β-Conglycinin對(duì)不同發(fā)育時(shí)期鯉魚消化酶活力的影響

邢秀蘋，賴紅娥，趙　晗，楊歡歡，吳莉芳，閆　磊

(1 吉林農(nóng)業(yè)大學(xué) 動(dòng)物科技學(xué)院，吉林 長(zhǎng)春 130118；2 廈門利洋水產(chǎn)科技有限公司，福建 廈門 361012)

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β-Conglycinin對(duì)不同發(fā)育時(shí)期鯉魚消化酶活力的影響

邢秀蘋1，賴紅娥1，趙晗1，楊歡歡2，吳莉芳1，閆磊1

(1 吉林農(nóng)業(yè)大學(xué) 動(dòng)物科技學(xué)院，吉林 長(zhǎng)春 130118；2 廈門利洋水產(chǎn)科技有限公司，福建 廈門 361012)

[摘要]【目的】 研究β-伴大豆球蛋白(β-Conglycinin)對(duì)鯉幼魚、稚魚蛋白酶和淀粉酶活力的影響?！痉椒ā?以初始體質(zhì)量為(10.06±0.14) g/尾的鯉稚魚和(110.23±0.23) g/尾的鯉幼魚為研究對(duì)象，以魚粉為動(dòng)物蛋白源，面粉、糊精為糖源，混合油脂(m(魚油)∶m(玉米油)=1∶1)為脂肪源，分別配制5種等氮(鯉幼魚和稚魚粗蛋白質(zhì)量分?jǐn)?shù)分別為36%和40%)、等能(鯉幼魚和稚魚總能分別是15.2和16.9 MJ/kg) 的半精制飼料，其β-Conglycinin的添加量(質(zhì)量分?jǐn)?shù))分別為0(CK)，2.0%，4.0%，6.0%和8.0%，每組飼料設(shè)3個(gè)重復(fù)，在控溫單循環(huán)養(yǎng)殖系統(tǒng)中進(jìn)行為期8周的飼養(yǎng)試驗(yàn)，試驗(yàn)結(jié)束后，取鯉幼魚、稚魚的前、中、后腸道和肝胰臟，分別用福林-酚試劑法和淀粉酶試劑盒法，測(cè)定腸道和肝胰臟蛋白酶及淀粉酶的活力?！窘Y(jié)果】 鯉幼魚肝胰臟蛋白酶活力各組之間差異不顯著(P>0.05)；β-Conglycinin 添加量為6.0%和8.0%組的前腸、中腸蛋白酶活力顯著低于對(duì)照組(P<0.05)；而β-Conglycinin添加量為8.0%組后腸蛋白酶活力顯著低于對(duì)照組(P<0.05)。在鯉稚魚肝胰臟和后腸，β-Conglycinin添加量為8.0%組的蛋白酶活力顯著低于對(duì)照組(P<0.05)；前腸蛋白酶活力則以2.0%，4.0%，6.0%和8.0%添加組顯著低于對(duì)照組(P<0.05)；中腸蛋白酶活力為4.0%，6.0%和8.0%添加組顯著低于對(duì)照組(P<0.05)。 β-Conglycinin對(duì)鯉幼魚和稚魚肝胰臟、前腸、中腸及后腸淀粉酶活力均無顯著影響(P>0.05)。【結(jié)論】 鯉幼魚配合飼料中β-Conglycinin的添加量不應(yīng)超過6.0%；鯉稚魚配合飼料中β-Conglycinin的添加量不應(yīng)超過2.0%。

