KR102386296B1 - Complex-fermented composition of Fabaton soybean leaves and Schisandra chinensis having enhanced anti-diabetic and anti-obesity effects and preparation method thereof - Google Patents
Complex-fermented composition of Fabaton soybean leaves and Schisandra chinensis having enhanced anti-diabetic and anti-obesity effects and preparation method thereof Download PDFInfo
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- KR102386296B1 KR102386296B1 KR1020190121298A KR20190121298A KR102386296B1 KR 102386296 B1 KR102386296 B1 KR 102386296B1 KR 1020190121298 A KR1020190121298 A KR 1020190121298A KR 20190121298 A KR20190121298 A KR 20190121298A KR 102386296 B1 KR102386296 B1 KR 102386296B1
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- pabaton
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Abstract
본 발명에서는 파바톤 콩잎과 오미자 혼합물을 특정 복합종균으로 복합발효하여 제조되어 소화효소 저해활성, 비당체 이소플라본, 오메가-6 지방산 및 항산화 활성이 증진되고, 식품가공적성(맛, 향, 색)이 증진된 파바톤 콩잎과 오미자 복합발효 조성물 및 그 제조방법이 제공된다.
본 발명에 따른 파바톤 콩잎과 오미자 복합발효 조성물은 알파-글루코시다아제 저해활성과 췌장 라이파아제 저해활성이 현저히 증진되어 당뇨 및 비만 개선효과가 우수하며, 파바톤 콩잎 특유의 향과 맛이 개선되어 기호성도 우수하다. 또한 본 발명에 따른 파바톤 콩잎과 오미자 복합발효 조성물은, 항당뇨 및 항비만 활성, 항산화 활성, 오메가-6 지방산, 총 페놀릭스 및 총 플라보노이드, 비당체 이소플라본 유도체가 강화되어 당뇨 및 비만 개선용 식품, 대사증후군 및 갱년기 증후군 개선용 식품으로도 유용하게 사용될 수 있다. In the present invention, it is prepared by complex fermentation of a mixture of Pabaton soybean leaves and Schisandra with a specific complex seed to enhance digestive enzyme inhibitory activity, non-saccharide isoflavones, omega-6 fatty acids and antioxidant activity, and food processing aptitude (taste, flavor, color) Provided are the enhanced Pabaton soybean leaf and Schisandra complex fermented composition and a method for manufacturing the same.
Fabaton bean leaf and Schisandra complex fermented composition according to the present invention have excellent antidiabetic and obesity improvement effects as alpha-glucosidase inhibitory activity and pancreatic lipase inhibitory activity are significantly improved, and the unique flavor and taste of pabaton bean leaf are improved. It also has excellent palatability. In addition, the compound fermented composition of Fabaton bean leaf and Schisandra Schisandra according to the present invention is fortified with anti-diabetic and anti-obesity activity, antioxidant activity, omega-6 fatty acids, total phenolics and total flavonoids, and non-saccharide isoflavone derivatives to improve diabetes and obesity. It can be usefully used as food, as a food for improving metabolic syndrome and menopausal syndrome.
Description
본 발명은 증진된 항당뇨 및 항비만 활성을 갖는 파바톤 콩잎과 오미자 복합발효 조성물 및 그 제조방법에 관한 것이다. 더욱 상세하게는 파바톤 콩잎과 오미자 혼합물을 특정 복합종균으로 복합발효하여 제조되어 소화효소 저해활성, 비당체 이소플라본, 오메가-6 지방산 및 항산화 활성이 증진되고, 식품가공적성(맛, 향, 색)이 증진된 파바톤 콩잎과 오미자 복합발효 조성물 및 그 제조방법에 관한 것이다. The present invention relates to a composite fermented composition of Pabaton soybean leaves and Schisandra Schisandra having enhanced antidiabetic and antiobesity activity, and a method for preparing the same. In more detail, it is manufactured by complex fermentation of a mixture of Pabaton bean leaves and Schisandra Schisandra with a specific complex spawn to enhance digestive enzyme inhibitory activity, non-saccharide isoflavones, omega-6 fatty acids and antioxidant activity, and food processing aptitude (taste, flavor, color ) relates to a composite fermented composition of Pabaton soybean leaves and Schisandra Schisandra, and a method for manufacturing the same.
콩잎은 콩(Glycine max. Merr L)과 비교했을 시 활용도는 다소 떨어지지만 비타민이 풍부하다고 알려져 있다. 콩잎을 식품으로 섭취하는 국가는 크게 알려진 바가 없으며 우리나라에서도 주로 경상도와 제주도에서만 장아찌, 반찬과 쌈용으로만 이용되고 있어 그 활용도가 매우 미비한 실정이었다. 콩잎과 관련하여 수행되어온 연구는 주로 콩잎김치 또는 장아찌와 관련된 기본적인 내용이며, 생리활성에 관한 연구로는 콩잎에 함유된 플라보놀, 폴리페놀 등이 수행되어 왔다. 이렇듯 오늘날까지 콩잎의 다양한 가공적성에 관한 연구는 아직 미흡한 실정이다.Compared to soybean (Glycine max. Merr L), soybean leaf is known to be rich in vitamins, although its utilization is somewhat lower. There is no known country that consumes soybean leaves as food, and in Korea, it is mainly used for pickles, side dishes, and ssamjang only in Gyeongsang-do and Jeju-do. Research conducted on soybean leaves is mainly related to bean leaf kimchi or pickled radish, and studies on physiological activity have been conducted on flavonols and polyphenols contained in soybean leaves. As such, studies on various processing aptitudes of soybean leaves are still insufficient.
이소플라본은 갱년기 또는 뼈 건강에 효능이 탁월하다고 이미 세계적으로 입증된 플라보놀 계열 화합물이다. 이소플라본 유도체들은 화학 구조상 식물성 에스트로겐과 매우 흡사한 구조를 형성함에 있어 기존의 식물성 에스트로겐과 견줄 만큼의 여러 생리학적 특성을 보인다. 특히 갱년기 증상완화와 완경기 이후 골다공증의 위험 감소 또는 콜레스테롤 감소효과로 인해 당뇨와 비만에도 효능이 있는 것으로 보고되었고 각종 항산화 활성도 우수한 것으로 알려져 있다. 한편 콩잎에 존재하는 이소플라본은 포도당 결합 형태의 배당체와 포도당과는 독립적인 형태의 비당체 형태로 존재한다. 콩과는 다르게 콩잎에는 비당체 이소플라본으로는 다이드제인(daidzein) 및 제니스테인(genistein)만이 존재하며, 배당체 이소플라본으로는 다이드진(daidzin), 말로닐 다이드진(malonyl daidzin)과 제니스틴(genistin), 말로닐 제니스틴(malonyl genistin)만이 존재한다. 이들 이소플라본 유도체들은 기본적으로 항산화 활성 뿐만 아니라 제니스테인은 유방암 등의 각종 항암 효과가 우수하며 다이드제인은 골다공증 개선에 우수하다 알려져 있다.Isoflavones are flavonol compounds that have already been proven worldwide to be effective for menopausal or bone health. Isoflavone derivatives show several physiological properties comparable to existing phytoestrogens in forming a structure very similar to that of phytoestrogens in chemical structure. In particular, it has been reported to be effective in diabetes and obesity due to the alleviation of menopausal symptoms and the reduction of the risk of osteoporosis after menopause or the reduction of cholesterol, and it is also known to have excellent antioxidant activity. On the other hand, isoflavones present in soybean leaves exist in the form of glucose-bound glycosides and non-saccharides independent of glucose. Unlike soybeans, only daidzein and genistein exist as non-saccharide isoflavones in soybean leaves, and daidzin, malonyl daidzin and genistine exist as glycoside isoflavones. (genistin) and malonyl genistin only exist. These isoflavone derivatives are known not only for their antioxidant activity, but also for genistein to have excellent anticancer effects such as breast cancer, and daidzein to improve osteoporosis.
최근 분자농법 기술을 이용하여 이소플라본 유도체 고함량 콩잎(파바톤 콩잎) 재배기술이 개발되었으나 (특허등록 10-1451298호), 이 콩잎은 식품가공적성(맛, 향, 색)이 좋지 않아 활용면에서 충분하지 않은 실정이었다. 이에 파바톤 콩잎의 가공적성 단점을 보완하고 기능성을 증진하여 파바톤 콩잎을 건강기능식품으로 활용될 수 있게 하는 소재와 기술의 개발이 요구되어 왔다.Recently, a technology for cultivating soybean leaves (Pabaton bean leaves) with a high content of isoflavone derivatives has been developed using molecular farming technology (Patent Registration No. 10-1451298). was not sufficient in Accordingly, there has been a demand for the development of materials and technologies that can be used as health functional foods by supplementing the disadvantages of processing aptitude and enhancing the functionality of Pabaton bean leaves.
