KR20230072664A - A composition for immune enhancement comprising citrus extract fermented by bacillus strains - Google Patents
A composition for immune enhancement comprising citrus extract fermented by bacillus strains Download PDFInfo
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- KR20230072664A KR20230072664A KR1020210159140A KR20210159140A KR20230072664A KR 20230072664 A KR20230072664 A KR 20230072664A KR 1020210159140 A KR1020210159140 A KR 1020210159140A KR 20210159140 A KR20210159140 A KR 20210159140A KR 20230072664 A KR20230072664 A KR 20230072664A
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- bacillus
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- A—HUMAN NECESSITIES
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- 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
- 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
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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
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
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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
- A23V2250/00—Food ingredients
- A23V2250/20—Natural extracts
- A23V2250/21—Plant extracts
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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
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Abstract
본 발명은 시트러스 발효 추출물을 유효성분으로 포함하는 면역 증진용 식품 조성물에 관한 것이다.The present invention relates to a food composition for enhancing immunity comprising a fermented citrus extract as an active ingredient.
Description
본 발명은 시트러스 발효 추출물을 유효성분으로 포함하는 면역 증진용 조성물에 관한 것으로, 의약품 및 식품 등 다양한 용도로서 활용 가능하다.The present invention relates to a composition for enhancing immunity comprising a fermented citrus extract as an active ingredient, and can be used for various purposes such as pharmaceuticals and foods.
모든 생물체는 생명을 영위하기 위해 끊임없이 많은 영양소를 필요로 하며, 영양소는 식품의 형태로 항상 외부로부터 공급되거나 일부는 체내에서 합성된다.All organisms constantly need a lot of nutrients to sustain life, and nutrients are always supplied from the outside in the form of food or some are synthesized in the body.
음식물 속에는 인간의 생명현상을 유지하는데 필요한 물질들이 포함되어 있으며, 인간은 음식물을 섭취하여 소화기관에서 소화시키고 흡수하여 체내의 조직세포에 공급함으로써 생명을 유지할 수 있다.Food contains substances necessary for maintaining human life, and humans can maintain life by ingesting food, digesting it in the digestive system, absorbing it, and supplying it to tissue cells in the body.
따라서 좋은 영양은 건강한 신체를 유지시켜주며, 건강한 신체는 건강한 정신을 보장한다.Therefore, good nutrition maintains a healthy body, and a healthy body guarantees a healthy mind.
영양은 직접, 간접으로 질병의 원인이 되기도 하며, 질병의 예방 및 치료에도 관여하는 것으로 알려져 있는데, 인체에 필요한 영양소의 균형섭취 제대로 이루어지지 않아 생기는 영양분의 부족도 문제이고, 과잉섭취 또한 심각한 문제이며, 이로 인해 각종 질병이 유발될 수 있다.Nutrition is known to directly or indirectly cause diseases, and is also involved in the prevention and treatment of diseases. , which can cause various diseases.
최근 생활 수준이 높아지고 식생활이 서구화되면서 영양과잉, 운동부족 등으로 인한 비만, 당뇨, 고혈압 및 심장병 등과 같은 성인병 발병률이 증가하고 있다. 또한 평균수명 연장으로 인하여 인구가 점차 노령화되면서 건강에 대한 관심이 점차 높아지고 있다.BACKGROUND OF THE INVENTION [0002] As living standards have recently increased and dietary habits have become westernized, the incidence of adult diseases such as obesity, diabetes, high blood pressure, and heart disease due to overnutrition and lack of exercise is increasing. In addition, as the population is gradually aging due to the extension of life expectancy, interest in health is gradually increasing.
건강증진(health promotion)은 질병, 특히 만성 퇴행성질환이나 감염증, 계속되는 스트레스에 대한 저항력의 증가, 일상의 활동력의 증대를 꾀하는 것으로 이를 위해 영양의 개선, 면역기능 강화, 적절한 운동과 휴식, 정신적 활동의 지속 등 생활양식 전반에 미치는 관리가 요구된다. Health promotion is to increase resistance to diseases, especially chronic degenerative diseases or infections, and to increase daily activity. Sustainability and overall lifestyle management are required.
면역은 체내에 존재하는 자기방어체계로서 인체가 외부에서 침입하는 각종 물질이나 생명체를 자기 자신에 대한 이물질로 인식하여 제거하고 대사시키는 과정이다.Immunity is a self-defense system that exists in the body, and is a process in which the human body recognizes various substances or organisms invading from the outside as foreign substances to itself, removes them, and metabolizes them.
외부 자극에 의한 손상이나 병원 미생물의 침입으로부터 자신을 방어하기도 하지만 염증반응 등과 같이 자기 자신의 조직에 손상을 줄 수도 있다.It defends itself from damage caused by external stimuli or the invasion of pathogenic microorganisms, but it can also damage its own tissue, such as an inflammatory response.
면역기능의 변화를 조절하여 정상으로 회복시키거나 변화의 폭을 줄여 주는 작용으로 면역기능 억제나 면역기능 증강으로 구분된다.It is an action that regulates changes in immune function to restore them to normal or reduce the width of changes, and is divided into suppression of immune function or enhancement of immune function.
면역 기능에 영향을 미치는 요인에는 유전적, 환경적 요인, 나이, 성별, 심리적 스트레스, 영양상태, 식이습관, 질병 등이 있다.Factors that affect immune function include genetic and environmental factors, age, gender, psychological stress, nutritional status, dietary habits, and diseases.
면역 기능의 조절 기능을 갖는 건강기능식품은 현재까지, 저하된 면역기능을 증진 시키는 기능성과 과도한 면역기능을 조절하는 기능성, 자기 성분에 대한 면역반응을 조절하는 자가면역기능 조절 기능성으로 구분된다.Functional health foods with the function of regulating immune function are classified into functionalities that promote reduced immune function, functions that control excessive immune function, and functions that regulate autoimmune function that regulate immune responses to self-components.
면역기능이 저하되면 과민반응을 일으키는 경우가 있는데 이러한 과민반응은 천식, 계절성 또는 동년성 비염, 알러지성 부비강염, 결막염, 식품과 약품에 의한 알러지, 아토피성 피부염, 두드러기, 벌침에 의한 알러지 등과 같이 다양하게 나타난다.When the immune function is weakened, hypersensitivity reactions may occur, and these hypersensitivity reactions are diverse, such as asthma, seasonal or winter rhinitis, allergic sinusitis, conjunctivitis, food and drug allergy, atopic dermatitis, hives, and allergy due to bee stings. appear
여러 원인으로 인한 면역기능 장애를 극복하기 위하여, 다양한 면역 증강제가 사용되고 있으나, 부작용 및 생체 안전성의 문제가 있으므로 장기 복용이 가능하고 예방용으로도 적합한 면역 증진 조성물이 요구되는 실정이다.In order to overcome immune dysfunction due to various causes, various immune enhancers are used, but there is a need for an immune enhancing composition that can be taken for a long time and is suitable for prevention because of side effects and biosafety problems.
본 발명은 전술한 종래 기술의 문제점을 해결하기 위한 것으로, 본 발명의 목적은 생체 안전성이 우수한 천연 식물 유래의 기능성 식의약품을 제공하는 것이다.The present invention is to solve the problems of the prior art described above, and an object of the present invention is to provide a functional food or drug derived from natural plants with excellent biosafety.
본 발명의 일 측면에 따르면, 시트러스(citrus) 발효 추출물을 유효성분으로 포함하는 면역 증진용 조성물이 제공된다.According to one aspect of the present invention, there is provided a composition for enhancing immunity comprising a fermented citrus extract as an active ingredient.
일 실시예에 있어서, 상기 시트러스(citrus)는 풋귤, 감귤, 자몽, 및 오렌지로 이루어진 군에서 하나 이상 선택된 것일 수 있다.In one embodiment, the citrus may be one or more selected from the group consisting of green tangerines, tangerines, grapefruits, and oranges.
일 실시예에 있어서, 상기 발효 추출물은 상기 발효 추출물은 바실러스 속(Bacillus spp.) 균주를 발효시켜 수득한 것일 수 있다.In one embodiment, the fermentation extract may be obtained by fermenting a Bacillus spp. strain.
일 실시예에 있어서, 상기 균주는 바실러스 서브틸리스(Bacillus subtilis), 바실러스 사펜시스(Bacillus safensis), 바실러스 아밀로리퀴페시언스(Bacillus amyloliquefaciens), 및 바실러스 메가테리(Bacillus megaterium)로 이루어진 군에서 하나 이상 선택된 것일 수 있다.In one embodiment, the strain is one from the group consisting of Bacillus subtilis , Bacillus sapensis, Bacillus safensis, Bacillus amyloliquefaciens , and Bacillus megaterium . It may be selected above.
본 발명의 다른 측면에 따르면, 시트러스(citrus) 발효 추출물을 유효성분으로 포함하는 면역 증진용 건강기능식품이 제공된다.According to another aspect of the present invention, there is provided a health functional food for enhancing immunity comprising a fermented citrus extract as an active ingredient.
