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4種豆粕替代魚粉對大黃魚生長、抗氧化及抗菌能力的影響

2016-03-06 02:03陳乃松華雪銘黃旭雄陳曉明朱偉星
海洋漁業(yè) 2016年5期
關(guān)鍵詞:大黃魚魚粉豆粕

吳 釗,陳乃松,華雪銘,黃旭雄,陳曉明,王 壇,王 剛,朱偉星,孔 純

(上海海洋大學農(nóng)業(yè)部水產(chǎn)種質(zhì)資源與利用重點開放實驗室,上海 201306)

4種豆粕替代魚粉對大黃魚生長、
抗氧化及抗菌能力的影響

吳 釗,陳乃松,華雪銘,黃旭雄,陳曉明,王 壇,王 剛,朱偉星,孔 純

(上海海洋大學農(nóng)業(yè)部水產(chǎn)種質(zhì)資源與利用重點開放實驗室,上海 201306)

分別用9種等氮等能的飼料投喂初始體質(zhì)量為(34.72±0.28)g的大黃魚(Pseudosciaena crocea)。其中1組投喂對照飼料(含50%魚粉,不含豆粕),另外8個試驗組分別投飼由去皮豆粕(DSM)、酶解豆粕(ESM)、發(fā)酵豆粕Ⅰ(FSMⅠ)和發(fā)酵豆粕Ⅱ(FSMⅡ)替代20%和40%的魚粉的飼料,9組分別命名為FM、DSM20、DSM40、ESM20、ESM40、FSMⅠ20、FSMⅠ40、FSMⅡ20、FSMⅡ40。在海水浮式網(wǎng)箱中進行7周的養(yǎng)殖實驗后,評定4種豆粕替代魚粉的可行性及適宜替代水平。結(jié)果顯示,試驗組與對照組魚存活率和特定生長率無顯著差異(P>0.05)。血清生化指標顯示,F(xiàn)M組和FSMⅡ20組超氧化物歧化酶(SOD)活性顯著高于其它試驗組(P<0.05),F(xiàn)M、DSM20、FSMⅠ40、FSMⅡ20組過氧化氫酶(CAT)活性顯著高于DSM40、ESM20、ESM40及FSMⅠ20組(P<0.05),不同試驗組的丙二醛(MDA)含量均不同程度高于對照組。酶解豆粕替代40%魚粉導致實驗魚的血清對哈維氏弧菌的抵抗能力下降,去皮豆粕替代20%魚粉導致血清對溶藻弧菌抵抗能力下降;但發(fā)酵豆粕不影響血清及黏液對3種菌的抵抗能力。研究表明,以特定生長率、飼料轉(zhuǎn)化率和抗菌能力為評價指標,發(fā)酵豆粕是魚粉的最佳替代源,發(fā)酵豆粕Ⅰ和Ⅱ均能替代20%~40%的魚粉,但存在抗氧化能力下降的風險,尤其是發(fā)酵豆粕Ⅰ40%替代組;去皮豆粕和酶解豆粕替代魚粉在抗菌能力和抗氧化能力方面無優(yōu)勢。

大黃魚;魚粉;豆粕;抗氧化能力;抗菌能力

魚粉因具有蛋白質(zhì)含量高、水產(chǎn)動物所必需的氨基酸豐富、易被水產(chǎn)動物消化吸收等優(yōu)點,在水產(chǎn)飼料中被廣泛應用。然而,隨著飼料行業(yè)的快速發(fā)展,導致魚粉供不應求,價格飚升[1]。目前,世界魚粉的供應已經(jīng)不能滿足日益增長的飼料業(yè)的需求,因此,尋找魚粉替代源成為國內(nèi)外研究的熱點。

植物性蛋白源因其價格低廉且供應穩(wěn)定,備受研究者的關(guān)注。其中,大豆蛋白源具有消化吸收率高、氨基酸組成較好、價格合理和資源量豐富等特點,是水產(chǎn)飼料應用最多的植物蛋白源之一。但是,大豆蛋白源在魚類飼料中過量使用,不僅影響魚類的攝食和生長[2-4],而且影響魚的健康和免疫功能[5-8]。其主要原因在于適口性不佳[9-11]、抗營養(yǎng)因子多[12-14]和氨基酸不平衡[15]等。通過使用復合植物蛋白源[16]、添加酶制劑[17]、微生物發(fā)酵[18-19]及添加晶體氨基酸[20-21]等方法可以緩解以上問題。

