KR20190114315A - Method for producing fermentative product of cultured mountain ginseng extract with increased small molecule ginsenoside content and fermentative product of cultured mountain ginseng extract with increased small molecule ginsenoside content produced by the same method - Google Patents
Method for producing fermentative product of cultured mountain ginseng extract with increased small molecule ginsenoside content and fermentative product of cultured mountain ginseng extract with increased small molecule ginsenoside content produced by the same method Download PDFInfo
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- KR20190114315A KR20190114315A KR1020180036832A KR20180036832A KR20190114315A KR 20190114315 A KR20190114315 A KR 20190114315A KR 1020180036832 A KR1020180036832 A KR 1020180036832A KR 20180036832 A KR20180036832 A KR 20180036832A KR 20190114315 A KR20190114315 A KR 20190114315A
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- wild ginseng
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
- A61K36/258—Panax (ginseng)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A23V2250/00—Food ingredients
- A23V2250/20—Natural extracts
- A23V2250/21—Plant extracts
- A23V2250/2124—Ginseng
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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Abstract
Description
본 발명은 저분자 진세노사이드 함량이 증가된 산삼배양근 추출물의 발효물의 제조방법 및 상기 방법에 의해 제조된 저분자 진세노사이드 함량이 증가된 산삼배양근 추출물의 발효물에 관한 것이다.The present invention relates to a method for preparing a fermented product of wild ginseng cultured root extract with increased low molecular weight ginsenoside content, and a fermented product of wild ginseng cultured root extract with increased low molecular weight ginsenoside content prepared by the above method.
산삼(山蔘)은 고려인삼의 시조로서 두릅나무과(Araliaceae), 인삼속(Panax)에 속하는 다년생 음지성 초본식물로서, 인삼(Panax ginseng C.A. Meyer)이 야생상태에서 자연 발아하여 성장한 삼(蔘)을 일컬으며 우리나라에서는 오래전부터 약용으로 사용되어 왔다. 특히 천연산삼은 발견되는 것이 더욱 희박해지고 있으며, 소수의 심마니들에 의해서 발견되는 신비의 양약으로 알려져있다. 산삼은 천종, 지종, 인종, 장뇌삼의 4가지로 분류되는데 천종, 지종, 인종은 야생 삼으로 조류가 종자를 먹은 뒤 산 속에 배설하여 자생한 것을 말한다. 장뇌산삼은 산삼의 종자를 야생에서 인위적으로 재배한 것을 말한다.Mountain ginseng is a perennial herbaceous herbaceous plant belonging to the family Araliaceae and Panax ginseng, which is the ancestor of Korean ginseng, and ginseng ( Panax ginseng CA Meyer) grown and grown naturally in the wild It has been used in Korea for a long time medicinal. Natural ginseng, in particular, is becoming thinner and less known, and is known as a mysterious medicine found by a few simnies. Mountain ginseng is classified into four types: cheonjong, jijong, race, and camphor ginseng, and cheonjong, jijong, and race are wild ginseng. Jangwangsan ginseng is an artificially grown seed of wild ginseng.
산삼은 사포닌(saponin), 항산화물질, 펩타이드, 다당류, 지방산, 알콜, 비타민 등을 함유하고 있으며, 이 중 트리테르페노이드(tripenoid) 계열의 화합물인 진세노사이드(ginsenoside)는 주요 생리활성물질로 보고되었다. 트리테르페노이드는 올레아난(oleanane) 계열과 다마란(dammarane) 계 사포닌으로 구분되며, 진세노사이드의 대부분은 다마란계 사포닌으로써 구조적 특징으로 구분되는데, 3, 12 및 20번 탄소에 수산기(hydroxyl group)가 있는 프로토파낙사디올(protopanaxadiol, PPD) 타입의 사포닌과 6, 12 및 20번 탄소에 수산기가 있는 프로토파낙사트리올(portpanaxatriol, PPT) 타입의 사포닌으로 구분된다. 프로토파낙사디올(PPD)계 진세노사이드로는 Rb1, Rb2, Rc 및 Rd 등이 있고, 프로토파낙사트리올(PPT)계 진세노사이드로는 Rh1, Rg2, Rg1 및 Re 등이 있으며, PPD계는 중추신경 진정에 효과가 있고, PPT계는 혈중 콜레스테롤 수치 감소에 효과가 있는 것으로 알려져있다. 이외에도 산삼은 항암, 항산화, 방사선 장해 방어작용, 항당뇨, 갱년기 장해 개선, 항스트레스, 항피로 작용, 항염증 작용 등에 효과가 있는 것으로 보고된 바 있다.Wild ginseng contains saponin, antioxidants, peptides, polysaccharides, fatty acids, alcohols, vitamins, etc. Among these, ginsenoside, a triterpenoid-based compound, is the main bioactive substance. Reported. Triterpenoids are divided into oleanane and dammarane saponins, and most of ginsenosides are structurally characterized as damascene saponins. It is divided into protopanaxadiol (PPD) type saponins with hydroxyl group) and saponins of type portpanaxatriol (PPT) with hydroxyl groups at
그러나 진세노사이드는, 삼에 함유된 채로 존재하는 상태에서는 당이 진세노사이드 기본골격에 붙어 있어서 다당류의 형태를 취하고, 이는 곧바로 체내 흡수가 되기 어려우며, 장내에서 다당류가 단당류로 가수분해된 후, 즉 고분자에서 저분자로 전환된 후에 비로소 흡수된다. 대표적인 저분자 진세노사이드로는 Rg3, Rk1, Rg5, Compound K 등이 있으며, Rg3는 혈압저하, 항암 효과를 지니며 Rk1 및 Rg5는 치매 예방, 아토피, 골다공증 예방 등의 효과가 보고되었으며, Compound K는 항염 및 간보호 효과 등이 보고되었다. 따라서, 체내의 흡수율을 높이기 위해서는, 진세노사이드의 저분자화가 선행될 필요가 있음이 알려져 있다.However, ginsenosides, when present in the hemp, have sugars attached to the ginsenoside backbone and take the form of polysaccharides, which are difficult to be absorbed directly into the body, and after the polysaccharides are hydrolyzed into the monosaccharides in the intestines, That is, it is only absorbed after being converted to low molecules in the polymer. Representative low molecular ginsenosides include Rg3, Rk1, Rg5, and Compound K. Rg3 has an effect on lowering blood pressure and anticancer, and Rk1 and Rg5 have been reported to prevent dementia, atopy, and osteoporosis. Anti-inflammatory and hepatoprotective effects have been reported. Therefore, in order to increase the absorption rate in the body, it is known that the low molecular weight of ginsenoside needs to be preceded.
