KR102053941B1 - Method for extracting phenolic compounds comprising resveratrol from peanut sprouts - Google Patents
Method for extracting phenolic compounds comprising resveratrol from peanut sprouts Download PDFInfo
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- KR102053941B1 KR102053941B1 KR1020190029271A KR20190029271A KR102053941B1 KR 102053941 B1 KR102053941 B1 KR 102053941B1 KR 1020190029271 A KR1020190029271 A KR 1020190029271A KR 20190029271 A KR20190029271 A KR 20190029271A KR 102053941 B1 KR102053941 B1 KR 102053941B1
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- peanut sprouts
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- peanut
- resveratrol
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- 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
-
- 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
-
- 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
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Botany (AREA)
- Mycology (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
Description
본 발명은 땅콩새싹으로부터 레스베라트롤을 비롯한 페놀성 화합물을 추출하는 방법에 관한 것으로, 구체적으로 땅콩새싹을 셀루클라스트(celluclast), 비스코자임(viscozyme), 아밀로글루코시다아제(amyloglucosidase), 프로모자임(promozyme), 말토게나제(maltogenase), 덱스트로자임(dextrozyme), 테르마밀(termamyl), 파파인(papain), 프로타멕스(protamex), 뉴트라아제(neutrase) 및 플라보르자임(flavourzyme)과 효소반응시켜 반응생성물을 수득하는 단계; 및 상기 반응생성물로부터 열수추출하는 단계;를 포함하는 땅콩새싹으로부터 레스베라트롤을 비롯한 페놀성 화합물을 추출하는 방법에 관한 것이다.The present invention relates to a method for extracting phenolic compounds including resveratrol from peanut sprouts, specifically peanut sprouts (celluclast), biscozyme (amycolucosidase), amyloglucosidase, promozyme ( enzymatic reaction with promozyme, maltogenase, dextrozyme, termamyl, papain, protamex, neutrase and flavorzyme To obtain a reaction product; It relates to a method for extracting phenolic compounds, including resveratrol from peanut sprouts comprising; and hot water extraction from the reaction product.
땅콩은 콩과에 속하는 일년생의 초본식물로 지방과 단백질을 많이 함유하고 있는 식품이다. 즉각 취식이나 버터, 마가린, 식용유 등 다양한 형태로 섭취되고 있다(Lee et al., 2004). 땅콩은 천연 폴리페놀 화합물인 레스베라트롤(resveratrol)이 함유되어 있는 대표적인 식물로, 발아 땅콩새싹은 기능성 영양성분이 풍부하고 수분함량이 높고 식미가 우수하며 식품소재로서의 이용 범위가 넓은 장점을 지니고 있다(Ikeda et al., 1984). 땅콩의 새싹을 발아하는 과정에서 지방과 칼로리는 낮아지고 땅콩에 미량으로 존재하는 천연 폴리페놀 화합물인 레스베라트롤의 함량이 증가된다(Lee et al., 2003; Kang et al., 2010; Kim et al., 2010). 이러한 땅콩새싹은 암, 당뇨, 심장병 예방, 항산화 작용, 염증 억제, 노화 방지, 동맥경화, 혈중 콜레스테롤 저하 및 기억력 증진 등의 효능이 알려져 있으며 특히 레스베라트롤은 적포도, 땅콩, 오디 등에도 함유되어 있어 다양한 약리효과들이 보고되고 있다(Kim, 2013).Peanuts are annual herbaceous plants belonging to the legumes and are high in fat and protein. It is consumed in various forms, such as immediate eating, butter, margarine and cooking oil (Lee et al., 2004). Peanut is a representative plant that contains resveratrol, a natural polyphenol compound. Germinated peanut sprouts have abundant functional nutrition, high water content, good taste, and a wide range of use as food materials (Ikeda). et al., 1984). During sprouting of peanuts, fat and calories are lowered and the content of resveratrol, a natural polyphenol compound present in trace amounts in peanuts, is increased (Lee et al., 2003; Kang et al., 2010; Kim et al. , 2010). These peanut sprouts are known to be effective in preventing cancer, diabetes, heart disease, antioxidant activity, inhibiting inflammation, preventing aging, hardening of arteries, lowering blood cholesterol and improving memory. Pharmacological effects are reported (Kim, 2013).