[關(guān)鍵詞]β-伴大豆球蛋白；鯉；幼魚；稚魚；蛋白酶；淀粉酶

隨著集約化水產(chǎn)養(yǎng)殖的發(fā)展，魚粉資源短缺，尋求魚粉蛋白源替代品已成為國際性研究課題。大豆蛋白源是水產(chǎn)飼料應(yīng)用最多的植物蛋白源之一。目前，國內(nèi)外學(xué)者在大豆蛋白源替代魚粉方面做了許多研究，涉及的魚類主要有虹鱒(Oncorhynchusmykiss)[1]、金頭鱒(SparusaurataL.)[2]、大西洋鮭(SalmosalarL.)[3]、雜交羅非魚(Oreochromisniloticus×O.aureus)[4]、草魚(Ctenopharyngondonidellus)[5]、大黃魚(PseudoscjaenacroceaR.)[6]、異育銀鯽 (Carassiusauratusgibelio)[7]、齊口裂腹魚(Schizothoraxprenanti)[8]、埃及胡子鲇(Clariaslazera)[9]、鯉魚(Cyprinuscarpio)[10]等，研究的內(nèi)容多集中在大豆蛋白源替代魚粉蛋白后對(duì)魚類攝食、消化、生長(zhǎng)、健康等的影響。這些研究表明，以過量的大豆蛋白替代魚粉蛋白，不僅會(huì)影響魚類腸上皮細(xì)胞增生及腸道組織形態(tài)，而且會(huì)影響魚類對(duì)飼料的消化及生長(zhǎng)性能。其主要原因是大豆蛋白中含有多種抗?fàn)I養(yǎng)因子，其中大豆抗原蛋白是大豆中主要的抗?fàn)I養(yǎng)因子之一。β-伴大豆球蛋白(β-Conglycinin)比大豆球蛋白(Glycinin)具有更強(qiáng)的抗原性[11]，普通的熱處理不能滅活β-伴大豆球蛋白的免疫活性，其能夠引起魚類消化道過敏，造成胃、腸道損傷，進(jìn)而引起消化吸收障礙，甚至死亡。但由于大豆抗原蛋白具有蛋白含量高、價(jià)格低廉、來源豐富等優(yōu)點(diǎn)，因此國內(nèi)外學(xué)者就大豆抗原蛋白對(duì)動(dòng)物的影響進(jìn)行了廣泛研究。目前，關(guān)于大豆抗原蛋白的研究主要集中在豬[12]、犢牛[13]、鼠[14]、羔羊[15]等陸生動(dòng)物上，而對(duì)水產(chǎn)動(dòng)物研究報(bào)道較少，僅見郭林英[16]研究了大豆β-伴球蛋白提取物對(duì)鯉魚腸上皮細(xì)胞增殖及其功能的影響。魚類消化道的消化酶是影響飼料消化吸收的主要因素，消化酶受體內(nèi)外多種因素的影響。本研究分別以鯉幼魚和稚魚為供試動(dòng)物，研究了β-Conglycinin對(duì)不同發(fā)育時(shí)期鯉魚消化酶活力的影響，旨在為合理開發(fā)利用大豆蛋白源及大豆抗原蛋白的去除提供依據(jù)。

1材料與方法

1.1β-Conglycinin的分離純化

β-Conglycinin采用簡(jiǎn)化膜中間試驗(yàn)方法[17]獲得。

1.2試驗(yàn)飼料

以魚粉為動(dòng)物蛋白源，面粉、糊精為糖源，混合油脂(m(魚油)∶m(玉米油)=1∶1)為脂肪源，分別配制5種等氮(鯉幼魚和稚魚的蛋白質(zhì)量分?jǐn)?shù)分別為36%和40%)、等能(鯉幼魚和稚魚總能分別為15.2和16.9 MJ/kg) 的半精制飼料，β-Conglycinin的添加量(質(zhì)量分?jǐn)?shù))分別為0(對(duì)照，CK)，2.0%，4.0%，6.0%和8.0%。各原料粉碎后過孔徑0.246 mm的篩，按配方準(zhǔn)確稱其質(zhì)量，在吉林農(nóng)業(yè)大學(xué)動(dòng)物水產(chǎn)實(shí)驗(yàn)室用電動(dòng)絞肉機(jī)制成粒徑1.5 和2.5 mm顆粒飼料。曬干后置于-20 ℃冰箱中保存?zhèn)溆?。鯉幼魚和稚魚的試驗(yàn)飼料組成及營養(yǎng)成分見表1和表2。

