KR102189342B1 - Bacillus subtilis SRCM103716 strain having excellent β-glucosidase and antioxidant activity and uses thereof - Google Patents
Bacillus subtilis SRCM103716 strain having excellent β-glucosidase and antioxidant activity and uses thereof Download PDFInfo
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- KR102189342B1 KR102189342B1 KR1020190116331A KR20190116331A KR102189342B1 KR 102189342 B1 KR102189342 B1 KR 102189342B1 KR 1020190116331 A KR1020190116331 A KR 1020190116331A KR 20190116331 A KR20190116331 A KR 20190116331A KR 102189342 B1 KR102189342 B1 KR 102189342B1
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- bacillus subtilis
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Abstract
Description
본 발명은 β-글루코시다아제(β-glucosidase) 활성이 우수하고, 유해미생물에 대한 항균활성 및 β-용혈 활성이 있고, 유해물질, 유해효소 및 바이오제닉 아민을 생성하지 않으면서, 참깨박 발효시 총 폴리페놀 및 총 플라보노이드 함량과 항산화 활성을 증가시키는 것을 특징으로 하는 바실러스 서브틸리스(Bacillus subtilis) SRCM103716 균주(KFCC11832P), 상기 균주 또는 이의 배양액을 유효성분으로 함유하는 참깨박 발효용 미생물 제제, 상기 균주를 이용하여 리그난 함량 및 항산화 활성이 증진된 참깨박 혼합 발효물의 제조방법 및 상기 방법으로 제조된 리그난 함량 및 항산화 활성이 증진된 참깨박 혼합 발효물에 관한 것이다.The present invention has excellent β-glucosidase activity, has antibacterial activity against harmful microorganisms and β-hemolytic activity, does not produce harmful substances, harmful enzymes and biogenic amines, fermenting sesame seeds Bacillus subtilis SRCM103716 strain (KFCC11832P), characterized in that it increases the total polyphenol and total flavonoid content and antioxidant activity, a microbial preparation for fermentation of sesame seeds containing the strain or a culture solution thereof as an active ingredient, It relates to a method for preparing a sesame seed mixture fermented product with improved lignan content and antioxidant activity using the strain, and to a sesame seed mixed fermented product with improved lignan content and antioxidant activity prepared by the above method.
참깨는 참깨과 참깨속(Pedaliaceae. Sesamum)에 속하는 1년생 초본으로 열대에서 냉온대에 걸쳐 재배되는 세계적인 유료작물이다. 참깨의 영양학적 성분을 살펴보면 지질 51%, 단백질 20%, 당질 15% 정도 함유하고 있으며, 쌀과 소맥에 비하여 칼슘, 인, 아연, 철, 비타민 B1, B2, 불포화지방산 등이 풍부한 식품이다. 이와 더불어, 참깨에는 항산화 효과를 나타내는 세사민(sesamin) 0.1~1.0%와 세사미놀(sesaminol)이 0.3~0.7%가 함유되어 있다.Sesame is a one-year herb belonging to the sesame family (Pedaliaceae. Sesamum), and is a worldwide paid crop grown from tropical to cold and temperate zones. When looking at the nutritional components of sesame seeds, it contains 51% lipid, 20% protein, and 15% sugar. Compared to rice and wheat, it is a food rich in calcium, phosphorus, zinc, iron, vitamins B1, B2, and unsaturated fatty acids. In addition, sesame seeds contain 0.1-1.0% sesamin and 0.3-0.7% sesamin, which have antioxidant effects.
또한, 참깨를 적정온도로 볶음 처리하고 압착법으로 착유한 다음, 필수적으로 수반되는 부산물인 착유박에는 인체에 유효한 성분들이 다량 함유되어 있다. 참깨박에 존재하는 주요 유효성분으로는 세사민(sesamin), 세사미놀(sesaminol), 세사몰리놀(sesamolinol), 피노레시놀(pinoresinol)과 같은 리그난(lignan) 성분들이 있다. 이들은 주로 포도당과 결합된 수용성 배당체의 형태로 존재하며, 그 함량은 약 1% 정도이다. 상기 리그난 배당체는 지질과산화 억제 효과를 가지고 있으며 그 첨가 농도에 따라 지질과산화 억제 효과가 증가함이 보고된 바 있다.In addition, after the sesame seeds are roasted at an appropriate temperature and milked by a compression method, the milking foil, which is an essential by-product, contains a large amount of ingredients effective for the human body. The main active ingredients present in sesame seeds include lignan components such as sesamin, sesaminol, sesamolinol, and pinoresinol. They mainly exist in the form of water-soluble glycosides bound to glucose, and their content is about 1%. The lignan glycosides have a lipid peroxidation inhibitory effect, and it has been reported that the lipid peroxidation inhibitory effect increases with the added concentration.
또한, 참깨박의 리그난 성분들은 저밀도 지단백질(low density lipoprotein; LDL)의 산화를 방지하는 것으로 알려져 있다. 특히, 상기 리그난 성분 중 세사미놀 배당체(sesaminol glucosides)는 강한 항산화력과 항동맥경화성이 보고된 바 있다.In addition, the lignan components of sesame seeds are known to prevent oxidation of low density lipoprotein (LDL). In particular, among the lignan components, sesaminol glucosides have been reported to have strong antioxidant power and antiarteriosclerosis.
한국등록특허 제1786938호에는 전통장류로부터 분리한 바실러스 서브틸리스 SY07 균주 및 이의 용도가 개시되어 있고, 한국공개특허 제2015-0101789호에는 복합 효소 생산능 및 항균 활성을 갖는 바실러스 서브틸리스 BK418 균주 및 이의 용도가 개시되어 있으나, 본 발명의 β-글루코시다아제 활성 및 항산화 활성이 우수한 바실러스 서브틸리스 SRCM103716 균주 및 이의 용도와는 상이하다.Korean Patent Registration No.1786938 discloses Bacillus subtilis SY07 strain isolated from traditional paste and its use, and Korean Patent Publication No. 2015-0101789 discloses Bacillus subtilis BK418 strain having complex enzyme-producing ability and antibacterial activity. And the use thereof, but are different from the Bacillus subtilis SRCM103716 strain having excellent β-glucosidase activity and antioxidant activity of the present invention and use thereof.
본 발명은 상기와 같은 요구에 의해 안출된 것으로서, 본 발명에서는 총 폴리페놀 및 총 플라보노이드 함량과 항산화 활성이 우수하고, 효소 분비능을 지니는 고초균을 선발하고자 하여, 전통식품으로부터 바실러스 서브틸리스(Bacillus subtilis) SRCM103716 균주를 분리하였고, 상기 균주는 참깨박 발효시 총 폴리페놀 및 총 플라보노이드 함량과 항산화 활성을 증가시키면서, 프로테아제(protease), 아밀라아제(amylase), 셀룰라제(cellulase) 및 β-글루코시다아제(β-glucosidase)에 대한 효소 활성이 있으며, 유해미생물에 대한 항균활성 및 β-용혈 활성이 있고, 유해물질, 유해효소 및 바이오제닉 아민을 생성하지 않음을 확인하였다. 따라서, 본 발명에서 분리한 바실러스 서브틸리스 SRCM103716 균주는 고품질의 참깨박 혼합 발효물 제조에 중요하게 사용될 수 있는 것을 확인함으로써, 본 발명을 완성하였다.The present invention was conceived by the above requirements, and in the present invention, Bacillus subtilis ( Bacillus subtilis) from traditional foods is intended to select Bacillus subtilis, which is excellent in total polyphenol and total flavonoid content and antioxidant activity, and has enzyme secretion ability. ) SRCM103716 strain was isolated, and the strain increased total polyphenol and total flavonoid content and antioxidant activity during sesame seed fermentation, while protease, amylase, cellulase, and β-glucosidase ( β-glucosidase), antimicrobial activity against harmful microorganisms, and β-hemolysis activity, and it was confirmed that no harmful substances, harmful enzymes, and biogenic amines were produced. Therefore, by confirming that the Bacillus subtilis SRCM103716 strain isolated in the present invention can be used importantly for the production of high-quality sesame seed mixed fermented products, the present invention was completed.
