KR102403615B1 - Manufacturing method for composition of gastrodia elata blume - Google Patents
Manufacturing method for composition of gastrodia elata blume Download PDFInfo
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- KR102403615B1 KR102403615B1 KR1020190159686A KR20190159686A KR102403615B1 KR 102403615 B1 KR102403615 B1 KR 102403615B1 KR 1020190159686 A KR1020190159686 A KR 1020190159686A KR 20190159686 A KR20190159686 A KR 20190159686A KR 102403615 B1 KR102403615 B1 KR 102403615B1
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- chunma
- bioconversion
- concentrate
- fermented
- cheonma
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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
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- 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
- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
- A23L19/01—Instant products; Powders; Flakes; Granules
-
- 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
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/065—Microorganisms
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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
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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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
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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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
- A23V2300/00—Processes
- A23V2300/14—Extraction
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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
- A23V2400/00—Lactic or propionic acid bacteria
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Abstract
본 발명은 천마 조성물의 제조 방법에 관한 것으로서, 더욱 상세히는 천마를 분쇄하여 얻어진 천마 분말에 미생물을 접종하여 발효시키는 생물 전환 기술을 통해 제조되는 천마 조성물의 제조 방법에 관한 것이다. 본 발명은 천마를 분쇄하여 얻어진 천마 분말에 미생물을 접종하여 발효시키는 생물 전환 기술을 적용하고, 상기 생물 전환 기술을 통해 얻어진 발효물로부터 천마생물전환 추출물을 추출한 후 농축하여 부원료를 배합하는 과정을 통해 제조된 천마 조성물을 제공함으로써, 생물 전환 기술을 이용하여 천마 특유의 쓴 맛을 감소시켜 맛과 향이 개선된 천마 조성물을 제공할 수 있으며, 이를 통해 천마 섭취에 대한 사용자의 거부감을 해소함과 아울러 천마 조성물의 용이한 섭취에 따라 천마가 가진 성분의 신체 흡수율을 증진시켜 혈액순환의 개선과 동맥경화, 고지혈증 등의 예방과 같은 효과를 제공할 수 있다.The present invention relates to a method for producing a cheonma composition, and more particularly, to a method for producing a cheonma composition produced through a bioconversion technique of inoculating and fermenting a cheonma powder obtained by pulverizing a cheonma. The present invention applies the bioconversion technology of inoculating and fermenting the Chunma powder obtained by pulverizing the Chunma, and extracting the Chunma bioconversion extract from the fermented product obtained through the bioconversion technology, and then concentrating it through the process of blending the auxiliary materials By providing the prepared Chunma composition, it is possible to provide a Chunma composition with improved taste and flavor by reducing the peculiar bitterness of Chunma using bioconversion technology, thereby resolving the user’s objection to Chunma ingestion, and the Chunma composition It can provide effects such as improvement of blood circulation and prevention of arteriosclerosis, hyperlipidemia, etc.
Description
본 발명은 천마 조성물의 제조 방법에 관한 것으로서, 더욱 상세히는 천마를 분쇄하여 얻어진 천마 분말에 미생물을 접종하여 발효시키는 생물 전환 기술을 통해 제조되는 천마 조성물의 제조 방법에 관한 것이다.The present invention relates to a method for producing a cheonma composition, and more particularly, to a method for producing a cheonma composition produced through a bioconversion technique of inoculating and fermenting a cheonma powder obtained by pulverizing a cheonma.
천마(天麻)는 참나무 종류의 썩은 그루터기에 나는 버섯의 균사에 붙어사는 여러해살이 기생식물로서, 굵고 긴 덩이줄기를 가지고 있으며 덩이줄기로부터 높이 1m쯤 되는 줄기가 자라난다.Cheonma (天麻) is a perennial parasitic plant that lives on the mycelium of mushrooms that grow on the rotten stump of an oak tree.
또한, 천마의 줄기의 빛깔은 주황빛이고 전혀 잎을 가지고 있지 않으며, 꽃은 줄기 끝에 곧게 선 이삭 꼴로 모여 핀다. 또한, 천마는 3장의 꽃잎이 서로 달라붙어 불룩한 단지모양을 이루는데 주둥이 부분은 세 개로 갈라져 있으며, 꽃의 길이는 2cm 안팎이고 빛깔은 노랗다.In addition, the color of the stem of Cheonma is orange, and it has no leaves at all, and the flowers bloom in a cluster at the tip of the stem in the shape of an ear. In addition, the three petals of Cheonma are attached to each other to form a bulging jar, and the spout is divided into three, and the length of the flower is about 2cm and the color is yellow.
천마에는 게스트로딘이라는 유효성분이 다량 함유돼 있다. 이 게스트로딘 성분이 혈관 내에 쌓여 있는 노폐물을 청소해주고 이를 통해 혈압안정과 혈류량을 늘려 원활한 혈액순환을 도와준다고 알려져 있다. 특히, 천마는 뇌혈관이나 심장혈관을 더욱 튼튼하게 해주며 겨울철 특히 많이 발생되는 뇌경색, 뇌출혈 등은 물론이고 심근경색에도 좋은 음식으로 전해지고 있다.Chunma contains a large amount of active ingredient called gestrodin. It is known that this guestrodin component cleans up waste products accumulated in the blood vessels, thereby stabilizing blood pressure and increasing blood flow to help smooth blood circulation. In particular, Chunma strengthens cerebral and cardiovascular blood vessels, and is said to be good food for myocardial infarction as well as cerebral infarction and cerebral hemorrhage, which occur a lot in winter.
또한, 천마는 또한 예부터 '풍을 다스리는 풀'이라고 해 '정풍초'라고도 불리며 중풍에 좋은 음식으로 알려져 있으며, 특히 혈액뇌장벽을 통과하는 물질이 함유된 유일한 한방약재라고 한다. 이 외에도, 천마는 몸 속 나쁜 콜레스테롤 수치는 낮춰 동맥경화, 고지혈증 예방에 도움을 준다.In addition, Chunma is also called 'Jungpungcho' as it has been called 'the grass that rules the wind' since ancient times, and is known as a food good for stroke. In addition to this, Chunma helps to prevent arteriosclerosis and hyperlipidemia by lowering the level of bad cholesterol in the body.
그러나, 이러한 천마의 뛰어난 효능에도 불구하고, 천마는 고유의 비린맛과 쓴맛을 가지는 동시에 점액질이 존재하여 섭취하는데 거부감이 있어 유통이 활발하게 이루어지고 있지 않다.However, despite the excellent efficacy of Chunma, Chunma has a unique fishy taste and bitter taste, and at the same time has a mucilage, so there is a feeling of reluctance to take it, so its distribution is not actively made.
따라서, 이러한 천마의 섭취를 용이하게 할 수 있도록 지원하기 위한 제품 개발이 요구되고 있다.Therefore, there is a need for product development to support such an easy ingestion of Chunma.
본 발명은 천마를 분쇄하여 얻어진 천마 분말에 미생물을 접종하여 발효시키는 생물 전환 기술을 적용하고, 상기 생물 전환 기술을 통해 얻어진 발효물로부터 천마생물전환 추출물을 추출한 후 농축하여 부원료를 배합하는 과정을 통해 제조된 천마 조성물을 제공하여, 천마의 맛과 향을 개선하여 식이편의성 개선함과 아울러 신체 흡수율을 증진시켜 다양한 효능을 가진 천마를 용이하게 섭취할 수 있도록 지원하는데 그 목적이 있다.The present invention applies the bioconversion technology of inoculating and fermenting the Chunma powder obtained by pulverizing the Chunma, and extracting the Chunma bioconversion extract from the fermented product obtained through the bioconversion technology, and then concentrating it through the process of blending the auxiliary materials The purpose of providing the prepared Chunma composition is to improve the taste and flavor of Chunma to improve dietary convenience and to support body absorption to facilitate the ingestion of Chunma having various effects.
본 발명의 실시예에 따른 천마 조성물의 제조 방법은, 천마를 분쇄하여 천마 분말을 포함하는 분쇄물을 준비하는 분말화 단계와, 상기 분쇄물과 물을 1:20의 중량 비율로 혼합한 혼합물을 제조한 후 상기 혼합물에 대한 당화 작업을 통해 천마 당화액을 준비하는 당화 단계와, 락토바실러스 브레비스(Lactobacillus brevis) 및 락토바실러스 플란타럼(Lactobacillus platarum)의 혼합 균주를 상기 천마 당화액의 3 ~ 7중량%로 상기 천마 당화액에 접종하여 발효하는 발효 단계 및 상기 발효 단계를 통해 얻어진 발효물로부터 천마생물전환 추출물을 추출한 후 농축하여 얻어진 천마생물전환 농축액에 하수오 농축액, 배 농축액, 홍삼 농축액 및 산양삼 추출액을 미리 설정된 비율로 첨가하여 배합한 후 살균하여 천마 조성물을 제조하는 제조 단계를 포함할 수 있다.The method for producing a cheonma composition according to an embodiment of the present invention comprises a powdering step of pulverizing a cheonma to prepare a comminuted product containing cheonma powder, and a mixture obtained by mixing the grinded product and water in a weight ratio of 1:20. After preparation, a saccharification step of preparing a saccharification solution for the saccharification of the mixture, and a mixed strain of Lactobacillus brevis and Lactobacillus
본 발명과 관련된 일 예로서, 상기 당화 단계는 상기 혼합물을 55℃ ~ 65℃에서 3 ~ 5 시간 당화작업하여 상기 천마 당화액을 준비하는 단계를 더 포함하는 것을 특징으로 할 수 있다.As an example related to the present invention, the saccharification step may further include the step of preparing the saccharification solution by saccharifying the mixture at 55° C. to 65° C. for 3 to 5 hours.
본 발명과 관련된 일 예로서, 상기 발효 단계는 상기 락토바실러스 브레비스 및 락토바실러스 플란타럼으로 이루어진 상기 혼합 균주를 맥아배지에 종균 배양한 다음 상기 천마 당화액에 접종하는 단계를 더 포함하는 것을 특징으로 할 수 있다.As an example related to the present invention, the fermentation step further comprises the step of culturing the mixed strain consisting of the Lactobacillus brevis and Lactobacillus plantarum in a malt medium and then inoculating the saccharification solution in the thousand horses. can do.
본 발명과 관련된 일 예로서, 상기 제조 단계는 상기 발효물로부터 80℃ ~ 90℃에서 23 ~ 25 시간동안 천마생물전환 추출물을 추출한 후 50Brix% ~ 55Brix%가 되도록 농축하여 천마생물전환 농축액을 제조하는 단계를 더 포함하는 것을 특징으로 할 수 있다.As an example related to the present invention, the manufacturing step extracts the Chunma bioconversion extract from the fermented product at 80°C to 90°C for 23 to 25 hours and then concentrates it to 50Brix% to 55Brix% to prepare a Chunma bioconversion concentrate It may be characterized in that it further comprises a step.
본 발명과 관련된 일 예로서, 상기 제조 단계는 상기 천마생물전환 농축액 25 ~ 35중량%에, 상기 하수오 농축액 15 ~ 25중량%, 상기 배 농축액 15 ~ 19중량%, 상기 홍삼 농축액 2~5%, 상기 산양삼 추출액 25 ~ 35중량%을 첨가하여 45Brix% ~ 50Brix%가 되도록 배합하는 단계를 더 포함하는 것을 특징으로 할 수 있다.As an example related to the present invention, the manufacturing step is 25 to 35% by weight of the Chunma bioconversion concentrate, 15 to 25% by weight of the sewage concentrate, 15 to 19% by weight of the pear concentrate, 2 to 5% of the red ginseng concentrate, It may be characterized in that it further comprises the step of adding 25 to 35% by weight of the wild ginseng extract and blending so as to be 45Brix% to 50Brix%.
