KR102178556B1 - Prebiotics composition for improving intestinal microflora comprising garlic peel extract and funtional food comprising the same - Google Patents
Prebiotics composition for improving intestinal microflora comprising garlic peel extract and funtional food comprising the same Download PDFInfo
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- KR102178556B1 KR102178556B1 KR1020190032160A KR20190032160A KR102178556B1 KR 102178556 B1 KR102178556 B1 KR 102178556B1 KR 1020190032160 A KR1020190032160 A KR 1020190032160A KR 20190032160 A KR20190032160 A KR 20190032160A KR 102178556 B1 KR102178556 B1 KR 102178556B1
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- Prior art keywords
- inulin
- garlic peel
- peel extract
- extract
- food
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Classifications
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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
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
- A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
- A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
- A23G3/48—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds containing plants or parts thereof, e.g. fruits, seeds, 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
- 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
- A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
- A23V2200/3202—Prebiotics, ingredients fermented in the gastrointestinal tract by beneficial microflora
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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
- A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
- A23V2200/3204—Probiotics, living bacteria to be ingested for action in the digestive tract
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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
- A23V2300/14—Extraction
Abstract
본 발명은 이눌린을 핵심 성분으로 함유하는 마늘껍질 추출물을 유효성분으로 포함하는 장내균총 개선용 프리바이오틱스 조성물 및 이를 포함함으로써 프로바이오틱스의 생육을 촉진시켜 장내균총을 개선시킬 수 있는 기능성 식품에 관한 것이다.The present invention relates to a prebiotic composition for improving intestinal flora including a garlic peel extract containing inulin as a core component as an active ingredient, and a functional food capable of improving the intestinal flora by promoting the growth of probiotics by including the same.
Description
본 발명은 마늘껍질 추출물을 포함하는 장내균총 개선용 프리바이오틱스 조성물 및 이를 포함하는 기능성 식품에 관한 것으로, 보다 상세하게는 이눌린(inulin)을 핵심 성분으로 함유하는 마늘껍질 추출물을 유효성분으로 포함하는 장내균총 개선용 프리바이오틱스 조성물 및 이를 포함함으로써 프로바이오틱스의 생육을 촉진시켜 장내균총을 개선시킬 수 있는 기능성 식품에 관한 것이다.The present invention relates to a prebiotic composition for improving intestinal flora including garlic peel extract and a functional food including the same, and more particularly, to a garlic peel extract containing inulin as a core component as an active ingredient. It relates to a prebiotic composition for improving intestinal flora, and a functional food capable of improving the intestinal flora by promoting the growth of probiotics by including the same.
프로바이오틱스(probiotics)는 장건강에 이로운 장내 미생물로서, 대부분은 유산균이며 장내 pH를 감소시켜 유해세균이 자라지 못하는 환경을 조성한다. 또한, 프로바이오틱스의 먹이성분은 프리바이오틱스(prebiotics)라고 불리는 난소화성 올리고당으로, 프로바이오틱스의 생육을 촉진한다. Probiotics are intestinal microbes that are beneficial to intestinal health, and most of them are lactic acid bacteria and reduce intestinal pH to create an environment where harmful bacteria cannot grow. In addition, the food component of probiotics is an indigestible oligosaccharide called prebiotics, which promotes the growth of probiotics.
프리바이오틱스는 장내에서 유익한 박테리아(프로바이오틱스)의 생장을 돕는 난소화성 성분으로서, 프로바이오틱스(probiotics)의 영양원이 되어 장내 환경을 개선하는 데 도움을 주는 물질이다.Prebiotics are indigestible ingredients that help the growth of beneficial bacteria (probiotics) in the intestine, and are substances that help improve the intestinal environment by becoming a nutrient source for probiotics.
이러한 프리바이오틱스는 올리고당(프락토올리고당, 자일로올리고당 이눌린)과 같은 다당류 탄수화물로 이루어져 있으며, 대부분이 식이섬유의 형태로 존재한다. These prebiotics consist of polysaccharide carbohydrates such as oligosaccharides (fructooligosaccharides, xyloligosaccharides inulin), and most of them exist in the form of dietary fiber.
농산물이나 식품으로부터 프라바이오틱스를 분리한 종래기술로는 더덕 추출물을 함유하는 장 건강 개선용 조성물(특허문헌 1), 홍국 추출물을 포함하는 장 건강 개선용 조성물(특허문헌 2), 락토바실러스 균주의 안전성 증진을 위한 프리바이오틱스 조성물 및 이를 이용한 안정화 증진방법(특허문헌 3) 등이 개시되어 있다.Conventional techniques for separating prabiotics from agricultural products or foods include a composition for improving intestinal health containing Deodeok extract (Patent Document 1), a composition for improving intestinal health including red yeast extract (Patent Document 2), and Lactobacillus strains. A prebiotic composition for improving safety and a method for improving stabilization using the same (Patent Document 3) are disclosed.
그러나, 프리바이오틱스를 마늘 껍질등과 같은 농업 부산물에서 분리할 수 있다면 부산물 처리는 물론 경제적 부가가치를 높일 수 있는 바, 마늘 껍질등과 같은 농업 부산물을 프리바이오틱스 소재로 활용하기 위한 연구개발이 절실히 요구되고 있다.However, if prebiotics can be separated from agricultural by-products such as garlic peels, it can increase economic added value as well as treatment of by-products. Therefore, research and development to utilize agricultural by-products such as garlic peels as prebiotics is urgently needed. Is required.
본 발명은 상기와 같은 종래기술의 문제점을 해결하기 위하여 안출된 것으로서, 본 발명의 목적은 부산물인 마늘 껍질로부터 얻은 추출물을 프리바이오틱스 소재로 활용하기 위한 프리바이오틱스 조성물을 제공하는 것이다.The present invention has been devised to solve the problems of the prior art as described above, and an object of the present invention is to provide a prebiotic composition for utilizing an extract obtained from a by-product garlic peel as a prebiotic material.
본 발명의 다른 목적은 상기 프리바이오틱스 조성물을 활용하여 프로바이오틱스의 성장을 촉진시킴으로써 장내균총을 개선시킬 수 있는 기능성 식품을 제공하는 것이다.Another object of the present invention is to provide a functional food capable of improving the intestinal flora by promoting the growth of probiotics using the prebiotic composition.
본 발명은 상기와 같은 목적을 달성하기 위하여, 마늘껍질 추출물을 유효성분으로 포함하는 장내균총 개선용 프리바이오틱스 조성물을 제공한다.The present invention provides a prebiotic composition for improving intestinal flora, comprising a garlic peel extract as an active ingredient in order to achieve the above object.
또한, 본 발명은 상기 프리바이오틱스 조성물을 포함하는 기능성 식품을 제공한다.In addition, the present invention provides a functional food comprising the prebiotics composition.
현재 대부분의 프리바이오틱스는 화학적으로 합성되고 있는데, 본 발명에 의하면 식품 부산물인 마늘 껍질로부터 프리바이오틱스를 추출하여 사용할 수 있기 때문에, 새로운 프리바이오틱스 소재를 제공할 수 있을 뿐만 아니라, 부산물 처리에 소요되는 비용을 절감할 수 있는 장점이 있다. Currently, most prebiotics are chemically synthesized, but according to the present invention, since prebiotics can be extracted and used from garlic skin, which is a food by-product, it is possible to provide a new prebiotic material, as well as to treat by-products. There is an advantage of reducing the cost required.
또한, 프리바이오틱스가 함유된 식품은 일상 생활에서 섭취하는 프리바이오틱스와 같은 의미를 지니기 때문에, 프리바이오틱스 소재로 본 발명에 의한 마늘껍질 추출물을 포함하는 쿠키 등과 같은 기능성 식품은 새로운 개념의 프리바이오틱스와 식품의 결합으로서, 건강 관리를 위한 시작점을 제공할 수 있다.In addition, since foods containing prebiotics have the same meaning as prebiotics consumed in daily life, functional foods such as cookies containing the garlic peel extract according to the present invention as prebiotics are a new concept of free As a combination of biotics and food, it can provide a starting point for health care.
도 1은 프로바이오틱스에 대한 마늘껍질 추출물의 프리바이오틱스 특성을 나타낸 것이다.
도 2는 마늘껍질 추출물과 표준 이눌린의 HPLC-RID 비교 분석 결과를 나타낸 것으로서, 추가로 미지의 피크(UK)가 미정 샘플에서 확인되었다.
도 3a는 마늘껍질 추출물과 비교한 표준 이눌린의 1H NMR 상대 분석 결과를 나타낸 것으로서, 시료에서 알려지지 않은 피크가 확인되었다.
도 3b는 마늘껍질 추출물과 비교한 표준 이눌린의 13C NMR 상대 분석 결과를 나타낸 것으로서, 시료에서 알려지지 않은 피크가 확인되었다.
도 4는 이눌린과 마늘껍질 조추출물의 정량 분석 및 HPLC 기반 비교 분석 결과를 나타낸 것이다.
도 5a는 이눌린과 마늘껍질 정제 추출물(GPPE)의 1H NMR 비교 분석 결과를 나타낸 것이다.
도 5b는 이눌린과 마늘껍질 정제 추출물(GPPE)의 13C NMR 비교 분석 결과를 나타낸 것이다.
도 6은 이눌린과 마늘껍질 정제 추출물(GPPE)의 FTIR 비교 분석 결과를 나타낸 것이다.
도 7은 이눌린과 마늘껍질 정제 추출물(GPPE)의 입자크기 분석 결과를 나타낸 것이다.
도 8은 이눌린과 마늘껍질 정제 추출물(GPPE)의 X선 회절패턴 분석 결과를 나타낸 것이다.
도 9는 이눌린과 마늘껍질 정제 추출물(GPPE)의 열 중량 분석 결과를 나타낸 것이다.
도 10은 투과전자현미경(TEM) 이미지를 나타낸 것으로서, A는 이눌린, B는 마늘껍질 추출물을 나타낸 것이다.
도 11은 상업용 이눌린과 마늘껍질 추출물의 프로바이오틱스 균주 생육 증진 효능 평가 결과를 나타낸 것이다.
도 12는 상업용 이눌린 및 마늘껍질 추출물 처리시 C. elegans 장관 내 Lactobacillus rhamnosus 집락 형성능을 나타낸 것이다.
도 13은 Lactobacillus rhamnosus에 대한 이눌린과 마늘껍질 추출물(GPE)을 결합시킨 쿠키의 생육증진 효능을 나타낸 것이다.
도 14는 투과전자현미경(TEM) 이미지를 나타낸 것으로서, a는 쿠키(대조군), b는 이눌린을 포함하는 쿠키에서 추출한 프리바이오틱스, c는 마늘껍질 추출물을 포함하는 쿠키에서 추출한 프리바이오틱스를 나타낸 것이다.Figure 1 shows the properties of the prebiotics of garlic peel extract for probiotics.
FIG. 2 shows the comparative analysis result of HPLC-RID between garlic peel extract and standard inulin, and an unknown peak (UK) was further confirmed in the undecided sample.
Figure 3a shows the relative 1 H NMR analysis of standard inulin compared to the garlic peel extract, an unknown peak was confirmed in the sample.