최근에는 첨단 생명공학기술을 응용하여 산삼을 실험실에서 조직배양하는 기술이 국내 연구자들에 의해 개발되었다. 생물배양기(bioreactor)를 이용하여 병충해, 계절변화, 토지조성 등에 영향을 받지 않으면서 재배가 어려운 약용식물의 조직이나 세포를 연중 안정적으로 생산할 수 있고, 유용성분의 표준화가 가능하다는 큰 장점이 있다. 산삼배양근은 야생산삼의 조직 일부를 떼어내어 무균상태로 기내도입하여 식물의 전형성능(totipotency)에 의해 형성되는 부정근(adventitious root)을 말한다.Recently, domestic researchers have developed a technology for tissue culture of wild ginseng in the laboratory by applying advanced biotechnology. Bioreactors can be used to stably produce tissues or cells of medicinal plants that are difficult to grow year round without being affected by pests, seasonal changes, land composition, etc., and can standardize useful components. The wild ginseng culture root is an adventitious root formed by the totipotency of the plant by removing a part of the wild ginseng tissue and introducing it into the cabin aseptically.
한편, 한국등록특허 제1564487호에는 발효효소와 알코올발효균주를 이용한 '저분자 진세노사이드의 제조방법'이 기재되어 있고, 한국등록특허 제1467522호에는 '감압 재순환 고속 증숙 공정장치를 이용한 인삼의 저분자 진세노사이드 함량 증진 방법'이 개시되어 있으나, 본 발명의 저분자 진세노사이드 함량이 증가된 산삼배양근 추출물의 발효물의 제조방법 및 상기 방법에 의해 제조된 저분자 진세노사이드 함량이 증가된 산삼배양근 추출물의 발효물에 대해서는 기재된 바가 없다.Meanwhile, Korean Patent No. 1544487 describes a 'method of preparing low molecular weight ginsenosides' using fermentation enzymes and alcohol fermenting strains, and Korean Patent No. 1447522' low molecular weight of ginseng using reduced-pressure recycling high-speed steaming process equipment. Method of enhancing ginsenoside content 'is disclosed, but a method for preparing a fermentation product of wild ginseng cultured root extract with increased low molecular weight ginsenoside content of the present invention and a ginseng cultured root extract with increased low molecular weight ginsenoside content prepared by the method Fermented products are not described.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 산삼배양근 추출물의 분획물에 열처리하고, 상기 열처리된 산삼배양근 분획물을 페디오코커스 펜토사세우스(Pediococcus pentosaceus) HLJG0702 균주로 발효시킨 후 산삼배양근 추출물의 발효물 내의 진세노사이드 함량을 분석한 결과, 열처리 및 발효처리 하지 않은 산삼배양근 추출물에 비해 고분자 진세노사이드 함량은 감소하고 저분자 진세노사이드 함량이 증가된 것을 확인함으로써, 본 발명을 완성하였다.The present invention is derived from the above requirements, the present inventors heat-treated the fraction of wild ginseng culture root extract, and fermented wild ginseng cultured root fraction with Pediococcus pentosaceus ( Pediococcus pentosaceus ) HLJG0702 strain As a result of analyzing the ginsenoside content in the fermentation product of the culture root extract, it was confirmed that the polymer ginsenoside content was decreased and the low molecular weight ginsenoside content was increased, compared to the wild ginseng cultured root extract without heat treatment and fermentation. It was.
상기 과제를 해결하기 위해, 본 발명은 (a) 조직배양한 산삼배양근을 동결건조하고 분쇄하여 산삼배양근 동결건조 분말을 얻는 단계; (b) 상기 (a) 단계의 산삼배양근 동결건조 분말을 에탄올로 추출하고 농축하여 농축액을 얻는 단계; (c) 상기 (b) 단계의 농축액에 물을 혼합한 후 에탄올을 가하여 분획하여 에탄올 분획물을 얻는 단계; (d) 상기 (c) 단계의 에탄올 분획물에 물을 혼합한 후 열처리하는 단계; 및 (e) 상기 (d) 단계의 열처리된 에탄올 분획물을 발효하는 단계;를 포함하는, 저분자 진세노사이드 함량이 증가된 산삼배양근 추출물의 발효물의 제조방법을 제공한다.In order to solve the above problems, the present invention comprises the steps of (a) freeze-drying and grinding the cultured wild ginseng cultured root; (b) extracting the ginseng cultured lyophilized powder of step (a) with ethanol and concentrating to obtain a concentrate; (c) mixing water with the concentrate of step (b) and then fractionating by adding ethanol to obtain an ethanol fraction; (d) heat-treating the ethanol fraction of step (c) after mixing water; And (e) fermenting the heat-treated ethanol fraction of step (d). It provides a method of producing a fermentation product of wild ginseng cultured root extract with increased low molecular weight ginsenoside content.
또한, 본 발명은 상기 방법에 의해 제조된 저분자 진세노사이드 함량이 증가된 산삼배양근 추출물의 발효물을 제공한다.In addition, the present invention provides a fermentation product of wild ginseng cultured root extract with increased low molecular ginsenoside content prepared by the above method.
본 발명에 따른 산삼배양근 추출물의 발효물의 제조방법은 간단한 공정을 통해 체내 흡수율이 높은 저분자 진세노사이드 함량을 증가시킬 수 있으며, 무균상태에서 조직 배양한 산삼배양근을 사용함으로써 건강식품 소재로 적용함에 있어 안정성이 높다.Fermented product of the ginseng cultured root extract according to the present invention can increase the low molecular weight ginsenoside content with high absorption in the body through a simple process, in the application to health food material by using wild ginseng cultured tissue culture in aseptic state High stability
도 1은 산삼배양근 추출물의 물, 30% 에탄올 및 60% 에탄올 분획물 내의 진세노사이드 함량을 나타내는 HPLC(high performance liquid chromatography) 크로마토그램이다.
도 2는 열처리 시간에 따른 산삼배양근 추출물의 분획물 내의 진세노사이드 함량을 나타내는 HPLC 크로마토그램이다.
도 3은 본 발명의 저분자 진세노사이드 함량이 증가된 산삼배양근 추출물의 발효물의 제조방법의 공정도이다.1 is a high performance liquid chromatography (HPLC) chromatogram showing ginsenoside content in water, 30% ethanol and 60% ethanol fractions of wild ginseng root extract.
Figure 2 is an HPLC chromatogram showing the ginsenoside content in the fraction of wild ginseng cultured root extract with heat treatment time.
Figure 3 is a process chart of the fermentation method of the fermented ginseng culture root extract with increased low molecular weight ginsenoside content of the present invention.