본 발명은 천마를 분쇄하여 얻어진 천마 분말에 미생물을 접종하여 발효시키는 생물 전환 기술을 적용하고, 상기 생물 전환 기술을 통해 얻어진 발효물로부터 천마생물전환 추출물을 추출한 후 농축하여 부원료를 배합하는 과정을 통해 제조된 천마 조성물을 제공함으로써, 생물 전환 기술을 이용하여 천마 특유의 쓴 맛을 감소시켜 맛과 향이 개선된 천마 조성물을 제공할 수 있으며, 이를 통해 천마 섭취에 대한 사용자의 거부감을 해소함과 아울러 천마 조성물의 용이한 섭취에 따라 천마가 가진 성분의 신체 흡수율을 증진시켜 혈액순환의 개선과 동맥경화, 고지혈증 등의 예방과 같은 효과를 제공할 수 있다.The present invention applies the bioconversion technology of inoculating and fermenting the Chunma powder obtained by pulverizing the Chunma, and extracting the Chunma bioconversion extract from the fermented product obtained through the bioconversion technology, and then concentrating it through the process of blending the auxiliary materials By providing the prepared Chunma composition, it is possible to provide a Chunma composition with improved taste and flavor by reducing the peculiar bitter taste of Chunma by using bioconversion technology, thereby resolving the user’s objection to the Chunma intake and the Chunma composition It can provide effects such as improvement of blood circulation and prevention of arteriosclerosis, hyperlipidemia, etc.
도 1은 본 발명의 실시예에 따른 천마 조성물의 제조 방법에 대한 순서도.
도 2 내지 도 7은 본 발명의 실시예에 따른 천마 조성물의 제조 방법 중 천마 당화액에 접종되는 혼합균주의 우수균주 분석 결과를 나타낸 도면.
도 8 내지 도 19는 본 발명의 실시예에 따른 천마 발효물의 실험 분석 결과를 나타낸 도면.
도 20 내지 도 23은 본 발명의 실시예에 따른 천마생물전환 추출물의 실험 분석 결과를 나타낸 도면.1 is a flowchart for a method of manufacturing a cheonma composition according to an embodiment of the present invention.
Figures 2 to 7 are views showing the analysis results of excellent strains of the mixed strain inoculated into the Chunma saccharification solution of the method for producing a Chunma composition according to an embodiment of the present invention.
8 to 19 is a view showing the experimental analysis results of the fermented Chunma according to an embodiment of the present invention.
20 to 23 are diagrams showing the experimental analysis results of the Chunma bioconversion extract according to an embodiment of the present invention.
본 발명에서 사용되는 기술적 용어는 단지 특정한 실시예를 설명하기 위해 사용된 것으로, 본 발명을 한정하려는 의도가 아님을 유의해야 한다. 또한, 본 발명에서 사용되는 기술적 용어는 본 발명에서 특별히 다른 의미로 정의되지 않는 한, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 의미로 해석되어야 하며, 과도하게 포괄적인 의미로 해석되거나, 과도하게 축소된 의미로 해석되지 않아야 한다. 또한, 본 발명에서 사용되는 기술적인 용어가 본 발명의 사상을 정확하게 표현하지 못하는 잘못된 기술적 용어일 때에는 당업자가 올바르게 이해할 수 있는 기술적 용어로 대체되어 이해되어야 할 것이다. 또한, 본 발명에서 사용되는 일반적인 용어는 사전에 정의되어 있는 바에 따라, 또는 전후 문맥상에 따라 해석되어야 하며, 과도하게 축소된 의미로 해석되지 않아야 한다.It should be noted that the technical terms used in the present invention are only used to describe specific embodiments, and are not intended to limit the present invention. In addition, the technical terms used in the present invention should be interpreted as meanings generally understood by those of ordinary skill in the art to which the present invention belongs, unless otherwise defined in the present invention. It should not be construed in the meaning of a human being or in an excessively reduced meaning. In addition, when the technical term used in the present invention is an incorrect technical term that does not accurately express the spirit of the present invention, it should be understood by being replaced with a technical term that can be correctly understood by those skilled in the art. In addition, the general terms used in the present invention should be interpreted according to the definition in the dictionary or according to the context before and after, and should not be interpreted in an excessively reduced meaning.
또한, 본 발명에서 사용되는 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한 복수의 표현을 포함한다. 본 발명에서 "구성된다" 또는 "포함한다" 등의 용어는 발명에 기재된 여러 구성 요소들 또는 여러 단계를 반드시 모두 포함하는 것으로 해석되지 않아야 하며, 그 중 일부 구성 요소들 또는 일부 단계들은 포함되지 않을 수도 있고, 또는 추가적인 구성 요소 또는 단계들을 더 포함할 수 있는 것으로 해석되어야 한다.Also, the singular expression used in the present invention includes the plural expression unless the context clearly dictates otherwise. In the present invention, terms such as “consisting of” or “comprising” should not be construed as necessarily including all of the various components or various steps described in the invention, and some components or some steps may not be included. It should be construed that it may further include additional components or steps.
또한, 본 발명에서 사용되는 제 1, 제 2 등과 같이 서수를 포함하는 용어는 구성 요소들을 설명하는데 사용될 수 있지만, 구성 요소들은 용어들에 의해 한정되어서는 안 된다. 용어들은 하나의 구성 요소를 다른 구성 요소로부터 구별하는 목적으로만 사용된다. 예를 들어, 본 발명의 권리 범위를 벗어나지 않으면서 제 1 구성 요소는 제 2 구성 요소로 명명될 수 있고, 유사하게 제 2 구성 요소도 제 1 구성 요소로 명명될 수 있다.Also, terms including ordinal numbers such as first, second, etc. used in the present invention may be used to describe the elements, but the elements should not be limited by the terms. The terms are used only for the purpose of distinguishing one component from another. For example, without departing from the scope of the present invention, a first component may be referred to as a second component, and similarly, the second component may also be referred to as a first component.
이하, 첨부된 도면을 참조하여 본 발명에 따른 바람직한 실시예를 상세히 설명하되, 도면 부호에 관계없이 동일하거나 유사한 구성 요소는 동일한 참조 번호를 부여하고 이에 대한 중복되는 설명은 생략하기로 한다.Hereinafter, a preferred embodiment according to the present invention will be described in detail with reference to the accompanying drawings, but the same or similar components are assigned the same reference numerals regardless of reference numerals, and redundant description thereof will be omitted.
또한, 본 발명을 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 본 발명의 요지를 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다. 또한, 첨부된 도면은 본 발명의 사상을 쉽게 이해할 수 있도록 하기 위한 것일 뿐, 첨부된 도면에 의해 본 발명의 사상이 제한되는 것으로 해석되어서는 아니 됨을 유의해야 한다.In addition, in the description of the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted. In addition, it should be noted that the accompanying drawings are only for easy understanding of the spirit of the present invention, and should not be construed as limiting the spirit of the present invention by the accompanying drawings.
이하, 도면을 참고하여 본 발명의 실시예에 따른 천마 조성물의 제조 방법에 대해 상세히 설명한다.Hereinafter, with reference to the drawings will be described in detail with respect to the manufacturing method of the composition according to the embodiment of the present invention.
도 1은 본 발명의 실시예에 따른 천마 조성물의 제조 방법에 대한 순서도이다.1 is a flowchart for a method of manufacturing a cheonma composition according to an embodiment of the present invention.
우선, 천마(天麻, Gastrodia elata Blume)를 증숙 건조한 후 분쇄하여 제조된 천마 분말을 포함하는 분쇄물을 준비한다(S1).First, prepare a pulverized product containing Chunma powder prepared by steaming and drying Chunma (天麻, Gastrodia elata Blume) and then pulverizing it (S1).
또한, 상기 분쇄물과 물을 1:20의 중량 비율로 혼합한 혼합물을 제조한 후 상기 혼합물에 대한 당화 작업을 통해 천마 당화액을 준비한다(S2).In addition, after preparing a mixture in which the pulverized product and water are mixed in a weight ratio of 1:20, a saccharification solution is prepared by saccharifying the mixture (S2).
이때, 상기 분쇄물을 부직포에 넣고 상기 분쇄물의 20배의 물을 넣어 혼합물을 제조한 후 상기 혼합물을 55℃ ~ 65℃에서 3 ~ 5 시간 당화작업하여 상기 천마 당화액을 준비한다.At this time, the pulverized product is put in a nonwoven fabric, and 20 times the water of the pulverized product is added to prepare a mixture, and then the mixture is saccharified at 55° C. to 65° C. for 3 to 5 hours to prepare the saccharified solution.
여기서, 상기 혼합물을 60℃에서 4시간 동안 당화작업하여 상기 천마 당화액을 준비하는 것이 바람직하다.Here, it is preferable to prepare the saccharification solution by saccharifying the mixture at 60° C. for 4 hours.
또한, 락토바실러스 브레비스(Lactobacillus brevis) 및 락토바실러스 플란타럼(Lactobacillus platarum)의 혼합 균주(또는 혼합균)를 상기 천마 당화액의 3 ~ 7중량%로 상기 천마 당화액에 접종하여 발효할 수 있으며, 이를 통해 발효물(또는 천마 발효물)을 얻을 수 있다(S3).In addition, a mixed strain (or mixed bacteria) of Lactobacillus brevis (Lactobacillus brevis) and Lactobacillus platarum is inoculated into the Chunma saccharified solution at 3 to 7% by weight of the Chunma saccharified solution and fermented, , through this, it is possible to obtain a fermented product (or a fermented product of Chunma) (S3).
이때, 상기 혼합 균주를 상기 천마 당화액의 5중량%로 준비한 후 상기 천마 당화액에 접종하는 것이 바람직하며, 상기 접종은 상기 천마 당화액에 상기 천마 당화액의 5중량%에 해당하는 상기 혼합균주를 첨가하여 교반하는 것을 의미할 수 있다.At this time, it is preferable to inoculate the mixed strain with 5% by weight of the saccharification solution of the thousand horses after preparing the mixture, and the inoculation is the mixed strain corresponding to 5% by weight of the saccharification solution of the thousand horses in the saccharification solution. It may mean adding and stirring.
또한, 혼합균주가 접종된 상기 천마 당화액을 발효하여 상기 발효물을 얻을 수 있다.In addition, the fermented product can be obtained by fermenting the cheonma saccharification solution inoculated with a mixed strain.
또한, 상기 락토바실러스 브레비스 및 락토바실러스 플란타럼으로 이루어진 상기 혼합 균주를 맥아배지에 종균 배양한 다음 상기 천마 당화액에 접종할 수 있다.In addition, the mixed strain consisting of the Lactobacillus brevis and Lactobacillus plantarum can be seed cultured in malt medium and then inoculated into the saccharification solution of the thousand horses.
또한, 상기 발효를 통해 얻어진 상기 발효물로부터 천마생물전환 추출물(또는 천마 추출물)을 추출한 후 농축하여(S4) 얻어진 천마생물전환 농축액(또는 천마 농축액)에 하수오 농축액, 배 농축액, 홍삼 농축액 및 산양삼 추출액을 미리 설정된 비율로 첨가하여 배합한 후 살균하여 천마 조성물을 제조한다(S5).In addition, after extracting the Chunma bioconversion extract (or Chunma extract) from the fermented product obtained through the fermentation and concentrating (S4), the Chunma bioconversion concentrate (or Chunma concentrate) obtained from the sewage concentrate, pear concentrate, red ginseng concentrate and wild ginseng extract is added in a preset ratio and then sterilized to prepare a cheonma composition (S5).