오미자(Schisandra chinensis)는 원형형태로 지름은 약 1cm이며 색상은 짙고 붉은 빛깔을 담은 과실로 그 속에는 빨간 즙과 적갈색 종자가 1개 내지 2개를 함유하고 있다. 단맛, 신맛, 쓴맛, 짠맛, 매운맛의 5가지 맛이 느껴진다하여 붙여진 이름의 오미자는 크게 기관지 개선, 간 기능 개선, 뇌 질환 예방, 신장 기능 강화, 피부 개선, 기력회복 등 6가지 효능을 보유하고 있으며, 특히 식품의약품안전처로부터 2016년 12월 여성 갱년기 증상 완화 기능성 원료 등록이 된 상태이다. 오미자는 북오미자, 남오미자 또는 흑오미자 등이 있으며 이는 주로 태백산 일대에 가장 많이 자라며 남오미자는 남부 섬지방, 흑오미자는 제주도에서 잘 자라는 것으로 학계에 보고되어 있다. 오미자의 주요 활성 성분은 리그난 화합물로 그 유도체로는 schisandrin, schisantherin, schisandrol, gomisin 계열 정도로 구별할수 있다. 이러한 오미자의 기능성 성분을 이용한 연구 결과로는 오미자 첨가 고추장(Kim et al. 2003. Korean J. Food Sci. Technol.), 두부(Kim et al. 2008. Korean J. Food Nutr.), 요구르트(Hong et al. 2004. Korean J. Food Nutr.) 등이 한 형태가 있으나, 파바톤 콩잎과 함께 복합발효 기술과 그로부터의 현저한 효과는 알려진 바 없다. Schisandra ( Schisandra ) chinensis ) is a round shape, about 1 cm in diameter, and a dark red fruit containing red juice and 1 to 2 reddish-brown seeds. Omija, so named because it tastes sweet, sour, bitter, salty, and spicy, has six effects, including bronchial improvement, liver function improvement, brain disease prevention, kidney function strengthening, skin improvement, and energy recovery. , In particular, it has been registered as a functional ingredient for relieving symptoms of menopausal women from the Ministry of Food and Drug Safety in December 2016. There are North Schisandra, South Schisandra, and Black Schisandra, which are mainly grown in the Taebaeksan area. It has been reported in academia that South Schisandra grows well in the southern island region and Black Schisandra grows well in Jeju Island. The main active ingredient of Schisandra is a lignan compound, and its derivatives can be classified into schisandrin, schisantherin, schisandrol, and gomisin series. As a result of the study using these functional ingredients of Schisandra, Gochujang with Schisandra (Kim et al. 2003. Korean J. Food Sci. Technol.), tofu (Kim et al. 2008. Korean J. Food Nutr.), and yogurt (Hong) et al. 2004. Korean J. Food Nutr.) has one form, but the complex fermentation technique with Pabaton bean leaf and the remarkable effect thereof are not known.
본 발명의 목적은 소화효소 저해활성, 항산화 활성 및 식품가공적성(맛, 향, 색)이 증진된 파바톤 콩잎과 오미자 복합발효 조성물을 제공하는 것이다, It is an object of the present invention to provide a complex fermented composition of Pabaton soybean leaf and Schisandra Schisandra with improved digestive enzyme inhibitory activity, antioxidant activity and food processing aptitude (taste, flavor, color),
본 발명의 또 다른 목적은, 소화효소 저해활성, 비당체 이소플라본, 오메가-6 지방산, 항산화 활성 및 식품가공적성(맛, 향, 색)이 증진된 파바톤 콩잎과 오미자 복합발효 조성물의 제조방법을 제공하는 것이다. Another object of the present invention is a method for producing a complex fermented composition of Pabaton soybean leaves and Schisandra with improved digestive enzyme inhibitory activity, non-saccharide isoflavones, omega-6 fatty acids, antioxidant activity and food processing aptitude (taste, flavor, color) is to provide
본 발명의 또 다른 목적은, 상기 파바톤 콩잎과 오미자 복합발효 조성물을 포함하는 당뇨 및/또는 비만 개선용 건강기능성 식품을 제공하는 것이다.Another object of the present invention is to provide a functional health food for improving diabetes and/or obesity comprising the complex fermented composition of Pabaton soybean leaves and Schisandra Schisandra.
상기 목적을 달성하기 위하여, 하기 단계들을 포함하는, 소화효소 저해활성, 비당체 이소플라본, 오메가-6 지방산, 항산화 활성 및 식품가공적성(맛, 향, 색)이 증진된 파바톤 콩잎과 오미자 복합발효 조성물의 제조방법을 제공한다:In order to achieve the above object, Pabaton bean leaf and Schisandra complex with improved digestive enzyme inhibitory activity, non-saccharide isoflavones, omega-6 fatty acids, antioxidant activity and food processing aptitude (taste, flavor, color), including the following steps A method for preparing a fermentation composition is provided:
ⅰ) 파바톤 콩잎과 오미자를 7 : 3의 중량비로 혼합한 혼합물을 준비하는 단계, 및 i) preparing a mixture of pabaton bean leaf and omija in a weight ratio of 7: 3, and
ⅱ) 상기 혼합물을 살균한 후 락토바실러스 플란타륨 P1201 및 락토바실러스 브레비스 WCP02의 복합종균을 접종하여 복합발효하는 단계. ii) After sterilizing the mixture, inoculating a complex seed of Lactobacillus plantarum P1201 and Lactobacillus brevis WCP02 and performing complex fermentation.
단계 ⅰ) Step i) 파바톤Pavaton 콩잎과 오미자 혼합물 준비 Preparation of bean leaf and Schisandra mixture
파바톤 콩잎과 오미자를 7 : 3의 중량비로 혼합한 혼합물을 준비한다. Prepare a mixture of pabaton bean leaf and omija in a weight ratio of 7:3.
본 발명에서 파바톤 콩잎은 재배 전·후에 식물생장호르몬인 에틸렌(ethylene), 에틸렌 공여체(에세폰) 또는 에틸렌 발생제를 처리하여 고 이소플라본 유도체를 함유하는 콩잎을 의미한다. 파바톤 콩잎은 상업적으로 입수할 수 있고 파바톤 콩잎에는 이소플라본 함량이 평균 약 2,000 ∼ 15,000 ㎍/g 정도의 고농도이다. In the present invention, Pavaton soybean leaves refer to soybean leaves containing high isoflavone derivatives by treating plant growth hormone ethylene, an ethylene donor (Esepone), or an ethylene generator before and after cultivation. Fabaton soybean leaves are commercially available, and the average isoflavone content in Pabaton soybean leaves is about 2,000 to 15,000 μg/g in high concentration.
파바톤 콩잎은 수확하여 세척한 후 바로 착즙하여 사용하거나 또는 건조하여 분말화한 것을 사용할 수 있다.Pabaton soybean leaves can be harvested and washed and then used immediately after squeezing, or dried and powdered.
본 발명에서 오미자는 세척한 후 바로 착즙하여 사용하거나 또는 건조하여 분말화한 것을 사용할 수 있다.In the present invention, omija may be used by juicing immediately after washing, or dried and powdered.
본 발명들은 파바톤 콩잎에 오미자를 7 : 3의 중량비로 혼합시 식품가공적성(맛, 향, 색)이 가장 적정하게 보완된다 (도 1 및 표 1). According to the present invention, food processing aptitude (taste, aroma, color) is most appropriately supplemented when omija is mixed with pabaton bean leaf in a weight ratio of 7: 3 (FIG. 1 and Table 1).
ⅱ) 복합발효ii) Complex Fermentation
상기 혼합물을 살균한 후 락토바실러스 플란타륨 P1201 및 락토바실러스 브레비스 WCP02의 복합종균을 접종하여 복합발효한다. After the mixture is sterilized, a complex seed of Lactobacillus plantarum P1201 and Lactobacillus brevis WCP02 is inoculated and subjected to complex fermentation.
본 발명에서 복합발효에 사용되는 복합종균은 수탁번호 KACC91848P로 기탁된 락토바실러스 플란타륨 P1201(Lactobacillus plantarum P1201) 균주와 수탁번호 KACC92159P로 기탁된 락토바실러스 브레비스 WCP02(Lactobacillus brevis WCP02) 균주를 포함한다. The complex spawn used in the complex fermentation in the present invention is Lactobacillus plantarum P1201 deposited under the accession number KACC91848P ( Lactobacillus plantarum ) P1201) strain and Lactobacillus brevis WCP02 deposited with accession number KACC92159P ( Lactobacillus brevis WCP02) strains.
원활한 발효를 위하여 파바톤 콩잎과 오미자 혼합물에 당류와 정제수를 첨가하여 발효할 수 있다. 당류와 정제수는 파바톤 콩잎과 오미자 혼합물 100 중량부에 대하여 각각 1~ 50 중량부 및 500~2,000 중량부로 첨가하는 것이 바람직하다. For smooth fermentation, sugar and purified water can be added to the mixture of Pabaton soybean leaves and Schisandra for fermentation. The sugar and purified water are preferably added in an amount of 1 to 50 parts by weight and 500 to 2,000 parts by weight, respectively, based on 100 parts by weight of the mixture of Pabaton bean leaf and Schisandra.
