KR101824147B1 - Soybean fermented composition having enhanced physiological activity and health functional food for alleviating climacteric or menopausal syndrome comprising the same - Google Patents
Soybean fermented composition having enhanced physiological activity and health functional food for alleviating climacteric or menopausal syndrome comprising the same Download PDFInfo
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- KR101824147B1 KR101824147B1 KR1020150157128A KR20150157128A KR101824147B1 KR 101824147 B1 KR101824147 B1 KR 101824147B1 KR 1020150157128 A KR1020150157128 A KR 1020150157128A KR 20150157128 A KR20150157128 A KR 20150157128A KR 101824147 B1 KR101824147 B1 KR 101824147B1
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- fermentation
- soybean
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- activity
- functional food
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- A—HUMAN NECESSITIES
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- A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
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- A—HUMAN NECESSITIES
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Abstract
본 발명에서는 락토바실러스 플란타륨 P1201 균주로 발효되고 총 페놀릭스 및 총 플라보노이드의 함량, 항산화 활성, 소화효소(탄수화물 및 지방 가수분해효소) 저해활성, 지방생성 억제효과, 및 비배당체-이소플라본(아글리콘)의 함량이 증진된 콩 발효조성물 및 이를 포함하는 갱년기 또는 폐경기 증상 개선용 건강기능성 식품이 제공된다.
본 발명에 따른 콩 발효조성물을 포함하는 건강기능성 식품은 안면홍조, 비만, 불면증, 우울증, 골다공증, 당뇨, 및 고혈압과 같은 갱년기 또는 폐경기 증상 개선에 효과적이다.In the present invention, the fermentation with Lactobacillus plantai P1201 strain and fermentation with total phenolic acid and total flavonoid content, antioxidative activity, digestive enzyme (carbohydrate and lipid hydrolyzing enzyme) inhibitory activity, lipogenesis inhibitory effect, and non-glycoside-isoflavone Aglycone) is enhanced, and a health functional food for improving menopausal symptoms or menopausal symptoms comprising the same.
The health functional food comprising the fermented soybean composition according to the present invention is effective for improving menopausal symptoms such as facial flushing, obesity, insomnia, depression, osteoporosis, diabetes, and hypertension.
Description
본 발명은 증진된 생리활성을 갖는 콩 발효조성물 및 이를 포함하는 갱년기 또는 폐경기 증상 개선용 건강기능성 식품에 관한 것으로, 더욱 상세하게는 락토바실러스 플란타륨 P1201 균주로 발효되고 총 페놀릭스 및 총 플라보노이드의 함량, 항산화 활성, 소화효소(탄수화물 및 지방 가수분해효소) 저해활성, 지방생성 억제효과, 및 비배당체-이소플라본(아글리콘)의 함량이 증진된 콩 발효조성물 및 이를 포함하는 갱년기 또는 폐경기 증상 개선용 건강기능성 식품에 관한 것이다.The present invention relates to a soybean fermentation composition having enhanced physiological activity and a health functional food for improving the symptoms of menopausal symptoms or menopausal symptoms comprising the same. More particularly, the present invention relates to a soybean fermentation composition having enhanced physiological activity and fermenting into a lactobacillus plantarium P1201 strain, Soybean fermentation composition with improved content, antioxidative activity, digestive enzyme (carbohydrate and lipid hydrolyzing enzyme) inhibitory activity, fat production inhibitory effect, and non-glycoside-isoflavone (aglycon) content and improvement of menopausal symptoms ≪ / RTI >
생활수준 향상 및 의학기술 발전으로 저출산 및 고령화 문제가 심화됨에 따라 각종 성인병 발생이 증가되고 있으며, 특히 갱년기 및 폐경기 여성의 경우 여성호르몬 감소에 의한 다양한 증상들이 발생되고 있으며 대사질환 (비만, 당뇨, 고혈압, 동맥경화 등)은 약 60% 이상 높은 것으로 알려져 이에 대한 근본적인 해결책이 요구되고 있다.As a result of increasing living standards and advancing medical technology, problems of low fertility and aging have been increasing, and various adult diseases have been increasing. Especially, in menopausal and postmenopausal women, various symptoms due to reduction of female hormone are being developed, and metabolic diseases (obesity, diabetes, hypertension , Arteriosclerosis, etc.) are known to be higher by about 60% or more, and a fundamental solution is required.
갱년기 증상은 남성 호르몬 또는 여성 호르몬의 분비 감소에 의해 유발되는 질환을 말한다. 특히, 여성의 경우 난소의 노화로 인한 에스트로겐의 분비 감소에 의해 폐경을 전후로 약 2 내지 10년에 걸쳐 나타나는 증상으로 고열, 안면홍조, 땀, 비만, 불면증, 우울증, 요실금, 통증, 골다공증, 당뇨, 및 고혈압과 같은 증상이 유발된다. 이러한 갱년기 증상의 개선을 위하여, 호르몬 대체요법, 비스테로이드계 제제 등의 약물 치료법이 개발되었으며, 현재 호르몬 대체요법이 가장 효과적인 방법으로 사용되고 있다. 그러나 호르몬을 장기 투여하는 경우에는 발암 위험성, 두통, 체중 증가와 같은 부작용 등이 발생하는 것으로 알려져 있다. 따라서 안전하고 효과적인 천연물 유래의 갱년기 및 폐경기 증상 개선제의 개발이 시도되고 있고, 여전히 요구되고 있다 (등록특허 제1087608호, 등록특허 제1272602호).Menopause is a disease caused by a decrease in the secretion of male hormones or female hormones. In particular, in women, ovarian senescence is caused by a reduction in the secretion of estrogen, which causes symptoms of menopausal symptoms for about 2 to 10 years before and after the onset of ovarian failure, such as high fever, facial flushing, sweating, obesity, insomnia, depression, urinary incontinence, And hypertension. In order to improve the symptoms of menopausal symptoms, hormone replacement therapy and non-steroid drugs have been developed and currently hormone replacement therapy is the most effective method. However, long-term administration of hormones is known to cause side effects such as cancer risk, headache, and weight gain. Therefore, development of safe and effective natural menopausal remedies for menopausal symptoms and postmenopausal symptoms has been and is still being demanded (Patent No. 1087608, Patent No. 1272602).
콩은 대표적인 식물성 단백질(35 ~ 45%) 식품일 뿐만 아니라 지방질(10 ~ 15%), 탄수화물(10%) 및 무기질(5%)의 균형적인 급원으로 특히 한국인에게는 두부, 장류, 두유, 콩기름 등의 식품의 형태로 소비되고 있다. 콩은 영양학적 측면뿐만 아니라 사포닌, 이소플라본, 레시틴과 같은 생리활성 물질에 관한 연구가 활발해지면서 기능성 식품의 재료로서 각광받고 있다. 특히 이소플라본은 식품의약품안전처 건강기능식품 고시형 원료로서 폐경기 이후 뼈 건강에 도움을 줄 수 있으며 일일 섭취권장량은 비배당체 이소플라본(aglycone, 아글리콘) 형태로 약 24 ~ 27 ㎍/g 으로 설정되어 있다. 그러나 콩 속의 이소플라본은 배당체인 β-글리코사이드가 25%, 말로닐-β-글리코사이드가 70 ~ 80%, 아세틸-β-글리코사이드가 5%이고, 비배당체인 아글리콘(aglycone)이 2% 정도만 존재한다 (Penalvo et al. 2004; Kim et al. 2005; Rostagno et al. 2004). 특히 콩의 다양한 생리활성은 대부분 콩에 함유된 아글리콘 형태(daizenin, gycitein, genistein의 3가지)의 이소플라본으로 보고되고 있다. 아글리콘 형태 중 제니스테인(genistein)은 항산화활성 뿐만 아니라 유방암 및 전립선암 등에 효과적이며 다이드제인(daidzein)은 폐경기 여성의 골다공증에 효과적인 것으로 알려져 있다. 따라서 콩의 이소플라본 함량을 증진시키고자 콩 품종, 재배 환경, 발아, 가공 처리 기술의 개발이 지속하여 진행되어 오고 있으며 아울러 비배당체인 아글리콘 형태로의 전환 기술 개발도 시도되고 있으나, 여전히 더 요구되고 있다(등록특허 1161231호, 등록특허 1302376호).Soybean is a balanced source of fat (10-15%), carbohydrate (10%) and minerals (5%) as well as representative vegetable protein (35-45%) food. Especially, soybean, soybean, soybean oil, soybean oil In the form of food. Soybean has been attracting attention as a material for functional foods as a result of research on physiologically active substances such as saponin, isoflavone, and lecithin as well as nutritional aspects. Especially, isoflavone is useful as a raw material for health food and functional food of the Food and Drug Administration, and it can help bone health after menopause. The daily recommended intake amount is about 24 ~ 27 ㎍ / g in the form of aglycone (aglycone) . However, in soybean isoflavones, 25% of the glycosides glycosides, 70% to 80% of the malonyl-β-glycosides, 5% of the acetyl-β-glycosides, and 2% of the aglycone, (Penalvo et al., 2004; Kim et al., 2005; Rostagno et al., 2004). Especially, various physiological activities of soybean have been reported as isoflavones of aglycon forms (daizenin, gycitein, genistein) contained in soybeans. Among the aglycone forms, genistein is effective for antioxidant activity as well as for breast cancer and prostate cancer, and daidzein is known to be effective for osteoporosis in postmenopausal women. Therefore, the development of soybean variety, cultivation environment, germination and processing technology has been proceeding in order to improve the soybean isoflavone content, and development of the non-glycosylated aglycon type conversion technology has been attempted, (Registered patent No. 1161231, registered patent No. 1302376).
