KR20050123002A - A fish-feed to containing an extract siberian-ginseng - Google Patents
A fish-feed to containing an extract siberian-ginseng Download PDFInfo
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- KR20050123002A KR20050123002A KR1020040048702A KR20040048702A KR20050123002A KR 20050123002 A KR20050123002 A KR 20050123002A KR 1020040048702 A KR1020040048702 A KR 1020040048702A KR 20040048702 A KR20040048702 A KR 20040048702A KR 20050123002 A KR20050123002 A KR 20050123002A
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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- Life Sciences & Earth Sciences (AREA)
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- Polymers & Plastics (AREA)
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- Zoology (AREA)
- Biotechnology (AREA)
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- Health & Medical Sciences (AREA)
- Insects & Arthropods (AREA)
- Marine Sciences & Fisheries (AREA)
- Birds (AREA)
- Medicines Containing Plant Substances (AREA)
- Fodder In General (AREA)
- Feed For Specific Animals (AREA)
Abstract
본 발명은 건강보조식품이나 약학제로 생산되는 가시오가피를 재추출하여 양식어류의 사료에 첨가하여 사용함으로서, 양식어류의 체액성 및 세포성 비특이적 면역을 자극하여 면역기능이 개선됨과 동시에 실제 병원세균의 감염실험을 실시한 결과에서 항병력이 증가되는 것으로 확인할 수 있었으며, 이는 그동안 재활용하지 못한 가시오가피 추출 부산물을 이용하여 해양생물용 면역기능 개선제로 개발이 가능한 가시오가피 추출물을 이용한 어류사료용 사료에 관한 것이다.The present invention is used by re-extracting the thorny opis produced by dietary supplements or pharmaceuticals and adding them to the feed of farmed fish, stimulating humoral and cellular nonspecific immunity of farmed fish to improve the immune function and at the same time infection of actual pathogens As a result of the experiment, it was confirmed that the anti-history is increased, and this relates to the feed for fish feed using prickly bark extract, which can be developed as an immune function improving agent for marine organisms using the prickly bark extract by-product that has not been recycled.
Description
본 발명은 가시오가피 추출물이 첨가된 어류양식용 사료에 관한 것으로, 좀 더 상세하게는 가시오가피의 추출물 및 상기 가시오가피를 추출 후 생성된 부산물을 재활용하여 사료의 구성성분으로 첨가함으로서 어류의 면역기능을 향상시킬 수 있는 어류양식용 사료에 관한 것이다.The present invention relates to a feed for fish cultures added to the thorny ogapi extract, more specifically to improve the immune function of the fish by adding the extract of the thorny opiaceae and by-products generated after the extraction by recycling as a component of the feed To aquaculture feed for fish.
어류양식은 짧은 기간동안 양식기술의 발전으로 급격한 양적 증가를 보여 왔으며, 1997년에 약 320만톤의 국내 수산물 생산량 중에서 수산양식에 의한 수산물 공급량은 약 100만톤에 이르고 있다. 최근에는 상기한 수산물은 바로 건강식품이라는 인식이 확산되면서 그 수요가 지속적으로 늘고 있다.Fish farming has seen a rapid quantitative increase due to the development of aquaculture technology for a short period of time, and out of about 3.2 million tons of domestic fishery production in 1997, the aquatic supply of aquaculture is about 1 million tons. In recent years, the demand for aquatic products is continuously increasing as the perception that the seafood is just a health food.
그러나, 최근 연안역의 오염과 양식생물의 질병 등으로 어류양식을 통한 수산물 공급량은 크게 늘지않고 있는 실정이며, 이러한 원인은 적극적으로 양식어장을 개발하지 못한 데에서 그 이유를 찾을 수 있으나, 근본적으로 연안어장의 오염이 수반된 양식환경의 악화로 연중 양식생물이 질병에 노출되면서 생산성이 크게 떨어졌기 때문이다. However, in recent years, the supply of marine products through fish farming has not increased significantly due to the pollution of coastal areas and diseases of farmed organisms. The reason for this can be found in the fact that the fisheries have not been actively developed. Due to the deterioration of the farming environment accompanied by the pollution of fisheries, productivity has decreased significantly due to the exposure of aquaculture organisms to diseases throughout the year.
이에 해상가두리나 육상 넙치양식장의 경우 연간 총생산 경비의 10%이상을 질병예방 및 치료목적으로 사용되고 있어 수산양식산업의 경제성을 크게 악화시키고 있다. 또한, 이와 같이 양식생물의 질병예방이나 치료목적으로 각종 약제를 남용함에 따라 양식생물의 식품안정성은 떨어지고 내병성도 나빠질 뿐만 아니라 국내외적으로 제기되고 있는 PCB나 다이옥신 등의 환경호르몬으로 식품안정성에 대한 의구심이 증폭되고 있어 양식생물에 대한 기호도가 크게 떨어지고 있기 때문에 적절한 대책이 없을 경우 한순간 양식업체가 쉽게 무너질 수 있다는 것이다.As a result, more than 10% of the annual production cost of marine cages and land flounder farms is being used for disease prevention and treatment. In addition, as a result of the abuse of various medicines for the purpose of preventing or treating diseases of aquaculture organisms, the food stability of aquaculture organisms is reduced and disease resistance is worsened, and there are doubts about food stability with environmental hormones such as PCB and dioxin, which are raised at home and abroad. As the amplification of the aquaculture products is amplifying, the acceptance of aquaculture organisms is greatly declining, so if a proper countermeasure is not taken, aquaculture companies can easily collapse.
