계속 당신생 반응에 의하여 당이 생성되며 혈당치가 간에 의하여 제거되지 못하는 것이고, 간에서의 당 항상성의 유지는 주로 효소에 대한 활성의 조절에 의해 달성되며, 글루코키나제 [glucokinase (GK)]는 간의 당 항상성 유지에 중심이 되는 효소이다. GK는 포도당을 glucose-6-phosphate (G-6-P)로 인산화시켜 해당작용(glycolysis)시키거나 글리코겐(glycogen)을 합성시켜서 혈당치를 낮춘다. Cytokine의 일종인 종양괴사인자 알파[tumor necrosis factor-α(TNF-α)]는 GK 활성을 낮춤. 인삼 발효물이 간에서의 인슐린 저항성 관련 기능에 미치는 영향을 평가하기 위하여 간세포에 TNF-α로 인슐린 저항성을 유발시킨 후 GK 활성에 미치는 영향 을 검증하였다.Insulin resistance in the liver is that sugar is produced by your response even though the blood sugar level is high, and the blood sugar level cannot be removed by the liver.Maintenance of sugar homeostasis in the liver is mainly achieved by regulation of enzyme activity. Glucokinase (GK) is an enzyme that is central to the maintenance of sugar homeostasis in the liver. GK phosphorylates glucose into glucose-6-phosphate (G-6-P) to either glycolyse or synthesize glycogen to lower blood sugar levels. Tumor necrosis factor-α (TNF-α), a type of cytokine, lowers GK activity. In order to evaluate the effect of ginseng fermentation on insulin resistance-related function in liver, the effect of TNF-α on insulin resistance in liver cells was examined.
-세포주 : 인간 원발성 간암 세포주(Human hepatocellular carcinomia cell line)인 HepG2 세포Cell line: HepG2 cell, a human primary hepatocellular carcinomia cell line
- 인슐린 저항성 유도 조건 : 세포가 자라서 세포 배양 용기에 90％ 정도 꽉 차면 종양괴사인자 알파 10ng/mL을 배지에 첨가하고, 24시간 동안 배양하였다. Insulin resistance induction condition: When cells grow and are 90% full in the cell culture vessel, tumor necrosis factor alpha 10ng / mL was added to the medium, and cultured for 24 hours.
- 인삼 발효 추출물의 첨가 방법: TNF-α와 동시에 첨가하였다.Addition method of ginseng fermented extract: It was added simultaneously with TNF-α.
- GK 활성 측정: 포도당이 GK에 의하여 G-6-P로 전환되어 G-6-P 디하이드로게나아제에 의하여 NAD가 NADH로 환원되는 원리를 이용하였다.Determination of GK activity: The principle is that glucose is converted to G-6-P by GK and that NAD is reduced to NADH by G-6-P dehydrogenase.
그 결과 본 발명의 백삼 주근의 K-643 발효물의 부탄올 추출물은 GK의 활성을 백삼주근에 비하여 33％ 증가시켰으며, 이를 표 3에 나타내었다.As a result, butanol extract of the K-643 fermentation of white ginseng root of the present invention increased the GK activity by 33% compared to white ginseng root, which is shown in Table 3.
표 3. 백삼 주근의 유산균 K-643 발효물의 부탄올 추출물이 간세포 글루코키나제 활성에 미치는 영향Table 3. Effect of Butanol Extract of Lactic Acid Bacteria K-643 Fermentation of White Ginseng Root on Hepatocellular Glucokinase Activity
(fold of TNF-α treated only)Glucokinase Activity *
(fold of TNF-α treated only)
(TNF-α-treated control)TN added control
(TNF-α-treated control)
*Values are mean± SEM (n=6).Values are mean ± SEM (n = 6).
*Significantly different from control group at *P<0.05.* Significantly different from control group at * P <0.05.
** 실시예 3-2에서 백삼 주근을 K-643 균주로 발효시킨 후 부탄올로 추출한 추출물이다.** Example 3-2 is an extract extracted with butanol after fermenting the white ginseng root with K-643 strain.