본 발명의 일 측면에 따른 조성물은 천연 추출물을 유효성분으로 포함하여 생체 안전성이 우수할 뿐만 아니라 면역 증진 활성이 우수하므로 건강기능식품뿐만 아니라 면역 관련 질환의 치료 용도로서 유용하게 활용할 수 있다.Since the composition according to one aspect of the present invention contains natural extracts as an active ingredient and has excellent biosafety and immune enhancing activity, it can be usefully used as a health functional food as well as for the treatment of immune-related diseases.
본 발명의 효과는 상기한 효과로 한정되는 것은 아니며, 본 발명의 상세한 설명 또는 청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다.The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the configuration of the invention described in the claims.
도 1은 본 발명의 일 실시예에 따른 시트러스 발효 추출물의 세포 독성을 평가한 결과이다.
도 2는 본 발명의 일 실시예에 따른 시트러스 발효 추출물의 면역 증진 활성을 평가(NO assay)한 결과이다.
도 3 및 4는 본 발명의 일 실시예에 따른 시트러스 발효 추출물의 면역 증진 활성(TNF-a)을 평가한 결과이다.1 is a result of evaluating the cytotoxicity of a fermented citrus extract according to an embodiment of the present invention.
Figure 2 is the result of evaluating the immune enhancing activity (NO assay) of the fermented citrus extract according to an embodiment of the present invention.
3 and 4 are results of evaluating the immune enhancing activity (TNF-a) of the fermented citrus extract according to an embodiment of the present invention.
본 명세서에서 사용되는 용어는 본 발명에서의 기능을 고려하면서 가능한 현재 널리 사용되는 일반적인 용어들을 선택하였으나, 이는 당 분야에 종사하는 기술자의 의도 또는 판례, 새로운 기술의 출현 등에 따라 달라질 수 있다. The terms used in this specification have been selected from general terms that are currently widely used as much as possible while considering the functions in the present invention, but these may vary depending on the intention of a person skilled in the art, precedent, or the emergence of new technologies.
또한, 특정한 경우는 출원인이 임의로 선정한 용어도 있으며, 이 경우 해당되는 발명의 설명 부분에서 상세히 그 의미를 기재할 것이다. 따라서 본 발명에서 사용되는 용어는 단순한 용어의 명칭이 아닌, 그 용어가 가지는 의미와 본 발명의 전반에 걸친 내용을 토대로 정의되어야 한다.In addition, in a specific case, there is also a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, not simply the name of the term.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다. Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in this application, it should not be interpreted in an ideal or excessively formal meaning. don't
수치 범위는 상기 범위에 정의된 수치를 포함한다. 본 명세서에 걸쳐 주어진 모든 최대의 수치 제한은 낮은 수치 제한이 명확히 쓰여져 있는 것처럼 모든 더 낮은 수치 제한을 포함한다. 본 명세서에 걸쳐 주어진 모든 최소의 수치 제한은 더 높은 수치 제한이 명확히 쓰여져 있는 것처럼 모든 더 높은 수치 제한을 포함한다. 본 명세서에 걸쳐 주어진 모든 수치 제한은 더 좁은 수치 제한이 명확히 쓰여져 있는 것처럼, 더 넓은 수치 범위 내의 더 좋은 모든 수치 범위를 포함할 것이다.Numerical ranges are inclusive of the values defined therein. Every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written. Every numerical limitation given throughout this specification will include every better numerical range within the broader numerical range, as if the narrower numerical limitations were expressly written.
이하, 본 발명의 실시예를 상세히 기술하나, 하기 실시예에 의해 본 발명이 한정되지 아니함은 자명하다.Hereinafter, embodiments of the present invention will be described in detail, but it is obvious that the present invention is not limited by the following examples.
본 발명의 일 측면에 따르면, 시트러스(citrus) 발효 추출물을 유효성분으로 포함하는 면역 증진용 조성물이 제공된다.According to one aspect of the present invention, there is provided a composition for enhancing immunity comprising a fermented citrus extract as an active ingredient.
상기 “시트러스(Citrus)”의 사전적 의미는 귤과의 귤속(Citrus), 귤속(Fortunella), 탱자속(Pontirus)의 3속에 걸친 총괄적 명칭이며, 상기 시트러스는 나리진(Narigin), 나르제닌(Naringenin), 헤스페리딘(Hesperidin), 탄제레틴(Tangeretin), 노비레틴(Nobiletin) 등 다양한 화합물을 포함할 수 있다.The dictionary meaning of “Citrus” is a general name for the three genera of Citrus, Fortunella, and Pontirus of the Tangerine family, and the Citrus is Narigin, Nargenin ( Naringenin), Hesperidin, Tangeretin, and Nobiletin.
상기 시트러스는 감귤, 금귤, 만다린, 탠저린, 스위티, 시트론, 영귤, 천혜향, 칼라만시, 포멜로, 한라봉, 오렌지, 레몬, 자몽, 라임, 유자 등의 시트러스 계열에 포함되는 과일 및 이들의 미숙과를 지칭하며, 귤속(Citrus) 식물에 속하는 한 특별히 제한되지 않는다.Citrus includes citrus, kumquat, mandarin, tangerine, sweetie, citron, tangerine, cheonhyehyang, calamansi, pomelo, hallabong, orange, lemon, grapefruit, lime, citron, etc. It refers to, and is not particularly limited as long as it belongs to the Citrus plant.
일 실시예에 있어서, 상기 시트러스(citrus)는 풋귤, 감귤, 자몽, 및 오렌지로 이루어진 군에서 하나 이상 선택될 수 있다.In one embodiment, the citrus (citrus) may be one or more selected from the group consisting of green tangerines, tangerines, grapefruits, and oranges.
상기 “풋귤”은 감귤의 미성숙과로 과피가 착색되기 전인 초록색 상태의 열매를 의미한다.The “green tangerine” is an immature citrus fruit and refers to a fruit in a green state before the skin is colored.
상기 미성숙과는 당도 8 브릭스 미만의 극조생 온주밀감 또는 9 브릭스 미만의 조생 및 보통 온주밀감을 지칭할 수 있다.The immature family may refer to an extremely early mandarin orange with a sugar content of less than 8 bricks or an early and common mandarin orange with a sugar content of less than 9 bricks.
상기 “감귤”은 운향과 감귤속에 속하는 상록 소교목의 총칭으로, 온주밀감(C. unshiu)을 비롯하여 홍귤나무(C. deliciosa), 왕귤나무(C. grandis), 유자나무(C. junos), 여름귤나무(C. natsudaidai), 시트론(C. medica), 라임(C. aurantifolia), 레몬(C. limon), 향귤(C. aurantium), 러프 멜론(C. jambhiri), 밤호박(C. maxima), 지각(C. aurantium), 편귤(C. tangerina), 빈귤(C. leiocarpa), 사두감(C. pseudogalgul TANAKA), 편귤(C. tangerrina TANAKA), 빈귤(C. leocarpa), 홍귤(C. tachibana TANAKA), 감자(C. benokoji TANAKA), 병귤(C. nippokoreana TANAKA), 동정귤(C. erythrosa TANAKA), 호랑이깡(C. sulcata HORT. Ex. Tan), 나스(C. natsudaidai HAY), 당유자(C. grandis OSBECK) 및 팔삭(C. hassaku HORT. Ex Y.Tan)으로 이루어진 군에서 선택될 수 있으나 이에 제한되는 것은 아니다.The "citrus" is a generic term for evergreen small trees belonging to the genus Rutaceae and Citrus, including Onju tangerine ( C. unshiu ), red tangerine tree ( C. deliciosa ), tangerine tree ( C. grandis ), citron tree ( C. junos ), Summer tangerine ( C. natsudaidai ), citron ( C. medica ), lime ( C. aurantifolia ), lemon ( C. limon ), tangerine ( C. aurantium ), rough melon ( C. jambhiri ), chestnut squash ( C. maxima ), crust ( C. aurantium ), tangerine ( C. tangerina ), tangerine ( C. leiocarpa ), persimmon ( C. pseudogalgul TANAKA ), tangerine ( C. tangerrina TANAKA ), tangerine ( C. leocarpa ), red tangerine ( C. tachibana TANAKA ), potato ( C. benokoji TANAKA ), bottled tangerine ( C. nippokoreana TANAKA ), dong tangerine ( C. erythrosa TANAKA ), tiger cracker ( C. sulcata HORT. Ex. Tan ), eggplant ( C. natsudaidai HAY) ), Dangyuja ( C. grandis OSBECK ) and Palsak ( C. hassaku HORT. Ex Y.Tan ), but may be selected from the group consisting of, but is not limited thereto.
상기 “자몽(grapefruit)”은 감귤속에 속하는 그레이프프루트 나무의 열매로서 모양은 귤과 같이 둥글고 지름은 10 내지 15 cm이며 겉껍질은 가죽질이고 표면이 매끄러우며 노란색이다. The "grapefruit" is a fruit of a grapefruit tree belonging to the genus Citrus, which has a round shape like a tangerine, a diameter of 10 to 15 cm, a leathery outer skin, a smooth surface, and a yellow color.
상기 “오렌지(Citrus sinensis)”는 귤속에 속하는 식물로 비타민 C, 비타민 B1, B2, 플라보노이드, 베타카토닌, 시트릭 애씨드, 팩틴 등의 유용한 성분을 풍부하게 함유하고 있다.The “orange ( Citrus sinensis )” is a plant belonging to the genus Tangerine and contains a wealth of useful ingredients such as vitamin C, vitamins B1 and B2, flavonoids, beta-catonin, citric acid, and pectin.