상기 과제를 해결하기 위해, 본 발명은 β-글루코시다아제(β-glucosidase) 활성이 우수하고, 유해미생물에 대한 항균활성 및 β-용혈 활성이 있고, 유해물질, 유해효소 및 바이오제닉 아민을 생성하지 않으면서, 참깨박 발효시 총 폴리페놀 및 총 플라보노이드 함량과 항산화 활성을 증가시키는 것을 특징으로 하는 바실러스 서브틸리스(Bacillus subtilis) SRCM103716 균주(KFCC11832P)를 제공한다.In order to solve the above problems, the present invention has excellent β-glucosidase activity, has antibacterial activity against harmful microorganisms and β-hemolytic activity, and generates harmful substances, harmful enzymes and biogenic amines. Without, it provides a Bacillus subtilis ( Bacillus subtilis ) SRCM103716 strain (KFCC11832P), characterized in that it increases the total polyphenol and total flavonoid content and antioxidant activity during sesame seed fermentation.
또한, 본 발명은 바실러스 서브틸리스(Bacillus subtilis) SRCM103716 균주 또는 이의 배양액을 유효성분으로 함유하는 참깨박 발효용 미생물 제제를 제공한다.In addition, the present invention provides a microbial preparation for fermentation of sesame seeds containing Bacillus subtilis SRCM103716 strain or a culture solution thereof as an active ingredient.
또한, 본 발명은 (1) 분쇄한 참깨박에 물을 가수하여 증자한 후 냉각한 참깨박에 바실러스 서브틸리스(Bacillus subtilis) SRCM103716 균주를 접종한 후 발효하여 총 폴리페놀 및 총 플라보노이드 함량과 항산화 활성 및 β-글루코시다아제(β-glucosidase) 활성이 증진된 참깨박 고초균 발효물을 제조하는 단계; (2) 분쇄한 참깨박에 물을 가수하여 증자한 후 냉각한 참깨박에 아스퍼질러스 오리재(Aspergillus oryzae) 균주를 접종한 후 발효하여 참깨박 발효물을 제조하는 단계; 및 (3) 상기 (1)단계의 제조한 참깨박 고초균 발효물과 상기 (2)단계의 제조한 참깨박 발효물을 혼합하는 단계를 포함하여 제조하는 것을 특징으로 하는 리그난 함량 및 항산화 활성이 증진된 참깨박 혼합 발효물의 제조방법을 제공한다.In addition, the present invention is (1) inoculated with Bacillus subtilis SRCM103716 strain in the cooled sesame seed after adding water to the pulverized sesame meal and fermented to increase the total polyphenol and total flavonoid content and antioxidant. Preparing a fermented product of sesame gourd Bacillus bacillus with improved activity and β-glucosidase activity; (2) adding water to the pulverized sesame meal to increase the steam, and then inoculating the cooled sesame seed with Aspergillus oryzae strain and fermenting to prepare a sesame seed fermented product; And (3) mixing the fermented sesame seed meal prepared in step (1) with the fermented sesame seed meal prepared in step (2), and enhancing antioxidant activity. It provides a method for producing fermented sesame seed mixture.
또한, 본 발명은 상기 방법으로 제조된 리그난 함량 및 항산화 활성이 증진된 참깨박 혼합 발효물을 제공한다.In addition, the present invention provides a sesame seed mixed fermented product with improved lignan content and antioxidant activity prepared by the above method.
본 발명에서는 전통 식품에서 분리한 바실러스 서브틸리스(Bacillus subtilis) SRCM103716 균주가 참깨박 발효시 총 폴리페놀 및 총 플라보노이드 함량과 항산화 활성을 현저하게 증가시키고, β-글루코시다아제(β-glucosidase) 활성이 우수하고, 유해미생물에 대한 항균활성 및 β-용혈 활성이 있고, 유해물질, 유해효소 및 바이오제닉 아민을 생성하지 않음을 확인하였다. 본 발명의 바실러스 서브틸리스 SRCM103716 균주를 이용하여 참깨박 발효물 제조 시 고품질의 발효물을 제조할 수 있어, 식품산업에 유용하게 이용될 수 있을 것으로 판단된다.In the present invention, Bacillus subtilis SRCM103716 strain isolated from traditional food significantly increases total polyphenol and total flavonoid content and antioxidant activity during sesame seed fermentation, and β-glucosidase activity It was confirmed that this was excellent, had antimicrobial activity against harmful microorganisms and β-hemolytic activity, and did not produce harmful substances, harmful enzymes and biogenic amines. It is believed that the Bacillus subtilis SRCM103716 strain of the present invention can be used to prepare a high-quality fermented product when producing a sesame seed fermented product, and thus can be usefully used in the food industry.
도 1은 본 발명의 바실러스 서브틸리스 SRCM103716의 계통수(phylogenetic tree)이다.
도 2는 참깨박 고초균 발효물의 제조공정을 도식화한 것이다.
도 3은 고초균으로 발효 전과 후, 참깨박 발효물의 생균수를 비교한 그래프이다.
도 4는 고초균으로 발효 전과 후, 참깨박 발효물의 DPPH 라디칼 소거능을 비교한 그래프이다.
도 5는 고초균으로 발효 전과 후, 참깨박 발효물의 총 폴리페놀 함량을 비교한 그래프이다.
도 6은 고초균으로 발효 전과 후, 참깨박 발효물의 총 플라보노이드 함량을 비교한 그래프이다.
도 7은 혼합비율에 따른 참깨박 발효 혼합물의 DPPH 라디칼 소거능을 비교한 그래프이다.
도 8은 참깨박 발효 혼합물을 이용한 간장의 제조공정을 도식화한 것이다.1 is a phylogenetic tree of Bacillus subtilis SRCM103716 of the present invention.
Figure 2 is a schematic diagram of the manufacturing process of sesame seed Bacillus fermented product.
3 is a graph comparing the number of viable cells of fermented sesame seeds before and after fermentation with Bacillus bacillus.
4 is a graph comparing DPPH radical scavenging ability of fermented sesame seeds before and after fermentation with Bacillus bacillus.
5 is a graph comparing the total polyphenol content of fermented sesame seeds before and after fermentation with Bacillus bacillus.
6 is a graph comparing the total flavonoid content of fermented sesame seeds before and after fermentation with Bacillus bacillus.
7 is a graph comparing the DPPH radical scavenging ability of a fermented sesame seed mixture according to the mixing ratio.
Figure 8 is a schematic diagram of the manufacturing process of soy sauce using a sesame seed fermentation mixture.
본 발명의 목적을 달성하기 위하여, 본 발명은 β-글루코시다아제(β-glucosidase) 활성이 우수하고, 유해미생물에 대한 항균활성 및 β-용혈 활성이 있고, 유해물질, 유해효소 및 바이오제닉 아민을 생성하지 않으면서, 참깨박 발효시 총 폴리페놀 및 총 플라보노이드 함량과 항산화 활성을 증가시키는 것을 특징으로 하는 바실러스 서브틸리스(Bacillus subtilis) SRCM103716 균주(KFCC11832P)를 제공한다.In order to achieve the object of the present invention, the present invention has excellent β-glucosidase activity, antibacterial activity against harmful microorganisms and β-hemolytic activity, and harmful substances, harmful enzymes and biogenic amines. It provides a Bacillus subtilis ( Bacillus subtilis ) SRCM103716 strain (KFCC11832P), characterized in that it increases the total polyphenol and total flavonoid content and antioxidant activity during fermentation of sesame seeds without generating.
본 발명의 일 구현 예에 따른 균주에서, 상기 유해미생물은 바실러스 세레우스(Bacillus cereus), 스타필로코커스 아우레우스(Staphylococcus aureus) 및 리스테리아 모노시토게네스(Listeria monocytogenes)이며, 상기 유해물질은 페닐알라닌(phenylalanine)이며, 상기 유해효소는 우레아제(urease) 및 β-글루쿠로니다아제(β-glucuronidase)이며, 상기 바이오제닉 아민은 티라민 및 히스타민일 수 있으나, 이에 제한되지 않는다. In the strain according to an embodiment of the present invention, the harmful microorganisms are Bacillus cereus , Staphylococcus aureus , and Listeria monocytogenes , and the harmful substances are phenylalanine. (phenylalanine), and the harmful enzymes are urease and β-glucuronidase, and the biogenic amine may be tyramine and histamine, but is not limited thereto.
또한, 본 발명의 일 구현 예에 따른 균주에서, 상기 균주는 프로테아제(protease), 아밀라아제(amylase) 및 셀룰라제(cellulase) 분비능을 추가로 가질 수 있으나, 이에 제한되지 않는다.In addition, in the strain according to an embodiment of the present invention, the strain may additionally have the ability to secrete protease, amylase, and cellulase, but is not limited thereto.