종래 기술에서의 요구에 부응하기 위해 연구를 지속한 결과, 본 발명자들이 선행 연구에서 발굴하여 분리/동정하였던, 생균제제에 적합하고 공액리놀레산 생산성이 우수한 락토바실러스 플란타륨(Lactobacillus plantarum) P1201 균주를 이용하여 콩을 발효할 경우, 놀랍게도 갱년기 또는 폐경기 증상 개선에 필요한 생리활성 즉, 총 페놀릭스 및 총 플라보노이드의 함량, 항산화 활성, 소화효소(탄수화물 및 지방 가수분해효소) 저해활성, 지방생성 억제효과, 및 비배당체-이소플라본(아글리콘)의 함량이 증진된 발효조성물을 얻을 수 있다는 것을 확인하여 본 발명을 완성하게 되었다. As a result of continuing the study to meet the demand in the prior art, the inventors of the present invention found that Lactobacillus plantarum P1201 strain, which is suitable for probiotics and excellent in conjugated linoleic acid productivity, The antioxidant activity, the digestive enzyme (carbohydrate and lipid hydrolyzing enzyme) inhibitory activity, the inhibitory effect on lipogenesis, the antioxidant activity, the antioxidant activity, And the non-glycoside-isoflavone (aglycon) can be obtained. Thus, the present invention has been completed.
따라서 본 발명의 목적은 락토바실러스 플란타륨(Lactobacillus plantarum) P1201 균주로 발효되어 증진된 생리활성을 갖는 콩 발효조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a soybean fermentation composition having physiological activity enhanced by fermentation with Lactobacillus plantarum strain P1201.
또한 본 발명의 목적은 본 발명의 콩 발효조성물을 포함하는 갱년기 또는 폐경기 증상 개선용 건강기능성 식품을 제공하는 것이다.It is also an object of the present invention to provide a health functional food for improving menopausal symptoms or menopausal symptoms comprising the fermented soybean composition of the present invention.
또한 본 발명의 목적은 콩 발효조성물에서 비배당체-이소플라본(아글리콘)의 함량을 증진시키는 방법을 제공하는 것이다.It is also an object of the present invention to provide a method for enhancing the content of non-glycoside-isoflavone (aglycon) in soybean fermentation composition.
상기 목적을 달성하기 위하여, 본 발명은 락토바실러스 플란타륨 P1201 균주로 발효되어 증진된 생리활성을 갖는 콩 발효조성물을 제공한다.In order to accomplish the above object, the present invention provides a fermented soybean composition having physiological activity enhanced by fermentation with Lactobacillus plantai P1201 strain.
본 발명에서 콩 발효조성물의 발효 '균주'로는 본 발명자들이 분리, 동정하여 국립농업과학원 농업유전자원센터(KACC)에 2013년 8월 13일에 기탁한(수탁번호: KACC 91848P) 락토바실러스 플란타륨 P1201 균주가 바람직하다.In the present invention, the fermented soybean fermentation composition is referred to as a " strain ", which is isolated and identified by the present inventors and deposited on August 13, 2013 (Accession No .: KACC 91848P) at the National Institute of Agricultural Science and Technology (KACC) Lt; RTI ID = 0.0 > P1201 < / RTI >
본 발명에서 '발효'는 콩에 락토바실러스 플란타륨 P1201 균주를 접종하여 20 ~ 40 ℃에서 12시간 이상 발효시키는 단계를 포함하여 이루어질 수 있다. 균주 접종은 1.0 ~ 10.0%(v/v)이 바람직하다.In the present invention, the 'fermentation' may include a step of inoculating the soybean with Lactobacillus plantai P1201 and fermenting the soybean at 20 to 40 ° C for 12 hours or more. The inoculum of the strain is preferably 1.0 to 10.0% (v / v).
본 발명에서 콩은 생콩 또는 증자콩이 사용될 수 있으나, 100 ~ 120 ℃에서 30 ~ 60분간 증자된 증자콩이 바람직하다. 또한 콩은, 발효 전에, 셀룰라제, 프로테아제 및/또는 에스테라제의 가수분해 효소를 처리하여 콩 가수분해물로 제조한 후, 발효를 수행할 수 있다. 가수분해 효소의 처리량은 1 ~ 10 unit가 바람직하며, 이와 같은 효소처리에 의해 생리활성 물질의 생산성을 증가시킬 수 있다.In the present invention, beans may be soybeans or expanded beans, but preferred are beans grown at 100 to 120 DEG C for 30 to 60 minutes. In addition, the soybean can be subjected to fermentation after being made into soybean hydrolyzate by treating hydrolytic enzymes of cellulase, protease and / or esterase before fermentation. The throughput of the hydrolytic enzyme is preferably 1 to 10 units, and the productivity of the physiologically active substance can be increased by such an enzyme treatment.
본 발명에서 '생리활성' 총 페놀릭스 및 총 플라보노이드의 함량, 항산화 활성, 소화효소(탄수화물 및 지방 가수분해효소) 저해활성, 지방생성 억제효과, 및 비배당체-이소플라본(아글리콘)의 함량 등을 포함한다 (실험예 2~7). 이들 생리활성의 증진은 안면홍조, 비만, 불면증, 우울증, 골다공증, 당뇨, 및 고혈압과 같은 갱년기 또는 폐경기 증상 개선에 효과적이다.In the present invention, the content of total phenolic acid and total flavonoid, antioxidative activity, inhibitory activity of digestive enzymes (carbohydrate and lipid hydrolyzing enzyme), inhibitory effect on lipogenesis, and content of unglycoside-isoflavone (aglycon) etc. (Experimental Examples 2 to 7). These enhanced physiological activities are effective in improving menopausal symptoms such as facial flushing, obesity, insomnia, depression, osteoporosis, diabetes, and hypertension.
본 발명의 또 다른 목적에 따라서, 본 발명에 따른 콩 발효조성물을 포함하는 갱년기 또는 폐경기 증상 개선용 건강기능성 식품을 제공한다.According to another object of the present invention, there is provided a health functional food for improving menopausal symptoms or symptoms of menopausal symptoms comprising the fermented soybean composition according to the present invention.
본 발명의 갱년기 및 폐경기 증상은 안면홍조, 비만, 불면증, 우울증, 골다공증, 당뇨, 및 고혈압 등을 포함한다.The menopausal and menopausal symptoms of the present invention include facial flushing, obesity, insomnia, depression, osteoporosis, diabetes, and hypertension.
본 발명에서 '식품'은 콩제품 (두유, 두부, 콩고기, 콩스택, 콩과자), 유제품 (우유, 치즈), 발효유 (액상 요구르트, 호상 요구르트), 발효식품 (김치류, 장류, 피클), 발효 음료, 기타 가공품(환, 과립, 분말) 등의 형태가 될 수 있으나 이에 제한되지는 않는다.The term " food " in the present invention refers to a food such as soybean products (soybean milk, tofu, soybean meal, soybean stack, soybean cake), dairy products (milk, cheese), fermented milk (liquid yogurt, yogurt yogurt), fermented foods (kimchi, Fermented beverages, and other processed products (such as rings, granules, and powders), but are not limited thereto.
본 발명의 또 다른 목적에 따라서, 콩 발효조성물에서 비배당체-이소플라본(아글리콘)의 함량을 증진시키는 방법으로, 콩에 락토바실러스 플란타륨 P1201 균주를 접종하여 20 ~ 40 ℃에서 12시간 이상 발효시키는 단계를 포함하여 이루어지는 방법을 제공한다.According to another object of the present invention, there is provided a method for enhancing the content of non-glycoside-isoflavone (aglycon) in a fermented soybean composition, wherein soybean is inoculated with Lactobacillus plantai P1201 strain and cultured at 20 to 40 DEG C for 12 hours or more And a step of fermenting.
균주 접종, 콩은 상기에서 정의된 바와 같다.Strain inoculation, soybeans are as defined above.
또한 콩은, 발효 전에, 셀룰라제, 프로테아제 및/또는 에스테라제의 가수분해 효소를 처리하여 콩 가수분해물로 제조한 후, 발효를 수행할 수 있다. In addition, the soybean can be subjected to fermentation after being made into soybean hydrolyzate by treating hydrolytic enzymes of cellulase, protease and / or esterase before fermentation.
효소처리는 상기에서 정의된 바와 같다. The enzyme treatment is as defined above.
본 발명의 상기 방법에 따라 제조된 콩 발효조성물은 비배당체-이소플라본(아글리콘)의 함량이 현저히 증진된다 (표 3 참조).The soybean fermentation composition prepared according to the method of the present invention significantly improves the content of non-glycoside-isoflavone (aglycon) (see Table 3).
