KR101206837B1 - Development of chinese herb food material with complex function of hematosis and osteoblast activation - Google Patents
Development of chinese herb food material with complex function of hematosis and osteoblast activation Download PDFInfo
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- KR101206837B1 KR101206837B1 KR1020100030785A KR20100030785A KR101206837B1 KR 101206837 B1 KR101206837 B1 KR 101206837B1 KR 1020100030785 A KR1020100030785 A KR 1020100030785A KR 20100030785 A KR20100030785 A KR 20100030785A KR 101206837 B1 KR101206837 B1 KR 101206837B1
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- red ginseng
- extract
- kelp
- hours
- ginseng extract
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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
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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
- A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
- A23L17/60—Edible seaweed
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- 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
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- 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 조골 및 조혈기능을 갖는 기능성 식품 조성물 및 그 제조 방법에 관한 것으로, 홍삼(4년근 이상)가루 1kg 기준으로 정제수 5~20L를 첨가하여 50~100℃에서 2~10시간 추출한 후 50~100℃에서 12 내지 48시간 숙성시켜 홍삼액을 제조하고, 상기한 홍삼액을 100~150℃에서 10~20분간 가열 후 실온상태로 냉각시키고 유산균 스타터를 초기균수 3~10x10 cfu/mL 접종하고 20~60℃에서 24~48시간 발효시켜 발효 홍삼액을 얻고, 상기 홍삼액을 동결건조하여 동결건조한 시료의 5~20배량의 50~100% 주정을 가해 추출하는 발효 홍삼액 추출 과정과, 건조한 다시마를 열풍 건조에서 100~150℃에서 10~60분간 열처리한 후 분쇄하여, 분쇄물 1kg 기준으로 5~20L의 정제수를 가하여 50~150℃에서 2~10시간 환류냉각 추출하여 상등액과 침전물을 분리하여 반복 추출 후, 유기산을 이용하여 가수분해하여 동결 건조하는 열처리 다시마 추출액 가수분해 과정과, 상기한 열처리 다시마 추출물의 유기산 가수분해한 동결 건조 시료와 발효 홍삼추출물 동결 건조 시료를 1~10:10~1의 비율로 혼합하는 과정을 포함하여 구성되는 것을 특징으로 하는 기능성 식품 및 그 제조 방법에 관한 것이다.The present invention relates to a functional food composition having osteoblastic and hematopoietic function and a method of manufacturing the same, by adding 5-20L of purified water based on 1kg of red ginseng (4 years old or more) powder and extracting it at 50-100 ° C. for 2-10 hours. Red ginseng solution was prepared by aging at 100 ° C. for 12 to 48 hours, and the red ginseng solution was heated at 100 to 150 ° C. for 10 to 20 minutes, then cooled to room temperature, and inoculated with lactic acid bacteria starter 3 to 10 × 10 cfu / mL and 20 to 60 Fermented red ginseng extract obtained by fermentation at 24 ° C. for 24 to 48 hours, fermented red ginseng extract extracted by adding 50-100% alcohol of 5-20 times the freeze-dried sample by freeze-drying the red ginseng extract, and dried kelp 100 in hot air drying. After heat treatment at ~ 150 ℃ for 10-60 minutes and then pulverized, 5 ~ 20L purified water on the basis of 1kg of crushed powder, reflux-cooled extraction at 50-150 ℃ for 2-10 hours to separate the supernatant and sediment, and then repeatedly extracted, organic acid Hydrolysing using Heat treatment kelp extract hydrolyzed by lyophilization and mixing the organic acid hydrolyzed lyophilized sample and fermented red ginseng extract lyophilized sample of the heat treatment kelp extract at a ratio of 1-10: 10-1 It relates to a functional food and a method for producing the same.
Description
본 발명은 조골 및 조혈기능을 갖는 기능성 식품 조성물 및 그 제조 방법에 관한 것이다.The present invention relates to a functional food composition having a osteoblast and a hematopoietic function and a method for producing the same.
임신을 유지하는 기간 동안 임산부는 태아 때문에 영양요구량과 미네랄 요구량이 많아지게 된다. 따라서 임신기간 중에 충분히 태아조직 형성에 도움이 되는 칼슘, 철분 혹은 그 유도체 등이 임산부에게 공급된다면 태아뿐 아니라 임산부도 최상의 상태로 건강을 유지할 수 있는데 도움이 될 것이다.During pregnancy, pregnant women increase their nutritional and mineral requirements because of the fetus. Therefore, if calcium, iron, or derivatives thereof, which are sufficient for the formation of fetal tissue during pregnancy, are supplied to pregnant women, it will help to maintain the best health as well as the fetus.
그리고, 산후에는 임신을 유지하면서 부족하게 된 철분과 출산 중 발생되는 다량의 출혈 등으로 인하여 빈혈이 생기기 쉬운 특징이 있다. 뿐만 아니라 산후에 모유 수유를 선호하는 환경에서, 100일 이후의 모유수유는 영아의 모유 섭취량이 늘어나면서 산모들의 영양과 체력저하가 현저하게 진행되는 경향이 있다. 이때 상당수의 산모들은 모유수유를 하면서 뼈의 주요 구성성분인 칼슘을 포함한 다량의 미네랄 손실로 인하여 뼈가 약해지고 어지럼증, 무기력증, 빈혈 등 증상을 호소하기 쉽다. 장기적으로 철분결핍이 지속되면 심한 경우에는 악성빈혈이나 골다공증의 가능성이 높아질 수 있으며 또한 저체중으로 심각한 건강장해를 일으킬 수 있다.In addition, the postpartum is characterized by a tendency to cause anemia due to iron deficiency while maintaining pregnancy and a large amount of bleeding generated during childbirth. In addition, in an environment where breastfeeding is preferred after birth, breastfeeding after 100 days tends to significantly progress in nutrition and stamina of mothers as the infant's breast milk intake increases. At this time, many mothers are breastfeeding, and the loss of a large amount of minerals, including calcium, the major constituent of bone, weakens the bones and makes it easy to complain of symptoms such as dizziness, lethargy and anemia. If iron deficiency persists in the long term, it can increase the likelihood of pernicious anemia or osteoporosis in severe cases and can cause serious health problems due to low weight.
이에 따라 시중에는 철분 보충제가 판매되고 있는데, 현재 시중에서 판매되고 있는 철분 보충제의 1일 함량은 성인 남자 권장량의 3-11배 범위로 장기간 복용할 경우 중독증을 유발할 수 있어 과잉섭취로 인한 독성과 소화 장애 및 변비 등의 부작용이 문제점으로 지적되고 있다.
As a result, commercially available iron supplements are sold on the market. The daily supplement of iron supplements on the market is 3-11 times the recommended amount of adult males. Side effects such as disorders and constipation are pointed out as problems.
한편, 골다공증(osteoporosis)이란 조직학적으로 골조성에는 변함없이 뼈의 감소된 상태를 말한다. 골다공증의 궁극적 치료목표는 칼슘과 비타민 D의 부족, 과다한 동물성 단백질, 나트륨(Na), 카페인, 섬유질 섭취 및 음주와 같은 유발인자를 제거하여 주는 한편 골흡수를 억제하고 골형성을 촉진하여 감소된 골량을 회복시키는 것인데 FDA에서 공인받은 것은 에스트로겐(estrogen)과 칼시토닌(calcitonin) 뿐으로 estrogen은 여러 가지 부작용과 투여시기를 적절하게 유지하기 어렵고 유방암, 자궁암, 간질환, 고혈압, 편두통 등을 가진 환자들은 금기시되어 여러 가지 불편함이 따른다.
On the other hand, osteoporosis (osteoporosis) refers to the reduced state of the bone intact in bone composition. The ultimate therapeutic goal for osteoporosis is to reduce bone mass by suppressing bone resorption and promoting bone formation while eliminating triggers such as calcium and vitamin D deficiency, excess animal protein, sodium (Na), caffeine, fiber intake and drinking. Estrogen and calcitonin have been approved by the FDA, and estrogen is difficult to maintain various side effects and timing, and is contraindicated in patients with breast cancer, uterine cancer, liver disease, high blood pressure, and migraine headaches. There are various inconveniences.
따라서 부작용이 적고, 기능성이 검증된 안전한 한방 식품재료로부터의 빈혈 및 골다공증 예방의 효과를 가지는 한방소재와 이를 이용한 기능성 식품의 개발이 요구되고 있는 실정이다.
Therefore, there is a demand for the development of herbal foods and functional foods using the same, which have an effect of preventing anemia and osteoporosis from safe herbal food ingredients with little side effects and proven functionality.
그리고, 산업이 발달되고, 소득이 높아질수록 전반적인 영양섭취가 풍부해져서 신장이 크게 향상되면서 사회적으로도 큰 신장이 선호되고 있는 추세이다. 이에 따라 비교적 신장이 작은 어린이 및 청소년들의 열등감은 상대적으로 가중되고 있어 성장 촉진을 위한 제품의 수요가 급증하고 있는 현실이다.
In addition, as the industry develops and the income increases, the overall nutrition intake is abundant, and the height is greatly improved. As a result, the inferiority of children and adolescents, who are relatively small, is aggravating, and the demand for products to promote growth is rapidly increasing.
상기한 바와 같은 인체에 무해한 기능성식품 소재 개발의 요구에 의한, 기능성식품의 선행기술로는, 빈혈 관련하여 한방소재를 이용한 특허로 대한민국 공개특허 10-2006-0012368호에 발효콩, 다시마 등을 포함한 혼합재료를 이용한 건강기능식품에 관한 기술이 공개되어있고, 공개특허 10-2004-0041867호에는 각종 생약재를 이용한 건강보조식품식품 및 그 제조방법 등이 개시되어 있다.As a prior art of functional foods, by the demand of functional food material development harmless to the human body as described above, including the fermented soybean, kelp, etc. in the Republic of Korea Patent Publication No. 10-2006-0012368 as a patent using an herbal material related to anemia A technology related to a health functional food using a mixed material is disclosed, and Patent Publication No. 10-2004-0041867 discloses a health supplement food using various herbal medicines and a method of manufacturing the same.
골생성 및 골다공증에 관한 특허로는 대한민국 등록특허 0740565호와 0718490호에서 파골세포 분화억제효과를 가지는 대추 추출물과 황금추출물 및 이를 포함하는 건강기능성 조성물이 있으며, 그 밖에 공개특허 특2002-0084360호에는 녹용 및 기타 한약재를 포함하는 한방음료 제조에 관한 특허가 있다.Patents related to bone formation and osteoporosis include jujube extract and golden extract having osteoclast differentiation effect in Korean Patent Nos. 0740565 and 0718490, and a health functional composition comprising the same, and in other Patent Publication No. 2002-0084360 There is a patent on the manufacture of herbal beverages including antler and other herbal medicines.
