KR20080036315A - Fermented products of phellinus linteus by lactic acid bacteria and preparation method thereof - Google Patents
Fermented products of phellinus linteus by lactic acid bacteria and preparation method thereof Download PDFInfo
- Publication number
- KR20080036315A KR20080036315A KR1020060102770A KR20060102770A KR20080036315A KR 20080036315 A KR20080036315 A KR 20080036315A KR 1020060102770 A KR1020060102770 A KR 1020060102770A KR 20060102770 A KR20060102770 A KR 20060102770A KR 20080036315 A KR20080036315 A KR 20080036315A
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- KR
- South Korea
- Prior art keywords
- lactic acid
- acid bacteria
- lactobacillus
- situation mushroom
- present
- Prior art date
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Abstract
Description
도 1은 본 발명의 일실시예에 따른 상황버섯 유산균 발효물을 제조하는 과정을 모식도로 나타낸 것이다.Figure 1 shows a schematic diagram showing the process of producing a situation mushroom lactic acid bacteria fermentation according to an embodiment of the present invention.
도 2a는 상황버섯 유산균 발효물의 멜라닌 합성 저해 효과를 확인하기 위하여 멜라닌 종양 세포에 알파-MSH, 코직산(800uM) 또는 상황버섯 유산균 발효물을 각각 0.2%, 0.4%의 농도로 처리한 후 멜라닌의 합성 정도를 디지털 카메라로 관찰한 그림이고,Figure 2a is the synthesis of melanin after treatment with melanin tumor cells in the concentration of 0.2%, 0.4% alpha-MSH, kojic acid (800uM) or S. mushroom lactic acid bacteria fermentation in the melanin tumor cells to confirm the inhibitory effect of melanin synthesis Is a picture of the degree observed with a digital camera,
도 2b는 상기 멜라닌의 합성 정도를 405nm에서 흡광도를 측정함으로써 그 결과를 그래프화하여 나타낸 것이다.Figure 2b is a graph showing the result of measuring the degree of synthesis of melanin by absorbance at 405nm.
도 3은 사람의 섬유아세포에 상황버섯 유산균 발효물을 각각 0.05, 0.2, 0.5 또는 1%의 농도로 처리한 후, 섬유아세포에서의 프로콜라겐 합성 정도를 광학농도계를 이용하여 측정한 결과를 나타낸 그래프이다.FIG. 3 is a graph showing the results of measuring the degree of procollagen synthesis in fibroblasts after treatment with human fibroblasts at concentrations of 0.05, 0.2, 0.5 or 1%, respectively. to be.
도 4는 사람의 섬유아세포에 상황버섯 유산균 발효물을 각각 0.05, 0.2, 0.5 또는 1%의 농도로 처리한 후, 각 처리군에서의 섬유아세포 세포생존율을 MTT 분석을 통해 나타낸 그래프이다.4 is a graph showing the fibroblast cell viability in each treatment group after treatment of human fibroblasts at concentrations of 0.05, 0.2, 0.5 or 1%, respectively, of the situation mushroom lactic acid bacteria fermentation.
도 5는 SOD 어세이 키트를 사용하여 상황버섯 유산균 발효물을 각각 0.083, 0.83 또는 8.3%의 농도로 처리한 후, SOD 활성도를 측정한 결과이며, 양성대조군으로는 SOD를 각각 0.1, 1 또는 10 unit/ml로 처리하여 SOD 활성도를 측정한 결과이다.5 is a result of measuring the SOD activity after treatment of the situation mushroom lactic acid bacteria fermentation at a concentration of 0.083, 0.83 or 8.3% using the SOD assay kit, respectively, 0.1, 1 or 10 as a positive control group SOD activity was measured by treatment with unit / ml.
도 6은 상황버섯 유산균 발효물의 면역반응효과를 확인하기 위하여 마우스의 대식세포에 상황버섯 유산균 발효물을 각각 0.2, 1 또는 2%의 농도로 처리한 후, MTT 분석을 통해 대식세포의 증식 정도를 나타낸 그래프이다.6 is treated with the concentration of 0.2, 1 or 2% of the situation mushroom lactic acid bacteria fermentation in the macrophages of mice in order to confirm the immune response effect of the situation mushroom lactic acid bacteria fermentation, MTT analysis of the degree of proliferation of macrophages The graph shown.
도 7은 상황버섯 유산균 발효물의 산화질소 생성 촉진 효과를 확인하기 위하여 마우스의 대식세포에 상황버섯 유산균 발효물을 각각 0.2, 1 또는 2%의 농도로 처리한 후, 생성된 산화질소의 양을 570nm에서 흡광도를 측정하여 나타낸 결과이다. FIG. 7 shows that the amount of nitric oxide produced after treating S. mushroom lactic acid bacteria fermentation at concentrations of 0.2, 1, or 2% in macrophages of mice to confirm the effect of promoting nitric oxide production of S. mushroom lactic acid bacteria fermentation. This is the result of measuring absorbance at.
본 발명은 상황버섯 유산균 발효물 및 이의 제조방법에 관한 것으로, 보다 구체적으로 본 발명은 탄소원으로 전분질 원료를 사용하여 당화시킨 당화액에 상황버섯 분말 및 효소를 첨가하여 당화시킨 후, 유산균을 첨가하여 상황버섯 분말을 발효시키는 것을 특징으로 하는 상황버섯 유산균 발효물의 제조방법 및 상기 방법에 의해 제조된 상황버섯 유산균 발효물에 관한 것이다. The present invention relates to fermented mushrooms lactic acid bacteria and a method for preparing the same, and more specifically, the present invention is added to the saccharified solution saccharified using a starch raw material as a carbon source, and added to the saccharified mushroom powder and enzyme, The present invention relates to a method for producing a situation mushroom lactic acid bacteria fermentation, and a situation mushroom lactic acid bacteria fermented product prepared by the method.
상황버섯은 예로부터 중국에서는 상이라고도 하여 뽕나무에 기생하는 버섯으로 조사되어 왔으나 우리나라에서 조사된 바에 의하면 뽕나무 이외에 일부 활엽수의 심재부에서도 야생되는 것을 볼 수 있는 것으로 알려져 있고(차동렬, 월간잠사, 12, 34-37, 1992), 소나무 비늘버섯과 (Hymenochaetaceae)의 진흙버섯속(Phellius Quel.em.Imaz)에 속하며, 우리나라에서 상황버섯이라 함은 목질 진흙버섯(Phellinus Linteus)을 지칭한다. 상황버섯이라 일컫는 목질 진흙버섯 속에는 전세계적으로 약 48종이 보고 되어 있으며, 우리나라에서는 녹슨 진흙버섯 (P. ferrginosus), 마른 진흙버섯 (P. gilvus), 말똥 진흙버섯 (P. igniarius), 가지 진흙버섯 (P. Laevigatus), 벚나무 진흙버섯 (P. pomaceus), 찰 진흙버섯 (P. robustus), 전나무 진흙버섯(P. hartigii ), 낙엽 진흙버섯 (P. pini)등 8종이 존재하는 것으로 알려져 있고, 항암효과, 면역강화기능, 종양저지효과, 자궁출혈 및 월경 불순 해소효과 등의 약리작용을 가지고 있다고 알려져 있다. Situation mushrooms have been investigated as mushrooms in the mulberry tree, also known as Sang in China. However, in Korea, it is known that wild mushrooms can be found in the heart of some hardwoods besides mulberry trees (Cha Dong-Ryul, Monthly Jamsa, 12, belong to the 34 to 37, 1992), the scales of pine mushrooms and mushroom mud (Hymenochaetaceae) (Phellius Quel.em.Imaz), referred to the situation in the country mushroom refers to the mud woody mushroom (Phellinus linteus). The situation has been referred to as wood mushrooms Mushrooms ln mud around the world reported about 48 paper refers, in our country rusty mud mushroom (P. ferrginosus), dry mud mushroom (P. gilvus), mud, dung mushrooms (P. igniarius), kind of muddy mushrooms ( P. Laevigatus ), Prunus mud mushroom ( P. pomaceus ), clay mud mushroom ( P. robustus ), fir mud mushroom (P. hartigii ) , deciduous mushroom ( P. pini ) It is known to have pharmacological effects such as anticancer effect, immune enhancing function, tumor suppression effect, uterine bleeding and menstrual impurity effect.
또한, 상황버섯 추출물에는 여러 종류의 당, 플라보노이드 성분, 섬유질, 이소플라본 등이 다량 함유되어 있어 화장료에서의 보습력과 피부노화 방지기능, 항산화 효과가 우수하고, 특히 피부 미백효과 면에서 효과가 매우 우수하다고 보고된 바 있다(T. lked, et al., Fragrance J., 6, 59, 1990). 이에 따라 최근에는 여러 가지 효능이 알려진 상황버섯을 식료품, 화장품 및 의약품 등에 응용하려는 노력들이 시도되고 있다. In addition, the situation mushroom extract contains a large amount of various kinds of sugars, flavonoids, fiber, isoflavones, etc., so it is excellent in moisturizing, anti-aging, and antioxidant effects in cosmetics, especially in terms of skin whitening effect. (T. lked, et al., Fragrance J. , 6, 59, 1990). Accordingly, efforts have recently been made to apply situation mushrooms, which are known to have various effects, to foodstuffs, cosmetics, and medicines.
유산균(lactic acid bacteria)은 탄수화물을 분해하고, 이를 이용하여 유산을 만드는 세균으로서 산소가 적은 곳에서 잘 증식하는 통성 혐기성균 또는 편성 혐기성균이다. 상기 유산균은 5개 속으로 구분할 수 있는데, 스트렙토코커스(Streptococcus), 락토바실러스(Lactobacillus), 루코노스탁(Leuconostoc), 비피도박테리아(Bifidobacteria) 및 페디오코커스(Pediococcus)로 분류 되어진다. 이러한 유산균은 장내 pH를 산성으로 유지시켜 대장균이나 클로스트리디움(Clostridium sp.)과 같은 유해균의 번식을 억제하고 설사와 변비를 개선할 뿐 아니라, 비타민 합성, 항암작용, 혈청 콜레스테롤 저하 등의 역할을 하는 것으로 알려져 있다. 특히, 유산균은 장의 점막과 상피세포에 강하게 결합할 수 있는 특정 단백질을 가지고 있어 유해 세균의 성장을 막는 정장 작용에 많은 도움을 준다. 또한, 유산균은 대식세포의 증식을 촉진하여 대식세포의 장내 유해 세균에 대한 인지능력, 살균능력 등을 강화시키고, 면역 관련 물질의 분비를 촉진하여 면역증강 효과를 나타내는 것으로 알려져 있다(Gabriela perdigon et al., J. of food Protection 53:404-410, 1990; Katsumasa sato et al., Microbiol . Immunol., 32(7):689-698, 1988). 그러므로 최근에는 건강에 대한 관심이 높아져 가면서 상기와 같은 인간에게 유익한 유산균을 건강식품, 의약품 및 화장품 등에 응용하여 개발하고자 하는 연구가 활발히 진행되고 있다. Lactic acid bacteria (lactic acid bacteria) are carbohydrates that break down carbohydrates and use them to make lactic acid bacteria. The lactic acid bacteria can be divided into five, and is classified as Streptococcus (Streptococcus), Lactobacillus bacteria (Lactobacillus), Lu Pocono Stark (Leuconostoc), bifidobacteria bacteria (Bifidobacteria) and Phedi O Rhodococcus (Pediococcus). These lactic acid bacteria maintain the acidic intestinal pH to inhibit the reproduction of harmful bacteria such as Escherichia coli and Clostridium sp. And improve diarrhea and constipation, as well as play a role in vitamin synthesis, anticancer action, and serum cholesterol lowering. It is known. In particular, lactic acid bacteria have a specific protein that can bind strongly to the intestinal mucosa and epithelial cells, help a lot of work to prevent the growth of harmful bacteria. In addition, lactic acid bacteria are known to promote the proliferation of macrophages to enhance the cognitive and bactericidal ability of macrophage intestinal harmful bacteria, and to promote the secretion of immune-related substances (Gabriela perdigon et al. , J. of food Protection 53: 404-410, 1990; Katsumasa sato et al., Microbiol . Immunol ., 32 (7): 689-698, 1988). Therefore, in recent years, as interest in health increases, studies are being actively conducted to develop lactic acid bacteria, which are beneficial to humans, by applying them to health foods, medicines, and cosmetics.
