KR102514059B1 - Fermented pomegranate composition for improving bone health or female menopausal symptoms, containing high content of ellagic acid and gallic acid with increased absorption into the body and its use having skin whitening, wrinkle improvement or antioxidant function, and method for manufacturing the same by using EFC method - Google Patents
Fermented pomegranate composition for improving bone health or female menopausal symptoms, containing high content of ellagic acid and gallic acid with increased absorption into the body and its use having skin whitening, wrinkle improvement or antioxidant function, and method for manufacturing the same by using EFC method Download PDFInfo
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- KR102514059B1 KR102514059B1 KR1020220085802A KR20220085802A KR102514059B1 KR 102514059 B1 KR102514059 B1 KR 102514059B1 KR 1020220085802 A KR1020220085802 A KR 1020220085802A KR 20220085802 A KR20220085802 A KR 20220085802A KR 102514059 B1 KR102514059 B1 KR 102514059B1
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- pomegranate
- fermented
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- juice
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Abstract
Description
본 발명은 체내흡수율이 증가된 엘라그산과 갈산을 고함량 포함하는 뼈건강 또는 여성갱년기 증상 개선용 설류 발효조성물 및 EFC 공법을 이용한 그의 제조방법에 관한 것으로서, 더욱 상세하게는 EFC 공법을 이용하여 석류를 특정 효소와 균주로 발효하여 갈산과 엘라그산 등 유효성분이 현저하게 증가되어 뼈건강 또는 여성갱년기 증상 개선용 석류 발효조성물과 그 제조방법에 관한 것이다.The present invention relates to a fermented semolina composition for improving bone health or women's menopausal symptoms containing a high content of ellagic acid and gallic acid with increased absorption in the body, and a method for manufacturing the same using the EFC method, and more particularly, to pomegranate using the EFC method It relates to a fermented pomegranate composition for improving bone health or female menopausal symptoms by significantly increasing active ingredients such as gallic acid and ellagic acid by fermenting with specific enzymes and strains and a manufacturing method thereof.
석류는 이도금양목, 부처꽃과, 석류나무속, 석류나무과, 석류나무종에 속하는 낙엽 활엽 소교목으로 약 500여종이 존재한다. 석류는 이란이 원산지이고 인도, 파키스탄 등 남아시아에서 주로 생산되며 우리나라에는 조선시대에 들어왔다.Pomegranate is a deciduous broad-leaved small arboreous tree belonging to the genus Ido Geumyang, Buddha flower family, Pomegranate genus, Pomegranate family, and Pomegranate species, and there are about 500 species. Pomegranates are native to Iran and are mainly produced in South Asia, including India and Pakistan, and were introduced to Korea during the Joseon Dynasty.
열매는 얇은 칸막이가 된 여섯 개의 작은 방으로 나뉘고 그 안에 수많은 씨앗을 품고 있다. 석류는 새콤달콤하면서 독특한 맛과 뛰어나고 다양한 건강상 이로움 때문에 세계적으로 가장 인기 있는 기능성 식품의 하나로 꼽히고 있으며, 그 소비가 급속히 확대되고 있다.The fruit is divided into six small chambers that have become thin partitions and contain numerous seeds in them. Pomegranate is considered one of the world's most popular functional foods because of its sweet and sour taste and excellent and various health benefits, and its consumption is rapidly expanding.
석류에는 탄수화물 단백질, 지방, 비타민 B군, C, E, K, 무기물 등 풍부한 영양성분을 함유하고 있다. 특히 항산화, 항암, 항균, 항염증 등 강력한 생리활성을 가진 페놀성 화합물을 비롯한 식물 estrogen 등 다양한 종류의 phytochemicals을 함유하고 있다. Pomegranate contains abundant nutrients such as carbohydrates, proteins, fats, vitamins B group, C, E, K, and minerals. In particular, it contains various types of phytochemicals such as plant estrogens as well as phenolic compounds with strong physiological activities such as antioxidant, anticancer, antibacterial, and anti-inflammatory properties.
석류의 대표적인 효능은 갱년기 장애 개선과 피부보호 기능이며, 이미 대한민국 식품의약품안전처에 건강기능식품 원료로 등재되어있다. 이와 같은 효능은 석류에 함유되어 있는 엘라그산으로부터 생성되는 urolithin과 같은 에스토로겐 유사물질(estrogenic substances)과 갈산과 같은 페놀성 화합물에 의한 강력한 항산화 물질에 기인한다.The representative efficacy of pomegranate is the improvement of menopausal disorders and skin protection, and it is already listed as a health functional food ingredient by the Ministry of Food and Drug Safety in Korea. Such efficacy is due to strong antioxidant substances by estrogenic substances such as urolithin generated from ellagic acid contained in pomegranate and phenolic compounds such as gallic acid.
그런데 이러한 두가지 대표적인 생리활성 물질은 실제로 석류즙에 함유되어 있기는 하지만 매우 미량 함유하고 있으며, 대부분 이들이 전구물질인 ellagtannin과 gallotannin 등 분해성 탄닌(hydrolyable tannins)으로 존재한다. However, although these two representative physiologically active substances are actually contained in pomegranate juice, they are contained in very small amounts, and most of them exist as hydrolyable tannins such as ellagtannin and gallotannin, which are precursors.
따라서 석류 과즙에서 유효성분의 활성을 개선하기 위해서는 엘라그산과 갈산을 증대시켜 체내흡수를 촉진할 수 있는 기능성 원료생산 기술개발이 필요하다.Therefore, in order to improve the activity of active ingredients in pomegranate juice, it is necessary to develop functional raw material production technology that can promote absorption in the body by increasing ellagic acid and gallic acid.
대부분의 여성은 폐경기 이후 여성 호르몬의 결핍으로 인해 다양한 정신적, 신체적 증상과 불편함을 경험하게 된다. 의학적으로 생리가 완전히 끝나고 1년이 지나면 폐경으로 진단하는데, 폐경이 진행되는 전 후 시기를 폐경 이행기라고 하고 여성이 가임기에서 폐경기로 이행되는 시기를 포괄적으로 갱년기라고 한다. 우리나라 여성들은 경우 폐경 연령은 평균 51~55세인 것으로 조사되고 있다.Most women experience various mental and physical symptoms and discomforts due to deficiency of female hormones after menopause. Medically, menopause is diagnosed one year after menstruation is completely over. In Korean women, the average age of menopause is 51 to 55 years old.
폐경기 이후 여성에서는 에스트로겐의 급속한 감소로 인하여, 심리학적 및 감성적 징후, 예컨대 피로, 흥분, 불면, 집중력 저하, 우울증, 기억 상실, 두통, 근심 및 신경과민이나 소심증 등이 발생할 우려가 현저히 높아지게 되며, 재발성 안면 홍조에 의한 수면 방해에 따른 피로와 흥분, 간헐성 현기증, 감각이상, 심계항진 및 빈맥, 메스꺼움, 변비, 설사, 관절통, 근육통, 수족 냉증 및 체중 증가 현상의 발생 우려 또한 현저히 높아지게 된다.Due to the rapid decrease in estrogen in postmenopausal women, the risk of experiencing psychological and emotional symptoms such as fatigue, excitement, insomnia, poor concentration, depression, memory loss, headache, anxiety, and nervousness or timidity is significantly increased, and recurrence Concerns about the occurrence of fatigue and excitement, intermittent dizziness, paresthesia, palpitations and tachycardia, nausea, constipation, diarrhea, joint pain, muscle pain, coldness of the limbs and weight gain due to sleep disturbance by hot flushes are also significantly increased.
또한, 골다공증이 쉽게 유발될 뿐 아니라, 심장병, 고혈압, 뇌졸중 등의 심혈관계 질환으로 인한 사망률도 증가하고, 피부 및 비뇨생식계통의 변화를 초래하며, 자가면역성 질환, 백내장 및 대장암 등의 발병 가능성이 높아지게 되는 등 여러 가지 변화를 격게된다.In addition, osteoporosis is easily induced, mortality due to cardiovascular diseases such as heart disease, high blood pressure, and stroke increases, causes changes in the skin and genitourinary system, and is likely to cause autoimmune diseases, cataracts, and colorectal cancer. It goes through many changes, such as getting higher.
현재, 이와 같은 갱년기의 여러 증상을 개선하는데 있어서 가장 효과적인 방법은 에스트로겐을 투여하여 결핍된 에스트로겐을 보충해 주는 것으로 알려져 있다. 그러나, 합성 에스트로겐의 장기간 투여는 유방암과 자궁암을 유발할 수 있다는 심각한 문제점이 있는 것으로 보고되고 있다. Currently, it is known that the most effective method for improving various symptoms of menopause is to supplement the lack of estrogen by administering estrogen. However, it has been reported that there is a serious problem that long-term administration of synthetic estrogens can cause breast cancer and uterine cancer.