한편, 상기한 선행기술에서도 사용되고 있는 다시마의 경우에는 고부가가치 칼슘공급원을 개발하기위한 기초연구로서 많은 연구가 진행되고 있는데 동물실험결과 다시마 식이가 대퇴골의 칼슘함량과 강도를 높여주는 것으로 나타나 다시마가 뼈의 칼슘흡착을 도와주는 것으로 보고되고 있으며, 이러한 다시마의 유효성분 중에서 특히 알긴산 추출에 관한 기술들이 다수 공개되어 있다.On the other hand, in the case of kelp used in the above-described prior art, many studies are being conducted as a basic research for developing a high value-added calcium source.As a result of animal experiments, the kelp diet increases the calcium content and strength of the femur. It has been reported to help the calcium adsorption of, and a number of techniques for extracting alginic acid among the active ingredients of kelp have been published.
그 예로서, 대한민국 등록특허 10-0367716호와 10-0367721호 및 10-0367722호에서는 다시마로부터 저분자 알긴산의 추출방법에 대하여 각각 고온가압 전처리, 초음파전처리, 마이크로파 전처리 기술이 공개되어 있다.
As an example, Korean Patent Nos. 10-0367716 and 10-0367721 and 10-0367722 disclose high pressure pretreatment, ultrasonic pretreatment, and microwave pretreatment techniques for the extraction method of low molecular weight alginic acid from kelp, respectively.
한편, 인삼은 뛰어난 약리효과로 인해 2,000년 전부터 동양에서는 이용되어 왔는데 그 중 인삼의 지용성 성분이 항암효과뿐만 아니라 적혈구 및 헤모글로빈 증가와 같은 빈혈예방 및 치료에 효과를 가지며 동시에 면역기능을 활성화하는 것으로 보고되고 있다.On the other hand, ginseng has been used in the Orient for 2,000 years ago because of its excellent pharmacological effect. Among them, the fat-soluble component of ginseng is reported to be effective in preventing and treating anemia such as erythrocyte and hemoglobin as well as anti-cancer effect and at the same time activating immune function. It is becoming.
본 발명의 목적은 한방 재료로부터 임산부/수유부용 빈혈예방과 골생성 촉진의 복합기능을 가지면서 그 효과가 상승되도록 하는 조골 및 조혈기능을 갖는 기능성 식품 조성물 및 그 제조 방법을 제공하는 데 있다.It is an object of the present invention to provide a functional food composition having a osteoblast and a hematopoietic function and a method for producing the same, having a combined function of preventing anemia for pregnant women and lactating mothers and promoting bone formation from herbal materials.
상기한 목적을 달성하기 위한 본 발명의 특징은, 홍삼(4년근 이상)가루 1kg 기준으로 정제수 5~20L를 첨가하여 50~100℃에서 2~10시간 추출한 후 50~100℃에서 12 내지 48시간 숙성시켜 홍삼액을 제조하고, 상기한 홍삼액을 100~150℃에서 10~20분간 가열 후 실온상태로 냉각시키고 유산균 스타터를 초기균수 3~10x10 cfu/mL 접종하고 20~60℃에서 24~48시간 발효시켜 발효 홍삼액을 얻고, 상기 홍삼액을 동결건조하여 동결건조한 시료의 5~20배량의 50~100% 주정을 가해 추출하는 발효 홍삼액 추출 과정과, 건조한 다시마를 열풍 건조에서 100~150℃에서 10~60분간 열처리한 후 분쇄하여, 분쇄물 1kg 기준으로 5~20L의 정제수를 가하여 50~150℃에서 2~10시간 환류냉각 추출하여 상등액과 침전물을 분리하여 반복 추출 후, 유기산을 이용하여 가수분해하여 동결 건조하는 열처리 다시마 추출액 가수분해 과정과, 상기한 열처리 다시마 추출물의 유기산 가수분해한 동결 건조 시료와 발효 홍삼추출물 동결 건조 시료를 1~10:10~1의 비율로 혼합하는 과정을 포함하여 구성되는 것을 특징으로 하는 기능성 식품 제조 방법에 있다.Features of the present invention for achieving the above object, red ginseng (4 years old or more) by adding 5 ~ 20L of purified water on the basis of 1kg powder extracted for 2 to 10 hours at 50 ~ 100
그리고, 본 발명의 다른 특징은 열처리 다시마 추출물의 유기산 가수분해한 동결 건조 시료와 발효 홍삼추출물 동결 건조 시료를 1~10:10~1의 비율로 혼합하여 구성되는 기능성 식품 조성물에 있다.In addition, another feature of the present invention is a functional food composition composed by mixing the organic acid hydrolyzed lyophilized sample and the fermented red ginseng extract lyophilized sample of the heat treatment kelp extract in a ratio of 1-10: 10-1.
상기한 본 발명에 의하면 다시마 및 홍삼은 조골, 조혈기능을 가지고 있어서, 임산부가 필요한 철분제의 공급을 대체할 수도 있으며, 특히 빈혈을 동반한 임산부에게는 빈혈을 조절해주는 기능과 함께 고기능성 골성장 특성이 있다.According to the present invention, the kelp and red ginseng have osteoblastic, hematopoietic function, and can replace the iron supply necessary for pregnant women, and especially for pregnant women with anemia, high functional bone growth characteristics along with the function of controlling anemia have.
또한, 산후에 산모가 먹는 기능성식품(예를 들면 기능성우유 등)을 통하여, 영유아기에 아이에게도 영양과 뼈를 구성하는 성분이 충분하게 포함된 모유를 제공할 수 있게 되고 이로 인하여 아이의 성장에도 도움이 된다.In addition, through functional foods (for example, functional milk, etc.) that mothers eat after giving birth, it is possible to provide breast milk containing enough ingredients for nourishment and bones to children even in infants and toddlers. Becomes
동시에 임산부/수유부뿐 아니라 성장이 약하고 면역기능이 저하되어 있는 영, 유아 및 어린이용 제품에도 즉시 활용이 가능하다.At the same time, it can be used not only for pregnant and lactating women, but also for infants, infants and children with weak growth and poor immune function.
또한, 본 발명의 조성물은 단순히 분/우유 등에 투입할 수 있을 뿐 아니라 개별적으로 제형에 따라서는 고가의 건강기능성 식품으로 만들 수 있으며, 건강기능성 식품 및 소재의 다양화로 수입대체효과가 있다.In addition, the composition of the present invention can be simply added to the powder / milk, etc., depending on the dosage form can be made of expensive health functional food, there is an import substitution effect by the diversification of health functional food and materials.
그리고, 상기한 다시마는 전처리로서 가열하게 되면 알긴산을 포함한 유효 성분의 추출수율이 향상되는 효과가 있고, 점도가 낮아지고 맛 등 종합적인 기호도가 향상되며, 분자량이 낮은 알긴산의 추출이 가능해지는 효과가 있다.In addition, when the kelp is heated as a pretreatment, the extraction yield of the active ingredient including alginic acid is improved, the viscosity is lowered, the overall acceptability of taste is improved, and the extraction of alginic acid having a low molecular weight is possible. have.
특히, 열처리 다시마추출물 가수분해물은 조혈, 조골 특성이 우수하며, 다시마 추출물을 열처리만 사용한 것과 홍삼 추출물만 사용한 것에 비하여 이들을 혼합할 경우 다시마와 홍삼의 효과를 동시에 얻을 수 있는 효과가 있고, 종합적인 기호도에 있어서도 각각 단독으로 사용하는 것에 비하여 향상된 효과가 있다.In particular, the heat treated kelp extract hydrolyzate is excellent in hematopoietic and osteoblastic properties, and the effect of kelp and red ginseng at the same time can be obtained when mixed with them, compared to those using only heat treatment and red ginseng extract. In addition, there is an improved effect compared to using each alone.
도 1은 열처리에 따른 다시마 추출물의 분자구조를 살펴보기 위해 FT-IR 측정 결과를 나타내는 도면.
도 2는 유기산을 이용한 다시마 추출물의 가수분해물에 있어서 FT-IR을 이용한 결합구조의 변화를 나타내는 도면.
도 3은 골수세포에 대한 발효홍삼 추출물과 열처리 다시마추출물의 가수분해물 독성을 알아보기 위하여 MTS assay 실험 결과를 나타내는 도면.
도 4는 골수세포를 11일 동안 배양 후 형성되는 집락군의 형태는 집락을 형성한 세포의 크기가 작고 밀집된 집락인 CFU-G와 세포들이 비교적 크고 산발적으로 분포한 집락인 CFU-M으로 정의하였으며, CFU-G과 CFU-M을 포함하는 집락인 경우인 CFU-GM의 집락수를 나타내는 도면.
도 5는 홍삼, 발효홍삼 및 열처리 다시마추출물 가수분해물의 조혈모세포 생성에 대한 집락수 측정 결과를 나타내는 도면.
도 6은 홍삼, 발효 홍삼 및 열처리 다시마추출물 가수분해물의 조혈모세포 생성에 대한 집락수 측정 결과를 나타내는 도면.
도 7은 복합 혼합물의 조혈모세포에 대한 집락군 형성을 나타내는 도면.
도 8은 발효 홍삼, 열처리 다시마 추출물 가수분해물, 복합 혼합물의 조골세포의 증식활성을 나타내는 도면
도 9는 발효 홍삼, 열처리 다시마 추출물 가수분해물, 복합 혼합물의 배양 72시간 후의 골아세포 증식 결과를 나타내는 도면1 is a view showing the results of the FT-IR measurement to look at the molecular structure of kelp extract according to the heat treatment.
2 is a view showing a change in the binding structure using FT-IR in the hydrolyzate of kelp extract using an organic acid.
Figure 3 is a view showing the results of the MTS assay experiments to determine the hydrolyzate toxicity of fermented red ginseng extract and heat-treated kelp extract against bone marrow cells.
4 is a colony group formed after culturing bone marrow cells for 11 days and defined as CFU-G, which is a small, dense colony of colony-forming cells, and CFU-M, which is a relatively large and sporadic distribution of cells. , Which shows the number of colonies of CFU-GM in the case of colonies containing CFU-G and CFU-M.
Figure 5 shows the results of colony count measurement for hematopoietic stem cell production of red ginseng, fermented red ginseng and heat treated kelp extract hydrolyzate.
Figure 6 shows the results of colony count measurement for hematopoietic stem cell production of red ginseng, fermented red ginseng and heat treated kelp extract hydrolyzate.
7 shows colony formation for hematopoietic stem cells of the complex mixture.
