KR102183814B1 - Preparation method of liquid culture of Lentinus edode mycelia and the liquid culture of Lentinus edode mycelia - Google Patents
Preparation method of liquid culture of Lentinus edode mycelia and the liquid culture of Lentinus edode mycelia Download PDFInfo
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Abstract
Description
본 발명은 효소활성이 우수한 표고버섯 균사체 배양액 조성물의 제조방법 및 그 배양액 조성물에 관한 것으로, 구체적으로는 표고버섯 자실체를 배양 배지로 사용하여 효소활성이 높은 표고버섯 균사체 배양액 조성물을 제조하고 이를 화장료 등의 제조에 이용하는 기술에 관한 것이다.The present invention relates to a method for preparing a shiitake mushroom mycelium culture solution composition having excellent enzyme activity, and to a culture solution composition thereof, and specifically, a shiitake mushroom mycelium culture solution composition having high enzyme activity is prepared by using the shiitake mushroom fruiting body as a culture medium. It relates to a technology used in the manufacture of.
천연물로부터 유용 성분 및 생리활성 물질 등의 추출 효율을 높이기 위한 방법으로는 고온추출법, 고압추출법, 초음파추출법, 초임계추출법, 효소 반응 추출법 등 다양한 추출 방법이 있다. 그중에서도 효소 반응 추출법(Enzymatic Extraction, Enzyme-assisted Extraction)은 효소를 이용하여 추출 효율을 높이는 추출법이다. 효소는 생물이 생성하는 단백질로 화학반응을 촉진시키는 촉매이다. 화학촉매는 유기용매의 사용, 높은 온도와 압력 사용 조건이 필요한데 비해 효소는 온건한 반응조건에서 촉매력과 기질특이성이 높고 부산물의 생성이 적다는 장점이 있다. 또한, 유기용매를 사용하지 않고 수용액에서 반응시키기에 환경 친화적이다. There are various extraction methods such as high temperature extraction, high pressure extraction, ultrasonic extraction, supercritical extraction, and enzyme reaction extraction as methods for increasing the extraction efficiency of useful components and physiologically active substances from natural products. Among them, Enzymatic Extraction (Enzyme-assisted Extraction) is an extraction method that improves extraction efficiency using enzymes. Enzymes are proteins produced by living organisms and are catalysts that accelerate chemical reactions. Chemical catalysts require the use of organic solvents and high temperature and pressure conditions, whereas enzymes have the advantage of having high catalytic power and substrate specificity under moderate reaction conditions and less generation of by-products. In addition, it is environmentally friendly to react in an aqueous solution without using an organic solvent.
버섯은 균류 중 자실체를 형성하는 생물로, 생물학적으로는 진균류에 속한다. 버섯류는 영양기관인 균사체(Mycelium)와 번식기관인 포자를 지닌 자실체(Fruit body)로 되어 있는데, 균사체는 식물의 뿌리, 줄기, 잎에 해당되며, 자실체는 꽃에 해당된다. 버섯은 뿌리, 줄기, 잎의 구별이 없고 엽록소가 없어 균사를 이용하여 다른 생물이 생성해놓은 양분을 흡수하여 살아간다. 버섯은 양분 흡수 방법에 따라 부생균, 목재부후균, 균근류, 기생균으로 나눌 수 있다. Mushrooms are organisms that form fruiting bodies among fungi, and biologically belong to fungi. Mushrooms are composed of mycelium, which is a nutritional organ, and a fruit body with spores, which is a breeding organ, and the mycelium corresponds to the roots, stems, and leaves of plants, and the fruiting body corresponds to flowers. Mushrooms have no distinction between roots, stems, and leaves, and do not have chlorophyll, so they use mycelium to absorb nutrients generated by other organisms. Mushrooms can be divided into by-products, wood decay bacteria, mycorrhizal fungi, and parasites according to the nutrient absorption method.