본 발명에 따른 발효조성물은 갱년기 또는 폐경기 증상 개선에 필요한 생리활성인, 총 페놀릭스 및 총 플라보노이드의 함량, 항산화 활성, 소화효소(탄수화물 및 지방 가수분해효소) 저해활성, 지방생성 억제효과, 및 비배당체-이소플라본(아글리콘)의 함량이 증진되어 있어서, 항산화, 체중 조절, 콜레스테롤 저하, 고지혈증 개선, 동맥경화 완화, 당뇨병 완화, 혈액순환 개선, 면역력 개선, 안면홍조 개선 및 골다공증 개선 등의 여성호르몬 불균형에 따른 갱년기 및 폐경기 증상 개선용 건강기능성 식품으로서 유용하다. The fermentation composition according to the present invention is useful as an antioxidant, a digestive enzyme (carbohydrate and lipid hydrolyzing enzyme) inhibiting activity, an inhibitory effect on fat production, and a ratio of total phenolic acid and total flavonoid, which are physiological activities necessary for improving menopausal symptoms or menopausal symptoms, The content of glycosides-isoflavones (aglycones) has been increased, and it is expected that the amount of female hormones such as antioxidants, weight control, cholesterol lowering, hyperlipidemia improvement, arteriosclerosis alleviation, diabetic relaxation, improvement of blood circulation, improvement of immunity, improvement of facial flushing and improvement of osteoporosis And is useful as a health functional food for improving menopausal symptoms and menopausal symptoms due to imbalance.
본 발명에 따른 콩 발효조성물을 포함하는 건강기능성 식품은 안면홍조, 비만, 불면증, 우울증, 골다공증, 당뇨, 및 고혈압과 같은 갱년기 또는 폐경기 증상 개선에 효과적이다.The health functional food comprising the fermented soybean composition according to the present invention is effective for improving menopausal symptoms such as facial flushing, obesity, insomnia, depression, osteoporosis, diabetes, and hypertension.
본 발명에 따라 제조된 콩 발효조성물은 비배당체-이소플라본(아글리콘)의 함량이 현저히 향상된다. The soybean fermentation composition prepared according to the present invention significantly improves the content of the non-glycoside-isoflavone (aglycon).
도 1은 본 발명에 따른 콩 발효조성물의 발효 전, 후의 이소플라본 유도체의 크로마토그램을 나타낸 그래프이다.
도 2는 본 발명에 따른 콩 발효조성물의 발효 전, 후의 이소플라본 유도체의 점유율 변화를 나타낸 그래프이다.
도 3은 본 발명에 따른 콩 발효조성물의 발효 전, 후의 농도별 처리에 따른 항산화 활성을 나타내는 그래프이다 (도 3a: DPPH 라디칼 소거활성, 도 3b: ABTS 라디칼 소거활성, 도 3c: 하드록실 라디칼 소거활성, 도 3d: FRAP 환원력).
도 4는 본 발명에 따른 콩 발효조성물의 발효 전, 후의 농도별 처리에 따른 소화효소 저해 활성을 나타내는 그래프이다 (도 4a: 알파-글루코시다아제 저해 활성, 도 4b: 알파-아밀라아제 저해 활성, 도 4c: 췌장 리파아제 저해 활성).
도 5는 3T3-L1 지방세포주에 분화를 유도하면서 본 발명에 따른 콩 발효조성물의 발효 전, 후의 농도별 처리 9일후 지방축적 정도를 나타낸 사진이다.
도 6은 3T3-L1 지방세포주에 분화를 유도하면서 본 발명에 따른 콩 발효조성물의 발효 전, 후의 농도별 처리 9일후 TG 생성량을 나타낸 그래프이다.
도 7은 3T3-L1 지방세포주에 분화를 유도하면서 본 발명에 따른 콩 발효조성물의 발효 전, 후의 농도별 처리 9일후 유전자 LPL (도 7a), C/EBP-α (도 7b), adiponectin (도 7c), aP2 (도 7d), FAS (도 7e) 및 ACC (도 7f)의 mRNA 발현 변화를 나타낸 사진이다.1 is a graph showing a chromatogram of an isoflavone derivative before and after fermentation of a fermented soybean composition according to the present invention.
2 is a graph showing changes in occupancy of isoflavone derivatives before and after fermentation of the fermented soybean composition according to the present invention.
FIG. 3 is a graph showing the antioxidative activity of the soybean fermentation composition according to the present invention before and after fermentation according to the concentration treatment (FIG. 3a: DPPH radical scavenging activity, FIG. 3b: ABTS radical scavenging activity, Activity, Figure 3d: FRAP reducing power).
FIG. 4 is a graph showing the digestive enzyme-inhibiting activity of the soybean fermentation composition according to the present invention before and after fermentation according to the concentration treatment (FIG. 4a: alpha-glucosidase inhibitory activity, FIG. 4b: alpha-amylase inhibitory activity, 4c: pancreatic lipase inhibitory activity).
FIG. 5 is a photograph showing the degree of fat accumulation after 9 days of treatment according to the concentration before and after fermentation of the soybean fermentation composition according to the present invention while inducing differentiation into 3T3-L1 fat cell line.
FIG. 6 is a graph showing the amount of TG produced after 9 days of treatment according to the concentration before and after fermentation of the soybean fermentation composition according to the present invention while inducing differentiation into 3T3-L1 adipocytic cell line. FIG.
FIG. 7 shows the results of the differentiation of the 3T3-L1 adipocyte lineage, the LPL (FIG. 7A), C / EBP-α (FIG. 7B) and adiponectin 7c), aP2 (Fig. 7d), FAS (Fig. 7e) and ACC (Fig. 7f).
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 will be described in more detail by the following examples. These examples are provided for illustrating the present invention, and the scope of the present invention should not be limited thereby.
실시예Example
참고예Reference example 1 : 접종 균주 준비 1: Inoculation strain preparation
본 발명의 콩 발효조성물의 발효를 위한 국립농업과학원 농업유전자원센터에 기탁된 락토바실러스 플란타륨 P1201 균주 (수탁번호: KACC 91848P)를 MRS 액체배지 (Difco사, USA)에서 24시간 배양한 후, 발아맥아 100 g에 5배의 정제수를 가하여 100℃에서 6시간 추출하여 거즈로 여과하여 제조한 맥아배지에 접종하여 48시간 혼합 배양하여 접종을 위한 종균으로 준비하였다.Lactobacillus plantarum P1201 strain (Accession No .: KACC 91848P) deposited at the National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology, for fermentation of the soybean fermentation composition of the present invention, was cultured in MRS liquid medium (Difco, USA) for 24 hours , 5 g of purified water was added to 100 g of germinated malt, and the mixture was extracted for 6 hours at 100 ° C for filtration through gauze. The mixture was inoculated for 48 hours to prepare seeds for inoculation.
제조예 : 콩 발효조성물 제조Preparation Example: Preparation of Soybean Fermentation Composition
농촌진흥청 국립식량과학원 남부작물부에서 2014년 수확하여 공급받은 대원콩 1kg을 흐르는 수돗물에 3회 세척하고 콩 무게의 약 3배 분량의 물을 첨가하여 25 ℃에서 6시간 수침한 후 100 ℃에서 30분간 증자하여 50 ~ 55 ℃에서 2 ~ 3일간 건조한 후 분쇄기에서 분쇄하여 분말화하였다.Rural Development Administration In the southern crop department of the National Institute of Food Science, it was washed 3 times in tap water flowing 1kg of the raw soybean which was harvested and supplied in 2014, and about three times as much as the weight of soybean was added. After watering at 25 ℃ for 6 hours, Minute, dried at 50 to 55 ° C for 2 to 3 days, pulverized in a pulverizer, and pulverized.
이와 같이 준비된 증자콩 분말 500 g에 설탕 5%(25 g)와 물 5 L를 첨가하여 혼합한 후, 121℃에서 15분간 살균하였고 상온으로 냉각한 후, 셀룰라아제, 프로테아제 및 에스테라제 (Sigma-Aldrich 사)를 최종 10 unit 되게 첨가하여 37 ℃에서 24시간 동안 처리하여 콩 가수분해물로 제조하여 사용하였다.5 g (25 g) of sugar and 5 L of water were added to 500 g of the thus-prepared soybean powder, and the mixture was sterilized at 121 ° C for 15 minutes. After cooling to room temperature, the cellulase, protease and esterase Aldrich) was added in a final volume of 10 units and treated at 37 ° C for 24 hours to prepare soybean hydrolyzate.
제조된 콩 가수분해물 5 L에 참고예에서 준비된 실시예 종균 (락토바실러스 플란타륨 P1201 균주) 배양액을 5%(v/v)로 접종한 후, 약 37℃에서 72시간 정치 발효시켜 콩 발효조성물을 완성하였다.5 L of the soybean hydrolyzate prepared in Reference Example was inoculated at 5% (v / v) in a culture medium of the Example prepared in Reference Example (Lactobacillus plantai P1201 strain), and then allowed to stand at 37 ° C for 72 hours for constant fermentation, .
실험예 1. 콩 발효조성물의 이화학적 특성Experimental Example 1. Physicochemical properties of soybean fermented composition
본 발명에 따른 콩 발효조성물의 발효 전, 후에 이화학적 특성을 분석하여 표 1에 나타냈다.Before and after fermentation, the soybean fermentation composition according to the present invention was analyzed for its physicochemical properties and shown in Table 1.
pH는 pH 측정기를 사용하여 측정하였고, 산도 측정은 콩 발효조성물 1 ml를 0.1N-NaOH 용액으로 pH 8.4±2까지 중화시키는데 소요된 0.1N-NaOH의 소비 ml수를 구하고 젖산(lactic acid)양으로 환산하여 %로 총산을 나타내었다. 생균수 측정은 콩 발효조성물 1 ml를 멸균생리식염수에 단계별로 희석하여 MRSA 배지에 도말하고 30 ℃에서 48시간 배양 후 나타난 젖산균 특유의 콜로니를 측정하여 생균수(log cfu/g)로 나타내었다.The pH was measured using a pH meter. The acidity was determined by measuring the consumption ml of 0.1 N NaOH required to neutralize 1 ml of soybean fermented composition with 0.1 N NaOH solution to pH 8.4 ± 2, and the amount of lactic acid In terms of%. The viable cell count was expressed as the number of viable cells (log cfu / g) after 1 ml of the fermented soybean composition was diluted in sterilized physiological saline stepwise, plated on MRSA medium, and cultured at 30 ° C for 48 hours for colonies characteristic of lactic acid bacteria.
