KR101952951B1 - Production method of black yeast culture liquid containing compound k, functional cosmetic and compound containing compound k - Google Patents
Production method of black yeast culture liquid containing compound k, functional cosmetic and compound containing compound k Download PDFInfo
- Publication number
- KR101952951B1 KR101952951B1 KR1020180142190A KR20180142190A KR101952951B1 KR 101952951 B1 KR101952951 B1 KR 101952951B1 KR 1020180142190 A KR1020180142190 A KR 1020180142190A KR 20180142190 A KR20180142190 A KR 20180142190A KR 101952951 B1 KR101952951 B1 KR 101952951B1
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- KR
- South Korea
- Prior art keywords
- wild ginseng
- callus
- compound
- weight
- black yeast
- Prior art date
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Abstract
Description
본 발명은 생물전환을 이용하여 산삼 캘러스로부터 컴파운드 케이가 함유된 흑효모 배양액을 수득하는 공정으로, 상세하게는 A. pullulans 균주를 이용하여 흑효모 배양시 산삼 캘러스 배양액을 첨가함으로써 미백과 주름개선의 효능을 가지는 베타글루칸 수득과 함께 첨가한 산삼 캘러스 배양액의 진세노사이드(proto panaxa diol, PPD)를 생물전환하여 항산화능이 높은 컴파운드 케이를 수득하는 공정에 관한 것이다. 더욱 상세하게는, 배양한 산삼 캘러스 착즙액을 기질로 하여 A. pullulans 균주를 통하여 생물전환한 배양액은 베타글루칸과 컴파운드 케이가 다량 함유되어 미백과 주름개선에 도움을 주는 기능성원료를 수득하여 화장품 및 식품에 사용할 수 있는 공정에 관한 것이다.The present invention is a process for obtaining a black yeast culture solution containing compound K from wild ginseng callus using bioconversion, in detail, by adding the ginseng callus culture when cultivating black yeast using A. pullulans strain of whitening and wrinkle improvement The present invention relates to a process for obtaining a compound k having high antioxidant capacity by bioconversion of ginsenoside (proto panaxa diol (PPD)) of wild ginseng callus culture added together with obtaining beta glucan. More specifically, the cultured bio-converted through the A. pullulans strain using the cultured wild ginseng callus juice as a substrate contains a large amount of beta glucan and compound K to obtain a functional raw material to help whitening and wrinkle improvement cosmetics and It relates to a process that can be used for food.
고려인삼(Panax ginseng C.A.Meyer)은 오가과 인삼속 다년생 작물이며, 동북아 지역에서 원기를 보충하는 약물로 널리 이용되어 온 중요한 한약재 중 하나이다.식품의약품안전처에 따르면, 인삼의 효능은 면역력개선, 피로회복, 기억력개선, 혈행개선, 항산화 효과를 가지고 있으며, 학술적인 측면에서 심혈관계, 면역계, 신경계에 대한 효능 및 항암, 항 당뇨 작용 등이 보고된 바 있다. 인삼은 재배형태에 따라 재배 인삼과 산삼으로 분류할 수 있는데, 야생에서 자생하는 산삼은 일반적인 인삼에 비해 그 생장속도가 느리다. 산삼을 상품적으로 사용하기 위해서는 약 10 년에서 15 년이 된 것을 사용하며 재배인삼에 비해서 약효가 뛰어나다고 보고되어 있다(Wang ZH, et al. Study on the wild ginseng and cultured ginseng in northeas of china. International symposium on ginseng. 1998:109-14) 이러한 이유로 산삼은 인삼에 비해 가격이 높고 연구가 많이 이루어지지 않은 실정이다.Korean ginseng (Panax ginseng CAMeyer) is a perennial crop of the genus Oga and ginseng, and is one of the important herbal medicines widely used as a refreshing drug in Northeast Asia. According to the Korea Food and Drug Administration, the efficacy of ginseng improves immunity and recovery of fatigue. It has the effect of improving memory, improving blood circulation, and antioxidation, and has been reported in terms of efficacy on the cardiovascular system, immune system, nervous system and anticancer and antidiabetic effects. Ginseng can be classified into cultivated ginseng and wild ginseng according to the cultivation type, and wild ginseng grows slower than general ginseng. The commercial use of wild ginseng is about 10 to 15 years old, and it is reported to be superior to cultivated ginseng (Wang ZH, et al. Study on the wild ginseng and cultured ginseng in northeas of china. International symposium on ginseng. 1998: 109-14) For this reason, wild ginseng is more expensive and less studied than ginseng.