한편, 피부는 자외선, 화학물질, 오염물질 등의 외부환경적 요인에 의해 자극을 받으면 피부 내에서 활성산소 및 자유라디칼이 생성되고 생성된 활성산소 및 자유라디칼에 의해 피부노화가 일어나거나 또는 멜라닌 색소침착에 의한 피부흑화가 발생하게 된다. 일상적으로 피부는 자외선을 받으면 자외선으로부터 피부 및 신체를 보호하기 위하여 멜라닌 생성을 촉진하게 되며 이러한 멜라닌은 피부 세포의 일종인 멜라노사이트에서 생성되고 이로 인하여 피부 곳곳에 멜라닌 색소침착이 일어나게 된다. 따라서 최근 생활수준의 향상 및 생활양식의 변화로 인해 남녀노소 깨끗한 피부에 관심을 가지게 되었고 궁극적으로 피부흑화예방 및 침착색소를 제거하는 등의 기술개발에 관심이 대두되었으며 이에 따라 색소침착 치료제나 이와 관련된 화장품 및 기능성 식품 등이 개발되고 있다. On the other hand, when the skin is stimulated by external environmental factors such as ultraviolet rays, chemicals and contaminants, free radicals and free radicals are generated in the skin, and skin aging occurs due to the free radicals and free radicals generated or melanin pigment. Skin darkening occurs due to deposition. In general, the skin stimulates melanin production in order to protect the skin and the body from the ultraviolet rays, which is produced by melanocytes, a kind of skin cells, and melanin pigmentation occurs throughout the skin. Therefore, the recent improvement in living standards and lifestyle changes has led to interest in clean skin for all ages, and ultimately the development of technologies such as prevention of skin blackening and removal of pigmented pigments has emerged. Cosmetics and functional foods are being developed.
현재까지 개발되어 사용되고 있는 피부 미백제로는 하이드로퀴논, 아스코르빅산, 코직산 및 알부틴 등이 미백 화장료로 이용되고 있다. 그러나 상기 화합물들은 쉽게 분해되거나 착색되고, 효능 및 효과가 불분명한 단점이 있어서 보다 우수한 효능을 가지면서 부작용이 없는 자연에서 추출한 유효성분의 발굴에 관심이 증대되고 있다. Hydroquinone, ascorbic acid, kojic acid, and arbutin are used as skin whitening agents that have been developed and used so far. However, the compounds are easily decomposed or colored, and the disadvantages of which the efficacy and the effect are unclear have increased interest in the discovery of an active ingredient extracted from nature having better efficacy and no side effects.
따라서 최근에는 피부미백 효능 및 피부노화를 억제하는 활성이 우수하다고 알려진 상황버섯을 이용한 상황버섯 추출물 정제 방법 및 상황버섯을 이용한 기능성 조성물 등이 개발되고 있는데, 이와 관련된 종래 기술을 살펴보면, 대한민국 등록특허 제0589484호에는 상황버섯으로부터 추출된 추출물 및 상기 추출물을 함유하는 화장료에 대해 개시되어 있으며, 대한민국 등록특허 제0345382호에는 항산화 효과에 의한 피부 노화 방지 기능을 갖는 자연산 상황버섯 추출물이 함유된 미백화장 료에 대해 개시되어 있고, 대한민국 등록특허 제0206538호에는 상황버섯 분말 추출물을 함유하는 피부 미백용 화장료 조성물에 대해 개시되어 있다. Therefore, recently, a method for purifying situational mushroom extracts using a situation mushroom and a functional composition using a situation mushroom have been developed, which are known to be excellent in skin whitening efficacy and an activity of inhibiting skin aging. No. 0589484 discloses an extract extracted from a situation mushroom and a cosmetic containing the extract. Korean Patent No. 045382 discloses a whitening cosmetic containing a natural situation mushroom extract having an anti-aging function due to an antioxidant effect. For example, Korean Patent No. 0206538 discloses a cosmetic composition for skin whitening containing a situation mushroom powder extract.
그러나 상기 기술된 종래기술의 상황버섯 추출물을 추출하는 방법은 열수추출 또는 알콜 등의 유기용매를 이용한 추출 방법으로써 고온 및 유기용매에 의하여 상황버섯 유효성분의 활성이 상실되거나, 추출물 등의 안정성이 매우 낮아 쉽게 분해 되는 단점이 있으며, 추출과정 중에 유효성분이 손실되는 등의 문제점이 있다. 따라서 여러 가지 효능을 많이 함유하고 있는 상황버섯으로부터 추출물을 수득하는데 있어서, 그 효능을 상실하지 않고 보다 개선된 효능을 함유할 수 있는 새로운 방법의 개발이 요구되고 있는 실정이다.However, the method of extracting the situation mushroom extract of the prior art described above is an extraction method using an organic solvent such as hot water extraction or alcohol, the activity of the situation mushroom active ingredient is lost by high temperature and organic solvent, or the stability of the extract is very There is a disadvantage that it is easily decomposed, and there is a problem such as loss of the active ingredient during the extraction process. Therefore, in obtaining an extract from a situation mushroom containing a lot of various effects, there is a need for the development of a new method that can contain more improved efficacy without losing its efficacy.
이에 본 발명자들은 보다 개선된 효능을 가지는 상황버섯의 유효성분을 수득하는 방법을 연구하던 중, 전분질 원료를 사용하여 당화시킨 당화액에 상황버섯 분말 및 효소를 첨가하여 당화시킨 후, 인체에 여러 가지 유익한 효능을 가지고 있는 유산균을 첨가하여 상황버섯 분말을 발효시킴으로써 상황버섯 유산균 발효물을 제조하였고, 상기 방법에 의해 제조된 상황버섯 유산균 발효물이 멜라닌 합성 저해, 프로콜라겐 합성 촉진, 섬유아세포의 증식 촉진, 항산화 효과, 대식세포의 증식 및 산화질소의 생성량을 증가시키는 효능을 모두 가지고 있음을 확인함으로써 본 발명을 완성하였다.Therefore, while the present inventors are studying a method of obtaining an active ingredient of a situation mushroom having improved efficacy, after adding a situation mushroom powder and an enzyme to a saccharified solution saccharified using starch raw material, various kinds of human body Lactic acid bacteria fermented by the addition of lactic acid bacteria having a beneficial effect was prepared by fermenting the situation mushroom lactic acid bacteria, the mushrooms lactic acid bacteria fermented by the method prepared by the above method inhibits melanin synthesis, promotes collagen synthesis, promotes the growth of fibroblasts The present invention was completed by confirming that it has all of the antioxidant effect, the effect of increasing the proliferation of macrophages and the production of nitric oxide.
따라서 본 발명의 목적은 상황버섯 유산균 발효물의 제조방법 및 상기 방법에 의해 제조된 상황버섯 유산균 발효물을 제공하는 것이다. Therefore, an object of the present invention is to provide a method for producing a situation mushroom lactic acid bacteria fermentation and a situation mushroom lactic acid bacteria fermented by the method.
또한, 본 발명의 다른 목적은 상기 상황버섯 유산균 발효물을 함유하는 피부 미백용 조성물, 피부노화 및 주름 개선용 조성물, 항산화용 조성물 및 면역증강용 조성물을 제공하는 것이다.In addition, another object of the present invention is to provide a composition for skin whitening, a composition for improving skin aging and wrinkles, an antioxidant composition, and an immuno-enhancing composition containing the situation mushroom lactic acid bacteria fermentation.
상기와 같은 본 발명의 목적을 달성하기 위해서, 본 발명은In order to achieve the object of the present invention as described above, the present invention
(a) 전분질 원료를 액화 및 당화하는 단계;(a) liquefying and saccharifying the starch raw material;
(b) 상기 (a) 단계로부터 수득한 당화액에 상황버섯 분말 및 효소를 추가로 첨가하여 당화하는 단계;(b) adding saccharified mushroom powder and enzyme to the saccharification solution obtained from step (a) to further saccharify;
(c) 상기 (b) 단계에서 수득한 당화액에 유산균 및 효모를 추가로 접종하여 발효시키는 단계를 포함하는 것을 특징으로 하는 상황버섯 유산균 발효물의 제조방법을 제공한다. (c) provides a method for producing a situation mushroom lactic acid bacteria fermentation, comprising the step of fermenting by further inoculating lactic acid bacteria and yeast in the saccharified solution obtained in step (b).
또한, 본 발명은 상기 방법에 의해 제조된 상황버섯 유산균 발효물을 제공한다.The present invention also provides a situation mushroom lactic acid bacteria fermented product prepared by the above method.
또한, 본 발명은 상기 상황버섯 유산균 발효물을 함유하는 피부 미백용 조성물을 제공한다.In another aspect, the present invention provides a composition for skin whitening containing the situation mushroom lactic acid bacteria fermentation.
또한, 본 발명은 상기 상황버섯 유산균 발효물을 함유하는 피부노화 및 주름 개선용 조성물을 제공한다. In addition, the present invention provides a composition for improving skin aging and wrinkles containing the situation mushroom lactic acid bacteria fermentation.
또한, 본 발명은 상기 상황버섯 유산균 발효물을 함유하는 항산화용 조성물을 제공한다.The present invention also provides an antioxidant composition containing the situation mushroom lactic acid bacteria fermentation.
나아가 본 발명은 상기 상황버섯 유산균 발효물을 함유하는 면역증강용 조성물을 제공한다.Furthermore, the present invention provides a composition for immuno-enhancing containing the situation mushroom lactic acid bacteria fermentation.
이하, 본 발명을 보다 상세히 설명하기로 한다.Hereinafter, the present invention will be described in more detail.
