KR102671355B1 - Enzymatic Hydrolysates of Defatted Perilla frutescens Residue with antioxidant activity and producing method thereof - Google Patents
Enzymatic Hydrolysates of Defatted Perilla frutescens Residue with antioxidant activity and producing method thereof Download PDFInfo
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- KR102671355B1 KR102671355B1 KR1020210017998A KR20210017998A KR102671355B1 KR 102671355 B1 KR102671355 B1 KR 102671355B1 KR 1020210017998 A KR1020210017998 A KR 1020210017998A KR 20210017998 A KR20210017998 A KR 20210017998A KR 102671355 B1 KR102671355 B1 KR 102671355B1
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- Prior art keywords
- enzyme
- perilla
- meal
- antioxidant activity
- decomposition product
- Prior art date
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Classifications
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
과제: 항산화 활성을 갖는 들깨박 효소분해물 및 그 제조방법을 제공하려는 것.
또한, 미생물의 배양배지로 사용 가능한 들깨박 효소분해물 및 그 제조방법을 제공하려는 것.
해결수단: 본 발명은 항산화 활성을 갖는 들깨박 효소분해물 및 그 제조방법에 관한 것으로서, 좀 더 자세히는 들깨박을 단백질 분해효소 알칼라아제(Alcalase) 및 탄수화물 분해효소 세레믹스(Ceremix)로 처리하여 수득되며 총당 8.5 mg/mL 이상, 환원당 5.0 mg/mL 이상, 가용성 단백질 2.8 mg/mL 이상 및 폴리페놀 0.8 mg/mL 이상의 함량으로 포함하는 항산화 활성을 갖는 들깨박 효소분해물 및 그 제조방법에 관한 것이다. 또한, 본 발명의 항산화 활성을 갖는 들깨박 효소분해물은 폴리페놀 증가, ABTS 양이온 라디칼 소거 효과, DPPH 유리라디칼 소거효과 등의 항산화 기능을 나타낸다. 또한, 본 발명의 항산화 활성을 갖는 들깨박은 유산균 배양을 위한 배지로써 효과를 나타낸다.Task: To provide perilla seed meal enzyme decomposition product with antioxidant activity and a method for producing the same.
In addition, the aim is to provide an enzyme decomposition product of perilla seed meal that can be used as a culture medium for microorganisms and a method for producing the same.
Solution: The present invention relates to an enzyme decomposition product of perilla meal with antioxidant activity and a method for producing the same. In more detail, perilla meal is treated with the proteolytic enzyme Alcalase and the carbohydrate decomposition enzyme Ceremix. It relates to an enzyme hydrolyzed product of perilla seed meal that has antioxidant activity and contains more than 8.5 mg/mL of total sugar, more than 5.0 mg/mL of reducing sugar, more than 2.8 mg/mL of soluble protein, and more than 0.8 mg/mL of polyphenol, and a method for producing the same. . In addition, the enzyme decomposition product of perilla meal with antioxidant activity of the present invention exhibits antioxidant functions such as increasing polyphenols, ABTS cation radical scavenging effect, and DPPH free radical scavenging effect. In addition, perilla meal with antioxidant activity of the present invention is effective as a medium for culturing lactic acid bacteria.
Description
본 발명은 항산화 활성을 갖는 들깨박 효소분해물 및 그 제조방법에 관한 것으로서, 좀 더 자세히는 들깨박을 단백질 분해효소 알칼라아제(Alcalase) 및 탄수화물 분해효소 세레믹스(Ceremix)로 처리하여 수득된 항산화 활성을 갖는 들깨박 효소분해물, 그 제조방법 및 그 이용에 관한 것이다. The present invention relates to an enzyme decomposition product of perilla meal with antioxidant activity and a method for producing the same. More specifically, it relates to an antioxidant product obtained by treating perilla meal with the proteolytic enzyme Alcalase and the carbohydrate decomposition enzyme Ceremix. It relates to an active enzyme decomposition product of perilla seed meal, its production method, and its use.
들깨(Perilla frutescens Britton)는 인도의 고지대와 중국 중남부를 원산지로 하는 1년생 초본으로 한국, 중국, 인도, 일본 등에서 재배되고 있다(1). 들깨는 종자와 잎을 식용으로 사용해 왔지만「향약집성방 향약초본」에서는 민간약으로도 소개하고 있다(2). 들깨 종자는 수분 3.9%, 단백질 16.0%, 지방 39.5%, 탄수화물 20.2%, 섬유 17.5%, 무기질 2.9%로 구성되어 있다. 무기질로는 인(P), 마그네슘(Mg), 칼슘(Ca), 철(Fe), 망간(Mn), 아연(Zn), 구리(Cu) 등을 함유하고 있다. 유지작물 중 땅콩, 홍화, 해바라기, 아마 등에는 함유되지 않은 마그네슘 성분이 들깨 종자에는 100 g당 275 mg이 들어있을 정도로 들깨 종자는 영양학적으로 우수하다(3). 들깨 종자에 40% 이상 함유되어 있는 지방산은 올레산, 리놀레산, 소량의 팔미트산 등으로 구성되어 있고(4), 특히 들깨 종자유는 오메가-3 지방산의 함량이 높다(5). 들깨 종자유의 불포화 지방산은 영양학적으로는 대단히 중요하지만, 들깨 종자유에서 지방산 산화가 쉽게 일어나는 원인이 된다(6). Perilla frutescens Britton is an annual herb native to the highlands of India and south-central China and is cultivated in Korea, China, India, and Japan (1). The seeds and leaves of perilla have been used for food, but it is also introduced as a folk medicine in the “Collected Herbal Medicines” (2). Perilla seeds are composed of 3.9% moisture, 16.0% protein, 39.5% fat, 20.2% carbohydrate, 17.5% fiber, and 2.9% minerals. It contains minerals such as phosphorus (P), magnesium (Mg), calcium (Ca), iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu). Among oil crops, perilla seeds are nutritionally excellent as they contain 275 mg of magnesium per 100 g, which is not contained in peanuts, safflower, sunflower, and flax (3). Fatty acids contained in more than 40% of perilla seeds consist of oleic acid, linoleic acid, and a small amount of palmitic acid (4), and perilla seed oil in particular has a high content of omega-3 fatty acids (5). The unsaturated fatty acids in perilla seed oil are very important nutritionally, but they cause fatty acid oxidation to easily occur in perilla seed oil (6).
2018년 유지작물 총생산량 중 들깨가 차지하는 비율은 62.8%일 정도로 들깨는 우리나라에서 가장 많이 생산되는 유지작물이다(7). 들깨는 종자를 직접 사용하기도 하지만, 들깨를 착유하여 얻어진 들깨유로도 많이 사용된다. 생들기름의 낮은 산화안정성 때문에 들깨는 주로 볶아서 착유하는데 이 과정에서 탈지 들깨박이 많이 발생한다(7). 들깨는 볶는 과정에서 조단백질, 당질 및 중성지질의 함량은 증가하나, 볶음 온도와 시간에 따라 인, 인지질, 아미노산, 당 등의 함량이 급격히 감소하는 경향이 있다(8). 들깨 종자의 착유로 발생되는 부산물인 들깨박의 주성분은 탄수화물과 단백질인데, 특히 단백질에는 함황 아미노산이면서 필수 아미노산인 메티오닌이 46%를 차지할 정도로 풍부하게 들어 있다. 또한 들깨박은 무기질, 폴리페놀 및 미량의 지질 성분을 함유하고 있고(9,10), 들깨박에 관한 연구로는 들깨유 성분(1), 지질대사(12) 및 항산화 활성(5,13) 등이 보고된 바 있다. 착유 후에도 남아있는 유용성분으로 보아 들깨박은 바이오매스 소재로서의 이용 가치가 높다고 볼 수 있지만 현재는 일부만 비료나 사료로 이용되고 대부분 폐기되고 있다(8). Perilla seeds account for 62.8% of the total oil crop production in 2018, making perilla seeds the most produced oil crop in Korea (7). Perilla seeds are used directly, but perilla oil obtained by extracting perilla seeds is also widely used. Due to the low oxidation stability of raw perilla oil, perilla seeds are mainly roasted and extracted, and during this process, a lot of defatted perilla meal is generated (7). The content of crude protein, carbohydrates, and neutral lipids in perilla seeds increases during the roasting process, but the content of phosphorus, phospholipids, amino acids, and sugars tends to decrease rapidly depending on the roasting temperature and time (8). The main components of perilla meal, a by-product from the extraction of perilla seeds, are carbohydrates and proteins. In particular, the protein is rich in methionine, a sulfur-containing amino acid and an essential amino acid, accounting for 46%. In addition, perilla meal contains minerals, polyphenols, and trace amounts of lipids (9,10), and studies on perilla seed meal include perilla oil components (1), lipid metabolism (12), and antioxidant activity (5,13). This has been reported. Considering the useful components remaining after milking, perilla meal can be considered to have high usability as a biomass material, but currently only some of it is used as fertilizer or feed and most of it is discarded (8).
최근 농수산물의 가공과정에서 다량 배출되는 부산물로부터 생리활성 물질을 분리하는 연구(14)가 지속적으로 이루어지고 있고, 게 껍질 속의 chitosan(15), 참깨박 속의 항산화 물질(16), 감귤 껍질 속의 아스코르브산(17) 등의 사례가 보고된 바 있다. 특히 농수산 가공 부산물에 함유되어 있는 폴리페놀 성분(18)은 생체 내에서의 질병 예방 효과와 항산화 활성으로 인해 크게 주목받고 있다. 농수산 가공 부산물 및 폐기물에는 상당량의 유용성분이 잔류하고 있지만 이를 활용하기 위한 연구는 여전히 부족한 실정이다. 이에 본 발명은 농수산 가공 부산물을 유용 자원화하는 연구의 일환으로, 들깨박의 바이오매스 소재로서의 활용 가능성을 확인하고자 하였다. 들깨박의 유용성분을 효과적으로 이용하기 위해서 들깨박의 불용성 물질을 가용화하여 생리활성 및 미생물 배양 배지로서의 가능성을 확인함으로써 바이오매스 폐기물의 산업적 활용을 위한 기초 자료를 제시하자고 하였다. Recently, research on the separation of bioactive substances from by-products released in large quantities during the processing of agricultural and marine products (14) has been continuously conducted, including chitosan in crab shells (15), antioxidants in sesame meal (16), and ascorbic acid in citrus peels. Cases such as (17) have been reported. In particular, polyphenol components (18) contained in agricultural and fisheries processing by-products are attracting great attention due to their disease-prevention effect and antioxidant activity in vivo. A significant amount of useful substances remain in agricultural and fisheries processing by-products and wastes, but research on utilizing them is still lacking. Accordingly, the present invention sought to confirm the possibility of utilizing perilla meal as a biomass material as part of research on turning agricultural and fisheries processing by-products into useful resources. In order to effectively use the useful components of perilla meal, we proposed to present basic data for industrial use of biomass waste by solubilizing the insoluble substances in perilla meal and confirming its physiological activity and potential as a microbial culture medium.
본 발명은 항산화 활성을 갖는 들깨박 효소분해물 및 그 제조방법을 제공하는 것을 목적으로 한다.The purpose of the present invention is to provide an enzyme decomposition product of perilla seed meal with antioxidant activity and a method for producing the same.
또한, 본 발명은 미생물의 배양배지로 사용 가능한 들깨박 효소분해물 및 그 제조방법을 제공하는 것을 목적으로 한다.Additionally, the purpose of the present invention is to provide an enzyme decomposition product of perilla seed meal that can be used as a culture medium for microorganisms and a method for producing the same.
또한, 본 발명은 아미노산 성분과 당류를 다량 포함하는 들깨박 효소분해물을 이용한 미생물 배양 방법을 제공하는 것을 목적으로 한다.Additionally, the present invention aims to provide a method for cultivating microorganisms using enzyme decomposition product of perilla meal containing a large amount of amino acids and sugars.