表 1　鯉幼魚飼料配方及營養(yǎng)水平(風(fēng)干基礎(chǔ))

表 2　鯉稚魚飼料配方及營養(yǎng)水平(風(fēng)干基礎(chǔ))

1.3飼養(yǎng)條件及管理

養(yǎng)殖試驗(yàn)在吉林農(nóng)業(yè)大學(xué)控溫單循環(huán)系統(tǒng)中進(jìn)行，試驗(yàn)期間連續(xù)充氣，水中氨氮質(zhì)量濃度低于0.5 mg/L，溶解氧高于5.0 mg/L，溫度為25～27 ℃，養(yǎng)殖試驗(yàn)持續(xù)8周。

試驗(yàn)魚來源于吉林省九臺(tái)市漁場(chǎng)，試驗(yàn)前飽食投喂對(duì)照組飼料，預(yù)飼15 d，預(yù)飼試驗(yàn)結(jié)束后，饑餓24 h，挑選鰭鱗完整、規(guī)格整齊、體質(zhì)健壯的鯉幼魚((110.23±0.23) g/尾) 300尾和鯉稚魚((10.06±0.14) g/尾) 450尾，分別隨機(jī)放養(yǎng)在15個(gè)玻璃缸中，鯉幼魚每缸放養(yǎng)20尾，鯉稚魚每缸放養(yǎng)30尾。放養(yǎng)前用質(zhì)量濃度為20 mg/L的高錳酸鉀溶液藥浴10 min，隨機(jī)安排3個(gè)玻璃缸為一個(gè)試驗(yàn)組。在試驗(yàn)過程中，每天稱取足量飼料，分2次投喂(09:00，16:00)，投餌方式為人工手撒，直至魚不再到水面攝食為止，日投餌率為體質(zhì)量的3%～5%，每天記錄每缸魚的攝食飼料質(zhì)量。

1.4樣品的收集與粗酶液的制備

參照吳莉芳等[18]的方法進(jìn)行樣品收集與粗酶液制備。(1)樣品的收集。飼養(yǎng)試驗(yàn)結(jié)束前停食24 h后，每缸活體解剖10尾魚，取出肝胰臟和其他內(nèi)臟，稱其質(zhì)量(精確到0.01 g)。取出腸道和肝胰臟，剔除附著物，用去離子水沖洗腸道內(nèi)容物，濾紙吸干，-20 ℃冰柜保存待測(cè)。腸道從第一個(gè)回折點(diǎn)以前為前腸，最后一個(gè)回折點(diǎn)以后為后腸，其間為中腸。(2)粗酶液的制備。稱樣品質(zhì)量，加入10倍體積的高純水勻漿，在4 ℃冰箱中靜置過夜，5 000 r/min離心10 min，取上清液即為粗酶液，4 ℃冰箱保存、待測(cè)。粗酶液需在24 h內(nèi)測(cè)定完畢。

1.5消化酶活力的測(cè)定

蛋白酶活力采用福林-酚試劑法(Folin-phenol)測(cè)定[18]；淀粉酶活力采用淀粉酶試劑盒(南京建成科技有限公司)測(cè)定[18]。

1.6數(shù)據(jù)統(tǒng)計(jì)分析

采用SPSS17.5軟件對(duì)鯉幼魚、稚魚蛋白酶及淀粉酶活力進(jìn)行方差分析，若差異顯著，進(jìn)一步進(jìn)行LSD和Duncan’s多重比較，分析組間差異顯著性。試驗(yàn)數(shù)據(jù)用“平均值±標(biāo)準(zhǔn)差”(Mean±SD)表示。顯著性水平設(shè)定為P<0.05。

2結(jié)果與分析

2.1β-Conglycinin對(duì)鯉幼魚和稚魚蛋白酶活力的影響

β-Conglycinin對(duì)鯉幼魚、稚魚蛋白酶活力的影響分別見表3和表4。

表 3　β-Conglycinin對(duì)鯉幼魚蛋白酶活力的影響

注：同列數(shù)據(jù)后標(biāo)不同小寫字母表示差異顯著(P<0.05)。下表同。

Note：Different lowercase letters in each column indicate significant difference(P<0.05).The same below.