Figure 3b shows the results of the 13 C NMR relative analysis of standard inulin compared to the garlic peel extract, an unknown peak was confirmed in the sample.
Figure 4 shows the results of quantitative analysis and HPLC-based comparative analysis of inulin and crude garlic peel extract.
Figure 5a shows the 1 H NMR comparative analysis results of inulin and garlic peel purified extract (GPPE).
Figure 5b shows the results of 13 C NMR comparative analysis of inulin and garlic peel purified extract (GPPE).
6 shows the FTIR comparative analysis results of inulin and garlic peel purified extract (GPPE).
Figure 7 shows the particle size analysis results of inulin and garlic peel purified extract (GPPE).
Figure 8 shows the results of X-ray diffraction pattern analysis of inulin and garlic peel purified extract (GPPE).
9 shows the results of thermogravimetric analysis of inulin and garlic peel purified extract (GPPE).
10 shows a transmission electron microscope (TEM) image, where A is inulin, B is a garlic peel extract.
11 shows the results of evaluation of the efficacy of commercial inulin and garlic peel extract for promoting probiotic strain growth.
12 is a commercial inulin and Lactobacillus rhamnosus in C. elegans intestinal tract when treated with garlic peel extract It shows the ability to form colonies.
Figure 13 shows the growth-promoting efficacy of cookies in which inulin and garlic peel extract (GPE) are combined with Lactobacillus rhamnosus .
14 shows a transmission electron microscope (TEM) image, where a is a cookie (control), b is a prebiotic extracted from a cookie containing inulin, and c is a prebiotic extracted from a cookie containing a garlic peel extract. will be.
본 발명의 일 구현예는 마늘껍질 추출물을 유효성분으로 포함하는 장내균총 개선용 프리바이오틱스 조성물에 관한 것이다.One embodiment of the present invention relates to a prebiotic composition for improving intestinal flora comprising a garlic peel extract as an active ingredient.
본 발명에서, "장내균총 개선용"이라 함은, 장내 유익균의 증식 또는 생장은 촉진하고 장내 유해균의 증식 또는 생장은 억제하되, 장내 유익균과 유해균의 균형을 유지하는 것을 의미한다.In the present invention, the term "for improving the intestinal flora" means promoting the proliferation or growth of beneficial bacteria in the intestine and inhibiting the proliferation or growth of harmful bacteria in the intestine, but maintaining a balance between beneficial bacteria in the intestine and harmful bacteria.
상기 "장내 유익균"이라 함은, 장내에 서식하고 있으면서 인체에 유익한 효능을 미치는 미생물을 총칭할 수 있다. 예컨대, 장내 유익균은 프로바이오틱스를 포함할 수 있다.The term "intestinal beneficial bacteria" may collectively refer to microorganisms that live in the intestine and have beneficial effects on the human body. For example, beneficial bacteria in the intestine may include probiotics.
상기 "장내 유해균"이라 함은, 장내에서 서식하고 있으면서 장염 등과 같이 인체에 해로운 효과를 미치는 미생물을 총칭할 수 있는데, 대장균(Escherichia coli), 클로스트리듐 속(Clostridium sp.), 스타필로코쿠스 속(Staphylococcus sp.) 등을 예로 들 수 있다.The term "intestinal harmful bacteria" may collectively refer to microorganisms that live in the intestine and have a harmful effect on the human body such as enteritis, etc., Escherichia coli ), Clostridium sp., Staphylococcus sp., and the like are exemplified.
또한 본 발명에서, "프로바이오틱스"라 함은, 체내에서 건강에 좋은 영향을 미치는 미생물을 의미할 수 있는데, 락토바실러스 속(Lactobacillus sp.), 락토코커스 속(Lactococcus sp.), 스트렙토코커스 속(Streptococcus sp.), 엔테로코커스 속(Enterococcus sp.), 페디오코커스 속(Pediococcus sp.), 비피도박테리움 비피둠 속(Bifidobacterium sp.) 등을 예로 들 수 있다. In addition, in the present invention, the term "probiotic" is, there can mean the microorganism on a good effect on health in the body, Lactobacillus genus (Lactobacillus sp.), Lactococcus genus (Lactococcus sp.), Streptococcus genus (Streptococcus sp.), Enterococcus sp.), Pediococcus sp.), Bifidobacterium sp., and the like.
본 발명에서, "프리바이오틱스"라 함은, 유익균을 포함하는 미생물에 의해 이용되어 미생물의 생육이나 활성을 촉진시킴으로써, 숙주의 건강에 좋은 효과를 나타내게 하는 성분을 의미할 수 있다.In the present invention, the term "prebiotics" may mean a component that is used by microorganisms including beneficial bacteria to promote growth or activity of microorganisms, thereby exhibiting a good effect on the health of a host.
본 발명의 상기 프리바이오틱스 조성물에서, 상기 마늘껍질 추출물은 탄소수 1 내지 4의 저급 알코올, 바람직하게는 에탄올일 수 있다.In the prebiotic composition of the present invention, the garlic peel extract may be a lower alcohol having 1 to 4 carbon atoms, preferably ethanol.
본 발명의 상기 프리바이오틱스 조성물에 포함되는 상기 마늘껍질 추출물은 이눌린을 유효성분으로 포함할 수 있다.The garlic peel extract included in the prebiotic composition of the present invention may contain inulin as an active ingredient.
본 발명의 상기 프리바이오틱스 조성물은 이눌린을 더 포함할 수 있는데, 마늘껍질 추출물과 상기 이눌린을 포함할 경우, 프로바이오틱스균의 생육을 촉진시킬 수 있다.The prebiotic composition of the present invention may further include inulin, and when the garlic peel extract and the inulin are included, the growth of probiotic bacteria may be promoted.
본 발명의 다른 구현예는 상기의 프리바이오틱스 조성물을 포함하는 기능성 식품에 관한 것이다.Another embodiment of the present invention relates to a functional food comprising the prebiotic composition.
본 발명에서, 상기 "기능성 식품"이라 함은, 건강에 유용한 효과를 주는 식품 소재나 성분을 사용하여 일반적인 형태로 제조 및 가공 과정을 거쳐 일반적인 방법에 의하여 섭취하거나, 식품으로서 일상적으로 섭취하는 것을 의미한다.In the present invention, the term "functional food" refers to ingestion by a general method or daily intake as a food through a manufacturing and processing process in a general form using food materials or ingredients that have a useful effect on health. do.
본 발명에서, 상기 기능성 식품은 장내균총 개선용일 수 있는데, 상기 장내균총이 개선됨으로써, 변비, 설사 또는 잘 관련 질환을 개선할 수 있다.In the present invention, the functional food may be for improving the intestinal flora, and by improving the intestinal flora, constipation, diarrhea or well-related diseases can be improved.
본 발명에서, 상기 식품은 가열 식품 또는 비가열 식품일 수 있는데, 이는 상기 프리바이오틱스 조성물에 포함되는 마늘껍질 추출물의 열 안정성이 우수하기 때문이다.In the present invention, the food may be a heated food or a non-heated food, because the heat stability of the garlic peel extract contained in the prebiotic composition is excellent.
본 발명에서, 상기 식품으로는 식물성 식품, 동물성 식품 등을 불문하고, 전분질 식품, 단백질 식품, 지방질 식품을 불문하며, 천연식품, 가공식품, 음료 등을 불문하고, 농산식품, 축산식품, 수산식품 등을 불문하며, 주식류, 부식류, 기호식품, 조미료 등을 불문하고, 통조림식품, 레토르트식품, 즉석식품, 건조식품, 냉동식품, 발효식품 등을 불문한다.In the present invention, the foods include plant foods, animal foods, etc., starchy foods, protein foods, fatty foods, natural foods, processed foods, beverages, etc., agricultural foods, livestock foods, fishery foods Regardless of whether or not, stocks, side dishes, favorite foods, seasonings, etc., canned food, retort food, instant food, dried food, frozen food, fermented food, etc.
본 발명에서, 상기 식품은 제과·제빵류, 유가공품, 식육제품, 어육제품, 두부류 또는 묵류, 식용유지류, 면류, 다류, 음료류, 특수영양식품, 건강보조식품, 조미식품, 인삼제품류, 김치절임식품, 건포류, 과일, 야채, 과일 또는 야채의 건조제품, 절단제품, 과일쥬스, 야채쥬스, 이들의 혼합쥬스, 면류, 축산가공식품, 수산가공식품, 유가공식품, 발효유식품, 두류식품, 곡류식품, 미생물발효식품, 제과제빵, 양념류, 육가공류, 음료 등을 예로 들 수 있으나, 이에 한정되는 것은 아니다. In the present invention, the food is confectionery and bakery, dairy products, meat products, fish meat products, tofu or jelly, edible oils and fats, noodles, teas, beverages, special nutritional foods, health supplements, seasoning foods, ginseng products, kimchi pickles , Raisins, fruits, vegetables, dried fruits or vegetables, cut products, fruit juices, vegetable juices, mixed juices thereof, noodles, processed livestock foods, processed seafood, dairy processed foods, fermented milk foods, bean foods, grain foods , Microbial fermented foods, confectionery, condiments, processed meats, beverages, etc. may be exemplified, but are not limited thereto.
본 발명에서, 상기 식품의 형태는 고형, 분말, 과립, 정제, 캡슐, 액상 또는 음료 형태일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the form of the food may be solid, powder, granule, tablet, capsule, liquid or beverage, but is not limited thereto.
본 발명에서, 상기 식품은 쿠키일 수 있다.In the present invention, the food may be a cookie.
이하, 본 발명을 실시예를 통하여 상세히 설명한다. 그러나 이들 실시예는 본 발명을 구체적으로 설명하기 위한 것일 뿐, 본 발명의 범위가 이들 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.
<실시예 1> <Example 1>
1. 재료 및 방법1. Materials and methods
(1). 부산물 시료 제조(Preparation of by-product samples)(One). Preparation of by-product samples
마늘 껍질을 그늘에서 잘 말린 뒤, 말린 샘플을 부수었다. 분쇄된 분말은50ml 코니칼 튜브(conical tube)에 보관하였다.After drying the garlic peel well in the shade, the dried sample was crushed. The pulverized powder was stored in a 50 ml conical tube.
(2). 부산물 추출(Extraction of by-product samples)(2). Extraction of by-product samples
시료로 사용한 마늘 껍질은 Soxylet 방법(Palaniappan, A et al 2017)으로 탈지하였다. 탈지된 시료 50g에 100ml 아세트산 나트륨 완충액(sodium acetate buffer)과 1mg α-아밀레이스(alpha amylase)를 첨가한 후 95℃에서 12시간 동안 냉각한 뒤, 100mg 글루코아밀라아제를 혼합하여 55℃에서 48시간 동안 가열하였다. Garlic peels used as samples were degreased by the Soxylet method (Palaniappan, A et al 2017). After adding 100 ml sodium acetate buffer and 1 mg α-amylase to 50 g of the degreased sample, cooling at 95° C. for 12 hours, then mixing 100 mg glucoamylase at 55° C. for 48 hours. Heated.