이때, 상기 발효물로부터 80℃ ~ 90℃에서 23 ~ 25 시간동안 천마생물전환 추출물을 추출한 후 50Brix% ~ 55Brix%가 되도록 농축하여 천마생물전환 농축액을 제조하는 것이 바람직하다.At this time, it is preferable to prepare a Chunma bioconversion concentrate by extracting the Chunma bioconversion extract from the fermented product at 80° C. to 90° C. for 23 to 25 hours and then concentrating it so that it becomes 50Brix% to 55Brix%.
더욱 바람직하게는, 상기 발효물로부터 85℃에서 24시간 동안 천마생물전환 추출물을 추출한 후 54Brix%가 되도록 농축하여 천마생물전환 농축액을 제조하는 것이 바람직하다.More preferably, it is preferable to extract the Chunma bioconversion extract from the fermented product at 85° C. for 24 hours and then concentrate it to 54Brix% to prepare the Chunma bioconversion concentrate.
또한, 상술한 구성에서, 상기 천마생물전환 농축액 25 ~ 35중량%에, 하수오 농축액 15 ~ 25중량%, 배 농축액 15 ~ 19중량%, 홍삼 농축액 2~5%, 산양삼 추출액 25 ~ 35중량%을 첨가하여 45Brix% ~ 50Brix%가 되도록 배합하는 것이 바람직하다.In addition, in the above-described configuration, 25 to 35% by weight of the Chunma bioconversion concentrate, 15 to 25% by weight of sewage concentrate, 15 to 19% by weight of pear concentrate, 2 to 5% of red ginseng concentrate, 25 to 35% by weight of wild ginseng extract It is preferable to add it and mix it so that it becomes 45Brix% - 50Brix%.
이때, 천마 생물전환 농축액 30중량%에 하수오 농축액 20중량%, 배 농축액 17중량%, 홍삼 농축액 3% 및 산양삼 추출액 30%을 첨가하여, 47Brix%가 되도록 배합하는 것이 더욱 바람직하다.At this time, it is more preferable to add 20% by weight of sewo concentrate, 17% by weight of pear concentrate, 3% of red ginseng concentrate and 30% of wild ginseng extract to 30% by weight of Chunma bioconversion concentrate, and blending so as to be 47Brix%.
상술한 구성을 통해, 본 발명은 천마를 분쇄하여 얻어진 천마 분말에 미생물을 접종하여 발효시키는 생물 전환 기술을 적용하고, 상기 생물 전환 기술을 통해 얻어진 발효물로부터 천마생물전환 추출물을 추출한 후 농축하여 부원료를 배합하는 과정을 통해 제조된 천마 조성물을 제공함으로써, 생물 전환 기술을 이용하여 천마 특유의 쓴 맛을 감소시켜 맛과 향이 개선된 천마 조성물을 제공할 수 있으며, 이를 통해 천마 섭취에 대한 사용자의 거부감을 해소함과 아울러 천마 조성물의 용이한 섭취에 따라 천마가 가진 성분의 신체 흡수율을 증진시켜 혈액순환의 개선과 동맥경화, 고지혈증 등의 예방과 같은 효과를 제공할 수 있다.Through the above-described configuration, the present invention applies the bioconversion technology of inoculating and fermenting the Chunma powder obtained by crushing the Chunma, extracting the Chunma bioconversion extract from the fermented product obtained through the bioconversion technology, and then concentrating the auxiliary material By providing the Chunma composition prepared through the process of mixing It can provide effects such as improvement of blood circulation and prevention of arteriosclerosis, hyperlipidemia, etc. by improving the body absorption rate of the components possessed by Chunma according to the easy intake of Chunma composition.
상술한 바와 같은 본 발명의 실시예에 따른 천마 조성물의 제조 방법을 통해 얻어진 천마 조성물에 대한 효과를 증명하기 위한 천마 조성물의 제조 과정에서 얻어지는 시료 또는 천마 조성물의 제조에 사용되는 시료를 이용하여 다양한 실험을 진행하였으며, 이를 이하에서 도면을 참고하여 상세히 설명한다.Various experiments using a sample obtained in the manufacturing process of the cheonma composition or a sample used for the preparation of the cheonma composition to prove the effect on the cheonma composition obtained through the manufacturing method of the cheonma composition according to the embodiment of the present invention as described above was carried out, and this will be described in detail with reference to the drawings below.
도 2 내지 도 7은 본 발명의 실시예에 따른 천마 조성물의 제조 방법 중 천마 당화액에 접종되는 혼합균주의 우수균주 분석 결과를 나타낸 도면이다.2 to 7 are diagrams showing the results of analysis of superior strains of the mixed strain inoculated into the Chunma saccharification solution in the method of manufacturing the Chunma composition according to an embodiment of the present invention.
우선, 천마 생물전환 추출물의 제조를 위한 최적 조건(균주, 온도, 기간 등) 확립을 위한 실험을 진행하여, 먼저 우수균주 확보를 위해 균을 접종하지 않은 천마, 맥아배지에 종균배양한 Lactobacillus brevis(KCTC 3102, 이하, LB와 동일), Lactobacillus platarum(KCTC 21024, 이하, LP와 동일)균주, 혼합균주(LB+LP)로 균 접종량 2.5%. 온도 35℃, 0 ~ 5일 발효로 동일조건하에 실험을 진행하였다.First, an experiment was conducted to establish optimal conditions (strain, temperature, period, etc.) for the production of Chunma bioconversion extract, and Lactobacillus brevis ( KCTC 3102, hereinafter, same as LB), Lactobacillus platarum (KCTC 21024, hereinafter, same as LP) strain, mixed strain (LB+LP) with 2.5% inoculation. The experiment was carried out under the same conditions with a temperature of 35 °C and fermentation for 0 to 5 days.
또한, 생물전환기술의 조건확립을 위해, 균을 접종하지 않은 천마에 선정된 혼합균주(LB+LP)를 2.5%, 5% 및 7%를 접종하고, 온도는 30℃, 35℃ 및 40℃로 설정하며, 0, 24, 48, 72, 96, 120 시간동안 조건별 생물전환 시킨 후 이화학적 특성 및 항산화 활성 등을 확인하였다.In addition, in order to establish the conditions for bioconversion technology, 2.5%, 5% and 7% of the selected mixed strain (LB+LP) was inoculated into the uninoculated Cheonma, and the temperature was 30℃, 35℃ and 40℃ After bioconversion for each condition for 0, 24, 48, 72, 96, and 120 hours, physicochemical properties and antioxidant activity were checked.
우선, 도 2는 미생물균주 및 발효시간에 따른 천마 발효물의 pH 및 산도 변화에 관한 도면이다.(천마 : 균을 첨가하지 않은 천마, 천마LB : Lactobacillus brevis균 이용 발효물, 천마LP : Lactobacillus plantarum 이용 발효물, 천마LB+LP : Lactobacillus plantarum 및 Lactobacillus plantarum 혼합균 이용 발효물)First, Figure 2 is a view regarding the change in pH and acidity of the fermented Chunma according to the microbial strain and fermentation time. Fermented product, Chunma LB+LP: Fermented product using Lactobacillus plantarum and Lactobacillus plantarum mixed bacteria)
발효 중 일어나는 가장 큰 변화는 젖산균에 의해 재료 내에 있는 당분이 젖산이나 유기산으로 변화하여 pH가 감소하고 산도가 증가하는 것이며 pH와 산도의 변화는 발효 정도를 짐작 할 수 있는 중요한 지표, 발효 중 pH는 초기에는 큰 차이가 없었으나 발효가 진행됨에 따라 LB:Lactobacillus brevis(KCTC 3102, LB)균, LP:Lactobacillus plantarum(KCTC 21024, LP)균을 혼합사용하여 발효한 천마가 낮아지는 결과를 나타내었다. The biggest change that occurs during fermentation is that the sugar in the material is changed to lactic acid or organic acid by lactic acid bacteria, leading to a decrease in pH and an increase in acidity. There was no significant difference at the beginning, but as the fermentation progressed, the fermented Chunma was lowered by using a mixture of LB: Lactobacillus brevis (KCTC 3102, LB) and LP: Lactobacillus plantarum (KCTC 21024, LP) bacteria.
발효가 진행됨에 따라 LB:Lactobacillus brevis(KCTC 3102, LB)균, LP:Lactobacillus plantarum(KCTC 21024, LP)균을 혼합사용하여 발효한 천마의 산도값이 높아지는 결과를 나타내었다. 실험 결과 pH가 낮아짐에 따라 산도가 증가하는 경향을 나타내는데 이는 숙성이 진행되면서 미생물에 의해 생성된 젖산 등의 다양한 유기산 생성에 의해 기인한 것으로 판단되어진다.As the fermentation progressed, the acidity value of Chunma fermented using a mixture of LB: Lactobacillus brevis (KCTC 3102, LB) and LP: Lactobacillus plantarum (KCTC 21024, LP) bacteria was increased. As a result of the experiment, the acidity tends to increase as the pH decreases, which is thought to be due to the production of various organic acids such as lactic acid produced by microorganisms as the aging progresses.
도 3은 미생물균주 및 발효시간에 따른 천마 발효물의 생균수 변화에 대한 도면이다.(천마 : 균을 첨가하지 않은 천마, 천마LB : Lactobacillus brevis균 이용 발효물, 천마LP : Lactobacillus plantarum 이용 발효물, 천마LB+LP : Lactobacillus plantarum 및 Lactobacillus plantarum 혼합균 이용 발효물)3 is a view of the change in the number of viable cells of the fermented Chunma according to the microbial strain and fermentation time. Chunma LB+LP: Fermented product using Lactobacillus plantarum and Lactobacillus plantarum mixed bacteria)
균수의 증가의 변화를 살펴본 결과, 발효시간에 따른 pH와 산도의 변화에 비례하여 생균수가 증가하였다고 보고되어져 있으며, 본 연구에서도 발효시간에 따른 pH와 산도의 변화에 비례하여 pH 감소(도 2) 및 산도가 증가(도 3)하였으며 특히 혼합균(LB+LP)에서 높은 함량을 나타내어 균의 증식이 촉진된 것으로 사료된다.As a result of examining the change in the increase in the number of bacteria, it is reported that the number of viable cells increased in proportion to the change in pH and acidity according to the fermentation time, and in this study, the pH decreased in proportion to the change in pH and acidity according to the fermentation time (Fig. 2) and acidity increased (FIG. 3), and in particular, a high content was shown in mixed bacteria (LB+LP), suggesting that the proliferation of bacteria was promoted.