상기 “면역(immunity)”은 생물체 내에서 병원체와 종양 세포 등을 탐지한 후 무력화하여 질병으로부터 생명체를 보호하는 기작을 의미하는 것으로, 상기 “면역 증진(Immunostimulation)”은 암 및 염증 등과 같은 다양한 질환에 대한 신체 방어 기전을 보강하는 일련의 치료학적 또는 예방학적 전략을 모두 포함할 수 있다.The “immunity” refers to a mechanism that detects pathogens and tumor cells in an organism and then neutralizes them to protect the organism from disease. The “immunostimulation” refers to various diseases such as cancer and inflammation. It can include any of a series of therapeutic or prophylactic strategies that reinforce the body's defense mechanisms against
상기 시트러스는 다양한 생리 활성을 가지는 것으로 알려져 있으나, 본 발명자들은 상기 시트러스에 대한 발효 공정을 통해 면역 증진 효과를 개선하고자 하였다.Citrus is known to have various physiological activities, but the present inventors tried to improve the immune enhancing effect through a fermentation process for the citrus.
상기 “발효”는 미생물이 자신이 가지고 있는 효소를 이용해 유기물을 분해시키는 과정을 의미한다. 상기 미생물을 통한 발효과정을 거친 원료는 발효 전보다 최소 2배에서 최대 수 십배의 활성이 증대되거나 기타 유효 성분의 함량이 현저히 변화될 수 있다.The "fermentation" means a process in which microorganisms decompose organic matter using their own enzymes. Raw materials that have undergone the fermentation process through the microorganisms may have at least two to several tens of times greater activity than before fermentation, or significantly change the content of other active ingredients.
상기 발효를 거친 대사물질은 각종 아미노산, 유기산, 항산화 물질을 함유할 수 있으며, 발효 과정에 의해 입자가 작아지고 독성이 분해되어 흡수율이 증진될 수 있으므로, 그로 인한 기능성이 개선될 수 있다.Metabolites that have undergone fermentation may contain various amino acids, organic acids, and antioxidants, and the absorption rate may be improved by reducing the particle size and decomposing toxicity through the fermentation process, thereby improving functionality.
상기 “발효 추출물”은 천연 원료를 자연적으로, 또는 회전감압농축기 및 동결건조기를 사용하여 건조시킨 후, 발효 균주를 접종하고 발효시켜 제조할 수 있다.The “fermented extract” may be prepared by inoculating and fermenting a fermented strain after drying the natural raw material naturally or using a rotary vacuum concentrator and a freeze dryer.
상기 발효 추출물은 바실러스 속(Bacillus spp.) 균주를 발효시켜 수득한 것일 수 있다.The fermented extract may be obtained by fermenting a Bacillus spp. strain.
상기 바실러스 속(bacillus spp.) 균주는 막대 모양의 그람 양성균 세균의 총칭으로, 후벽균류의 한 종류이며, 바실러스 서브틸리스(Bacillus subtilis), 바실러스 사펜시스(Bacillus safensis), 바실러스 아밀로리퀴페시언스(Bacillus amyloliquefaciens), 및 바실러스 메가테리(Bacillus megaterium)로 이루어진 군에서 하나 이상 선택될 수 있으나, 이에 제한되는 것은 아니다.The Bacillus genus ( bacillus spp. ) strain is a generic term for rod-shaped Gram-positive bacteria, and is a type of wall fungus, Bacillus subtilis , Bacillus sapensis ( Bacillus safensis ), Bacillus amyloliquipesciens ( Bacillus amyloliquefaciens ), and Bacillus megateri ( Bacillus megaterium ), but may be one or more selected from the group consisting of, but is not limited thereto.
상기 “유효성분으로 포함하는”은 투여하고자 하는 개체에 대한 의도하는 효과를 제공하기에 충분한 양을 포함하는 것을 의미하며, 상기 시트러스 발효 추출물은 생체 안전성이 우수하므로 당업자가 양적 상한을 적절히 조정할 수 있다.The "contained as an active ingredient" means to include an amount sufficient to provide the intended effect on the subject to be administered, and since the citrus fermented extract has excellent biosafety, those skilled in the art can appropriately adjust the upper limit of the amount. .
상기 “추출물”은 용매와 추출 원료를 특정 조건 하에서 접촉시킴으로써 추출 원료에 함유된 유효성분이 전이된 용매를 지칭하는 것으로, 천연물로부터 원료에 함유된 성분을 분리해낸 물질이라면, 추출 방법이나 성분의 종류와 무관하게 모두 포함할 수 있다. 예컨대, 물이나 유기용매를 이용하여 천연물로부터 용매에 용해되는 성분을 추출한 것, 천연물의 특정 성분, 예컨대 오일과 같은 특정 성분만을 추출하여 얻어진 것 등을 모두 포함할 수 있다.The "extract" refers to a solvent in which the active ingredient contained in the extraction material is transferred by contacting the solvent and the extraction material under specific conditions. Anything can be included regardless. For example, those obtained by extracting a component soluble in a solvent from a natural product using water or an organic solvent, and those obtained by extracting a specific component of a natural product, for example, only a specific component such as oil, may be included.
상기 추출물은 시트러스 원료(감귤류)를 건조 후 분말화하거나, 열수추출법, 에탄올 추출법 등 종래 알려진 다양한 추출법에 의해 수득할 수 있고, 과피나 열매를 모두 추출 원료로서 사용할 수 있다.The extract can be obtained by pulverizing citrus raw materials (citrus fruits) after drying, or by various extraction methods known in the art, such as hot water extraction or ethanol extraction, and both peels and fruits can be used as extraction raw materials.
상기 추출물은 물, 알코올, 에틸아세테이트, 아세톤, 핵산, 디클로로메탄 또는 이들의 혼합 용매로 추출될 수 있고, 바람직하게는 30 내지 70%(w/w) 농도의 에탄올에 의해 추출될 수 있다.The extract may be extracted with water, alcohol, ethyl acetate, acetone, nucleic acid, dichloromethane or a mixed solvent thereof, preferably with ethanol at a concentration of 30 to 70% (w/w).
상기 원료에 포함된 유효성분은 용매의 극성에 따라 추출 비율이 상이해질 수 있으므로, 용매에 따른 생리 활성 물질의 선택성을 고려하여 달리할 수 있다.Since the active ingredient included in the raw material may have a different extraction ratio depending on the polarity of the solvent, it may be different in consideration of the selectivity of the physiologically active substance according to the solvent.
상기 추출물은 추출 원료를 물로 수세한 후 건조 및 분쇄하여, 원료 중량의 8 내지 12배에 달하는 부피의 용매로 약 1 내지 24시간 동안 환류 순환 추출, 가압 추출, 초음파 추출 등 통상적인 방법으로 추출 및 여과하여 제조할 수 있다.The extract is obtained by washing the extraction raw material with water, then drying and pulverizing, extracting by a conventional method such as reflux circulation extraction, pressure extraction, ultrasonic extraction, etc. for about 1 to 24 hours with a solvent in a volume of 8 to 12 times the weight of the raw material. It can be prepared by filtration.
상기 추출물은 감압 증류 또는 동결 건조 등과 같은 추가적인 공정에 의해 분말 상태로 수득할 수 있다.The extract may be obtained in powder form by additional processes such as distillation under reduced pressure or freeze drying.
상기 추출물은 통상적인 정제 과정을 거친 추출물도 포함할 수 있다. 예컨대, 상기 추출물은 일정한 분자량 컷-오프 값을 갖는 한외 여과막을 이용한 분리, 다양한 크로마토그래피(크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의한 분리 등 추가적으로 실시된 다양한 정제 방법을 통해 얻어진 분획물을 포함할 수 있다.The extract may also include an extract that has undergone a conventional purification process. For example, the extract is subjected to various additional purification methods such as separation using an ultrafiltration membrane having a certain molecular weight cut-off value and separation by various chromatography (made for separation according to size, charge, hydrophobicity or affinity). It may include fractions obtained through
상기 면역 증진용 조성물은 식품 조성물, 예컨대 면역 증진을 위한 건강기능식품으로서 사용될 수 있다.The composition for enhancing immunity can be used as a food composition, such as a health functional food for enhancing immunity.
상기 건강기능식품은 건강기능식품에 관한 법률에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미하며, 상기 기능성은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미할 수 있다.The health functional food refers to food manufactured and processed using raw materials or ingredients having functional properties useful for the human body in accordance with the Health Functional Food Act, and the functional food regulates nutrients with respect to the structure and function of the human body or physiologically It may mean ingestion for the purpose of obtaining useful effects for health purposes such as action.
상기 식품 조성물은 통상의 식품 첨가물을 포함할 수 있으며, 상기 식품 첨가물은 다른 규정이 없는 한 식품의약품안전처에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 적합성 여부를 판단할 수 있다. The food composition may include conventional food additives, and the food additives are in accordance with the standards and standards for the item in accordance with the general rules of the Food Additive Code and general test methods approved by the Ministry of Food and Drug Safety, unless otherwise specified. suitability can be judged.