인삼은 다양한 특이 성분들을 함유하고 있으며, 그 중에서도 진세노사이드는 많은 약리 활성을 나타내며 인삼의 주요 지표성분으로 알려져 있다. 알려진 진세노사이드는 총 38종이 있으며, 인삼에 24종, 홍삼에 32종이 존재하고, 그 중 18종이 인삼과 홍삼에 공통적으로 존재함이 알려져 있다. 수삼에는 Rb1, Rg1 등의 인삼 사포닌이 존재하나, 비교적 분자량이 크고 체내 흡수율이 낮다고 알려져 있다.Ginseng contains a variety of specific ingredients, among which ginsenosides have many pharmacological activities and are known as major indicators of ginseng. There are a total of 38 known ginsenosides, 24 species in ginseng and 32 species in red ginseng, and 18 kinds of ginsenosides are known to exist in common in ginseng and red ginseng. Although ginseng saponins such as Rb1 and Rg1 exist in fresh ginseng, it is known to have relatively high molecular weight and low body absorption rate.
인삼의 다양한 특이 성분 중 주요 지표성분인 진세노사이드는 담마린 골격 배당체에 결합하는 당의 개수와 위치에 따라 디올계와 트리올계 진세노사이드로 분류되며, 알려진 진세노사이드 총 38종 중 인삼에는 24종, 홍삼에는 32종의 진세노사이드가 있다. 가공하지 않은 수삼에 포함된 대표적인 진세노사이드인 Rb1, Rg1 등의 인삼 사포닌은 비교적 분자량이 크고 체내 흡수율이 낮다고 알려져 있다.Ginsenosides, which are the main indicators of various ginsengs, are classified into diol and triol ginsenosides according to the number and position of sugars bound to the dammarin skeletal glycosides. There are 32 kinds of ginsenosides in species and red ginseng. Ginseng saponins, such as Rb1 and Rg1, which are representative ginsenosides contained in raw ginseng, are known to have relatively high molecular weight and low body absorption.
반면, 진세노사이드 중 자연 상태의 인삼에 함유되어 있지 않은 컴파운드 케이 는 진세노사이드 중 유일하게 체내에서 전량 흡수되며, 장내 미생물의 생물전환에 의해 Rb1으로부터 생성되는 것으로 알려져 있다. 컴파운드 케이 의 효능에 대해 대한민국 등록특허 10-2013-0025926, 10-2009-0022928, 10-1897005 등의 다양한 특허가 개시되어 있으며, 항염증, 항알러지, 신경보호 등의 다양한 효능연구가 진행되어 있다.On the other hand, compound ginseng, which is not contained in ginseng in its natural state, is the only ginsenoside absorbed in the body and is known to be produced from Rb1 by bioconversion of intestinal microorganisms. Various patents such as Republic of Korea Patent Registration No. 10-2013-0025926, 10-2009-0022928, 10-1897005 have been disclosed for the efficacy of the compound K, and various efficacy studies such as anti-inflammatory, anti-allergic, neuroprotection, etc. have been conducted. .
이러한 생리활성을 가지는 컴파운드 케이를 이용하여 다양한 용도의 기능성 원료를 얻을 수 있으나, 자연 상태의 인삼에 함유되어 있지 않아 일반적으로 열처리, 산처리, 소화효소 처리 등의 가공을 통환 전환으로 컴파운드 케이를 획득한다. 인삼 원물의 비용 문제와 과 낮은 저해율로 인해, 컴파운드 케이의 높은 생리활성에도 불구하고 효율적인 원료 소재로써 평가받지 못하고 있는 실정이다.The compound k having the physiological activity can be used to obtain functional raw materials for various uses. However, since it is not contained in natural ginseng, the compound k is generally obtained by conversion through processing such as heat treatment, acid treatment, and digestive enzyme treatment. do. Due to the cost problem and low inhibition rate of ginseng raw materials, despite the high physiological activity of compound k, it has not been evaluated as an effective raw material.
또한, 대한민국 등록특허 제10-1676607호에는 야생에서 자생하는 산삼의 캘러스를 배양하여 초고압 유화기 추출을 통한 자외선 보호, 항염, 항산화 효능에 대한 특허가 개시되어 있다. 그러나 산삼 캘러스 배양만을 통하여 산삼 추출물의 유효성분 증대를 일으키고자 할 경우 특정한 진세노사이드의 선택적인 증대 효과를 기대하기는 어려운 단점이 있다.In addition, the Republic of Korea Patent No. 10-1676607 discloses a patent for ultraviolet protection, anti-inflammatory, antioxidant efficacy through the extraction of ultra-high pressure emulsifier by culturing wild ginseng callus growing in the wild. However, if you want to increase the active ingredient of wild ginseng extract only through the culture of wild ginseng callus, it is difficult to expect the selective increase effect of a particular ginsenoside.
이에 본 발명은 상기와 같은 문제점을 해결하기 위해 안출된 것으로서, 본 발명의 목적은 기능성 화장품을 비롯하여 식의약용 물질로 사용 가능한 유용 진세노사이드인 컴파운드 케이를 제어 가능한 환경에서 선택적으로 대량 획득하는 데 그 목적이 있다. Accordingly, the present invention has been made to solve the above problems, an object of the present invention is to selectively obtain a large amount of compound g, which is a useful ginsenoside that can be used as a food and pharmaceutical material in functional cosmetics in a controllable environment. The purpose is.