도 4는 미생물균주 및 발효시간에 따른 천마발효물의 총 페놀함량에 관한 도면이다.(W : 균을 첨가하지 않은 천마, LB : Lactobacillus brevis균 이용 발효물, LP : Lactobacillus plantarum 이용 발효물, LB+LP : Lactobacillus plantarum 및 Lactobacillus plantarum 혼합균 이용 발효물)4 is a view of the total phenol content of the fermented Chunma according to the microbial strain and fermentation time. LP: Fermented product using Lactobacillus plantarum and Lactobacillus plantarum mixed bacteria)
발효하여 비교해 본 결과 L. brevis(Lactobacillus brevis)와 L. plantarum(Lactobacillus plantarum)의 혼합균주 사용했을 때 가장 높은 함량을 보였고, 발효가 진행됨에 3일차에 각각 527.49±65.86 mg GAE/100g(천마), 4839.82±69.33 mg GAE/100g(LB), 4963.45±13.48 mg GAE/100g(LP), 4782.51±18.97 mg GAE/100g(LB+LP), 4일차에 4508.65±66.73 mg GAE/100g(천마), 4473.04±59.51 mg GAE/100g(LB), 5169.36±66.45 mg GAE/100g(LP), 5331.05±15.63 mg GAE/100g(LB+LP)으로 총페놀 함량이 높아짐을 나타내었다. As a result of fermentation and comparison, the highest content was shown when a mixed strain of L. brevis (Lactobacillus brevis) and L. plantarum (Lactobacillus plantarum) was used. , 4839.82±69.33 mg GAE/100g(LB), 4963.45±13.48 mg GAE/100g(LP), 4782.51±18.97 mg GAE/100g(LB+LP), 4508.65±66.73 mg GAE/100g(Cheonma) on
도 5는 미생물균주 및 발효시간에 따른 천마발효물의 총 플라보노이드함량을 나타낸 도면이다. (W : 균을 첨가하지 않은 천마, LB : Lactobacillus brevis균 이용 발효물, LP : Lactobacillus plantarum 이용 발효물, LB+LP : Lactobacillus plantarum 및 Lactobacillus plantarum 혼합균 이용 발효물)5 is a view showing the total flavonoid content of the fermented Chunma according to the microbial strain and fermentation time. (W: Cheonma without added bacteria, LB: Fermented product using Lactobacillus brevis bacteria, LP: Fermented product using Lactobacillus plantarum, LB+LP: Fermented product using Lactobacillus plantarum and Lactobacillus plantarum mixed bacteria)
발효하여 비교해 본 결과 L. brevis와 L. plantarum의 혼합균주를 사용했을 때 가장 높은 함량을 보였고 0h-21.46±1.48 mg CE/100g, 48h-37.76±2.94 mg CE/100g으로 120h 까지 발효가 진행됨에 따라 총플라보노이드 함량이 높아짐을 나타내었다.As a result of fermentation and comparison, the highest content was shown when a mixed strain of L. brevis and L. plantarum was used, and the fermentation proceeded up to 120h at 0h-21.46±1.48 mg CE/100g, 48h-37.76±2.94 mg CE/100g. It was shown that the total flavonoid content was increased accordingly.
도 6은 미생물균주 및 발효시간에 따른 천마발효물의 항산화활성(DPPH, IC50(㎍/mL))을 나타낸 도면이다.Figure 6 is a view showing the antioxidant activity (DPPH, IC 50 (㎍ / mL)) of the fermented Chunma according to the microbial strain and fermentation time.
대조군인 ascorbic acid의 IC50은 11.46±0.76 ㎍/mL이었으며 DPPH radical 소거능의 IC50값을 비교해 본 결과 0시간에서 214.77±1.86㎍/mL (천마), 209.11±2.05 ㎍/mL (LB), 200.46±2.04㎍/mL (LP), 199.46±1.94 ㎍/mL (LB+LP)으로 나타났으며 발효가 진행됨에 따라 IC50값이 점점 낮아져서 3일에는 104.45±0.62 ㎍/mL(천마), 108.02±2.04 ㎍/mL(LB), 109.71±2.77 ㎍/mL(LP), 102.01±1.74 ㎍/mL(LB+LP)이고 4일에는 90.46±1.96 ㎍/mL(천마), 97.74±1.83 ㎍/mL(LB), 108.07±2.04 ㎍/mL(LP), 103.79±2.13 ㎍/mL(LB+LP)로 IC50값이 낮아져 DPPH radical 소거활성이 가장 좋았으며 이후 IC50값이 증가하면서 120시간에서 120.91±1.83 ㎍/mL(천마), 128.13±2.70 ㎍/mL(LB), 140.17±0.91 ㎍/mL(LP), 137.43±0.86 ㎍/mL(LB+LP)로 나타났다. 미생물 균주별로 발효하여 비교해 본 결과 L. brevis와 L. plantarum의 혼합균주를 사용하여 72h까지 발효가 진행됨에 따라 0h-199.46±1.94 ㎍/mL, 48h-159.76±1.62 ㎍/mL으로 IC50값이 가장 낮았으며 DPPH radical 소거활성이 가장 좋았다.The IC 50 of ascorbic acid, a control group, was 11.46±0.76 μg/mL. As a result of comparing the IC 50 values of DPPH radical scavenging activity, at 0 hours, 214.77±1.86 μg/mL (Chunma), 209.11±2.05 μg/mL (LB), 200.46 It was found to be ±2.04㎍/mL (LP), 199.46± 1.94㎍ /mL (LB+LP), and as the fermentation progressed, the IC50 value gradually decreased. 2.04 μg/mL (LB), 109.71±2.77 μg/mL (LP), 102.01±1.74 μg/mL (LB+LP), and on
도 7은 미생물균주 및 발효시간에 따른 천마발효물의 항산화활성(ABTS, IC50(㎍/mL))를 나타낸 도면이다.7 is a view showing the antioxidant activity (ABTS, IC 50 (㎍ / mL)) of the fermented Chunma according to the microbial strain and fermentation time.
대조군인 Ascorbic acid의 IC50은 10.80±1.24 ㎍/mL이었으며, ABTS radical 소거능의 IC50값을 비교해 본 결과 0시간에서 182.14±1.82 ㎍/mL (천마), 184.17±2.64 ㎍/mL (LB), 186.45±2.83 ㎍/mL (LP), 180.46±2.08 ㎍/mL (LB+LP)으로 나타났으며 발효가 진행됨에 따라 IC50값이 점점 낮아져서 3일에는 100.45±1.03 ㎍/mL(천마), 93.78±0.36 ㎍/mL(LB), 92.41±0.27 ㎍/mL(LP), 88.76±0.75 ㎍/mL(LB+LP)이고, 4일에는 90.78±1.79 ㎍/mL(천마), 80.17±078 ㎍/mL(LB), 79.46±3.71 ㎍/mL(LP), 78.46±1.46 ㎍/mL(LB+LP)로 IC50값이 낮아져 DPPH radical 소거활성이 가장 좋았으며, 이후 IC50값이 증가하면서 120시간에서 132.45±0.90 ㎍/mL(천마), 129.46±0.69 ㎍/mL(LB), 130.78±0.34 ㎍/mL(LP), 111.76±1.11 ㎍/mL(LB+LP)으로 나타났다. 미생물 균주별로 발효하여 비교해 본 결과 L. brevis와 L. plantarum의 혼합균주 사용이 0h-180.46±2.08 ㎍/mL, 48h-161.78±124 ㎍/mL으로 IC50값이 가장 낮았으며 DPPH radical 소거활성이 가장 좋았다.Ascorbic acid, a control group, had an IC 50 of 10.80±1.24 μg/mL, and as a result of comparing the IC 50 values of ABTS radical scavenging activity, 182.14±1.82 μg/mL (Chunma), 184.17±2.64 μg/mL (LB), and 184.17±2.64 μg/mL (LB), It was found to be 186.45±2.83 μg/mL (LP), 180.46±2.08 μg/mL (LB+LP), and as the fermentation progressed, the IC 50 value gradually decreased. ±0.36 μg/mL (LB), 92.41±0.27 μg/mL (LP), 88.76±0.75 μg/mL (LB+LP), and on
상술한 바와 같이, 우수균주 확보를 위해 균을 접종하지 않은 천마, 맥아배지에 종균배양한 Lactobacillus brevis(KCTC 3102, LB), Lactobacillus platarum(KCTC 21024, LP)균주, 혼합균주(LB+LP)로 균 접종량 2.5%. 온도 35℃, 0 ~ 5일 발효로 동일조건하에 실험을 진행하여 천마의 이화학적 특성 및 생리활성 측정 결과 단일 균을 사용 하는 것 보다는 복합균주(혼합균주)를 사용한 것이 효과가 전반적 우수하게 나타나는 것을 확인 할 수 있었다.As described above, in order to secure excellent strains, Lactobacillus brevis (KCTC 3102, LB), Lactobacillus platarum (KCTC 21024, LP) strains, mixed strains (LB + LP), which were seed cultured on uninoculated Chunma, malt medium 2.5% fungal inoculum. Experiments were carried out under the same conditions with a temperature of 35℃ and fermentation for 0 to 5 days, and as a result of measuring the physicochemical properties and physiological activity of Chunma, it was confirmed that the effect of using a complex strain (mixed strain) rather than using a single strain was generally excellent. could check
도 8은 미생물접종량과 온도에 따른 천마발효물의 pH 변화를 나타낸 도면이고, 도 9는 미생물접종량과 온도에 따른 천마발효물의 산도 변화를 나타낸 도면이고, 도 10은 미생물접종량과 온도에 따른 천마발효물의 생균수 변화를 나타낸 도면이다. (1)LB+LP:Lactobacillus brevis 및 Lactobacillus plantarum의 혼합균 이용 발효물, 2)Control:균 첨가 하지않은 천마발효물, 3)2.5% : 혼합균2.5%를 첨가한 천마발효물, 4)5% : 혼합균5%를 첨가한 천마발효물, 5)7% : 혼합균7%를 첨가한 천마발효물)8 is a view showing the change in pH of the fermented Chunma according to the microbial inoculation amount and temperature, FIG. 9 is a view showing the change in the acidity of the fermented Chunma according to the microbial inoculation amount and temperature, and FIG. It is a diagram showing the change in the number of viable cells. ( 1) LB+LP: Fermented product using a mixture of Lactobacillus brevis and Lactobacillus plantarum, 2) Control: Fermented Chunma without adding bacteria, 3) 2.5%: Fermented product with 2.5% of mixed bacteria, 4) 5 %: Chunma fermented product with 5% mixed bacteria added, 5) 7%: Chunma fermented product with 7% mixed bacteria added)
도 8에 나타난 바와 같이, 균을 첨가하지 않은 천마와 선정된 혼합균주(LB+LP)를 2.5%, 5% 및 7% 접종한 천마를 각각 30℃, 35℃ 및 40℃에서 0, 24, 48, 72, 96, 120시간동안 발효하여 측정한 결과 유의적인 차이를 보인 pH는 0, 72, 120h이었으며 각 실험군 사이의 pH에서는 큰 차이는 없었으나 35℃에서 발효한 천마발효물이 그 중 가장 pH의 변화가 크며 낮았고, 발효가 진행됨에 따라 공통적으로 3일째에서 가장 낮은 pH를 나타냈고, 그 후 서서히 올라 5일째에는 높은 값을 나타냈다.As shown in FIG. 8, Chunma inoculated with 2.5%, 5% and 7% of the selected mixed strain (LB+LP) with no bacteria added was inoculated at 30℃, 35℃ and 40℃ respectively at 0, 24, As a result of fermenting for 48, 72, 96, and 120 hours, the pHs showing significant differences were 0, 72, and 120 h. There was no significant difference in pH between each experimental group, but the fermented Chunma fermented at 35℃ was the most The change in pH was large and low, and as the fermentation progressed, the lowest pH was commonly shown on the 3rd day, and then gradually increased to a high value on the 5th day.
