KR101097838B1 - A method for preparing fermented soymilk having improved good sensory property and Lipid lowering ability by Bacillus subtilis and Corni fructus, and fermented soymilk for prevention atherosclerosis obtained from this method - Google Patents
A method for preparing fermented soymilk having improved good sensory property and Lipid lowering ability by Bacillus subtilis and Corni fructus, and fermented soymilk for prevention atherosclerosis obtained from this method Download PDFInfo
- Publication number
- KR101097838B1 KR101097838B1 KR1020090029299A KR20090029299A KR101097838B1 KR 101097838 B1 KR101097838 B1 KR 101097838B1 KR 1020090029299 A KR1020090029299 A KR 1020090029299A KR 20090029299 A KR20090029299 A KR 20090029299A KR 101097838 B1 KR101097838 B1 KR 101097838B1
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- South Korea
- Prior art keywords
- bacillus subtilis
- fermented
- soymilk
- milk
- lipid
- Prior art date
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Abstract
본 발명은 고초균 (Bacillus subtilis)과 산수유(Corni fructus)를 이용하여 관능성과 지질저하능이 개선된 발효두유를 제조하는 방법 및 이로부터 얻어진 동맥경화 예방용 발효두유에 관한 것으로, 본 발명에 의하여 처리한 발효두유의 경우 혈장지질 저하, 간지질 저하, 동맥의 지질침착 억제효과로 인하여, 결과적으로 동맥경화를 예방하는 건강기능식품으로 이용할 수 있다.The present invention relates to a method for producing fermented soy milk with improved functionality and lipid lowering ability using Bacillus subtilis and Corni fructus , and to fermented soy milk for preventing atherosclerosis obtained therefrom. Fermented soy milk may be used as a health functional food that prevents arteriosclerosis due to plasma lipid lowering, liver lipid lowering, and arterial lipid suppression effect.
발효두유, 관능성, 지질저하능, 혈장지질, 간지질, 지질침착 Fermented soy milk, functional, lipid lowering, plasma lipid, liver lipid, lipid deposition
Description
본 발명은 고초균과 산수유를 이용하여 관능성과 지질저하능이 개선된 발효두유를 제조하는 방법 및 이로부터 얻어진 동맥경화 예방용 발효두유에 관한 것으로, 구체적으로는 고초균과 산수유에 의하여 처리한 발효두유에 대한 혈장지질 저하, 간지질 저하, 동맥의 지질침착 억제효과를 확인하고 동맥경화를 예방하는 건강기능식품으로 제공하는 기술에 관한 것이다. The present invention relates to a method for producing fermented soy milk with improved functionalities and lipid lowering ability using Bacillus subtilis and cornus milk, and to a fermented soy milk for preventing atherosclerosis, specifically, to fermented soy milk treated with Bacillus subtilis and cornus milk. The present invention relates to a technology for confirming plasma lipid lowering, liver lipid lowering, and inhibiting arterial lipid deposition and providing a functional food for preventing atherosclerosis.
청국장은 Bacillus natto나 Bacillus subtilis류가 생산하는 효소의 작용으로 대두 단백질이 분해되어 가용성 질소화합물인 펩톤, 펩타이드, 아미노산 등이 생성된 발효식품으로 좋은 단백질 급원이다. 최근에는 청국장이 단백질 급원으로 뿐만 아니라 건강기능성이 있음이 알려지고 있고, 이러한 기능으로는 혈전 형성 억제능(1), 체중감소 및 혈압강하효과(2) 혈당강하(3) 항산화 효과(3) 그리고 돌연변 이 억제(4) 등이 보고되고 있다. 그러나 청국장은 끓여서 먹는 식품이라 일상생활에서 항상 섭취하기에는 한계가 있다. 이에 최근에는 분말 또는 환 등의 형태로 청국장 제품이 개발되어 사람들이 이들 제품을 물 또는 우유와 함께 섭취하고 있다. 그러나 청국장 분말이 우유에 잘 녹지 않아 이러한 문제점을 해결한 제품을 원하고 있다.Cheonggukjang is a fermented food source of fermented foods in which soy protein is decomposed by the action of enzymes produced by Bacillus natto or Bacillus subtilis . Recently, it is known that Cheonggukjang has not only a protein source but also a health function, and these functions include antithrombotic activity (1), weight loss and blood pressure lowering effect (2) blood sugar lowering (3) antioxidant effect (3) and stone Mutagen suppression (4) and the like have been reported. However, Cheonggukjang is boiled and eaten, so there is a limit to always intake in everyday life. Recently, Cheonggukjang products have been developed in the form of powder or pills, and people are taking these products with water or milk. However, because the Cheonggukjang powder is not soluble in milk, we want a product that solves this problem.
한편, 두유는 우유에 비해 콜레스테롤, 유당, 글루텐 등이 함유되어 있지 않아 우유 섭취에 따른 문제가 있는 사람들에게 각광을 받고 있으나 콩의 비린 냄새 때문에 우유보다 선호도가 떨어지고 있다. 발효우유의 장점은 장내에서 프로바이오틱스로 작용하여 건강을 증진시켜 장수할 수 있는 것을 보고되고 있고, 이에 발효두유 역시 프로바이오틱스 효과를 지닐 수 있는 것으로 생각되고 많은 학자들에 의해 락토바실러스나 비피도박테리아로 단독 또는 혼합 발효시켜(2,5) 발효두유 제조에 성공하였고, 이들 발효두유는 항산화효과 (6), 콜레스테롤 저하효과(7), 항돌연변이 및 항암효과(8), 그리고 면역조절 효과(1) 등이 있는 것으로 보고되었다. 이에 본 발명에서는 청국장분말 또는 청국장 환 제품을 우유와 함께 섭취하는 번거로움을 없애고, 이와 동일한 효능을 갖는 기능성 청국두유를 제조하고자 하려는 것이다. On the other hand, soy milk does not contain cholesterol, lactose, gluten, etc. compared to milk has attracted the attention of people who have problems with milk intake, but because of the fishy smell of soybeans are less preferred than milk. The benefits of fermented milk have been reported to act as a probiotics in the intestine to promote health and longevity. Therefore, fermented soy milk is also thought to have a probiotic effect, and many scholars say it is Lactobacillus or Bifidobacteria alone. Or fermented soymilk with mixed fermentation (2,5), and these fermented soymilk have antioxidant effects (6), cholesterol lowering effects (7), antimutagenic and anticancer effects (8), and immunomodulatory effects (1). It is reported that there is. Therefore, in the present invention, to eliminate the hassle of ingesting the Cheonggukjang powder or Cheonggukjang pill product with milk, and to prepare a functional Cheongguk soy milk having the same efficacy.
한편, 종래 발효두유와 관련된 기술로는 대한민국 특허출원 제 2006-0052516호, 대한민국 특허공개 제 2006-0001656호, 제 2005-0095770호, 제 2005-0053555 호, 제 2003-0097264호, 제 2001-0023143호, 제 1998-032749호, 제 1998-070641호 등이 있다. 그러나 이 기술들은 모두 발효두유의 제조법에 초점이 맞추어져 있으며, 상기 문헌 어디에도 발효두유의 지질저하 효과에 대한 교시나 개시된 바가 없다.Meanwhile, technologies related to conventional fermented soy milk include Korean Patent Application No. 2006-0052516, Korean Patent Publication No. 2006-0001656, 2005-0095770, 2005-0053555, 2003-0097264, 2001-0023143 No. 1998-032749, 1998-070641, and the like. However, all of these techniques are focused on the preparation of fermented soymilk, and none of the literature teaches or discloses the lipid lowering effects of fermented soymilk.