최근 식물의 세포벽을 효소로 분해함으로서 식물체 유용물질의 생산효율을 높이거나, 공정개선과 제품 품질향상을 높이려는 시도를 하고 있다. 사과, 배, 당근, 배추, 망고, 바나나 등 여러 식물체를 대상으로 이루어지고 있는데, 아직까지 땅콩새싹을 활용한 연구는 이루어지고 있지 않다. 또한 한 가지 효소를 통해 유용물질의 생산효율을 높이는 것이 아니라 여러 종류의 효소를 통해 보다 빠르게 세포벽을 분해하고 많은 양의 유용물질과 생산효율을 높이고자 한다. 땅콩나물이 식품소재로 이용성이 증가하고 있고 국제시장에서도 수요가 증가하고 있음에도 불구하고 땅콩나물의 생산방식은 효율적이지 못한 부분이 많았다(Kang et al., 2011). 우리나라의 땅콩 및 땅콩새싹 연구는 생산성 향상을 위한 육종(Pae et al., 2004), 재배개선(Cheong et al., 2001), 유용성분(Wang et al., 2005; Lee et al., 2003) 등에 연구가 있었으나, 추출 효율과 관련된 연구는 미흡하였다.Recently, by decomposing plant cell walls with enzymes, attempts have been made to increase the production efficiency of plant useful substances or to improve process and product quality. Apples, pears, carrots, cabbages, mangoes, bananas, and other plants are being studied, but peanut sprouts have not been studied. In addition, rather than improving the production efficiency of useful substances through one enzyme, the various types of enzymes will be used to break down the cell wall more quickly and increase the amount of useful substances and production efficiency. Although peanut sprouts are increasing in availability as food materials and demand is increasing in the international market, the production method of peanut sprouts has been inefficient (Kang et al., 2011). Korean Peanut and Peanut Sprout Research has been conducted for breeding to improve productivity (Pae et al., 2004), cultivation improvement (Cheong et al., 2001), useful ingredients (Wang et al., 2005; Lee et al., 2003) There have been studies on the back, but studies related to extraction efficiency have been insufficient.
이에 본 발명자는 땅콩새싹으로부터 레스베라트롤을 비롯한 페놀성 화합물과 같은 유용성분을 효율적으로 추출할 수 있는 방법을 개발하고자 하였으며, 특히 에탄올과 같은 유기용매를 사용하지 않고 인체에 안전한 방법을 사용함으로써 별도의 가공공정 없이도 직접 식품 등에 활용할 수 있도록 하여 활용성을 높이고자 하였다.Therefore, the present inventors have attempted to develop a method for efficiently extracting useful components such as resveratrol and phenolic compounds from peanut sprouts, and in particular, by using a method safe for the human body without using an organic solvent such as ethanol. It was intended to increase the usability by allowing it to be used directly in food without a process.
따라서 본 발명의 주된 목적은 에탄올 등의 유기용매를 사용하지 않고 인체에 안전한 방법으로 땅콩새싹으로부터 레스베라트롤을 비롯한 페놀성 화합물과 같은 유용성분을 효율적으로 추출할 수 있는 방법을 제공하는데 있다.Therefore, the main object of the present invention is to provide a method for efficiently extracting useful components such as resveratrol and phenolic compounds from peanut sprouts in a safe manner to the human body without using an organic solvent such as ethanol.
본 발명의 한 양태에 따르면, 본 발명은 땅콩새싹을 셀루클라스트(celluclast), 비스코자임(viscozyme), 아밀로글루코시다아제(amyloglucosidase), 프로모자임(promozyme), 말토게나제(maltogenase), 덱스트로자임(dextrozyme), 테르마밀(termamyl), 파파인(papain), 프로타멕스(protamex), 뉴트라아제(neutrase) 및 플라보르자임(flavourzyme)과 효소반응시켜 반응생성물을 수득하는 단계; 및 상기 반응생성물로부터 열수추출하는 단계;를 포함하는 땅콩새싹으로부터 레스베라트롤을 비롯한 페놀성 화합물을 추출하는 방법을 제공한다.According to one aspect of the present invention, the present invention is a peanut sprout cellulast (celluclast), biscozyme (amycolucosidase), amyloglucosidase (promozyme), maltogenase (dextozyme), dex Enzymatic reaction with dextrozyme, termamyl, papain, papain, protamex, neutrase and flavorzyme to obtain a reaction product; It provides a method for extracting phenolic compounds, including resveratrol from peanut sprouts comprising; and extracting hot water from the reaction product.
본 발명의 방법에 있어서, 상기 땅콩새싹은 열풍건조한 것이 바람직하다.In the method of the present invention, the peanut sprout is preferably hot air dried.
본 발명의 방법에 있어서, 상기 효소반응을 pH 5.3 내지 5.7의 pH조건 및 50 내지 60℃의 온도조건에서 10 내지 20시간 동안 수행하는 것이 바람직하다.In the method of the present invention, the enzyme reaction is preferably carried out for 10 to 20 hours at pH conditions of pH 5.3 to 5.7 and temperature conditions of 50 to 60 ℃.
본 발명의 방법에 있어서, 상기 열수추출을 90 내지 100℃에서 30분 내지 2시간 동안 수행하는 것이 바람직하다.In the method of the present invention, it is preferable to perform the hot water extraction for 30 minutes to 2 hours at 90 to 100 ℃.