<시험예 3> 지방세포에서의 당항상성 관련 기능 평가Experimental Example 3 Evaluation of Functions Related to Glucose Tolerance in Adipocytes
- 실험재료 - Materials
Insulin, Dexamethasone, IBMX (이상 Sigma)Insulin, Dexamethasone, IBMX (Sigma or higher)
phospho-AMPK, ACC antibody, beta actin antibody (Cell signaling)phospho-AMPK, ACC antibody, beta actin antibody (Cell signaling)
- 지방세포 분화Adipocyte differentiation
3T3-L1-preadipocyte cell은 12-well plate에 seeding하였다. 이틀 후에 adipocyte differentiation 은 hormone cocktail containing 1M dexamethasone, 5g/mL insulin, and 0.5mM IBMX과 함께 유도하였다. 이틀 후에, medium 은 normal medium containing insulin (5g/mL)으로 바꿔주었고 약 6일 후 80％이상의 지방 세포로의 전환을 관찰하였다.3T3-L1-preadipocyte cells were seeded on a 12-well plate. Two days later adipocyte differentiation was induced with hormone cocktail containing 1M dexamethasone, 5g / mL insulin, and 0.5mM IBMX. After two days, the medium was changed to normal medium containing insulin (5 g / mL) and after about 6 days conversion to more than 80% fat cells was observed.
- Glucose uptake assayGlucose uptake assay
지방전구세포인 3T3-Ll에 호르몬을 처리하여 지방세포로 분화시킨 후, PBS로 세척하고 시료 물질과 0.1％ BSA가 첨가된 DMEM 배지를 넣고 12시간 전배양하였다. 배지를 제거하고 시료와 인슐린이 첨가된 Krebs-Ringer-Hepes (KRP) 완충액으로 교환하여 37℃에서 30분간 배양하였다. 이 후, 0.1 μCi 2-deoxy-D-[3H] glucose와 1mM glucose를 첨가하고 10분간 더 배양한 후, scintillation counter로 radioactivity를 측정함으로 세포내로 유입된 포도당 양을 측정하였다. Hormones were treated with adipocytes, 3T3-Ll, to differentiate into adipocytes, washed with PBS, and added to DMEM medium containing 0.1% BSA and pre-cultured for 12 hours. The medium was removed, exchanged with the sample and Krebs-Ringer-Hepes (KRP) buffer to which insulin was added, and incubated at 37 ° C. for 30 minutes. Thereafter, 0.1 μCi 2-deoxy-D- [ 3 H] glucose and 1 mM glucose were added thereto, followed by further incubation for 10 minutes, and then the amount of glucose introduced into the cells was measured by measuring radioactivity with a scintillation counter.
- PPAR-gamma assayPPAR-gamma assay
HEK293 cell을 10％ FBS가 든 DME 배지에 부유시키고 28-well plate에 분주하여 60-70％ confluent 할 때까지 배양하였다. SuperFect(Qiagen)를 이용하여 PPRE reporter plasmid와 PPAR-γ plasmid를 co-transfection시켜 3시간 후 동일한 배지로 교환한 후 24시간 더 배양하고 시료를 사용할 경우, 세포를 lysis하기 3시간 전에 시료를 처리하였다. Well 당 Passive lysis buffer 100㎕를 첨가하여 세포를 lysis시키고, Luciferase assay system(Promega)으로 luciferase 발현 정도를 조사하였다. 이때 β-gal plasmid를 transfection의 internal standard로 사용하였다. 먼저 분화를 유도하고 6일 후 PPRE reporter plasmid를 transfection시켰다. 3시간 후에 시료를 4시간 처리하였다. HEK293 세포와 같은 방법으로 luciferase assay를 실시하였다. HEK293 cells were suspended in DME medium containing 10% FBS, aliquoted in 28-well plates and incubated until 60-70% confluent. After co-transfection of PPRE reporter plasmid and PPAR-γ plasmid using SuperFect (Qiagen), they were exchanged with the same medium after 3 hours, and further incubated for 24 hours, and samples were treated 3 hours before cell lysis. . Cells were lysed by adding 100 μl of passive lysis buffer per well, and luciferase expression level was examined by Luciferase assay system (Promega). At this time, β-gal plasmid was used as an internal standard of transfection. First, differentiation was induced and PPRE reporter plasmid was transfected 6 days later. After 3 hours the sample was treated for 4 hours. Luciferase assay was performed in the same manner as HEK293 cells.