8 is a view showing the proliferative activity of osteoblasts of fermented red ginseng, heat treated kelp extract hydrolyzate, complex mixture
9 shows osteoblast proliferation results after 72 hours of culture of fermented red ginseng, heat-treated kelp extract hydrolyzate and complex mixture
이하 본 발명의 실시예를 첨부한 도면을 참조하여 상세히 설명한다.
Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.
I. 홍삼 및 다시마의 전처리와 추출물 제조 및 유효성분추출 I. Pretreatment of Red Ginseng and Kelp, Preparation of Extracts and Extraction of Active Ingredients
1. 전처리1. Pretreatment
1) One) 홍삼액Red ginseng 제조 Produce
홍삼(4년근 이상, 국내산)을 전문점에서 구입하여 80 mesh 정도로 분쇄하였다. 홍삼가루 1kg에 정제수 10L를 첨가하여 80℃에서 5시간 추출한 후 70℃에서 24시간 숙성시켜 홍삼액을 제조, 동결 건조하여 -23℃에서 보관하면서 시료로 사용하였다.
Red ginseng (4 years old or more, domestic) was purchased from a specialty store and ground to about 80 mesh. 10 g of purified water was added to 1 kg of red ginseng powder, extracted at 80 ° C. for 5 hours, and aged at 70 ° C. for 24 hours to prepare red ginseng solution, freeze-dried, and stored as -23 ° C. as a sample.
2) 발효 홍삼의 제조2) Preparation of Fermented Red Ginseng
발효 홍삼은 상기 홍삼액을 121℃ 오토클레이브(autoclave)에서 15분간 가열 후 실온상태로 냉각시키고 발효홍삼 제조용 복합 유산균 스타터(Lactobacillus bulgaricus(한국종균협회, 한국, 2006) + Lactobacillus acidophilus (한국종균협회, 한국, 2006))를 각각 초기균수 4.5x10 cfu/mL 접종하고 40℃에서 36시간 발효시켜 발효 홍삼액을 얻고 이를 동결 건조하여 -23℃에 보관하면서 실험 시료로 사용하였다.
Fermented red ginseng was heated for 15 minutes in an autoclave at 121 ° C. in an autoclave, cooled to room temperature, and mixed with Lactobacillus bulgaricus ( Lactobacillus bulgaricus ( Korean spawn association, Korea, 2006 ) + Lactobacillus ) acidophilus ( Korean spawn association, Korea, 2006)) was inoculated with 4.5x10 cfu / mL of the initial bacterial count and fermented at 40 ° C for 36 hours to obtain fermented red ginseng solution, which was freeze-dried and stored at -23 ° C.
3) 다시마 전처리3) Kelp pretreatment
열처리 다시마의 처리는 건조한 다시마를 1회 살수 세척하고 열풍 건조기(50℃)에서 건조한 후 121℃에서 30분간 열처리하였다. 이 후 열풍 건조기(50℃)에서 건조 하여 80 mech로 분쇄한 후 -23℃에서 냉동 보관하면서 추출시료로 사용하였으며, 무처리 다시마는 건조한 다시마를 1회 살수 세척하고 열풍 건조기(50℃)에서 건조한 후 80 mesh로 분쇄한 후 -23℃에서 냉동 보관하면서 추출시료로 사용하였다.
Heat treatment kelp was sprayed once dried kelp, dried in a hot air dryer (50 ℃) and heat-treated at 121
2. 추출물 제조2. Extract Manufacturer
1) 홍삼 및 발효 홍삼의 추출1) Extraction of Red Ginseng and Fermented Red Ginseng
홍삼 및 발효 홍삼 시료 각각의 10배 양의 70% 주정을 첨가하여 실온에서 24시간 동안 침지 추출한 후 상등액과 침전물을 분리하는 방법으로 3회 반복하였다. 추출물은 12,000rpm으로 10분간 원심분리한 후 Whatman No. 1 여과지로 여과한 후 농축하였으며, 동결건조 후 냉동실(-23℃)에 보관하면서 본 실험의 시료로 사용하였다.
Ten times the amount of red ginseng and fermented red ginseng samples 70% ethanol was added and immersed for 24 hours at room temperature and then repeated three times by separating the supernatant and precipitate. The extract was centrifuged at 12,000 rpm for 10 minutes and then Whatman No. After filtration with 1 filter paper and concentrated, and freeze-dried and stored in the freezer (-23 ℃) was used as a sample of this experiment.
2) 다시마 및 열처리 다시마 추출2) Kelp and Heat Treatment Kelp Extract
다시마 및 열처리 다시마 분말 시료로부터 알긴산의 추출은 100g에 증류수 10배 양을 가하여 100℃에서 4시간 환류 냉각 추출하여 상등액과 침전물을 분리하여 3회 반복 추출하였다.
Extraction of alginic acid from kelp and heat treated kelp powder samples was performed by extracting 10 times the amount of distilled water to 100 g and reflux cooling under reflux at 100 ° C. for 4 hours to separate the supernatant and the precipitate and extract three times.
3. 추출물의 유효성분 추출3. Extraction of Active Ingredients of Extracts
1) 발효 홍삼 추출물의 유효성분 추출1) Extraction of Active Ingredients from Fermented Red Ginseng Extract
i. i. 진세노사이드Gin Senocide
시료에 80% methanol을 넣고 1시간 환류 추출한 다음 여과하여 잔사를 반복 추출하고 여액을 감압 농축하였으며 이를 증류수로 정용하고 분액 깔대기에 옮겨 diethyl ether로 2회 반복 추출하여 불순물을 제거하였다. 이후 남아있는 물 층에 수포화부탄올 용액을 첨가하여 반복 추출한 다음 수포화 부탄올층을 증류수로 세척하였다. 그 후 감압 하에서 농축 후 methanol로 용해하여 0.45㎛ membrane filter로 여과하고 그 여과액을 HPLC(Jasco PU-980, Tokyo, Japan)를 사용하여 사포닌의 주요성분인 진세노사이드의 분석에 사용하였다. 이때 컬럼은 Merck chromolith RP-18e Column(4.6x100 ㎜, Germany)을, 검출기는Jasco UV detector(203 nm)를 사용하였으며 이동상으로는 10% methanol과 80% acetonitrile을 사용하였고 이동상의 유속은 분당 2.5 mL, 시료 주입량은 20 ㎕ 이었다.
80% methanol was added to the sample, and the mixture was extracted under reflux for 1 hour, filtered, and the residue was repeatedly extracted. The filtrate was concentrated under reduced pressure. The filtrate was purified by distilled water, transferred to a separatory funnel, and extracted twice with diethyl ether to remove impurities. Then, the saturated butanol solution was repeatedly added to the remaining water layer, and the saturated butanol layer was washed with distilled water. Thereafter, the mixture was concentrated under reduced pressure, dissolved in methanol, filtered through a 0.45 μm membrane filter, and the filtrate was used for analysis of ginsenoside, a main component of saponin, using HPLC (Jasco PU-980, Tokyo, Japan). The column used Merck chromolith RP-18e Column (4.6x100 mm, Germany), the detector used Jasco UV detector (203 nm), the mobile phase used 10% methanol and 80% acetonitrile, and the mobile phase flow rate was 2.5 mL / min. The sample injection amount was 20 µl.
iiii . 산성 다당체. Acidic polysaccharides
산성 다당체의 비색 측정은 시료추출액에 carbazole 0.25 mL와 C-H2SO4 3 mL를 넣은 후 80℃ 수욕 상에서 5분간 반응시킨 다음 실온에서 방치한 것을 525 nm에서 흡광도를 측정하였으며 대조구는 carbazole 대신에 ethanol을 사용하였다.
For colorimetric measurement of acidic polysaccharides, 0.25 mL of carbazole and 3 mL of CH 2 SO 4 were added to the sample extract, reacted for 5 minutes in an 80 ° C water bath, and then absorbed at 525 nm. The control was ethanol instead of carbazole. Used.
iiiiii . . 총페놀성Total phenolic 화합물함량 Compound content
시료를 methanol로 추출한 여과액을 0.1mL를 시험관에 넣고 증류수와 Forlinciocalteu's reagent를 첨가하였다. 실온에서 3분 동안 방치 후 sodium carbonate solution을 첨가하고 혼합하여 30분 동안 방치 후 spectrophotometer(Optizen 2120UV, Mecasys, Korea)를 이용하여 724 nm에서 흡광도를 측정하였다. 표준품으로는 gallic acid(Sigma Chemical, Co.,)를 사용하여 검량선을 작성하여 함량을 산출하였다.
0.1 mL of the filtrate from which the sample was extracted with methanol was placed in a test tube, and distilled water and Forlincio calteu's reagent were added thereto. After standing at room temperature for 3 minutes, sodium carbonate solution was added and mixed for 30 minutes, and then absorbance was measured at 724 nm using a spectrophotometer (Optizen 2120UV, Mecasys, Korea). As a standard product, a calibration curve was prepared using gallic acid (Sigma Chemical, Co.,) to calculate the content.
2) 다시마 추출물 유효 성분 추출2) Kelp Extract Active Ingredient Extraction
i. 고형분 함량i. Solid content
고형분 함량은 refractometer(Atago hand refractometer, Atago Co., LTD., Japan)를 이용하여 Brix를 측정하였다. Brix 고형분 농도 환산은 105℃ 상압건조법에 의한 고형분 측정과 Brix간의 비례관계 직선식에서 구하였으며, 이를 추출액 100 mL에 대한 고형분량으로 나타내었다.
Solid content was measured by Brix using a refractometer (Atago hand refractometer, Atago Co., LTD., Japan). The conversion of Brix solids concentration was calculated from the linear relationship between Brix measurement and Brix measurement by 105 ° C atmospheric pressure drying, and this was expressed as solids content for 100 mL of extract.
iiii . 알긴산(. Alginic acid ( AlginicAlginic acidacid ) 함량) content
추출 공정에 의해 추출된 다시마 추출액 일정량에 CaCl2용액을 첨가하여 알긴산 칼슘으로 석출한 후 묽은 염산을 첨가하여 잔존 Ca을 제거하고 원심분리하여 수분을 제거하였다. 이를 다시 NaOH를 함유한 methyl alcohol에 넣고 밀폐, 교반하여 탈수 및 알긴산나트륨으로 전환시킨 후 methyl alcohol에 담긴 알긴산나트륨을 압착하여 alcohol을 분리하고 열풍건조하여 항량을 구하였으며, 이를 추출액 100 mL에 대한 알긴산 함량으로 나타내었다.
CaCl 2 solution was added to a predetermined amount of kelp extract extracted by the extraction process to precipitate calcium alginate, and then diluted hydrochloric acid was added to remove residual Ca and centrifuged to remove water. The resultant was put into methyl alcohol containing NaOH, sealed and stirred to be dehydrated and converted to sodium alginate. After pressing sodium alginate contained in methyl alcohol, alcohol was separated and hot air dried to obtain a specific amount. The content is shown.