상기 식품 첨가물 공전에 기재된 품목은 예컨대 케톤류, 글리신, 구연산칼륨, 니코틴산, 계피산 등의 화학적 합성물, 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물, L-글루타민산나트륨 제제, 면류첨가알칼리제, 보존료제제, 타르색소제제 등의 혼합제제류를 들 수 있다.Items described in the food additives code include, for example, ketones, glycine, potassium citrate, chemical compounds such as nicotinic acid and cinnamic acid, natural additives such as persimmon pigment, licorice extract, crystalline cellulose, goyang pigment, guar gum, sodium L-glutamate preparations, and noodles. and mixed preparations such as added alkali agent, preservative preparation, and tar color preparation.
상기 식품 조성물은 면역 증진을 위한 식품 및 음료 등에 다양하게 이용될 수 있으며, 예컨대, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강기능성 보조 식품, 식품 첨가제 등에 사용될 수 있다.The food composition can be used in a variety of foods and beverages for enhancing immunity, for example, various foods, beverages, gum, tea, vitamin complexes, health functional supplements, food additives, and the like.
또한, 상기 건강기능식품은 면역 증진을 목적으로 정제, 과립, 분말, 캅셀, 액상의 용액 및 환으로 이루어진 군에서 선택된 어느 하나의 제형으로 제조 및 가공될 수 있다.In addition, the health functional food may be prepared and processed into any one formulation selected from the group consisting of tablets, granules, powders, capsules, liquid solutions, and pills for the purpose of enhancing immunity.
구체적으로 상기 정제 형태의 건강기능식품은 시트러스 발효 추출물, 부형제, 결합제, 붕해제 및 다른 첨가제와의 혼합물을 통상의 방법으로 과립화한 다음, 활택제 등을 넣어 압축 성형하거나, 상기 혼합물을 직접 압축 성형하여 제조할 수 있다. 또한, 상기 정제 형태의 건강기능식품은 필요에 따라 교미제 등을 함유할 수 있으며, 필요에 따라 적당한 제피제로 제피할 수도 있다.Specifically, the health functional food in the form of a tablet is obtained by granulating a mixture of a citrus fermented extract, an excipient, a binder, a disintegrant, and other additives in a conventional manner, and then compression molding by adding a lubricant or the like, or directly compressing the mixture It can be manufactured by molding. In addition, the health functional food in the form of a tablet may contain a corrigent, etc., if necessary, and may be coated with an appropriate coating agent, if necessary.
상기 캅셀 형태의 건강기능식품 중 경질캅셀제는 통상의 경질캅셀에 시트러스 발효 추출물 및 부형제 등의 첨가제와의 혼합물 또는 그의 입상물 또는 제피한 입상물을 충진하여 제조할 수 있으며, 연질캅셀제는 시트러스 발효 추출물 및 부형제 등의 첨가제와의 혼합물을 젤라틴 등 캅셀기제에 충진하여 제조할 수 있다. 상기 연질캅셀제는 필요에 따라 글리세린 또는 솔비톨 등의 가소제, 착색제, 보존제 등을 함유할 수 있다.Among the health functional foods in the form of capsules, hard capsules can be prepared by filling ordinary hard capsules with a mixture of citrus fermented extract and additives such as excipients, granular material thereof, or coated granular material, and soft capsules are fermented citrus extracts. And it can be prepared by filling a mixture with additives such as excipients into a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, and a preservative, if necessary.
상기 환 형태의 건강기능식품은 시트러스 발효 추출물, 부형제, 결합제, 붕해제 등의 혼합물을 적당한 방법으로 성형하여 조제할 수 있으며, 필요에 따라 백당이나 다른 적당한 제피제로 제피를, 또는 전분, 탈크 또는 적당한 물질로 환의를 입힐 수도 있다.The health functional food in the form of a ring can be prepared by molding a mixture of fermented citrus extract, excipients, binders, disintegrants, etc. in an appropriate way, and if necessary, it is coated with white sugar or other suitable coating agent, or starch, talc, or a suitable coating agent. You can also wear clothes with materials.
상기 과립형태의 건강기능식품은 시트러스 발효 추출물, 부형제, 결합제, 붕해제 등의 혼합물을 적당한 방법으로 입상으로 제조할 수 있으며, 필요에 따라 착향제, 교미제 등을 함유할 수 있다. The health functional food in the form of granules can be granularly prepared from a mixture of fermented citrus extract, excipients, binders, disintegrants, etc. by an appropriate method, and may contain flavoring agents, flavoring agents, etc., if necessary.
또한, 상기 부형제, 결합제, 붕해제, 활택제, 교미제, 착향제 등에 대한 용어 정의는 당업계에 공지된 문헌에 기재된 것으로 그 기능 등이 동일 내지 유사한 것들을 포함할 수 있다.In addition, definitions of terms for excipients, binders, disintegrants, lubricants, corrigents, flavoring agents, etc. are described in literature known in the art, and may include those having the same or similar functions.
본 발명의 다른 측면에 따르면, 시트러스 발효 추출물을 유효성분으로 포함하는 면역력 개선용 약학적 조성물이 제공된다.According to another aspect of the present invention, there is provided a pharmaceutical composition for improving immunity comprising a fermented citrus extract as an active ingredient.
상기 조성물은 면역 활성을 증진시킴으로써, 면역결핍, 면역저하 또는 면역계 손상으로 인한 질환의 예방, 개선 또는 치료에 유용하게 이용될 수 있다The composition can be usefully used for preventing, improving or treating diseases caused by immunodeficiency, immunosuppression or immune system damage by enhancing immune activity.
상기 “예방”은 병리학적 현상의 발생 빈도 또는 정도를 감소시키는 모든 행위를 의미한다. 예방은 완전할 수 있으며 또는 부분적일 수도 있다. 이 경우에는 개체 내의 염증에 의해 발생되는 증상이 상기 조성물을 사용 하지 않은 경우와 비교하여 감소하는 현상을 의미할 수 있다.The "prevention" means any action that reduces the frequency or severity of pathological phenomena. Prevention can be complete or partial. In this case, it may mean a phenomenon in which symptoms caused by inflammation in the subject are reduced compared to the case where the composition is not used.
상기 “치료”는 치료하고자 하는 대상 또는 세포의 천연 과정을 변경시키기 위하여 임상적으로 개입하는 모든 행위를 의미하며, 임상 병리 상태가 진행되는 동안 또는 이를 예방하기 위하여 수행할 수 있다. 목적하는 치료 효과는 질병의 발생 또는 재발을 예방하거나, 증상을 완화시키거나, 질병에 따른 모든 직접 또는 간접적인 병리학적 결과를 저하시키거나, 전이를 예방하거나, 질병 진행 속도를 감소시키거나, 질병 상태를 경감 또는 일시적 완화시키거나, 예후를 개선시키는 것을 포함할 수 있다.The "treatment" means any action that clinically intervenes to change the natural process of a target or cell to be treated, and can be performed while a clinical pathology is progressing or to prevent it. The desired therapeutic effect is to prevent the occurrence or recurrence of the disease, alleviate the symptoms, reduce any direct or indirect pathological consequences of the disease, prevent metastasis, reduce the rate of disease progression, or reduce the rate of disease progression. alleviating or palliating the condition, or improving the prognosis.
상기 조성물은 경구적 전달, 비경구적 전달의 형태로 투여될 수 있다. 상기 조성물은 전신 또는 국소 투여될 수 있으며, 상기 투여는 경구 투여 및 비경구 투여를 포함할 수 있다. 상기 조성물은 적절한 투여 형태를 제공하도록 적합한 양의 약학적으로 허용되는 비히클 또는 담체와 함께 제형화될 수 있다.The composition may be administered in the form of oral delivery or parenteral delivery. The composition may be administered systemically or locally, and the administration may include oral administration and parenteral administration. The composition may be formulated with a pharmaceutically acceptable vehicle or carrier in suitable amounts to provide an appropriate dosage form.
상기 조성물은 약학 조성물의 제조에 사용되는 담체, 부형제 및 희석제를 더 포함할 수 있다. 상기 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유를 들 수 있으나, 이에 제한되는 것은 아니다.The composition may further include carriers, excipients and diluents used in the preparation of pharmaceutical compositions. The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.
또한, 상기 조성물은 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제제화하여 사용할 수 있다.In addition, the composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions.
경구 투여를 위한 고형제제는 정제, 환제, 산제, 과립제, 캡슐제 등이 사용될 수 있고, 상기 고형제제는 상기 시트러스 발효 추출물과 이의 분획물들에 적어도 하나 이상의 부형제, 예컨대, 전분, 칼슘카보네이트, 수크로스, 락토오스, 또는 젤라틴 등을 혼합하여 조제할 수 있다. 또한, 상기 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제가 사용될 수 있다.Solid preparations for oral administration may be tablets, pills, powders, granules, capsules, etc., and the solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose, to the fermented citrus extract and its fractions. , lactose, gelatin, etc. can be mixed and prepared. In addition to the above excipients, lubricants such as magnesium styrate and talc may be used.
경구 투여를 위한 액상 제제는 현탁제, 내용액제, 유제, 시럽제 등이 사용될 수 있고, 단순희석제인 물, 리퀴드 파라핀 외에 여러 가지 부형제, 예컨대 습윤제, 감미제, 방향제, 보존제 등이 사용될 수 있다.Liquid preparations for oral administration may include suspensions, internal solutions, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives in addition to simple diluents such as water and liquid paraffin.