참고문헌references
1.Heo. S., Lee. SK. and Joo. H.K. : Isolation and identification of fibrinolytic bacteria from Korean traditional cheonggukjang. Agric. Chem. Biotechnol. 41:119-124 (1998)1.Heo. S., Lee. SK. and Joo. H.K. : Isolation and identification of fibrinolytic bacteria from Korean traditional cheonggukjang. Agric. Chem. Biotechnol. 41: 119-124 (1998)
2.Yang. J.L., Lee. S.H. and Song. Y.S. : Improving effect of powders of cooked soybean and cheongguljang on blood pressure and lipid metabolism in spontaneously hypertensive rats. J. Korean Soc. Food Sci Nutr. 32:899-905 (2003)2.Yang. J.L., Lee. S.H. and Song. Y.S. : Improving effect of powders of cooked soybean and cheongguljang on blood pressure and lipid metabolism in spontaneously hypertensive rats. J. Korean Soc. Food Sci Nutr. 32: 899-905 (2003)
3.Kim. M.H. and Lee. J.H. : Antioxidant materials in domestic Meju and Doenjang. J. Korea Soc. Food Nutr. 23:604-613 (1994)3.Kim. M.H. and Lee. J.H. : Antioxidant materials in domestic Meju and Doenjang. J. Korea Soc. Food Nutr. 23: 604-613 (1994)
4.Park. K.Y., Moon. S.H., Cheigh. H.S. and Baik. H.S. : Antimutagenic effects of Doenjang. J. Food Sci. Nutri. 1:151-158 (1996)4.Park. K.Y., Moon. S.H., Cheigh. H.S. and Baik. H.S. : Antimutagenic effects of Doenjang. J. Food Sci. Nutri. 1: 151-158 (1996)
5.Ryu Sh. : Studies on antioxidative effects and antioxidative components of soybean and chingkukjang. Doctoral thesis Inje University of Korea. pp 23-122 (2002)5.Ryu Sh. : Studies on antioxidative effects and antioxidative components of soybean and chingkukjang. Doctoral thesis Inje University of Korea. pp 23-122 (2002)
6.Ryu. S.H., Lee. Y.S. and Moon. G.S. : Effects of salt and soysauce condiment on lipid oxidation in broloed mackerel(Scomber japonicus). Korean J. Food Sci. Technol. 34(6):1030-1035 (2003)6.Ryu. S.H., Lee. Y.S. and Moon. G.S. : Effects of salt and soysauce condiment on lipid oxidation in broloed mackerel (Scomber japonicus). Korean J. Food Sci. Technol. 34 (6): 1030-1035 (2003)
7.Kim. K.J., Ryu. M.K. and Kim. S.S. : Chungkook-jang koji fermentation with rice straw. Korean J. Food Sci. Technol. 14(4):301-308 (1982)7.Kim. K.J., Ryu. M.K. and Kim. S.S. : Chungkook-jang koji fermentation with rice straw. Korean J. Food Sci. Technol. 14 (4): 301-308 (1982)
8.Kim. S.H., Yang. J.L. and Song. Y.S. : Physiological functions of Chongkukjang. Food Industry and Nutrition, 4(2):40-46 (1999)8.Kim. S.H., Yang. J.L. and Song. Y.S. Physiological functions of Chongkukjang. Food Industry and Nutrition, 4 (2): 40-46 (1999)
이에 본 발명자들은 관능성과 지질저하능이 모두 우수한 발효두유를 제조하기 위하여 연구한 결과, 고초균으로 발효두유를 제조하는데 성공하였을 뿐만 아니라, 더불어 산수유를 첨가함에 의해 관능성과 지질저하 기능성이 증가된 음료를 제조할 수 있음을 확인하였을 뿐 아니라 동맥경화 유발 마우스에 있어 혈당지질, 간지질, 동맥내 지질 침착 효과를 규명하고 본 발명을 완성하기에 이르렀다. Therefore, the present inventors have studied to prepare fermented soy milk with excellent functional and lipid lowering ability, and have not only succeeded in preparing fermented soy milk with Bacillus subtilis, but also produced beverages with increased functionality and lipid lowering function by adding cornus milk. In addition to confirming that it is possible to determine the effects of blood lipid lipid, hepatic lipid, intraarterial lipid deposition in atherosclerosis mice, the present invention was completed.
즉, 본 발명의 일 목적은 관능성과 지질저하능을 모두 갖는 발효두유를 제조하는 방법을 제공하는데 있다.That is, one object of the present invention to provide a method for producing a fermented soy milk having both functional and lipid lowering ability.
본 발명의 다른 목적은 동맥경화 예방용 건강기능식품으로서 발효두유를 제공하는데 있다. Another object of the present invention to provide a fermented soy milk as a health functional food for preventing atherosclerosis.
본 발명의 제1 견지에 있어서, In a first aspect of the invention,
고초균 (Bacillus subtilis)과 산수유(Corni fructus)를 이용한 피브린 저해 혈성을 갖고 관능성과 지질저하능이 개선된 발효두유를 제조하는 방법을 제공한다. Provided is a method for preparing fermented soymilk with fibrin inhibitory activity using Bacillus subtilis and Corni fructus and having improved functionality and lipid lowering ability.
본 발명의 제2 견지에 있어서, In the second aspect of the present invention,
상기 제1 견지의 방법에 의해 제조되며, 혈장지질 저하, 간지질 저하, 동맥의 지질침착 억제효과에 의하여 동맥경화를 예방하는 건강기능식품으로서 발효두유 를 제공한다. Produced by the method of the first aspect, provides a fermented soy milk as a health functional food to prevent atherosclerosis by the plasma lipid lowering, liver lipid lowering, inhibiting the lipid deposition of the arteries.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명에서는 고초균 (Bacillus subtilis)과 산수유(Corni fructus)를 이용함으로써 피브린 저해 활성을 갖고 관능성과 지질저하능이 개선된 발효두유를 제조할 수 있다. In the present invention, by using Bacillus subtilis and Corni fructus , fermented soybean milk with fibrin inhibitory activity and functional and lipid lowering ability can be prepared.
이때 고초균과 산수유는 포도당 함량이 전체 중량 대비 1%가 되도록 첨가한 다음 121-150℃에서 15분간 증자시켜 멸균처리한 두유에 산수유와 고초균의 순서로 첨가하는 것이 바람직하다. At this time, the subtilis bacteria and cornus milk are added so that the glucose content is 1% of the total weight, and then added to the sterilized soymilk for 15 minutes at 121-150 ° C. to be added in the order of cornus and subtilis bacteria.
즉, 상기 산수유의 농도는 10°Brix 이고, 함량은 최종 농도 기준으로 1%가 되도록 첨가하여 피브린 분해능을 개선시키고 관능성 또한 최적으로 극대화시킬 수 있다. That is, the concentration of the cornus oil is 10 ° Brix, the content is added to be 1% on the basis of the final concentration to improve the fibrin resolution and can also maximize the functionality.
또한, 사용되는 두유는 시판되는 두유를 사용할 수도 있으며, 혹은 대두를 정선하여 열처리하고 대두 중량의 8 내지 10배의 열수를 첨가하여 열수마쇄하고, 상기 대두 마쇄물을 열처리하여 추출한 다음, 이를 여과하여 두유를 수득한 다음 사용할 수도 있다. In addition, the soymilk used may be commercially available soymilk, or selected soybeans, heat-treated, and hydrothermal grinding by adding hot water of 8 to 10 times the weight of soybean, heat-processing and extracting the soybean grinding, and then filtering it. Soymilk can also be obtained and then used.
또한, 상기 고초균 7-8 logCFU/ml이 되도록 고초균 배양액을 첨가하여 접종함으로써 피브린 분해능을 개선시키고 지질 저하능 또한 최적으로 극대화시킬 수 있다. 상술한 고초균 조건은 발효두유의 유산균수가 최저 6 logCFL/mL이 되어야 하고 냉장보관 시 1주일 이상 이 균의 수를 유지하여야 하는, 발효두유가 프로바이오틱스 효과를 지니기 위한 조건을 만족하기 위하여 설정된 것이다. In addition, by inoculating the Bacillus subtilis culture medium so that the Bacillus subtilis 7-8 logCFU / ml to improve the fibrin degradation and lipid lowering capacity can also be maximized. The above-described subtilis bacterium conditions are set to satisfy the conditions for fermented soy milk having a probiotic effect, in which the number of lactic acid bacteria of fermented soy milk should be at least 6 logCFL / mL and the number of these bacteria should be maintained for at least one week when refrigerated.
이때 상기 고초균 배양액은 맛과 항산화 기능성이 우수한 청국장(청국장, 순창, 김용순)에서 분리한 Bacillus subtilis 2829PNU015를 0.8% sodium chloride 함유 배양배지에 접종시켜 37℃에서 24시간동안 수조에서 진탕 배양하여 수득하는 것이 바람직하다. 구체적으로는, 하기 실시예에서 규명한 바와 같이, 상기 Bacillus subtilis 2829PNU015는 순창지역의 청국장 6가지 제품으로부터 항산화능과 관능성이 높은 청국장을 선발한 후 이로부터 균을 분리하였다. 이들 Bacillus subtilis균으로 발효두유를 제조하는 과정을 확립하고, 피브린 분해능이 높은 3종류의 Bacillus subtilis를 선정하여 발효두유를 제조하고 이들의 항산화능 및 피브린 분해능을 비교하였다. 상기 Bacillus subtilis 2829PNU015는 2009년 3월 26일자로 기탁하였다(기탁번호 KCCM 42923).The Bacillus subtilis 2829PNU015 isolated from Cheonggukjang (Cheonggukjang, Sunchang, Kim Yong-soon) with excellent taste and antioxidant function was inoculated in a culture medium containing 0.8% sodium chloride to obtain shaking culture in a water bath at 37 ° C for 24 hours. desirable. Specifically, as described in the following examples, Bacillus subtilis 2829PNU015 was selected from the six products of Cheonggukjang in the Sunchang region was selected from the Cheonggukjang with high antioxidant capacity and functionalities after separating the bacteria. The process of producing fermented soymilk with Bacillus subtilis bacteria was established, and three kinds of Bacillus subtilis with high fibrin degradability were selected to prepare fermented soymilk and their antioxidant and fibrin degradability were compared. The Bacillus subtilis 2829PNU015 was deposited on March 26, 2009 (Accession No. KCCM 42923).
