KR100852984B1 - 90 A Fermented Bacillus sp. CS90 green tea bamboo salt soup prepared with fermented soybeans it's making method - Google Patents

90 A Fermented Bacillus sp. CS90 green tea bamboo salt soup prepared with fermented soybeans it's making method Download PDF

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KR100852984B1
KR100852984B1 KR1020070060251A KR20070060251A KR100852984B1 KR 100852984 B1 KR100852984 B1 KR 100852984B1 KR 1020070060251 A KR1020070060251 A KR 1020070060251A KR 20070060251 A KR20070060251 A KR 20070060251A KR 100852984 B1 KR100852984 B1 KR 100852984B1
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서원택
강수태
최철용
조계만
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이승희
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
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    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/10—Preserving against microbes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/302—Foods, ingredients or supplements having a functional effect on health having a modulating effect on age

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Abstract

A method of manufacturing fermented soybeans containing green tea and bamboo salt using Bacillus sp. CS90 with antimicrobial activity against various food microorganisms, acid-resistant property and high phenolic substance producing capacity by beta-glucosidase activity is provided to obtain fermented soybeans with high antioxidant capacity, improved flavor and functionality. Fermented soybeans containing green tea and bamboo salt is prepared by the steps of: (1) inoculating 50g heated soybeans with Bacillus sp. CS90 and culturing at 39deg.C for 48hr; (2) inoculating 4kg heated soybeans which are obtained by soaking in water at room temperature for 12hr, dehydrating and heating at 120deg.C for 2.5hr in an autoclave with 50g strains of the cultured Bacillus sp. CS90 and culturing; (3) drying the obtained raw fermented soybeans to a moisture content of 5 to 10% and grinding to 180meshes; and (4) adding 0.5 to 1.0% by weight of bamboo salt with 230meshes and 0.5 to 1.0% by weight of green tea powder with 230meshes to the fermented soybeans.

Description

바실러스 속 씨에스 90 균주를 이용하여 제조된 녹차죽염청국장 및 그의 제조방법 {A Fermented Bacillus sp. CS90 green tea bamboo salt soup prepared with fermented soybeans it's making method}Green Tea Bamboo Salt Cheonggukjang prepared using Bacillus C. sp. 90 strain and its preparation method {A Fermented Bacillus sp. CS90 green tea bamboo salt soup prepared with fermented soybeans it's making method}

도 1은 본 발명인 바실러스 속 (Bacillus sp.) 씨에스 90 (CS90)균주를 이용하여 제조된 녹차죽염청국장 및 그의 제조방법의 흐름도1 is a flow chart of green tea porridge salt soup prepared using Bacillus sp.

본 발명은 바실러스 속 (Bacillus sp.) 씨에스 90 (CS90)균주를 이용하여 제조된 녹차죽염청국장 및 그의 제조방법에 관한 것으로, 이를 더욱 상세하게 설명하면, 내산성, 내담즙산성, 여러 가지 식중독 미생물에 대한 항균활성, 및 베타-글루코시다제(β-glucosidase) 활성이 우수한 신규 바실러스 속 (Bacillus sp.) CS90 균주[한국농업생명공학연구원, 2007년 5월 11일자 기탁, (기탁번호: KACC91315P)]를 삶은 대두에 접종 발효시켜 제조된 생리활성이 우수한 청국장에 관한 것이며, 상기 바실러스 CS90 균주는 인체의 장에서 생존가능성이 높을 뿐만 아니라, 식중독을 예방하는데 도움을 줄 수 있고, 균주가 가지고 있는 베타-글루코시다제(β-glucosidase) 활성으로 인하여 제조된 청국장에 다량의 폴리페놀을 생성시킴으로서 항산화능력이 높은 청국장 제조를 가능하게 한다. 이러한 특성은 우수한 생균제제로서의 기능을 하게하며. 제조된 청국장의 분말에 적당량의 죽염과 녹차가루를 첨가함으로서 그 기능성을 활용할 수 있을 뿐만 아니라 청국장의 풍미를 개선시키는 효과를 제공하는 것으로서 바실러스 속 (Bacillus sp.) 씨에스 90 (CS90)균주를 이용하여 제조된 녹차죽염청국장 및 그의 제조방법에 관한 것이다. The present invention relates to green tea porridge salt soup prepared using Bacillus sp. CS 90 (CS90) strain, and a method for preparing the same. More specifically, the present invention relates to acid resistance, bile acid resistance, Novel Bacillus sp. CS90 strain having excellent antimicrobial activity and beta-glucosidase activity [Korea Agricultural Research Institute of Agriculture and Biotechnology, deposited on May 11, 2007, (Accession Number: KACC91315P)] It relates to Cheonggukjang excellent in physiological activity produced by fermentation in boiled soybeans, the Bacillus CS90 strain is not only high viability in the intestine of the human body, but also can help prevent food poisoning, and has a beta- Glucosidase (β-glucosidase) activity by producing a large amount of polyphenols in the produced Cheonggukjang makes it possible to produce a high antioxidant capacity. These properties make it a good probiotic. By adding an appropriate amount of bamboo salt and green tea powder to the powder of Cheonggukjang prepared, it can not only utilize its functionality, but also improve the flavor of Cheonggukjang, using Bacillus sp. 90 strain (CS90) strain. It relates to a green tea porridge salt soup prepared and a method for producing the same.