表 4　β-Conglycinin對(duì)鯉稚魚蛋白酶活力的影響

由表3可以看出，鯉幼魚肝胰臟蛋白酶活力各組之間差異不顯著(P>0.05)。2.0%和4.0% β-Conglycinin 添加組前腸、中腸蛋白酶活力與對(duì)照組差異不顯著(P>0.05)，6.0%和8.0% β-Conglycinin添加組前腸、中腸蛋白酶活力顯著低于對(duì)照組(P<0.05)；8.0% β-Conglycinin添加組前腸蛋白酶活力顯著低于2.0%，4.0%和6.0%添加組(P<0.05)，6.0% β-Conglycinin添加組前腸蛋白酶活力顯著低于2.0%和4.0%添加組(P<0.05)；6.0%和8.0%β-Conglycinin添加組中腸蛋白酶活力顯著低于2.0%及4.0%添加組(P<0.05)。2.0%，4.0%，6.0%β-Conglycinin添加組后腸蛋白酶活力與對(duì)照組差異不顯著(P>0.05)，8.0%添加組后腸蛋白酶活力顯著低于對(duì)照組(P<0.05)；2.0%，4.0%，6.0%和8.0%添加組之間后腸蛋白酶活力無顯著差異(P>0.05)。

由表4可知，在鯉稚魚肝胰臟和后腸中，2.0%， 4.0%和6.0% β-Conglycinin添加組蛋白酶活力與對(duì)照組差異不顯著(P>0.05)，8.0%β-Conglycinin添加組蛋白酶活力顯著低于對(duì)照組(P<0.05)，2.0%，4.0%，6.0%和8.0%β-Conglycinin添加組之間蛋白酶活力無顯著差異(P>0.05)；在鯉稚魚前腸中，2.0%， 4.0%，6.0%和8.0% β-Conglycinin添加組蛋白酶活力顯著低于對(duì)照組(P<0.05)，2.0%和4.0% β-Conglycinin添加組蛋白酶活力顯著高于6.0%及8.0%添加組(P<0.05)；在鯉稚魚中腸中，2.0% β-Conglycinin添加組蛋白酶活力與對(duì)照組差異不顯著(P>0.05) ， 而4.0%，6.0%和8.0%β-Conglycinin添加組蛋白酶活力顯著低于對(duì)照組(P<0.05)，2.0%β-Conglycinin添加組蛋白酶活力顯著高于4.0%，6.0%和8.0%添加組(P<0.05)，4.0%β-Conglycinin添加組蛋白酶活力顯著高于6.0%和8.0%添加組(P<0.05)。

2.2β-Conglycinin對(duì)鯉幼魚和稚魚淀粉酶活力的影響

表5和表6表明，在本試驗(yàn)條件下，2.0%， 4.0%，6.0%和8.0%β-Conglycinin添加組鯉幼魚與稚魚肝胰臟、前腸、中腸、后腸淀粉酶活力與對(duì)照組差異均不顯著(P>0.05)。