표고버섯(Lentinus edodes)은 식물의 분류학상 진균문, 담자균강, 주름버섯목, 느타리과에 속하며, 넓은잎나무인 밤나무, 졸참나무, 상수리나무 등의 마른나무에 자라며, 자연의 임야와 인공재배에 의해 모두 생산 할 수 있다. 표고버섯은 목재부후균으로 균사체가 가지고 있는 효소를 이용하여 배지를 부패시키고 분해시켜 필요한 영양분을 섭취하여 성장한다. 표고버섯의 균사체는 소화되지 않는 다당류인 셀룰로오스, 헤미셀룰로오스, 리그닌 등을 스스로의 효소에 의하여 대사에 이용할 수 있는 성분으로 분해한다.Shiitake (Lentinus edodes) belongs to the family Fungi, Basidiomyces, Pleurotus oysteraceae, oyster family, and grows on dry trees such as broad-leaf trees such as chestnut, Japanese oak, and oak, by natural forestry and artificial cultivation. All can be produced. Shiitake mushrooms are wood decay bacteria and grow by ingesting necessary nutrients by decaying and decomposing the medium using enzymes of mycelium. The mycelium of shiitake mushrooms decomposes indigestible polysaccharides such as cellulose, hemicellulose, and lignin into components that can be used for metabolism by their own enzymes.
한편, 표고버섯 자실체에는 단백질과 지질, 탄수화물, 회분, 칼슘, 인, 철, 나트륨, 칼륨 등의 무기질과 비타민(B1, B2) 등이 많이 들어있어 영양소가 풍부하다.On the other hand, the fruiting body of shiitake mushrooms contains a lot of minerals such as protein, lipid, carbohydrate, ash, calcium, phosphorus, iron, sodium, potassium, and vitamins (B1, B2), and is rich in nutrients.
본 발명자들은 표고버섯 균사체 유래 효소의 생산 효율을 높이는 방법에 대하여 연구하던 중, 표고버섯 자실체 추출물을 배양 배지로 사용하여 특정 방법으로 제조된 표고버섯 균사체 배양액 조성물이 화장품 등의 원료로 사용되는 천연물의 추출 효율성 증대 및 활성 증대 효과를 나타내어 천연물의 추출 촉매제 등으로 유용하게 사용될 수 있음을 발견하여 본 발명을 완성하였다. The inventors of the present invention were studying a method to increase the production efficiency of enzymes derived from shiitake mushroom mycelium, and the shiitake mushroom mycelium culture solution composition prepared by a specific method using the shiitake fruiting body extract as a culture medium is a natural product used as a raw material for cosmetics, etc. The present invention was completed by discovering that it can be usefully used as a catalyst for extraction of natural products by showing an effect of increasing extraction efficiency and increasing activity.
본 발명은 표고버섯을 배양 배지로 사용하여 효소활성이 높은 표고버섯 균사체 배양액 조성물을 제조하는 방법을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a method for preparing a shiitake mushroom mycelium culture solution composition having high enzyme activity by using shiitake mushrooms as a culture medium.
또한, 본 발명은 상기 제조방법에 의하여 제조되어 천연물의 추출 촉매제 또는 추출용매로 사용될 수 있는 표고버섯 균사체 배양액 조성물을 제공하는 것을 다른 목적으로 한다.In addition, another object of the present invention is to provide a shiitake mushroom mycelium culture solution composition that can be used as an extraction catalyst or extraction solvent of natural products by the above production method.
상기 목적을 달성하기 위하여 본 발명에 따르면, (A) 표고버섯 자실체를 추출하여 액체 배양배지를 제조하는 단계; (B) 표고버섯 균사체를 접종하는 단계; (C) 20~30℃, 진탕 배양(100~150rpm) 조건으로, 7~21일 동안 배양하는 단계; (D) 배양 종료 후 배양액 전체를 블렌더(blender) 처리하여 세포벽을 파쇄하는 단계; 및 (E) 블렌더(blender) 처리한 배양액을 45~55℃에서 24~72시간 동안 배양하며 자가 소화시키는 단계를 포함하는 표고버섯 균사체 배양액 조성물의 제조 방법이 제공된다.According to the present invention to achieve the above object, (A) preparing a liquid culture medium by extracting the shiitake fruiting body; (B) inoculating shiitake mushroom mycelium; (C) 20 ~ 30 ℃, in the shaking culture (100 ~ 150rpm) conditions, culturing for 7 ~ 21 days; (D) crushing the cell wall by processing the entire culture medium after completion of the culture with a blender; And (E) there is provided a method for producing a shiitake mushroom mycelium culture solution composition comprising the step of self-digesting and incubating the culture solution treated with a blender at 45 to 55°C for 24 to 72 hours.