본 발명은 상황버섯 유산균 발효물을 제조함에 있어서 탄소원으로 전분질 원료를 사용하여 당화시킨 당화액에 상황버섯 분말 및 효소를 첨가하여 당화시킨 후, 유산균을 첨가하여 상황버섯 분말을 발효시켰다는 점에 특징이 있다. 상기 본 발명에 따른 상황버섯 발효물의 제조는 기존의 당업계에 알려진 천연으로부터 유효성분을 추출하는 방법인 열수추출 또는 유기용매에 의한 추출 방법이 아닌 상황버섯 분말을 당화시킨 후, 면역 활성 및 여러 가지 생리학적 기능을 가지고 있는 유산균을 첨가하여 상황버섯 분말을 발효시킴으로써 여러 가지 개선된 효능을 가지고 있는 상황버섯 발효물이 제조되었다. The present invention is characterized by the fact that in the preparation of the situation mushroom lactic acid bacteria fermented by adding starch mushroom powder and enzyme to the saccharified solution saccharified using a starch raw material as a carbon source, and then added lactic acid bacteria to ferment the situation mushroom powder have. The preparation of the situation mushroom fermentation according to the present invention is not a method of extracting the active ingredient from the known natural water by hot water extraction or an organic solvent extraction method after glycosylating the situation mushroom powder, immune activity and various The situation mushroom fermented product having various improved efficacy was prepared by fermenting the situation mushroom powder by adding lactic acid bacteria having a physiological function.
따라서 본 발명에서는 상황버섯 분말을 유산균을 이용하여 발효시킴으로써 상황버섯 유산균 발효물을 제조하였으며 제조방법을 단계별로 설명하면 다음과 같다.Therefore, in the present invention, the situation mushroom lactic acid bacteria fermented product was prepared by fermenting the situation mushroom powder using lactic acid bacteria.
제1단계: 전분질 원료를 액화 및 First step: liquefying starch raw materials 당화하는Saccharified 단계 step
본 발명에 따른 상황버섯 유산균 발효물을 제조하기 위하며 먼저 전분질 원료에 증류수를 첨가하여 전분질 원료를 호화하고 당화시켜 당화액을 제조한다. 상기 단계는 탄소원으로 전분질 원료를 사용하여 상기 전분질 원료에 증류수를 첨가하고 80~90℃까지 온도를 높여 호화시킨 후, 액화효소를 첨가하여 80~90℃에서 1~2 시간 동안 반응시킨 다음, 종균을 사용하여 제조한 코지(입국)를 당화용기에 넣고 당화효소를 첨가하여 55~65℃에서 2 ~3시간 동안 반응시킬 수 있다.In order to prepare the situation mushroom lactic acid bacteria fermentation according to the present invention, first, distilled water is added to the starch raw material to prepare the saccharified solution by gelatinizing and saccharifying the starch raw material. The step is to add the distilled water to the starch raw material using a starch raw material as a carbon source and to increase the temperature up to 80 ~ 90 ℃, gelatinized, and then reacted for 1 to 2 hours at 80 ~ 90 ℃ by adding a liquefied enzyme, Cozy (entry) prepared using the put into the saccharification vessel can be reacted for 2 to 3 hours at 55 ~ 65 ℃ by adding a saccharifying enzyme.
상기 전분질 원료는 쌀을 곱게 갈아 분말로 만들어 사용할 수 있고, 바람직하게는 미강을 사용할 수 있다. 상기 미강은 현미를 백미로 정미하는 과정에서 발생하는 부산물로서 쌀겨와 쌀눈으로 이루어진 분말을 말한다. 이러한 미강에는 쌀의 영양성분 대부분이 포함되어 있고, 풍부한 양의 지질, 비타민 B군, 양질의 단백질, 섬유질 및 인 등도 풍부하게 함유하고 있다. The starch raw material may be used to finely grind the rice into a powder, preferably rice bran. The rice bran refers to a powder consisting of rice bran and rice snow as by-products generated in the process of refining brown rice with white rice. The rice bran contains most of the nutritional ingredients of rice, and is rich in lipids, vitamin B group, high quality protein, fiber and phosphorus.
상기 액화효소는 전분의 내부 결합을 무작위적으로 가수분해해서 효모가 이용할 수 있는 발효성 당인 소량의 포도당(glucose), 말토스(maltose), 저분자 덱스트린(dextrin)등을 생성하여 전분 현탁액을 빠른 속도로 맑은 용액으로 전환시킬 수 있는 효소를 말한다. 대표적인 액화효소로는 α-아밀라아제(α-amylase)가 있으며 시중에서 구입이 가능하다. 본 발명에서는 노보자임사의 α-아밀라아제(α-amylase)를 사용하였다.The liquefied enzyme randomly hydrolyzes the internal bonds of starch to produce small amounts of glucose, maltose, and low molecular weight dextrin, which are fermentable sugars that can be used by the yeast. Refers to an enzyme that can be converted to a clear solution. A representative liquefied enzyme is α-amylase and is commercially available. In the present invention, α-amylase of Novozyme was used.
상기 종균으로는 아스퍼질러스 나이거(Aspergillus niger), 아스퍼질러스 우사미(Aspergillus Usamii), 아스퍼질러스 오리제(Aspergillus Oryzae), 아스퍼질러 스 가와치(Aspergillus kawachi), 아스퍼질러스 아와모리(Aspergillus awamori) 및 리조푸스 오리제(Rhizopus oryzae) 균주를 사용할 수 있다. 그러나 바람직하게는 아스퍼질러스 오리제(Aspergillus Oryzae)를 사용할 수 있다.The seed as the Aspergillus and this (Aspergillus niger), Usami Aspergillus (Aspergillus Usamii), Aspergillus duck claim (Aspergillus Kawachi (Aspergillus kawachi) Oryzae), Aspergillus, Aspergillus awamori (Aspergillus awamori ) and Rhizopus oryzae strains can be used. However, preferably Aspergillus Oryzae can be used.
또한, 상기 당화효소는 액화효소에 의해 무작위적으로 일부 분해된 전분을 가수분해함으로써 당으로 전환시키는 효소를 말한다. 당화효소로는 특별히 한정되지 않으며, β-아밀라아제 (β- amylase)와 글루코-아밀라아제(gluco-amylase)등이 대표적이다. 당화효소는 시중에서 용이하게 구입하여 사용할 수 있으며, 본 발명의 일실시예에서는 노보자임사의 글루코-아밀라아제를 사용하였다. In addition, the glycosylase refers to an enzyme that is converted into a sugar by hydrolyzing starch randomly partially degraded by liquefied enzyme. The glycosylase is not particularly limited, and β-amylase and β-amylase and gluco-amylase are typical. The glycosylase can be easily purchased and used commercially, in one embodiment of the present invention was used gluco-amylase of Novozyme.
제2단계: 상황버섯 분말 및 효소를 첨가하여 Second step: adding mushroom powder and enzyme 당화하는Saccharified 단계 step
상기 제1단계에서 수득한 당화액에 상황버섯 분말 및 효소를 추가로 첨가하여 상황버섯 분말을 당화시킴으로써 당화액을 제조한다. 이때, 상기 상황버섯 분말 및 효소는 상기 제1단계에서 수득한 당화액 총중량에 대하여 각각 0.1~2.0 중량% 및 0.1~0.5 중량%로 첨가하고 50~60℃에서 2~5시간 동안 반응시킬 수 있다. 그러나 바람직하게는 상기 상황버섯 분말 및 효소를 상기 제1단계에서 수득한 당화액 총중량에 대하여 각각 1.0~2.0 중량% 및 0.1~0.2 중량%로 첨가하고 60℃에서 2시간 동안 반응시킬 수 있다. 상기 상황버섯은 항암효과, 면역증강 효과, 노화억제 및 성인병 예방과 치료에 효험이 있는 것으로 알려지면서 식용뿐만 아니라 약용으로도 그 이용성이 날로 증대되고 있다. 본 발명에 따른 상기 상황버섯은 특별한 제한 없 이 시중에서 판매되고 있는 것을 용이하게 구입하여 사용할 수 있으며, 본 발명에서는 상기 상황버섯을 분쇄기로 곱게 갈아 분말로 만든 것을 사용하였다.The saccharified liquid is prepared by saccharifying the turmeric mushroom powder by additionally adding the turmeric mushroom powder and enzyme to the saccharified liquid obtained in the first step. At this time, the situation mushroom powder and enzyme may be added in 0.1 ~ 2.0% by weight and 0.1 ~ 0.5% by weight relative to the total weight of the saccharified solution obtained in the first step and reacted for 2 to 5 hours at 50 ~ 60 ℃ . Preferably, however, the situation mushroom powder and enzyme may be added in an amount of 1.0 to 2.0% by weight and 0.1 to 0.2% by weight based on the total weight of the saccharified solution obtained in the first step, and reacted at 60 ° C. for 2 hours. The situation mushroom is known to be effective in anti-cancer effect, immune enhancing effect, aging inhibitory and adult disease prevention and treatment, and its use is increasing day by day as well as edible. The situation mushroom according to the present invention can be easily purchased and used in the market without particular limitation, in the present invention was used to grind the situation mushroom finely with a grinder made of powder.
또한, 상기 효소는 당 분해 효소로써 셀룰라아제, 글루카나아제, 아밀라아제, 크실라아제, 펜토사나아제, 프로타아제 및 크실로시다아제 등을 사용할 수 있으며, 또한 이들의 혼합물을 사용할 수 있다. 본 발명의 일실시예에서는 노보자임사의 셀룰라아제, 글루카나아제, 아밀라아제 및 크실라아제로 이루어진 혼합효소를 사용하였다.In addition, the enzyme may be used as a glycolysis enzyme cellulase, glucanase, amylase, xylase, pentosanase, protase and xylosidase, and mixtures thereof. In one embodiment of the present invention was used a mixed enzyme consisting of cellulase, glucanase, amylase and xylase of Novozyme.
제3단계: 발효 단계Stage 3: Fermentation stage
상기 제2단계에서 수득한 당화액에 유산균 및 효모를 접종하여 발효시킴으로써 본 발명의 상황버섯 유산균 발효물을 제조한다. 이 단계에서는 상기 제2단계에서 수득한 당화액 총중량에 대하여 유산균 및 효모를 각각 1~1.5 중량% 및 1~2 중량%로 접종하고 15℃에서 10~15일간 발효시키거나 또는 20~25℃에서 24~26시간 동안 발효시킬 수 있다. 바람직하게는 상기 제2단계에서 수득한 당화액 총중량에 대하여 유산균 및 효모를 각각 1 중량%로 접종하고 15℃에서 10~15일 동안 발효시킬 수 있다. Lactobacillus lactic acid bacteria of the present invention is prepared by inoculating and fermenting lactic acid bacteria and yeast in the saccharified solution obtained in the second step. In this step, 1 to 1.5% by weight and 1 to 2% by weight of lactic acid bacteria and yeast are inoculated with respect to the total weight of the saccharified solution obtained in the second step, and fermented at 15 ° C. for 10 to 15 days or at 20 to 25 ° C. It can be fermented for 24 to 26 hours. Preferably, the total amount of saccharified liquid obtained in the second step may be inoculated with lactic acid bacteria and yeast at 1% by weight, respectively, and fermented at 15 ° C. for 10 to 15 days.
