CN112369353B - 一种提高大菱鲆幼鱼植物蛋白部分替代饲料效果的养殖方法 - Google Patents
一种提高大菱鲆幼鱼植物蛋白部分替代饲料效果的养殖方法 Download PDFInfo
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Abstract
本发明提供一种促进大菱鲆幼鱼生长的桑叶添加剂，能够有效的缓解高植物蛋白饲料替代鱼粉对大菱鲆的不良影响，促进大菱鲆的生长。本发明首先提供一种能够促进大菱鲆幼鱼生长的桑叶添加剂，是将桑叶用复合蛋白酶进行酶解制备的。本发明中的饲料添加剂在大菱鲆幼鱼饲料中显著促进了大菱鲆的生长，降低了全鱼粗脂肪的含量、血清中一些脂肪代谢指标的含量且调控了脂质代谢相关的基因的表达。且不影响大菱鲆的饲料效率、肝体比、脏体比和肥满度；不影响鱼体水分、灰分和蛋白的含量。
Description
技术领域
本发明属于鱼类养殖技术领域，具体涉及一种提高大菱鲆幼鱼植物蛋白饲料效率的养殖方法，即提高大菱鲆饲料植物蛋白替代效果，降低大菱鲆脂肪含量并促进大菱鲆幼鱼生长的方法。
背景技术
大菱鲆又叫多宝鱼，属于鲽形目，鲆科，菱鲆属，是一种肉食性海水鱼类，是我国重要的经济鱼类之一。大菱鲆具有生长迅速，刺少肉质肥美，腥味小，高蛋白质，低脂肪，富含不饱和脂肪酸及微量元素等诸多优点而受到消费者喜爱。大菱鲆对饲料中蛋白质含量要求较高(理想的蛋白含量在50％左右)，而鱼粉是饲料中蛋白质的主要来源。但是，鱼粉长期供不应求，且其价格高昂，因此需要用来源广泛、价格低廉的植物蛋白源来替代适量的鱼粉以降低饲料成本。但在养殖过程中发现，过高的植物蛋白替代鱼粉会导致大菱鲆生长滞缓，免疫力下降，脏器损伤(肝脏损伤和肠道炎症)，进而会导致鱼类死亡率升高。因此，寻找免疫调节剂已经引起了水产研究者的广泛关注。此外在工厂化养殖过程中，脂肪的过度积累会导致生产损失，肉质改变，和鱼类的脂肪肝，因此脂质代谢的调节已成为水产养殖研究的一个值得关注的重点。
桑叶是蚕桑行业的主要的原料，仅中国一个国家桑叶年产量就有1197万吨，每年都会有大量多余叶子被浪费掉或用作陆地养殖动物的原料。桑叶因其营养成分丰富，活性物质含量众多，价格低廉且极易获得而受到广泛关注。桑叶的成分非常多样，其中常量营养素中蛋白质含量最高，大概在15-28％之间，其氨基酸种类也非常丰富，尤其是缬氨酸，谷氨酰胺，亮氨酸，甘氨酸和赖氨酸。桑叶中维生素和矿物质的含量也极高，且桑叶还含有多种有效的生物活性成分，如黄酮类和多酚类、多糖、类固醇、萜类物质、酚酸、生物碱和有机酸等。但是，由于桑叶中还有较高的纤维素，这些纤维素不仅不能被鱼类利用，而且还会对鱼类的生长产生不利的作用。抗营养因子的存在也影响了桑叶在大菱鲆养殖中的应用。
发明内容
本发明目的是提供一种提高大菱鲆幼鱼植物蛋白饲料效率的养殖方法，将桑叶作为大菱鲆饲料功能性添加剂使用，以提高大菱鲆饲料植物蛋白替代效果，降低大菱鲆脂肪含量，提高大菱鲆幼鱼免疫力，有效的缓解高植物蛋白饲料替代鱼粉对大菱鲆的不良影响。