상기 (A) 단계에서의 배지는 표고버섯 자실체를 추출용매와 혼합하고 90~100℃에서 2시간동안 추출하여 제조되는 것임을 특징으로 한다. 상기 (D) 단계에서 블렌더(blender) 처리는 10,000~20,000 rpm의 속도로 3~5분간 이루어지는 것임을 특징으로 한다.The medium in step (A) is characterized in that it is prepared by mixing the shiitake fruiting bodies with an extraction solvent and extracting them at 90 to 100°C for 2 hours. In the step (D), the blender treatment is characterized in that it is performed for 3 to 5 minutes at a speed of 10,000 to 20,000 rpm.
상기 다른 목적을 달성하기 위하여 본 발명에 따르면 상기 제조방법에 의하여 제조된 표고버섯 균사체 배양액 조성물이 제공된다. 상기 균사체 배양액 조성물은 천연물의 추출 촉매제 또는 추출용매로 사용되는 것임을 특징으로 한다.In order to achieve the above other object, according to the present invention there is provided a shiitake mushroom mycelium culture solution composition prepared by the above manufacturing method. The mycelium culture liquid composition is characterized in that it is used as an extraction catalyst or extraction solvent of natural products.
본 발명에 따라 제조되는 표고버섯 균사체 배양액 조성물은 효소활성이 우수하여 화장품 원료 등으로 사용되는 천연물의 추출 촉매제 또는 추출 용매로써 유용하게 사용될 수 있다. The shiitake mushroom mycelium culture solution composition prepared according to the present invention has excellent enzyme activity and can be usefully used as an extraction catalyst or extraction solvent for natural products used as raw materials for cosmetics.
이하, 본 발명을 더욱 구체적으로 설명한다.Hereinafter, the present invention will be described in more detail.
종래 표고버섯 균사체는 천연물의 발효균주로 사용되어 왔다. 대한민국 공개특허 제10-2017-0078900호 "앵무새나무 발효 추출물을 유효성분으로 함유하는 화장료 조성물"에는 앵무새나무의 발효물 제조에 표고버섯 균사체 등을 사용하는 것이 개시되어 있다.Conventionally, shiitake mushroom mycelium has been used as a natural fermentation strain. Republic of Korea Patent Laid-Open Patent No. 10-2017-0078900 "Cosmetic composition containing fermented parrot tree fermentation extract as an active ingredient" discloses the use of shiitake mushroom mycelium in the manufacture of fermented parrot tree.
또한 대한민국 공개특허 제10-2012-0078327호 "표고버섯 균사체 배양물에서 추출된 다당체를 유효성분으로 함유하는 보습 및/또는 미백용 화장료 조성물"에는 표고버섯 균사체 배양물 자체가 미백 또는 보습 화장료로 사용될 수 있다는 것이 개시되어 있다.In addition, Korean Patent Application Publication No. 10-2012-0078327 "Moisturizing and/or whitening cosmetic composition containing polysaccharides extracted from shiitake mushroom mycelium cultures as an active ingredient" includes the shiitake mushroom mycelium culture itself to be used as a whitening or moisturizing cosmetic. It is disclosed that you can.
본 발명은 표고버섯 자실체를 배양배지로 사용하고, 2단계의 효소 생성 증대 과정을 거쳐 제조되는 표고버섯 균사체 배양액 조성물이 우수한 효소활성을 나타낸다는 것을 기술적 특징으로 한다.The present invention is a technical feature that the shiitake mushroom mycelium culture solution composition prepared by using the shiitake fruiting body as a culture medium and undergoing a two-step enzyme production enhancement process exhibits excellent enzyme activity.