상기 유산균은 사람이나 동물의 소화관이나 수십종의 농산물에 이르기까지 자연계에 널리 분포되어 있으며, 특히, 장내 상피세포에 부착하여 유산, 지방산, 항생물질 및 과산화수소 등을 분해하여 유해균을 억제하는 활성이 있고, 면역력을 증진시킬 뿐만 아니라 음식물의 영양학적 가치를 증진시킨다고 알려져 있다. 한편, 이러한 유산균에 속하는 세균으로는 연쇄상 구균, 테디오코카스, 류코노스톡, 유산간균 및 비피더스균 등 수십 종이 있다. 본 발명에 사용할 수 있는 상기 유산균의 종류로는 이에 제한되지는 않으나, 락토바실러스 애시도필루스(Lactobacillus acidophilus), 락토바실러스 불가리쿠스(Lactobacillus bulgaricus), 락토바실러스 카세이(Lactobacillus casei), 락토바실러스 플란타룸(Lactobacillus plantarum), 락토바실러스 델브루에키 (Lactobacillus delbruekii), 락토바실러스 존슨니(Lactobacillus johnsonii), 락토바실러스 람노서스(Lactobacillus rhamnosus ), 락토바실러스 사케이 (Lactobacillus sakei), 스트렙토코커스 써모필루스(Streptococcus thermophilus), 스트렙토코커스 피시움(Streptococcus pythium), 비피도박테리움 비피둠(Bifidobacterium bifidum ), 비피도박테리움 인판티스 (Bifidobacterium infantis), 비피도박테리움 롱검(Bifidobacterium longum), 페디오코커스 펜토사세우스(Pediococcus pentosaceus), 류코노스톡 메센테로이데스 (Leuconostoc mesenteroides)가 있을 수 있고, 바람직하게는 락토바실러스 플란타룸일 수 있다. The lactic acid bacteria are widely distributed in the natural world up to the digestive tract or dozens of agricultural products of humans or animals, and in particular, they are attached to the intestinal epithelial cells and decompose lactic acid, fatty acids, antibiotics, hydrogen peroxide, and the like to inhibit harmful bacteria. In addition, it is known to enhance the nutritional value of food as well as to boost immunity. On the other hand, the bacteria belonging to such lactic acid bacteria, there are dozens of species such as streptococcus, Tediococcus, leuconosstock, lactic acid bacilli and bifidus. Examples of the lactic acid bacteria that can be used in the present invention are not limited thereto, but are not limited thereto, Lactobacillus acidophilus , Lactobacillus bulgaricus , Lactobacillus casase ( Lactobacillus casei ), Lactobacillus plantarta Lactobacillus plantarum , Lactobacillus delbruekii , Lactobacillus Johnson you (Lactobacillus johnsonii), Lactobacillus ramno Saskatchewan (Lactobacillus rhamnosus), Lactobacillus four K (Lactobacillus sakei), Streptococcus spent a brush Ruth (Streptococcus thermophilus), Streptococcus fish Stadium (Streptococcus pythium), Bifidobacterium Bifidobacterium bifidum ) , Bifidobacterium Infante Tees (Bifidobacterium infantis), ronggeom Bifidobacterium (Bifidobacterium longum ), Pediococcus pentosaceus , Leuconostoc mesenteroides , preferably Lactobacillus plantarum.
또한, 상기 효모로는 특별히 한정되지는 않으며, 사카로마이세스 세레비지애(Saccharomyces cerevisiae), 사카로마이세스 사케이 (Saccharomyces sakei), 잔토필로마이세스 덴드로로우스 (Xanthophyllomyces dendrorhous)를 사용할 수 있으며, 본 발명의 일실시예에서는 사카로마이세스 세레비지애(Saccharomyces cerevisiae)를 사용하였다.In addition, the yeast is not particularly limited, Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Saccharomyces sakei , Xanthophyllomyces ( Xanthophyllomyces) dendrorhous ), and Saccharomyces cerevisiae was used in one embodiment of the present invention.
따라서 본 발명은 상기와 같은 방법에 따라 본 발명의 상황버섯 유산균 발효물을 제조할 수 있다.Therefore, the present invention can produce a situation mushroom lactic acid bacteria fermented product of the present invention according to the above method.
제4단계: 불순물 제거 및 농축 단계Step 4: remove impurities and concentrate
상기와 같은 방법에 의해 제조된 본 발명의 상황버섯 유산균 발효물은 발효물 내에 존재하는 불순물을 제거하는 단계 및 농축하는 단계를 추가로 수행할 수 있다.The situation mushroom lactic acid bacterium fermentation product of the present invention prepared by the method as described above may further perform the step of removing the impurities present in the fermentation and concentration.
상기 발효물 내에 존재하는 불필요한 용존 물질 및 부유고형물질과 같은 불순물을 제거하는 방법은 당업계에 공지된 방법으로 수행할 수 있으며, 대표적인 방법으로는 여과, 응집침전 및 활성탄을 이용한 흡착 등이 있을 수 있다. 상기 여과는 원심 분리를 하거나 여과기를 이용하여 수행할 수 있으며, 상기 응집침전은 화학약품, 즉, 응집제를 처리하여 불순물들을 침전시킬 수 있고, 상기 활성탄을 이용한 흡착은 발효물을 활성탄을 통과시킴으로써 발효물 내의 불순물들이 활성탄에 흡착되어 제거되는 방법이다. 본 발명의 일실시예에서는 상기 발효단계를 거쳐 제조된 본 발명의 상황버섯 유산균 발효물의 불순물을 제거하기 위하여 원심 분리 및 여과지를 이용하여 여과한 후, 활성 탄소, 사케라이트 및 감즙을 첨가하여 교반한 다음, 정치하는 과정을 추가로 수행하였다. 상기 활성 탄소는 활성 탄소의 우수한 흡착작용에 의하여 탈색 또는 탈취의 효과가 있으며, 상기의 사케라이트 및 감즙은 여과액 내에서 앙금을 형성할 수 있는 단백성분들을 응집 및 침전시켜 혼탁을 방지할 수 있는 효과가 있다.A method for removing impurities such as unnecessary dissolved substances and suspended solids present in the fermentation may be performed by a method known in the art, and representative methods may include filtration, flocculation and adsorption using activated carbon. have. The filtration may be carried out by centrifugation or using a filter, and the flocculation sedimentation may process chemicals, that is, flocculants to precipitate impurities, and the adsorption using the activated carbon may be carried out by passing the fermentation product through the activated carbon. Impurities in water are adsorbed and removed by activated carbon. In one embodiment of the present invention, to remove the impurities of the situation mushroom lactic acid bacteria fermentation of the present invention prepared through the fermentation step filtered using centrifugation and filter paper, and then stirred by adding activated carbon, sakelite and juice Next, the process of standing still further. The activated carbon has the effect of decolorization or deodorization by the excellent adsorption of activated carbon, the sakelite and juice can prevent the clouding by agglomeration and precipitation of protein components that can form sediment in the filtrate It works.
상기와 같은 후처리가 끝난 상황버섯 유산균 발효물은 여과지 또는 여과기를 이용하여 여과 후 감압증발기를 이용하여 2~5배로 농축한 발효물을 수득할 수 있다. 본 발명의 일실시예에서는 부치사 제품의 감압증발기인 Rotavapor R-114를 이용하여 본 발명의 상황버섯 유산균 발효물을 농축하였다. As described above, after finishing the situation mushroom lactic acid bacteria fermented product can be obtained by using a filter paper or a filter and then concentrated by 2 to 5 times using a reduced pressure evaporator. In an embodiment of the present invention, the situation mushroom lactic acid bacterium fermentation product of the present invention was concentrated using Rotavapor R-114, which is a decompression evaporator manufactured by an auxiliary product.
따라서 본 발명은 상황버섯 유산균 발효물의 제조방법 및 상기 방법에 의해 제조된 상황버섯 유산균 발효물을 제공한다.Therefore, the present invention provides a method for producing a situation mushroom lactic acid bacteria fermentation and a situation mushroom lactic acid bacteria fermented by the method.
본 발명자들은 본 발명의 상황버섯 유산균 발효물이 미백효과가 있는지 확인하기 위하여 멜라닌 종양 세포에 본 발명의 상황버섯 유산균 발효물을 처리한 후 합성되는 멜라닌의 양을 확인하였다(실시예 3 참조). 그 결과, 본 발명의 상황버섯 유산균 발효물을 처리한 멜라닌 종양 세포에서 합성된 멜라닌의 양은 멜라닌 합성을 유도하는 것으로 알려진 알파-MSH(멜라노 사이트 자극 호르몬)을 처리한 멜라닌 종양 세포에서 합성된 멜라닌의 양보다 현저하게 감소된 것으로 나타났고, 이는 미백효과가 있는 것으로 알려진 코직산을 처리한 것과 거의 유사한 멜라닌 양을 보이는 것을 확인할 수 있었다(도 2a 및 2b 참조). The present inventors confirmed the amount of melanin synthesized after treating the situation mushroom lactic acid bacteria fermentation of the present invention to melanin tumor cells to determine whether the situation mushroom lactic acid bacteria fermentation of the present invention has a whitening effect (see Example 3). As a result, the amount of melanin synthesized from the melanocyte tumor cells treated with the situation mushroom lactic acid bacteria fermentation of the present invention is the melanin synthesized from melanin tumor cells treated with alpha-MSH (melanosite stimulating hormone) known to induce melanin synthesis. It was found to be significantly reduced than the amount, which was confirmed to show a melanin amount almost similar to the treatment with kojic acid known to have a whitening effect (see Figs. 2a and 2b).
따라서 본 발명의 상황버섯 유산균 발효물이 멜라닌 합성을 저해하는 효능이 있어 피부의 미백 효과 효능이 있음을 알 수 있었다. Therefore, it can be seen that the lactic acid bacteria fermented mushroom of the present invention has an effect of inhibiting melanin synthesis and thus has a whitening effect of skin.
인간의 피부는 나이가 들면서 여러 가지 내적 또는 외적인 요인에 의하여 그 기능이 저하된다. 특히, 피부의 한 부분인 진피의 대부분을 차지하는 콜라겐의 양이 감소하고 콜라겐을 합성하는 섬유아세포의 활성 또한 감소하여 새로운 콜라겐의 합성도 줄어들면서 피부 노화가 진행되고, 표피의 두께가 전체적으로 얇아지고 세포와 조직에 탈수현상이 일어나 건조해지며 잔주름이 늘어나고 점차적으로 주름이 깊어진다. As human skin ages, its function is degraded by various internal or external factors. In particular, the amount of collagen that occupies most of the dermis, which is a part of the skin, decreases, and the activity of the fibroblasts that synthesize collagen decreases, thereby reducing the synthesis of new collagen, leading to aging of the skin, thinning of the epidermis, and thinning of the epidermis. Dehydration occurs in tissues and tissues, resulting in increased wrinkles and deeper wrinkles.
