KR102536627B1 - Composition for preventing or improving alcoholic liver injury comprising fermented kiwi - Google Patents
Composition for preventing or improving alcoholic liver injury comprising fermented kiwi Download PDFInfo
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- KR102536627B1 KR102536627B1 KR1020220125023A KR20220125023A KR102536627B1 KR 102536627 B1 KR102536627 B1 KR 102536627B1 KR 1020220125023 A KR1020220125023 A KR 1020220125023A KR 20220125023 A KR20220125023 A KR 20220125023A KR 102536627 B1 KR102536627 B1 KR 102536627B1
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- South Korea
- Prior art keywords
- composition
- fermented
- kiwi
- liver damage
- alcohol
- Prior art date
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Abstract
Description
본 발명은 대한민국 중소벤처기업부 지원하에 과제번호 S3260875에 의해 이루어진 것으로서, 상기 과제의 관리전문기관은 "중소기업기술정보진흥원", 연구사업명은 "지역특화산업육성+(R&D)", 연구과제명은 "신규 유산균을 이용한 발효골드키위의 알콜성 간손상 억제 건강기능식품 소재 개발 및 사업화", 과제수행기관은 "(주)바이텍", 연구기간은 2022.04.01 ~ 2023.03.31이다.
본 발명은 키위 발효물을 포함하는 알코올성 간손상 예방 또는 개선용 조성물에 관한 것으로, 더욱 구체적으로 유산균으로 발효한 키위 발효물을 유효성분으로 포함하는 알코올성 간손상 예방 또는 개선용 조성물에 관한 것이다.The present invention was made under the support of the Ministry of SMEs and Startups of the Republic of Korea under task number S3260875, and the management specialized institution for the task is "Small and Medium Business Technology Information Promotion Agency", the research project name is "Regional Specialized Industry Fostering + (R&D)", and the research task name is " Development and commercialization of health functional food material for suppression of alcoholic liver damage of fermented gold kiwifruit using new lactic acid bacteria", the project executing agency is "Bytech Co., Ltd.", and the research period is 2022.04.01 ~ 2023.03.31.
The present invention relates to a composition for preventing or improving alcoholic liver damage containing fermented kiwifruit, and more particularly, to a composition for preventing or improving alcoholic liver damage containing fermented kiwifruit fermented with lactic acid bacteria as an active ingredient.
장에서 흡수된 알코올의 90%는 체내에서 대사되고, 나머지 10%는 소변, 날숨, 땀 등을 통해 대사되지 않은 채로 체외로 배출된다. 흡수된 알코올의 대부분은 간에서 대사되며, 뇌, 췌장, 위에서도 알코올이 대사되기도 하지만 간에 비하면 매우 소량에 불과하다. 이와 같이 간은 알코올을 대사하는 주요 장기이므로, 만성적으로 알코올을 섭취하는 사람에서 간손상이 잘 발생하는 것으로 알려져 있다. 간 손상을 일으키는 원인은 다양하나 어떤 원인이든 조절되지 않고 오랜 시간 염증을 유발하여 간 손상이 지속되면 간 섬유화가 누적되어 결국 간경변증과 간암으로 진행할 위험이 커진다.90% of the alcohol absorbed in the intestine is metabolized in the body, and the remaining 10% is excreted from the body without being metabolized through urine, exhalation, sweat, etc. Most of the alcohol absorbed is metabolized in the liver, and alcohol is also metabolized in the brain, pancreas, and stomach, but only a very small amount compared to the liver. As described above, since the liver is a major organ that metabolizes alcohol, it is known that liver damage often occurs in people who chronically consume alcohol. There are various causes of liver damage, but any cause causes inflammation for a long time without control, and if liver damage continues, liver fibrosis accumulates, eventually increasing the risk of progressing to cirrhosis and liver cancer.
알코올성 간손상은 일반적으로 알코올로 인한 간 내 지방축적 증가와 알코올 대사로 인한 산화스트레스 및 염증반응 증가에 의해 유발된다. 알코올로 인한 간 내 지방축적은 알코올 과량 섭취에 의해 간에 지방이 과량 공급되고 지방조직에서 유리 지방산 분해를 촉진하여 간 내 유리지방산 유입을 증가시킨다는 점에서는 영양 과잉으로 인한 간 내 지방축적 과정과 공통적인 부분이 존재하나, 알코올 대사 과정에서 nicotinamide adenine dinucleotide(NAD)가 NADH(환원형 NAD)로 환원되고, 이에 따라 NADH/NAD 비율이 증가되면서 탄수화물과 지방 대사에 불균형이 나타나게되어 당 신생(gluconeogenesis)은 감소되고 지방산 합성은 증가된다는 점에서 차이가 있다. 알코올로 인한 간 내 지방축적은 알코올성 지방간을 유발하게 되며, 일반적으로 술을 끊으면 회복될 수 있는 질환이지만 증상이 거의 나타나지 않는다는 점에서 한편으로는 주기적으로 관리가 필요한 질환이라고 볼 수 있다. 알코올 대사로 인한 산화스트레스 및 염증반응은 알코올 대사 과정에서 생성된 상당량의 활성산소가 지질을 과산화시키고, 이러한 반응을 통해 생성된 지질과산화물이 혈중 세포와 세포막을 손상시킴으로써 유발되며, 지속적인 염증반응은 결국에는 간섬유화를 촉진시키는 반응성 알데히드 생성을 유발하게 된다. 알코올 대사과정에서 생성된 아세트알데히드는 반응성이 큰 물질로 단백질이나 DNA와 다양한 부가 생성물(adduct)을 형성함으로써 간 내 글루타치온을 고갈시켜 미토콘드리아에 존재하는 글루타치온을 선택적으로 감소시켜 항산화능을 저하시키고, TNF-α를 포함한 염증성 사이토카인의 생성을 촉진시킴으로써 간세포에 독성을 유발하게 되며, 결론적으로는 간손상을 유발하게 된다.Alcohol-induced liver damage is generally caused by increased fat accumulation in the liver and increased oxidative stress and inflammatory responses due to alcohol metabolism. Fat accumulation in the liver due to alcohol is similar to the process of fat accumulation in the liver caused by excessive nutrition in that excessive fat is supplied to the liver by excessive alcohol intake and promotes the decomposition of free fatty acids in adipose tissue to increase the inflow of free fatty acids into the liver. However, in the process of alcohol metabolism, nicotinamide adenine dinucleotide (NAD) is reduced to NADH (reduced NAD), and as a result, the NADH/NAD ratio increases, resulting in an imbalance in carbohydrate and fat metabolism, leading to gluconeogenesis. It differs in that it is reduced and fatty acid synthesis is increased. Fat accumulation in the liver caused by alcohol causes alcoholic fatty liver disease, which is generally a disease that can be recovered by quitting alcohol, but on the other hand, it can be seen as a disease that requires periodic management in that symptoms rarely appear. Oxidative stress and inflammatory reactions caused by alcohol metabolism are caused by a significant amount of active oxygen generated during alcohol metabolism that peroxidizes lipids, and lipid peroxides generated through this reaction damage blood cells and cell membranes. causes the production of reactive aldehydes that promote hepatic fibrosis. Acetaldehyde, generated during alcohol metabolism, is a highly reactive substance and forms various adducts with proteins or DNA to deplete glutathione in the liver and selectively reduce glutathione present in mitochondria, lowering antioxidant activity and reducing TNF By promoting the production of inflammatory cytokines, including -α, it induces toxicity to hepatocytes, and consequently causes liver damage.
현재 우리나라의 알콜성 간질환 발생 빈도가 상대적으로 증가함에 따라 간질환에서 차지하는 중요성 또한 증가하고 있으며, 실제 간경변증환자의 18.6%가 알코올과 연관이 있는 것으로 보고되고 있는 만큼 알코올성 간손상을 예방 및 개선함으로써 다양한 간질환이 유발되지 않도록 할 필요가 있다.Currently, as the incidence of alcoholic liver disease in Korea is relatively increasing, the importance of liver disease is also increasing. It is necessary to prevent the occurrence of various liver diseases.