본 발명에 따르면 에탄올과 같은 유기용매를 사용하지 않고 효소처리 및 열수추출과 같은 인체에 안전한 방법으로 땅콩새싹으로부터 레스베라트롤을 비롯한 페놀성 화합물을 효율적으로 추출할 수 있다. 따라서 본 발명의 방법은 식품 등 인체에 적용하기 위한 목적으로 땅콩새싹으로부터 유효성분을 추출함에 있어서 재료인 땅콩새싹의 낭비를 줄여 원료비를 절감할 수 있는 장점이 있다. 또한 본 발명의 방법으로 수득된 땅콩새싹 가공산물은 용매를 제거하기 위한 별도의 공정없이 바로 식품 등의 소재로 안전하게 활용할 수 있는 장점도 있다.According to the present invention, it is possible to efficiently extract phenolic compounds including resveratrol from peanut sprouts in a human-safe method such as enzyme treatment and hot water extraction without using an organic solvent such as ethanol. Therefore, the method of the present invention has the advantage of reducing raw material costs by reducing the waste of peanut sprouts as a material in extracting the active ingredient from peanut sprouts for the purpose of applying to humans such as food. In addition, the peanut sprout processed product obtained by the method of the present invention has the advantage that it can be safely used as a material such as food immediately without a separate process for removing the solvent.
도 1은 땅콩새싹의 열수추출물, 에탄올추출물 및 단일효소반응산물의 총 페놀성 화합물 함량을 분석한 결과이다. 그래프 상단의 알파벳이 서로 다른 경우 통계적으로 유의한 차이(P<0.05)가 있다는 것을 의미한다.
도 2는 땅콩새싹의 복합효소반응산물의 총 페놀성 화합물 함량을 분석한 결과이다. 그래프 상단의 알파벳이 서로 다른 경우 통계적으로 유의한 차이(P<0.05)가 있다는 것을 의미한다.
도 3은 땅콩새싹의 복합효소반응 후 열수추출물의 총 페놀성 화합물 함량을 분석한 결과이다. 그래프 상단의 알파벳이 서로 다른 경우 통계적으로 유의한 차이(P<0.05)가 있다는 것을 의미한다.1 is a result of analyzing the total phenolic compound content of the hot water extract, ethanol extract and homoenzyme reaction product of peanut sprout. If the alphabet at the top of the graph is different, it means that there is a statistically significant difference ( P <0.05).
Figure 2 shows the results of analyzing the total phenolic compound content of the complex enzyme reaction product of peanut sprout. If the alphabet at the top of the graph is different, it means that there is a statistically significant difference ( P <0.05).
Figure 3 shows the results of analyzing the total phenolic compound content of the hot water extract after the complex enzyme reaction of peanut sprout. If the alphabet at the top of the graph is different, it means that there is a statistically significant difference ( P <0.05).
본 발명의 방법은 땅콩새싹을 셀루클라스트(celluclast), 비스코자임(viscozyme), 아밀로글루코시다아제(amyloglucosidase), 프로모자임(promozyme), 말토게나제(maltogenase), 덱스트로자임(dextrozyme), 테르마밀(termamyl), 파파인(papain), 프로타멕스(protamex), 뉴트라아제(neutrase) 및 플라보르자임(flavourzyme)과 효소반응시켜 반응생성물을 수득하는 단계; 및 상기 반응생성물로부터 열수추출하는 단계;를 포함하는 것을 특징으로 한다.The method of the present invention comprises peanut sprouts (celluclast), biscozyme (amycolucosidase), amyloglucosidase (promozyme), maltogenase (maltogenase), dextrozyme (dextrozyme), Enzymatic reaction with termamyl, papain, protamex, neutrase and flavorzyme to obtain a reaction product; And extracting hot water from the reaction product.
땅콩새싹은 땅콩을 약 1주일 정도 발아시켜 싹을 틔운 것을 의미하며, 일반적으로 콩나물과 같이 물로만 재배할 수 있다. 본 발명의 방법은 이러한 땅콩새싹으로부터 레스베라트롤을 비롯한 페놀성 화합물을 효율적으로 추출할 수 있다. 본 발명에서 상기 땅콩새싹은 열풍건조한 상태로 사용하는 것이 바람직하다.Peanut sprouts mean that the sprouts are sprouted by germinating peanuts for about a week, and generally can be grown only with water, such as bean sprouts. The method of the present invention can efficiently extract phenolic compounds including resveratrol from these peanut sprouts. The peanut sprout in the present invention is preferably used in a hot air dry state.
본 발명에서 사용하는 상기 효소들은 식품 등에 활용할 수 있는 산업적으로 생산 및 판매되는 효소제로, 시중에서 쉽게 구입할 수 있다.The enzymes used in the present invention are industrially produced and sold enzymes that can be utilized in foods, etc., and can be easily purchased on the market.
셀루클라스트는 진균류인 Trichoderma resei로부터 생산되는 효소제로 셀룰로오스(cellulose)를 포도당(glucose)로 분해하는 것으로 알려져 있다. Cellulast is an enzyme produced from the fungus Trichoderma resei , which is known to break down cellulose into glucose.