大黃魚(Pseudosciaena crocea),屬鱸形目,石首魚科,黃魚屬,又名黃魚、黃瓜魚,為我國特有的地方性種類,主要分布在福建和浙江沿海地區(qū),因其生長迅速、肉質(zhì)鮮美而深受消費者的青睞。研究表明,0.57 g的大黃魚飼料中所需蛋白質(zhì)水平在47%以上[22],且飼料的蛋白質(zhì)主要來源于魚粉。鑒于目前魚粉的供求矛盾和價格情況,尋找魚粉的替代源成為大黃魚配合飼料研制中需要重點解決的問題之一。目前,大黃魚飼料中魚粉替代的研究已有報道[23-25],主要集中在蛋白源選擇以及替代水平方面,涉及肉骨粉、各種植物蛋白及復合蛋白等,但尚未見幾種不同加工工藝的豆粕對魚粉替代效果的比較研究。另有研究表明,添加晶體氨基酸可以緩解豆粕中氨基酸不平衡;去皮可以消除種皮中的大部分單寧;酶解可以消除豆粕中的植酸以及大豆低聚糖等;微生物發(fā)酵可降低豆粕中胰蛋白酶抑制因子、皂甙、植酸等抗營養(yǎng)因子的抗營養(yǎng)效應,從而提高魚類的攝食及生長[26]。因此,本研究擬在添加包膜晶體氨基酸的條件下,用去皮豆粕、酶解豆粕以及兩種不同發(fā)酵程度的豆粕分別替代配方中20%和40%的魚粉,從生長性能、抗氧化以及抗菌能力等方面綜合判斷4種豆粕替代魚粉的可行性,以期為豆粕在大黃魚人工配合飼料中的應用提供參考。

1 材料與方法

1.1 實驗飼料

以魚粉、4種不同豆粕(去皮豆粕、酶解豆粕、發(fā)酵豆粕Ⅰ、發(fā)酵豆粕Ⅱ)、玉米蛋白粉、噴干血球粉以及谷朊粉為主要蛋白源、魚油和大豆磷脂油為主要脂肪源,配制9種等氮(48%)等脂(12%)等無氮浸出物(13%)的實驗飼料。對照組為50%魚粉組,試驗組以4種不同豆粕分別替代20%和40%的魚粉,在替代組中添加蛋氨酸、賴氨酸以及蘇氨酸以確保此3種氨基酸含量與對照組一致。飼料配方及成分分析見表1。

1.2 養(yǎng)殖實驗的設計和飼養(yǎng)管理

養(yǎng)殖實驗于上海農(nóng)好飼料有限公司養(yǎng)殖基地(位于福建省寧德市蕉城區(qū)三都澳海區(qū))的網(wǎng)箱中進行。在開始正式實驗前,將大黃魚放入4 m×4 m×3 m的網(wǎng)箱中暫養(yǎng)、馴化2周。經(jīng)24 h饑餓,挑選體格健壯、大小均勻的大黃魚(34.72 g ±0.28 g)隨機分為9組。每組隨機分配3個浮式海水網(wǎng)箱(長×寬×深:1.2 m×1 m×2 m),每個網(wǎng)箱放養(yǎng)40 ind,每天飽食投喂2次(06∶00和16∶00)。養(yǎng)殖周期為49 d,養(yǎng)殖期間每天記錄投喂量以及死亡情況。

1.3 樣品采集和分析

養(yǎng)殖結(jié)束后,將實驗魚饑餓24 h,從網(wǎng)箱中全部撈起,用丁香酚麻醉后計數(shù)并稱重。分別從每個網(wǎng)箱中隨機取12 ind魚,尾靜脈抽血;將血樣置于4℃冰箱靜置4h后,離心(836×g)10 min,分離取得血清,并于-20℃保存。對抽取血液的6 ind魚取其肝臟,并稱重用于計算肝體比。分離背部側(cè)線上方的肌肉,于-20℃保存用于測定肌肉成分;參考PALAKSHA等[28]的方法,用細胞刮棒在魚背側(cè)面輕輕刮取體表黏液。剩余的魚置于-20℃保存用于體組成分析。