- AMPK 웨스턴블롯팅-AMPK Western Blotting
12-well plate에 seeding된 3T3-L1 지방세포에 각각의 시료를 직접 100ug/ml의 농도로 3시간 처리 하였다. 3시간 후에 Media를 버린 후 Ice-cold phosphate- buffered saline (PBS)에 두 번 세척하였다, 그 후 lysis buffer (50 Tris??HCl [pH 7.4], 1％ NP-40, 0.25％ sodium deoxycholate, 150NaCl, 1EDTA, 1PMSF, 1sodium orthovanadate, 1NaF, 1g/mL aprotinin, 1g/mL leupeptin, and 1g/mL pepstatin)를 이용하여 세포를 모은 후 SDS-PAGE로 단백질을 분리, 겔을 nitrocellulose paper에 transfer한 후 AMPK, AMPK substrate ACC의 인산화 및 β-actin 단백질 발현을 western blot으로 확인하였다. Each sample was directly treated with 3 T3-L1 adipocytes seeded in a 12-well plate at a concentration of 100 ug / ml for 3 hours. After 3 hours, the media was discarded and washed twice with Ice-cold phosphate-buffered saline (PBS), and then lysis buffer (50 Tris ?? HCl [pH 7.4], 1% NP-40, 0.25% sodium deoxycholate, 150NaCl). , 1EDTA, 1PMSF, 1sodium orthovanadate, 1NaF, 1g / mL aprotinin, 1g / mL leupeptin, and 1g / mL pepstatin) to collect the cells, isolate proteins with SDS-PAGE, transfer gel to nitrocellulose paper, and then use AMPK , AMPK substrate ACC phosphorylation and β-actin protein expression was confirmed by western blot.
그 결과 본 발명의 백삼 주근의 K-643 발효물의 에탄올 추출물은 발효시키지 않은 백삼 주근 추출물에 비하여 당흡수가 증진되었으며 이를 도 1에 나타내었다. As a result, the ethanol extract of the fermented K-643 of white ginseng root of the present invention was improved in glucose uptake compared to the white ginseng root extract which was not fermented.
본 발명의 백삼 주근의 K-643 발효물의 에탄올 추출물은 발효시키지 않은 백삼 주근 추출물에 비하여 PPARγ 활성을 증진시켰으며 이를 도 2에 나타내었다. Ethanol extract of K-643 fermentation of white ginseng root of the present invention enhanced PPARγ activity compared to white ginseng root extract which was not fermented and is shown in FIG. 2.
본 발명의 백삼 주근의 K-643 발효물의 에탄올 추출물은 발효시키지 않은 백삼 주근 추출물에 비하여 인산화된 ACC 및 인산화된 AMPK의 발현을 증진시켰으며 이를 도 3에 나타내었다. Ethanol extract of K-643 fermentation of white ginseng root of the present invention enhanced the expression of phosphorylated ACC and phosphorylated AMPK compared to white ginseng root extract which was not fermented, and is shown in FIG. 3.
<시험예 4> 췌장 소도에서의 인슐린 분비에 미치는 영향Test Example 4 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 이용함. 떼어낸 췌장은 피콜 (ficoll) 경사를 이용하여 소도를 분리한다. 소도를 크렙스 링거 이탄화 버퍼(KRB; Krebs-Ringer bicarbonate buffer, pH 7.2)에 담아 37℃, 5％ CO2 배양기에서 1일간 배양 후 사용한다.Isolation of pancreatic islets from Sprague-Dawley rats was by direct injection of collagenase into the bile duct. The detached pancreas is isolated using a ficoll gradient. Sodium is contained in Krebs-Ringer bicarbonate buffer (KRB, pH 7.2) and used after incubation in a 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 측정하고, 인슐린 농도의 측정은 효소면역방법(EIA; enzyme immunoassay) 이용한 키트를 사용한다. Pancreatic islets incubated for 24 hours were washed twice with KRB, and then three of them were fixed in size per well. The secretion ability according to the glucose concentration, the secretion ability according to the culture time, and the insulin secretion ability by the bioconverter were measured. The measurement is performed using a kit using enzyme immunoassay (EIA).