비스코자임은 Aspergillus로부터 생산되는 다효소복합체로 아라바나아제(arabanase), 셀룰라아제(cellulase), 베타-글루카나아제(β-glucanase), 헤미셀룰라아제(hemicellulase) 및 자일라나아제(xylanase)를 포함하는 탄수화물가수분해효소들을 함유하는 것으로 알려져 있다.Biscozyme is a multi-enzyme complex produced from Aspergillus and contains carbohydrates including arabanase, cellulase, beta-glucanase, hemicellulase and xylanase It is known to contain hydrolases.
아밀로글루코시다아제는 Aspergillus niger로부터 생산되는 효소제로 녹말(starch)의 1,4- 및 1,6-알파 결합을 가수분해하는 것으로 알려져 있다.Amyloglucosidase is an enzyme produced from Aspergillus niger and is known to hydrolyze starch's 1,4- and 1,6-alpha bonds.
프로모자임은 Bacillus acidopullulyticus로부터 생산되는 풀루라나아제(pullulanase)로 풀루란(pullulan)이나 알파-아밀라아제(α-amylase)로 부분적으로 분해된 아밀로펙틴(amylopectin) 중에 있는 1,6-알파 결합을 분해하는 것으로 알려져 있다. Promozyme is a pullulanase produced from Bacillus acidopullulyticus that breaks down 1,6-alpha bonds in amylopectin partially degraded by pullulan or alpha-amylase. Known.
말토게나제는 Bacillus stearothermophilus의 유전자를 함유한 Bacillus subtilisamyloliquefaciens로부터 생산되는 열안정성이 높은 말토게닉 아밀라아제(maltogenic amylase)로 부분적으로 분해된 전분과 말토트리오스(maltotriose)를 포함한 저분자량의 올리고당의 1,4-알파 글리코시드 결합을 가수분해하여 비환원성 말단으로부터 말토오스(maltose)를 생성하는 것으로 알려져 있다.Maltogenase is a low-molecular-weight oligosaccharide containing 1,4 starch and maltotriose partially decomposed with high-maltogenic amylase produced from Bacillus subtilisamyloliquefaciens containing Bacillus stearothermophilus gene. It is known to hydrolyze alpha glycoside bonds to produce maltose from non-reducing ends.
덱스트로자임은 Aspergillus niger의 유전적 변형 균주로부터 생산되는 글루코아밀라아제(glucoamylase) 및 Bacillus의 유전적 변형 균주로부터 생산되는 풀루라나아제(pullulanase)의 혼합효소제로 녹말의 1,4- 및 1,6-알파 결합을 가수분해하는 것으로 알려져 있다.Dextrozyme is a mixed enzyme of glucoamylase produced from a genetically modified strain of Aspergillus niger and pullulanase produced from a genetically modified strain of Bacillus and is a 1,4- and 1,6-starch of starch. It is known to hydrolyze alpha bonds.
테르마밀은 Bacillus licheniformis의 유전적 변형 균주로부터 생산되는 열안정성 알파 아밀라아제(amylase)로 아밀로오스(amylose) 및 아밀로펙틴(amylopectin)의 1,4-알파 글리코시드 결합을 가수분해하는 것으로 알려져 있다.Thermamil is a thermostable alpha amylase produced from a genetically modified strain of Bacillus licheniformis and is known to hydrolyze the 1,4-alpha glycoside bonds of amylose and amylopectin.
파파인은 Carica papaya로부터 생산되는 펩티다아제 C1 패밀리(peptidase C1 family)에 속하는 시스테인 프로테아제(cysteine protease)로 단백질, 펩타이드, 아마이드, 및 아미노산 및 펩타이드의 에스터를 가수분해하는 것으로 알려져 있다.Papain is a cysteine protease belonging to the peptidase C1 family produced from Carica papaya and is known to hydrolyze proteins, peptides, amides, and esters of amino acids and peptides.
프로타멕스는 Bacillus로부터 반응 표면 방법(response surface methodology)을 사용한 배치 발효(batch fermentation)로 생산되는 프로테아제(protease)로 알려져 있다.Protamex is known as protease produced from batch fermentation from the response surface methodology from Bacillus .
뉴트라아제는 Bacillus amyloliquefaciens로부터 생산되는 엔도프로테아제(endoprotease)로 알려져 있다. Neutraase is known as an endoprotease produced from Bacillus amyloliquefaciens .
플라보르자임은 Aspergillus oryzae로부터 생산되는 프로테아제(protease)로 엔도(endo-) 및 엑소(exo-) 프로테아제 활성을 나타내는 것으로 알려져 있다.Flavorzyme is a protease produced from Aspergillus oryzae and is known to exhibit endo- and exo-protease activity.
본 발명에서 상기 효소반응은 식물재료를 효소반응시키는 통상적인 방법을 통해 달성할 수 있으나, 수용액 중에서 상기 땅콩새싹과 상기 효소들이 접촉할 수 있는 상태로 유지하는 방법을 사용하는 것이 바람직하다. 예를 들어, 물에 땅콩새싹과 상기 효소들을 첨가하고 효소가 작용할 수 있는 범위 내의 pH 및 온도 조건으로 일정시간 유지하는 방법을 사용할 수 있다.In the present invention, the enzymatic reaction may be achieved through a conventional method of enzymatically reacting plant material, but it is preferable to use a method of keeping the peanut sprout and the enzyme in contact with each other in an aqueous solution. For example, a method of adding peanut sprouts and the enzymes to water and maintaining them at a pH and temperature condition within a range in which the enzymes can act may be used.