따라서 본 발명자들은 본 발명의 상황버섯 유산균 발효물이 피부노화 및 주름을 개선하는 효과가 있는지 사람의 섬유아세포를 통해 확인하였다. 즉, 본 발명의 일실시예에서는 사람의 섬유아세포에 본 발명의 상황버섯 유산균 발효물을 처리한 후, 콜라겐의 전구물질인 프로콜라겐의 합성을 측정하였다. 그 결과, 본 발명의 상황버섯 유산균 발효물을 처리한 섬유아세포에서의 프로콜라겐 합성이 상황버섯 유산균 발효물을 처리하지 않은 대조군에 비해 월등히 증가한 것을 확인할 수 있었고, 특히, 상황버섯 유산균 발효물의 처리 농도가 증가할수록 합성되는 프로콜라겐의 양도 증가하는 것을 확인할 수 있었다(도 3 참조). 또한, 본 발명의 상황버섯 유산균 발효물이 섬유아세포의 세포 증식을 촉진하는지 확인하기 위하여, 사람의 섬유아세포에 상황버섯 유산균 발효물을 처리한 후, MTT 분석을 통해 세포 증식 효과를 확인하였다. 그 결과, 본 발명의 상황버섯 유산균 발효물을 처리하지 않은 대조군에 비해 본 발명의 상황버섯 유산균 발효물을 처리한 섬유아세포의 세포 증식이 촉진되었음을 확인할 수 있었다(도 4 참조). 따라서 본 발명의 상황버섯 유산균 발효물이 섬유아세포의 세포증식 촉진 및 프로콜라겐 합성 촉진을 통해 피부의 노 화 및 주름을 개선하는 효과가 있을 것으로 추정할 수 있었다. Therefore, the present inventors confirmed through human fibroblasts whether the situation mushroom lactic acid bacteria fermentation of the present invention has an effect of improving skin aging and wrinkles. That is, in one embodiment of the present invention, after treating the fibrous cells of the present invention, the lactic acid bacteria fermented mushroom of the present invention, the synthesis of procollagen, a precursor of collagen, was measured. As a result, it was confirmed that the procollagen synthesis in fibroblasts treated with the situation mushroom lactic acid bacterium fermentation product of the present invention was significantly increased compared to the control group not treated with the situation mushroom lactic acid bacteria fermentation product, in particular, the concentration of the situation mushroom lactic acid bacteria fermentation product As was increased, the amount of procollagen synthesized was also increased (see FIG. 3). In addition, in order to confirm whether the situation mushroom lactic acid bacteria fermentation of the present invention promotes cell proliferation of fibroblasts, after treating the situation mushroom lactic acid bacteria fermentation to human fibroblasts, the cell proliferation effect was confirmed through MTT analysis. As a result, it was confirmed that the cell proliferation of fibroblasts treated with the situation mushroom lactic acid bacteria fermentation of the present invention was promoted compared to the control group not treated with the situation mushroom lactic acid bacteria fermentation of the present invention (see FIG. 4). Therefore, the situation mushroom lactic acid bacteria fermentation of the present invention could be estimated to have the effect of improving the aging and wrinkles of the skin through promoting the proliferation of fibroblasts and the promotion of procollagen synthesis.
나아가, 본 발명자들은 본 발명의 일실시예에서 본 발명의 상황버섯 유산균 발효물이 항산화 효과가 있는지 확인하기 위하여 SOD(superoxide dismutase) 활성도를 측정하였다. 항산화 효과를 측정하는 방법은 당업계에서 널리 사용되고 있는 SOD 어세이 키트를 이용하여 수행하였다(실시예 7 참조). 그 결과, 본 발명의 상황버섯 유산균 발효물의 농도가 높아질수록 SOD 활성도도 높아지는 것을 확인할 수 있었다(도 5 참조). 따라서 상기의 결과로 본 발명의 상황버섯 유산균 발효물이 우수한 SOD(superoxide dismutase) 활성도를 가지고 있어 항산화 효과가 있음을 알 수 있었다. Furthermore, the present inventors measured the SOD (superoxide dismutase) activity to determine whether the situation mushroom lactic acid bacteria fermentation of the present invention has an antioxidant effect in one embodiment of the present invention. The method of measuring the antioxidant effect was performed using a SOD assay kit which is widely used in the art (see Example 7). As a result, the higher the concentration of lactic acid bacteria fermented mushrooms of the present invention was confirmed to increase the SOD activity (see Fig. 5). Therefore, as a result of the above situation, it was found that the lactic acid bacteria fermented mushroom of the present invention has an excellent SOD (superoxide dismutase) activity and has an antioxidant effect.
그러므로 본 발명은 상기 본 발명의 방법에 의해 제조된 상황버섯 유산균 발효물을 함유하는 피부 미백용 조성물, 피부노화 및 주름 개선용 조성물을 제공한다.Therefore, the present invention provides a composition for skin whitening, a skin aging and wrinkle improvement composition containing the situation mushroom lactic acid bacteria fermented product prepared by the method of the present invention.
또한, 본 발명은 상기 상황버섯 유산균 발효물을 함유하는 항산화용 조성물을 제공한다.The present invention also provides an antioxidant composition containing the situation mushroom lactic acid bacteria fermentation.
본 발명자들은 유산균을 이용하여 상황버섯 분말을 발효시킨 본 발명의 상황버섯 유산균 발효물이 면역 증강 효과가 있는지 확인하기 위하여 대식세포의 증식 촉진 효과 및 대식세포에서의 산화질소 생성량을 측정하였다(실시예 7 및 8 참조). 그 결과, 체내에서 병원균 등 이물질을 감지하여 면역반응을 담당하는 대식세포의 증식이 상황버섯 유산균 발효물을 처리하지 않은 대조군에 비해 상황버섯 유산균 발효물을 처리한 실험군에서 월등하게 촉진된 것을 확인할 수 있었고, 발효물의 처리 농도가 증가할수록 대식세포의 증식 촉진 효과도 증가하는 것을 확인할 수 있었다(도 6 참조). 또한, 본 발명의 상황버섯 유산균 발효물에 의한 대식세포의 중요한 매개인자인 산화질소의 생성량을 확인한 결과, 상황버섯 유산균 발효물을 처리하지 않은 대조군에 비해 상황버섯 유산균 발효물을 처리한 실험군에서 생성된 산화질소의 양이 증가하였음을 확인할 수 있었다. 특히, 생성된 산화질소의 양은 처리한 상황버섯 유산균 발효물의 농도 의존적으로 증가하는 것을 확인할 수 있었다(도 7 참조). 따라서 상기의 결과로 본 발명의 상황버섯 유산균 발효물은 면역 기능을 증진시키는 효능을 가지고 있음을 알 수 있었다. The present inventors measured the effect of promoting the proliferation of macrophages and the amount of nitric oxide produced in macrophages in order to determine whether the situation mushroom lactic acid bacteria fermentation of the present invention fermenting the situation mushroom powder using lactic acid bacteria has an immune enhancing effect (Example 7 and 8). As a result, it was confirmed that the macrophage cells responsible for the immune response by detecting foreign substances such as pathogens in the body were significantly promoted in the experimental group treated with the situation mushroom lactic acid bacterium fermentation product compared to the control group which did not process the situation mushroom lactic acid bacteria fermentation product. As the concentration of the fermented product increased, the effect of promoting the proliferation of macrophages also increased (see FIG. 6). In addition, as a result of confirming the production amount of nitric oxide which is an important mediator of macrophages by the situation mushroom lactic acid bacteria fermentation product of the present invention, compared to the control group not treated with the situation mushroom lactic acid bacteria fermentation product produced in the experimental group It was confirmed that the amount of nitrogen oxides increased. In particular, it was confirmed that the amount of nitric oxide produced increased in a concentration-dependent manner of the treated situation mushroom lactic acid bacteria (see FIG. 7). Therefore, as a result, it was found that the situation mushroom lactic acid bacteria fermentation of the present invention has an effect of enhancing immune function.
그러므로 본 발명은 상기 본 발명의 방법에 의해 제조된 상황버섯 유산균 발효물을 함유하는 면역증강용 조성물을 제공한다. Therefore, the present invention provides a composition for immuno-enhancing containing the situation mushroom lactic acid bacteria fermented product prepared by the method of the present invention.
상기 본 발명에 따른 피부 미백용 조성물, 피부노화 및 주름 개선용 조성물, 항산화용 조성물 및 면역증강용 조성물은 식품 조성물, 약학적 조성물 또는 화장품 조성물의 형태일 수 있다.The composition for skin whitening, the composition for improving skin aging and wrinkles, the composition for antioxidant and the composition for immuno-enhancing according to the present invention may be in the form of a food composition, a pharmaceutical composition or a cosmetic composition.
상기 식품 조성물의 경우에는 본 발명의 상황버섯 유산균 발효물과 함께 식품 조성물의 제조 분야에서 일반적으로 사용되는 하나 이상의 부형제 및 첨가제를 포함하여 당 분야의 공지 방법에 따라 용이하게 다양한 형태로 제조될 수 있다. 상기에서 본 발명의 식품 조성물은 기능성 식품(functional food), 영양 보조제(nutritional supplement), 건강식품(health food) 및 식품 첨가제(food additives) 등의 모든 형태를 포함한다. 예를 들면, 건강식품으로는 본 발명의 상황버섯 유산균 발효물 자체를 차, 쥬스 및 드링크의 형태로 제조하여 음용하도록 하거나, 과립화, 캡슐화 및 분말화하여 섭취할 수 있다. 또한, 본 발명의 상황버섯 유산균 발효물을 식품 첨가제의 형태로 사용하기 위해서는 분말 또는 농축액 형태로 제조하여 사용할 수 있다.In the case of the food composition, one or more excipients and additives generally used in the field of preparing a food composition together with the situation mushroom lactic acid bacteria fermentation of the present invention may be easily prepared in various forms according to known methods in the art. . In the above, the food composition of the present invention includes all forms such as functional food, nutritional supplement, health food and food additives. For example, as a health food can be prepared by drinking the situation mushroom lactic acid bacteria fermentation itself of the present invention in the form of tea, juice and drinks, or granulated, encapsulated and powdered. In addition, in order to use the situation mushroom lactic acid bacteria fermented product of the present invention in the form of food additives can be prepared in powder or concentrate form.
본 발명의 식품 조성물 중 본 발명의 상황버섯 유산균 발효물의 바람직한 함유량으로는 식품 100g 당 약 0.1 ~ 10 mg일 수 있다.A preferred content of the situation mushroom lactic acid bacteria fermentation of the present invention in the food composition of the present invention may be about 0.1 to 10 mg per 100 g of food.
상기 약학적 조성물의 경우에는 본 발명의 방법에 따라 제조된 상황버섯 유산균 발효물을 단독으로 포함하거나 또는 하나 이상의 약학적으로 허용되는 담체, 부형제 또는 희석제를 추가로 포함할 수 있다. 상기에서 '약학적으로 허용되는'이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다.In the case of the pharmaceutical composition, it may include the situation mushroom lactic acid bacteria fermented product prepared according to the method of the present invention alone or may further include one or more pharmaceutically acceptable carriers, excipients or diluents. As used herein, 'pharmaceutically acceptable' refers to a composition that is physiologically acceptable and does not normally cause an allergic or similar reaction when administered to a human.
상기 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그 네슘 스테아레이트 및 광물유를 들 수 있다. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
상기 약학적 조성물은 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다. 또한, 본 발명의 조성물은 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 사용하여 제형화될 수 있다. 제형은 분말, 과립, 정제, 에멀젼, 시럽, 에어로졸, 연질 또는 경질 젤라틴 캅셀, 멸균 주사용액, 멸균 분말의 형태일 수 있다. The pharmaceutical composition may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives. In addition, the compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.