그 결과, 본 발명의 백삼 주근의 K-643 발효물의 에탄올 추출물은 인삼 추출물을 단독으로 사용한 것에 비하여 배지 농도 3.3mM에서의 (기저) 인슐린 분비에는 큰 영향주지 않았으나, 배지 농도 16.7mM에서의 인슐린 분비 (포도당 자극)는 인삼 추출물을 단독으로 사용한 것에 비하여 크게 증가시켰으며 이를 표 4에 나타내었다. As a result, the ethanol extract of the K-643 fermentation of white ginseng root of the present invention did not have a significant effect on the (base) insulin secretion at the medium concentration of 3.3 mM, but the insulin secretion at the medium concentration of 16.7 mM compared with the ginseng extract alone. (Glucose stimulation) was significantly increased compared to the ginseng extract alone was shown in Table 4.
백삼 주근의 K-643 발효물의 부탄올 추출물은 인삼 추출물을 단독으로 사용한 것에 비하여 기저 및 포도당 자극 인슐린 분비는 증가시켰으며 이를 표 5에 나타내었다. Butanol extract of K-643 fermentation of white ginseng root increased basal and glucose-stimulated insulin secretion compared to ginseng extract alone and is shown in Table 5.
백삼 미삼의 K-643 발효물의 부탄올 추출물은 인삼 추출물을 사용한 것에 비하여 기저 인슐린 분비에는 영향이 없었으나 포도당 자극 인슐린 분비는 증가시켰으며 이를 표 6에 나타내었다. Butanol extract of K-643 fermentation of white ginseng did not affect basal insulin secretion compared to ginseng extract but increased glucose-stimulated insulin secretion and are shown in Table 6.
즉 미삼의 K-643 발효물의 부탄올 추출물은 그 자체만으로는 인슐린을 분비시키지 않았으나 포도당에 의한 인슐린 분비는 증폭시키는 효과를 보였다.In other words, butanol extract of K-643 fermentation of misam did not secrete insulin by itself, but amplified insulin secretion by glucose.
표 4. 백삼 주근의 유산균 K-643 발효물의 에탄올 추출물의 췌장 소도에서의 인슐린 분비 효과Table 4. Insulin Secretion Effects of Ethanol Extracts of Lactic Acid Bacteria K-643 Fermentation of Panax ginseng CA Meyer
Item
White ginseng root
K-643 Fermentation Material **
*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.
**실시예 3-1에서 백삼 주근을 K-643 균주로 발효시킨 후 에탄올로 추출한 추출물이다. ** Example 3-1 is an extract extracted with ethanol after fermenting the white ginseng root with K-643 strain.
표 5. 백삼 주근의 유산균 K-643 발효물의 부탄올 추출물이 췌장 소도에서의 인슐린 분비에 미치는 영향Table 5. Effect of Butanol Extract of Lactic Acid Bacteria K-643 Fermentation of Panax ginseng Root on Insulin Secretion in Pancreatic Islets
Item
*Values are mean± SEM (n=6).Values are mean ± SEM (n = 6).
*Significantly different from control group at ***P<0.001.* Significantly different from control group at *** P <0.001.
실시예 3-2에서 백삼 주근을 K-643 균주로 발효시킨 후 부탄올로 추출한 추출물이다.In Example 3-2, the white ginseng root was fermented with K-643 strain and then extracted with butanol.
표 6. 미삼의 유산균 K-643 발효물의 부탄올 추출물이 췌장 소도에서 인슐린 분비에 미치는 영향Table 6. Effect of Butanol Extract of Lactic Acid Bacteria K-643 Fermentation of Panax ginseng on Insulin Secretion in Pancreatic Islets
Item
K-643 Fermentation Material **
*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.
**실시예 4-2에서 백삼 미삼을 K-643 균주로 발효시킨 후 부탄올로 추출한 추출물이다.** In Example 4-2, white ginseng is an extract extracted with butanol after fermentation with K-643 strain.