본 발명자들은 들깨박의 불용성 성분을 가용화하기 위해 효소분해법을 사용하였다. 들깨박의 주성분인 탄수화물과 단백질의 효율적인 분해를 위하여 최적의 탄수화물 분해효소와 단백질 분해효소를 선정하였다. 불용성 탄수화물을 당화하는데 효소분해를 이용(19)하는 이유는 강산을 이용한 가수분해에서는 미생물 대사에 영향을 미치는 독성물질이 생성(20)될 수 있기 때문이다. 효소분해에 의해 불용성 단백질이 가용화되면 유리 아미노산이 다량 생성되고 이는 미생물 배양에서 질소원으로 작용하여 발효 속도를 향상시킬 수 있을 것이다(21). 본 발명자들은 들깨박의 가용화를 위한 효소를 선정하였고, 최적의 효소반응 조건을 확립하였다. 또한, 효소분해로 얻어진 들깨박 효소분해물의 항산화 소재 및 유용미생물 배양배지로서의 활용가능성을 확인하였다.The present inventors used an enzymatic digestion method to solubilize the insoluble components of perilla meal. The optimal carbohydrate-decomposing enzyme and proteolytic enzyme were selected for efficient decomposition of carbohydrates and proteins, which are the main components of perilla meal. The reason why enzymatic digestion is used to saccharify insoluble carbohydrates (19) is because hydrolysis using strong acids can produce toxic substances that affect microbial metabolism (20). When insoluble proteins are solubilized by enzymatic digestion, a large amount of free amino acids are produced, which can act as a nitrogen source in microbial culture and improve the fermentation rate (21). The present inventors selected enzymes for solubilization of perilla meal and established optimal enzyme reaction conditions. In addition, the feasibility of using the enzyme decomposition material of perilla seed meal obtained through enzymatic decomposition as an antioxidant material and a culture medium for useful microorganisms was confirmed.
본 발명은 들깨박을 분쇄하여 들깨박 분말을 제조하는 단계; 상기 들깨박 분말을 증류수 또는 완충액 등의 용매에 현탁하는 단계: 상기 들깨박 현탁액에 단백질 분해효소 및 탄수화물 분해효소를 첨가하는 단계; 상기 단백질 분해효소 및 탄수화물 분해효소를 첨가한 들깨박 현탁액을 항온수조 등에서 반응시키는 단계; 상기 반응을 완료하는 단계; 상기 반응이 완료된 반응물을 원심분리하는 단계;로부터 수득되며 총당 8.5 mg/mL 이상, 바람직하게는 8.5~12 mg/mL, 더욱 바람직하게는 9~11 mg/mL, 환원당 5.0 mg/mL 이상, 바람직하게는 5~8 mg/mL, 더욱 바람직하게는 6.5~7 mg/mL, 가용성 단백질 2.8 mg/mL 이상, 바람직하게는 2.8~5 mg/mL, 더욱 바람직하게는 3~4 mg/mL 및 폴리페놀 0.8 mg/mL 이상, 바람직하게는 0.8~3 mg/mL, 더욱 바람직하게는 1~2 mg/mL의 함량으로 포함하는 항산화 활성을 갖는 들깨박 효소분해물을 제공한다. The present invention includes the steps of grinding perilla meal to produce perilla meal powder; Suspending the perilla meal powder in a solvent such as distilled water or a buffer solution: adding a proteolytic enzyme and a carbohydrate decomposition enzyme to the perilla meal suspension; Reacting the perilla meal suspension to which the proteolytic enzyme and carbohydrate decomposition enzyme were added in a constant temperature water bath, etc.; completing the reaction; Centrifuging the reaction product upon completion of the reaction; obtained from the total sugar content of 8.5 mg/mL or more, preferably 8.5 to 12 mg/mL, more preferably 9 to 11 mg/mL, reducing sugar 5.0 mg/mL or more, preferably Preferably 5 to 8 mg/mL, more preferably 6.5 to 7 mg/mL, soluble protein 2.8 mg/mL or more, preferably 2.8 to 5 mg/mL, more preferably 3 to 4 mg/mL and poly Provided is an enzyme decomposition product of perilla meal with antioxidant activity containing phenol in a content of 0.8 mg/mL or more, preferably 0.8 to 3 mg/mL, and more preferably 1 to 2 mg/mL.
또한, 본 발명의 단백질 분해효소는 알칼라아제(Alcalase)인 것을 특징으로 한다. Additionally, the proteolytic enzyme of the present invention is characterized as Alcalase.
또한, 본 발명의 탄수화물 분해효소는 세레믹스(Ceremix)인 것을 특징으로 한다. 세레믹스는 Novozymes 사의 복합 효소 브랜드이며, Ceremix® Flex는 말토제닉 아밀레이즈 (maltogenic amylase), 풀룰라네이즈 (pullulanase) 및 알파 아밀레이즈 (alpha-amylase)의 혼합물이다.In addition, the carbohydrate decomposing enzyme of the present invention is characterized as Ceremix. Ceremix is a brand of complex enzymes from Novozymes. Ceremix ® Flex is a mixture of maltogenic amylase, pullulanase and alpha-amylase.
또한, 본 발명의 단백질 분해효소 및 탄수화물 분해효소는 0.5~3:0.5~3의 비율로 들깨박 분말 기준 0.5~5%(w/w)로 첨가하는 것을 특징으로 한다. In addition, the proteolytic enzyme and carbohydrate-degrading enzyme of the present invention are characterized in that they are added at a ratio of 0.5 to 3:0.5 to 3 and 0.5 to 5% (w/w) based on perilla meal powder.
또한, 본 발명의 항산화 활성을 갖는 들깨박 효소분해물은 미생물 배양배지, 바람직하게는 유산균 배양배지로 사용하는 것을 특징으로 한다. In addition, the enzyme decomposition product of perilla meal with antioxidant activity of the present invention is characterized by being used as a microbial culture medium, preferably a lactic acid bacteria culture medium.
또한, 본 발명의 상기 유산균은 락토바실러스(Lactobacillus sp.) 속, 비피도박테리움(Bifidobacterium sp.) 속, 스트렙토코커스(Streptococcus sp.) 속, 락토코커스(Lactococcus sp.) 속, 엔테로코커스(Enterococcus sp.) 속, 페디오코커스(Pediococcus sp.) 속, 류코노스톡(Leuconostoc sp.) 속, 및 비셀라(Weissella sp.) 속으로 이루어진 군에서 선택되는 하나 이상의 균주인 것을 특징으로 한다.In addition, the lactic acid bacteria of the present invention include Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Lactococcus sp., and Enterococcus. sp.), Pediococcus sp., Leuconostoc sp., and Weissella sp.
이뿐만 아니라, 본 발명은 들깨박을 분쇄하여 들깨박 분말을 제조하는 단계; 상기 들깨박 분말을 멸균하는 단계; 상기 멸균한 들깨박 분말 5~15%(w/w)을 증류수, 완충액 등의 적절한 용매에 현탁하는 단계: 상기 들깨박 분말 현탁액에 단백질 분해효소 알칼라아제(Alcalase) 및 탄수화물 분해효소 세레믹스(Ceremix)를 0.5~3:0.5~3의 비율로 건조 들깨박 분말 기준 0.5~5%(w/w)로 첨가하는 단계; 상기 단백질 분해효소 및 탄수화물 분해효소를 첨가한 들깨박 분말 현탁액을 pH 5~8에서 항온수조 등에서 온도를 유지하며 0.5~24시간, 바람직하게는 1~5시간 반응시키는 단계; 상기 반응물을 70~100℃에서 2~10분간 열처리하는 등의 방법으로 반응을 완료하는 단계; 상기 반응 완료된 반응물을 원심분리하는 단계;를 포함하는 항산화 활성을 갖는 들깨박 효소분해물 제조방법에 관한 것이다. In addition, the present invention includes the steps of grinding perilla meal to produce perilla meal powder; Sterilizing the perilla meal powder; Suspending 5-15% (w/w) of the sterilized perilla meal powder in an appropriate solvent such as distilled water or buffer solution: Adding the proteolytic enzyme Alcalase and carbohydrate decomposition enzyme Cermix to the perilla meal powder suspension ( Adding 0.5 to 5% (w/w) of Ceremix) based on dried perilla seed meal powder at a ratio of 0.5 to 3:0.5 to 3; reacting the perilla meal powder suspension to which the proteolytic enzyme and carbohydrate decomposition enzyme were added for 0.5 to 24 hours, preferably 1 to 5 hours, while maintaining the temperature in a constant temperature water bath at pH 5 to 8; Completing the reaction by heat treating the reactant at 70 to 100°C for 2 to 10 minutes; It relates to a method for producing an enzyme decomposition product of perilla meal with antioxidant activity, comprising the step of centrifuging the reaction product after completion of the reaction.
또한, 본 발명은 상기 항산화 활성을 갖는 들깨박 효소분해물을 포함하는 유산균 배양배지에 관한 것이다.In addition, the present invention relates to a lactic acid bacteria culture medium containing the enzyme decomposition product of perilla meal having the above antioxidant activity.
또한, 본 발명은 상기 유산균이 락토바실러스(Lactobacillus sp.) 속, 비피도박테리움(Bifidobacterium sp.) 속, 스트렙토코커스(Streptococcus sp.) 속, 락토코커스(Lactococcus sp.) 속, 엔테로코커스(Enterococcus sp.) 속, 페디오코커스(Pediococcus sp.) 속, 류코노스톡(Leuconostoc sp.) 속 및 비셀라(Weissella sp.) 속으로 이루어진 군에서 선택되는 하나 이상의 균주인 것을 특징으로 하는 항산화 활성을 갖는 들깨박 효소분해물을 포함하는 유산균 배양배지에 관한 것이다.In addition, the present invention relates to the lactic acid bacteria being of the genus Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Lactococcus sp., and Enterococcus. sp.), Pediococcus sp., Leuconostoc sp., and Weissella sp.). It relates to a lactic acid bacteria culture medium containing enzyme decomposition product of perilla meal.
또한, 본 발명은 In addition, the present invention
항산화 활성을 갖는 들깨박 효소분해물을 포함하는 배양 배지를 준비하는 단계; Preparing a culture medium containing enzyme decomposition product of perilla seed meal with antioxidant activity;
상기 배양 배지에 유산균 현탁액 2~10%(v/v)를 접종하는 단계; 및 Inoculating 2-10% (v/v) of lactic acid bacteria suspension into the culture medium; and
상기 유산균을 32~38℃에서 1~20시간 동안 진탕 배양하는 단계;를 포함하는 들깨박 효소분해물을 이용한 유산균 배양방법에 관한 것이다.It relates to a method of cultivating lactic acid bacteria using an enzyme decomposition product of perilla meal, comprising the step of shaking and culturing the lactic acid bacteria at 32-38°C for 1-20 hours.
또한, 본 발명은 상기 항산화 활성을 갖는 들깨박 효소분해물을 포함하는 건강 기능성 식품 조성물에 관한 것이다. 본 발명으로 얻어진 항산화 활성을 갖는 들깨박 효소분해물을 이용한 건강 기능성 식품 조성물은 음료 또는 차로 음용할 수 있다. 또한, 상기 살균처리 완료된 추출물을 건조하고 곱게 분쇄하여 분말로 사용할 수도 있다. 또한, 상기 추출물과 상기 분말을 2:8 중량비로 교반기 또는 용기에 투입하여 균일하게 혼합하고 과립으로 제조하여 사용할 수도 있다. In addition, the present invention relates to a health functional food composition containing the enzyme decomposition product of perilla seed meal having the above antioxidant activity. The health functional food composition using the enzyme decomposition product of perilla seed meal with antioxidant activity obtained by the present invention can be consumed as a beverage or tea. Additionally, the sterilized extract may be dried, finely ground, and used as a powder. Additionally, the extract and the powder may be added to a stirrer or container at a weight ratio of 2:8, mixed uniformly, and manufactured into granules for use.
본 발명으로 얻어진 항산화 활성을 갖는 들깨박 효소분해물을 이용한 건강 기능성 식품 조성물은 각 원료의 영양학적 및 기능적 성분을 지속적으로 섭취할 수 있다. 또한, 소비자의 기호에 맞게 차로 마실 수 있으며, 우유, 요구르트 등에 첨가하여 음용하기에도 적당하다. 이뿐만 아니라, 각종 국, 반찬 등 요리에 감칠맛을 내게 하는 조미료로서 사용하는 것도 가능하다.The health functional food composition using the enzyme decomposition product of perilla seed meal with antioxidant activity obtained by the present invention can continuously consume the nutritional and functional ingredients of each raw material. In addition, it can be drunk as tea according to the consumer's preference, and is also suitable for drinking by adding it to milk, yogurt, etc. In addition, it can be used as a seasoning to add flavor to various dishes such as soups and side dishes.