시료를 3000rpm에서 20분간 원심 분리하여 파괴시킨 뒤 추출한 후, 시료 100g과 DDW 250ml를 섞고 실온에서 2시간 동안 보관하였다. 본 용액을 3000rpm에서 20분간 원심 분리한 후, 상층액을 제거하고 99% 에탄올을 3배 넣어 잘 섞은 다음, 에탄올을 증발시켜 추출물을 얻었다.The sample was centrifuged at 3000 rpm for 20 minutes and then extracted, and then 100 g of the sample and 250 ml of DDW were mixed and stored at room temperature for 2 hours. After centrifuging this solution at 3000 rpm for 20 minutes, the supernatant was removed, 99% ethanol was added 3 times to mix well, and the ethanol was evaporated to obtain an extract.
(3). 생육증진 효능(3). Growth promotion effect
생육증진 시험은 액체 배지에서 배양한 8종의 균주(Pediococcus acidilactici SDL1405, Pediococcus acidilactici SDL1406, Pediococcus acidilactici SDL1402, Streptococcus thermophilus SCML300, Streptococcus thermophilus SCML337, Weissella cibaria SCCB2306, Enterococcus faecium SC54, Lactobacillus rhamnosus JDFM6)를 사용하였다. Growth promotion test used a strain of 8 species cultured in liquid medium (Pediococcus acidilactici SDL1405, Pediococcus acidilactici SDL1406 , Pediococcus acidilactici SDL1402, Streptococcus thermophilus SCML300, Streptococcus thermophilus SCML337, Weissella cibaria SCCB2306, Enterococcus faecium SC54, Lactobacillus rhamnosus JDFM6).
분광 광도계를 사용하여 600nm에서 12시간 동안 12-웰 셀 플레이트를 사용하여 배양한 후 흡광도를 측정하였다. 프리바이오틱스 용액과 10- 1으로 희석한 배양액을 혼합한 것들과, 프리바이오틱스 용액과 일반적인 영양 배지(대조군)를 섞은 것을 비교 분석하였다.After incubation using a 12-well cell plate at 600 nm using a spectrophotometer for 12 hours, the absorbance was measured. It was compared to those mixed with 1 a mixture of a culture liquid diluted and, prebiotics solution and common nutrient medium (control) - prebiotics solution and 10.
a) 상업용 a) commercial 프리바이오틱스Prebiotics 이눌린 처리 Inulin treatment
영양배지(펩톤 10.0g/L, 비프 익스트랙트 10.0g/L, 염화칼슘 5.0g/L, 멸균후의 pH 7.3±0.1) 1ml, 상업용 이눌린 용액(TCI - CJ3TK- CE - 25G) 1ml, 희석 배양액 1ml씩 12웰 셀 플레이트에 넣었고 이눌린 수용액 농도는 100, 150, 200 mg/ml를 사용하였다. 대조군은 2ml의 영양 배지와 1ml의 배양액을 첨가하였으며 3ml 멸균 증류수(DW)를 블랭크로 사용하였다. Nutritional medium (Peptone 10.0g/L, Beef Extract 10.0g/L, calcium chloride 5.0g/L, pH 7.3±0.1 after sterilization) 1ml, commercial inulin solution (TCI-CJ3TK- CE-25G) 1ml, diluted culture solution 1ml each It was placed in a 12-well cell plate, and the concentration of inulin aqueous solution was 100, 150, 200 mg/ml. As a control group, 2 ml of nutrient medium and 1 ml of culture solution were added, and 3 ml of sterile distilled water (DW) was used as a blank.
흡광도는 분광 광도계를 사용해 600nm에서 측정했으며, 초기 값을 측정한 후 12시간 동안 37℃의 배양기에 보관하면서 3시간마다 흡광도를 측정하였다. 본 실험은 3 반복했고, 결과는 3 반복의 평균 및 표준 편차를 사용하였다. The absorbance was measured at 600 nm using a spectrophotometer, and after measuring the initial value, the absorbance was measured every 3 hours while being stored in an incubator at 37°C for 12 hours. This experiment was repeated 3 times, and the results used the mean and standard deviation of 3 replicates.
b) 마늘껍질 추출물 처리b) Garlic peel extract treatment
12-웰 셀 플레이트에 영양배지 1ml, 마늘껍질 추출물 용액 1ml, 희석 배양액 1ml씩 넣었다. 마늘껍질 추출물 용액의 농도는 1, 2.5, 5, 7.5, 10 mg/ml를 사용하였다. In a 12-well cell plate, 1 ml of nutrient medium, 1 ml of garlic peel extract solution, and 1 ml of diluted culture solution were added each. The concentrations of the garlic peel extract solution were 1, 2.5, 5, 7.5, and 10 mg/ml.
이눌린 처리 실험과 같이, 영양 배지 2ml와 배양액 1ml를 대조군에 첨가하였고, 멸균 증류수(DW) 3ml를 blank로 사용하였다. 분광 광도계를 사용하여 600nm에서의 흡광도를 측정하고, 초기 값을 측정한 뒤 12시간 동안 37℃의 배양기에 보관하면서 3시간마다 흡광도를 측정하였다. 본 실험은 3 반복하였고, 결과는 3 반복의 평균 및 표준 편차를 사용하였다. As in the inulin treatment experiment, 2 ml of nutrient medium and 1 ml of culture solution were added to the control group, and 3 ml of sterile distilled water (DW) was used as a blank. The absorbance was measured at 600 nm using a spectrophotometer, and after measuring the initial value, the absorbance was measured every 3 hours while being stored in an incubator at 37° C. for 12 hours. This experiment was repeated 3 times, and the results used the mean and standard deviation of 3 replicates.
(4). 마늘껍질 추출물의 분리, 정제 및 특성규명(4). Isolation, purification and characterization of garlic peel extract
a) 고성능 액체 크로마토그래피 - 증발 a) High performance liquid chromatography-evaporation 광산란Light scattering 검출기( Detector( HPLCHPLC -- ELSDELSD ))
설탕 함량은 Agilent 1260액체크로마토그라피 시스템(1260 RID, Agilent, USA)을 사용하여 분석하였다. 이동상은 1 mL min-1의 유속에서 80% 아세토니트릴을 사용했고, 농도는 100g FW 당 mg으로 표시하였다. Sugar content was analyzed using an Agilent 1260 liquid chromatography system (1260 RID, Agilent, USA). The mobile phase used 80% acetonitrile at a flow rate of 1 mL min -1 and the concentration was expressed in mg per 100 g FW.
이눌린에 존재하는 당의 동정과 정량은 Isocratic solvent acetonitrile : 물(75:25)을 이동체로 사용하였다. 30℃에서 Ashahipak NH2P-504E(250 mm x 5.0 mm, Shodex, Japan) 컬럼을 이용해 HPLC(SPD 20A, Shimadzu, Japan)로 실험하였고, 1 mL min-1의 유속으로 상을 분리시켰다. Isocratic solvent acetonitrile: water (75:25) was used as a vehicle for identification and quantification of sugars present in inulin. The experiment was performed by HPLC (SPD 20A, Shimadzu, Japan) using an Ashahipak NH2P-504E (250 mm x 5.0 mm, Shodex, Japan) column at 30°C, and the phases were separated at a flow rate of 1 mL min -1 .
폴리테트라플루오르에틸렌(PTFE) 막(0.45 ㎛)을 이용해 여과시켜 분석 전에 이눌린 이외의 것들을 제거하였다. 설탕은 유도된 이눌린 20㎕를 주입하여 증발 광산란 검출기(ELSD, Varian, UK) (nebulizer = 50℃ 유지, 증발기 온도 = 90℃, 질소 가스 = 1.6 bar)를 통해 검출하였다. 표준 이눌린과 마늘껍질 추출물, 시판 이눌린도 HPLC 분석을 실행하였으며, 농도는 mg/g으로 표시하였다.Filtering using a polytetrafluoroethylene (PTFE) membrane (0.45 μm) to remove other than inulin prior to analysis. Sugar was detected through an evaporative light scattering detector (ELSD, Varian, UK) (nebulizer = 50° C. maintenance, evaporator temperature = 90° C., nitrogen gas = 1.6 bar) by injecting 20 μl of induced inulin. Standard inulin, garlic peel extract, and commercial inulin were also subjected to HPLC analysis, and the concentration was expressed in mg/g.
b) b) 핵 자기Nuclear magnetism 공명( resonance( 1One H NMR 및 H NMR and 1313 C NMR)C NMR)
preparative HPLC에서 분리된 미지의 화합물을 CDCl3에 용해시켜 NMR 분광 분석을 실시하였다. 1H, 13C, NMR스펙트럼은 600 MHz FT-NMR 분광기(Bruker Avance II-600, Ettlingen, Germany)를 사용해 조사하였다. An unknown compound isolated by preparative HPLC was dissolved in CDCl3 and analyzed by NMR spectroscopy. 1 H, 13 C, and NMR spectrum were investigated using a 600 MHz FT-NMR spectrometer (Bruker Avance II-600, Ettlingen, Germany).
13Ccross polarization/total side band suppression(CP/TOSS) 실험은 7kHz의 회전 속도로 진행하여 sideband free spectrum을 얻었으며, 5 초 이완 지연을 갖는 샘플의 경우, 768 건의 스캔을 획득하였다. The 13 C cross polarization/total side band suppression (CP/TOSS) experiment was conducted at a rotation speed of 7 kHz to obtain a sideband free spectrum. In the case of a sample with a 5 second relaxation delay, 768 scans were obtained.
3분의 접촉시간 및 4ms의 a1H90 펄스 길이를 사용하였으며, SPINAL-64 decoupling sequence는 83 kHz의 고주파 전계 강도를 사용하여 양성자를 분리하였다. 13C의 화학적 이동은 176.2 ppm에서 글리신의 카르보닐 신호를 고려해 외부에서 참고하였다.The contact time of 3 minutes and the a1H90 pulse length of 4 ms were used, and the SPINAL-64 decoupling sequence separated protons using a high frequency electric field intensity of 83 kHz. The chemical shift of 13 C was externally referenced considering the carbonyl signal of glycine at 176.2 ppm.
c) 푸리에 변환 적외선 분광법(c) Fourier transform infrared spectroscopy ( FTIRFTIR ))
FT-IR 분광학 탄수화물(이눌린 & 바나나 정제 추출물)에 대한 FT-IR 스펙트럼은 Perkin-Elmer Frontier FT-IR 분광계 장비에 기록하였다. 모든 시험시료는 KBr 펠렛으로 준비하고 해상도 4.0 cm-1, 파수 범위 4000-400 cm-1에서 빈 KBr 펠렛 배경에 대해 스캔하였다.FT-IR Spectroscopy FT-IR spectra for carbohydrates (inulin & banana purified extract) were recorded on a Perkin-Elmer Frontier FT-IR spectrometer instrument. All test samples were prepared as KBr pellets and scanned against a blank KBr pellet background at a resolution of 4.0 cm -1 and a wavenumber range of 4000-400 cm -1 .
d) 크기 및 제타 전위d) magnitude and zeta potential
나노 입자의 크기 및 표면 제타 전위는 입자 분석기 SZ 100(Horiba, Japan)로 분석하였으며, 나노 입자의 희석된 수성 분산액을 측정하였다. 입자 크기 데이터는 Z-평균 직경으로, 입자 전하 데이터는 z 전위로 판별되었다.The size and surface zeta potential of the nanoparticles were analyzed with a particle analyzer SZ 100 (Horiba, Japan), and the diluted aqueous dispersion of the nanoparticles was measured. The particle size data were determined by the Z-average diameter and the particle charge data by the z potential.
e) X-ray 분말 회절법(e) X-ray powder diffraction method ( XRDXRD ))
회절 패턴은 실온에서 X-ray 회절계(XRD) (Rigaku miniflex IIC, USA)로 기록하였다. Diffraction patterns were recorded with an X-ray diffractometer (XRD) (Rigaku miniflex IIC, USA) at room temperature.