4. 다시마 추출물의 가수분해4. Hydrolysis of Kelp Extract
건조한 알긴산은 탈이온수에 녹여 1% 용액으로 제조하고 citric acid (Sigma No. 251275, USA)로 pH를 5.0으로 조정한 후 121℃에서 1시간 동안 가수분해하였다.
Dry alginic acid was dissolved in deionized water to prepare a 1% solution, and the pH was adjusted to 5.0 with citric acid (Sigma No. 251275, USA) and hydrolyzed at 121 ° C. for 1 hour.
5. 5. 발효홍삼Fermented Red Ginseng 및 다시마의 복합 혼합물 제조 And preparation of complex mixture of kelp
열처리 다시마 추출물의 유기산 가수분해한 동결건조 시료와 발효 홍삼 추출물 동결건조 시료를 1:1의 비율로 혼합한 것과 열처리 다시마 추출물의 유기산 가수분해 동결건조 시료와 발효 홍삼 추출물 동결 건조 시료를 10:1의 비율로 혼합한 것의 2종류로 제조하였다.
The organic acid hydrolyzed lyophilized sample of fermented kelp extract and the fermented red ginseng extract lyophilized sample were mixed at a ratio of 1: 1 and the organic acid hydrolyzed lyophilized sample and fermented red ginseng extract lyophilized sample of heat treated kelp extract It produced with two types of things mixed by the ratio.
IIII . 실험 방법. Experimental Method
(1) 물리/화학적 성질 실험 방법(1) Physical / chemical property test method
i. 수분함량 i. Moisture content
수분함량은 AOAC법에 따라 105℃ 상압가열건조법을 이용하여 측정하였다.
Moisture content was measured using a 105 ° C atmospheric pressure drying method according to the AOAC method.
iiii . . pHpH
건조 시료 0.1g에 증류수 20ml를 가하여 혼합한 후 실온에서 pH meter(Model520A, ORION, USA)를 사용하여 측정하였다.
20 ml of distilled water was added to 0.1 g of the dry sample, mixed, and measured using a pH meter (Model520A, ORION, USA) at room temperature.
iiiiii . 색상. color
색상은 색차계(Minolta Co. Japan)로 Hunter값을 조사하여 L*,a*, b*로 나타내었다.
The color was expressed as L *, a *, b * by examining Hunter value with a color difference meter (Minolta Co. Japan).
iviv . 분자량. Molecular Weight
0.02N NaNO3 용액에 용해한 각각의 시료를 겔크로마토그래피(gel chromatography)에 의하여 측정하였다. 즉 장치는 Gel chromatography system(USA), 수지는 Sepharose CL-6B, Column은 Waters Ultra-hydrogel 500, Waters Ultra-hydrogel 250, Waters Ultra-hydrogel 120, 표준품은 MP기준으로 PEG(polyethylene glycol) 23,000, 12,000, 6,240, 4,450, 1,500, 970, 600,106, flow rate 0.8 ml/min, 온도 25℃의 조건으로 측정하였다. 각 시료의 용출속도에 대한 표준 물질의 분자량 대수값으로 작성한 검량선으로부터 시료의 분자량을 계산하였다.
Each sample dissolved in 0.02N NaNO 3 solution was measured by gel chromatography. In other words, the device is Gel chromatography system (USA), the resin is Sepharose CL-6B, the column is
v. 관능적 특성 v. Sensual characteristic
홍삼 및 발효 홍삼 추출물 시료의 관능적 특성은 단맛(sweety), 쓴맛(bitter taste), 냄새(odor), 색상기호도(color acceptability) 및 전체적인 품질(overall quality)에 대하여 5점 척도법으로 나타내었다. Sensory characteristics of red ginseng and fermented red ginseng extract samples were expressed on a five-point scale for sweetness, bitter taste, odor, color acceptability and overall quality.
다시마 추출물 및 다시마 추출물 가수분해물의 관능적 특성은 단맛(sweety), 감칠맛(savory), 냄새(odor), 색상기호도(color acceptability) 및 전체적인 품질(overall quality)에 대하여 5점법으로 나타내었다.The organoleptic properties of kelp extract and kelp extract hydrolyzate are represented by a five-point method for sweetness, savory, odor, color acceptability and overall quality.
복합 혼합물의 관능적 특성은 단맛, 쓴맛, 이취, 색상, 입안에서의 느낌 및 종합적 품질에 대하여 5점법으로 나타내었다.The organoleptic properties of the complex mixtures were expressed by the five-point method for sweetness, bitterness, odor, color, mouthfeel, and overall quality.
즉, 1점; 매우 나쁘다, 또는 매구 약하다, 2점; 나쁘다, 또는 약하다, 3점; 보통, 4점; 좋다, 또는 강하다, 5점; 매우 좋다, 또는 매우 강하다.
That is, 1 point; Very bad or very weak; Bad or weak, 3 points; Usually 4 points; Good or strong, 5 points; Very good, or very strong.
vivi 점도 Viscosity
다시마, 다시마 추출물 가수분해물 및 복합 혼합물의 점도는 Brookfield viscometer(model-DV Ⅱ Brookfield Engineering Labs.)를 사용하여 5.0%의 추출액을 25℃에서 spindle No.4로 100rpm에서 60초간 회전시키면서 측정하였다.
The viscosity of the kelp, kelp extract hydrolyzate and the complex mixture was measured using a Brookfield viscometer (model-DV II Brookfield Engineering Labs.) While rotating 5.0% of the extract at 25 ° C. with spindle No. 4 at 100 rpm for 60 seconds.
viivii . 결합형태(. Combined form ( FTFT -- IRIR ) )
다시마 및 다시마 추출물 가수분해물의 결합형태 측정은 Genesis Ⅱ FT-IR TM spectrophotometer (Mattson, USA)를 사용하여 측정하였으며, NaCl disc(25×4 mm, Spectra-Tech Inc., USA)에 시료 물질을 가압 정제하여 측정한다.
The binding form of the kelp and kelp extract hydrolyzate was measured using a Genesis II FT-IR ™ spectrophotometer (Mattson, USA) and pressurized the sample material to NaCl disc (25 × 4 mm, Spectra-Tech Inc., USA). Purify and measure.
viiiviii . . 흡습Hygroscopic 안정성 stability
복합 혼합물의 흡습 안정성은 조성물 시료 1g 을 취하여 포화상태로 조절된 데시케이터 내에 방치하면서 2시간 단위로 10시간 동안 무게변화를 측정하여 시료의 흡습정도를 알아보았다.The hygroscopic stability of the composite mixture was taken to determine the hygroscopicity of the sample by measuring the change in weight for 10 hours every 2 hours while leaving the desiccator adjusted to the saturated state by taking 1g of the composition sample.
Water uptake(g)= Final weight - Initial weight
Water uptake (g) = Final weight-Initial weight
viiiiviiii . . 수용화Acceptance 정도 Degree
복합 혼합물의 수용화 정도는 수분 용해 지수(water solubility index : WSI)를 구하였다. 즉, 시료 10g에 물 100mL를 가하여 상온에서 10분간 교반 한 후 2,000 x g에서 10분간 원심분리 하였다. 이 때 상등액 10mL를 취하여 수분 칭량병에 넣은 후 105℃에서 4시간 동안 건조하여 고형분 함량을 측정한 후 WSI를 다음 식으로 결정하였다.The water solubility index (WSI) of the complex mixture was calculated. That is, 100 mL of water was added to 10 g of the sample, stirred at room temperature for 10 minutes, and centrifuged at 2,000 x g for 10 minutes. At this time, 10mL of the supernatant was added to a water weighing bottle, dried at 105 ° C. for 4 hours, and the solid content was measured. WSI was determined by the following equation.
WSI = (soluble solids g/10 ml) x 100ml/10g x 100%
WSI = (soluble solids g / 10 ml) x 100ml / 10g x 100%
(2) 단일 추출물과 복합 혼합물의 배양 (2) Cultivation of single extract and complex mixture 세포계에서In the cell system 조혈기능 검토( Hematopoietic function review inin vitrovitro 평가) evaluation)
1) 실험샘플 1) Experiment Sample
① 홍삼추출물① Red Ginseng Extract
② 젖산발효홍삼추출물② Lactic acid fermented red ginseng extract
③ 열처리 다시마 추출물의 유기산 가수분해물③ Organic Acid Hydrolyzate of Heat Treatment Kelp Extract
④ 젖산발효홍삼추출물 : 열처리 다시마 추출물의 유기산 가수분해물= 1:1④ Lactic acid fermented red ginseng extract: organic acid hydrolyzate of heat treated kelp extract = 1: 1
⑤ 젖산발효홍삼추출물 : 열처리 다시마 추출물의 유기산 가수분해물= 1:10⑤ Lactic acid fermented red ginseng extract: organic acid hydrolyzate of heat treated kelp extract = 1:10
⑥ 젖산발효홍삼추출물 : 열처리 다시마 추출물의 유기산 가수분해물= 10:1
⑥ Lactic acid fermented red ginseng extract: organic acid hydrolyzate of heat treated kelp extract = 10: 1
2) 5-(3-2) 5- (3- caroboxymethcaroboxymeth -- oxyphenyloxyphenyl )-2H-) -2H- tetratetra -- zoliumzolium innerinner saltsalt (( MTSMTS ) ) assayassay 를 이용한 독성 실험Toxicity test
홍삼추출물과 열처리 다시마 추출물의 유기산 가수분해물의 세포에 대한 독성여부는 MTS assay 방법으로 분석하였다. 이는 mitochondrial dehydrogenases 에 의하여 MTS가 formazan 으로 전환되는 것을 측정하는 것이다. 96 well plate 에 2×105cells/well/100㎕의 골수세포를 분주하고, 홍삼추출물과 열처리 다시마 추출물의 유기산 가수분해물을 일정농도로 처리하였다. 이 plate를 37℃, 5% CO2 배양기에서 16시간 배양한 후 MTS 용액을 20㎕씩 넣고 배양기에서 4시간 동안 반응시킨 후, ELISA reader 로 490nm에서 흡광도를 측정하여, 대조군에 대한 세포생존율을 백분율로 표시하였다.
Toxicity of cells from organic acid hydrolysates of red ginseng extract and heat-treated kelp extracts was analyzed by MTS assay. This measures the conversion of MTS to formazan by mitochondrial dehydrogenases. 2 × 10 5 cells / well / 100 μl of bone marrow cells were dispensed into a 96 well plate, and the organic acid hydrolyzate of red ginseng extract and heat treated kelp extract was treated at a constant concentration. After incubating the plate for 16 hours at 37 ° C. and 5% CO 2 incubator, 20 μl of MTS solution was added and reacted for 4 hours in the incubator. The absorbance was measured at 490 nm using an ELISA reader to determine the percentage of cell viability for the control group. Marked as.