본 발명에 따르면, 상기 효소반응의 pH 조건을 pH 5.3 ~ 5.7로 하고 온도 조건을 50 ~ 60℃로 하여 10 ~ 20시간 동안 효소반응을 수행하는 것이 레스베라트롤을 비롯한 페놀성 화합물의 추출 효율을 높이기 위해 바람직하다. 보다 바람직하게는 pH 5.4 ~ 5.6, 53 ~ 57℃로 14 내지 18시간 효소반응하는 것이 좋다.According to the present invention, the pH conditions of the enzyme reaction to pH 5.3 ~ 5.7 and the temperature conditions to 50 ~ 60 ℃ to perform the enzyme reaction for 10 to 20 hours to increase the extraction efficiency of phenolic compounds, including resveratrol desirable. More preferably, the enzyme reaction is performed at pH 5.4 to 5.6 and 53 to 57 ° C. for 14 to 18 hours.
본 발명에서 상기 열수추출은 물을 용매로 사용하고 가열하여 식물재료로부터 유용성분을 추출하기 위한 통상적인 방법을 통해 달성할 수 있으나, 레스베라트롤을 비롯한 페놀성 화합물의 추출 효율을 높이기 위해 추출 온도를 90 ~ 100℃로 하여 30분 ~ 2시간 동안 추출하는 것이 바람직하다. 보다 바람직하게는 93 ~ 97℃로 40분 ~ 80분 동안 추출하는 것이 좋다. 수용액 중에서 효소반응을 수행한 다음 별도로 물을 첨가하지 않고 가열하는 방법으로 상기 열수추출을 수행할 수 있다.In the present invention, the hot water extraction may be achieved through a conventional method for extracting useful components from plant materials by using water as a solvent and heating, but the extraction temperature is increased to 90 to increase the extraction efficiency of phenolic compounds, including resveratrol It is preferable to extract for 30 minutes-2 hours as ~ 100 degreeC. More preferably it is extracted for 40 minutes to 80 minutes at 93 ~ 97 ℃. The hot water extraction may be carried out by performing an enzymatic reaction in an aqueous solution and then heating without adding water separately.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.
[실시예]EXAMPLE
1. 방법1. How to
1-1. 재료1-1. material
국내산 땅콩을 직접 재배한 땅콩새싹을 50℃에서 24시간 열풍 건조시키고 3 ~ 5cm로 세절하여 효소처리 또는 추출에 시료로 사용하였다.Peanut sprouts cultivated directly from domestic peanuts were hot-air dried at 50 ° C. for 24 hours, and cut into 3 to 5 cm and used as samples for enzyme treatment or extraction.
1-2. 열수추출 및 효소처리1-2. Hot water extraction and enzyme treatment
열수추출, 에탄올추출, 단일효소처리, 복합효소처리, 복합효소처리+열수추출의 크게 다섯 가지 방법으로 구분하여 수행하였다.Hot water extraction, ethanol extraction, single enzyme treatment, complex enzyme treatment, complex enzyme treatment + hot water extraction were performed in five ways.
1-2-1. 열수 또는 에탄올추출1-2-1. Hot water or ethanol extraction
상기 1-1의 땅콩새싹 시료에 증류수를 1 : 50의 중량비로 첨가하고, 95±5℃에서 1, 2, 4시간으로 추출시간을 달리하여 열수추출하였다.Distilled water was added to the peanut sprout sample of 1-1 in a weight ratio of 1:50, and hot water was extracted by varying the extraction time at 95 ± 5 ° C. for 1, 2 and 4 hours.
에탄올추출은 증류수 대신 같은 비율로 에탄올을 첨가하고 2시간 동안 환류냉각 추출하였다.Ethanol extraction was added to the same ratio instead of distilled water and extracted with reflux cooling for 2 hours.
1-2-2. 단일효소처리1-2-2. Single enzyme treatment
상기 1-1의 땅콩새싹 시료에 증류수를 1 : 50의 중량비로 첨가하고, 표 1에서 선택된 임의의 효소를 첨가(효소반응액 중 최종 2중량%가 되도록 첨가)하여 각 pH 및 온도 조건에 따라 쉐이커 수조(shaker water bath)에서 교반하면서 8시간 또는 16시간 동안 효소반응시킨 다음 효소활성을 억제시키기 위해 105℃에서 15분간 열처리하였다.Distilled water was added to the peanut sprout sample of 1-1 in a weight ratio of 1:50, and any enzyme selected in Table 1 was added (added to be the final 2% by weight in the enzyme reaction solution) according to each pH and temperature conditions. Enzyme reaction was carried out for 8 hours or 16 hours while stirring in a shaker water bath, and then heat-treated at 105 ° C. for 15 minutes to inhibit enzyme activity.