(%, 젖산 기준)Acidity
(%, Based on lactic acid)
(log cfu/g)Viable cell count
(log cfu / g)
표 1에 나타난 바와 같이, 발효 시작 전에는 pH가 6.01, 산도는 0.43%를 나타내었으며, 발효 후에는 젖산 생성에 기인하여 pH는 4.17로 감소하고 산도는 1.12%로 상승하였다. 생균수 역시 발효 전에 9.21 log cfu/g이었고 발효 후에는 10.49 log cfu/g으로 발효가 진행됨에 따라 증가하는 경향을 나타내었다. 이들 결과로부터 본 발효조성물은 발효식품에 적합한 이화학적 특성을 가짐을 알 수 있다.As shown in Table 1, the pH was 6.01 and the acidity was 0.43% before the start of fermentation. After fermentation, the pH decreased to 4.17 and the acidity rose to 1.12% due to lactic acid production. The viable cell count was also increased to 9.21 log cfu / g before fermentation and to 10.49 log cfu / g after fermentation. From these results, it can be seen that the present fermentation composition has suitable physicochemical properties for the fermented food.
참고예Reference example 2 : 생리활성 분석시료 준비 2: Analysis of physiological activity Sample preparation
제조예에서 발효 전 (발효 0시간), 발효 후 (발효 72시간)에 각각의 콩 발효조성물을 500 ml씩 취하여 -70℃에서 동결건조시킨 후 분말화하였다.500 ml of each soybean fermentation composition was taken at the pre-fermentation time (
동결건조된 분말 시료 10 g에 50% 메탄올 200 ml을 가한 후 20℃, 300 rpm에서 12시간 추출한 후 여과지로 1차 여과한 후 0.45 μm 여과필터로 2차 여과하여 얻은 여과액 시료를 하기 총 페놀릭스 함량, 총 플라보노이드 함량과 이소플라본 함량의 측정에 사용하였다.To 10 g of the lyophilized powder sample, 200 ml of 50% methanol was added, and the mixture was extracted at 20 ° C and 300 rpm for 12 hours. Then, the filtrate was subjected to primary filtration through a filter paper, followed by secondary filtration through a 0.45 μm filtration filter. Rix content, total flavonoid content and isoflavone content.
동결건조된 분말 시료 20 g에 50% 메탄올을 200 ml 가하여 상온에서 12시간 동안 교반하여 추출하였다. 이 후 원심분리하여 상등액을 모은 후 여과지(No. 2, Whatman, Tokyo Roshi Kaisha, Ltd., Tokyo, Japan)로 여과하여 모은 후 여과액을 감압농축기(EYELA, Tokyo, Rikakikai Co., Tokyo, Japan)를 이용하여 60℃에서 감압 농축 후 제조하였다. 농축된 시료는 추출용매로 녹여 각각 0.25, 0.50 및 1.0 mg/ml 농도별 분석 시료를 제조하여 항산화 활성 및 소화효소 저해활성을 평가하였다.200 ml of 50% methanol was added to 20 g of the lyophilized powder sample, and the mixture was stirred at room temperature for 12 hours. After centrifugation, the supernatant was collected and filtered through a filter paper (No. 2, Whatman, Tokyo Roshi Kaisha, Tokyo, Japan), and the filtrate was concentrated in a vacuum condenser (EYELA, Tokyo, Rikakikai Co., Tokyo, Japan ) At 60 < [deg.] ≫ C. Concentrated samples were dissolved in extraction solvent to prepare analytical samples at 0.25, 0.50 and 1.0 mg / ml, respectively. Antioxidant activity and digestive enzyme inhibitory activity were evaluated.
50% 메탄올 추출물을 건조시켜 제공된 분말 시료를 DMSO에 녹여 100, 500 및 1,000 ㎍/ml 농도별 분석 시료를 제조하여 지방생성 억제능, TG 생성량 감소능을 평가하였다.The 50% methanol extracts were dried, and the powder samples were dissolved in DMSO to prepare analytical samples of 100, 500 and 1,000 ㎍ / ml concentration. The ability to inhibit lipogenesis and reduce TG production was evaluated.
실험예 1. 본 발명에 따른 콩 발효조성물의 총 페놀릭스 및 총 플라보노이드 함량 분석Experimental Example 1. Analysis of total phenolics and total flavonoid contents of soybean fermentation composition according to the present invention
총 페놀릭스 함량은 Folin Denis법(1912)으로 측정하였다.Total phenolic content was measured by Folin Denis method (1912).
구체적으로는 참고예 2에서 준비된 본 발명의 콩 발효조성물의 각각의 여과액 시료를 시험관에 0.5 ml 분주하고 여기에 25% Na2CO3 용액 0.5 ml를 첨가하여 3분간 정치시켰다. 다시 2N-Folin-Ciocalteu 페놀 시약 0.25 ml를 첨가하여 혼합한 다음 30 ℃에서 1시간 동안 정치시킨 후 750 nm에서 흡광도를 측정하였다. 이때 총 페놀릭스 함량은 갈산을 이용하여 작성한 표준곡선으로부터 함량을 구하여 갈산에 상당하는 양으로 계산하였고 그 결과를 표 2에 나타냈다.Specifically, 0.5 ml of each of the filtrate samples of the soybean fermentation composition of the present invention prepared in Reference Example 2 was put into a test tube, and 25% Na 2 CO 3 0.5 ml was added and allowed to stand for 3 minutes. After addition of 0.25 ml of 2N-Folin-Ciocalteu phenol reagent, the mixture was allowed to stand at 30 ° C for 1 hour, and the absorbance was measured at 750 nm. The content of total phenolics was calculated from the standard curve prepared using gallic acid, and the content was calculated as the amount equivalent to gallic acid. The results are shown in Table 2.
총 플라보노이드 함량은 Davis 법(1999)을 변형하여 측정하였다. The total flavonoid content was measured by modifying the Davis method (1999).
구체적으로는 참고예 2에서 준비된 본 발명의 콩 발효조성물의 각각의 여과액 시료 0.5 ml를 음성대조구와 측정용액으로 나누어 두 개의 시험관에 취하고 여기에 디에틸렌 글리콜 1.0 ml 및 1 N-NaOH 0.01 ml를 가하여 미리 예열해놓은 37℃ 항온수조에서 1시간 방치시켰다. 그 후 분광광도계(Spectronic 2D)를 이용하여 420 nm에서 흡광도를 측정하였고 루틴(rutin)을 이용하여 작성한 표준 검량곡선으로부터 값을 산출하여 상기 표 2에 나타냈다.Specifically, 0.5 ml of each of the filtrate samples of the soybean fermentation composition of the present invention prepared in Referential Example 2 was divided into a negative control and a measurement solution and taken in two test tubes. Then, 1.0 ml of diethylene glycol and 0.01 ml of 1 N NaOH And allowed to stand in a preheated 37 ° C water bath for 1 hour. Then, the absorbance was measured at 420 nm using a spectrophotometer (Spectronic 2D), and the value was calculated from a standard calibration curve prepared using a rutin. The results are shown in Table 2 above.
표 2에서 확인되는 바와 같이, 발효가 진행됨에 따라 총 페놀릭스 함량 및 총 플라보노이드 함량은 증가하였고, 72시간 발효 후 총 페놀릭스 함량은 약 24% 증가하였고 특히 총 플라보노이드 함량은 약 2.7배 증가하였음을 알 수 있다.As shown in Table 2, the total phenolic content and the total flavonoid content were increased with fermentation, and the total phenolic content was increased by about 24% after 72 hours of fermentation. Especially, the total flavonoid content was increased about 2.7 times Able to know.
실험예 2. 이소플라본 함량 분석Experimental Example 2. Analysis of isoflavone content
이소플라본 함량은 Cho 등(2011)의 방법에 따라 HPLC로 분석하였다.Isoflavone content was analyzed by HPLC according to the method of Cho et al. (2011).