본 발명의 항산화 활성을 갖는 들깨박 효소분해물 제조방법을 좀 더 자세히 설명하면, 먼저 들깨박을 분쇄하여 분말화하고 체로 거른 후 고압증기멸균기에서 멸균하였다. 상기 멸균한 들깨박 분말 5~10%(w/w)을 증류수에 현탁한 다음, 단백질 분해효소 알칼라아제(Alcalase) 및 탄수화물 분해효소 세레믹스(Ceremix)를 0.5~3:0.5~3의 비율로 건조 들깨박 분말 기준 0.5~5%(w/w)로 첨가하였다. 단백질 분해효소와 탄수화물 분해효소의 첨가량이 0.5%(w/w) 미만인 경우에는 가용성 단백질 및 환원당의 함량 증가가 충분하지 않으며, 5%(w/w)를 초과하는 경우에는 경제적이지 않다.To describe in more detail the method of producing an enzyme decomposition product of perilla meal with antioxidant activity of the present invention, first, perilla meal was ground, powdered, sieved, and sterilized in a high-pressure steam sterilizer. 5-10% (w/w) of the sterilized perilla meal powder was suspended in distilled water, and then the protease Alcalase and carbohydrate-decomposition enzyme Ceremix were added at a ratio of 0.5-3:0.5-3. It was added at 0.5 to 5% (w/w) based on dried perilla seed meal powder. If the amount of proteolytic enzyme and carbohydrate decomposition enzyme added is less than 0.5% (w/w), the increase in the content of soluble protein and reducing sugar is not sufficient, and if it exceeds 5% (w/w), it is not economical.
다음으로는 상기 단백질 분해효소 및 탄수화물 분해효소를 첨가한 들깨박 현탁액을 pH 5~8에서 항온수조에서 0.5시간 이상, 바람직하게는 0.5~5시간, 더욱 바람직하게는 0.5~3시간 반응시켰다. 반응 시간이 0.5 시간 미만인 경우에는 단백질 및 탄수화물 분해 반응이 충분히 일어나지 않으며, 3시간을 초과하는 경우에는 아미노산 등의 가용성 단백질 및/또는 환원당 함량 증가 정도가 미미하여 경제적이지 못하다.Next, the perilla meal suspension to which the proteolytic enzyme and carbohydrate decomposition enzyme were added was reacted in a constant temperature water bath at pH 5 to 8 for at least 0.5 hours, preferably 0.5 to 5 hours, more preferably 0.5 to 3 hours. If the reaction time is less than 0.5 hours, protein and carbohydrate decomposition reactions do not sufficiently occur, and if it exceeds 3 hours, the increase in the content of soluble proteins such as amino acids and/or reducing sugars is minimal, making it uneconomical.
다음으로는 상기 반응물을 70~100 ℃에서 2~10분간 열처리하는 등의 방법으로 효소분해 반응을 완료하였다. 반응이 완료된 반응물을 원심분리하여 들깨박 효소분해물을 제조하였다. 이렇게 제조된 본 발명의 효소분해물은 항산화 활성을 나타내며, 항산화 활성 기능성 식품이나 의약품 보조제, 미생물 배지 등의 다양한 용도로 이용할 수 있다.Next, the enzymatic decomposition reaction was completed by heat-treating the reactant at 70-100°C for 2-10 minutes. After the reaction was completed, the reactant was centrifuged to prepare perilla meal enzyme decomposition product. The enzyme decomposition product of the present invention prepared in this way exhibits antioxidant activity and can be used for various purposes such as antioxidant-active functional foods, pharmaceutical supplements, and microbial media.
본 발명의 효소분해물은 항산화 활성을 현저하게 증진하여, 약제학적 분야에서 공지의 방법에 의해 건강 기능성 식품 등으로 제제화할 수 있고, 조성물 자체 또는 약제학적으로 허용되는 담체, 부형제 등과 혼합하여 통상의 약학적 제제, 예를 들면 드링크제와 같은 액제, 시럽제, 캡슐제 등으로 제제화될 수 있으며, 이들은 경구 또는 비경구로 투여될 수 있다. The enzyme decomposition product of the present invention significantly improves antioxidant activity, and can be formulated into health functional foods, etc. by methods known in the pharmaceutical field, and can be used as a composition in itself or by mixing with pharmaceutically acceptable carriers, excipients, etc. It can be formulated as a liquid preparation, for example, a drink-like liquid, syrup, capsule, etc., and can be administered orally or parenterally.
또한, 본 발명의 항산화 활성을 갖는 들깨박 효소분해물은 유산균 배양배지로 이용 가능하다. 환원당과 가용성 단백질 함량이 높아 유산군 증식이 잘 일어났으며 이에 따라 젖산을 많이 생산하여 산도도 크게 증가하기에 들깨박 효소분해물은 미생물 배양배지로 충분히 활용 가능하다. In addition, the enzyme decomposition product of perilla meal with antioxidant activity of the present invention can be used as a culture medium for lactic acid bacteria. Due to its high reducing sugar and soluble protein content, lactic acid group proliferates easily, and as a result, a lot of lactic acid is produced and acidity increases significantly, so the enzyme decomposition product of perilla meal can be fully utilized as a microbial culture medium.
본 발명의 항산화 활성을 갖는 들깨박 효소분해물은 수율, 총당, 환원당, 가용화 단백질 함량 모두 효소처리하지 않은 대조군보다 매우 높게 나타났다.The yield, total sugar, reducing sugar, and solubilized protein content of the perilla meal enzyme digestion product having antioxidant activity of the present invention were all significantly higher than the control group that was not treated with enzyme.
또한, 본 발명의 항산화 활성을 갖는 들깨박 효소분해물은 폴리페놀 증가, ABTS (2, 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid) 양이온 라디칼 소거 효과, DPPH (2,2-diphenyl-1-picrylhydrazyl radical) 유리라디칼 소거효과 등의 항산화 기능을 나타내었다.In addition, the enzyme decomposition product of perilla seed meal with antioxidant activity of the present invention increases polyphenol, ABTS (2, 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid) cation radical scavenging effect, and DPPH (2,2-diphenyl) -1-picrylhydrazyl radical) showed antioxidant functions such as free radical scavenging effect.
따라서, 본 발명의 항산화 활성을 갖는 들깨박 효소분해물은 건강 기능성 식품으로 이용할 수 있다. Therefore, the enzyme decomposition product of perilla meal with antioxidant activity of the present invention can be used as a health functional food.
또한, 본 발명의 항산화 활성을 갖는 들깨박 효소분해물은 유산균 등의 미생물 배양을 위한 배지로 유용하다. In addition, the enzyme decomposition product of perilla meal with antioxidant activity of the present invention is useful as a medium for culturing microorganisms such as lactic acid bacteria.
도 1은 들깨박 효소분해물에 다양한 효소를 처리하여 생성된 환원당 함량을 비교한 것이다. 데이터 = 평균 ± 표준편차 (n=3)
도 2는 들깨박 효소분해물에 다양한 효소를 처리하여 생성된 가용성 단백질 함량을 비교한 것이다. 데이터 = 평균 ± 표준편차 (n=3)
도 3은 알칼라아제 및 세레믹스 효소를 동시에 처리하여 들깨박 효소분해물 생성에 최적인 pH를 탐색한 결과이다. 데이터 = 평균 ± 표준편차 (n=3)
도 4는 들깨박 효소분해물에 효소 가수 분해시 최적의 효소 첨가량을 알아본 결과이다. 데이터 = 평균 ± 표준편차 (n=3)
도 5는 들깨박 효소분해물의 효소 가수 분해시 효소 첨가량에 따라 얻어지는 가용성 단백질 함량을 나타낸 것이다. 데이터 = 평균 ± 표준편차 (n=3)
도 6은 들깨박 효소분해물의 효소 가수 분해시 반응 시간에 따른 당 함량을 비교한 것이다. 데이터 = 평균 ± 표준편차 (n=3)
도 7은 들깨박 효소분해물의 효소 가수 분해 반응 시간에 따른 가용성 단백질 함량 결과이다. 데이터 = 평균 ± 표준편차 (n=3)
도 8은 들깨박 효소분해물(-○-) 및 미처리 대조군 (-□-)의 ABTS 양이온 라디칼 소거 활성을 비교한 것이다. 데이터 = 평균 ± 표준편차 (n=3)
도 9는 들깨박 효소분해물(-○-) 및 미처리 대조군 (-□-)의 DPPH 유리라디칼 소거 활성을 비교한 것이다. 데이터 = 평균 ± 표준편차 (n=3)
도 10은 들깨박 효소분해물 및 미처리 대조군에서 L. mesenteroides 310-12 유산균을 배양하고 시간에 따른 당 변화를 비교한 것이다. 데이터 = 평균 (n=2)
도 11은 들깨박 효소분해물 및 미처리 대조군에서 적정 산도 및 pH 변화를 비교한 것이다. 데이터 = 평균 (n=2) Figure 1 compares the reducing sugar content produced by treating perilla meal enzyme decomposition products with various enzymes. Data = mean ± standard deviation (n=3)
Figure 2 compares the soluble protein content produced by treating perilla meal enzyme decomposition products with various enzymes. Data = mean ± standard deviation (n=3)
Figure 3 shows the results of exploring the optimal pH for producing perilla meal enzyme decomposition product by simultaneously treating alkalase and Ceremic enzyme. Data = mean ± standard deviation (n=3)
Figure 4 shows the results of determining the optimal amount of enzyme addition during enzymatic hydrolysis of perilla meal enzyme decomposition product. Data = mean ± standard deviation (n=3)
Figure 5 shows the soluble protein content obtained according to the amount of enzyme added during enzymatic hydrolysis of perilla meal enzymatic digestion. Data = mean ± standard deviation (n=3)
Figure 6 compares the sugar content according to reaction time during enzymatic hydrolysis of perilla meal enzyme decomposition product. Data = mean ± standard deviation (n=3)
Figure 7 shows the results of soluble protein content according to the enzymatic hydrolysis reaction time of the perilla meal enzyme decomposition product. Data = mean ± standard deviation (n=3)
Figure 8 compares the ABTS cation radical scavenging activity of perilla seed enzyme decomposition product (-○-) and untreated control (-□-). Data = mean ± standard deviation (n=3)
Figure 9 compares the DPPH free radical scavenging activity of perilla meal enzyme decomposition product (-○-) and untreated control (-□-). Data = mean ± standard deviation (n=3)
Figure 10 shows the culture of L. mesenteroides 310-12 lactic acid bacteria in perilla meal enzymatic digestion and untreated control and comparing the changes in sugar over time. data = mean (n=2)
Figure 11 is a comparison of titratable acidity and pH changes in perilla meal enzyme decomposition product and untreated control group. data = mean (n=2)
아래에서는 구체적인 실시예를 들어 본 발명의 구성을 좀 더 자세히 설명한다. 그러나, 본 발명의 범위가 실시예의 기재에 의하여 한정되는 것이 아님은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 자명하다.Below, the configuration of the present invention will be described in more detail through specific examples. However, it is obvious to those skilled in the art that the scope of the present invention is not limited by the description of the examples.
재료 및 시약Materials and Reagents
경상남도 창원시 마산합포구 진전면에서 수확한 들깨에서 들깨유를 착유하고 남은 들깨박을 사용하였다. 들깨박 효소분해를 위해 사용한 단백질 분해효소인 알칼라아제, 플라보르자임, 뉴트라제, 프로타멕스와 탄수화물 분해효소인 세레믹스, 펙티나아제, 비스코자임은 Novozyme사(Bagsvaerd, Denmark)의 제품이었다. D-글루코스, 3,5-DNS (dinitrosalicylic acid), 브래드포드 용액, BSA (bovine serum albumin), Folin-Ciocalteu 시약, 갈산 (gallic acid), 2,2-ABTS (azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), 2,2-DPPH (diphenyl-picrylhydrazyl), 과황산칼륨 (Potassium Persulfate), 페놀프탈레인 (phenolphthalein)은 Sigma-Aldrich사(St. Louis, MO, USA)의 제품을 사용하였다. 유산균은 청운대학교 화학공학과의 보유 균주인 Leuconostoc mesenteroides 310-12 (22)를, 균주 배양을 위한 Lactobacilli MRS broth는 Difco사(Detroit, MI, USA)의 제품을 사용하였다. 그 외 시약은 1급 이상의 제품을 사용하였다. Perilla oil was extracted from perilla seeds harvested in Jinjin-myeon, Masan Happo-gu, Changwon-si, Gyeongsangnam-do, and the remaining perilla seed meal was used. The proteolytic enzymes Alcalase, Flavorzyme, Neutrase, and Protamex and the carbohydrate decomposition enzymes Ceremix, Pectinase, and Viscozyme used for enzymatic decomposition of perilla meal were products of Novozyme (Bagsvaerd, Denmark). . D-glucose, 3,5-DNS (dinitrosalicylic acid), Bradford solution, BSA (bovine serum albumin), Folin-Ciocalteu reagent, gallic acid, 2,2-ABTS (azino-bis(3-ethylbenzothiazoline- 6-sulfonic acid), 2,2-DPPH (diphenyl-picrylhydrazyl), Potassium Persulfate, and phenolphthalein were used from Sigma-Aldrich (St. Louis, MO, USA). As lactic acid bacteria, Leuconostoc mesenteroides 310-12 (22), a strain owned by the Department of Chemical Engineering at Cheongwoon University, was used, and Lactobacilli MRS broth for strain culture was from Difco (Detroit, MI, USA). For other reagents, products of grade 1 or higher were used.