그런 다음 청국장두유의 관능성 및 기능성을 개선하기 위하여 기능성 소재로서 발굴한 산수유를 첨가한 두유에 고초균을 접종한 다음 37-40℃에서 pH 5.5±0.2에 도달하도록 대략 8시간동안 발효시킴으로써 혈장지질 저하, 간지질 저하, 동맥의 지질침착 억제효과가 개선된 동맥경화 예방용 발효두유를 수득하게 된다. 즉, apoE(-/-) 마우스에 항동맥경화 청국장두유를 8주간 섭취시킨 후 지질저하 및 동맥경 화 억제효과를 청국장 분말을 첨가시킨 두유와 대비한 결과, 탁월한 효과를 규명하게 되었다. Then, in order to improve the functionality and functionality of Cheonggukjang soybean milk, inoculated with Bacillus subtilis soymilk added as a functional material, and lowered plasma lipid by fermenting for approximately 8 hours to reach pH 5.5 ± 0.2 at 37-40 ℃ The fermented soy milk for the prevention of atherosclerosis is improved, which lowers liver lipid and suppresses lipid deposition of arteries. In other words, apoE (-/-) mice were ingested with anti-arterial cured soybean soymilk for 8 weeks, and the effects of lipid lowering and atherosclerosis were compared with soymilk added with Chungkookjang powder.
이상 살펴본 바와 같이, 본 발명에서 제조된 발효두유는 혈장지질 저하, 간지질 저하, 동맥의 지질침착 억제효과로 인하여, 결과적으로 동맥경화를 예방하는 건강기능식품으로 이용할 수 있다.As described above, the fermented soymilk prepared in the present invention may be used as a health functional food that prevents arteriosclerosis due to the effect of lowering lipid lipids, lowering liver lipids, and inhibiting lipid deposition of arteries.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of examples. However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.
<재료><Material>
- 청국장 -Cheonggukjang
청국장은 2007년 8월 순창 지역에서 시판하고 있는 완제품 6종류를 구입하여 이화학적 분석 및 Bacillus 균 분리를 위한 시료로 사용하였다. 하기표 1에서 보듯이, 각 제조업자는 S1: Moon Ok Rae, S2 : Hyang Guk Woon, S3 : Kim Young Sun, S4 : Moon Neung Ja, S5 : Oh Bok, S6 : Oh Sun E 이었다. Cheonggukjang purchased 6 finished products from Sunchang in August 2007 and used them as samples for physicochemical analysis and Bacillus isolates. As shown in Table 1, each manufacturer was S1: Moon Ok Rae, S2: Hyang Guk Woon, S3: Kim Young Sun, S4: Moon Neung Ja, S5: Oh Bok, S6: Oh Sun E.
- 두유 -Soymilk
청국장으로부터 분리한 Bacillus sp.와 상업적 균주로 사용하고 있는 MYCO(Innobiz Myco), KCCM(KCCM 11316)균을 사용하여 발효두유 제조를 위해 동화식품(양산, 한국)에서 구입하여 재료로 사용하였다. Bacillus sp. MYCO (Innobiz Myco) and KCCM (KCCM 11316), which are used as commercial strains, were purchased from Donghwa Food (Yangsan, Korea) for fermented soymilk production and used as ingredients.
- Bacillus 균 - Bacillus bacteria
순창지역에서 제조된 6종류의 청국장에서 분리된 Bacillus 균(하기표 1 참조) 중 K제조사 청국장으로 분리한 Bacillus subtilis 2805 PNU 014, Bacillus subtilis 2829 PNU 015, Bacillus subtilis 2825 PNU 016를 부산대로부터 분주받아 본 연구에 사용하였다. Bacillus subtilis 2805 PNU 014, Bacillus subtilis 2829 PNU 015, Bacillus subtili s 2825 PNU 016 isolated from Bacillus bacteria (see Table 1) isolated from six kinds of Cheonggukjang prepared in Sunchang area. Used in this study.
productsproducts
- 기능성 소재 -Functional material
동맥경화 예방 기능성이 증진된 맞춤형 발효두유를 생산하기 위하여 공동연구를 수행하고 있는 천호식품(Busan, Korea)에서 생산하는 자색고구마, 흑마늘, 양파, 강화사자발쑥, 마늘, 산수유의 추출액을 공급받아 사용하였다.It is supplied with extracts of purple sweet potato, black garlic, onion, fortified lion cucurbita, garlic, and cornus milk produced by Cheonho Foods (Busan, Korea), which is conducting joint research to produce customized fermented soy milk with enhanced function to prevent atherosclerosis. It was.
<실험 방법> Experimental Method
실시예 1. 발효두유 제조Example 1 Preparation of Fermented Soymilk
청국장으로부터 분리한 Bacillus subtilis 2805 PNU 014, Bacillus subtilis 2829 PNU 015, Bacillus subtilis 2825 PNU 016 (P Univ., Busan, Korea)와 상업적으로 사용되고 있는 MYCO (Innobiz Myco, Gyeong Ju, Korea) 및 KCCM (KCCM 11316, Seoul, Korea)을 0.8% sodium chloride를 함유하는 nutrient broth에 접종시켜 37℃에서 24시간 water bath에서 배양시킨 후 1% glucose를 포함한 멸균된 두유에 1% (CFU 107~108) 접종시켜 40℃에서 pH 5.5±0.2가 될 때까지 약 8시간 동안 진탕 배양하였다. Bacillus subtilis 2805 PNU 014, Bacillus subtilis 2829 PNU 015, Bacillus subtilis 2825 PNU 016 (P Univ., Busan, Korea) isolated from Cheonggukjang and MYCO (Innobiz Myco, Gyeong Ju, Korea) and KCCM (KCCM 11316) , Seoul, Korea) was inoculated in nutrient broth containing 0.8% sodium chloride, incubated in water bath at 37 ℃ for 24 hours, and then inoculated with 1% (
기능성 발효두유제조는 멸균된 두유에 10oBrix 산수유가 최종농도 1% 되도록 첨가한 후 여기에 동일량의 고초균을 접종한다. 이들 발효용액을 40℃에서 pH 5.5±0.2가 될 때까지 진탕 배양한다. 일반적으로 소요되는 시간은 약 8시간이었다. 생성된 발효두유의 성상은 두유보다 농도가 짙은 액상이었다. Functional fermented soymilk preparation is added to the sterilized soymilk so that the final concentration of 10 o Brix cornus milk is 1% and then inoculated with the same amount of Bacillus subtilis. The fermentation solutions are shaken at 40 ° C. until the pH reaches 5.5 ± 0.2. Normal time was about 8 hours. The resulting fermented soy milk was in a liquid phase with a higher concentration than soy milk.
실시예 2. Example 2. BacillusBacillus 균의 fibrin 분해 효과 Fibrin Degradation Effect of Bacteria
S3 청국장으로부터 분리된 Bacillus subtilis 2805 PNU 014, Bacillus subtilis 2829 PNU 015 그리고 Bacillus subtilis 2825 PNU 016 및 상업성균 2종 중에서 혈전 용해능이 가장 우수한 균을 선정하고자 fibrin plate 법으로 Bacillus 균주의 fibrin 분해능을 알아보았다(Table 6). The fibrin degradability of Bacillus strains was determined by fibrin plate method to select the best bacterium from among Bacillus subtilis 2805 PNU 014, Bacillus subtilis 2829 PNU 015 and Bacillus subtilis 2825 PNU 016 and two commercial bacteria isolated from S3 Cheonggukjang. Table 6).