또한, 본 발명은 바실러스 서브틸리스(Bacillus subtilis) SRCM103716 균주(KFCC11832P) 또는 이의 배양액을 유효성분으로 함유하는 참깨박 발효용 미생물 제제를 제공한다.In addition, the present invention provides a microbial preparation for fermentation of sesame seeds containing Bacillus subtilis SRCM103716 strain (KFCC11832P) or a culture solution thereof as an active ingredient.
본 발명에 따른 참깨박 발효용 미생물 제제는 용액, 분말, 현탁액, 분산액, 에멀젼, 유성 분산액, 페이스트, 분진, 흩뿌림 물질 또는 과립제로 제조할 수 있으나, 이에 제한되지는 않는다.The microbial preparation for fermentation of sesame seeds according to the present invention may be prepared as a solution, powder, suspension, dispersion, emulsion, oily dispersion, paste, dust, scattering material or granules, but is not limited thereto.
본 발명은 또한, The present invention also,
(1) 분쇄한 참깨박에 물을 가수하여 증자한 후 냉각한 참깨박에 바실러스 서브틸리스(Bacillus subtilis) SRCM103716 균주를 접종한 후 발효하여 총 폴리페놀 및 총 플라보노이드 함량과 항산화 활성 및 β-글루코시다아제(β-glucosidase) 활성이 증진된 참깨박 고초균 발효물을 제조하는 단계;(1) After adding water to the crushed sesame seed meal, water was added, and then Bacillus subtilis SRCM103716 strain was inoculated in the cooled sesame seed meal, and fermented to obtain total polyphenol and total flavonoid content, antioxidant activity, and β-glucose. Preparing a fermented product of sesame gourd Bacillus bacillus with enhanced sidase (β-glucosidase) activity;
(2) 분쇄한 참깨박에 물을 가수하여 증자한 후 냉각한 참깨박에 아스퍼질러스 오리재(Aspergillus oryzae) 균주를 접종한 후 발효하여 참깨박 발효물을 제조하는 단계; 및(2) adding water to the pulverized sesame meal to increase the steam, and then inoculating the cooled sesame seed with Aspergillus oryzae strain and fermenting to prepare a sesame seed fermented product; And
(3) 상기 (1)단계의 제조한 참깨박 고초균 발효물과 상기 (2)단계의 제조한 참깨박 발효물을 혼합하는 단계를 포함하여 제조하는 것을 특징으로 하는 리그난 함량 및 항산화 활성이 증진된 참깨박 혼합 발효물의 제조방법을 제공한다.(3) The lignan content and antioxidant activity are enhanced, comprising the step of mixing the fermented sesame seed meal prepared in step (1) and the fermented sesame seed meal prepared in step (2). It provides a method of manufacturing a sesame seed mixed fermented product.
본 발명의 참깨박 혼합 발효물의 제조방법은, 보다 구체적으로는The method for producing a fermented sesame seed mixture of the present invention,
(1) 분쇄한 참깨박에 물을 1.2~1.8배 가수하여 110~130℃에서 10~20분 동안 증자한 후 냉각한 참깨박에 바실러스 서브틸리스(Bacillus subtilis) SRCM103716 균주를 접종한 후 34~40℃에서 20~28시간 동안 발효하여 총 폴리페놀 및 총 플라보노이드 함량과 항산화 활성 및 β-글루코시다아제(β-glucosidase) 활성이 증진된 참깨박 고초균 발효물을 제조하는 단계;(1) Water is added 1.2 to 1.8 times to the crushed sesame seed, and then increased at 110 to 130°C for 10 to 20 minutes, and then Bacillus subtilis SRCM103716 strain was inoculated into the cooled sesame seed meal. Fermenting at 40° C. for 20 to 28 hours to prepare a fermented sesame gourd Bacillus fermented product with enhanced total polyphenol and total flavonoid content, antioxidant activity, and β-glucosidase activity;
(2) 분쇄한 참깨박에 물을 0.4~0.6배 가수하여 110~130℃에서 10~20분 동안 증자한 후 냉각한 참깨박에 아스퍼질러스 오리재(Aspergillus oryzae) 균주를 접종한 후 28~32℃에서 68~76시간 동안 발효하여 참깨박 발효물을 제조하는 단계; 및(2) After adding water 0.4 to 0.6 times to the crushed sesame seed, add water at 110 to 130°C for 10 to 20 minutes, and inoculate the cooled sesame seed with Aspergillus oryzae strain. Fermenting at 32° C. for 68 to 76 hours to prepare a sesame seed fermented product; And
(3) 상기 (1)단계의 제조한 참깨박 고초균 발효물과 상기 (2)단계의 제조한 참깨박 발효물을 4~6:94~96 중량비율로 혼합하는 단계를 포함할 수 있으며,(3) mixing the fermented sesame seed meal prepared in step (1) and the fermented sesame seed meal prepared in step (2) in a weight ratio of 4 to 6: 94 to 96,
더욱 구체적으로는More specifically
(1) 분쇄한 참깨박에 물을 1.5배 가수하여 121℃에서 15분 동안 증자한 후 냉각한 참깨박에 바실러스 서브틸리스(Bacillus subtilis) SRCM103716 균주를 접종한 후 37℃에서 24시간 동안 발효하여 총 폴리페놀 및 총 플라보노이드 함량과 항산화 활성 및 β-글루코시다아제(β-glucosidase) 활성이 증진된 참깨박 고초균 발효물을 제조하는 단계;(1) After adding water 1.5 times to the pulverized sesame seed, it was increased for 15 minutes at 121°C, and then Bacillus subtilis SRCM103716 strain was inoculated into the cooled sesame seed and fermented at 37°C for 24 hours. Preparing a fermented product of sesame gourd Bacillus with enhanced total polyphenol and total flavonoid content, antioxidant activity and β-glucosidase activity;
(2) 분쇄한 참깨박에 물을 0.5배 가수하여 121℃에서 15분 동안 증자한 후 냉각한 참깨박에 아스퍼질러스 오리재(Aspergillus oryzae) 균주를 접종한 후 30℃에서 72시간 동안 발효하여 참깨박 발효물을 제조하는 단계; 및(2) After adding water 0.5 times to the pulverized sesame meal and increasing it at 121°C for 15 minutes, inoculating the cooled sesame seed with Aspergillus oryzae strain and fermenting it at 30°C for 72 hours. Preparing a fermented sesame seed meal; And
(3) 상기 (1)단계의 제조한 참깨박 고초균 발효물과 상기 (2)단계의 제조한 참깨박 발효물을 5:95 중량비율로 혼합할 수 있다.(3) The fermented sesame seed meal prepared in step (1) and the fermented sesame seed meal prepared in step (2) may be mixed in a weight ratio of 5:95.
본 발명은 또한, 상기 방법으로 제조된 리그난 함량 및 항산화 활성이 증진된 참깨박 혼합 발효물을 제공한다.The present invention also provides a sesame seed mixed fermented product with improved lignan content and antioxidant activity prepared by the above method.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.
제조예Manufacturing example 1. One. 참깨박Sesame seeds 발효 혼합물 Fermentation mixture
(1) 분쇄한 참깨박에 물을 1.5배(v/w) 가수하여 121℃에서 15분 동안 증자한 후 60℃로 냉각한 참깨박에 바실러스 서브틸리스(Bacillus subtilis) SRCM103716 균주를 초기균수가 3~4 log CFU/mL가 되도록 접종한 후 37℃에서 24시간 동안 발효하여 참깨박 고초균 발효물을 제조하였다.(1) Add 1.5 times (v/w) water to the pulverized sesame meal, increase the water at 121℃ for 15 minutes, and then add Bacillus subtilis SRCM103716 strain to the sesame leaf cooled to 60℃. After inoculation to a concentration of 3-4 log CFU/mL, fermentation was performed at 37° C. for 24 hours to prepare a fermented product of Sesame Bacteria Bacillus.
(2) 분쇄한 참깨박에 물을 0.5배(v/w) 가수하여 121℃에서 15분 동안 증자한 후 60℃로 냉각한 참깨박에 아스퍼질러스 오리재(Aspergillus oryzae) 균주를 0.3%(v/w) 접종한 후 30℃에서 72시간 동안 발효하여 참깨박 발효물을 제조하였다.(2) 0.5 times (v/w) of water was added to the crushed sesame meal, and then increased at 121°C for 15 minutes, and then 0.3% ( Aspergillus oryzae ) strain was added to the sesame seeds cooled to 60°C. v/w) After inoculation, fermentation was performed at 30° C. for 72 hours to prepare a sesame seed fermented product.