따라서, 상기 호르몬 대체 요법에 사용되는 합성 에스트로겐의 보다 안전한 대체물로서 식물성 에스트로겐인 즉, 피토에스트로겐(phytoestrogen)을 찾고자 하는 연구가 최근 활발히 진행되고 있는 추세에 있다. 피토에스트로겐은 동물의 체내에서 에스트로겐성 효과를 발휘하는, 식물 중에 존재하는 비스테로이드성 화합물을 지칭하며, 항암 작용과 심장병에 효과가 있는 것으로 알려져 있는 곡물, 과일, 채소들의 대부분은 피토에스트로겐을 미량 포함하고 있다. Therefore, as a safer substitute for synthetic estrogens used in the hormone replacement therapy, studies to find phytoestrogens, that is, phytoestrogens, are currently being actively conducted. Phytoestrogen refers to non-steroidal compounds present in plants that exert an estrogenic effect in the body of animals, and most of the grains, fruits, and vegetables known to have anticancer and heart disease effects contain trace amounts of phytoestrogens. are doing
즉, 여성의 갱년기 증상을 개선하는데 효과적이면서도, 유방암, 자궁암 등을 유발하지 않는 등 안전한 약품 또는 건강기능식품에 대한 필요성이 꾸준히 제기되어 오고 있다.That is, the need for safe drugs or health functional foods, such as effective in improving women's menopausal symptoms and not causing breast cancer, uterine cancer, etc., has been steadily raised.
갱년기 장애 개선 효능이 있는 식물성 estrogenic 물질로는 대두, 알팔파 등의 이소플라본, 살구, 견과류 등의 리그난, 땅콩의 라스베라트롤, 딸기, 블랙베리, 호두 및 석류 등의 엘라그산 등이 알려져 있다.Plant estrogenic substances that are effective in improving menopausal disorders are known, such as isoflavones from soybeans and alfalfa, lignans from apricots and nuts, resveratrol from peanuts, and ellagic acid from strawberries, blackberries, walnuts and pomegranates.
이들 중 대표적인 식물성 phytoestrogen 함유 자원으로 석류를 들수 있다. 석류는 아토시아닌, 갈산 등 다양한 페놀성화합물과 엘라그산과 같은 phytochemicals을 함유하고 있다. 특히, 엘라그산은 사람이 섭취 후 장내 미생물에 의해 분해되어 urolithins 이라는 물질로 변환되고 특히 urolithin A, B는 강력한 에스토로겐 효과를 나타낸다. Among them, pomegranate is a representative plant phytoestrogen-containing resource. Pomegranate contains various phenolic compounds such as athocyanin and gallic acid, and phytochemicals such as ellagic acid. In particular, ellagic acid is decomposed by intestinal microorganisms after human consumption and converted into substances called urolithins, and urolithin A and B, in particular, exhibit strong estrogenic effects.
그러나 석류에 들어 있는 엘라그산은 대부분 먹지 못하는 껍질에 들어 있고 일상 섭취하는 석류즙에는 그 함량이 극히 낮아 갱년기 증상 개선에 유효한 효능을 얻기 위해서는 대단히 많은 량을 섭취해야 한다. 일부에서는 여러 품종의 석류 중 본래부터 에라그산의 함량이 높은 품종을 사용하는 방법을 제시하고 있으나 이정도로 충분한 효과를 보기 어렵다. However, most of the ellagic acid contained in pomegranate is in the inedible peel, and the content of pomegranate juice consumed daily is extremely low, so a very large amount must be consumed to obtain effective efficacy in improving menopausal symptoms. Some suggest a method of using a variety of pomegranate with a high content of ellagic acid, but it is difficult to achieve sufficient effect.
따라서 석류의 품종에 관계없이 석류 즙에 엘라그산의 함량을 근본적으로 증가시키는 기술개발이 필요하다. Therefore, it is necessary to develop technology to fundamentally increase the content of ellagic acid in pomegranate juice regardless of the variety of pomegranate.
기존에 석류에서 유용 생리활성 성분을 증가시키는 방법으로 한국공개특허 제10-2020-0064501호에서 갱년기 증상 개선용 석류 발효물, 한국등록특허 제10-1272602호에서 고함량 엘라그산 함유 갱년기 개선용 석류추출물 등이 제안되어 있다.Korean Patent Publication No. 10-2020-0064501, a fermented pomegranate product for menopausal symptom improvement, and Korean Patent No. 10-1272602, a pomegranate for menopausal improvement containing high content of ellagic acid, are conventional methods for increasing useful physiologically active components in pomegranate. Extracts and the like are proposed.
또한, 한국특허등록 제10-0542478호에서는 석류 유래 피토에스트로겐을 함유하는 성류 추출물에 대한 갱년기 관련 건강기능식품이 제안되어 있으며, 한국영양학회지에 발표된 연구논문에서도 석류 농축액이 갱년기 증상 개선에 효과가 있음을 제안하고 있다.[위지향 외, J Korean Soc Food Sci Nutr 44(4), 506~515(2015)]In addition, Korean Patent Registration No. 10-0542478 proposes a menopausal-related health functional food for Semolina extract containing pomegranate-derived phytoestrogens. [Wei Ji-hyang et al., J Korean Soc Food Sci Nutr 44(4), 506~515(2015)]
그러나 이러한 종래의 기술에서는 원래부터 함량이 높은 품종의 석류를 사용하거나 석류 추출물로부터 갱년기 증상 개선의 효과에 대해 알려져 있기는 하지만, 활성분의 함량이 제한적이고 체내 흡수도 한계가 있어서 그 효과를 충분하게 기대하기 어려운 문제가 있었다.However, in this conventional technique, although the pomegranate of a high content variety is known from the beginning or the effect of improving menopausal symptoms from a pomegranate extract is known, the active ingredient content is limited and the absorption in the body is limited, so that the effect is sufficiently effective. There was an unexpected problem.
본 발명은 상기와 같은 문제 해결을 위하여, 석류에 함유된 엘라그산, 갈산 등 유효성분의 함량을 현격하게 증대시키고, 체내흡수율을 향상시켜 그 적용 효과를 현격하게 향상시키는 것을 해결과제로 한다.In order to solve the above problems, the present invention has as a problem to significantly increase the content of active ingredients such as ellagic acid and gallic acid contained in pomegranate, and to significantly improve the application effect by improving the absorption rate in the body.
따라서 본 발명의 목적은 석류에 함유된 유효성분의 전구물질에 직접 작용하는 효소와 균주를 이용하여 유효성분의 함량을 고함량으로 함유하는 뼈건강 또는 여성 갱년기 증상 개선용 석류 발효조성물을 제공하는데 있다.Therefore, an object of the present invention is to provide a fermented pomegranate composition for improving bone health or female menopausal symptoms containing a high content of active ingredients using enzymes and strains that directly act on precursors of active ingredients contained in pomegranate. .
상기와 같은 본 발명의 과제해결을 위하여, 본 발명은 석류를 탄네이즈와 Lactobacillus vespulae DCY75로 효소발효시켜 얻은 발효물을 유효성분으로 함유하는 뼈건강 또는 여성 갱년기 증상 개선용 석류 발효조성물을 제공한다.In order to solve the problems of the present invention as described above, the present invention provides a fermented pomegranate composition for improving bone health or female menopausal symptoms containing a fermented product obtained by enzymatically fermenting pomegranate with tannaise and Lactobacillus vespulae DCY75 as an active ingredient.
본 발명의 바람직한 실시예에 의하면, 상기 발효물은 석류즙, 석류쥬스 또는 석류농축액 중 하나이상을 발효한 발효물을 포함할 수 있다.According to a preferred embodiment of the present invention, the fermented product may include a fermented product obtained by fermenting one or more of pomegranate juice, pomegranate juice or pomegranate concentrate.
본 발명의 바람직한 실시예에 의하면, 상기 탄네이즈는 0.01∼1 중량% 사용하고, Lactobacillus vespulae DCY75는 0.5x107cfu/ml 내지 3x107cfu/ml 로 발효된 발효물을 포함할 수 있다.According to a preferred embodiment of the present invention, the tannaise is used in an amount of 0.01 to 1% by weight, and Lactobacillus vespulae DCY75 may contain a fermented product fermented at 0.5x10 7 cfu/ml to 3x10 7 cfu/ml.
본 발명의 뼈건강 또는 여성갱년기 증상 개선용 조성물은 건강식품으로 유용하다.The composition for improving bone health or female menopausal symptoms of the present invention is useful as a health food.
또한, 본 발명의 뼈건강 또는 여성갱년기 증상 개선용 조성물은 약학조성물로 유용하다.In addition, the composition for improving bone health or female menopausal symptoms of the present invention is useful as a pharmaceutical composition.
또한, 본 발명의 뼈건강 또는 여성갱년기 증상 개선용 조성물은 동물용 사료첨가제 또는 동물용 영양제 조성물로 유용하다.In addition, the composition for improving bone health or female menopausal symptoms of the present invention is useful as an animal feed additive or animal nutrient composition.
또한, 본 발명은 In addition, the present invention
석류 과육으로부터 석류즙, 석류쥬스 또는 석류농축액 중 하나이상의 석류 원액을 제조하는 단계; Preparing at least one pomegranate juice from pomegranate pulp, pomegranate juice or pomegranate concentrate;
석류 원액을 5-10brix로 희석하여 탄네이즈 0.01∼1 중량%와 Lactobacillus vespulae DCY75의 유산균 0.5x107cfu/ml 내지 3x107cfu/ml 로 처리하고 30~40℃에서 2~48시간 동안 진탕 배양하는 단계; 및The pomegranate stock solution was diluted with 5-10 brix and treated with tannaise 0.01-1% by weight and Lactobacillus vespulae DCY75 lactic acid bacteria 0.5x10 7 cfu / ml to 3x10 7 cfu / ml and cultured with shaking at 30-40 ° C for 2-48 hours step; and
상기 진탕 배양하여 얻은 효소반응액을 70∼80℃에서 효소를 실활하는 단계Inactivating the enzyme reaction solution obtained by the shaking culture at 70 to 80 ° C.