키위(kiwi fruit)는 다래나무과(Acinidiaceae) 다래나무속(Actinidia)에 속하는 자웅이주의 넝쿨성 낙엽과수로 주로 온대지역에서 자란다. 열매 형태가 갈색 털로 덮여 있어 뉴질랜드에 서식하는 '키위'라는 새와 닮아 키위라고 이름이 붙여졌다고 하며, 우리나라에서는 양다래, 참다래라 부르기도 한다. 키위는 식품의 맛, 향미뿐만 아니라 다양한 생리활성에 기여한다고 알려진 페놀성 화합물을 함유되어 있고, 비타민 C, 비타민 E가 풍부하며, 엽산, 칼륨, 칼슘, 인 등의 무기질이 다량 함유되어 있을 뿐만 아니라 클로로필, 카로티노이드와 같은 건강에 유익한 생리활성물질을 함유되어 있다. 이와 같이 키위에는 다양한 생리활성물질 등이 포함되어 있어 키위를 이용한 다양한 연구가 계속해서 진행되고 있다. 구체적으로, 한국등록특허 제1081910호는 키위 추출물을 함유하는 피부톤 개선 및 피부노화방지 효과가 있는 화장료 조성물에 관한 것으로, 키위 추출물이 피부의 당화반응을 효과적으로 억제함으로써 피부톤을 개선하고 피부 주름을 완화시킬 수 있음이 개시되어 있다. 한국등록특허 제2027798는 골드키위 유산균 발효물을 유효성분으로 함유하는 항산화 조성물에 관한 것으로, 락토바실러스 플란타룸(Lactobacillus plantarum) 균주 배양액으로 발효하여 제조된 골드키위 유산균 발효물은 발효하지 않은 골드키위에 비해 총 페놀 및 플라보노이드의 함량이 현저히 증가되어 항산화 활성이 우수함이 개시되어 있다. 또한, 키위에 함유된 액티니딘은 단백질을 분해하는 효소로 소화 촉진에 효과적이기 때문에 위 건강에 도움이 되는 과일로서 알려져 있다. 그러나 종래 기술에는 키위 발효물이 알코올성 간손상을 개선하는 효과에 대해서는 언급된 바 없다.The kiwi fruit is a dioecious vine deciduous tree belonging to the genus Actinidia of the family Acinidiaceae and grows mainly in temperate regions. It is said that the fruit shape is covered with brown hairs and resembles a bird called 'Kiwi' that lives in New Zealand, so it was named kiwi. Kiwi contains phenolic compounds known to contribute to various physiological activities as well as the taste and flavor of food, is rich in vitamins C and E, and contains a large amount of minerals such as folic acid, potassium, calcium, and phosphorus. It contains physiologically active substances beneficial to health, such as chlorophyll and carotenoids. As such, since kiwifruit contains various physiologically active substances, various studies using kiwifruit are continuously being conducted. Specifically, Korean Patent Registration No. 1081910 relates to a cosmetic composition containing a kiwi extract that has skin tone improvement and skin aging prevention effects, and the kiwi extract effectively inhibits the glycation reaction of the skin to improve skin tone and relieve skin wrinkles. It has been disclosed that Korean Patent No. 2027798 relates to an antioxidant composition containing fermented gold kiwi lactic acid bacteria as an active ingredient . It is disclosed that the content of total phenols and flavonoids is significantly increased compared to the antioxidant activity. In addition, actinidin contained in kiwi is an enzyme that decomposes protein and is effective in promoting digestion, so it is known as a fruit that is helpful for stomach health. However, in the prior art, there is no mention of the effect of fermented kiwi to improve alcoholic liver damage.
이에, 본 발명자들은 상기 종래기술들의 문제점들을 극복하기 위하여 예의 연구노력한 결과, 키위를 유산균으로 발효한 키위 발효물을 유효성분으로 포함하는 알코올성 간손상 예방 또는 개선용 조성물의 경우, 간에서의 알코올 대사를 촉진시켜 알코올 대사가 활성화됨에 따라 혈중 에탄올 및 알데하이드의 농도가 감소되고, 간 내 지방축적 및 염증성 사이토카인의 생성을 억제할 뿐만 아니라 콜레스테롤 및 중성지방 축적을 억제함으로써 알코올성 간손상을 예방 또는 개선할 수 있음을 확인하고, 본 발명을 완성하게 되었다.Accordingly, the inventors of the present invention have made intensive research efforts to overcome the problems of the prior art, and as a result, in the case of a composition for preventing or improving alcoholic liver damage containing fermented kiwifruit as an active ingredient, alcohol metabolism in the liver As the alcohol metabolism is activated, the concentration of ethanol and aldehyde in the blood is reduced, and the accumulation of fat in the liver and the production of inflammatory cytokines are suppressed, as well as the accumulation of cholesterol and triglycerides, thereby preventing or improving alcoholic liver damage. It was confirmed that it could be done, and the present invention was completed.
따라서, 본 발명의 주된 목적은 간 내 지방축적 및 염증성 사이토카인의 생성을 억제하고, 간에서의 알코올 대사를 촉진시켜 혈중 에탄올 및 알데하이드의 농도를 감소시키며, 콜레스테롤 및 중성지방 축적을 억제함으로써 알코올성 간손상을 예방 또는 개선할 수 있는 키위 발효물을 포함하는 알코올성 간손상 예방 또는 개선용 조성물을 제공하는 데 있다.Therefore, the main object of the present invention is to inhibit the accumulation of fat in the liver and the production of inflammatory cytokines, promote alcohol metabolism in the liver to reduce the concentration of ethanol and aldehyde in the blood, and suppress the accumulation of cholesterol and triglyceride to prevent alcoholic liver disease. It is to provide a composition for preventing or improving alcoholic liver damage comprising fermented kiwi fruit capable of preventing or improving damage.
본 발명의 다른 목적은 간에서의 알코올 대사를 촉진시켜 알코올 대사가 활성화됨에 따라 혈중 에탄올 및 알데하이드의 농도를 감소시킴으로써 숙취를 해소할 수 있는 키위 발효물을 포함하는 숙취 해소용 조성물을 제공하는 데 있다.Another object of the present invention is to provide a composition for relieving a hangover containing a fermented kiwi fruit capable of relieving a hangover by reducing the concentration of ethanol and aldehyde in the blood as alcohol metabolism is activated by promoting alcohol metabolism in the liver. .
본 발명의 다른 목적은 숙취 해소용 조성물을 포함하는 음료 및 식품을 제공하는 데 있다.Another object of the present invention is to provide a beverage and food containing a composition for relieving hangover.
본 발명의 한 양태에 따르면, 본 발명은 키위를 유산균으로 발효한 키위 발효물을 유효성분으로 포함하는 알코올성 간손상 예방 또는 개선용 조성물을 제공한다.According to one aspect of the present invention, the present invention provides a composition for preventing or improving alcoholic liver damage comprising fermented kiwifruit as an active ingredient.
본 발명에서의 용어 ‘알코올성 간손상 예방 또는 개선’은 알코올 섭취로 인해 발생되는 간에 손상을 줄 수 있는 요인을 저해 또는 억제함으로써 간손상을 예방 또는 개선하여 결론적으로는 간손상에 의한 간질환을 예방하는 것으로, 구체적으로는 간에서의 알코올 대사를 촉진시켜 알코올 대사가 활성화됨에 따라 혈중 에탄올 및 알데하이드의 농도가 감소되고, 간 내 지방축적 및 염증성 사이토카인의 생성을 억제할 뿐만 아니라 콜레스테롤 및 중성지방 축적을 억제함으로써 알코올성 간손상을 예방 또는 개선하는 것을 의미한다. In the present invention, the term 'prevention or improvement of alcoholic liver damage' means to prevent or improve liver damage by inhibiting or suppressing factors that may cause liver damage caused by alcohol consumption, and consequently, to prevent liver disease caused by liver damage. Specifically, by promoting alcohol metabolism in the liver, alcohol metabolism is activated, thereby reducing the concentration of ethanol and aldehyde in the blood, suppressing fat accumulation in the liver and the production of inflammatory cytokines, as well as cholesterol and triglyceride accumulation It means to prevent or improve alcoholic liver damage by suppressing.