따라서, 각종 수산약품 및 항생제를 사용하지 않고 식품안정성이 확보된 신선한 양식어류를 공급하기 위해서는 질병이 발생되기 이전에 특정 병원체에 대한 백신을 적극 활용하거나 면역증강물질의 투여로 어체의 면역기능을 강화시키는 것이 필수적이다.Therefore, in order to supply fresh farmed fish with food stability without the use of various aquatic medicines and antibiotics, vaccination against specific pathogens is actively used before the disease occurs, or the immune function of the fish is strengthened by the administration of immune enhancing substances. It is essential.
이와 같이 불특정 질병에 저항할 수 있도록 어류 자체의 면역능을 증강시켜 주는 물질에 관한 연구가 폭넓게 연구되고 있으며, 이러한 물질로는 β-glucan(Won et al., 2004), 기생충 구제제인 levamisole(Kajita et al., 1990), 달걀 유래 산생물인 EF203(Yoshida et al., 1993) 등 많은 연구가 진행되고 있는 물질부터, 감귤 발효액(Song et al., 2002), 녹차 (Park et al., 1999), 그리고 미역, 다시마, 파래와 같은 각종 해조류(Choi et al., 1995; Nakagawa et al., 1985; Yone et al., 1996)에 이르기까지 매우 다양하게 연구된 바 있다. As such, research on a substance that enhances the immunity of the fish itself to resist an unspecified disease has been widely studied. Such substances include β-glucan (Won et al., 2004) and levamisole (Kajita et al., A parasite control agent). al., 1990), and many other researches, including egg-derived EF203 (Yoshida et al., 1993), citrus fermentation broth (Song et al., 2002), green tea (Park et al., 1999). And various seaweeds such as seaweed, kelp and seaweed (Choi et al., 1995; Nakagawa et al., 1985; Yone et al., 1996).
특히 최근에는 각종 생약제를 사료에 첨가하여 사료 효율 개선 및 비특이적 면역 기능 증가 효과가 있다는 보고가 이어지고 있으나(Hwang et al., 1999; Kwon et al., 1999), 아직까지는 식품안정성을 보장하면서 어류 자체의 면역능을 증강시켜 줄 수 있는 물질에 대한 연구는 미비한 형편이다.In particular, recently, various herbal supplements have been reported to improve feed efficiency and increase nonspecific immune function (Hwang et al., 1999; Kwon et al., 1999). Studies on substances that can enhance the immune system is poorly done.
한편, 가시오가피는 드룹나무과에 속하는 낙엽관목으로 시베리안 인삼(Siberian ginseng)이라고도 불리우며, 원산지는 남동쪽 러시아, 북 중국, 북 한국, 북 일본 등의 동쪽 타이가(Taiga)지역에서 재배되고 있다.On the other hand, Prickly Pear is a deciduous shrub belonging to the Druphaceae, also called Siberian ginseng, and its origin is cultivated in the eastern Taiga region of southeastern Russia, North China, North Korea, and North Japan.
상기한 가시오가피의 주요성분은 엘레우테로사이드(eleutherosides)로 7개의 주요 엘레우테로사이드가 규명되었으며 그 중 엘레우테로사이드 B와 E가 집중적으로 연구되어지고 있다. 엘레우테로사이드 B는 진정, 불면증, 건망증, 성선 자극, 혈압강하, 자율신경 조정, 당뇨병의 당대사 개선, 당뇨병의 합병증 예방, 간기능 개선 등의 작용을 하고, 엘레우테로사이드 E는 항피로, 항스트레스, 성선자극, 학습향상, 지구력, 스태미너, 집중력, 항류머티스 등의 작용을 하는 것으로 알려져 있다. 또한, 가시오가피는 복합다당류를 함유하고 있어 이러한 성분은 면역기능을 향상시키는데 중요한 역할을 한다고 보고된 바 있다.The main components of the thorn ogapi are eleutherosides, and seven major eleuterosides have been identified, among which eleuterosides B and E have been intensively studied. Eleutheside B acts as sedation, insomnia, forgetfulness, gonadotropin, lowering blood pressure, autonomic nerve adjustment, improving glucose metabolism, preventing complications of diabetes, and improving liver function. It is known to act as anti-stress, gonadotropin, learning improvement, endurance, stamina, concentration, antirheumatic. In addition, it has been reported that these components play an important role in improving immune function because the bar contains complex polysaccharides.