최근 식품을 통해 질병을 예방하고 삶의 질을 향상시키고자 하는 소위 “웰빙”을 전통 발효식품을 통하여 찾고자 하는 노력이 증가하고 있다. 이와 관련하여 우리의 전통 발효식품인 청국장은 이를 실현하는데 가장 적합한 식품의 하나가 될 것이다. 또한, 청국장은 대두발효식품 중 가장 짧은 시간에 제조할 수 있으면서 영양학적인 측면이나 경제적인 측면에서 대두를 가장 효과적으로 먹는 방법의 하나로 인식되고 있다.  Recently, there has been an increasing effort to find so-called “well-being” through traditional fermented foods to prevent diseases and improve the quality of life through food. In this regard, Cheonggukjang, our traditional fermented food, will be one of the most suitable foods to realize this. In addition, Cheonggukjang is recognized as one of the most effective way to eat soybeans in terms of nutritional and economical while being produced in the shortest time of soybean fermented food.

청국장은 삶은 대두에 고초균(Bacillus subtilis) 등을 접종하여 40℃ 부근의 온도에서 2 - 3일 발효시킨 것으로 고초균이 생산하는 아밀라아제, 프로테아제, 리파아제, 및 베타-글루코시다아제 등과 같은 효소의 작용으로 대두에 함유되어 있는 성분들이 우리 인체에서 소화흡수 되기 쉬운 상태로 변화하여 영양이 풍부하고 기능적으로 우수한 각종 페놀산, 이소플라본, 사포닌, 피트산, 유리아미노산, 생리활성 펩타이드, 비타민 B 복합체, 레반이나 글루타민산의 중합체 등과 같은 점액성물질 등을 다량 함유하고 있다. 이들 물질들은 각종 성인병을 예방하고 신진대사를 촉진하여 신체기능이 원활하도록 도와 건강유지 및 증진에 도움을 줄 수 있다.Cheonggukjang was inoculated with Bacillus subtilis on boiled soybeans and fermented for 2 to 3 days at a temperature around 40 ℃. Nutrients, isoflavones, saponins, phytic acid, free amino acids, bioactive peptides, vitamin B complex, levan or glutamic acid And a large amount of viscous substances such as polymers. These substances can help to maintain and promote health by preventing various adult diseases and promoting metabolism to help the body function smoothly.

청국장의 풍부한 영양과 다양한 기능의 우수성에도 불구하고 청국장에 대한 선호도는 매우 낮아 실생활에서는 일부 계층에서만 활용되고 있는 실정에 있다. 청국장 제품의 가장 큰 불만은 불쾌취와 보존기간 동안의 품질저하에 문제점이 있다. In spite of the rich nutrition and superiority of various functions of Cheonggukjang, the preference for Cheonggukjang is so low that it is used only in some classes in real life. The biggest complaint of Cheonggukjang products is the problem of unpleasantness and deterioration during the storage period.

본 발명은 상기 설명한 바와 같은 종래 기술의 문제점을 더욱 효율적으로 해결하기 위하여 제공된 것으로서, 본 발명에서는 재래식 청국장이 갖는 단점을 개선하고 프로바오틱스(probiotics)로서의 기능 등을 기대하기 위해 각종 식품위생미생물에 대한 항균활성과 내산성 및 내담즙산성, 점질물 형성능이 우수하며, 베타글루코시다제에 의한 높은 페놀성 물질 생성능력과 대두 발효 시, 악취발생이 낮은 신규 바실러스 [바실러스 속 (Bacillus sp.) 씨에스 90 (CS90) : 한국농업생명공학연구원, 2007년 5월 11일자로 기탁, (기탁번호: KACC91315P)]를 전통장류로부터 선별 분리하였으며, 이와 같이 선별 분리된 균주를 삶은 대두에 접종하여 청국장을 발효 제조고 건조 분말화한 후, 죽염과 녹차분말을 첨가함으로서 맛과 풍미를 높였다. 이렇게 함으로서 기존의 청국장이 갖는 기능성을 더욱 효율적으로 활용할 수 있는 녹차죽염청국장을 제공하고자 함에 그 목적이 있다.The present invention is provided to more efficiently solve the problems of the prior art as described above, in the present invention to improve the disadvantages of conventional Cheonggukjang and to expect the function as probiotics (probiotics), etc. It is excellent in antibacterial activity, acid resistance, bile acid resistance, and viscous substance formation ability, and high phenolic substance generation ability by betaglucosidase and low odor during soybean fermentation [ Bacillus sp. CS 90 ( CS90): Korea Agricultural Research Institute of Bioscience and Biotechnology, deposited on May 11, 2007, (Accession No .: KACC91315P)] was isolated from traditional soybeans. After dry powdering, the taste and flavor were enhanced by adding bamboo salt and green tea powder. By doing so, its purpose is to provide green tea porridge salted soybean soup that can more efficiently utilize the functionality of the existing Cheonggukjang.