本发明首先提供一种能够促进大菱鲆幼鱼生长的桑叶添加剂，是将桑叶用复合蛋白酶进行酶解制备的；
所述的复合蛋白酶，包含有纤维素酶、果胶酶、淀粉酶、蛋白酶、β-葡聚糖酶和木聚糖酶；
更进一步的，所述的桑叶添加剂，是先用复合蛋白酶进行酶解，酶解产物再用混合菌进行发酵制备的；
所述的混合菌，其中包含有酵母菌、乳酸菌和芽孢杆菌
本发明所提供的桑叶添加剂用于制备大菱鲆饲料；
所述的大菱鲆饲料，其中使用植物蛋白替代了45％或以上的鱼粉；
所述的桑叶添加剂在大菱鲆饲料中的添加量为1-2％。
本发明还提供一种大菱鲆幼鱼的养殖方法，是使用添加了桑叶添加剂的植物蛋白饲料来饲喂大菱鲆幼鱼。
本发明解决了桑叶无法直接添加到饲料中的问题。本发明中的饲料添加剂在大菱鲆幼鱼饲料中显著促进了大菱鲆的生长，提高大菱鲆饲料植物蛋白添加比例，降低了全鱼粗脂肪的含量、血清中一些脂肪代谢指标的含量且调控了脂质代谢相关的基因的表达。且不影响大菱鲆的饲料效率、肝体比、脏体比和肥满度；不影响鱼体水分、灰分和蛋白的含量。
附图说明
图1：大菱鲆幼鱼照片图；
图2：大菱鲆幼鱼养殖实验照片图；
图3：大菱鲆肝脏hmgcor，Cyp7a1，LPL和PPAR-γmRNA的表达量。
具体实施方式
大菱鲆饲料中蛋白质含量较高，而饲料中蛋白质的主要来源是鱼粉，常规大菱鲆饲料中鱼粉含量高达60％。但是，由于近十几年来，鱼粉供应严重不足，且其价格高昂，在这种情况下，必须尽快找到合适的植物蛋白源以降低饲料成本。目前单一植物蛋白源一般可替代大菱鲆饲料中20％左右的鱼粉而对大菱鲆生长不产生显著影响(如豆粕，玉米蛋白粉)，复合蛋+白源替代比例一般不会超过30％。当配合饲料中含有植物蛋白比例较高时，会影响饲料适口性，损害大菱鲆肠道健康，影响养殖效果。
为了解决这一问题，本发明筛选获得了能够有效解决高植物蛋白对大菱鲆带来的影响的添加剂，从而促成了本发明。
下面结合实施例对本发明进行详细的描述。
实施例1制备酶解桑叶
复合酶酶解桑叶(F)：使用的复合酶购自山东苏柯汉生物工程股份有限公司，复合酶包含有纤维素酶、果胶酶、淀粉酶、蛋白酶、β-葡聚糖酶和木聚糖酶。每千克桑叶用1g的复合酶，复合酶首先加水稀释40倍，再加入桑叶中拌匀后装入封口袋，密封后在50℃酶解3小时，在75℃放置2分钟以终止酶作用。然后将酶解的桑叶铺在托盘上在烘箱(50℃)内烘干，得到酶解桑叶(F)。
将酶解桑叶作为添加剂，与大菱鲆幼鱼饲料的其它组分按从小到大的比例逐级混匀，制成大菱鲆幼鱼饲料。
实施例2：酶解桑叶进行发酵
复合酶酶解后发酵桑叶(FF)：
在上一步酶解得到的酶解桑叶(F)的基础上再用混合菌进行发酵，混合菌购自沧州市中信生物科技有限公司，包含有酵母菌、乳酸菌和芽孢杆菌(按1:1:1的比例混合)。
首先进行菌种活化，具体步骤是将以菌种：红糖：水＝1:1:50的比例放入锥形瓶，密封后在37℃活化4小时备用。每1kg酶解桑叶加入4-5g的菌种，酶解桑叶和活化菌液混匀后，放入封口袋密封在37℃酶解7天，然后将酶解后发酵的桑叶铺在托盘上在烘箱(50℃)内烘干。酶解桑叶后再用混合菌进行发酵得到酶解后发酵的桑叶(FF)；与大菱鲆幼鱼饲料的其它组分按从小到大的比例逐级混匀制成饲料。