(3) 상기 (1)단계의 제조한 참깨박 고초균 발효물과 상기 (2)단계의 제조한 참깨박 발효물을 5:95 중량비율로 혼합하였다.(3) The fermented sesame seed meal prepared in step (1) and the fermented sesame seed meal prepared in step (2) were mixed at a weight ratio of 5:95.
실시예Example 1. 고초균별 특성 1. Characteristics of Bacillus bacteria
간장, 고추장, 된장, 젓갈 등에서 분리한 진흥원 기보유중인 바실러스 200여 종 중 β-글루코시다아제 활성을 보유한 고초균 15종을 선발하여 실험을 진행하였다. 실험 균주 리스트는 표 1과 같다. The experiment was conducted by selecting 15 species of Bacillus Bacillus having β-glucosidase activity out of 200 species of Bacillus previously owned by Jinheungwon isolated from soy sauce, red pepper paste, soybean paste, and salted fish. Table 1 shows a list of experimental strains.
1) 세포외분비 효소활성(Amylase, Protease, Cellulase) 분석1) Analysis of extracellular secretion enzyme activity (Amylase, Protease, Cellulase)
한천 확산법 방법에 준하여 바실러스 균주를 5 ml의 LB broth에 접종하여 24시간 동안 30℃, 150 rpm으로 진탕 배양기에서 배양하였으며, 배양액을 13,000 rpm에서 원심분리한 후 0.45 ㎛ 필터를 이용하여 제균한 시료를 시험액으로 사용하였다. According to the agar diffusion method, Bacillus strains were inoculated into 5 ml of LB broth and cultured in a shaking incubator at 30°C and 150 rpm for 24 hours, and the culture solution was centrifuged at 13,000 rpm and then sterilized using a 0.45 μm filter. It was used as a test solution.
바실러스 균주의 균체 외로 방출하는 세포의 효소 중 아밀라아제(amylase), 프로테아제(protease), 셀룰라아제(cellulase) 활성을 검증하기 위해 한천 확산법(agar diffusion method)를 사용하였으며, 각 효소와 특이적으로 반응하는 기질의 성분이 포함된 선발배지를 제조하여 사용하였다. 아밀라아제 활성을 측정하기 위해 가용성 전분(starch soluble, Junsei)를 기질로 선택하여 가용성 전분 1%가 포함된 2% 한천을 첨가한 배지를 사용하였으며, 프로테아제 활성을 측정하기 위해 2% 탈지유(BD, Difco)가 첨가된 1.5% 한천 배지를 제조하여 사용하였다. 셀룰라아제 활성을 검증하기 위해 CMC(carboxylmethyl cellulose, Junsei)를 기질로 선택하여 1% CMC가 첨가된 1.5% 한천 배지를 제조하였다. 각 기질이 포함된 배지에 8 mm의 홀(hole)을 형성한 후 각 웰(well)에 균주 시험액 100 ㎕씩을 분주하여 37℃에서 24시간 배양하여 형성된 클리어 존(clear zone)의 크기를 측정하여 균주의 효소활성을 측정하였으며, 아밀라아제 활성을 측정하기 위해서 Lugol staining 방법을, 셀룰라아제 활성을 측정하기 위해서는 congo red staining을 이용하였다.Agar diffusion method was used to verify the activity of amylase, protease, and cellulase among the enzymes of cells released outside the cells of the Bacillus strain, and a substrate that reacts specifically with each enzyme A selection medium containing the ingredients of was prepared and used. In order to measure amylase activity, a medium containing 2% agar containing 1% soluble starch was used by selecting soluble starch (starch soluble, Junsei) as a substrate, and 2% skim milk (BD, Difco) was used to measure the protease activity. ) Was added to prepare and use 1.5% agar medium. In order to verify the cellulase activity, CMC (carboxylmethyl cellulose, Junsei) was selected as a substrate to prepare a 1.5% agar medium to which 1% CMC was added. After forming an 8 mm hole in the medium containing each substrate, 100 μl of the strain test solution was dispensed into each well, and the size of the clear zone formed by incubating at 37°C for 24 hours was measured. Enzymatic activity of the strain was measured, Lugol staining was used to measure amylase activity, and congo red staining was used to measure cellulase activity.
*+; positive, -; negative * +; positive, -; negative
2) 식품유해미생물에 대한 항균활성 측정2) Measurement of antibacterial activity against food harmful microorganisms
한천 확산법 방법에 준하여 바실러스 균주를 5 ml의 LB broth에 접종하여 24시간 동안 30℃, 150 rpm 진탕 배양기에서 배양하였으며, 배양액을 13,000 rpm에서 원심분리한 후 0.45 ㎛ 필터를 이용하여 제균한 시료를 시험액으로 사용하였다.According to the agar diffusion method, Bacillus strain was inoculated into 5 ml of LB broth and cultured in a shaking incubator at 30°C and 150 rpm for 24 hours, and the culture solution was centrifuged at 13,000 rpm and then sterilized using a 0.45 μm filter as a test solution. Was used as.
식품유해활성을 측정하기 위해 지시균주를 KCCM(Korean Culture Center of Microorganisms)와 KCTC(Korean Collection for Type Cultures)에서 분양받아 사용하였다. 각 지시균주를 적절한 배지에 배양하여 O.D(optical density)가 600 nm에서 0.4~0.6이 되도록 배양한 후 배지에 0.8% 한천 배지에 1% 혼합하여 배지를 제조하였으며, 한천 확산법(agar diffusion method)에 준하여 각 지시균주가 포함된 배지에 8 mm의 홀(hole)을 형성한 후 각 웰(well)에 균주 시험액 100 ㎕씩을 분주하여 30~37℃에서 24시간 배양한 후 홀(hole) 주변에 형성된 억제환의 크기를 측정하여 선발된 균주의 항균활성을 측정하였다.In order to measure the food harmful activity, the indicator strains were sold and used in KCCM (Korean Culture Center of Microorganisms) and KCTC (Korean Collection for Type Cultures). Each indicator strain was cultivated in an appropriate medium, cultivated so that the OD (optical density) was 0.4 to 0.6 at 600 nm, and then mixed with 0.8% agar medium to 1% to prepare a medium, followed by agar diffusion method. According to this, after forming an 8 mm hole in the medium containing each indicator strain, 100 µl of the strain test solution was dispensed into each well, cultured at 30-37°C for 24 hours, and then formed around the hole. The size of the inhibitory ring was measured to measure the antibacterial activity of the selected strain.
그 결과, SRCM103716 균주는 바실러스 세레우스(Bacillus cereus), 스타필로코커스 아우레우스(Staphylococcus aureus) 및 리스테리아 모노시토게네스(Listeria monocytogenes)에 대한 항균 활성을 확인하였다.As a result, SRCM103716 strain was confirmed antimicrobial activity against Bacillus cereus , Staphylococcus aureus , and Listeria monocytogenes .
3) 선별균주의 용혈활성(Hemolysis type) 분석3) Analysis of hemolytic activity (Hemolysis type) of selected strains
바실러스 균주의 용혈활성을 분석하기 위해 Blood agar(Kisanbio, Korea)를 구입하여 각 blood agar 배지에 선별 균주를 획선도말한 후 24시간 동안 30℃에서 배양하였으며, 콜로니 주변 한천에 변화가 없는 경우 γ-hemolysis, 적혈구를 완전히 파괴하여 집락 주변으로 투명한 환(clear zone)이 형성된 경우 β-hemolysis, 일부 적혈구만 파괴하여 콜로니 주변에 녹색으로 관찰되는 경우 α-hemolysis로 판정하였다.To analyze the hemolytic activity of Bacillus strains, Blood agar (Kisanbio, Korea) was purchased and the selected strain was streaked on each blood agar medium, and then cultured at 30°C for 24 hours. If there is no change in the agar around the colony Hemolysis was evaluated as β-hemolysis, when red blood cells were completely destroyed and a clear zone was formed around the colony, and α-hemolysis was determined when only some red blood cells were destroyed and green around the colony.
그 결과, SRCM103716 균주는 β-용혈 활성이 있으면서, 페닐알라닌(phenylalanine), 우레아제(urease) 및 β-글루쿠로니다아제(β-glucuronidase)의 유해대사산물을 생성하지 않음을 확인하였다.As a result, it was confirmed that the SRCM103716 strain had β-hemolytic activity, and did not produce harmful metabolites of phenylalanine, urease, and β-glucuronidase.