이와 같은 효능을 갖는 가시오가피는 열수추출하여 얻어진 추출액이 이미 건강보조식품이나 약학제와 같이 다양한 형태로 제품화되고 있으나, 이러한 가시오가피의 용도는 대부분 인체용 약제성분으로 사용되고 있는 바, 그 사용용도가 매우 한정되어 있었다.Prickly Ogapi, which has such efficacy, has been commercialized in various forms, such as health supplements and pharmaceuticals, but the extracts obtained by hot water extraction are mostly used as pharmaceutical ingredients for humans. It was.
이에 본 발명자는 인체용으로 생산되는 가시오가피 추출물을 양식어류의 사료에 첨가하고 양식어류의 비특이적인 면역능과 실제 항병력을 조사한 결과 뛰어난 효과가 있음을 확인하고 본 발명을 완성하기에 이르렀다.Therefore, the present inventors added the thorn stem extract produced for the human body to the feed of farmed fish and examined the non-specific immunity and actual anti-history of the farmed fish, and confirmed that there was an excellent effect and completed the present invention.
따라서 본 발명은 상기와 같은 문제점을 해결하기 위한 것으로, 건강보조식품이나 약학제로 생산되는 가시오가피의 추출물을 양식어류의 사료에 첨가하여 사용함으로서, 양식어류의 비특이적인 면역능과 실제 항병력을 향상시킬 수 있는 어류양식용 사료를 제공하는 것을 목적으로 한다.Therefore, the present invention is to solve the problems as described above, by using the extract of the thorn ogapi produced as a dietary supplement or pharmaceutical supplement to the feed of farmed fish, it is possible to improve the non-specific immunity of the farmed fish and the actual anti-history The purpose is to provide fish feed.
상기와 같은 목적을 달성하기 위하여 본 발명은,The present invention to achieve the above object,
가시오가피를 열수추출하여 얻어진 추출물을 양식어류의 사료 첨가제로 사용하는 것을 특징으로 하는 가시오가피 추출물을 이용한 어류양식용 사료를 제공함으로서 달성된다.It is achieved by providing a feed for fish farming using the barbed oak extract, characterized in that the extract obtained by hydrothermal extraction of the barbed oak skin is used as a feed additive for farmed fish.
이하에서는 본 발명에 대하여 좀 더 상세하게 설명하기로 한다.Hereinafter, the present invention will be described in more detail.
먼저 가시오가피는 7개의 주요 엘레우테로사이드(eleutherosides)로 구성되어 있으며, 그 중 엘레우테로사이드 B는 진정, 불면증, 건망증, 성선 자극, 혈압강하, 자율신경 조정, 당뇨병의 당대사 개선, 당뇨병의 합병증 예방, 간기능 개선 등의 작용을 하고, 엘레우테로사이드 E는 항피로, 항스트레스, 성선자극, 학습향상, 지구력, 스태미너, 집중력, 항류머티스 등의 작용을 할 뿐만 아니라 가시오가피는 복합다당류를 함유하고 있어 면역기능을 향상시키는데 중요한 역할을 한다고 앞에서 밝힌 바 있다.First of all, googapi consists of seven major eleutherosides, among which eleuteroside B is sedation, insomnia, forgetfulness, gonadotropin, lowering blood pressure, autonomic nervous system, improving diabetes metabolism, diabetes It prevents complications and improves liver function, and Eleutheside E not only acts as anti-fatigue, antistress, gonadotropin, learning improvement, endurance, stamina, concentration, antirheumatic, but also polysaccharides. As it contains, it has played an important role in improving immune function.
이와 같은 가시오가피를 선별하여 깨끗이 세척하고 건조한 후, 10배량의 정제수와 함께 밀폐형 고압추출기에 넣고 2기압, 100 ~ 110℃의 온도에서 24시간동안 열수추출하여 가시오가피 추출물을 얻게 된다. 또한, 상기와 같이 1차 추출하고 남은 가시오가피 부산물을 다시 6배량의 물과 함께 고압추출기에 넣고 상술한 방법과 동일한 조건에서 다시 재추출하여 2차 추출물을 얻게 된다.After sorting the thorny skin such as washed and dried, and put into a sealed high-pressure extractor with 10 times the purified water and 2 atm, hot water extraction for 24 hours at a temperature of 100 ~ 110 ℃ to obtain the thorny ivy extract. In addition, as the above-mentioned after extracting the primary by-products of the remaining oogapi by 6 times the amount of water in the high-pressure extractor again and re-extracted under the same conditions as described above to obtain a second extract.
이와 같이 가시오가피의 1차 추출물과 2차 추출물을 혼합하고 냉장보관하면서 어류양식의 사료에 첨가제로 혼합사용함으로서 양식어류의 비특이적인 면역능과 실제 항병력을 향상시킬 수 있음을 실험적으로 확인하였다.Thus, by mixing and refrigerated the primary and secondary extracts of prickly thorns and using them as an additive to the feed of fish culture experimentally confirmed that the non-specific immunity of the cultured fish and the actual anti-history can be improved.