飼料、肌肉、全魚體組成分析參照AOAC(1993)[27]的方法。水分測定在105℃烘箱中烘至恒重;粗灰分在馬弗爐中550℃灼燒測定;粗蛋白質(zhì)采用凱氏定氮法測定;粗脂肪采用氯仿—甲醇法測定。

血清、黏液以及肝臟中超氧化物歧化酶(SOD)、溶菌酶(LZM)、過氧化氫酶(CAT)、丙二醛(MDA)均采用南京建成生物工程研究所試劑盒測定,血清以及組織勻漿中的蛋白濃度采用考馬斯亮藍法測定,血清和肝臟中的谷草轉(zhuǎn)氨酶(AST)、谷丙轉(zhuǎn)氨酶(ALT)采用邁瑞生物醫(yī)療電子股份有限公司試劑盒測定。

血清及黏液抗菌活力測定根據(jù)SUNYER等[29]方法稍作修改。將100μL稀釋的菌液(哈維氏弧菌、溶藻弧菌、嗜水氣單孢菌)加入96孔板,并加入等體積的血清或黏液,另取不加菌作陰性對照,不加血清和黏液作陽性對照,將其置于28℃條件下恒溫孵育24 h,在620 nm波長條件讀取OD值,每2 h讀一次,最后結(jié)果根據(jù)前后兩次穩(wěn)定的OD值來計算。

1.4 計算公式

增重率(WG)(%)=(Wt-W0)/W0×100

特定生長率(SGR)=(LnWt-LnW0)×100/t

攝食量(FI)=Dd/[(N0+Nt)/2]

飼料轉(zhuǎn)化率(FCR)=(Wt-W0)/Dd

肝體比(HSI)(%)=100×WL/Wt

肥滿度(CF)(%)=100×Wt/L3

抗菌活力(ATB)=1-(OD2-OD1)/OD0上述各公式中,Wt、W0分別為實驗大黃魚的終末平均個體質(zhì)量和初始平均個體質(zhì)量;N0、Nt分別為實驗大黃魚的初始尾數(shù)和終末尾數(shù);WL為肝臟濕重;t為實驗天數(shù);L是魚的體長(cm);Dd為總攝食量;OD0、OD1、OD2分別為陽性對照、陰性對照及試驗組OD值。

1.5 數(shù)據(jù)處理與統(tǒng)計分析

采用SPSS 17.0對數(shù)據(jù)進行單因素方差分析(One-way ANOVA),用Duncan氏法進行多重差異顯著性比較,顯著水平P<0.05。

表1 實驗飼料配方及成分分析(%風干物質(zhì))Tab.1 Analysis on composition and proximate of the experimental diets(%air-dry matter)

2 結(jié)果與分析

2.1 不同豆粕替代魚粉對大黃魚生長和存活率的影響

表2顯示,試驗組與對照組在存活率、增重率與特定生長率上無顯著差異(P>0.05)。由表3可見,DSM20組攝食量顯著高于對照組(P<0.05),而其它試驗組攝食量與對照組無顯著差異(P>0.05),且各組飼料轉(zhuǎn)化率無顯著差異(P>0.05)。肝體比以及肥滿度在各組之間同樣無顯著差異(P>0.05)。

表2 不同豆粕替代魚粉試驗組中大黃魚生長和存活狀況Tab.2 Grow th and survival of large yellow croaker in different experimental groups

表3 各組的飼料轉(zhuǎn)化率、攝食量、肝體比以及肥滿度Tab.3 Feed conversion ratio,feed intake,HSI and CF in experimental groups

2.2 不同豆粕替代魚粉對大黃魚體組成的影響

各組魚全魚及肌肉組成見表4。不同豆粕替代魚粉影響大黃魚全魚以及肌肉中的蛋白質(zhì)和脂肪含量,隨著替代水平增加,肌肉和全魚中蛋白含量有下降的趨勢。不同豆粕在40%替代水平時,全魚中蛋白含量顯著低于對照組,而脂肪含量呈相反的趨勢;肌肉中粗蛋白與粗脂肪亦呈類似的趨勢。