도 9에 나타난 바와 같이, 균을 첨가하지 않은 천마와 선정된 혼합균주(LB+LP)를 2.5%, 5% 및 7% 접종한 천마를 각각 30℃, 35℃ 및 40℃에서 0, 24, 48, 72, 96, 120시간동안 발효하여 측정한 결과 유의적인 차이를 보인 산도는 0, 72, 120h이었으며 각 실험 군 사이의 산도에서는 큰 차이는 없었으나 35℃에서 발효한 천마발효물이 그 중 가장 산도의 변화가 크며 높았고 발효가 진행됨에 따라 공통적으로 3일째에서 가장 높은 산도를 나타냈고 그 후 서서히 낮아져 5일째에는 낮은 값을 나타냈다. 35℃ 천마발효물의 0일에서 각각 0.72±0.83(천마), 0.70±1.14(2.5%), 0.74±0.79(5%) 및 0.74±0.79(7%)였으며 3일에서 각각 1.06±1.92(천마), 1.44±1.68(2.5%), 1.4±1.32(5%) 및 1.32±0.89(7%)으로 낮아졌으며, 그 후 5일째까지 서서히 올라 0.68±1.22(천마), 0.99±0.44(2.5%), 0.86±0.65(5%) 및 0.82±0.52(7%)을 나타내었다.As shown in FIG. 9, Chunma inoculated with 2.5%, 5% and 7% of the selected mixed strain (LB+LP) with no bacteria added was inoculated at 30℃, 35℃ and 40℃ respectively at 0, 24, As a result of fermenting for 48, 72, 96, and 120 hours, the significant differences in acidity were 0, 72, and 120 h. There was no significant difference in acidity between each experimental group. The change in acidity was the largest and highest, and as fermentation progressed, it showed the highest acidity in common on the 3rd day, and then gradually decreased to show a low value on the 5th day. On
도 10에 나타난 바와 같이, 발효시간에 따른 pH와 산도의 변화에 비례하여 생균수가 증가하였다고 보고되어져 있으며, 본 연구에서도 발효시간에 따른 pH와 산도의 변화에 비례하여 pH 감소 및 산도가 증가하였으며 특히 35℃에서 2.5% 접종한 발효물에서 높은 함량을 나타내어 균의 증식이 촉진된 것으로 사료된다.As shown in FIG. 10 , it is reported that the number of viable cells increased in proportion to the change in pH and acidity according to the fermentation time, and in this study, the pH decreased and the acidity increased in proportion to the change in pH and acidity according to the fermentation time. It is considered that the growth of bacteria was promoted by showing a high content in the fermented product inoculated with 2.5% at 35°C.
또한, 도 11은 미생물접종량과 온도에 따른 천마발효물의 총 페놀함량을 나타낸 도면이고, 도 12는 미생물접종량과 온도에 따른 천마발효물의 총 플라보노이드함량을 나타낸 도면이며, 도 13은 미생물균주 및 발효시간에 따른 천마발효물의 항산화활성((DPPH radical scavening activity)-IC50(㎍/mL))을 나타낸 도면이고, 도 14는 미생물균주 및 발효시간에 따른 천마발효물의 항산화활성((ABTS radical scavening activity)-IC50(㎍/mL))을 나타낸 도면이다. (Control:균 첨가 하지않은 천마발효물, 2.5% : 혼합균2.5%를 첨가한 천마발효물, 5% : 혼합균5%를 첨가한 천마발효물, 7% : 혼합균7%를 첨가한 천마발효물)In addition, Figure 11 is a view showing the total phenol content of the fermented Chunma according to the microbial inoculation amount and temperature, Figure 12 is a view showing the total flavonoid content of the fermented Chunma according to the microbial inoculum and temperature, Figure 13 is a microbial strain and fermentation time It is a view showing the antioxidant activity ((DPPH radical scavening activity)-IC 50 (㎍ / mL)) of the fermented cheonma according to, Figure 14 is the antioxidant activity of the fermented cheonma according to the microbial strain and fermentation time ((ABTS radical scavening activity) -IC 50 (μg/mL)) is shown. (Control: Chunma fermented product without added bacteria, 2.5%: Chunma fermented product with 2.5% mixed bacteria added, 5%: Chunma fermented product with 5% mixed bacteria added, 7%: Chunma with 7% mixed bacteria added fermented product)
도 11에 나타난 바와 같이, 발효하여 비교해 본 결과 온도 35℃에서 2.5% 혼합균주를 첨가한 천마발효물에서 가장 높은 함량을 나타내었으며 0h-2894.54±1.6 mg GAE/100g(2.5%)에서 72h-5538.51±1.97 mg GAE/100g(2.5%)을 나타내었다.As shown in FIG. 11, as a result of fermenting and comparing, the fermented Chunma fermented with 2.5% mixed strain at a temperature of 35°C showed the highest content, and at 0h-2894.54±1.6 mg GAE/100g (2.5%), 72h-5538.51 ±1.97 mg GAE/100 g (2.5%).
또한, 도 12에 나타난 바와 같이, 발효하여 비교해 본 결과 온도 35℃에서 2.5% 혼합균주를 첨가한 천마 발효물에서 가장 높은 함량을 나타내었으며 0h-211.98±1.57 mg CE/100g (2.5%)에서 72h-25.11±2.05 mg CE/100g (2.5%)을 나타내었다.In addition, as shown in FIG. 12, as a result of fermenting and comparing, the fermented Chunma fermented with 2.5% mixed strain at a temperature of 35°C showed the highest content, and 0h-211.98±1.57 mg CE/100g (2.5%) for 72h. It showed -25.11±2.05 mg CE/100g (2.5%).
또한, 도 13에 나타난 바와 같이, 발효하여 비교해 본 결과 온도 35℃에서 2.5% 혼합균주를 첨가한 천마 발효물에서 발효가 진행됨에 따라 0h-279.55±0.75 ㎍/mL (2.5%)에서 72h-94.35±1.52 ㎍/mL(2.5%)로 IC50값이 가장 낮았으며 DPPH radical 소거활성이 가장 좋았다.In addition, as shown in FIG. 13, as the fermentation proceeds in the fermented Chunma fermented with 2.5% mixed strain at a temperature of 35°C, as a result of fermentation, 0h-279.55±0.75 μg/mL (2.5%) at 0h-279.55±0.75 μg/mL (2.5%) for 72h-94.35 The IC 50 value was the lowest at ±1.52 ㎍/mL (2.5%) and the DPPH radical scavenging activity was the best.
또한, 도 14에 나타난 바와 같이, 발효하여 비교해 본 결과 온도 35℃에서 2.5% 혼합균주를 첨가한 천마 발효물에서 발효가 진행됨에 따라 0h-183.62±1.44 ㎍/mL (2.5%)에서 72h-175.69±0.64㎍/mL(2.5%)로 IC50값이 가장 낮았으며 DPPH radical 소거활성이 가장 좋았다.In addition, as shown in FIG. 14, as the fermentation progressed in the fermented Chunma fermented with 2.5% mixed strain at a temperature of 35 ° C. The IC 50 value was the lowest at ±0.64㎍/mL (2.5%) and the DPPH radical scavenging activity was the best.
상술한 바와 같이, 생물전환기술 조건확립을 위해 균을 접종하지 않은 천마, 선정된 혼합균주(LB+LP)를 2.5%, 5% 및 7%를 접종하고, 온도는 30℃, 35℃ 및 40℃로 설정하고, 0, 24, 48, 72, 96, 120시간 동안 조건별 생물전환 시킨 후 이화학적 특성 및 항산화 활성 등을 확인하였다. pH는 35℃에서 3일째 발효한 천마발효물이 가장 pH의 변화가 크며 낮은 값을 나타내었고 산도는 35℃에서 3일째 발효한 천마발효물이 0일-0.70±1.14(2.5%), 3일-1.44±1.68(2.5%)로 그 중 가장 산도의 변화가 크며 높았다. 생균수는 발효시간에 따른 pH와 산도의 변화에 비례하여 35℃에서 2.5% 접종한 발효물에서 높은 함량을 나타내어 균의 증식이 촉진된 것으로 사료된다. 총 페놀, 플라보노이드 함량은 L. brevis와 L. plantarum의 혼합균주를 2.5% 사용하여 35℃에서 3일 발효한 천마발효물에서 높은 함량을 나타내었고 항산화활성에서도 총 페놀, 플라보노이드 함량과 비례적인 관계를 나타내었다. 측정 결과 단일 균 2.5%를 첨가한 발효물을 35℃에서 3일동안 발효한 것이 효과가 전반적 우수하게 나타나는 것을 확인 할 수 있었다.As described above, 2.5%, 5%, and 7% of the selected mixed strain (LB+LP) was inoculated, and the temperature was 30℃, 35℃ and 40 It was set at ℃, and after bioconversion for each condition for 0, 24, 48, 72, 96, and 120 hours, physicochemical properties and antioxidant activity were checked. As for the pH, the fermented Chunma fermented on the 3rd day at 35℃ showed the greatest change in pH and the lowest value, and the acidity of the fermented cheonma fermented at 35℃ on the 3rd day was 0 days - 0.70±1.14 (2.5%), 3 days. It was -1.44±1.68 (2.5%), and among them, the change in acidity was the largest and highest. The number of viable cells was high in the fermented product inoculated with 2.5% at 35°C in proportion to the change in pH and acidity according to the fermentation time, so it is considered that the proliferation of bacteria was promoted. The total phenol and flavonoid contents showed a high content in the fermented Chunma fermented for 3 days at 35°C using 2.5% of a mixed strain of L. brevis and L. plantarum, and the antioxidant activity showed a proportional relationship with the total phenol and flavonoid contents. indicated. As a result of the measurement, it was confirmed that the overall effect was excellent when the fermented product added with 2.5% of a single bacteria was fermented at 35°C for 3 days.
한편, 천마 발효 조건의 확립을 위한 실험 분석 결과를 이하 도면을 참고하여 설명한다.On the other hand, the results of the experimental analysis for the establishment of the fermentation conditions of Cheonma will be described with reference to the drawings below.
우선, 증숙 건조하여 분말화 한 천마 200kg에 20배수의 물을 넣어 85℃에서 4시간동안 당화한 후 식혀 천마 당화액을 제조하고, 맥아배지에 종균 배양한 Lactobacillus brevis(KCTC 3102, LB)균주와 Lactobacillus platarum(KCTC 21024, LP)균주를 혼합한 혼합균주인 (LB+LP)균을 상기 천마 당화액의 5중량% 준비한 후 상기 천마 당화액에 첨가하여 교반한 뒤 발효탱크에서 35℃에서 3일간 발효하여 천마 발효물(또는 생물전환 천마 발효물)을 시료로서 제조한다.First, steam-dried and powdered Chunma 200kg was put in 20-fold water, saccharified at 85℃ for 4 hours, cooled to prepare Chunma saccharified solution, and Lactobacillus brevis (KCTC 3102, LB) strain cultured in malt medium and After preparing 5% by weight of the Lactobacillus platarum (KCTC 21024, LP) strain mixed with the strain Lactobacillus platarum (KCTC 21024, LP), 5% by weight of the Chunma saccharified solution was added and stirred, and then stirred in a fermentation tank at 35℃ for 3 days. Fermentation to prepare a fermented cheonma (or a fermented fermented fermented fermented horsetail) as a sample.
상기 천마 발효물의 pH 측정을 위해, 시료로부터 원심분리기(CF-10, DAIHAN Scientific Co., Ltd. Korea)를 이용하여 얻은 상등액을 pH meter (model 3510, Jenway, UK)를 사용하여 측정하였다. 각 실험은 3회 반복하여 평균값으로 나타내었다. For measuring the pH of the fermented Chunma, the supernatant obtained from the sample using a centrifuge (CF-10, DAIHAN Scientific Co., Ltd. Korea) was measured using a pH meter (model 3510, Jenway, UK). Each experiment was repeated three times and expressed as an average value.