상기 목적을 달성하기 위하여, 본 발명은 전통 장류로부터 각종 바실러스 균주를 분리하고 이로부터 대두발효 시, 낮은 악취발생, 높은 점성물질 형성, 내산성, 내담즙산성, 및 각종 식중독미생물에 대한 항균활성이 우수한 균주를 선별하여 균주를 동정한다. In order to achieve the above object, the present invention isolates the various Bacillus strains from traditional Jangjang, and when soybean fermentation, low odor generation, high viscosity material formation, acid resistance, bile acid resistance, and excellent antibacterial activity against various food poisoning microorganisms Strains are selected to identify strains.

동정된 균주를 삶은 대두에 접종하고 39℃에서 48시간 발효시켜 청국장을 제조하고, 청국장의 저장성을 높이기 위하여 50℃의 열풍 건조기에서 24시간 건조한 후, 분말청국장을 제조할 수 있는 통상의 입도로 분말하여 청국장분말을 제조하며, 그 분쇄입도는 제한하지 않는다.  Identified strains were inoculated in boiled soybeans and fermented at 39 ° C. for 48 hours to produce Cheonggukjang, and dried in a hot air dryer at 50 ° C. for 24 hours in order to increase the shelf life of Cheonggukjang. To prepare the Cheonggukjang powder, and its particle size is not limited.

분말 청국장의 풍미개선과 죽염과 녹차가 갖는 기능성을 부과하기 위하여 분말청국장 중량에 대하여 0.1 ~ 1.5% 내외의 죽염과 0.1 ~ 1.5% 내외의 녹차분말을 첨가하고 잘 혼합하여 죽염녹차 청국장을 제조한다. In order to improve the flavor of powdered Cheonggukjang and impart the functionality of bamboo salt and green tea, bamboo salt of 0.1 ~ 1.5% and green tea powder of about 0.1 ~ 1.5% are added to the weight of powdered soybean paste and mixed well to make bamboo salt green tea.

이하, 본 발명의 구성을 도 1에 나타낸 구체적인 방법과 실시예를 들어 상세히 설명하면, 다음과 같다. Hereinafter, the configuration of the present invention will be described in detail with reference to the specific method and embodiment shown in FIG.

[실시예 1] 미생물 분리Example 1 Microbial Separation

1. 대두 발효균주 선별1. Selection of Soybean Fermented Strains

각 지역에서 수집한 재래식 된장 1 g 을 9 ml의 멸균생리식염수에 현탁시키고 강하게 진탕 후 5분간 방치시켜 얻어진 상등액을 바실러스 분리를 위한시료로 사용하였다. 평판 당 30~300여 집락이 나타나도록 멸균생리식염수로 적당히 희석한 후, Tryticase Soy Agar(DIFCO사, USA) 평판배지에 도말하고 30℃의 항온기에서 48~72시간 배양한 후, 나타난 다양한 바실러스 특유의 집락을 같은 조성의 평판배지에서 순수분리하였다. 분리된 균주는 20%(v/v)의 글리세롤(glycerol)이 들어있는 Trytic Soy Broth(DIFCO사, USA)에 현탁시켜 냉동보존 하였으며, 필요에 따라 Tryticase Soy Agar배지에서 활성화하여 사용하였다.       A supernatant obtained by suspending 1 g of conventional soybeans collected in each region in 9 ml of sterile saline solution and shaking for 5 minutes was used as a sample for the separation of Bacillus. After proper dilution with sterile physiological saline solution to show 30 to 300 colonies per plate, plated on Tryticase Soy Agar (DIFCO, USA) plate medium and incubating for 48 to 72 hours in a 30 ℃ thermostat, Colonies were separated purely from plate media of the same composition. The isolated strain was suspended and cryopreserved in Trytic Soy Broth (DIFCO, USA) containing 20% (v / v) of glycerol, and activated in Tryticase Soy Agar medium as needed.