비경구 투여를 위한 제제는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 사용될 수 있다.Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories.
상기 비수성용제, 현탁제는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르가 사용될 수 있다. 상기 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴이 사용될 수 있다.Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used as the non-aqueous solvent or suspending agent. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin fat, and glycerogeratin may be used.
상기 약학적 조성물은 약제학적으로 유효한 양이 대상체에 투여될 수 있다. 상기 “약제학적으로 유효한 양”은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.The pharmaceutical composition may be administered to a subject in a pharmaceutically effective amount. The "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is based on the type and severity of the patient's disease, the activity of the drug, and the drug It may be determined according to factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical arts.
상기 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와 순차적으로 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 바람직하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is preferable to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.
이하 실시예를 통해, 본 발명을 더욱 상술하나 하기 실시예에 의해 본 발명이 제한되지 아니함은 자명하다.Through the following examples, the present invention is further detailed, but it is obvious that the present invention is not limited by the following examples.
제조예 1 : 바실러스 서브틸리스(Preparation Example 1: Bacillus subtilis ( Bacillus subtilisBacillus subtilis ) 발효 추출물 제조) Preparation of fermented extract
제조예 1-1. 귤미성숙과(7월수확분) 발효(추출)물(HR1903-F7C-BS_CJH101-W)Preparation Example 1-1. Unripe tangerine fruits (harvested in July) fermented (extracted) water (HR1903-F7C-BS_CJH101-W)
7월에 수확한 귤미성숙과 분쇄물과 정제수를 일정비율(1:1 내지 8)로 혼합한 후 pH를 조절(6.0 내지 7.5)하여 발효기에 넣고 멸균하였다.Unripe mandarin oranges harvested in July and pulverized water were mixed at a certain ratio (1: 1 to 8), and then the pH was adjusted (6.0 to 7.5) and put into a fermenter and sterilized.
미리 준비된 종균 Bacillus subtilis 5 내지 10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다. Bacillus subtilis 5 to 10% (w/w) prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 Bacillus subtilis 균주는 미국 국립생물공학정보센터(NCBI)에 등록된 Bacillus subtilis CJH 101(MW063656) 균주를 사용하였다.As the Bacillus subtilis strain, Bacillus subtilis CJH 101 (MW063656) strain registered with the National Center for Biotechnology Information (NCBI) was used.
상기 귤미성숙과 발효물에 일정 비율(1 내지 5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the unripe mandarin orange and fermented product, extracted by heating at 120 ° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 1-2. 귤미성숙과(8월수확분) 발효(추출)물(HR1903-F8C-BS_CJH101-W)Preparation Example 1-2. Unripe tangerine fruit (harvested in August) fermented (extracted) water (HR1903-F8C-BS_CJH101-W)
8월에 수확한 귤미성숙과 분쇄물과 정제수를 일정비율(1:1 내지 8)로 혼합한 후 pH를 조절(6.0 내지 7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus subtilis 5 내지 10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다. Unripe tangerines harvested in August and pulverized water were mixed at a certain ratio (1: 1 to 8), and then the pH was adjusted (6.0 to 7.5) and put into a fermentor and sterilized. Bacillus subtilis 5 to 10% (w/w) prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 귤미성숙과 발효물에 일정 비율(1 내지 5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the unripe mandarin orange and fermented product, extracted by heating at 120 ° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 1-3. 귤(12월수확분) 발효(추출)물(HR1903-FC-BS_CJH101-W)Preparation Example 1-3. Tangerine (December harvest) fermented (extracted) water (HR1903-FC-BS_CJH101-W)
12월에 수확한 귤 분쇄물과 정제수를 일정비율(1:1 내지 8)로 혼합한 후 pH를 조절(6.0 내지 7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus subtilis 5 내지 10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다. After mixing the tangerine pulverized material harvested in December and purified water at a certain ratio (1:1 to 8), the pH was adjusted (6.0 to 7.5) and put into a fermenter and sterilized. Bacillus subtilis 5 to 10% (w/w) prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 귤 발효물에 일정 비율(1 내지 5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the fermented tangerine, extracted by heating at 120 ° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 1-4. 자몽 발효(추출)물(HR1903-FGF-BS_CJH101-W)Preparation Example 1-4. Grapefruit fermented (extracted) water (HR1903-FGF-BS_CJH101-W)
자몽 분쇄물과 정제수를 일정비율(1:1 내지 8)로 혼합한 후 pH를 조절(6.0 내지 7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus subtilis 5 내지 10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다. Grapefruit pulverized material and purified water were mixed at a certain ratio (1:1 to 8), and the pH was adjusted (6.0 to 7.5) and put into a fermentor and sterilized. Bacillus subtilis 5 to 10% (w/w) prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 자몽 발효물에 일정 비율(1 내지 5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the fermented grapefruit product, extracted by heating at 120° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 1-5. 오렌지 발효(추출)물(HR1903-FOR-BS_CJH101-W)Preparation Example 1-5. Orange fermented (extracted) water (HR1903-FOR-BS_CJH101-W)
오렌지 분쇄물과 정제수를 일정비율(1:1 내지 8)로 혼합한 후 pH를 조절(6.0 내지 7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus subtilis 5 내지 10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다. Orange pulverized material and purified water were mixed at a certain ratio (1:1 to 8), and then the pH was adjusted (6.0 to 7.5) and put into a fermentor and sterilized. Bacillus subtilis 5 to 10% (w/w) prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 오렌지 발효물에 일정 비율(1 내지 5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the orange fermented product, extracted by heating at 120 ° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 2 : 바실러스 사펜시스(Preparation Example 2: Bacillus sapensis ( Bacillus safensisBacillus safensis ) 발효 추출물 제조) Preparation of fermented extract
제조예 2-1. 귤미성숙과(7월수확) 발효(추출)물(HR1903-F7C-BSaf_CJH102-W)Preparation Example 2-1. Unripe tangerine fruits (harvested in July) fermented (extracted) water (HR1903-F7C-BSaf_CJH102-W)
7월에 수확한 귤미성숙과 분쇄물과 정제수를 일정비율(1:1 내지 8)로 혼합한 후 pH를 조절(6.0 내지 7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus safensis 5 내지 10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다. Unripe mandarin oranges harvested in July and pulverized water were mixed at a certain ratio (1: 1 to 8), and then the pH was adjusted (6.0 to 7.5) and put into a fermenter and sterilized. Bacillus safensis 5 to 10% (w/w) prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 Bacillus safensis 균주는 미국 국립생물공학정보센터(NCBI)에 등록된 Bacillus safensis CJH 102(MW0188550) 균주를 사용하였다.As the Bacillus safensis strain, Bacillus safensis CJH 102 (MW0188550) strain registered with the National Center for Biotechnology Information (NCBI) was used.