본 발명의 목적을 달성하기 위하여, 본 발명은In order to achieve the object of the present invention, the present invention
(a) 조직배양한 산삼배양근을 동결건조하고 분쇄하여 산삼배양근 동결건조 분말을 얻는 단계;(a) freeze-drying and grinding the cultured wild ginseng cultured root to obtain a wild ginseng cultured lyophilized powder;
(b) 상기 (a) 단계의 산삼배양근 동결건조 분말을 에탄올로 추출하고 농축하여 농축액을 얻는 단계;(b) extracting the ginseng cultured lyophilized powder of step (a) with ethanol and concentrating to obtain a concentrate;
(c) 상기 (b) 단계의 농축액에 물을 혼합한 후 에탄올을 가하여 분획하여 에탄올 분획물을 얻는 단계;(c) mixing water with the concentrate of step (b) and then fractionating by adding ethanol to obtain an ethanol fraction;
(d) 상기 (c) 단계의 에탄올 분획물에 물을 혼합한 후 열처리하는 단계; 및(d) heat-treating the ethanol fraction of step (c) after mixing water; And
(e) 상기 (d) 단계의 열처리된 에탄올 분획물을 발효하는 단계;를 포함하는, 저분자 진세노사이드 함량이 증가된 산삼배양근 추출물의 발효물의 제조방법을 제공한다.(e) fermenting the heat-treated ethanol fraction of step (d); provides a method of producing a fermentation product of wild ginseng culture root extract with increased low molecular ginsenoside content.
본 발명의 일 구현 예에 따른 저분자 진세노사이드 함량이 증가된 산삼배양근 추출물의 발효물의 제조방법에 있어서, 상기 (e) 단계의 발효는 페디오코커스 펜토사세우스(Pediococcus pentosaceus) HLJG0702 균주(기탁번호: KACC81017BP)를 이용하여 발효하는 것일 수 있으나, 이에 제한되지 않는다. 상기 기탁균주는 한국등록특허공보 제1816596호에 개시된 균주이다.In the method for producing a fermentation product of wild ginseng culture root extract with increased low molecular weight ginsenoside content according to one embodiment of the present invention, the fermentation of step (e) is Pediococcus pentosaceus ( Pediococcus pentosaceus ) HLJG0702 strain (deposited) No .: may be fermented using KACC81017BP), but is not limited thereto. The deposited strain is a strain disclosed in Korean Patent Publication No. 1816596.
또한, 본 발명의 상기 제조방법에 있어서, 상기 저분자 진세노사이드는 Rg2, Rg3, Rg5, Rg6, Rh1, Rh4 및 Rk1으로 이루어진 군으로부터 선택된 하나 이상일 수 있으나, 이에 제한되지 않는다.In addition, in the production method of the present invention, the low molecular ginsenoside may be one or more selected from the group consisting of Rg2, Rg3, Rg5, Rg6, Rh1, Rh4 and Rk1, but is not limited thereto.
본 발명의 일 구현 예에 따른 저분자 진세노사이드 함량이 증가된 산삼배양근 추출물의 발효물의 제조방법은 구체적으로는,Specifically, the method for preparing a fermentation product of wild ginseng cultured root extract with increased low molecular weight ginsenoside content according to one embodiment of the present invention,
(a) 조직배양한 산삼배양근을 동결건조하고 분쇄하여 산삼배양근 동결건조 분말을 얻는 단계;(a) freeze-drying and grinding the cultured wild ginseng cultured root to obtain a wild ginseng cultured lyophilized powder;
(b) 상기 (a) 단계의 산삼배양근 동결건조 분말에 65~75%(v/v) 에탄올을 첨가하여 45~55℃에서 7~9시간씩 2~3회 반복 추출한 후 농축하여 농축액을 얻는 단계;(b) 65 to 75% (v / v) ethanol was added to the wild ginseng root lyophilized powder of step (a), followed by repeated
(c) 상기 (b) 단계의 농축액에 물을 동량으로 첨가하여 혼합한 후 55~65%(v/v) 에탄올을 가하여 분획하여 에탄올 분획물을 얻는 단계;(c) adding the same amount of water to the concentrate of step (b), followed by mixing to obtain 55-65% (v / v) ethanol and fractionating to obtain an ethanol fraction;
(d) 상기 (c) 단계의 에탄올 분획물에 물을 첨가하여 혼합한 혼합액을 115~125℃에서 25~125분 동안 열처리하는 단계; 및(d) heat-treating the mixed solution mixed with water by adding water to the ethanol fraction of step (c) for 25-125 minutes at 115-125 ° C; And
(e) 상기 (d) 단계의 열처리된 혼합액에 페디오코커스 펜토사세우스(Pediococcus pentosaceus) HLJG0702 균주(기탁번호: KACC81017BP)를 접종하여 28~32℃에서 22~26시간 동안 발효하는 단계를 포함할 수 있고, 보다 구체적으로는,(e) inoculating Pediococcus pentosaceus ( Pediococcus pentosaceus ) HLJG0702 strain (Accession Number: KACC81017BP) on the heat-treated mixture of step (d) and fermenting at 28-32 ° C. for 22-26 hours. You can do that, more specifically,
(a) 조직배양한 산삼배양근을 -40℃에서 동결건조한 후, 건조된 산삼배양근 1 kg을 100 메쉬(mesh)의 크기로 분쇄하여 분말을 얻는 단계;(a) lyophilizing the cultured wild ginseng cultured root at −40 ° C., and then grinding 1 kg of the dried wild ginseng cultured root to a size of 100 mesh to obtain a powder;
(b) 상기 (a) 단계의 산삼배양근 동결건조 분말에 70%(v/v) 에탄올을 10배(v/w) 첨가하여 50℃에서 8시간씩 3회 반복 추출한 후, 농축하여 농축액을 얻는 단계;(b) 10 times (v / w) of 70% (v / v) ethanol was added to the wild ginseng root lyophilized powder of step (a), extracted three times at 50 ° C. for 8 hours, and concentrated to obtain a concentrate. step;
(c) 상기 (b) 단계의 농축액에 물을 동량으로 첨가하여 50 brix 현탁액을 제조한 후, 60%(v/v) 에탄올을 가하여 에탄올 분획물을 얻는 단계;(c) adding 50 parts of the same amount of water to the concentrate of step (b) to prepare a 50 brix suspension, and then adding 60% (v / v) ethanol to obtain an ethanol fraction;
(d) 상기 (c) 단계의 에탄올 분획물의 동결건조 분말에 물을 20배(v/w) 혼합하여 5 brix 현탁액을 제조한 후, 120℃에서 120분 동안 열처리하는 단계; 및(d) mixing 5 times (v / w) of water with the lyophilized powder of the ethanol fraction of step (c) to prepare a 5 brix suspension, and then heat-treating at 120 ° C. for 120 minutes; And
(e) 상기 (d) 단계의 열처리된 현탁액에 페디오코커스 펜토사세우스(Pediococcus pentosaceus) HLJG0702 균주(기탁번호: KACC81017BP) 배양액을 5%(v/v) 접종하여 30℃에서 24시간 동안 발효하는 단계를 포함할 수 있으나, 이에 제한되지 않는다.(e) 5% (v / v) of Pediococcus pentosaceus HLJG0702 strain (Accession Number: KACC81017BP) inoculated in the heat-treated suspension of step (d) and fermented at 30 ° C. for 24 hours. It may include, but is not limited to.