본 발명의 해결하고자 하는 과제는 이상에서 언급된 것들에 한정되지 않으며, 언급되지 아니한 다른 해결과제들은 아래의 기재로부터 당업자에게 명확하게 이해되어 질 수 있을 것이다.The problem to be solved of the present invention is not limited to those mentioned above, other problems not mentioned will be clearly understood by those skilled in the art from the following description.
이러한 본 발명의 목적을 달성하기 위한 수단으로서, 본 발명에 따른 컴파운드 케이의 획득 방법은 당사에서 개발한 A. pullulans 균주가 glucose를 이용하여 베타글루칸을 생성하는 과정에서 당 중합체에서 당을 이용한다는 착안점으로 산삼의 캘러스 배양을 통한 수득한 산삼 캘러스 착즙액에 포함된 진세노사이드를 다량으로 획득하는 단계와, 상기 산삼 캘러스 착즙액을 기질로 이용하여 A. pullulans 균주를 배양하는 단계 그리고 진세노사이드를 컴파운드케이로 생물전환을 유도하는 단계를 포함하는 것을 특징으로 한다.As a means for achieving the object of the present invention, the method of obtaining a compound K according to the present invention is an A. pullulans strain developed by the company focuses on the use of sugar in the sugar polymer in the process of producing beta glucan using glucose Obtaining a large amount of ginsenosides contained in wild ginseng callus juice obtained through the callus culture of wild ginseng, culturing the A. pullulans strain using the ginseng callus juice as a substrate and ginsenosides It is characterized in that it comprises the step of inducing bioconversion into compound k.
보다 구체적으로, 본 발명에 따른 컴파운드 케이를 함유하는 흑효모 배양액의 생산 방법은 산삼 캘러스를 배양하는 S1단계와; 배지에 흑효모(A. pullulans) 균주를 접종한 다음 배양하여 흑효모 균주발효액을 마련하고, 상기 흑효모 균주발효액에 상기 S1단계에서 배양된 산삼 캘러스를 첨가한 다음 배양하여 상기 산삼 캘러스에 함유된 진세노사이드를 컴파운드 케이로 생물전환하는 S2단계와; 상기 S2단계에서 생물전환된 흑효모 균주발효 배양액을 여과하고 열처리하는 S3단계;를 포함하는 것을 특징으로 한다.More specifically, the production method of the black yeast culture solution containing the compound K according to the present invention comprises the step S1 of culturing wild ginseng callus; Inoculated with a strain of black yeast (A. pullulans) in the medium and cultured to prepare a black yeast strain fermentation broth, and added to the black yeast strain fermentation broth cultured in the step S1 and then cultured contained in the wild ginseng callus S2 step of bioconversion ginsenoside to the compound K; Characterized in that it comprises a; S3 step of filtering and heat-treating the fermentation culture of the black yeast strain bioconverted in the step S2.
또한, 본 발명에 따른 컴파운드 케이를 함유하는 흑효모 배양액의 생산 방법에 있어서, S1단계는 산삼을 형성층이 드러나도록 절편 형태로 만들고 상기 절편을 고체배지 위에 치상하여 캘러스를 유도하는 단계와, 유도된 산삼 캘러스를 생물반응기를 이용하여 1차 배양 후 엘리시터 처리하는 단계와, 엘리시터 처리 후 2차 배양하여 수득한 산삼 캘러스 배양체를 착즙하여 산삼 캘러스 착즙액을 마련하며, 상기 S2단계에서 상기 흑효모 균주발효액에 첨가하는 산삼 캘러스는 상기 산삼 캘러스 착즙액이며, 상기 산삼 캘러스 착즙액의 첨가량은 상기 흑효모 균주발효액 총 중량의 10±2중량%인 것을 특징으로 한다.In addition, in the production method of the black yeast culture solution containing the compound K according to the present invention, step S1 is a step of inducing callus by making the wild ginseng in the form of fragments so that the forming layer is exposed and placing the sections on a solid medium. Preparing wild ginseng callus juice solution by extracting wild ginseng callus from the culture of wild ginseng callus obtained by the second culture after the primary culture using the bioreactor and the second culture after the eliminator treatment, and preparing the juice of the wild ginseng callus in the step S2. The wild ginseng callus added to the strain fermentation broth is the wild ginseng callus juice, the addition amount of the wild ginseng callus juice is characterized in that 10 ± 2% by weight of the total weight of the black yeast strain fermentation solution.