본 발명에 따르면, (A) 표고버섯 자실체를 추출하여 액체 배양배지를 제조하는 단계; (B) 표고버섯 균사체를 접종하는 단계; (C) 20~30℃, 진탕 배양(100~150rpm) 조건으로, 7~21일 동안 배양하는 단계; (D) 배양 종료 후 배양액 전체를 블렌더(blender) 처리하여 세포벽을 파쇄하는 단계; 및 (E) 블렌더(blender) 처리한 배양액을 45~55℃에서 24~72시간 동안 배양하며 자가 소화시키는 단계를 포함하는 표고버섯 균사체 배양액 조성물의 제조 방법이 제공된다. According to the present invention, (A) preparing a liquid culture medium by extracting the fruiting body of shiitake mushrooms; (B) inoculating shiitake mushroom mycelium; (C) 20 ~ 30 ℃, in the shaking culture (100 ~ 150rpm) conditions, culturing for 7 ~ 21 days; (D) crushing the cell wall by processing the entire culture medium after completion of the culture with a blender; And (E) there is provided a method for producing a shiitake mushroom mycelium culture solution composition comprising the step of self-digesting and incubating the culture solution treated with a blender at 45 to 55°C for 24 to 72 hours.
상기 (A) 단계에서의 배지는 표고버섯 자실체를 추출용매와 혼합하고 90~100℃에서 2시간동안 추출하여 제조되는 것이다. 추출용매로는 물을 사용할 수 있고, 에탄올, 메탄올, 아세톤 등과 같이 표고버섯 자실체 성분들을 유용하게 추출할 수 있는 용매를 사용할 수도 있다.The medium in the step (A) is prepared by mixing the shiitake fruiting bodies with an extraction solvent and extracting them at 90 to 100°C for 2 hours. Water may be used as the extraction solvent, and a solvent capable of usefully extracting the fruiting components of shiitake mushrooms, such as ethanol, methanol, and acetone, may be used.
상기 표고버섯 자실체는 상기 혼합물 전체 중량에 대하여 1~10중량%의 비율로 혼합되는 것이 바람직하다. The shiitake mushroom fruit body is preferably mixed in a ratio of 1 to 10% by weight based on the total weight of the mixture.
상기 (D) 단계에서 블렌더(blender) 처리는 10,000~20,000 rpm의 속도로 3~5분간 처리하는 것이 바람직하다.In the step (D), the blender treatment is preferably performed for 3 to 5 minutes at a speed of 10,000 to 20,000 rpm.
영양성분이 풍부한 표고버섯 자실체를 배지로 하고, 블렌더 처리를 통한 세포벽 파쇄 및 자가소화 유도의 2단계 공정을 통하여 제조되는 본 발명의 표고버섯 균사체 배양액 조성물은 효소활성이 크게 증대되는 것을 확인할 수 있었다(시험예). It was confirmed that the enzyme activity of the shiitake mushroom mycelium culture solution composition of the present invention prepared through a two-step process of cell wall disruption and induction of self-digestion through a blender treatment, using a nutrient-rich shiitake fruiting body as a medium ( Test example).
그러므로 본 발명의 표고버섯 균사체 배양액 조성물은 화장품, 의약품, 식품 등의 원료로 사용되는 천연물의 추출 촉매제 또는 추출용매로 유용하게 사용될 수 있다.Therefore, the shiitake mushroom mycelium culture solution composition of the present invention can be usefully used as an extraction catalyst or extraction solvent of natural products used as raw materials for cosmetics, pharmaceuticals, and foods.
[실시예][Example]
이하, 본 발명을 하기 실시예 및 시험예에 의거하여 더욱 상세하게 설명하고자 한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명은 하기 실시예에 의해 한정되는 것이 아니다.Hereinafter, the present invention will be described in more detail based on the following Examples and Test Examples. However, the following examples are for illustrative purposes only, and the present invention is not limited by the following examples.
실시예 1 ~ 5: 표고버섯 균사체 배양액 조성물 제조Examples 1 to 5: Preparation of shiitake mushroom mycelium culture solution composition
표고버섯 균사체를 하기 표 1의 비율로 용매인 정제수에 혼합한 후 90~100℃에서 2시간동안 추출한 다음 여과하여 여액을 수득하였다. 수득한 여액을 121℃에서 20분간 멸균처리하여 액체 배양배지를 제조하였다. Shiitake mushroom mycelium was mixed with purified water as a solvent in the ratio of Table 1, extracted for 2 hours at 90 to 100°C, and filtered to obtain a filtrate. The obtained filtrate was sterilized at 121° C. for 20 minutes to prepare a liquid culture medium.