본 발명에서의 키위는 Actinidia chinensis 속(골드키위)의 키위일 수 있으며, 세부 품종으로는 제시골드(Actinidia chinensis Planch var. chinensis ‘Jecy Gold’), 한라골드(Actinidia chinensis Planch var. chinensis ‘Halla Gold’), 해금(Actinidia chinensis Planch var. chinensis ‘Haegum'), 제스프리골드 Zespri®Gold (Actinidia chinensis Planch var. chinensis ‘Hort16A’), 제스프리썬골드 Zespri®SunGold (Actinidia chinensis Planch var. chinensis ‘Zesy002’), 제스프리 제시003 Zespri®Zesy003 (Actinidia chinensis Planch var. chinensis ‘Zesy003’), 제스프리 제쉬004 Zespri®ZESH004 (Actinidia chinensis Planch var. chinensis ‘Zesh004’), 도리 Consorzio Dori Europe®Dori (Actinidia chinensis Planch var. chinensis 'AC 1536') 및 진골드 Jingold® (Actinidia chinensis Planch 'Jintao')로 구성된 군에서 선택되는 키위일 수 있으나, 이에 제한되지 않는다.Kiwi in the present invention may be a kiwi of the genus Actinidia chinensis (gold kiwi), and detailed varieties include Jessie Gold (Actinidia chinensis Planch var. chinensis 'Jecy Gold'), Halla Gold (Actinidia chinensis Planch var. chinensis 'Halla Gold') '), Haegeum (Actinidia chinensis Planch var. chinensis 'Haegum'), Zespri®Gold (Actinidia chinensis Planch var. chinensis 'Hort16A'), Zespri®SunGold (Actinidia chinensis Planch var. chinensis 'Zesy002') Jessure Jesse 003 ZESPRI®ZESY003 (Actinidia chinensis planch var. China Zesy003) ESH004 '), Dori Consorzio Dori Europe®Dori 'AC 1536') and Jingold® (Actinidia chinensis Planch 'Jintao'), but is not limited thereto.
본 발명의 알코올성 간손상 예방 또는 개선용 조성물에 있어서, 상기 유산균은 키위 유래 유산균으로, 상기 키위 유래 유산균은 락토코쿠스 락티스(Lactococcus Lactis) VI-01 KCTC 14351BP 및 락토바실러스 파라카세이(Lactobacillus paracasei) VI-02 KCTC 14352BP 중 하나 이상인 것을 특징으로 한다.In the composition for preventing or improving alcoholic liver damage of the present invention, the lactic acid bacteria are kiwi-derived lactic acid bacteria, and the kiwi-derived lactic acid bacteria are Lactococcus lactis VI-01 KCTC 14351BP and Lactobacillus paracasei It is characterized in that at least one of VI-02 KCTC 14352BP.
본 발명자들은 키위로부터 분리된 균주들로부터 유산균을 분리하고 선별된 균주를 16s rRNA 분석을 통해 유전체 염기서열을 해독하였다. 또한, 유전체 염기서열 해독을 통해 선별된 균주를 “락토코쿠스 락티스(Lactococcus Lactis) VI-01” 및 “락토바실러스 파라카세이(Lactobacillus paracasei) VI-02”로 명명하고, 생명공학연구원 생명자원센터(KCTC)에 2020년 11월 3일자로 기탁하고, 기탁번호 KCTC14351BP, KCTC14352BP를 각각 부여받았다.The present inventors isolated lactic acid bacteria from strains isolated from kiwi and decoded the genome sequences of the selected strains through 16s rRNA analysis. In addition, the strains selected through genome sequencing were named “ Lactococcus Lactis VI-01” and “ Lactobacillus paracasei VI-02”, and the Bioscience and Biotechnology Research Center (KCTC) on November 3, 2020, and were given accession numbers KCTC14351BP and KCTC14352BP, respectively.
본 발명의 알코올성 간손상 예방 또는 개선용 조성물에 있어서, 상기 유산균은 당업계에서 일반적으로 발효에 이용하는 유산균일 수 있으며, 바람직하게는 락토바실러스 파라카제이(Lactobacillus paracasei), 락토바실러스 엑시도필러스(Lactobacillus acidophilus), 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 헬베티커스(Lactobacillus helveticus), 락토바실러스 가세리(Lactobacillus gasseri), 락토바실러스 델브루에키이 서브스피시즈 불가리쿠스(Lactobacillus delbrueckii ssp. bulgaricus), 락토바실러스 퍼멘텀(Lactobacillus fermentum), 락토바실러스 플란타륨(Lactobacillus plantarum), 락토바실러스 루테리(Lactobacillus reuteri), 락토바실러스 람노서스(Lactobacillus rhamnosus) 및 락토바실러스 살리바리우스(Lactobacillus salivarius)로 구성된 군으로부터 선택되는 하나 이상의 균주, 더욱 바람직하게는 락토바실러스 엑시도필러스(Latobacillus acidophilus), 락토바실러스 카제이(Lactobacillus casei) 및 락토바실러스 헬베티커스(Lactobacillus Helveticus)로 구성된 군으로부터 선택되는 하나 이상의 균주를 포함하는 것을 특징으로 한다.In the composition for preventing or improving alcoholic liver damage of the present invention, the lactic acid bacteria may be lactic acid bacteria commonly used for fermentation in the art, preferably Lactobacillus paracasei, Lactobacillus acidophilus ( Lactobacillus acidophilus) , Lactobacillus casei, Lactobacillus helveticus, Lactobacillus gasseri, Lactobacillus delbrueckii subspecies bulgaricus (Lactobacillus delbrueckii ssp. bulgaricus), lacto Bacillus fermentum, Lactobacillus plantarum , Lactobacillus reuteri , Lactobacillus rhamnosus and At least one strain selected from the group consisting of Lactobacillus salivarius, more preferably Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus Helveticus ) It is characterized by comprising one or more strains selected from the group consisting of.
본 발명에서의 키위 발효물은 제1 배지에 균주를 각각 배양(종균배양)하여 제1 배양산물(종배양액)을 제조하고, 제1 배양산물을 제2 배지에 접종 및 배양(대량배양)하여 제2 배양산물(대량배양액)을 제조한 다음, 이를 원심분리하여 상등액을 제거하여 수득한 배양액을 키위퓨레와 혼합 및 발효하여 수득할 수 있다. 좀 더 상세하게는 상기 제1 배양산물(종배양액)은 균주를 각각의 배지의 0.01 내지 0.5%로 접종한 뒤 35℃ 내지 37℃에서 18시간 내지 28시간 배양하여 제조하고, 상기 제2 배양산물(대량배양액)은 균주를 배지의 0.01 내지 1%씩 접종한 뒤, 35℃ 내지 37℃에서 7시간 내지 10시간 배양하여 제조할 수 있다. 또한, 키위 퓨레와 배양액의 혼합 비율은 7.5~8.5:2.5~1.5 일 수 있으며, 바람직하게는 8:2 비율로 혼합할 수 있다.In the fermented product of kiwifruit in the present invention, each strain is cultured (seed culture) in a first medium to prepare a first culture product (seed culture medium), and the first culture product is inoculated and cultured in a second medium (mass culture) After preparing the second culture product (mass culture medium), it can be obtained by centrifuging it to remove the supernatant, and then mixing and fermenting the obtained culture medium with kiwi puree. More specifically, the first culture product (species culture medium) is prepared by inoculating the strain with 0.01 to 0.5% of each medium and culturing at 35 ° C to 37 ° C for 18 to 28 hours, and the second culture product (Massive culture) can be prepared by inoculating the strain at 0.01 to 1% of the medium and culturing at 35 ° C to 37 ° C for 7 to 10 hours. In addition, the mixing ratio of kiwi puree and culture medium may be 7.5 to 8.5:2.5 to 1.5, preferably 8:2.
본 발명의 알코올성 간손상 예방 또는 개선용 조성물에 있어서, 상기 키위 발효물은 조성물 총 중량 대비 30 내지 80 중량%, 바람직하게는 30 내지 60 중량% 포함되는 것을 특징으로 한다. 상기 키위 발효물의 함량이 30 중량% 미만일 경우 충분한 알코올성 간손상 예방 또는 개선 효과를 나타내기 어려우며, 80 중량% 초과할 경우 함량 대비 알코올성 간손상 예방 또는 개선 효과가 충분하지 못하여 경제적인 측면에서 바람직하지 못하다.In the composition for preventing or improving alcoholic liver damage of the present invention, the fermented kiwi product is characterized in that it is included in 30 to 80% by weight, preferably 30 to 60% by weight, based on the total weight of the composition. When the content of the fermented kiwi product is less than 30% by weight, it is difficult to sufficiently prevent or improve alcoholic liver damage, and when it exceeds 80% by weight, the effect of preventing or improving alcoholic liver damage is not sufficient compared to the content, which is not preferable from an economic point of view. .
본 발명의 알코올성 간손상 예방 또는 개선용 조성물에 있어서, 상기 조성물은 건강기능식품 조성물인 것을 특징으로 한다.In the composition for preventing or improving alcoholic liver damage of the present invention, the composition is characterized in that it is a health functional food composition.