상기 귤미성숙과 발효물에 일정 비율(1 내지 5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the unripe mandarin orange and fermented product, extracted by heating at 120 ° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 2-2. 귤미성숙과(8월수확) 발효(추출)물(HR1903-F8C-BSaf_CJH102-W)Preparation Example 2-2. Unripe tangerine fruit (harvested in August) fermented (extracted) water (HR1903-F8C-BSaf_CJH102-W)
8월에 수확한 귤미성숙과 분쇄물과 정제수를 일정비율(1:1 내지 8)로 혼합한 후 pH를 조절(6.0 내지 7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus safensis 5 내지 10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다. Unripe tangerines harvested in August and pulverized water were mixed at a certain ratio (1: 1 to 8), and then the pH was adjusted (6.0 to 7.5) and put into a fermentor and sterilized. Bacillus safensis 5 to 10% (w/w) prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 귤미성숙과 발효물에 일정 비율(1 내지 5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the unripe mandarin orange and fermented product, extracted by heating at 120 ° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 2-3. 귤(12월수확) 발효(추출)물(HR1903-FC-BSaf_CJH102-W)Preparation Example 2-3. Tangerine (harvest in December) fermented (extracted) water (HR1903-FC-BSaf_CJH102-W)
12월에 수확한 귤 분쇄물과 정제수를 일정비율(1:1 내지 8)로 혼합한 후 pH를 조절(6.0 내지 7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus safensis 5 내지 10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다. After mixing the tangerine pulverized material harvested in December and purified water at a certain ratio (1:1 to 8), the pH was adjusted (6.0 to 7.5) and put into a fermenter and sterilized. Bacillus safensis 5 to 10% (w/w) prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 귤 발효물에 일정 비율(1 내지 5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the fermented tangerine, extracted by heating at 120 ° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 2-4. 자몽 발효(추출)물(HR1903-FGF-BSaf_CJH102-W)Preparation Example 2-4. Grapefruit fermented (extracted) water (HR1903-FGF-BSaf_CJH102-W)
자몽 분쇄물과 정제수를 일정비율(1:1 내지 8)로 혼합한 후 pH를 조절(6.0 내지 7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus safensis 5 내지 10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다.Grapefruit pulverized material and purified water were mixed at a certain ratio (1:1 to 8), and the pH was adjusted (6.0 to 7.5) and put into a fermentor and sterilized. Bacillus safensis 5 to 10% (w/w) prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 자몽 발효물에 일정 비율(1 내지 5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the fermented grapefruit product, extracted by heating at 120° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 2-5. 오렌지 발효(추출)물(HR1903-FOR-BSaf_CJH102-W)Preparation Example 2-5. Orange fermented (extracted) water (HR1903-FOR-BSaf_CJH102-W)
오렌지 분쇄물과 정제수를 일정비율(1:1 내지 8)로 혼합한 후 pH를 조절(6.0 내지 7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus safensis 5 내지 10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다. Orange pulverized material and purified water were mixed at a certain ratio (1:1 to 8), and then the pH was adjusted (6.0 to 7.5) and put into a fermentor and sterilized. Bacillus safensis 5 to 10% (w/w) prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 오렌지 발효물에 일정 비율(1 내지 5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the orange fermented product, extracted by heating at 120 ° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 3 : 바실러스 아밀로리퀴페시언스(Preparation Example 3: Bacillus amyloriquipesciance ( Bacillus amyloliquefaciensBacillus amyloliquefaciens ) 발효 추출물 제조) Preparation of fermented extract
제조예 3-1. 귤미성숙과(7월수확) 발효(추출)물(HR1903-F7C-BA-W)Preparation Example 3-1. Unripe tangerine fruits (harvested in July) fermented (extracted) water (HR1903-F7C-BA-W)
7월에 수확한 귤미성숙과 분쇄물과 정제수를 일정비율(1:1 내지 8)로 혼합한 후 pH를 조절(6.0 내지 7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus amyloliquefaciens 5 내지 10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다. Unripe mandarin oranges harvested in July and pulverized water were mixed at a certain ratio (1: 1 to 8), and then the pH was adjusted (6.0 to 7.5) and put into a fermenter and sterilized. 5 to 10% (w/w) of Bacillus amyloliquefaciens prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 귤미성숙과 발효물에 일정 비율(1 내지 5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the unripe mandarin orange and fermented product, extracted by heating at 120 ° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 3-2. 귤미성숙과(8월수확) 발효(추출)물(HR1903-F8C-BA-W)Preparation Example 3-2. Unripe tangerine fruits (harvested in August) fermented (extracted) water (HR1903-F8C-BA-W)
8월에 수확한 귤미성숙과 분쇄물과 정제수를 일정비율(1:1 내지 8)로 혼합한 후 pH를 조절(6.0 내지 7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus amyloliquefaciens 5 내지 10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다. Unripe tangerines harvested in August and pulverized water were mixed at a certain ratio (1: 1 to 8), and then the pH was adjusted (6.0 to 7.5) and put into a fermentor and sterilized. 5 to 10% (w/w) of Bacillus amyloliquefaciens prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 귤미성숙과 발효물에 일정 비율(1 내지 5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the unripe mandarin orange and fermented product, extracted by heating at 120 ° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 3-3. 귤(12월수확) 발효(추출)물(HR1903-FC-BA-W)Preparation Example 3-3. Tangerine (harvest in December) fermented (extracted) water (HR1903-FC-BA-W)
귤 분쇄물과 정제수를 일정비율(1:1 내지 8)로 혼합한 후 pH를 조절(6.0 내지 7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus amyloliquefaciens 5 내지 10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다. After mixing the tangerine pulverized material and purified water at a certain ratio (1:1 to 8), the pH was adjusted (6.0 to 7.5) and put into a fermentor and sterilized. 5 to 10% (w/w) of Bacillus amyloliquefaciens prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 귤 발효물에 일정 비율(1 내지 5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the fermented tangerine, extracted by heating at 120 ° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 3-4. 자몽 발효(추출)물(HR1903-FGF-BA-W)Preparation Example 3-4. Grapefruit fermented (extracted) water (HR1903-FGF-BA-W)
자몽 분쇄물과 정제수를 일정비율(1:1내지8)로 혼합한 후 pH를 조절(6.0내지7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus amyloliquefaciens 5 내지 10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다. Grapefruit pulverized material and purified water were mixed at a certain ratio (1:1 to 8), and the pH was adjusted (6.0 to 7.5) and put into a fermentor and sterilized. 5 to 10% (w/w) of Bacillus amyloliquefaciens prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 자몽 발효물에 일정 비율(1내지5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the fermented grapefruit product, extracted by heating at 120° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 3-5. 오렌지 발효(추출)물(HR1903-FOR-BA-W)Preparation Example 3-5. Orange fermented (extracted) water (HR1903-FOR-BA-W)
오렌지 분쇄물과 정제수를 일정비율(1:1내지8)로 혼합한 후 pH를 조절(6.0내지7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus amyloliquefaciens 5내지10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다. Orange pulverized material and purified water were mixed at a certain ratio (1:1 to 8), and then the pH was adjusted (6.0 to 7.5) and put into a fermentor and sterilized. 5 to 10% (w/w) of Bacillus amyloliquefaciens prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 오렌지 발효물에 일정 비율(1내지5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the fermented orange product, extracted by heating at 120 ° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 4 : 바실러스 메가테리(Preparation Example 4: Bacillus megateri ( Bacillus megateriumBacillus megaterium ) 발효 추출물 제조) Preparation of fermented extract
제조예 4-1. 귤미성숙과(7월수확) 발효(추출)물(HR1903-F7C-BM-W)Preparation Example 4-1. Unripe tangerine fruits (harvested in July) fermented (extracted) water (HR1903-F7C-BM-W)
7월에 수확한 귤미성숙과 분쇄물과 정제수를 일정비율(1:1 내지 8)로 혼합한 후 pH를 조절(6.0 내지 7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus megaterium 5 내지 10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다. Unripe mandarin oranges harvested in July and pulverized water were mixed at a certain ratio (1: 1 to 8), and then the pH was adjusted (6.0 to 7.5) and put into a fermenter and sterilized. Bacillus megaterium 5 to 10% (w/w) prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 귤미성숙과 발효물에 일정 비율(1 내지 5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the unripe mandarin orange and fermented product, extracted by heating at 120 ° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 4-2. 귤미성숙과(8월수확) 발효(추출)물(HR1903-F8C-BM-W)Preparation Example 4-2. Unripe tangerine fruits (harvested in August) fermented (extracted) water (HR1903-F8C-BM-W)
8월에 수확한 귤미성숙과 분쇄물과 정제수를 일정비율(1:1 내지 8)로 혼합한 후 pH를 조절(6.0 내지 7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus megaterium 5 내지 10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다.Unripe tangerines harvested in August and pulverized water were mixed at a certain ratio (1: 1 to 8), and then the pH was adjusted (6.0 to 7.5) and put into a fermentor and sterilized. Bacillus megaterium 5 to 10% (w/w) prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 귤미성숙과 발효물에 일정 비율(1 내지 5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the unripe mandarin orange and fermented product, extracted by heating at 120 ° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 4-3. 귤(12월수확) 발효(추출)물(HR1903-FC-BM-W)Preparation Example 4-3. Tangerine (harvest in December) fermented (extracted) water (HR1903-FC-BM-W)
귤 분쇄물과 정제수를 일정비율(1:1 내지 8)로 혼합한 후 pH를 조절(6.0 내지 7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus megaterium 5 내지 10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다.After mixing the tangerine pulverized material and purified water at a certain ratio (1:1 to 8), the pH was adjusted (6.0 to 7.5) and put into a fermentor and sterilized. Bacillus megaterium 5 to 10% (w/w) prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 귤 발효물에 일정 비율(1 내지 5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the fermented tangerine, extracted by heating at 120 ° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 4-4. 자몽_발효(추출)물(HR1903-FGF-BM-W)Preparation Example 4-4. Grapefruit_fermented (extracted) water (HR1903-FGF-BM-W)
자몽 분쇄물과 정제수를 일정비율(1:1내지8)로 혼합한 후 pH를 조절(6.0내지7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus megaterium 5내지10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다.Grapefruit pulverized material and purified water were mixed at a certain ratio (1:1 to 8), and the pH was adjusted (6.0 to 7.5) and put into a fermentor and sterilized. Bacillus megaterium 5 to 10% (w/w) prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 자몽 발효물에 일정 비율(1내지5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the fermented grapefruit product, extracted by heating at 120° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
제조예 4-5. 오렌지_발효(추출)물(HR1903-FOR-BM-W)Preparation Example 4-5. Orange_fermented (extracted) water (HR1903-FOR-BM-W)
오렌지 분쇄물과 정제수를 일정비율(1:1내지8)로 혼합한 후 pH를 조절(6.0내지7.5)하여 발효기에 넣고 멸균하였다. 미리 준비된 종균 Bacillus megaterium 5내지10%(w/w)를 접종하고 32±2℃에서 36시간 동안 발효를 진행하였다.Orange pulverized material and purified water were mixed at a certain ratio (1:1 to 8), and then the pH was adjusted (6.0 to 7.5) and put into a fermentor and sterilized. Bacillus megaterium 5 to 10% (w/w) prepared in advance was inoculated and fermentation was performed at 32±2° C. for 36 hours.