또한, 상기한 가시오가피를 열수추출시 두충, 대추, 감초, 당귀 등과 같은 한약재를 가시오가피에 대해 1 ~ 10중량% 더 첨가하게 되면 좀 더 바람직한 효과를 가져오게 되는데, 이는 상기한 한약재의 약효성분은 혈액순환개선, 해독작용에 탁월한 효과가 있는 것으로 알려져 있으므로 어류의 면역능과 항병력 향상에 도움을 줄 수 있기 때문이다.In addition, when adding 1 ~ 10% by weight of the medicinal herbs, such as worms, jujube, licorice, Angelica, etc. during the hot water extraction of the thorny oakpi, the medicinal ingredients of the medicinal herbs are blood It is known that it has an excellent effect on improving circulation and detoxification, so it can help to improve immunity and anti-competence of fish.
이와 같이 재추출된 가시오가피 추출액은 사료에 대하여 1 ~ 3중량%가 포함되도록 하는 것이 바람직한데, 이는 상기 가시오가피 추출액이 1중량% 미만으로 첨가되면 그 양이 너무 적어 본 발명이 기대하는 어류의 면역능과 항병력 향상을 얻을 수 없으며, 반대로 가시오가피 추출액을 3중량%를 초과하여 첨가하게 되면 어류의 면역능과 항병력에 대한 향상은 미비하게 나타나며 오히려 감소하는 경향을 보임을 확인하였다.Thus, the re-extracted thorny oocyte extract is preferably included in an amount of 1 to 3% by weight based on the feed. If the thorny oocyte extract is added in an amount of less than 1% by weight, the amount is too small, and the immunity of the fish expected by the present invention It was confirmed that the anti-history improvement could not be obtained. On the contrary, the addition of more than 3% by weight of the extracts showed that the immunity and anti-history of fish were insignificant and tended to decrease.
이하 본 발명을 하기한 실시예를 통하여 보다 상세하게 설명하기는 하나 이는 본 발명의 이해를 돕기 위하여 제시된 것일 뿐, 본 발명이 하기의 실시예 만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples, which are only presented to aid the understanding of the present invention, and the present invention is not limited to the following examples.
<실시예 1><Example 1>
생물학적 여과조와 포말분리기가 설치되어 있는 순환여과식 사육시설내 700ℓ사육조에 평균체중이 약 60g 되는 넙치 육성어 50마리를 각각 수용하고, 이와 더불어 동일한 700ℓ사육조에 평균체중이 약 3.2g의 넙치 치어를 각각 수용하였다. In a circulating filtration breeding facility equipped with a biological filtration tank and a foam separator, 50 flounder larvae with an average weight of about 60 grams are housed, respectively.In addition, larvae with a mean weight of 3.2 grams are placed in the same 700 ℓ breeding tank. Each was accommodated.
실험군으로 중량비로 가시오가피 90%와 두충 3.5%, 대추 3.5%, 감초 1.5%, 당귀 1.5%로 구성된 혼합물을 10배량의 정제수와 함께 밀폐형 고압추출기에 넣고 2기압, 110℃의 온도에서 24시간동안 열수추출하여 얻어진 가시오가피 추출물과 상기와 같이 1차 추출하고 남은 가시오가피 부산물을 다시 6배량의 물과 함께 고압추출기에 넣고 상술한 방법과 동일한 조건에서 다시 재추출하여 얻어진 2차 추출물을 혼합하여 냉장보관하면서 사료공급 1시간 전에 사료에 각각 3%와 10%씩 흡착시킨 다음, 오전과 오후에 1일 2회 반복공급하면서 넙치 육성어와 치어를 8주간 실험사육하였다.In the experimental group, a mixture consisting of 90% of thorns, 3.5% of jujube, 3.5% of jujube, 1.5% of licorice, and 1.5% of Angelica was added to a closed high-pressure extractor with 10 times of purified water and heated for 24 hours at a temperature of 2 atmospheres and 110 ℃. After extracting the thorny opi extract obtained by extraction and the remaining thorny opi by-products as described above, the mixture was re-extracted in the high-pressure extractor with 6 times the amount of water and re-extracted again under the same conditions as described above. One hour before feeding, the feed was adsorbed by 3% and 10%, respectively, and the flounder grown and fry were fed for 8 weeks while feeding twice daily in the morning and afternoon.
또한, 대조군으로서 실험군과 동일한 조건의 넙치 육성어와 치어에 사료공급 1시간 전에 10%의 증류수를 흡착시킨 사료를 실험군과 동일한 방법으로 8주간 실험사육하였다. In addition, as a control group, the feed was adsorbed with 10% distilled water 1 hour before feeding to flounder larvae and fry under the same conditions as the experimental group, and was fed for 8 weeks in the same manner as the experimental group.
본 실험에 사용된 사료는 중량비로 조단백질 48%, 조지방 8%, 탄수화물 38%, 비타민 premix 3%, 미네랄 premix 3%가 혼합된 것을 사용하였다.For the feed used in this experiment, 48% crude protein, 8% crude fat, 38% carbohydrate, 3% vitamin premix and 3% mineral premix were used.
<실험예 1>Experimental Example 1
실시예 1의 실험군과 대조군의 넙치를 8주간 사육한 후 하기 수학식 1을 통해 사료계수를 측정하여 첨부된 도 1에 나타내었다.After raising the flounder of the experimental group and the control group of Example 1 for 8 weeks, the feed coefficient was measured through Equation 1 and shown in FIG. 1.