분석 컬럼은 Lichrophore 100 RP C18 컬럼 (4.6 × 250 mm, 5 μm, Merck, Germany)을 사용하였고 이동상 용매는 0.2% 글라시얼 아세트산 수용액 (A)와 0.2% 아세토니트릴 수용액 (B)으로 분석하였다. 이동상 조건으로는 A 용매 기준으로 0분-100%, 15분-90%, 25분-80%, 35분-75%, 45분-65% 및 50분-65%로 유지시켰다. 참고예 2에서 준비된 본 발명의 콩 발효조성물의 각각의 여과액 시료 20 ㎕를 주입하였으며 이동상 속도는 30℃에서 1 ml/min로 유지하였다. 검출기는 디오드 어레이 디텍터 (Agilent 1200 series, Agilent Co.)를 사용하였으며 흡광도 254 nm에서 정량하여, 각각의 결과를 도 1, 도 2 및 표 3에 나타냈다.The analytical column was a
도 1에 나타낸 본 발명에 따른 콩 발효조성물의 발효 전, 후의 이소플라본 유도체의 크로마토그램에서 확인되는 바와 같이, 콩 발효 추출물은 발효 전후, 공통적으로 9종류의 이소플라본 유도체(daidzin, glycitin, genistin, malonyldaidzin, malonylglycitin, malonylgenistin, daidzein, glycitein, 및 genistein)가 검출되었으며, 발효 전에 검출된 주요 유도체로는 배당체 형태의 다이드진(daidzin), 글라이시틴(glycitin) 및 제니스틴(genistin) (피크 1, 2 및 3)이 높은 함량으로 측정되었으나, 발효 후에는 이러한 배당체 형태가 대부분 감소하였고 이에 상응하여 비배당체 형태의 다이드제인(daidzein) 및 제니스테인(genistein) (피크 9 및 12)이 크게 증가하여 주요 이소플라본 유도체로 검출됨을 알 수 있다.As shown in the chromatogram of the isoflavone derivative before and after fermentation of the soybean fermentation composition according to the present invention shown in FIG. 1, the soybean fermentation extract commonly contains nine kinds of isoflavone derivatives (daidzin, glycitin, genistin, malonylglycitin, malonylgenistin, daidzein, glycitein, and genistein were detected. The major derivatives detected before fermentation were glycosides such as daidzin, glycitin and genistin (
(㎍/g)Isoflavone content
(/ / G)
**tr: trace(<0.002 ㎍/g)
***nd: 검출되지 않음* All experiments were repeated three times
** tr: trace (<0.002 / / g)
*** nd: Not detected
상기 표 3에 나타난 바와 같이, 본 발명에 따른 콩 발효조성물의 비배당체 형태의 이소플라본인 아글리콘의 함량 (339.58 ㎍/g)은 발효 전 (72.91 ㎍/g)에 비하여 약 4.7배 증가하였고, 특히 다이드제인(daidzein) 및 제니스테인 (genistein)의 함량은 발효 전과 비교했을 때 약 10배 증가하였음을 확인할 수 있다.As shown in Table 3, the content of aglycon (339.58 占 퐂 / g), an isoflavone of the non-glycosidic form of the soybean fermentation composition according to the present invention, increased about 4.7 times as compared with that before fermentation (72.91 占 퐂 / Especially, the contents of daidzein and genistein increased about 10 times as compared with those before fermentation.
또한 도 2에 나타낸 본 발명에 따른 콩 발효조성물의 발효 전, 후의 이소플라본 유도체의 점유율 변화에서 확인할 수 있듯이, 배당체인 글리코사이드 이소플라본은 발효 전 89.21%이었던 것이 발효 후 20.52%로 감소하였고 비배당체인 아글리콘 이소플라본은 발효 전 8.11%였던 것이 74.85%로 점유율이 크게 증가한 바, 본 발명에 따른 발효에 의해 아글리콘으로의 전환이 매우 높게 이루어졌음을 알 수 있다.As can be seen from the change in the occupancy of the isoflavone derivative before and after fermentation of the soybean fermentation composition according to the present invention shown in FIG. 2, glycoside isoflavone, which is a glycoside, was decreased from 89.21% before fermentation to 20.52% The aglycon isoflavone, which was 8.11% before fermentation, increased to 74.85%, which shows that the conversion to aglycon was very high due to fermentation according to the present invention.
실험예 3. 콩 발효조성물의 항산화 활성 검정Experimental Example 3. Antioxidant Activity Test of Soybean Fermentation Composition
본 발명에 따른 콩 발효조성물의 항산화 활성은 DPPH 라디칼 소거 활성, ABTS 라디칼 소거 활성 및 하이드록실 라디칼 소거 활성, 및 환원력(FRAP)의 4가지 방법으로 측정하였다.The antioxidative activity of the soybean fermentation composition according to the present invention was measured by four methods of DPPH radical scavenging activity, ABTS radical scavenging activity and hydroxyl radical scavenging activity, and reducing power (FRAP).
<DPPH 라디칼 소거 활성>≪ DPPH radical scavenging activity >
DPPH(1,1-diphenyl-2-picrylhydrazyl, Sigma D9132, FW 393.4, C18H12N5O6)는 매우 안정한 라디칼로서 525 nm에서 특정 흡광도를 나타내는 보라색 화합물이다. DPPH 라디칼 소거활성을 측정하여 항산화성을 확인하였다.DPPH (1,1-diphenyl-2- picrylhydrazyl, Sigma D9132, FW 393.4, C 18 H 12 N 5 O 6) is a purple compound showing a specific absorption at 525 nm as an extremely stable radical. DPPH radical scavenging activity was measured to confirm antioxidant activity.
구체적으로는 DPPH 용액 0.8 ml와 참고예 2에서 준비된 발효 전후 농도별 각각의 분석 시료 0.2 ml를 가하고 이를 혼합하여 암실에서 30분간 방치시켰으며 반응 후 분광광도계를 사용하여 525 nm에서 흡광도를 측정하였다. 음성대조구 실험은 시료 대신에 PBS 완충액(NaCl 8.76 g, NaH2PO4 0.11 g, Na2HPO4 0.596 g)을 사용하였고 라디칼 소거활성은 시료용액의 첨가구와 무첨가구 사이의 흡광도 값을 구하여 아래와 같은 식에 대입하여 백분율(%)로 표시하였으며 그 결과를 도 3a에 나타냈다:Specifically, 0.8 ml of the DPPH solution and 0.2 ml of each analytical sample prepared before and after the fermentation prepared in Reference Example 2 were added, and the mixture was allowed to stand in the dark room for 30 minutes. After the reaction, the absorbance was measured at 525 nm using a spectrophotometer. In the negative control experiment, PBS buffer (NaCl 8.76 g, NaH 2 PO 4 0.11 g, Na 2 HPO 4 0.596 g) was used instead of the sample, and the radical scavenging activity was determined by the absorbance value between the addition of the sample solution and the no addition, (%) ≪ / RTI > and the results are shown in Figure 3a: < RTI ID =
DPPH 라디칼 소거활성(%) =DPPH radical scavenging activity (%) =
[1-(음성대조구 흡광도 ÷ 실험구 흡광도)] × 100 [1- (negative control absorbance / experimental absorbance)] × 100
도 3a에 의하면, DPPH 라디칼 소거활성은 발효 전에 비하여 발효 후에, 분석 시료의 농도의존적으로 증가했음을 확인할 수 있다.3A, it can be confirmed that the DPPH radical scavenging activity was increased in a concentration-dependent manner after the fermentation compared with before the fermentation.
<ABTS 라디칼 소거활성><ABTS radical scavenging activity>
ABTS 라디칼 소거활성은 2-azino-bis의 색을 띤 라디칼의 감소정도에 따라 항산화능을 검사하는 방법이다. 즉 이 방법은 시료와 표준물질(Trolox, 6-hydroxy -2,5,7,8-tetramethylchroman-2-carboxylic acid)의 값과 비교하여 항산화능을 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.The ABTS radical scavenging activity is a method of examining the antioxidant ability of 2-azino-bis according to the degree of reduction of colored radicals. In other words, this method is a method to measure the antioxidant ability by comparing with the value of sample and reference material (Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) The cations (ABTS + ) are erased and decolorized into cyan. At this time, the degree of ABTS cations (ABTS + ) can be detected by measuring the absorbance and the decoloring reaction is completed within 1 minute.
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 참고예 2에서 준비된 발효 전후 농도별 각각의 분석 시료 1.0 ml를 첨가하여 3분간 반응시켜 732 nm에서 흡광도를 측정하였다. 음성대조구 실험은 시료 대신에 PBS 완충액(NaCl 8.76 g, NaH2PO4 0.11 g, Na2HPO4 0.596 g)을 사용하였고 라디칼 소거활성은 시료용액의 첨가구와 무첨가구 사이의 흡광도 값을 구하여 아래와 같은 식에 대입하여 백분율(%)로 표시하였으며, 그 결과를 도 3b에 나타냈다.Specifically, ABTS + radicals were formed by mixing 5 ml of 7 mM ABTS + 5 ml of 2.45 mM K 2 S 2 O 8 in a dark room for 12 to 16 hours. After incubation at 732 nm, the absorbance of ABTS + 0.9 ml was adjusted to 0.7 ± 0.02, and 1.0 ml of each sample was added to the sample before and after fermentation for 3 min. The absorbance was measured at 732 nm . In the negative control experiment, PBS buffer (NaCl 8.76 g, NaH 2 PO 4 0.11 g, Na 2 HPO 4 0.596 g) was used instead of the sample, and the radical scavenging activity was determined by the absorbance value between the addition of the sample solution and the no addition, (%) In the formula, The results are shown in FIG. 3B.
ABTS 라디칼 소거활성(%) =ABTS radical scavenging activity (%) =
[1-(음성대조구 흡광도 ÷ 실험구 흡광도)] × 100 [1- (negative control absorbance / experimental absorbance)] × 100
도 3b에 나타난 바와 같이, ABTS 라디칼 소거활성은 발효 전과 비교했을 때 발효 후에 훨씬 증가하였고 특히 본 발명의 콩 발효조성물의 0.25 mg/g 농도의 시료 처리시 51.30%를, 1.0 mg/g의 고농도 처리 시 67.30%의 우수한 라디칼 소거활성을 보였다.As shown in FIG. 3B, the ABTS radical scavenging activity was significantly increased after fermentation compared to before fermentation. In particular, 51.30% of 0.25 mg / g of the soybean fermentation composition of the present invention was treated at a high concentration of 1.0 mg / g And showed an excellent radical scavenging activity of 67.30%.