타겟 : Cu (l¼1.54 Å); 필터 : Kbfoil 필터; 전압 : 40 kV; 전류 : 30 mA; 시정 수 : 10 mm s-1; scanning 범위 : 10e80 rom; 전체 scale : 200.2.2.4.7. Target: Cu (l ¼ 1.54 Å); Filter: Kbfoil filter; Voltage: 40 kV; Current: 30 mA; Time constant: 10 mm s -1 ; scanning range: 10e80 rom; Overall scale: 200.2.2.4.7.
f) 열적 성능저하(Thermal degradation)f) Thermal degradation
열중량 분석기(TGAQ50, USA)를 사용하여 온도에 따른 물질의 물리적 특성 변화를 측정하였다. TGA 분석을 위해 2.0 mg의 시료를 채취해 800℃까지 10 min- 1 의 속도로 가열하였으며, 분석을 위해 20 mL/min- 1 의 유속에서 퍼지 가스로 질소를 사용하였다.A thermogravimetric analyzer (TGAQ50, USA) was used to measure the change in physical properties of the material with temperature. For TGA analysis sampling of 2.0 mg to 10 min up to 800 ℃ - were heated at a rate of 1, 20 mL / min for the analysis was used as the nitrogen purge gas at a flow rate of 1.
g) 투과전자 현미경(g) Transmission electron microscope ( TEMTEM ))
세포를 실온에서 1시간 동안 2% 글루타르알레하이드(glutaraldehyde), 2% 파라포름알데하이드(paraformaldehyde)를 혼합한 0.1 M of carcodylate 완충액으로 고정시켰다. Cells were fixed with 0.1 M of carcodylate buffer mixed with 2% glutaraldehyde and 2% paraformaldehyde for 1 hour at room temperature.
0.1M carcodylate 완충액으로 수 회 세척한 후, 세포를 에탄올(50% ,60%, 70%, 80%, 90%에서 각각 20분씩, 100% 20분씩 두 번)과 프로필렌 옥사이드(2회)로 탈수한 다음, 60℃에서 2일 동안 점차적으로 농도를 증가시킨 eponate 812에서 중합반응을 일으켰다. After washing several times with 0.1M carcodylate buffer, the cells were dehydrated with ethanol (20 minutes each in 50%, 60%, 70%, 80%, 90%, twice each for 100% 20 minutes) and propylene oxide (2 times). Then, polymerization reaction was initiated in eponate 812, which was gradually increased in concentration for 2 days at 60°C.
Ultra microtome(Ultracut UCT, Leica)을 사용하여 시료를 절단하고 우라닐아세테이트와 구연산납(lead citrate)으로 염색한 후, 강원대학교 기초과학연구센터의 전계방출 TEM(JEM-2100F, JEOL)을 이용하여 분석을 하였다.The sample was cut using an ultra microtome (Ultracut UCT, Leica), stained with uranyl acetate and lead citrate, and then the field emission TEM (JEM-2100F, JEOL) of the Basic Science Research Center of Kangwon National University was used. Analysis was done.
(5). (5). 꼬마선충(Little nematode ( C. eleganceC. elegance )모델을A) the model 이용한 Used in in vivovivo 효능분석 Efficacy analysis
a) 선충류의 성장 단계(Nematode and stage of development)a) Nematode and stage of development
C. elegans는 4개의 성장단계(L1, L2, L3, L4)로 나누어지며 알에서 부화 직후의 단계를 L1단계, 알을 낳기 직전의 성인 단계를 L4 단계라고 한다. 선충은 직경 35mm의 NGM(선충 육성 배지) 아가 배지에 50μL의 E. coli OP50을 도포한 후 20℃에서 보관하였다. C. elegans is divided into four growth stages (L1, L2, L3, L4). The stage immediately after hatching from the egg is called the L1 stage, and the adult stage just before laying eggs is called the L4 stage. Nematodes were stored at 20° C. after applying 50 μL of E. coli OP50 to a 35 mm diameter NGM (nematode growth medium) agar medium.
C. elegans(N2) 균주는 미네소타의 Caenorhabditis Genetic Center(CGC)에서 제공받았다. The C. elegans (N2) strain was provided by the Caenorhabditis Genetic Center (CGC) in Minnesota.
b) 균주 및 성장배지 (Bacterial strains and growth media) b) strain and the growth medium (Bacterial strains and growth media)
메틸렌 저항성 Staphylococcus aureus와 Escherichia coli OP50 균주는 US Food Fermentation Laboratory Culture Collection(Wyndmoor, PA., USA)에서 제공받았으며, Trypticase soy broth(TSB, BD Biosciences, San Jose, CA, USA)에서 증식한 균주를 -80℃에서 보관하였다. 각 배양은 TSA 및 MRS(BD Biosciences)를 사용하여 37℃에서 24시간 동안 배양 후 실험에 사용하였다.Methylene resistant Staphylococcus aureus and Escherichia coli OP50 strain was provided by the US Food Fermentation Laboratory Culture Collection (Wyndmoor, PA., USA), and the strain grown in Trypticase soy broth (TSB, BD Biosciences, San Jose, CA, USA) was stored at -80°C. Each culture was used for experiments after incubation at 37° C. for 24 hours using TSA and MRS (BD Biosciences).
c) 주화성 분석(c) Analysis of chemotaxis ( ChemotaxisChemotaxis assay) assay)
주화성 분석은 Martin et al.(2017) 방법을 사용하였다. 즉, 세균(OP50 10㎕)을 배양한 후에 초기 성장(L4) 단계의 선충을 별도로 씨딩(seeding)하였다. 50마리의 young adult 단계의 선충을 S-basal 완충액으로 두 번 세척한 후 플레이트(5cm)의 중앙에 위치시켰다. For chemotaxis analysis, Martin et al. (2017) method was used. That is, after culturing the bacteria (OP50 10µl), nematodes in the initial growth (L4) stage were separately seeded. 50 young adult stage nematodes were washed twice with S-basal buffer and placed in the center of the plate (5 cm).
플레이트에 원형 2개를 표시한 후, 0.5 M 소디움 아자이드(sodium azide) 용액을 20μl 첨가하여 해당 구역에서 비 운동성 선충을 제외시켰다. 이사분원 시스템을 사용하여 시작점에 관계없이 처리구와 대조구 시료를 같은 거리에서 선충의 움직임을 모니터링하였다. After marking two circles on the plate, 20 μl of 0.5 M sodium azide solution was added to exclude non-motile nematodes from the area. The movement of the nematodes was monitored at the same distance between the treatment and control samples regardless of the starting point using the quadrilateral system.
이 시스템에서 선충은 유인물질(냄새)에 반응하며, 유인물질은 선충이 더 접근하도록 유도하였다. 두 장소에서 선충의 수는 25℃에서 90분 후 확인한 다음 4℃에서 더 배양하였다. In this system, the nematodes react to the attractant (odor), and the attractant induced the nematode to gain more access. The number of nematodes in both places was checked after 90 minutes at 25℃ and then further cultured at 4℃.
주화성 분석은 각 조건에서 3회 반복 수행하였다. 각 실험에서 주화성 지수(CI)는 다음 공식을 사용하여 계산하였다. The chemotactic analysis was performed three times in each condition. In each experiment, the coincidence index (CI) was calculated using the following formula.
주화성 지수(CI) = [MOESE의 웜 수 - OP50(control)의 선충수 / 총 선충수] Coincidence Index (CI) = [Number of worms in MOESE-Number of nematodes in OP50(control) / Number of total nematodes]
점수 +1.0은 표적을 향한 최대 접근도를 나타내며, 화학물질을 포함하는 사분면에 도착하는 선충이 100%임을 나타내며, 지수 -1.0은 최대 반발도를 나타낸다.A score of +1.0 represents the maximum degree of access to the target, 100% of nematodes arriving in the quadrant containing the chemical, and the index -1.0 represents the maximum degree of repulsion.
4) 점착성 분석(Colonization test)4) Adhesion analysis (Colonization test)
점착성 실험은 L1 단계의 선충을 균일하게 도포한 후에 수행되었다. L1 단계의 선충을 M9 완충액으로 세척하고 상업용 이눌린 및 마늘껍질 추출물 용액으로 분무된 NGM 아가 배지에 넣었다. The adhesion test was performed after evenly applying the L1 stage nematodes. The L1 stage nematodes were washed with M9 buffer and placed in NGM agar medium sprayed with commercial inulin and garlic peel extract solution.
NGM 아가 배지에 분무한 수용액의 농도는 프로바이오틱스 균주의 성장이 증가하기 시작하는 농도로 설정하였다(이눌린 10 mg/ml, 마늘 껍질시료 10 mg/ml). 선충은 하루 동안 상업용 이눌린 및 마늘껍질 추출물 용액을 섭취하게 한 후 다음날 프로바이오틱스 균주를 접종한 NGM 아가 배지에 옮겨졌다. The concentration of the aqueous solution sprayed on the NGM agar medium was set to a concentration at which the growth of the probiotic strain begins to increase (
프로바이오틱스 균주는 Lactobacillus rhamnosus JDFM6을 사용하였으며, 실험은 총 7일 동안 진행하였다. 선충을 1, 3, 5, 7 일 배양한 후 꺼내어 분말화한 후 플레이트에서 점착성을 확인하였으며, 선충은 실험기간 동안 매일 새로운 배지에서 옮겨 증식시켰다. 프리바이오틱스 수용액을 섭취하지 않은 선충을 대조군으로 사용하였다. Lactobacillus rhamnosus JDFM6 was used as the probiotic strain, and the experiment was carried out for a total of 7 days. After culturing the nematodes for 1, 3, 5, 7 days, they were taken out and powdered, and the adhesion was confirmed on the plate, and the nematodes were transferred from a new medium every day during the experiment to grow. Nematodes that did not take an aqueous solution of prebiotics were used as a control.
(6). 마늘껍질 추출물을 이용한 (6). Using garlic peel extract 프리바이오틱스Prebiotics 쿠키의 특성 규명 Characterization of cookies
a) 쿠키의 제조a) preparation of cookies
쿠키 반죽은 무염 버터(20.5%), 설탕(17.6%), 달걀(9.9%), 밀가루(51.6%) 그리고 베이킹 파우더(0.4%)로 제조하였다. 프리바이오틱스와 마늘껍질 추출물이 들어간 쿠키는 설탕 10 mg을 대체하여 첨가하였다. Cookie dough was prepared with unsalted butter (20.5%), sugar (17.6%), eggs (9.9%), flour (51.6%) and baking powder (0.4%). Cookies containing prebiotics and garlic peel extract were added by replacing 10 mg of sugar.