살균은 통상의 방법에 따라 수행할 수 있다. Sterilization can be performed according to a conventional method.
접종은 복합종균 배양액을 1∼10%(v/v)으로 접종하며, 바람직하게는 5%(v/v)로 접종한다. 발효는 20∼40℃에서 12시간 이상 수행한다. For inoculation, 1 to 10% (v/v) of the complex seed culture medium is inoculated, preferably 5% (v/v). Fermentation is carried out at 20-40° C. for more than 12 hours.
접종이 1%(v/v) 미만으로 수행되면 발효속도가 지연될 수 있고 10%(v/v) 초과로 수행되면 경제적 효용성이 떨어진다. 발효온도가 20℃ 미만이면 발효속도가 지연되며 40℃ 초과이면 균 생육이 저지되어 발효속도가 지연되고 이상 발효가 발생할 수 있다. 발효시간이 12시간 미만일 경우는 생물전환률이 50% 미만으로 미흡하다.If the inoculation is performed at less than 1% (v/v), the fermentation rate may be delayed, and if it is performed at more than 10% (v/v), the economical effectiveness is lowered. If the fermentation temperature is less than 20℃, the fermentation rate is delayed, and if it exceeds 40℃, the growth of bacteria is inhibited, thereby delaying the fermentation rate and abnormal fermentation may occur. If the fermentation time is less than 12 hours, the bioconversion rate is less than 50%.
상기 균주들을 복합종균으로 사용하여 특정 중량비로 혼합된 파바톤 콩잎과 오미자 혼합물을 복합발효 시, 발효 전 그리고 각각의 단독 발효에 비하여, 놀랍게도 알파-글루코시다아제(α-glucosidase) 저해활성(항당뇨)과 췌장 라이파아제(lipase) 저해활성(항비만)이 현저히 증진되었다(도 2a, 도 2b). When the Pabaton bean leaf and Schisandra Schisandra mixture mixed in a specific weight ratio using the above strains as a complex seed strain were subjected to complex fermentation, it was surprisingly alpha-glucosidase inhibitory activity (anti-diabetic ) and pancreatic lipase inhibitory activity (anti-obesity) were significantly enhanced (FIGS. 2a, 2b).
본 발명의 또 다른 목적에 따라서, 상기 제조방법에 의해 제조된, 파바톤 콩잎과 오미자 복합발효 조성물을 제공한다. According to another object of the present invention, there is provided a composite fermented composition of Pabaton bean leaf and Schisandra Schisandra, prepared by the above manufacturing method.
본 발명에 따른 파바톤 콩잎과 오미자 복합발효 조성물은 75% 이상의 알파-글루코시다아제 저해활성과 46% 이상의 췌장 라이파아제 저해활성을 가져서 당뇨 개선 및 비만 개선용 식품소재로 유용하다(도 2a, 도 2b). Fabaton bean leaf and Schisandra complex fermented composition according to the present invention has alpha-glucosidase inhibitory activity of 75% or more and pancreatic lipase inhibitory activity of 46% or more, so it is useful as a food material for improving diabetes and obesity (Fig. 2a, 2b).
또한 본 발명에 따른 파바톤 콩잎과 오미자 복합발효 조성물은 오메가-6 지방산인 올레산과 리놀레산이 각각 337mg/100 g 이상과 1720 mg/100 g 이상으로 현저히 증진되었다(표 2). In addition, in the Pabaton bean leaf and Schisandra complex fermented composition according to the present invention, oleic acid and linoleic acid, which are omega-6 fatty acids, were significantly improved to 337 mg/100 g or more and 1720 mg/100 g or more, respectively (Table 2).
본 발명에 따른 파바톤 콩잎과 오미자 복합발효 조성물은 환원당과 단백질 함량이 증진되고(표 3), GABA(gamma-aminobutyric acid)와 필수 아미노산 함량이 증진되고(표 4), 총 페놀릭스와 총 플라보노이드 함량도 증진되었다(표 5).The Fabaton bean leaf and Schisandra complex fermented composition according to the present invention has improved reducing sugar and protein content (Table 3), GABA (gamma-aminobutyric acid) and essential amino acid content (Table 4), total phenolics and total flavonoids The content was also increased (Table 5).
본 발명에 따른 파바톤 콩잎과 오미자 복합발효 조성물의 제조방법은 상기와 같은 특정 복합균주를 이용하여 복합발효함에 의해, 파바톤 콩잎에 함유된 고 이소플라본 유도체를 비당체 이소플라본 유도체 즉, 다이드제인과 제니스테인으로 40% 이상 전환하였다(표 6). The manufacturing method of the Pabaton bean leaf and Schisandra complex fermented composition according to the present invention is a non-saccharide isoflavone derivative, i.e., daid, by performing complex fermentation using the specific complex strain as described above. More than 40% conversion to zein and genistein (Table 6).
본 발명에 따른 파바톤 콩잎과 오미자 복합발효 조성물은 다이드제인을 348 μg/g 이상 그리고 제니스테인을 119 μg/g 이상 함유하여 비당체 이소플라본 유도체가 현저히 강화되었다(표 6).Fabaton bean leaf and Schisandra complex fermented composition according to the present invention contained 348 μg/g or more of daidzein and 119 μg/g or more of genistein, so that non-saccharide isoflavone derivatives were remarkably strengthened (Table 6).
본 발명에 따른 파바톤 콩잎과 오미자 복합발효 조성물은 또한 항산화 활성이 증진되었다(표 7).Fabaton bean leaf and Schisandra complex fermented composition according to the present invention also enhanced antioxidant activity (Table 7).
본 발명의 또 다른 목적에 따라서, 본 발명은 상기 파바톤 콩잎과 오미자 복합발효 조성물을 포함하는 당뇨 및/또는 비만 개선용 식품을 제공하는 것이다.According to another object of the present invention, it is an object of the present invention to provide a food for improving diabetes and/or obesity comprising the Pabaton bean leaf and Schisandra complex fermented composition.
본 발명에 따른 식품은 파바톤 콩잎과 오미자 복합발효 조성물을 그대로 첨가하거나 이의 추출물을 활용하여 다른 식품 성분과 혼합되어 제조될 수 있고, 통상적인 방법에 따라 적절하게 제조될 수 있다.The food according to the present invention may be prepared by adding the Pabaton bean leaf and Schisandra complex fermented composition as it is or by using an extract thereof and mixing it with other food ingredients, and may be appropriately prepared according to a conventional method.
본 발명에서 상기 식품의 종류는 특별히 제한되지 않으며, 콩잎제품(환제, 정제제, 캡슐제 등), 발효차, 분말과 과립제, 발효음료(파우치제, 드링크제 등) 등 일 수 있다. In the present invention, the type of the food is not particularly limited, and may be soybean leaf products (pills, tablets, capsules, etc.), fermented tea, powders and granules, fermented beverages (pouches, drinks, etc.).
본 발명에 따른 식품은 파바톤 콩잎과 오미자 복합발효 조성물을 포함하여, 비당체 이소플라본 유도체를 매우 고함량으로 함유함에 따라서 갱년기 완화용 식품으로 활용될 수 있다.The food according to the present invention contains a very high content of non-saccharide isoflavone derivatives, including the complex fermented composition of Pabaton soybean leaves and Schisandra, so it can be used as a food for relieving menopause.
본 발명의 파바톤 콩잎과 오미자 복합발효 조성물은 알파-글루코시다아제 저해활성과 췌장 라이파아제 저해활성이 현저히 증진되어 당뇨 및 비만 개선효과가 우수하다. 아울러 본 발명에 따른 파바톤 콩잎과 오미자 복합발효 조성물은 파바톤 콩잎 특유의 향과 맛이 개선되어 기호성도 우수하다.Fabaton bean leaf and Schisandra complex fermented composition of the present invention is excellent in the improvement of diabetes and obesity as the alpha-glucosidase inhibitory activity and pancreatic lipase inhibitory activity are significantly enhanced. In addition, the Pabaton bean leaf and Schisandra complex fermented composition according to the present invention has improved palatability and flavor unique to the Pabaton bean leaf.
또한 본 발명에 따른 파바톤 콩잎과 오미자 복합발효 조성물은, 항당뇨, 항비만 활성, 비당체 이소플라본 유도체, 오메가-6 지방산, 항산화 활성, 총 페놀릭스 및 총 플라보노이드가 강화되어 당뇨 및 비만 개선용 건강식품, 대사증후군 및 갱년기 증후군 개선용 건강기능성식품으로도 유용하게 사용될 수 있다. In addition, the compound fermented composition of Fabaton bean leaf and Schisandra Schisandra according to the present invention has anti-diabetic, anti-obesity activity, non-saccharide isoflavone derivatives, omega-6 fatty acids, antioxidant activity, total phenolics and total flavonoids for improving diabetes and obesity. It can be usefully used as a health food, as a health functional food for improving metabolic syndrome and menopausal syndrome.