<시험예 5> 췌장 베타세포 사멸에 미치는 영향Test Example 5 Effect on Pancreatic Beta-cell Death
- 세포주 : MIN6N8 세포주 Cell line: MIN6N8 cell line
- 세포사멸 유도 조건-Apoptosis induction condition
ㆍ사이토카인에 의한 유도 : 인터페론-γ(IFN-γ10 ng/mL), 종양괴사인자(TNF)-α(10 ng/mL), 인터루이킨-1β(10 ng/mL)의 혼합물을 48시간 배지에 첨가함. 본 발명의 인삼 발효 추출물인 백삼 주근의 K-643 발효물의 에탄올 추출물, 백삼 미삼의 K-643 발효물의 에탄올 추출물을 사이토카인과 동시에 처리하였다.Induction by cytokines: A mixture of interferon-γ (IFN-
ㆍ유리지방산에 의한 유도 : 팔미트산 50mM을 50％ 에탄올에 용해하여 0.5mM 이 되도록하고, 48시간 후 배지에 첨가하였다. 본 발명의 인삼 발효 추출물인 백삼 주근의 K-643 발효물의 에탄올 추출물, 백삼 주근의 K-643 발효물의 부탄올 추출물, 백삼 미삼의 K-643 발효물의 에탄올 추출물, 백삼 미삼의 K-643 발효물의 부탄올 추출물을 팔미트산과 동시에 처리하였다.Induction by free fatty acid: 50 mM of palmitic acid was dissolved in 50% ethanol to 0.5 mM and added to the medium after 48 hours. Ethanol extract of K-643 fermentation of white ginseng root, ginseng fermentation extract of the present invention, butanol extract of K-643 fermentation of white ginseng root, ethanol extract of K-643 fermentation of white ginseng, butanol extract of K-643 fermentation of white ginseng 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.
상기의 결과로부터, 본 발명의 백삼 주근의 K-643 발효물의 에탄올 추출물은 사이토카인 유도 베타세포 사멸을 주근 추출물에 비하여 더 억제하는 효과를 보였다(표 7 참조). From the above results, the ethanol extract of the K-643 fermentation of white ginseng root of the present invention showed more inhibitory effect on cytokine induced beta cell death compared to the root extract (see Table 7).
본 발명의 백삼 미삼의 K-643 발효물의 에탄올 추출물은 사이토카인 유도 베타세포 사멸을 미삼 추출물에 비하여 더 억제하는 효과를 보였다(표 8 참조). Ethanol extract of the K-643 fermentation of white ginseng rice ginseng showed more inhibitory effect on cytokine-induced beta cell death compared to rice ginseng extract (see Table 8).
본 발명의 백삼 주근의 K-643 발효물의 에탄올 추출물은 팔미틴산 유도 베타세포 사멸을 주근 추출물에 비하여 더 억제하는 효과를 보였다(표 9 참조). Ethanol extract of the K-643 fermentation of white ginseng root of the present invention showed more inhibiting palmitic acid-induced beta cell death compared to the root extract (see Table 9).
본 발명의 백삼 주근의 K-643 발효물의 부탄올 추출물은 팔미틴산 유도 베타세포 사멸을 주근 추출물에 비하여 더 억제하는 효과를 보였다(표 10 참조). Butanol extract of the K-643 fermentation of white ginseng root of the present invention showed more inhibiting palmitic acid-induced beta cell death compared to the root extract (see Table 10).
본 발명의 백삼 미삼의 K-643 발효물의 에탄올 추출물은 팔미틴 유도 베타세포 사멸을 미삼 추출물에 비하여 더 억제하는 효과를 보였다(표 11 참조). Ethanol extract of the K-643 fermentation of white ginseng misam of the present invention showed a more inhibitory effect compared to palmitin-induced beta cell death compared to the rice extract (see Table 11).
본 발명의 백삼 미삼의 K-643 발효물의 부탄올 추출물은 사이토카인 유도 베타세포 사멸을 미삼 추출물에 비하여 더 억제하는 효과를 보였다(표 12 참조).Butanol extract of K-643 fermentation of white ginseng misam showed more inhibitory effect on cytokine-induced beta cell death as compared to misam extract (see Table 12).
표 7. 백삼 주근의 유산균 K-643 발효물의 에탄올 추출물이 췌장베타세포에서 사이토카인 유도 세포사멸에 미치는 영향Table 7. Effect of Ethanol Extract of Lactic Acid Bacteria K-643 Fermentation of White Ginseng Root on Cytokine-induced Apoptosis in Pancreatic Beta Cells
White ginseng root
Freckle K-643 fermented product **
*Values are mean± SEM (n=6).Values are mean ± SEM (n = 6).