3) 3) CFUCFU -- GMGM 측정 Measure
쥐의 대퇴골 박리 후 IMDM (2% FBS) 을 주입하여 골수 세포 부유액을 만든 후 세포 수를 측정하여 골수세포의 최종 농도를 2×105/ml이 되도록 한 다음, methyl-cellulose 배지(StemCell Technologies, Vancouver, Canada; MethocultTM GF M3534) 1mL당 2×104/ml 조혈모세포를 넣어 37℃, 5% CO2의 습기가 조절되는 세포 배양기에서 배양하였다. 이때 실험군은 농도별로 추출물을 배양액에 혼합하였고, 대조군은 distilled water(D.W.)를 배양액에 혼합하였다. 배양액은 methycellulose, FBS, bovine serum albumin, 고 insulin, human transferrin, 2-mercaptoethanol, rm stem cell factor, rm IL-3, rm IL-6, IMDM 및 supplements 의 구성물을 포함하고 있다.After femoral dissection of rats, IMDM (2% FBS) was injected to make bone marrow cell suspension, and then the number of cells was measured so that the final concentration of bone marrow cells was 2 × 10 5 / ml, followed by methyl-cellulose medium (StemCell Technologies, 2 × 10 4 / ml hematopoietic stem cells were added per 1 mL of Vancouver, Canada; Methocult TM GF M3534) and cultured in a cell incubator at 37 ° C. and 5% CO 2 . At this time, the experimental group was mixed with the extract by the concentration, the control group was mixed with distilled water (DW) in the culture. The culture contains methycellulose, FBS, bovine serum albumin, high insulin, human transferrin, 2-mercaptoethanol, rm stem cell factor, rm IL-3, rm IL-6, IMDM and supplements.
이를 11일간 배양 시킨 후 도립현미경(inverted microscope)을 통하여 CFU-GM 집락수를 측정하였다. 집락의 측정은 150배의 현미경시야에서 30개 이상의 세포가 모인 경우에 1개의 집락 형성(colony formation)으로 간주하였다. CFU-G와 CFU-M를 모두 포함하는 집락인 경우를 CFU-GM으로 정의하고, 이들 집락수는 dish에 형성된 일정 조혈모세포를 기준으로 계산하였으며, 모든 측정값은 평균±표준편차로 하였다.After 11 days of incubation, the number of CFU-GM colonies was measured by an inverted microscope. The measurement of colonies was regarded as one colony formation when 30 or more cells gathered at 150 times the microscopic field of view. Colonies containing both CFU-G and CFU-M were defined as CFU-GM, and these colonies were calculated based on the hematopoietic stem cells formed in the dish, and all measured values were mean ± standard deviation.
이때 대조군은 D.W.를 배양액에 혼합하였으며, 실험군은 홍삼, 발효 홍삼추출물 및 열처리 다시마 추출물의 유기산 가수분해물을 D.W.에 녹여 배양액에 혼합하여 실험에 사용하였다.
At this time, the control group was mixed with DW culture medium, the experimental group was dissolved in the organic acid hydrolyzate of red ginseng, fermented red ginseng extract and heat treatment kelp extract in DW was used for the experiment.
(3)단일 추출물 및 복합 혼합물 배양 (3) single extract and complex mixture culture 세포계에서In the cell system 골아세포Osteoblast 증식활성 측정 Proliferation activity measurement
1) 실험샘플 1) Experiment Sample
① 홍삼추출물① Red Ginseng Extract
② 젖산발효홍삼추출물② Lactic acid fermented red ginseng extract
③ 열처리 다시마 추출물의 유기산 가수분해물③ Organic Acid Hydrolyzate of Heat Treatment Kelp Extract
④ 젖산발효홍삼추출물 : 열처리 다시마 추출물의 유기산 가수분해물= 1:1④ Lactic acid fermented red ginseng extract: organic acid hydrolyzate of heat treated kelp extract = 1: 1
⑤ 젖산발효홍삼추출물 : 열처리 다시마 추출물의 유기산 가수분해물= 1:10⑤ Lactic acid fermented red ginseng extract: organic acid hydrolyzate of heat treated kelp extract = 1:10
⑥ 젖산발효홍삼추출물 : 열처리 다시마 추출물의 유기산 가수분해물= 10:1
⑥ Lactic acid fermented red ginseng extract: organic acid hydrolyzate of heat treated kelp extract = 10: 1
2) 2) 골아세포의Osteoblast 증식 촉진 활성 Proliferation promoting activity
MTT assay - for Ez-CyTox를 이용하여 활성을 측정하였다. 즉, ① cell을 seeding 한 plate 에 농도에 맞게 준비한 시료를 처리한다. ② 시료 처리 후 24h 동안 배양한 뒤 MTT 시약을 media 양의 1/10 을 처리해 준다 (96well에서는 well 당 media 를 100ul 처리 하므로 10ul 의 MTT시약을 처리 한다. ③ MTT 시약을 처리 한 뒤 다시 37℃ CO2 incubator에서 3시간동안 반응 시킨 다음 Microplate reader 를 이용하여 460nm에서 흡광도를 측정한다. ④ 시료 처리 후 72h 후에도 2, 3과 동일하게 MTT 시약을 처리 한 뒤 3시간 후에 460nm에서 흡광도를 측정 한다.Activity was measured using MTT assay-for Ez-CyTox. That is, ① process the sample prepared according to the concentration in the cell seeded plate. ② Incubate for 24h after sample processing, and
일반적인 MTT 시약의 경우 insoluble formazan crystal 을 형성하여 이것을 녹이기 위해서는 Media 를 제거 한 후 DMSO 등의 유기용매를 처리하여 formazan 을 녹인 후 측정을 하는데 Ez-CyTox 는 수용성 Tetrazolium salt 로 되어있어 media에 잘 녹기 때문에 따로 유기용매로 녹여서 측정할 필요 없이 media를 그대로 둔 상태로 흡광도 측정이 가능하다.
In the case of general MTT reagent, insoluble formazan crystal is formed to dissolve it. After removing the media, the organic solvent such as DMSO is dissolved and formazan is dissolved and measured. Ez-CyTox is water-soluble Tetrazolium salt, so it is well dissolved in the media. Absorbance can be measured with the media intact without dissolving in organic solvents.
IIIIII . 결과. result
1. 물리/화학적 성질1. Physical / chemical properties
(1) 홍삼 및 (1) red ginseng and 발효홍삼Fermented Red Ginseng
1) One) 추출수율Extraction yield , 수분함량 및 , Moisture content and pHpH
80℃에서 5시간 열탕처리로 얻은 홍삼액과 이를 복합 유산균으로 발효시켜 얻은 발효 홍삼액을 동결 건조한 실험시료를 70% 주정으로 추출한 홍삼 추출물의 수율과 수분함량 및 pH를 표 1에 나타내었다. Table 1 shows the yield, water content and pH of the red ginseng extract obtained by fermenting the red ginseng solution obtained by boiling water treatment at 80 ° C. for 5 hours and the fermented red ginseng solution obtained by fermenting the same with lactic acid bacteria.
그 결과 추출물의 수율은 홍삼추출물 25.20%, 발효 홍삼 추출물 24.72%이었으며, 수분함량과 pH는 홍삼 추출물 2.38%, 4.83, 발효 홍삼 추출물 2.99%, 4.03이었다.As a result, the yield of the extract was 25.20% of red ginseng extract and 24.72% of fermented red ginseng extract, and the water content and pH were 2.38%, 4.83, 2.99% of fermented red ginseng extract and 4.03.
2) 색상2) color
각 추출물의 헌터(hunter)값에 의한 색상은 표 2에 나타내는 바와 같이 명도를 나타내는 L*값의 경우는 발효 홍삼 추출물이 높았으며, 적색도를 나타내는 a*값은 홍삼 추출물이 높게 나타났다. 황색도를 나타내는 b*값이 경우는 발효 홍삼 추출물이 높게 나타나 전체적인 추출물의 색상은 발효 홍삼 추출물이 홍삼 추출물에 비해 밝은 갈색을 나타내었다. As the color of Hunter (hunter) value of each extract, as shown in Table 2, the L * value indicating the lightness was higher in the fermented red ginseng extract, the a * value indicating the redness was higher in the red ginseng extract. In the case of b * value indicating yellowness, the fermented red ginseng extract was high, and the overall color of the extract was light brown than the red ginseng extract.
3) 분자량3) molecular weight
Gel chromatography로 측정한 각 추출물의 분자량은 표 3에 나타내는 바와 같이 홍삼 추출물이 Mn 1,643 daltons, Mw 1,791 daltons 이었으며, 발효 홍삼 추출물은 Mn 1,059 daltons, Mw 1,066 daltons 으로 발효 홍삼 추출물의 분자량이 낮게 나타났다.As shown in Table 3, the molecular weight of each extract measured by gel chromatography was red ginseng extract Mn 1,643 daltons, Mw 1,791 daltons, fermented red ginseng extract was Mn 1,059 daltons, Mw 1,066 daltons showed low molecular weight of fermented red ginseng extract.
4) 관능적 특성4) sensory characteristics
각 추출물의 관능적 특성은 단맛, 쓴맛, 냄새, 색상 기호도 및 종합적 품질에 대해서 5점 척도법으로 나타내었다.Sensory properties of each extract were expressed on a five-point scale for sweetness, bitterness, odor, color acceptability, and overall quality.
그 결과 표 4에 나타내는 바와 같이 발효 홍삼 추출물이 단맛, 냄새, 색상기호도에서 홍삼 추출물보다 높게 나타났으며, 쓴맛의 경우는 홍삼 추출물이 평점 4.1로 발효 홍삼 추출물(3.1)보다 높게 나타났다. 종합적인 품질의 경우는 발효 홍삼 추출물이 평점 3.8로 홍삼 추출물 평점 3.1보다 높게 나타났다. 발효 홍삼 추출물의 맛은 쓴맛이 보통보다 약하면서 단맛이 느껴지고 홍삼 특유의 냄새에 약한 달콤한 향기가 있었다. 그리고 발효 홍삼 추출물의 색상은 명도가 높아 색상 기호도가 높았으며 이와 같은 관능적 특성으로 인해 전체적인 품질이 높게 평가된 것으로 판단된다. As a result, as shown in Table 4, the fermented red ginseng extract was higher than the red ginseng extract in sweetness, odor and color symbol. The bitter taste of red ginseng extract was higher than the fermented red ginseng extract (3.1) with a rating of 4.1. In terms of overall quality, fermented red ginseng extract scored 3.8, higher than red ginseng extract score 3.1. The taste of fermented red ginseng extract was weaker than normal, but it was sweet and had a sweet scent. In addition, the color of fermented red ginseng extract was high in color and high in color preference.