도 1은 본 발명의 파바톤 콩잎과 오미자 복합발효 조성물 제조를 위해 콩잎과 오미자의 최적 혼합비율 선정 과정을 보여주는 사진이다.
도 2는 본 발명의 파바톤 콩잎과 오미자 복합발효 조성물의 알파-글루코시다아제 저해활성(도 2a)과 췌장 라이파아제 저해활성(도 2b)을 보여주는 그래프이다.
도 3은 복합발효 전의 파바톤 콩잎과 오미자 혼합물의 HPLC 크로마토그램을 나타낸 것이다.
도 4는 본 발명에 따른 파바톤 콩잎과 오미자 복합발효 조성물의 HPLC 크로마토그램을 나타낸 것이다.1 is a photograph showing the process of selecting the optimal mixing ratio of soybean leaves and Schisandra for the preparation of the Pabaton bean leaf and Schisandra complex fermented composition of the present invention.
Figure 2 is a graph showing the alpha-glucosidase inhibitory activity (Fig. 2a) and the pancreatic lipase inhibitory activity (Fig. 2b) of the combination fermented composition of Fabaton bean leaf and Schisandra of the present invention.
Figure 3 shows the HPLC chromatogram of the mixture of Pabaton soybean leaves and Schisandra omija before complex fermentation.
Figure 4 shows the HPLC chromatogram of the composition of Pabaton soybean leaf and Schisandra Schisandra complex fermented according to the present invention.
다음의 실시예들에 의해 본 발명이 더 상세히 설명된다. 이들 실시예는 본 발명을 예시하기 위한 것이며, 본 발명의 범위가 이들에 의해 제한되어서는 안 된다. The present invention is further illustrated by the following examples. These examples are for the purpose of illustrating the present invention, and the scope of the present invention should not be limited thereto.
제조예production example : : 파바톤Pavaton 콩잎과 오미자 복합발효 조성물 제조 Manufacture of composite fermented composition of bean leaf and Schisandra Schisandra
발효를 위해 사용된 종균 락토바실러스 플란타륨 P1201 혹은 락토바실러스 브레비스 WCP02은 한국유전자은행(KCTC)에서 분양받아 본 발명에서 종균으로 각각 단독 혹은 복합적으로 사용하였다. The seed strains Lactobacillus plantarum P1201 or Lactobacillus brevis WCP02 used for fermentation were purchased from the Korea Genetic Bank (KCTC) and used individually or in combination as a seed strain in the present invention.
파바톤 콩잎은 경상대학교 천연물화학연구실에서 에세폰 처리하여 재배 및 수확한 것을 제공받아 열풍건조하여 분말화한 것을 사용하였다. 오미자는 경상남도 거창군에서 재배하여 건조된 것을 구입하여 분말화한 것을 사용하였다.Pabaton soybean leaves were grown and harvested by Esepone treatment at Gyeongsang National University's Natural Product Chemistry Lab, and dried with hot air and powdered were used. Omija was grown in Geochang-gun, Gyeongsangnam-do, dried, and powdered was used.
파바톤 콩잎과 오미자 혼합 발효 조성물 제조에 앞서 각각의 파바톤 콩잎 및 오미자를 각각 9:1, 8:2, 7:3, 6:4 비율로 혼합하고 이를 티백에 나눠 담아 뜨거운 물에 우려내었다(도 1). 이를 경남과학기술대학교 학부생 30명을 대상으로 하여 관능평가를 실시하고 그 결과, 9:1 비율과 8:2 비율의 경우 파바톤 콩잎 첨가량이 많으며 오미자 첨가량이 적음에 의해 신맛, 쓴맛, 혹은 색감에서 낮은 점수를 획득하였고 강한 떫은맛으로 인해 최적 혼합비율에서 제외되었다. 파바톤 콩잎과 오미자 소재의 7:3 비율의 경우 오미자의 신맛을 적당히 느낄 수 있었고 떫은맛은 느낄 수 없었으며 전체적인 기호도 평이 우수하여 총점 10점 중 8점을 부여받았다. 이 혼합 비율로 혼합시 파바톤 콩잎 특유의 향과 떫은맛을 상기시키고 적당한 신맛과 향을 낼 수 있었던바 식품가공적성에 올바른 혼합비율은 파바톤 콩잎과 오미자 7:3 비율로 최종 선정되었다(표 1).Prior to the preparation of the Pabaton bean leaf and Schisandra mixed fermentation composition, each Pabaton bean leaf and Schisandra leaf were mixed in a ratio of 9:1, 8:2, 7:3, 6:4, respectively, and divided into tea bags and brewed in hot water ( Fig. 1). This sensory evaluation was conducted on 30 undergraduate students at Kyungnam University of Science and Technology, and as a result, in the case of 9:1 and 8:2 ratios, the amount of Pabaton soybean leaf added was high and the amount of Schisandra Schisandra was low. It obtained a low score and was excluded from the optimal mixing ratio due to its strong astringent taste. In the case of 7:3 ratio of Pabaton bean leaf and Schisandra material, the sour taste of Omija could be felt moderately, and the astringent taste could not be felt. When mixing at this mixing ratio, the unique aroma and astringency of Pabaton bean leaves were recalled and a suitable acidity and flavor could be produced. The correct mixing ratio for food processing aptitude was finally selected as a 7:3 ratio of Pabaton bean leaves and Schisandra (Table 1). ).
최종 선정된 혼합비율(7:3)을 토대로 파바톤 콩잎과 오미자 혼합물의 발효 조성물을 제조하였다. Based on the final selected mixing ratio (7:3), a fermented composition of Pabaton bean leaf and Schisandra Schisandra mixture was prepared.
구체적으로는 파바톤 콩잎과 오미자의 혼합물(5 g)과 설탕(2 g)을 250 ml 유리병에 담고 정제수 95 ml를 첨가 및 혼합하고 121℃에서 15분 살균처리 과정을 수행한 후, 락토바실러스 플란타륨 P1201 균주와 락토바실러스 브레비스 WCP02 균주 를 복합하여 MRS 액체배지(Difco사, USA)에서 48시간 동안 배양한 것을 5%(v/v)로 접종한 후 37℃에서 72시간 정치 발효시켜서 본 발명에 따른 복합발효 조성물 (실시예 1)을 제조하였다. Specifically, a mixture (5 g) of Pabaton soybean leaves and omija (5 g) and sugar (2 g) are placed in a 250 ml glass bottle, 95 ml of purified water is added and mixed, and the sterilization process is performed at 121°C for 15 minutes, followed by Lactobacillus Plantarium P1201 strain and Lactobacillus brevis WCP02 strain were combined and cultured for 48 hours in MRS liquid medium (Difco, USA), inoculated with 5% (v/v), and then fermented for 72 hours at 37 ° C. A composite fermentation composition according to the invention (Example 1) was prepared.
비교를 위하여, 각각, 파바톤 콩잎과 오미자의 혼합물(5 g)과 설탕(2 g)을 250 ml 유리병에 담고 정제수 95 ml를 첨가 및 혼합하고 121℃에서 15분 살균처리 과정을 수행한 후, 락토바실러스 플란타륨 P1201 균주 단독 또는 락토바실러스 브레비스 WCP02 균주 단독을 MRS 액체배지(Difco사, USA)에서 48시간 동안 배양한 것을, 5%(v/v)로 접종한 후 37℃에서 72시간 정치 발효시켜서, 락토바실러스 플란타륨 P1201 단독 발효 조성물 (비교예 1) 및 락토바실러스 브레비스 WCP02 균주 단독 발효 조성물 (비교예 2)를 제조하였다. For comparison, each, put a mixture (5 g) of Pabaton bean leaf and Schisandra leaf and sugar (2 g) in a 250 ml glass bottle, add and mix 95 ml of purified water, and perform a sterilization process at 121°C for 15 minutes , Lactobacillus plantarum P1201 strain alone or Lactobacillus brevis WCP02 strain alone was cultured for 48 hours in MRS liquid medium (Difco, USA), 5% (v/v) after inoculation at 37°C for 72 hours By stationary fermentation, Lactobacillus plantarum P1201 single fermentation composition (Comparative Example 1) and Lactobacillus brevis WCP02 strain single fermentation composition (Comparative Example 2) were prepared.