또한, 본 발명에 따른 약학적 조성물은 경구, 경피, 피하, 정맥 또는 근육을 포함한 여러 경로를 통해 투여될 수 있다. 본 발명에 따른 상황버섯 유산균 발효물의 약학적으로 유효한 양은 0.1 ~ 100 mg/day/체중kg, 바람직하게는 1 ~ 10 mg/day/체중kg 일 수 있다. 그러나 상기 약학적으로 유효한 양은 암의 종류 및 이의 중증정도, 환자의 연령, 체중, 건강상태, 성별, 투여 경로 및 치료기간 등에 따라 적절히 변화될 수 있다.In addition, the pharmaceutical compositions according to the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. Pharmaceutically effective amount of the situation mushroom lactic acid bacteria fermentation according to the present invention may be 0.1 ~ 100 mg / day / kg body weight, preferably 1 ~ 10 mg / day / weight kg. However, the pharmaceutically effective amount may be appropriately changed depending on the type of cancer and its severity, the age, weight, health condition, sex, route of administration and duration of treatment.
상기 화장품 조성물의 경우에는 본 발명의 방법에 따라 제조된 상황버섯 유산균 발효물을 유효성분으로 포함하고 이외에 화장품 조성물에 통상적으로 이용되는 성분들을 포함하며, 예컨대 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제 및 담체를 포함할 수 있다. 그러나 본 발명에 따른 화장품 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 이에 제한되는 것은 아니나 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린싱, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있다. 보다 상세하게는, 유연 화장수, 영양 화장수, 영양 크림, 마사지 크림, 에센스, 아이 크림, 클렌징 크림, 클렌징 포옴, 클렌징 워터, 팩, 스프레이 또는 파우더의 제형으로 제조될 수 있다. In the case of the cosmetic composition includes a mushroom mushroom lactic acid bacteria fermented product prepared according to the method of the present invention as an active ingredient, in addition to the components commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, Conventional adjuvants and carriers such as pigments and perfumes. However, the cosmetic composition according to the present invention may be prepared in any formulation conventionally prepared in the art, for example, but not limited to, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, Soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, sprays and the like. More specifically, it may be prepared in the form of a flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.
또한, 본 발명에 따른 화장료 조성물은 전체 화장료 조성에 대하여 본 발명의 상황버섯 유산균 발효물을 0.01~30 중량%의 함량으로 배합할 수 있다.In addition, the cosmetic composition according to the present invention may be blended with the content of 0.01 ~ 30% by weight of the situation mushroom lactic acid bacteria fermentation of the present invention with respect to the total cosmetic composition.
이하, 본 발명을 실시예에 의해 상세히 설명하기로 한다. 그러나 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. Hereinafter, the present invention will be described in detail by way of examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.
<< 실시예Example 1> 1>
본 발명에 따른 상황버섯 유산균 Situary mushroom lactic acid bacteria according to the present invention 발효물의Fermented product 제조 Produce
본 발명에 따른 상황버섯 유산균 발효물을 제조하기 위하여 먼저, 탄소원으로 전분질 원료인 미강분을 액화 및 당화하여 당화액을 제조하였다. 즉, 미강분 272g(청주공장 부산물)에 증류수 1680g을 첨가하고 90℃까지 온도를 높이면서 호화시킨 후, α-아밀라아제(Termamyl 120L, 노보자임사) 0.39mg을 첨가하여 90℃에서 1.5시간 동안 반응시킨 후, 아스퍼질러스 오리제(Aspergillus oryzae)로 제국한 코지 48g을 당화용기에 넣고, 글루코아밀라아제 0.39mg(Spirizyme, 노보자임사)을 첨 가하여 60℃에서 2.5시간 동안 반응시켜 당화액 2ℓ을 제조하였다.In order to prepare a situation mushroom lactic acid bacteria fermentation according to the present invention, first, liquefied and saccharified rice bran powder as a carbon source to prepare a saccharified solution. That is, 1680 g of distilled water was added to 272 g of rice bran powder (by-product of Cheongju plant), and the mixture was gelatinized while raising the temperature to 90 ° C. 48 g of koji empire impregnated with Aspergillus oryzae was added to a saccharification container, and 0.39 mg of glucoamylase (Spirizyme, Novozyme) was added and reacted at 60 ° C. for 2.5 hours to prepare 2 L of saccharified solution. It was.
이후, 상기 당화액에 상황버섯 분말 20g을 첨가하고 셀룰라아제, 글루카나아제, 아밀라아제, 크실라아제 등의 혼합 효소 0.5g(ceremix MG, 노보자임사)을 첨가하여 60℃에서 2시간 동안 상황버섯 분말을 당화하였다. 그런 뒤, 상기 당화액에 김치로부터 분리한 유산균의 한 종류인 락토바실러스 플란타룸(Lactobacillus plantarum) 및 효모인 사카로마이세스 세레비지애(Saccharomyces cerevisiae)를 상기 당화액의 총 부피에 대하여 각각 1중량%(w/v) 접종하여 15℃에서 12일간 정치배양 하면서 발효시켜 본 발명의 상황버섯 유산균 발효물을 제조하였다. Then, 20 g of mushroom mushroom powder was added to the saccharified solution, and 0.5 g (ceremix MG, Novozyme) mixed enzymes such as cellulase, glucanase, amylase, and xylase were added to the mushroom mushroom powder at 60 ° C. for 2 hours. Glycosylated. Thereafter, Lactobacillus plantarum , a type of lactic acid bacteria isolated from kimchi, and Saccharomyces cerevisiae , a yeast Saccharomyces cerevisiae , were added to the total volume of the saccharified solution. Fermented to the situation mushroom lactic acid bacteria of the present invention by fermentation by incubation by weight incubation at 15 ℃ 12% by weight (w / v).
<< 실시예Example 2> 2>
본 발명에 따른 상황버섯 유산균 Situary mushroom lactic acid bacteria according to the present invention 발효물의Fermented product 제조 Produce
상기 실시예 1에서 제조된 상황버섯 유산균 발효물을 불순물 제거 및 농축하는 단계를 추가로 수행하였다. 이를 위해 먼저 상기 실시예 1에서 제조된 상황버섯 유산균 발효물을 원심분리 및 0.45um 여과지를 이용하여 여과하였고, 이취 제거와 침전물 생성에 대한 안정성을 위하여 500ppm의 활성탄소를 첨가하고 20분 교반 후 30분 정치하였으며, 150ppm의 사케라이트와 30ppm의 감즙을 첨가하여 10분간 교반 후 10시간 동안 방치하였다. 상기와 같은 후처리가 끝난 발효액은 0.45um 여과지로 여과 후 감압증발기(Rotavapor R-114, Buchi)를 이용하여 2 ~ 5배 농축하여 상황버섯 유산균 발효물 1.5ℓ을 수득하였다. 상기 본 발명의 실시예 2에 따른 상황버섯 유산균 발효물을 제조하는 과정에 대한 모식도를 도 1에 나타내었다. The step of removing the impurities and concentration of the situation mushroom lactic acid bacteria fermentation prepared in Example 1 was further performed. To this end, first, the situation mushroom lactic acid bacteria fermented product prepared in Example 1 was filtered by centrifugation and 0.45um filter paper, and 500ppm of activated carbon was added for stability against odor removal and precipitate formation, followed by 30 minutes of stirring. After 150 minutes of sakelite and 30ppm of juice was added, the mixture was stirred for 10 minutes and left for 10 hours. After the post-treatment of the fermentation broth was filtered to 0.45um filter paper and concentrated 2 to 5 times using a reduced pressure evaporator (Rotavapor R-114, Buchi) to obtain 1.5 l of the mushrooms lactic acid bacteria fermentation. Figure 1 shows a schematic diagram for the process of producing a situation mushroom lactic acid bacteria fermentation according to Example 2 of the present invention.
<< 실시예Example 3> 3>
멜라닌 합성 저해 효과Melanin Synthesis Inhibitory Effect
상기 실시예 1에 의해 제조된 본 발명의 상황버섯 유산균 발효물이 멜라닌 종양 세포에서 멜라닌 합성 저해 효과가 있는지 알아보기 위하여 하기와 같은 실험을 수행하였다. In order to determine whether the situation mushroom lactic acid bacteria fermentation of the present invention prepared by Example 1 has an inhibitory effect on melanin synthesis in melanin tumor cells, the following experiment was performed.
먼저, 마우스 유래 악성 멜라닌 종양 세포주 B16F1(KCLB 8007, 한국세포주은행)에 페니실린(100 IU/mL), 스트렙토마이신(100 g/mL) 및 10% FBS(fetal bovine serum)를 함유하는 DMEM(Dulbecco's Modified Eagle's Medium) 배지를 첨가하여 37℃, 5% 이산화탄소를 포함하는 배양기내에서 배양하였다. 상기 배양된 멜라닌 종양 세포주 B16F1을 24웰 플레이트(well plate)에 1 X 104 의 세포수로 분주하여 1일 배양하였다. 배양 후 배지를 제거한 다음, 새로운 DMEM 배지에 멜라닌 합성을 유도하는 것으로 알려진 알파-MSH (melanocyte stimulating hormone; 멜라노사이트 자극 호르몬, 시그마)을 50nM의 농도가 되도록 첨가하여 멜라닌 합성을 유도하였고, 동시에 상황버섯 유산균 발효물을 각각 0.2% 또는 0.4%의 농도가 되도록 첨가하여 3일간 배양하였다. 이때, 아무것도 처리하지 않은 것을 음성대조군으로 사용하였고, 양성대조군으로는 미백효과가 있는 것으로 알려진 코직산(Kojic acid, 시그마)을 800uM의 농도가 되도록 첨가한 것을 사용하였다. 각 실험군 및 대조군에서 합성된 멜라닌의 양을 육안으로 확인하였고 그 변화를 디지털 카메라(Nikkon, 일본)로 촬 영하였으며 멜라닌 종양 세포에서 합성되어 분비된 멜라닌 양의 정량화는 각 실험군 및 대조군의 배지를 96웰 플레이트에 100ul씩 옮긴 후, 405nm에서 흡광도를 측정하였고 그 결과를 도 2에 나타내었다. First, DMEM (Dulbecco's Modified) containing penicillin (100 IU / mL), streptomycin (100 g / mL) and 10% FBS (fetal bovine serum) in mouse-derived malignant melanin tumor cell line B16F1 (KCLB 8007, Korea Cell Line Bank) Eagle's Medium) medium was added and cultured in an incubator containing 37 ° C., 5% carbon dioxide. The cultured melanin tumor cell line B16F1 was dispensed in a 24-well plate at a cell number of 1 × 10 4 and cultured for 1 day. After incubation, the medium was removed, and then, melanin synthesis was induced by adding melanocyte stimulating hormone (melanocyte stimulating hormone, sigma), which is known to induce melanin synthesis, to a concentration of 50 nM in a new DMEM medium. Lactic acid bacteria fermentation was added to a concentration of 0.2% or 0.4%, respectively, and incubated for 3 days. At this time, the untreated was used as a negative control group, the positive control group was used to add kojic acid (Kojic acid, sigma) known to have a whitening effect to a concentration of 800uM. The amount of melanin synthesized in each experimental group and the control group was visually confirmed, and the change was photographed by a digital camera (Nikkon, Japan), and the quantification of the amount of melanin synthesized and secreted by melanin tumor cells was measured in each group. After transferring 100ul to the well plate, the absorbance at 405nm was measured and the result is shown in FIG.