본 발명에 따른 제조방법은, 상기 (e) 단계의 발효 종료 후 여과하는 단계를 추가로 포함할 수 있으나, 이에 제한되지 않는다.The production method according to the present invention may further include a step of filtering after the fermentation of step (e), but is not limited thereto.
본 발명은 또한, 상기 방법에 의해 제조된 저분자 진세노사이드 함량이 증가된 산삼배양근 추출물의 발효물을 제공한다.The present invention also provides a fermentation product of wild ginseng cultured root extract having an increased content of low molecular ginsenosides prepared by the above method.
본 발명의 산삼배양근 추출물의 발효물은 산삼배양근 추출물의 분획물을 열처리하고 상기 열처리된 산삼배양근 분획물을 펜토사세우스(Pediococcus pentosaceus) HLJG0702 균주로 발효시켜 대조구(산삼배양근 추출물)에 비해 저분자 진세노사이드 함량이 증가된 발효물로, 저분자 진세노사이드 Rg2, Rg3, Rg5, Rg6, Rh1, Rh4 및 Rk1의 함량이 증가된 발효물이다.Fermentation of wild ginseng baeyanggeun extract of the present invention water is a low molecular weight ginsenosides than by heating the fractions of ginseng baeyanggeun extract and fermenting said thermally processed ginseng baeyanggeun fractions with pen soil three mouse (Pediococcus pentosaceus) HLJG0702 strain control (wild ginseng baeyanggeun extract) Fermented products with increased content are fermented products with increased content of low molecular weight ginsenosides Rg2, Rg3, Rg5, Rg6, Rh1, Rh4 and Rk1.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.
재료 및 방법Materials and methods
1. 식물재료1. Plant Materials
실험에 사용된 산삼배양근은 2017년 10월 주식회사 화진바이오코스메틱 생명공학연구소에서 배양한 것으로, 양구군에서 채취한 35년근 이상으로 추정되는 야생산삼을 기내도입하여 배양한 것이다. 산삼배양근의 배양에 사용된 배지는 SH(Schenk and Hildebrandt Medium) 47.25 g과 수크로오스 450 g을 정제수 15 L에 혼합한 후, pH 5.75로 보정하고 18L 생물반응기(Bio-reactor)에 투입하여 제조하였다. 이후 고온·고압(127℃, 60분, 0.15 mPa)에서 멸균한 후 냉각시키고, 전배양(pre-incubation)된 산삼 세포 중 우량한 세포주를 선별하여 30 g을 접종하고 온도 25.0±0.5℃, 습도 40.0±5.0%에서 8~9주간 배양하여 사용하였다.The wild ginseng root used in the experiment was cultured at the Hwajin Biocosmetic Biotechnology Research Institute in October 2017, and wild ginseng, which is estimated to be more than 35 years old, collected from Yanggu-gun, was introduced and incubated. The medium used for the culture of wild ginseng cultured muscle was prepared by mixing 47.25 g of Schenk and Hildebrandt Medium (SH) and 450 g of sucrose in 15 L of purified water, calibrating to pH 5.75, and adding an 18L bioreactor. After sterilization at high temperature and high pressure (127 ° C, 60 minutes, 0.15 mPa), the cells were cooled and inoculated with 30 g of pre-incubated wild ginseng cells, inoculated with 30 g, temperature of 25.0 ± 0.5 ° C, and humidity of 40.0. Cultures were used for 8-9 weeks at ± 5.0%.
2. 공시균주2. Test strain
균주는 농업유전자원정보센터(KACC)에 기탁한 그람 양성균인 페디오코커스 펜토사세우스(Pediococcus pentosaceus) HLJG0702(기탁번호: KACC81017BP)를 사용하였다.The strain was Gram-positive bacteria Pediococcus pentosaceus HLJG0702 (Accession Number: KACC81017BP) deposited in the Agricultural Genetic Resource Information Center (KACC).
균주 배양에 사용된 배지는 MRS(Man-Rogosa-Sharpe) 55.25 g을 정제수 1 L에 혼합한 후, pH 6.2로 보정하였다. 이후 고온·고압(121℃, 15분, 0.12 mPa)에서 멸균한 후 냉각시키고, 초저온 냉장고에 보관중인 스타터 유산균 분말 페디오코커스 펜토사세우스 HLJG0702 10 g을 MRS 배지 100 ㎖과 혼합하여 30℃에서 48시간 동안 배양하여 사용하였다.The medium used for strain culture was mixed with 55.25 g of MRS (Man-Rogosa-Sharpe) in 1 L of purified water, and then adjusted to pH 6.2. After sterilization at high temperature and high pressure (121 ° C., 15 minutes, 0.12 mPa) and cooled, 10 g of Starter Lactobacillus powder Pediococcus pentosaceus HLJG0702 stored in an ultra low temperature refrigerator was mixed with 100 ml of MRS medium at The culture was used for 48 hours.
3. 시약 및 기기3. Reagents and Instruments
분석시약은 물(HPLC grade, JT Baker, 미국), 아세토나이트릴(HPLC grade, acetonitrile; JT Baker), 에탄올(HPLC grade, JT Baker)을 사용하였고, 표준품은 진세노사이드 Rg1, Re, Rb2, Rb3, Rh1, Rd, Rg6, Rh4, Rg3, Rk1, Rg5, Rh2 및 Rh3(Sigma-aldrich, 미국); Rg2(Ambo institute, 한국); Rb1(HWI ANALYTIK GmbH, 독일); 및 화합물 K(Compound K) 및 Rc(Chemfaces, 중국)를 사용하였다.Reagents were water (HPLC grade, JT Baker, USA), acetonitrile (HPLC grade, acetonitrile; JT Baker), ethanol (HPLC grade, JT Baker), and standard products were ginsenosides Rg1, Re, Rb2, Rb3, Rh1, Rd, Rg6, Rh4, Rg3, Rk1, Rg5, Rh2 and Rh3 (Sigma-aldrich, USA); Rg2 (Ambo institute, Korea); Rb1 (HWI ANALYTIK GmbH, Germany); And compound K (Compound K) and Rc (Chemfaces, China).