아을러 실시예 1, 비교예 1 및 비교예 2 각각의 발효 0시간(무접종구)의 시료를 37℃에서 72시간 유지하였다 (참조예). In addition, the samples of each of Example 1, Comparative Example 1 and Comparative Example 2
참고예: 항당뇨 및 항비만 효능 분석 시료 준비Reference Example: Preparation of samples for analysis of anti-diabetic and anti-obesity efficacy
제조예에서 제조된 각각의 파바톤 콩잎과 오미자 발효 조성물 (실시예 1, 비교예 1, 비교예 2, 참조예들)을 동결건조시킨 후 분말화하였다. 동결건조 분말 시료 1 g에 80% 발효주정 40 ml를 첨가한 후 20℃, 300 rpm에서 12시간 추출한 후 원심분리하고 상등액을 0.45 ㎛ 여과필터로 여과하여 얻은 여과액을 분석시료로 사용하였다.Each of the Pabaton bean leaves and Schisandra fermented compositions (Example 1, Comparative Example 1, Comparative Example 2, Reference Examples) prepared in Preparation Example were freeze-dried and then powdered. After adding 40 ml of 80% fermented alcohol to 1 g of the freeze-dried powder sample, extraction was performed at 20° C. and 300 rpm for 12 hours, centrifuged, and the filtrate obtained by filtering the supernatant through a 0.45 μm filter was used as an analysis sample.
시험예test example 1: One: 항당뇨antidiabetic 및 and 항비만anti-obesity 효능 측정 Efficacy measurement
항당뇨와 항비만 효능은 효소모델을 기반으로 실시하였다. 항당뇨 효소모델은 알파-글루코시다아제이며 항비만 효소모델은 췌장 라이파아제이다. 이들 항당뇨와 항비만 효능은 파라-니트로페놀법(para-nitrophenol method)으로 검정하였다.Anti-diabetic and anti-obesity efficacy was conducted based on the enzyme model. The anti-diabetic enzyme model is alpha-glucosidase and the anti-obesity enzyme model is pancreatic lipase. These anti-diabetic and anti-obesity effects were tested by the para-nitrophenol method.
<알파-글루코시다아제 저해활성><alpha-glucosidase inhibitory activity>
참고예에서 준비된 각각의 분석시료 50 ㎕, 알파-글루코시다아제 (0.5 U/ml) 효소용액 50 ㎕, 200 mM 인산나트륨 완충용액(pH 6.8) 50 ㎕를 혼합하여 37℃에서 10분간 예비반응 하였다. 이후 인산나트륨 완충용액(pH 6.8)에 녹인 p-NPG (5 mM) 100 ㎕를 가하여 다시 37℃에서 10분 반응시켰으며 반응액에 Na2CO3 (100 mM) 0.75 ml를 가해 최종반응을 정지시킨 후 420 nm에서 분광광도계를 이용하여 흡광도를 측정하였다. 음성대조구는 시료 대신에 증류수를 취하였으며 분석 시료 첨가구와 무첨가구 사이의 흡광도 차이를 백분율(%)로 나타내어 그 결과를 도 2a에 나타냈다.50 μl of each analysis sample prepared in Reference Example, 50 μl of alpha-glucosidase (0.5 U/ml) enzyme solution, and 50 μl of 200 mM sodium phosphate buffer (pH 6.8) were mixed and pre-reacted at 37° C. for 10 minutes. . After that, 100 μl of p-NPG (5 mM) dissolved in sodium phosphate buffer (pH 6.8) was added and reacted at 37° C. for 10 minutes, and 0.75 ml of Na 2 CO 3 (100 mM) was added to the reaction solution to stop the final reaction. Then, the absorbance was measured at 420 nm using a spectrophotometer. For the negative control, distilled water was taken instead of the sample, and the difference in absorbance between the group with and without the addition of the analysis sample was expressed as a percentage (%), and the results are shown in FIG. 2A.
도 2a에 도시된 바와 같이, 비교예 1 및 비교예 2의 발효 조성물(각각 락토바실러스 플란타륨 P1201과 락토바실러스 브레비스 WCP02 단독 균주로 발효)의 알파-글루코시다아제 저해활성은 참조예(무접종구)에 비하여 약간 증가하였으나 실시예 1의 파바톤 콩잎과 오미자 복합발효 조성물(락토바실러스 플란타륨 P1201과 락토바실러스 브레비스 WCP02 복합균주로 발효)에서는 2배 가량(75% 이상) 현저히 증진되었다. As shown in Fig. 2a, the alpha-glucosidase inhibitory activity of the fermentation compositions of Comparative Examples 1 and 2 (fermented with Lactobacillus plantarium P1201 and Lactobacillus brevis WCP02 single strains, respectively) was a reference example (no inoculation). Although slightly increased compared to the previous), in the Pabaton bean leaf and Schisandra complex fermented composition of Example 1 (fermented with Lactobacillus plantarium P1201 and Lactobacillus brevis WCP02 complex strains), it was significantly improved by about 2 times (75% or more).
따라서 본 발명에 따른 파바톤 콩잎과 오미자 복합발효 조성물은 알파-글루코시다아제 저해활성이 현저히 증진되어 당뇨 개선효과가 우수함을 알 수 있다.Therefore, it can be seen that the Fabaton bean leaf and Schisandra fermented composition according to the present invention significantly improved the alpha-glucosidase inhibitory activity and thus had an excellent diabetes improvement effect.
<췌장 <pancreas 라이파아제lipase 저해활성> inhibitory activity>
참고예에서 준비된 각각의 분석시료 50 ㎕, 췌장 리파아제 (1.0 U/ml) 효소용액 50 ㎕, 및 200 mM 인산나트륨 완충용액(pH 6.8) 50 ㎕를 혼합하여 37 ℃에서 10분간 예비반응시켰다. 반응 후 인산나트륨 완충용액에 녹인 p-NPB(5 mM) 100 ㎕를 가하여 동일하게 10분간 반응시킨 후 100 mM Na2CO3 0.75 ml를 가해 반응을 종결시켜 420 nm에서 흡광도를 측정하였다. 음성대조구는 시료 대신에 증류수를 취하였으며 시료용액의 첨가구와 무첨가구 사이의 흡광도 차이를 백분율(%)로 나타내어 그 결과를 도 2b에 나타냈다.50 μl of each assay sample prepared in Reference Example, 50 μl of pancreatic lipase (1.0 U/ml) enzyme solution, and 50 μl of 200 mM sodium phosphate buffer (pH 6.8) were mixed and pre-reacted at 37° C. for 10 minutes. After the reaction, 100 μl of p-NPB (5 mM) dissolved in sodium phosphate buffer was added and reacted for 10 minutes. Then, 0.75 ml of 100 mM Na 2 CO 3 was added to terminate the reaction, and absorbance was measured at 420 nm. For the negative control, distilled water was taken instead of the sample, and the difference in absorbance between the addition and the non-addition group of the sample solution was expressed as a percentage (%), and the results are shown in FIG. 2B.
도 2b에 도시된 바와 같이, 비교예 1 및 비교예 2의 발효 조성물의 췌장 라이파아제 저해활성은 각각 37% 및 35%를 나타내었으나, 실시예 1의 복합발효 조성물의 경우는 46% 이상의 저해활성을 보여 현저히 증진되었다. As shown in FIG. 2b , the pancreatic lipase inhibitory activity of the fermentation compositions of Comparative Examples 1 and 2 was 37% and 35%, respectively, but in the case of the complex fermentation composition of Example 1, inhibition of 46% or more activity was markedly improved.
따라서 본 발명에 따른 파바톤 콩잎과 오미자 복합발효 조성물은 췌장 라이파아제 저해활성이 증진됨에 따라 비만 개선효과가 있음을 알 수 있다. Therefore, it can be seen that the Fabaton soybean leaf and Schisandra complex fermented composition according to the present invention has an effect of improving obesity as the pancreatic lipase inhibitory activity is enhanced.
시험예test example 2: 2: 파바톤Pavaton 콩잎과 오미자 복합발효 조성물의 지방산 조성 Fatty acid composition of soybean leaf and Schisandra complex fermented composition
제조예의 방법과 동일한 방식으로 파바톤 콩잎과 오미자 혼합물을 1 kg 복합발효하여 제조한 파바톤 콩잎과 오미자 복합발효 조성물(실시예 2)와 파바톤 콩잎과 오미자 혼합물 원료(비교예 3)을 대상으로 지방산 함량 비교분석 과정을 수행하였다. The Pabaton bean leaf and Schisandra complex fermented composition (Example 2) and the Pabaton bean leaf and Schisandra mixture raw material (Comparative Example 3), which were prepared by complex fermentation of 1 kg of Pabaton bean leaf and Schisandra mixture in the same manner as in Preparation Example A fatty acid content comparative analysis process was performed.
지방산 분석은 12 N HCl를 이용한 지방산 메틸에스테르화를 실시하였다. 각각의 동결 건조된 비교예 3의 분말 그리고 실시예 2의 분말 100 mg에 각각 6 N HCl 염산 5 ml를 첨가하여 100℃에서 10분간 가수분해 과정을 포함한다. 그리고 나서 삼불화붕소(BF3) 2 ml를 가하고 교반 후 30분 다시 가온하여 지방산 메틸에스테르화를 진행하였다. 반응 종결 후 이소옥탄 1 ml를 첨가하여 층 분리가 일어나게끔 하고 무수아황산나트륨과 함께 탈수시켜 0.45 ㎛ 필터로 여과하고 가스크로마토그래피로 식품공전에 따라 분석하여 그 결과를 표 2에 나타냈다..Fatty acid analysis was carried out by methyl esterification of fatty acids using 12 N HCl. Each of the freeze-dried powders of Comparative Example 3 and Example 2 was hydrolyzed at 100° C. for 10 minutes by adding 5 ml of 6 N HCl hydrochloric acid to 100 mg of each. Then, 2 ml of boron trifluoride (BF 3 ) was added, stirred and heated again for 30 minutes to proceed with fatty acid methyl esterification. After completion of the reaction, 1 ml of isooctane was added to cause layer separation, dehydrated with anhydrous sodium sulfite, filtered through a 0.45 μm filter, and analyzed by gas chromatography according to Food Standards. The results are shown in Table 2.