1-2-3. 복합효소처리1-2-3. Complex enzyme treatment
상기 1-1의 땅콩새싹 시료에 증류수를 1 : 50의 중량비로 첨가하고, 표 2와 같이 각 실험군에 따른 효소 조성별로 효소를 첨가(실험군 당 첨가되는 각각의 효소를 동일한 중량비로 하고, 효소반응액 중 각 효소들을 합한 중량이 최종 2중량%가 되도록 첨가)하여 각 pH 및 온도 조건에 따라 쉐이커 수조(shaker water bath)에서 교반하면서 16시간 동안 효소반응시킨 다음 효소활성을 억제시키기 위해 105℃에서 15분간 열처리하였다.Distilled water was added to the peanut sprout sample of 1-1 in a weight ratio of 1:50, and enzymes were added for each enzyme composition according to each experimental group as shown in Table 2 (each enzyme added per experimental group in the same weight ratio, and the enzyme reaction The total weight of each enzyme in the solution was added to the final 2% by weight), followed by enzymatic reaction for 16 hours with stirring in a shaker water bath according to each pH and temperature conditions, and then at 105 ° C to inhibit enzymatic activity. Heat treatment was performed for 15 minutes.
실험군 7 및 8의 경우, 복합효소처리 이후 효소활성을 억제시키기 위해 105℃에서 15분간 열처리하고, 95℃로 낮추어 1시간 또는 2시간 동안 가열하는 방법으로 추출을 병행하였다.In the case of Experimental Groups 7 and 8, the mixture was heat-treated at 105 ° C. for 15 minutes in order to inhibit enzymatic activity after the complex enzyme treatment, followed by extraction by heating at 95 ° C. for 1 hour or 2 hours.
열수추출시간Enzyme Treatment Time /
Hot water extraction time
1-3. 총 페놀성 화합물 분석1-3. Total Phenolic Compound Analysis
상기 1-2에서 열수추출, 에탄올추출, 단일효소처리, 복합효소처리, 복합효소처리+열수추출로 수득된 용액 1㎖과 3차 증류수 9㎖를 혼합한 후 폴린 & 시오칼토 페놀 시약(Folin & Ciocalteu's phenol reagent) 1㎖를 넣고 혼합하여 실온에서 5분간 반응시켰다. 이후 반응용액에 7% Na2CO3 용액 10㎖를 넣은 다음 3차 증류수로 25㎖로 정용하였다. 이 혼합 용액을 23℃에서 2시간 동안 정치한 후 UV-분광광도계(UV-spectrophotometer)(UV-1201, Shimadzu, Tokyo, Japan)로 760㎚에서 흡광도를 측정하였다. 갈산(gallic acid)을 이용하여 작성된 검량선에 각각 측정된 흡광도를 적용하여 총 페놀 화합물 함량을 계산하였다(Kim et al., 2003).In 1 to 2, 1 ml of the solution obtained by hot water extraction, ethanol extraction, single enzyme treatment, complex enzyme treatment, complex enzyme treatment + hot water extraction and 9 ml of tertiary distilled water were mixed, 1 ml of Ciocalteu's phenol reagent) was added and mixed for 5 minutes at room temperature. Thereafter, 10 ml of a 7% Na 2 CO 3 solution was added to the reaction solution, followed by 25 ml of distilled water. The mixed solution was allowed to stand at 23 ° C. for 2 hours, and then absorbance was measured at 760 nm with a UV-spectrophotometer (UV-1201, Shimadzu, Tokyo, Japan). The total phenolic compound content was calculated by applying absorbance measured to the calibration curve prepared using gallic acid (Kim et al., 2003).
1-4. 레스베라트롤 정량 분석1-4. Resveratrol Quantitative Analysis
상기 1-2에서 열수추출, 에탄올추출, 복합효소처리, 복합효소처리+열수추출로 수득된 용액을 각각 여과 및 농축 과정을 거친 후 동결건조하여 냉동 보관하면서 시료로 사용하였다.The solution obtained by hot water extraction, ethanol extraction, complex enzyme treatment, complex enzyme treatment + hot water extraction in 1-2 was subjected to filtration and concentration, respectively, and lyophilized to be used as a sample while being frozen and stored.
HPLC shimadzu LC-20AT 펌프, SPD-M20A 포토다이오드 어레이 디텍터(photodiode array detector)(shimadzu, Tokyo, Japan)를 사용하여 상기 1-2의 효소처리 또는 열수추출로 수득된 동결건조물의 레스베라트롤(resveratrol)을 정량 분석하였다.Lyophilized resveratrol obtained by the enzymatic treatment or hydrothermal extraction of the above 1-2 using an HPLC shimadzu LC-20AT pump, SPD-M20A photodiode array detector (shimadzu, Tokyo, Japan). Quantitative analysis.