를 포함하는 뼈건강 또는 여성 갱년기 증상 개선용 석류 발효조성물을 제조하는 방법을 제공한다.It provides a method for producing a pomegranate fermented composition for improving bone health or female menopausal symptoms comprising a.
본 발명의 바람직한 실시예에 의하면, 상기 진탕 배양은 80~200rpm으로 교반 하에 이루어질 수 있다.According to a preferred embodiment of the present invention, the shaking culture may be performed under agitation at 80 to 200 rpm.
본 발명의 바람직한 실시예에 의하면, 상기 효소를 실활하는 단계는 5~30분간 수행될 수 있다.According to a preferred embodiment of the present invention, the step of inactivating the enzyme may be performed for 5 to 30 minutes.
본 발명은 석류 추출물에 존재하는 가수분해성 탄닌을 EFC공법으로 발효시켜 엘라그산과 갈산 및 페놀성 화합물 등의 유효성분을 짧은 시간에 선택적로 다량 생성할 수 있는 효과가 있다.The present invention has the effect of selectively producing a large amount of active ingredients such as ellagic acid, gallic acid and phenolic compounds in a short time by fermenting the hydrolyzable tannins present in the pomegranate extract by the EFC method.
본 발명은 상기와 같은 유효성분이 고함량으로 함유되어 있으므로, 이러한 유효성분 강화로 인해 항산화 효과가 우수하고 뼈건강 또는 여성 갱년기 증상 개선 등의 효능이 현저하게 증가되는 효과가 있다.Since the present invention contains a high content of the above active ingredients, the antioxidant effect is excellent due to the strengthening of these active ingredients, and the efficacy such as bone health or female menopausal symptom improvement is remarkably increased.
또한, 반응을 통해 석류즙, 석류쥬스 또는 석류농축액 중의 tannin이 감소되어 떫은 맛이 줄어 맛을 개선하는 등 품질을 현저하게 향상시킬 수 있는 효과도 있다. In addition, through the reaction, tannin in pomegranate juice, pomegranate juice, or pomegranate concentrate is reduced, thereby reducing astringent taste and improving taste.
도 1은 EFC 공법으로 발효한 석류 발효조성물의 갈산과 엘라그산의 증가를 나타낸 그림이다.
도 2는 EFC 공법으로 발효한 석류 발효조성물에 대한 갈산과 엘라그산의 함량변화를 보여주는 HPLC profile 이다.
도 3a는 MCF세포에서 estradiol이 세포성장에 미치는 영향을 나타낸 그래프이다.
도 3b는 MCF세포에서 발효 전(대조군)과 비교에 1 및 실시예 시료인 발효 후 석류즙이 세포성장에 미치는 영향을 비교하여 나타낸 그래프이다.
도 4은 발표 처리 전 후 석류즙이 estrogen receptor 발현에 미치는 영향을 나타낸 PCR 사진이다.
도 5는 발효 전(Con)과 비교예 1(Tan) 및 실시예(Tan+L)의 발효 후 석류즙이 estrogen receptor 발현에 미치는 영향을 정량적으로 나타낸 그래프이다.
도 6은 발효 전(Control)과 비교예 1(Tanase) 및 실시예(Tan+L)의 발효 후 석류즙이 LPS에 의해 유도된 NO생성 억제효과를 비교하여 나타낸 그래프이다. 1 is a diagram showing an increase in gallic acid and ellagic acid in a pomegranate fermentation composition fermented by the EFC method.
Figure 2 is an HPLC profile showing changes in the contents of gallic acid and ellagic acid in the fermented pomegranate composition fermented by the EFC method.
Figure 3a is a graph showing the effect of estradiol on cell growth in MCF cells.
Figure 3b is a graph showing the comparison of the effect of pomegranate juice on cell growth after fermentation, which is a sample of Example 1 and Example 1, compared to before fermentation (control group) in MCF cells.
4 is a PCR photograph showing the effect of pomegranate juice on estrogen receptor expression before and after release treatment.
5 is a graph quantitatively showing the effect of pomegranate juice on estrogen receptor expression before fermentation (Con) and after fermentation of Comparative Example 1 (Tan) and Example (Tan+L).
6 is a graph showing a comparison of the NO production inhibitory effect induced by LPS of pomegranate juice before fermentation (Control) and after fermentation of Comparative Example 1 (Tanase) and Example (Tan+L).
이하, 본 발명을 하나의 구현예로서 더욱 상세하게 설명하면 다음과 같다.Hereinafter, the present invention will be described in more detail as one embodiment.
본 발명은 석류를 이른바 EFC(효소발효공법)을 이용하여 뼈건강 또는 여성 갱년기 증상 개선에 특히 효과가 우수한 석류 발효조성물로 제조한 것을 특징으로 한다.The present invention is characterized in that the pomegranate is prepared as a pomegranate fermented composition that is particularly effective in improving bone health or female menopausal symptoms using a so-called EFC (enzymatic fermentation method).
본 발명의 바람직한 실시예에 의하면, 석류를 효소 발효함으로써, 석류즙, 석류쥬스 또는 석류농축액 등(이를 ‘석류즙’ 또는 ‘석류 원액’으로 통칭할 수 있음)에 존재하는 가수분해성 탄닌(hydrolyable tannins)을 탄네이즈와 새로운 유산균(Lactobacillus vespulae DCY75)로 효소 발효시켜 엘라그산과 갈산의 함량을 획기적으로 증가시킨 것이다. According to a preferred embodiment of the present invention, hydrolyzable tannins present in pomegranate juice, pomegranate juice, pomegranate concentrate, etc. (which may be collectively referred to as 'pomegranate juice' or 'pomegranate undiluted solution') by enzymatic fermentation of pomegranate ) was enzymatically fermented with tannaise and a new lactic acid bacteria ( Lactobacillus vespulae DCY75) to dramatically increase the content of ellagic acid and gallic acid.
본 발명의 바람직한 실시예에 의하면, 이러한 본 발명의 석류 발효조성물은 항산화 및 갱년기 증상 개선 효과가 우수한 엘라그산과 갈산의 함량이 현저한 증가된 석류발효액으로 제공될 수 있다. 바람직하게는 갈산은 5배 이상, 엘라그산은 1.5배 이상 증가된 효과를 얻을 수 있으며, 항산화 활성 및 에스트로겐 활성을 월등하게 증가시킬 수 있다.According to a preferred embodiment of the present invention, the fermented pomegranate composition of the present invention can be provided as a fermented pomegranate solution with significantly increased contents of ellagic acid and gallic acid, which are excellent in antioxidant and menopausal symptom improvement effects. Preferably, the effect of gallic acid and ellagic acid is increased by 5 times or more and ellagic acid by 1.5 times or more, and antioxidant activity and estrogenic activity can be remarkably increased.
이렇게 본 발명의 방법으로 석류의 주요 유효성분의 함량을 현저하게 향상시킬 수 있는 것은, 석류를 발효함에 있어서 특정 조건의 발효를 수행함으로써 가능한 것이다. 이를 위해 본 발명에서는 석류를 탄네이즈와 유산균으로 발효처리하여 석류즙에서 생리활성 성분을 증가시켜서 현저하게 우수한 효과를 나타낸다.In this way, it is possible to significantly improve the content of the main active ingredient of pomegranate by the method of the present invention by performing fermentation under specific conditions in fermenting pomegranate. To this end, in the present invention, pomegranate is fermented with tannaise and lactic acid bacteria to increase physiologically active components in pomegranate juice, thereby exhibiting remarkably excellent effects.
본 발명의 바람직한 실시예에 의하면, 석류를 효소발효공법을 이용하여 발효처리할 수 있다. 여기서 효소발효공법(EFC; Enzyme Fermentation Control System)이라 함은 효소 존재하에 발효 처리하는 공법으로서, 이러한 공법을 이용해 생리활성 물질을 구조를 전환하면 체내흡수가 현저하게 잘 이루어지고, 또 약리효능이 높은 유효성분을 대량 생산할 수 있도록 해주는 특화된 공법이라 할 수 있다.According to a preferred embodiment of the present invention, pomegranate can be fermented using an enzyme fermentation method. Here, the Enzyme Fermentation Control System (EFC) is a method of fermenting in the presence of an enzyme, and when the structure of a physiologically active substance is converted using this method, absorption into the body is remarkably well achieved and pharmacological efficacy is high. It can be said to be a specialized method that enables mass production of active ingredients.