<하이드록실 라디칼 소거활성><Hydroxyl 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 참고예 2에서 준비된 발효 전후 농도별 각각의 분석 시료 1.4 ml를 혼합하여 37℃에서 4시간 반응시켰다. 이 혼합액에 1% 티오바르비투르산(thiobarbituric acid)/증류수와 2.8% 트리클로로아세트산 (trichloroaceric acid)/증류수를 각각 1 ml를 가하여 100 ℃에서 20분간 가열하여 발색시키고 적당히 냉각시킨 후 520 nm에서 흡광도를 측정하였다. 음성대조구 실험은 시료 대신에 PBS 완충액(NaCl 8.76 g, NaH2PO4 0.11 g, Na2HPO4 0.596 g)을 사용하였고 하이드록실 라디칼 소거활성은 시료용액의 첨가구와 무첨가구 사이의 흡광도 차이를 백분율(%)로 나타냈으며 그 결과를 도 3c에 도시했다.Hydroxyl radical scavenging activity was measured 10 mM FeSO 4. 0.2 ml of 7H 2 O-EDTA, 0.2 ml of 10 mM 2-deoxyribose, 0.2 ml of 10 mM H 2 O 2 and 1.4 ml of each analytical sample prepared before and after fermentation prepared in Reference Example 2 were mixed and incubated at 37 ° C for 4 hours Lt; / RTI > To this mixture was added 1 ml of 1% thiobarbituric acid / distilled water and 2.8% trichloroaceric acid / distilled water, and the mixture was heated at 100 ° C for 20 minutes to develop color. After cooling appropriately, absorbance Were measured. In the negative control experiment, PBS buffer solution (8.76 g of NaCl, 0.11 g of NaH 2 PO 4 , and 0.596 g of Na 2 HPO 4 ) was used instead of the sample, and the hydroxyl radical scavenging activity was determined by the percentage of absorbance difference between the addition of the sample solution and the non- (%). The results are shown in Fig. 3C.
도 3c에 나타난 바와 같이, 발효 전에는 처리 농도에 따른 하이드록실 라디칼 소거활성이 큰 차이 없이 유사하였으나, 발효 후에는 하이드록실 라디칼 소거활성이 0.25 mg/g 농도에서 46.68%, 1.0 mg/g 농도에서 57.23%로 농도의존적으로 크게 증진됨을 확인할 수 있다.As shown in FIG. 3C, the hydroxyl radical scavenging activity was similar to that of the fermentation broth without fermentation, but the hydroxyl radical scavenging activity was 46.68% at the concentration of 0.25 mg / g and 57.23% at the concentration of 1.0 mg / %, Respectively, in a concentration-dependent manner.
<FRAP 환원력><FRAP reducing power>
FRAP (Ferric reducing antioxidant power) 환원력 분석은 화합물의 환원력을 측정하는 방법으로 Fe3+를 Fe2+로 환원시키는 힘을 측정하는 방법이다.FRAP (Ferric reducing antioxidant power) The reduction power analysis is a method of measuring the reducing power of Fe 3+ to Fe 2+ by measuring the reducing power of a compound.
구체적으로는 FeⅢ-TPTZ(ferric tripyridyl triazine)가 시료의 환원력에 의하여 푸른색의 FeⅡ-TPTZ(ferrous tripyridyl triazine)으로 환원될 때 흡광도를 측정하여 항산화성을 알아보는 것이다. FRAP 환원력 분석에서 반응액으로는 30mM 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 준비하였으며, 아세테이트 완충액, TPTZ 용액 및 FeCl3 용액을 10:1:1 (v/v/v)로 혼합하여 37 ℃에서 15분간 예비반응을 시켜두었다. 참고예 2에서 준비된 발효 전후 농도별 각각의 분석 시료 50 ㎕와 FRAP 시약 950 ㎕를 시험관에 분주한 후 약 15분간 반응시키고 마이크로플레이트 리더 (Biorad 3055, Sweden)를 사용하여 593 nm에서 흡광도를 측정하여 그 결과를 도 3d에 나타냈다.Specifically, when Fe III -TPTZ (ferric tripyridyl triazine) is reduced to blue Fe II -TPTZ (ferrous tripyridyl triazine) by the reducing power of the sample, the antioxidant activity is measured by measuring the absorbance. FRAP analysis reaction solution to the reducing power is 2,4,6- 10 mM dissolved in 30mM acetate buffer (pH 3.6), 40 mM HCl in a tree-pyridyl -s- triazine (TPTZ, T1253, C 18 H 12 N 6, MW312 (PH 7.0) and 20 mM FeCl 3 (F7134, MW 162.20, in DW) were prepared, and the acetate buffer, the TPTZ solution and the FeCl 3 solution were mixed at a ratio of 10: 1: 1 (v / v / v) Preliminary reaction was allowed. 50 μl of each analytical sample and 950 μl of FRAP reagent prepared in Referential Example 2 were placed in a test tube, reacted for about 15 minutes, and the absorbance was measured at 593 nm using a microplate reader (Biorad 3055, Sweden) The results are shown in Fig.
도 3d에 도시된 바와 같이, FRAP 환원력도 발효 전(0.13)과 비교했을 때 발효 후(0.58)에 훨씬 증가하였고 특히 본 발명의 콩 발효조성물의 농도가 높아짐에 따라서 환원력이 크게 증가하였으며, 1.0 mg/g의 고농도 처리시 환원력이 약 3배 이상 증가함을 보였다.As shown in FIG. 3D, the FRAP reducing power was significantly increased after fermentation (0.58) as compared with before fermentation (0.13). In particular, as the soybean fermentation composition of the present invention was increased, the reducing power was greatly increased and 1.0 mg / g, the reducing power increased about 3 times.
따라서 상기 결과들로부터 본 발명에 따른 콩 발효조성물은 항산화 활성이 크게 증진된다는 것을 알 수 있다.Therefore, it can be seen from the above results that the soybean fermentation composition according to the present invention greatly enhances the antioxidant activity.
실험예 4. 콩 발효조성물의 소화효소 저해 활성 검정Experimental Example 4. Digestive enzyme inhibitory activity assay of soybean fermented composition
본 발명에 따른 콩 발효조성물의 소화효소 저해활성은 당뇨병 개선효과의 지표인 알파-글루코시다아제(α-glucosidase) 저해활성, 알파-아밀라제(α-amylase) 저해활성, 및 비만 개선효과의 지표인 췌장 리파아제(pancreatic lipase) 저해활성의 3가지 방법으로 측정하였다.The digestive enzyme inhibitory activity of the fermented soybean composition according to the present invention is an indicator of the inhibitory effect of alpha-glucosidase, alpha -amylase, and obesity, which are indicators of the diabetes-improving effect Pancreatic lipase inhibitory activity was measured by three methods.
<알파-글루코시다아제 저해 활성>≪ Alpha-Glucosidase Inhibitory Activity >
알파-글루코시다아제 저해 활성은 참고예 2에서 준비된 발효 전후 농도별 각각의 분석 시료 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 최종 반응을 정지시킨 후 420 nm에서 분광광도계를 이용하여 흡광도를 측정하였다. 음성대조구는 시료 대신에 추출용매를 취하였으며 시료용액의 첨가구와 무첨가구 사이의 흡광도 차이를 백분율(%)로 나타내어 그 결과를 도 4a에 나타냈다.The α-glucosidase inhibitory activity was determined by measuring the concentration of each of 50 μl of the assay sample, 50 μl of the α-glucosidase (0.5 U / ml) enzyme solution, 200 mM sodium phosphate buffer solution (pH 6.8) Were mixed and preliminarily reacted at 37 占 폚 for 10 minutes. Subsequently, 100 μl of p -NPG (5 mM) dissolved in sodium phosphate buffer solution (pH 6.8) was added, and the mixture was further reacted at 37 ° C for 10 minutes. To the reaction mixture was added Na 2 CO 3 (100 mM) was added to stop the final reaction and the absorbance was measured at 420 nm using a spectrophotometer. The negative control was taken as the extraction solvent instead of the sample. The difference in absorbance between the addition of the sample solution and the non-addition solution was expressed as a percentage (%), and the results are shown in FIG. 4A.
도 4a에 도시된 바와 같이, 알파-글루코시다아제 저해 활성은 발효 전에 비하여 발효 후에 증가하였고, 특히 0.50 mg/g 농도 처리시 알파-글루코시다아제 저해 활성은 발효 전에는 23.57%이었으나 발효 후에는 41.22%로 약 2배 향상된 것을 알 수 있다.As shown in FIG. 4A, the alpha-glucosidase inhibitory activity was increased after fermentation, and the alpha-glucosidase inhibitory activity at the concentration of 0.50 mg / g was 23.57% before fermentation, but 41.22% Which is about twice as high as that of the first embodiment.