본 발명의 알코올성 간손상 예방 또는 개선용 조성물에 있어서, 상기 건강기능식품 조성물은 섭취와 활용에 편리한 제형을 가질 수 있고, 바람직하게는 캡슐, 정제, 분말, 과립, 액상, 환, 편상, 페이스트상, 시럽, 겔, 음료, 젤리 및 바로 구성된 군으로부터 선택되는 하나 이상의 제형인 것을 특징으로 한다. 이러한 제형으로 제조하기 위하여 통상의 부형제, 안정제, 증점제 등을 더 포함할 수 있다. 또한, 식품첨가물을 추가로 포함할 수 있으며, "식품첨가물" 으로서의 적합 여부는 다른 규정이 없는 한 식품의약품안전처에 승인된 식품첨가물공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다.In the composition for preventing or improving alcoholic liver damage of the present invention, the health functional food composition may have a convenient dosage form for ingestion and utilization, preferably in the form of capsules, tablets, powders, granules, liquids, pills, slices, and pastes. , It is characterized in that at least one formulation selected from the group consisting of syrups, gels, beverages, jellies and bars. In order to prepare such a formulation, conventional excipients, stabilizers, thickeners, and the like may be further included. In addition, food additives may be additionally included, and the suitability as a “food additive” shall be determined according to the general rules of the Food Additive Code and general test methods approved by the Ministry of Food and Drug Safety, unless otherwise specified. judged by
본 발명의 건강기능식품 조성물이 분말형태로 제형화 될 경우, 액체질소동결기(liquid nitrogen freezer, LNF)를 이용하여 키위 발효물을 -180℃ 내지 -195℃에서, 바람직하게는 -195℃에서 급속동결한 후, 급속동결된 발효물을 -35℃ 내지 -45℃에서, 바람직하게는 -40℃에서 동결건조하여 수득한 건조된 원료를 분쇄함으로써 제형화할 수 있다.When the health functional food composition of the present invention is formulated in powder form, the fermented product of kiwi is stored at -180 ° C to -195 ° C, preferably at -195 ° C, using a liquid nitrogen freezer (LNF). After quick-freezing, the quick-frozen fermented product can be formulated by grinding the dried raw material obtained by lyophilization at -35°C to -45°C, preferably at -40°C.
본 발명의 알코올성 간손상 예방 또는 개선용 조성물에 있어서, 상기 조성물은 식품 조성물인 것을 특징으로 한다.In the composition for preventing or improving alcoholic liver damage of the present invention, the composition is characterized in that it is a food composition.
본 발명의 알코올성 간손상 예방 또는 개선용 조성물에 있어서, 상기 식품 조성물은 키위 발효물을 주성분으로 이용 가능한 식품으로 제형화될 수 있으며, 바람직하게는 유제품, 음료, 소스류, 잼류 및 제과류로 구성된 군에서 선택되는 하나 이상의 제형인 것을 특징으로 한다.In the composition for preventing or improving alcoholic liver damage of the present invention, the food composition may be formulated as a food that can use fermented kiwi as a main component, preferably in the group consisting of dairy products, beverages, sauces, jams and baked goods Characterized in that it is one or more formulations selected.
본 발명의 일 실험예에 따르면, 본 발명에 따른 키위 발효물은 알코올 유도된 마우스(EtOH)에서 증가한 염증성 사이토카인을 감소시켰으며(실험예 2 참조), 알코올 유도된 마우스(EtOH)에서 증가한 에탄올 및 알데하이드를 감소시켰고(실험예 3 참조), 알코올 유도된 마우스(EtOH)에서 증가한 콜레스테롤 및 중성지방을 감소시켰다(실험예 4 참조). 이러한 결과는, 본 발명에 따른 키위 발효물은 알코올에 의해 발생되는 간에 손상을 줄 수 있는 요인을 저해 또는 억제함으로써 간손상을 예방 또는 개선할 수 있음을 시사한다.According to an experimental example of the present invention, the kiwi fermented product according to the present invention reduced the increased inflammatory cytokines in alcohol-induced mice (EtOH) (see Experimental Example 2), and increased ethanol in alcohol-induced mice (EtOH). and aldehyde (see Experimental Example 3), and increased cholesterol and triglyceride levels in alcohol-induced mice (EtOH) were reduced (see Experimental Example 4). These results suggest that the fermented kiwi product according to the present invention can prevent or improve liver damage by inhibiting or suppressing factors that can cause liver damage caused by alcohol.
본 발명의 다른 한 양태에 따르면, 본 발명은 키위를 유산균으로 발효한 키위 발효물을 유효성분으로 포함하는 숙취 해소용 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a composition for relieving hangover comprising, as an active ingredient, fermented kiwifruit fermented with lactic acid bacteria.
본 발명에서의 용어 ‘숙취 해소’는 섭취한 알코올 및 이의 대사 산물인 알데하이드에 의해 유발되는 두통, 피로감, 근육통, 안구 충혈, 갈증, 전신권태, 복부팽만감, 구토, 속쓰림, 인지기능 감소 등과 같은 신체적 숙취 증상들을 개선하는 것으로, 구체적으로는 간에서의 알코올 대사를 촉진시켜 알코올 대사가 활성화됨에 따라 혈중 에탄올 및 알데하이드의 농도를 감소시킴으로써 에탄올 및 알데하이드에 의해 유발되는 숙취 증상들을 해소하는 것을 의미한다.In the present invention, the term 'relief from hangover' refers to physical symptoms such as headache, fatigue, muscle pain, eye congestion, thirst, general malaise, abdominal distension, vomiting, heartburn, and cognitive decline caused by ingested alcohol and its metabolite, aldehyde. Improvement of hangover symptoms, specifically means to relieve hangover symptoms caused by ethanol and aldehyde by reducing the concentration of ethanol and aldehyde in the blood as alcohol metabolism is activated by promoting alcohol metabolism in the liver.
본 발명의 다른 한 양태에 따르면, 본 발명은 키위를 유산균으로 발효한 키위 발효물을 유효성분으로 포함하는 숙취 해소용 조성물을 포함하는 숙취 해소용 음료 및 식품에 관한 것이다.According to another aspect of the present invention, the present invention relates to a drink and food for relieving a hangover, including a composition for relieving a hangover containing fermented kiwi fruit obtained by fermenting kiwi with lactic acid bacteria as an active ingredient.
본 발명에 따른 상기 식품은 음료 이외에도 분말, 과립, 정제, 캡슐 및 환으로 구성된 군으로부터 선택되는 하나 이상의 제형일 수 있다.The food according to the present invention may be one or more formulations selected from the group consisting of powders, granules, tablets, capsules and pills in addition to beverages.
전술한 바와 같이, 본 발명에 따른 키위 발효물을 포함하는 조성물은 간에서의 알코올 대사를 촉진시켜 알코올 대사가 활성화됨에 따라 혈중 에탄올 및 알데하이드의 농도가 감소되고, 간 내 지방축적 및 염증성 사이토카인의 생성을 억제할 뿐만 아니라 콜레스테롤 및 중성지방 축적을 억제함으로써 알코올성 간손상을 예방 또는 개선할 수 있으며, 다양한 숙취 증상들을 해소할 수 있다.As described above, the composition containing the fermented kiwifruit according to the present invention promotes alcohol metabolism in the liver, and as the alcohol metabolism is activated, the concentration of ethanol and aldehyde in the blood is reduced, and the accumulation of fat in the liver and the level of inflammatory cytokines are reduced. It can prevent or improve alcoholic liver damage and relieve various hangover symptoms by suppressing cholesterol and triglyceride accumulation as well as inhibiting production.
도 1은 본 발명에 따른 기탁 균주를 그람 염색한 도면이다.
도 2는 본 발명에 따른 기탁 균주의 탄산칼슘 분해에 따른 투명환 형성을 나타내는 도면이다.
도 3은 본 발명에 따른 기탁 균주 Lactococcus Lactis VI-01 KCTC 14351BP의 형태를 관찰한 도면이다.
도 4는 본 발명에 따른 기탁 균주 Lactobacillus paracasei VI-02 KCTC 14352BP의 형태를 관찰한 도면이다.
도 5는 본 발명에 따른 기탁 균주 Lactococcus Lactis VI-01 KCTC 14351BP의 계통수이다.
도 6은 본 발명에 따른 기탁 균주 Lactobacillus paracasei VI-02 KCTC 14352BP의 계통수이다.
도 7은 알코올로 유도된 마우스에 키위 발효물을 처리하였을 때의 체중 변화를 측정한 결과이다(Normal; 알코올 무처리, EtOH; 알코올 처리, FGL; 알코올 처리+키위 발효물(50 mg/kg), FGM; 알코올 처리+키위 발효물(125 mg/kg), FGH; 알코올 처리+키위 발효물(250 mg/kg)).