또한, 상기 천마 발효물의 총산 측정을 위해, 총산은 시료를 원심분리 후 얻은 상등액 1 mL를 0.1N-NaOH 용액으로 pH 8.2±2까지 중화시키는데 소요된 0.1N-NaOH의 소비 mL 수를 구하고 아래 식에서와 같이 젖산(lactic acid)양으로 총산을 환산하였다. 각 실험은 3회 반복하여 평균값으로 나타내었다.In addition, for the total acid measurement of the Chunma fermented product, the number of mL consumption of 0.1N-NaOH required to neutralize 1 mL of the supernatant obtained after centrifugation of the total acid silver sample to pH 8.2±2 with a 0.1N-NaOH solution is obtained from the formula below Total acid was converted to the amount of lactic acid as shown. Each experiment was repeated three times and expressed as an average value.
젖산 % =0.9×0.1N-NaOH 소비량(mL)/시료 채취량(1 mL)Lactic acid % =0.9×0.1N-NaOH consumption (mL)/sample amount (1 mL)
또한, 상기 천마 발효물의 생균수 측정을 위해, 생균수 측정은 각각의 시료를 생리식염수에 단계별로 희석하여 MRSA 배지에 도말하고 30℃에서 48시간 배양 후 나타난 젖산균 특유의 colony를 측정하여 생균수(log cfu/mL)로 나타내었다. 각 실험은 3회 반복하여 수행하였으며 평균값으로 나타내었다.In addition, in order to measure the number of viable cells in the fermented Chunma, the number of viable cells is measured by diluting each sample step-by-step in physiological saline, spreading it on MRSA medium, and culturing at 30° C. for 48 hours. log cfu/mL). Each experiment was repeated three times, and the average value was expressed.
도 15는 생물전환 천마발효물의 pH, 산도 및 생균수 변화를 나타낸 도면이다.(LB+LP:Lactobacillus brevis 및 Lactobacillus plantarum 5% 혼합균 이용 발효물)15 is a view showing changes in pH, acidity and number of viable cells of the bioconversion fermented Chunma. (LB + LP: Lactobacillus brevis and
앞 실험 결과에 따라 혼합균주 5%를 첨가하여 35℃에서 72h 발효한 천마를 생물전환 최적조건으로 확립하였으며. 천마(LB+LP-5%)를 첨가하여 35℃에서 0, 24, 48, 72, 96, 120시간 동안 발효하여 측정한 결과 pH는 각각 4.86±0.24(0h), 4.54±0.82(24h), 4.00±0.16 48h), 3.94±049(72h),4.06±0.64(96h) 및 4.09±0.27 (120h)으로 72시까지 서서히 감소하여 발효 중 가장 낮은 값을 나타내었고 그 이후 발효 120시간까지 점차 높아짐을 나타내었다. 도는 각각 0.92±0.15(0h), 1.36±0.43(24h), 1.5±0.43(48h), 1.46±0.58(72h), 1.43±0.85(96h) 및 1.38±0.54(120h)으로 72시까지 서서히 높아져 발효 중 가장 높은 값을 나타내었고 그 이후 발효 120시간까지 점차 낮아짐을 나타내었다. 생균 수는 각각 8.86±0.55 log cfu/g(0h), 11.26±0.8 log cfu/g(24h), 12.11±0.37 log cfu/g(48h), 12.30±0.91 log cfu/g(72h), 11.02±0.11 log cfu/g(96h) 및 10.64±1.00 log cfu/g(120h)으로 발효가 진행되면서 발효 3일째인 72시간에 12.30±0.91 log cfu/g(72h)로 최대균수를 나타낸 후 10.64 log cfu/g(120h)으로 감소하였다.According to the results of the previous experiment, Chunma, which was fermented for 72 h at 35°C with the addition of 5% of the mixed strain, was established as the optimal condition for biotransformation. Chunma (LB+LP-5%) was added and fermented at 35℃ for 0, 24, 48, 72, 96, and 120 hours. As a result, the pH was 4.86±0.24(0h), 4.54±0.82(24h), respectively. 4.00±0.16 48h), 3.94±049(72h), 4.06±0.64(96h), and 4.09±0.27(120h) until 72 hours, showing the lowest value during fermentation, and then gradually increasing until 120 hours of fermentation. indicated. Degrees are 0.92±0.15(0h), 1.36±0.43(24h), 1.5±0.43(48h), 1.46±0.58(72h), 1.43±0.85(96h), and 1.38±0.54(120h), respectively, which gradually rise until 72 hours and ferment. It showed the highest value among them, and thereafter, it gradually decreased until 120 hours of fermentation. The number of viable cells was 8.86±0.55 log cfu/g (0h), 11.26±0.8 log cfu/g (24h), 12.11±0.37 log cfu/g (48h), 12.30±0.91 log cfu/g (72h), 11.02±, respectively. As fermentation proceeded at 0.11 log cfu/g (96h) and 10.64±1.00 log cfu/g (120h), the maximum number of bacteria was exhibited at 12.30±0.91 log cfu/g (72h) at 72 hours, the third day of fermentation, followed by 10.64 log cfu /g (120 h).
상기 천마 발효물의 총페놀 함량 측정을 위해, 추출 및 발효물 10 ㎕에 2% Na2CO3 용액 200 ㎕ 첨가하여 3분간 정치시켰다. 그 다음 2N Folin-Ciocalteu phenol 시약 10 ㎕ 첨가 및 혼합한 후 30℃ incubator에서 27분 동안 발색시켰다. 발색된 시료는 microplate reader (SpectraMax M5, Molecular Devices, CA, USA)를 사용하여 750 nm에서 흡광도를 측정하였다. 총 페놀 함량은 gallic acid를 이용하여 작성한 표준 검량곡선에 의해 값을 산출하였다.To measure the total phenol content of the fermented Chunma, 200 μl of a 2% Na 2 CO 3 solution was added to 10 μl of extraction and fermentation, and allowed to stand for 3 minutes. Then, 10 μl of 2N Folin-Ciocalteu phenol reagent was added and mixed, and the color was developed in an incubator at 30° C. for 27 minutes. The absorbance of the colored sample was measured at 750 nm using a microplate reader (SpectraMax M5, Molecular Devices, CA, USA). The total phenol content was calculated using a standard calibration curve prepared using gallic acid.
도 16은 생물전환 천마발효물의 총 페놀함량을 나타낸 도면으로서, 각각 3558.53±45.8 mg GAE/100g(0h), 4527.55±98.95 mg GAE/100g(24h), 5544.59±9.71 mg GAE/100g(48h), 5161.34±75.46 mg GAE/100g(72h), 4257.41±49.64 mg GAE/100g(96h) 및 3140.92±89.32 mg GAE/100g(120h)으로 발효가 진행되면서 발효 3일째인 72시간까지 활성이 점차 높아져 5161.34±75.46 mg GAE/100g(72h)로 가장 높은 활성을 나타내었으며, 점차 낮아서 발효 120시간에서 3140.92±89.32 mg GAE/100g(120h)으로 나타났다.Figure 16 is a view showing the total phenol content of the bioconversion Chunma fermented product, 3558.53±45.8 mg GAE/100g (0h), 4527.55±98.95 mg GAE/100g (24h), 5544.59±9.71 mg GAE/100g (48h), respectively; As fermentation proceeded with 5161.34±75.46 mg GAE/100g (72h), 4257.41±49.64 mg GAE/100g (96h) and 3140.92±89.32 mg GAE/100g (120h), the activity gradually increased until 72 hours, the 3rd day of fermentation, and 5161.34± It showed the highest activity at 75.46 mg GAE/100g (72h), and gradually decreased to 3140.92±89.32 mg GAE/100g (120h) at 120 hours of fermentation.
상기 천마 발효물의 총플라보노이드 함량 측정을 위해, 추출물 및 발효물 10 ㎕, 증류수 117 ㎕, 5% NaNO2 27.5 ㎕를 37℃에서 5분간 반응시킨 다음 10% Al3·6H2O 15㎕를 첨가하여 37℃에서 6분간 반응하였다. 그 후 1N NaOH 50㎕를 첨가하여 37℃에서 11분간 반응정지 시켰다. 반응정지된 시료를 microplate reader를 사용하여 510nm에서 흡광도를 측정하였다. 총플라보노이드 함량은 카테킨(catechins)을 이용하여 작성한 표준검량곡선에 의해 값을 산출하였다.To measure the total flavonoid content of the Chunma fermented product, 10 μl of extract and fermented product, 117 μl of distilled water, and 27.5 μl of 5% NaNO 2 were reacted at 37° C. for 5 minutes, and then 15 μl of 10% Al 3 6H 2 O was added. The reaction was carried out at 37°C for 6 minutes. After that, 50 μl of 1N NaOH was added to stop the reaction at 37° C. for 11 minutes. The absorbance was measured at 510 nm using a microplate reader for the stopped reaction sample. The total flavonoid content was calculated using a standard calibration curve prepared using catechins.
도 17은 생물전환 천마발효물의 총 플라보노이드함량을 나타낸 도면으로서, 각각 16.27±0.69 mg CE/100g(0h), 15.29±0.71 mg CE/100g(24h), 18.35±0.24 mg CE/100g(48h), 18.22±0.99 mg CE/100g(72h), 1698±0.40 mg CE/100g(96h) 및 14.50±1.15 mg CE/100g(120h)으로 발효가 진행되면서 발효 2일째와 발효 3일째가 유의적인 차이는 없었으나 발효 3일째인 72시간까지 활성이 점차 높아져 18.22±0.99 mg CE/100g(72h)으로 가장 높은 활성을 나타내었으며, 점차 낮아서 발효 120시간에서 14.50±1.15 mg CE/100g(120h)으로 나타났다.17 is a view showing the total flavonoid content of the bioconverted Chunma fermented product, respectively, 16.27±0.69 mg CE/100g (0h), 15.29±0.71 mg CE/100g (24h), 18.35±0.24 mg CE/100g (48h), As fermentation proceeded at 18.22±0.99 mg CE/100g (72h), 1698±0.40 mg CE/100g (96h), and 14.50±1.15 mg CE/100g (120h), there was no significant difference between the 2nd day and the 3rd day of fermentation. However, the activity gradually increased until 72 hours, the 3rd day of fermentation, showing the highest activity at 18.22±0.99 mg CE/100g (72h), and gradually decreased to 14.50±1.15 mg CE/100g (120h) at 120 hours of fermentation.
상기 천마 발효물의 DPPH 소거활성 측정을 위해, 추출물 및 발효물 시료를 96-well plate에 10 ㎕ 와 사용 직전 만든 0.2 mM DPPH용액 190 ㎕를 섞은 다음, 실온의 암실에서 30분간 반응시켜 microplate reader (SpectraMax M5, Molecular Devices, CA, USA)를 사용하여 520 nm에서 흡광도를 측정하였다. 양성대조군으로는 Ascorbic acid를 사용하여 비교하였으며 DPPH radical 소거활성은 [1-(시료첨가구의 흡광도 / 음성대조구의 흡광도)]×100으로 계산하여 백분율로 나타내었다. To measure the DPPH scavenging activity of the fermented Chunma, 10 μl of the extract and the fermented product sample were mixed with 190 μl of the 0.2 mM DPPH solution prepared just before use in a 96-well plate, and then reacted in the dark at room temperature for 30 minutes with a microplate reader (SpectraMax M5, Molecular Devices, CA, USA) was used to measure the absorbance at 520 nm. Ascorbic acid was used as a positive control for comparison, and the DPPH radical scavenging activity was calculated as [1-(absorbance of sample added / absorbance of negative control)]×100 and expressed as a percentage.