**백삼 주근의 K-643 발효물의 에탄올 추출물을 나타낸다.** Represents the ethanol extract of K-643 fermentation of white ginseng root.
표 8. 미삼의 유산균 K-643 발효물의 에탄올 추출물이 췌장 베타세포에서 사이토카인 유도 세포사멸에 미치는 영향Table 8. Effect of Ethanol Extracts of Lactic Acid Bacteria K-643 Fermentation of Panax ginseng on Cytokine-induced Apoptosis in Pancreatic Beta Cells
K-643 Fermentation Material **
*Values are mean± SEM (n=6).Values are mean ± SEM (n = 6).
**미삼의 K-643 발효물의 에탄올 추출물을 나타낸다.** Represents the ethanol extract of the fermented K-643 fermented rice.
표 9. 백삼 주근의 유산균 K-643 발효물의 에탄올 추출물이 췌장베타세포에 서 팔미틴산 유도 세포사멸에 미치는 영향Table 9. Effect of Ethanol Extract of Lactic Acid Bacteria K-643 Fermentation of Panax ginseng Root on Palmitate-induced Apoptosis in Pancreatic Beta Cells
White ginseng root
K-643 Fermentation Material **
*Values are mean± SEM (n=6).Values are mean ± SEM (n = 6).
**백삼 주근의 K-643 발효물의 에탄올 추출물을 나타낸다.** Represents the ethanol extract of K-643 fermentation of white ginseng root.
표 10. 백삼 주근의 유산균 K-643 발효물의 부탄올 추출물이 췌장 베타세포에서 팔미틴산 유도 세포사멸에 미치는 영향Table 10. Effect of Butanol Extract of Lactic Acid Bacteria K-643 Fermentation of Panax ginseng Root on Palmitate-induced Apoptosis in Pancreatic Beta Cells
White ginseng root
K-643 Fermentation Material **
*Values are mean± SEM (n=6).Values are mean ± SEM (n = 6).
**백삼 주근의 K-643 발효물의 부탄올 추출물을 나타낸다.** Represents the butanol extract of K-643 fermentation of white ginseng root.
표 11. 미삼의 유산균 K-643 발효물의 에탄올 추출물이 췌장 베타세포에서 팔미틴산 유도 세포사멸에 미치는 영향Table 11. Effect of Ethanol Extracts of Lactic Acid Bacteria K-643 Fermentation of Panax ginseng on Palmitate-induced Apoptosis in Pancreatic Beta Cells
Misam
K-643 Fermentation Material **
*Values are mean± SEM (n=6).Values are mean ± SEM (n = 6).
**미삼의 K-643 발효물의 에탄올 추출물을 나타낸다.** Represents the ethanol extract of the fermented K-643 fermented rice.
표 12. 미삼의 유산균 K-643 발효물의 부탄올 추출물이 췌장 베타세포에서 팔미틴산 유도 세포사멸에 미치는 영향Table 12. Effect of Butanol Extract of Lactic Acid Bacteria K-643 Fermentation of Panax ginseng on Palmitate-induced Apoptosis in Pancreatic Beta Cells
Misam
K-643 Fermentation Material **
*Values are mean± SEM (n=6).Values are mean ± SEM (n = 6).
**미삼의 K-643 발효물의 부탄올 추출물을 나타낸다.** Represents the butanol extract of K-643 fermented rice.
이상의 결과를 종합하면, 본 발명의 백삼 주근의 K-643 발효물의 에탄올 추출물은 당흡수를 증진시키고, PPARγ 활성을 증진시키며, 인산화된 ACC 및 인산화된 AMPK의 발현을 증진시킨다. 또한 포도당 자극 인슐린 분비를 증가시키며, 사이 토카인 유도 베타세포 사멸을 억제하였으며, 팔미틴산 유도 베타세포 사멸도 억제하는 효과를 나타낸다. In summary, the ethanol extract of the K-643 fermentation of white ginseng root of the present invention enhances glucose uptake, enhances PPARγ activity, and enhances expression of phosphorylated ACC and phosphorylated AMPK. It also increases glucose-stimulated insulin secretion, inhibits cytokine-induced beta cell death, and inhibits palmitic acid-induced beta cell death.