실시예 1: 들깨박 효소분해를 위한 효소 선정Example 1: Selection of enzymes for enzymatic degradation of perilla seed meal
들깨박은 분쇄하여 분말화하고 200 mesh 체로 거른 후 실험에 필요한 중량으로 나누고 고압증기멸균기(121 ℃, 20 min)에서 멸균하여 사용하였다. 들깨박 효소분해에 적합한 효소를 선정하기 위해 단백질 분해효소인 알칼라아제, 플라보르자임, 뉴트라제, 프로타멕스와 탄수화물 분해효소인 세레믹스, 펙티나아제, 비스코자임을 사용하였다. 건조 중량 기준으로 10%(w/w)의 들깨박을 증류수에 현탁한 다음, 각각의 효소를 들깨박의 1%(w/w)가 되게 가하여 항온수조(50℃, 180 rpm)에서 2시간 반응시키고 90℃에서 5분간 열처리로 반응을 정지시킨 후, 원심분리(3000 × g, 10분)하여 상등액을 얻었다. 대조군은 효소를 첨가하지 않고 같은 조건에서 방치한 들깨박 현탁액을 원심분리하여 얻어진 상등액을 사용하였다. 반응액의 pH는 각 효소 반응의 최적 pH로 조정하였다. 비스코자임, 세레믹스와 펙티나아제는 pH 5.0으로, 뉴트라제와 대조군은 pH 7.0으로, 플라보르자임과 프로타멕스는 pH 7.5로, 알칼라아제는 pH 8.0으로 조정한 후 효소반응을 진행하였다. Perilla meal was ground, powdered, sieved through a 200 mesh sieve, divided into the weight required for the experiment, and sterilized in an autoclave (121°C, 20 min) before use. To select enzymes suitable for enzymatic decomposition of perilla meal, proteolytic enzymes Alcalase, Flavorzyme, Neutrase, and Protamex and carbohydrate decomposition enzymes Ceremics, Pectinase, and Viscozyme were used. 10% (w/w) of perilla meal based on dry weight was suspended in distilled water, then each enzyme was added to 1% (w/w) of perilla meal and incubated in a constant temperature water bath (50°C, 180 rpm) for 2 hours. After the reaction was stopped by heat treatment at 90°C for 5 minutes, the reaction was centrifuged (3000 × g, 10 minutes) to obtain a supernatant. As a control group, the supernatant obtained by centrifuging a perilla meal suspension left under the same conditions without adding enzymes was used. The pH of the reaction solution was adjusted to the optimal pH for each enzyme reaction. Viscozyme, Cermix, and Pectinase were adjusted to pH 5.0, Neutrase and control group were adjusted to pH 7.0, Flavorzyme and Protamex were adjusted to pH 7.5, and Alcalase was adjusted to pH 8.0 before the enzymatic reaction was performed. .
들깨박의 효소 처리 조건에 따라 얻어진 효소분해 현탁액과 효소를 첨가하지 않고 단순 추출된 미분해 현탁액을 각각 원심분리하였고, 침전을 제거한 후 얻어진 상등액을 각각 효소분해물과 미처리 대조군으로 정의하였다. The enzymatically digested suspension obtained according to the enzyme treatment conditions of perilla meal and the undigested suspension simply extracted without adding enzymes were centrifuged, respectively, and the supernatant obtained after removing the precipitate was defined as the enzymatically digested product and the untreated control, respectively.
실시예Example 2: 2: 들깨박Perilla seed meal 효소분해의 최적 반응 조건 Optimal reaction conditions for enzymatic digestion
1) 최적 pH 1) Optimum pH
효소는 건조 들깨박 분말 기준으로 1%(w/w) 첨가하였으며, 이때 탄수화물 분해효소와 단백질 분해효소의 비율은 1(0.5%):1(0.5%)로 하였다. 10%(w/w)의 들깨박 현탁액에 효소를 첨가한 시료를 다양한 pH (pH 5.0, 6.0, 7.0, 8.0, 5.0~8.0) 조건에서 항온수조(50℃, 180 rpm)를 사용하여 2시간 반응시키고, 90℃에서 5분간 열처리로 반응을 정지시킨 후 원심분리(3000 × g, 10분)하여 상등액을 얻었다.Enzyme was added at 1% (w/w) based on dried perilla meal powder, and the ratio of carbohydrate-decomposing enzyme and proteolytic enzyme was 1 (0.5%):1 (0.5%). Samples containing enzymes added to a 10% (w/w) perilla meal suspension were incubated for 2 hours using a constant temperature water bath (50°C, 180 rpm) under various pH conditions (pH 5.0, 6.0, 7.0, 8.0, 5.0~8.0). The reaction was stopped by heat treatment at 90°C for 5 minutes and then centrifuged (3000 × g, 10 minutes) to obtain a supernatant.
2) 최적 효소 첨가량2) Optimal enzyme addition amount
효소는 건조 들깨박 분말 기준으로 0.5∼3%(w/w)로 첨가량을 달리하여 들깨박 현탁액에 첨가하였고, 탄수화물 분해효소와 단백질 분해효소의 비율은 1:1로 하였다. 10%(w/w)의 들깨박 현탁액에 각각 첨가량을 달리하여 효소를 첨가하고 시료의 pH를 7.0으로 조정한 후 항온수조(50℃, 180 rpm)에서 2시간 반응시키고, 90℃에서 5분간 열처리로 반응을 정지시킨 후 원심분리(3000 × g, 10분)하여 상등액을 얻었다.Enzymes were added to the perilla meal suspension in varying amounts of 0.5 to 3% (w/w) based on dried perilla meal powder, and the ratio of carbohydrate-decomposing enzyme and proteolytic enzyme was 1:1. Enzymes were added in different amounts to a 10% (w/w) perilla meal suspension, the pH of the sample was adjusted to 7.0, and then reacted in a constant temperature water bath (50°C, 180 rpm) for 2 hours, then at 90°C for 5 minutes. After the reaction was stopped by heat treatment, the supernatant was obtained by centrifugation (3000 × g, 10 minutes).
3) 적정 반응 시간3) Appropriate reaction time
효소는 건조 들깨박 분말 기준으로 2%(w/w) 첨가하였으며, 이때 탄수화물 분해효소와 단백질 분해효소의 비율은 1(1%):1(1%)로 하였다. 10%(w/w)의 들깨박 현탁액에 효소를 첨가한 시료의 pH를 7.0으로 조정한 후 항온수조(50℃, 180 rpm)에서 0~3시간 반응시키고, 90℃에서 5분간 열처리로 반응을 정지시킨 후 원심분리(3000 × g, 10분)하여 상등액을 얻었다.Enzyme was added at 2% (w/w) based on dried perilla meal powder, and the ratio of carbohydrate-decomposing enzyme and proteolytic enzyme was 1 (1%):1 (1%). The pH of the sample in which enzyme was added to a 10% (w/w) perilla meal suspension was adjusted to 7.0, then reacted in a constant temperature water bath (50°C, 180 rpm) for 0 to 3 hours, followed by heat treatment at 90°C for 5 minutes. After stopping, centrifugation (3000 × g, 10 minutes) was performed to obtain the supernatant.
실시예Example 3: 3: 들깨박Perilla seed meal 효소분해물의enzyme decomposition product 성분 분석 Ingredient analysis
1) 수율1) Yield
효소분해물과 대조군을 일정량 실험용 접시에 담은 뒤 60℃에서 충분히 건조하여 얻어진 고형분을 기준으로 수율을 구하였다(수학식 1). A certain amount of the enzyme digest and control group were placed in a laboratory dish and dried sufficiently at 60°C, and the yield was calculated based on the solid content obtained (Equation 1).
2) 총당2) Total per person
들깨박 효소분해물과 대조군의 총당 함량은 총당 정량법(Phenol sulfuric acid method)으로 측정하였다(23). 100배 희석한 시료 0.5 mL에 5% 페놀 0.5 mL와 황산 2.5 mL를 혼합하여 상온에서 20분간 방치한 후 470 nm에서 흡광도를 측정하였다. 표준물질로 D-글루코스를 이용하여 총당 함량을 정량하였다.The total sugar content of perilla meal enzymatic digestion and the control group was measured using the total sugar quantification method (phenol sulfuric acid method) (23). 0.5 mL of 100-fold diluted sample was mixed with 0.5 mL of 5% phenol and 2.5 mL of sulfuric acid, left at room temperature for 20 minutes, and the absorbance was measured at 470 nm. Total sugar content was quantified using D-glucose as a standard substance.
3) 환원당3) reducing sugar
들깨박 효소분해물과 대조군의 환원당 함량은 DNS법으로 측정하였다(24). 5배 희석한 시료 1 mL에 DNS 시약 2 mL와 증류수 7 mL를 혼합하여 90℃ 이상의 수조에서 5분간 열처리하고 얼음물에서 10분간 정치시킨 후 540 nm에서 흡광도를 측정하였다. 표준물질로 D-글루코스를 사용하여 환원당의 함량을 구하였다. The reducing sugar content of perilla meal enzymatic digestion and the control group was measured by the DNS method (24). 1 mL of the 5-fold diluted sample was mixed with 2 mL of DNS reagent and 7 mL of distilled water, heat-treated in a water bath above 90°C for 5 minutes, placed in ice water for 10 minutes, and the absorbance was measured at 540 nm. The content of reducing sugar was determined using D-glucose as a standard substance.
4) 가용성 단백질4) Soluble protein
들깨박 효소분해물과 대조군의 가용성 단백질 함량은 Bradford법으로 분석하였다(25). 5배 희석한 시료 0.1 mL와 브래드포드 시약 3 mL를 혼합하여 상온에서 20분간 방치한 다음 595 nm에서 흡광도를 측정하였다. 표준물질로 BSA를 사용하여 가용화 단백질 함량을 구하였다. The soluble protein content of perilla meal enzymatic digest and control group was analyzed by the Bradford method (25). 0.1 mL of the 5-fold diluted sample was mixed with 3 mL of Bradford reagent, left at room temperature for 20 minutes, and then the absorbance was measured at 595 nm. Solubilized protein content was determined using BSA as a standard material.
5) 폴리페놀 5) Polyphenols
들깨박 효소분해물과 대조군의 폴리페놀 함량은 Folin-Denis 방법으로 분석하였다(26). 20배 희석한 시료 0.2 mL에 증류수 0.8 mL와 Folin-Ciocalteu 시약 1 mL를 넣고 상온에서 1시간 방치한 후 725 nm에서 흡광도를 측정하였다. 표준물질로 갈산 (gallic acid)을 사용하여 폴리페놀 함량을 구하였다.The polyphenol content of perilla meal enzymatic digest and control group was analyzed by the Folin-Denis method (26). To 0.2 mL of the 20-fold diluted sample, 0.8 mL of distilled water and 1 mL of Folin-Ciocalteu reagent were added, left at room temperature for 1 hour, and the absorbance was measured at 725 nm. Polyphenol content was determined using gallic acid as a standard substance.