순수분리된 균주는 약 50 g의 삶은 대두가 들어 있는 250 ml Erlenmeyer flask에 접종하고 39℃의 항온기에서 발효를 진행시켜 점질물형성 정도와 악취발생 정도를 관능적으로 검토하여 가능한 악취발생이 적은 고 점성 균주를 1차적으로 선별하였다.  Purely isolated strains were inoculated into 250 ml Erlenmeyer flasks containing about 50 g of boiled soybeans and fermented in a thermostat at 39 ° C to examine the formation of viscous substances and odor incidence. Was screened primarily.

1차 선별된 균주는 Trytic Soy Broth 20 ml가 들어 있는 100 ml Erlenmeyer flask에 접종하고 항온진탕배양기에서 150 rpm의 속도로 48시간 진탕배양한 후, 내산성 검사를 실시하였다. 내산성 시험은 pH 2~3 범위에서 약 106 cells/ml 농도로 현탁시킨 후 정해진 시간 처리 시킨 후의 생균수를 처리 전의 생균수와 비교하여 생존율로 표현하였다. pH 2에서 3시간 경과 시 대부분의 균주는 사멸하였으나 일부 균주에서는 다양한 생존능력을 나타내고 있었다. The primary screened strains were inoculated into 100 ml Erlenmeyer flasks containing 20 ml of Trytic Soy Broth and shaken for 48 hours at 150 rpm in a constant temperature incubator, followed by acid resistance test. In the acid resistance test, the number of viable cells after suspending at a concentration of about 10 6 cells / ml in the range of pH 2-3 was expressed as viability compared to the number of viable cells before treatment. After 3 hours at pH 2, most of the strains were killed, but some strains showed various viability.

표 1은 분리균주의 내산성을 나타낸 것으로, 표 1 에서 보는바와 같이 CS90 균주는 pH 2에서 3시간 처리 시 42%의 가장 높은 생존율을 나타내고 있었으며, pH 2에서 6시간 처리 시 23%의 가장 높은 생존율을 나타내고 있었다. Table 1 shows the acid resistance of the isolate. As shown in Table 1, the CS90 strain showed the highest survival rate of 42% at 3 hours of treatment at pH 2, and the highest survival rate of 23% at 6 hours of treatment at pH 2. Was showing.

Figure 112007044463462-pat00001
Figure 112007044463462-pat00001

선별균주의 내담즙산성을 검토하기 위해 분리균주를 0.1~1%의 담즙산이 함유되어 있는 Tryticase Soy Agar배지에 접종 배양하여 집락형성 능력으로부터 최소저지농도를 구하였는데, 표 2는 분리균주의 내담즙산성을 나타낸 것으로, 표 2에서 보는 바와 같이, 1%의 담즙산이 들어 있는 배지에서도 집락을 형성하는 것으로 보아 최소 저지농도는 1% 이상이므로 내담즙산성이 높은 것으로 판단되었다.  In order to examine the bile acidity of the selected strains, the isolates were inoculated and cultured in a Tryticase Soy Agar medium containing 0.1-1% bile acid to obtain the minimum inhibitory concentration from colony forming ability. As shown in Table 2, as shown in Table 2, it was determined that colonies were formed even in a medium containing 1% bile acid, and therefore, the minimum stopping concentration was 1% or more, and thus, bile acid resistance was determined to be high.

Figure 112007044463462-pat00002
Figure 112007044463462-pat00002

선별균주의 식품위생미생물에 대한 항균활성은 공시균주가 접종되어 있는 평판에 분리균주를 접종하고 30℃의 항온기에서 배양함으로서 나타나는 생육저지환의 발생유무로부터 판단하였다. 표 3은 분리균주의 식품위생미생물에 대한 항균활성을 나타내는 것으로, 표 3에서 보는바와 같이, 선별균주는 다양한 항균범위를 나타내고 있었다. The antimicrobial activity against the food hygiene microorganisms of the selected strains was determined by inoculation of the isolated strains on the plate inoculated with the test strains and incubated in a thermostat at 30 ° C. Table 3 shows the antimicrobial activity against the food hygiene microorganisms of the isolated strain, as shown in Table 3, the selected strain showed a variety of antibacterial range.