본 발명의 바람직한 실시예에 의하면, 본 발명에서는 석류 추출물에서 핵심 phytoestrogen인 엘라그산과 갈산을 이들의 전구물질에 직접 작용하는 효소와 특수균주를 사용하여 효소발효공법을 이용함으로 인해 단기간에 엘라그산과 갈산 등 유효성분을 획기적으로 증가시킬 수 있는 것이다.According to a preferred embodiment of the present invention, in the present invention, ellagic acid and gallic acid, which are key phytoestrogens in pomegranate extract, are produced in a short period of time by using an enzyme fermentation method using enzymes and special strains that act directly on their precursors. Active ingredients such as gallic acid can be dramatically increased.
본 발명의 바람직한 실시예에 의하면, 상기 발효물인 석류 추출물인 석류즙, 석류쥬스 또는 석류농축액 중 하나이상을 발효한 발효물을 포함할 수 있다.According to a preferred embodiment of the present invention, a fermented product obtained by fermenting at least one of pomegranate extract, pomegranate juice, pomegranate juice, or pomegranate concentrate, which is the fermented product, may be included.
본 발명의 바람직한 실시예에 의하면, 상기 탄네이즈는 0.01∼1 중량%와 Lactobacillus vespulae DCY75 0.5x107cfu/ml 내지 3x107cfu/ml 이 동시에 적용되어 발효된 것을 특징으로 한다, 여기서 효소만 적용하거나 유산균만 적용하는 경우는 본 발명에 따른 현저한 생리활성 효과를 얻기 어렵다.According to a preferred embodiment of the present invention, the tannase is characterized in that 0.01 to 1% by weight and Lactobacillus vespulae DCY75 0.5x10 7 cfu / ml to 3x10 7 cfu / ml are simultaneously applied and fermented. When only lactic acid bacteria are applied, it is difficult to obtain a significant physiological activity according to the present invention.
특히, 본 발명에 따르면 상기 효소는 탄네이즈가 바람직하게 사용될 수 있고, 유산균으로서는 Lactobacillus vespulae DCY75가 선택적으로 바람직하게 사용되는데, 여기서 유산균이 다른 종류로 사용되는 경우 본 발명에 따른 효과를 기대할 수 없다.In particular, according to the present invention, tannaise can be preferably used as the enzyme, and Lactobacillus vespulae DCY75 is selectively and preferably used as the lactic acid bacterium. However, when other types of lactic acid bacteria are used, the effect according to the present invention cannot be expected.
이와 같이, 본 발명의 바람직한 실시예에 의하면, 본 발명은 탄네이즈를 단독 사용하거나 Lactobacillus vespulae DCY75를 단독으로 사용하여 발효하는 경우는 본 발명의 목적달성이 어렵다. 또한, 탄네이즈 사용 후 Lactobacillus vespulae DCY75를 사용하거나 Lactobacillus vespulae DCY75 유산균 사용 후 탄네이즈를 사용하는 경우에도 본 발명의 목적달성이 어렵다. As described above, according to a preferred embodiment of the present invention, it is difficult to achieve the object of the present invention when fermentation is performed using tannaise alone or Lactobacillus vespulae DCY75 alone. In addition, even when Lactobacillus vespulae DCY75 is used after using tannaise or tannaise is used after using Lactobacillus vespulae DCY75 lactic acid bacteria, it is difficult to achieve the object of the present invention.
따라서 본 발명은 탄네이즈와 Lactobacillus vespulae DCY75를 동시에 사용하여 발효처리하여 상승효과를 얻는 것에 기술적 의의가 있다. 이러한 두 성분을 동시에 사용하는 경우 본 발명의 우수한 효과를 얻을 수 있다. 그러므로 탄네이즈와 Lactobacillus vespulae DCY75를 동시에 사용하는 경우 선택적으로 활성성분인 갈산이나 엘라그산 등의 생리활성성분이 다른 경우에 비해 상승효과로 현저하게 우수한 결과를 나타낼 수 있다. 또한 체내 흡수율도 현저학 개선된다.Therefore, the present invention has technical significance in obtaining a synergistic effect by fermentation treatment using tannaise and Lactobacillus vespulae DCY75 at the same time. When these two components are used simultaneously, excellent effects of the present invention can be obtained. Therefore, when tannaise and Lactobacillus vespulae DCY75 are used simultaneously, the selectively active ingredients such as gallic acid and ellagic acid can show remarkably superior results due to synergistic effects compared to other cases. In addition, the absorption rate in the body is significantly improved.
본 발명의 바람직한 실시예에 의하면, 상기와 같은 효소발효공법을 적용하기 위해서, 본 발명에서는 우선 석류과육으로부터 압착방법에 의한 석류 추출물을 준비할 수 있다. 이러한 석류 추출물은 예컨대 석류즙, 석류쥬스 또는 석류농축액 등으로 준비할 수 있다.According to a preferred embodiment of the present invention, in order to apply the enzyme fermentation method as described above, in the present invention, pomegranate extract can be prepared by a compression method from pomegranate pulp. Such a pomegranate extract can be prepared with, for example, pomegranate juice, pomegranate juice or pomegranate concentrate.
이렇게 준비된 석류 추출물은 특정 효소와 특정 유산균으로 발효처리하는데, 예컨대 석류 추출물을 5-10brix로 희석하여 탄네이즈 0.01∼1 중량%. 더 좋기로는 0.05~0.5 중량%와 Lactobacillus vespulae DCY75의 유산균 0.5x107cfu/ml 내지 3x107cfu/ml. 더 좋기로는 0.8x107cfu/ml 내지 1.5x107cfu/ml로 처리하여 발효하고 30~40℃에서 2~48시간 동안 진탕 배양하는 단계를 거칠 수 있다.The pomegranate extract prepared in this way is fermented with specific enzymes and specific lactic acid bacteria. More preferably 0.05-0.5% by weight and Lactobacillus vespulae DCY75 0.5x10 7 cfu / ml to 3x10 7 cfu / ml. More preferably, it may undergo fermentation by treatment with 0.8x10 7 cfu/ml to 1.5x10 7 cfu/ml, followed by shaking culture at 30-40°C for 2-48 hours.
본 발명의 바람직한 실시예에 의하면, 상기와 같이 발효처리하여 진탕 배양하여 얻은 효소반응액을 70∼80℃에서 효소를 실활하는 단계를 거쳐서 목적하는 석류 발효조성물을 제조할 수 있다.According to a preferred embodiment of the present invention, the desired pomegranate fermentation composition can be prepared by inactivating the enzyme at 70 to 80 ° C. in the enzyme reaction solution obtained by fermentation and shaking culture as described above.
본 발명의 바람직한 실시예에 의하면, 상기 진탕 배양은 80~200rpm, 더 바람직하게는 100~150rpm으로 교반 하에 이루어질 수 있다. 또한, 상기 발효 배양 후 효소를 실활하는 단계는 5~30분간 수행될 수 있다.According to a preferred embodiment of the present invention, the shaking culture may be performed under agitation at 80 to 200 rpm, more preferably 100 to 150 rpm. In addition, the step of inactivating the enzyme after the fermentation culture may be performed for 5 to 30 minutes.
본 발명의 바람직한 실시예에 의하면, 상기와 같이 제조된 석류 발효조성물은 엘라그산이나 갈산 등의 생리활성성분이 현저하게 향상된 결과물로 얻어질 수 있으며, 이러한 석류 발효조성물은 항산화 효과는 물론 뼈건간이나 여성갱년기 증상 개선에 매우 우수한 효과를 나타낸다.According to a preferred embodiment of the present invention, the fermented pomegranate composition prepared as described above can be obtained as a result of significantly improved physiologically active ingredients such as ellagic acid and gallic acid, and such a fermented pomegranate composition has antioxidant effects as well as bone health and It shows a very good effect in improving female menopausal symptoms.
따라서 본 발명은 상기와 같은 석류 발효조성물을 유효성분으로 하는 뼈건강 또는 여성갱년기 증상 개선용 조성물을 특징으로 한다.Therefore, the present invention is characterized by a composition for improving bone health or women's menopausal symptoms comprising the above fermented pomegranate composition as an active ingredient.
본 발명의 바람직한 실시예에 의하면, 이러한 본 발명의 뼈건강 또는 여성갱년기 증상 개선용 조성물은 건강식품으로 매우 유용하다. 특히 이러한 본 발명의 조성물을 상기 용도로 사용하는 경우 생체 흡수율이 우수하여 우수한 사용 효과를 발휘할 수 있다.According to a preferred embodiment of the present invention, the composition for improving bone health or female menopausal symptoms of the present invention is very useful as a health food. In particular, when the composition of the present invention is used for the above purpose, it has an excellent bioabsorption rate and can exhibit excellent use effects.
본 발명의 바람직한 실시예에 의하면, 본 발명의 조성물을 건강식품으로 이용하는 경우에는 정제, 캅셀제, 과립, 그래뉼, 환제, 겔제, 분말, 젤리, 액제, 드링크, 탄산음료, 청량음료 등 다양한 형태로 제조하여 사용할 수 있다.According to a preferred embodiment of the present invention, when the composition of the present invention is used as a health food, it is prepared in various forms such as tablets, capsules, granules, granules, pills, gels, powders, jellies, liquids, drinks, carbonated drinks, soft drinks, etc. and can be used.
또한, 본 발명의 조성물은 상기와 유사한 제형으로 경구투여용 약학 조성물로 유용하다.In addition, the composition of the present invention is useful as a pharmaceutical composition for oral administration in a dosage form similar to the above.