2.3 不同豆粕替代魚粉對大黃魚生化指標的影響

如表5和表6所示,血清中FM和FSMⅡ20組魚SOD活性顯著高于其它替代組;而肝臟中SOD活性是在DSM20組和FSMⅡ20組最高。血清中CAT活性同樣是在FSMⅡ20組最高;而肝臟中CAT活性是在FSMⅠ40組最高。血清中溶菌酶活性在ESM40組最低;而血清中MDA在ESM20組和ESM40組最高,肝臟中MDA水平在ESM40組最高。血清中總蛋白(TP)、AST、ALT以及肝臟中AST、ALT在各組之間沒有差異。由表7可知,試驗組魚體表黏液中SOD活性顯著高于對照組,黏液中MDA在酶解豆粕20%替代組顯著高于對照組。

表4 各組全魚及肌肉組成Tab.4 Proximate composition of fish and muscle in experimental groups (%)

表5 各組魚血清生化指標含量Tab.5 Contents of biochemical indexs in serum of fish in experimental groups

表6 各組魚肝臟生化指標含量Tab.6 Contents of biochemical indexs in liver of fish in experimental groups

2.4 不同豆粕替代魚粉對大黃魚血清及黏液抗菌能力的影響

血清及體表黏液對哈維氏弧菌、溶藻弧菌以及嗜水氣單孢菌的抵抗能力如表8。ESM40組血清對哈維氏弧菌抵抗能力顯著低于FM組,其它試驗組與對照組無顯著差異;而DSM20組血清對溶藻弧菌抵抗能力顯著低于FM組,黏液對溶藻弧菌抵抗能力在ESM20組和ESM40組最低。不同組魚血清及黏液對嗜水氣單孢菌的抵抗能力沒有明顯差異。

表7 各組魚黏液-生化指標含量Tab.7 Contents of biochemical mucus in liver of fish in experimental groups

表8 血清及黏液抗菌活力Tab.8 Antimicrobial activity of serum and mucus

3 討論

3.1 不同豆粕替代魚粉對大黃魚生長和存活率的影響

本實驗中試驗組與對照組相比,存活率、增重率與特定生長率均無顯著差異,表明在一定的范圍內(nèi)豆粕對魚粉的替代效果不受替代水平的影響[26]。同樣的實驗在虹鱒(Oncorhynchus mykiss)[30-31]、奧尼羅非魚(Oreochromis niloticus ×O.aureus)[32]上也均顯示用適量的豆粕替代魚粉不會對魚生長造成負面影響。LI等[33]在大黃魚的研究中發(fā)現(xiàn),大黃魚幼魚飼料中豆粕替代魚粉的最適替代比例為30%,而本研究在40%的替代水平下依然不會對大黃魚生長造成負面影響,這可能與本研究所使用豆粕的抗營養(yǎng)因子減少[26,34-35]以及添加復合包膜晶體氨基酸有關(guān)。在虹鱒[36]、紅擬石首魚(Sciaenops ocellatus)[37]的研究中已證實添加晶體氨基酸可以改善豆粕替代魚粉的效果。

3.2 不同豆粕替代魚粉對大黃魚體組成的影響

ZHANG等[24]在大黃魚飼料中用植物蛋白替代魚粉的研究中發(fā)現(xiàn),隨著替代水平的增加,魚體粗蛋白質(zhì)含量出現(xiàn)降低趨勢,而本實驗中也出現(xiàn)相同的趨勢,這可能是由于植物蛋白替代魚粉雖能滿足大黃魚生長所需的蛋白質(zhì)量,但仍存在包膜晶體氨基酸用于合成魚體蛋白質(zhì)的利用率不如蛋白質(zhì)來源的結(jié)合態(tài)氨基酸,游離氨基酸的不同步吸收導致魚體內(nèi)蛋白質(zhì)的合成下降的可能[24,38]。隨著替代比例的增加,魚體肌肉中蛋白含量出現(xiàn)下降,脂肪含量出現(xiàn)上升的趨勢,這與陳乃松等[38]在大口黑鱸(Micropterus salmoides)中的研究結(jié)果一致。