4) 바이오제닉 아민 생성 유무 확인4) Confirmation of production of biogenic amine
바이오제닉 아민 생성 유무를 확인한 결과, BCH3520 균주를 제외한 모든 균주에서 티라민 및 히스타민을 생성하지 않음을 확인하였다.As a result of checking the presence or absence of biogenic amine production, it was confirmed that all strains except for the BCH3520 strain did not produce tyramine and histamine.
ND: 검출되지 않음ND: not detected
5) β-글루코시다아제(β-glucosidase) 활성5) β-glucosidase activity
실험 균주 15종의 β-글루코시다아제(β-glucosidase) 활성을 측정하기 위하여 β-glucosidase activity assay kit(Sigma)를 이용하였다. 실험 균주 15종을 전배양한 다음, 본배양액은 LB broth(Difco)에 1%(v/v) 접종하여 37℃에서 24시간 진탕 배양하였다. 본배양액의 상등액을 수거한 다음, 균체를 50 mM phosphate buffer(pH 7.0)에 풀어준 후, 이를 시료로 이용하였다. 1 웰에 220 ㎕ 증류수, 1 웰에는 20 ㎕ 증류수, 200 ㎕ calibrator를 첨가하였고, 샘플의 각각의 웰에 assay buffer 200 ㎕, β-NPG substrate 8 ㎕를 넣어준 다음, 시료를 20 ㎕ 첨가하였다. 이후 초기 흡광도를 405 nm에서 측정한 다음 37℃에서 20분 동안 방치한 다음 최종 흡광도를 405 nm에서 측정하였다. 이 후, 아래의 계산식을 이용하여 β-글루코시다아제 활성 값을 확인하였다. 실험 균주 15종에 대한 β-글루코시다아제 활성값은 표 5에 나타내었다.A β-glucosidase activity assay kit (Sigma) was used to measure the β-glucosidase activity of 15 strains of the experiment. After pre-culture of 15 strains of the experiment, the main culture solution was inoculated with 1% (v/v) in LB broth (Difco) and cultured with shaking at 37°C for 24 hours. After collecting the supernatant of the main culture solution, the cells were dissolved in 50 mM phosphate buffer (pH 7.0), and then used as a sample. 220 µl distilled water, 20 µl distilled water, and 200 µl calibrator were added to 1 well, 200 µl of assay buffer and 8 µl of β-NPG substrate were added to each well of the sample, and then 20 µl of the sample was added. Thereafter, the initial absorbance was measured at 405 nm, and then left at 37°C for 20 minutes, and the final absorbance was measured at 405 nm. Thereafter, the β-glucosidase activity value was confirmed using the following calculation formula. The β-glucosidase activity values for 15 experimental strains are shown in Table 5.
β-글루코시다아제 활성(Unit/L) = (최종 흡광도 - 최초 흡광도 × 250 units/L)/(calibrator 흡광도 - 물 흡광도)β-glucosidase activity (Unit/L) = (final absorbance-initial absorbance × 250 units/L)/(calibrator absorbance-water absorbance)
β-글루코시다아제 활성은 BCH3711, SRCM103716에서 각각 9.63 unit/L, 12.6 unit/L로 가장 높게 나타났다. β-glucosidase activity was highest in BCH3711 and SRCM103716 at 9.63 unit/L and 12.6 unit/L, respectively.
실시예Example 2. 고초균 종류에 따른 2. According to the type of Bacillus 참깨박Sesame seeds 발효물의Fermented 품질특성 Quality characteristics
참깨박의 고초균 발효를 위하여 도 2와 같은 방법으로 발효를 진행하였다.Fermentation was carried out in the same manner as in FIG. 2 for fermentation of sesame seed meal.
1) 참깨박 소재 고초균 15종 발효물 생균수 1) Number of fermented products of 15 kinds of Bacillus bacteria in sesame seed meal
발효하기 전과 후의 생균수의 결과는 도 3에 나타내었다. 그 결과, 전체 발효한 시료에서 8 log CFU/mL 이상 측정되었다. 15종 모두 참깨박 소재에서 잘 자라는 것을 확인하였다.The results of the number of viable cells before and after fermentation are shown in FIG. 3. As a result, 8 log CFU/mL or more was measured in all fermented samples. All 15 species were confirmed to grow well on sesame seed material.
2) β-글루코시다아제 활성 2) β-glucosidase activity
균주에 따른 참깨박 발효물에 대하여 β-글루코시다아제 활성을 측정하였다. 시료는 상등액을 수거하여 50 mM phosphate buffer (pH 7.0)에 풀어준 후, 이를 시료로 이용하였다. β-glucosidase activity assay kit (Sigma)을 이용하여 전과 같은 방법으로 측정한 결과는 표 6과 같다. 그 결과, 바실러스 서브틸리스 SRCM103716로 발효한 참깨박에서 β-글루코시다아제 활성이 16.88 unit/L로 가장 높게 측정되었다.Β-glucosidase activity was measured for the sesame seed fermented product according to the strain. As a sample, the supernatant was collected, dissolved in 50 mM phosphate buffer (pH 7.0), and used as a sample. Table 6 shows the results measured in the same manner as before using the β-glucosidase activity assay kit (Sigma). As a result, Bacillus subtilis In sesame seeds fermented with SRCM103716, β-glucosidase activity was the highest at 16.88 unit/L.
3) DPPH 라디칼 소거능 측정3) DPPH radical scavenging ability measurement
DPPH (1,1-diphenyl-2-picrylhydrazyl) 라디칼 소거능은 Blois (1958)의 방법을 변형하여 전자공여작용(electron donationg abilities, EDA)에 대한 효과로 각 시료의 환원력을 측정하였다. 즉, 시료 50 ㎕에 0.15 mM DPPH 용액(99% MeOH에 용해) 150 ㎕을 가한 후 30분간 상온에서 방치한 후 분광광도계를 사용하여 흡광도 517 nm에서 흡광도의 변화를 측정하였다. 각 시료의 라디칼 소거능은 아래의 식에 의해 시료 첨가구 및 무첨가구간의 흡광도 차이를 백분율로 나타내었다.DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging ability was measured by modifying the method of Blois (1958) to measure the reducing power of each sample as an effect on electron donation capabilities (EDA). That is, 150 µl of a 0.15 mM DPPH solution (dissolved in 99% MeOH) was added to 50 µl of a sample, and then allowed to stand at room temperature for 30 minutes, and then the change in absorbance at 517 nm was measured using a spectrophotometer. The radical scavenging ability of each sample was expressed as a percentage of the difference in absorbance between the addition and no addition of the sample by the following equation.
전자공여능(%) = (1 - 시료첨가구의 흡광도/무처리구의 흡광도) × 100Electron donating ability (%) = (1-absorbance of sample addition/absorbance of untreated group) × 100
참깨박 소재의 발효 전과 후에 따른 DPPH 라디칼 분석 결과는 도 4와 같다. 발효전에는 43.24~45.34%의 활성값을 가졌으나, 발효 후 모두 활성이 증가하였다. 이 중 BCH3711, SRCM103716를 이용한 참깨박 발효물이 각각 63.82%, 66.45%로 가장 높게 증가하였다.The DPPH radical analysis results of the sesame seed material before and after fermentation are shown in FIG. 4. Before fermentation, it had an activity value of 43.24~45.34%, but after fermentation, all activity increased. Among them, fermented sesame seeds using BCH3711 and SRCM103716 increased the highest at 63.82% and 66.45%, respectively.