实施例3养殖实验
实验实施地点位于山东省胶南市，使用上述实施例1的酶解桑叶(F)和实施例2的发酵桑叶(FF)中作为添加剂添加到大菱鲆配合饲料作为实验组，以含有60％的鱼粉的饲料组为正对照组(FM)，以复合植物蛋白(豆粕：玉米蛋白粉：谷朊粉：花生粕＝49:25:16:10)替代45％的鱼粉的饲料组为负对照组(CON)。饲料具体配方见表1。
表1：实验饲料配方表
注：1FM:鱼粉组；CON:用复合植物蛋白源替代45％的鱼粉；F1:在CON组饲料中添加1％的复合酶酶解的桑叶；F2:在CON组饲料中添加2％的复合酶酶解的桑叶；FF1:在CON组饲料中添加1％的复合酶酶解后再发酵的桑叶；FF2：在CON组饲料中添加2％的复合酶酶解后再发酵的桑叶。
a购自青岛七好生物技术有限公司。鱼粉：粗蛋白69.23％，粗脂肪7.56％；小麦粉：粗蛋白20.01％，粗脂肪2.50％；啤酒酵母：粗蛋白54.65％，粗脂肪3.04％。
b复合植物蛋白是按豆粕：玉米蛋白粉：谷朊粉：花生粕＝49:25:16:10的比例混合所得。植物蛋白原料购自青岛七好生物技术有限公司。豆粕：粗蛋白51.81％，粗脂肪2.43％；玉米蛋白粉：粗蛋白67.41％，粗脂肪2.56％；谷朊粉：粗蛋白86.00％，粗脂肪0.90％；花生粕：粗蛋白54.58％，粗脂肪1.17％。
c维生素预混料(mg/kg diet)：维生素B1，25；维生素B2，45；维生素B6，20；维生素K，10；维生素B12，10；肌醇，800；酸钙，60；烟酸，200；叶酸，20；生物素，60；维生素A，32；维生素D，5；维生素E，240；泛维生素磷酸酯，2000；微晶纤维素,16473。
d矿物质预混料(mg/kg diet)：CoCl2(1％)，50；CuSO4·5H2O，10；FeSO4·7H2O，80；ZnSO4·H2O，50；MnSO4·H2O，45；MgSO4·7H2O，1,200；H2NaOSe(1％)，20；沸石粉，3485。
e桑叶：购自成都桑果果食品有限公司。
f其他：磷酸二氢钙0.50％，氯化胆碱0.25％，诱食剂1％(甜菜碱：硫代甜菜碱：苏氨酸：甘氨酸：5-磷酸肌苷＝4:2:2:1:1)；丙酸钙0.05％；乙氧基喹啉0.05％；海藻酸钠1％；三氧化二钇0.1％。
选取初重6.50±0.01g的大菱鲆幼鱼，分进18个桶(桶体积为300L)，每桶30条鱼，每个处理设置三个平行组，进行9周的养殖实验。养殖期间水温控制在18.0-21.0℃之间，盐度控制在29-32‰之间，溶氧高于7mg/l，氨氮和亚硝酸盐的含量低于0.1mg/l。
养殖实验结束后，称鱼重，根据鱼重和摄食的饲料量计算饲料效率、增重率和特定生长率。解剖鱼体，取出内脏团和肝脏称量，计算肝体比、脏体比和肥满度。以烘干法测定鱼体水分，以索氏抽提法测量鱼体粗脂肪，以凯氏定氮法测定鱼体粗蛋白，以马弗炉燃烧法测定鱼体灰分。取大菱鲆的肝脏组织和血清，测定血清中总胆固醇、甘油三酯、高密度脂蛋白及低密度脂蛋白的含量。并测定与脂肪代谢相关的基因的表达量。