컬럼(column)은 C18 컬럼(250㎜×4.6㎜, 5.0㎛, Simpek, shimadzu, Tokyo, Japan)을 사용하였고, 이동상은 A : 아세토나이트릴(acetonitrile) + 0.1% 포름산(formic acid), B : 물 + 0.1% 포름산을 사용하였다. 이동상 A : B를 시간에 따라 0 : 100에서 100 : 0으로 달리하여 40분까지 검출하였다. 이때 이동상 속도는 1.2㎖/min, 시료 주입량은 20㎕이고, 파장은 310㎚에서 분석하였다. 표준물질 레스베라트롤은 메탄올에 용해하여 사용하였다(Kang et al., 2010).As a column, a C18 column (250 mm × 4.6 mm, 5.0 μm, Simpek, shimadzu, Tokyo, Japan) was used, and the mobile phase was A: acetonitrile + 0.1% formic acid, B: Water + 0.1% formic acid was used. Mobile phases A: B were detected from 40 to 100: 0 to 100 over time. In this case, the mobile phase velocity was 1.2 ml / min, the sample injection amount was 20 µl, and the wavelength was analyzed at 310 nm. Standard resveratrol was used by dissolving in methanol (Kang et al., 2010).
1-5. 통계처리1-5. Statistical processing
모든 실험은 3회 반복 실시하여 평균과 표준편차(mean±SD)로 나타내었으며, 각 평균값에 대한 검증은 SAS® version 9.11(SAS institute, Cary, NC, USA)를 이용하여 던컨의 다중검정(Duncan's multiple range test)으로 검증하였다.All experiments were repeated three times and expressed as mean and standard deviation (mean ± SD), and each mean value was tested by Duncan's multiple test (Duncan's) using SAS® version 9.11 (SAS Institute, Cary, NC, USA). multiple range test).
2. 결과2. Results
2-1. 페놀성 화합물 함량2-1. Phenolic Compound Content
열수추출, 에탄올추출, 단일효소처리, 복합효소처리, 복합효소처리+열수추출의 총 페놀성 화합물의 함량을 분석한 결과는 도 1 내지 3과 같다.The results of analyzing the total phenolic compound content of hot water extraction, ethanol extraction, single enzyme treatment, complex enzyme treatment, complex enzyme treatment + hot water extraction are as shown in Figs.
열수 1시간 추출의 경우 약 17.5㎎ GAE/g으로 나타났고, 열수 2시간 추출의 경우 18.75㎎ GAE/g, 열수 4시간 추출의 경우 약 20.25㎎ GAE/g으로 추출시간이 증가할수록 총 페놀성 화합물 함량이 증가하는 것으로 나타났다. 에탄올 2시간 추출에서는 25㎎ GAE/g으로 나타났다. 단일효소처리는 임의의 효소 2가지를 선택하여 이중 1가지 씩 효소처리에 사용하였으며, 8시간 효소처리하였을 때 각각 22.75, 24㎎ GAE/g으로 나타났고, 16시간 효소처리하였을 때 25.25, 24.5㎎ GAE/g으로 8시간보다 16시간 효소처리한 경우 총 페놀성 화합물 함량이 증가하는 것으로 나타났다. 이 결과들로 보아 효소처리는 열수추출보다는 페놀성 화합물의 함량을 높일 수 있으며, 유기용매인 에탄올추출과 유사한 정도로 페놀성 화합물의 함량을 높일 수 있는 것으로 판단된다.The extraction time was about 17.5mg GAE / g for 1 hour hot water extraction, 18.75mg GAE / g for 2 hours hot water extraction and about 20.25mg GAE / g for 4 hours hot water extraction. It was found that the content increased. Ethanol 2-hour extraction showed 25 mg GAE / g. In the single enzyme treatment, two random enzymes were selected and used for enzyme treatment one by one. When the enzyme treatment was carried out for 8 hours, it was 22.75 and 24 mg GAE / g, respectively. When the enzyme treatment was carried out for 16 hours, 25.25 and 24.5 mg. The total phenolic compound content was increased when the enzyme was treated with GAE / g for 16 hours rather than 8 hours. These results show that the enzyme treatment can increase the content of phenolic compounds rather than hot water extraction, and can increase the content of phenolic compounds to a degree similar to ethanol extraction, which is an organic solvent.