그 결과, 하기 도 1에서와 같이 가시오가피 추출액이 흡착된 사료를 사용한 실험군 중에서 10% 첨가구의 사료계수가 1.17로 1.05의 사료계수를 나타낸 대조구에 비해 다소 높았으나, 3% 및 10% 실험구 모두 대조구와 비교하여 유의하게 차이가 없음을 확인할 수 있었다.As a result, as shown in FIG. 1, the feed coefficient of the 10% addition group was 1.17, which was slightly higher than that of the control group which showed the feed coefficient of 1.05, but both 3% and 10% of the control group were used. Compared with, it was confirmed that there is no significant difference.
<실험예 2>Experimental Example 2
-혈청 라이소자임의 활성조사-Serum Lysozyme Activity Study
실시예 1의 실험군과 대조군의 넙치에 대하여 Parry et al. (1995)의 turbidimetric method를 이용하여 혈청 라이소자임(lysozyme) 활성을 측정하였다. 즉, Micrococcus lysodeikticus (0.2㎎/㎖) 현탁액 950㎕와 혈청 50㎕를 혼합하여 25℃에서 30초 및 4분 30초간 반응시킨 후 530nm에서 흡광도를 측정하여 첨부된 도 2에 나타내었다. 라이소자임 활성은 units/㎖로 나타내었으며, 1unit는 흡광도 값이 0.001 감소한 값으로 표시하였다.For the flounder of the experimental group and the control group of Example 1 Parry et al. Serum lysozyme activity was measured using the turbidimetric method of (1995). That is, 950 μl of Micrococcus lysodeikticus (0.2 mg / ml) suspension and 50 μl of serum were mixed and reacted at 25 ° C. for 30 seconds and 4 minutes 30 seconds, and the absorbance at 530 nm was measured and shown in FIG. 2. Lysozyme activity was expressed in units / ml, and 1 unit was expressed with a 0.001 decrease in absorbance.
그 결과, 하기 도 2와 같이 가시오가피 추출액이 흡착된 사료를 사용한 실험군의 혈청 라이소자임은 무처리된 대조군과 비교하여 첨가 사료 투여구에서 높게 나타났으며, 특히 실험군에서도 3% 첨가구는 4주와 8주 모두 유의하게 높은 증가를 보였고, 최고치는 4주째에 나타났다. 점액 lysozyme은 8주째에 3% 첨가구에서만 유의적인 증가를 보임을 확인할 수 있었다. As a result, the serum lysozyme of the experimental group using the feed adsorbed prickly ginseng extract as shown in Figure 2 was higher in the added feed administration compared to the untreated control, especially in the experimental group 3% added 4 weeks and 8 weeks All showed significantly higher gains, with a peak at 4 weeks. Mucus lysozyme showed a significant increase only at 3% addition at 8 weeks.
<실험예 3>Experimental Example 3
-식세포의 활성산소 측정-Determination of free radicals in phagocytes
실시예 1의 실험군과 대조군에 대한 넙치의 두신을 무균적으로 분리하여 2% fetal calf serum(FCS), 1 % penicillin/streptomycin 및 0.2% heparin이 함유된 L-15 medium에서 nylon mesh를 통과시켜 세포 현탁액을 준비하고, 상기 세포 현탁액을 각각 27%, 36%, 45% 및 54% 농도의 Percoll에 중층시킨 후 450g에서 30분간 원심 분리하여 백혈구를 분리하였다. 이와 같이 분리된 백혈구는 0.1% tryphan blue로 viability를 확인한 후 2×106 cells/㎖ 농도로 조정하여 96 well tissue culture plate에 분주한 다음, 18℃에서 2시간 부착시켜 상층액을 제거한 후 3회 세척하여 식세포를 준비하였다. 또한, FCS로 옵소닌화된 zymosan을 nitroblue tetrazolium(NBT) 용액에 현탁시킨 후 식세포가 부착된 well에 100㎕씩 첨가하고 18℃에서 30분간 반응시킨 다음 100% methanol로 세포를 고정시킨 다음, 각 well에 2M KOH 용액 120㎕와 dimethyl sulphoxide(DMSO) 140㎕를 첨가한 후 630nm에서 흡광도를 측정하여 첨부된 도 3에 나타내었다. 이 때 blank는 2M KOH와 DMSO의 혼합액을 사용하였다.Aseptic isolation of the head of the flounder for the experimental group and the control group of Example 1 was passed through a nylon mesh in L-15 medium containing 2% fetal calf serum (FCS), 1% penicillin / streptomycin and 0.2% heparin The suspension was prepared and the cell suspension was layered in Percoll at 27%, 36%, 45% and 54% concentrations, respectively, and centrifuged at 450 g for 30 minutes to separate leukocytes. The leukocytes separated in this way were checked for viability with 0.1% tryphan blue, adjusted to a concentration of 2 × 10 6 cells / ml, and dispensed into 96 well tissue culture plates, and then attached at 18 ° C. for 2 hours to remove supernatant. Washing prepared phagocytes. In addition, zymosan opsonized with FCS was suspended in nitroblue tetrazolium (NBT) solution, and then 100 μl of each well was added to the wells to which phagocytes were attached, reacted at 18 ° C. for 30 minutes, and the cells were fixed with 100% methanol. 120 μl of 2M KOH solution and 140 μl of dimethyl sulphoxide (DMSO) were added to the wells, and the absorbance was measured at 630 nm. In this case, a blank mixture of 2M KOH and DMSO was used.