HPLC-UVD(High Performance Liquid Chromatography-Ultraviolet Detector)는 Ultimate 3000 series(Thermo, 미국)를 사용하였으며, 그 외에도 회전감압농축기(rotary vacuum evaporator, Eyela, 일본), 저울(balance, Mettler toledo, 스위스), 초음파분해(sonication, Jeio tech, 한국), 교반기(vortex mixer, Scientific Industries, 미국) 등을 사용하였다. HPLC-UVD (High Performance Liquid Chromatography-Ultraviolet Detector) used the Ultimate 3000 series (Thermo, USA), as well as a rotary vacuum evaporator (Eyela, Japan), balance (balance, Mettler toledo, Switzerland), Sonication (Jeio tech, Korea), agitator (vortex mixer, Scientific Industries, USA) and the like were used.
4. 산삼배양근 추출 조건별 시료 준비4. Preparation of Samples for Wild Ginseng Cultured Root Extraction Conditions
8~9주 동안 배양한 산삼배양근 100 kg을 동결건조(-40℃)한 후, 실험에 사용하였다.100 kg of wild ginseng cultured cultured for 8-9 weeks were lyophilized (-40 ° C.) and used for experiments.
4-1. 추출용매 조건별 시료 준비4-1. Sample Preparation by Extraction Solvent Conditions
건조된 산삼배양근 1 kg을 100 메쉬(mesh)의 크기로 분쇄한 분말에 물, 20% 에탄올, 40% 에탄올, 60% 에탄올, 70% 에탄올, 80% 에탄올 및 100% 에탄올을 10배(v/w)씩 각각 혼합한 후 2시간 동안 진탕 추출하고, 0.2 ㎛ 필터로 여과한 후 농축하여 동결건조하였다.1 kg of dried ginseng cultured roots were pulverized to a size of 100 mesh, and water, 20% ethanol, 40% ethanol, 60% ethanol, 70% ethanol, 80% ethanol and 100% ethanol were 10 times (v / Each w) was mixed and shaken for 2 hours, filtered through a 0.2 μm filter, concentrated and lyophilized.
4-2. 추출시간 조건별 시료 준비4-2. Sample Preparation by Extraction Time Conditions
건조된 산삼배양근 1 kg을 100 메쉬(mesh)의 크기로 분쇄한 분말에 70% 에탄올을 10배(v/w) 혼합하여 2, 4, 6, 8, 16 및 32시간 동안 각각 추출하고, 0.2 ㎛ 필터로 여과한 후 농축하여 동결건조하였다.1 kg of dried ginseng cultured roots were mixed to a powder size of 100 mesh and 10% (v / w) of 70% ethanol was extracted for 2, 4, 6, 8, 16 and 32 hours, respectively, 0.2 The filtrate was filtered through a 탆 filter, concentrated and lyophilized.
4-3. 추출온도 조건별 시료 준비4-3. Sample Preparation by Extraction Temperature Condition
건조된 산삼배양근 1 kg을 100 메쉬(mesh)의 크기로 분쇄한 분말에 70% 에탄올을 10배(v/w) 혼합하여 30, 40, 50, 60, 70 및 80℃에서 2시간 동안 각각 추출하고, 0.2 ㎛ 필터로 여과한 후 농축하여 동결건조하였다.1 kg of dried ginseng cultured roots were mixed to a powder of 100 mesh and 10% (v / w) of 70% ethanol was extracted for 2 hours at 30, 40, 50, 60, 70 and 80 ° C, respectively. Filtered through a 0.2 μm filter, concentrated and lyophilized.
4-4. 추출횟수 조건별 시료 준비4-4. Sample preparation by condition
건조된 산삼배양근 1 kg을 100 메쉬(mesh)의 크기로 분쇄한 분말에 70% 에탄올을 10배(v/w) 혼합하여 50℃에서 2시간 동안 각각 1, 2, 3, 4회 반복 추출하고, 0.2 ㎛ 필터로 여과한 후 농축하여 동결건조하였다.1 kg of dried ginseng cultured roots were mixed to a powder of 100 mesh, and 10% (v / w) of 70% ethanol was mixed and extracted 1, 2, 3 and 4 times at 50 ° C. for 2 hours. It was filtered through a 0.2 μm filter, concentrated and lyophilized.
5. HP-20 칼럼 크로마토그래피를 이용한 산삼배양근 추출물의 분획물 제조5. Preparation of Fractions of Wild Ginseng Cultured Root Extract by HP-20 Column Chromatography
건조된 산삼배양근 2 kg을 100 메쉬(mesh)의 크기로 분쇄한 분말에 70% 에탄올을 10배(v/w) 혼합하고 50℃에서 8시간 동안 3회 반복 추출한 후 농축하여 동결건조하였으며, 상기 동결건조 분말에 정제수를 1:1(v/w) 비율로 혼합하여 50 brix 현탁액 1 L를 제조하였다. 상기 현탁액의 정제를 위해 Diaion HP-20 칼럼 크로마토그래피를 이용하였으며 물, 30% 에탄올 및 60% 에탄올을 순차적으로 20배(v/v) 용출(elution)시켜 3개의 분획물을 수득한 후 농축 및 동결건조하였다.2 kg of dried ginseng cultured roots were mixed in a powder ground to a size of 100 mesh, mixed with 10% (v / w) of 70% ethanol, extracted three times for 8 hours at 50 ° C, concentrated and lyophilized. 1 L of 50 brix suspension was prepared by mixing purified water with lyophilized powder at a ratio of 1: 1 (v / w). Diaion HP-20 column chromatography was used to purify the suspension. Water, 30% ethanol and 60% ethanol were sequentially 20 times (v / v) eluted to yield three fractions which were then concentrated and lyophilized.
6. 산삼배양근 추출물의 분획물에 대한 열처리 6. Heat treatment of fractions of wild ginseng cultured root extract
상기 분획물 중 진세노사이드 함량이 높은 60% 에탄올 분획물에 정제수를 20배(v/w) 혼합하여 제조된 5 brix 혼합액을 120℃에서 각각 30, 60 및 120분 동안 열처리하였다.The 5 brix mixture prepared by mixing purified
7. 열처리된 산삼배양근 분획물의 발효 7. Fermentation of Heat-treated Wild Ginseng Root Fractions
스타터 유산균 분말 페디오코커스 펜토사세우스(Pediococcus pentosaceus) HLJG0702 균주 10 g을 MRS 배지 100 ㎖에 혼합하여 30℃에서 48시간 동안 전배양한 후 2,000 rpm 조건으로 5분 동안 원심분리하였다. 원심분리를 통해 수득한 유산균 펠렛을 정제수와 혼합한 후 열처리한 산삼배양근 추출물의 분획물에 5%(v/v) 접종하여 30℃에서 24시간 동안 발효하였고, 발효가 끝난 후 5 ㎛ 카트리지 필터로 여과하고 고형분 60%로 농축하였다.Starter Lactobacillus powder Pediococcus pentosaceus ( Pediococcus pentosaceus ) HLJG0702 strain 10 g was mixed in 100 ml of MRS medium, pre-incubated for 48 hours at 30 ℃ and centrifuged for 5 minutes at 2,000 rpm conditions. Lactobacillus pellets obtained through centrifugation were mixed with purified water and then inoculated with 5% (v / v) of the heat-treated wild ginseng cultured root extract, fermented at 30 ° C. for 24 hours, and filtered through a 5 μm cartridge filter. And concentrated to 60% solids.