본 발명의 조성물은 다양한 형태의 통상적인 첨가제, 부형제 등을 혼합하여 사용할 수 있다.The composition of the present invention may be used by mixing various types of conventional additives, excipients, and the like.
본 발명에 따른 조성물의 건강식품이나 약학조성물은 유효성분 성인 기준으로 10~1,000mg의 량으로 1일 2~3회 경구투여 할 수 있다.The health food or pharmaceutical composition of the composition according to the present invention can be orally administered 2 to 3 times a day in an amount of 10 to 1,000 mg based on the active ingredient for adults.
또한, 상기와 같은 뼈건강 또는 여성갱년기 증상 개선용 조성물은 통상의 방법으로, 또는 통상의 동물용 사료첨가제용 또는 동물용 영양제용 첨가제 등을 첨가하여 동물용 사료첨가제 또는 동물용 영양제 등으로 유용하다. 이 경우 동물에 투여하기 위한 투여 제형 역시 상기와 같은 다양한 방법으로 적용할 수 있다.In addition, the composition for improving bone health or female menopausal symptoms as described above is useful as an animal feed additive or animal nutrient by adding a conventional method or a conventional animal feed additive or animal nutrient additive. . In this case, dosage forms for administration to animals can also be applied in various ways as described above.
이와 같이, 본 발명에 따른 석류 발효조성물은 본 발명은 석류 추출물에 존재하는 가수분해성 탄닌을 EFC공법으로 발효시켜 엘라그산과 갈산 및 페놀성 화합물을 짧은 시간에 선택적으로 다량 생성할 수 있는 것이다. 그러므로 본 발명은 엘라그산과 갈산 등 유효성분을 단기간에 획기적으로 증가시킬 수 있는 효과가 있다.As described above, the pomegranate fermentation composition according to the present invention can selectively produce a large amount of ellagic acid, gallic acid, and phenolic compounds in a short time by fermenting the hydrolyzable tannins present in the pomegranate extract by the EFC method. Therefore, the present invention has the effect of dramatically increasing active ingredients such as ellagic acid and gallic acid in a short period of time.
이러한 본 발명의 석류 발효조성물은 생리활성이 우수한 유효성분 강화로 인해 뼈건강 또는 여성갱년기 증상 개선 등의 효능이 현저하게 증가되는 효과가 있다.The fermented pomegranate composition of the present invention has an effect of remarkably increasing efficacy such as bone health or improvement of female menopausal symptoms due to the strengthening of active ingredients having excellent physiological activity.
또한, 본 발명은 석류즙, 석류쥬스 또는 석류농축액 중 하나이상의 석류 원액에 대하여 효소발효 반응을 통해 석류즙, 석류쥬스 또는 석류농축액 중의 tannin이 감소되어 떫은 맛이 크게 감소하여 맛도 향상시킬 수 있어서, 석류 추출물이나 이를 이용한 건강식품, 의약품 등에 대하여 품질을 향상시킬 수 있는 효과도 있다. In addition, according to the present invention, tannin in pomegranate juice, pomegranate juice, or pomegranate concentrate is reduced through an enzyme fermentation reaction for at least one pomegranate undiluted solution selected from pomegranate juice, pomegranate juice, or pomegranate concentrate, thereby significantly reducing astringent taste and improving taste. , there is also an effect of improving the quality of pomegranate extract, health food, medicine, etc. using it.
이하, 본 발명의 실시예에 의거 상세하게 설명하겠는바, 본 발명이 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited by the examples.
실시예 : 석류 발효조성물의 제조Example: Preparation of pomegranate fermentation composition
(1) 석류과육으로부터 압착방법에 의한 석류착즙 원액(석류즙)을 제조하는 단계를 거친다.(1) A step of preparing pomegranate juice (pomegranate juice) from pomegranate pulp by a pressing method is performed.
(2) 상기 (1)단계에서 제조한 석류착즙 원액을 8brix로 희석하여 탄네이즈0.2 중량%와 Lactobacillus vespulae DCY75(1x107cfu/ml)로 처리하고 34℃에서 24시간 발효하고, 120rpm으로 교반하에 진탕 배양하는 단계를 거쳐서 효소반응액을 얻는다. (2) The pomegranate juice stock solution prepared in step (1) was diluted with 8brix, treated with tannaise 0.2% by weight and Lactobacillus vespulae DCY75 (1x10 7 cfu / ml), fermented at 34 ° C for 24 hours, and stirred at 120 rpm. An enzyme reaction solution is obtained through shaking culture.
(3) 상기 (2)단계에서 얻은 효소반응액을 70∼80℃에서 10분간 처리하여 효소를 실활시킨다.(3) The enzyme reaction solution obtained in step (2) is treated at 70-80 ° C for 10 minutes to inactivate the enzyme.
(4) 상기 단계에서 실활한 효소 반응액을 석류 농축액으로 조제하여 석류 발효조성물을 제조한다.(4) A pomegranate fermentation composition is prepared by preparing the enzyme reaction solution deactivated in the above step as a pomegranate concentrate.
비교예 1Comparative Example 1
상기 실시예와 동일한 비교 조건으로 실시하되, 효소로 탄네이즈(TAH; Tannin acylhydrolase)를 단독으로 사용하되 유산균을 사용하지 아니한 다음 발효하고 진탕 배양하여 석류 발효조성물을 제조하였다.It was carried out under the same comparative conditions as in the above example, but tannin acylhydrolase (TAH) was used alone as an enzyme, but lactic acid bacteria were not used, followed by fermentation and shaking culture to prepare a fermented pomegranate composition.
비교예 2Comparative Example 2
상기 실시예와 동일한 비교 조건으로 실시하되, 효소로 탄네이즈(TAH)를 사용한 후, 이어서 유산균으로 Lactobacillus vespulae DCY75를 사용하여 발효하고 진탕 배양하여 석류 발효조성물을 제조하였다.It was carried out under the same comparative conditions as in the above example, but after using tannase (TAH) as an enzyme, followed by fermentation using Lactobacillus vespulae DCY75 as a lactic acid bacterium and shaking culture to prepare a fermented pomegranate composition.
비교예 3Comparative Example 3
상기 실시예와 동일한 비교 조건으로 실시하되, 유산균으로 Lactobacillus vespulae DCY75를 사용하여 발효하고, 이어서 효소로 탄네이즈(TAH)를 사용한 후 진탕 배양하여 석류 발효조성물을 제조하였다.It was carried out under the same comparative conditions as in the above example, but fermentation was performed using Lactobacillus vespulae DCY75 as a lactic acid bacterium, followed by shaking culture after using tannase (TAH) as an enzyme to prepare a fermented pomegranate composition.
비교예 4Comparative Example 4
상기 실시예와 동일한 비교 조건으로 실시하되, 유산균으로 Lactobacillus vespulae DCY75를 사용하여 발효하고 진탕 배양하여 석류 발효조성물을 제조하였다.It was carried out under the same comparative conditions as in the above example, but fermented using Lactobacillus vespulae DCY75 as a lactic acid bacterium and cultured with shaking to prepare a fermented pomegranate composition.
실험예 1 : 유산균 선발을 위한 발효효과 비교Experimental Example 1: Comparison of fermentation effects for selection of lactic acid bacteria
유산균의 발효 효과를 비교하기 위해, 상기 실시예와 동일한 원료를 사용하여 발효시키되, 효소를 사용하지 아니하고 유산균으로 Lactobacillus brevis, Lactobacillus vespulae DCY75, Lactobacillus panaxibrevis DCY65, Bacillus amyloliquefaciens, Saccharomyces cerevisiae 를 각각 단독으로 사용하여 발효하여 석류 발효조성물을 제조하였다.In order to compare the fermentation effect of lactic acid bacteria, fermentation was performed using the same raw materials as in the above example, but without using enzymes , Lactobacillus brevis, Lactobacillus vespulae DCY75, Lactobacillus panaxibrevis DCY65, Bacillus amyloliquefaciens, Saccharomyces cerevisiae were used alone, respectively. Fermented to prepare a pomegranate fermentation composition.
상기 각 유산균 사용 발효조성물에 대하여 갈산 생성량을 대조군과 비교하여 측정하였다. 그 결과로, 하기 표 1은 발효를 위한 균주선발을 위해 갈산 생성량을 기준으로 나타낸 결과이다.For the fermentation composition using each of the lactic acid bacteria, the amount of gallic acid produced was measured by comparison with the control group. As a result, Table 1 below shows the results based on the amount of gallic acid produced for the selection of strains for fermentation.
상기 표 1에서 보면, 갈산 생성량에서 Lactobacillus vespulae DCY75가 다른 균주에 비하여 월등하에 우수한 효과를 보인다. 따라서 유산균으로 Lactobacillus vespulae DCY75를 이용하는 것이 갈산 등 유효생리활성 물질의 증가에 선택적으루 우수한 효과가 있음을 보여준다.As shown in Table 1, Lactobacillus vespulae DCY75 shows a superior effect compared to other strains in terms of gallic acid production. Therefore, it shows that using Lactobacillus vespulae DCY75 as a lactic acid bacterium has a selectively excellent effect on increasing effective physiologically active substances such as gallic acid.