5) 홍삼 추출물의 유효성분 비교5) Comparison of Active Ingredients of Red Ginseng Extract
i. i. 진세노사이드Gin Senocide
HPLC로 분석한 ginsenoside의 분석 결과는 표 5에 나타내었다. 그 결과 total ginsenoside 함량은 홍삼 추출물이 31,621 mg/kg으로 발효 홍삼 추출물의 19,750.03 mg/kg보다 높았다. 홍삼 특유의 성분인 ginsenoside Rg3, Rh1, Rh2는 홍삼 추출물과 발효 홍삼 추출물에서 모두 분석되었는데 함량은 Rh2의 경우는 발효 홍삼 추출물에서 높게 나타났으며 Rg3와 Rh1의 경우는 홍삼 추출물에서 높게 나타났다. 그러나 각각의 total ginsenoside에 대한 백분율로 비교할 경우에는 홍삼 추출물과 발효 홍삼 추출물의 ginsenoside함량은 큰 차이를 보이지 않는 것으로 사료된다.The results of analysis of ginsenosides analyzed by HPLC are shown in Table 5. As a result, the total ginsenoside content of red ginseng extract was 31,621 mg / kg, which was higher than 19,750.03 mg / kg of fermented red ginseng extract. The ginsenosides Rg3, Rh1, and Rh2, which are unique to red ginseng, were analyzed in both red ginseng extract and fermented red ginseng extract. However, the ginsenoside content of red ginseng extract and fermented red ginseng extract did not show a significant difference in the percentage of total ginsenosides.
반면에 인삼사포닌중의 하나인 compound K의 경우에는 발효 홍삼 추출물에 213.43 mg/kg(1.08%)이 함유되어 있었으며, 홍삼 추출물에서는 함유되지 않은 것으로 나타났다.In contrast, compound K, one of ginseng saponins, contained 213.43 mg / kg (1.08%) in fermented red ginseng extract, but not red ginseng extract.
iiii . . 산성다당체와Acid polysaccharides and 총 페놀성 화합물 함량 Total Phenolic Compound Content
홍삼의 산성 다당체는 thrombin에 의한 fibrinogen의 응고를 저해, 동물실험에서 조혈을 촉진하며, 75% 에탄올에 침전하는 분획에서 임파구 증식활성을 보였다는 연구보고서가 있으며, 생쥐를 이용한 생체실험에서 골수세포 비장세포 및 GM-CFU가 증가되는 결과를 얻어 화학요법제나 방사선 치료 시 보조 요법제로 사용할 수 있는 가능성이 제시되고 있는 홍삼의 유효성분이다. 따라서 본 발명에서도 홍삼 및 발효 홍삼의 유효성분으로 관찰하였다. 그 결과 표 6에 나타내는 바와 같이 발효 홍삼추출물에서 산성 다당체의 함유량이 8.21%로 홍삼 추출물의 그것보다 높게 관찰되었다. 이는 발효에 의해 세포벽 성분 등의 분해에 의해 용출이 쉬워진 것에 원인이 있는 것으로 사료된다. 한편 홍삼의 주요 항산화성분으로 알려진 총 페놀성 화합물은 홍삼 추출물에는 0.68%, 발효 홍삼 추출물에는 2.17% 함유된 것으로 나타났다. The acidic polysaccharide of red ginseng inhibits the coagulation of fibrinogen by thrombin, promotes hematopoiesis in animal experiments, and has shown lymphocyte proliferation activity in fractions precipitated in 75% ethanol. It is an active ingredient of red ginseng that has been shown to increase the cells and GM-CFU, which can be used as adjuvant therapy in chemotherapy or radiation therapy. Therefore, the present invention was observed as an active ingredient of red ginseng and fermented red ginseng. As a result, as shown in Table 6, the content of acidic polysaccharide in the fermented red ginseng extract was 8.21%, higher than that of the red ginseng extract. This is thought to be due to the elution being easy due to the decomposition of cell wall components and the like by fermentation. Meanwhile, as the main antioxidant component of red ginseng Known total phenolic compounds contained 0.68% in red ginseng extract and 2.17% in fermented red ginseng extract.
(2) 다시마 추출물(2) Kelp Extract
1) 추출 수율, 수분함량 및 1) extraction yield, moisture content and pHpH
다시마와 같은 해조류는 대부분 비소화성 복합다당류로서 두꺼운 세포막으로 이루어져 있어 산이나 알카리에 비교적 안정하고 특수한 세균효소에 의하지 않고서는 분해가 어렵다. 앞서 선행연구와 예비실험을 한 결과 특수효소에 의한 분석 및 알긴산의 추출은 수율에 있어서 재현성이 매우 낮았으며, EDTA-2Na을 첨가하는 방법은 추출수율은 높으나 식품 첨가물로서 제한이 있어 배제하였다. 따라서 본 발명에 있어서 다시마로부터 알긴산의 추출은 현장에서 직접 응용할 수 있으며 식품에 직접적인 활용에 용이한 열수추출에 의하였다.Algae such as kelp are mostly non-digestible polysaccharides, which are composed of thick cell membranes, which are relatively stable to acids and alkalis, and are difficult to decompose without special bacterial enzymes. As a result of previous studies and preliminary experiments, the analysis by special enzyme and extraction of alginic acid showed very low reproducibility in yield, and the method of adding EDTA-2Na was excluded because of high extraction yield but limited as a food additive. Therefore, the extraction of alginic acid from kelp in the present invention can be applied directly in the field and by the hot water extraction that is easy to use directly in food.
열수 추출에 의한 알긴산의 추출수율, 수분함량 및 pH는 표 7에 나타내었다.The extraction yield, water content and pH of alginic acid by hot water extraction are shown in Table 7.
그 결과 수율은 열처리 다시마의 경우 20.48%, 무처리 다시마의 경우 15.55%이었으며, 수분함량은 무처리 다시마 추출물 5.59%, 열처리 다시마 추출물 4.29%, pH는 무처리 다시마 추출물 5.92, 열처리 다시마 추출물 5.50으로 조사되었다.As a result, the yield was 20.48% for heat treated kelp and 15.55% for untreated kelp. The water content was 5.59% untreated kelp extract, 4.29% untreated kelp extract, 5.92% untreated kelp extract, 5.92, 5.50 treated kelp extract, 5.50 It became.
2) 수용성 고형분 함량과 알긴산 함량2) water soluble solid content and alginic acid content
열수 추출에 의한 다시마의 수용성 고형분 함량은 표 8에 나타내는 바와 같이 열처리 다시마 추출물에서 6.157 g/100 mL로 무처리 다시마 추출물의 3.982 g/100 mL 보다 높게 나타났으며, 알긴산 함량의 경우도 열처리 다시마 추출물에서 1.964 g/100 mL로 무처리 다시마 추출물보다 높게 나타났다. 이와 같은 결과는 표 7의 알긴산 추출 수율과 마찬가지로 열처리로 인해 두꺼운 다시마 세포벽이 손상을 입으면서 조체의 흡수 및 팽윤이 증가되어 수용성 성분의 추출이 보다 용이해진 결과로 사료된다. As shown in Table 8, the water-soluble solid content of kelp by hot water extraction was higher than 3.982 g / 100 mL of untreated kelp extract in the heat treated kelp extract, and also in the case of alginic acid. Was 1.964 g / 100 mL higher than untreated kelp extract. These results are the same as the alginic acid extraction yield of Table 7, as the heat treatment damages the thick sea tangle cell wall increased the absorption and swelling of the granules, which makes it easier to extract the water-soluble components.
3) 다시마 추출물의 물리화학적 특성3) Physicochemical Properties of Kelp Extract
i. 점도i. Viscosity
열처리 다시마와 무처리 다시마의 열수 추출물의 점도는 표 9에 나타내는 바와 같이 무처리 다시마 추출물이 76.6 cps인데 비해 열처리 다시마 추출물의 경우 63.3 cps로 낮게 나타났다.As shown in Table 9, the viscosity of the hot water extracts of heat treated kelp and untreated kelp was lower than that of untreated kelp extract of 66.6 cps compared to that of untreated kelp extract.
iiii . 색상. color
열수 추출 다시마 추출물의 색상은 표 10에 나타내는 바와 같이 열처리 다시마 추출물이 L*값이 높고, a* 및 b*값은 낮게 나타나 무처리 다시마 추출물에 비해 밝은 회백색을 나타내었다.As shown in Table 10, the color of the hot-water extracted kelp extract showed high L * value and low a * and b * values, indicating a light grayish white color compared to the untreated kelp extract.
iiiiii . 분자량. Molecular Weight
열수 추출물의 분자량은 gel permeation chromatograph system에 의해 분석하였다(표 11). 그 결과 무처리 다시마 추출물의 경우 Mw 20,789 daltons, 열처리 다시마 추출물의 경우는 Mw 11,283 daltons으로 분석되어 열처리에 의해 다시마 추출물의 분자량이 감소하는 것으로 판단된다.The molecular weight of the hydrothermal extract was analyzed by gel permeation chromatograph system (Table 11). As a result, Mw 20,789 daltons for the untreated kelp extract and Mw 11,283 daltons for the heat treated kelp extract were determined to decrease the molecular weight of the kelp extract by heat treatment.
iviv . . FTFT -- IRIR
열처리에 따른 다시마 추출물의 분자구조를 살펴보기 위해 FT-IR을 측정하였다. 그 결과 도 1에 나타내는 바와 같이 열처리에 의해 알긴산이 저분자화 되어도 분자구조의 특징적인 변화는 관찰할 수 없었다.
FT-IR was measured to examine the molecular structure of kelp extracts after heat treatment. As a result, as shown in FIG. 1, even if alginic acid became low molecular weight by heat processing, the characteristic change of molecular structure could not be observed.
v. 관능적 특성v. Sensual characteristic
열처리 다시마 추출물과 무처리 다시마 추출물의 관능적 특성의 비교는 표 12에 나타내었다. 그 결과 5점법으로 평가한 단맛, 감칠 맛 및 냄새항목에서 열처리 다시마의 평점이 높게 나타났으며 색상기호도의 경우에도 열처리 다시마에서 높게 평가 되었다. 이와 같은 결과는 색상이 표 10에서 나타난 것과 같이 열처리 다시마의 L*값이 높은 결과 밝은색을 띄어 기호도가 높게 나타난 것으로 판단되며, 전체적인 품질에서도 열처리 다시마가 평점 3.8로 무처리 다시마(평점 2.1)보다 양호한 것으로 평가되었다.The sensory characteristics of the treated kelp extract and the untreated kelp extract are shown in Table 12. As a result, the score of heat treated kelp was high in sweet, umami, and odor items evaluated by the 5-point method. As shown in Table 10, it was judged that the L * value of the heat treated kelp was high in color, and the preference was high.In addition, the quality of the heat treated kelp was higher than the untreated kelp (Grade 2.1). It was evaluated as good.