8. 수율 측정8. Yield Measurement
수율은 초기 투입한 시료의 무게 대비 최종 제조된 시료의 무게를 하기 계산식에 대입하여 계산하였다. Yield was calculated by substituting the weight of the final sample compared to the weight of the sample initially put into the following formula.
수율(%) = B / A x 100Yield (%) = B / A x 100
(A : 초기 사용된 시료의 무게, B : 최종 제조된 시료의 무게)(A: weight of sample used initially, B: weight of finished sample)
9. 총 진세노사이드 함량 분석9. Total Ginsenoside Content Analysis
시료 약 2 g을 50 ㎖ 부피의 플라스크에 취하고 증류수를 표선까지 채운 후 완전하게 용해시켜 시린지 필터(syringe filter, 0.45 μm)로 여과하였다.About 2 g of the sample was taken in a 50 ml volumetric flask, distilled water was filled to the mark, completely dissolved, and filtered through a syringe filter (0.45 μm).
총 진세노사이드는 고성능 액체크로마토그래피(high performance liquid chromatography, HPLC) 분석으로 실시하였다. 컬럼은 Capcell pak C18 column(Shiseido, 5 μm, 250x4.6 mm), 이동상은 아세토나이트릴(acetonitrile)과 물을 사용하였다. 시료 주입량은 10 ㎕, 유속은 1.0 ㎖/분, 온도는 30℃, UV 검출기는 203 nm에서 하기 표 1과 같은 이동상 조건으로 측정하였다.Total ginsenosides were performed by high performance liquid chromatography (HPLC) analysis. The column was a Capcell pak C18 column (Shiseido, 5 μm, 250 x 4.6 mm), and the mobile phase was acetonitrile and water. The sample injection amount was 10 μl, the flow rate was 1.0 ml / min, the temperature was 30 ° C., and the UV detector was measured under mobile phase conditions as shown in Table 1 below at 203 nm.
시험물질의 정량값은 측정값의 피크면적을 검량선 (y = ax + b) 식에 대입한 후 아래의 계산식에 의해 산출하였다. 또한 검량선은 x축에 농도를 y축에 피크면적을 적용하여 최소자승법으로 계산하였다. The quantitative value of the test substance was calculated by the following equation after substituting the peak area of the measured value into the calibration curve (y = ax + b) equation. The calibration curve was calculated by the least-squares method by applying the peak area to the y-axis and the concentration on the x-axis.
정량값 (mg/g) = C x (a x b)/S x 1/1,000Quantitative value (mg / g) = C x (a x b) /
(C : 시험 용액 중 개별 진세노사이드 농도(㎍/㎖),(C: individual ginsenoside concentration (μg / mL) in the test solution,
a : 시험 용액의 전량 (㎖), b : 희석배수a: total amount of test solution (ml), b: dilution factor
S : 시료 채취량 (g), 1/1,000 : 단위 환산 계수)S: sampling amount (g), 1 / 1,000: unit conversion factor)
실시예 1. 추출 조건별 수율 및 총 진세노사이드 함량 분석Example 1. Analysis of yield and total ginsenoside content according to extraction conditions
1-1. 추출 용매별 비교1-1. Comparison by Extraction Solvent
산삼배양근의 용매별 수율 및 총 진세노사이드 함량을 측정한 결과, 수율은 물 및 에탄올(20%, 40%, 60%, 70%, 80% 및 100%)의 전 구간에서 1.4% 이상인 것으로 확인되었고, 특히 70% 에탄올로 추출하였을 때 2.794±0.163%로 가장 높은 수율을 나타내었다.Solvent yield and total ginsenoside content of wild ginseng culture root were determined to be 1.4% or more in all sections of water and ethanol (20%, 40%, 60%, 70%, 80% and 100%). The highest yield was obtained with 2.794 ± 0.163%, especially when extracted with 70% ethanol.
또한, 총 진세노사이드 함량은 물 추출에서 26.28 mg/g이었고, 에탄올 농도가 증가할수록 총 진세노사이드 함량이 각각 30.46 mg/g, 35.39 mg/g, 48.38 mg/g, 48.08 mg/g, 55.96 mg/g, 66.83 mg/g으로 증가하였다. 모든 추출 용매별 조건에서 저분자 진세노사이드인 Rh4, Rg3, Rk1, Rg5 및 화합물 K(compound K)는 검출되지 않았지만, 20% 에탄올 추출부터 에탄올 농도가 증가할수록 Rb1, Rb2, Rd 함량이 증가하여 총 진세노사이드 함량이 증가된 것을 알 수 있었다(표 2). In addition, the total ginsenoside content was 26.28 mg / g in water extraction, and as the ethanol concentration increased, the total ginsenoside content was 30.46 mg / g, 35.39 mg / g, 48.38 mg / g, 48.08 mg / g, 55.96, respectively. mg / g, increased to 66.83 mg / g. The low molecular weight ginsenosides Rh4, Rg3, Rk1, Rg5 and compound K (compound K) were not detected under all extraction solvent conditions, but the contents of Rb1, Rb2 and Rd increased as the ethanol concentration increased from 20% ethanol extraction. It was found that the ginsenoside content was increased (Table 2).
1-2. 추출 시간별 비교1-2. Comparison by Extraction Time
산삼배양근의 추출 시간별 수율 및 총 진세노사이드 함량을 측정한 결과, 수율은 2시간 추출에서 2.270±0.094%로 가장 낮았고, 추출 시간이 증가할수록 수율도 증가하여 32시간 추출에서 3.018±0.037%로 가장 높았다. 이는 추출 시간이 증가할수록 용출되는 양이 증가하기 때문인 것으로 사료되었다.As a result of measuring the yield and total ginsenoside content of wild ginseng cultivated roots by the extraction time, the yield was the lowest as 2.270 ± 0.094% after 2 hours extraction, and the yield also increased as the extraction time increased to 3.018 ± 0.037% after 32 hours extraction. High. It is thought that this is because the amount eluted increases with increasing extraction time.