도 8a 내지 도 8c는 알코올로 유도된 마우스에 키위 발효물을 처리하였을 때의 혈중 사이토카인을 확인한 결과이다(8a; TNF-α, 8b; IL-1β, 8c; IL-6, Normal; 알코올 무처리, EtOH; 알코올 처리, FGL; 알코올 처리+키위 발효물(50 mg/kg), FGM; 알코올 처리+키위 발효물(125 mg/kg), FGH; 알코올 처리+키위 발효물(250 mg/kg)).
도 9a 및 도 9b는 알코올로 유도된 마우스에 키위 발효물을 처리하였을 때의 혈중 에탄올 및 알데하이드를 확인한 결과이다(9a; 에탄올, 9b; 알데하이드, Normal; 알코올 무처리, EtOH; 알코올 처리, FGL; 알코올 처리+키위 발효물(50 mg/kg), FGM; 알코올 처리+키위 발효물(125 mg/kg), FGH; 알코올 처리+키위 발효물(250 mg/kg)).
도 10a 및 도 10b는 알코올로 유도된 마우스에 키위 발효물을 처리하였을 때의 혈중 콜레스테롤 및 중성지방을 확인한 결과이다(10a; 콜레스테롤, 10b; 중성지방, Normal; 알코올 무처리, EtOH; 알코올 처리, FGL; 알코올 처리+키위 발효물(50 mg/kg), FGM; 알코올 처리+키위 발효물(125 mg/kg), FGH; 알코올 처리+키위 발효물(250 mg/kg)).1 is a diagram showing Gram staining of deposited strains according to the present invention.
Figure 2 is a view showing the formation of a transparent ring according to the calcium carbonate decomposition of the deposited strain according to the present invention.
Figure 3 is a view of observing the morphology of the deposited strain Lactococcus Lactis VI-01 KCTC 14351BP according to the present invention.
Figure 4 is a view of observing the morphology of the deposited strain Lactobacillus paracasei VI-02 KCTC 14352BP according to the present invention.
5 is a phylogenetic tree of the deposited strain Lactococcus Lactis VI-01 KCTC 14351BP according to the present invention.
Figure 6 is a phylogenetic tree of the deposited strain Lactobacillus paracasei VI-02 KCTC 14352BP according to the present invention.
7 is a result of measuring body weight change when alcohol-induced mice were treated with fermented kiwifruit (Normal; alcohol untreated, EtOH; alcohol treated, FGL; alcohol treated + fermented kiwifruit (50 mg/kg)). , FGM; alcohol treatment+kiwi fermented product (125 mg/kg), FGH; alcohol treatment+kiwi fermented product (250 mg/kg)).
8a to 8c are results of confirming blood cytokines when alcohol-induced mice were treated with fermented kiwifruit (8a; TNF-α, 8b; IL-1β, 8c; IL-6, Normal; no alcohol). Treatment, EtOH; Alcohol treatment, FGL; Alcohol treatment+Kiwi fermentation product (50 mg/kg), FGM; Alcohol treatment+Kiwi fermentation product (125 mg/kg), FGH; Alcohol treatment+Kiwi fermentation product (250 mg/kg) )).
9a and 9b are results of confirming blood ethanol and aldehyde when alcohol-induced mice were treated with kiwi fermented product (9a; ethanol, 9b; aldehyde, Normal; alcohol untreated, EtOH; alcohol treated, FGL; Alcohol treatment+kiwi fermented product (50 mg/kg), FGM; alcohol treatment+kiwi fermented product (125 mg/kg), FGH; alcohol treatment+kiwi fermented product (250 mg/kg)).
10a and 10b are results of confirming blood cholesterol and triglyceride levels when alcohol-induced mice were treated with kiwi fermented product (10a; cholesterol, 10b; neutral fat, Normal; alcohol untreated, EtOH; alcohol treated, FGL; alcohol treatment+fermented kiwifruit (50 mg/kg), FGM; alcohol treatment+fermented kiwifruit (125 mg/kg), FGH; alcohol treatment+fermented kiwifruit (250 mg/kg)).
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail through examples. Since these examples are intended to illustrate the present invention only, the scope of the present invention is not to be construed as being limited by these examples.
준비예 1: 미생물의 분리 및 확인Preparation Example 1: Isolation and identification of microorganisms
1) 분리원 및 시료 전처리1) Separation Source and Sample Pretreatment
골드키위로부터 유산균 분리를 위해 골드키위 생과와 퓨레를 사용하였다. 골드키위 생과는 뉴질랜드에서 수입된 제스프리 골드키위를 2020년 9월에 마트에서 구매하였으며, 3차 증류수로 세척하여 이물을 제거한 후 껍질을 포함하여 잘게 잘라 사용하였다. 골드키위 퓨레는 ㈜남양냉동식품의 골든키위퓨레를 2020년 5월에 구매하였고, -20℃에 보관하며 사용 시 실온에 완전히 녹여 고르게 혼합하여 실험에 사용하였다.Gold kiwi fruit and puree were used to separate lactic acid bacteria from gold kiwi. Gold kiwi fruit was purchased from Zespri Gold Kiwi imported from New Zealand at a mart in September 2020, washed with tertiary distilled water to remove foreign substances, and then cut into small pieces, including the peel, and used. Gold kiwi puree was purchased from Namyang Frozen Food Co., Ltd. in May 2020, stored at -20 ° C, completely melted at room temperature and mixed evenly before use in the experiment.
2) 자연 발효 2) Natural fermentation
잘게 자른 골드키위와 퓨레는 염장 조건과 탈지 조건으로 발효하였다. 염장 조건은 각각의 시료에 8%의 첨일염을 첨가해 3시간 동안 염장한 후 멸균한 1% 프락토올리고당 용액을 혼합하였고, 탈지 조건은 각각의 시료에 멸균한 2% 탈지와 1% 프락토올리고당 용액을 혼합하였다. 혼합 한 모든 시료는 수산화나트륨 용액을 사용해 pH를 6.0 이상으로 보정하였고, 37℃ 배양기에서 약 3일 동안 발효하였다. Chopped gold kiwi and puree were fermented under salting and degreasing conditions. For salting conditions, each sample was salted for 3 hours by adding 8% of cheil salt, and then sterilized 1% fructooligosaccharide solution was mixed. Oligosaccharide solutions were mixed. All mixed samples were corrected to pH 6.0 or higher using sodium hydroxide solution, and fermented for about 3 days in a 37°C incubator.
3) 유산균 선별 및 동정3) Selection and identification of lactic acid bacteria
발효한 시료는 고르게 혼합한 후, 발효액 일부를 취하여 BCP plate count agar(EIKEN, Japan)를 사용해 접종하여 37℃에서 48시간 배양하였다. 이후 배지가 노란색을 띠며 phenotype이 서로 colony를 대상으로 MRS agar (Difco, USA) 평판배지를 사용하여 순수분리 하였다. 순수분리한 균주는 colony의 특성을 확인하고, 현미경 관찰(×1000)을 통해 세포의 형태를 확인하였으며, 그람염색을 통해 그람양성을 나타내는 균주만을 1차 선별하였다(도 1). 이후 탄산칼슘 (DAEJUNG, Korea)이 1% 함유된 MRS agar 배지에 접종하여 37℃에서 48시간 배양하여 유기산에 의해 탄산칼슘을 분해해 투명환 (clear zone)이 크게 형성된 균주 2종을 최종 선별하였다(도 2).After mixing the fermented sample evenly, a portion of the fermentation broth was inoculated using BCP plate count agar (EIKEN, Japan) and incubated at 37 ° C for 48 hours. Afterwards, the medium was yellow and the phenotypes were purified using MRS agar (Difco, USA) plate medium for colony. For the pure isolated strain, the characteristics of the colony were confirmed, the cell morphology was confirmed through microscopic observation (×1000), and only the strains showing Gram-positive were first screened through Gram staining (FIG. 1). Thereafter, it was inoculated into MRS agar medium containing 1% calcium carbonate (DAEJUNG, Korea) and cultured at 37 ° C for 48 hours to decompose calcium carbonate with organic acid to finally select two strains with large clear zones. (Fig. 2).