또한, 본 발명에 따른 컴파운드 케이를 함유하는 흑효모 배양액의 생산 방법에 있어서, S2단계의 배지는 설탕 5-20 중량%, 효모엑기스 1~2 중량%, 질산암모늄( NH4NO3) 0.05~2.0 중량%, 염화칼륨(KCl) 1.0~4.0 중량%, 제2인산칼륨(KH2PO4) 1.0~2.0 중량%, 황산마그네슘(MgSO4) 0.5~1.0 중량%, 황산제일철(FeSO4) 0.001~0.01 중량%와 잔량의 정제수로 이루어지는 것을 특징으로 한다.In addition, in the production method of the black yeast culture solution containing the compound K according to the present invention, the medium of step S2 is 5-20% by weight of sugar, 1-2% by weight of yeast extract, ammonium nitrate (NH 4 NO 3 ) 0.05 ~ 2.0% by weight, potassium chloride (KCl) 1.0-4.0% by weight, dibasic potassium phosphate (KH 2 PO 4 ) 1.0-2.0% by weight, magnesium sulfate (MgSO 4 ) 0.5-1.0% by weight, ferrous sulfate (FeSO 4 ) 0.001- It is characterized by consisting of 0.01% by weight and the residual amount of purified water.
이상과 같은 구성의 본 발명에 따른 A. pullulans 균주를 이용한 산삼 캘러스 생물전환에 따른 컴파운드 케이 생산 공정에 의하면, 화장품을 비롯하여 식의약용물질로 사용 가능한 컴파운드 케이를 소량의 산삼 원물로부터 대량으로 생산하여 사용할 수 있다.According to the compound k production process according to the wild ginseng callus bioconversion using the A. pullulans strain of the present invention as described above, by producing a large amount of compound k, which can be used as a food and pharmaceutical material, such as cosmetics from a small amount of raw ginseng raw materials Can be used.
본 발명의 효과는 이상에서 언급된 것들에 한정되지 않으며, 언급되지 아니한 다른 효과들은 아래의 기재로부터 당업자에게 명확하게 이해되어 질 수 있을 것이다.The effects of the present invention are not limited to those mentioned above, and other effects that are not mentioned will be clearly understood by those skilled in the art from the following description.
도 1 내지 3은 실시예 1의 산삼 캘러스의 생물전환 전,후 항산화능, 멜라닌 합성저해, 콜라겐 합성량을 비교한 그래프들이다.1 to 3 are graphs comparing the antioxidant capacity, melanin synthesis inhibition, collagen synthesis amount before and after bioconversion of wild ginseng callus of Example 1.
이하, 본 발명의 실시 예를 상세히 설명하면 다음과 같다.Hereinafter, an embodiment of the present invention will be described in detail.
본 발명을 설명함에 있어서, 관련된 공지기능 혹은 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우 그 상세한 설명은 생략한다. 또한, 후술되는 용어들은 본 발명에서의 기능을 고려하여 정의된 용어들로서 이는 사용자의 의도 또는 판례 등에 따라 달라질 수 있다. 그러므로 그 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다.In describing the present invention, if it is determined that the detailed description of the related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted. In addition, terms to be described below are terms defined in consideration of functions in the present invention, which may vary according to the intention or precedent of the user. Therefore, the definition should be made based on the contents throughout the specification.
본 발명은 소량의 산삼 원물로부터 대량의 산삼 캘러스를 배양한 뒤 A. pullulans 균주를 이용한 발효시 생물전환을 이용하여 다른 유효성분에 최대한 영향을 주지 않으면서 특정한 유효물질인 컴파운드 케이를 생물전환을 이용하여 대량으로 획득하면서 베타글루칸을 동시에 생산하는 방법을 제공함에 있다.The present invention is to cultivate a large amount of wild ginseng callus from a small amount of raw ginseng raw material and then use the bio conversion to the compound K, which is a specific active substance, without affecting the other active ingredients as much as possible using bio conversion in fermentation using A. pullulans strains. It provides a method of producing beta glucan at the same time while obtaining in large quantities.
본 발명에 따른 컴파운드 케이를 함유하는 흑효모 배양액의 생산 방법은 크게 산삼 캘러스를 배양하는 S1단계와, 배지에 흑효모 균주를 접종한 다음 배양하여 흑효모 균주발효액을 마련하고, 상기 흑효모 균주발효액에 상기 S1단계에서 배양된 산삼 캘러스를 첨가한 다음 배양하여 상기 산삼 캘러스에 함유된 진세노사이드를 컴파운드 케이로 생물전환하는 S2단계와, 상기 S2단계에서 생물전환된 흑효모 균주발효 배양액을 여과하고 열처리하는 S3단계를 포함하고 있다.The production method of the black yeast culture solution containing the compound K according to the present invention is largely S1 step of cultivating wild ginseng callus, inoculated with the black yeast strain in the medium and then cultured to prepare a black yeast strain fermentation solution, the black yeast strain fermentation solution S2 step of bioconversion of ginsenosides contained in the ginseng callus to compound K and culturing the black yeast strain fermentation broth bioconverted in step S2 were filtered after adding the wild ginseng callus cultured in step S1 to S3 step of heat treatment is included.