表 5　 β-Conglycinin對(duì)鯉幼魚淀粉酶活力的影響

表 6　β-Conglycinin對(duì)鯉稚魚淀粉酶活力的影響

3討論

3.1β-Conglycinin對(duì)鯉幼魚和稚魚蛋白酶活力的影響

本研究結(jié)果表明，β-Conglycinin對(duì)鯉幼魚和稚魚的前腸、中腸、后腸蛋白酶活力的影響存在一定的差異。在鯉幼魚的配合飼料中，β-Conglycinin添加量為6.0%和8.0%組前腸、中腸蛋白酶活力顯著低于對(duì)照組(P<0.05)；在鯉稚魚的配合飼料中，β-Conglycinin 添加量為2.0%，4.0%，6.0%和 8.0% 組前腸蛋白酶活力顯著低于對(duì)照組(P<0.05)，而中腸蛋白酶活力則以4.0%，6.0%和8.0% β-Conglycinin添加組顯著低于對(duì)照組(P<0.05)。這可能是由于鯉幼魚和稚魚消化道結(jié)構(gòu)發(fā)育程度不同，對(duì)β-Conglycinin的敏感性不同所致。鯉稚魚消化系統(tǒng)發(fā)育尚不成熟，消化器官不發(fā)達(dá)，消化機(jī)能不完善，消化道中酶的分泌量不足，使大量未消化的營養(yǎng)物質(zhì)進(jìn)入了腸道。因此，β-Conglycinin也可大量進(jìn)入腸道，引起腸道損傷，從而導(dǎo)致消化酶活力降低。張帆等[6]研究了飼料中豆粕替代魚粉對(duì)大黃魚消化酶活性的影響，結(jié)果表明，大黃魚腸道胰蛋白酶的活性隨豆粕替代水平的升高而顯著降低。Burrells等[19]研究表明，在飼料中添加一定量的大豆蛋白，會(huì)引起虹鱒的后腸結(jié)構(gòu)形態(tài)變化，并降低刷狀緣的酶活性。Krogdahl等[20]研究發(fā)現(xiàn)，豆粕能夠引起虹鱒中腸與后腸上皮刷狀緣胞外酶堿性磷酸酶、亮氨酸氨肽酶以及麥芽糖酶、乳糖酶、蔗糖酶活性下降。吳莉芳等[9]研究了去皮豆粕替代魚粉對(duì)埃及胡子鲇消化酶活力的影響，結(jié)果表明，當(dāng)去皮豆粕替代魚粉蛋白的45%和60%時(shí)，埃及胡子鲇前腸和后腸的蛋白酶活力顯著下降。Ksudhik 等[21]在大西洋鮭的飼料中添加一定量的大豆蛋白，引起其后腸結(jié)構(gòu)發(fā)生形態(tài)變化，刷狀緣的酶活性降低。關(guān)于不同添加量的β-Conglycinin引起不同發(fā)育時(shí)期鯉魚腸道組織結(jié)構(gòu)的變化，需進(jìn)一步通過組織學(xué)方法進(jìn)行研究。

3.2β-Conglycinin對(duì)鯉幼魚和稚魚淀粉酶活力的影響

魚類的淀粉酶是碳水化合物水解酶類的一種，活性較低，同種魚類不同消化器官淀粉酶的活力不同，另外隨著魚類年齡的增加，其淀粉酶活力也發(fā)生改變，淀粉酶活性在同一消化器官不同部位也會(huì)有所差異。彭翔等[22]在黑鯛魚飼料中用發(fā)酵豆粕替代0～50%的魚粉蛋白質(zhì)，研究結(jié)果表明，飼料中各組淀粉酶的活性差異不顯著。吳莉芳等[23]研究了不同大豆蛋白源替代魚粉對(duì)鯉魚蛋白酶和淀粉酶活力的影響，結(jié)果表明，不同大豆蛋白源對(duì)鯉魚淀粉酶活力影響不顯著。錢曦等[24]研究報(bào)道，在翹嘴紅鲌的飼料中，當(dāng)豆粕替代魚粉蛋白的13%和27%時(shí)，對(duì)其肝胰臟和腸道淀粉酶活力影響不顯著。在本試驗(yàn)條件下，β-Conglycinin 的添加量對(duì)鯉幼魚和稚魚肝胰臟、前腸、中腸、后腸淀粉酶活力影響均不顯著。這主要是由于魚類的淀粉酶對(duì)食物類型和飼料組成有明顯的適應(yīng)性[25]。鯉魚屬于雜食性魚類，在天然的食譜中存在一定量的植物蛋白源，而β-Conglycinin就屬于植物蛋白源。因此，鯉魚肝胰臟和腸道淀粉酶對(duì)β-Conglycinin具有一定的適應(yīng)性。

