劉姣,韓華,孫飛雪,張叢堯,曹淑青,趙乃錫,馬悅欣

(1.大連海洋大學農(nóng)業(yè)部北方海水增養(yǎng)殖重點實驗室,遼寧大連116023;2.大連海洋學校,遼寧大連116023)

餌料中添加芽孢桿菌BC26對刺參幼參消化酶、免疫反應和抗病力的影響

劉姣1,韓華2,孫飛雪1,張叢堯1,曹淑青1,趙乃錫1,馬悅欣1

(1.大連海洋大學農(nóng)業(yè)部北方海水增養(yǎng)殖重點實驗室,遼寧大連116023;2.大連海洋學校,遼寧大連116023)

將濃度為105、107、109CFU/g(餌料)的芽孢桿菌BC26添加到基礎(chǔ)餌料中,用每一濃度的餌料投喂一組刺參幼參(1.374 g±0.558 g),對照組投喂基礎(chǔ)餌料,每組均設(shè)3個平行,試驗在12個盛有100 L過濾海水的塑料桶中進行,每桶放50頭幼參,飼養(yǎng)試驗共進行45 d。試驗結(jié)束后,分析芽孢桿菌BC26對幼參腸道消化酶活力、免疫反應和抗病力的影響。結(jié)果表明:投喂芽孢桿菌BC26餌料的各組幼參腸道中的胰蛋白酶、脂肪酶活力,以及體腔液中的酚氧化酶、總一氧化氮合酶活力均極顯著高于對照組(P＜0.01);投喂?jié)舛葹?07、109CFU/g(餌料)的芽孢桿菌BC26組幼參體腔細胞的吞噬活力極顯著高于對照組(P＜0.01);用病原菌燦爛弧菌NB13攻毒各組幼參,投喂芽孢桿菌BC26組的幼參發(fā)病率和死亡率明顯低于對照組。研究表明,餌料中添加芽孢桿菌BC26可促進刺參消化和增強免疫功能,并提高其抗病力。

刺參;芽孢桿菌BC26;消化酶;免疫反應

刺參Apostichopus japonicus隸屬于棘皮動物門、海參綱,又名仿刺參,是中國北方地區(qū)重要的海珍品養(yǎng)殖種類。隨著其養(yǎng)殖密度的迅速增加,刺參傳染性疾病經(jīng)常發(fā)生,如稚參培育階段的細菌性潰爛病,幼參培育、養(yǎng)成階段的皮膚潰爛病和急性口圍腫脹癥等,給刺參養(yǎng)殖業(yè)造成巨大的經(jīng)濟損失,限制了該產(chǎn)業(yè)的可持續(xù)發(fā)展[1-5]。近年來,環(huán)境友好的生物控制疾病方法(如飼喂益生菌)已成為水產(chǎn)養(yǎng)殖研究的重要課題,Qi等[6]對水產(chǎn)養(yǎng)殖中有關(guān)益生菌種類、效果、機制的研究應用以及存在的問題進行了詳細的綜述。益生菌在刺參養(yǎng)殖業(yè)中的應用已受到國內(nèi)一些學者的關(guān)注,張濤等[7]將從刺參腸道中分離的乳酸菌L-2和芽孢桿菌K-3混合添加到餌料中養(yǎng)殖刺參,發(fā)現(xiàn)幼參腸道蛋白酶和淀粉酶活力明顯提高。Zhao等[8]發(fā)現(xiàn),用添加枯草芽孢桿菌T13的餌料飼養(yǎng)刺參,幼參的體腔細胞吞噬作用、呼吸爆發(fā)活力、總一氧化氮合酶活力和抗病力均顯著提高。Ma等[9]和Liu等[10]將從野生刺參腸道中分離的仙人掌有孢漢遜酵母C21和梅奇酵母C14[11]分別添加到餌料中投喂刺參,結(jié)果均可刺激幼參的免疫反應,提高其抗病力。楊志平等[12]從健康刺參腸道中篩選出潛在產(chǎn)酶益生菌芽孢桿菌BC26,本研究中,筆者用保存在農(nóng)業(yè)部北方海水增養(yǎng)殖重點實驗室的該菌株對幼參進行了消化酶活力、免疫反應和抗病力影響的試驗,旨在為BC26菌株在刺參養(yǎng)殖中的應用提供參考。