버터에 설탕을 넣고 상온에서 혼합한 후 달걀을 첨가하여 혼합하였다. 밀가루와 베이킹 파우더를 첨가하여 주걱으로 혼합한 후, 균일한 크기로 분할하여 플런저(plunger)에 옮겼다. Sugar was added to the butter, mixed at room temperature, and then mixed by adding eggs. Flour and baking powder were added, mixed with a spatula, divided into uniform sizes, and transferred to a plunger.
이후 각 반죽에 프리바이오틱스와 추출물을 첨가하고, 무게 13~14g의 반죽을 가로 50mm, 세로 10mm 로 잘라 일정형태의 모양을 만든 후, 180℃로 예열된 오븐에 15분간 구워 사용하였다. Thereafter, prebiotics and extracts were added to each dough, and the dough weighing 13-14 g was cut into 50 mm in width and 10 mm in length to form a shape, and then baked in an oven preheated to 180° C. for 15 minutes and used.
b) 쿠키의 크기b) size of the cookie
직경, 높이 및 무게를 다음과 같이 측정하였다. 직경은 각 쿠키의 중심부를 지나는 양끝 점을 이어 측정하였으며, 높이는 쿠키의 바닥에서 위쪽 표면까지의 길이를 측정하였다.The diameter, height and weight were measured as follows. The diameter was measured by connecting both end points passing through the center of each cookie, and the height was measured from the bottom of the cookie to the upper surface.
각 측정값은 mm로 표시하였다. 쿠키의 중량은 저울로 측정하였고, 단위는 g으로 표시하였다. 직경, 높이, 무게는 각 3회 반복하여 측정하였다. Each measured value is expressed in mm. The weight of the cookie was measured with a balance, and the unit was expressed in grams. The diameter, height, and weight were measured by repeating each of three times.
c) 색도 측정c) chromaticity measurement
쿠키의 색도는 색차계로 측정하여 밝기, 적색도, 황색도 값을 3회 반복 측정하고, 그 평균값으로 나타내었다.The chromaticity of the cookie was measured with a color difference meter, and the values of brightness, redness, and yellowness were repeatedly measured three times, and expressed as an average value.
d) 조직감 분석d) texture analysis
제조한 쿠키의 조직감은 조직감 측정기(texture analyzer)를 사용하여 경도를 3회 반복 측정하여 평균값을 구하였으며, 경도 단위는 N으로 표시하였다.The texture of the prepared cookie was measured by repeating the hardness three times using a texture analyzer to obtain an average value, and the hardness unit was expressed as N.
e) 일반성분 분석e) General component analysis
쿠키의 수분, 회분, 조단백질, 조지방, 조섬유 함유량은 Association of Official Analytical Chemists(AOAC, 2000)에 의하여 3회 반복 실험하였으며, 총탄수화물은 Southgate (1991)에 의하여 측정하였다. 각 측정값은 g/100g으로 표시하였다. The moisture, ash, crude protein, crude fat, and crude fiber content of cookies were tested three times by the Association of Official Analytical Chemists (AOAC, 2000), and total carbohydrates were measured by Southgate (1991). Each measured value was expressed in g/100g.
수분을 측정하기 위해 5g의 쿠키 샘플을 항량에 달할 때까지 105℃의 오븐에서 3시간 동안 건조시킨 후 사용하였다(925.10). 회분 함량은 쿠키 샘플 5g을 570℃의 전기로에서 잿빛으로 변할때까지 회화시킨 후 분석하였다(923.03). To measure the moisture, 5 g of a cookie sample was dried in an oven at 105° C. for 3 hours until a constant weight was reached, and then used (925.10). Ash content was analyzed after 5 g of a cookie sample was incinerated in an electric furnace at 570° C. until it turned gray (923.03).
조단백질 함량은 킬달법으로 측정하였으며, 총 단백질량은 질소계수 6.25를 배하여 산출하였다(984.13). 조지방 함량은 속실렛 추출기를 이용하였으며, 석유에테르를 사용하여 60-80℃에서 5시간 동안 추출한 후 분석하였다(922.06). The crude protein content was measured by the Kjeldahl method, and the total protein content was calculated by multiplying the nitrogen coefficient of 6.25 (984.13). The crude fat content was analyzed after extraction at 60-80°C for 5 hours using a Soxhlet extractor and using petroleum ether (922.06).
조섬유 함량은 산ㆍ알칼리 (2.5N) 소화에 의해 측정하였으며, 5g의 탈지된 쿠키 샘플을 산ㆍ알칼리로 소화시킨 후, 전기로에서 회화하여 회분의 최종 중량을 조섬유량으로 환산하였다(ISO 5498-1981, reaffirmed 2010). Crude fiber content was measured by digestion with acid and alkali (2.5N), and 5 g of degreased cookie sample was digested with acid and alkali, and then incinerated in an electric furnace to convert the final weight of the ash into crude fiber (ISO 5498-1981 , reaffirmed 2010).
총 식이섬유 함량은 수용성 섬유(993.19)와 불용성 섬유(991.42)의 합으로 하였으며, 효소처리법으로 분석하였다.The total dietary fiber content was determined as the sum of water-soluble fiber (993.19) and insoluble fiber (991.42), and was analyzed by enzyme treatment.
f) 저장성 테스트f) storage test
개발된 쿠키는 금속성 폴리프로필렌(PP) 파우치에 각 5개씩 포장하여 실온 25℃의 찬장에 21일간 저장하였다. 주단위로 파우치를 개봉하여 수분함량, 과산화물값(PV), 색도, 조직도를 3회 반복 측정하였으며, 신선한 쿠키와 비교분석하였다.The developed cookies were packaged in a metallic polypropylene (PP) pouch, 5 each, and stored in a cupboard at room temperature for 21 days. The pouch was opened on a weekly basis, and the moisture content, peroxide value (PV), chromaticity, and organization were repeatedly measured three times, and compared with fresh cookies.
g) 수분함량g) moisture content
대조구와 이눌린 첨가 쿠키의 수분함량은 105℃에서 습도 측정기(Sartorius MA 35, Goettingen, Germany)를 이용하여 측정하였다. 쿠키 샘플 1g을 분석하여 결과값을 백분율(%)로 나타내었다. The moisture content of the control and inulin-added cookies were measured at 105°C using a humidity meter (Sartorius MA 35, Goettingen, Germany). 1 g of a cookie sample was analyzed and the result was expressed as a percentage (%).
h) 쿠키의 h) of cookies 프리바이오틱스Prebiotics 효능 efficacy
개발된 쿠키를 분쇄한 후 추출하여 샘플을 제조하였다. 추출 방법은 식품 부산물로부터 프리바이오틱스를 추출한 방법과 동일하게 진행하였으며, 추출물은 프로바이오틱스 생육증진 효능 실험에 사용하였다. The developed cookies were crushed and extracted to prepare a sample. The extraction method was carried out in the same manner as the method of extracting prebiotics from food by-products, and the extract was used in the experiment of probiotic growth promoting efficacy.
생육증진 효능 평가를 위해 Lactobacillus rhamnosus JDFM6을 사용하였으며, 각 샘플은 10 mg/ml 농도로 첨가하였다. 37℃에서 12시간 배양하였으며, 3시간 간격으로 분광계를 이용하여 600nnm에서 흡광도를 측정하였다. 각 실험은 3회 반복 진행하였다. Lactobacillus rhamnosus JDFM6 was used to evaluate the growth promoting efficacy, and each sample was added at a concentration of 10 mg/ml. Incubated at 37°C for 12 hours, and absorbance was measured at 600nnm using a spectrometer at 3 hour intervals. Each experiment was repeated three times.
(7). 통계 분석(7). Statistical analysis
유의성 분석은 One-way ANOVA(일원 분산분석)검정을 실시하였으며, Tukey's test로 평균을 구하였고, 각 처리구간의 최소 유의차(p <0.05) 수준에서 통계처리 하였다. 모든 시험분석은 평균과 표준편차를 산출하기 위해 2회 이상 반복실험 하였다.For significance analysis, a one-way ANOVA (one-way analysis of variance) test was performed, and the average was obtained by Tukey's test, and statistical processing was performed at the level of the least significant difference (p <0.05) between each treatment section. All test analyzes were repeated two or more times to calculate the mean and standard deviation.
2. 실험 결과2. Experiment result
(1). 마늘껍질 추출물의 (One). Of garlic peel extract 프로바이오틱스Probiotics 생육증진 효능 평가 Evaluation of growth promotion efficacy
8종의 프로바이오틱스 균주를 대상으로 마늘껍질 추출물의 생육증진 효능을 분석한 결과는 도 1과 같다. 도 1에서 버는 바와 같이, 추출물 2.5 mg 이상의 농도에서는 대조군에 비하여 8종 모두 생육이 증가하는 것으로 나타났다. The results of analyzing the growth promoting effect of the garlic peel extract targeting 8 kinds of probiotic strains are shown in FIG. 1. As shown in Figure 1, it was found that growth of all eight species increased compared to the control at a concentration of 2.5 mg or more of the extract.
특히, Enterococcus faecium SC54 균주 및 Weissella cibaria SCCB2306 균주는 OD값이 2.0 이상으로 나타났으며, 추출물을 5mg/㎖ ~ 10mg/㎖으로 처리한 경우 대조군과 비교하여 우세한 성장률을 보였다. In particular, Enterococcus faecium SC54 strain and Weissella cibaria SCCB2306 strain had an OD value of 2.0 or higher, and when the extract was treated with 5mg/ml to 10mg/ml showed a dominant growth rate compared to the control group.
(2). 특정 탄수화물 정량에 따른 탄수화물 정제 및 특성 규명(2). Carbohydrate purification and characterization according to specific carbohydrate quantitation
a) a) HPLCHPLC -RID 분석-RID analysis
HPLC는 당을 유도할 필요가 없으므로 올리고당 분리에 널리 사용된다(Katapodis et al., 2003). ELSD 병합 HPLC는 올리고당 측정을 위한 기본 도구인 굴절률(RI) 기반 검출기보다 우수한 안정성을 제공하고 더 민감한 것으로 보고되었다. HPLC is widely used for oligosaccharide separation because it does not need to induce sugar (Katapodis et al., 2003). It has been reported that ELSD combined HPLC provides superior stability and is more sensitive than the refractive index (RI) based detector, which is a basic tool for measuring oligosaccharides.