그 결과, 멜라노사이트 자극 호르몬인 알파-MSH를 처리한 군은 알파-MSH에 의해 멜라닌 합성이 크게 증가하여 배지색이 진한 갈색으로 변한 것을 확인할 수 있었고, 알파-MSH 및 상황버섯 유산균 발효물을 동시에 처리한 실험군의 배지색은 알파-MSH만을 처리한 군에 비해 배지색이 옅음을 확인할 수 있었으며 이는 코직산을 단독으로 처리한 양성대조군의 배지색과 유사한 정도로 옅음을 확인할 수 있었다(도 2a 참조). 또한, 상기 실험군 및 대조군의 배지에 분비된 멜라닌의 양을 정량 분석 한 결과, 알파-MSH만을 처리한 군은 멜라닌 함량이 비처리군에 비해 약 230%까지 증가한 것을 확인할 수 있었다. 반면, 알파-MSH 및 상황버섯 유산균 발효물을 동시에 처리한 실험군의 멜라닌 함량은 약 120%인 것으로 나타났다. 이는 알파-MSH를 단독 처리한 군의 멜라닌 함량보다 매우 많이 감소한 양이며 코직산을 처리한 양성 대조군과 거의 유사한 양인 것을 확인할 수 있었다(도 2b 참조). 따라서 상기의 결과로, 본 발명의 상황버섯 유산균 발효물은 알파-MSH에 의해 유도된 멜라닌 합성을 강력하게 저해하는 작용이 있음을 알 수 있었다.As a result, the group treated with alpha-MSH, a melanocyte stimulating hormone, showed a significant increase in melanin synthesis by alpha-MSH and the medium color turned to dark brown. The medium color of the treated experimental group was confirmed to be lighter in color than the alpha-MSH-only group, which was similar to that of the positive control group treated with kojic acid alone (see FIG. 2A). In addition, as a result of quantitative analysis of the amount of melanin secreted in the medium of the experimental group and the control group, it was confirmed that the group treated with alpha-MSH only increased the melanin content by about 230% compared to the untreated group. On the other hand, the melanin content of the experimental group treated with alpha-MSH and S. mushroom lactic acid fermentation at the same time was about 120%. This was found to be much lower than the melanin content of the group treated with alpha-MSH alone and almost similar to the positive control treated with kojic acid (see FIG. 2b). Therefore, as a result of the above, it can be seen that the situation mushroom lactic acid bacteria fermentation of the present invention has a strong inhibitory action on the synthesis of melanin induced by alpha-MSH.
<< 실시예Example 4> 4>
프로콜라겐의 합성 촉진효과Pro-collagen Synthesis Promoting Effect
본 발명의 상황버섯 유산균 발효물이 사람의 섬유아세포에서 프로콜라겐의 합성을 촉진하는 효과가 있는지 알아보기 위하여 하기와 같은 실험을 수행하였다. In order to determine whether the situation mushroom lactic acid bacteria fermentation of the present invention has an effect of promoting the synthesis of procollagen in human fibroblasts, the following experiment was performed.
먼저, 사람 섬유아세포(normal human dermal fibroblasts; 동국대학교 박정극 교수 제공)에 페니실린(100 IU/mL), 스트렙토마이신(100 ug/mL) 및 10% FBS(fetal bovine serum)를 함유하는 DMEM(Dulbecco's Modified Eagle's Medium) 배지를 넣고 37℃, 5% 이산화탄소를 포함하는 배양기 내에서 배양하였다. 상기 배양된 섬유아세포를 24 웰 플레이트(well plate)에 각 웰 당 5× 104 개의 세포수로 분주한 다음, 상기 세포 배양조건에서 24시간 배양하였다. 배양 완료 후, 배지를 제거한 다음, 상기 실시예 1에서 제조된 상황버섯 유산균 발효물을 각각 0.05%, 0.2%, 0.5% 또는 1% 농도로 포함한 혈청이 제거된(serum free) 배지에서 72시간 동안 37℃에서 배양하였다. 이때, 대조군으로는 상황버섯 유산균 발효물을 처리하지 않은 것을 사용하였다. 이후, 각 처리군의 배양액을 수거하여 단백질 전기영동을 위한 샘플로 사용하였다. 단백질 전기영동을 위해 수거한 배지와 샘플 버퍼를 1:1로 혼합하여 약 30ul의 전기영동 시료를 준비하였다. 상기 준비한 시료는 가열하여 단백질들을 변형 시킨 후, 10% SDS-PAGE에서 단백질 전기영동을 수행하였다. 이후, PVDF 막(바이오 래드사)으로 단백질을 옮기고 항-프로콜라겐 타입 1 항체(서울대학교 정진호 교수 제공)를 1차 항체로 사용하고, 항-마우스 항체-HRP(시그마)를 2차 항체로 사용하여, 엑스레이 필름(아그파) 에서 가시화 하였다. 또한, 가시화 된 필름의 웨스턴 밴드를 광학농도측정법(Quantity one software program, 바이오 래드 사)을 이용하여 정량화 하였다.First, DMEM (Dulbecco's Modified) containing penicillin (100 IU / mL), streptomycin (100 ug / mL) and 10% FBS (fetal bovine serum) in normal human dermal fibroblasts (provided by Professor Park Jung-keok, Dongguk University) Eagle's Medium) medium was added and cultured in an incubator containing 37 ° C., 5% carbon dioxide. From the busiest the cultured fibroblast cells in a 24-well plate (well plate) be 5 × 10 4 cells per well in the following, wherein said cell culture condition were cultured for 24 hours. After completion of the culture, the medium was removed, and then, for 72 hours in a serum-free medium containing the situation mushroom lactic acid bacteria fermentation product prepared in Example 1 at a concentration of 0.05%, 0.2%, 0.5% or 1%, respectively. Incubated at 37 ° C. At this time, the control was used that did not process the situation mushroom lactic acid bacteria fermentation. Then, the culture solution of each treatment group was collected and used as a sample for protein electrophoresis. About 30ul of electrophoretic samples were prepared by mixing 1: 1 with the collected medium and the sample buffer for protein electrophoresis. The prepared samples were heated to deform proteins, and protein electrophoresis was performed at 10% SDS-PAGE. Subsequently, the protein was transferred to a PVDF membrane (Bio Rad), and an
그 결과, 도 3에 나타낸 바와 같이, 상황버섯 유산균 발효물을 처리한 실험군에서의 프로콜라겐 합성이 상황버섯 유산균 발효물을 처리하지 않은 대조군에 비해 증가한 것으로 나타났으며, 상황버섯 유산균 발효물의 양을 0.05, 0.2, 0.5 및 1%로 증가하면서 처리하였을 경우, 합성되는 프로콜라겐의 양도 1.08, 1.14, 1.17 및 1.25로 증가하는 것을 확인할 수 있었다. 따라서 상기의 결과로 본 발명의 상황버섯 유산균 발효물이 프로콜라겐 합성을 촉진하는 효과가 있음을 확인할 수 있었다.As a result, as shown in Figure 3, it was shown that the procollagen synthesis in the experimental group treated with the situation mushroom lactic acid bacteria fermentation increased compared to the control group not treated with the situation mushroom lactic acid bacteria, the amount of the situation mushroom lactic acid bacteria fermentation When treated with an increase of 0.05, 0.2, 0.5 and 1%, the amount of synthesized procollagen was found to increase to 1.08, 1.14, 1.17 and 1.25. Therefore, as a result, it was confirmed that the situation mushroom lactic acid bacteria fermentation of the present invention has an effect of promoting procollagen synthesis.
<< 실시예Example 5> 5>
세포증식 촉진효과Cell growth promoting effect
본 발명의 상황버섯 유산균 발효물이 사람 섬유아세포에서 세포 증식을 촉진하는 효과가 있는지 확인하기 위하여 먼저, 사람 섬유아세포(normal human dermal fibroblasts; 동국대학교 박중극 교수 제공)에, 페니실린(100 IU/mL), 스트렙토마이신(100 ug/mL) 및 10% FBS(fetal bovine serum)를 함유하는 DMEM(Dulbecco's Modified Eagle's Medium) 배지를 넣고 37℃, 5% 이산화탄소를 포함하는 배양기 내에서 배양하였다. 상기 배양된 섬유아세포를 24 웰 플레이트(well plate)에 각 웰 당 5× 104 개의 세포수로 분주한 다음, 상기와 같은 세포 배양조건에서 24시간 배 양하였다. 배양 완료 후, 배지를 제거한 다음, 상기 실시예 1에서 제조된 상황버섯 유산균 발효물을 0.05%, 0.2%, 0.5% 또는 1% 농도로 포함하는 혈청이 제거된(serum free) 배지에서 72시간 동안 37℃에서 배양하였고, 이때, 대조군으로는 상황버섯 유산균 발효물을 처리하지 않은 것을 사용하였다. 각 처리군의 세포 수를 정량적으로 확인하기 위하여 MTT 분석을 수행하여 세포 생존률을 측정하였고, MTT 분석은 5 mg/ml 농도의 MTT 용액(시그마)을 1/10 희석한 배지로 세포에 2-4 시간 처리한 후, 세포에서의 발색 변화를 측정함으로써 수행하였다. First of all, in order to confirm whether the situation mushroom lactic acid bacteria fermentation product of the present invention has an effect of promoting cell proliferation in human fibroblasts, penicillin (100 IU / mL) is provided to normal human dermal fibroblasts (provided by Professor Dongguk University). ), Dulbecco's Modified Eagle's Medium (DMEM) medium containing streptomycin (100 ug / mL) and 10% fetal bovine serum (FBS) was added thereto, and cultured in an incubator containing 37 ° C and 5% carbon dioxide. The cultured fibroblasts were aliquoted into 5 wells of 10 4 cells per well in a 24-well plate, and then cultured for 24 hours under the same cell culture conditions. After completion of the culture, the medium was removed, and then, for 72 hours in a serum-free medium containing 0.05%, 0.2%, 0.5%, or 1% of the situation mushroom lactic acid bacteria fermented product prepared in Example 1 above. Incubated at 37 ℃, at this time, the control was used that did not process the situation mushroom lactic acid bacteria fermentation. In order to quantitatively check the number of cells in each treatment group, MTT assay was performed to measure cell viability, and MTT assay was performed on cells 2-4 in medium diluted 1/10 of MTT solution (Sigma) at 5 mg / ml concentration. After time treatment, this was done by measuring the change in color development in the cells.
그 결과, 상황버섯 유산균 발효물을 처리한 실험군의 섬유아세포 세포수가 상황버섯 유산균 발효물을 처리하지 않은 대조군의 세포수에 비해 월등히 증가한 것을 확인할 수 있었고, 또한, 상황버섯 유산균 발효물의 양을 많이 처리할수록 섬유아세포의 세포수도 점차적으로 증가하는 것으로 나타났다(도 4 참조). 따라서 본 발명의 상황버섯 유산균 발효물이 사람 섬유아세포의 세포 증식을 촉진하는 효과가 있음을 알 수 있었다. As a result, it was confirmed that the number of fibroblast cells of the experimental group treated with the situation mushroom lactic acid bacteria fermentation increased significantly compared to the number of cells of the control group not treated with the situation mushroom lactic acid bacteria fermentation, and also, the amount of the situation mushroom lactic acid bacteria fermentation was treated a lot. As the number of fibroblasts gradually increased, it was shown (see FIG. 4). Therefore, it was found that the situation mushroom lactic acid bacteria fermentation of the present invention has an effect of promoting cell proliferation of human fibroblasts.
