KR20200081948A - Method for producing soybean paste and soy sauce adding Rhus verniciflua fermented extract - Google Patents
Method for producing soybean paste and soy sauce adding Rhus verniciflua fermented extract Download PDFInfo
- Publication number
- KR20200081948A KR20200081948A KR1020180172003A KR20180172003A KR20200081948A KR 20200081948 A KR20200081948 A KR 20200081948A KR 1020180172003 A KR1020180172003 A KR 1020180172003A KR 20180172003 A KR20180172003 A KR 20180172003A KR 20200081948 A KR20200081948 A KR 20200081948A
- Authority
- KR
- South Korea
- Prior art keywords
- lacquer
- fermented
- prepared
- soy sauce
- miso
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Images
Classifications
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- A23L11/20—
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
- A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/50—Soya sauce
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2250/00—Food ingredients
- A23V2250/20—Natural extracts
- A23V2250/21—Plant extracts
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2300/00—Processes
- A23V2300/10—Drying, dehydrating
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2300/00—Processes
- A23V2300/14—Extraction
Abstract
Description
본 발명은 옻나무 껍질에 장수버섯 균사체를 접종하여 발효한 후 건조하여 발효 옻을 준비하는 단계; 및 상기 준비한 발효 옻에 물을 첨가한 후 추출하고 여과하여 제조한 옻 발효 추출액을 이용하여 제조하는 것을 특징으로 하는 옻 된장 및 간장의 제조방법 및 상기 방법으로 제조된 옻 된장 및 간장에 관한 것이다.The present invention is a step of preparing a fermented lacquer by inoculating the lacquer bark and fermenting it by inoculating longevity mushroom mycelia; And it relates to a method of manufacturing lacquered miso and soy sauce, and lacquered miso and soy sauce prepared by the above method, characterized in that it is prepared by using a fermented lacquer extract prepared by extraction and filtration after adding water to the prepared fermented lacquer.
옻은 옻나무과(Anacardiaceae)의 낙엽교목으로 학명은 Rhus vernichiflua Stokes이며 원산지는 중국에서 히말라야에 걸친 지역뿐 아니라 한반도 각처에서 자라거나 재배되어 전 세계적으로도 약 600여 종으로 다양한 종류가 있다. 우리나라 등 동북아시아에 자생하고 있는 종류는 주로 참옻나무이며 보통 옻은 수액을 채취하여 도료의 옻칠로 사용하거나 열매로부터 왁스를 얻는다. 약리효과는 옻나무 껍질 바로 안쪽에서 나오는 옻 진액성분에서 우루시올이나 후라보노이드 등의 각종 약성 물질이 추출되며 옻의 가장 중요한 성분인 우루시올은 웅담의 주성분인 우루소데옥시콜린산과 분자구조가 99% 일치한다. 한방에서의 옻은 '맵고 따뜻하며 독이 있고 간과 비에 들어가 근육과 뼈를 강하게 하며 상처를 치유하고 오장육부를 안정시켜주며 어혈을 풀어 기침을 멎게 해주며 속이 결리고 맺힌 것을 풀어주고 나쁜 독을 풀어준다'라고 하여 예로부터 섭취에 의한 효과에 대해 많이 알려졌다. 옻은 수포, 가려움, 발진 등의 접촉성 피부염을 유발하는 우루시올 성분을 가지고 있으나 당뇨병과 위장병치료를 위해 전통적으로 닭과 오리 등을 조리할 때 첨가하여 먹어왔다. 옻에 함유된 폴리페놀 및 플라보노이드 성분은 항산화, 항돌연변이, 항염증 및 항균과 α-글루코시다아제 억제 효과 등이 있는 것으로 보고되어 있다. 최근 옻이 고부가가치 산업으로 인식되면서 식의약소재로 이용하려는 시도가 증가하고 있다. 식품의약품안전처에서는 2004년 알레르기(allergy) 유발 성분인 우루시올이 제거된 옻 추출물로 옻닭 및 옻오리에 식용으로 사용할 수 있도록 규제를 완화하였다. 2013년에는 아까시재목버섯(장수버섯, Fomitella fraxinea )을 이용하여 우루시올 성분을 제거한 옻나무 추출물로 장류, 발효 식초, 탁주, 약주, 청주 및 과실주에 한하여 발효공정 전에만 사용할 수 있도록 옻의 기준과 규격이 개정되었다. 현재 온오프라인에서 무독화 옻 추출물을 이용한 다양한 형태의 제품들이 판매되고 있다.Sumac is a deciduous tree of the family Anacardiaceae, whose scientific name is Rhus vernichiflua It is a Stokes and originated from China to the Himalayas, as well as from around the Korean Peninsula. The species native to Northeast Asia, such as Korea, is mainly oak, and lacquer is usually collected from sap to be used as lacquer for paint or to obtain wax from fruits. The pharmacological effect is that various medicinal substances such as urushiol and flavonoids are extracted from the lacquer extract component immediately coming from the inside of the lacquer bark. Urushiol, the most important component of lacquer, has a 99% molecular structure that matches the main component of Wootdam, urusodeoxycholic acid. The lacquer from the oriental medicine'is spicy, warm, poisonous, enters the liver and rain, strengthens muscles and bones, heals wounds, stabilizes the five genitals, relieves coughing, relieves coughing, releases condensation and looses bad poison. It has been known a lot about the effects of ingestion since ancient times. Lacquer contains urushiol, which causes contact dermatitis such as blisters, itchiness, and rash, but has been traditionally added to cook chickens and ducks to treat diabetes and gastrointestinal diseases. The polyphenol and flavonoid components contained in lacquer have been reported to have antioxidant, antimutagenic, anti-inflammatory and antibacterial and α-glucosidase inhibitory effects. Recently, as lacquer is recognized as a high value-added industry, attempts to use it as a food and drug material are increasing. In 2004, the Ministry of Food and Drug Safety loosened regulations to allow edible use for lacquer chicken and lacquer duck as a lacquer extract from which urushiol, an allergy-causing ingredient, was removed. In 2013, Ashwood lumber mushroom (longevity mushroom, Fomitella fraxinea ) , and the standards and specifications of lacquer have been revised so that only lacquer, fermented vinegar, takju, yakju, cheongju and fruit wine can be used only before the fermentation process. Currently, various types of products are sold online and offline using detoxified lacquer extract.
된장은 우리나라의 대표적인 전통발효 식품으로 된장국은 한국인의 가장 선호하는 음식 중 하나로 자주 섭취하는 음식이다. 된장은 콩을 주원료로 한 우수한 단백질의 공급식품이고, 저장성이 뛰어난 조미식품이다. 또한 된장은 발효, 숙성 과정 중 생성되는 각종 펩타이드, 아미노산, 유리당 등에 의해 구수한 맛과 향을 가진 것이 특징이며, 페놀화합물과 멜라노이딘(melanoidin) 종류의 항산화 물질을 가지는 것으로 알려져 있다. 된장의 발효는 콩의 유용한 성분을 인체에 흡수가 잘 되는 물질로 변화시켜, 항염증, 항산화, 항암기능 등을 가지는 것으로 알려졌다. Miso is one of Korea's representative traditional fermented foods, and miso soup is one of the most favored foods of Koreans. Soybean paste is an excellent protein-supplied food with soy as the main ingredient, and a seasoned food with excellent storage. In addition, miso is characterized by having a taste and aroma that is obtained by various peptides, amino acids, and free sugars produced during fermentation and aging, and is known to have antioxidants of phenolic compounds and melanoidin types. Fermentation of miso is known to have anti-inflammatory, antioxidant, and anti-cancer functions by converting useful components of soybeans into substances that are easily absorbed by the human body.
간장은 발효 및 숙성과정 중 기능성 물질인 펩타이드, 마이야르(Maillard) 반응에 의한 갈변물질, 폴리페놀 화합물, 이소플라본과 같은 물질들의 생성 및 함량이 변화가 되며, 이러한 물질들이 항산화, 항암, 항고혈압 등 여러 가지 생리작용이 확인되면서 단순 조미식품이 아닌 기능성 식품으로 재조명되고 있다. 대두중에는 갈변 물질 이외에도 α-토코페롤, 이소플라본(isoflavones) 및 페놀산 등의 항산화 물질이 함유되어 있는 것으로 알려져 있는데, 이러한 항산화성은 발효과정 중에 형성된 갈변물질에 의하여 더 강하게 일어날 수 있으며, 여러 가지 종류의 인지방질, α-토코페롤, 아미노산, 펩티드 등도 함께 기여할 것으로 판단된다. 간장을 비롯한 된장 및 고추장 등의 장류는 예로부터 조미를 목적으로 널리 애용된 대두 발효 식품으로, 육류 자원이 풍부하지 못한 우리 민족에게 곡류 단백질에서 부족 되기 쉬운 필수 아미노산과 필수 지방산 등의 영양성분을 공급해주는 중요한 기능을 가지고 있는 전통식품이다. 아미노산에 의한 구수한 맛, 유리당에 의한 단맛, 유기산에 의한 신맛, 그리고 소금에 의한 짠맛이 어우러져 기본 맛을 내고, 유리아미노산인 글루탐산에 의한 지미, 아르기닌에 의한 고미, 아스파르트산에 의한 감미 등이 혼합되어 맛을 향상시킨다. 대두를 발효한 간장은 다양한 맛 성분과 함께 항산화, 항암 효능을 가지는 펩타이드 및 이소플라본 등과 같은 폴리페놀 화합물, 간 해독작용이 있는 메티오닌 및 콜레스테롤을 용해하여 동맥경화 예방과 고혈압 개선에 도움을 주는 레시틴과 같은 기능성 물질들이 함유되어 있어 음식의 조미료뿐만 아니라 기능성 소재로서의 활용 가능성도 보고되고 있다. 최근에는 간장의 기능성과 관련하여 시판 간장에서 추출한 다당류의 항염증 효과, 항균 및 항암활성, 장관면역등과 관련한 연구가 진행된 바 있다. 이러한 다양한 기능성에도 불구하고, 대표적인 전통 슬로우 푸드인 간장의 섭취를 제한하는 이유 중 하나는 제조 시 첨가되는 높은 식염의 함량 때문이다. 나트륨의 과다 섭취는 고혈압과 같은 심혈관계 질환에 대한 위험을 증가시키는 인자로 지적되고 있다. 한편으로, 저염화에 따른 보존성의 유지를 위해서 기능성이 있는 천연 소재들을 발굴하여 부재료로 활용함으로써 전통식품의 저염화 및 기능성 강화를 모색하는 연구들이 진행되고 있다. 이처럼 전통 장류식품의 기능성을 강화하기 위한 연구들이 진행되어 왔으나 아직까지 옻을 첨가한 장류의 생리활성 규명에 관한 연구는 수행된 바 없다.During the fermentation and fermentation process, the production and content of substances such as functional substances such as peptides, browning substances by the Maillard reaction, polyphenol compounds, and isoflavones are changed, and these substances are antioxidant, anti-cancer, and anti-hypertensive. As various physiological effects have been confirmed, it is being re-illuminated as a functional food rather than a simple seasoning food. It is known that soybeans contain antioxidants such as α-tocopherol, isoflavones, and phenolic acid in addition to browning substances, and these antioxidants can be more strongly caused by browning substances formed during fermentation, and various kinds Phospholipids, α-tocopherol, amino acids, and peptides are also expected to contribute. Soy sauce, soybean paste, and red pepper paste, etc., are soybean fermented foods that have been widely used for seasoning purposes since ancient times, and provide nutrients such as essential amino acids and essential fatty acids that are prone to lack in grain protein to our nation, which is not rich in meat resources. Haeju is a traditional food with important functions. The basic taste is achieved by combining the sweet taste by amino acid, sweet taste by free sugar, sour taste by organic acid, and salty taste by salt, and Jimmy by glutamic acid, free amino acid, sweetness by arginine, and sweetness by aspartic acid are mixed. Improves taste Soy sauce fermented soybean dissolves polyphenol compounds, such as peptides and isoflavones, which have antioxidant and anti-cancer effects, along with various taste ingredients, and lecithin, which helps to prevent arteriosclerosis and improve hypertension by dissolving methionine and cholesterol, which have liver detoxification effects. Since it contains the same functional substances, the possibility of utilization as a functional material as well as a seasoning of food has been reported. Recently, studies have been conducted on the anti-inflammatory effect, antibacterial and anticancer activity, and intestinal immunity of polysaccharides extracted from commercial soy sauce in relation to the functionality of the liver. Despite these various functionalities, one of the reasons for limiting the intake of soy sauce, a typical traditional slow food, is the high salt content added during manufacture. Excess sodium intake has been pointed out as a factor that increases the risk for cardiovascular disease, such as hypertension. On the other hand, research is underway to find low-chlorination and functional enhancement of traditional foods by discovering functional natural materials and using them as subsidiary materials in order to maintain the preservability due to low-chlorination. As described above, studies have been conducted to enhance the functionality of traditional jangjang foods, but no research has been conducted on the identification of physiological activity of jangjang with lacquer.