PDA(Potato Dextrose Agar) 평판배지에 계대 배양하여 준비한 접종용 표고버섯 균사체를 1cm X 1cm의 크기로 잘라 상기 제조된 액체 배양배지에 접종하여, 25℃의 온도에서, 14일간 진탕 배양(100rpm)하였다. 이후 여과 및 원심분리하여 균사체를 제거하여 표고버섯 균사체 배양액 조성물을 제조하였다. The shiitake mushroom mycelium for inoculation prepared by subculturing on PDA (Potato Dextrose Agar) plate medium was cut into a size of 1cm X 1cm and inoculated in the prepared liquid culture medium, and cultured with shaking (100rpm) for 14 days at a temperature of 25°C. . Thereafter, the mycelium was removed by filtration and centrifugation to prepare a shiitake mushroom mycelium culture solution composition.
실시예 6 ~ 10: 표고버섯 균사체 배양액 조성물 제조Examples 6 to 10: Preparation of Shiitake Mushroom Mycelium Culture Solution Composition
하기 표 2의 조성으로 상기 실시예 1~5와 동일한 방법으로 액체 배양배지를 제조한 후, PDA(Potato Dextrose Agar) 평판배지에 계대 배양하여 준비한 접종용 표고버섯 균사체를 1cm X 1cm의 크기로 잘라 상기 제조된 액체 배양배지에 접종하여, 25℃의 온도에서, 14일간 진탕 배양(100rpm)하였다. After preparing a liquid culture medium in the same manner as in Examples 1 to 5 with the composition of Table 2, cut the shiitake mushroom mycelium for inoculation prepared by subculturing on a PDA (Potato Dextrose Agar) plate medium to a size of 1 cm X 1 cm. It was inoculated into the prepared liquid culture medium and cultured with shaking (100 rpm) for 14 days at a temperature of 25°C.
배양액을 waring blender로 3분간 처리하여 균사체의 세포벽을 파쇄하였다. 이후 여과 및 원심 분리하여 균사체를 제거하여 표고버섯 균사체 배양액 조성물을 제조하였다.The culture medium was treated with a waring blender for 3 minutes to disrupt the cell walls of the mycelium. Thereafter, the mycelium was removed by filtration and centrifugation to prepare a shiitake mushroom mycelium culture solution composition.
실시예 11 ~ 15: 표고버섯 균사체 배양액 조성물 제조Examples 11 to 15: Preparation of Shiitake Mushroom Mycelium Culture Solution Composition
하기 표 3의 조성으로 상기 실시예 1~5와 동일한 방법으로 액체 배양배지를 제조한 후, PDA(Potato Dextrose Agar) 평판배지에 계대 배양하여 준비한 접종용 표고버섯 균사체를 1cm X 1cm의 크기로 잘라 상기 제조된 액체 배양배지에 접종하여, 25℃의 온도에서, 14일간 진탕 배양(100rpm)하였다. 배양액을 waring blender로 3분간 처리하여 균사체의 세포벽을 파쇄하였다. After preparing a liquid culture medium in the same manner as in Examples 1 to 5 with the composition of Table 3, cut the shiitake mushroom mycelium for inoculation prepared by subculturing on a PDA (Potato Dextrose Agar) plate medium to a size of 1 cm X 1 cm. It was inoculated into the prepared liquid culture medium and cultured with shaking (100 rpm) for 14 days at a temperature of 25°C. The culture medium was treated with a waring blender for 3 minutes to disrupt the cell walls of the mycelium.
이어서 세포벽이 파쇄된 배양액을 45℃에서 60시간 동안 배양하며 자가 소화를 유도하였다. 이후 여과 및 원심 분리하여 균사체를 제거하여 표고버섯 균사체 배양액 조성물을 제조하였다.Subsequently, the culture medium having the cell wall crushed was cultured at 45° C. for 60 hours to induce self-digestion. Thereafter, the mycelium was removed by filtration and centrifugation to prepare a shiitake mushroom mycelium culture solution composition.
비교예 1: 표고버섯 균사체 배양액 조성물 제조Comparative Example 1: Preparation of Shiitake Mushroom Mycelium Culture Solution Composition
일반적인 액체 배양배지인 PDB(Potato Dextrose Broth) 배지를 2.4% 사용한 것을 제외하고는 상기 실시예 12와 동일한 방법으로 하여 표고버섯 균사체 배양액 조성물을 제조하였다.A shiitake mushroom mycelium culture solution composition was prepared in the same manner as in Example 12, except that 2.4% of a general liquid culture medium, PDB (Potato Dextrose Broth) medium was used.