Bacillus subtilis 2829 PNU 015의 피브린분해능은 27.2 mU plasmin/hr로 실험한 5종의 Bacillus 균주 중 혈전 용해능이 가장 높은 것으로 나타났으며 혈전 용해능이 가장 낮은 것은 청국장으로부터 분리된 PNU 014 였다. 상기 피브린 분해능이 높은 Bacillus subtilis 2829 PNU 015을 2009년 3월 26일자로 기탁하였다(기탁번호 KCCM 42923). Fibrin resolution of Bacillus subtilis 2829 PNU 015 was the highest among the five Bacillus strains tested with 27.2 mU plasmin / hr. The lowest thrombolytic ability was PNU 014 isolated from Cheonggukjang. Bacillus subtilis 2829 PNU 015 with high fibrin resolution was deposited on March 26, 2009 (Accession No. KCCM 42923).
실시예 3. 피브린 분해능이 높은 기능성 재료의 선정Example 3 Selection of Functional Material with High Fibrin Resolution
건강기능식품으로 제조되어 사용되고 있는 산수유, 마늘, 숙성마늘, 자색고구마, 강화사자발쑥의 추출물의 피브린 분해능을 관찰하여 하기표 2에 정리하였다. Fibrin degradability of the extracts of cornus, garlic, ripened garlic, purple sweet potato, and fortified wormwood, which were prepared and used as health functional foods, was observed and summarized in Table 2 below.
구체적으로는, 산수유, 마늘, 숙성마을, 자색고구마, 그리고 강화사자발쑥 농축액을 천호식품(부산, 한국)으로부터 공급받았다. 이들의 농도를 10oBrix로 맞추어 실험에 사용하였다. Specifically, cornus, garlic, aged village, purple sweet potato, and fortified mugwort concentrate were supplied from Cheonho Foods (Busan, Korea). Their concentration was adjusted to 10 o Brix and used for the experiment.
Fibrin 분해활성은 Astrup등(Lee. S.J., Lee. K.I., Rhee. S.H. and Park. K.Y. : Phsiological activity in Doenjang added with various Mushrooms. Korean J. Food Cookery SCI. 20(4):365-370 (2004))의 피브린 평판법을 응용하여 사용하였다. 즉, 10mM 인상완충 용액 1 mL에 fibrinogen 0.03g을 넣어 37℃에서 완전히 녹인 용액에 동일 buffer에 녹인 100 NIH thrombin 50㎕를 넣어 균일하게 흔들어 petridish에 분주하여 fibrinogen이 fibrin으로 서서히 굳어지게 하였다. 제조한 plate는 실온에서 40분간 방치한 후 실험에 사용하였다. Fibrin degrading activity is described by Astrup et al. (Lee.SJ, Lee.KI, Rhee.SH and Park.KY: Phsiological activity in Doenjang added with various Mushrooms.Korean J. Food Cookery SCI. 20 (4): 365-370 (2004) Fibrin plate method of That is, 0.03 g of fibrinogen was added to 1 mL of 10 mM impression buffer solution, and 50 µl of 100 NIH thrombin dissolved in the same buffer was added to the solution completely dissolved at 37 ° C., and the mixture was shaken uniformly to allow the fibrinogen to gradually solidify with fibrin. The prepared plate was used for the experiment after leaving for 40 minutes at room temperature.
각 시료의 피브린 분해 활성 측정은 미리 제조된 피브린 평판에 시료 50㎕을 떨어뜨려 37℃에서 18시간동안 배양한 후 각 시료의 분해된 면적을 측정하였다. 대조구로서는 정제된 혈전용해효소인 plasmin(0.5U/mL)을 사용하였다. 각 시료의 fibrin 분해 활성은 plasmin의 분해 면적에 비례하여 plasmin의 활성으로 나타내었다. Fibrin degradation activity of each sample was measured by dropping 50 μl of the sample on a previously prepared fibrin plate and incubating at 37 ° C. for 18 hours to determine the degraded area of each sample. As a control, plasmin (0.5 U / mL), a purified thrombolytic enzyme, was used. Fibrin degrading activity of each sample was represented by plasmin activity in proportion to the area of plasmin degradation.
상기표 2에서 보듯이, 마늘, 숙성마늘, 자색고구마, 강화사자발쑥 추출물보다는 산수유의 피브린 분해능이 가장 높았다.As shown in Table 2, the highest fibrin degradability of cornus milk than garlic, ripened garlic, purple sweet potato, and fortified wormwood extract.
실시예 4. 산수유 첨가량 결정Example 4 Determination of Cornus Amount Added
실시예 3에서 선정된 산수유의 발효시 첨가할 농도를 결정하기 위하여 10OBrix 농도의 산수유를 발효두유에 농도별로 첨가하여 30명을 대상으로 관능검사를 실시하고 하기표 3에 정리하였다. In order to determine the concentration to be added during the fermentation of the selected corn cob in Example 3, corn cob at a concentration of 10 O Brix was added to the fermented soy milk by concentration, and the sensory test was performed on 30 people and summarized in Table 3 below.
참고로, 관능검사는 부산대 대학원생 30명을 대상으로 두유, 발효두유, 청국장분말에 대한 훈련을 시킨 다음 랜덤화된 블락디자인으로 실시하였다. 외관, 맛, 향, 질감, 그리고 선호도에 대하여 9점 척도법을 사용하여 검사하였다. 1점은 각 항목별 평가가 가장 나쁜 경우이며 9점은 해당항목의 관능 결과가 가장 좋을 때이다. For reference, the sensory test was conducted on 30 soybean milk, fermented soy milk, and Cheonggukjang powder for 30 graduate students in Pusan National University. Appearance, taste, aroma, texture, and preference were examined using a 9-point scale. One point is the worst case evaluation of each item and nine points is the best sensory result of the item.
acceptanceOverall
acceptance
Values are mean±SD (n=30).Values are mean ± SD (n = 30).
a-cData were significantly different with one-way ANOVA followed by Duncan'smultiplerangetestatthe0.05levelofsignificance. ac Data were significantly different with one-way ANOVA followed by Duncan'smultiplerangetestatthe0.05levelofsignificance.
NS : not significant NS: not significant
상기표 3에서 보듯이, 10° Brix 농도의 산수유를 전체 중량 대비 1% 농도로 첨가시 외관, 맛, 그리고 종합평가에서 가장 좋은 점수를 얻었다.As shown in Table 3, when the 10 ° Brix concentration of cornus oil was added at a concentration of 1% of the total weight, the best score was obtained in appearance, taste, and comprehensive evaluation.
실시예 5. 산수유 첨가 방법Example 5 Cornus Milk Addition Method
산수유를 발효두유에 첨가하는 방법을 결정하기 위하여, 두유와 산수유를 함께 고초균으로 발효시킨 제품 (Fermented soymilk containing Corni fructus, FSCC), 발효두유에 산수유를 첨가한 제품 (Corni fructus extract added fermented soymilk. CAFS)의 관능을 발효두유(Fermented soymilk)와 현재 일반인들이 흔히 사용하고 있는 청국장분말을 우유에 타서 먹는 제품 (chungkukjang powder (5%) added soybean milk CPAS)과 비교 관찰하고 결과를 하기표 4에 정리하였다.Fermented soymilk containing Corni fructus (FSCC) and Corni fructus extract added fermented soymilk. ) Were compared with fermented soymilk and fermented soymilk (Cookkukjang powder (5%) added soybean milk CPAS), which is commonly used by the general public, and summarized in Table 4 below. .
SoymilkFermented
Soymilk
AcceptanceOverall
Acceptance
Values were mean±SD (n=30).Values were mean ± SD (n = 30).
a,bData were significantly different with one-way ANOVA followed by Duncan's multiple range test at the 0.05l evel of significance. a, b Data were significantly different with one-way ANOVA followed by Duncan's multiple range test at the 0.05l evel of significance.
NS: not significantNS: not significant
FS: fermented soymilk with Bacillus subtilis 2829PNU015FS: fermented soymilk with Bacillus subtilis 2829PNU015
FSCC: Fermented soymilk containing Corni fructus. The mixture solution of Corni fructus extract (1%, 10oBrix) and soymilk fermented with Bacillus subtilis 2829PNU015FSCC: Fermented soymilk containing Corni fructus . The mixture solution of Corni fructus extract (1%, 10 o Brix) and soymilk fermented with Bacillus subtilis 2829
CAFS: Corni fructus extract added fermented soymilk. 1% Corni fructus extracts was added to the fermented soymilk with Bacillus subtilis 2829PNU015.CAFS: Corni fructus extract added fermented soymilk. 1% Corni fructus extracts was added to the fermented soymilk with Bacillus subtilis 2829PNU015.