3.3 不同豆粕替代魚粉對大黃魚健康的影響

生物有機體在正常的生理生化反應中需要氧自由基的參與,并通過生物體內(nèi)酶和非酶性抗氧化系統(tǒng)清除過量自由基,維持機體內(nèi)氧化與抗氧化的動態(tài)平衡,這種動態(tài)平衡向氧化移動,則會導致生物有機體的氧化應激損傷[39]??寡趸到y(tǒng)中,SOD是生物體內(nèi)唯一以自由基為底物的酶,負責抑制超氧化物自由基的生成,催化超氧陰離子發(fā)生歧化反應,轉(zhuǎn)變?yōu)镠2O2和O2[40],隨后由CAT催化H2O2分解為H2O和O2[41],從而使細胞免于遭受H2O2的毒害。若多余的活性氧自由基不能被及時清除,則產(chǎn)生大量的脂質(zhì)過氧化產(chǎn)物MDA,引起組織和細胞氧化損傷[42]。本研究中血清SOD和CAT在各組之間有顯著差異,表明大黃魚的抗氧化系統(tǒng)受到豆粕種類和使用量的影響;而肝臟作為機體的代謝中心,粘液作為非特異免疫系統(tǒng)的第一道防線,SOD和CAT在這兩個組織的活性在各組間也有不同程度的變化,說明肝臟和粘液的抗氧化作用也受到豆粕種類和使用量的影響,但平衡氧自由基的表現(xiàn)方式與血液稍有不同。MDA在對照組血清中的含量顯著低于試驗組,說明不同試驗組的魚體內(nèi)抗氧化能力相對弱于對照組。同樣,酶解豆粕20%替代組、酶解豆粕40%替代組及發(fā)酵豆粕Ⅰ40%替代組血清MDA顯著高于其它試驗組,這也說明不同豆粕對大黃魚體內(nèi)抗氧化能力的影響也是有差異的,而這可能與不同工藝豆粕中的抗營養(yǎng)因子的差異相關(guān),具體原因有待進一步分析。由ALT和AST結(jié)果表明,不同豆粕替代魚粉不影響魚體肝臟氨基酸代謝活動,肝功能未受到影響,這可能是由于魚體內(nèi)有天然的抗氧化系統(tǒng)得以維持正常的生理功能[43],但一旦超過魚體自身調(diào)節(jié)的范圍,就會對機體造成損傷。

魚體血清及黏液含有一些抵御病菌的分子[44-45],其抗菌能力是魚體免疫機能強弱最直接的一種表現(xiàn)。本實驗中不同豆粕替代魚粉對大黃魚血清及黏液抵抗嗜水氣單孢菌的能力沒有影響,酶解豆粕替代40%的魚粉會導致大黃魚血清對哈維氏弧菌抵抗力下降,酶解豆粕替代20%或40%魚粉會導致大黃魚體表黏液對溶藻弧菌的抵抗力下降,而兩種發(fā)酵豆粕分別替代20%及40%魚粉均沒有導致大黃魚血清及黏液對3種菌的抵抗能力顯著下降,這可能是由于在發(fā)酵過程中,大豆異黃酮中的葡萄糖苷會轉(zhuǎn)化為葡萄糖苷元,從而使得苷元成分大增,因此抗菌性能提高[46]。以上結(jié)果說明,大黃魚血清及黏液的抗菌能力因病原菌種類而異,SANGEETHA等[47]發(fā)現(xiàn)河鱒(Salvelinus alpinus)、黑線鱈(Melanogrammus aeglefinus)、黏液鰻(Myxineglutinosa)的酸性黏液提取物對魚和人類的病原具有較強的特殊抗菌活性,這與魚體表黏液中的絲氨酸和半胱氨酸蛋白水解酶類溶解寄生物有關(guān)[48-49]。

4 小結(jié)

本研究表明,以特定生長率、飼料轉(zhuǎn)化率和抗菌能力為評價指標,發(fā)酵豆粕Ⅰ和Ⅱ能替代20%~40%的魚粉,但存在抗氧化能力下降的風險,尤其是發(fā)酵豆粕Ⅰ40%替代組;去皮豆粕和酶解豆粕替代魚粉在抗菌能力和抗氧化能力方面均無優(yōu)勢;4種豆粕替代魚粉,全魚和肌肉的蛋白質(zhì)含量、脂肪含量分別呈下降和上升趨勢。

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Effects of replacement of dietary fish meal by four kinds of soybean meal on grow th,antioxidant and antibacterial ability of Pseudosciaena crocea R.