도 18은 생물전환 천마발효물의 항산화활성((DPPH radical scavening activity)-IC50(㎍/mL))을 나타낸 도면이다.(LB+LP:Lactobacillus brevis 및 Lactobacillus plantarum 5%혼합균 이용 발효물)18 is a view showing the antioxidant activity ((DPPH radical scavening activity)-IC 50 (㎍ / mL)) of the bioconverted fermented horseradish. (LB + LP: Lactobacillus brevis and
대조군인 Ascorbic acid의 IC50은 11.46±0.76 ㎍/mL이었으며 DPPH radical 소거능의 IC50값을 비교해 본 결과 각각 252.91±2.49 ㎍/mL (0h), 189.80±2.40 ㎍/mL(24h), 186.93±4.08 ㎍/mL(48h), 163.20±4.82 ㎍/mL(72h), 237.22±8.31 ㎍/mL(96h) 및 229.45±7.80 ㎍/mL(120h)으로 3일 째에서 IC50값이 가장 낮아 DPPH radical 소거활성이 가장 좋았으며, 발효가 진행됨에 따라 IC50값이 증가하면서 120시간에서 229.45±7.80 ㎍/mL(120h)로 나타났다. 발효 3일째가 163.20±4.82 ㎍/mL(72h)로 IC50값이 가장 낮았으며 DPPH radical 소거활성이 가장 좋았다.Ascorbic acid, a control group, had an IC 50 of 11.46±0.76 μg/mL, and as a result of comparing the IC 50 values of DPPH radical scavenging activity, 252.91±2.49 μg/mL (0h), 189.80±2.40 μg/mL (24h), and 186.93±4.08, respectively. At
상기 천마 발효물의 ABTS 소거활성 측정을 위해, 7 mM ABTS 용액에 2.45 mM의 potassium persulfate를 1:1로 혼합한 다음 실온의 암실에서 24시간 방치시킨 후 ABTS radical (ABTS+·)을 만들고 732 nm에서 흡광도 값이 0.7±0.02가 되도록 PBS(phosphate buffer saline, pH 7.4) buffer로 희석하여 사용하였다. 96-well plate에 ABTS radical 용액 190 ㎕와 추출물 및 발효물 시료 10 ㎕를 첨가하여 1분동안 정치시킨 다음 732 nm에서 흡광도를 측정하였다. 양성대조군으로는 Ascorbic acid를 사용하였으며 ABTS radical 소거활성은 [1-(시료첨가구의 흡광도/음성대조구의 흡광도)]×100으로 계산하여 백분율로 표시하였다.To measure the ABTS scavenging activity of the Chunma ferment, 2.45 mM potassium persulfate was mixed 1:1 with 7 mM ABTS solution and left in the dark at room temperature for 24 hours to form ABTS radical (ABTS+·) and absorbance at 732 nm It was diluted with PBS (phosphate buffer saline, pH 7.4) buffer so that the value was 0.7±0.02. 190 μl of ABTS radical solution and 10 μl of extract and fermented product samples were added to a 96-well plate, left for 1 minute, and absorbance was measured at 732 nm. Ascorbic acid was used as a positive control group, and the ABTS radical scavenging activity was calculated as [1-(absorbance of sample added/absorbance of negative control)]×100 and expressed as a percentage.
도 19는 생물전환 천마발효물의 항산화활성((ABTS radical scavening activity)-IC50(㎍/mL))을 나타낸 도면이다.19 is a view showing the antioxidant activity ((ABTS radical scavening activity)-IC 50 (㎍ / mL)) of the bioconverted fermented horseradish.
대조군인 Ascorbic acid의 IC50은 10.41±1.20 ㎍/mL이었으며 ABTS radical 소거능의 IC50값을 비교해 본 결과 각각 169.94±6.18 ㎍/mL (0h), 152.10±3.21 ㎍/mL(24h), 133.25±5.32 ㎍/mL(48h), 125.79±4.51 ㎍/mL(72h), 136.26±6.34 ㎍/mL(96h) 및 188.33±2.37 ㎍/mL(120h)으로 3일 째에서 IC50값이 가장 낮아 ABTS radical 소거활성이 가장 좋았으며, 발효가 진행됨에 따라 IC50값이 증가하면서 120시간에서 188.33±2.37 ㎍/mL((120h)로 나타났다. 발효 3일째가 125.79±4.51 ㎍/mL(72h)로 IC50값이 가장 낮았으며 ABTS radical 소거활성이 가장 좋았다.Ascorbic acid, a control group, had an IC 50 of 10.41±1.20 μg/mL, and as a result of comparing the IC 50 values of ABTS radical scavenging activity, 169.94±6.18 μg/mL (0h), 152.10±3.21 μg/mL (24h), and 133.25±5.32, respectively. ABTS radical scavenging with the lowest IC 50 values at
이하 도면은 상기 천마 발효물에서 추출한 천마생물전환 추출물(이하, 천마 추출물)의 실험 분석 결과를 나타낸 도면이다.The following figure is a view showing the experimental analysis results of the Chunma bioconversion extract (hereinafter, Chunma extract) extracted from the Chunma fermented product.
우선, 상기 천마 추출물의 염증활성 및 성분 분석을 위해, 본 실험에 사용된 마우스의 대식세포주인 RAW264.7 세포는 세포배양을 위해 10% FBS (fetal bovine serum)와 1% P/S (penicilin-streptomycin)가 첨가된 DMEM (Dulbecco's modified eagle medium)배지를 사용하였다. 배양된 RAW264.7 세포가 80% confluent 되었을 때 PBS로 세척한 후 Cell Scraper를 이용해 세포를 탈착시켜 6,000 rpm에서 5분 동안 원심분리한 후 2일 간격으로 계대배양하였다.First, for the analysis of the inflammatory activity and components of the Chunma extract, RAW264.7 cells, a macrophage of the mouse used in this experiment, were prepared with 10% FBS (fetal bovine serum) and 1% P/S (penicilin-) for cell culture. DMEM (Dulbecco's modified eagle medium) medium with streptomycin) was used. When the cultured RAW264.7 cells became 80% confluent, they were washed with PBS, detached using a cell scraper, centrifuged at 6,000 rpm for 5 minutes, and subcultured every 2 days.
또한, 상기 천마 추출물의 Raw 264.7 cell 세포생존율 측정 (MTS assay)을 위해, 천마 추출물에 대한 세포 독성은 5-(3-carboxyme thoxyphenyl)-2H-tetraza lium inner salt(MTS)의 방법을 약간 변형하여 측정하였다. 96-well plate에 RAW 264.7 세포를 1×105 cells/mL가 되도록 분주한 다음 24시간 동안 CO2 incubator (37℃, 5% CO2)에서 예비 배양한 후, 각 시료를 최종 농도(1000 ㎍/mL)로 세포를 처리하여 18시간 동안 배양하였다. 이후 well당 MTS solution 20 ㎕씩 가하고 CO2 incubator에서 4시간 반응시킨 뒤 microplate reader (SpectraMax M5, Molecular Devices, CA, USA)를 이용하여 490 nm에서 흡광도를 측정하였다. In addition, for the Raw 264.7 cell viability measurement (MTS assay) of the Chunma extract, the cytotoxicity to the Chunma extract was slightly modified by the method of 5-(3-carboxyme thoxyphenyl)-2H-tetrazalium inner salt (MTS). measured. Raw 264.7 cells were aliquoted to 1×105 cells/mL in a 96-well plate, and then pre-incubated in a CO 2 incubator (37° C., 5% CO 2 ) for 24 hours. mL) and incubated for 18 hours. After that, 20 μl of MTS solution was added per well and reacted in a CO 2 incubator for 4 hours, and then absorbance was measured at 490 nm using a microplate reader (SpectraMax M5, Molecular Devices, CA, USA).
도 20은 천마 추출물의 날짜별 Raw 264.7 대식세포에서 세포생존율 측정을 나타낸 도면으로서, MTS assay를 이용한 세포 독성 결과 천마 추출물 0~7일 모두에서 90%이상의 생존율을 보였다. 따라서 LPS는 천마 추출물 1000㎍/mL 농도에서 세포에 독성이 나타나지 않음을 확인하였다.20 is a view showing the measurement of cell viability in Raw 264.7 macrophages of the Chunma extract by date. As a result of cytotoxicity using the MTS assay, the Chunma extract showed a survival rate of 90% or more in 0-7 days. Therefore, it was confirmed that LPS did not appear to be toxic to cells at a concentration of 1000 μg/mL of Chunma extract.
또한, 상기 천마 추출물의 산화질소(Nitric oxide) 함량을 측정하기 위해, NO의 농도는 96-well plate에 1×105 cells/mL가 되도록 분주하여 24시간 동안 CO2 incubator(37℃, 5% CO2)에서 배양한 후, 각 시료를 1000 ㎍/mL의 농도로 2시간 전처리한 다음 LPS(Lipopolysaccharide, 400 ng/mL)를 처리하여 18시간 동안 배양하였다. 배양액과 동일한 양의 Griess reagent를 넣은 후 microplate reader (SpectraMax M5, Molecular Devices, CA, USA)를 이용하여 540 nm에서 흡광도를 측정하였다. NO의 농도는 NaNO2의 농도별 표준곡선을 기준으로 계산하였다.In addition, in order to measure the nitric oxide content of the Chunma extract, the concentration of NO was dispensed to 1×105 cells/mL in a 96-well plate, and a CO 2 incubator (37° C., 5% CO 2 ) for 24 hours. ), each sample was pretreated at a concentration of 1000 μg/mL for 2 hours, then treated with LPS (Lipopolysaccharide, 400 ng/mL) and incubated for 18 hours. After adding the same amount of Griess reagent as the culture medium, absorbance was measured at 540 nm using a microplate reader (SpectraMax M5, Molecular Devices, CA, USA). The concentration of NO was calculated based on the standard curve for each concentration of NaNO 2 .
도 21은 천마 추출물의 조건별 Raw 264.7 대식세포에서 LPS에 의한 산화질소 생성 효과를 나타낸 도면으로서, RAW264.7 세포에 1000 ㎍/mL 농도의 추출물을 넣은 후 2시간 후 LPS를 첨가하여 처리하였다. NO assay 결과는 도 21에 나타난 바와 같이 LPS 단독으로 처리된 세포에서는 10.15±0.31 μM 의 NO 생성을 나타내었는데 1000 ㎍/mL 농도에서는 3일과 7일이 비교적 높은 억제율을 보였다. LPS 처리를 하지 않은 세포의 경우 0.65 μM의 NO 분비량을 나타내었으며, 72, 76%의 NO 생성 억제 효과를 나타내었다. RAW 세포에서 LPS로 유도로 증가한 NO생성물이 천마 추출물로 인해 억제되는 것을 확인하였다. 7일이 경과한 천마추출물은 다른 날에 비해 높은 NO를 억제 하였으나 강한 신맛으로 사용이 불가하였고, 3일 처리한 것을 추출물을 사용하였다.21 is a view showing the nitric oxide production effect by LPS in Raw 264.7 macrophages of Chunma extract by condition. After adding the extract at a concentration of 1000 μg/mL to RAW264.7 cells, 2 hours later, LPS was added and treated. As shown in FIG. 21 , the NO assay results showed that 10.15±0.31 μM of NO was produced in cells treated with LPS alone. At a concentration of 1000 μg/mL, 3 and 7 days showed relatively high inhibition rates. In the case of cells not treated with LPS, the amount of NO secretion was 0.65 μM, and the NO production inhibitory effect of 72 and 76% was shown. It was confirmed that the NO product increased by induction with LPS in RAW cells was inhibited by Chunma extract. Chunma extract after 7 days suppressed higher NO compared to other days, but it was not available due to strong sour taste. The extract was used after 3 days of treatment.