실험에 2 : 갈산 생성량 비교를 통한 효과 비교실험Experiment 2: Effect comparison experiment through comparison of gallic acid production
상기 실시예와 비교예 1 ~ 4에 대하여 각 시료에 대해 발효처리 후 최종 생성된 석류 발효조성물에 대하여 각각 갈산 생성량을 측정하여 비교하였다.For each of the above examples and Comparative Examples 1 to 4, the amount of gallic acid produced in the final pomegranate fermentation composition after fermentation was measured and compared.
그 결과는, 하기 표에 나타내었다. 하기 표 2는 탄네이즈(TAH) 및 Lactobacillus vespulae DCY75의 각각 단독 사용과 순차적 사용(화살표로 표시), 그리고 동시 혼합 사용(+로 표시)에 따른 동일 조건에서의 갈산 생성량의 결과를 비교하여 나타낸 것이다.The results are shown in the table below. Table 2 below compares the results of gallic acid production under the same conditions according to the use of tannaise (TAH) and Lactobacillus vespulae DCY75 alone, sequential use (indicated by arrows), and simultaneous mixed use (indicated by +), respectively. .
상기 표 2에서 보면, 탄네이즈(TAH)와 Lactobacillus vespulae DCY75를 동시에 사용한 실시예의 경우가 다른 비교군에 비하여 월등하게 우수한 갈산 생성량의 증가을 보여준다. 이 결과로부터, 탄네이즈(TAH)와 Lactobacillus vespulae DCY75를 각각 단독으로 사용하거나 순차적으로 사용하는 경우에 비해 이들을 동시에 사용하는 경우가 현저하게 우수한 상승효과가 있음을 확인할 수 있다.As shown in Table 2, the example in which tannaise (TAH) and Lactobacillus vespulae DCY75 were simultaneously used showed a significantly superior increase in gallic acid production compared to other comparative groups. From this result, it can be confirmed that there is a remarkably superior synergistic effect when tannaise (TAH) and Lactobacillus vespulae DCY75 are used simultaneously compared to the case where they are used alone or sequentially.
실험예 3 : 갈산과 엘라그산의 함량변화 실험Experimental Example 3: Content change test of gallic acid and ellagic acid
상기 실시예의 시료에 대한 엘라그산(Ellaguc acid)과 갈산(Gallic acid) 분석은 HPLC시스템으로 분석하였다. HPLC system은 Agilent 1260 Infinity Quaternary 펌프(G1311B), Agilent 1260 Infinity이었으며, 표준자동 시료 주입기(G1329B), Agilent 1260 Infinity 컬럼 온도 조절 장치 구획(G1316A), 및 Agilent 1260 Infinity Variable Wavelength Detector(G1314F)가 기기로 사용되었다. The analysis of Ellaguc acid and Gallic acid for the samples of the above examples was performed using an HPLC system. The HPLC system was an Agilent 1260 Infinity Quaternary Pump (G1311B), an Agilent 1260 Infinity, a standard autosampler (G1329B), an Agilent 1260 Infinity column thermostat compartment (G1316A), and an Agilent 1260 Infinity Variable Wavelength Detector (G1314F) as instruments. has been used
사용한 컬럼은 ZORBAX Eclipse Plus C18 컬럼(250mm × 4.6mm, 5μm 입자 크기)(Mil ford, MA, USA)이 고정상으로 선택되었다. 이동상은 농도구배로 용리액 조성은 다음과 같다. (0-16분, 15-30% B, 16-34분, 30-100% B, 34-40분, 100-100% B, 40-41분, 100-15% B; 41-42분 15-15% B)The column used was a ZORBAX Eclipse Plus C18 column (250 mm × 4.6 mm, 5 μm particle size) (Mil Ford, MA, USA) was selected as the stationary phase. The mobile phase is a concentration gradient, and the composition of the eluent is as follows. (0-16 min, 15-30% B, 16-34 min, 30-100% B, 34-40 min, 100-100% B, 40-41 min, 100-15% B; 41-42 min 15 -15% b)
상기 실험으로 석류즙의 발효 전(대조군)과 실시예에 따른 발효 후의 시료에 대하여 갈산 및 엘라그산 함량변화를 측정하여 본 결과는 다음 표 3 및 도 1과 같다.In the above experiment, the contents of gallic acid and ellagic acid were measured for samples before fermentation of pomegranate juice (control group) and after fermentation according to Examples, and the results are shown in Table 3 and FIG. 1 below.
상기 표에서의 결과에 대해, 함량변화를 보여주는 HPLC profile은 도 2와 같다.For the results in the table above, the HPLC profile showing the change in content is shown in FIG. 2.
이 결과로부터 실시에와 같은 방법으로 발효처리한 석류발효물의 경우 발효 전에 비해 갈산과 엘라그산 모두에서 월등하게 증가한 것으로 확인되었다.From this result, it was confirmed that in the case of the fermented pomegranate product fermented in the same way as in the example, both gallic acid and ellagic acid increased significantly compared to before fermentation.
실험에 4 : 항산화활성 변화Experiment 4: change in antioxidant activity
실시예의 발효추출물의 항산화활성은 DPPH 이용한 자유 라디칼 소거 활성 측정법으로 실시하였다. 즉, 에탄올에 용해시킨 DPPH 용액을 495㎕씩 시험관에 분주하고, 여기에 상기 시료 및 동충하초 추출물을 5㎕씩 첨가하여, 최종 반응용액이 500㎕가 되도록 하였다. 이를 일정시간 동안 반응시킨 후, 96-wall plate에 200㎕씩 2개의 wall에 분주한 뒤 517nm에서의 흡광도 변화를 측정하였다. 활성도는 gallic acid, Rutin 및 Ascorbic acid 대등량으로 각각 표시하였다.Antioxidant activity of the fermented extracts of Examples was carried out by a free radical scavenging activity measurement method using DPPH. That is, 495 μl of the DPPH solution dissolved in ethanol was dispensed into the test tube, and 5 μl of the sample and Cordyceps sinensis extract were added thereto to make the
항산화 활성 척도의 하나인 reducing power는 Nayan 등(2013)의 방법에 따라 다음과 같이 측정하였다. 즉, 10-100ug/ml 농도의 시료와 표준용액을 1.0ml의 탈이온 증류수에 녹인 후 2.5ml의 인산염완충액(pH 6.5)과 2.5ml의 potassium ferricyanide 용액과 혼합하였다. 이 용액을 50℃에서 20분간 반응시킨 후 식힌 다음 2.5ml의 10% TCA용액을 천가하고 잘 섞은 후 3,000rpm에서 10분간 원심분리하였다. 상등액 2.5ml 취한 다음 2.5ml의 증류수와 신선한 0.1% ferric chloride용액을 첨가하고 700nm에서 흡광도 변화를 측정하였다. Reducing power 계산은 gallic acid, rutin 및 ascorbic acid를 대조로 측정하여 나타내었다.Reducing power, one of the measures of antioxidant activity, was measured according to the method of Nayan et al. (2013) as follows. That is, samples and standard solutions having a concentration of 10-100ug/ml were dissolved in 1.0 ml of deionized distilled water, and then mixed with 2.5 ml of phosphate buffer (pH 6.5) and 2.5 ml of potassium ferricyanide solution. This solution was reacted at 50 ° C. for 20 minutes, cooled, and then 2.5 ml of 10% TCA solution was added, mixed well, and centrifuged at 3,000 rpm for 10 minutes. After taking 2.5 ml of the supernatant, 2.5 ml of distilled water and fresh 0.1% ferric chloride solution were added, and the absorbance change was measured at 700 nm. Reducing power calculations were performed by measuring gallic acid, rutin and ascorbic acid as controls.
하기 표 4는 시료인 석류즙의 발효 전(대조군)과 비교예 1 및 실시예에 의한 발효 후 항산화활성 변화를 나타낸 것이다.Table 4 below shows the change in antioxidant activity of pomegranate juice as a sample before fermentation (control group) and after fermentation according to Comparative Example 1 and Examples.
상기 표 4레서 보면, 실시예의 시료의 경우 다른 시료에 비해 월등하게 우수한 항산화 활성을 나타내는 것으로 확인된다.From Table 4 above, it is confirmed that the samples of the examples show significantly superior antioxidant activity compared to other samples.
실험예 5 ; 세포성장에 미치는 영향 측정Experimental Example 5; Measurement of effect on cell growth
세포성장에 미치는 영향 측정을 위하여 MCF-7 세포주를 이용하였다. ER-양성 인간 유방암 세포주인 MCF-7은 American Type Culture Collection(ATCC)에서 구입하였으며, 10%(V/V)의 소 태아 혈청과 페니실린-스트렙토마이신 용액이 보충된 DMEM 배지에서, 95% 공기/5% CO2의 가습된 대기, 37°C 온도에서 배양하였다.To measure the effect on cell growth, the MCF-7 cell line was used. The ER-positive human breast cancer cell line, MCF-7, was purchased from the American Type Culture Collection (ATCC) and grown in DMEM medium supplemented with 10% (V/V) fetal bovine serum and penicillin-streptomycin solution in 95% air/ Incubated in a humidified atmosphere of 5% CO 2 at a temperature of 37°C.