Figure 112007044463462-pat00003
Figure 112007044463462-pat00003

베타글루코시다제 활성은 Trytic Soy Broth 50 ml가 들어 있는 250 ml Erlenmeyer flask에 접종하고 항온진탕배양기에서 150 rpm의 속도로 48시간 진탕배양한 후, 원심분리에 의한 상등액을 효소액으로 사용하여 측정하였다. 효소활성은 김 등의 방법(한국식품과학회지 31권: 1405-1409, 1999)에 따라 측정하여 U/mg protein 또는 U/g 시료로 표시하였다.  Betaglucosidase activity was measured by inoculating 250 ml Erlenmeyer flask containing 50 ml of Trytic Soy Broth, shaking culture for 48 hours at 150 rpm in a constant temperature incubator, and using the supernatant by centrifugation as enzyme solution. Enzyme activity was measured according to the method of Kim et al. (Korean Journal of Food Science and Technology No. 31: 1405-1409, 1999) and expressed as U / mg protein or U / g sample.

표 4는 분리균주의 베타-글루코시다아제 활성을 나타내는 것으로, 그 결과는 표 4에서 보는바와 같이, CS90 균주에서 베타-글루코시다아제 활성이 2.4(unit/mg protein)로 가장 높은 활성이 확인되었다. Table 4 shows the beta-glucosidase activity of the isolate, and the results showed that the highest beta-glucosidase activity was 2.4 (unit / mg protein) in the CS90 strain. .

Figure 112007044463462-pat00004
Figure 112007044463462-pat00004

상기의 표 1, 표 2, 표 3, 및 표 4의 결과로부터 CS90 균주는 내산성과 내담즙산성이 우수할 뿐만 아니라, 각종 식품위생미생물에 대해서도 우수한 항균활성과 높은 베타-글루코시다제 활성을 나타내고 있어 프로바이오틱스로서 활용이 가능할 것으로 판단되어 최종적으로 선별하였다. From the results of Table 1, Table 2, Table 3, and Table 4, the CS90 strain not only has excellent acid resistance and bile acid resistance, but also shows excellent antibacterial activity and high beta-glucosidase activity against various food sanitary microorganisms. Therefore, it was finally selected as it could be used as a probiotic.

2. 최종 선별균주 CS90의 미생물학적 동정2. Microbiological Identification of Final Selected Strain CS90

최종 선별한 CS90 균주를 Tryticase Soy Agar 평판배지에 접종하고 30℃에서 2일간 배양한 결과 직경 3 mm 정도의 표면이 거친 불투명한 고초균 특유의 집락이 형성되었다. CS90 균주는 그람 양성, 카탈라아제 양성의 포자형성 간균으로 15℃~45℃의 온도 범위에서 증식하였으며, 최적온도는 35℃이었다. 식염농도 15.0W/V%에서도 증식이 가능하여 비교적 내염성이 강한 바실러스로 판단되었다.The final screened CS90 strain was inoculated on Tryticase Soy Agar plate medium and incubated at 30 ° C. for 2 days, resulting in colony-specific opaque subtilis of roughly 3 mm diameter. The CS90 strain was a Gram-positive, catalase-positive spore-forming bacterium that grew in the temperature range of 15 ° C. to 45 ° C., and the optimum temperature was 35 ° C. Proliferation was possible even at the salt concentration of 15.0 W / V %, which was considered to be relatively bacillus resistant.

API 50 NE kit(BioMeriux)를 사용하여 균주 CS90의 당 이용성 조사를 통한 균 동정을 실시한 결과 표 5와 같았다. 이 같은 결과는 Bacillus subtilis와 유사성이 98.2%로 가장 높은 것으로 나타났다.Using the API 50 NE kit (BioMeriux) to identify the bacteria through the investigation of sugar availability of strain CS90 as shown in Table 5. These results showed the highest similarity with Bacillus subtilis (98.2%).

Figure 112007044463462-pat00005
Figure 112007044463462-pat00005

MIDI [Microbial Identification System (Hewlett Packard)]를 이용하여 CS90 균주의 세포 지방산을 분석한 결과는 표 6에서 보는 바와 같이, Bacillus 속에 특이적으로 많이 존재하는 Iso-C15:0(25.44%)와 Anteiso-C15:0(39.84%)가 절반 이상을 차 지하여 CS90 균주는 Bacillus 속에 속함을 알 수 있었다. 한편, DNA G+C 조성 분석결과 47.17 mol%로써 시 Bacillus 속의 mol% 비와 유사한 결과를 보였다.As a result of analyzing cellular fatty acids of CS90 strain using MIDI [Microbial Identification System (Hewlett Packard)], as shown in Table 6, Iso-C 15: 0 (25.44%) and Anteiso which are specifically present in Bacillus -C 15: 0 (39.84%) accounted for more than half, indicating that the CS90 strain belongs to the genus Bacillus . On the other hand, DNA G + C composition analysis showed 47.17 mol%, similar to the mol% ratio in the genus Bacillus .