상기 오렌지 발효물에 일정 비율(1내지5배) 물을 넣고 120℃에 4시간 동안 가열하여 추출한 후 여과하였다. 농축기로 10 내지 20 brix로 농축한 후 동결건조기를 이용하여 분말화하였다.A certain ratio (1 to 5 times) of water was added to the fermented orange product, extracted by heating at 120 ° C. for 4 hours, and then filtered. After concentrating to 10 to 20 brix with a concentrator, it was powdered using a freeze dryer.
실험예 1 : 세포 배양Experimental Example 1: Cell culture
세포주인 RAW264.7 macrophage cell은 American Type Culture Collection(ATCC, Manassas. USA)에서 구입하여 사용하였다.A cell line, RAW264.7 macrophage cell, was purchased from American Type Culture Collection (ATCC, Manassas. USA) and used.
세포의 배양을 위하여 10% Fetal Bovin Serum(FBS, Hyclone USA), 100 units/mL penicillin/Streptoycin(Hyclone, USA)이 함유된 DMEM(Dulbecco's modified of Eagle's medium, Hyclone,USA) 배지를 사용하였으며, 37℃, 5% CO2 존재하의 incubator(Thermo, Forma, Germany)에서 배양하였다.For cell culture, DMEM (Dulbecco's modified of Eagle's medium, Hyclone, USA) medium containing 10% Fetal Bovin Serum (FBS, Hyclone USA) and 100 units/mL penicillin/Streptoycin (Hyclone, USA) was used. It was cultured in an incubator (Thermo, Forma, Germany) in the presence of ℃, 5% CO 2 .
실험예 2 : 독성 평가(MTT assay)Experimental Example 2: Toxicity evaluation (MTT assay)
살아있는 세포 내 미토콘드리아의 탈수소효소에 의해 Tetrazolium salt(WST-1)에서 formazan으로 변화하는 것을 이용하여, 생성된 formazan의 농도를 spectrophotometer로 측정함으로써 세포 생존률을 측정하였다.Using the change from Tetrazolium salt (WST-1) to formazan by mitochondrial dehydrogenase in living cells, cell viability was measured by measuring the concentration of formazan produced using a spectrophotometer.
Ez-cytox(Dozen, Korea)을 이용하여 프로토콜에 따라 MTT assay를 수행하였다.MTT assay was performed according to the protocol using Ez-cytox (Dozen, Korea).
Raw264.7 세포를 5x103 cells/100μL로 96 well에 분주한 후 37℃, 5% CO2 배양기에서 24시간 동안 배양하였다.Raw264.7 cells were dispensed into 96 wells at 5x10 3 cells/100 μL and then cultured for 24 hours in a 37°C, 5% CO 2 incubator.
농도별로 시료를 처리한 후 24시간 동안 배양하였다. 배양 후 10% MTT 시약이 포함된 배지로 교체하고 37℃에서 1시간 동안 반응시킨 후 450nm 파장에서 microplate reader(Epoch, Biotek)로 측정하였다.Samples were treated for each concentration and then incubated for 24 hours. After incubation, the medium was replaced with a medium containing 10% MTT reagent, reacted at 37° C. for 1 hour, and measured with a microplate reader (Epoch, Biotek) at a wavelength of 450 nm.
각 시료에 대한 세포생존율에 의한 독성 평가는 3회 반복하여 측정하였으며, 그에 대한 평균값과 표준편차를 산출하였다.Toxicity evaluation by cell viability for each sample was measured three times, and the average value and standard deviation thereof were calculated.
도 1을 참조하면, 제조예 1 내지 4의 시료는 모든 농도 범위(10, 100, 1000 μg/mL)에서 세포 독성이 나타나지 않았다.Referring to Figure 1, the samples of Preparation Examples 1 to 4 did not show cytotoxicity in all concentration ranges (10, 100, 1000 μg / mL).
실험예 1 : 세포 배양Experimental Example 1: Cell culture
세포주인 RAW264.7 macrophage cell은 American Type Culture Collection(ATCC, Manassas. USA)에서 구입하여 사용하였다.A cell line, RAW264.7 macrophage cell, was purchased from American Type Culture Collection (ATCC, Manassas. USA) and used.
세포의 배양을 위하여 10% Fetal Bovin Serum(FBS, Hyclone USA), 100 units/mL penicillin/Streptoycin(Hyclone, USA)이 함유된 DMEM(Dulbecco's modified of Eagle's medium, Hyclone,USA) 배지를 사용하였으며, 37℃, 5% CO2 존재하의 incubator(Thermo, Forma, Germany)에서 배양하였다.For cell culture, DMEM (Dulbecco's modified of Eagle's medium, Hyclone, USA) medium containing 10% Fetal Bovin Serum (FBS, Hyclone USA) and 100 units/mL penicillin/Streptoycin (Hyclone, USA) was used. It was cultured in an incubator (Thermo, Forma, Germany) in the presence of ℃, 5% CO 2 .
실험예 2 : 독성 평가(MTT assay)Experimental Example 2: Toxicity evaluation (MTT assay)
살아있는 세포 내 미토콘드리아의 탈수소효소에 의해 Tetrazolium salt(WST-1)에서 formazan으로 변화하는 것을 이용하여, 생성된 formazan의 농도를 spectrophotometer로 측정함으로써 세포 생존률을 측정하였다.Using the change from Tetrazolium salt (WST-1) to formazan by mitochondrial dehydrogenase in living cells, cell viability was measured by measuring the concentration of formazan produced using a spectrophotometer.
Ez-cytox(Dozen, Korea)을 이용하여 프로토콜에 따라 MTT assay를 수행하였다.MTT assay was performed according to the protocol using Ez-cytox (Dozen, Korea).
Raw264.7 세포를 5x103 cells / 100μL로 96 well에 분주한 후 37℃, 5% CO2 배양기에서 24시간 동안 배양하였다.Raw264.7 cells were dispensed into 96 wells at 5x10 3 cells / 100 μL and then cultured for 24 hours in a 37°C, 5% CO 2 incubator.
농도별로 시료를 처리한 후 24시간 동안 배양하였다. 배양 후 10% MTT 시약이 포함된 배지로 교체하고 37℃에서 1시간 동안 반응시킨 후 450 nm 파장에서 microplate reader(Epoch, Biotek)로 측정하였다.Samples were treated for each concentration and then incubated for 24 hours. After incubation, the medium was replaced with a medium containing 10% MTT reagent, reacted at 37° C. for 1 hour, and measured with a microplate reader (Epoch, Biotek) at a wavelength of 450 nm.
각 시료에 대한 세포생존율에 의한 독성 평가는 3회 반복하여 측정하였으며, 그에 대한 평균값과 표준편차를 산출하였다.Toxicity evaluation by cell viability for each sample was measured three times, and the average value and standard deviation thereof were calculated.
도 1을 참조하면, 제조예 1 내지 4의 추출물은 모든 농도(10, 100, 1000 μg/mL) 범위에서 세포 독성이 나타나지 않았다.Referring to Figure 1, the extracts of Preparation Examples 1 to 4 did not show cytotoxicity in all concentrations (10, 100, 1000 μg / mL) range.
실험예 3 : 면역 효능 평가(NO assay)Experimental Example 3: Immune efficacy evaluation (NO assay)
Nitric Oxide(NO)의 농도 측정을 위해 그리스 반응(griess reaction)을 이용하여 540nm에서 흡광도를 측정하였다.Absorbance was measured at 540 nm using a grease reaction to measure the concentration of nitric oxide (NO).
N-(1-naphthyl)-ethylenediamine, sulfanilamide, 및 NO2 -가 반응하여 azo couling을 이루는데, 두 개의 링 형태가 540nm의 파장에서 최대 흡광도 값을 가진다.N-(1-naphthyl)-ethylenediamine, sulfanilamide, and NO 2 - react to form azo couling, and the two ring shapes have the maximum absorbance value at a wavelength of 540 nm.
Raw264.7 세포를 5x104 cells / 250μL로 24 well plate 에 분주하고 37℃, 5% CO2 배양기에서 24시간 동안 배양하였다.Raw264.7 cells were dispensed into a 24 well plate at 5x10 4 cells / 250 μL and cultured for 24 hours in a 37°C, 5% CO 2 incubator.
시료를 농도별로 처리하고 48시간 동안 CO2 배양기에 배양하였다. 세포배양 상등액 50 μL를 취해 N1 buffer 50μL를 혼합하고 5 내지 10분 상온에 반응시켰다. Samples were treated by concentration and incubated in a CO 2 incubator for 48 hours. 50 μL of the cell culture supernatant was mixed with 50 μL of N1 buffer and reacted at room temperature for 5 to 10 minutes.
N2 buffer 50 μL를 혼합하여 10분간 반응시킨 후 microplate reader(Epoch, Biotek)를 이용하여 560nm 흡광도를 측정하였다.After mixing 50 μL of N2 buffer and reacting for 10 minutes, the absorbance at 560 nm was measured using a microplate reader (Epoch, Biotek).
동일한 방법으로 3회 측정하였으며, 그에 대한 평균값과 표준편차를 구하였다.It was measured three times in the same way, and the average value and standard deviation thereof were obtained.
도 2A를 참조하면, 제조예 1-1의 경우, 대조군 대비 NO 생성능이 100 μg/mL에서 약 9%, 1000 μg/mL에서 약 42% NO 생성능이 증가하였다.Referring to Figure 2A, in the case of Preparation Example 1-1, compared to the control group, NO production ability increased by about 9% at 100 μg / mL and about 42% at 1000 μg / mL.