실시예Example 4: 항산화 활성 4: Antioxidant activity
1) One) ABTSABTS 양이온 라디칼 cation radical 소거능scavenging ability
들깨박 효소분해물과 대조군의 ABTS 양이온 라디칼 소거능은 Re등의 방법으로 측정하였다(27). 7.4 mM ABTS와 2.6 mM 과황산 칼륨 (potassium persulfate)이 들어있는 ABTS 용액을 제조한 후 어두운 곳에서 15시간 정치하여 ABTS 양이온 라디칼을 생성시켰고 414 nm에서 흡광도가 1.5±0.2가 되도록 희석하여 사용하였다. 20배 희석한 시료 0.1 mL와 ABTS 용액 3 mL를 혼합하여 상온에서 1시간 30분 방치한 후 414 nm에서 흡광도를 측정하였다. 양이온 라디칼 소거능은 다음의 수식으로 계산하였다(수학식 2). The ABTS cation radical scavenging ability of perilla meal enzymatic digest and control group was measured by the method of Re et al. (27). An ABTS solution containing 7.4mM ABTS and 2.6mM potassium persulfate was prepared and left in the dark for 15 hours to generate ABTS cation radicals. The solution was diluted to have an absorbance of 1.5 ± 0.2 at 414 nm. 0.1 mL of the 20-fold diluted sample was mixed with 3 mL of the ABTS solution, left at room temperature for 1 hour and 30 minutes, and the absorbance was measured at 414 nm. The cation radical scavenging ability was calculated using the following formula (Equation 2).
2) DPPH 유리라디칼 소거능2) DPPH free radical scavenging ability
들깨박 효소분해물과 대조군의 DPPH 유리라디칼 소거능은 Blois의 방법으로 확인하였다(28). 20배 희석한 시료 0.2 mL에 에탄올 2 mL와 DPPH 용액 0.8 mL를 혼합하여 상온에서 30분 방치한 후 517 nm에서 흡광도를 측정하였다. 유리라디칼 소거능은 양이온 라디칼 소거능과 같은 수식을 이용하여 계산하였다(수학식 2).The DPPH free radical scavenging ability of perilla meal enzymatic digest and control group was confirmed by Blois' method (28). 0.2 mL of the 20-fold diluted sample was mixed with 2 mL of ethanol and 0.8 mL of DPPH solution, left at room temperature for 30 minutes, and then the absorbance was measured at 517 nm. The free radical scavenging ability was calculated using the same formula as the cation radical scavenging ability (Equation 2).
실시예 5: 들깨박 효소분해물을 이용한 유산균 배양Example 5: Lactic acid bacteria culture using enzyme decomposition product of perilla meal
1) 배양방법1) Culture method
사면 배지에 배양한 Leuconostoc mesenteroides 310-12 균주를 Lactobacilli MRS broth에 접종하고 36 ℃에서 19시간 동안 진탕 배양했다. 배양액 30 mL를 원심분리(3000 × g, 30분)한 후 균체는 회수하여 멸균 식염수로 2회 세척하고 균체를 배양액과 동일 부피의 멸균 식염수에서 현탁하였다. 들깨박 효소분해물에 균주 현탁액 5%(v/v)를 접종하고 36 ℃에서 180 rpm으로 15시간 동안 진탕 배양하였다. 배양하면서 3시간 간격으로 샘플을 채취하여 생육, 적정산도, 환원당, pH 측정을 통해 유산균 생장을 확인하였다. 대조군으로는 들깨박 미분해물을 사용하였다. Leuconostoc cultured on slope medium. mesenteroides 310-12 strain was inoculated into Lactobacilli MRS broth and cultured with shaking at 36°C for 19 hours. After centrifuging 30 mL of the culture medium (3000 × g, 30 minutes), the cells were recovered, washed twice with sterile saline solution, and the cells were suspended in the same volume of sterile saline solution as the culture solution. Perilla meal enzymatic digestion was inoculated with 5% (v/v) of the strain suspension and cultured at 36°C with shaking at 180 rpm for 15 hours. During cultivation, samples were collected at 3-hour intervals to confirm the growth of lactic acid bacteria by measuring growth, titratable acidity, reducing sugar, and pH. As a control group, undecomposed perilla seed meal was used.
2) 유산균 생장, 적정 산도, 환원당, pH 측정2) Measurement of lactic acid bacteria growth, titratable acidity, reducing sugar, and pH
Leuconostoc mesenteroides 310-12 균주의 생육 정도는 시간별로 채취한 배양액을 생리 식염수로 10배 희석하여 610 nm에서 흡광도를 측정함으로써 확인하였다. 배양액의 pH는 pH 미터(model 700, Eutech instruments, Singapore)로 직접 측정하였다. Leuconostoc The growth level of the mesenteroides 310-12 strain was confirmed by diluting the culture medium collected over time 10 times with physiological saline and measuring the absorbance at 610 nm. The pH of the culture medium was directly measured with a pH meter (model 700, Eutech instruments, Singapore).
적정 산도는 시간별로 채취한 배양액 1 mL와 멸균 증류수 9 mL를 잘 혼합한 후 0.01 N NaOH로 적정하고 NaOH 소모량은 젖산으로 환산하였다(29). 지시약은 페놀프탈레인을 사용하였고, 적정 산도 계산은 수학식 3과 같다.The titratable acidity was titrated with 0.01 N NaOH after mixing 1 mL of the culture medium collected at each hour with 9 mL of sterilized distilled water, and the NaOH consumption was converted to lactic acid (29). Phenolphthalein was used as an indicator, and the titratable acidity was calculated as shown in Equation 3.
환원당 정량은 시간별로 채취한 배양액을 10배 희석한 후 1 mL를 취해 증류수 7 mL과 DNS 시약 2 mL를 혼합하여 실시예 3의 3)의 환원당 정량법으로 확인하였다.Quantification of reducing sugar was confirmed by the reducing sugar quantification method in Example 3, 3) by diluting the culture medium collected over time by 10 times, taking 1 mL, mixing 7 mL of distilled water and 2 mL of DNS reagent.
결과 1: Result 1: 들깨박Perilla seed meal 효소분해를 위한 효소 선정 결과 Enzyme selection results for enzymatic digestion
건조 중량 기준으로 10%(w/w)의 들깨박을 증류수에 현탁시킨 후 각각의 효소를 들깨박의 1%(w/w)로 첨가하여 반응시킨 후 얻은 상등액의 환원당과 가용성 단백질 함량을 비교한 결과를 도 1 및 도 2에 나타내었다. 대조군은 효소를 첨가하지 않고 단순 추출된 시료로 하였다. 10% (w/w) of perilla meal based on dry weight was suspended in distilled water, then each enzyme was added at 1% (w/w) of perilla meal and reacted, and the reducing sugar and soluble protein contents of the supernatant obtained were compared. The results are shown in Figures 1 and 2. The control group was a sample simply extracted without adding enzymes.
환원당 정량 결과, 세레믹스 > 알칼라아제 > 비스코자임 > 프로타멕스 > 플라보르자임 > 뉴트라제 > Pectinase > Control 순으로 시료의 환원당 함량이 높게 나타났다(도 1). 탄수화물 분해효소인 세레믹스 처리시 환원당 함량이 1.39±0.00 mg/mL로 가장 높은 값을 보였다. 특이하게도 단백질 분해효소인 알칼라아제를 처리하였을 때 환원당 함량이 두 번째로 높게 나타났고, 또한 다른 단백질 분해효소인 프로타멕스와 플라보르자임 처리시에도 환원당 함량이 다소 높게 나타났다. 이는 식물 세포벽에 존재하는 peptidoglycan의 단백질 부분을 단백질 분해효소가 가수 분해하여 다당류와 단백질의 용해도를 증가시키는데 기인한 것으로 여겨진다(30).As a result of reducing sugar quantification, the reducing sugar content of the samples was found to be high in the following order: Ceremix > Alcalase > Viscozyme > Protamex > Flavorzyme > Neutrase > Pectinase > Control (Figure 1). When treated with Ceremix, a carbohydrate-decomposing enzyme, the reducing sugar content showed the highest value of 1.39 ± 0.00 mg/mL. Uniquely, when treated with Alcalase, a proteolytic enzyme, the reducing sugar content was the second highest, and also when treated with Protamex and Flavorzyme, which are other proteolytic enzymes, the reducing sugar content was also slightly higher. This is believed to be due to proteolytic enzymes hydrolyzing the protein portion of peptidoglycan present in plant cell walls, thereby increasing the solubility of polysaccharides and proteins (30).
단백질 추출시 산과 알칼리를 사용하면 독성 물질이 발생하고 중화 과정이 필요하기 때문에 단백질 분해효소를 사용하는 것이 친환경적이며 안전하다(31,32). 가용화 단백질 정량 결과, 알칼라아제 > 프로타멕스 > 뉴트라제 > Control > 세레믹스 > 비스코자임 > 플라보르자임 순으로 시료의 가용성 단백질 함량이 높게 나타났다(도 2). 단백질 분해효소인 알칼라아제 처리시 가용성 단백질 함량이 1.14±0.15 mg/mL로 가장 높은 값을 보였다.Since the use of acids and alkalis during protein extraction generates toxic substances and requires a neutralization process, using proteolytic enzymes is environmentally friendly and safe (31,32). As a result of solubilized protein quantification, the soluble protein content of the sample was found to be high in the following order: Alcalase > Protamex > Neutrase > Control > Ceremic > Viscozyme > Flavorzyme (Figure 2). When treated with Alcalase, a proteolytic enzyme, the soluble protein content showed the highest value of 1.14±0.15 mg/mL.
효소분해물의 환원당과 가용성 단백질 분석 결과, 단백질 분해효소인 알칼라아제의 단독 사용으로도 상대적으로 높은 들깨박의 가수분해를 기대할 수 있으나, 본 발명에서는 들깨박 효소분해의 주된 목적이 미생물의 배양배지로서의 가능성을 확인하는 것이기 때문에 미생물의 탄소원으로 사용되는 환원당이 매우 중요하게 고려되었고, 따라서 탄수화물 분해효소인 세레믹스를 같이 사용하는 것이 바람직하다고 보았다. 또한, 본 연구에서 들깨박 효소분해물로 유산균을 배양할 때 탄소원인 환원당이 많이 필요하기 때문에 들깨박 효소분해물 제조에 사용할 효소로 단백질 분해효소 중에서는 알칼라아제를, 탄수화물 분해효소 중에서는 세레믹스를 선정하였다. As a result of analysis of reducing sugars and soluble proteins of the enzymatic digestion, relatively high hydrolysis of perilla meal can be expected even with the sole use of alcalase, a proteolytic enzyme. However, in the present invention, the main purpose of enzymatic digestion of perilla meal is to provide a culture medium for microorganisms. Because the purpose was to confirm the possibility, the reducing sugar used as a carbon source for microorganisms was considered very important, and therefore it was considered desirable to use Ceremics, a carbohydrate decomposing enzyme, together. In addition, in this study, when cultivating lactic acid bacteria with enzyme decomposition product of perilla meal, a lot of reducing sugars, which are carbon sources, were required, so the enzymes used for producing enzyme decomposition product from perilla meal were alcalase among proteolytic enzymes and Ceremix among enzymes against enzyme decomposition of carbohydrates. selected.
결과 2: Result 2: 들깨박Perilla seed meal 효소분해의 최적 반응 조건 Optimal reaction conditions for enzymatic digestion
1) 최적 pH 1) Optimum pH
들깨박 효소분해의 최적 반응 조건을 결정하기 위한 실험은 표 1과 같이 실시되었다. 효소는 알칼라아제와 세레믹스를 동시에 사용하였고, 반응 시간, 온도, 효소 첨가량을 고정한 상태에서 pH에 따른 효소분해 효율을 평가하였다. 최적 pH가 결정된 다음에는 pH, 반응 시간, 온도를 고정한 상태에서 알칼라아제와 세레믹스의 첨가량에 따른 효소분해 효율을 평가하였고, 효소의 첨가량을 결정한 다음에는 pH, 온도, 효소첨가량을 고정한 상태에서 효소반응 시간에 따른 효소분해 효율을 평가하였다. 이러한 결과를 종합하여 들깨박 효소분해의 최적 반응 조건을 결정하였다. An experiment to determine the optimal reaction conditions for enzymatic degradation of perilla meal was conducted as shown in Table 1. Alcalase and Ceremix were used simultaneously as enzymes, and the enzyme decomposition efficiency was evaluated according to pH while fixing the reaction time, temperature, and enzyme addition amount. After the optimal pH was determined, the enzymatic decomposition efficiency was evaluated according to the amount of alcalase and Ceremix added while fixing the pH, reaction time, and temperature. After determining the amount of enzyme added, the pH, temperature, and enzyme addition amount were fixed. The enzyme decomposition efficiency was evaluated according to the enzyme reaction time. By combining these results, the optimal reaction conditions for enzymatic degradation of perilla seed meal were determined.