Figure 112007044463462-pat00006
Figure 112007044463462-pat00006

CS90 균주의 좀 더 정확한 동정을 위하여, 16S rDNA 염기서열 분석을 수행하였다. 균주 CS90의 16S rDNA 서열 분석결과 염기서열은 1,517 bp로 확인되었고 그 서열은 표 7과 같았다. 이 염기서열을 미국 진뱅크 (GenBank) 데이터베이스로부터 얻은 세균학적으로 연관되어 있을 것으로 추정되는 연관종의 16S rDNA 염기서열과 크러스탈 더블류(CLUSTAL W) 소프트웨어를 사용하여 계통발생학적 분석을 한 결과, CS90 균주는 Bacillus에 속함을 알 수 있었다. For more accurate identification of the CS90 strain, 16S rDNA sequencing was performed. As a result of 16S rDNA sequence analysis of strain CS90, the nucleotide sequence was found to be 1,517 bp, and the sequence thereof was shown in Table 7. Phylogenetic analysis of the nucleotide sequence using 16S rDNA sequencing and CLUSTAL W software of related species presumed to be bacteriologically related from the US GenBank database. CS90 strain was found to belong to Bacillus .

16S rDNA 염기서열에 기초하여 CS90 균주의 염기서열을 계통발생학적 유사성을 나 타내는 균주와 비교한 결과는 표 8에서 보는바와 같이, 16S rDNA 서열 유사성의 수준은 90.8-99.7% 있었다. 이들 균주 중 Bacillus subtilis MA139(DQ415893)와 99.7%의 가장 높은 16S rDNA 서열 유사성은 나타내고 있었다.Comparing the nucleotide sequence of the CS90 strain with the strain showing phylogenetic similarity based on the 16S rDNA nucleotide sequence, as shown in Table 8, the level of 16S rDNA sequence similarity was 90.8-99.7%. Among these strains, the highest 16S rDNA sequence similarity with Bacillus subtilis MA139 (DQ415893) was 99.7%.

Figure 112007044463462-pat00007
Figure 112007044463462-pat00007

Figure 112007044463462-pat00008
Figure 112007044463462-pat00008

이상의 결과를 토대로 CS90 균주는 바실러스 섭티리스(Bacillus subtilis)에 속하는 새로운 균주임을 알 수 있었으며, 바실러스 섭티리스 씨에스 90(Bacillus subtilis CS90)이라 명하여 한국농업생명공학연구원에 2007년 5월 11일자로 기탁하였다(기탁번호: KACC91315P).Based on the above results, the CS90 strain was found to be a new strain belonging to Bacillus subtilis, and was named Bacillus subtilis CS90 and was deposited on May 11, 2007 by the Korea Research Institute of Agriculture and Biotechnology. (Accession Number: KACC91315P).

[실시예 2] 대두 발효에 의한 청국장 제조Example 2 Preparation of Cheonggukjang by Soybean Fermentation

청국장 제조를 위해, 종배양하는 종배양과정(1)으로, 먼저 50 g의 삶은 대두가 250 ml Erlenmeyer flask에 CS90 균주를 접종하여 39℃에서 48시간 종배양 하였다. For the production of Cheonggukjang, as a seed culture process (1) to seed culture, 50 g boiled soybean was inoculated with CS90 strain in a 250 ml Erlenmeyer flask and incubated at 39 ° C. for 48 hours.

생청국장을 제조하는 생청국장제조과정(2)으로, 상기의 종배양한 CS90 균주 50 g을 4 Kg의 삶은 대두에 접종하고 39℃에서 발효를 진행시켜 생청국장을 제조하였다. 이때, 대두는 실온에서 12시간 침지하여 물을 빼고 120℃의 압력솥에서 2.5시간 삶은 것을 사용하였다.  In the production process of the fresh soybean soup (2) to prepare the raw cheonggukjang, 50 g of the above cultured CS90 strain was inoculated in 4 Kg of boiled soybean and fermented at 39 ° C. to prepare the raw cheonggukjang. At this time, the soybeans were immersed for 12 hours at room temperature, water was used, and boiled for 2.5 hours in a 120 ℃ pressure cooker.

발효시간 경과에 따른 청국장의 생균수 및 물리화학적 변화는 표 9와 같았다. 발효시간 경과에 따라 생균수가 증가하여 48시간 경과에서 1011cells/g에 달하였으며, 글루코시다제 활성에 의한 가용성 페놀성 물질이 증가하였고 이에 기인하는 것으로 판단되는 라디칼 소거활성도 증가하였다. The viable cell counts and physicochemical changes of Cheonggukjang with the fermentation time were as shown in Table 9. As the fermentation time increased, the number of viable cells increased to 10 11 cells / g after 48 hours, and the soluble phenolic substances due to glucosidase activity increased, and the radical scavenging activity which was thought to be due to this increased.