제조예 1-4의 경우, 대조군 대비 NO 생성능이 100 μg/mL에서 약 13%, 1000 μg/mL에서 약 71% NO 생성능이 증가하였다.In the case of Preparation Examples 1-4, NO production ability increased by about 13% at 100 μg / mL and about 71% at 1000 μg / mL compared to the control group.
도 2B를 참조하면, 제조예 2-1의 경우, 대조군 대비 NO 생성능이 100 μg/mL에서 약 20%, 1000 μg/mL에서 약 48% NO 생성능이 증가하였다.Referring to Figure 2B, in the case of Preparation Example 2-1, compared to the control group, NO production ability increased by about 20% at 100 μg / mL and about 48% at 1000 μg / mL.
제조예 2-4의 경우, 대조군 대비 NO 생성능이 100 μg/mL에서 약 11%, 1000 μg/mL에서 약 42% NO 생성능이 증가하였다.In the case of Preparation Examples 2-4, NO production ability increased by about 11% at 100 μg / mL and about 42% at 1000 μg / mL compared to the control group.
제조예 3-1의 경우, 대조군 대비 NO 생성능이 100 μg/mL에서 약 18%, 1000 μg/mL에서 약 43% NO 생성능이 증가하였다.In the case of Preparation Example 3-1, NO production ability increased by about 18% at 100 μg / mL and about 43% at 1000 μg / mL compared to the control group.
제조예 3-5의 경우, 대조군 대비 NO 생성능이 100 μg/mL에서 약 15%, 1000 μg/mL에서 약 36% NO 생성능이 증가하였다.In the case of Preparation Examples 3-5, NO production ability increased by about 15% at 100 μg / mL and about 36% at 1000 μg / mL compared to the control group.
제조예 4-1의 경우, 대조군 대비 NO 생성능이 100 μg/mL에서 약 8%, 1000 μg/mL에서 약 23% NO 생성능이 증가하였다.In the case of Preparation Example 4-1, NO production ability increased by about 8% at 100 μg/mL and about 23% at 1000 μg/mL compared to the control group.
상기 각 발효 추출물은 발효 시간에 따라 면역 활성이 일부 변화하였으나, 발효하지 않은 시트러스 추출물 소재 대비 NO 생성능이 현저히 증진되었다.Although the immune activity of each of the fermented extracts was partially changed according to the fermentation time, the NO production ability was significantly improved compared to the non-fermented citrus extract material.
상기 결과는 시트러스 추출물의 면역 증진 활성은 간균 균주의 발효에 의해 현저히 증대될 수 있음을 시사한다.These results suggest that the immune-enhancing activity of the citrus extract can be remarkably enhanced by fermentation of the bacillus strain.
실험예 4 : 면역 효능 평가(TNF-a 생성량 평가)Experimental Example 4: Immunity efficacy evaluation (TNF-a production evaluation)
면역 증가 효능을 알아보기 위해 Raw264.7 세포를 5x104 cells/250μL로 24 well plate 에 분주하고 37℃, 5% CO2 배양기에서 24시간 동안 배양하였다.In order to examine the efficacy of increasing immunity, Raw264.7 cells were dispensed into a 24 well plate at 5x10 4 cells/250 μL and cultured for 24 hours in a 37°C, 5% CO 2 incubator.
시료를 농도별로 처리하고 4시간동안 CO2 배양기에 배양한 후 세포 부유액을 원심 분리하여 세포들은 침전시켜 상층액을 수집하였다.Samples were treated for each concentration and cultured in a CO 2 incubator for 4 hours. Then, the cell suspension was centrifuged to precipitate the cells, and the supernatant was collected.
상층액 내 TNF-a 및 IL-6 생성량을 ELISA kit(R&D systems Inc., Minneapolis, MN, USA를 이용하여 사용자 매뉴얼에 기재된 방법대로 정량하였다.The amount of TNF-a and IL-6 produced in the supernatant was quantified using an ELISA kit (R&D systems Inc., Minneapolis, MN, USA) according to the method described in the user manual.
도 3A를 참조하면, 제조예 1-1의 경우, 대조군 대비 TNF-a 양이 10 μg/mL에서 7 pg/mL, 100 μg/mL에서 78 pg/mL, 1000 μg/mL에서 323 pg/mL 증가하였다.3A, in the case of Preparation Example 1-1, the amount of TNF-a compared to the control group was 7 pg/mL at 10 μg/mL, 78 pg/mL at 100 μg/mL, and 323 pg/mL at 1000 μg/mL. increased.
제조예 1-4의 경우, 대조군 대비 TNF-a 양이 10 μg/mL에서 38 pg/mL, 100 μg/mL에서 271 pg/mL, 1000 μg/mL에서 475 pg/mL 증가하였다.In the case of Preparation Examples 1-4, the amount of TNF-a increased by 38 pg/mL at 10 μg/mL, 271 pg/mL at 100 μg/mL, and 475 pg/mL at 1000 μg/mL compared to the control group.
도 3B를 참조하면, 제조예 2-1의 경우, 대조군 대비 TNF-a 양이 100 μg/mL에서 65 pg/mL, 1000 μg/mL에서 309 pg/mL 증가하였다.Referring to Figure 3B, in the case of Preparation Example 2-1, compared to the control group, the amount of TNF-a increased by 65 pg/mL at 100 μg/mL and by 309 pg/mL at 1000 μg/mL.
제조예 2-4의 경우, 대조군 대비 TNF-a 양이 100 μg/mL에서 72 pg/mL, 1000 μg/mL에서 297 pg/mL 증가하였다.In the case of Preparation Examples 2-4, the amount of TNF-a increased by 72 pg/mL at 100 μg/mL and 297 pg/mL at 1000 μg/mL compared to the control group.
제조예 3-1의 경우, 대조군 대비 TNF-a 양이 100 μg/mL에서 58 pg/mL, 1000 μg/mL에서 326 pg/mL 증가하였다.In the case of Preparation Example 3-1, the amount of TNF-a compared to the control group increased by 58 pg/mL at 100 μg/mL and by 326 pg/mL at 1000 μg/mL.
제조예 3-5의 경우, 대조군 대비 TNF-a 양이 100 μg/mL에서 43 pg/mL, 1000 μg/mL에서 198 pg/mL 증가하였다.In the case of Preparation Examples 3-5, the amount of TNF-a compared to the control group increased by 43 pg/mL at 100 μg/mL and by 198 pg/mL at 1000 μg/mL.
상기 각 발효 추출물은 발효 시간에 따라 면역 활성이 일부 변화하였으나, 발효하지 않은 시트러스 추출물 소재 대비 TNF-a 생성능이 현저히 증진되었다.Although the immune activity of each of the fermented extracts was partially changed according to the fermentation time, the ability to produce TNF-a was significantly improved compared to the non-fermented citrus extract material.
상기 결과는 시트러스 추출물의 면역 증진 활성은 간균 발효에 의해 현저히 증대될 수 있음을 시사한다.These results suggest that the immune-enhancing activity of the citrus extract can be remarkably enhanced by fermentation with bacilli.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 예를 들어, 단일형으로 설명되어 있는 각 구성 요소는 분산되어 실시될 수도 있으며, 마찬가지로 분산된 것으로 설명되어 있는 구성 요소들도 결합된 형태로 실시될 수 있다.The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.
본 발명의 범위는 후술하는 청구범위에 의하여 나타내어지며, 청구범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.
Claims (7)
상기 시트러스(citrus)는 풋귤, 감귤, 자몽, 및 오렌지로 이루어진 군에서 하나 이상 선택된 것인, 조성물.According to claim 1,
The composition of claim 1, wherein the citrus is at least one selected from the group consisting of green tangerines, tangerines, grapefruits, and oranges.
상기 발효 추출물은 바실러스 속(Bacillus spp.) 균주를 발효시켜 수득한 것인, 조성물.According to claim 1,
The fermented extract is obtained by fermenting a Bacillus spp. strain, the composition.
상기 균주는 바실러스 서브틸리스(Bacillus subtilis), 바실러스 사펜시스(Bacillus safensis), 바실러스 아밀로리퀴페시언스(Bacillus amyloliquefaciens), 및 바실러스 메가테리(Bacillus megaterium)로 이루어진 군에서 하나 이상 선택된 것인, 조성물.According to claim 3,
The strain is Bacillus subtilis ( Bacillus subtilis ), Bacillus sapensis ( Bacillus safensis ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), and Bacillus megateri ( Bacillus megaterium ) One or more selected from the group consisting of, a composition .
상기 시트러스(citrus)는 풋귤, 감귤, 자몽, 및 오렌지로 이루어진 군에서 하나 이상 선택된 것인, 건강기능식품.According to claim 5,
The citrus (citrus) is at least one selected from the group consisting of green tangerines, tangerines, grapefruits, and oranges, health functional food.
상기 발효 추출물은 바실러스 속(Bacillus spp.) 균주를 발효시켜 수득한 것인 건강기능식품.According to claim 5,
The fermented extract is a health functional food obtained by fermenting a strain of Bacillus spp .
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