<알파-아밀라제 저해 활성>≪ Alpha-amylase inhibitory activity >
알파-아밀라제 저해 활성은 참고예 2에서 준비된 발효 전후 농도별 각각의 분석 시료 50 ㎕를 시험관에 분주한 후 α-아밀라제(1.0 U/ml) 효소용액 50 ㎕와 200 mM 인산나트륨 완충용액(pH 6.8) 50 ㎕를 첨가하여 37 ℃에서 10분간 예비 반응을 시켰다. 반응액에 200 mM 인산나트륨 완충용액(pH 6.8)에 녹인 1% 전분용액을 0.25 ml를 가하여 다시 37 ℃에서 10분간 반응시켰다. 그리고 나서 0.5 N-NaOH 및 30% 타르타르산나트륨칼륨을 용해한 48 mM DNS 시약을 0.25 ml 첨가하여 100 ℃에서 20분간 끓여 발색 및 냉각시킨 후 570 nm에서 흡광도를 측정하였다. 음성대조구는 시료 대신에 추출용매를 취하였으며 시료용액의 첨가구와 무첨가구 사이의 흡광도 차이를 백분율(%)로 나타내어 그 결과를 도 4b에 나타냈다.The α-amylase inhibitory activity was determined by dividing 50 μl of each analytical sample prepared before and after fermentation prepared in Reference Example 2 into a test tube, adding 50 μl of α-amylase (1.0 U / ml) enzyme solution and 200 mM sodium phosphate buffer solution ) Was added and preliminary reaction was carried out at 37 占 폚 for 10 minutes. 0.25 ml of a 1% starch solution dissolved in 200 mM sodium phosphate buffer solution (pH 6.8) was added to the reaction solution, followed by reaction at 37 ° C for 10 minutes. Then, 0.25 ml of a 48 mM DNS reagent in which 0.5 N NaOH and 30% sodium tartarate were dissolved was added, and the mixture was boiled at 100 ° C for 20 minutes, developed and cooled, and absorbance was measured at 570 nm. As a negative control, the extraction solvent was used instead of the sample. The difference in absorbance between the addition of the sample solution and the non-addition solution was expressed as a percentage (%), and the results are shown in FIG.
도 4b에 도시된 바와 같이, 알파-아밀라제 저해 활성도 발효 전에 비하여 발효 후에 증가하였고, 특히 발효 전에는 저농도 처리시 저해활성이 없었으나 발효 후에는 저농도에서도 저해활성을 보였고, 농도가 높아짐에 따라서 0.50 mg/g 처리 시 50.63% 및 1.0 mg/g 처리 시에는 59.41%로 알파-아밀라제 저해 활성은 크게 증진됨을 확인할 수 있다. As shown in FIG. 4B, the inhibitory activity of alpha-amylase was increased after fermentation compared with before fermentation. In particular, there was no inhibitory activity at low concentration before fermentation, but inhibition activity at low concentration after fermentation was 0.50 mg / 50.63% at the g treatment and 59.41% at the 1.0 mg / g treatment, the alpha-amylase inhibiting activity is greatly enhanced.
<췌장 리파아제 저해활성><Pancreatic Lipase Inhibitory Activity>
췌장 리파아제 저해활성은 참고예 2에서 준비된 발효 전후 농도별 각각의 분석 시료 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 추출용매를 취하였으며 시료용액의 첨가구와 무첨가구 사이의 흡광도 차이를 백분율(%)로 나타내어 그 결과를 도 4c에 나타냈다.The pancreatic lipase inhibitory activity was determined by mixing 50 μl of each analytical sample, 50 μl of pancreatic lipase (1.0 U / ml) enzyme solution and 50 μl of 200 mM sodium phosphate buffer solution (pH 6.8) prepared before and after fermentation prepared in Reference Example 2 And preliminary reaction was carried out at 37 ° C for 10 minutes. After reacting, 100 μl of p -NPB (5 mM) dissolved in sodium phosphate buffer solution was added and reacted for 10 minutes in the same manner. Then, 0.75 ml of 100 mM Na 2 CO 3 was added to terminate the reaction and the absorbance was measured at 420 nm. The negative control was obtained by taking the extraction solvent instead of the sample, and the difference in absorbance between the addition of the sample solution and the non-addition solution was expressed as a percentage (%), and the result is shown in FIG.
도 4c에 도시된 바와 같이, 췌장 리파아제 저해 활성은 발효 전에는 저해활성이 없거나 낮은 저해활성(최대 11.5%)을 나타낸 반면에, 발효 후에는 농도 의존적으로 췌장 리파아제 효소 저해활성이 증가하였으며, 0.25 mg/g 농도 처리시 37.49%, 0.50 mg/g 농도 처리시 54.56%, 1.0 mg/g 농도 처리시 62.82%의 췌장 리파아제 저해 활성을 나타내어 본 발명의 콩 발효조성물의 췌장 리파아제 저해 활성은 약 6배 이상 향상되었음을 확인할 수 있다.As shown in FIG. 4C, the pancreatic lipase inhibitory activity showed no inhibitory activity or low inhibitory activity (up to 11.5%) before fermentation, whereas the pancreatic lipase enzyme inhibitory activity was increased in a concentration-dependent manner after fermentation, and 0.25 mg / the inhibitory activity against pancreatic lipase of the soybean fermentation composition of the present invention was improved by about 6 times or more when the concentration was 37.49%, 54.56% at 0.50 mg / g, and 62.82% at 1.0 mg / .
따라서 상기 결과들로부터 본 발명에 따른 콩 발효조성물은 소화효소 저해 활성이 크게 증진된다는 것을 알 수 있다.Therefore, it can be seen from the above results that the soybean fermentation composition according to the present invention greatly enhances digestive enzyme inhibitory activity.
실험예 5. 지방 생성 억제능 검정Experimental Example 5. Test for inhibiting lipogenesis
본 발명에 따른 콩 발효조성물의 지방 생성 억제능은 3T3-L1 지방전구세포 모델에서 측정하였다.The ability of the soybean fermentation composition according to the present invention to inhibit lipogenesis was measured in a 3T3-L1 lipid precursor cell model.
3T3-L1 지방 전구세포는 미국세포은행 (American Type Culture Collection, ATCC)을 통하여 구입하여 10% 소태아혈청(FBS, Gibco Co., USA)와 1% 페니실린-스트렙토마이신(P/S, Gibco Co., USA)이 첨가된 DMEM 배지(Dulbecco's modified Eagle's medium)를 이용하여 37 ℃, 5% CO2조건에서 배양하였다. 3T3-L1 지방전구세포의 분화 유도하기 위하여 60cm 플레이트에 5×105cells/plate로 분주하고 이틀에 한번씩 배지를 교환하면서 세포가 완전히 융합상태가 될때까지 배양하였다. 융합상태시 첫날에는 1.5 ㎍/ml 인슐린, 1μM 덱사메타손, 500 μM IBMX, 와 1μM 로시글리타존 (3T3-L1 분화키트, Biovision, CA, USA)이 첨가된 DMEM 배지를 처리하여 분화를 유도하였고 지방세포분화에 미치는 영향을 관찰하기 위해 분화 시작시 참고예2에서 준비된 발효 전후 농도별(100, 500 및 1,000㎍/ml) 각각의 분석 시료를 처리하였다. 분화 유도 3일 후, 시료와 1.5㎍/ml 인슐린이 포함된 DMEM으로 이틀에 한 번씩 교환해 주면서 6일간 더 배양하였고, 총 9일후 오일 레드 염색법으로 염색한 결과를 도 5에 나타냈다. 대조구는 추출 시료 대신에 시료를 녹인 용매 DMSO를 처리하였다.3T3-L1 adipose precursor cells were purchased from American Type Culture Collection (ATCC) and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS, Gibco Co., USA) and 1% penicillin-streptomycin (P / S, Gibco Co The cells were cultured in Dulbecco's modified Eagle's medium at 37 ° C and 5% CO 2 . To induce the differentiation of 3T3-L1 adipose precursor cells, the cells were plated at 5 × 10 5 cells / plate on a 60 cm plate and cultured for two days until the cells were completely fused. On the first day of fusion, differentiation was induced by treatment with DMEM supplemented with 1.5 μg / ml insulin, 1 μM dexamethasone, 500 μM IBMX, and 1 μM rosiglitazone (3T3-L1 differentiation kit, Biovision, CA, USA) (100, 500 and 1,000 μg / ml) prepared at the start of differentiation before and after fermentation prepared in Referential Example 2, respectively. After 3 days of induction of differentiation, samples were incubated for 6 days with DMEM supplemented with 1.5 占 퐂 / ml insulin once every other day, and the results were stained with oil red staining for 9 days in total. The results are shown in Fig. The control was treated with solvent DMSO in which the sample was dissolved in place of the extraction sample.
도 5에 도시된 바와 같이, 발효 전의 분석 시료를 처리한 경우에는 3T3-L1 지방세포의 분화가 억제되지 않고 지방이 생성되어 축적됨을 확인할 수 있는 반면에, 발효 후의 본 발명에 따른 콩 발효조성물의 분석 시료를 처리한 경우는 3T3-L1 지방세포의 분화가 억제되고 지방 축적이 감소된 것을 확인할 수 있다. As shown in FIG. 5, it can be confirmed that when the analytical sample before fermentation is treated, the differentiation of 3T3-L1 adipocytes is not inhibited and fat is produced and accumulated, while the fermented soybean fermentation composition according to the present invention after fermentation When the analytical sample was treated, it was confirmed that the differentiation of 3T3-L1 adipocytes was inhibited and the fat accumulation was reduced.