표 2에 나타낸 바와 같이, 비교예 3(원료)에 비하여 실시예(복합발효 조성물)에서 오메가-6 지방산인 올레산과 리놀레산이 각각 337mg 이상과 1720 mg/100 g이상으로 현저히 증가하였다As shown in Table 2, the omega-6 fatty acids oleic acid and linoleic acid in Example (complex fermentation composition) were significantly increased to 337 mg or more and 1720 mg/100 g or more, respectively, compared to Comparative Example 3 (raw material).
시험예test example 3: 3: 파바톤Pavaton 콩잎과 오미자 복합발효 조성물의 이화학적 특성 평가 Evaluation of Physicochemical Characteristics of Soybean Leaf and Schisandra Schisandra Complex Fermentation Composition
시험예 2에서 제조된 각각의 조성물 (비교예 3 및 실시예 2)의 이화학적 특성 평가는 pH, 산도, 환원당, 단백질 함량과 생균수 측정으로 실시하였다. Physicochemical properties of each composition prepared in Test Example 2 (Comparative Examples 3 and 2) were evaluated by measuring pH, acidity, reducing sugar, protein content, and number of viable cells.
pH는 pH 미터기(MP 220, London, UK)를 사용하여 각각의 시료를 전극봉에 접촉시켜 나타난 수치를 기입하였다. 산도는 시료 10 ml에 대하여 0.1 N NaOH 용액으로 pH 8.3까지 중화시키는 데 소요된 ml수를 구하고 최종 젖산 양으로 환산하여 백분율(%)로 나타내었다. 환원당은 각각의 시료를 일정량 원심분리하고 적당히 희석한 용액 0. 1 ml에 DNS 시약 1 ml를 첨가하고 100℃에서 30분간 발색시키고 얼음물에 담궈 급속히 냉각시킨 후 570 nm에서 흡광도를 측정하여 나타난 흡광도 수치를 포도당을 이용하여 작성한 검량곡선에 대입하여 g/L로 나타내었다. 단백질 함량은 동결건조 분말 0.1 g에 증류수 1 ml를 첨가하여 혼합하고 biuret 시약 4 ml를 가해 37℃에서 20분 반응을 진행하였다. 반응이 끝난 후 적당히 원심분리하고 상등액만을 540 nm에서 흡광도 값을 측정하였고 이때 소혈청알부민을 이용하여 작성된 표준검량선에 대입하여 mg/g으로 나타내었다. 생균수는 멸균생리식염수로 각각의 시료를 단계별 희석하여 MRS 유산균 전용검출 배지에 평판도말하고 37℃ 24시간 배양 후 나타난 집락을 계수기로 측정하여 cfu로 나타내었다. 각각의 측정 결과를 표 3에 나타냈다. The pH was recorded by contacting each sample with an electrode using a pH meter (MP 220, London, UK). The acidity was calculated as the number of ml required to neutralize to pH 8.3 with 0.1 N NaOH solution for 10 ml of the sample, and converted into the final amount of lactic acid and expressed as a percentage (%). For reducing sugar, a certain amount of each sample is centrifuged, 1 ml of DNS reagent is added to 0.1 ml of an appropriately diluted solution, color developed at 100° C. for 30 minutes, rapidly cooled by immersion in ice water, and absorbance measured at 570 nm. was substituted into the calibration curve prepared using glucose and expressed as g/L. For protein content, 1 ml of distilled water was added to 0.1 g of lyophilized powder, mixed, and 4 ml of biuret reagent was added, followed by reaction at 37° C. for 20 minutes. After the reaction was completed, centrifugation was carried out appropriately, and the absorbance value of only the supernatant was measured at 540 nm. At this time, it was substituted into the standard calibration curve prepared using bovine serum albumin and expressed as mg/g. The number of viable cells was obtained by diluting each sample step-by-step with sterile physiological saline, plated on a MRS lactic acid bacteria-only detection medium, and the colonies that appeared after incubation at 37°C for 24 hours were measured with a counter and expressed as cfu. Each measurement result is shown in Table 3.
(젖산, %)total acidity
(lactic acid, %)
(g/L)reducing sugar
(g/L)
(mg/g)protein
(mg/g)
pH는 발효 조성물인 실시예 2에서 3.82로 감소하였고 산도는 약간 증가하였다. 환원당은 비교예 3(1.82 g/L) 보다 실시예 2에서 2.75 g/L로 증가하였다. 단백질 함량은 발효 처리에 따른 차이 없이 비교예 3과 유사한 함량을 보였고 생균수는 증가하였다.The pH decreased to 3.82 in Example 2, the fermentation composition, and the acidity slightly increased. The reducing sugar increased to 2.75 g/L in Example 2 than in Comparative Example 3 (1.82 g/L). The protein content showed a similar content to Comparative Example 3 without any difference according to the fermentation process, and the number of viable cells was increased.
시험예test example 4: 4: 파바톤Pavaton 콩잎과 오미자 복합발효 조성물의 Soybean leaf and Schisandra complex fermented composition 유리아미노산free amino acids 조성 분석 compositional analysis
유리아미노산 분석은 아미노산 자동 분석기로 수행하였다. 상세하게는, 시험예 2에서 제조된 각각의 비교예 3 분말 그리고 실시예 2의 분말 100 mg을 정확히 시험관에 칭량하고 여기에 증류수 4 ml를 가하고 60℃에서 1시간 가수분해 과정을 수행하였다. 여기에 5-술포설라살산(sulfosalicylic acid) 2 ml를 첨가하고 2시간 방치시켜 단백질을 침전시킨 뒤 약 3분간 원심분리한 뒤에 상등액만을 60℃의 감압조건하에 완전 증발을 시켰다. 이후 남은 여액에 대해 리튬 시트레이트 버퍼(pH 2.2) 2 ml를 첨가 및 용해하고 0.45 ㎛ 필터로 여과한 여액을 아미노산 자동 분석기에 주입하여 유리아미노산을 분석하였고, 그 결과를 표 4에 나타냈다.Free amino acid analysis was performed with an automatic amino acid analyzer. Specifically, each of the powder of Comparative Example 3 prepared in Test Example 2 and 100 mg of the powder of Example 2 were precisely weighed in a test tube, 4 ml of distilled water was added thereto, and a hydrolysis process was performed at 60° C. for 1 hour. To this, 2 ml of 5-sulfosalicylic acid was added, and the protein was precipitated by allowing it to stand for 2 hours. After centrifugation for about 3 minutes, only the supernatant was completely evaporated under reduced pressure at 60°C. To the remaining filtrate, 2 ml of lithium citrate buffer (pH 2.2) was added and dissolved, and the filtrate filtered through a 0.45 μm filter was injected into an automatic amino acid analyzer to analyze free amino acids, and the results are shown in Table 4.
표 4에 나타낸 바와 같이, 비 필수아미노산 5종과 필수아미노산 5종이 검출되었고 비교예 3에 비하여 실시예 2의 아미노산 함량이 증가하는 결과를 나타내었다. 비 필수아미노산에서는 GABA(γ-aminobutyric acid)가 실시예 2에서 126.01 mg/100 g으로 증가되었고, 필수아미노산 중에서는 발린(valine)이 마찬가지로 비교예 3에 비하여 실시예 2(80.06 mg/100 g)에서 크게 증진되었다.As shown in Table 4, 5 types of non-essential amino acids and 5 types of essential amino acids were detected, and the amino acid content of Example 2 was increased compared to Comparative Example 3. In non-essential amino acids, GABA (γ-aminobutyric acid) was increased to 126.01 mg/100 g in Example 2, and among essential amino acids, valine was similarly increased in Example 2 (80.06 mg/100 g) compared to Comparative Example 3 has been greatly improved in
시험예test example 5: 5: 파바톤Pavaton 콩잎과 오미자 복합발효 조성물의 생리활성물질 분석 Analysis of physiologically active substances of soybean leaf and Schisandra complex fermented composition
생리활성물질 분석은 참고예에서 제조한 방법을 토대로 비교예 3과 실시예 2의 분석시료를 제조하였다. 분석시료 각각에 대하여 750 및 420 nm에서 흡광도 수치를 측정하는 비색법을 통해 총 페놀릭스와 총 플라보노이드 함량을 측정하였다.For the analysis of physiologically active substances, the analytical samples of Comparative Examples 3 and 2 were prepared based on the method prepared in Reference Example. Total phenolics and total flavonoid content were measured through a colorimetric method of measuring absorbance values at 750 and 420 nm for each of the analytes.