WU Zhao,CHEN Nai-song,HUA Xue-ming,HUANG Xu-xiong,CHEN Xiao-ming,WANG Tan,WANG Gang,ZHU Wei-xing,KONG Chun
(Key Laboratory of Freshwater Fishery Germplasm Resources,Ministry of Agriculture,Shanghai Ocean University,Shanghai 201306)

Due to the high protein content and rich essential amino acids for aquatic animals,fish meal has been widely used in aquaculture feed industry.However the worldwide fish meal supply can not meet thegrowing demand for aquaculture,it is necessary to look for alternative sources for fish meal.Soybean meal is easily digested and absorbed by aquatic animals,so it is one of the most widely used plant protein sources in aquatic feed.While excessive use of soybean meal with amino acid imbalance and many antinutritional factors in feed,affects not only feeding and growth of fish,but also the health and immune function.It has been demonstrated that the problem of antinutritional factors and amino acid imbalance can be eased by enzyme additives,microbial fermentation and crystalline amino acids additive.Large yellow croakerPseudosciaena croceaR.is an important commercial marine fish species in China.It is favored by consumers due to its high nutritional value.As other carnivores,the high protein requirement of large yellow croaker is mainly from fish meal,so substitution of fish meal in the feed is also a key problem in their culture.In this study,a total of1080Pseudosciaena croceaR.with an average body weight of(34.72g±0.28g)were randomly divided into 9 groups with 3 replicates each,and each replicate contained 40 fish.Fish were fed with 9 isonitrogenous and isoenergetic diets,the control group(50%fishmeal,without soybean meal)and 8 experimental groups formulated by replacing 20%or 40%fish meal with dehulled,enzyme-treated,fermented soybean mealⅠor fermented soybean mealⅡwith crystalline amino acids,respectively.The 9 groups was respectively named as FM,DSM20,DSM40,ESM20,ESM40,F(xiàn)SMⅠ20,F(xiàn)SMⅠ40,F(xiàn)SMⅡ20,F(xiàn)SMⅡ40.A 7-week trial was conducted in floating nets to evaluate the feasibility of the replacement of fishmeal by four different kinds of soybean meal,and to measure the appropriate replacement level.Results showed that there was no significant difference in the survival rate,special growth rate between the control group and experimental groups.And there was no significant difference on feed conversion ratio,hepatosmatic index and condition factor among all groups.The superoxide dismutase activity and catalase in serum,liver and mucus were significantly different among groups.The superoxide dismutase activity of serum in FM and FSMⅡ20 was higher than other experimental groups.The catalase of serum in FM,DSM20,F(xiàn)SMⅠ40 and FSMⅡ20 was higher than that in DSM40,ESM20,ESM40 and FSMⅠ20.The malondialdehyde in serum and liver of replacement groups were higher than that in the control group.Serum showed significantly lower capacity to killV.harveyiin the group of 40%fish meal replaced by enzyme-treated soybean meal and lower capacity to killV.alginolyticusin the group of 20%fish meal replaced by dehulled soybean meal.Serum and mucus showed no significant difference on the capacity to killA.hydrophilaamong all groups.Fermented soybean meal did not affect serum and mucus to kill the three kinds of bacteria.In conculsion,among the tested soybean meal,fermented soybean meal was the best substitution for fish meal,it could replace 20%-40%fish meal based on the specific growth rate,feed conversion ratio and antibacterial ability,but there was a risk of reduced antioxidation ability especially in the group of40%fish meal replaced by fermented soybean meal I.While dehulled and enzyme-treated soybean meal had inferior position on antibacterial ability as well as antioxidation ability.

Pseudosciaena crocea;fish meal;soybean meal;antioxidant ability;antibacterial ability

S 963.31

A

1004-2490(2016)05-0495-12

2015-11-19

上海市科技興農(nóng)重點攻關(guān)項目(2013第5-8號);水產(chǎn)動物遺傳育種中心上海市協(xié)同創(chuàng)新中心(ZF1206)

吳 釗,男,碩士研究生,研究方向為水產(chǎn)動物營養(yǎng)學。

華雪銘,副教授,主要研究方向為水產(chǎn)動物營養(yǎng)。E-mail:xmhua@shou.edu.cn,Tel:021-61900416

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