그 결과, 하기 도 3과 같이 가시오가피 추출액이 흡착된 사료를 사용한 실험군의 3% 첨가구에서는 시간이 소요될수록 활성산소가 증가되었으며, 특히 8주째에 매우 높은 활성을 나타내었다. 그러나 10% 첨가구에서는 오히려 활성이 감소하였고, 8주째에는 오히려 무처리된 대조군 보다 유의하게 감소한 결과를 보임을 확인할 수 있었다.As a result, in the 3% addition of the experimental group using the feed adsorbed prickly pear extract as shown in Figure 3, the active oxygen increased with time, especially showed a very high activity at 8 weeks. However, the activity was decreased in the 10% added group, and at 8 weeks, the activity was significantly reduced than the untreated control group.
<실험예 4>Experimental Example 4
-식세포의 식균능 조사-Phagocytosis of Phagocytes
실시예 1의 실험군과 대조군에 대하여 넙치의 미부정맥을 헤파린 처리된 주사기로 0.25㎖를 채혈한 후, 실리콘 처리된 유리시험관에 옮기고 Escherichia coli formalin killed cell(FKC) 부유액 0.025㎖를 넣어 27℃에서 반응시킨 다음. 상기 반응 3시간 후, 반응시킨 혈액을 슬라이드 글라스에 도말하여 May-Giemsa 염색법으로 염색하였다. 식작용은 식세포 100개에 대한 식균율과 식균지수을 하기 수학식 2와 3을 통해 측정하여 첨부된 도 4에 나타내었다. 단, 식포를 형성한 식세포라도 식포 내에 세균이 관찰되지 않으면 식작용을 하지 않은 것으로 하였다.For the experimental group and the control group of Example 1, 0.25 ml of the flounder was collected by a heparinized syringe, and then transferred to a siliconized glass test tube, and 0.025 ml of Escherichia coli formalin killed cell (FKC) suspension was added and reacted at 27 ° C. Then. After 3 hours, the reacted blood was plated on slide glass and stained by May-Giemsa staining. Phagocytosis is shown in Figure 4 attached to measure the phagocytosis rate and phagocytosis index for 100 phagocytes through the following equations (2) and (3). However, even if phagocytic cells formed phenotype were not observed in bacteria, phagocytosis was not performed.
그 결과, 하기 도 4와 같이 가시오가피 추출액이 흡착된 실험군 중에서 3% 첨가구의 경우, 식균율과 식균지수 모두 대조구에 비해 현저히 증가하였고 (p < 0.05), 특히 8주째에는 최고치를 보였다. 그러나 실험군 중에서 10% 첨가구의 경우는 식세포 한 개당 식작용된 세균의 수를 의미하는 식균지수는 유의하게 증가하였으나, 전체 식세포 중 식작용을 일으킨 식세포 수의 비율을 의미하는 식균율에서의 변화는 일어나지 않음을 확인할 수 있었다.As a result, as shown in Figure 4, in the experimental group adsorbed prickly pear extract, 3% addition group, both the phagocytosis rate and the phagocytosis index increased significantly compared to the control (p <0.05), especially at 8 weeks. However, in the 10% addition group of the experimental group, the phagocytosis index, which means the number of phagocytosis bacteria per phagocytosis, increased significantly, but there was no change in phagocytosis, which means the ratio of phagocytosis that caused phagocytosis among all phagocytes. I could confirm it.
<실험예 5>Experimental Example 5
-혈액세포 수의 변화 조사-Investigation of changes in blood cell count
실시예 1의 실험군과 대조군에 대한 넙치의 혈액세포의 수적 변화를 혈액 도말 표본법으로 조사하였다. 즉, 넙치의 미부정맥에서 채혈한 혈액을 곧바로 슬라이드 글라스에 도말하여, May-Giemsa 염색법으로 염색한 후 광학 현미경으로 관찰하면서 적혈구 5,000세포당 임파구, 전구, 그리고 호중구의 수를 계수하여 그 결과를 첨부된 도 5에서 나타내었다. The numerical changes of blood cells of the olive flounder for the experimental group and the control group of Example 1 were examined by blood smear sampling method. In other words, blood collected from the arrhythmia of the flounder was immediately smeared on slide glass, stained with May-Giemsa staining, and observed under an optical microscope, and counted the number of lymphocytes, bulbs, and neutrophils per 5,000 red blood cells. 5 is shown.