CPAS: Chungkukjang powder (5%) added soymilk CPAS: Chungkukjang powder (5%) added soymilk
상기표 4에서 보듯이, 두유와 산수유를 함께 발효시킨 제품의 종합평가 점수가 가장 높았다. 외관의 경우 발효두유에 비해 어두워져서 나쁜 편이었으나 맛이 산수유 첨가에 의해 개선되어 청국장 냄새가 적게 났으며 따라서 종합평가가 가장 좋았다. 결과적으로, 산수유를 첨가하는 방법은 발효 시 첨가하는 것이 발효 후 첨가하는 것 보다 냄새 및 맛에서 유의적으로 좋은 것으로 나타났다. 그리고 현재 사용하고 있는 청국장 분말 첨가 우유보다 냄새와 맛이 유의적으로 개선된 것도 확인할 수 있었다. As shown in Table 4, the comprehensive evaluation score of the product fermented soy milk and cornus milk together was the highest. In appearance, it was darker than fermented soymilk, but the taste was improved by the addition of Cornus soybean, and the smell of Cheonggukjang was less. Therefore, the overall evaluation was the best. As a result, the method of adding cornus oil was found to be significantly better in smell and taste than in fermentation than in fermentation. In addition, it was confirmed that the odor and taste were significantly improved than the current Cheonggukjang powdered milk.
실시예 6. 기능성 발효두유의 DPPH 소거 효과Example 6 DPPH Scavenging Effect of Functional Fermented Soymilk
10oBrix 산수유를 두유에 1% 첨가하여 고초균으로 발효시킨 기능성 발효두유의 유리기 소거능을 산수유를 첨가하지 않은 발효두유와 대비하고 얻어진 결과를 도 1에 정리하였다. The result obtained by comparing free radical scavenging ability of functional fermented soy milk fermented with Bacillus subtilis by adding 1% of 10 o Brix Cornus milk to soymilk compared to fermented soymilk without adding cornus milk is summarized in FIG. 1.
구체적으로는, 발효두유 및 기능성 발효두유의 메탄올 추출물 100 μL와 60 μM 1,1-diphenyl-2-picrylhydrazyl (DPPH) 100 μL를 96 well plate에 넣은 후 혼합하여 암소에서 30분간 방치시킨 후 ELISA reader를 사용하여 540 nm에서 흡광도를 측정하였다(10). 발효두유의 메탄올 추출물은 동결건조한 시료를 10배의 메탄올로 실온에서 추출한 후 용매를 완전히 날려 사용하였다. 실험을 위해 얻어진 메탄올 추출물을 에탄올에 농도별로 용해시켜 사용하였다. DPPH 소거 효과는 시료를 첨가하지 않은 대조군과 비교하여 백분율(%)로 나타내었다.Specifically, 100 μL of methanol extracts of fermented soy milk and functional fermented soy milk and 100 μL of 60 μM 1,1-diphenyl-2-picrylhydrazyl (DPPH) were added to a 96 well plate, mixed and left for 30 minutes in a cow, followed by ELISA reader. Absorbance was measured at 540 nm using (10). Methanol extract of fermented soy milk was used to extract the
도 1에서 보듯이, 기능성 발효두유의 DPPH 소거 효과는 농도 의존적으로 증가하였으며 각 실험 농도에서 발효두유보다 유의적으로 높은 것으로 나타나 산수유 첨가 발효두유의 기능성을 확인하였다.As shown in Figure 1, the DPPH scavenging effect of the functional fermented soy milk increased concentration-dependently and significantly higher than the fermented soy milk at each experimental concentration, confirming the functionality of fermented soymilk added with cornus milk.
실시예 7. 기능성 발효두유의 동맥경화유발 마우스에서 지질저하 효과Example 7 Lipid Lowering Effect of Functional Fermented Soymilk in Atherosclerotic Mice
(1) 실험동물(1) experimental animals
청국장 두유가 혈장 지질저하를 통한 동맥경화 예방 효과에 미치는 영향을 살펴보기 위하여 형질 변형된 동맥경화유발 동물 모델인 apoprotein E deficient (apo E(-/-)) 마우스를 (주)중앙실험동물 (서울, 한국)에서 구입하여 실험에 사용하였다. To investigate the effects of Chungkookjang soymilk on the prevention of atherosclerosis through plasma lipid reduction, apoprotein E deficient (apo E (-/-) ) mouse, a transformed atherosclerosis-induced animal model, was used as a central laboratory animal (Seoul). , Korea) and used for the experiment.
실험동물은 1주일 동안 실험실 환경에 적응시킨 다음 각 군의 체중이 동일하게 조정하여 대조군(Control), 기능성발효두유(fermented soybean milk containing Corni fructus, FSCC)군, 청국장분말첨가두유(chungkukjang power added soybean milk, CPAS)군으로 나누었다. 청국장 분말 첨가 두유는 기능성 발효두유의 지질저하 및 항동맥경화 효과를 비교하기 위하여 긍정대조군으로 사용하였다. 각 실험군의 동물은 각 7마리씩이었다.The experimental animals were acclimated to the laboratory environment for 1 week, and then the weight of each group was adjusted equally so that the control group, fermented soybean milk containing Corni fructus (FSCC) group, and chungkukjang power added soybean were added. milk, CPAS) group. Cheonggukjang powder added soymilk was used as a positive control to compare the lipid-lowering and anti-arterial effects of functional fermented soymilk. There were seven animals in each experimental group.
(2) 식이조제 및 사육(2) Diet and Breeding
본 실험에 사용한 식이는 고콜레스테롤혈증을 유발하기 위한 동맥경화유발식이 (Atherogenic diet, AD)로 AIN-76 diet 조제법에 기준하여 제조하여 대조군 식이로 사용하였다. The diet used in the experiment was an atherosclerotic diet (Atherogenic diet, AD) to induce hypercholesterolemia and was prepared according to the AIN-76 diet preparation and used as a control diet.
동물실험을 위하여 제조한 기능성발효두유 (Fermented soymilk containing Corni furctus, FSCC)와 청국장 가루를 5% 첨가한 두유 (Chungkukjang powder added soymilk, CPAS)를 동결 건조 후 첨가하였다. FSCC와 CPAS 첨가량은 20.8 mL/100g body weight/day로 성인(60 Kg)의 우유 1회 섭취기준인 200mL의 10배에 해당하는 양이다. 식이는 1주 간격으로 조제하였다(100 g기준, 표 5 참조). Functional fermented soymilk (Fermented soymilk containing Corni furctus (FSCC)) prepared for animal experiments and soymilk (Chungkukjang powder added soymilk, CPAS) containing 5% of Cheonggukjang powder were added after freeze drying. The amount of FSCC and CPAS added is 20.8 mL / 100 g body weight / day, which is 10 times the 200 mL of adult (60 Kg) milk daily. The diet was prepared at weekly intervals (100 g standard, see Table 5).
동물 사육실의 온도는 20±1℃, 상대습도는 55±5%를 유지하였으며, 명암의 주기는 12시간 간격으로 조절하였다. 동물이 사료와 식수는 자유롭게 섭취하도록 충분히 공급하였으며 식이는 매일 같은 시간에 공급하였다. 동물실험기간은 8주이었다. 8주 사육 후, 마우스는 희생 전 24시간 절식시킨 다음 ether로 마취시킨 후 70% 에탄올로 복부를 소독한 후 개복하였다. 복부를 개복한 후 헤파린으로 처리한 멸균 주사기를 시용하여 하대정맥 및 심장에서 혈액을 재취하였다. 채혈 후 PBS 용액으로 관류하여 장기 내 남은 혈액을 제거하였고 간, 심장, 신장, 폐, 비장, 고환 등의 각 장기들을 적출하였다. 적출된 장기들을 PBS로 여러 차례 씻은 후 여과지를 이용하여 수분을 완전히 제거하였다. 각 장기의 무게를 측정한 다음 -70℃ 냉동고에 보관하였다. The temperature of the animal breeding room was maintained at 20 ± 1 ℃ and relative humidity at 55 ± 5%. Animals were supplied with sufficient feed and drinking water, and diets were fed at the same time each day. The animal duration was 8 weeks. After 8 weeks of breeding, mice were fasted for 24 hours before sacrifice, anesthetized with ether, and then disinfected with 70% ethanol and then opened. After the abdomen was opened, blood was taken from the inferior vena cava and heart using a sterile syringe treated with heparin. After the blood was collected, the remaining blood in the organ was removed by perfusion with PBS solution. The collected organs were washed several times with PBS, and then water was completely removed using filter paper. Each organ was weighed and stored in a -70 ° C. freezer.