백삼 주근의 K-643 발효물의 부탄올 추출물은 간세포 글루코키나제 활성을 증가시키고, 기저 및 포도당 자극 인슐린 분비는 증가시키며, 팔미틴산 유도 베타세포 사멸을 억제하는 효과를 나타낸다. Butanol extract of K-643 fermentation of white ginseng root extract increases the hepatocellular glucokinase activity, increases basal and glucose-stimulated insulin secretion, and inhibits palmitic acid induced beta cell death.
백삼 미삼의 K-643 발효물의 에탄올 추출물은 사이토카인 유도 베타세포 사멸을 억제하며, 팔미틴 유도 베타세포 사멸을 억제하는 효과를 나타낸다. Ethanol extracts of K-643 fermentation of white ginseng and ginseng inhibit cytokine-induced beta cell death and suppress palmitine-induced beta cell death.
백삼 미삼의 K-643 발효물의 부탄올 추출물은 포도당 자극 인슐린 분비를 증가시키며,사이토카인 유도 베타세포 사멸을 억제하는 효과를 보였다. Butanol extract of K-643 fermentation of white ginseng increased glucose secretion insulin secretion and suppressed cytokine-induced beta cell death.
상기 결과를 토대로, 본 발명의 당항상성 개선용 조성물은 약제학적 조성물로 사용되는 경우 당항상성을 유지하는 기능을 회복시킬 수 있으므로, 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.Based on the above results, the composition for improving glycemic improvement of the present invention can restore the function of maintaining glycemicity when used as a pharmaceutical composition, and therefore, those with impaired fasting glucose, impaired glucose tolerance, those diagnosed with prediabetes, and diabetic patients. It was confirmed that there is an effect that can be used as a pancreatic beta cell protectant and insulin resistance improver. 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.
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 K-643은 인삼 또는 인삼 추출물을 발효시킬 수 있어 인삼 발효 추출물을 얻을 수 있다.K-643 of the present invention can ferment ginseng or ginseng extract to obtain a ginseng fermented extract.
본 발명의 신규한 K-643은 생체 이용률이 낮은 진세노사이드 Rg1, Rb1, Rg1, Re 등을 진세노사이드 Rh1, Rg3, Rh2, C-K 등으로 전환시킬 수 있어 본 발명에 의한 인삼 발효 추출물이 함유된 약제학적 제제, 건강기능식품에 적용할 수 있다. The novel K-643 of the present invention can convert ginsenosides Rg1, Rb1, Rg1, Re and the like having low bioavailability into ginsenosides Rh1, Rg3, Rh2, CK, etc. It can be applied to pharmaceutical preparations, health foods.
또한 본 발명의 신규한 K-643을 이용하여 제조한 인삼 또는 인삼 추출물을 발효시킨 인삼 발효 추출물은 포도당 항상성 개선용에 사용할 수 있어 이를 약제학 및/또는 건강기능식품에 사용하는 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 있다. In addition, the ginseng or ginseng fermented extracts fermented with ginseng or ginseng extract prepared using the novel K-643 of the present invention can be used for improving the homeostasis of glucose, so that when used in pharmaceuticals and / or health functional foods, It can restore the function, there is an effect that can be used as a fasting glucose impaired person, impaired glucose tolerance, a person diagnosed with pre-diabetes, pancreatic beta cell protector and insulin resistance improver in diabetic patients.
도 1은 백삼 주근의 K-643 발효물의 에탄올 추출물에 의한 지방세포에서의 포도당 흡수 증진 작용을 보여주는 도면이다.1 is a diagram showing the effect of glucose uptake in adipocytes by the ethanol extract of K-643 fermentation of white ginseng root.
도 2는 백삼 주근의 K-643 발효물의 에탄올 추출물의 지방세포에서의 PPARγ 증진 작용을 보여주는 도면이다.Figure 2 is a diagram showing the PPARγ enhancing effect of the ethanol extract of K-643 fermentation of white ginseng root in adipocytes.
도 3은 백삼 주근의 K-643 발효물의 에탄올 추출물의 지방세포에서의 인산화된 ACC 및 인산화된 AMPK 발현 증진 작용을 보여주는 도면이다.3 is a diagram showing the phosphorylated ACC and phosphorylated AMPK expression enhancing action of the ethanol extract of K-643 fermentation of white ginseng root.