상기 S1단계의 산삼 캘러스 착즙액에는 진세노사이드 Re, 진세노사이드 Rb1, 진세노사이드 Gp17 등이 포함되어 있다.The wild ginseng callus juice of step S1 includes ginsenoside Re, ginsenoside Rb1, ginsenoside Gp17, and the like.
상기 S1단계는 산삼을 형성층이 드러나도록 절편 형태로 만들고 상기 절편을 고체배지 위에 치상하여 캘러스를 유도하는 단계와, 유도된 산삼 캘러스를 생물반응기를 이용하여 1차 배양 후 엘리시터 처리하는 단계와, 엘리시터 처리 후 2차 배양하여 수득한 산삼 캘러스 배양체를 착즙하여 산삼 캘러스 착즙액을 마련한다.The step S1 is a step of forming a ginseng to form a section to reveal the forming layer to induce callus by placing the section on a solid medium, and the first step of incubating the induced ginseng callus using a bioreactor, after the eliminator treatment; The wild ginseng callus culture obtained by the secondary culture after the eliminator treatment is juiced to prepare a ginseng callus juice solution.
상기 S2단계에 이용되는 균주인 A. pullulans 균주는 자연계에 존재하는 효모균의 일종으로, 세포증식을 촉진하고 손상된 세포를 회복시키며 콜라겐 분해를 저해하는 기능을 가진 베타글루칸을 다량으로 분비하며 이 균주를 이용한 생물전환 반응을 유도할 경우 베카글루칸을 포함한 산삼 캘러스 착즙액으로부터 컴파운드 케이를 수득할 수 있다. A. pullulans strain, which is a strain used in the step S2, is a kind of yeast that exists in nature, and secretes a large amount of beta glucan having a function of promoting cell proliferation, repairing damaged cells, and inhibiting collagen degradation. Compound k may be obtained from wild ginseng callus juice containing becaglucan when inducing a bioconversion reaction.
상기 S2단계에서 상기 흑효모 균주발효액에 첨가하는 산삼 캘러스는 상기 산삼 캘러스 착즙액이다. 그리고 상기 산삼 캘러스 착즙액의 첨가량은 상기 흑효모 균주발효액 총 중량의 10±2중량%인 것을 예시할 수 있다.The wild ginseng callus added to the black yeast strain fermentation solution in step S2 is the wild ginseng callus juice. And the addition amount of the wild ginseng callus juice may be exemplified that 10 ± 2% by weight of the total weight of the black yeast strain fermentation solution.
상기 A. pullulans 균주 발효를 72시간 내지 144시간 진탕 배양한 다음, 산삼 캘러스 착즙액 10%가 되도록 첨가하고 24시간 내지 144시간 jar fermentor 배양하여 생물전환이 이루어진다.The A. pullulans strain fermentation was carried out for 72 hours to 144 hours shaking culture, and then added to 10% wild ginseng callus juice solution and the culture of the jar fermentor for 24 hours to 144 hours.
더욱 바람직하게는 산삼 캘러스 착즙액 10% 첨가 후 멸균 작업을 거쳐 A. pullulans 균주 발효액에 접종 후 72시간 jar fermentor에서 96시간 배양함을 예시로 한다.More preferably, after inoculating the fermentation broth of A. pullulans strain after adding 10% of wild ginseng callus juice solution to sterilization, incubating 96 hours in a jar fermentor for 72 hours.
상기 S3단계로 A. pullulans 균주에 의한 생물전환 배양이 완료된 후 잔여물을 제거하기 위해 생물전환 처리한 흑효모 균주발효 배양액을 필터프레스로 여과하고, 잔류하는 균의 활성을 제거하기 위해 열처리한다.After the bioconversion culture by the A. pullulans strain is completed in step S3, the black yeast strain fermentation broth subjected to the bioconversion treatment is filtered through a filter press to remove the residue, and heat-treated to remove the remaining bacteria.