한국공개특허 제2018-0082173호에는 감초를 이용한 기능성 된장 및 간장의 제조방법이 개시되어 있고, 한국등록특허 제1913347호에는 생강 메주를 이용한 간장 및 된장의 제조방법이 개시되어 있으나, 본 발명의 옻 발효 추출액을 첨가한 된장 및 간장의 제조방법과는 상이하다.Korean Patent Publication No. 2018-0082173 discloses a method of manufacturing functional miso and soy sauce using licorice, and Korean Registered Patent No. 1913347 discloses a method of manufacturing soy sauce and miso using ginger meju, but the lacquer of the present invention It is different from the manufacturing method of soybean paste and soy sauce to which fermentation extract is added.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명의 목적은 된장 및 간장 제조 시 식품첨가물은 일체 사용하지 않고, 옻을 이용한 된장 및 간장을 제조하기 위해, 최적의 옻 전처리, 배합비, 발효 및 숙성조건 등으로 제조하여, 옻 유래 영양성분이 가미되고 면역 활성이 증진되어 건강에 유익할 뿐만 아니라, 품질 및 기호도가 높은 기능성 된장 및 간장의 제조방법을 제공하는 데 있다.The present invention was derived by the above-described demands, and the object of the present invention is to use the food additives in the manufacture of miso and soy sauce, and in order to prepare miso and soy sauce using lacquer, optimal lacquer pretreatment, mixing ratio, and fermentation And it is to provide a method for producing functional miso and soy sauce, which is manufactured under the aging conditions, has lactose-derived nutritional components and promotes immune activity, which is not only beneficial to health, but also has high quality and preference.
상기 과제를 해결하기 위해, 본 발명은 (1) 옻나무 껍질에 장수버섯 균사체를 접종하여 발효한 후 건조하여 발효 옻을 준비하는 단계; (2) 상기 (1)단계의 준비한 발효 옻에 물을 첨가한 후 추출하고 여과하여 옻 발효 추출액을 제조하는 단계; (3) 불린 대두를 증자한 후 고초균 및 황국균을 접종하여 메주로 성형하고, 건조한 후 발효하여 메주를 제조하는 단계; (4) 상기 (3)단계의 제조한 메주를 소금물에 침지하여 발효한 발효물로부터 된장을 분리하는 단계; 및 (5) 상기 (4)단계의 분리한 된장에 상기 (2)단계의 제조한 옻 발효 추출액과 소금을 첨가한 후 숙성시키는 단계를 포함하여 제조하는 것을 특징으로 하는 옻 된장의 제조방법을 제공한다.In order to solve the above problems, the present invention comprises the steps of (1) preparing a fermented lacquer by inoculating a lacquer bark and fermenting it by inoculating the mycelia of longevity mushrooms; (2) adding water to the fermented lacquer prepared in the step (1), followed by extraction and filtration to prepare a lacquer fermentation extract; (3) after soaking soybean soybeans, inoculating archaea and Hwanggukgyun to form a meju, drying and fermenting to prepare meju; (4) separating the fermented soybean paste by dipping the meju prepared in the step (3) in salt water; And (5) adding the lacquer fermentation extract prepared in the step (2) and salt to the separated miso in the step (4), followed by aging to provide a method for producing the lacquered miso. do.
또한, 본 발명은 상기 방법으로 제조된 옻 된장을 제공한다.In addition, the present invention provides a lacquer miso prepared by the above method.
또한, 본 발명은 (1) 옻나무 껍질에 장수버섯 균사체를 접종하여 발효한 후 건조하여 발효 옻을 준비하는 단계; (2) 상기 (1)단계의 준비한 발효 옻에 물을 첨가한 후 추출하고 여과하여 옻 발효 추출액을 제조하는 단계; (3) 불린 대두를 증자한 후 고초균 및 황국균을 접종하여 메주로 성형하고, 건조한 후 발효하여 메주를 제조하는 단계; (4) 상기 (3)단계의 제조한 메주를 소금물에 침지하여 발효한 발효물로부터 간장을 분리하는 단계; 및 (5) 상기 (4)단계의 분리한 간장에 상기 (2)단계의 제조한 옻 발효 추출액과 소금을 첨가한 후 숙성시키는 단계를 포함하여 제조하는 것을 특징으로 하는 옻 간장의 제조방법을 제공한다.In addition, the present invention (1) lactose bark inoculated with longevity mushroom mycelium, fermented and dried to prepare fermented lacquer; (2) adding water to the fermented lacquer prepared in the step (1), followed by extraction and filtration to prepare a lacquer fermentation extract; (3) after soaking soybean soybeans, inoculating archaea and Hwanggukgyun to form a meju, drying and fermenting to prepare meju; (4) separating the soy sauce from the fermented fermentation by dipping the meju prepared in the step (3) in salt water; And (5) adding the lacquer fermentation extract prepared in the step (2) and salt to the separated soy sauce in the step (4), followed by aging, thereby providing a method for producing the lacquer soy sauce. do.
또한, 본 발명은 상기 방법으로 제조된 옻 간장을 제공한다.In addition, the present invention provides a lacquer soy sauce prepared by the above method.
본 발명의 옻 발효 추출액을 이용한 된장 및 간장은 옻의 맛과 향미로 인해 기호도가 높고, 옻의 다양한 영양성분과 기능성이 가미되어 종래의 된장 및 간장에 비해 소비자들의 영양적 요구에 부합할 수 있고, 감칠맛, 깊은맛 및 풍미가 더욱 증진되어 기호도가 우수한 된장 및 간장을 제공할 수 있다.The miso and soy sauce using the lacquer fermentation extract of the present invention have a high preference due to the taste and flavor of the lacquer, and various nutritional components and functionalities of the lacquer are added to meet the nutritional needs of consumers compared to the conventional miso and soy sauce. , Umami, deep taste and flavor are further enhanced to provide miso and soy sauce having excellent palatability.
도 1은 RAW 264. 7 세포에 옻 추출물의 농도별 처리 시 세포 생존율을 비교한 그래프이다.
도 2는 RAW 264. 7 세포에 옻 첨가량 및 발효기간에 따른 된장 추출물의 농도별 처리 시 세포 생존율을 비교한 그래프이다.
도 3은 RAW 264. 7 세포에 옻 첨가량 및 발효기간에 따른 간장 추출물의 농도별 처리 시 세포 생존율을 비교한 그래프이다.
도 4는 옻 추출물의 농도별 처리 시 NO 생성량을 비교한 그래프이다.
도 5는 옻 첨가량 및 발효기간에 따른 된장 추출물의 농도별 처리 시 NO 생성량을 비교한 그래프이다.
도 6은 옻 첨가량 및 발효기간에 따른 간장 추출물의 농도별 처리 시 NO 생성량을 비교한 그래프이다.
도 7은 옻 추출물의 농도별 처리 시 염증성 사이토카인(TNF-α, IL-1β, IL-6) 억제 효능을 비교한 그래프이다.
도 8은 옻 첨가량 및 발효기간에 따른 된장 추출물의 농도별 처리 시 IL-6 억제 효능을 비교한 그래프이다.
도 9는 옻 첨가량 및 발효기간에 따른 간장 추출물의 농도별 처리 시 IL-6 억제 효능을 비교한 그래프이다.
도 10은 옻 추출물의 농도별 처리 시 PGE2 억제 효능을 비교한 그래프이다.
도 11은 옻 첨가량 및 발효기간에 따른 된장 추출물의 농도별 처리 시 PGE2 억제 효능을 비교한 그래프이다.
도 12는 옻 첨가량 및 발효기간에 따른 간장 추출물의 농도별 처리 시 PGE2 억제 효능을 비교한 그래프이다.
도 8 및 도 11의 0.1 0% 0w: 옻 0% 첨가+발효 0주 된장을 0.1 ㎍/mL 농도로 처리, 10 0% 0w: 옻 0% 첨가+발효 0주 된장을 10 ㎍/mL 농도로 처리, 0.1 0% 8w: 옻 0% 첨가+발효 8주 된장을 0.1 ㎍/mL 농도로 처리, 10 0% 8w: 옻 0% 첨가+발효 8주 된장을 10 ㎍/mL 농도로 처리, 0.1 5% 8w: 옻 5% 첨가+발효 8주 된장을 0.1 ㎍/mL 농도로 처리, 10 5% 8w: 옻 5% 첨가+발효 8주 된장을 10 ㎍/mL 농도로 처리, 0.1 10% 8w: 옻 10% 첨가+발효 8주 된장을 0.1 ㎍/mL 농도로 처리, 10 10% 8w: 옻 10% 첨가+발효 8주 된장을 10 ㎍/mL 농도로 처리한 시료를 의미한다.
도 9 및 도 12의 0.1 0% 0w: 옻 0% 첨가+발효 0주 간장을 0.1 ㎍/mL 농도로 처리, 10 0% 0w: 옻 0% 첨가+발효 0주 간장을 10 ㎍/mL 농도로 처리, 0.1 0% 8w: 옻 0% 첨가+발효 8주 간장을 0.1 ㎍/mL 농도로 처리, 10 0% 8w: 옻 0% 첨가+발효 8주 간장을 10 ㎍/mL 농도로 처리, 0.1 5% 8w: 옻 5% 첨가+발효 8주 간장을 0.1 ㎍/mL 농도로 처리, 10 5% 8w: 옻 5% 첨가+발효 8주 간장을 10 ㎍/mL 농도로 처리, 0.1 10% 8w: 옻 10% 첨가+발효 8주 간장을 0.1 ㎍/mL 농도로 처리, 10 10% 8w: 옻 10% 첨가+발효 8주 간장을 10 ㎍/mL 농도로 처리한 시료를 의미한다.1 is a graph comparing cell viability when treated by concentration of lacquer extract on RAW 264. 7 cells.
Figure 2 is a graph comparing the cell viability during treatment by concentration of miso extract according to the amount of lacquer and fermentation period in RAW 264. 7 cells.
Figure 3 is a graph comparing the cell viability during treatment by concentration of soy extract according to the amount of lacquer and fermentation period in RAW 264. 7 cells.
4 is a graph comparing the amount of NO produced during treatment by concentration of lacquer extract.
5 is a graph comparing the amount of NO produced during treatment by concentration of miso extract according to the amount of lacquer addition and fermentation period.
6 is a graph comparing the amount of NO produced during treatment by concentration of soy extract according to the amount of lacquer added and the fermentation period.
7 is a graph comparing the inhibitory efficacy of inflammatory cytokines (TNF-α, IL-1β, IL-6) upon treatment by concentration of lacquer extract.
FIG. 8 is a graph comparing IL-6 inhibitory efficacy when treating by concentration of miso extract according to the amount of lacquer added and fermentation period.
9 is a graph comparing the IL-6 inhibitory efficacy when treating by concentration of soy extract according to the amount of lacquer added and fermentation period.
10 is PGE 2 when treated by concentration of lacquer extract It is a graph comparing the inhibitory efficacy.
11 is PGE 2 during treatment by concentration of miso extract according to the amount of lacquer added and fermentation period It is a graph comparing the inhibitory efficacy.
12 is PGE 2 during treatment by concentration of soy extract according to the amount of lacquer added and fermentation period It is a graph comparing the inhibitory efficacy.
8 and 11 0.1 0% 0w: 0% lacquer + fermented 0 weeks miso is treated at a concentration of 0.1 µg/mL, 10 0% 0w: 0% lacquer + 0 weeks fermented miso is added at a concentration of 10 µg/mL Treatment, 0.1 0% 8w: Add 0% lacquer + fermented 8 weeks miso at a concentration of 0.1 µg/mL, 10 0% 8w: Add 0% lacquer + fermented 8 weeks miso at a concentration of 10 µg/mL, 0.1 5 % 8w: added 5% lacquer + fermented 8 weeks miso at a concentration of 0.1 μg/mL, 10 5% 8w: added 5% lacquer + fermented 8 weeks miso at a concentration of 10 μg/mL, 0.1 10% 8w: lacquer 10% addition + fermented 8-week miso is treated at a concentration of 0.1 µg/mL, 10 10% 8w: refers to a sample treated with 10% lacquer + fermented 8-week miso at a concentration of 10 µg/mL.
9 and 12, 0.1 0% 0w: 0% lacquer + fermented 0 weeks soy sauce treated at a concentration of 0.1 µg/mL, 10 0% 0w: 0% lacquer + 0 weeks fermented soy sauce at a concentration of 10 µg/mL Treatment, 0.1 0% 8w: added 0% lacquer + fermented 8 weeks soy sauce at a concentration of 0.1 µg/mL, 10 0% 8w: added 0% lacquer + fermented 8 weeks soy sauce treated at a concentration of 10 µg/mL, 0.1 5 % 8w: added 5% lacquer + fermented 8 weeks soy sauce at a concentration of 0.1 µg/mL, 10 5% 8w: added 5% lacquer + fermented 8 weeks soy sauce at a concentration of 10 µg/mL, 0.1 10% 8w:
본 발명의 목적을 달성하기 위하여, 본 발명은In order to achieve the object of the present invention, the present invention
(1) 옻나무 껍질에 장수버섯 균사체를 접종하여 발효한 후 건조하여 발효 옻을 준비하는 단계;(1) preparing the fermented lacquer by inoculating the lacquer bark with fermentation by inoculating the mycelia of longevity mushrooms and drying;
(2) 상기 (1)단계의 준비한 발효 옻에 물을 첨가한 후 추출하고 여과하여 옻 발효 추출액을 제조하는 단계;(2) adding water to the fermented lacquer prepared in the step (1), followed by extraction and filtration to prepare a lacquer fermentation extract;
(3) 불린 대두를 증자한 후 고초균 및 황국균을 접종하여 메주로 성형하고, 건조한 후 발효하여 메주를 제조하는 단계;(3) after soaking soybean soybeans, inoculating archaea and Hwanggukgyun to form a meju, drying and fermenting to prepare meju;
(4) 상기 (3)단계의 제조한 메주를 소금물에 침지하여 발효한 발효물로부터 된장을 분리하는 단계; 및(4) separating the fermented soybean paste by dipping the meju prepared in the step (3) in salt water; And
(5) 상기 (4)단계의 분리한 된장에 상기 (2)단계의 제조한 옻 발효 추출액과 소금을 첨가한 후 숙성시키는 단계를 포함하여 제조하는 것을 특징으로 하는 옻 된장의 제조방법을 제공한다.(5) It provides a method of manufacturing lacquered miso, comprising adding and fermenting the lacquer fermentation extract prepared in the step (2) and salt to the separated miso in the step (4). .