<110> KOREA FOOD RESEARCH INSTITUTE <120> Novel Pediococcus pentosaceus K-643 and extracts from ginseng-fermented products using Pediococcus pentosaceus K-643 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 sequencing 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> 946 <212> DNA <213> Pediococcus pentosaceus K-643 <400> 5 ccgacttcgt gtaggcgagt tgcagcctac agtccgaact gagaatggtt ttaagagatt 60 agcttaacct cgcggtctcg cgactcgttg taccatccat tgtagcacgt gtgtagccca 120 ggtcataagg ggcatgatga tttgacgtcg tccccacctt cctccggttt gtcaccggca 180 gtctcactag agtgcccaac ttaatgctgg caactagtaa taagggttgc gctcgttgcg 240 ggacttaacc caacatctca cgacacgagc tgacgacaac catgcaccac ctgtcattct 300 gtccccgaag ggaacctcta atctcttaga ctgtcagaag atgtcaagac ctggtaaggt 360 tcttcgcgta gcttcgaatt aaaccacatg ctccaccgct tgtgcgggcc cccgtcaatt 420 cttttgagtt tcaaccttgc ggtcgtactc cccaggcgga ttacttaatg cgttagctgc 480 agcactgaag ggcggaaacc ctccaacact tagtaatcat cgtttacggc atggactacc 540 agggtatcta atcctgttcg ctacccatgc tttcgagcct cagcgtcagt tgcagaccag 600 acagccgcct tcgccactgg tgttcttcca tatatctacg catttcaccg ctacacatgg 660 agttccactg tcctcttctg cactcaagtc tcccagtttc caatgcactt cttcggttga 720 gccgaaggct ttcacattag acttaaaaga ccgcctgcgc tcgctttacg cccaataaat 780 ccggataacg cttgccacct acgtattacc gcggctgctg gcacgtagtt agccgtggct 840 ttctggttaa ataccgtcac tgggtaaaca gttactctta cccacgttct tctttaacaa 900 cagagcttta cgagccgaaa cccttcttca ctcacgcggc gttgct 946 <110> KOREA FOOD RESEARCH INSTITUTE <120> Novel Pediococcus pentosaceus K-643 and extracts from ginseng-fermented products using Pediococcus pentosaceus K-643 for improving glucose homeostasis and manufacturing method the <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 sequencing 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> 946 <212> DNA <213> Pediococcus pentosaceus K-643 <400> 5 ccgacttcgt gtaggcgagt tgcagcctac agtccgaact gagaatggtt ttaagagatt 60 agcttaacct cgcggtctcg cgactcgttg taccatccat tgtagcacgt gtgtagccca 120 ggtcataagg ggcatgatga tttgacgtcg tccccacctt cctccggttt gtcaccggca 180 gtctcactag agtgcccaac ttaatgctgg caactagtaa taagggttgc gctcgttgcg 240 ggacttaacc caacatctca cgacacgagc tgacgacaac catgcaccac ctgtcattct 300 gtccccgaag ggaacctcta atctcttaga ctgtcagaag atgtcaagac ctggtaaggt 360 tcttcgcgta gcttcgaatt aaaccacatg ctccaccgct tgtgcgggcc cccgtcaatt 420 cttttgagtt tcaaccttgc ggtcgtactc cccaggcgga ttacttaatg cgttagctgc 480 agcactgaag ggcggaaacc ctccaacact tagtaatcat cgtttacggc atggactacc 540 agggtatcta atcctgttcg ctacccatgc tttcgagcct cagcgtcagt tgcagaccag 600 acagccgcct tcgccactgg tgttcttcca tatatctacg catttcaccg ctacacatgg 660 agttccactg tcctcttctg cactcaagtc tcccagtttc caatgcactt cttcggttga 720 gccgaaggct ttcacattag acttaaaaga ccgcctgcgc tcgctttacg cccaataaat 780 ccggataacg cttgccacct acgtattacc gcggctgctg gcacgtagtt agccgtggct 840 ttctggttaa ataccgtcac tgggtaaaca gttactctta cccacgttct tctttaacaa 900 cagagcttta cgagccgaaa cccttcttca ctcacgcggc gttgct 946
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