이는 열처리로 인해 다양한 수용성 성분의 용출이 보다 쉬워져서 단맛과 감칠맛이 많아진 반면에 강한 해조취가 부드러워진 것에 원인이 있는 것으로 사료된다. It is believed that this is due to the tendency of the soft seaweed to soften while the elution of various water-soluble components becomes easier due to the heat treatment.
(3) 다시마 추출물의 가수분해(3) Hydrolysis of Kelp Extract
i. 점도i. Viscosity
유기산을 이용하여 다시마 추출물을 가수분해하여 측정한 점도는 표 13과 같다. 즉, 열처리 다시마의 점도는 1.25로 무처리 다시마의 점도보다 낮았다.The viscosity measured by hydrolyzing the kelp extract using an organic acid is shown in Table 13. That is, the viscosity of the heat treated kelp was 1.25, which was lower than that of the untreated kelp.
iiii . 분자량. Molecular Weight
유기산 가수분해에 의해 저분자화 한 다시마 추출물의 분자량은 표 14와 같이 열처리 다시마의 경우 Mn 137 daltons, Mw 138 daltons이었으며, 무처리 다시마는 Mn 1,058, Mw 1,073으로 분석되었다.The molecular weight of the kelp extract, which was low molecular weight by organic acid hydrolysis, was Mn 137 daltons and Mw 138 daltons for heat treated kelp as shown in Table 14, and the untreated kelp was analyzed as Mn 1,058 and Mw 1,073.
iiiiii . . FTFT -- IRIR
유기산을 이용한 다시마 추출물의 가수분해물에 있어서 FT-IR을 이용한 결합구조의 변화를 관찰하였다. 도 2에 나타내는 바와 같이 근적외선 신축진동흡수위치는 carboxylic acid의 수산기(OH)(3,420cm-1 부근), CH 신축진동(2,100cm-1 부근), C=O 신축진동(1,640cm-1 부근), C-O 신축진동(1,420cm-1 부근) 및 anti-symmetrical ring(1,030cm-1 부근)으로 열처리 다시마에서 확인되었으나 무처리 다시마에서는 C-O 신축진동, 730cm-1 부근, 400cm-1 부근의 결합을 볼 수 없었다.
The change of binding structure using FT-IR was observed in the hydrolyzate of kelp extract using organic acid. As shown in Fig. 2, the near-infrared elastic vibration absorption positions include hydroxyl group (OH) of carboxylic acid (near 3,420 cm -1 ), CH elastic vibration (near 2,100 cm -1 ), and C = O elastic vibration (near 1,640 cm -1 ). , CO stretching vibration (1,420cm -1 vicinity) and anti-symmetrical ring (1,030cm -1 vicinity) in but make the heat treatment in the non-treatment kelp seaweed see the combination of the CO stretching vibration, 730cm -1 near, 400cm -1 near the Could not.
iviv . 관능적 특성. Sensual characteristic
유기산을 이용하여 가수분해한 다시마 추출물의 관능적 특성은 표 15에 나타내는 바와 같이 열처리 다시마 추출물이 무처리 다시마 추출물보다 단맛, 감칠맛, 냄새, 색상 기호도 및 전반적인 품질의 전체 항목에서 높게 평가 되었다. 이는 citric acid를 이용한 높은 온도(121℃)에서 가수분해한 결과 올리고당이 생성되어 단맛과 감칠맛이 상승되어 기호도가 양호하게 평가 된 것으로 추측한다. 더불어 차기년도에는 다시마 가수분해물의 당질조성을 확인할 필요가 있는 것으로 사료된다. The organoleptic properties of kelp extracts hydrolyzed using organic acids were higher in the whole items of sweetness, flavor, smell, color taste and overall quality than the untreated kelp extracts, as shown in Table 15. It is assumed that the result of hydrolysis at high temperature (121 ℃) using citric acid resulted in the production of oligosaccharides, which resulted in an increase in sweetness and umami. In addition, it is necessary to confirm the sugar composition of the hydrolyzate of kelp in the next year.
(4) 발효 홍삼 및 (4) fermented red ginseng and 저분자Low molecule 다시마 추출물의 복합 혼합물 Complex mixture of kelp extract
젖산 발효한 홍삼 추출물의 80 mesh 동결 건조시료와 열처리 다시마 추출물의 유기산 가수분해물 80 mesh 동결 건조시료를 준비하여 복합 혼합물을 제조하고 이를 실험에 사용하였다.The 80 mesh lyophilized sample of lactic acid fermented red ginseng extract and the
1) One) pHpH 와 색상And color
발효 홍삼과 가수분해한 다시마 추출물의 복합 혼합물에 있어 표 16에 나타내는 바와 같이 pH는 각각 무게비 1:1로 혼합한 FRGA1은 4.73, 1:10의 비율로 조성한 FRGA2는 5.05였으며, 색상은 FRGA2가 L*값이 높고 a* 및 b*값이 낮아서 밝은 색상을 띄었다.In the composite mixture of fermented red ginseng and hydrolyzed kelp extract, as shown in Table 16, the FRGA1, which had a pH ratio of 1: 1, had a ratio of 4.73 and 1:10, and the ratio of FRGA2 was 5.05, and the color of FRGA2 was L. High * values and low a * and b * values resulted in bright colors.
2) 2) 흡습안정성Moisture Stability
혼합물 시료 1g 을 취하여 포화상태로 조절된 데시케이터 내에 방치하면서 2시간 단위로 10시간 동안 무게변화를 측정하여 시료의 흡습정도를 알아보았다(표 17). 그 결과 FRGA1의 경우에 10시간 경과 후에 0.03g의 무게변화가 있었으며, FRGA2는 0.05g 정도의 무게변화가 있는 것으로 관찰되어 FRGA1의 경우가 보다 흡습안정성이 높은 것으로 나타났다.1 g of the mixture sample was taken and placed in a saturator controlled saturator, and the weight change was measured for 10 hours every 2 hours to determine the hygroscopicity of the sample (Table 17). As a result, in the case of FRGA1, there was a change in weight of 0.03g after 10 hours and FRGA2 showed a change in weight of about 0.05g, which shows that the moisture absorption stability of FRGA1 is higher.
3) 3) 수용화Acceptance 정도 Degree
수분용해지수로 나타낸 복합 혼합물의 수용화 정도는 69.3-71.7%로 나타났으며 FRGA1의 수용화 정도가 큰 것으로 관찰되었다.(표 18)The degree of solubility of the complex mixture represented by the water solubility index was 69.3-71.7%, and the degree of solubility of FRGA1 was observed (Table 18).
4) 점도4) viscosity
복합 혼합물의 점도는 2.17-13.03 cps 범위였으며 FRGA2의 점도가 높게 관찰되었다.(표 19)The viscosity of the composite mixture was in the range of 2.17-13.03 cps and a high viscosity of FRGA2 was observed (Table 19).
5) 관능적 특성5) sensory characteristics
복합 혼합물의 관능적 특성은 단맛, 쓴맛, 감칠 맛, 냄새, 색상 기호도 및 전체적인 품질에 대해서 5점 척도법으로 평가 되었다.Sensory properties of the composite mixtures were evaluated on a five-point scale for sweetness, bitterness, umami, odor, color acceptability and overall quality.
그 결과 단맛과 쓴맛은 FRGA1이, 감칠맛은 FRGA2가 높게 평가되었으며, 냄새는 FRGA1이 높게 평가되었다. 색상 기호도는 FRGA2가, 전체적인 품질은 FRGA1이 높게 평가되어 전반적으로 발효 홍삼 추출물의 함유량이 많은 샘플이 기호도가 높은 것으로 평가되었다.(표 20)As a result, FRGA1 was evaluated for sweetness and bitterness, FRGA2 for umami, and FRGA1 for odor. The color acceptability was high in FRGA2 and the overall quality was high in FRGA1. Overall, the samples with high content of fermented red ginseng extract were evaluated as having high acceptability (Table 20).
2. 단일 추출물 및 복합 혼합물의 배양 2. Cultivation of single extracts and complex mixtures 세포계에서In the cell system 조혈기능 검토( Hematopoietic function review inin vitrovitro 평가) evaluation)
실험동물의 골수세포를 배양하여 colony-forming unit for granulocyte macrophage(CFU-GM)의 집락수를 측정하는 방법을 이용하여 홍삼, 발효 홍삼 및 열처리 다시마추출물의 유기산 가수분해물(제 5절의 표과 도에 tangleweed extrcat로 표기됨)의 조혈모세포 생성에 끼치는 영향을 확인하였다.Organic acid hydrolysates of red ginseng, fermented red ginseng, and heat-treated kelp extracts by culturing bone marrow cells of experimental animals and measuring colony-forming units of colony-forming unit for granulocyte macrophage (CFU-GM). The effects of hematopoietic stem cell production on the stem cells (marked as extrcat) were identified.
(1) (One) 발효홍삼Fermented Red Ginseng 및 다시마추출물 And kelp extract 가수분해물이Hydrolyzate 골수세포에 미치는 영향 Impact on bone marrow cells
골수세포에 대한 발효홍삼 추출물과 열처리 다시마추출물의 가수분해물 독성을 알아보기 위하여 MTS assay를 수행하였다. 그 결과 도 3에 나타내는 바와 같이 발효홍삼 추출물과 열처리 다시마추출물 가수분해물의 경우 500ug/ml 농도에서도 독성은 나타나지 않았다.
MTS assay was performed to investigate the hydrolyzate toxicity of fermented red ginseng extract and heat-treated kelp extracts against bone marrow cells. As a result, as shown in FIG. 3, the fermented red ginseng extract and the heat treated kelp extract hydrolyzate did not show toxicity even at the concentration of 500 ug / ml.
(2) (2) CFUCFU -G, -G, CFUCFU -M 및 -M and CFUCFU -- GMGM 의 of 집락군Colony 형성 관찰 Formation observation
골수세포를 11일 동안 배양 후 형성되는 집락군의 형태는 집락을 형성한 세포의 크기가 작고 밀집된 집락인 CFU-G와 세포들이 비교적 크고 산발적으로 분포한 집락인 CFU-M으로 정의하였으며, CFU-G과 CFU-M을 포함하는 집락인 경우인 CFU-GM의 집락수를 본 실험에서 측정하였다(도 4).