또한, 총 진세노사이드 함량은 6시간 추출에서 52.38 mg/g로 가장 낮았고 8시간 추출에서 60.56 mg/g로 가장 높았으며, 총 진세노사이드 함량은 추출 수율(용출되는 양)과 관련이 없는 것으로 판단되었다(표 3).In addition, the total ginsenoside content was the lowest as 52.38 mg / g at 6 hours extraction and the highest as 60.56 mg / g at 8 hours extraction, and the total ginsenoside content was not related to the extraction yield (eluted amount). Judgment was made (Table 3).
1-3. 추출 온도별 비교1-3. Comparison by Extraction Temperature
산삼배양근의 추출 온도별 수율 및 총 진세노사이드 함량을 측정한 결과, 수율은 온도가 높아질수록 점점 증가하다가 70℃에서 3.906±0.077%로 가장 높은 수율을 보였으며, 80℃에서는 감소하는 것으로 확인되었다.As a result of measuring the yield and total ginsenoside content of wild ginseng cultivated roots at different extraction temperatures, the yield increased with increasing temperature and showed the highest yield at 3.906 ± 0.077% at 70 ℃ and decreased at 80 ℃. .
또한, 총 진세노사이드 함량은 50℃에서 62.43 mg/g로 가장 높았으며, Rg1, Rb1 및 Rb2가 각각 12.26 mg/g, 19.26 mg/g 및 17.02 mg/g로 함유되어 있어 전체의 77%를 차지하는 것으로 확인되었다. 70℃에서는 총 진세노사이드 함량은 58.78 mg/g을 나타내었지만, 50℃에서 함량이 높았던 Rb1, Rb2가 감소하였고 50℃ 이하의 온도에서 검출되지 않았던 저분자 진세노사이드인 Rg2, Rh1, Rh4 및 Rk1이 검출되었다(표 4). 상기 결과를 통해, 고온의 추출 조건에 의해 고분자 진세노사이드가 저분자화됨을 유추할 수 있었다.In addition, the total ginsenoside content was the highest at 50.degree. 62.43 mg / g, and Rg1, Rb1 and Rb2 were 12.26 mg / g, 19.26 mg / g and 17.02 mg / g, respectively, representing 77% of the total. It was confirmed to occupy. The total ginsenoside content was 58.78 mg / g at 70 ° C, but the low molecular weight ginsenosides Rg2, Rh1, Rh4 and Rk1, which were high at 50 ° C, decreased Rb1, Rb2 and were not detected at temperatures below 50 ° C. Was detected (Table 4). Through the above results, it can be inferred that the polymer ginsenoside is low molecular weight by the high temperature extraction conditions.
1-4. 추출 횟수별 비교1-4. Comparison by Extraction Count
산삼배양근의 추출 횟수별 수율 및 총 진세노사이드 함량을 측정한 결과, 수율은 추출 횟수가 증가할수록 감소하였고, 총 진세노사이드 함량은 47.95~52.57 mg/g의 범위 내에서 비슷한 수준으로 관찰되었으며, 특히 3회 추출에서 가장 높은 것으로 확인되었다. As a result of measuring the yield and total ginsenoside content of wild ginseng cultivated roots, the yield decreased as the number of extraction increased, and total ginsenoside content was observed to be similar within the range of 47.95 ~ 52.57 mg / g. In particular, it was confirmed that the highest in the three extraction.
각각의 진세노사이드 함량을 비교해보면, 1회 추출과 2~4회 추출에서 서로 다른 경향을 보여주었는데, 진세노사이드 Rg1 및 Re의 함량은 추출 횟수가 증가할수록 감소하였고 Rb2는 3회 추출까지 증가하였으며, Rd 및 Rg6는 매회 증가하였고, 4회 추출에서는 이전에 검출되지 않았던 Rk1 및 Rg5가 각각 0.73 mg/g, 1.22 mg/g로 검출되었다(표 5). 이러한 결과는, 추출 횟수 증가에 따라 반복적인 고온 조건(50℃)이 가해짐에 따라 1회 추출에서 용출된 고분자 진세노사이드가 저분자화된 것으로 사료되었다.Comparing each ginsenoside content showed different trends in one extraction and two to four extractions. The contents of ginsenosides Rg1 and Re decreased with increasing extraction times and Rb2 increased up to three extractions. Rd and Rg6 increased each time, and Rk1 and Rg5, which were not detected before in four extractions, were detected at 0.73 mg / g and 1.22 mg / g, respectively (Table 5). These results suggest that the high molecular weight ginsenosides eluted in one extraction were applied as the repetitive high temperature condition (50 ° C.) was applied as the number of extractions increased.
실시예 2. 산삼배양근 추출물의 분획물 내의 진세노사이드 함량 및 수율 분석Example 2 Ginsenoside Content and Yield Analysis in Fractions of Wild Ginseng Cultured Root Extracts
산삼배양근 추출물의 Diaion HP-20 칼럼 크로마토그래피를 통해 수득된 물, 30% 에탄올 및 60% 에탄올 분획물의 수율은 각각 60.231±1.512%, 24.374±1.451%, 15.395±0.942%로 확인되었으나, 총 진세노사이드 함량은 물, 30% 에탄올 및 60% 에탄올 분획물이 각각 1.52±0.03 mg/g, 4.57±0.05 mg/g, 372.63±3.11 mg/g인 것으로 나타나, 60% 에탄올 분획물에서 총 진세노사이드 함량이 가장 높은 것을 확인하였다(표 6 및 도 1).Yields of water, 30% ethanol and 60% ethanol fractions obtained through Diaion HP-20 column chromatography of wild ginseng root extract were 60.231 ± 1.512%, 24.374 ± 1.451% and 15.395 ± 0.942%, respectively. The side content indicated that the water, 30% ethanol and 60% ethanol fractions were 1.52 ± 0.03 mg / g, 4.57 ± 0.05 mg / g and 372.63 ± 3.11 mg / g, respectively, resulting in a total ginsenoside content in the 60% ethanol fraction. The highest was confirmed (Table 6 and FIG. 1).
실시예 3. 산삼배양근 분획물의 열처리에 따른 진세노사이드 함량 분석Example 3 Analysis of Ginsenoside Content According to Heat Treatment of Wild Ginseng Cultured Root Fractions
산삼배양근 추출물의 60% 에탄올 분획물을 120℃에서 30분, 60분, 120분동안 각각 열처리하여 총 진세노사이드 함량을 분석한 결과, 각각 196.38±2.19 mg/g, 298.85±5.10 mg/g, 269.81±3.75 mg/g로 검출되었다(표 7 및 도 2). 60% ethanol fraction of wild ginseng cultured root extract was heat-treated at 120 ° C for 30, 60, and 120 minutes to analyze total ginsenoside content, respectively, 196.38 ± 2.19 mg / g, 298.85 ± 5.10 mg / g, 269.81 It was detected at ± 3.75 mg / g (Table 7 and Figure 2).