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Effects of β-Conglycinin on activities of protease and amylase in juvenile and larval common carps

XING Xiu-ping1,LAI Hong-e1,ZHAO Han1,YANG Huan-huan2,WU Li-fang1,YAN Lei1

(1FacultyofAnimalScienceandTechnology,JilinAgriculturalUniversity,Changchun,Jilin130118,China;2LiyangXiamenAquaticTechnologyCompanyLimited,Xiamen,Fujian361012,China)

Abstract：【Objective】 The research investigated the effects of β-Conglycinin on activities of protease and amylase in juvenile and larval common carps.【Method】 Larval and juvenile common carps with the initial weights of (10.06±0.14) g/tail and (110.23±0.23) g/tail were used as experimental objects for eight-week feeding trial at controlled temperature in single recirculating system.Five diets with identical nitrogen (total crude protein contents for juvenile and larval were 36% and 40%,respectively) and energy (total energies for juvenile and larval were 15.2 and 16.9 MJ/kg,respectively) as well as different β-Conglycinin contents (0(CK),2.0%,4.0%,6.0%,and 8.0%) were provided.Fish meal was animal protein source,dextrin and flour were carbohydrate source,and mixed oil (m(corn oil)∶m(fish oil)=1∶1) was fat source.Each group had three repetitions.The activities of protease and amylase in foregut,midgut and hepatopancreas were detected using Folin-phenol method and Amylase kit.【Result】 No significant effects of β-Conglycinin on activities of protease in hepatopancreas of juvenile common carps were observed (P>0.05).The activities of protease in foregut and midgut of juvenile common carps in 6.0% and 8.0% groups were significantly lower than that of the control group (P<0.05),and that in hindgut of 8.0% group were significantly lower than that of the control group (P<0.05).The activities of protease in hepatopancreas and hindgut of larval common carps in 8.0% groups were significantly lower than that of the control group (P<0.05),that of 2.0%,4.0%,6.0%,and 8.0% groups were significantly lower (P<0.05) in foregut,and that of 4.0%,6.0%,and 8.0% groups were significantly lower (P<0.05) in midgut.In addition,no significant effects of β-Conglycinin on activities of amylase in hepatopancreas and tract of both juvenile and larval common carps were observed (P>0.05).【Conclusion】 The amount of β-Conglycinin should be less than 6.0% and 2.0% in the diet of juvenile and larval common carp,respectively.

Key words：β-Conglycinin;Cyprinus carpio;juvenile common carp;larval common carp;protease;amylase

[文章編號(hào)]1671-9387(2016)01-0019-06

[中圖分類號(hào)]S965.116.31+2

[文獻(xiàn)標(biāo)志碼]A

[作者簡(jiǎn)介]邢秀蘋(1989-)，女，吉林農(nóng)安人，在讀碩士，主要從事水產(chǎn)動(dòng)物營養(yǎng)與飼料研究。E-mail:461934406@qq.com[通信作者]吳莉芳(1970-)，女，吉林農(nóng)安人，教授，博士，主要從事水產(chǎn)動(dòng)物營養(yǎng)與飼料研究。

[基金項(xiàng)目]吉林省教育廳項(xiàng)目(2012043)

[收稿日期]2014-04-10

DOI：網(wǎng)絡(luò)出版時(shí)間:2015-12-0214:2510.13207/j.cnki.jnwafu.2016.01.004

網(wǎng)絡(luò)出版地址:http://www.cnki.net/kcms/detail/61.1390.S.20151202.1425.008.html