상기 천마 추출물의 성분 분석을 위해, 도 22에 도시된 바와 같이, 표준물질(Gastrodin, 4-hydroxybenzyl alcohol) HPLC 분석을 실시 하였으며, 시료의 제조는 추출물 분말을 정확한 양을 취하여 증류수에 용해시켜 membrane filter로 여과하여 사용하였으며 본 과제에서는 50 mg/mL 농도로 제작하여 시료로 사용하였다. 지표성분은 1000, 500, 250, 125, 62.5, 31.25, 15.625 ㎍/mL의 농도로 MeOH에 녹여 만든 후 표준곡선 작성 및 정량시 표준용액으로 사용하였다. HPLC 분석기기는 Waters allience e2695 system(2998 photodiode array detector)을 사용하였고, 컬럼은 Luna(5um, 4.6×250mm, phenomenex Co.)를 사용하였으며, 이동상은 용매 A는 acetonitrile을 사용하였고, 용매 B는 증류수와 0.1% formic acid를 혼합하여 gradient program으로 하여 UV 흡광도 280 nm에서 측정하였다. 정량분석은 표준물질 Gastrodin, 4-hydroxybenzyl alcohol의 peak area와 retention time을 비교하였다.For component analysis of the Chunma extract, as shown in FIG. 22, HPLC analysis was performed on a standard substance (Gastrodin, 4-hydroxybenzyl alcohol), and for the preparation of the sample, an exact amount of the extract powder was taken and dissolved in distilled water, followed by a membrane filter It was filtered and used, and in this project, it was prepared at a concentration of 50 mg/mL and used as a sample. The indicator components were prepared by dissolving them in MeOH at concentrations of 1000, 500, 250, 125, 62.5, 31.25, and 15.625 μg/mL, and then used as standard solutions for standard curve preparation and quantification. As HPLC analysis equipment, Waters allience e2695 system (2998 photodiode array detector) was used, the column was Luna (5um, 4.6 × 250mm, phenomenex Co.), and as the mobile phase, acetonitrile was used for solvent A, and distilled water for solvent B. and 0.1% formic acid were mixed, and the UV absorbance was measured at 280 nm using a gradient program. For quantitative analysis, the peak area and retention time of standard substances Gastrodin and 4-hydroxybenzyl alcohol were compared.
도 23은 천마 추출물의 표준물질 함량비교를 나타낸 도면으로서, 천마 추출물의 날짜별 표준 물질 함량 변화를 보면 Gastrodin의 경우 3일까지 함량이 조금씩 증가하였고 이후에는 함량이 감소하였다. 4-hydroxybenzyl alcohol 4일을 제외하고는 조금씩 증가하는 경향을 보였다.23 is a view showing a comparison of the standard substance content of the Chunma extract. Looking at the change in the standard substance content by date of the Chunma extract, the content of Gastrodin increased little by little until 3 days, and the content decreased thereafter. 4-hydroxybenzyl alcohol showed a tendency to increase little by little except for 4 days.
상술한 바를 통해, 혼합균주 5%를 첨가하여 35℃에서 72h 발효한 천마를 생물전환 최적조건으로 확립하였으며. 천마(LB+LP, 5%)를 첨가하여 35℃에서 0, 24, 48, 72, 96, 120시간 동안 발효하여 측정한 결과 pH는 서서히 감소하여 3일째 3.94±049로 가장 낮은 값을 나타내었고 산도는 3일 째 1.46±0.58로 가장 높은 값을 나타내었다. 생균수 역시 산도와 비례하여 3일에 12.30±0.91 log cfu/g의 값을 보여 최대균수를 나타내었다.Through the above-mentioned bar, Chunma fermented for 72 h at 35 ° C with the addition of 5% of the mixed strain was established as the optimal condition for bioconversion. As a result, the pH gradually decreased and showed the lowest value at 3.94±049 on the 3rd day, as a result of fermenting for 0, 24, 48, 72, 96, and 120 hours at 35°C by adding Chunma (LB+LP, 5%). The pH value was 1.46±0.58 on the 3rd day, showing the highest value. The number of viable cells also showed a value of 12.30±0.91 log cfu/g on
총 페놀과 플라보노이드 함량을 비교해 본 결과 35℃에서 혼합균주 5%를 사용하여 발효한 발효물은 3일째때 가장 높은 함량을 보였고 ABTS radical와 DPPH radical 역시 3일 째에서 IC50값이 가장 낮아 소거활성이 가장 좋았다.As a result of comparing the total phenol and flavonoid contents, the fermented product fermented at 35°C using 5% of the mixed strain showed the highest content on the 3rd day, and the ABTS radical and DPPH radical also had the lowest IC 50 values on the 3rd day, resulting in scavenging activity. this was the best
전술된 내용은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자라면 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 수정 및 변형이 가능할 것이다. 따라서, 본 발명에 개시된 실시예들은 본 발명의 기술 사상을 한정하기 위한 것이 아니라 설명하기 위한 것이고, 이러한 실시예에 의하여 본 발명의 기술 사상의 범위가 한정되는 것은 아니다. 본 발명의 보호 범위는 아래의 청구범위에 의하여 해석되어야 하며, 그와 동등한 범위 내에 있는 모든 기술 사상은 본 발명의 권리범위에 포함되는 것으로 해석되어야 할 것이다.Those of ordinary skill in the art to which the present invention pertains may modify and modify the above-described contents without departing from the essential characteristics of the present invention. Accordingly, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but to explain, and the scope of the technical spirit of the present invention is not limited by these embodiments. The protection scope of the present invention should be construed by the following claims, and all technical ideas within the scope equivalent thereto should be construed as being included in the scope of the present invention.
Claims (5)
상기 분쇄물과 물을 1:20의 중량 비율로 혼합한 혼합물을 제조한 후 상기 혼합물을 55℃ ~ 65℃에서 3 ~ 5 시간 당화작업하여 상기 천마 당화액을 준비하는 당화 단계;
락토바실러스 브레비스(Lactobacillus brevis) 및 락토바실러스 플란타럼(Lactobacillus platarum)의 혼합 균주를 상기 천마 당화액의 5중량%로 상기 천마 당화액에 접종하여 35℃로 72시간 발효하는 발효 단계; 및
상기 발효 단계를 통해 얻어진 발효물로부터 천마생물전환 추출물을 추출한 후 농축하여 얻어진 천마생물전환 농축액에 하수오 농축액, 배 농축액, 홍삼 농축액 및 산양삼 추출액을 미리 설정된 비율로 첨가하여 배합한 후 살균하여 천마 조성물을 제조하는 제조 단계를 포함하되,
상기 제조 단계는 상기 발효물로부터 85℃에서 24 시간동안 천마생물전환 추출물을 추출한 후 50Brix% ~ 55Brix%가 되도록 농축하여 천마생물전환 농축액을 제조하고,
상기 천마생물전환 농축액 25 ~ 35중량%에, 상기 하수오 농축액 15 ~ 25중량%, 상기 배 농축액 15 ~ 19중량%, 상기 홍삼 농축액 2~5%, 상기 산양삼 추출액 25 ~ 35중량%을 첨가하여 45Brix% ~ 50Brix%가 되도록 배합하는 단계를 포함하는 천마 조성물의 제조 방법.
A pulverization step of preparing a pulverized product containing Chunma powder by pulverizing the Chunma;
A saccharification step of preparing a mixture of the pulverized product and water in a weight ratio of 1:20, then saccharifying the mixture at 55°C to 65°C for 3-5 hours to prepare the saccharification solution;
A fermentation step of inoculating a mixed strain of Lactobacillus brevis and Lactobacillus platarum into the Chunma saccharified solution at 5% by weight of the Chunma saccharified solution and fermenting at 35° C. for 72 hours; and
After extracting and concentrating the Chunma bioconversion extract from the fermented product obtained through the fermentation step, sewage concentrate, pear concentrate, red ginseng concentrate and wild ginseng extract in a preset ratio were added to the Chunma bioconversion concentrate in a preset ratio and then sterilized to obtain a Chunma composition Including a manufacturing step of manufacturing,
The manufacturing step extracts the Chunma bioconversion extract from the fermented product at 85° C. for 24 hours and then concentrates it to 50Brix% ~ 55Brix% to prepare a Chunma bioconversion concentrate,
45Brix by adding 25 to 35% by weight of the Chunma bioconversion concentrate, 15 to 25% by weight of the sewage concentrate, 15 to 19% by weight of the pear concentrate, 2 to 5% of the red ginseng concentrate, and 25 to 35% by weight of the wild ginseng extract % ~ 50. Method for producing a composition comprising the step of blending so as to be Brix%.
상기 발효 단계는
상기 락토바실러스 브레비스 및 락토바실러스 플란타럼으로 이루어진 상기 혼합 균주를 맥아배지에 종균 배양한 다음 상기 천마 당화액에 접종하는 단계를 더 포함하는 것을 특징으로 하는 천마 조성물의 제조 방법.
The method according to claim 1,
The fermentation step
The method for producing a cheonma composition, characterized in that it further comprises the step of culturing the mixed strain consisting of the Lactobacillus brevis and the Lactobacillus plantarum in malt medium and then inoculating the saccharification solution in the cheonma.
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| KR1020190159686A KR102403615B1 (en) | 2019-12-04 | 2019-12-04 | Manufacturing method for composition of gastrodia elata blume |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100625610B1 (en) * | 2005-03-28 | 2006-09-20 | 무주군약초영농조합법인 | Chunma beverage of paste type and manufacturing method thereof |
| KR100870139B1 (en) | 2007-12-20 | 2008-11-24 | 주식회사 참선진 종합식품 | Beverage comprising gastrodia elata extract, brown rice-phellinus linteus extract, raw material of herb medicine extract and preparation method thereof |
| KR101918942B1 (en) * | 2017-11-14 | 2018-11-15 | 재단법인경남한방항노화연구원 | Fermented drink using glycine max (L.) merrill and herbal ingredients and manufacturing method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101201528B1 (en) | 2010-01-28 | 2012-11-15 | (사)무주천마사업단 | Ramyun Containing Gastrodiae and Manufacturing method thereof |
| KR20180049682A (en) * | 2016-11-03 | 2018-05-11 | 전라북도(농업기술원) | Method for fermented Gastrodia elata BL using lactic acid bacteria |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100625610B1 (en) * | 2005-03-28 | 2006-09-20 | 무주군약초영농조합법인 | Chunma beverage of paste type and manufacturing method thereof |
| KR100870139B1 (en) | 2007-12-20 | 2008-11-24 | 주식회사 참선진 종합식품 | Beverage comprising gastrodia elata extract, brown rice-phellinus linteus extract, raw material of herb medicine extract and preparation method thereof |
| KR101918942B1 (en) * | 2017-11-14 | 2018-11-15 | 재단법인경남한방항노화연구원 | Fermented drink using glycine max (L.) merrill and herbal ingredients and manufacturing method thereof |
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