1 材料與方法

1.1 材料

試驗用健康幼參購買于大連某刺參養(yǎng)殖場。試驗用基礎(chǔ)餌料配方同文獻 [12]。

1.2 方法

1.2.1 含菌餌料的制備與試驗設(shè)計 將芽孢桿菌BC26種子接種于胰蛋白胨大豆肉湯(TSB)培養(yǎng)基中,于25℃下振蕩培養(yǎng)16 h,將菌懸液以4 500 r/min離心15 min,細胞用生理鹽水重懸,平板菌落計數(shù),再將細胞懸液添加到刺參基礎(chǔ)餌料中,制成濃度為105、107、109CFU/g(餌料)的含菌餌料。

將刺參幼參在水溫約14℃的條件下暫養(yǎng)2周后開始正式試驗。選擇大小為(1.374±0.558)g的健康幼參隨機分配到12個盛有100 L過濾海水的塑料桶中,每桶放50頭幼參,用3個濃度的含菌餌料和基礎(chǔ)餌料分別投喂一組刺參幼參,每組均設(shè)3個平行,每日投喂一次,投喂量為刺參體質(zhì)量的5%,每2天換水1/2,并吸底去除殘餌及糞便。試驗期間,溫度為7～14℃,pH為7.8～8.2,鹽度為33～34。

1.2.2 指標的測定與計算

1)腸道消化酶活力。將刺參幼參養(yǎng)殖45 d后,從每桶隨機取5頭移至僅有海水的桶中16 h,以便幼參將腸道內(nèi)容物排空。解剖取其腸道,用濾紙吸干,稱重后以9倍體積的冰冷生理鹽水勻漿,在4℃下以6 000 r/min離心10 min,取上清液備用。

依據(jù)Holm等[13]的方法,使用南京建成科技有限公司胰蛋白酶試劑盒測定胰蛋白酶活力。胰蛋白酶活力單位定義為:在pH 8.0、37℃條件下,每毫克蛋白中含有的胰蛋白酶每分鐘使吸光度變化0.003即為一個酶活力單位(U)。依據(jù) Shihabi等[14]的方法,使用南京建成科技有限公司脂肪酶試劑盒測定脂肪酶活力。脂肪酶活力單位定義為:在37℃條件下,每克組織蛋白在本反應體系中與底物反應1 min,每消耗1 μmol底物為一個酶活力單位(U)。

2)免疫指標。幼參養(yǎng)殖45 d后,先饑餓16 h,從每桶隨機取5頭幼參,以滅菌海水沖洗幼參體表,解剖取其體腔液500 μL,加入至盛有等體積抗凝劑(0.02 mol/L EGTA,0.34 mol/L NaCl, 0.019 mol/L KCl,0.068 mol/L Tris-HCl,pH 8.0)[15]的離心管中,混合均勻后,取300 μL用于吞噬試驗,剩余的體腔液在6 000 r/min下離心10 min,取上清液,用于測定酚氧化酶和總一氧化氮合酶活力。

依據(jù)Hannam等[16]的方法,通過測定中性紅染過的釀酒酵母顆粒確定體腔細胞的吞噬活力,體腔細胞對酵母顆粒的吞噬吸收通過標準曲線計算,以50 μL樣品中每毫克蛋白吞噬的酵母細胞數(shù)量表示吞噬活力。根據(jù) Smith等[17]的方法,測定酚氧化酶活力,以L-dopa為底物,胰蛋白酶為誘導因子。將50 μL待測樣品和50 μL CAC緩沖液(含有0.1%的胰蛋白酶)混勻后在25℃下孵育10 min,然后加入100 μL L-dopa(3 mg/mL)混勻,讀取490 nm處的吸光值。酚氧化酶活力單位定義為:在試驗條件下,每分鐘 OD490nm值增加0.001為一個酶活力單位(U)。使用南京建成科技有限公司總一氧化氮合酶試劑盒測定總一氧化氮合酶活力,總一氧化氮合酶活力單位定義為:每毫升體腔液每分鐘生成1 nmol NO為一個酶活力單位(U)。

1.2.3 蛋白含量的測定及攻毒試驗 采用考馬斯亮藍法,以牛血清白蛋白為標準[18],測定幼參腸道勻漿液上清液、體腔液中總蛋白的含量。

飼養(yǎng)試驗結(jié)束后,從每組分別取10頭幼參進行攻毒試驗,對每頭幼參注射燦爛弧菌NB13菌株0.1 mL(密度為107CFU/mL),該菌株是幼參急性口圍腫脹癥的病原菌[2],觀察并記錄幼參被攻毒后一個月中的發(fā)病和死亡情況。

1.3 數(shù)據(jù)處理

試驗數(shù)據(jù)均用平均值±S.D.表示,用 SPSS 19.0軟件進行單因素方差分析(One-way ANOVA),用Dunncan法進行多重比較,顯著性水平設(shè)為0.05。