도 2는 표준 정제 이눌린, 마늘껍질 추출물의 표준 당을 분석한 결과이다. 이늘린은 주로 24.9 mgml-1로 구성되어 있는 것을 확인하였으며, 정제된 표준 이눌린과 비교한 결과, 마늘껍질 추출물은 주요물질이 이눌린인 것으로 확인하였다. 또한, Prep-HPLC 로 분획한 경우, 정제된 피크와 유사한 것으로 나타나 핵심 주요성분이 이눌린인 것으로 나타났다.2 is a result of analysis of standard sugars of standard purified inulin and garlic peel extract. It was confirmed that inulin was mainly composed of 24.9 mgml -1 , and as a result of comparison with purified standard inulin, it was confirmed that the main substance of garlic peel extract was inulin. In addition, when fractionated by Prep-HPLC, it appeared similar to the purified peak, indicating that the core main component was inulin.
b) b) 1One H NMR 및 H NMR and 1313 C NMRC NMR
① ① 1One H NMRH NMR
마늘껍질 추출물의 1H NMR 스펙트럼을 분석하였으며, 그 결과는 도 3a와 같다. 이눌린의 링 양성자는 3.5~4.5 ppm 범위 내에서 신호를 나타내었으며, 이눌린과 마늘껍질 추출물에서 동일하게 나타난 3.851, 4.021, 4.245, 4.767 ppm의 피크는 D-자일로피라노실(D-xylopyranosyl)의 β-글리코사이드 결합에 의한 것으로 사료된다. The 1 H NMR spectrum of the garlic peel extract was analyzed, and the results are shown in FIG. 3A. Ring protons of inulin exhibited signals within the range of 3.5 to 4.5 ppm, and peaks of 3.851, 4.021, 4.245, and 4.767 ppm, which were the same in inulin and garlic peel extracts, were β of D-xylopyranosyl. -It is thought to be due to glycoside binding.
이 결과는 이눌린 샘플의 FTIR 스펙트럼에서 보이는 3291 cm-1, 3274 cm-1 범위의 피크와 일치한다. 이와 유사하게, 2807.31 cm- 1 과 2251.72 cm-1 범위에서 보이는 피크는 무수 자일로오스 양성자(anhydroxylose proton)의 존재와 특정 글루쿠론산(glucuronic acid)의 화학적 이동을 의미한다(Sun et al., 2004). This result is consistent with the peaks in the range of 3291 cm -1 and 3274 cm -1 seen in the FTIR spectrum of the inulin sample. Similarly, the peaks seen in the 2807.31 cm - 1 and 2251.72 cm -1 ranges indicate the presence of anhydrous xylose protons and chemical shifts of certain glucuronic acids (Sun et al., 2004).
② ② 1313 C NMR C NMR
핵자기 공명장치를 이용하여 이눌린 및 마늘껍질 추출물의 물질 구조를 규명하고 특성을 분석하였으며, 결과는 도 3b와 같다. 두 샘플 모두 5가지의 주요 신호가 관찰되었으며, 이는 아라비노오스(arabinose)에 상응하는 62.15, 64.33(C-5) ppm, 이눌린에 상응하는 76.65, 81.16(C-2, 3, 4) ppm으로 관찰되었다. Material structures of inulin and garlic peel extract were identified and characteristics were analyzed using a nuclear magnetic resonance device, and the results are shown in FIG. 3B. Five major signals were observed in both samples, 62.15, 64.33 (C-5) ppm corresponding to arabinose and 76.65, 81.16 (C-2, 3, 4) ppm corresponding to inulin. Was observed.
페룰산(fellulic acid)은 α-L-아라비노퓨라노실 잔기의 C-5에 연결되어 있다. 81.19, 103.33 (C-1) ppm에서 관찰되는 신호는 마늘껍질 추출물에 함유되어 있는 이눌린의 α-L-아라비노퓨라노오스와 β-D-1,4-자일로피라노오스의 아노머 탄소 원자에 상응하는 것으로 사료된다. Ferulic acid is linked to C-5 of the α-L-arabinofuranosyl residue. The signals observed at 81.19 and 103.33 (C-1) ppm were the anomer carbons of α-L-arabinofuranose and β-D-1,4-xylopyranose of inulin contained in garlic peel extract. It is believed to correspond to the atom.
모든 이눌린의 기본 골격은 선형 구조로 Nandini and Salinathe (2001)의 연구와 유사한 결과를 보였다. 이러한 신호들은 밀배유에 관한 연구결과와 유사한 것으로 나타났다(Hoffman et al., 1992). 또한 마늘껍질 추출물의 경우 18.12, 20.25, 23.36, 25.45, 29.81, 59.49, 181.44 ppm등 탄소기반의 미지의 탄수화물 피크가 관찰되었다. All of the basic skeletons of inulin showed a linear structure, which was similar to that of Nandini and Salinathe (2001). These signals were found to be similar to the results of studies on wheat endosperm (Hoffman et al., 1992). In the case of garlic peel extract, unknown carbohydrate peaks based on carbon such as 18.12, 20.25, 23.36, 25.45, 29.81, 59.49, 181.44 ppm were observed.
(3). 마늘껍질 (3). Garlic peel 조추출물의Crude extract 이눌린 정제 및 특성 규명 Inulin tablets and characterization
a) a) ELSDELSD -- HPLCHPLC 분석 analysis
HPLC는 당 유도체화 과정이 필요하지 않기 때문에 올리고당 분리를 위해 널리 사용되는 분석법이다(Katapodis et al.,). HPLC-ELSD는 민감도가 높아 과당류 측정을 위해 쓰이는 굴절도 기반 검출기보다 더욱 안정적인 기준점을 제공하는 것으로 알려져 있다. HPLC is a widely used assay for oligosaccharide separation because it does not require a sugar derivatization process (Katapodis et al.,). HPLC-ELSD has high sensitivity and is known to provide a more stable reference point than the refractive index-based detector used for fructose measurement.
도 4는 이눌린 및 마늘껍질 추출물의 표준 당분을 나타낸 결과이다. 마늘껍질 추출물의 가수분해물을 HPLC로 분석하여 이눌린 농도를 분석하였으며, 표준물질로 상업용 이눌린을 사용하였다. Figure 4 is a result showing the standard sugar content of inulin and garlic peel extract. The hydrolyzate of the garlic peel extract was analyzed by HPLC to analyze the concentration of inulin, and commercial inulin was used as a standard material.
이눌린은 주로 1.511 mg ml-1 농도로 존재하는 것으로 확인하였다. 프리바이오틱스를 화학적으로 합성시킬 경우, 모노머 및 올리고머의 방출을 심화 유도하는 것으로 알려져 있으며(Aachary and Prapylla, 2009), 유도된 이눌린을 농축시킴으로써 식품의 프리바이오틱스 첨가제로서 사용될 수 있을 것으로 사료된다.It was confirmed that inulin was mainly present at a concentration of 1.511 mg ml -1 . When prebiotics are chemically synthesized, it is known to induce the release of monomers and oligomers deeply (Aachary and Prapylla, 2009), and it is thought that it can be used as a prebiotic additive in foods by concentrating the induced inulin.
b) b) 1One H NMR 및 H NMR and 1313 C NMRC NMR
① ① 1One H NMR 분석H NMR analysis
마늘껍질 정제 추출물(GPPE)의 1H NMR 분석 결과는 도 5a 와 같다. 이눌린의 경우 양성자 봉우리가 3.4-5.4 ppm 에서 나타난 것을 확인하였다. 표준 이눌린 및 마늘껍질 추출물에서 동일하게 나타난 5.392, 4.700, 4.182, 4.000, 3.825, 3.658, 2.658, 3.492, 3.412 ppm 범위의 양성자 봉우리는 이눌린 구조의 D-자일로피라노실 단위 간 β-글리코사이드 결합을 나타내는 것을 알 수 있다. The results of 1 H NMR analysis of the purified garlic peel extract (GPPE) are shown in FIG. 5A. In the case of inulin, it was confirmed that the proton peak appeared at 3.4-5.4 ppm. Proton peaks in the range of 5.392, 4.700, 4.182, 4.000, 3.825, 3.658, 2.658, 3.492, and 3.412 ppm, which were identical in standard inulin and garlic peel extracts, showed β-glycoside bonds between D-xylopyranosyl units of inulin I can see what it represents.
이러한 결과는 이눌린 샘플의 FTIR 분석 스펙트럼에서 나타난 2807.31 cm-1, 2251.72 cm-1 범위의 밴드와 상응하는 것으로 사료된다. 이와 유사하게 4.182, 3.782 ppm에서 나타난 양성자 봉우리는 무수 자일로오스 양성자(anhydroxylose proton)의 존재와 글루쿠론산 화합물의 이동의 결과로 나타난 것으로 사료된다 (Sun et al., 2004). These results are considered to correspond to the bands in the range of 2807.31 cm -1 and 2251.72 cm -1 in the FTIR spectrum of the inulin sample. Similarly, the proton peaks at 4.182 and 3.782 ppm are thought to be a result of the presence of anhydrous xylose protons and the transfer of glucuronic acid compounds (Sun et al., 2004).
3.00, 3.43 ppm에서 나타난 피크는 α-L-아라비노퓨라노오스 잔기의 아노머릭 양성자(anomeric proton)의 존재를 의미하며, 본 결과에 의하여 마늘껍질 추출물의 이눌린과 아라비노오스 잔류물의 존재를 확인하였다. The peaks at 3.00 and 3.43 ppm indicate the presence of an anomeric proton of the α-L-arabinofuranose residue, and the presence of inulin and arabinose residues in the garlic peel extract was confirmed by this result. I did.
또한, 이눌린 기본구조에 부착되어 있는 아세틸 그룹의 메틸 양성자를 나타내는 4.2-5.00ppm의 공통된 피크가 관찰되었으며, 이와 관련하여 옥수수대의 아라비노실란의 존재가 1H NMR 분석에 의해 보고된 바 있다(Egues et al., 2013). 표준 이눌린의 구조는 NMR 분석을 통해 규명하였으며, 치커리 추출 이눌린과 구조적 특성이 90% 이상 일치하는 것으로 확인하였다.In addition, a common peak of 4.2-5.00 ppm representing the methyl proton of the acetyl group attached to the inulin basic structure was observed, and in this regard, the presence of arabinosilane of corn stalk was reported by 1 H NMR analysis (Egues et al., 2013). The structure of standard inulin was identified through NMR analysis, and it was confirmed that the structural characteristics of inulin extracted from chicory were more than 90% identical.
② ② 1313 C NMR 분석C NMR analysis
마늘껍질 정제 추출물(GPPE)의 13C NMR 분석 결과는 도 5b와 같으며, 이는 이눌린과 GPPE의 구조를 추가적으로 입증하였다. 각 샘플의 아라비노오스와 이눌린 존재를 나타내는 61.02, 62.23(C-5)와 77.16(C-2, 3, 4) ppm 에서 주요 5가지 신호를 관찰하였다. The 13 C NMR analysis results of the garlic peel purified extract (GPPE) are shown in Fig. 5b, which further demonstrates the structures of inulin and GPPE. Five major signals were observed at 61.02, 62.23 (C-5) and 77.16 (C-2, 3, 4) ppm indicating the presence of arabinose and inulin in each sample.