분리한 2종 균주는 임의로 VI-01, VI-02로 명명하였고, 30% glycerol을 사용해 stock으로 제조하여 -80℃에 보관하였다. 2종 균주의 당분해능, arginin과 esculin 분해능 등의 생화학적 특성 확인을 위해 API 50 CHL kit (Biomerieux, France)를 사용해 medium의 색 변화를 apiweb program (http://apiweb.biomerieux.com)을 이용하여 동정하였다.The two separated strains were arbitrarily named VI-01 and VI-02, and were prepared as stocks using 30% glycerol and stored at -80 ° C. To confirm the biochemical characteristics of the two strains, such as glycolysis, arginin and esculin resolution,
16s rRNA 분석은 ㈜바이오팩트에 의뢰하여 27F와 1492R primer를 사용한 PCR 수행 및 sequencing 분석을 진행하였다. 확인된 각 균주의 염기서열은 NCBI의 blast program을 사용하여 GenBank에 등록된 16S ribosomal RNA gene sequence와 상동성을 비교하였고, neighbor-joining method를 적용한 ClustalX 2.1 프로그램을 사용해 alignment 하였다. 계통수는 ClustalX 2.1의 bootstrap N-J tree 방법을 사용하였고 NJplot 프로그램으로 확인하였다.For 16s rRNA analysis, PCR was performed using 27F and 1492R primers and sequencing analysis was performed by requesting Biofact Co., Ltd. The nucleotide sequence of each identified strain was compared for homology with the 16S ribosomal RNA gene sequence registered in GenBank using the blast program of NCBI, and aligned using the ClustalX 2.1 program using the neighbor-joining method. The phylogenetic tree used the bootstrap N-J tree method of ClustalX 2.1 and was confirmed with the NJplot program.
4) 유전체 염기서열 해독4) Genome sequence decoding
Strain VI-01 균주의 16S rRNA 유전자 분석을 통해 1,414bp 크기의 염기서열을 확인한 결과, Lactococcus lactis NBRC 100933 strain과 100%의 상동성을 나타내었다. 따라서 VI-01 균주를 Lactococcus lactis VI-01이라 명명하였다(서열번호 1).As a result of confirming the nucleotide sequence of 1,414 bp through 16S rRNA gene analysis of strain VI-01 strain, it showed 100% homology with Lactococcus lactis NBRC 100933 strain. Therefore, strain VI-01 was named Lactococcus lactis VI-01 (SEQ ID NO: 1).
Strain VI-02 균주의 16S rRNA 유전자 분석을 통해 1,441bp 크기의 염기서열을 확인한 결과, Lactobacillus paracasei R094 strain과 99%의 상동성을 나타내었다. 따라서 VI-02 균주를 Lactobacillus paracasei VI-02라 명명하였다(서열번호 2).As a result of confirming the nucleotide sequence of 1,441 bp through 16S rRNA gene analysis of strain VI-02 strain, it showed 99% homology with Lactobacillus paracasei R094 strain. Therefore, the VI-02 strain was named Lactobacillus paracasei VI-02 (SEQ ID NO: 2).
5) 당 이용성조사를 통한 동정5) Identification through glucose availability survey
분리균주의 API 50 CHL kit를 사용하여 당 이용성 조사를 통한 균 동정 결과 strain VI-01은 Lactococcus lactis와 98.4%의 유사성이 확인되었으며, Strain VI-02는 Lactobacillus paracasei와 99.6%의 유사성이 확인되었다.As a result of strain identification through sugar utilization
준비예 2: 균주의 생화학적 및 형태학적 특성Preparation Example 2: Biochemical and Morphological Characteristics of Strains
1) 생화학적 특성 1) Biochemical characteristics
분리 균주의 생화학적 특성은 API 50 CHL kit로 측정하였다. MRS agar 배지에 순수배양한 colony를 API 50 CHL medium에 적정농도로 희석하여 API 50 CHL kit에 접종한 후, 37℃에서 24~48시간 동안 배양하며 접종된 medium의 색 변화를 관찰하였다. Biochemical characteristics of the isolated strains were measured using
Lc. lactis VI-01 균주는 49개의 당 중 galactose, D-glucose, D-fructose, D-mannose, mannitol, maltose, lactose등 총 19개의 당을 이용하였으나, sorbitol이나 xylitol 등은 이용하지 못하는 것으로 확인되었다(표 1). Lc. lactis VI-01 strain used a total of 19 sugars, including galactose, D-glucose, D-fructose, D-mannose, mannitol, maltose, and lactose among 49 sugars, but it was confirmed that sorbitol or xylitol could not be used ( Table 1).
L.paracasei VI-02 균주는 당 49개 중 galactose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol 등을 포함하여 22개의 당을 이용하였고, lactose와 xylitol 등은 이용하지 못하였다(표 2). L. paracasei VI-02 strain used 22 sugars, including galactose, D-glucose, D-fructose, D-mannose, mannitol, and sorbitol among 49 sugars, but lactose and xylitol were not used ( Table 2).
2) 형태학적 특성2) Morphological characteristics
형태학적 특성은 MRS agar 배지에서 배양한 균주의 콜리니 형태와 색 등을 확인하였으며, 현미경으로 균주 형태를 관찰하였다.For morphological characteristics, the shape and color of colonies of the strain cultured in MRS agar medium were confirmed, and the strain shape was observed under a microscope.
Lc.lactis VI-01 균주의 colony는 0.5-1.5 mm의 작고 둥근 형태이며, 흰색의 광택이 있는 형태이다. 현미경으로 균주의 형태를 관찰한 결과 구균으로 확인되었다(도 3).The colony of strain Lc.lactis VI-01 is small, round, 0.5-1.5 mm, and glossy white. As a result of observing the morphology of the strain under a microscope, it was confirmed as cocci (FIG. 3).
L.paracasei VI-02 균주의 colony 형태는 1.5-2.5 mm의 둥글고 볼록한 형태이며, 흰색 또는 크림색의 광택이 있다. 현미경 관찰 결과 간균으로 long chain을 형성하였다(도 4).The colony of strain L. paracasei VI-02 is 1.5-2.5 mm round and convex, and has white or creamy luster. As a result of microscopic observation, long chains were formed as bacilli (FIG. 4).
준비예 3: 균주기탁Preparation Example 3: Deposit of Strains
16S RNA sequence분석과 형태학적인 특성을 확인하고 최종적으로 균주 확인 작업을 진행한 뒤 생물자원센터에 기탁절차를 진행하여 기탁번호를 부여받았다(표 3).After confirming the 16S RNA sequence analysis and morphological characteristics, and finally confirming the strain, the deposit procedure was performed at the Center for Biological Resources and a deposit number was assigned (Table 3).
실시예 : 유산균을 이용한 키위 발효물의 제조Example: Preparation of kiwi fermented product using lactic acid bacteria
1) 키위입고1) wearing kiwi
다양한 품종의 키위 사용이 가능하나, 본 발명에서는 Actinidia chinensis 속(골드키위)의 키위를 사용하였다.Although various types of kiwifruit can be used, in the present invention, kiwifruit of the genus Actinidia chinensis (gold kiwifruit) was used.
2) 전처리과정(키위 퓨레제조)2) Pretreatment process (manufacturing of kiwi puree)
키위 과실을 수회 세척하고, 원료로 사용할 수 있는 과실 키위를 선별한다. 선별기준은 육안으로 관찰하였을 때 과실에 상처가 없는 것, 썩거나 균에 오염되지 않는 것, 당도가 13 Brix% 이상(넘지 않을 시 후숙과정을 거침)인 것을 선별한다. 선별된 과실의 과피를 벗기고 과육을 세절한다.Kiwi fruit is washed several times, and fruit kiwi fruit that can be used as a raw material is selected. The selection criteria are to select fruits that have no damage when observed with the naked eye, that are not rotten or contaminated by bacteria, and that have a sugar content of 13 Brix% or more (if not exceeded, go through a post-ripening process). Peel the skin of the selected fruit and cut the flesh.
3) 마쇄·씨분리과정(키위 퓨레제조)3) Grinding and seed separation process (making kiwi puree)
마쇄·씨분리는 세절된 과육을 원심분리 마쇄기에 투입하여 마쇄하고, 거름망에 걸러진 씨를 제거 후 교반하여 균질한다.Grinding·seed separation puts the cut flesh into a centrifugal crusher, crushes it, removes the seeds filtered through a sieve, and then stirs to make it homogeneous.
4) 유산균 배양과정(종배양-탱크배양-원심분리)4) Lactic acid bacteria cultivation process (species culture - tank culture - centrifugation)
4-1) 유산균 균종4-1) Lactic acid bacteria species
키위 발효물을 제조하기 위해 이용하는 유산균은 이전 실험에서 분리한 Lc. lactis VI-01 KCTC14351BP 및 L.paracasei VI-02 KCTC14352BP 균주에 락토바실러스 엑시도필러스(Latobacillus acidophilus), 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 헬베티커스(Lactobacillus Helveticus) 3종의 락토바실러스 균종을 혼합하여 발효를 진행하였고, 3종의 락토바실러스 균종의 경우 Sacco사 제품을 사용하였다.The lactic acid bacteria used to prepare the fermented kiwi product were Lc. lactis VI-01 KCTC14351BP and L.paracasei VI-02 KCTC14352BP strains, Lactobacillus acidophilus, Lactobacillus casei , Lactobacillus helveticus Lactobacillus Helveticus was mixed to proceed with fermentation, and in the case of three types of Lactobacillus strains, products from Sacco were used.