시험예 1: 표고버섯 균사체 배양액 조성물의 단백질 정량Test Example 1: Quantification of protein in shiitake mushroom mycelium culture solution composition
표고버섯 균사체 배양액 조성물에 포함된 단백질 함량을 확인하기 위해 Bradford 법을 수행하였다. 효소는 단백질의 일종으로 단백질 함량이 높을수록 생성된 효소의 양도 높다는 것을 의미한다. 그 결과를 표 4에 나타내었다.The Bradford method was performed to check the protein content contained in the shiitake mushroom mycelium culture composition. An enzyme is a type of protein, meaning that the higher the protein content, the higher the amount of enzyme produced. The results are shown in Table 4.
시험예 2: 표고버섯 균사체 배양액 조성물의 효소 활성 확인Test Example 2: Enzyme Activity Confirmation of Shiitake Mushroom Mycelium Culture Solution Composition
상기 실시예 및 비교예의 표고버섯 균사체 배양액 조성물의 효소 활성을 확인하기 위해 API ZYM kit를 이용하여 분석하였다. API ZYM kit는 19종의 가수분해효소 기질을 포함하고 있는 enzyme assay kit이다. API ZYM kit의 각 큐플에 시료를 65㎕씩 분주하여 37℃에서 4시간동안 배양 한 후 반응시약을 각 큐플에 한 방울씩 첨가한 다음 5분 후 반응색의 변화정도를 확인하여 효소 활성을 측정하였다. 각 큐플의 색의 변화정도에 따라 0~5까지의 값으로 표시할 수 있으며, 0은 음성반응, 5는 최대 강도의 반응이고 1~4는 중간 반응 값이다. 반응색의 변화정도가 높을수록 효소 활성이 높다는 것을 의미하며, 그 결과를 하기 표 5에 나타내었다. 대조군은 효소기질(enzyme substrate)이 없는 큐플에 시료를 넣고 반응시약을 첨가한 것을 사용하였다.In order to confirm the enzyme activity of the shiitake mushroom mycelium culture solution compositions of the above Examples and Comparative Examples, the API ZYM kit was used to analyze. API ZYM kit is an enzyme assay kit containing 19 kinds of hydrolase substrates. After dispensing 65 µl of a sample into each cuple of API ZYM kit, incubating at 37°C for 4 hours, add a drop of reaction reagent to each cuple, and then measure the enzyme activity by checking the degree of change in reaction color after 5 minutes. I did. Depending on the degree of color change of each cuple, it can be displayed as a value from 0 to 5, where 0 is the negative response, 5 is the maximum intensity response, and 1-4 is the intermediate response value. The higher the degree of change of the reaction color is, the higher the enzyme activity is, and the results are shown in Table 5 below. As a control group, a sample was placed in a Qple without an enzyme substrate and a reaction reagent was added.
상기 표 5에서 확인되는 바와 같이 표고버섯 자실체를 배지로 하고, 블렌더 처리 및 자가소화 유도의 2단계 처리를 통하여 제조되는 상기 실시예 11~15의 표고버섯 균사체 배양액 조성물은 PDB 배지를 사용하여 제조된 배양액 조성물(비교예 1), 블렌더 처리 및 자가소화 유도 처리를 거치지 않은 배양액 조성물(실시예 1~5) 및 자가소화 유도 처리를 거치지 않은 배양액 조성물(실시예 6~10)에 비하여 효소활성이 매우 높게 나타남을 알 수 있다.As shown in Table 5 above, the shiitake mushroom mycelium culture composition of Examples 11 to 15 prepared through a two-stage treatment of blender treatment and self-digestion induction using shiitake fruit bodies as a medium was prepared using PDB medium. Enzymatic activity is very much compared to the culture solution composition (Comparative Example 1), the culture solution composition not subjected to the blender treatment and the self-digestion induction treatment (Examples 1 to 5), and the culture solution composition (Examples 6 to 10) not subjected to the autodigestion induction treatment. It can be seen that it appears high.