페룰산은 α-L-아라비노퓨라노실 잔기의 5번 탄소와 결합하여 있으며, 81.19, 103.33(C-1) ppm의 피크는 각각 α-L-아라비노퓨라노실의 아노머 탄소 양성자와 이눌린의 β-D-1,4-자일로피라노오스를 나타낸다. Ferulic acid is bound to
모든 이눌린의 기본 골격은 선형 구조로 확인하였으며, 이는 Nandini and Salimate (2001)과 유사한 결과를 보였다. 이와 같은 결과는 밀 배유(Hoffman et al., 1992), 손가락조 껍질(Subba Rao and Muralikrishna, 2004)에 관한 연구에 기반하였다. 이눌린의 기본 구조는 분자간 혹은 다당류와의 상호작용으로 이루어져 있으므로, 각 분자의 기능적 특성에 크게 관여할 수 있다. All of the basic skeletons of inulin were identified as linear structures, which showed similar results to Nandini and Salimate (2001). These results were based on studies on wheat endosperm (Hoffman et al., 1992) and fingernail skin (Subba Rao and Muralikrishna, 2004). Since the basic structure of inulin consists of interactions with molecules or polysaccharides, it can be largely involved in the functional properties of each molecule.
c) c) FTIRFTIR
마늘껍질 정제 추출물(GPPE)과 표준 이눌린의 FTIR 스펙트럼에서 도 6에 나타난 바와 같이 공통 작용기 그룹이 존재하는 것을 확인하였다. 두 FTIR 스펙트럼 모두 헤미셀룰로오스(이눌린)의 대표적인 결합인 O-H, C=O 진동에 해당하는 피크를 나타내었다. 특히, 1600~1000cm-1 범위에서 특정 대역의 최대치를 보여 특징적인 신호 패턴을 보였다. 가장 강렬한 광대역 범위는 3245cm-1과 3267cm- 1 의 OH 기로 나타났다. It was confirmed that a common functional group exists as shown in FIG. 6 in the FTIR spectrum of the garlic peel purified extract (GPPE) and standard inulin. Both FTIR spectra showed peaks corresponding to OH and C=O vibrations, which are representative bonds of hemicellulose (inulin). In particular, it showed a characteristic signal pattern by showing the maximum value of a specific band in the range of 1600~1000cm -1 . The most intense broadband range 3245cm -1 and 3267cm - OH groups were: 1.
마늘껍질 추출물 샘플에서 1654cm-1, 1624cm-1 범위에서 흡수 대역을 보였으며, 이는 아세틸 그룹의 존재를 나타낸다. Samata et al., (2012)은 옥수수대의 알칼리 추출 과정에서 자일란과 XOS의 당 잔기의 탈아세틸화를 관찰하였다. The garlic peel extract sample showed an absorption band in the range of 1654cm -1 and 1624cm -1 , indicating the presence of an acetyl group. Samata et al., (2012) observed deacetylation of sugar residues in xylan and XOS during the alkaline extraction of corn stalks.
물 추출은 이눌린의 아세틸 그룹을 보존하고, 고부가가치 기능성 식품 성분 생산을 위한 효율적인 효소 가수분해에서 중요한 역할을 하는 마늘 추출물의 용해도를 향상시킨다. CH 결합에 상응하는 1397cm-1 및 1395cm-1의 흡수대역은 셀룰로오스 및 헤미셀룰로오스 화학 구조의 존재를 나타낸다(도 6 참조).Water extraction preserves the acetyl group of inulin and improves the solubility of garlic extract, which plays an important role in efficient enzymatic hydrolysis for the production of high-value functional food ingredients. Absorption band of 1397cm -1 and 1395cm -1 corresponding to the CH bond indicates the presence of the cellulose and hemicellulose chemical structure (see Fig. 6).
마늘 추출물의 존재는 1150cm-1과 900cm-1 범위에서 뚜렷이 나타났으며, 1500~800cm-1의 밴드는 4-O-메틸글루쿠로노자일란(4-O-methylglucuronoxylans)과 이눌린을 기반으로 한 마늘 추출물의 전형적인 특성이다. The presence of garlic extract was evident in the range of 1150cm -1 and 900cm -1 , and the band of 1500~800cm -1 was based on 4-O-methylglucuronoxylans and inulin. This is a typical characteristic of garlic extract.
마늘 추출물 샘플 모두 표준 이눌린과 유사한 것으로 확인하였으며, 일부 스펙트럼의 변화는 곁사슬의 당 잔기에 의한 것으로 사료된다.All samples of garlic extract were confirmed to be similar to standard inulin, and some spectral changes are believed to be due to sugar residues in the side chain.
(d) 입자 크기 및 제타 전위(d) particle size and zeta potential
프로바이오틱스의 입자 크기는 물과 반응하는 표면적의 변화를 반영하여 다당류의 수화 및 점도에 영향을 미치는 주요인이며, 분해율과 정반대의 연관성을 갖는다. 증류수에 분산된 샘플의 입자를 분석하기 위해 레이저 굴절을 이용한다. The particle size of probiotics reflects the change in the surface area reacting with water and is a major factor influencing the hydration and viscosity of polysaccharides, and has an opposite relationship with the decomposition rate. Laser refraction is used to analyze the particles in the sample dispersed in distilled water.
그 결과 마늘껍질 추출물에서 두 개의 강도가 관찰되었지만, 입자 크기는 매우 다양한 것으로 확인하였다. 이와 대조적으로, 이눌린 입자는 GPPE의 입자에 비해 상당히 컸으며(도 7 참조), 이는 SEM을 이용한 형태학적 분석결과와 상응하는 결과이다(도 10 참조). As a result, two strengths were observed in the garlic peel extract, but it was confirmed that the particle size was very variable. In contrast, inulin particles were significantly larger than those of GPPE (see Fig. 7), which corresponds to the result of morphological analysis using SEM (see Fig. 10).
마늘껍질 추출물의 형태학 및 입자 크기의 변화는 잔류물과 관련 물질에 기인하는 구조 변화로 인해 발생하는 것으로 사료되며, 이는 식품과 다른 용도의 기능성에도 영향을 미칠 수 있다. Changes in the morphology and particle size of the garlic peel extract are thought to be caused by structural changes caused by residues and related substances, which may affect the functionality of food and other uses.
e) X-ray 회절e) X-ray diffraction
마늘껍질 추출물과 이눌린의 X선 회절(XRD) 패턴은 각각 803cps, 809cps로 치커리 추출 이눌린(880cps)과 비교하여 20°에서 보다 넓은 피크를 보여 이눌린 구조가 완전히 비형상적임을 확인하였다(도 8 참조).X-ray diffraction (XRD) patterns of the garlic peel extract and inulin were 803 cps and 809 cps, respectively, showing a wider peak at 20° compared to the chicory extract inulin (880 cps), confirming that the inulin structure was completely non-shape (see FIG. 8 ). ).
이눌린과 GPPE를 첨가한 쿠키 샘플의 경우 뚜렷한 피크가 관찰되지 않았으며, 이는 정제도에 의한 것으로 사료된다. 따라서 마늘껍질 추출물은 비결정형의 구조로 셀룰로오스 관련 가수 분해에 더 유용할 것으로 사료된다. Kawai et al. (2013)는 완전 비정형 상태를 보고한 바 있다.In the case of the cookie sample to which inulin and GPPE were added, no distinct peak was observed, which is thought to be due to the degree of purification. Therefore, garlic peel extract is considered to be more useful in hydrolysis related to cellulose due to its amorphous structure. Kawai et al. (2013) reported a completely atypical state.
f) f) 열중량Thermal weight 분석 analysis
이눌린 가열 과정에서의 중량 변화와 특성을 파악하기 위해 열중량분석(TGA)을 이용하였다. 마늘껍질 추출물의 TGA 곡선은 이눌린과 비교하여 상당한 분해량을 보였다(도 9 참조). Thermogravimetric analysis (TGA) was used to determine the weight change and characteristics during the heating process of inulin. The TGA curve of the garlic peel extract showed a significant amount of decomposition compared to inulin (see FIG. 9).
마늘껍질 추출물의 중량 손실은 127~140℃, 이눌린은 160~162℃ 범위에서 발생하였으며, 200~400℃ (65-70%)와 400-500℃(10-12%)의 온도 범위에서 이눌린이 분해되는 것을 확인하였다. The weight loss of garlic peel extract was 127~140℃, and inulin occurred in the range of 160~162℃, and inulin was found in the temperature range of 200~400℃ (65-70%) and 400-500℃ (10-12%). It was confirmed that it was decomposed.
헤미셀룰로오스는 셀룰로오스나 리그닌보다 열적 안정성이 낮고, 이눌린은 225-350℃에서 분해되는 것으로 알려져 있다. Hemicellulose is known to have lower thermal stability than cellulose or lignin, and inulin is known to decompose at 225-350°C.
g) 전자현미경 관찰g) electron microscope observation
전자현미경 관찰 결과, 이눌린은 구면 형태와 거친 표면을 갖는 입자가 혼합되어 있는 불규칙한 형태를 보이는 반면(도 10의 A 참조), 마늘껍질 추출물은 큰 입자로 구성되어 있는 것을 확인하였다(도 10의 B 참조).As a result of observation with an electron microscope, it was confirmed that inulin exhibits an irregular shape in which particles having a spherical shape and a rough surface are mixed (see Fig. 10A), whereas the garlic peel extract is composed of large particles (Fig. 10B. Reference).
(4). 상업용 이눌린과 마늘껍질 추출물의 생육증진 효능 평가 (4). Evaluation of the efficacy of commercial inulin and garlic peel extract for promoting growth
이눌린 및 마늘껍질 추출물의 프로바이오틱스 생육 증진 효능을 젖산균 8종에 대하여 진행하였다. 프리바이오틱스 유효성이 우수한 균주는 Pediococcus acidilactici SDL1405, Pediococcus acidilactici SDL 1406, Weissella cibaria SCCB2306, Lactobacillus rhamnosus JDFM6인 것으로 확인하였다. Efficacy of promoting probiotic growth of inulin and garlic peel extract was performed on 8 lactic acid bacteria. The strains with excellent prebiotic efficacy were Pediococcus acidilactici SDL1405, Pediococcus acidilactici SDL 1406, Weissella cibaria SCCB2306, and Lactobacillus rhamnosus JDFM6.
항생제 사용시 시간이 경과함에 따라 성장이 월등하게 증가하는 것으로 확인하였다. 반면, 대조구의 경우 시간경과에 따라 생육이 급격히 감소하였으며, 마늘껍질 추출물은 보다 일관되고 우수한 결과를 보였다(도 11 참조). When using antibiotics, it was confirmed that the growth was remarkably increased over time. On the other hand, in the case of the control, the growth rapidly decreased with the passage of time, and the garlic peel extract showed more consistent and excellent results (see FIG. 11).
일반적으로 Enterococcus faecium SC54와 Weissella cibaria SCCB2306 균주의 경우 1.0 O.D 값이 증가하여 2.0 O.D값을 보이는 것을 확인하였으며, 5mg 이상 투여시 대조구와 비교하여 생육이 우세하게 증가하는 것을 확인하였다.In general, in the case of Enterococcus faecium SC54 and Weissella cibaria SCCB2306 strains, it was confirmed that 1.0 OD value was increased to show 2.0 OD value, and it was confirmed that when administered more than 5 mg, growth predominantly increased compared to control.
(5). 이눌린 및 마늘껍질 추출물의 효능 평가 (5). Efficacy evaluation of inulin and garlic peel extract in in vivovivo
장관내 집락 형성능은 C. elegans 모델을 이용하여 평가하였으며, 프리바이오틱스를 투여하지 않은 것을 대조구로 하였다. 그 결과, 프리바이오틱스 비섭취 그룹은 섭취 그룹에 비하여 집락 형성이 낮게 나타났으며, 3일째 가장 높은 집락을 형성한 뒤 시간경과에 따라 감소하는 경향을 보였다. Intestinal colonization ability was evaluated using the C. elegans model, and those without prebiotics were used as controls. As a result, the colony formation in the non-prebiotics group was lower than that of the ingestion group, and after the formation of the highest colony on the 3rd day, there was a tendency to decrease with time.