그 결과, 하기 도 5에서와 같이 가시오가피 추출액이 흡착된 사료를 사용한 실험군을 무처리된 대조군과 비교하여 임파구와 전구는 크게 유의차를 보이지 않았으나, 호중구는 현저하게 증가하는 것으로 나타났다. 다만 실험군 중에서 3% 첨가구는 4주와 8주 모두에서 유의한 증가를 보였으나, 10% 첨가구는 4주째에만 유의적인 증가를 보이다가 8주째에는 오히려 감소하는 것으로 나타났다. 최고치는 8주째 3% 첨가구에서 관찰되었다.As a result, the experimental group using the feed adsorbed prickly pear extract as shown in FIG. 5 showed no significant difference between lymphocytes and progenitors, but neutrophils were significantly increased. In the experimental group, the 3% group showed a significant increase in both 4 and 8 weeks, but the 10% group showed a significant increase only in the 4th week and then decreased in the 8th week. Peaks were observed at 3% addition at 8 weeks.
<실험예 6>Experimental Example 6
-병원세균의 공격실험--Attack test of hospital bacteria-
실시예 1의 실험군과 대조군의 넙치에 대하여 어병 세균인 Edwardsiella tarda, FSW910410과 Vibrio anguillarum으로 공격 실험을 한 후 생존율을 구하였다. 실험에 사용한 세균은 어체 통과를 2회 실시한 후, TSA 배지에서 재분리된 집락을 24시간째에 수집하여 사용하였다.Survival rate was determined after the challenge experiment with fish disease bacteria Edwardsiella tarda, FSW910410 and Vibrio anguillarum on the flounder of the experimental group and the control group of Example 1. The bacteria used in the experiment were subjected to two passages of the fish body, and the colonies re-separated from TSA medium were collected and used at 24 hours.
상기 실험은 넙치 치어와 성어 두 그룹으로 나누어 실시하였고, 각 실험에 사용된 넙치의 수는 20마리씩 사용하였다. 먼저, 넙치 치어의 공격 실험은 먼저 E. tarda와 V. anguillarum이 4×1010/2ℓ의 농도로 현탁된 3ℓ삼각플라스크 2개에 10 마리씩 나누어 넣고, 이를 1시간 동안 방치한 후 현탁액을 깨끗한 해수로 교체하고 7일간 폐사율을 관찰하여 첨부된 도 6에 나타내었다. 또한, 넙치 성어의 공격 실험은 먼저 100ℓ 원형 FRP수조 2개에 10마리씩 나누어 넣고, 각각의 세균이 2×108/㎖로 준비된 균액을 0.1㎖씩 어체에 복강 주사하면서 7일간 폐사율을 측정하여 첨부된 도 7에 나타내었다. 이 때 공격 실험 중 사육수온은 26℃를 유지하였고, 사육수는 1일 2회 절반씩 교체하였다.The experiment was divided into two groups of flounder fry and eel, and the number of flounder used in each experiment was 20 animals. First, the halibut fry attack experiments are first and E. tarda V. anguillarum is 4 × 10 10 / suspended at a concentration of 3ℓ 2ℓ Erlenmeyer flask 2 divided into 10 rats in dogs, this pure water and the suspension was allowed to stand for an hour Replaced with and observed mortality for 7 days is shown in Figure 6 attached. In addition, an attack experiment of the flounder tuna was first divided into two 100 l round FRP tanks, and the mortality was measured for 7 days while intraperitoneally injecting 0.1 ml of the bacterial solution prepared for each bacteria into 2 × 10 8 / ml. 7 is shown. At this time, the breeding water temperature was maintained at 26 ℃ during the attack experiment, the breeding water was replaced by half twice a day.
그 결과, 하기 도 6에서와 같이 넙치 치어의 Edwardsiella tarda에 대한 방어력은 가시오가피 추출액이 흡착된 사료를 사용한 실험군인 3%와 10% 첨가구 둘 다 증가율을 나타냈으며, 특히 3% 첨가구에서의 방어력이 현저히 높은 것으로 관찰되었다. Vibrio anguillarum에 대한 방어력에 대해서도 비슷한 결과를 보이지만, 역시 3% 첨가구의 방어력이 대조구에 비해 매우 높은 것으로 나타났다.As a result, as shown in Figure 6 below, the protection against the flounder larvae Edwardsiella tarda showed an increase rate of both 3% and 10% added group, which was the experimental group using the feed adsorbed spinach extract, especially the 3% added group This was observed to be significantly higher. Similar results were found for defense against Vibrio anguillarum , but again The 3% added group showed much higher protection than the control group.
또한, 하기 도 7에서와 같이 넙치 성어의 Edwardsiella tarda에 대한 방어력은 3% 첨가구가 다소 증가한 것으로 나타났으나, 10% 첨가구에서는 대조구와 그다지 차이를 보이지 않았다. Vibrio anguillarum에 대한 방어력에 대해서도 3% 첨가구의 넙치는 방어력의 증가를 보였으나, 10% 첨가구에서는 오히려 대조구보다 낮은 방어력을 보이는 것으로 관찰되었다.In addition, as shown in FIG. 7 below, the protection of the halibut sperm Edwardsiella tarda was slightly increased by 3% addition, but did not show much difference with the control in 10% addition. On defense against Vibrio anguillarum The flounder of the 3% added group showed an increase in defensiveness, but the 10% added flounder showed a lower protective effect than the control group.