4-2) 배양과정4-2) Cultivation process
4-2-1) 종균배양4-2-1) Seed culture
종균배양은 MRS Broth로 배양하였다. 구체적인 배양 방법은 각 균주를 각각의 배지의 0.1%로 접종한 뒤, 37℃에서 24시간 배양하여 제1 배양산물(균주)을 수득하였다. 멸균은 121℃에서 15분 동안 실시하였다.Seed culture was cultured with MRS Broth. In the specific culture method, each strain was inoculated with 0.1% of each medium and then cultured at 37° C. for 24 hours to obtain a first culture product (strain). Sterilization was performed at 121° C. for 15 minutes.
4-2-2) 대량배양4-2-2) Mass culture
대량배양은 상기 제1 배양산물을 이용하여 실시하였다. MRS 배지에 제1 배양산물을 접종한 후 37℃에서 9시간 배양하여 제2 배양산물을 수득하였다. 멸균은 121℃에서 20분 동안 실시하였다. Mass culture was carried out using the first culture product. After inoculating the first culture product in the MRS medium, the second culture product was obtained by culturing at 37° C. for 9 hours. Sterilization was performed at 121° C. for 20 minutes.
4-2-3) 원심분리 및 농축4-2-3) Centrifugation and concentration
배양이 완료된 유산균을 원심분리하여 배양액과 유산균주를 농축한다.The cultured lactic acid bacteria are centrifuged to concentrate the culture medium and lactic acid bacteria strain.
5) 혼합5) mix
원료(키위퓨레)와 유산균 배양액을 8:2 비율로 혼합한다. 구체적인 구성 및 접종량은 하기 표 4과 같다.Mix the raw material (kiwi puree) and the lactobacillus culture medium at a ratio of 8:2. The specific composition and inoculation amount are shown in Table 4 below.
6) 발효6) fermentation
상기 혼합한 원료를 37℃에서 8~12시간 배양한다.The mixed raw materials are incubated at 37°C for 8 to 12 hours.
7) 분말화7) Powdering
실험에 이용하기 위하여 상기 제조된 발효물을 급속동결 및 동결건조한 후, 분쇄하여 분말화하여 키위 발효물 분말을 제조하였다.For use in experiments, the prepared fermented product was rapidly frozen and freeze-dried, and then pulverized and powdered to prepare fermented kiwi powder.
준비예 4: 동물 모델의 준비Preparation Example 4: Preparation of animal model
본 실험에 사용한 실험동물은 생후 8주령 ICR 수컷 마우스 40마리(8마리/군)를 다물사이언스(대전, 한국)으로부터 구입하여 1주일 간 사육 환경에 적응시켰다. 사육환경에 적응이 완료된 마우스는 체중을 측정한 후, 난괴법에 따라 8마리씩 총 5군으로 분류하였다: 각 군은 정상군(Normal), EtOH 5 g/kg BW로 경구 투여한 군(EtOH), 5 g/kg EtOH와 발효 골드키위 분말 50 mg/kg BW 투여군(FGL), 5 g/kg EtOH와 발효 골드키위 분말 125 mg/kg BW 투여군(FGM), 5 g/kg EtOH와 발효 골드키위 분말 250 mg/kg 투여군(FGH)으로 나누었다. 실험 시작 2주는 Normal 군과 EtOH군은 DW만 경구투여 하였으며, 발효 골드키위 분말 투여군은 각 농도의 시료를 1회/일로 경구투여했다. 그 후, 2주간은 Normal 군에는 DW만 2회 공급하였으며, EtOH군에는 에탄올과 DW를 공급하였다. 발효 골드키위 분말 투여군에는 에탄올과 체중 1 kg당 50, 125, 250 mg의 분말을 녹여 2주간 추가 경구투여를 진행하였다. 실험동물 사육실의 온도는 22±2℃, 상대 습도 50±10%, 환기횟수 10~20회/시간이었으며, 조명 사이클은 12시간 간격으로 조정하였다. 실험이 진행되는 동안 마우스들은 물과 사료를 자유롭게 공급하였다. 모든 동물실험 절차는 전북대학교 동물실험 윤리위원회의 승인을 받아 수행하였다(JBNU 2022-095).Experimental animals used in this experiment were 40 ICR male mice (8 mice/group), 8 weeks old, purchased from Damul Science (Daejeon, Korea) and adapted to the breeding environment for one week. Mice that had completed adaptation to the rearing environment were weighed and classified into 5 groups of 8 mice each according to the egg mass method: each group was a normal group (Normal), and a group orally administered with EtOH 5 g/kg BW (EtOH) , 5 g/kg EtOH and fermented
실험예 1: 알코올 유도된 마우스의 체중 측정Experimental Example 1: Weight measurement of alcohol-induced mice
알코올성 간손상은 체중 증가를 유발하기 때문에 알코올로 유도된 마우스 체중 측정을 통해 키위 발효물의 알코올성 간손상 개선 효과를 확인하였다.Since alcohol-induced liver damage causes weight gain, the alcohol-induced liver damage improvement effect of fermented kiwi fruit was confirmed by measuring the weight of mice induced by alcohol.
구체적으로는, 난괴법에 따라 군별로 동물들을 나누고 체중을 측정하여 기록하였고, 매주 1회씩 체중을 개체별로 측정하고 평균값을 구하여 나타내였다.Specifically, the animals were divided into groups according to the egg mass method, and the body weight was measured and recorded.
그 결과 도 7에서 확인할 수 있듯이, 키위 발효물을 처리하지 않은 알코올 유도된 마우스(EtOH)에서 증가한 체중이 키위 발효물을 농도별로(FGL(50mg/kg), FGM(125mg/kg), FGH(250mg/kg)) 처리하였을 때 감소하였다.As a result, as can be seen in Figure 7, the increased body weight in alcohol-induced mice (EtOH) that were not treated with kiwi fermented product was determined by concentration (FGL (50 mg / kg), FGM (125 mg / kg), FGH ( 250 mg/kg))).
실험예 2: 알코올 유도된 마우스의 혈중 사이토카인 확인Experimental Example 2: Identification of cytokines in the blood of alcohol-induced mice
알코올 유도된 마우스 혈중에서의 사이토카인 농도를 확인함으로써 키위 발효물의 알코올성 간손상 개선 효과를 확인하였다.The alcohol-induced liver damage improvement effect of fermented kiwifruit was confirmed by checking the cytokine concentration in the blood of alcohol-induced mice.
구체적으로, 각 사이토카인을 측정할수 있는 ELISA kit를 이용하여 측정하였다. 각 사이토카인 항체가 부착되어 있는 96-well plate에 혈청과 사이토카인 antibody cocktail을 넣고 1시간 실온에서 반응한 후, wash buffer를 이용하여 세척하였다. 그 후 각 well에 TMB buffer를 넣고 10분의 반응 시간을 거친 뒤, stop buffer를 추가하여 반응을 정지시키고 microplate reader를 이용하여 450 nm에서 흡광도를 측정하였다. 각 사이토카인의 농도는 ELSIA kit (abcam, London, UK)에 포함되어 있는 표준 용액에서 산출된 곡선으로부터 계산하였다.Specifically, it was measured using an ELISA kit capable of measuring each cytokine. Serum and cytokine antibody cocktail were added to a 96-well plate to which each cytokine antibody was attached, reacted at room temperature for 1 hour, and washed using wash buffer. Then, TMB buffer was added to each well, and after a reaction time of 10 minutes, stop buffer was added to stop the reaction, and absorbance was measured at 450 nm using a microplate reader. The concentration of each cytokine was calculated from the curve calculated from the standard solution included in the ELSIA kit (abcam, London, UK).
그 결과 도 8a 내지 도 8c에서 확인할 수 있듯이, 키위 발효물을 처리하지 않은 알코올 유도된 마우스(EtOH)에서 증가한 염증성 사이토카인이 키위 발효물을 농도별로 처리하였을 때 감소하였으며, 특히 IL-6가 현저하게 감소하였다.As a result, as can be seen in FIGS. 8a to 8c, inflammatory cytokines increased in alcohol-induced mice (EtOH) not treated with fermented kiwifruit were reduced when fermented kiwifruit was treated by concentration, in particular, IL-6 was significantly decreased considerably.