시험예 3: 추출 촉매 효과 시험(1)Test Example 3: Extraction catalyst effect test (1)
상기 실시예의 표고버섯 균사체 배양액 조성물의 추출 촉매효과를 시험하기 위하여 천연물을 추출하여 추출 효율을 확인하였다. 천연물은 화장료로 널리 쓰이는 15종을 선정하여 사용하였다.In order to test the extraction catalytic effect of the shiitake mushroom mycelium culture solution composition of the above example, natural products were extracted and extraction efficiency was confirmed. 15 kinds of natural products widely used as cosmetics were selected and used.
먼저, 하기 표 6과 같이 각 천연물 50g을 정제수 1Kg에 넣고 냉각 콘덴서가 달린 추출기에서 50℃로 24시간 동안 추출한 후 여과하여 여액을 수득하였다. 여액은 감압증발기로 용매를 증발시켜 추출물을 제조하였으며, 수득량을 표 6에 나타내었다. First, as shown in Table 6 below, 50g of each natural product was put into 1Kg of purified water, extracted for 24 hours at 50°C in an extractor equipped with a cooling condenser, and filtered to obtain a filtrate. The filtrate was prepared by evaporating the solvent with a vacuum evaporator, and the yields are shown in Table 6.
상기 실시예 12에서 제조된 표고버섯 균사체 배양액 조성물의 추출 촉매 활성을 평가하기 위해 추출용매에 혼합하여 천연물을 추출하였다. In order to evaluate the extraction catalytic activity of the shiitake mushroom mycelium culture solution composition prepared in Example 12, a natural product was extracted by mixing it with an extraction solvent.
각 천연물 50g을 정제수 900g에 넣고 실시예 12의 배양액 조성물 100g을 첨가한 다음 냉각 콘덴서가 달린 추출기에서 50℃로 24시간동안 추출한 후 여과하여 여액을 수득하였다. 여액은 감압증발기로 용매를 증발시켜 추출물을 제조하였으며 그 결과를 하기 표 7에 나타내었다. 50 g of each natural product was added to 900 g of purified water, and 100 g of the culture solution composition of Example 12 was added, followed by extraction at 50° C. for 24 hours in an extractor equipped with a cooling condenser, followed by filtration to obtain a filtrate. The filtrate was prepared by evaporating the solvent with a vacuum evaporator, and the results are shown in Table 7 below.
상기 표 7에서 확인되는 바와 같이, 표고버섯 자실체를 배지로 하고, 블렌더 처리 및 자가소화 유도의 2단계 공정을 통하여 제조되는 상기 실시예 12의 표고버섯 균사체 배양액 조성물을 추출보조제로 첨가하여 천연물을 추출하는 경우에 천연물의 추출 수율이 크게 증대됨을 알 수 있다. 그러므로 본 발명의 표고버섯 균사체 배양액 조성물은 천연물 추출에 있어서, 추출 촉매제나 추출용매로서 효과적으로 사용될 수 있음을 알 수 있다.As shown in Table 7 above, a natural product was extracted by adding the shiitake mushroom mycelium culture solution composition of Example 12 prepared through a two-step process of blender treatment and self-digestion induction as a medium using shiitake fruit bodies as an extraction aid. In this case, it can be seen that the extraction yield of natural products is greatly increased. Therefore, it can be seen that the shiitake mushroom mycelium culture solution composition of the present invention can be effectively used as an extraction catalyst or extraction solvent in the extraction of natural products.
시험예 4: 추출 촉매 효과 시험(2)Test Example 4: Extraction catalyst effect test (2)
상기 시험예 3에서 수득한 천연 추출물의 성분 중 폴리페놀 및 플라보노이드의 함량을 측정하여 상기 실시예의 표고버섯 균사체 배양액 조성물의 유용성분 증대 및 추출 촉매효과를 확인하였다.The content of polyphenols and flavonoids in the components of the natural extract obtained in Test Example 3 was measured to confirm the increase of useful components and the catalytic effect of extraction of the shiitake mushroom mycelium culture solution composition of the above example.