<총 <Gun 페놀릭스phenolics 및 총 플라보노이드> and total flavonoids>
총 페놀릭스 함량 측정은 널리 알려진 것으로 Folin-Denis법을 조금 변형하였다. 각각의 분석시료 0.5 ml를 시험관에 분주하고 25% Na2CO3 용액 0.5 ml를 첨가하고 3분간 반응 후 2 N Folin-Ciocalteu phenol 용액을 0.25 ml 분주하고 37℃에서 1시간 발색반응을 진행하였다. 발색된 각각의 샘플들은 750 nm에서 흡광도를 측정하고 이때 갈산(gallic acid)으로부터 작성된 표준검량곡선에 대입하여 mg/g으로 값을 산출하였고, 그 결과를 표 5에 나타냈다.Determination of total phenolics content is widely known, and the Folin-Denis method is slightly modified. 0.5 ml of each analysis sample was dispensed into a test tube, 0.5 ml of a 25% Na 2 CO 3 solution was added, and after reaction for 3 minutes, 0.25 ml of 2 N Folin-Ciocalteu phenol solution was dispensed and the color reaction was performed at 37° C. for 1 hour. The absorbance of each colored sample was measured at 750 nm, and the value was calculated in mg/g by substituting it into a standard calibration curve prepared from gallic acid, and the results are shown in Table 5.
표 5에 나타낸 바와 같이, 총 페놀릭스 함량은 비교예 3에 비하여 실시예 2에서 2.74 mg/g으로 증가하였다.As shown in Table 5, the total phenolics content was increased to 2.74 mg/g in Example 2 compared to Comparative Example 3.
총 플라보노이드 함량 측정은 각각의 분석시료 0.5 ml를 시험관에 취하고 여기에 디에틸렌글리콜 용액 1.0 ml를 첨가하고 또한 여기에 1 N NaOH 0.01 ml를 분주하여 37℃에서 1시간 반응시켰다. 그 후 420 nm에서 흡광도를 측정하였고 이때 루틴(rutin)으로부터 작성된 표준검량곡선에 대입하여 mg/g으로 그 값을 산출하였고, 그 결과를 표 5에 나타냈다.To measure the total flavonoid content, 0.5 ml of each assay sample was taken in a test tube, 1.0 ml of diethylene glycol solution was added thereto, and 0.01 ml of 1 N NaOH was dispensed thereto and reacted at 37° C. for 1 hour. Thereafter, absorbance was measured at 420 nm, and the value was calculated in mg/g by substituting it into a standard calibration curve prepared from rutin, and the results are shown in Table 5.
표 5에 나타낸 바와 같이, 총 플라보노이드 함량 또한 비교예 3에 비하여 실시예 2에서는 2.87 mg/g으로 그 함량이 크게 증진됨을 알 수 있다.As shown in Table 5, it can be seen that the total flavonoid content is also significantly increased to 2.87 mg/g in Example 2 compared to Comparative Example 3.
시험예test example 6: 6: 파바톤Pavaton 콩잎과 오미자 복합발효 조성물의 이소플라본 함량 분석 Analysis of Isoflavone Content of Soybean Leaf and Schisandra Schisandra Complex Fermentation Composition
이소플라본 함량 분석은 HPLC 크로마토그램을 이용하여 분석하였다. 구체적으로는 참고예를 기초로 제조한 비교예 3 및 실시예 2의 분석시료를 사용하였고, 분석 컬럼은 Lichrophore 100 RP C18 column(4.6×250 mm, 5 ㎛, Merck, Germany)을 사용하였다. 이동상 용매는 HPLC 등급 H20(A)와 아세토니트릴(B)로 분석하였고, 이동상 조건은 B 기준으로 각각 20, 10, 10 및 10분 동안 10%, 20%, 25% 및 35%로 유지시켰다. 각각의 분석시료 20 ㎕를 주입하였고 이동상의 속도는 30℃에서 분당 1 ml의 흐름률을 적용시켰다. 이소플라본은 diode array UV detector(Agilent 1200 series, Agilent사, USA)의 흡광도 254 nm에서 정량하여 표준품의 검량곡선과 비교하여 함량(㎍/g)을 계산하였다. 각각의 결과를 표 6 및 도 3(비교예 3) 및 도 4(실시예 2)에 나타냈다.Isoflavone content analysis was analyzed using HPLC chromatogram. Specifically, the analytical samples of Comparative Examples 3 and 2 prepared based on Reference Examples were used, and the
표 6에 나타낸 바와 같이. 비교예 3의 배당체인 글리코사이드 함량은 1,185.33 ㎍/g이었으나 실시예 2에서는 897.48 ㎍/g으로 감소하였다. 배당체인 말로닐글리코사이드 역시 실시예 2에서 976.97 ㎍/g으로 감소하였다. As shown in Table 6. The glycoside content of the glycoside of Comparative Example 3 was 1,185.33 μg/g, but decreased to 897.48 μg/g in Example 2. The glycoside malonylglycoside was also reduced to 976.97 μg/g in Example 2.
이에 비하여 비당체 형태의 아글리콘 함량은 164.82 ㎍/g(비교예 3)에서 467.68 ㎍/g으로 약 2.8배 이상 비당체 이소플라본 함량이 증진된 것으로 확인된다. 특히 실시예 2는 다이드제인은 348 ㎍/g 이상, 제니스테인은 119 ㎍/g 이상 함유하였다.On the other hand, it is confirmed that the aglycone content of the aglycone form is increased by about 2.8 times or more, from 164.82 μg/g (Comparative Example 3) to 467.68 μg/g. In particular, Example 2 contained 348 µg/g or more of daidzein and 119 µg/g or more of genistein.
도 3 및 도 4의 크로마토그램에서 도시되는 바와 같이, 비교예 3(도 3)과 그리고 실시예 2(도 4)에서는 공통적으로 4종의 배당체 이소플라본(다이드진, 제니스틴, 말로닐다이드진, 말로닐제니스틴)과 2종의 비당체 이소플라본(다이드제인, 제니스테인) 유도체가 검출되었다. 실시예 2의 복합발효 조성물에서는 피크 5번과 6번인 비당체 형태의 다이드제인과 제니스테인이 크게 증가하였음을 확인할 수 있다. As shown in the chromatograms of FIGS. 3 and 4 , in Comparative Example 3 ( FIG. 3 ) and Example 2 ( FIG. 4 ), four types of glycoside isoflavones (daidzine, genistine, malonyldaidzine) , malonylgenistine) and two non-saccharide isoflavone (daidzein, genistein) derivatives were detected. In the complex fermentation composition of Example 2, it can be seen that the
따라서 락토바실러스 플란타륨 P1201 및 락토바실러스 브레비스 WCP02로 이루어지는 2종의 복합균주로 복합발효된 본 발명에 따른 파바톤 콩잎과 오미자 복합발효 조성물은 비당체 이소플라본(다이드제인과 제니스테인)의 함량이 현저히 강화됨을 알 수 있다. Therefore, the Pabaton soybean leaf and Schisandra complex fermented composition according to the present invention, which is complex fermented with two complex strains consisting of Lactobacillus plantarum P1201 and Lactobacillus brevis WCP02, has a higher content of non-saccharide isoflavones (daidzein and genistein). It can be seen that it is significantly strengthened.
시험예test example 7: 7: 파바톤Pavaton 콩잎과 오미자 복합발효 조성물의 항산화 활성 분석 Analysis of antioxidant activity of soybean leaf and omija complex fermented composition
항산화 활성 분석은 DPPH, ABTS, 하이드록실 라디칼 소거활성과 FRAP 환원력의 방법으로 수행하였다.Antioxidant activity analysis was performed by methods of DPPH, ABTS, hydroxyl radical scavenging activity and FRAP reducing power.
<< DPPHDPPH 라디칼 소거활성> Radical scavenging activity>
DPPH(1,1-diphenyl-2-picrylhydrazyl)는 매우 안정한 라디칼로서 517∼525 nm에서 최대 흡광도를 나타내는 보라색 화합물이다. DPPH (1,1-diphenyl-2-picrylhydrazyl) is a very stable radical and is a purple compound exhibiting maximum absorbance at 517-525 nm.