본 발명의 옻 된장의 제조방법은, 보다 구체적으로는Method of manufacturing the lacquer miso of the present invention, more specifically
(1) 옻나무 껍질에 장수버섯 균사체를 접종하여 18~24℃에서 25~35일 동안 발효한 후 45~55℃에서 건조하여 발효 옻을 준비하는 단계;(1) preparing the fermented lacquer by inoculating the lacquer bark with longevity mushroom mycelium and fermenting at 18-24°C for 25-35 days, followed by drying at 45-55°C;
(2) 상기 (1)단계의 준비한 발효 옻에 발효 옻 대비 8~12배(v/w) 물을 첨가한 후 90~110℃에서 5~7시간 동안 추출한 후 여과하여 옻 발효 추출액을 제조하는 단계;(2) After adding 8-12 times (v/w) water compared to the fermented lacquer to the fermented lacquer prepared in the step (1), extracting it at 90-110°C for 5-7 hours and filtering it to prepare a lacquer fermentation extract. step;
(3) 12~18시간 동안 불린 대두를 110~120℃에서 8~12분 동안 증자한 후 고초균 및 황국균을 접종하고 메주로 성형하고, 55~65℃에서 20~28시간 동안 건조한 후 28~32℃에서 10~14일 동안 발효하여 메주를 제조하는 단계;(3) Soybeans soaked for 12-18 hours are increased at 110-120°C for 8-12 minutes, inoculated with Bacillus and Bacillus bacteria, molded into meju, dried at 55-65°C for 20-28 hours, and then 28-32 Preparing meju by fermenting at 10° C. for 10-14 days;
(4) 상기 (3)단계의 제조한 메주를 22~28%(w/v) 소금물에 0.8~1.2:2~2.5 중량비율로 침지하여 25~30℃에서 1~3개월 동안 발효한 발효물로부터 된장을 분리하는 단계; 및(4) Fermented product fermented at 25~30℃ for 1-3 months by immersing the meju prepared in the step (3) in a ratio of 0.8~1.2:2~2.5 in 22~28% (w/v) brine Separating miso from; And
(5) 상기 (4)단계의 분리한 된장에 상기 (2)단계의 제조한 옻 발효 추출액과 소금을 85~93:5~10:2~4(w:v:w) 비율로 첨가한 후 20~25℃에서 7~9주 동안 숙성시키는 단계를 포함할 수 있으며,(5) After adding the lacquer fermentation extract and salt prepared in step (2) in the ratio of 85 to 93:5 to 10:2 to 4 (w:v:w) to the separated miso in step (4) It may include a step of aging for 7 to 9 weeks at 20 ~ 25 ℃,
더욱 구체적으로는More specifically
(1) 옻나무 껍질에 장수버섯 균사체를 접종하여 21℃에서 30일 동안 발효한 후 50℃에서 건조하여 발효 옻을 준비하는 단계;(1) preparing a fermented lacquer by inoculating the lacquer bark with longevity mushroom mycelia and fermenting at 21°C for 30 days, followed by drying at 50°C;
(2) 상기 (1)단계의 준비한 발효 옻에 발효 옻 대비 10배(v/w) 물을 첨가한 후 100℃에서 6시간 동안 추출한 후 여과하여 옻 발효 추출액을 제조하는 단계;(2) adding 10 times (v/w) water compared to fermented lacquer to the fermented lacquer prepared in the step (1), extracting it at 100° C. for 6 hours, and filtering to prepare a lacquer fermentation extract;
(3) 15시간 동안 불린 대두를 115℃에서 10분 동안 증자한 후 고초균 및 황국균을 접종하고 메주로 성형하고, 60℃에서 24시간 동안 건조한 후 30℃에서 12일 동안 발효하여 메주를 제조하는 단계;(3) After soaking soybeans for 15 hours at 115° C. for 10 minutes, inoculating archaebacteria and Hwanggukgyun, shaping with meju, drying at 60° C. for 24 hours, fermenting at 30° C. for 12 days to prepare meju ;
(4) 상기 (3)단계의 제조한 메주를 25%(w/v) 소금물에 1:2.25 중량비율로 침지하여 25~30℃에서 2개월 동안 발효한 발효물로부터 된장을 분리하는 단계; 및(4) separating the miso prepared from step (3) in 25% (w/v) brine at a 1:2.25 weight ratio to separate miso from fermented products fermented at 25-30° C. for 2 months; And
(5) 상기 (4)단계의 분리한 된장에 상기 (2)단계의 제조한 옻 발효 추출액과 소금을 86.29~92.04:5~10:2.96~3.71(w:v:w) 비율로 첨가한 후 20~25℃에서 8주 동안 숙성시키는 단계를 포함할 수 있다.(5) After the lacquer fermentation extract and salt prepared in step (2) are added to the separated miso in step (4) in the ratio of 86.29~92.04:5~10:2.96~3.71(w:v:w) It may include a step of aging at 20 ~ 25 ℃ for 8 weeks.
본 발명은 또한, The present invention also
(1) 옻나무 껍질에 장수버섯 균사체를 접종하여 발효한 후 건조하여 발효 옻을 준비하는 단계;(1) preparing the fermented lacquer by inoculating the lacquer bark with fermentation by inoculating the mycelia of longevity mushrooms and drying;
(2) 상기 (1)단계의 준비한 발효 옻에 물을 첨가한 후 추출하고 여과하여 옻 발효 추출액을 제조하는 단계;(2) adding water to the fermented lacquer prepared in the step (1), followed by extraction and filtration to prepare a lacquer fermentation extract;
(3) 불린 대두를 증자한 후 고초균 및 황국균을 접종하여 메주로 성형하고, 건조한 후 발효하여 메주를 제조하는 단계;(3) after soaking soybean soybeans, inoculating archaea and Hwanggukgyun to form a meju, drying and fermenting to prepare meju;
(4) 상기 (3)단계의 제조한 메주를 소금물에 침지하여 발효한 발효물로부터 간장을 분리하는 단계; 및(4) separating the soy sauce from the fermented fermentation by dipping the meju prepared in the step (3) in salt water; And
(5) 상기 (4)단계의 분리한 간장에 상기 (2)단계의 제조한 옻 발효 추출액과 소금을 첨가한 후 숙성시키는 단계를 포함하여 제조하는 것을 특징으로 하는 옻 간장의 제조방법을 제공한다.(5) It provides a method for producing a lacquer soy sauce, characterized in that it comprises the step of aging after adding the lacquer fermentation extract and salt prepared in the step (2) to the separated soy sauce in the step (4). .
본 발명의 옻 간장의 제조방법은, 보다 구체적으로는The manufacturing method of lacquer soy sauce of the present invention, more specifically
(1) 옻나무 껍질에 장수버섯 균사체를 접종하여 18~24℃에서 25~35일 동안 발효한 후 45~55℃에서 건조하여 발효 옻을 준비하는 단계;(1) preparing the fermented lacquer by inoculating the lacquer bark with longevity mushroom mycelium and fermenting at 18-24°C for 25-35 days, followed by drying at 45-55°C;
(2) 상기 (1)단계의 준비한 발효 옻에 발효 옻 대비 8~12배(v/w) 물을 첨가한 후 90~110℃에서 5~7시간 동안 추출한 후 여과하여 옻 발효 추출액을 제조하는 단계;(2) After adding 8-12 times (v/w) water compared to the fermented lacquer to the fermented lacquer prepared in the step (1), extracting it at 90-110°C for 5-7 hours and filtering it to prepare a lacquer fermentation extract. step;
(3) 12~18시간 동안 불린 대두를 110~120℃에서 8~12분 동안 증자한 후 고초균 및 황국균을 접종하고 메주로 성형하고, 55~65℃에서 20~28시간 동안 건조한 후 28~32℃에서 10~14일 동안 발효하여 메주를 제조하는 단계;(3) Soybeans soaked for 12-18 hours are increased at 110-120°C for 8-12 minutes, inoculated with Bacillus and Bacillus bacteria, molded into meju, dried at 55-65°C for 20-28 hours, and then 28-32 Preparing meju by fermenting at 10° C. for 10-14 days;
(4) 상기 (3)단계의 제조한 메주를 22~28%(w/v) 소금물에 0.8~1.2:2~2.5 중량비율로 침지하여 25~30℃에서 1~3개월 동안 발효한 발효물로부터 간장을 분리하는 단계; 및(4) Fermented product fermented at 25~30℃ for 1-3 months by immersing the meju prepared in the step (3) in a ratio of 0.8~1.2:2~2.5 in 22~28% (w/v) brine Separating the liver from; And
(5) 상기 (4)단계의 분리한 간장에 상기 (2)단계의 제조한 옻 발효 추출액과 소금을 84~92:5~10:3.5~5(v:v:w) 비율로 첨가한 후 20~25℃에서 7~9주 동안 숙성시키는 단계를 포함할 수 있으며,(5) After the lactose fermentation extract and salt prepared in step (2) are added to the separated soy sauce in step (4) at 84~92:5~10:3.5~5(v:v:w) It may include a step of aging for 7 to 9 weeks at 20 ~ 25 ℃,
더욱 구체적으로는More specifically
(1) 옻나무 껍질에 장수버섯 균사체를 접종하여 21℃에서 30일 동안 발효한 후 50℃에서 건조하여 발효 옻을 준비하는 단계;(1) preparing a fermented lacquer by inoculating the lacquer bark with longevity mushroom mycelia and fermenting at 21°C for 30 days, followed by drying at 50°C;
(2) 상기 (1)단계의 준비한 발효 옻에 발효 옻 대비 10배(v/w) 물을 첨가한 후 100℃에서 6시간 동안 추출한 후 여과하여 옻 발효 추출액을 제조하는 단계;(2) adding 10 times (v/w) water compared to fermented lacquer to the fermented lacquer prepared in the step (1), extracting it at 100° C. for 6 hours, and filtering to prepare a lacquer fermentation extract;
(3) 15시간 동안 불린 대두를 115℃에서 10분 동안 증자한 후 고초균 및 황국균을 접종하고 메주로 성형하고, 60℃에서 24시간 동안 건조한 후 30℃에서 12일 동안 발효하여 메주를 제조하는 단계;(3) After soaking soybeans for 15 hours at 115° C. for 10 minutes, inoculating archaebacteria and Hwanggukgyun, shaping with meju, drying at 60° C. for 24 hours, fermenting at 30° C. for 12 days to prepare meju ;
(4) 상기 (3)단계의 제조한 메주를 25%(w/v) 소금물에 1:2.25 중량비율로 침지하여 25~30℃에서 2개월 동안 발효한 발효물로부터 간장을 분리하는 단계; 및(4) separating the soy sauce from the fermented product fermented for 2 months at 25-30° C. by immersing the meju prepared in the step (3) in 25% (w/v) brine at a weight ratio of 1:2.25; And
(5) 상기 (4)단계의 분리한 간장에 상기 (2)단계의 제조한 옻 발효 추출액과 소금을 85.26~91.18:5~10:3.82~4.74(v:v:w) 비율로 첨가한 후 20~25℃에서 8주 동안 숙성시키는 단계를 포함할 수 있다.(5) After the lacquer fermentation extract and salt prepared in step (2) are added to the separated soy sauce in step (4) at 85.26~91.18:5~10:3.82~4.74(v:v:w) It may include a step of aging at 20 ~ 25 ℃ for 8 weeks.
본 발명의 옻 된장 또는 옻 간장의 제조방법에서, 상기 (1) 및 (2)단계에 거쳐 옻 발효 추출액을 제조하는 것이 옻에 함유된 독성을 중화시키면서 간장 및 된장 제조 시 적정량 첨가할 경우 장류와 향미 및 맛이 잘 어우러지면서 부드러운 맛으로 인해 품질 및 기호도가 우수한 간장 및 된장 제조에 적합한 옻 발효 추출액으로 제조할 수 있었다.In the method of manufacturing lacquer miso or lactose of the present invention, preparing lactose fermentation extract through steps (1) and (2) neutralizes the toxicity contained in lacquer while adding an appropriate amount when producing soy sauce and miso. The flavor and taste were well blended, and due to the soft taste, it could be prepared with a lacquer fermentation extract suitable for soy sauce and miso production, which has excellent quality and preference.
또한, 본 발명의 옻 된장 또는 옻 간장의 제조방법에서, 상기 (3)단계의 조건으로 제조된 메주는 메주 겉면의 수분이 충분히 말라 유해한 세균 및 곰팡이가 번식하지 않으면서 이로운 곰팡이가 충분히 번식하여 된장 및 간장 제조에 적합한 메주로 제조할 수 있었다.In addition, in the method of manufacturing the lacquered miso or lactose of the present invention, the meju produced under the conditions of step (3) is sufficiently dry on the outer surface of the meju so that the beneficial fungi multiply without causing harmful bacteria and fungi to multiply. And meju suitable for soy sauce production.
또한, 본 발명의 옻 된장 또는 옻 간장의 제조방법에서, 상기 (4)단계의 조건으로 발효하는 것이 된장 및 간장 특유의 이미 및 이취가 나지 않으면서 충분히 발효가 되어 감칠맛 및 깊은맛이 증진된 장류 제조에 적합하도록 발효할 수 있었다.In addition, in the method of manufacturing the lacquered miso or lactose of the present invention, the fermentation that is fermented under the conditions of step (4) is sufficiently fermented without the odor and odor peculiar to the miso and soy sauce, and the umami and deep taste are enhanced. It could be fermented to suit manufacturing.