- 산삼 캘러스 배양-Wild ginseng callus culture
산삼 캘러스 배양은 통상적으로 사용되는 식물의 캘러스 유도 배양 및 생물배양기 배양을 통하여 배양을 진행하였다. 산삼 캘러스를 배양하기 위하여 필요한 산삼 원물은 덕유산에서 채취한 산삼을 구하여 사용하였다. 산삼 원물을 세척 후 산삼의 줄기 부분만을 절개한 뒤 겉껍질을 벗겨내어 원통형으로 가공한 산삼 원물을 절단면 방향으로 잘라내어 산삼의 형성층이 드러나도록 절편을 만들었다. 산삼 캘러스 유도를 위한 배지는 정제수에 1% MS(Murashige and Skoog Medium) powder와 3% 설탕, 1mg/L 디클로로페녹시아세트산, 2.1g/L gelite를 첨가한 뒤 pH를 5.8로 맞추고 autoclave 에 121℃, 15 분 처리 후 온도를 낮춘 고체배지를 사용하였다. 산삼 원물 절편을 배지 위에 치상한 후 캘러스를 유도하였다. 유도된 캘러스는 2주에 한번 다른 고체배지에 계대하여 배양하였다. 고체배지로 배양한 캘러스를 생물반응기를 이용한 액체배지에 배양하였다. 액체배양을 위한 생물배양기에 1% MS(Murashige and Skoog Medium) powder와 3% 설탕, 1mg/L 디클로로페녹시아세트산이 첨가된 정제수를 넣고 생물배양기를 멸균한 후, 배지 1L 당 60g의 산삼 캘러스를 접종한 뒤 5주간 공기를 주입하며 배양하였다. 이후 배양기의 입구를 개봉한 뒤 엘리시터를 처리하고 1주 배양을 진행한 뒤 배양을 종료하고 산삼 캘러스를 수득하였다. 수득한 산삼 캘러스는 착즙 형태로 제조하였다. 상기 캘러스 배양법은 통상적인 방법으로 진행하였다.Wild ginseng callus culture was carried out through the callus-induced culture and the culture culture of the commonly used plants. The wild ginseng raw material needed to cultivate wild ginseng callus was obtained by using wild ginseng collected from Deogyusan. After washing the raw ginseng raw material, only the stem portion of the wild ginseng was cut and then the outer shell was peeled off to cut the raw ginseng raw material processed into a cylindrical shape in the direction of the cutting surface to make a section to reveal the forming layer of the wild ginseng. The medium for induction of wild ginseng callus was 1% MS (Murashige and Skoog Medium) powder, 3% sugar, 1mg / L dichlorophenoxyacetic acid, 2.1g / L gelite in purified water, adjusted to pH 5.8, and 121 ℃ in autoclave. The solid medium was lowered after 15 minutes treatment. Wild ginseng original sections were placed on the medium and then callus was induced. Induced callus was incubated in another solid medium once every two weeks. Callus cultured in a solid medium was cultured in a liquid medium using a bioreactor. In a bioculture for liquid culture, 1% MS (Murashige and Skoog Medium) powder, 3% sugar, and 1mg / L dichlorophenoxyacetic acid were added to the purified water, sterilized the bioculture medium, and 60g of wild ginseng callus per 1L of medium was added. After inoculation, the cells were incubated with air for 5 weeks. Thereafter, after opening the inlet of the incubator, the eliminator was treated, and the culture was completed for one week. Then, the culture was terminated to obtain wild ginseng callus. The obtained wild ginseng callus was prepared in the form of juice. The callus culture method was performed in a conventional manner.
- 산삼 캘러스의 생물전환-Biotransformation of wild ginseng callus
A. pullulans 균주발효액으로부터 산삼 캘러스의 생물전환을 위하여 제조한 배지는 설탕 5-20 중량%, 효모엑기스 1-2 중량%, 질산암모늄 0.05-2.0 중량%, 염화칼륨 1.0-4.0 중량%, 제2인산칼륨 1.0-2.0 중량%, 황산마그네슘 0.5-1.0 중량%, 황산제일철 0.001-0.01 중량%와, 잔량의 정제수로 이루어지는 조성의 배지를 만들고, pH를 6.0으로 조정한다. 상기 배지를 통상의 방법으로 후라스크 배양과 5.0L의 Jar Frmentor배양을 하였다. 후라스크 배양은 100ml배지를 500ml 후라스크에 넣고 멸균 후 흑효모 균주(Aureobasidium pullulans)를 접종하여 28℃, 150rpm으로 5일간 배양하였으며, Jar Fermentor배양은 3.0L의 생산용 배지를 만들고 pH 6.0, 28℃, 500rpm, air 1.0vvm으로 4일간 배양하였다. 배양 후 산삼 캘러스 10중량% 첨가 후 28℃, 500rpm, air 1.0vvm으로 3일간 배양하였다.The medium prepared for the bioconversion of wild ginseng callus from A. pullulans strain fermentation broth was 5-20% by weight of sugar, 1-2% by weight of yeast extract, 0.05-2.0% by weight of ammonium nitrate, 1.0-4.0% by weight of potassium chloride, diphosphate A medium having a composition consisting of 1.0-2.0% by weight of potassium, 0.5-1.0% by weight of magnesium sulfate, 0.001-0.01% by weight of ferrous sulfate and the remaining amount of purified water was made, and the pH was adjusted to 6.0. The medium was cultured in a flask and cultured with 5.0 L Jar Frmentor in a conventional manner. In 100 ml medium, 100 ml medium was inoculated into a 500 ml glass flask and sterilized, inoculated with black yeast strain (Aureobasidium pullulans), and incubated at 28 ° C. and 150 rpm for 5 days, and Jar Fermentor culture produced 3.0 L of medium for production of pH 6.0, 28 Incubated for 4 days at ℃, 500rpm, air 1.0vvm. After incubation, 10 wt% of wild ginseng callus was added, and then incubated at 28 ° C., 500 rpm, and 1.0 vvm for 3 days.