4) 총 폴리페놀(polyphenol) 함량4) Total polyphenol content
총 페놀 함량은 Folin-Denis 변법에 따라 추출된 시료를 농도별로 적절히 희석한 후 측정하였다. 각 농도별 시료 200 ㎕에 Folin-Ciocalteu 시약 12.5 ㎕를 혼합하여 교반한 뒤, 120분 동안 상온에서 방치하여 반응시켰다. 반응액의 흡광도 값은 UV-Vis 분광광도계(DU 800, Beckman coulter, Fullerton, CA, USA)를 사용하여 750 nm에서 분석하였다. 총 페놀 함량은 갈산을 분석시료와 동일한 방법으로 분석하여 얻은 표준 검량선으로부터 시료 추출물의 총 페놀 함량을 산출하였고, 총 폴리페놀 함량은 1 L 중의 mg gallic acid equivalent로 나타내었다.The total phenol content was measured after appropriately diluting the sample extracted according to the Folin-Denis variant for each concentration. After mixing and stirring 12.5 µl of Folin-Ciocalteu reagent to 200 µl of each concentration sample, the mixture was allowed to stand at room temperature for 120 minutes to react. The absorbance value of the reaction solution was analyzed at 750 nm using a UV-Vis spectrophotometer (
참깨박 소재의 발효 전과 후에 따른 폴리페놀 함량을 측정한 결과는 도 5와 같다. 발효 후 대조구를 제외한 모든 균주에서 폴리페놀 함량이 증가하는 것을 확인하였으며, 이 중 특히 BCH3637, BCH3678, SRCM103716의 3종은 각각 2490.2, 2559.4, 2803.3 mg/L로 가장 높게 증가하였다.The results of measuring the polyphenol content before and after fermentation of the sesame seed material are shown in FIG. 5. After fermentation, it was confirmed that the polyphenol content increased in all strains except for the control, and among them, the three species, BCH3637, BCH3678, and SRCM103716, increased the highest at 2490.2, 2559.4, and 2803.3 mg/L, respectively.
5) 총 플라보노이드(flavonoid) 함량5) Total flavonoid content
총 플라보노이드 함량은 Davis 변법에 따라 추출된 시료를 농도별로 적절히 희석한 후 측정하였다. 각 농도별 시료 0.5 mL에 10% 질산알루미늄(aluminum nitrate) 0.1 mL, 1M 초산 칼륨(potassium acetate) 0.1 mL 및 에탄올 4.3 mL를 차례로 가하여 혼합한 후 상온에서 40분간 방치하여 반응시켜 450 nm에서 흡광도를 측정하였다. 표준물질로는 갈산을 시료와 동일한 방법으로 분석하여 얻은 표준 검량선으로부터 시료 추출물의 총 플라보노이드 함량을 산출하였고, 총 플라보노이드 함량은 1 L 중의 mg gallic acid equivalent로 나타내었다.The total flavonoid content was measured after appropriately diluting the sample extracted according to the Davis variant for each concentration. To 0.5 mL of each concentration sample, 0.1 mL of 10% aluminum nitrate, 0.1 mL of 1M potassium acetate, and 4.3 mL of ethanol were sequentially added and mixed, and then allowed to stand at room temperature for 40 minutes to react, thereby increasing the absorbance at 450 nm. Measured. As a standard material, the total flavonoid content of the sample extract was calculated from a standard calibration curve obtained by analyzing gallic acid in the same manner as the sample, and the total flavonoid content was expressed as mg gallic acid equivalent in 1 L.
참깨박 소재의 발효 전과 후에 따른 플라보노이드 함량을 측정한 결과는 도 6과 같다. 발효 후 대조구를 제외한 모든 균주에서 플라보노이드 함량이 증가하는 것을 확인하였으며, 이 중 특히 BCH3637, BCH3678, SRCM103716의 3종은 각각 1423.27, 1688.67, 1553.08 mg/L로 가장 높게 증가하는 것으로 확인되었다.The results of measuring the flavonoid content before and after fermentation of the sesame seed material are shown in FIG. 6. After fermentation, it was confirmed that the content of flavonoids increased in all strains except for the control, and among them, the three species BCH3637, BCH3678, and SRCM103716 were found to increase the highest at 1423.27, 1688.67 and 1553.08 mg/L, respectively.
5. 참깨박 고초균 발효 균주 선발5. Selection of sesame seeds Bacillus fermentation strains
이에 참깨박 소재의 발효에 대한 균주로 β-글루코시다아제 활성 및 DPPH 라디칼 소거능 활성이 우수하고 발효 이후 총 폴리페놀 함량과 플라보노이드 함량의 증가율이 높은 바실러스 서틸리스(Bacillus subtilis) SRCM103716로 선발하였으며(16S rRNA 서열은 서열번호 1임), 선발한 균주의 계통도는 도 1과 같으며, 2019년 07월 10일 한국미생물보존센터(KCCM)에 기탁하여 기탁번호 KFCC11832P를 부여받았다.Therefore, Bacillus subtilis SRCM103716 was selected as a strain for fermentation of sesame seed material, which has excellent β-glucosidase activity and DPPH radical scavenging activity, and has a high increase rate of total polyphenol content and flavonoid content after fermentation ( The 16S rRNA sequence is SEQ ID NO: 1), and the schematic diagram of the selected strain is as shown in FIG. 1, and was deposited with the Korea Microbial Conservation Center (KCCM) on July 10, 2019, and was given the accession number KFCC11832P.
실시예Example 3. 3. 참깨박Sesame seeds 발효물의Fermented 혼합비율 설정 Mixing ratio setting
참깨박 바실러스 서브틸리스(Bacillus subtilis) SRCM103716 발효물과 참깨박 아스퍼질러스 오리재(Aspergillus oryzae) 발효물의 혼합 비율을 설정하기 위하여 표 7과 같이 샘플을 제조하였다. DPPH 라디칼 소거능을 확인하여 가장 높은 항산화활성을 갖는 비율로 참깨박 발효 간장 제조 구간을 설정하기로 하였다.Sesame Seed Bacillus subtilis SRCM103716 Fermented Product and Sesame Seed Aspergillus Duck ( Aspergillus oryzae ) Samples were prepared as shown in Table 7 in order to set the mixing ratio of the fermented product. By confirming the DPPH radical scavenging ability, it was decided to set the sesame meal fermented soy sauce production section with the ratio having the highest antioxidant activity.
1) One) 참깨박Sesame seeds 발효물Fermentation 혼합비율에 따른 According to the mixing ratio DPPHDPPH 라디칼Radical 소거능Scavenging ability 결과 result
참깨박 발효물 혼합비율에 따른 DPPH 라디칼 소거능 결과는 도 7과 같다. 아스퍼질러스 오리재(A. oryzae) 95%, 바실러스 서브틸리스(B. subtilis) SRCM103716 5% 혼합 비율에서 DPPH 라디칼 활성 결과 84.12%로 가장 높게 측정되었으며, 다음으로 아스퍼질러스 오리재(A. oryzae) 100%에서 77.28%로 측정되었다. 이에 참깨박 발효 간장 제조를 위하여 아스퍼질러스 오리재(A. oryzae) 95%, 바실러스 서브틸리스(B. subtilis) SRCM103716 5%가 가장 적합함을 확인하였다.The DPPH radical scavenging ability results according to the mixing ratio of fermented sesame seeds are shown in FIG. 7. Aspergillus duck material ( A. oryzae ) 95%, Bacillus subtilis ( B. subtilis ) SRCM103716 DPPH radical activity at a mixing ratio of 5% was measured the highest as 84.12%, followed by Aspergillus duck material ( A. oryzae ) It was measured from 100% to 77.28%. Therefore, it was confirmed that 95% of Aspergillus ducks ( A. oryzae ) and 5% of Bacillus subtilis ( B. subtilis ) SRCM103716 are most suitable for the production of sesame seed fermented soy sauce.
2) 2) 참깨박Sesame seeds 비발효물과Non-fermented products 참깨박Sesame seeds 발효 혼합물의 Fermentation mixture DPPHDPPH 라디칼Radical 소거활성 및 Scavenging activity and 리그League 난 성분을 측정I measure the ingredients
최종적으로 참깨박 아스퍼질러스 오리재 발효물 95%와 바실러스 서브틸리스 SRCM103716 발효물 5%를 혼합하여 간장 제조에 사용하기로 결정하였다. 이에 참깨박 비발효물과 참깨박 발효 혼합물의 DPPH 라디칼 소거활성 및 리그난 성분을 측정하였다.Finally, 95% of sesame seed Aspergillus duckweed fermented product and 5% of Bacillus subtilis SRCM103716 fermented product were mixed and decided to be used in the production of soy sauce. Accordingly, DPPH radical scavenging activity and lignan components of the mixture of non-fermented sesame seeds and sesame seeds were measured.