또한, 본 발명의 옻 된장 또는 옻 간장의 제조방법에서, 상기 (5)단계와 같은 재료 배합비 및 숙성조건으로 제조된 된장 및 간장은 적절한 옻의 향 및 맛으로 인해 기호도를 더욱 증진시키면서 항염증 활성의 기능성이 증진된 된장 및 간장으로 제조할 수 있었다.In addition, in the method of manufacturing the lacquer miso or lactose of the present invention, the miso and soy sauce prepared in the material mixing ratio and aging conditions as in step (5) further enhances the palatability due to the proper flavor and taste of lacquer, while anti-inflammatory activity It could be prepared with miso and soy sauce with improved functionality.
본 발명은 또한, 상기 방법으로 제조된 옻 된장을 제공한다.The present invention also provides lacquer miso prepared by the above method.
본 발명은 또한, 상기 방법으로 제조된 옻 간장을 제공한다.The present invention also provides lacquer soy sauce prepared by the above method.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, the content of the present invention is not limited to the following examples.
제조예Manufacturing example 1. 옻 된장 1. lacquer miso
(1) 옻나물 껍질 300 kg을 2×2 ㎝ 크기로 자른 다음 수분이 통할 수 있는 포대에 나누어 담았다. 옻나무 껍질에 수분을 흡수시키기 위해 1일 동안 수침시키고 물빼기를 하였으며, 이때 옻나무 껍질의 수분함량은 63%이었다. 이후 옻나무 껍질을 꺼내 5 L 버섯재배 봉투에 옮겨 담은 후 필터를 달아 121℃에서 100분 동안 살균하여 전처리하였다.(1) 300 kg of lacquered shells were cut into 2×2 cm pieces, and then divided into bags that could pass moisture. To absorb moisture in the lacquer bark, it was immersed and drained for 1 day, and the moisture content of the lacquer bark was 63%. After that, the bark was removed, transferred to a 5 L mushroom cultivation bag, and then filtered and pretreated by sterilization at 121°C for 100 minutes.
(2) 상기 (1)단계의 전처리한 옻나무 껍질에 장수버섯 균사체를 접종하여 21℃에서 30일간 배양한 후, 50℃에서 열풍 건조시켜 발효 옻을 제조하였다. 이후 믹서로 잘게 간 발효 옻 2 kg에 20 L의 증류수를 첨가하고 중탕기에 넣어 100℃에서 6시간 동안 열수 추출하고 무게를 재어 증발된 수분량은 보정하였으며 고형물을 제거한 상등액을 취하여 옻 발효 추출액을 제조하였다.(2) Inoculated with longevity mushroom mycelium on the pretreated lacquer bark of step (1), incubated at 21°C for 30 days, dried by hot air at 50°C to prepare fermented lacquer. Subsequently, 20 L of distilled water was added to 2 kg of fermented lacquer finely chopped with a mixer, placed in a water bath, extracted with hot water at 100° C. for 6 hours, weighed, and the amount of evaporated water was corrected. .
(3) 선별한 대두를 3회 세척한 후 대두와 물을 1:3 비율로 15시간 동안 침지한 후 2시간 동안 탈수하여 NK증자기에서 115℃에서 10분 동안 증자하였다. 삶은 대두는 컨베이어벨트를 이용하여 이동된 후 자연 냉각시켜 고초균(Bacillus subtilis)은 증자 전 대두 무게의 0.05%를 접종시키고, 황국균(Aspergillus oryzae)는 증자 전 대두 무게의 0.1%씩 각각 접종하여 성형하였다. 만들어진 사각 메주는 24시간 동안 60℃의 건조실에 건조한 후 발효온도 30℃, 발효습도 80%의 발효실로 옮겨 12일간 발효하여 메주를 제조하였다.(3) After washing the selected soybeans three times, soybean and water were immersed in a 1:3 ratio for 15 hours, and then dehydrated for 2 hours, and then increased at 115°C for 10 minutes in NK steam. Boiled soybeans were transported using a conveyor belt, and then cooled naturally. Bacillus subtilis was inoculated with 0.05% of the soybean weight before steaming, and Hwanggukgyun ( Aspergillus oryzae ) was inoculated and molded by 0.1% of soybean weight before steaming. . The prepared square meju was dried in a drying room at 60°C for 24 hours, and then moved to a fermentation room at a fermentation temperature of 30°C and a fermentation humidity of 80% to ferment for 12 days to prepare meju.
(4) 상기 (3)단계의 제조한 메주를 25%(w/v) 소금물에 1:2.25 중량비율로 침지하여 25~30℃에서 2개월 동안 발효한 발효물로부터 된장을 분리하였다.(4) The meju prepared in the step (3) was immersed in 25% (w/v) brine at a 1:2.25 weight ratio to separate miso from the fermented product fermented at 25-30°C for 2 months.
(5) 상기 (4)단계의 분리한 된장에 상기 (2)단계의 제조한 옻 발효 추출액과 소금을 92.04:5:2.96(w:v:w) 또는 86.29:10:3.71(w:v:w) 비율로 첨가한 후 20~25℃에서 8주 동안 숙성시켰다.(5) 92.04:5:2.96 (w:v:w) or 86.29:10:3.71 (w:v:) of the lacquer fermentation extract and salt prepared in step (2) above in the separated miso in step (4). w) After adding at a rate, aged at 20-25°C for 8 weeks.
제조예Manufacturing example 2. 옻 간장 2. lacquer soy sauce
(1) 상기 제조예 1의 (3)의 단계의 제조한 메주를 25%(w/v) 소금물에 1:2.25 중량비율로 침지하여 25~30℃에서 2개월 동안 발효한 발효물로부터 간장을 분리하였다.(1) Soy sauce from the fermented product fermented for 2 months at 25-30° C. by immersing the meju prepared in the step (3) of Preparation Example 1 in 25% (w/v) brine at a weight ratio of 1:2.25. Separated.
(2) 상기 (1)단계의 분리한 간장에 상기 제조예 1의 (2)단계의 제조한 옻 발효 추출액과 소금을 91.18:5:3.82(v:v:w) 또는 85.26:10:4.74(v:v:w) 비율로 첨가한 후 20~25℃에서 8주 동안 숙성시켰다.(2) To the separated soy sauce in the step (1), the lacquer fermentation extract and the salt prepared in step (2) in Preparation Example 1 are 91.18:5:3.82 (v:v:w) or 85.26:10:4.74( v:v:w) and then aged at 20-25℃ for 8 weeks.
실험방법Experiment method
(1) 옻 발효 추출액의 제조(1) Preparation of lacquer fermentation extract
발효 옻은 2017년 4월 전북 임실에서 채취한 옻나물 껍질 300 kg을 2×2 ㎝ 크기로 자른 다음 수분이 통할 수 있는 포대에 나누어 담았다. 옻나무 껍질에 수분을 흡수시키기 위해 1일 동안 수침시키고 물빼기를 하였으며, 이때 옻나무 껍질의 수분함량은 63%이었다. 이후 옻나무 껍질을 꺼내 5 L 버섯재배 봉투에 옮겨 담은 후 필터를 달아 121℃에서 100분 동안 살균하였다. 미리 제조된 장수버섯 균사체를 접종하여 21℃에서 30일간 배양한 후, 50℃에서 열풍 건조시켜 발효 옻을 제조하였다. 이후 믹서로 잘게 간 발효 옻 2 kg에 20 L의 증류수를 첨가하고 중탕기에 넣어 100℃에서 6시간 동안 열수 추출하고 무게를 재어 증발된 수분량은 보정하였으며 고형물을 제거한 상등액을 옻 발효 추출액으로써 사용하였다. 현재 식품의약품안전처에 의거한 옻발효식품 즉 장류 및 발효식초의 경우 옻나무 중량을 기준으로 최종제품 중량의 10.0% 이하에서 사용이 가능하며 본 실험에 사용한 발효옻 추출액 100 g내에는 옻나무 중량으로 10 g이 함유되어 있다고 판단하였다.Fermented lacquer was cut into 2×2 cm sized 300 kg of lacquer bean shells collected from Imsil, Jeollabuk-do in April 2017, and then divided into sacks that could penetrate moisture. To absorb moisture in the lacquer bark, it was immersed and drained for 1 day, and the moisture content of the lacquer bark was 63%. Thereafter, the bark was taken out, transferred to a 5 L mushroom cultivation bag, and then filtered and sterilized at 121°C for 100 minutes. After inoculating the mycelium of the long-lived mushrooms prepared in advance, and cultured at 21°C for 30 days, dried by hot air at 50°C to prepare fermented lacquer. Subsequently, 20 L of distilled water was added to 2 kg of fermented lacquer chopped with a mixer, placed in a water bath, extracted with hot water at 100° C. for 6 hours, weighed, and the amount of evaporated water was corrected. The supernatant from which solids were removed was used as the lacquer fermentation extract. In the case of lacquer fermented foods according to the Ministry of Food and Drug Safety, ie, berry and fermented vinegar, it can be used at 10.0% or less of the final product weight based on the weight of lacquer, and 10 g of lacquer weight is used in 100 g of fermented lacquer extract used in this experiment. It was judged that g was contained.
(2) 옻 된장 및 옻 간장 제조(2) lacquer miso and lacquer soy sauce production
선별한 대두를 3회 세척한 후 대두와 물을 1:3 비율로 15시간 침지한 후 2시간 동안 탈수하여 NK증자기에서 115℃에서 10분 동안 증자하였다. 삶은 대두는 컨베이어벨트를 이용하여 이동된 후 자연 냉각시켜 고초균은 증자 전 대두 무게의 0.05%를 접종시키고, 곰팡이는 증자 전 대두 무게의 0.1%씩 각각 접종하여 성형하였다. 만들어진 사각 메주는 24시간 동안 60℃의 건조실에 건조한 후 발효온도 30℃, 발효습도 80% 발효실로 옮겨 12일간 발효하였다. 이후 발효가 완료된 사각메주를 염수 25%에 메주:염수=1:2.25(중량대비) 비율로 침지하고 2개월 동안 1차 발효하였다. 이후 된장과 간장으로 분리하여 옻 발효 추출액을 된장과 간장 시료 각 무게에 5%, 10%가 되도록 첨가하여 옻된장의 경우 최종염도가 15%가 되게 동일하게 하였으며, 옻간장의 경우도 최종염도를 18%로 일정하게 하여 배합비를 설계하였다. 이후 8주간 상온에서 숙성하였으며 옻 발효 추출액을 첨가하지 않은 시료를 대조구로 하였다.After washing the selected soybeans 3 times, the soybean and water were immersed in a 1:3 ratio for 15 hours, and then dehydrated for 2 hours, and then increased at 115°C for 10 minutes in NK steam. Boiled soybeans were transported using a conveyor belt, and then cooled naturally, so that the Bacillus spp. was inoculated with 0.05% of the soybean weight before steaming, and mold was molded by inoculating 0.1% of the soybean weight before steaming, respectively. The dried square meju was dried in a drying room at 60°C for 24 hours, and then fermented to a fermentation temperature of 30°C and a fermentation humidity of 80% for 12 days. Subsequently, the fermented square meju was immersed in 25% saline at a ratio of meju:salt=1:2.25 (by weight) and fermented first for 2 months. Subsequently, the fermented extract of lacquer was separated into miso and soy sauce to add 5% and 10% to each weight of the miso and soy sauce samples, so that the final salinity was 15%. The mixing ratio was designed to be constant at 18%. Subsequently, the sample was aged at room temperature for 8 weeks and the sample without the lacquer fermentation extract was used as a control.
(3) 일반성분 분석(3) General component analysis
(가) 수분함량 및 pH 측정(A) Moisture content and pH measurement
수분함량은 적외선 수분측정기(FD-720, Kett Co., Japan)를 이용하여 측정하였고, pH는 무게 5 g을 취하고 증류수를 45 mL에 희석하여 진탕시킨 후 pH 미터(Mettler Toledo Gmbh, Switzerland)를 이용하여 측정하였다.The moisture content was measured using an infrared moisture meter (FD-720, Kett Co., Japan), the pH was 5 g, the distilled water was diluted with 45 mL and shaken to shake the pH meter (Mettler Toledo Gmbh, Switzerland). It was measured using.
(나) 염도 측정(B) Salinity measurement
시료 10 g에 증류수 90 mL를 가하여 1분 동안 교반한 후 염도계(ATAGO, TM-30D)로 측정하였다.90 mL of distilled water was added to 10 g of the sample, stirred for 1 minute, and then measured with a salinometer (ATAGO, TM-30D).
(다) 아미노태 질소(amino-type nitrogen) 측정(C) Measurement of amino-type nitrogen
아미노태 질소는 Formol법으로 측정하였다. 시료 1 g을 비커에 취하고 증류수 50 mL를 가하고 240초 동안 진탕 혼합하여 충분히 용해한 다음 0.1N NaOH 용액으로 pH 8.4로 적정하였다. 여기에 중성 포르말린 20 mL를 가하고 20초 동안 진탕 혼합한 뒤 다시 0.1N NaOH 용액으로 pH 8.4가 되도록 중화·적정하였다.The amino nitrogen was measured by the Formol method. 1 g of the sample was taken into a beaker, 50 mL of distilled water was added, shaken and mixed for 240 seconds, sufficiently dissolved, and then titrated to pH 8.4 with a 0.1N NaOH solution. To this, 20 mL of neutral formalin was added, shake-mixed for 20 seconds, and then neutralized and titrated to pH 8.4 again with a 0.1N NaOH solution.
아미노태 질소(mg%)=(0.1N NaOH 적정량×1.401×F×희석배수×100)/시료량(g)Amino nitrogen (mg%) = (0.1N NaOH titration × 1.401 × F × dilution multiple × 100) / sample volume (g)
(라) 총 질소(Total Nitrogen) 측정(D) Total Nitrogen Measurement
총 질소 함량은 시료 1 g을 분해장치(Foss Digester 2020)로 분해시키고 켈텍 장치(Foss Kjeltec System 2400)를 이용하여 증류한 후 적정하여 0.1N-HCl의 ml수를 총 질소로 환산하여 양을 구하였다.The total nitrogen content is determined by decomposing 1 g of the sample with a decomposition device (Foss Digester 2020), distilling it using a Keltec device (Foss Kjeltec System 2400), and titrating to convert the amount of 0.1N-HCl into total nitrogen. Did.