효소는 단백질이기 때문에 pH에 따라 입체 구조와 활성 부위의 정전기적 성질이 달라지므로 촉매 작용을 위한 최적의 pH가 존재한다(34). 들깨박에 알칼라아제와 세레믹스의 두 효소를 다양한 pH에서 처리하여 효소분해물을 얻었고, 환원당과 가용성 단백질을 분석한 결과를 도 3에 나타내었다. 세레믹스의 최적 pH는 5.0이고, 알칼라아제의 최적 pH는 8.0이기 때문에 두 효소를 동시에 사용하는 경우의 적정 pH를 결정하는 것은 쉽지 않다. pH 조건 중 pH 5.0~8.0의 경우, 세레믹스와 알칼라아제의 최적 pH가 서로 다르기 때문에 두 효소를 처리하고 먼저 pH 5에서 1시간 반응시킨 후 pH 8로 조정하여 1시간 더 반응시키는 방법을 택하였다(35). 대조군은 효소 처리하지 않은 들깨박 현탁액을 pH 7.0로 조절하여 단순 추출한 것이다. 효소반응액의 pH에 따른 환원당과 가용성 단백질 분석 결과는 도 3에 나타내었다. 상대적인 환원당 함량은 pH 7의 조건에서 가장 높게 나타났고, pH 6과 8은 비슷한 수준으로 그 다음 높았다(도 3). 상대적인 가용성 단백질 함량은 pH 8에서 가장 높았고, pH 7이 두 번째로 높게 나타났다(도 3). 기대와는 달리 pH를 5.0에서 8.0으로 순차적으로 처리한 시험군에서 환원당과 가용성 단백질 함량이 가장 낮게 나타났다. 이는 두 효소를 넣고 pH 5에서 먼저 반응을 시키는 동안 알칼라아제의 변성이 발생하였고 세레믹스 또한 1시간 반응으로는 충분히 탄수화물을 분해시키지 못한 것으로 여겨지며, 이후 pH 8로 올렸을 때 세레믹스는 불활성화되고 알칼라아제는 기능을 회복하지 못한데 기인한 것으로 판단된다. 이상의 결과로 보아 들깨박 효소분해에 있어 알칼라아제는 주된 역할을, 세레믹스는 탄수화물의 분해를 조금 더 향상시키는 보조적인 역할을 한다는 것을 알 수 있었다. 들깨박 효소분해물을 미생물 배양에 사용할 경우, 탄소원으로 이용되는 환원당이 중요하기 때문에 pH 7과 8 중에 환원당이 많이 얻어지는 pH 7을 효소반응의 최적 pH로 결정하였다.Since enzymes are proteins, the three-dimensional structure and electrostatic properties of the active site vary depending on pH, so there is an optimal pH for catalytic action (34). Perilla meal was treated with two enzymes, Alcalase and Ceremix, at various pH to obtain enzymatic digests, and the results of analyzing reducing sugars and soluble proteins are shown in Figure 3. Since the optimal pH of Ceremix is 5.0 and the optimal pH of Alcalase is 8.0, it is not easy to determine the optimal pH when using both enzymes simultaneously. In the case of pH 5.0~8.0, the optimal pH of Ceremics and Alcalase are different, so the method of treating the two enzymes and reacting first at pH 5 for 1 hour, then adjusting to pH 8 and reacting for another hour was chosen. (35). The control group was a simple extraction of perilla seed meal suspension without enzyme treatment by adjusting the pH to 7.0. The results of reducing sugar and soluble protein analysis according to the pH of the enzyme reaction solution are shown in Figure 3. The relative reducing sugar content was highest at pH 7, and pH 6 and 8 were the next highest at similar levels (Figure 3). The relative soluble protein content was highest at pH 8, and the second highest at pH 7 (Figure 3). Contrary to expectations, the test group treated sequentially with pH from 5.0 to 8.0 showed the lowest reducing sugar and soluble protein contents. This is because denaturation of alkalase occurred while the two enzymes were added and reacted at pH 5, and Cermix was also believed to not have sufficiently decomposed carbohydrates in the 1-hour reaction. When the pH was raised to 8, Cermix was inactivated. It is believed that this is due to the failure of alcalase to recover its function. Based on the above results, it was found that alcalase plays a main role in the enzymatic decomposition of perilla meal, and Ceremics plays an auxiliary role in slightly improving the decomposition of carbohydrates. When using perilla meal enzyme decomposition product for microbial culture, reducing sugar used as a carbon source is important, so pH 7, which yields the most reducing sugar among pH 7 and 8, was determined as the optimal pH for the enzyme reaction.
2) 최적 효소 첨가량2) Optimal enzyme addition amount
효소 첨가량을 들깨박 대비 0%, 0.5%, 1%, 2%, 3%(w/w)로 달리하여 들깨박 현탁액에 첨가하고 pH 7.0으로 조정하여 효소분해 반응을 수행하였다. 효소반응액의 효소 첨가량에 따른 환원당과 가용성 단백질 분석 결과는 도 4와 5에 나타내었다. 들깨박 효소분해 반응에서 2%(w/w)의 효소 첨가량까지는 환원당과 가용성 단백질이 효소 첨가량에 비례하여 증가하였다. 환원당 함량은 알칼라아제와 세레믹스를 1:1로 혼합한 효소를 건조 들깨박 대비 2%(w/w) 첨가하여 효소반응을 수행하였을 경우 가장 높게 나타났고, 그 이상 첨가하였을 때는 거의 변화가 없었다(도 4). 가용성 단백질 함량도 효소를 건조 들깨박 대비 2%(w/w) 첨가하였을 경우 가장 높게 나타났고, 그 이상 첨가하였을 때는 거의 변화가 없었다(도 5). The amount of enzyme added was changed to 0%, 0.5%, 1%, 2%, and 3% (w/w) compared to perilla meal, and the enzyme was added to the perilla meal suspension and adjusted to pH 7.0 to perform an enzyme decomposition reaction. The results of reducing sugar and soluble protein analysis according to the amount of enzyme added to the enzyme reaction solution are shown in Figures 4 and 5. In the enzyme decomposition reaction of perilla meal, reducing sugars and soluble proteins increased in proportion to the amount of enzyme added up to 2% (w/w). The reducing sugar content was highest when the enzyme reaction was performed by adding 2% (w/w) of enzyme mixed with Alcalase and Ceremix in a 1:1 ratio compared to dried perilla meal, and when more was added, there was almost no change. There was none (Figure 4). The soluble protein content was also highest when 2% (w/w) of enzyme was added compared to dried perilla meal, and there was almost no change when more was added (Figure 5).
환원당은 0.5%(w/w)의 효소 첨가에서 2.88±0.00 mg/mL로, 1%(w/w)의 효소 첨가에서는 4.11±0.02 mg/mL로 급격히 증가하였으나 그 이상에서는 상승률이 크게 둔화되는 경향을 보였다. 2%(w/w)의 효소 첨가 시 4.33±0.04 mg/mL로 환원당을 가장 많이 얻을 수 있었고 그 이상에서는 차이가 나지 않았다. 가용성 단백질은 0.5%(w/w)의 효소 첨가에서 1.16±0.15 mg/mL로 급격히 증가하였으나 그 이상에서는 상승률이 크게 둔화되는 경향을 보였다. 2%(w/w)의 효소 첨가 시 1.36±0.05 mg/mL로 가용성 단백질을 가장 많이 얻을 수 있었고 그 이상에서는 차이가 나지 않았다. 따라서 효소반응액의 환원당과 가용성 단백질 함량으로 보아 2%(w/w)의 효소 첨가량이 가장 적절한 것으로 여겨진다. 이는 효소분해에 사용된 단백질 분해효소의 종류가 달라도 2%첨가했을 때 수율이 가장 높았다는 보고(36)와도 일치하였고, 또한 효소분해 반응에서효소 농도를 높이면 분해량이 증가하지만, 일정 농도 이후부터는 분해량이 증가하지 않는다는 보고(37)와도 일치하였다. 본 연구에서도 알칼라아제와 세레믹스를 1:1로 혼합한 효소의 첨가량을 건조 들깨박 대비 2%(w/w)로 결정하였다. Reducing sugars increased rapidly to 2.88 ± 0.00 mg/mL with the addition of 0.5% (w/w) enzyme and to 4.11 ± 0.02 mg/mL with the addition of 1% (w/w) enzyme, but the rate of increase slowed significantly beyond that. showed a trend. When 2% (w/w) of enzyme was added, the highest amount of reducing sugar was obtained at 4.33 ± 0.04 mg/mL, and there was no difference beyond that. Soluble protein increased rapidly to 1.16 ± 0.15 mg/mL at the addition of 0.5% (w/w) enzyme, but the rate of increase tended to slow down significantly beyond that. When 2% (w/w) of enzyme was added, the highest amount of soluble protein was obtained at 1.36 ± 0.05 mg/mL, and there was no difference beyond that. Therefore, considering the reducing sugar and soluble protein content of the enzyme reaction solution, an enzyme addition amount of 2% (w/w) is considered most appropriate. This was consistent with the report (36) that the yield was highest when 2% was added even if the type of proteolytic enzyme used for enzymatic digestion was different. In addition, as the enzyme concentration in the enzymatic digestion reaction was increased, the amount of decomposition increased, but after a certain concentration, decomposition occurred. This was consistent with the report (37) that the amount did not increase. In this study, the amount of enzyme added in a 1:1 ratio of Alcalase and Ceremix was determined to be 2% (w/w) compared to dried perilla meal.
3) 적정 효소반응 시간 3) Appropriate enzyme reaction time
효소는 촉매 작용에 있어 반응 시간이 길수록 생성물을 많이 얻을 수 있지만 경제성을 고려하여 생성물의 증가가 정체되는 시점을 적정 반응시간으로 결정하는 것이 바람직하다(38). 최적 반응 시간을 결정하기 위해, 효소 첨가량을 들깨박 대비 2%(w/w) 들깨박 현탁액에 첨가하고 pH 7.0으로 조정하여 0, 0.5, 1, 2, 3시간 효소분해 반응을 수행하였다. 효소반응의 시간에 따른 환원당과 가용성 단백질 분석 결과는 도 6과 7에 나타내었다. 들깨박 효소분해 반응에서 환원당은 반응 3시간까지도 반응시간에 비례하여 지속적으로 증가하였으나(도 6), 가용성 단백질은 효소반응 2시간까지 시간에 비례하여 지속적으로 증가하였으나 그 이상의 시간에서는 오히려 감소하는 것으로 나타났는데 이는 실험 오차로 보이고 2시간 이후에는 단백질 분해반응이 정체되는 것으로 여겨진다(도 7).In the case of enzyme catalysis, the longer the reaction time, the more products can be obtained. However, considering economics, it is desirable to determine the appropriate reaction time at the point when the increase in products plateaus (38). To determine the optimal reaction time, an enzyme addition amount of 2% (w/w) compared to perilla meal was added to the perilla meal suspension, the pH was adjusted to 7.0, and the enzymatic digestion reaction was performed for 0, 0.5, 1, 2, and 3 hours. The results of reducing sugar and soluble protein analysis according to the enzyme reaction time are shown in Figures 6 and 7. In the perilla meal enzymatic decomposition reaction, reducing sugars continued to increase in proportion to the reaction time up to 3 hours of reaction (Figure 6), but soluble proteins continued to increase in proportion to time until 2 hours of enzymatic reaction, but decreased at longer times. This appears to be an experimental error, and the protein degradation reaction is believed to be stagnant after 2 hours (Figure 7).