장기는 homogenizer(Polytron, PT-MR 3100, Switzerland)로 균질화 하여 시료로 사용하였다.Organs were homogenized with a homogenizer (Polytron, PT-MR 3100, Switzerland) and used as samples.
1)Atherogenic diet was prepared according to the guideline of AIN-76A to which 1.25% cholesterol and 10% lard added 1) Atherogenic diet was prepared according to the guideline of AIN-76A to which 1.25% cholesterol and 10% lard added
2)FSCC: Fermented soymilk containing Corni fructus. The mixture solution of Corni fructus extract (1%, 10oBrix) and soymilk fermented with Bacillus subtilis 2829PNU015 2) FSCC: Fermented soymilk containing Corni fructus . The mixture solution of Corni fructus extract (1%, 10 o Brix) and soymilk fermented with Bacillus subtilis 2829
3)CPAS : Chungkukjang powder was added to the soymilk to be 5%. 3) CPAS: Chungkukjang powder was added to the soymilk to be 5%.
4)The amount of sample added to the diet was 10 times of the one serving of soymilk (200 mL) consumed by adults (60kg) 4) The amount of sample added to the diet was 10 times of the one serving of soymilk (200 mL) consumed by adults (60kg)
(3) 혈장 지질 저하 효과(3) plasma lipid lowering effect
in vitro 실험에서 DPPH 소거 효과가 현저히 높은 것으로 확인된 기능성 청국두유를 제조하여 유전 형질 변경된 동맥경화유발 마우스 (Apo E knockout mice)에게 8주간 섭취시키고, 체중 및 주요장기의 무게를 대비 관찰하여 하기표 6에 정리하였다. Functional in vitro soybean milk, which was found to have a significantly high DPPH scavenging effect in vitro , was prepared and fed to genetically modified Apo E knockout mice for 8 weeks, and compared with the weight and weight of major organs. It summarized in 6.
group1) Experimentalal
group 1)
gain (%)2) Body weight
gain (%) 2)
Values are mean ± SD Values are mean ± SD
NS : not significantNS: not significant
FSCC: Fermented soymilk containing Corni fructus. The mixture solution of Corni fructus extract (1%, 10oBrix) and soymilk fermented with Bacillus subtilis 2829PNU015FSCC: Fermented soymilk containing Corni fructus . The mixture solution of Corni fructus extract (1%, 10 o Brix) and soymilk fermented with Bacillus subtilis 2829
CPAS: Chungkukjang powder(5%) added soymilkCPAS: Chungkukjang powder (5%) added soymilk
1)Control group: mouse fed atherogenic diet (AD, 1.25% cholesterol diet), 1) Control group: mouse fed atherogenic diet (AD, 1.25% cholesterol diet),
FSCC group: mouse fed AD containing FSCC (33.3 mL/kg BW), FSCC group: mouse fed AD containing FSCC (33.3 mL / kg BW),
CPAS group: mouse fed AD containing CPAS (33.3 mL/kg BW)CPAS group: mouse fed AD containing CPAS (33.3 mL / kg BW)
2)(final weight-initial weight)/final weight × 100 2) (final weight-initial weight) / final weight × 100
상기표 6에서 보듯이, 마우스의 체중 및 중요장기의 무게는 동맥경화식이를 섭취한 대조군 및 청국장분말을 5% 첨가한 두유를 섭취한 긍정대조군 마우스에 비해 유의적인 차이가 없어 본 연구에서 제조한 기능성 발효두유는 섭취에 문제가 없음을 확인하였다. As shown in Table 6, the weight of the mice and the weight of the organs were not significantly different from those of the control group fed the atherosclerosis diet and the positive control group fed the soymilk containing 5% of the Cheonggukjang powder. Functional fermented soymilk was confirmed to have no problems with intake.
나아가, 기능성 발효두유를 섭취한 마우스의 혈중지질 농도를 청국장 분말을 첨가한 두유를 섭취한 마우스와 비교한 결과를 하기표 7에 정리하였다. Furthermore, the results of comparing the lipid lipid concentrations of the mice ingesting functional fermented soymilk with the mice ingesting soymilk added with Cheonggukjang powder are summarized in Table 7 below.
구체적으로는, 채혈한 혈액은 4℃에서 3,000rpm으로 15분간 원심분리하여 혈장을 분리하였다. Specifically, the collected blood was centrifuged at 3,000 rpm for 15 minutes at 4 ° C to separate plasma.
① 총 콜레스테롤 농도 측정① Total cholesterol concentration measurement
혈장의 총 콜레스테롤 농도는 효소법에 의한 정량용 키트 시약(Total cholesterol AM 202-K, 아산제약, 한국)으로 측정하였다(12). 시료 0.02 mL에 조제한 반응시약(효소시약 즉, AM 157S-2 1vail을 효소시약 용해액 1병에 용해) 3 mL씩을 첨가하여 vortex로 혼합하고 37℃의 water bath에서 5분간 반응시켜 490 nm에서 흡광도를 측정하였다. 콜레스테롤 표준 용액을 이용하여 위의 방법과 동일하게 발색시켜 흡광도를 측정한 후, 시료의 흡광도 값을 다음 식에 대입하여 혈장의 콜레스테롤 농도를 구하였다.Plasma total cholesterol concentration was measured by a kit reagent (Total cholesterol AM 202-K, Asan Pharmaceutical, Korea) for quantification by enzyme method (12). 3 mL each of the reaction reagents prepared (0.01 mL of enzyme reagent, ie AM 157S-2 dissolved in 1 bottle of enzyme reagent solution) were added to 0.02 mL of the sample, mixed with vortex, and reacted for 5 minutes in a water bath at 37 ° C. Was measured. Using the standard cholesterol solution, the absorbance was measured by the same method as described above, and then the absorbance value of the sample was substituted into the following equation to determine the cholesterol concentration of plasma.
② 중성지방 농도 측정② Triglyceride concentration measurement
중성지방 농도는 중성지방 측정용 키트 시약(Triglyceride AM 157S-K, 아산제약)으로 측정하였다(13). 실험방법은 총 콜레스테롤 측정을 위한 시료조제와 동일한 방법으로 준비하였다. 중성지방 표준 용액을 이용하여 위의 방법과 동일하게 10분간 발색시켜 540 nm에서 흡광도를 측정한 후, 시료의 흡광도 값을 다음식에 대입하여 혈장의 중성지방 농도를 계산하였다.Triglyceride concentration was measured with a kit reagent for triglyceride measurement (Triglyceride AM 157S-K, Asan Pharmaceutical) (13). Experimental method was prepared in the same manner as the sample preparation for measuring total cholesterol. Using a triglyceride standard solution, the color was measured for 10 minutes in the same manner as above, and the absorbance at 540 nm was measured.
③ 고밀도지단백질의 콜레스테롤 농도 측정 ③ Measurement of cholesterol concentration of high density lipoprotein
고밀도지단백질 (HDL cholesterol)내 콜레스테롤 농도는 효소법에 의한 키트시약 (HDL Cholesterol AM 203-K, 아산제약)을 이용하여 측정하였다. 실험방법은 분리한 혈장 0.2 mL에 분리시액 0.2 mL을 넣어 잘 혼합하여 10분간 실온에 방치한 후 4℃에서 3,000rpm으로 10분간 원심분리 후 상층액을 0.1 mL을 취하여 조제한 반응시약 3 mL씩 첨가하여 만들고 표준액은 콜레스테롤과 동일한 방법으로 만들어 vortex mixter로 혼합하고 37℃에서 5분간 반응시켜 60분 이내에 파장 490 nm에서 흡광도를 측정하고, 시료의 흡광도 값을 다음 식에 대입하여 HDL 콜레스테롤 농도를 구하였다.Cholesterol concentration in HDL cholesterol was measured using a kit reagent (HDL Cholesterol AM 203-K, Asan Pharmaceutical) by enzyme method. In the experimental method, 0.2 mL of the separated solution was added to 0.2 mL of the separated plasma, mixed well, and allowed to stand at room temperature for 10 minutes. After centrifugation at 3,000 rpm for 10 minutes at 4 ° C, 3 mL of the reaction reagents prepared by taking 0.1 mL of the supernatant were added. The standard solution was prepared in the same way as cholesterol, mixed with a vortex mixter, reacted at 37 ° C for 5 minutes, and absorbance was measured at wavelength 490 nm within 60 minutes. .