실험예 3: 알코올 유도된 마우스의 혈중 에탄올 및 알데하이드 확인Experimental Example 3: Identification of ethanol and aldehyde in the blood of alcohol-induced mice
알코올 유도된 마우스 혈중에서의 에탄올 및 알데하이드의 농도를 확인함으로써 키위 발효물의 알코올성 간손상 개선 효과를 확인하였다.The alcohol-induced liver damage improvement effect of fermented kiwifruit was confirmed by checking the concentrations of ethanol and aldehyde in the blood of alcohol-induced mice.
구체적으로, 혈중 알코올 농도 측정은 ethanol assay kit(abcam, ab65343)을 사용하였다. 수득한 혈청과 reaction mix를 혼합한 후, 37℃에서 30분간 반응시킨 뒤 1시간 상온에서 추가 반응을 진행하였다. 이때, 반응과정 중 빛을 차단하여 반응하였다. 반응이 끝난 후에는 microplate reader를 사용하여 570nm에서 흡광도를 측정하였다.Specifically, blood alcohol concentration was measured using an ethanol assay kit (abcam, ab65343). After mixing the obtained serum and reaction mix, reaction was performed at 37°C for 30 minutes, followed by an additional reaction at room temperature for 1 hour. At this time, the reaction was performed by blocking light during the reaction process. After the reaction was completed, absorbance was measured at 570 nm using a microplate reader.
혈중 알데히드 농도 측정은 aldehyde quantification assay kit(abcam, ab112113)을 사용하였다. 수득한 혈청과 2x yellow mixture를 혼합한 후, 상온에서 30~60분간 빛을 차단하여 반응시킨 후 microplate reader를 사용하여 550 nm에서 흡광도를 측정하였다.The concentration of aldehyde in blood was measured using an aldehyde quantification assay kit (abcam, ab112113). After mixing the obtained serum and 2x yellow mixture, reacting at room temperature by blocking light for 30 to 60 minutes, the absorbance was measured at 550 nm using a microplate reader.
그 결과 도 9a 및 도 9b에서 확인할 수 있듯이, 키위 발효물을 처리하지 않은 알코올 유도된 마우스(EtOH)에서 증가한 에탄올 및 알데하이드가 키위 발효물을 농도별로 처리하였을 때 감소하였다.As a result, as can be seen in FIGS. 9a and 9b, the increased ethanol and aldehyde in the alcohol-induced mice (EtOH) not treated with the fermented kiwifruit decreased when the fermented kiwifruit was treated by concentration.
실험예 4: 알코올 유도된 마우스의 혈중 콜레스테롤 및 중성지방 확인Experimental Example 4: Confirmation of Blood Cholesterol and Triglyceride in Alcohol-Induced Mice
알코올 유도된 마우스 혈중에서의 콜레스테롤 및 중성지방의 농도를 확인함으로써 키위 발효물의 알코올성 간손상 개선 효과를 확인하였다.The alcohol-induced liver damage improvement effect of fermented kiwifruit was confirmed by checking the concentrations of cholesterol and triglyceride in the blood of alcohol-induced mice.
구체적으로, 실험 종료 후 수득한 혈액을 1,500 rpm, 20 min, 4℃로 원심분리하여 혈청을 수득하였으며, 수득한 혈청은 본 실험에 사용하였다. 혈액 중 total cholesterol 및 triglyceride 분석을 위하여 BIOMAX사의 assay kit를 이용하여 측정하였으며, 각 TC와 TG의 농도는 kit에 포함되어 있는 표준 용액에서 산출된 곡선으로부터 계산하였다.Specifically, blood obtained after completion of the experiment was centrifuged at 1,500 rpm, 20 min, 4° C. to obtain serum, and the obtained serum was used in this experiment. For the analysis of total cholesterol and triglyceride in the blood, it was measured using an assay kit from BIOMAX, and the concentration of each TC and TG was calculated from the curve calculated from the standard solution included in the kit.
그 결과 도 10a 및 도 10b에서 확인할 수 있듯이, 키위 발효물을 처리하지 않은 알코올 유도된 마우스(EtOH)에서 증가한 콜레스테롤 및 중성지방이 키위 발효물을 농도별로 처리하였을 때 감소하였다.As a result, as can be seen in FIGS. 10a and 10b, cholesterol and triglyceride increased in alcohol-induced mice (EtOH) not treated with fermented kiwifruit were decreased when fermented kiwifruit was treated by concentration.
Claims (12)
A composition for preventing or improving alcoholic liver damage comprising fermented kiwifruit fermented with lactic acid bacteria as an active ingredient, wherein the lactic acid bacteria are Lactococcus lactis VI-01 KCTC 14351BP and Lactobacillus para Casei ( Lactobacillus paracasei ) VI-02 A composition for preventing or improving alcoholic liver damage, characterized in that at least one of KCTC 14352BP.
상기 유산균은 락토바실러스 엑시도필러스(Latobacillus acidophilus), 락토바실러스 카제이(Lactobacillus casei) 및 락토바실러스 헬베티커스(Lactobacillus Helveticus)로 구성된 군으로부터 선택되는 하나 이상의 균주를 포함하는 것을 특징으로 알코올성 간손상 예방 또는 개선용 조성물.
According to claim 1,
The lactic acid bacteria include one or more strains selected from the group consisting of Lactobacillus acidophilus , Lactobacillus casei and Lactobacillus Helveticus Alcoholic liver damage A composition for prevention or improvement.
상기 키위 발효물은 조성물 총 중량 대비 30 내지 80 중량% 포함되는 것을 특징으로 하는 알코올성 간손상 예방 또는 개선용 조성물.
According to claim 1,
The fermented kiwi product is a composition for preventing or improving alcoholic liver damage, characterized in that it is included in 30 to 80% by weight relative to the total weight of the composition.
상기 조성물은 건강기능식품 조성물인 것을 특징으로 하는 알코올성 간손상 예방 또는 개선용 조성물.
According to claim 1,
The composition is a composition for preventing or improving alcoholic liver damage, characterized in that the health functional food composition.
상기 건강기능식품 조성물은 캡슐, 정제, 분말, 과립, 액상, 환, 편상, 페이스트상, 시럽, 겔, 음료, 젤리 및 바로 구성된 군으로부터 선택되는 하나 이상의 제형인 것을 특징으로 하는 알코올성 간손상 예방 또는 개선용 조성물.
According to claim 6,
The health functional food composition is one or more formulations selected from the group consisting of capsules, tablets, powders, granules, liquids, pills, flakes, pastes, syrups, gels, drinks, jellies and bars. composition for improvement.
상기 조성물은 식품 조성물인 것을 특징으로 하는 알코올성 간손상 예방 또는 개선용 조성물.
According to claim 1,
The composition is a composition for preventing or improving alcoholic liver damage, characterized in that the food composition.
상기 식품 조성물은 유제품, 음료, 소스류, 잼류 및 제과류로 구성된 군에서 선택되는 하나 이상의 제형인 것을 특징으로 하는 알코올성 간손상 예방 또는 개선용 조성물.
According to claim 8,
The food composition is a composition for preventing or improving alcoholic liver damage, characterized in that at least one formulation selected from the group consisting of dairy products, beverages, sauces, jams and baked goods.
A composition for relieving hangover containing, as an active ingredient, fermented kiwifruit fermented with lactic acid bacteria, wherein the lactic acid bacteria are Lactococcus lactis VI-01 KCTC 14351BP and Lactobacillus paracasei, which are lactic acid bacteria derived from kiwi. ) A composition for relieving hangover, characterized in that at least one of VI-02 KCTC 14352BP.
A beverage for relieving hangover comprising the composition according to claim 10.
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| KR20210063728A (en) * | 2019-11-25 | 2021-06-02 | (주) 바이텍 | Fermented kiwi powder for improving bowel function and method of preparing the same |
| KR20220049661A (en) * | 2020-10-14 | 2022-04-22 | (주) 바이텍 | Kiwi fermentation composition using lactic acid bacteria, preparation method thereof, and use thereof |
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| Han et al., Relationships among alcoholic liver disease, antioxidants, and antioxidant enzymes. World J Gastroenterol. 2016, Vol. 22, No. 1, pp. 37-49 1부.* * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024071624A1 (en) * | 2022-09-30 | 2024-04-04 | (주)바이텍 | Composition for preventing or relieving alcoholic liver damage comprising kiwi-fermented product |
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| Publication number | Publication date |
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| WO2024071624A1 (en) | 2024-04-04 |
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