이와 대조적으로 프리바이오틱스 투여 그룹의 경우, 시간 경과 후에도 유사수준의 집락을 형성하는 것을 확인하였다(도 12 참조).In contrast, in the case of the prebiotic administration group, it was confirmed that colonies at a similar level were formed even after the passage of time (see FIG. 12).
3. 쿠키에의 활용3. Use of cookies
(1). 쿠키의 크기 측정(One). Measuring the size of cookies
쿠키를 제조한 후 무게, 지름, 높이를 측정한 결과를 하기의 표 1에 나타내었다. 표 1에서 보는 바와 같이, 쿠키 제조 전·후의 무게는 13~14g으로 유사하였다. 또한, 제조 후의 직경은 50~51mm으로, 대조구 대비 큰 변화를 보이지 않았다. After preparing the cookie, the results of measuring the weight, diameter, and height are shown in Table 1 below. As shown in Table 1, the weight before and after the preparation of cookies was similar, ranging from 13 to 14 g. In addition, the diameter after manufacture was 50 to 51 mm, and there was no significant change compared to the control.
따라서, 무게, 지름, 높이 측정 결과 프리바이오틱스를 첨가한 경우 유의적인 변화는 관찰되지 않았으며, 부정적인 영향을 미치지 않을 것으로 사료된다.Therefore, as a result of measuring weight, diameter, and height, when prebiotics were added, no significant change was observed, and it is believed that there is no negative effect.
(2). 색도 측정(2). Chromaticity measurement
색차계를 이용하여 쿠키의 색도를 측정한 결과를 하기의 표 2에 나타내었다. 표 2에서 보는 바와 같이, 이눌린 및 마늘껍질 추출물이 첨가된 쿠키의 경우 대조군 대비 어둡게 측정되었다. The results of measuring the chromaticity of the cookie using a color difference meter are shown in Table 2 below. As shown in Table 2, in the case of cookies to which inulin and garlic peel extract were added were measured darker than the control.
마늘껍질 추출물 첨가 쿠키의 경우 대조구 대비 가장 큰 차이를 보였으며, 이는 추출물의 색상이 짙은 갈색으로 쿠키의 색상에 영향을 미친것으로 사료된다.Cookies added with garlic peel extract showed the greatest difference compared to the control, which is thought to have an effect on the color of cookies as the color of the extract was dark brown.
(3). 조직도 분석(3). Organization chart analysis
쿠키의 조직도를 분석한 결과를 하기 의 표 3에 나타내었다. 표 3에서 보는 바와 같이, 이눌린 및 마늘껍질 추출물 첨가 쿠키의 경우 파괴 응력과 가능성은 대조구보다 유의적으로 낮게 나타났으며, 쿠키의 질감이 더 부드럽게 나타난 것으로 확인하였다. The results of analyzing the organizational chart of the cookies are shown in Table 3 below. As shown in Table 3, in the case of cookies added with inulin and garlic peel extract, the fracture stress and probability were significantly lower than that of the control, and the texture of the cookie was found to be more smooth.
이눌린 5% 첨가 쿠키와 마늘껍질 추출물 첨가 쿠키는 대조구와 유사한 질감을 나타내었으며, 슈크로오스를 대체하여 첨가할 경우 쿠키의 흡인력이 감소하는 경향을 보여 질감이 부드러워지는 것을 확인하였다.Cookies with 5% inulin added and cookies with garlic peel extract showed similar texture to the control, and when added by replacing sucrose showed a tendency to decrease the suction power of the cookie, it was confirmed that the texture became soft.
앞선 실험에서 쿠키의 높이가 증가하고 직경이 감소한 것을 고려하여, 대조구와 처리구 간의 유의적인 차이는 나타나지 않았으나, 색도의 경우 처리구에서 감소하는 경향을 나타내었다.Considering that the height of the cookie increased and the diameter decreased in the previous experiment, there was no significant difference between the control and treatment, but the chromaticity showed a tendency to decrease in the treatment.
(4). 쿠키의 일반성분 조성(4). Composition of general ingredients of cookies
하기 표 4에서 보는 바와 같이, 이눌린과 마늘껍질 추출물을 첨가한 쿠키는 수분 함량이 증가하였으며, 이는 이눌린과 추출물로 인해 수분 결합이 강화되어 나타난 것으로 사료된다. 이눌린과 추출물을 15mg씩 첨가한 쿠키의 수분 함량은 대조구와 유사하게 나타났으나, 5, 10, 15% 농도로 처리한 경우 수분함량이 증가하는 것으로 확인하였다. As shown in Table 4 below, the moisture content of the cookies added with inulin and garlic peel extract increased, which is believed to be due to the enhanced water binding due to inulin and extract. The moisture content of cookies with 15 mg of inulin and extract added at a time was similar to that of the control, but it was confirmed that the moisture content increased when treated at concentrations of 5, 10, and 15%.
또한, 조섬유 함량의 경우 대조구와 비교하여 3배 가량 증가한 것을 확인하였다. 이외 단백질, 지방, 탄수화물, 회분의 경우 대조구와 유사한 것을 확인하였다.In addition, in the case of the crude fiber content, it was confirmed that it was increased by about 3 times compared to the control. Other proteins, fats, carbohydrates, and ash were confirmed to be similar to those of the control.
(5). 저장 안전성(5). Storage safety
수분, 미생물, 효소의 변화 및 산화 등 다양한 요인들이 제품의 저장 수명에 영향을 미친다(Adegoke et al., 1993). 저장 안전성 실험 결과, 포장된 쿠키는 실온(25℃)에서 21일간 안정적으로 유지되었으며, 15%의 이눌린, 마늘 추출물 첨가 시 보관 기간 동안의 수분 함량, 색도, 질감에 변화가 없는 것으로 확인하였다. 또한, 21일 보관 후에도 과산화물이 검출되지 않는 것으로 확인하였다. Various factors, such as moisture, microorganisms, changes in enzymes, and oxidation, affect the shelf life of products (Adegoke et al., 1993). As a result of storage safety testing, the packaged cookies were stably maintained for 21 days at room temperature (25°C), and it was confirmed that there was no change in moisture content, color, and texture during storage period when 15% of inulin and garlic extract were added. In addition, it was confirmed that no peroxide was detected even after storage for 21 days.
그러나 10, 15% 첨가된 쿠키는 저장기간 중 수분 변화가 급격히 이루어졌으며, 이눌린과 마늘 추출물 농도가 높을수록 수분 흡수량이 증가하여 텍스처가 유의적으로 변화하는 것을 확인하였다. However, it was confirmed that the moisture content of the cookies added with 10 or 15% changed rapidly during the storage period, and the moisture absorption increased as the concentration of inulin and garlic extract increased, and the texture changed significantly.
21일 경과 후 5% 이눌린 및 마늘껍질 추출물 첨가 쿠키의 경우 물화학적 특성이 대조구와 비교하여 유사한 결과를 보였지만, 10, 15% 첨가 쿠키의 경우 물리·화학적 특성이 크게 변화하였다.After 21 days, in the case of cookies with 5% inulin and garlic peel extract, the water-chemical properties were similar compared to that of the control, but the physical and chemical properties of cookies with 10 and 15% were significantly changed.
(6). (6). 프리바이오틱스Prebiotics 특성(이눌린 및 마늘껍질 추출물 첨가 쿠키의 생육 증진 효능) Characteristics (Efficacy of promoting growth of cookies with inulin and garlic peel extract)
이눌린과 마늘껍질 추출물을 첨가한 쿠키를 사용해 프로바이오틱스 생육 증진 효능을 평가한 결과를 도 13에 나타내었다. Fig. 13 shows the results of evaluating the probiotic growth-promoting efficacy using cookies containing inulin and garlic peel extract.
추출물을 첨가하지 않은 대조구의 경우 6시간 후 성장을 마친 반면, 이눌린 및 마늘껍질 추출물을 첨가한 쿠키의 경우 12시간 후에도 성장이 지속되는 것을 확인하였다. 이눌린과 마늘껍질 추출물 처리구 간의 유의적인 차이는 나타나지 않았다.In the case of the control to which the extract was not added, growth was completed after 6 hours, whereas the cookie to which inulin and garlic peel extract were added continued to grow after 12 hours. There was no significant difference between inulin and garlic peel extract treatment groups.
(7). 전자 현미경 관찰(7). Electron microscope observation
집락 형성능을 평가하기 위해, C. elegans에 프리바이오틱스를 먹이로 공급하여 실험을 진행한 결과, 도 14에서 보는 바와 같이, 프리바이오틱스를 섭취하지 않은 대조구와 비교하여 높은 군체 형성을 나타내었다. In order to evaluate the colony-forming ability, C. elegans was supplied with prebiotics as food and the experiment was conducted. As shown in FIG. 14, the colony formation was higher than that of the control without prebiotics.
대조구 및 처리구 모두 3일째 가장 높게 형성하는 것을 확인하였으며, 대조구와 이눌린 처리구의 경우 시간이 경과함에 따라 점진적으로 감소하는 결과를 보였다. 이와 대조적으로, 프리바이오틱스를 섭취하는 선충은 비슷한 수준을 유지하는 것을 확인하였다.It was confirmed that both the control group and the treatment group formed the highest on the 3rd day, and the control group and the inulin-treated group showed a gradual decrease as time passed. In contrast, it was confirmed that nematodes ingesting prebiotics maintained similar levels.
상술한 바와 같이 본 발명의 바람직한 실시예를 참조하여 설명하였지만, 본 발명의 기술 분야에서 통상의 지식을 가진 통상의 기술자라면 하기의 청구범위에 기재된 본 발명의 사상 및 영역으로부터 벗어나지 않는 범위 내에서 본 발명을 다양하게 수정 및 변경시킬 수 있음을 이해할 수 있을 것이다. As described above, the present invention has been described with reference to the preferred embodiments of the present invention, but those skilled in the art of the present invention have the present invention within the scope not departing from the spirit and scope of the present invention described in the following claims. It will be appreciated that various modifications and changes can be made to the invention.
Claims (9)
상기 마늘껍질 추출물은 마늘껍질 에탄올 추출물이고,
상기 마늘껍질 추출물의 농도는 5 mg/㎖ 내지 10 mg/㎖이며,
상기 장내균총은 프로바이오틱스를 포함하고, 상기 프로바이오틱스는 엔테로코커스 패시움(Enterococcus faecium) 및 바이셀라 시바리아(Weissella cibaria)를 포함하는 것을 특징으로 하는 장내균총 개선용 프리바이오틱스 조성물.As a prebiotic composition for improving intestinal flora containing garlic peel extract as an active ingredient,
The garlic peel extract is a garlic peel ethanol extract,
The concentration of the garlic peel extract is 5 mg/ml to 10 mg/ml,
The intestinal flora includes probiotics, and the probiotics include Enterococcus faecium and Weissella cibaria . A prebiotic composition for improving intestinal flora.
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