④ 저밀도지단백질의 콜레스테롤 농도 측정④ Measurement of cholesterol concentration of low density lipoprotein
저밀도지단백질의 콜레스테롤 농도는 Friedewald에 의해 하기 수학식에 근거하여 계산하였다. Cholesterol concentration of low density lipoprotein was calculated by Friedewald based on the following equation.
group3) Experimentalal
group 3)
Values were mean ± SD Values were mean ± SD
NS : not significantNS: not significant
1)See the legend of Table 6 1) See the legend of Table 6
a,bData were significantly different with one-way ANOVA followed by Duncan's multiple range test at the 0.05 level of significance. a, b Data were significantly different with one-way ANOVA followed by Duncan's multiple range test at the 0.05 level of significance.
상기표 7에서 보듯이, 기능성 발효 두유군의 혈장 총 콜레스테롤 농도는 동맥경화 식이를 섭취한 대조군에 비해 약 26.2% 감소하였으며(p<0.05), 긍정대조군인 청국장 분말 첨가군에 비해 약 15.0% 유의적(p<0.05)으로 감소하였다. 따라서 저밀도지단백질 (low density lipoprotein)의 농도도 기능성 발효두유는 대조군에 비해 30.0% 감소하였고 긍정대조군인 청국장 분말 첨가군에 비해 16.7% 감소하였음을 확인할 수 있었다. 기능성 발효두유군의 LDL-C 저하 효과는 대조군에 비해 유의적(P<0.05)이었으나 청국장분말 첨가 두유와는 유의적인 차이가 없었다. 따라서 청국발효두유는 혈장 콜레스테롤을 낮추고 동맥경화의 주 원인인 저밀도지단백질의 농도를 유의적으로 낮추었다. As shown in Table 7, the plasma total cholesterol concentration of the functional fermented soymilk group was reduced by 26.2% compared to the control group who consumed the atherosclerosis diet (p <0.05), and about 15.0% significantly compared to the positive control group added to the Chungkookjang powder. It decreased to the enemy (p <0.05). Therefore, the concentration of low density lipoprotein also decreased functional fermented soy milk by 30.0% compared with the control group and 16.7% compared with the addition of Cheonggukjang powder as a positive control. The LDL-C lowering effect of the functional fermented soy milk group was significantly (P <0.05) compared with the control group, but there was no significant difference with the soymilk added with the Cheonggukjang powder. Thus, fermented soybean milk lowered plasma cholesterol and significantly lowered the concentration of low-density lipoprotein, a major cause of atherosclerosis.
(4) 간지질 저하 효과 (4) hypolipidemic effect
기능성 청국두유의 간지질 저하 효과를 유전 형질 변경된 동맥경화유발 마우스 (Apo E knockout mice)에게 8주간 섭취시킨 다음 관찰하고 하기표 8에 정리하였다. The hepatic lipid lowering effect of functional Chungkook soybean milk was observed after 8 weeks ingestion in genetically modified Apo E knockout mice and summarized in Table 8 below.
구체적으로, 간은 chloroform과 methanol을 2:1로 섞은 용액을 첨가하여 homogenizer로 균질화한 뒤 2시간 동안 방치한 후 3,000rpm에서 30분간 원심분리하여 그 상층액을 지질 추출액으로서 사용하였다. 간의 총콜레스테롤과 중성지방 농도의 측정은 혈장 지질 측정 시 사용한 방법과 동일한 방법으로 실시하였다.Specifically, liver was added to a mixture of chloroform and methanol 2: 1, homogenized with a homogenizer, left for 2 hours, and centrifuged at 3,000 rpm for 30 minutes to use the supernatant as a lipid extract. The total cholesterol and triglyceride concentrations of the liver were measured by the same method as that used for the measurement of plasma lipids.
1)See the legend of Table 6 1) See the legend of Table 6
a,bData were significantly different with one-way ANOVA followed by Duncan's multiple range test at the 0.05 level of significance. a, b Data were significantly different with one-way ANOVA followed by Duncan's multiple range test at the 0.05 level of significance.
상기표 8에서 보듯이, 혈장과 마찬가지로 콜레스테롤 저하 효과는 관찰되었으나 중성지질 저하 효과는 관찰되지 않았다. 본 효과는 청국장 분말을 두유에 첨가하여 섭취하는 것과 유사한 효과를 나타내었다. As shown in Table 8, the cholesterol lowering effect was observed as in the plasma, but the neutral lipid lowering effect was not observed. This effect showed a similar effect to the intake of the Chungkukjang powder added to soy milk.
(5) 동맥의 지질 침착 억제 효과(5) inhibiting lipid deposition in arteries
기능성 청국두유의 동맥의 지질 침착정도를 유전 형질 변경된 동맥경화유발 마우스 (Apo E knockout mice)에게 8주간 섭취시키고 살펴본 결과를 하기표 9에 정리하였다. The lipid deposition of arterial soybean soybean milk was ingested into genetically modified Apo E knockout mice for 8 weeks and the results are summarized in Table 9 below.
구체적으로는, apoE-/- 마우스로부터 얻어진 심장을 10분동안 PBS에 넣은 다음 5분간 4% paraformaldehyde에 넣은 후 즉시 제거하였다. 그리고 하루동안 10% 중성 formalin에 고정 시켜 OCT compound를 이용하여 심장을 -70℃에서 보관하였다. 이 심장은 -20℃로 유지되는 저온 유지 장치를 이용하여 대동맥으로부터 연속하여 8㎛ 3조간으로 절편하였다. 이렇게 얻어진 지방병변은 Oil red O를 이용하여 염색하였으며 hematoxylin을 대비염색제로 사용하였다. 염색된 지방병변은 computer-assisted morphometry system(Axio Vision, Carl Zeiss, Canada)으로 측정하였으며 면적 계산은 image analysis program을 이용하였다.Specifically, the heart obtained from apoE-/-mice was placed in PBS for 10 minutes, then in 4% paraformaldehyde for 5 minutes, and immediately removed. And the heart was stored at -70 ℃ using OCT compound fixed in 10% neutral formalin for one day. The heart was sectioned in 3 trillion 8 μm series from the aorta with a cryostat maintained at -20 ° C. The resulting fat lesions were stained with Oil red O and hematoxylin was used as a counterstain. Stained lip lesions were measured using a computer-assisted morphometry system (Axio Vision, Carl Zeiss, Canada) and the area was calculated using an image analysis program.
1)Oil red O staining 으로 염색된 부분은 컴퓨터가 장착된 면적 분석기로 확인 1) The area stained with Oil red O staining is confirmed by the area analyzer equipped with a computer.
2,3)See the legend of Table 6 2,3) See the legend of Table 6
***p<0.001 by t-test between control and FSCC*** p <0.001 by t-test between control and FSCC
#p<0.05 by t-test between FSCC and CPAS# p <0.05 by t-test between FSCC and CPAS
상기표 9에서 보듯이, 지질 침착면적이 대조군에 비해 유의적으로 억제되었으며, 이 효과는 청국장 분말 첨가 두유보다 높았으나 유의적인 차이는 없었다. As shown in Table 9, the lipid deposition area was significantly suppressed compared to the control, the effect was higher than the soymilk added with Chungkookjang powder, but there was no significant difference.
상술한 바와 같은 본 발명을 정리하면 다음과 같다:In summary, the present invention as described above is as follows:
1. 순창지역 청국장 6제품의 이화학 특성 및 항산화성 1. Physicochemical Characteristics and Antioxidant Activity of 6 Products of Cheonggukjang, Sunchang Area
순창지역 청국장 6제품의 pH, 산도, 총균수, 아미노태 질소(NH3-N)의 농도 및 관능성은 S1,S2,S3에서 높았으며 이중 S3의 유리기 소거능이 다른 제품에 비해 유의적으로 가장 높았다. The pH, acidity, total bacterial count, amino nitrogen (NH 3 -N) concentration and functionalities of 6 products of Cheonggukjang in Sunchang were high in S1, S2, and S3, and the free radical scavenging ability of S3 was significantly higher than that of other products. .
2. 항동맥경화 기능성 발효두유 제조 조건 확립2. Establishment of conditions for producing anti-arterial functional fermented soymilk
1) S3 청국장으로부터 분리 동정된 3종류의 Bacillus 균, Bacillus subtilis 2805 PNU 014, Bacillus subtilis 2829 PNU 015, Bacillus subtilis 2825 PNU 016과 상업적으로 사용되고 있는 2종의 균, MYCO(Innobiz Myco, Gyeong Ju, Korea), KCCM(KCCM 11316, Seoul, Korea) 등 총 5종류의 Bacillus 균 중 Bacillus subtilis 2829 PNU 015의 피브린 분해능이 27.2 mU plasmin/hr로 가장 높았다.1) Three types of Bacillus isolates isolated from S3 Cheonggukjang, Bacillus subtili s 2805 PNU 014, Bacillus subtilis 2829 PNU 015, Bacillus subtilis 2825 PNU 016 and two commercially available bacteria, MYCO (Innobiz Myco, Gyeong Ju, The highest fibrin resolution of Bacillus subtilis 2829 PNU 015 was found to be 27.2 mU plasmin / hr among the five Bacillus strains (KCCM 11316, Seoul, Korea).