총 페놀함량Total phenol content
상기 시험예 3의 추출물 1~15를 1mg/mL의 농도로 제조한 추출액 1mL에 정제수 5mL를 첨가한 다음, Folin-Ciocalteau′s phenol reagent(Sigma-Aldrich)을 1mL 가하여 혼합한 후 실온에서 5분간 반응시켰다. 이후 1mL의 Na2CO3 포화 용액을 첨가하여 혼합한 후 실온에서 1시간동안 반응시킨 다음 760nm에서 흡광도를 측정하였다. 표준물질은 탄닌산(tannin acid)을 이용하여 검량선을 작성한 후 총 페놀함량을 구하였다. 5 mL of purified water was added to 1 mL of the extract prepared from the extracts 1 to 15 of Test Example 3 at a concentration of 1 mg/mL, and then 1 mL of Folin-Ciocalteau's phenol reagent (Sigma-Aldrich) was added and mixed, and then at room temperature for 5 minutes. Reacted. Thereafter, 1 mL of a saturated Na 2 CO 3 solution was added and mixed, followed by reacting at room temperature for 1 hour, and absorbance was measured at 760 nm. The total phenol content was calculated after preparing a calibration curve using tannin acid as a standard material.
총 플라보노이드함량Total flavonoid content
상기 시험예 3의 추출물 1~15를 1mg/1mL의 농도로 제조한 추출액 0.5mL에 에탄올 1.5mL, 10% AlCl3 용액 0.1mL, 1M Potassium acetate 용액 0.1mL, 정제수 2.8mL를 가하여 혼합한 후, 실온에서 40분간 반응시킨 후 415nm에서 흡광도를 측정하였다. 표준품은 케르세틴(quercetin)을 이용하여 검량선을 작성한 후 총 플라보노이드 함량을 구하였다.The extracts 1 to 15 of Test Example 3 were mixed by adding 1.5 mL of ethanol, 0.1 mL of 10% AlCl 3 solution, 0.1 mL of 1M Potassium acetate solution, and 2.8 mL of purified water to 0.5 mL of the extract prepared at a concentration of 1 mg/1 mL, After reacting at room temperature for 40 minutes, absorbance was measured at 415 nm. As a standard, a calibration curve was prepared using quercetin, and the total flavonoid content was calculated.
상기 표 8, 9에서 확인되는 바와 같이, 표고버섯 자실체를 배지로 하고, 블렌더 처리 및 자가소화 유도의 2단계 공정을 통하여 제조되는 상기 실시예 12의 표고버섯 균사체 배양액 조성물을 추출보조제로 첨가하여 천연물을 추출하는 경우에 천연물의 유용성분인 페놀 및 플라보노이드의 추출 수율이 크게 증대됨을 알 수 있다. 그러므로 본 발명의 표고버섯 균사체 배양액 조성물은 천연물 추출에 있어서, 유용성분을 증대시키는 추출 촉매제나 추출용매로서 효과적으로 사용될 수 있음을 알 수 있다.As shown in Tables 8 and 9, a natural product by adding the shiitake mushroom mycelium culture solution composition of Example 12 prepared through a two-step process of blender treatment and self-digestion induction using shiitake fruit bodies as a medium In the case of extracting, it can be seen that the extraction yield of phenol and flavonoids, which are useful components of natural products, is greatly increased. Therefore, it can be seen that the shiitake mushroom mycelium culture solution composition of the present invention can be effectively used as an extraction catalyst or extraction solvent to increase useful components in the extraction of natural products.
Claims (6)
(B) 표고버섯 균사체를 접종하는 단계;
(C) 20~30℃, 진탕 배양(100~150rpm) 조건으로, 7~21일 동안 배양하는 단계;
(D) 배양 종료 후 배양액 전체를 블렌더(blender) 처리하여 세포벽을 파쇄하는 단계; 및
(E) 블렌더(blender) 처리한 배양액을 45~55℃에서 24~72시간 동안 배양하며 자가 소화시키는 단계를 포함하는 추출 촉매제 또는 추출용매용 표고버섯 균사체 배양액 조성물의 제조 방법.(A) preparing a liquid culture medium by extracting the fruiting bodies of shiitake mushrooms;
(B) inoculating shiitake mushroom mycelium;
(C) 20 ~ 30 ℃, in the shaking culture (100 ~ 150rpm) conditions, culturing for 7 ~ 21 days;
(D) crushing the cell wall by processing the entire culture medium after completion of the culture with a blender; And
(E) A method for producing a shiitake mushroom mycelium culture solution composition for an extraction catalyst or an extraction solvent comprising the step of incubating the culture solution treated with a blender at 45 to 55° C. for 24 to 72 hours and self-digesting.
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