试验数据以平均值±标准误(X±SD),数据分析使用SPSS18.0软件，通过ANOVA分析数据间的显著性差异，对于存在显著性差异(P<0.05)的数值以英文上标字母标记。
结果显示，添加1％和2％的酶解桑叶(F)及添加2％的酶解后发酵的桑叶(FF)的饲料投喂大菱鲆，增重率显著高于对照组，显著促进了大菱鲆的生长(表2)。
表2饲料中添加桑叶对大菱鲆生长性能和饲料效率的影响
注：增重率(％)＝100×(终末体重-初始体重)/初始体重
特定生长率(％d-1)＝100×(Ln终末体重-Ln初始体重)/养殖天数
饲料效率(FE,％)＝鱼体增重/摄食饲料量
另外，桑叶添加剂表现出良好的降脂作用添加2％的FML和FFML显著降低了大菱鲆全鱼粗脂肪的含量(表3)，添加2％的FFML显著降低了大菱鲆血清中总胆固醇(T-CHO)的含量(表4)，且抑制了脂蛋白脂肪酶(LPL)mRNA的表达(图1)。2％的FML降低了高密度脂蛋白(HDL-C)的含量，而添加桑叶都显著降低了低密度脂蛋白(LDL-C)的含量(表4)。此外，添加2％的FML和FFML显著抑制了肝脏3-羟基-3-甲基戊二酰基辅酶A还原酶(Hmgcor)mRNA的表达(图1)。
表3：饲料中添加桑叶对大菱鲆形体指标和体成分的影响
注：肝体比(％)＝100％×肝脏的重量(g)/鱼体总重(g)
脏体比(％)＝100％×内脏团的重量(g)/鱼体总重(g)
肥满度(％)＝100％×鱼体总重(g)/体长(cm)3
表4：饲料中添加桑叶添加剂对大菱鲆血清脂质代谢指标的影响
注：T-CHO，总胆固醇；TG，甘油三酯；HDL-C，高密度脂蛋白胆固醇，LDL-C，低密度脂蛋白胆固醇。T-CHO，TG，HDL-C和LDL-C的单位是mmol/L。
本实验植物蛋白选取的是复合植物蛋白，经实验验证，将酶解桑叶和酶解后发酵处理的桑叶对单一植物蛋白替代鱼粉的饲料中，也有明显改善养殖效果的作用。
综上指标所述，用复合植物蛋白源替代45％鱼粉蛋白显著降低了大菱鲆的生长性能、饲料效率，并且造成了氧化应激。而在高植物蛋白饲料中添加2％的酶解桑叶或酶解+发酵桑叶显著促进大菱鲆生长，而且起到了良好的降脂作用。
Claims (2)
1.一种大菱鲆幼鱼的养殖方法，其特征在于，所述的方法是使用大菱鲆幼鱼饲料来饲喂大菱鲆幼鱼；所述的大菱鲆幼鱼饲料中使用植物蛋白替代了40%或以上的鱼粉；且大菱鲆幼鱼饲料中还添加了桑叶添加剂；所述的桑叶添加剂在大菱鲆饲料中的添加量为1-2%；
所述的桑叶添加剂先用复合蛋白酶进行酶解，酶解产物再用混合菌进行发酵制备的；
所述的复合蛋白酶包含有纤维素酶、果胶酶、淀粉酶、蛋白酶、β-葡聚糖酶和木聚糖酶；所述的混合菌中包含有酵母菌、乳酸菌和芽孢杆菌。
2.如权利要求1所述的养殖方法，其特征在于，所述的植物蛋白为豆粕、玉米蛋白粉、谷朊粉、花生粕的混合物，其中豆粕：玉米蛋白粉：谷朊粉：花生粕的质量份数比为49:25:16:10。
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