(2-1) (2-1) 참깨박Sesame seeds 발효 혼합물의 Fermentation mixture DPPHDPPH 라디칼Radical 소거활성 결과 Scavenging activity result
바실러스 서틸리스(Bacillus subtilis) SRCM103716 균주와 아스퍼질러스 오리재(Aspergillus oryzae)를 이용한 참깨박 발효 혼합물과 비발효물의 DPPH 라디칼 소거활성 결과는 표 8에 나타내었다. 측정 결과 100배 희석에서 참깨박 비발효물과 참깨박 발효 혼합물이 각각 28.4%, 73.3%로 발효 후 약 160% 항산화 활성이 증가하는 것으로 확인되었다.Table 8 shows the results of DPPH radical scavenging activity of the fermented mixture and non-fermented sesame seeds using Bacillus subtilis SRCM103716 strain and Aspergillus oryzae . As a result of the measurement, it was confirmed that at 100 times dilution, the non-fermented sesame seed and the sesame seed fermented mixture increased about 160% after fermentation to 28.4% and 73.3% respectively.
(2-2) (2-2) 참깨박Sesame seeds 발효 혼합물의 Fermentation mixture 리그난Lignan 성분 분석 결과 Component analysis result
바실러스 서틸리스(Bacillus subtilis) SRCM103716 균주와 아스퍼질러스 오리재(Aspergillus oryzae)를 이용한 참깨박 발효 혼합물과 비발효물의 리그난 함량 분석 결과를 표 9에 나타내었다. 분석 결과 전체 리그난 함량이 57.94% 증가하였으며, 각각의 리그난 조성의 변화로는 세사미놀 3 배당체(Sesaminol triglucoside)의 함량이 발효 후 18.93% 감소하였고, 세사미놀 2 배당체(Sesaminol diglucoside) 및 세사미놀(Sesaminol)의 함량은 각각 1340.44%, 2071.43% 증가하는 것으로 확인되었다. 이는 전체적인 리그난의 함량 증가뿐만 아니라 발효대사에 의해 세사미놀 배당체 결합이 분해되어 고분자 배당체 결합은 감소하는 반면 저분자 세사미놀 배당체의 함량은 증가하는 것으로 확인되었다.Table 9 shows the results of the analysis of the lignan content of the fermented mixture and non-fermented sesame seeds using Bacillus subtilis SRCM103716 strain and Aspergillus oryzae . As a result of the analysis, the total lignan content increased by 57.94%, and the content of sesaminol triglucoside decreased by 18.93% after fermentation due to changes in each lignan composition, and sesaminol diglucoside and sesaminol ) Was found to increase by 1340.44% and 2071.43%, respectively. It was confirmed that the sesaminol glycoside bond was decomposed by fermentation metabolism as well as the increase of the overall lignan content, so that the polymeric glycoside bond decreased, while the content of the low molecular sesaminol glycoside increased.
triglucosideSesaminol
triglucoside
diglucosideSesaminol
diglucoside
실시예Example 4. 4. 참깨박Sesame seeds 발효 혼합물을 이용한 간장의 품질특성 Quality Characteristics of Soy Sauce Using Fermented Mixture
참깨박 발효 혼합물을 이용한 간장의 제조공정은 도 8과 같다.The manufacturing process of soy sauce using the sesame seed fermentation mixture is shown in FIG.
1) One) 참깨박Sesame seeds 간장의 향기성분 분석 Analysis of fragrance components of soy sauce
참깨박 간장으로부터 휘발성 향기 성분을 분리, 동정하였으며, 분석 조건은 표 10과 같다.Volatile fragrance components were separated and identified from sesame seed soy sauce, and the analysis conditions are shown in Table 10.
(60m length × 0.25mm I.D × 0.50㎛)DB-WAX (Agilent)
(60m length × 0.25mm ID × 0.50㎛)
4℃/min ~ 150℃ - 5min
5℃/min ~ 230℃ - 10min40℃-3min
4℃/min ~ 150℃-5min
5℃/min ~ 230℃-10min
바실러스 서틸리스(Bacillus subtilis) SRCM103716 균주와 아스퍼질러스 오리재(Aspergillus oryzae)를 이용한 참깨박 발효 혼합물 비율에 따른 참깨박 간장의 향기성분 분석 결과를 표 11 내지 13에 나타내었다. 참깨박 발효 간장의 휘발성 성분을 분석한 결과 총 61종 화합물이 동정되었다. 이 중에서 알데히드류 5종, 방향족화합물 10종, 알코올류 10종, 산류 3종, 케톤류 10종, 에스테르류 4종, 기타 화합물 19종이 동정되었다. 특징적으로는 단일균주를 이용한 간장에 비해 복합균주를 이용한 간장에서 과일향, 엿기름향을 가진 3-메틸 부타날(3-Methyl butanal)의 함량이 5.28%, 훈연, 나무향, 베이컨향을 갖는 2-메톡시 페놀(2-methoxy phenol)이 17.01%, 과일향, 크림향, 버터향을 갖는 2,3-부탄디올(2,3-Butanediol)은 1.19% 각각 증가함을 확인하였다. 또한, 방향성을 보이지 않는 기타 휘발성 화합물의 함량이 34.3%에서 20.83%로 13.47% 감소하는 경향을 보여 결과적으로 SRCM103716 균주와 아스퍼질러스 오리재(Aspergillus oryzae)를 활용한 복합균주의 간장에서 아스퍼질러스 오리재(Aspergillus oryzae)만을 접종한 단일균주의 간장보다 향기성분의 화합물이 증가하는 것으로 확인되었다.Tables 11 to 13 show the results of analysis of fragrance components of sesame seed soy sauce according to the ratio of the sesame seed fermentation mixture using Bacillus subtilis SRCM103716 strain and Aspergillus oryzae . A total of 61 compounds were identified as a result of analyzing the volatile components of sesame seed fermented soy sauce. Among them, 5 aldehydes, 10 aromatic compounds, 10 alcohols, 3 acids, 10 ketones, 4 esters, and 19 other compounds were identified. Characteristically, compared to soy sauce using a single strain, the content of 3-methyl butanal with fruit and malt flavor was 5.28% in soy sauce using complex strains, and 2 with smoking, wood flavor and bacon flavor. -It was confirmed that 2-methoxy phenol increased by 17.01%, and 2,3-butanediol (2,3-Butanediol) having fruit flavor, cream flavor, and butter flavor increased by 1.19%, respectively. In addition, the content of other volatile compounds that do not show aroma showed a tendency to decrease by 13.47% from 34.3% to 20.83%. As a result, Aspergillus in the liver of a complex strain using SRCM103716 strain and Aspergillus oryzae It was confirmed that the fragrant compound was increased compared to the liver of a single strain inoculated with duck material ( Aspergillus oryzae ) alone.
2) 2) 참깨박Sesame seeds 간장의 유리 아미노산 측정 Measurement of free amino acids in soy sauce
유리 아미노산은 시료 2 g을 취하여 3차 증류수 30 mL를 넣고 교반하고 50 mL로 정용한 후 초음파를 이용하여 20분간 추출한 후 원심분리(3000 rpm, 10분)한 다음 상등액 2 mL에 5% TCA 2 mL를 넣고 원심분리(10,000 rpm, 10분)한 후 상등액을 취하여 0.02N-HCl로 희석한 후 0.2 ㎛ 실린지 필터에 통과시킨 후 하기 표 14의 분석조건으로 아미노산 분석기로 분석하였다.For free amino acids, take 2 g of a sample, add 30 mL of tertiary distilled water, stir, dilute to 50 mL, extract for 20 minutes using ultrasound, centrifuge (3000 rpm, 10 minutes), and add 2 mL of 5% TCA to 2 mL of the supernatant. Was then centrifuged (10,000 rpm, 10 minutes), the supernatant was taken, diluted with 0.02N-HCl, passed through a 0.2 μm syringe filter, and analyzed by an amino acid analyzer under the analysis conditions shown in Table 14 below.