2 結(jié)果

2.1 芽孢桿菌BC26對刺參幼參腸道消化酶活力的影響

從表1可見,試驗結(jié)束后,投喂芽孢桿菌BC26的各組幼參腸道的胰蛋白酶和脂肪酶活力均較對照組極顯著提高(P＜0.01),說明添加該菌能增強幼參腸道消化酶的活力,從而促進幼參對食物中蛋白質(zhì)和脂肪的消化利用。

表1 芽孢桿菌BC26對幼參腸道胰蛋白酶和脂肪酶活力的影響(n=3)Tab.1 Effects of dietary Bacillus sp.BC26 on activities of trypsin and lipase in intestine of sea cucumber Apostichopus japonicus(n=3)

2.2 芽孢桿菌BC26對刺參幼參免疫指標的影響

從表2可見:試驗結(jié)束后,投喂?jié)舛葹?07和109CFU/g(餌料)芽孢桿菌BC26的試驗組幼參的體腔細胞的吞噬活力均極顯著高于對照組(P＜0.01);投喂芽孢桿菌BC26的各組幼參體腔液中酚氧化酶活力和總一氧化氮合酶活力均極顯著高于對照組(P＜0.01)。

表2 芽孢桿菌BC26對幼參體腔細胞的吞噬活力以及體腔液中酚氧化酶和總一氧化氮合酶活力的影響(n=3)Tab.2 Effects of dietary Bacillus sp.BC26 on phagocytic activity of coelomocytes,and activities of phenoloxidase and total nitric oxide synthase in coelomic fiuid of sea cucumber Apostichopus japonicus(n=3)

2.3 芽孢桿菌BC26對刺參幼參抗燦爛弧菌NB13感染能力的影響

攻毒試驗結(jié)果表明,餌料中添加芽孢桿菌BC26可提高幼參抗燦爛弧菌 NB13感染的能力(表3),用燦爛弧菌NB13攻毒后的一個月內(nèi),對照組幼參的發(fā)病率為70%,死亡率為30%,而投喂添加芽孢桿菌BC26餌料的試驗組幼參發(fā)病率僅為10%,且無死亡現(xiàn)象。

表3 用燦爛弧菌NB13攻毒后各組幼參的累計發(fā)病率和死亡率Tab.3 Cumulative incidence and mortality of sea cucumber Apostichopus japonicus challenged with Vibrio splendidus NB13 in each group

3 討論

適宜濃度的益生菌在水生動物的消化過程中起著有益的作用。Liu等[19]用添加產(chǎn)蛋白酶益生菌枯草芽孢桿菌E20[108CFU/kg(餌料)]的餌料投喂凡納濱對蝦,對蝦腸道及內(nèi)含物中的蛋白酶活力顯著增加。本試驗中,用添加不同濃度芽孢桿菌BC26的餌料投喂幼參后,幼參腸道胰蛋白酶和脂肪酶活力均顯著提高,不過很難區(qū)分是由刺參合成的酶還是由細菌合成的酶,因為芽孢桿菌BC26可分泌胞外蛋白酶和脂肪酶[12,20]。另外,不同濃度的芽孢桿菌BC26對脂肪酶活力的影響也不同,其中添加107CFU/g(餌料)的芽孢桿菌BC26時效果最佳。

一般認為,刺參缺乏適應性免疫,完全依賴其先天性免疫來防御外來物質(zhì)。近年來,免疫刺激劑和益生菌等能增強宿主免疫力和抗病力的制品正被應用于刺參疾病的預防中[8-10,21-22]。 本研究證明,添加芽孢桿菌BC26的餌料對幼參具有免疫刺激效果。體腔細胞是棘皮動物抵御感染和損傷的第一道防線,起著吞噬、誘捕和包裹入侵微生物的作用[23]。Zhao等[8]和Zhang等[21]分別用添加內(nèi)源枯草芽孢桿菌T13[109CFU/g(餌料)]的餌料投喂刺參30 d,用添加外源枯草芽孢桿菌 [107CFU/g(餌料)]的餌料投喂刺參56 d,均提高了刺參體腔細胞的吞噬活力。本試驗中,用添加芽孢桿菌BC26[107、109CFU/g(餌料)]的餌料投喂幼參45 d后,其體腔細胞的吞噬活力顯著增強。類似的報道還有:用添加枯草芽孢桿菌E20[108CFU/kg(餌料)]的餌料投喂凡納濱對蝦98 d[24]和用添加芽孢桿菌S11[約1010CFU/g(餌料)]的餌料投喂斑節(jié)對蝦60 d后[25],對蝦血細胞的吞噬活力均顯著增強;用添加枯草芽孢桿菌AB1[107cells/g(餌料)][26]和芽孢桿菌 JB-1[108cells/g(餌料)][27]分別投喂虹鱒14 d后,其頭腎白細胞的吞噬活力均顯著提高。