상술한 바와 같이 본 발명의 가시오가피 추출물을 이용한 어류사료용 사료는 건강보조식품이나 약학제로 생산되는 가시오가피를 재추출하여 양식어류의 사료에 첨제로 사용함으로서, 양식어류의 체액성 및 세포성 비특이적 면역을 자극하여 면역기능이 개선됨과 동시에 실제 병원세균의 감염실험을 실시한 결과에서 항병력이 증가되는 것으로 확인할 수 있었으며, 이는 그동안 재활용하지 못한 가시오가피 추출 부산물을 이용하여 해양생물용 면역기능 개선제로 개발이 가능하다는 효과를 가져오는 것이다. As described above, the feed for fish feed using the thorny ogapi extract of the present invention re-extracts the thorny opis produced as a dietary supplement or pharmaceuticals and is used as an additive to the feed of farmed fish, thereby stimulating humoral and cellular nonspecific immunity of cultured fish As a result of the improvement of immune function, the result of the actual infection test of the pathogen was confirmed that the anti-history was increased, which can be developed as an immune function improving agent for marine organisms by using the by-products of thorny skin extract that have not been recycled. To bring.
또한, 본 발명은 각종 수산약품 및 항생제를 사용하지 않고 식품안정성이 확보된 상태에서 어체의 면역기능을 강화시킬 수 있으며, 따라서 수산양식산업의 경제성을 크게 향상시킬 수 있고 아울러 생산성과 상품성에서도 좋은 효과를 가져온다는 것이다.In addition, the present invention can enhance the immune function of the fish body in the state of food stability without using various aquatic medicines and antibiotics, and thus can greatly improve the economics of the aquaculture industry, and also good effect on productivity and commerciality Will bring.
도 1은 본 발명의 실험예에 대한 넙치의 사료계수를 측정한 도면1 is a view measuring the feed coefficient of the flounder for the experimental example of the present invention
도 2는 본 발명의 실험예에 대한 넙치의 혈청 라이소자임의 활성을 측정한 도면2 is a diagram measuring the activity of serum lysozyme of the olive flounder for the experimental example of the present invention
도 3은 본 발명의 실험예에 대한 넙치 식세포의 활성산소를 측정한 도면Figure 3 is a measure of the free radicals of halibut phagocytes for the experimental example of the present invention
도 4는 본 발명의 실험예에 대한 넙치의 식세포의 식균능을 측정한 도면4 is a view measuring the phagocytosis of phagocytic cells of the olive flounder for the experimental example of the present invention
도 5는 본 발명의 실험예에 대한 넙치의 혈액세포 수의 변화를 측정한 도면5 is a view measuring the change in the number of blood cells of the flounder for the experimental example of the present invention
도 6은 본 발명의 실험예에 대한 넙치 치어의 병원세균의 공격성을 측정한 도면Figure 6 is a measure of the aggression of pathogens of halibut fry for the experimental example of the present invention
도 7은 본 발명의 실험예에 대한 넙치 성어의 병원세균의 공격실험을 측정한 도면7 is a view measuring the attack experiment of the pathogen of the flatfish in the experimental example of the present invention
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100861456B1 (en) * | 2007-08-23 | 2008-10-02 | 안종만 | Feed Additives Using Wild Plants and Manufacturing Method Thereof |
| KR101039620B1 (en) * | 2009-05-25 | 2011-06-09 | 농업회사법인 제우 주식회사 | Liquid Feed Additives for Aquaculture Flounder |
| CN103070929A (en) * | 2013-02-01 | 2013-05-01 | 中国水产科学研究院黑龙江水产研究所 | Immunopotentiator for oncorhynchus masou masou |
| CN103549149A (en) * | 2013-10-31 | 2014-02-05 | 王馨洁 | Freshwater fish phagostimulant |
| KR102389270B1 (en) * | 2021-08-26 | 2022-04-22 | 주식회사 코팩스 | Feed additive for eels culture comprising Eucommia ulmoides extract as effective component and uses thereof |
-
2004
- 2004-06-26 KR KR1020040048702A patent/KR20050123002A/en not_active Ceased
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100861456B1 (en) * | 2007-08-23 | 2008-10-02 | 안종만 | Feed Additives Using Wild Plants and Manufacturing Method Thereof |
| KR101039620B1 (en) * | 2009-05-25 | 2011-06-09 | 농업회사법인 제우 주식회사 | Liquid Feed Additives for Aquaculture Flounder |
| CN103070929A (en) * | 2013-02-01 | 2013-05-01 | 中国水产科学研究院黑龙江水产研究所 | Immunopotentiator for oncorhynchus masou masou |
| CN103549149A (en) * | 2013-10-31 | 2014-02-05 | 王馨洁 | Freshwater fish phagostimulant |
| KR102389270B1 (en) * | 2021-08-26 | 2022-04-22 | 주식회사 코팩스 | Feed additive for eels culture comprising Eucommia ulmoides extract as effective component and uses thereof |
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