<< 실시예Example 6> 6>
SOD(SOD ( superoxidesuperoxide dismutasedismutase ) 활성도 측정 Activity measurement
본 발명의 상황버섯 유산균 발효물이 활성산소를 제거하는 활성인 SOD 활성이 있는지 확인하는 실험을 수행하였다. 이를 위해 먼저, 상황버섯 유산균 발효물의 최종농도를 증류수로 희석하여 0.083%, 0.83% 또는 8.3% 가 되도록 준비하고, 양성대조군으로 사용할 SOD(S-2515, 시그마)를 0.1, 1 또는 10 unit/ml 이 되도록 준비하였다. SOD 활성의 측정은 당업계에 잘 알려진 SOD 어세이 키트(도진도 분자 기술, INC.)를 사용하였다. 상기 SOD 어세이 키트의 원리는 유해한 활성산소 (O2-)를 무해한 과산화수소로 전환시키는 대표적인 항산화 효소인 SOD의 활성도를 측정하는 것으로써 키트에 포함되어 있는 크산틴 산화효소(xanthine oxidase)가 작용하여 활성산소가 만들어졌을 때, 시료가 보유하는 SOD 활성도가 높을수록 만들어진 활성산소가 WST-1이라는 기질을 WSH-1 포르마잔(formazan)으로 변화시키지 않기 때문에 색의 변화가 없게 된다. 반면, 시료에 SOD 활성도가 없으면 크산틴 산화효소에 의해 생성된 활성산소를 통하여 WSH-1이 WSH-1 포르마잔으로 쉽게 변화하기 때문에 색의 변화도가 커지게 된다. Situary mushroom lactic acid bacteria fermentation of the present invention was carried out to determine whether there is SOD activity that is active to remove the active oxygen. To this end, first, the final concentration of the situation mushroom lactic acid bacteria fermentation is diluted with distilled water to prepare 0.083%, 0.83% or 8.3%, 0.1, 1 or 10 unit / ml SOD (S-2515, Sigma) to be used as a positive control group It was prepared to be. Measurement of SOD activity was performed using the SOD Assay Kit (Dojindo Molecular Technology, INC.) Well known in the art. The principle of the SOD assay kit is to measure the activity of SOD, a representative antioxidant enzyme that converts harmful free radicals (O 2 − ) to harmless hydrogen peroxide, thereby acting as a function of xanthine oxidase included in the kit. When oxygen is produced, the higher the SOD activity held by the sample, the more active oxygen does not change the substrate of WST-1 to WSH-1 formazan. On the other hand, if there is no SOD activity in the sample, the change in color is increased because WSH-1 easily changes to WSH-1 formazan through the active oxygen produced by xanthine oxidase.
그 결과, 도 5에 나타난 바와 같이, 본 발명에 따른 상황버섯 유산균 발효물의 SOD 활성도는 발효물의 처리 농도가 높아짐에 따라 SOD 활성도가 증가하는 것을 확인할 수 있었다. 특히, 상황버섯 유산균 발효물을 8.3%의 농도로 처리한 경우에는 10 unit/ml의 SOD를 처리한 양성대조군의 SOD 활성도 보다 월등히 높은 것으로 나타났다. 상기의 결과, 본 발명자들은 본 발명의 상황버섯 유산균 발효물이 높은 SOD 활성도를 가지고 있어 항산화 작용 효과가 있음을 알 수 있었다. As a result, as shown in Figure 5, the SOD activity of the situation mushroom lactic acid bacteria fermentation according to the present invention was confirmed that the SOD activity increases as the concentration of the fermentation product increases. In particular, when the Lactobacillus Lactobacillus fermentate was treated at a concentration of 8.3%, the SOD activity of the positive control group treated with 10 unit / ml of SOD was significantly higher. As a result, the present inventors have found that the situation mushroom lactic acid bacteria fermentation of the present invention has a high SOD activity and has an antioxidant effect.
<< 실시예Example 8> 8>
산화질소(NO)의 생성량 측정Measurement of Nitric Oxide Production
대식세포에서 본 발명의 상황버섯 유산균 발효물에 의해 생성되는 산화질소(nitric oxide:NO)의 양을 측정하기 위하여 먼저, 대식세포주인 Raw 264.7 세포주(KCLB 40071)에 페니실린(100 IU/mL), 스트렙토마이신 (100 g/mL) 및 10% FBS (fetal bovine serum)를 함유하는 DMEM(Dulbecco's Modified Eagle's Medium) 배지를 넣고 37℃, 5% 이산화탄소를 포함하는 배양기내에서 배양하였다. 대식세포로부터 산화질소의 생성량을 확인하기 위하여 상기 배양된 대식세포를 96-웰 플레이트에 각 웰당 1× 105 개의 세포수로 분주한 다음 상기와 같은 세포배양 조건에서 24시간 동안 배양하였다. 배양 후 배지를 제거한 다음 상황버섯 유산균 발효물의 농도가 각각 0.2, 1 또는 2%가 되도록 배지에 첨가하여 24시간 동안 배양하였고 상층액을 수득하였다. 이때, 대조군으로는 상황버섯 유산균 발효물을 처리하지 않은 것을 사용하였다. 상기 배양액 중에 존재하는 대식세포주로부터 생성된 산화질소의 양은 그리에스(Griess) 시약(시그마)을 이용하여 측정하였다. 즉, 세포 배양 상등액 100㎕와 그리에스 100㎕를 혼합하여 96웰 플레이트에서 10분간 반응하여 570nm에서의 흡광도를 측정하였고, 생성된 산화질소의 농도는 소듐 니트레이트(sodium nitrate)를 희석하고 흡광도를 측정하여 표준곡선을 얻음으로써 이를 이용하여 측정하였다.In order to measure the amount of nitric oxide (NO) produced by the situation mushroom lactic acid bacteria fermentation of the present invention in macrophages, penicillin (100 IU / mL), DMEM (Dulbecco's Modified Eagle's Medium) medium containing streptomycin (100 g / mL) and 10% FBS (fetal bovine serum) was added thereto and cultured in an incubator containing 37 ° C and 5% carbon dioxide. In order to confirm the production amount of nitric oxide from macrophages, the cultured macrophages were dispensed into 96-well plates at 1 × 10 5 cells per well and incubated for 24 hours under the above cell culture conditions. After incubation, the medium was removed, and the cultured lactic acid bacteria fermented product was added to the medium so that the concentrations were 0.2, 1, or 2%, respectively, and cultured for 24 hours to obtain a supernatant. At this time, the control was used that did not process the situation mushroom lactic acid bacteria fermentation. The amount of nitric oxide produced from the macrophage line present in the culture was measured using Griess reagent (Sigma). That is, 100 μl of the cell culture supernatant and 100 μl of Gries were mixed and reacted for 10 minutes in a 96-well plate, and the absorbance at 570 nm was measured. The measurement was performed using a standard curve to obtain a standard curve.
그 결과, 본 발명에 따른 상황버섯 유산균 발효물에 의한 대식세포의 중요한 매개자로 알려진 산화질소의 생성량은 상황버섯 유산균 발효물을 농도 의존적으로 증가하여 처리하였을 경우, 대식세포에서의 산화질소 생성량도 점차적으로 증가하는 것으로 나타났다. 특히, 상황버섯 유산균 발효물을 처리하지 않은 대조군의 산화질소 생성량은 16.69% 인 것에 비해 2%의 농도로 상황버섯 유산균 발효물을 처리한 실험군의 산화질소 생성량은 66.19%로 나타났으며(도 7 참조), 이는 산화질소 생성량이 약 4배 가까이 증가한 것이다. 따라서 상기의 결과로 본 발명의 상황버섯 유산균 발효물이 면역기능을 증진시키는 효능이 있는 것을 알 수 있었다.As a result, the production of nitric oxide, which is known as an important mediator of macrophages by the situation mushroom lactic acid bacteria fermentation according to the present invention, when the situation mushroom lactic acid bacteria fermentation was increased in a concentration-dependent manner, the production of nitric oxide in the macrophages also gradually increased. Appeared to increase. In particular, the amount of nitric oxide produced in the control group treated with the situation mushroom lactic acid bacteria fermentation at 2% concentration was 66.19% compared to 16.69% in the control group not treated with the situation mushroom lactic acid bacteria fermentation (FIG. 7). This is an increase of about four times the amount of nitric oxide produced. Therefore, as a result, it was found that the situation mushroom lactic acid bacteria fermentation of the present invention has the effect of enhancing the immune function.
본 발명에 따른 상황버섯 유산균 발효물의 제조방법은 탄소원으로 전분질 원료를 사용하여 당화시킨 당화액에 상황버섯 분말 및 효소를 첨가하여 당화시킨 후, 유산균을 첨가하여 상황버섯 분말을 발효시켜 제조한 것으로써 상기 방법에 의해 제조된 본 발명의 상황버섯 유산균 발효물은 멜라닌 합성 저해, 프로콜라겐 합성 촉진, 섬유아세포의 증식 촉진, 항산화 효과, 대식세포의 증식 및 산화질소의 생성량을 증가시키는 효능을 모두 가지고 있어서 피부 미백용 조성물, 피부 노화 및 주름 개선용 조성물, 항산화용 조성물 및 면역 증강용 조성물에 유용하게 사용할 수 있는 효과가 있다.According to the present invention, a method for preparing a situation mushroom lactic acid bacteria fermented product is prepared by fermenting a situation mushroom powder by adding lactic acid bacteria after saccharifying by adding a mushroom mushroom powder and an enzyme to a saccharified solution saccharified using starch raw material as a carbon source. The situation mushroom lactic acid bacteria fermented product of the present invention prepared by the above method has all the effects of inhibiting melanin synthesis, promoting procollagen synthesis, promoting fibroblast proliferation, antioxidant effect, increasing macrophage proliferation and nitric oxide production. There is an effect that can be usefully used in the composition for skin whitening, the composition for improving skin aging and wrinkles, the composition for antioxidant and the composition for enhancing immunity.
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| KR101056351B1 (en) * | 2008-11-12 | 2011-08-12 | 정을권 | Method of preparing barberry extract with enhanced immune function |
| US20140045240A1 (en) * | 2010-11-12 | 2014-02-13 | Allergan, Inc. | Process for preparing metabolized conditioned growth media |
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| CN106119313A (en) * | 2016-06-29 | 2016-11-16 | 上海市农业科学院 | A kind of liquid fermentation method of phellinus igniarius mycelium |
| WO2020256297A1 (en) * | 2019-06-18 | 2020-12-24 | 주식회사 라비오 | Anti-aging composition comprising albatrellus confluens, and preparation method for same |
| WO2021261975A1 (en) * | 2020-06-26 | 2021-12-30 | 고려홍삼원 주식회사 | Vegetable extract comprising vegetable collagen and vegetable mucin, and method for preparing same |
| KR102432924B1 (en) * | 2021-04-02 | 2022-08-12 | 조기환 | Extract and composition comprising plant-derived collagen and mucin |
| KR102517371B1 (en) * | 2022-10-17 | 2023-04-04 | 주식회사 소담에프앤비 | Yogurt powder and the method of making yogurt drinks using thereof |
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