(4) 세포 독성 실험(4) Cytotoxicity experiment
(가) 옻 첨가 된장 및 간장 추출물 제조 (A) Preparation of miso and soy sauce extracts with lacquer
옻 추출물 0, 5, 10% 첨가 된장은 8주간 발효·숙성시킨 후 실험 재료로 사용하였다. 된장은 시료 무게의 10배에 해당하는 증류수를 첨가하여 24시간 실온에서 추출하였다. 여과지로 여과한 후 추출액은 -70℃에서 동결건조하여 세포실험에 사용하였다. The miso with 0, 5 and 10% lacquer extract was used as an experimental material after fermentation and aging for 8 weeks. Miso was extracted at room temperature for 24 hours by adding distilled water corresponding to 10 times the weight of the sample. After filtering with filter paper, the extract was lyophilized at -70°C and used for cell experiments.
옻 추출물 0, 5, 10% 첨가 간장은 8주 발효·숙성시킨 후 실험 재료로 사용하였다. 간장은 시료 무게의 10배에 해당하는 100% 에탄올을 첨가하여 24시간 실온에서 추출하였다.Soy sauce with 0, 5, and 10% lacquer extract was used as an experimental material after fermentation and aging for 8 weeks. Soy sauce was extracted at room temperature for 24 hours by adding 100% ethanol corresponding to 10 times the weight of the sample.
(나) 옻 첨가 간장 추출물의 탈염(B) Desalination of soy sauce extract with lacquer
추출액을 -20℃에서 하루 방치한 후 간장에 포함된 소금이 석출되면 상등액만 여과지(Whatman No 2)로 여과하여 여과액을 얻었다. 소금을 제거한 여과액을 회전 농축액(N-1000S, EYELA, Tokyo, Japan)에 넣고 45℃에서 에탄올을 완전히 증발시킨 후, -70℃에서 동결건조하여 세포실험에 사용하였다.After the extract was left at -20°C for one day, when the salt contained in the liver precipitated, only the supernatant was filtered with a filter paper (Whatman No 2) to obtain a filtrate. The filtrate from which the salt was removed was placed in a rotary concentrate (N-1000S, EYELA, Tokyo, Japan), and ethanol was completely evaporated at 45°C, followed by freeze-drying at -70°C for cell experimentation.
(다) 세포 배양(C) Cell culture
실험에 사용된 마우스 대식 세포 RAW 264.7은 한국 세포주은행에서 구입하였다. 세포는 습윤한 5% CO2, 37℃ 배양기(SANYO)에서 배양하였으며 배양액은 5% FBS(Fetal Bovine Serum), 1% Antibiotic-Antibiotic을 함유한 DMEM 배지를 사용하였다. FBS와 Antibiotic-Antibiotic는 Gibco, 배지는 Hyclone에서 구입하였다. 배지는 2~3일마다 교환하였으며 세포가 80% 이상 자랐을 때 PBS(Phosphate buffered saline solution)(Gibco)로 세척한 후 셀 스크레이퍼(cell scraper)를 사용하여 계대 배양하였다.The mouse macrophage RAW 264.7 used in the experiment was purchased from the Korea Cell Line Bank. Cells were cultured in a wet 5% CO 2 , 37° C. incubator (SANYO), and the culture medium was DMEM medium containing 5% FBS (Fetal Bovine Serum) and 1% Antibiotic-Antibiotic. FBS and Antibiotic-Antibiotic were purchased from Gibco and medium from Hyclone. The medium was changed every 2-3 days, and when the cells grew more than 80%, they were washed with PBS (Phosphate buffered saline solution) (Gibco), and then cultured by using a cell scraper.
(라) 세포 독성 평가(MTT assay)(D) Cytotoxicity evaluation (MTT assay)
살아있는 세포 내 미토콘드리아 내부에서 MTT가 탈수소효소 반응에 의해 포르마잔(formazan)으로 환원되면서 보라색을 나는 반응으로 식품 및 처리 약물에 대한 세포의 생존율을 확인하는 방법이다. RAW 264.7 세포를 24 웰 플레이트에 5×104 cell/well의 농도로 분주하고 5% CO2 인큐베이터에 24시간 동안 배양하였다. 배양된 세포에 옻 추출물, 된장 및 간장 추출물을 농도별로 처리하여 24시간 반응시킨 후 MTT 용액을 5 mg/mL 농도로 제조하여 각각 40 ㎕ 처리한 후 8시간 이상 배양하였다. 배양 후 DMSO 600 ㎕ 첨가하여 포르마잔을 완전히 용해한 후, 흡광도 측정기를 사용하여 540 nm의 파장에서 그 흡광도를 측정하여 생존율은 백분율로 계산하였다.It is a method to check the viability of cells for food and processed drugs by reacting with purple color while MTT is reduced to formazan by dehydrogenase reaction in mitochondria in living cells. RAW 264.7 cells were dispensed at a concentration of 5×10 4 cells/well in a 24-well plate and cultured in a 5% CO 2 incubator for 24 hours. After treating the cultured cells with lacquer extract, miso and soy sauce extracts by concentration for 24 hours, MTT solutions were prepared at a concentration of 5 mg/mL, treated with 40 μl each, and cultured for 8 hours or more. After the culture, 600 μl of DMSO was added to completely dissolve formazan, and the absorbance was measured at a wavelength of 540 nm using an absorbance meter to calculate the survival rate as a percentage.
(5) NO(Nitric oxide) 생성 억제 효능(5) Effect of inhibiting NO (Nitric oxide) production
RAW 264.7 세포주로부터 생성된 NO의 양은 세포 배양액 중에 존재하는 NO의 형태로서 염증반응을 완화시키는 효과가 있는지 평가하는 방법으로 실시하였다. RAW 264.7 세포를 24 웰 플레이트에 5×104 cell/well의 농도로 분주하고 5% CO2 인큐베이터에 24시간 동안 배양하였다. 배양된 세포에 약물을 농도별로 처리하여 4시간 배양 후, LPS(lipopolysaccharides, 1 ㎍/ml)를 처리하여 48시간 자극시킨 후 배양 상층액을 취하여 그리스 시약을 사용하여 96-웰 플레이트에 처리하고, 상온에서 15분간 반응한 후 540 nm 파장으로 측정하였다The amount of NO generated from the RAW 264.7 cell line was performed as a method of evaluating whether or not there is an effect of alleviating the inflammatory response in the form of NO present in the cell culture. RAW 264.7 cells were dispensed at a concentration of 5×10 4 cells/well in a 24-well plate and cultured in a 5% CO 2 incubator for 24 hours. After incubating for 4 hours by treating the cultured cells with concentrations, the cells were treated with LPS (lipopolysaccharides, 1 μg/ml) for 48 hours, and then the culture supernatant was taken and treated in a 96-well plate using a grease reagent. After reacting at room temperature for 15 minutes, it was measured at a wavelength of 540 nm.
(6) 염증성 사이토카인 억제 효능 (6) Efficacy in suppressing inflammatory cytokines
사이토카인을 측정하기 위하여 RAW 264.7 세포를 24 웰 플레이트에 5×104 cell/well의 농도로 분주하고 인큐베이터에 24시간 배양하였다. 배양된 세포에 옻 추출물, 된장 및 간장 추출물을 농도별로 처리하여 4시간 동안 배양 후, LPS(1 ㎍/mL)을 처리하여 18시간 배양하였다. 18시간 경과 후 배양 상층액을 e-튜브에 취하여 12,000 rpm, 10분 동안 원심분리하여 실험용 시료로 사용하였다. TNF-α, IL-1β, IL-6 염증성 사이토카인은 ELISA 키트를 사용하여 측정하였다. 실험 시작 하루 전 코팅 버퍼(coating buffer)에 1차 항체를 250:1 비율로 희석하여 100 ㎕씩 분주하고 4℃에서 하룻밤 동안 방치하였다. 다음날, 30분간 안정화 후 PBS로 3번 반복세척한 뒤 block buffer를 200 ㎕씩 분주하여 1시간 30분 동안 상온에 보관하였다. 0.05% PBST 3번 세척한 후 스텐다드(standard)와 시료(sample)를 100 ㎕씩 분주하고 2시간 동안 상온에 보관하였다. 0.05% PBST 5번 세척한 후 2차 항체와 발색효소를 block buffer와 희석하여 100 ㎕씩 분주하고 1시간 30분 동안 상온에 보관하였다. 0.05% PBST 6번 세척한 후 TMB 기질액을 100 ㎕씩 분주하고 30분 이내로 450~480 nm 흡광도로 측정하였다.To measure cytokines, RAW 264.7 cells were dispensed at a concentration of 5×10 4 cells/well in a 24 well plate and cultured in an incubator for 24 hours. The cultured cells were treated with lacquer extract, miso and soy sauce extracts by concentration, and then cultured for 4 hours, followed by LPS (1 µg/mL) treatment and cultured for 18 hours. After 18 hours, the culture supernatant was taken into an e-tube and centrifuged at 12,000 rpm for 10 minutes, and used as an experimental sample. TNF-α, IL-1β, and IL-6 inflammatory cytokines were measured using an ELISA kit. One day before the start of the experiment, the primary antibody was diluted in a 250:1 ratio in a coating buffer, dispensed 100 μl, and left at 4° C. overnight. The next day, after stabilization for 30 minutes, washed repeatedly with PBS three times, and 200 μl of block buffer was dispensed and stored at room temperature for 1 hour and 30 minutes. After washing 0.05% PBST three times, 100 µl of standard and sample were dispensed and stored at room temperature for 2 hours. After washing with 0.05
(7) 프로스타글란딘 E(PGE2) 생성 억제 효능 (7) Prostaglandin E (PGE 2 ) production inhibitory effect
활성화된 대식세포로부터 분비되는 염증물질인 PGE의 양을 세포배양액으로부터 ELISA kit를 이용하여 측정하였다. RAW 264.7 세포를 24 웰 플레이트에 5×104 cell/well의 농도로 분주하고 인큐베이터에 24시간 동안 배양하였다. 배양된 세포에 옻 추출물, 된장 및 간장 추출물을 농도별로 처리하여 4시간 배양 후, LPS(1 ㎍/mL)을 처리하여 24시간 배양하였다. 24시간 경과 후 배양 상층액을 e-튜브에 취하여 12,000 rpm으로 10분 동안 원심분리하여 실험을 위한 시료로 사용하였다. 시약들을 실험조건에 맞게 희석하여 준비하고, 스텐다드(standard)와 시료(sample)를 100 ㎕씩, assay buffer 50 ㎕, conjugate 50 ㎕, antibody 50 ㎕씩 분주하였다. 2시간 후, washing buffer로 4번 세척 후 conjugate 5 ㎕, pNpp substrate solution 200 ㎕씩 넣어주고 45분 후 405~480 nm 흡광도를 측정하였다.The amount of PGE, an inflammatory substance secreted from activated macrophages, was measured from the cell culture medium using an ELISA kit. RAW 264.7 cells were dispensed at a concentration of 5×10 4 cells/well in a 24-well plate and cultured in an incubator for 24 hours. The cultured cells were treated with lacquer extract, miso and soy sauce extracts by concentration, and then cultured for 4 hours, followed by treatment with LPS (1 µg/mL) and cultured for 24 hours. After 24 hours, the culture supernatant was taken into an e-tube and centrifuged at 12,000 rpm for 10 minutes, and used as a sample for the experiment. Reagents were prepared by diluting them according to the experimental conditions, and standard and sample samples were dispensed at 100 μl,
(8) 유전자 발현 분석 (8) Gene expression analysis
Nanostring 기법을 활용한 유전자 발현 분석은 RAW 264.7 세포를 6 cm 디쉬에 5×106 cell/well의 농도로 분주하고 인큐베이터에서 24시간 동안 배양하였다. 배양된 세포에 옻 추출물, 된장 및 간장 추출물을 농도별로 처리하여 1시간 배양 후, LPS(1 ㎍/mL)을 처리하여 24시간 동안 배양하였다. 이후 상층액을 제거한 뒤 PBS로 2회 세척한 후 PBS 1 mL를 넣어 스크레퍼로 취하여 e-튜브에 옮긴 뒤 4000 rpm, 5분 원심분리하여 샘플을 -70℃ 보관하여 유전자 분석에 사용하였다. 총 RNA를 분리하여 100 ng/5 ㎕가 되도록 RNA 양을 정량하였다. 냉동고에서 reporter codeset과 probeset을 꺼내서 얼음 위에서 녹인 후, 몇 차례 인버트(invert)해서 섞어주고, spin down시킨다. 70 ㎕의 혼성화 완충용액(hybridization buffer)을 reporter codeset tube에 넣어 master mix을 만들었다. 캡쳐 프로브(Capture probe)는 해당 튜브에 섞지 않고 8 ㎕를 각각의 웰에 분주하였다. 5 ㎕의 RNA를 각각의 웰에 분주하였다. 캡쳐 프로브(Capture probe)는 인버트한 후 각각의 웰에 2 ㎕씩 분주하였다. 반응이 시작되기 전에 서모사이클러(thermocycler)를 65℃에 reaction volume을 15 ㎕가 되게 pre-heating시켰다. 혼성화(hybridization)는 최소 12시간 이상 해주어야 하고, 반응이 끝나도 계속 65℃를 유지하고, 최대 혼성화 시간(maximum hybridization time)은 30시간을 초과하지 않게 하였다. 일단 서모사이클러(thermocycler)에서 꺼낸 시료는 재빨리 nCounter Prep-station 실시하였다. nCounter prep-station에 hybridization 과정이 완료된 샘플을 로딩하여 세척(washing) 및 고정화(immobilization) 과정을 수행하였다. Prep station에서 위의 과정이 완료된 카트리지를 디지털 분석기에 올린 후 counting한 후 nSolver software를 이용하여 발현 정도를 분석하였다. 타겟 유전자에 대한 정보는 표 3과 같다.In the gene expression analysis using the nanostring technique, RAW 264.7 cells were dispensed at a concentration of 5×10 6 cells/well in a 6 cm dish and cultured in an incubator for 24 hours. The cultured cells were treated with lactose extract, miso and soy sauce extracts by concentration, followed by incubation for 1 hour, followed by treatment with LPS (1 μg/mL) for 24 hours. Then, after removing the supernatant and washing twice with PBS, 1 mL of PBS was added, taken as a scraper, transferred to an e-tube, and centrifuged at 4000 rpm for 5 minutes to store the sample at -70°C for use in gene analysis. Total RNA was isolated and RNA was quantified to be 100 ng/5 μl. Remove the reporter codeset and probeset from the freezer, melt on ice, invert several times, mix, and spin down. A master mix was prepared by adding 70 μl of hybridization buffer to the reporter codeset tube. 8 μl was dispensed into each well without mixing the probe with the corresponding tube. 5 μl of RNA was dispensed into each well. After the capture probe was inverted, 2 µl was dispensed into each well. Before the reaction was started, the thermocycler was pre-heated at 65° C. to a reaction volume of 15 μl. Hybridization should be performed for at least 12 hours, and the reaction was maintained at 65°C, and the maximum hybridization time was not exceeded 30 hours. Once the sample was taken out from the thermocycler, nCounter Prep-station was performed quickly. The washing and immobilization were performed by loading the sample with the hybridization process completed on the nCounter prep-station. At the prep station, the cartridge with the above process was put on a digital analyzer, counted, and then the expression level was analyzed using nSolver software. Table 3 shows target gene information.