Hitachi 4.6×40mm (Ammonia filtering)Hitachi 4.6×60mm (speration)
Hitachi 4.6×40mm (Ammonia filtering)
발효과정 중에 효소작용으로 생성되는 유리 아미노산의 함량이 높을수록 맛과 영양이 뛰어난 우수식품으로 평가된다. 본 실험의 바실러스 서브틸리스(Bacillus subtilis) SRCM103716 균주와 아스퍼질러스 오리재(Aspergillus oryzae)를 이용한 참깨박 발효 혼합물 비율에 따른 발효 간장의 유리 아미노산 결과는 표 15와 같다. 아스퍼질러스 오리재 100% 발효 간장과 아스퍼질러스 오리재 95%, 바실러스 서브틸리스 SRCM103716 5% 발효 간장의 전체 유리 아미노산 함량은 각각 319.3 mg/L, 333.6 mg/L로 측정되었고, 장류의 글루탐산(glutamic acid)은 아스퍼질러스 오리재 100% 발효 간장과 아스퍼질러스 오리재 95%+바실러스 서브틸리스 SRCM103716 5% 발효 간장에서 각각 47.82 mg/L, 70.44 mg/L로 나타났다. 또한, 아스파르트산(aspartic acid)의 함량은 20.99 mg/L, 25.29 mg/L로 측정되어 구수한 맛, 간장 고유의 맛에 밀접한 영향을 미치는 성분들이 아스퍼질러스 오리재 95%+바실러스 서브틸리스 SRCM103716 5% 발효 간장에서 더 높게 나타났다. 쓴맛을 나타내는 유리 아미노산인 이소류신(isoleucine), 류신(leucine), 페닐알라닌(phenylalanine) 3종 모두 결과적으로 SRCM103716 균주+아스퍼질러스 오리재(Aspergillus oryzae)를 활용한 복합 균주의 간장에서 아스퍼질러스 오리재(Aspergillus oryzae)만을 접종한 단일 균주의 간장보다 낮게 나타나, 구수한 맛, 간장 고유의 맛이 증가하고, 쓴맛이 감소함을 확인하였다.The higher the content of free amino acids produced by enzyme action during fermentation, the better it is evaluated as an excellent food with excellent taste and nutrition. Table 15 shows the results of free amino acids in fermented soy sauce according to the ratio of the fermented sesame seed mixture using Bacillus subtilis SRCM103716 strain and Aspergillus oryzae of this experiment. Aspergillus duck jae 100% fermented soy sauce and Aspergillus duck jae 95%, Bacillus subtilis SRCM103716 The total free amino acid content of 5% fermented soy sauce was measured to be 319.3 mg/L and 333.6 mg/L, respectively, and glutamic acid in the paste was 100% of Aspergillus duck material, and 95% of fermented soy sauce and Aspergillus duck material. + Bacillus subtilis SRCM103716 5% fermented soy sauce was found to be 47.82 mg/L and 70.44 mg/L, respectively. In addition, the content of aspartic acid was measured as 20.99 mg/L and 25.29 mg/L, and the ingredients that closely affect the taste and the unique taste of soy sauce are Aspergillus ducks 95% + Bacillus subtilis. SRCM103716 was higher in 5% fermented soy sauce. All three kinds of free amino acids that exhibit bitter taste, such as isoleucine, leucine, and phenylalanine, resulted in SRCM103716 strain + Aspergillus duckweed ( Aspergillus oryzae ) in the liver of a complex strain utilizing Aspergillus duck material ( Aspergillus oryzae ) appeared lower than the soy sauce of a single strain inoculated only, and it was confirmed that the savory taste, the unique taste of soy sauce increased, and the bitter taste decreased.
<110> Microbial Institute for Fermentation Industyry <120> Bacillus subtilis SRCM103716 strain having excellent beta-glucosidase and antioxidant activity and uses thereof <130> PN19311 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 478 <212> DNA <213> Bacillus subtilis <400> 1 cttctcagga cgaacgctgg cggcgtgcct aatacatgca agtcgagcgg acagatggga 60 gcttgctccc tgatgttagc ggcggacggg tgagtaacac gtgggtaacc tgcctgtaag 120 actgggataa ctccgggaaa ccggggctaa taccggatgg ttgtttgaac cgcatggttc 180 aaacataaaa ggtggcttcg gctaccactt acagatggac ccgcggcgca ttagctagtt 240 ggtgaggtaa cggctcacca aggcaacgat gcgtagccga cctgagaggg tgatcggcca 300 cactgggact gagacacggc ccagactcct acgggaggca gcagtaggga atcttccgca 360 atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg aaggttttcg gatcgtaaag 420 ctctgttgtt agggaagaac aagtaccgtt cgaatagggc ggtaccttga cggtacct 478 <110> Microbial Institute for Fermentation Industyry <120> Bacillus subtilis SRCM103716 strain having excellent beta-glucosidase and antioxidant activity and uses thereof <130> PN19311 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 478 <212> DNA <213> Bacillus subtilis <400> 1 cttctcagga cgaacgctgg cggcgtgcct aatacatgca agtcgagcgg acagatggga 60 gcttgctccc tgatgttagc ggcggacggg tgagtaacac gtgggtaacc tgcctgtaag 120 actgggataa ctccgggaaa ccggggctaa taccggatgg ttgtttgaac cgcatggttc 180 aaacataaaa ggtggcttcg gctaccactt acagatggac ccgcggcgca ttagctagtt 240 ggtgaggtaa cggctcacca aggcaacgat gcgtagccga cctgagaggg tgatcggcca 300 cactgggact gagacacggc ccagactcct acgggaggca gcagtaggga atcttccgca 360 atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg aaggttttcg gatcgtaaag 420 ctctgttgtt agggaagaac aagtaccgtt cgaatagggc ggtaccttga cggtacct 478
Claims (7)
(2) 분쇄한 참깨박에 물을 가수하여 증자한 후 냉각한 참깨박에 아스퍼질러스 오리재(Aspergillus oryzae) 균주를 접종한 후 발효하여 참깨박 발효물을 제조하는 단계; 및
(3) 상기 (1)단계의 제조한 참깨박 고초균 발효물과 상기 (2)단계의 제조한 참깨박 발효물을 혼합하는 단계를 포함하여 제조하는 것을 특징으로 하는 리그난 함량 및 항산화 활성이 증진된 참깨박 혼합 발효물의 제조방법.(1) After adding water to the pulverized sesame seed meal, water was added and then inoculated with the Bacillus subtilis SRCM103716 strain of Clause 1 in the cooled sesame seed meal, and fermented to obtain total polyphenol and total flavonoid content and antioxidant activity. Preparing a fermented product of sesame gourd Bacillus bacillus with enhanced β-glucosidase activity;
(2) adding water to the pulverized sesame meal to increase the steam, and then inoculating the cooled sesame seed with Aspergillus oryzae strain and fermenting to prepare a sesame seed fermented product; And
(3) The lignan content and antioxidant activity are enhanced, comprising the step of mixing the fermented sesame seed meal prepared in step (1) and the fermented sesame seed meal prepared in step (2). Method for producing fermented sesame seed mixture.
(1) 분쇄한 참깨박에 물을 1.2~1.8배 가수하여 110~130℃에서 10~20분 동안 증자한 후 냉각한 참깨박에 제1항의 바실러스 서브틸리스(Bacillus subtilis) SRCM103716 균주를 접종한 후 34~40℃에서 20~28시간 동안 발효하여 총 폴리페놀 및 총 플라보노이드 함량과 항산화 활성 및 β-글루코시다아제(β-glucosidase) 활성이 증진된 참깨박 고초균 발효물을 제조하는 단계;
(2) 분쇄한 참깨박에 물을 0.4~0.6배 가수하여 110~130℃에서 10~20분 동안 증자한 후 냉각한 참깨박에 아스퍼질러스 오리재(Aspergillus oryzae) 균주를 접종한 후 28~32℃에서 68~76시간 동안 발효하여 참깨박 발효물을 제조하는 단계; 및
(3) 상기 (1)단계의 제조한 참깨박 고초균 발효물과 상기 (2)단계의 제조한 참깨박 발효물을 4~6:94~96 중량비율로 혼합하는 단계를 포함하여 제조하는 것을 특징으로 하는 리그난 함량 및 항산화 활성이 증진된 참깨박 혼합 발효물의 제조방법.The method of claim 5,
(1) Water was added 1.2 to 1.8 times to the pulverized sesame seed, and then increased at 110 to 130°C for 10 to 20 minutes, and then Bacillus subtilis SRCM103716 strain of Paragraph 1 was inoculated into the cooled sesame seed meal. After fermentation at 34-40° C. for 20-28 hours to prepare a sesame gourd Bacillus fermented product with improved total polyphenol and total flavonoid content, antioxidant activity, and β-glucosidase activity;
(2) After adding water 0.4 to 0.6 times to the crushed sesame seed, add water at 110 to 130°C for 10 to 20 minutes, and inoculate the cooled sesame seed with Aspergillus oryzae strain. Fermenting at 32° C. for 68 to 76 hours to prepare a sesame seed fermented product; And
(3) characterized by comprising the step of mixing the fermented sesame seed meal prepared in step (1) and the fermented sesame seed meal prepared in step (2) at a weight ratio of 4 to 6:94 to 96 Method for producing a mixed fermented product of sesame seed meal with improved lignan content and antioxidant activity.
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