더욱 상세하게는 DPPH 용액 0.8 ml와 참고예와 같이 준비된 비교예 3과 실시예 2의 분석시료 0.2 ml를 각각 첨가하고 이를 암실에서 30분간 반응 후 분광광도계를 사용하여 525 nm에서 흡광도를 측정하였다. 음성대조구로 증류수를 사용하였고 흡광도 차이를 구하여 하기 식에 대입하여 백분율(%)로 나타냈으며 그 결과는 표 7에 나타냈다:In more detail, 0.8 ml of the DPPH solution and 0.2 ml of the analysis samples of Comparative Example 3 and Example 2 prepared as in Reference Example were added, respectively, and reacted in the dark for 30 minutes. Absorbance was measured at 525 nm using a spectrophotometer. Distilled water was used as a negative control, and the difference in absorbance was obtained and expressed as a percentage (%) by substituting it in the following formula, and the results are shown in Table 7:
라디칼 소거활성(%) = [1-(음성대조구 흡광도 ÷ 실험구 흡광도)] × 100Radical scavenging activity (%) = [1-(absorbance of negative control ÷ absorbance of experimental group)] × 100
그 결과, 비교예 3의 DPPH 라디칼 소거활성은 38%였고 실시예 2는 55%로 크게 증가함을 알 수 있다.As a result, it can be seen that the DPPH radical scavenging activity of Comparative Example 3 was 38% and that of Example 2 was significantly increased to 55%.
<< ABTSABTS 라디칼 소거활성> Radical scavenging activity>
ABTS 라디칼 소거활성은 2-azino-bis의 색을 띤 라디칼의 감소정도에 따라 항산화 활성을 검사하는 방법이다. 이 방법은 시료와 표준물질(Trolox, 6-hydroxyl -2,5,7,8-tetramethylchroman-2-carboxylic acid)의 값과 비교하여 항산화 활성을 측정하는 방법으로서, 시료의 항산화력에 의해 ABTS 양이온(ABTS+)이 소거되어 청록색으로 탈색되는데, 이때 ABTS 양이온(ABTS+)의 제거 정도를 흡광도를 측정함으로서 알 수 있고, 탈색반응이 1분 내에 종료되므로 짧은 시간에 측정이 가능하다.ABTS radical scavenging activity is a method of examining antioxidant activity according to the degree of reduction of the colored radical of 2-azino-bis. This method is a method of measuring the antioxidant activity by comparing the value of the sample and the standard material (Trlox, 6-hydroxyl -2,5,7,8-tetramethylchroman-2-carboxylic acid). (ABTS+) is removed and the color is decolorized into cyan. At this time, the degree of removal of the ABTS cation (ABTS+) can be known by measuring the absorbance.
구체적으로는 7 mM ABTS+ 5 ml과 2.45 mM K2S2O8 5 ml를 섞어 암실에서 12∼16시간 방치시켜 ABTS+ 라디칼을 형성시켰다. 이후 732 nm에서 대조구의 흡광도 값이 0.7±0.02가 되도록 조절한 ABTS+ 0.9 ml와 각각의 분석시료 1.0 ml를 첨가하여 3분간 반응시켜 732 nm에서 흡광도를 측정하였다. 음성대조구로 증류수를 사용하였고 흡광도 차이를 구하여 상기 식에 대입하여 백분율(%)로 나타냈으며 그 결과는 표 7에 나타냈다.Specifically, 7
표 7에 나타낸 바와 같이 비교예 3에 비하여 실시예 2는 ABTS 라디칼 소거활성이 67%로 크게 증가함을 알 수 있다.As shown in Table 7, it can be seen that the ABTS radical scavenging activity of Example 2 is greatly increased to 67% compared to Comparative Example 3.
<< 하이드록실hydroxyl 라디칼 소거활성> Radical scavenging activity>
하이드록실 라디칼 소거활성 측정은 10 mM FeSO4.7H20-EDTA 0.2 ml, 10 mM 2-데옥시리보스 0.2 ml, 10 mM H2O2 0.2 ml와 각각의 분석시료 1.4 ml를 혼합하여 37℃에서 4시간 반응시켰다. 이 혼합액에 1% thiobarbituric acid(TBA)와 2.8% trichloroaceric acid(TCA)를 각각 1 ml 첨가하여 100 ℃에서 20분간 가열하여 발색시키고 적당히 냉각시킨 후 520 nm에서 흡광도를 측정하였다. 음성대조구로 증류수를 사용하였고 흡광도 차이를 구하여 상기 식에 대입하여 백분율(%)로 나타냈으며 그 결과는 표 7에 나타냈다.Hydroxyl radical scavenging activity was measured by mixing 0.2 ml of 10 mM FeSO4.7H20-EDTA, 0.2 ml of 10 mM 2-deoxyribose, 0.2 ml of 10 mM H2O2, and 1.4 ml of each assay sample, followed by reaction at 37° C. for 4 hours. To this mixture, 1 ml of each of 1% thiobarbituric acid (TBA) and 2.8% trichloroaceric acid (TCA) was added, heated at 100° C. for 20 minutes to develop color, and after cooling appropriately, absorbance at 520 nm was measured. Distilled water was used as a negative control, and the difference in absorbance was obtained and expressed as a percentage (%) by substituting it in the above formula, and the results are shown in Table 7.
표 7에 나타낸 바와 같이, 하이드록시 라디칼 소거활성도 실시예 2에서 증진되었다.As shown in Table 7, the hydroxy radical scavenging activity was also enhanced in Example 2.
<FRAP(ferric reducing antioxidant power) 환원력><FRAP (ferric reducing antioxidant power) reducing power>
FRAP 환원력 측정은 30 mM 아세테이트 완충액(pH 3.6), 40 mM 염산에 녹인 10 mM 2,4,6-트리피리딜-s-트리아진(TPTZ, T1253, C18H12N6, MW312.33) 및 20mM FeCl3(F7134, MW 162.20, in DW)를 먼저 준비하였으며, 아세테이트 완충액, TPTZ 용액 및 FeCl3 용액을 10:1:1 (v/v/v)로 혼합하여 37 ℃에서 15분간 예비반응을 실시하였다. 각각의 분석시료 50 ㎕와 FRAP 시약 950 ㎕를 시험관에 분주한 후 37℃에서 15분간 반응시키고 항산화 활성과 마찬가지로 593 nm에서 분광광도계를 사용하여 흡광도 값을 측정하였다. 음성대조구로 증류수를 사용하였고 흡광도 차이를 구하여 상기 식에 대입하여 백분율(%)로 나타냈으며 그 결과는 표 7에 나타냈다.FRAP reducing power was measured in 30 mM acetate buffer (pH 3.6), 10
표 7에 나타낸 바와 같이, 비교예 3(1.36)에 비하여 실시예 2(1.83)는 환원력도 증진되었다. As shown in Table 7, compared to Comparative Example 3 (1.36), Example 2 (1.83) also improved reducing power.
상기 결과들로부터 본 발명에 따른 파바톤 콩잎과 오미자 복합발효 조성물은 항산화 활성도 증진된다는 것을 알 수 있다.From the above results, it can be seen that the complex fermented composition of Pabaton bean leaf and Schisandra Schisandra according to the present invention also enhances antioxidant activity.
상기 결과들을 종합해 보면, 본 발명에 따른 파바톤 콩잎과 오미자 복합발효 조성물은 생리활성 물질(총 페놀릭스, 총 플라보노이드, 비당체 이소플라본)과 항산화 활성 및 소화효소 저해활성이 증대됨으로 인해 갱년기와 대사증후군 예방 식품으로 유용하게 이용될 수 있다.Combining the above results, the Pabaton bean leaf and Schisandra complex fermented composition according to the present invention have physiologically active substances (total phenolics, total flavonoids, non-saccharide isoflavones), antioxidant activity and digestive enzyme inhibitory activity. It can be usefully used as a food for preventing metabolic syndrome.
Claims (7)
ⅰ) 파바톤 콩잎과 오미자를 7 : 3의 중량비로 혼합한 혼합물을 준비하는 단계, 및
ⅱ) 상기 혼합물을 살균한 후 수탁번호 KACC91848P로 기탁된 락토바실러스 플란타륨 P1201 균주 및 수탁번호 KACC92159P로 기탁된 락토바실러스 브레비스 WCP02 균주의 복합종균을 접종하여 복합발효하는 단계를 포함하고,
상기 복합발효 조성물은 75%의 알파-글루코시다아제 저해활성과 46%의 췌장-라이파아제 저해활성을 갖고, 올레산 337mg/100 g과 리놀레산 1720 mg/100 g을 함유하는 것을 특징으로 하는 제조방법.
A method for producing a complex fermented composition of Pabaton soybean leaves and Schisandra, the method comprising:
i) preparing a mixture of pabaton bean leaf and omija in a weight ratio of 7: 3, and
ii) After sterilizing the mixture, inoculating a complex seed of Lactobacillus plantarum P1201 strain deposited with accession number KACC91848P and Lactobacillus brevis WCP02 strain deposited with accession number KACC92159P and performing complex fermentation,
The complex fermentation composition has 75% alpha-glucosidase inhibitory activity and 46% pancreatic-lipase inhibitory activity, and contains 337 mg/100 g of oleic acid and 1720 mg/100 g of linoleic acid. .
The method of claim 1, wherein the glycoside isoflavones contained in Fabaton soybean leaves are converted to 40% non-saccharide isoflavones.
A food comprising the complex fermented composition of Pabaton soybean leaves and Schisandra Schisandra produced by the method according to claim 1 .
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