가용성 페놀은 Folin-Ciocalteu 시약을 사용하는 Dewanto 등 (J. Agric. Food Chem 50: 4959-4964, 2002)의 방법에 따라 측정하였으며, 라디칼 소거활성은 Blois의 방법(우관식 등, 한국식품과학회지 38권: 355-360, 2006)을 변형한 DPPH에 의한 전자공여능으로서 측정하였다. Soluble phenol was measured according to the method of Dewanto et al. (J. Agric. Food Chem 50: 4959-4964, 2002) using Folin-Ciocalteu reagent, and radical scavenging activity was determined by Blois' method 38: 355-360, 2006) was measured as electron donating ability by the modified DPPH.

Figure 112007044463462-pat00009
Figure 112007044463462-pat00009

[실시예 3] 죽염 녹차 청국장 제조Example 3 Preparation of Bamboo Salt Green Tea Cheonggukjang

[실시 예 2]에서와 같이 제조된 생청국장의 저장성과 기능성 및 관능적성을 증가시키기 위해 죽염녹차청국장을 제조하였다. Bamboo salt green tea soybean soup was prepared to increase the shelf life, functionality and organoleptic properties of the raw saengcheongjang prepared as in [Example 2].

먼저, 상기 실 시예 2에서 제조된 생청국장을 분말화하는 분말청국장제조과정(3) 으로, 생청국장을 수분함량이 5~10%가 되도록 50℃의 열풍건조기에서 24시간 건조한 후 약 180 메시(mesh) 크기로 분쇄하여 분말청국장을 제조하였다.  First, in the powdered cheongukjang manufacturing process (3) to powder the raw cheonggukjang prepared in Example 2, after drying for 24 hours in a hot air dryer at 50 ℃ so that the moisture content of 5 ~ 10% about 180 mesh (mesh) Powder Cheonggukjang was prepared by pulverizing to size.

죽염녹차청국장(4)을 제조하기 위하여, 죽염과 녹차분말은 230 메시로 분쇄한 것을 사용하였으며, 분말청국장(3) 중량에 대하여 각각 0.5 ~1.5% 첨가 혼합하여 죽염녹차청국장을 제조하여 맛과 이취정도를 검사하여 표 10과 같은 결과를 얻었다. In order to manufacture bamboo salt green tea Cheonggukjang (4), bamboo salt and green tea powder were used as pulverized with 230 mesh, and 0.5 ~ 1.5% of the powdered cheonggukjang (3) was added and mixed to prepare bamboo salt green tea soup. The degree was examined to obtain the results shown in Table 10.

그 결과, 죽염과 녹차의 첨가량이 각각 0.5 - 1.0w/w% 첨가에서 청국장 특유의 이취가 제거되고 죽염과 녹차의 조화로운 맛을 엇을 수 있었다.As a result, the addition of bamboo salt and green tea added 0.5-1.0 w / w %, respectively, removed the peculiar odor of Cheonggukjang and the harmonious taste of bamboo salt and green tea.

따라서, 최적의 죽염녹차청국장(4)을 제조하기 위하여, 상기 제조된 분말청국장(3) 중량에 대하여 분쇄한 죽염 0.5~1.0 중량 %와 녹차분말 0.5~1.0 중량%를 첨가 혼합하여 죽염녹차청국장(4)을 제조하였다. Therefore, in order to produce an optimum bamboo salt green tea Chunggukjang (4), 0.5 to 1.0% by weight of the milled bamboo salt and 0.5 to 1.0% by weight of green tea powder was added to the weight of the prepared powder Chunggukjang (3) and mixed with bamboo salt green tea Chunggukjang ( 4) was prepared.

Figure 112007044463462-pat00010
Figure 112007044463462-pat00010

이상에서와 같이 본원 발명은 비록 상기의 실시예에 한하여 설명하였지만 반드시 여기에만 한정되는 것은 아니며 본 발명의 범주와 사상을 벗어나지 않는 범위 내에 서 다양한 변형실시가 가능함은 물론이다. As described above, although the present invention has been described with reference to the above embodiments, it is not necessarily limited thereto, and various modifications may be made without departing from the scope and spirit of the present invention.