실시예Example 1.일반성분 분석 1.General component analysis
옻 첨가 된장 숙성기간은 8주로 하였으며, 일반성분 분석으로는 수분, 염도, pH, 그리고 아미노태 질소를 분석하였다(표 4). 옻 된장 시료는 숙성기간 동안 수분 함량은 55.4~56.6%로 표준규격 60% 이하로 모두 적정 수준이었고, 각 된장의 염도는 숙성기간 중 큰 변화가 없었다. pH는 수소이온 농도로 식품의 산성과 알칼리성 정도를 나타내는 수치로, 숙성 초기 5.4~5.5 수준에서 숙성과정을 거치면서 5.2로 낮아졌으나 시료 간의 큰 차이는 없었다. 아미노태 질소는 발효식품의 숙성도를 판단하는 성분으로 된장의 제조와 숙성 과정 중에 단백질이 효소작용으로 가수분해 되어 맛을 내는 아미노산을 생성하는데, 대조구에 비해 옻 첨가 실험구의 아미노태 질소 함량이 낮았고 8주 숙성 후에 초기 수준보다 대조구는 48.1 mg%, 5% 옻된장은 31.6 mg%, 10% 옻된장은 36.1 mg% 증가하였으며, 모두 표준규격인 300 mg% 이상이었다. 일반적으로 수분의 전통식품표준규격은 60% 이하이며, 숙성정도의 지표로 사용되는 있는 아미노태 질소는 전통식품표준규격은 300 mg이다. The maturation period of miso added with lacquer was 8 weeks, and moisture, salinity, pH, and amino nitrogen were analyzed for general component analysis (Table 4). During the fermentation period, lacquered miso samples had a moisture content of 55.4 to 56.6%, which was appropriate to below 60% of the standard, and the salinity of each miso was not significantly changed during the fermentation period. The pH is a hydrogen ion concentration that indicates the acidity and alkalinity of the food. It was lowered to 5.2 during the aging process from the initial level of 5.4 to 5.5, but there was no significant difference between samples. Amino nitrogen is a component that determines the fermented food's maturity, and during the manufacturing and maturation process of the miso, the protein hydrolyzes through enzymatic action to produce an amino acid that tastes. The amino nitrogen content of the experimental group added with lacquer was lower than that of the control. After 8 weeks of maturation, the control increased by 48.1 mg%, 5% lacquered soybean paste 31.6 mg%, and 10% lacquered soybean paste increased by 36.1 mg% compared to the initial level, all of which exceeded the standard standard of 300 mg%. In general, the standard standard of food in water is 60% or less, and the amino nitrogen that is used as an indicator of maturity is 300 mg in the standard of traditional food.
옻 첨가 간장 숙성기간은 8주로 하였으며, 일반성분 분석으로는 총질소, 염도, pH, 그리고 아미노태질소를 분석하였다. 간장에 용해되어 있는 총 질소(TN)는 간장의 숙성과정 중 효소 및 미생물에 의해 콩에 함유되어 있는 질소성분이 분해되어 유리되면서 증가되며, 간장의 품질 지표로 이용되고 있다. 대조구, 옻 5% 간장 그리고 옻 10% 간장의 숙성기간 동안 총 질소함량은 0.96~0.99%, 0.88~0.91% 그리고 0.82~0.87%로 나타났으며 모든 간장시료에서 8주 후의 총 질소 함량은 숙성초기보다 증가되었으나 증가폭은 크지 않았다(표 5). 식품공전상 한식간장의 총 질소 기준은 0.7% 이상이며, 옻 간장의 경우 식품공전상 기준을 모두 충족하였다. 염도의 경우 모든 실험조건에서 18.2~18.4%로 유지되었으며, 재래식 및 개량식 간장의 염분 함량의 변화는 수분 손실량과 유사하여 간장의 염도는 수분의 증발량과의 관련성이 높다고 하였는데 본 시료에서는 유의적 차이가 적은 것으로 보아 수분 증발량이 적은 것으로 판단된다. pH의 변화는 모든 시료에서 변화가 적었으며, 숙성 8주 후의 pH는 약간 증가하였는데 이러한 결과는 숙성기간동안 총 질소함량이 증가한 것으로 보아 암모니아태 질소의 증가로 사료된다. 아미노태 질소는 발효식품의 숙성도를 판단하는 성분으로 간장의 제조와 숙성 과정 중에 단백질이 효소작용으로 가수 분해되어 맛을 내는 아미노산을 생성하는데, 숙성기간 동안 대조구(471.9~522.9 mg%), 옻 5% 간장(474.3~489.6 mg%) 그리고 옻 10% 간장(451.8~476.1 mg%)로 나타나, 대조구에 비해 옻 첨가 실험구의 아미노태 질소 함량이 낮았고 8주 숙성 후에 초기 수준보다 대조구는 43.8 mg%, 옻 5% 간장은 15.3 mg%, 옻 10% 간장은 24.3 mg% 증가하였다(표 5). 아미노태 질소는 단백질이 효소작용으로 가수분해되어 맛을 내는 아미노산을 생성하게 되며 아미노태 질소 함량 변화로 유리 아미노산의 변화를 간접적으로 추정할 수 있다. 본 실험 결과에서 간장의 아미노태 질소함량은 옻 발효 추출액의 첨가에 따른 유의적인 차이가 없는 것으로 추정된다.The soybean ripening period with lacquer was 8 weeks, and total nitrogen, salinity, pH, and amino nitrogen were analyzed by general component analysis. The total nitrogen (TN) dissolved in the liver is increased as the nitrogen component contained in soybeans is broken down and released by enzymes and microorganisms during the ripening process of the liver, and is used as a quality indicator for the liver. During the maturation period of the control,
실시예Example 2. 세포 독성 실험 2. Cytotoxicity experiment
RAW 264. 7 세포에서 옻 추출물, 된장, 간장 추출물을 농도별로 처리한 결과, 세포사멸에 영향을 주지 않아 독성이 없는 것으로 확인되었다. 옻 추출물(Rhus vernichiflua Stokes: RV)을 각각의 농도 (1~100 ㎍/mL)로 처리한 결과 RAW 264.7 세포의 세포생존율에는 영향을 미치지 않는 것으로 확인되었다(도 1). 옻 첨가 농도 0%, 5%, 10% 된장 및 간장의 발효 0주, 4주, 8주 추출물은 각각의 농도(0.1~10 ㎍/mL)로 처리한 결과 세포 생존율에 영향을 미치지 않아 실험 농도로 설정하였다(도 2, 3). 한편 옻 첨가 된장 및 간장 100 ㎍/mL 처리 시 세포생존율이 94% 수준으로 감소하여 실제 실험에 사용한 농도는 0.1~10 ㎍/mL로 정하였다.RAW 264. 7 cells were treated with lacquer extract, miso, and soy extract by concentration, and were found to be non-toxic because they did not affect cell death. As a result of treatment with lacquer extract ( Rhus vernichiflua Stokes: RV) at each concentration (1-100 μg/mL), it was confirmed that it did not affect the cell viability of RAW 264.7 cells (FIG. 1). 0%, 5%, 10% concentration of
실시예Example 3. NO(Nitric oxide) 생성 억제 효능 3. NO(Nitric oxide) production inhibitory effect
NO는 활성 산소종의 하나이며, 일반적으로 종양을 제거하는 면역반응에 관여하지만 과량 존재할 경우 인체에 염증반응을 조절하여 다양한 병리적인 반응을 유도한다. NO 저해에 대한 옻 추출물, 옻 첨가 된장 및 간장 추출물의 효과를 알아보기 위해 RAW 264.7 세포에 농도별 옻 추출물, 옻 첨가 된장 및 간장 추출물을 4시간 동안 처리한 후 LPS(1 ㎍/mL)를 48시간 동안 처리하여 NO 생성량을 측정하였다. 옻 추출물을 각각의 농도(1~100 ㎍/mL)로 처리한 결과, RAW 264.7 세포에서 LPS 처리로 증가된 NO 농도를 농도 의존적으로 억제하였다(도 4). 된장 추출물은 0.1~10 ㎍/mL로 처리한 결과 LPS에 의해 증가된 NO 생성을 모든 농도에서 억제하였다(도 5). 발효하지 않은 된장(발효 0주 된장)에서 옻 첨가 농도에 따른 NO 생성억제 효과는 나타나지 않았지만, 발효 8주 된장에서는 옻 첨가 농도가 증가할수록 NO 생성은 억제되는 것으로 나타났다. 특히 옻 추출물이 10% 함유된 발효 8주차 된장에서 NO 생성 억제 효능이 가장 뛰어났다. 옻 5% 및 10% 첨가 된장의 경우 발효하지 않은 된장(발효 0주 된장)에 비해 발효 8주 된장에서 NO 생성 억제 효능이 크게 나타났다. 간장 추출물 또한 농도별 0.1~10 ㎍/mL로 처리한 결과 LPS에 의해 증가되는 NO 생성을 억제하는 것으로 나타났다(도 6). 발효하지 않은 간장(발효 0주 간장)에서 옻 첨가 농도에 따른 NO 생성억제 효과는 나타나지 않았지만, 발효 8주 간장에서는 옻 첨가 농도가 증가할수록 NO 생성은 억제되는 것으로 나타났다. 특히 옻 추출물이 10% 함유된 발효 8주차 간장에서 NO 생성 억제 효능이 가장 뛰어났다.NO is one of the reactive oxygen species, and is generally involved in the immune response to remove the tumor, but in the presence of an excess, induces various pathological responses by regulating the inflammatory response in the human body. In order to investigate the effects of lacquer extract, lacquered miso and soy sauce extracts on NO inhibition, LPS (1 µg/mL) was treated after treatment with RAW 264.7 cells for each concentration for 4 hours. The amount of NO produced was measured by treating for a period of time. As a result of treatment with each concentration (1-100 μg/mL) of the lacquer extract, the NO concentration increased by LPS treatment in RAW 264.7 cells was suppressed in a concentration-dependent manner (FIG. 4 ). As a result of treatment with miso extract 0.1~10 μg/mL, NO production increased by LPS was suppressed at all concentrations (FIG. 5 ). In the non-fermented miso (fermented 0-week miso), NO production was suppressed according to the concentration of lacquer, but in the 8-week fermented miso, NO production was inhibited as the concentration of lacquer increased. In particular, in the 8th week of fermentation containing 10% lacquer extract, the efficacy of inhibiting NO production was the best. In the case of miso added with 5% and 10% lacquer, the NO production inhibitory effect was significantly higher in the fermented 8-week miso than the unfermented miso (fermented 0-week miso). Soy sauce extract was also treated with 0.1 to 10 µg/mL according to concentration, and it was found to suppress the NO production increased by LPS (FIG. 6). In non-fermented soy sauce (fermented soy sauce at 0 weeks), NO production was not inhibited according to the concentration of lacquer, but in 8 weeks of fermentation soy sauce, NO was inhibited as the concentration of lacquer was increased. In particular, in the 8th week of fermentation containing 10% lacquer extract, soy sauce was the most effective in inhibiting NO production.