특히, 120℃에서 120분 동안 열처리했을 경우 저분자 진세노사이드인 Rg2, Rh1, Rg6, Rh4, Rg3, Rk1 및 Rg5의 함량이 열처리하지 않은 조건(표 6 참고)에 비해 현저히 증가한 것을 알 수 있었다.In particular, when the heat treatment for 120 minutes at 120 ℃ was found that the content of the low molecular weight ginsenosides Rg2, Rh1, Rg6, Rh4, Rg3, Rk1 and Rg5 significantly increased compared to the unheated condition (see Table 6).
실시예 4. 열처리된 산삼배양근 분획물의 발효물 내 진세노사이드 함량 분석Example 4 Analysis of Ginsenosides Content in Fermented Fermented Wild Ginseng Cultured Fractions
산삼배양근 분획물을 120℃에서 120분 동안 열처리하고, 페디오코커스 펜토사세우스(Pediococcus pentosaceus) HLJG0702 균주(기탁번호: KACC81017BP)를 이용하여 30℃에서 24시간 동안 발효시킨 후 진세노사이드 함량을 분석하였다.The wild ginseng root fraction was heat-treated at 120 ° C. for 120 minutes and fermented at 30 ° C. for 24 hours using Pediococcus pentosaceus HLJG0702 strain (Accession No .: KACC81017BP), followed by analysis of ginsenoside content. It was.
그 결과, 발효 단계를 거치지 않은 열처리된 산삼배양근 분획물(표 7)에 비해 열처리된 산삼배양근 분획물의 발효물 내의 고분자 진세노사이드(Rg1, Re, Rb2)의 함량이 감소한 반면, 저분자 진세노사이드 중 특히 Rg3, Rk1 및 Rg5의 함량은 현저하게 증가된 것으로 확인되었다(표 8). 특히, 열처리 및 발효 단계 전의 산삼배양근 에탄올 추출물에 비해 저분자 진세노사이드 Rg2, Rh1, Rg6, Rh4, Rg3, Rk1 및 Rg5의 함량이 현저히 증가된 것을 알 수 있었다(표 8).As a result, the content of the polymer ginsenosides (Rg1, Re, Rb2) in the fermented product of the heat treated wild ginseng root fraction (Rg1, Re, Rb2) decreased compared to the heat treated wild ginseng root fraction (Table 7) that did not undergo the fermentation step, In particular, the contents of Rg3, Rk1 and Rg5 were found to be significantly increased (Table 8). In particular, the content of low molecular weight ginsenosides Rg2, Rh1, Rg6, Rh4, Rg3, Rk1 and Rg5 was significantly increased compared to wild ginseng root ethanol extract before the heat treatment and fermentation step (Table 8).
상기 결과를 통해, 본 발명의 산삼배양근 추출물의 발효물의 제조방법은 열처리 및 발효 단계에 의해 진세노사이드의 저분자화를 유도하는 것으로 판단할 수 있었다.Through the above results, the production method of the fermentation product of wild ginseng cultured root extract of the present invention was determined to induce the low molecular weight of ginsenoside by the heat treatment and fermentation step.
Claims (5)
(b) 상기 (a) 단계의 산삼배양근 동결건조 분말을 에탄올로 추출하고 농축하여 농축액을 얻는 단계;
(c) 상기 (b) 단계의 농축액에 물을 혼합한 후 에탄올을 가하여 분획하여 에탄올 분획물을 얻는 단계;
(d) 상기 (c) 단계의 에탄올 분획물에 물을 혼합한 후 열처리하는 단계; 및
(e) 상기 (d) 단계의 열처리된 에탄올 분획물을 발효하는 단계;를 포함하는, 저분자 진세노사이드 함량이 증가된 산삼배양근 추출물의 발효물의 제조방법.(a) freeze-drying and grinding the cultured wild ginseng cultured root to obtain a wild ginseng cultured lyophilized powder;
(b) extracting the ginseng cultured lyophilized powder of step (a) with ethanol and concentrating to obtain a concentrate;
(c) mixing water with the concentrate of step (b) and then fractionating by adding ethanol to obtain an ethanol fraction;
(d) heat-treating the ethanol fraction of step (c) after mixing water; And
(e) fermenting the heat-treated ethanol fraction of step (d); comprising, fermented product of wild ginseng cultured root extract with increased ginsenoside content of low molecular weight.
(a) 조직배양한 산삼배양근을 동결건조하고 분쇄하여 산삼배양근 동결건조 분말을 얻는 단계;
(b) 상기 (a) 단계의 산삼배양근 동결건조 분말에 65~75%(v/v) 에탄올을 첨가하여 45~55℃에서 7~9시간씩 2~3회 반복 추출한 후 농축하여 농축액을 얻는 단계;
(c) 상기 (b) 단계의 농축액에 물을 동량으로 첨가하여 혼합한 후 55~65%(v/v) 에탄올을 가하여 분획하여 에탄올 분획물을 얻는 단계;
(d) 상기 (c) 단계의 에탄올 분획물에 물을 첨가하여 혼합한 혼합액을 115~125℃에서 25~125분 동안 열처리하는 단계; 및
(e) 상기 (d) 단계의 열처리된 혼합액에 페디오코커스 펜토사세우스(Pediococcus pentosaceus) HLJG0702 균주(기탁번호: KACC81017BP)를 접종하여 28~32℃에서 22~26시간 동안 발효하는 단계를 포함하는, 저분자 진세노사이드 함량이 증가된 산삼배양근 추출물의 발효물의 제조방법.The method of claim 1,
(a) freeze-drying and grinding the cultured wild ginseng cultured root to obtain a wild ginseng cultured lyophilized powder;
(b) 65 to 75% (v / v) ethanol was added to the wild ginseng root lyophilized powder of step (a), followed by repeated extraction 2 to 3 times at 45 to 55 ° C. for 2 to 3 times to obtain a concentrated solution. step;
(c) adding the same amount of water to the concentrate of step (b), followed by mixing to obtain 55-65% (v / v) ethanol and fractionating to obtain an ethanol fraction;
(d) heat-treating the mixed solution mixed with water by adding water to the ethanol fraction of step (c) for 25-125 minutes at 115-125 ° C; And
(e) inoculating Pediococcus pentosaceus ( Pediococcus pentosaceus ) HLJG0702 strain (Accession Number: KACC81017BP) on the heat-treated mixture of step (d) and fermenting at 28-32 ° C. for 22-26 hours. To, the method of producing a fermentation product of wild ginseng cultured root extract with increased low molecular weight ginsenosides content.
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KR20220134270A (en) * | 2021-03-26 | 2022-10-05 | 주식회사 화진바이오코스메틱 | Method for producing fermentative product of cultured mountain ginseng extract with increased small molecule ginsenoside content through methyl jasmonate treatment |
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