상기 실시예를 통해 살펴본 바와 같이, 본 발명의 바실러스 속 CS90 균주는 우수한 내산성 및 내담즙산성을 지니고 있으며, 각종 식품위생미생물에 대한 항균활성과 베타-글루코시다제 활성에 의한 높은 페놀성 물질 생성 능력을 가지고 있어 CS90 균주는 인체의 장에서 생존가능성이 높을 뿐만 아니라, 식중독을 예방하는데 도움을 줄 수 있고, 청국장에 다량의 폴리페놀을 생성시킴으로서 항산화능력이 높은 청국장 제조를 가능하게 한다. 이러한 특성은 기능성이 우수한 생균제제로서의 청국장을 가능하게 할 것으로 판단된다. 또한, 제조된 청국장의 분말에 적당량의 죽염과 녹차가루를 첨가함으로서 그 기능성을 활용할 수 있을 뿐만 아니라 청국장의 풍미를 개선시키는 효과를 제공함으로서 누구나가 거부감 없이 편리하게 청국장을 소비할 수 있을 것으로 기대된다.As described through the above examples, the Bacillus genus CS90 strain of the present invention has excellent acid resistance and bile acid resistance, and ability to generate high phenolic substances by antibacterial activity and beta-glucosidase activity against various food hygiene microorganisms. Since the CS90 strain has a high viability in the intestine of the human body, it can help to prevent food poisoning, and by producing a large amount of polyphenols in Cheonggukjang enables the production of Cheonggukjang with high antioxidant capacity. This characteristic is believed to enable Cheonggukjang as a probiotic with excellent functionality. In addition, by adding an appropriate amount of bamboo salt and green tea powder to the powder of the produced Cheonggukjang not only can utilize its functionality, but also improve the flavor of Cheonggukjang so that anyone can conveniently consume Cheonggukjang without objection. .

서열목록 전자파일 첨부 Attach sequence list electronic file  

Claims (3)

녹차죽염청국장의 제조방법에 있어서.In the manufacturing method of green tea bamboo salt soup. 청국장 제조를 위해 먼저 50 g의 삶은 대두에 바실러스 속(Bacillus sp.) 씨에스 90 (CS90)균주를 접종하여, 39℃에서 48시간 종배양하는 종배양과정(1)과, For the production of Cheonggukjang, seed culture process (1) was first inoculated with 50 g of boiled soybean Bacillus sp. 90 (CS90) strain, followed by incubation at 39 ° C. for 48 hours. 상기 종배양(1)한 바실러스 속(Bacillus sp.) 씨에스 90(CS90) 균주 50 g을, 실온에서 12시간 침지하여 물을 뺀 후 120℃의 압력솥에서 2.5시간 삶은 4 Kg의 대두에 접종하고, 39℃에서 발효를 진행시켜 생청국장을 제조하는 생청국장제조과정(2)과,50 g of the strain (1) Bacillus sp. 90 (CS90) strain was immersed at room temperature for 12 hours to remove water, and then inoculated in 4 Kg of soybeans boiled for 2.5 hours in a pressure cooker at 120 ° C. Manufacturing process of fresh soybean soup (2) to proceed with fermentation at 39 ℃ 상기 제조된 생청국장(2)을 수분함량이 5~10%가 되도록 건조한 후, 180 메시(mesh) 크기로 분쇄하여 생청국장을 분말화하는 분말청국장제조과정(3)과, Drying the prepared soybean soup (2) to 5 to 10% moisture content, and then pulverized to 180 mesh (mesh) size to make the powdered soybean soup (3) and, 죽염과 녹차를 230 메시로 분쇄하여, 상기 제조된 분말청국장(3) 중량에 대하여 분쇄한 죽염 0.5~1.0 중량%와 녹차분말 0.5~1.0 중량%를 첨가 혼합하여 죽염녹차청국장(4)을 제조하는 것을 특징으로 하는 바실러스 속(Bacillus sp.) 씨에스 90을 이용하여 제조된 녹차죽염청국장의 제조방법.Bamboo salt and green tea were pulverized to 230 mesh, and 0.5 to 1.0% by weight of pulverized bamboo salt and 0.5 to 1.0% by weight of green tea powder were added to and mixed with the prepared powder Chungkukjang (3) to prepare bamboo salt green tea chunggukjang (4). Bacillus sp. ( Bacillus sp.) A method for preparing green tea porridge salt soup prepared using CS 90. 제 1항의 제조방법에 의하여 죽염녹차청국장이 제조되어지는 것을 특징으로 하는 바실러스 속(Bacillus sp.) 씨에스 90을 이용한 죽염녹차청국장.Bamboo salt green tea chungguk using Bacillus sp. CS 90, characterized in that the bamboo salt green tea jangguk prepared by the manufacturing method of claim 1. 제 2항에 있어서, 바실러스 속(Bacillus sp.) 씨에스 90을 이용하여 제조된 죽염녹차청국장은, 내산성과 내담즙산성, 항균활성, 항산화활성의 효능을 가지는 것을 특징으로 하는 죽염녹차청국장.The bamboo salt green tea soybean paste of claim 2, wherein the bamboo salt green tea soybean paste prepared using Bacillus sp. 90 has acid and bile acid resistance, antimicrobial activity, and antioxidant activity.
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