세포 독성 측정을 위해 MCF-7 세포를 무호르몬 배지에서 배양하고 96-웰 플레이트에 1 x 104 cells/well의 밀도로 시딩하고 5% CO2 인큐베이터에서 37℃에서 밤새 부착되도록 하였다. 배지를 버린 후 세포에 17β-Estradol과 석류추출물를 따로 처리한 다음 세포를 24시간 동안 배양하고 MTT(3-[4,5-디메틸티아졸-2-일]-2,5-디페닐테트라졸륨 브로마이드) 용액(20 μL/웰)을 사용하여 2~3시간 동안 세포 생존을 검출하였다. 마지막으로 100 μL DMSO를 사용하여 세포를 염색하여 포르마잔 결정을 유색 용액으로 발색하고 흡광도는 마이크로플레이트 리더(BioTek Instruments, Inc., Winooski, VT, USA)를 사용하여 570 nm에서 측정하였다.To measure cytotoxicity, MCF-7 cells were cultured in a hormone-free medium, seeded in a 96-well plate at a density of 1 x 10 4 cells/well, and allowed to adhere overnight at 37°C in a 5% CO 2 incubator. After discarding the medium, the cells were separately treated with 17β-Estradol and pomegranate extract, and then the cells were cultured for 24 hours, and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide was added to the cells. ) solution (20 μL/well) was used to detect cell viability for 2-3 hours. Finally, cells were stained with 100 μL DMSO to develop formazan crystals in a colored solution, and absorbance was measured at 570 nm using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).
첨부도면, 도 3a는 MCF세포에서 estradiol이 세포성장에 미치는 영향을 나타낸 그림이다. In the accompanying drawings, Figure 3a is a diagram showing the effect of estradiol on cell growth in MCF cells.
또한, 도 3b는 MCF세포에서 발효 전(대조군)과 비교에 1 및 실시예 시료인 발효 후 석류즙이 세포성장에 미치는 영향을 비교하여 나타낸 그림이다. 여기서 보면 실시예(TAN+L)의 경우가 일정 농도 이후에 현저하게 우수한 효과가 있음이 확인된다.In addition, Figure 3b is a diagram showing the comparison of the effect of pomegranate juice on cell growth after fermentation, which is a sample of Example 1 and Example 1, compared to before fermentation (control group) in MCF cells. Here, it is confirmed that the case of Example (TAN + L) has a remarkably excellent effect after a certain concentration.
실험예 6 : 에스트로젠 리셉터의 유전자 발현에 미치는 영향Experimental Example 6: Effect on estrogen receptor gene expression
역전사중합효소반응(RT-PCR)을 분석하기 위해 석류추출물이 처리된 MCF7 세포에서 TriZol LS 시약(Invitrogen, Carlsbad, CA, USA)으로 총 RNA를 추출하였다.For RT-PCR analysis, total RNA was extracted from pomegranate extract-treated MCF7 cells with TriZol LS reagent (Invitrogen, Carlsbad, CA, USA).
그 후 cDNA는 상업용 cDNA 합성 키트(Onebio, Lithuania, EU)의 권장 프로토콜에 따라 합성하였으며 이때 cDNA 합성을 위해 사용 된 RNA는 1㎍이었다.Then, cDNA was synthesized according to the recommended protocol of a commercial cDNA synthesis kit (Onebio, Lithuania, EU), and 1 μg of RNA was used for cDNA synthesis.
도 4은 발효처리 전 후 석류즙이 estrogen receptor 발현에 미치는 영향을 나타낸 PCR 사진이다.4 is a PCR photograph showing the effect of pomegranate juice on estrogen receptor expression before and after fermentation.
또한, 도 5는 발효 전(Con)과 비교예 1(Tan) 및 실시예(Tan+L)의 발효 후 석류즙이 estrogen receptor 발현에 미치는 영향을 정량적으로 나타낸 그림이다.5 is a picture quantitatively showing the effect of pomegranate juice on estrogen receptor expression before fermentation (Con) and after fermentation of Comparative Example 1 (Tan) and Example (Tan+L).
이 실험결과로부터, 실시예의 시료가 월등하게 우수한 효과가 있음을 확인할 수 있다.From these experimental results, it can be confirmed that the sample of Example has a significantly superior effect.
실험예 7 : NO생성에 미치는 영향Experimental Example 7: Effect on NO production
NO 생성 및 억제활성은 Griess 방법으로 측정하였다. 즉, RAW 264.7 세포에서 NO를 측정하기 위하여 먼저 RAW264.7 cell을 well 당 1x104 세포를 seeding하고 24시간 배양하고 1ug/ml의 LPS와 함께 다양한 농도의 석류농축물(PG) 및 석류발효농축물(PG-F0 전처리하였다. 세포에서 생성되어 배양액 중에 유리된 NO를 측정하기 위하여 100ul의 배양액에 동량의 Griess resgent를 첨가하고 540nm에서 흡광도를 측정하였으며 sod. nitrite를 표준용액으로 사용하였다. NO production and inhibitory activity were measured by the Griess method. That is, in order to measure NO in RAW 264.7 cells, first, RAW264.7 cells were seeded with 1x104 cells per well, cultured for 24 hours, and various concentrations of pomegranate concentrate (PG) and pomegranate fermented concentrate (with 1ug/ml LPS) PG-F0 was pretreated In order to measure the NO produced in the cells and released in the culture medium, the same amount of Griess resist was added to 100ul of the culture medium, and the absorbance was measured at 540 nm, and sod.nitrite was used as a standard solution.
도 6은 발효 전(Control)과 비교예 1(Tanase) 및 실시예(Tan+L)의 발효 후 석류즙이 LPS에 의해 유도된 NO생성 억제효과를 비교하여 나타낸 그림이다. 이 실험결과에서, 실시예의 시료다 다르 비교군에 비해 월등하게 우수한 NO 생성 억제 활성 효과가 확인된다.6 is a diagram showing a comparison of the NO production inhibitory effect induced by LPS of pomegranate juice before fermentation (Control) and after fermentation of Comparative Example 1 (Tanase) and Example (Tan+L). From the results of this experiment, it is confirmed that the NO production inhibitory activity effect of the samples of the Examples is significantly better than that of the other comparison groups.
실험예 8 : 조골세포 분화 활성 평가Experimental Example 8: Evaluation of osteoblast differentiation activity
뼈 건강 효능을 알아보기 위해 조골세포(MC3T3-E1)를 5x103 cells/100μL로 96 well에 분주하고 24시간 동안 안정화시켰다.To investigate the bone health effect, osteoblasts (MC3T3-E1) were dispensed into 96 wells at 5x10 3 cells/100 μL and stabilized for 24 hours.
실시예와 비교예 시료를 농도별로 처리하고 48시간 CO2배양기에 배양하였다. 배양 후 MTT(EZ-cytox, Daillab) 용액을 처리하고 2시간 동안 CO2배양 후 450 nm 파장에서 microplate reader(Epoch, Biotek)로 분석하였다. Samples of Examples and Comparative Examples were treated by concentration and cultured in a CO 2 incubator for 48 hours. After incubation, treatment with MTT (EZ-cytox, Daillab) solution, CO 2 incubation for 2 hours, and analysis with a microplate reader (Epoch, Biotek) at 450 nm wavelength.
그 분석결과는다음 표와 같다. 여기서 대조군은 비발효 석류즙을 이용하였다. The analysis results are shown in the following table. Here, non-fermented pomegranate juice was used as a control group.
실시예
Example
비교예 1
Comparative Example 1
상기 실험결과 실시예의 석류 발효조성물이 주골세포 분화를 촉진하여 뼈건강과 골형성에 현저한 효과가 있는 것으로 확인된다.As a result of the above experiments, it was confirmed that the fermented pomegranate composition of Example promotes osteoblast differentiation and has a remarkable effect on bone health and bone formation.
실험예 9 : ALP 활성 평가Experimental Example 9: ALP activity evaluation
세포 분화 효과 및 뼈건강 효과를 확인하고자 조골세포에 특이적으로 발현하는 ALP(alkaline phosphatase) 활성도를 측정하였다.In order to confirm the cell differentiation effect and the bone health effect, the activity of ALP (alkaline phosphatase) specifically expressed in osteoblasts was measured.
심험은 MC3T3-E1(조골세포)를 5x103 cells/250μL로 48 well에 분주하고 24시간 동안 안정화시켰다. For experimentation, MC3T3-E1 (osteoblasts) was dispensed into 48 wells at 5x10 3 cells/250μL and stabilized for 24 hours.
농도별로 시료를 처리하고 7일 동안 CO2배양기에서 배양하였다. PBS로 세척하고 Lysis buffer를 첨가하여 용해시킨 후 12,000rpm에서 15분간 원심 분리하였다. 세포막 성분 등을 침전시켜 상층액을 수집하고, ALP assay kit를 이용하여 상층액 내 ALP 활성을 정량하였다.Samples were processed for each concentration and cultured in a CO2 incubator for 7 days. After washing with PBS, dissolving by adding Lysis buffer, and centrifuging at 12,000 rpm for 15 minutes. The supernatant was collected by precipitating cell membrane components, etc., and the ALP activity in the supernatant was quantified using an ALP assay kit.