2) 청국균 발효두유의 최적 제조 조건은 두유에 Bacillus subtilis를 1%(CFU 107~108)농도로 접종 시켜 40℃에서 pH 5.5±0.2가 될 때까지 약 8시간 동안 진탕 배양하였다.2) Optimum conditions for the production of fermented soymilk were inoculated with 1% (
3) 청국균 발효두유의 DPPH 소거능, 총항산화능(TEAC), LDL 산화 억제능은 미 발효두유 보다 유의적으로 높았다(p<0.05). 이 중에서 Bacillus subtilis 2829 PNU 015 의 항산화 효과가 가장 우수한 것으로 나타났다(p<0.05).3) DPPH scavenging activity, total antioxidant activity (TEAC), and LDL oxidation inhibition activity of fermented soybean milk were significantly higher than that of unfermented soymilk (p <0.05). Among them, Bacillus subtilis 2829 PNU 015 showed the best antioxidant effect (p <0.05).
4) 청국균 발효두유의 기능성 및 관능성 증진을 위해 6가지 기능성 소재(마늘, 산수유, 자색고구마, 흑마늘, 양파, 강화사자발쑥)를 비교하였을 때 산수유의 피브린 분해능이 33.4 mU plasmin/hr로 가장 높았고 1% 첨가 농도에서 기호도가 가장 높았다. 4) The fibrin resolution of cornus milk was 33.4 mU plasmin / hr when the six functional materials (garlic, cornus, purple sweet potato, black garlic, onion, fortified lion cucurbita) were compared to enhance the functionality and functionality of fermented soymilk. It was high and the highest in the 1% addition concentration.
5) 산수유 1%를 첨가하여 발효한 청국균 발효두유는 산수유 미 첨가 청국균 발효두유에 비해 1,000 ㎍/mL 농도에서 DPPH 소거능 28.0%, O2 -소거능 22.8% 그리고 nitrite 소거능이 21.6% 높아져(p<0.001) 기능성 및 관능성이 증진 되었다. 5) The fermented soybean milk fermented with 1% cornus milk increased DPPH scavenging activity 28.0%, O 2 - scavenging activity 22.8% and nitrite scavenging activity at a concentration of 1,000 ㎍ / mL compared to the uncooked sorghum fermented soymilk (p). <0.001) Functionality and functionality were enhanced.
3. ApoE3. ApoE (-/-)(-/-) 마우스에서 항동맥경화 청국균 발효두유의 지질 저하 효과 Lipid-lowering Effect of Fermented Soymilk with Anti-arteriosclerosis in Mice
산수유 1% 첨가하여 Bacillus subtilis 2829 PNU 015(기탁번호 KCCM 42923)으로 발효시킨 두유를 동결건조하여 동맥경화사료에 첨가하여 8주 동안 섭취시킨 후 지질 저하 효과를 살펴보았다. 실험군은 동맥경화식이를 섭취한 대조군, 항동맥경화 기능성 발효유군, 그리고 긍정대조군으로 사용된 청국장분말을 첨가한 두유군 3군이었으며 군당 동물은 7마리이었다. Soymilk fermented with Bacillus subtilis 2829 PNU 015 (Accession No. KCCM 42923) by adding 1% cornus oil was lyophilized and added to atherosclerosis feed for 8 weeks to examine the lipid lowering effect. The experimental group consisted of the control group fed the atherosclerosis diet, the anti-arteriosclerosis functional fermented milk group, and the soymilk group 3 added with the Cheonggukjang powder used as a positive control group, and there were 7 animals per group.
1) 체중 증가량 및 간, 신장, 폐, 고환 그리고 비장의 무게는 대조군과 기능성 발효두유군 및 청국장 분말 첨가두유군 간에 유의적인 차이를 나타나지 않았다.1) There were no significant differences in weight gain and liver, kidney, lung, testis and spleen weights between the control group and the functional fermented soy milk group and Cheonggukjang powdered soy milk group.
2) 혈장 지질 농도는 콜레스테롤이 1.25% 첨가된 동맥경화식이에 의해 총콜레스테롤 및 LDL-C이 1013.7 mg/dL 와 930.3 mg/dl 이상 높아져 고콜레스테롤혈증이 유발되었으나 기능성 발효두유 첨가에 의해 각각 26.2%, 30.3% 그리고 청국장 분말 첨가에 의해 각각 15.4%, 16.4% 낮아졌다(p<0.05). 그러나 혈장 중성지방 및 HDL-C 농도를 개선하는 효과는 관찰되지 않았다. 2) Plasma lipid concentrations were increased by more than 1013.7 mg / dL and 930.3 mg / dl of total cholesterol and LDL-C by arteriosclerosis diet with 1.25% cholesterol, but hypercholesterolemia was induced by addition of functional fermented soymilk, respectively. , 30.3% and Chungkookjang powder decreased by 15.4% and 16.4%, respectively (p <0.05). However, no effect of improving plasma triglyceride and HDL-C levels was observed.
3) 간의 지질 농도는 기능성 발효두유군 및 청국장 분말첨가군의 콜레스테롤 농도는 대조군에 비해 각각 19.7%와 19.4% 낮아졌다(p<0.05). 그러나 중성 지방 농도는 군간 유의적인 차이가 없었다. 3) The lipid concentrations of liver were 19.7% and 19.4% lower than those of the control fermented soymilk group and Chunggukjang powder added group, respectively (p <0.05). However, triglyceride levels did not differ significantly between groups.
4) 간의 지질 산화정도(TBARS)는 기능성 발효두유군 및 청국장 분말 첨가군에서 대조군에 비해 각각 23.6%, 15.4% 유의적으로 낮아져 산화억제효과가 관찰되었다(p<0.05).4) The lipid oxidation degree (TBARS) of liver was significantly lower in functional fermented soymilk group and cheongukjang powder added group, 23.6% and 15.4%, respectively, than the control group ( p <0.05).
5) 분변의 지방 농도는 기능성 발효두유군군의 콜레스테롤 및 중성지방의 농도가 대조군에 비해 7.2% 및 9.5% 낮았졌으나 유의적인 차이는 없었다.5) Fecal fat concentrations in the functional fermented soymilk group were 7.2% and 9.5% lower than those of the control group, but there was no significant difference.
이상의 실험결과에서 청국균 발효두유는 두유에 비해 유리기 소거능 및 피브린 분해능이 높은 것으로 나타났으며 산수유를 첨가하여 제조하였을 때 관능성 및 지질저하 효과가 증진되었다. 이러한 산수유첨가 청국균발효두유의 효과는 청국장분말첨가두유보다 우수하여 장기 섭취 시 혈장 콜레스테롤 농도를 낮춤으로써 항동맥경화 효과가 있을 것으로 생각된다.In the above experiments, the fermented soybean milk was found to have higher free radical scavenging ability and fibrin resolution than soymilk. The effect of the agglomerated milk added fermented soybean milk is superior to that of the soybean milk added to the soybean paste powder, which is thought to have an anti-arteriosclerosis effect by lowering the plasma cholesterol concentration during long-term intake.
본 발명은 교육과학기술부와 한국산업기술재단의 지역혁신인력양성사업 (M-020-20070131134352)으로 수행된 연구결과이다. The present invention is the result of research conducted by the Ministry of Education, Science and Technology and the Korea Industrial Technology Foundation (M-020-20070131134352).
도 1은 본 발명의 일 실시예에 의한 산수유를 첨가하고 고초균으로 접종 발효시킨 두유에 대한 DPPH 유리기 활성능을 측정한 그래프이다. 도면내에서 약어 FS는 Bacillus subtilis 2829PNU015 접종 발효한 두유를 의미하며, FSCC는 FS에 Corni fructus extracts (1%, 10oBrix)를 추가하여 접종 발효한 두유를 의미한다. 1 is a graph measuring the DPPH free radical activity of soy milk added to Cornus milk and inoculated and fermented with Bacillus subtilis according to an embodiment of the present invention. In the drawings, the abbreviation FS refers to soymilk fermented with Bacillus subtilis 2829PNU015, and FSCC refers to soymilk inoculated and fermented by adding Corni fructus extracts (1%, 10 o Brix) to FS.
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