酚氧化酶系統(tǒng)是無脊椎動物免疫防御的重要組成部分。給斑節(jié)對蝦和凡納濱對蝦分別投喂芽孢桿菌S11[約1010CFU/g(餌料)][25]和枯草芽孢桿菌E20[108CFU/kg(餌料)][24]的餌料,其血細胞裂解液上清液中酚氧化酶活力明顯增加。Canicatti等[28]研究發(fā)現(xiàn),在刺參體腔細胞裂解液中酚氧化酶以酶原形式存在,外源的胰蛋白酶可激活酚氧化酶活力,在寄生蟲封入過程中,酚氧化酶負責黑色素沉積。本研究中,用添加芽孢桿菌 BC26 [107、109CFU/g(餌料)]的餌料投喂幼參45 d后,其體腔液的酚氧化酶活力明顯提高,這可能是酚氧化酶從體腔細胞分泌到胞外的緣故。Li等[29]也有類似的發(fā)現(xiàn),給凡納濱對蝦投喂含芽孢桿菌OJ[1010CFU/g(餌料)]的餌料28 d后,其血清中的酚氧化酶活力顯著高于對照組。

軟體動物和甲殼動物血細胞中存在一氧化氮合酶[30-32]。由一氧化氮合酶催化L-精氨酸與氧分子經(jīng)多步氧化還原反應生成一氧化氮,軟體動物和甲殼動物免疫細胞利用一氧化氮作為殺細菌分子[32-33],刺參體腔細胞裂解液的高一氧化氮合酶活力可以由枯草芽孢桿菌 T13[109CFU/g(餌料),30 d]誘導產(chǎn)生[8]。本研究表明,投喂含芽孢桿菌BC26的餌料45 d后,幼參體腔液的一氧化氮合酶活力明顯增加。

已有資料證明,餌料中添加芽孢桿菌能通過刺激細胞和體液免疫功能提高對蝦[24-25,29,34]和魚[26-27]對病原細菌或病毒的抵抗力。本研究結(jié)果表明,口服芽孢桿菌BC26可以降低刺參幼參被燦爛弧菌NB13攻毒后的累計死亡率,這可能是刺參細胞免疫和體液免疫反應增強的緣故。

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Effects of dietary live Bacillus sp.BC26 on digestive enzyme activity, immune response and disease resistance against Vibrio splendidus infection in juvenile sea cucumber Apostichopus japonicus

LIU Jiao1,HAN Hua2,SUN Fei-xue1,ZHANG Cong-yao1,CAO Shu-qing1,ZHAO Nai-xi1,MA Yue-xin1
(1.Key Laboratory of Mariculture&Stock Enhancement in North China's Sea,Ministry of Agriculture,Dalian Ocean University,Dalian 116023, China;2.Dalian Ocean School,Dalian 116023,China)

Juvenile sea cucumber Apostichopus japonicus was randomly divided into 12 plastic tanks of each filled with 100 L filtered sea water at a rate of 50 individuals per tank and fed basal diet(control group)and the basal diets containing bacterium Bacillus sp.BC26 at a dose of 105,107and 109CFU/g diet for 45 d with triplication. At the end of the feeding trial,effects of Bacillus sp.BC26 on digestive enzyme activity,immune response and disease resistance against Vibrio splendidus infection were studied in the sea cucumber fed the various diets.Results showed that there were significantly higher trypsin and lipase activities in the intestine,and phenoloxidase and total nitric oxide synthase activities in the coelomic fluid in the sea cucumber in the experimental groups than those in the control group(P＜0.01).The sea cucumber fed the diet containing Bacillus sp.BC26 at 107and 109CFU/g diet had significantly higher phagocytic activity than the sea cucumber in the control group(P＜0.01).Cumulative incidence and mortality of the sea cucumber fed the Bacillus sp.BC26 were found to be lower than those in the control group when 10 sea cucumber samples in each group were challenged with V.splendidus NB13.The findings show the supplementation of Bacillus sp.BC26 lead to improvement of digestive enzyme activity and the immune response of the sea cucumber juveniles,thus enhancing their resistance against V.splendidus.

Apostichopus japonicus;Bacillus sp.BC26;digestive enzyme;immune response

Q939.1;S968.9

A

2013-03-26

國家 “863”計劃重大項目(2012AA10A412)

劉姣(1989-),女,碩士研究生。E-mail:jiaojiao.123.456@163.com

馬悅欣(1963-),女,教授。E-mail:mayuexin@dlou.edu.cn

2095-1388(2013)06-0568-05