실험결과는 다음 표에 나타내었다. 여기서 대조군은 비발효 석류즙을 이용하였다.The experimental results are shown in the following table. Here, non-fermented pomegranate juice was used as a control group.
상기 실험결과 실시예의 시료가 월등하게 우수한 ALP 활성 효과가 있는 것으로 확인되었다.As a result of the above experiment, it was confirmed that the sample of Example had a significantly superior ALP activity effect.
실험예 10 : 골 재생 유도 활성 평가Experimental Example 10: Evaluation of bone regeneration induction activity
in vivo에서 실시예 조성물에 대한 골 재생 유도 효과를 확인하기 위해 두개골이 결손된 생쥐에 실시예, 비교예 1 및 대조군(비발효)을 처리한 후, 마이크로 CT(μCT) 및 조직학 분석을 수행하였다.In order to confirm the in vivo bone regeneration inducing effect of the Example compositions, mice with skull defects were treated with Examples, Comparative Example 1, and the control group (non-fermented), followed by micro CT (μCT) and histological analysis. .
마이크로 CT(μCT) 분석에 따르면, 실시예의 시료 조성물(1000 μg/mL) 은 6주 전 구간을 걸쳐 골 형성을 증가시킨 반면, 비교예 1은 일부, 대조군에서 극소의 골 형성이 유도되는 것으로 확인되었다.According to the micro-CT (μCT) analysis, the sample composition (1000 μg/mL) of Example increased bone formation over the entire 6 weeks, whereas Comparative Example 1 was found to induce minimal bone formation in some and control groups. It became.
조직학적 시험에 따르면, 실시예 조성물을 처리한 경우 대부분 섬유상 조직인 대조군 영역과 달리 결손 부위의 골 형성이 확인되었으며, 특히 비교예1의 조성물에 비해 결손 부위의 골 형성이 현저하게 등진되는 것으로 확인되었다.According to the histological test, when the composition of Example was treated, unlike the control region, which was mostly fibrous tissue, bone formation was confirmed in the defect area, and in particular, compared to the composition of Comparative Example 1, bone formation in the defect area was significantly reversed. .
실험예 11 : 가수분해성 탄닌 함량 실험Experimental Example 11: Hydrolyzable tannin content test
상기 실시예와 비교예 1에 따른 시료에 대하여, 동일 조건에서 통상의 방법으로 가수분해성 탄닌의 함량을 측정하여 비교하였다.With respect to the samples according to Example and Comparative Example 1, the content of hydrolysable tannin was measured and compared under the same conditions by a conventional method.
이에 대한 대조군(발효나 효소처리 전)과의 비교는 다음 표 7에 나타내었다.A comparison with the control group (before fermentation or enzyme treatment) is shown in Table 7 below.
상기 표는 EFC 공법으로 발효한 석류 발효조성물에 대한 가수분해성 탄닌 함량을 비교하여 나타낸 것이다. 이 실험결과로부터 탄네이즈(TAH)와 Lactobacillus vespulae DCY75를 동시에 사용한 실시예의 경우가 다른 비교군에 비하여 월등하게 우수한 탄닌 함량 감소 효과가 있음을 보여준다. 이 결과로부터, 탄네이즈(TAH)와 Lactobacillus vespulae DCY75를 혼합 사용하는 경우 탄네이드 단독 사용에 비해 탄닌 감소에도 월등하게 우수한 상승효과가 있음을 확인할 수 있다.The above table shows a comparison of the hydrolyzable tannin content of the fermented pomegranate composition fermented by the EFC method. From these experimental results, it is shown that the case of the example using tannase (TAH) and Lactobacillus vespulae DCY75 at the same time has a significantly superior tannin content reduction effect compared to other comparative groups. From this result, it can be confirmed that when tannaise (TAH) and Lactobacillus vespulae DCY75 are used in combination, there is a significantly superior synergistic effect in reducing tannin compared to the use of tannaid alone.
실험예 12 : 관능평가Experimental Example 12: Sensory evaluation
상기 실시예와 비교예 1에 따른 시료에 대하여, 동일 조건에서 맛을 비교하는 관능평가를 시행하였다.For the samples according to the above Example and Comparative Example 1, sensory evaluation was performed to compare the taste under the same conditions.
관능평가는 5점 평가 방법으로 대학생(남 36명, 여 13명)과 주부(11명) 등 60명을 대상으로 하여 떫은 맛과 단맛을 비교하여 평가하도록 하였다.The sensory evaluation was evaluated by comparing astringency and sweetness with 60 subjects, including university students (36 males, 13 females) and housewives (11 people), using a 5-point evaluation method.
이에 대한 대조군(발효나 효소처리 전)과의 비교는 다음 표에 나타내었다.A comparison with the control group (before fermentation or enzyme treatment) is shown in the following table.
상기 표 8은 EFC 공법으로 발효한 석류 발효조성물에 대한 떫은 맛과 단맛에 대한 관능을 비교하여 나타낸 것이다. 이 실험결과로부터 탄네이즈(TAH)와 Lactobacillus vespulae DCY75를 동시에 사용한 실시예의 경우가 다른 비교군에 비하여 우수한 맛의 개선 효과가 있음을 보여준다. 이 결과로부터, 탄네이즈(TAH)와 Lactobacillus vespulae DCY75를 혼합 사용하는 경우 탄네이드 단독 사용에 비해 맛에서도 우수한 상승효과가 있음을 확인할 수 있다. Table 8 shows a comparison of the astringency and sweetness of the fermented pomegranate composition fermented by the EFC method. From these experimental results, it is shown that the case of the example using tannaise (TAH) and Lactobacillus vespulae DCY75 at the same time has an excellent taste improvement effect compared to other comparative groups. From this result, it can be confirmed that when tannaise (TAH) and Lactobacillus vespulae DCY75 are used in combination, there is an excellent synergistic effect in taste compared to the use of tannaid alone.
상기 실험들의 결과로부터, 본 발명에 따른 실시예의 EFC 공법으로 발효한 석류 발효조성물은 그 특정 효소와 특정 유산균의 선택과 혼합 사용으로 인한 상승효과의 기술적 의의가 있는 것으로 확인되며, 체내흡수율이 증가된 엘라그산, 갈산 고함량 포함하는 뼈건강 또는 여성갱년기 증상에 대하여 현저하게 개선된 효과가 있음을 확인할 수 있었다. 또한, 이러한 조성물을 체내흡수율이 매우 높고 떫은 맛과 단맛 등 맛에서도 뚜렷한 개선 효과가 있음이 확인되었다.From the results of the above experiments, it was confirmed that the fermented pomegranate composition fermented by the EFC method of the embodiment according to the present invention has technical significance of the synergistic effect due to the selection and mixed use of the specific enzyme and specific lactic acid bacteria, and the absorption rate in the body is increased It was confirmed that there was a remarkably improved effect on bone health or female menopausal symptoms containing a high content of ellagic acid and gallic acid. In addition, it was confirmed that this composition had a very high absorption rate in the body and had a distinct improvement effect in taste such as astringency and sweetness.
Claims (9)
석류 원액을 5-10brix로 희석하여 탄네이즈 0.01∼1 중량%와 Lactobacillus vespulae DCY75의 유산균 0.5x107cfu/ml 내지 3x107cfu/ml 로 처리하고 30~40℃에서 2~48시간 동안 진탕 배양하는 단계; 및
상기 진탕 배양하여 얻은 효소반응액을 70∼80℃에서 효소를 실활하는 단계
를 포함하는 골다공증 또는 골감소증에 대한 뼈건강 또는 여성갱년기 증상 개선용 석류 발효조성물의 제조방법.Preparing at least one pomegranate juice from pomegranate pulp, pomegranate juice or pomegranate concentrate;
The pomegranate stock solution was diluted with 5-10 brix and treated with tannaise 0.01-1% by weight and Lactobacillus vespulae DCY75 lactic acid bacteria 0.5x10 7 cfu / ml to 3x10 7 cfu / ml and cultured with shaking at 30-40 ° C for 2-48 hours step; and
Inactivating the enzyme reaction solution obtained by the shaking culture at 70 to 80 ° C.
Method for producing a fermented pomegranate composition for improving bone health or female menopausal symptoms for osteoporosis or osteopenia, comprising a.
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| KR100542478B1 (en) | 2002-08-28 | 2006-01-11 | 한경대학교 산학협력단 | Functional health foodstuffs containing phytoestrogens derived from pomegranate |
| KR20110113122A (en) * | 2010-04-08 | 2011-10-14 | 이해련 | Pomegranate extract containing a high content of ellagic acid, and a method for improving effect of pomegranate extract related to postmenopausal syndrome |
| WO2019163437A1 (en) * | 2018-02-21 | 2019-08-29 | 株式会社ダイセル | Osteoclast differentiation inhibitor containing urolithin |
| KR20200064501A (en) | 2018-11-29 | 2020-06-08 | 씨제이제일제당 (주) | Fermented Pomegranate Having Enhanced Antioxidant Activity for Relieving Menopausal Symptom |
| KR20210114929A (en) * | 2018-11-05 | 2021-09-24 | 마블바이옴 인코포레이티드 | Microbial compositions comprising ellagitannins and methods of use |
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