본 실시예의 흑효모 균주(Aureobasidium pullulans)는 KJ 141로 명명된 균주를 사용하였으며 한국생명공학연구원 생물자원센터(KCTC)에 'KCTC 13548BP'로 등재되어 있다. The black yeast strain (Aureobasidium pullulans) of the present embodiment was used as a strain named KJ 141 and is listed as 'KCTC 13548BP' in the Korea Institute of Bioscience and Biotechnology (KCTC).
- 여과 및 열처리-Filtration and heat treatment
상기 A. pullulans 균주발효액으로부터 산삼 캘러스의 생물전환 발효액을 필터 여과를 통하여 균체를 제거한 생물전환된 배양액을 수득하였다. 교반 완료 후, 세균 및 효소의 활성을 제거하기 위하여 90℃에 10분 동안 열처리하였다.From the A. pullulans strain fermentation broth, the bioconverted fermentation broth of wild ginseng callus was obtained through filter filtration to obtain a bioconverted culture medium from which the cells were removed. After stirring was complete, heat treatment was carried out at 90 ℃ for 10 minutes to remove the activity of bacteria and enzymes.
[실험예 1]Experimental Example 1
상기 실시예 1의 흑효모 균주발효 배양액을 Agilent LC/MS 를 이용하여 컴파운드 케이 및 주요 진세노사이드의 함량 그리고 발효액에서의 베타글루칸 함량을 확인하였으며, 그 결과를 아래 표 1에 기재하였다.The black yeast strain fermentation broth of Example 1 was confirmed the content of the compound K and the main ginsenosides and the beta glucan content in the fermentation broth using Agilent LC / MS, the results are shown in Table 1 below.
표 1을 통해 확인할 수 있듯이, 생물전환 처리 시 Compound K의 함량이 증가된다는 것을 확인하였다. As can be seen from Table 1, it was confirmed that the content of Compound K is increased during the bioconversion treatment.
표 2를 통해서 알 수 있듯이, A. pullulans 균주 발효액에서의 베타글루칸 함량의 변화도 없었다. 따라서 A. pullulans 균주발효액으로부터 산삼 캘러스의 생물전환 과정을 통하여 컴파운드 케이를 효율적으로 얻을 수 있으면서 베타글루칸도 동시에 생산할 수 있음을 확인하였다.As can be seen from Table 2, there was no change of beta glucan content in A. pullulans strain fermentation broth. Therefore, it was confirmed that beta glucan can be produced at the same time as compound k can be efficiently obtained through the bioconversion process of wild ginseng callus from A. pullulans strain fermentation.
[실험예 2]Experimental Example 2
상기 실시예 1에서 생산된 처리 전 샘플과 생물전환된 샘플의 항산화능을 ABTS assay를 이용하여 확인하였다. 생쥐 흑색종 세포주 B16BL6 의 세포내 멜라닌 저해율을 통하여 미백효능을 확인하였다, 인간 섬유아세포 세포주 CCD986sk 세포주의 procollagen type 1 생성량을 ELISA로 측정하여 주름개선 효능(collagen 합성)을 확인하였으며, 그 결과를 도 2, 도 3 및 도 4에 각각 나타내었다.The antioxidant capacity of the pre-treated samples and the bioconverted samples produced in Example 1 was confirmed using the ABTS assay. The whitening efficacy of the mouse melanoma cell line B16BL6 was confirmed by the intracellular melanin inhibition rate. The procollagen type 1 production of the human fibroblast cell line CCD986sk cell line was measured by ELISA to confirm the antiwrinkle efficacy (collagen synthesis). 3 and 4, respectively.
도 1, 도 2, 도 3를 통해 확인할 수 있듯이, 생물전환 처리물의 화장품 원료로서의 기능성이 처리 전에 비해 전체적으로 증가하였음을 알 수 있다. 또한 생물전환이 일어나지 않은 처리 전 시료군 역시 미백 및 주름개선 효능이 확인됨에 따라, 생물전환을 통해 생산된 배양액 시료는 전환 전에 비해 기능적으로 진보한 기능성 화장품 원료로서의 가능성을 확인할 수 있다.As can be seen from FIG. 1, FIG. 2, and FIG. 3, it can be seen that the functionality of the bioconversion treatment product as a cosmetic raw material is increased as compared with before the treatment. In addition, as the sample group before the bioconversion does not occur also whitening and wrinkle improvement efficacy is confirmed, the culture sample produced through the bioconversion can confirm the potential as a functional cosmetic material functionally advanced than before the conversion.