실험예 6. 트리아실글리세라이드(TG) 생성 억제 검정Experimental Example 6: Triacylglyceride (TG) production inhibition assay
실험예 5에서와 같이, 참고예 2에서 준비된 발효 전후 농도별(100, 500 및 1,000㎍/ml) 각각의 분석 시료를 처리하여 9일 배양되어 준비된 3T3-L1 지방 전구세포에서 배지를 제거하고 PBS로 3번 세척 후 러버 폴리스만(rubber policeman)을 이용하여 세포를 수거하였다. 수거한 세포에 적당량의 TG 표준희석액 (Triglyceride colorimetric 분석 키트, cayman chemicals, Ann Arbor, MI, USA)을 첨가하여 현탁시킨 후, 초음파 (1초, 20×)로 처리하고 12,000 rpm 으로 15분간 원심분리 하였다. 원심분리 후 상층액에 있는 TG를 Triglyceride colorimetric 분석 키트(cayman chemicals, Ann Arbor, MI, USA)로 측정하였다. 결과는 대조구를 100으로 준해서 계산하여 도 6에 나타내었다.As in Experimental Example 5, each of the analytical samples of each concentration (100, 500 and 1,000 / / ml) prepared before and after the fermentation prepared in Reference Example 2 was treated, and the medium was removed from the prepared 3T3-L1 adipose precursor cells for 9 days, And then the cells were collected using a rubber policeman. The collected cells were suspended in an appropriate amount of TG standard dilution (Triglyceride colorimetric assay kit, cayman chemicals, Ann Arbor, MI, USA), treated with ultrasonic waves (1 second, 20x) and centrifuged at 12,000 rpm for 15 minutes Respectively. After centrifugation, TG in the supernatant was measured with a Triglyceride colorimetric assay kit (cayman chemicals, Ann Arbor, MI, USA). The results are shown in FIG.
도 6에 도시된 바와 같이, 발효 전의 분석 시료를 처리한 경우에는 3T3-L1 지방세포의 TG 생성량이 유의적으로 증가하였으나, 발효 후의 본 발명에 따른 콩 발효조성물의 분석 시료를 처리한 경우는 3T3-L1 지방세포의 TG 생성이 유의적으로 억제되는 것을 확인할 수 있다.As shown in FIG. 6, the amount of TG produced in the 3T3-L1 adipocytes was significantly increased in the case of treating the sample before fermentation. However, in the case of treating the soybean fermented composition according to the present invention after fermentation, -L1 adipocyte was significantly inhibited by TG production.
실험예 7. 지방세포 분화에 관여하는 유전자들의 mRNA 발현 억제 검정Experimental Example 7. Inhibition of mRNA expression of genes involved in adipocyte differentiation Assay
실험예 5에서와 같이, 참고예 2에서 준비된 발효 전후 농도별(100, 500 및 1,000㎍/ml) 각각의 분석 시료를 처리하여 9일 배양되어 준비된 3T3-L1 지방 전구세포에서 트리졸(Trizol) (Ambion, Carisbad, CA, USA)을 이용하여 RNA를 추출하였고 총 RNA는 정량 후, 지방세포 분화에 관여하는 유전자인 LPL, C/EBP-α, adiponectin, aP2, FAS 및 ACC의 mRNA 발현변화를 하기 표 4에 나타낸 PCR 프라이머를 사용하여 RT-PCR 및 qPCR로 상대정량하여 분석하여 그 결과를 각각 도 7a ~ 도 7f에 나타내었다. cDNA 합성과 RT-PCR(Reverse transcriptase polymerase chain reaction), qPCR(quantitative polymerase chain reaction)은 M-mlV Reverse transcriptase kit (Enzynomics, Daejeon, Korea), Maxime RT-PCR PreMix Kit (Intron Biotechnology, Seoul, Korea)과 ToprealTM qPCR 2×PreMIX PCR kit(Enzynomics, Daejeon, Korea)을 이용하였다.As in Experimental Example 5, the analytical samples of each of the concentrations (100, 500 and 1,000 占 퐂 / ml) prepared before and after fermentation prepared in Reference Example 2 were treated and cultured for 9 days. Trizol was added to the prepared 3T3- (LPL, C / EBP-α, adiponectin, aP2, FAS, and ACC), which are involved in adipocyte differentiation, The PCR primers shown in Table 4 were analyzed by RT-PCR and qPCR, respectively, and the results are shown in FIGS. 7A to 7F, respectively. cDNA synthesis and RT-PCR (reverse transcriptase polymerase chain reaction) and qPCR (quantitative polymerase chain reaction) were performed using M-mlV reverse transcriptase kit (Enzynomics, Daejeon, Korea), Maxime RT- PCR PreMix Kit (Intron Biotechnology, And
도 7a ~ 도 7f에 도시된 바와 같이, 발효 전의 분석 시료(BF)를 처리한 경우에는 유전자 LPL, C/EBP-α, adiponectin, aP2, FAS 및 ACC의 mRNA 발현이 농도 의존적으로 증가한 반면에, 발효 후의 본 발명에 따른 콩 발효조성물의 분석 시료(AF)를 처리한 경우는 유전자 LPL, C/EBP-α, adiponectin, aP2, FAS 및 ACC의 mRNA 발현이 농도 의존적으로 억제되었음을 확인할 수 있다. As shown in FIGS. 7A to 7F, mRNA expression of the genes LPL, C / EBP-alpha, adiponectin, aP2, FAS and ACC was increased in a concentration-dependent manner when the pretreated assay sample (BF) Analysis of the soybean fermented composition according to the present invention after fermentation showed that mRNA expression of the genes LPL, C / EBP- ?, adiponectin, aP2, FAS and ACC was inhibited in a concentration-dependent manner.
따라서 상기 결과들로부터 본 발명에 따른 콩 발효조성물은 지방세포 분화/생성의 억제효과가 크게 증진시킨다는 것을 알 수 있다. Therefore, it can be seen from the above results that the soybean fermentation composition according to the present invention greatly enhances the inhibitory effect on adipocyte differentiation / production.
또한 상기 실험예들의 검정 결과들로부터 본 발명에 따른 콩 발효조성물은 안면홍조, 비만, 불면증, 우울증, 골다공증, 당뇨, 및 고혈압과 같은 갱년기 또는 폐경기 증상 개선용 건강기능성 식품으로서 유용하게 사용될 수 있다는 것을 알 수 있다.From the test results of the above experimental examples, the soybean fermented composition according to the present invention can be effectively used as a health functional food for improving menopausal symptoms or postmenopausal symptoms such as facial flushing, obesity, insomnia, depression, osteoporosis, diabetes and hypertension Able to know.
Claims (8)
콩에 수탁번호 KACC91848P로 기탁된 락토바실러스 플란타륨 (Lactobacillus plantarum) P1201 균주를 접종하여 20~ 40 ℃에서 12시간 이상 발효시키는 단계를 포함하고,
상기 비배당체-이소플라본은 다이드제인(daidzein) 및 제니스테인(genistein)이고, 증진된 함량은 다이드제인 및 제니스테인 각각 164 ㎍/g 이상인 것을 특징으로 하는 방법.
As a method for enhancing the content of non-glycoside-isoflavone in soybean fermentation composition,
Comprising the step of inoculating Lactobacillus plantarum strain P1201 deposited with soybean under accession number KACC91848P and fermenting at 20 to 40 DEG C for at least 12 hours,
Wherein said unglycosylated-isoflavones are daidzein and genistein, and wherein the enhanced content is at least 164 ug / g of both daidzein and zenithaine.
[7] The method of claim 6, wherein the soybean is an expanded bean having a boiling point of 100 to 120 DEG C for 30 to 60 minutes.
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| KR102044602B1 (en) * | 2017-08-21 | 2019-11-13 | 경남과학기술대학교 산학협력단 | Preparation method for soybean-fermented composition having enhanced GABA, Conjugated linoleic acid and Aglycone isoflavones, combined probiotics for its fermentation and functional food of anti-diabetic and anti-obesity effects containing the soybean-fermented composition |
| WO2019088379A1 (en) * | 2017-11-02 | 2019-05-09 | 경희대학교 산학협력단 | Novel lactic acid bacteria and use thereof |
| KR102027798B1 (en) * | 2018-02-08 | 2019-10-02 | 제주대학교 산학협력단 | Composition for antioxidant comprising golden kiwi fruit lactic acid bacteria fermentation product as effective component |
| KR101946813B1 (en) | 2018-05-14 | 2019-02-12 | 주식회사 메타포뮬러 | Compositions for preventing or treating andropause-related symptoms |
| KR101946811B1 (en) | 2018-05-14 | 2019-02-12 | 주식회사 메타포뮬러 | Compositions for preventing or treating andropause-related symptoms |
| KR102107443B1 (en) * | 2018-08-31 | 2020-05-07 | (주)바이오리듬 | Lactobacillus probiotics preparation for preventing or relieving the female menopausal symptoms |
| KR102620793B1 (en) * | 2022-05-25 | 2024-01-04 | 천현수 | Composition for preventing, ameliorating or treating woman menopausal symptom comprising mixture of fermented soybean and fermented lettuce as effective component |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20240011288A (en) | 2022-07-18 | 2024-01-26 | 전남대학교산학협력단 | Composition for improving female menopause disorder by decreased secretion of estrogen and method for preparing the same |
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