환원당은 효소반응 3시간에 6.72±0.45 mg/mL로 시간에 따라 지속적으로 증가하였으나 그 증가의 폭은 시간이 갈수록 조금씩 둔화되는 양상을 보여주었다. 가용성 단백질은 효소반응 2시간까지 2.44±0.07 mg/mL로 시간에 비례하여 증가하는 경향을 보였으나 그 이상에서는 오히려 소폭 감소 즉 정체되는 양상을 보여주었다. 본 실험에서 환원당으로 보면 효소반응 시간을 3시간으로 하는 것이 적절하고, 가용성 단백질로 보면 효소반응 시간을 2시간으로 정하는 것이 적절한 것으로 나타났다. 반응 공정에서 반응 시간은 경제성과 밀접한 상관관계가 있기 때문에 이러한 측면을 고려하여 알칼라아제와 세레믹스로 들깨박을 효소분해시 적정 반응 시간은 2시간으로 결정하였다. Reducing sugar continued to increase over time to 6.72±0.45 mg/mL at 3 hours of enzyme reaction, but the extent of the increase gradually slowed down over time. Soluble protein tended to increase proportionally with time to 2.44±0.07 mg/mL up to 2 hours after the enzyme reaction, but showed a slight decrease, or stagnation, beyond that. In this experiment, it was found that for reducing sugars, it was appropriate to set the enzyme reaction time to 3 hours, and for soluble proteins, it was found to be appropriate to set the enzyme reaction time to 2 hours. Since the reaction time in the reaction process is closely correlated with economic efficiency, taking this aspect into consideration, the appropriate reaction time for enzymatic decomposition of perilla meal with alkalase and Ceremix was determined to be 2 hours.
본 발명의 들깨박 효소분해의 최적 반응 조건을 결정하였고, 그 결과를 표 2에 요약하였다. 10%(w/w)의 들깨박 현탁액에 알칼라아제와 세레믹스 효소를 건조 들깨박 분말 기준 2%(w/w) 첨가하여 pH를 7.0으로 조정한 후 항온수조(50℃, 180 rpm)에서 2시간 반응시키고, 90℃에서 5분간 열처리로 반응을 정지시킨 후 원심분리(3000 × g, 10분)하여 상등액을 얻는 효소분해 최적 조건을 확립하였다. 확립된 효소반응 조건으로 들깨박을 대량 처리하여 효소분해물을 얻었고, 그 효소분해물의 성분 분석, 항산화 생리활성 측정 및 유산균 배양 등의 실험을 수행하였다.The optimal reaction conditions for enzymatic decomposition of perilla meal of the present invention were determined, and the results are summarized in Table 2. Add 2% (w/w) of alcalase and Cermix enzyme based on dried perilla meal powder to 10% (w/w) perilla seed meal suspension to adjust the pH to 7.0, then place in a constant temperature water bath (50°C, 180 rpm). The reaction was incubated for 2 hours, the reaction was stopped by heat treatment at 90°C for 5 minutes, and then centrifuged (3000 × g, 10 minutes) to establish the optimal conditions for enzymatic digestion to obtain the supernatant. Enzyme decomposition products were obtained by processing large quantities of perilla meal under established enzyme reaction conditions, and experiments such as analysis of the components of the enzymatic decomposition products, measurement of antioxidant physiological activity, and cultivation of lactic acid bacteria were performed.
결과 3: Result 3: 들깨박Perilla seed meal 효소분해물의enzyme decomposition product 성분 분석 Ingredient analysis
들깨박 효소분해물의 수율, 총당, 환원당, 가용성 단백질, 폴리페놀 함량을 효소를 처리하지 않은 대조군과 비교한 결과를 표 3에 나타내었다. 들깨박 효소분해물의 수율은 39.57±1.55%로 대조군의 12.48±2.08%에 비해 3배 이상 높게 나타나 효소분해로 불용성 성분을 효과적으로 가용화할 수 있음을 보여주었다. 추출 수율의 증가는 바이오매스 소재로부터 유용성분을 다량 회수할 수 있음을 의미하기 때문에 들깨박 효소분해물의 높은 수율은 들깨박의 유용성분의 활용도 증가로 이어질 것으로 사료된다.Table 3 shows the results of comparing the yield, total sugar, reducing sugar, soluble protein, and polyphenol content of perilla meal enzymatic digestion with the control group that was not treated with enzyme. The yield of perilla meal enzymatic digestion was 39.57 ± 1.55%, which was more than three times higher than the control group's 12.48 ± 2.08%, showing that insoluble components can be effectively solubilized through enzymatic digestion. Because an increase in extraction yield means that a large amount of useful components can be recovered from biomass materials, it is believed that a high yield of perilla meal enzyme decomposition product will lead to an increase in the utilization of useful components of perilla meal.
총당은 환원당과 비환원당을 모두 포함하는 당을 나타낸다. 들깨박 효소분해물의 총당 함량은 9.80±0.33 mg/mL로 대조군의 8.27±0.06 mg/mL에 비해 18.5% 증가하는 것으로 나타나 효소분해로 불용성 탄수화물의 가용화가 이루어짐을 알 수 있었다. 이는 효소분해에 의해 들깨박의 불용성 다당류의 용출이 증가한 것으로 판단된다.Total sugar refers to a sugar that includes both reducing sugars and non-reducing sugars. The total sugar content of the perilla meal enzymatically digested product was 9.80±0.33 mg/mL, an 18.5% increase compared to the control group's 8.27±0.06 mg/mL, indicating that insoluble carbohydrates were solubilized through enzymatic digestion. This is believed to be due to increased elution of insoluble polysaccharides from perilla seed meal due to enzymatic decomposition.
환원당이란 헤미아세탈을 형성할 수 있는 알데하이드기 또는 케톤기를 보유하고 있어서 환원력을 가진 당이다(46). 들깨박 효소분해물의 환원당 함량은 6.87±0.25 mg/mL로 대조군의 4.36±0.09 mg/mL에 비해 56.5% 증가하는 것으로 나타나 효소분해에 의해 불용성 탄수화물의 가용화가 효과적으로 이루어짐을 알 수 있었다. 환원당은 미생물 배양시 탄소원으로 매우 중요하기 때문에 환원당 함량이 높다는 것은 미생물 배양 배지로서의 활용 가능성이 높음을 의미한다. 따라서 탄소원인 환원당으로 보아 들깨박 효소분해물은 미생물 배양 배지로서 충분히 활용 가능할 것으로 사료된다.A reducing sugar is a sugar that has reducing power because it possesses an aldehyde group or ketone group that can form a hemiacetal (46). The reducing sugar content of the enzymatic digestion of perilla meal was 6.87 ± 0.25 mg/mL, a 56.5% increase compared to 4.36 ± 0.09 mg/mL in the control group, indicating that insoluble carbohydrates were effectively solubilized by enzymatic digestion. Because reducing sugars are very important as a carbon source when cultivating microorganisms, a high reducing sugar content means that they have a high possibility of being used as a microbial culture medium. Therefore, considering the reducing sugar as a carbon source, it is believed that the enzyme decomposition product of perilla meal can be sufficiently utilized as a microbial culture medium.
들깨박 효소분해물의 가용성 단백질 함량은 3.22±0.15 mg/mL로 대조군의 2.67±0.02 mg/mL에 비해 20.6% 증가하는 것으로 나타나 효소분해에 의해 불용성 단백질의 가용화가 효과적으로 이루어짐을 알 수 있었다. 가용성 단백질, 펩타이드 및 아미노산은 미생물 배양 시에 질소원으로 이용되기 때문에 이들의 함량이 높다는 것은 미생물 배양 배지로서의 활용 가능성이 높음을 의미한다. 따라서 질소원인 가용성 단백질로 보아 들깨박 효소분해물은 미생물 배양 배지로서 충분히 활용 가능할 것으로 사료된다.The soluble protein content of the enzyme digested product of perilla meal was 3.22 ± 0.15 mg/mL, a 20.6% increase compared to 2.67 ± 0.02 mg/mL in the control group, indicating that insoluble proteins were effectively solubilized by enzyme digestion. Since soluble proteins, peptides, and amino acids are used as nitrogen sources when cultivating microorganisms, their high content means that they have a high possibility of being used as a microbial culture medium. Therefore, considering the soluble protein, which is a nitrogen source, it is believed that the enzyme decomposition product of perilla meal can be sufficiently utilized as a microbial culture medium.
식물에 들어있는 폴리페놀 화합물은 주로 항산화 활성을 갖는 것으로 알려져 있다(49). 폴리페놀 화합물은 천연 항산화제로 사용되고 있고 체내에서 건강 유지 및 항암, 콜레스테롤 저하 등 질병 예방에 효과적이다(50,51). 들깨박 효소분해물의 폴리페놀 함량은 1.64±0.06 mg/mL로 대조군의 0.69±0.01 mg/mL에 비해 2배 이상 높게 나타났고, 이는 효소분해로 들깨박의 식물 조직이 파괴되어 조직 내부에 있던 폴리페놀 화합물이 효과적으로 용출되었음을 의미한다. 탈지 들깨박을 에탄올로 추출하여 들깨의 항산화력과 비교한 결과, 들깨에 비해 탈지 들깨박의 항산화력은 약 1/20 정도로 나타난 것(53)으로 보아 들깨박을 단순히 용매 추출하여 항산화 물질을 회수하는 것에 비해 들깨박을 효소분해하는 것이 더 많은 양의 폴리페놀 화합물을 얻을 수 있다고 사료된다. 들깨박 효소분해물의 폴리페놀 함량이 대조군과 비교하여 현저히 증가한 것을 고려하면 들깨박 효소분해물은 우수한 항산화 활성을 나타낼 것으로 기대할 수 있다. Polyphenol compounds contained in plants are mainly known to have antioxidant activity (49). Polyphenol compounds are used as natural antioxidants and are effective in maintaining health in the body and preventing diseases such as anti-cancer and lowering cholesterol (50,51). The polyphenol content of the perilla meal enzymatically digested product was 1.64±0.06 mg/mL, which was more than twice as high as the control group's 0.69±0.01 mg/mL. This was due to the destruction of the plant tissue of the perilla meal through enzyme decomposition, resulting in polyphenol content inside the tissue. This means that the phenol compound was effectively eluted. As a result of extracting defatted perilla seed meal with ethanol and comparing it to the antioxidant power of perilla seeds, the antioxidant power of defatted perilla seed meal was found to be about 1/20 of that of perilla seeds (53). Therefore, it was found that antioxidant substances were recovered by simply solvent extracting perilla seed meal. It is believed that a greater amount of polyphenol compounds can be obtained by enzymatically decomposing perilla seed meal compared to hydrolysis. Considering that the polyphenol content of the perilla meal enzyme hydrolyzate was significantly increased compared to the control group, the perilla meal enzyme hydrolyzate can be expected to exhibit excellent antioxidant activity.
결과 4: Result 4: 들깨박Perilla seed meal 효소분해물의enzyme decomposition product 항산화 활성 antioxidant activity
ABTS 양이온 라디칼을 이용하여 들깨박 효소분해물의 양이온 라디칼 소거능을 확인하였다. ABTS와 과황산칼륨을 혼합하여 어두운 곳에 두면 ABTS 양이온 라디칼이 생성되고, 항산화 물질에 의해 양이온 라디칼이 소거되면서 청록색이 탈색되어 흡광도의 변화로 항산화 능력을 계산할 수 있다(27). 들깨박 효소분해물과 대조군의 들깨박 농도에 따른 ABTS 양이온 라디칼 소거능을 도 8에 나타냈다. 들깨박의 농도가 13.9 mg/mL일 때 들깨박 효소분해물의 양이온 라디칼 소거능은 67.39±0.70%였고, 대조군의 양이온 라디칼 소거능은 48.37±0.27%로 나타나 20% 정도의 차이를 보였다. 들깨박의 농도에 비례하여 ABTS 양이온 라디칼 소거능은 증가했지만 효소분해물의 양이온 라디칼 소거능이 훨씬 우수하였다. 이는 들깨박의 효소분해로 인해 폴리페놀 화합물이 다량 용출된 것에 기인한 것으로 여겨진다. 이는 폴리페놀 함량과 항산화 활성이 비례한다는 기존 보고(54.55)와 일치하는 결과이다.The cation radical scavenging ability of the enzyme decomposition product of perilla seed meal was confirmed using ABTS cation radicals. When ABTS and potassium persulfate are mixed and placed in the dark, ABTS cation radicals are generated, and as the cation radicals are eliminated by antioxidants, the blue-green color is discolored, and the antioxidant capacity can be calculated based on the change in absorbance (27). The ABTS cation radical scavenging ability according to the concentration of perilla meal enzyme decomposition product and control group is shown in Figure 8. When the concentration of perilla meal was 13.9 mg/mL, the cation radical scavenging ability of the enzyme decomposition product of perilla meal was 67.39 ± 0.70%, and the cation radical scavenging ability of the control group was 48.37 ± 0.27%, showing a difference of about 20%. The ABTS cation radical scavenging ability increased in proportion to the concentration of perilla seed meal, but the cation radical scavenging ability of the enzyme decomposition product was much superior. This is believed to be due to the leaching of a large amount of polyphenol compounds due to enzymatic decomposition of perilla meal. This result is consistent with the previous report (54.55) that polyphenol content and antioxidant activity are proportional.