복합효소처리는 당 분해 효소 4종과 6종, 단백질 분해 효소 4종, 당 분해 효소와 단백질 분해효소 11종을 혼합하여 사용하였으며 조합은 표 2와 같다. 효소처리시간을 16시간으로 했을 때 총 페놀성 화합물의 함량 분석 결과는 도 2와 같다. 복합효소처리에서 가장 높은 총 페놀성 화합물 함량을 보인 것은 실험군 6으로 나타났고 이때 함량은 24.25㎎ GAE/g이며, 다음으로 높은 함량을 보인 것은 실험군 2, 실험군 3으로 나타났고 함량은 각각 23.25, 22㎎ GAE/g으로 나타났다. 당 분해나 단백질 분해 효소만으로 조성된 복합효소보다 당 분해 효소와 단백질 분해 효소가 혼합적으로 조성된 복합효소에서 페놀성 화합물의 함량을 더 높일 수 있는 것으로 판단된다.The complex enzyme treatment used a mixture of four and six types of glycolytic enzymes, four kinds of proteolytic enzymes, and 11 types of glycolytic enzymes and proteolytic enzymes. The combinations are shown in Table 2. When the enzyme treatment time is 16 hours, the content analysis result of the total phenolic compound is shown in FIG. 2. Experimental group 6 showed the highest total phenolic compound content in the complex enzyme treatment, and the content was 24.25mg GAE / g, and the next highest content was experimental group 2, experimental group 3, and the contents were 23.25, 22, respectively. Mg GAE / g. It is believed that the content of phenolic compounds can be increased in the complex enzyme composed of a mixture of glycoprotein and proteolytic enzyme rather than the complex enzyme composed of sugar or protease alone.
복합효소처리 후 열수추출한 시료의 총 페놀성 함량을 측정한 결과는 도 3과 같다. 여기서 사용된 복합효소는 실험군 6으로 효소처리는 16시간을 진행한 후 즉시 반응종결, 1시간 열수추출, 2시간 열수추출로 나누어 실험을 진행하였다. 총 페놀성 화합물 함량 측정 결과는 즉시 반응종결 24.25㎎ GAE/g 보다 1시간 열수추출을 진행하였을 때 33.5㎎ GAE/g으로 약 38% 더 높게 나왔고, 2시간 열수추출을 진행하였을 때 28.4㎎ GAE/g으로 즉시 반응종결 했을 때 보다는 높지만 1시간 열수추출 했을 때 보다 낮은 함량을 보였다.The result of measuring the total phenolic content of the hydrothermally extracted sample after the complex enzyme treatment is shown in FIG. 3. The complex enzyme used here was experimental group 6, and the enzyme treatment was carried out for 16 hours, and then the reaction was terminated, 1 hour hydrothermal extraction, and 2 hours hydrothermal extraction were conducted. The result of measuring total phenolic compound content was about 38% higher than 33.5mg GAE / g when hot water extraction was performed for 1 hour than 24.25mg GAE / g, and 28.4mg GAE / g when hot water extraction was performed for 2 hours. It was higher than when the reaction was terminated immediately with g, but showed a lower content than when hot water was extracted for 1 hour.
2-2. 레스베라트롤 함량2-2. Resveratrol content
열수추출, 에탄올추출, 복합효소처리, 복합효소처리+열수추출의 레스베라트롤 함량을 분석한 결과는 표 3과 같다.Table 3 shows the results of resveratrol content of hot water extraction, ethanol extraction, complex enzyme treatment, complex enzyme treatment + hot water extraction.
에탄올 추출 방법이 112.94㎍/g로 가장 높은 함량을 보였고, 다음으로는 실험군 7에서 91.57㎍/g으로 높게 나타났다. 열수추출 및 나머지 실험군에서는 레스베라트롤 함량이 상대적으로 낮은 것으로 나타났다.The ethanol extraction method showed the highest content of 112.94 ㎍ / g, followed by 91.57 ㎍ / g in Experimental Group 7. The resveratrol content was relatively low in the hydrothermal extraction and the rest of the experimental groups.
이러한 결과들로 보아 실험군 7과 같은 복합효소처리 후 1시간 동안 열수추출을 병행할 경우 에탄올추출에 준할 정도로 땅콩새싹으로부터 레스베라트롤을 효율적으로 추출할 수 있는 것으로 판단된다.These results suggest that resveratrol can be efficiently extracted from peanut sprouts in the same degree as ethanol extract when hot water extraction is performed for 1 hour after complex enzyme treatment as in Experimental Group 7.
Claims (4)
상기 반응생성물로부터 열수추출하는 단계;를 포함하고,
상기 효소반응을 pH 5.3 내지 5.7의 pH조건 및 50 내지 60℃의 온도조건에서 10 내지 20시간 동안 수행하며,
상기 열수추출을 90 내지 100℃에서 30분 내지 2시간 동안 수행하는 것을 특징으로 하는, 땅콩새싹으로부터 레스베라트롤을 비롯한 페놀성 화합물을 추출하는 방법.Peanut sprouts are made from celluclast, biscozyme, amyloglucosidase, promozyme, maltogenase, dextrozyme, termamyl Enzymatic reaction with papain, protamex, neutrase and flavorzyme to obtain a reaction product; And
And extracting hot water from the reaction product.
The enzyme reaction is carried out for 10 to 20 hours at a pH of pH 5.3 to 5.7 and a temperature of 50 to 60 ℃,
The hot water extraction is characterized in that for 30 minutes to 2 hours at 90 to 100 ℃, extracting phenolic compounds, including resveratrol from peanut sprouts.
상기 땅콩새싹은 열풍건조한 것임을 특징으로 하는 방법.The method of claim 1,
The peanut sprout is characterized in that the hot air dried.
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