한편, 본 발명의 상세한 설명에서는 구체적인 실시예에 관해 설명하였으나, 본 발명은 개시된 실시예에 한정되지 않고 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 있어 본 발명의 기술적 사상을 벗어나지 않는 범위 내에서 여러 가지 치환, 변형 및 변경이 가능하다. 따라서, 본 발명의 범위는 설명된 실시예에 국한되어 정해져서는 안되며 후술하는 특허청구범위뿐만 아니라 이 특허청구범위와 균등한 것들을 포함하는 것으로 해석되어야 할 것이다.On the other hand, in the detailed description of the present invention has been described with respect to specific embodiments, the present invention is not limited to the disclosed embodiments, but a range that does not depart from the spirit of the present invention for those skilled in the art to which the present invention pertains. Various substitutions, modifications and variations are possible within the scope of the invention. Therefore, the scope of the present invention should not be limited to the described embodiments, but should be construed as including not only the claims below but also equivalents thereof.
Claims (5)
배지에 흑효모(A. pullulans) 균주를 접종한 다음 배양하여 흑효모 균주발효액을 마련하고, 상기 흑효모 균주발효액에 상기 S1단계에서 배양된 산삼 캘러스를 첨가한 다음 배양하여 상기 산삼 캘러스에 함유된 진세노사이드를 컴파운드 케이로 생물전환하는 S2단계와;
상기 S2단계에서 생물전환된 흑효모 균주발효 배양액을 여과하고 열처리하는 S3단계;
를 포함하는 것을 특징으로 하는 컴파운드 케이를 함유하는 흑효모 배양액의 생산 방법.
S1 step of culturing wild ginseng callus;
Inoculated with a strain of black yeast (A. pullulans) in the medium and then cultured to prepare a black yeast strain fermentation solution, and added to the black yeast strain fermentation broth cultured in the step S1 and then cultured contained in the wild ginseng callus S2 step of bioconversion ginsenoside to the compound K;
Step S3 of filtering and heat-treating the fermentation broth of the black yeast strain bioconverted in step S2;
Method for producing a black yeast culture solution containing a compound K, characterized in that it comprises a.
상기 S1단계는,
산삼을 형성층이 드러나도록 절편 형태로 만들고 상기 절편을 고체배지 위에 치상하여 캘러스를 유도하는 단계와, 유도된 산삼 캘러스를 생물반응기를 이용하여 1차 배양 후 엘리시터 처리하는 단계와, 엘리시터 처리 후 2차 배양하여 수득한 산삼 캘러스 배양체를 착즙하여 산삼 캘러스 착즙액을 마련하며,
상기 S2단계에서 상기 흑효모 균주발효액에 첨가하는 산삼 캘러스는 상기 산삼 캘러스 착즙액이며,
상기 산삼 캘러스 착즙액의 첨가량은 상기 흑효모 균주발효액 총 중량의 10±2중량%인 것을 특징으로 하는 컴파운드 케이를 함유하는 흑효모 배양액의 생산 방법.
The method of claim 1,
The step S1,
Inducing ginseng to form a section to reveal the formation layer of wild ginseng, and inducing the callus by placing the section on a solid medium, and after the primary culture of the induced ginseng callus using a bioreactor, after the eliminator treatment The wild ginseng callus culture obtained by the secondary culture is juiced to prepare a wild ginseng callus juice,
The wild ginseng callus added to the black yeast strain fermentation solution in step S2 is the wild ginseng callus juice,
The addition amount of the wild ginseng callus juice solution is a method for producing a black yeast culture solution containing a compound K, characterized in that 10 ± 2% by weight of the total weight of the black yeast strain fermentation broth.
상기 S2단계의 배지는 설탕 5-20 중량%, 효모엑기스 1~2 중량%, 질산암모늄( NH4NO3) 0.05~2.0 중량%, 염화칼륨(KCl) 1.0~4.0 중량%, 제2인산칼륨(K2HPO4) 1.0~2.0 중량%, 황산마그네슘(MgSO4) 0.5~1.0 중량%, 황산제일철(FeSO4) 0.001~0.01 중량%와 잔량의 정제수로 이루어지는 것을 특징으로 하는 컴파운드 케이를 함유하는 흑효모 배양액의 생산 방법.
The method of claim 1,
The medium of step S2 is 5-20% by weight of sugar, 1-2% by weight of yeast extract, 0.05-2.0% by weight of ammonium nitrate (NH 4 NO 3 ), 1.0-4.0% by weight of potassium chloride (KCl), dibasic potassium phosphate ( K 2 HPO 4 ) 1.0 to 2.0% by weight, magnesium sulfate (MgSO 4 ) 0.5 to 1.0% by weight, ferrous sulfate (FeSO 4 ) 0.001 to 0.01% by weight and the remaining amount of purified water containing a black black Method of producing yeast culture.
Functional cosmetics containing a compound k, characterized in that it contains a black yeast culture solution produced by any one of claims 1 to 3.
A compound containing a compound k, characterized in that it contains a black yeast culture solution produced by any one of claims 1 to 3.
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