실시예Example 4. 염증성 사이토카인 억제 효능 4. Inflammatory cytokine suppression effect
TNF-α는 급성염증반응을 주로 매개하는 사이토카인이며, 주로 활성화된 대식세포에 의해 분비된다. TNF-α의 중요한 역할은 면역세포의 조절이며, IL-1과 IL-6의 분비를 자극할 뿐만 아니라 NF-kB와 AP-1과 같은 전사인자들을 활성화시키거나 세포사멸을 유발한다. 옻 추출물이 RAW 264.7 세포에서 LPS에 의한 TNF-α 생성에 미치는 영향을 살펴 본 결과, 모든 옻 추출물 처리 군(1~100 ㎍/mL)에서 LPS만 처리한 대조군에 비해 TNF-α 생성을 농도 의존적으로 감소시켰다(도 7). IL-1β는 저농도의 경우 국소적 염증반응에 관여하고, 고농도 시 혈류로 들어가 내분비 기능을 나타내며, NF-kB와 AP-1 등의 전사인자를 활성화시키고, MAP Kinase 활성과 연관된다고 알려져 있다. 옻 추출물이 RAW 264.7 세포에서 LPS에 의한 IL-1β 생성에 미치는 영향을 살펴 본 결과, 모든 옻 추출물 처리 군(1~100 ㎍/mL)에서 LPS에 의해 증가된 IL-1 생성을 효과적으로 억제하였다. IL-6는 급성염증반응에 중요한 사이토카인이며, 국소적 및 전신적 범위에서 작용하고, 단핵세포, 혈관내피세포, 섬유모세포 외 여러 세포에서 IL-1과 TNF-α의 반응에 의해 생성되며, 적응성 면역에 영향을 주고 B세포 계열의 증식과 항체 분비에 관여하는 것으로 알려져 있다. 옻 추출물이 RAW 264.7 세포에서 LPS에 의한 IL-6 생성에 미치는 영향을 살펴 본 결과, 모든 옻 추출물 처리 군(1~100 ㎍/mL)에서 LPS에 의해 증가된 IL-6 생성을 농도 의존적으로 감소시켰다. 또한 옻 첨가 된장의 경우, LPS에 의하여 IL-6 생성량은 235.67±8.73 pg/mL로 LPS를 처리하지 않은 정상대조군에 비해 약 14배 증가하였다. 발효하지 않은 된장에 비해 발효 8주차 된장에서 IL-6 생성 억제효능이 크게 나타났다(도 8). 또한 옻 첨가 농도가 높을수록 IL-6 생성 억제 효능이 증가하는 경향을 보였다. 특히 10%의 옻 추출물이 첨가된 발효 8주차 된장 10 ㎍/mL 군에서 IL-6 농도는 159.32±4.4 pg/mL로 가장 크게 감소시켰다. 옻 첨가 간장의 경우, LPS에 의하여 IL-6 생성량은 525.01±31.89 pg/mL로 LPS를 처리하지 않은 정상대조군에 비해 약 37배 증가하였다. 발효하지 않은 간장에 비해 발효 8주차 간장에서 IL-6 생성 억제효능이 크게 나타났다(도 9). 또한 옻 첨가 농도가 높을수록 IL-6 생성 억제 효능이 증가하였다. 10%의 옻 추출물이 첨가된 발효 8주차 간장 10 ㎍/mL 군에서 IL-6 농도는 393.15±41.26 pg/mL로 가장 효과가 좋았다.TNF-α is a cytokine that mediates acute inflammatory reactions and is secreted mainly by activated macrophages. The important role of TNF-α is the regulation of immune cells, stimulates the secretion of IL-1 and IL-6, as well as activates transcription factors such as NF-kB and AP-1 or causes apoptosis. As a result of examining the effect of lacquer extract on TNF-α production by LPS in RAW 264.7 cells, TNF-α production was concentration-dependent compared to the control group treated with LPS in all lacquer extract-treated groups (1-100 μg/mL). (Fig. 7). It is known that IL-1β is involved in local inflammatory reactions at low concentrations, enters the bloodstream at high concentrations, exhibits endocrine function, activates transcription factors such as NF-kB and AP-1, and is associated with MAP Kinase activity. As a result of examining the effect of lacquer extract on IL-1β production by LPS in RAW 264.7 cells, the increased IL-1 production by LPS was effectively suppressed in all lacquer extract treated groups (1-100 μg/mL). IL-6 is an important cytokine for acute inflammatory reactions, acts in the local and systemic range, and is produced by the reaction of IL-1 and TNF-α in monocytes, vascular endothelial cells, fibroblasts, and many other cells. It is known to affect immunity and to participate in the proliferation and secretion of B cells. As a result of examining the effect of lacquer extract on IL-6 production by LPS in RAW 264.7 cells, the concentration of IL-6 increased by LPS in all lacquer extract treated groups (1-100 μg/mL) was decreased in a concentration-dependent manner. Ordered. In addition, in the case of miso added with lacquer, the amount of IL-6 produced by LPS was 235.67±8.73 pg/mL, which was increased about 14 times compared to the normal control without LPS. Compared to unfermented miso, the inhibitory effect of IL-6 production was significantly higher in fermented miso week 8 (FIG. 8). In addition, the higher the concentration of lacquer, the higher the inhibitory effect of IL-6 production. In particular, in the 10 μg/mL group of fermented 8-week miso with the addition of 10% lacquer extract, the concentration of IL-6 was most decreased to 159.32±4.4 pg/mL. In the case of soy sauce added with lacquer, the amount of IL-6 produced by LPS was 525.01±31.89 pg/mL, which was about 37 times higher than that of the normal control group without LPS. Compared to the non-fermented soy sauce, IL-6 production inhibitory effect was significantly increased in the soy sauce at week 8 (FIG. 9). In addition, the higher the concentration of lacquer, the higher the inhibitory effect of IL-6 production. The IL-6 concentration in the 10 μg/mL group of 8 weeks of fermentation with 10% lacquer extract was the most effective at 393.15±41.26 pg/mL.
실시예Example 5. 프로스타글란딘 E( 5. Prostaglandin E ( PGEPGE 22 ) 생성 억제 효능 ) Production inhibition efficacy
옻 추출물이 RAW 264.7 세포에서 염증성 인자인 PGE2 생성에 미치는 영향을 조사하기 위하여 ELISA 방법을 활용하여 측정하였다. TPA, LPS, TNF-α, ROI 등 전염증성 자극에 의해 생성되는 COX-2는 염증을 유발하는 물질인 프로스타글란딘 합성을 증가시켜 염증반응에 있어 중추적인 역할을 한다고 알려져 있다. COX-2의 선택적인 억제제의 개발은 염증의 치료에 target molecule이 되고 있으며 PGE2는 염증반응과 혈관확장을 일으키고, 과발현되었을 때 종양을 생성하기도 한다. 대식세포에 LPS 처리 시 PGE2의 수준이 1178.667±23.333 pg/mL로 LPS를 처리하지 않은 정상대조군에서 460.888±16.777 pg/mL로 나타나 LPS 처리에 의해 PGE2 생성이 약 2.6배 증가됨을 확인하였다. LPS만 처리한 대조군에 비해 모든 옻 추출물 처리군에서 PGE2 농도가 감소하였으며, 특히 옻 추출물 100 ㎍/mL군(907.555±51.675 pg/mL)에서 PGE 농도가 가장 크게 감소하였다(도 10). 옻 첨가 된장 및 간장 추출물의 PGE2 생성 억제 효능은 관찰되지 않았다(도 11 및 12).To investigate the effect of lacquer extract on the production of inflammatory factor PGE 2 in RAW 264.7 cells, it was measured using an ELISA method. It is known that COX-2 produced by pro-inflammatory stimuli such as TPA, LPS, TNF-α, and ROI plays a pivotal role in the inflammatory response by increasing the synthesis of prostaglandin, an agent that causes inflammation. The development of selective inhibitors of COX-2 has become a target molecule for the treatment of inflammation, and PGE 2 causes inflammatory reactions and vasodilation, and also produces tumors when overexpressed. The level of PGE 2 si LPS treated macrophages 1178.667 ± 23.333 appears in not treated with LPS in pg / mL normal control to 460.888 ± 16.777 pg / mL was confirmed that PGE 2 produced a 2.6 fold increased by LPS treatment. Compared to the LPS-only control group, the concentration of PGE 2 was decreased in all lacquer extract-treated groups, and in particular, the concentration of PGE was significantly reduced in the
실시예Example 6. 유전자 발현 분석 6. Gene expression analysis
LPS 유도 염증성 대식세포에서 옻 추출물, 옻 첨가 된장 및 간장 추출물의 처리가 항염증, 산화 스트레스 및 항산화시스템 유전자 발현에 미치는 영향을 살펴보았다. LPS를 처리한 대조군은 정상대조군(Blank)에 비해 염증관련 유전자인 COX-2, iNOS, 염증성 사이토카인, MMPs(matrix metalloproteinases), STAT5a, p-JAK2의 발현이 유의적으로 증가하였다(p<0.001~0.05). 반면 항산화관련 유전자인 Nrf2, Keap1, GCLC, GCLM, NQO1, GST는 유의적인 변화는 없었지만 HO-1, Mn-SOD은 정상군에 비해 대조군에서 유의적으로 증가하였으며 Cu,zn-SOD, catalase는 유의적으로 감소하는 경향을 보였다(p<0.05). 그러나, LPS 유도 염증상태인 대식세포에 옻 추출물을 1~100 ㎍/mL 처리하였을 때, 염증관련 유전자 발현 정도는 감소하는 경향을 보였다(표 6). 이러한 결과는 앞에서 살펴본 NO 및 염증성 사이토카인 생성량 억제와 일치하는 결과라 할 수 있다. LPS의 외부적인 자극에 의해 사이토카인 생성이 증가되면 JAK가 활성화되고, 인산화 과정을 거쳐 STATs를 활성화시키게 된다. 활성화된 STATs는 핵 내로 이동하게 되고 이는 타겟 유전자의 발현을 조절하는 핵 내 전사인자로 작용하게 된다. 따라서 옻 추출물의 항염증 기전은 핵 내 STATs의 translocation 억제를 통한 관련 유전자들의 발현 억제에 의한 것으로 사료된다. 염증반응은 외부의 자극에 의한 생체조직의 국소적인 방어보호 반응으로, 대식세포인 macrophage 등이 NO와 프로스타글란딘(prostaglandin, PG), 그리고 TNF-α와 같은 전염증성 사이토카인(pro-inflammatory cytokine)과 같은 염증매개물질의 생성에 관여하고 이를 조절한다. NO는 염증반응에서 중요한 역할을 하는 염증매개조절 인자로, NOS(nitric oxide synthases)에 의해 L-아르지닌(L-arginine)으로부터 생성되며, iNOS(inducible nitric oxide synthase)는 염증반응에 관여하는 대식세포에서 NO를 생성하여 염증반응을 촉진하는 역할을 한다. 또한, COX-2(cyclooxygenase-2)는 아라키돈산(arachidonic acid)을 프로스타글란딘 E2(PGE2)로 전환시키는 효소로 PG류의 다양한 염증반응 매개체들을 생성한다. 옻 추출물의 처리는 항산화관련 유전자 발현에는 변화를 일으키지 않아 옻 추출물은 LPS 유도 염증상태에서 항산화시스템에는 영향을 미치는 않은 것으로 사료된다. 항산화시스템에서 핵 내 전사인자인 Nrf2(nuclear factor erythroid 2-related factor 2)는 LPS 처리에 의해 거의 변화가 없었다. Nrf2는 세포의 종류, LPS의 처리 농도 및 시간 등에 의해 영향을 받는 것으로 알려져 있다.The effect of lacquer extract, lactose-added miso and soy sauce extract on LPS-induced inflammatory macrophages on the anti-inflammatory, oxidative stress and antioxidant system gene expression was examined. The control group treated with LPS was the expression of inflammation-related genes, COX-2, iNOS, inflammatory cytokines, MMPs (matrix metalloproteinases) compared with the control (Blank), STAT5a, p- JAK2 significantly increased (p <0.001 ~0.05). On the other hand, the antioxidant-related genes Nrf2, Keap1, GCLC, GCLM, NQO1, and GST were not significantly changed, but HO-1 and Mn-SOD were significantly increased in the control group than in the normal group, and Cu,zn-SOD and catalase were significant. It showed a tendency to decrease ( p <0.05). However, when the lactose extract was treated with 1-100 μg/mL of macrophages in LPS-induced inflammatory state, the degree of expression of inflammation-related genes tended to decrease (Table 6). These results can be said to be consistent with NO and inflammatory cytokine production inhibition. When cytokine production is increased by external stimulation of LPS, JAK is activated, and STATs are activated through phosphorylation. Activated STATs move into the nucleus, which acts as a transcription factor in the nucleus that regulates the expression of the target gene. Therefore, the anti-inflammatory mechanism of lacquer extract is thought to be due to the suppression of expression of related genes through inhibition of translocation of STATs in the nucleus. Inflammatory response is a local protective protective response of biological tissue by external stimulation. Macrophage, such as macrophages, contains NO, prostaglandin (PG), and pro-inflammatory cytokine such as TNF-α. It is involved in and regulates the production of the same inflammatory mediator. NO is an inflammatory mediator that plays an important role in the inflammatory reaction. It is produced from L-arginine by NOS (nitric oxide synthases), and iNOS (inducible nitric oxide synthase) is involved in the inflammatory response. It plays a role in promoting NO by producing NO in phagocytes. In addition, COX-2 (cyclooxygenase-2) is an enzyme that converts arachidonic acid to prostaglandin E 2 (PGE 2 ), which produces various inflammatory mediators of PG. It is thought that the treatment of lacquer extract does not change the expression of antioxidant-related genes, so lacquer extract does not affect the antioxidant system in the LPS-induced inflammatory state. In the antioxidant system, Nrf2 (nuclear factor erythroid 2-related factor 2), a nuclear transcription factor, was hardly changed by LPS treatment. Nrf2 is known to be affected by cell type, LPS treatment concentration and time.