유리라디칼은 강한 반응성을 갖고 있어 체내 단백질, 핵산, 지질 등을 손상시키는 원인이 된다(1). 본 발명에서는 DPPH 유리라디칼을 이용하여 들깨박 효소분해물의 유리라디칼 소거능을 확인하였다. 안정한 유리라디칼인 DPPH는 보라색 물질로 라디칼이 소거되면 노란색으로 바뀌게 되어 흡광도의 변화로 항산화 능력을 계산할 수 있다(57). 들깨박 효소분해물과 대조군의 들깨박 농도에 따른 DPPH 유리라디칼 소거능을 도 9에 나타냈다. 들깨박의 농도가 11.1 mg/mL일 때 들깨박 효소분해물의 유리라디칼 소거능은 86.52±0.48%였고, 대조군의 유리라디칼 소거능은 39.93±1.02%로 나타나 2배 이상의 차이를 보였다. 들깨박의 농도에 비례하여 DPPH 유리라디칼 소거능은 증가했지만 효소분해물의 유리라디칼 소거능이 2배 이상 우수하였다. 이 역시 들깨박의 효소분해로 인해 폴리페놀 화합물이 다량 용출된 것에 기인한 것으로 여겨진다. 이상의 결과로 보아 들깨박 효소분해물은 효소 처리하지 않은 대조군에 비해 양이온 라디칼 및 유리라디칼에 대한 소거활성이 크게 증가하였고 특히 유리라디칼 소거 능력이 매우 뛰어난 것으로 나타났다. 들깨박 효소분해물은 항산화 생리활성 소재로서 활용 가능성이 높다고 여겨진다. Free radicals have strong reactivity and cause damage to proteins, nucleic acids, and lipids in the body (1). In the present invention, the free radical scavenging ability of perilla seed enzyme decomposition product was confirmed using DPPH free radicals. DPPH, a stable free radical, is a purple substance that changes to yellow when the radical is eliminated, so the antioxidant ability can be calculated based on the change in absorbance (57). The DPPH free radical scavenging ability according to the concentration of perilla meal enzyme decomposition product and control group is shown in Figure 9. When the concentration of perilla meal was 11.1 mg/mL, the free radical scavenging ability of the enzyme decomposition product of perilla meal was 86.52 ± 0.48%, and the free radical scavenging ability of the control group was 39.93 ± 1.02%, showing a difference of more than two times. The DPPH free radical scavenging ability increased in proportion to the concentration of perilla meal, but the free radical scavenging ability of the enzyme decomposition product was more than two times better. This is also believed to be due to the leaching of a large amount of polyphenol compounds due to enzymatic decomposition of perilla meal. Based on the above results, the scavenging activity for cation radicals and free radicals of the perilla seed enzyme decomposition product was significantly increased compared to the control group that was not treated with enzymes, and the free radical scavenging ability was particularly excellent. It is believed that the enzyme decomposition product of perilla meal has high potential for use as an antioxidant bioactive material.
결과 5: Result 5: 들깨박Perilla seed meal 효소분해물을enzyme decomposition product 이용한 유산균 배양 Lactic acid bacteria culture using
들깨박 효소분해물과 대조군을 배양 배지로 하여 Leuconostoc mesenteroides 310-12 유산균을 배양하였다. 15시간 동안 배양하면서, 3시간마다 배양액을 채취하여 생장, 적정산도, 환원당, pH를 측정하였다. 배양 시간에 따른 유산균 증식곡선과 환원당 변화는 도 10에 나타내었다. 들깨박 효소분해물에서 유산균을 배양한 경우, 배양 초기부터 균의 증식이 급격히 증가하였고 15시간까지도 균의 증식을 관찰할 수 있었다. 대조군의 경우, 배양 6시간까지는 균의 증식이 거의 일어나지 않았고, 6시간 이후부터 12시간까지는 균의 증식이 활발하게 일어났으나, 그 이후에는 균의 증식이 중단되는 현상을 나타내었다. 들깨박 효소분해물은 대조군에 비해 탄소원인 환원당과 질소원인 가용성 단백질 함량이 높기 때문에 유산균 증식이 잘 일어남을 알 수 있었다. 효소분해물의 환원당 함량이 대조군에 비해 높았고, 배양 2시간 이후부터 6시간까지 소폭 증가하다가 그 이후부터는 일정량을 유지하는 경향을 나타내었다. 환원당은 유산균 배양에서 탄소원으로 사용되기도 하지만 들깨박에 잔류하는 탄수화물이 유산균에 의해 가수분해되면서 환원당 함량이 높아진 것이라 사료된다. Leuconostoc using perilla meal enzymatic digest and control as culture medium mesenteroides 310-12 lactic acid bacteria were cultured. While culturing for 15 hours, culture fluid was collected every 3 hours to measure growth, titratable acidity, reducing sugar, and pH. The growth curve of lactic acid bacteria and changes in reducing sugar according to culture time are shown in Figure 10. When lactic acid bacteria were cultured in enzyme decomposition product of perilla meal, bacterial growth increased rapidly from the beginning of culture, and bacterial growth could be observed up to 15 hours. In the case of the control group, almost no growth of bacteria occurred until 6 hours of incubation, and active growth occurred from 6 hours to 12 hours, but after that, the growth of bacteria stopped. It was found that the perilla meal enzymatically digested product had a higher content of reducing sugar, a carbon source, and soluble protein, a nitrogen source, than the control group, so lactic acid bacteria growth occurred well. The reducing sugar content of the enzymatic digest was higher than that of the control group, and increased slightly from 2 hours to 6 hours of incubation, but tended to remain at a constant level thereafter. Reducing sugars are also used as a carbon source in culturing lactic acid bacteria, but it is believed that the carbohydrates remaining in perilla meal are hydrolyzed by lactic acid bacteria, increasing the reducing sugar content.
유산균의 배양 시간에 따른 적정 산도와 pH는 도 11에 나타내었다. 유산균 증식에 따라 젖산이 생성되므로 유산균 증식에 비례하여 적정 산도도 증가하는 경향을 보였다. 들깨박 효소분해물에서는 유산균 증식이 잘 일어나기 때문에 이와 비례하여 산도가 증가하고, pH는 감소하게 된다. 따라서 유산균을 효소분해물에서 배양하는 경우, 대조군에 비해 산도는 높고 pH는 낮게 나타난다.The titratable acidity and pH according to the culture time of lactic acid bacteria are shown in Figure 11. Since lactic acid is produced as lactic acid bacteria grow, titratable acidity also tends to increase in proportion to the growth of lactic acid bacteria. Since lactic acid bacteria proliferate easily in perilla meal enzyme decomposition products, acidity increases and pH decreases in proportion. Therefore, when lactic acid bacteria are cultured in enzyme digests, the acidity is high and the pH is low compared to the control group.
들깨박 효소분해물은 대조군에 비해 환원당과 가용성 단백질 함량이 높아 유산균 증식이 잘 일어났으며 이에 따라 젖산을 많이 생성하여 산도도 크게 증가하였다. 따라서 들깨박 효소분해물은 미생물 배양 배지로 충분히 활용 가능하다고 여겨진다. Compared to the control group, the enzyme hydrolyzed product of perilla meal had higher reducing sugar and soluble protein content, leading to good growth of lactic acid bacteria. As a result, a lot of lactic acid was produced, and acidity also increased significantly. Therefore, it is believed that perilla meal enzyme decomposition product can be sufficiently utilized as a microbial culture medium.
Claims (8)
상기 들깨박 분말을 현탁하는 단계:
상기 들깨박 현탁액에 단백질 분해효소로서 알칼라아제(Alcalase) 및 탄수화물 분해효소로서 말토제닉 아밀레이즈 (maltogenic amylase), 풀룰라네이즈 (pullulanase) 및 알파 아밀레이즈 (alpha-amylase)의 혼합물을 첨가하는 단계;
상기 단백질 분해효소 및 탄수화물 분해효소를 첨가한 들깨박 현탁액을 반응시키는 단계; 및
상기 반응물을 원심분리하는 단계;로부터 수득되며 총당 8.5 mg/mL 이상, 환원당 5.0 mg/mL 이상, 가용성 단백질 2.8 mg/mL 이상 및 폴리페놀 0.8 mg/mL 이상을 포함하는 항산화 활성을 갖는 들깨박 효소분해물.
Preparing perilla meal powder by grinding perilla meal;
Step of suspending the perilla meal powder:
Adding a mixture of Alcalase as a proteolytic enzyme and maltogenic amylase, pullulanase, and alpha-amylase as carbohydrate decomposition enzymes to the perilla meal suspension. ;
Reacting the perilla meal suspension to which the proteolytic enzyme and carbohydrate decomposition enzyme were added; and
Centrifuging the reactant; perilla seed enzyme having antioxidant activity, which is obtained from and contains more than 8.5 mg/mL of total sugar, more than 5.0 mg/mL of reducing sugar, more than 2.8 mg/mL of soluble protein, and more than 0.8 mg/mL of polyphenol resolvent.
상기 단백질 분해효소 및 탄수화물 분해효소는 0.5~3:0.5~3의 비율로 들깨박 분말 기준 0.5~5%(w/w)로 첨가하는 것을 특징으로 하는 항산화 활성을 갖는 들깨박 효소분해물.
In claim 1,
An enzyme decomposition product of perilla meal with antioxidant activity, characterized in that the proteolytic enzyme and carbohydrate decomposition enzyme are added at a ratio of 0.5 to 3:0.5 to 3 and 0.5 to 5% (w/w) based on perilla meal powder.
상기 항산화 활성을 갖는 들깨박 효소분해물은 유산균 배양배지로 사용하는 것을 특징으로 하는 항산화 활성을 갖는 들깨박 효소분해물.
In claim 1,
An enzyme decomposition product of perilla meal with antioxidant activity, characterized in that the enzyme decomposition product of perilla meal with antioxidant activity is used as a culture medium for lactic acid bacteria.
상기 유산균은 락토바실러스(Lactobacillus sp.) 속, 비피도박테리움(Bifidobacterium sp.) 속, 스트렙토코커스(Streptococcus sp.) 속, 락토코커스(Lactococcus sp.) 속, 엔테로코커스(Enterococcus sp.) 속, 페디오코커스(Pediococcus sp.) 속, 류코노스톡(Leuconostoc sp.) 속 및 비셀라(Weissella sp.) 속으로 이루어진 군에서 선택되는 하나 이상의 균주인 것을 특징으로 하는 항산화 활성을 갖는 들깨박 효소분해물.
In claim 5,
The lactic acid bacteria include Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Lactococcus sp., Enterococcus sp., An enzyme digest of perilla meal with antioxidant activity, characterized in that it is one or more strains selected from the group consisting of the genus Pediococcus sp., Leuconostoc sp., and Weissella sp. .
A lactic acid bacteria culture medium containing the enzyme decomposition product of perilla seed meal having the antioxidant activity of claim 1 or claim 4.
상기 유산균은 락토바실러스(Lactobacillus sp.) 속, 비피도박테리움(Bifidobacterium sp.) 속, 스트렙토코커스(Streptococcus sp.) 속, 락토코커스(Lactococcus sp.) 속, 엔테로코커스(Enterococcus sp.) 속, 페디오코커스(Pediococcus sp.) 속, 류코노스톡(Leuconostoc sp.) 속 및 비셀라(Weissella sp.) 속으로 이루어진 군에서 선택되는 하나 이상의 균주인 것을 특징으로 하는 항산화 활성을 갖는 들깨박 효소분해물을 포함하는 유산균 배양배지.In claim 7,
The lactic acid bacteria include Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Lactococcus sp., Enterococcus sp., An enzyme decomposition product of perilla meal with antioxidant activity, characterized in that it is one or more strains selected from the group consisting of the genus Pediococcus sp., Leuconostoc sp., and Weissella sp. A lactic acid bacteria culture medium containing.
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