The colonies formed after culturing bone marrow cells for 11 days were defined as CFU-G, which is a small and dense colony of colony-forming cells, and CFU-M, which is a relatively large and sporadic distribution of cells. The colony count of CFU-GM, which is a colony containing G and CFU-M, was measured in this experiment (FIG. 4).
(3) 홍삼, (3) red ginseng, 발효홍삼Fermented Red Ginseng 및 열처리 다시마추출물 And heat treated kelp extract 가수분해물의Hydrolyzate 조혈모세포 생성에 대한 효과 비교 Comparison of effects on hematopoietic stem cell production
먼저 3가지 종류 추출물의 조혈모세포 생성에 대한 집락수를 측정한 결과, D.W.를 첨가한 배양액으로 증식시킨 대조군의 집락수를 기준으로 한 %변화에서는 열처리 다시마추출물 가수분해물 전체 농도(62.6-500 ug/mL)에서 집락수를 증가시켰으나 반면에 발효홍삼 및 홍삼추출물은 대조군과 유의적인 차이가 없었다. 그러나 홍삼 추출물은 대조군과 유의한 차이는 없었으나 조혈모 세포 집락수가 감소하는 경향을 보였다(도 5).First, the number of colonies for hematopoietic stem cell production of three kinds of extracts was measured, and the total concentration of heat treated kelp extract hydrolyzate (62.6-500 ug / mL) increased the number of colonies, while fermented red ginseng and red ginseng extracts were not significantly different from the control group. However, the red ginseng extract showed no significant difference from the control group, but showed a decrease in the number of hematopoietic stem cell colonies (FIG. 5).
한편 도 5의 재현성을 검토하고자 반복실험을 한 결과 홍삼 혹은 발효홍삼 추출물에 비해 열처리 다시마 추출물의 가수분해물을 처리한 실험군에서 조혈모세포의 집락수가 증가하는 비슷한 결과를 보여 주었으며, 농도가 높을수록 집락군 형성수가 증가되었다. 반면 홍삼 및 발효홍삼 추출물의 경우 조혈모 세포의 집락형성에 있어서 농도별(250-31.25 ug/ml)에 따른 유의적인 변화를 관찰할 수 없었다(도 6).
On the other hand, as a result of repeated experiments to examine the reproducibility of FIG. 5, the experimental group treated with hydrolyzate of the heat-treated kelp extract showed similar results of increasing the colony count of hematopoietic stem cells compared to the red ginseng or fermented red ginseng extract. The number of formations increased. On the other hand, in the red ginseng and fermented red ginseng extract, no significant change was observed according to the concentration (250-31.25 ug / ml) in the colony formation of hematopoietic stem cells (FIG. 6).
(4) 홍삼, 발효 홍삼 및 열처리 다시마 추출물의 (4) of red ginseng, fermented red ginseng and heat treated kelp extract 가수분해물의Hydrolyzate 복합 혼합물의 조혈모세포 생성에 대한 효과 비교 Comparison of Effects of Complex Mixtures on Hematopoietic Stem Cell Production
복합 혼합물의 조혈모세포에 대한 집락군 형성의 비교는 도 7에 나타내었다.그 결과 복합 혼합물(발효홍삼 추출물과 열처리 다시마 추출물 가수분해물의 1:1, 과 1:10)의 조혈모세포의 집락군 증식은 62.5-125 ug/mL의 농도에서는 대조군과 유의한 차이가 없었으나, 다시마 추출물 가수분해물의 함량이 높은 복합 혼합물(발효홍삼 추출물과 열처리 다시마 추출물 가수분해물의 1:10)에 있어서는 250 ug/mL의 농도에서는 조혈모세포의 집락군 증식이 높게 나타났다. 한편 열처리 다시마 추출물의 가수분해물 단독의 경우는 조혈모세포의 집락군 증식이 농도가 높을수록 높게 나타났다.
The comparison of colony formation to hematopoietic stem cells of the complex mixture is shown in FIG. 7. As a result, colony proliferation of hematopoietic stem cells of the complex mixture (1: 1, and 1:10 of fermented red ginseng extract and heat treated kelp extract hydrolysates) The concentration of 62.5-125 ug / mL was not significantly different from that of the control group, but 250 ug / mL was found in the complex mixture (1:10 of fermented red ginseng extract and heat treated kelp extract hydrolyzate) with high content of kelp extract hydrolyzate. At the concentration of, hematopoietic cell proliferation was high. On the other hand, in the case of the hydrolyzate of the heat treatment kelp extract alone, colony group proliferation of hematopoietic stem cells was higher with higher concentration.
3. 단일 추출물 및 복합 혼합물의 배양 3. Cultivation of single extracts and complex mixtures 세포계에서In the cell system 골아세포Osteoblast 증식활성 측정 Proliferation activity measurement
(1) (One) 골아세포의Osteoblast 증식활성-24 시간배양 Proliferative activity-24-hour culture
조골세포의 증식활성을 MTT-assay를 이용하여 세포 증식을 확인하였다(도 8). 24h 배양 후의 골아세포의 증식 결과는 발효 홍삼, 열처리 다시마 추출물 가수분해물(DAHST), 복합 혼합물-1(발효홍삼 추출물(FRG)과 열처리 다시마 추출물 가수분해물(DAHST)의 1:1 혼합물) 및 복합 혼합물-2(발효홍삼 추출물(FRG)과 열처리 다시마 추출물 가수분해물(DAHST)의 10:1 혼합물)에서 모두 대조군에 비해 높은 증식율을 나타내었으며, 특히 열처리 다시마 추출물 가수분해물의 경우 1000 ug/ mL농도에서는 120%의 골아세포 증식률을 보였다. 그리고 발효 홍삼과 복합 혼합물-1(발효홍삼 추출물(FRG)과 열처리 다시마 추출물 가수분해물(DAHST)의 1:1 혼합물)을 비교하면 복합 혼합물-1(발효홍삼 추출물(FRG)과 열처리 다시마 추출물 가수분해물(DAHST)의 1:1 혼합물)의 경우에서 골아세포 증식율이 높은 것으로 나타났다.
The proliferative activity of osteoblasts was confirmed by cell proliferation using MTT-assay (FIG. 8). The results of proliferation of osteoblasts after 24h culture were as follows: fermented red ginseng, heat treated kelp extract hydrolyzate (DAHST), complex mixture-1 (1: 1 mixture of fermented red ginseng extract (FRG) and heat treated kelp extract hydrolysate (DAHST)) and complex mixture -2 (10: 1 mixture of fermented red ginseng extract (FRG) and heat treated kelp extract hydrolyzate (DAHST)) showed higher proliferation rate than the control group, especially 120 mg / mL of heat treated kelp extract hydrolyzate. Osteoblast proliferation was shown. Comparing the fermented red ginseng and the complex mixture-1 (fermented red ginseng extract (FRG) and the heat treated kelp extract hydrolyzate (DAHST) 1: 1 mixture), the complex mixture-1 (fermented red ginseng extract (FRG) and the heat treated kelp extract hydrolyzate (A 1: 1 mixture of DAHST) showed high osteoblast proliferation.
(2) (2) 골아세포의Osteoblast 증식활성-72 시간 배양 Proliferative activity-72 hours incubation
배양 72시간 후의 골아세포 증식 결과는 도 9에 나타내었다. 이 실험에서 세포의 양이 많아서 인지 고농도에서 세포가 포화되어 공간 협소에 따른 스트레스로 세포가 사멸하는 모습이 발견되었다.Osteoblast proliferation after 72 hours of culture is shown in FIG. 9. In this experiment, it was found that the cells were saturated due to the high amount of cells, and the cells were killed by stress due to the narrow space.
발효 홍삼, 열처리 다시마 추출물 가수분해물, 복합 혼합물-1(발효홍삼 추출물(FRG)과 열처리 다시마 추출물 가수분해물(DAHST)의 1:1 혼합물) 및 복합 혼합물-2(발효홍삼 추출물(FRG)과 열처리 다시마 추출물 가수분해물(DAHST)의 10:1 혼합물)의 125 ug/ mL이하의 농도에서 대조군의 세포 증식 보다 유의하게 높은 활성을 나타내었다.
Fermented red ginseng, heat treated kelp extract hydrolyzate, complex mixture-1 (fermented red ginseng extract (FRG) and heat treated kelp extract hydrolyzate (DAHST) 1: 1 mixture) and complex mixture-2 (fermented red ginseng extract (FRG) and heat treated kelp) At a concentration of less than 125 ug / mL of the extract hydrolysate (10: 1 mixture of DAHST), it showed significantly higher activity than the cell proliferation of the control.
상기에서 수율은 추출 후에 얻은 동결건조물의 양을 추출에 사용된 시료의 무게로 나누어 백분율로 나타내었다. The yield is expressed as a percentage by dividing the amount of lyophilisate obtained after extraction by the weight of the sample used for extraction.
Claims (4)
다시마 추출액을 유기산을 이용하여 가수분해하여 동결 건조하는 다시마 추출물 제조 과정과,
상기한 다시마 추출물과 홍삼추출물을 중량비로 1~10:10~1의 비율로 혼합하는 과정을 포함하여 구성되는 것을 특징으로 하는 조골 및 조혈기능을 갖는 기능성 식품 제조 방법.After aging red ginseng powder extract at 50 ~ 100 ℃ for 12 to 48 hours, inoculated with lactic acid bacteria starter and fermented at 20 ~ 60 ℃ for 24 to 48 hours to obtain a red ginseng extract,
A process of preparing kelp extract, which is hydrolyzed by using an organic acid to freeze-dry the kelp extract;
Method for producing a functional food having osteoblast and hematopoietic function, characterized in that comprising the step of mixing the kelp extract and red ginseng extract in a ratio of 1 to 10:10 ~ 1 by weight.
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|---|---|---|---|---|
| KR100583435B1 (en) | 2004-11-23 | 2006-05-25 | 금산군 | Beverage composition containing ginseng for the prevention of happertension |
| KR100877348B1 (en) | 2007-04-16 | 2009-01-09 | 성인환 | concentrated liquid of ginseng fruit, manufacturing method thereof and health functional food |
-
2010
- 2010-04-05 KR KR1020100030785A patent/KR101206837B1/en not_active IP Right Cessation
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100583435B1 (en) | 2004-11-23 | 2006-05-25 | 금산군 | Beverage composition containing ginseng for the prevention of happertension |
| KR100877348B1 (en) | 2007-04-16 | 2009-01-09 | 성인환 | concentrated liquid of ginseng fruit, manufacturing method thereof and health functional food |
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| KR20110111618A (en) | 2011-10-12 |
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