정상 생리 상태에서 활성산소종(reactive oxygen species, ROS)은 세포 내 존재하는 다양한 항산화시스템에 의해 제거될 수 있지만, 과도한 ROS의 생성은 항산화시스템의 항상성 소실로 산화적 스트레스를 유발하며 미토콘드리아를 매개한 세포자멸사 신호의 활성화를 통하여 세포 독성을 나타낸다. 따라서 세포 내 발생한 과도한 산화적 스트레스를 제어할 수 있는 다양한 세포 내 기전에 대한 연구가 활발히 진행되고 있으며, 이중 Nrf2는 항산화 관련 단백질의 발현 증가를 유도하여 산화적 스트레스로부터 세포를 보호하는 핵심 전사인자 중 하나로 알려져 있다. Nrf2는 주요 항산화 효소들의 전사를 촉진하는 상위 신호 분자로 세포 내 산화적 스트레스를 조절하는 중요 전사인자로 알려져 있다. 정상 상태에서 Nrf2는 세포질에 Keap1 단백질과 결합되어 세포 내 단백질 분해 시스템(ubiquitin proteasome system)에 의해 분해된다. 세포에 유발된 산화적 스트레스는 Keap1으로부터 Nrf2를 해리시키고, 세포질에 존재하던 Nrf2가 핵으로 이동하여 표적유전자 프로모터 내 ARE에 결합하여 sestrin-2, glutamate-cysteine ligase catalytic subunits, heme oxygenase 1, NAD(P)H quinone oxidoreductase 1 등을 포함한 항산화 관련 유전자의 전사를 활성화시킨다. Nrf2의 활성화 과정은 산화적 스트레스에 의한 직접적인 Nrf2/Keap1 복합체 변형을 통한 Nrf2의 유리, protein kinase C, p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, 또는 phosphatidylinositol 3-kinase 등에 의한 Nrf2 인산화가 관여하는 것으로 보고되었다. Under normal physiological conditions, reactive oxygen species (ROS) can be eliminated by various antioxidant systems in the cell, but excessive ROS production causes oxidative stress due to loss of antioxidant system homeostasis and mediates mitochondria. It exhibits cytotoxicity through activation of apoptosis signals. Therefore, research on various intracellular mechanisms capable of controlling excessive oxidative stress generated in cells is being actively conducted, and Nrf2 is one of the key transcription factors that protect cells from oxidative stress by inducing an increase in the expression of antioxidant-related proteins. It is known as one. Nrf2 is a high-level signaling molecule that promotes the transcription of major antioxidant enzymes and is known to be an important transcription factor that regulates oxidative stress in cells. Under normal conditions, Nrf2 is bound to the Keap1 protein in the cytoplasm and is degraded by the intracellular proteosome system (ubiquitin proteasome system). Cell-induced oxidative stress dissociates Nrf2 from Keap1, and Nrf2 present in the cytoplasm moves to the nucleus and binds to ARE in the target gene promoter, sestrin-2, glutamate-cysteine ligase catalytic subunits,
옻 첨가 된장 추출물의 항염증 및 항산화 효과를 유전자 발현으로 분석한 결과, 옻을 첨가하지 않은 된장의 경우 발효가 진행됨에 따라 염증성 사이토카인의 유전자 발현이 감소되는 경향을 보였으며, 항산화관련 유전자 발현에는 영향을 미치지 않은 것으로 사료된다. 옻 첨가 농도에 따라, 발효 유무에 따른 유의적인 변화는 관찰되지 않았다(표 7).As a result of analyzing the anti-inflammatory and antioxidant effects of the extract of miso added with lacquer as a gene expression, in the case of miso without lactose, the gene expression of inflammatory cytokines tended to decrease as fermentation progressed. It is believed to have no effect. According to the concentration of lacquer, no significant change was observed with or without fermentation (Table 7).
옻 첨가 간장 추출물의 항염증 및 항산화 효과를 유전자 발현으로 분석한 결과, 옻을 첨가하지 않은 간장의 경우 발효가 진행됨에 따라 항산화관련 유전자 발현이 증가하는 경향을 보였으나, 염증관련 유전자 발현에는 변화가 없는 것으로 나타났다. 따라서, LPS 유도 염증성 상태에서 옻 추출물은 항염증 효과를 나타내지만 된장 및 간장에 첨가했을 경우 그 효과는 미미한 것으로 나타났다(표 8).As a result of analyzing the anti-inflammatory and anti-oxidative effects of lacquer-added soy sauce extract with gene expression, in the case of soy sauce without lacquer, the expression of antioxidant-related gene tended to increase as fermentation progressed, but there was no change in inflammation-related gene expression Appeared to be missing. Therefore, in the LPS-induced inflammatory state, lacquer extract showed anti-inflammatory effects, but when added to miso and soy sauce, the effect was minimal (Table 8).
Claims (6)
(2) 상기 (1)단계의 준비한 발효 옻에 물을 첨가한 후 추출하고 여과하여 옻 발효 추출액을 제조하는 단계;
(3) 불린 대두를 증자한 후 고초균 및 황국균을 접종하여 메주로 성형하고, 건조한 후 발효하여 메주를 제조하는 단계;
(4) 상기 (3)단계의 제조한 메주를 소금물에 침지하여 발효한 발효물로부터 된장을 분리하는 단계; 및
(5) 상기 (4)단계의 분리한 된장에 상기 (2)단계의 제조한 옻 발효 추출액과 소금을 첨가한 후 숙성시키는 단계를 포함하여 제조하는 것을 특징으로 하는 옻 된장의 제조방법.(1) preparing the fermented lacquer by inoculating the lacquer bark with fermentation by inoculating the mycelia of longevity mushrooms and drying;
(2) adding water to the fermented lacquer prepared in the step (1), followed by extraction and filtration to prepare a lacquer fermentation extract;
(3) after soaking soybean soybeans, inoculating archaea and Hwanggukgyun to form a meju, drying and fermenting to prepare meju;
(4) separating the fermented soybean paste by dipping the meju prepared in the step (3) in salt water; And
(5) A method of manufacturing lacquered miso, comprising adding and fermenting the lacquer fermentation extract prepared in the step (2) and salt to the separated miso in the step (4).
(1) 옻나무 껍질에 장수버섯 균사체를 접종하여 18~24℃에서 25~35일 동안 발효한 후 45~55℃에서 건조하여 발효 옻을 준비하는 단계;
(2) 상기 (1)단계의 준비한 발효 옻에 발효 옻 대비 8~12배(v/w) 물을 첨가한 후 90~110℃에서 5~7시간 동안 추출한 후 여과하여 옻 발효 추출액을 제조하는 단계;
(3) 12~18시간 동안 불린 대두를 110~120℃에서 8~12분 동안 증자한 후 고초균 및 황국균을 접종하고 메주로 성형하고, 55~65℃에서 20~28시간 동안 건조한 후 28~32℃에서 10~14일 동안 발효하여 메주를 제조하는 단계;
(4) 상기 (3)단계의 제조한 메주를 22~28%(w/v) 소금물에 0.8~1.2:2~2.5 중량비율로 침지하여 25~30℃에서 1~3개월 동안 발효한 발효물로부터 된장을 분리하는 단계; 및
(5) 상기 (4)단계의 분리한 된장에 상기 (2)단계의 제조한 옻 발효 추출액과 소금을 85~93:5~10:2~4(w:v:w) 비율로 첨가한 후 20~25℃에서 7~9주 동안 숙성시키는 단계를 포함하여 제조하는 것을 특징으로 하는 옻 된장의 제조방법.According to claim 1,
(1) preparing the fermented lacquer by inoculating the lacquer bark with longevity mushroom mycelium and fermenting at 18-24°C for 25-35 days, followed by drying at 45-55°C;
(2) After adding 8-12 times (v/w) water compared to the fermented lacquer to the fermented lacquer prepared in the step (1), extracting it at 90-110°C for 5-7 hours and filtering it to prepare a lacquer fermentation extract. step;
(3) Soybeans soaked for 12-18 hours are increased at 110-120°C for 8-12 minutes, inoculated with Bacillus and Bacillus bacteria, molded into meju, dried at 55-65°C for 20-28 hours, and then 28-32 Preparing meju by fermenting at 10° C. for 10-14 days;
(4) Fermented product fermented at 25~30℃ for 1-3 months by immersing the meju prepared in the step (3) in a ratio of 0.8~1.2:2~2.5 in 22~28% (w/v) brine Separating miso from; And
(5) After adding the lacquer fermentation extract and salt prepared in step (2) in the ratio of 85 to 93:5 to 10:2 to 4 (w:v:w) to the separated miso in step (4) Method of producing lacquer miso, characterized in that it comprises a step of aging for 7 to 9 weeks at 20 ~ 25 ℃.
(2) 상기 (1)단계의 준비한 발효 옻에 물을 첨가한 후 추출하고 여과하여 옻 발효 추출액을 제조하는 단계;
(3) 불린 대두를 증자한 후 고초균 및 황국균을 접종하여 메주로 성형하고, 건조한 후 발효하여 메주를 제조하는 단계;
(4) 상기 (3)단계의 제조한 메주를 소금물에 침지하여 발효한 발효물로부터 간장을 분리하는 단계; 및
(5) 상기 (4)단계의 분리한 간장에 상기 (2)단계의 제조한 옻 발효 추출액과 소금을 첨가한 후 숙성시키는 단계를 포함하여 제조하는 것을 특징으로 하는 옻 간장의 제조방법.(1) preparing the fermented lacquer by inoculating the lacquer bark with fermentation by inoculating the mycelia of longevity mushrooms and drying;
(2) adding water to the fermented lacquer prepared in the step (1), followed by extraction and filtration to prepare a lacquer fermentation extract;
(3) after soaking soybean soybeans, inoculating archaea and Hwanggukgyun to form a meju, drying and fermenting to prepare meju;
(4) separating the soy sauce from the fermented fermentation by dipping the meju prepared in the step (3) in salt water; And
(5) A method of manufacturing lacquer soy sauce, characterized in that it is prepared by adding and fermenting the lacquer fermentation extract prepared in step (2) and salt to the separated soy sauce in step (4).
(1) 옻나무 껍질에 장수버섯 균사체를 접종하여 18~24℃에서 25~35일 동안 발효한 후 45~55℃에서 건조하여 발효 옻을 준비하는 단계;
(2) 상기 (1)단계의 준비한 발효 옻에 발효 옻 대비 8~12배(v/w) 물을 첨가한 후 90~110℃에서 5~7시간 동안 추출한 후 여과하여 옻 발효 추출액을 제조하는 단계;
(3) 12~18시간 동안 불린 대두를 110~120℃에서 8~12분 동안 증자한 후 고초균 및 황국균을 접종하고 메주로 성형하고, 55~65℃에서 20~28시간 동안 건조한 후 28~32℃에서 10~14일 동안 발효하여 메주를 제조하는 단계;
(4) 상기 (3)단계의 제조한 메주를 22~28%(w/v) 소금물에 0.8~1.2:2~2.5 중량비율로 침지하여 25~30℃에서 1~3개월 동안 발효한 발효물로부터 간장을 분리하는 단계; 및
(5) 상기 (4)단계의 분리한 간장에 상기 (2)단계의 제조한 옻 발효 추출액과 소금을 84~92:5~10:3.5~5(v:v:w) 비율로 첨가한 후 20~25℃에서 7~9주 동안 숙성시키는 단계를 포함하여 제조하는 것을 특징으로 하는 옻 간장의 제조방법.According to claim 4,
(1) preparing the fermented lacquer by inoculating the lacquer bark with longevity mushroom mycelium and fermenting at 18-24°C for 25-35 days, followed by drying at 45-55°C;
(2) After adding 8-12 times (v/w) water compared to the fermented lacquer to the fermented lacquer prepared in the step (1), extracting it at 90-110°C for 5-7 hours and filtering it to prepare a lacquer fermentation extract. step;
(3) Soybeans soaked for 12-18 hours are increased at 110-120°C for 8-12 minutes, inoculated with Bacillus and Bacillus bacteria, molded into meju, dried at 55-65°C for 20-28 hours, and then 28-32 Preparing meju by fermenting at 10° C. for 10-14 days;
(4) Fermented product fermented at 25~30℃ for 1-3 months by immersing the meju prepared in the step (3) in a ratio of 0.8~1.2:2~2.5 in 22~28% (w/v) brine Separating the liver from; And
(5) After the lactose fermentation extract and salt prepared in step (2) are added to the separated soy sauce in step (4) at 84~92:5~10:3.5~5(v:v:w) Method of manufacturing a lacquer soy sauce, characterized in that it comprises a step of aging for 7 to 9 weeks at 20 ~ 25 ℃.
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| KR100931526B1 (en) * | 2009-09-11 | 2009-12-14 | 이성호 | Fermented food produced by fomes formentarius and a method for producing the same |
| KR20130066058A (en) * | 2011-12-12 | 2013-06-20 | 대한민국(농촌진흥청장) | Soybean paste comprising an urushiol free lacquer tree bark extract and the manufacturing method thereof |
| KR20160107989A (en) * | 2015-03-06 | 2016-09-19 | 원진식 | Method for manufacturing fermented lacquer soybeans using extract of Rhus verniciflura with Fomitella fraxinea and manufactured fermented lacquer soybeans by the method |
| KR20180037346A (en) * | 2016-10-04 | 2018-04-12 | 농업회사법인 고산 주식회사 | Sauces manufactured by the manufacturing method using a lacquer and a method for preparing sauces |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR100931526B1 (en) * | 2009-09-11 | 2009-12-14 | 이성호 | Fermented food produced by fomes formentarius and a method for producing the same |
| KR20130066058A (en) * | 2011-12-12 | 2013-06-20 | 대한민국(농촌진흥청장) | Soybean paste comprising an urushiol free lacquer tree bark extract and the manufacturing method thereof |
| KR20160107989A (en) * | 2015-03-06 | 2016-09-19 | 원진식 | Method for manufacturing fermented lacquer soybeans using extract of Rhus verniciflura with Fomitella fraxinea and manufactured fermented lacquer soybeans by the method |
| KR20180037346A (en) * | 2016-10-04 | 2018-04-12 | 농업회사법인 고산 주식회사 | Sauces manufactured by the manufacturing method using a lacquer and a method for preparing sauces |
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