CN109601761A - 一种大菱鲆肠炎病的营养性修复剂 - Google Patents
一种大菱鲆肠炎病的营养性修复剂 Download PDFInfo
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Abstract
本发明提供一种大菱鲆肠炎病的营养性修复剂，包含有谷氨酰胺、精氨酸和苏氨酸；其中谷氨酰胺、精氨酸和苏氨酸质量百分比的组成为：谷氨酰胺40％‑60％，精氨酸20％‑30％，苏氨酸20％‑30％。所述的营养性修复剂在检测到抗营养因子及植物蛋白源引起的大菱鲆肠道发生炎症后，使用该营养性修复剂，时间为4周‑8周。本发明的大菱鲆营养性肠炎修复剂在投喂含有引起大菱鲆肠炎的抗营养因子以及植物蛋白源的饲料后使用，能够显著减轻大菱鲆肠炎的发生、修复肠道。包括显著提高肠道绒毛高度、降低肠道绒毛粘连、降低固有层及黏膜下层炎性细胞的浸润、增加上皮细胞核上吸收空泡。而且相比采用使用抗生素等药物治疗，本发明的大菱鲆营养性肠炎修复剂更加安全、可靠。
Description
技术领域
本发明属于水产养殖饲料技术领域，具体涉及一种大菱鲆肠炎病的营养性修复剂。
背景技术
鱼类为人类提供了优质的蛋白源，对改善膳食营养结构、保障人类健康非常重要。随着社会发展，人民生活水平提高，健康意识日益增强，鱼产品需求量和消费量剧增。但鱼类养殖在蓬勃发展的同时，养殖水体污染和生态环境破坏问题不断凸显，造成鱼类疾病频繁暴发。究其原因在于鱼类生活在水中，为低等脊椎动物，消化能力和抗病力弱。其中肠道健康是鱼类健康养殖的关键，肠道是鱼类与外界环境接触面积最大的器官，因此是鱼类感染疾病的主要途径之一。鱼类肠道不仅是重要的消化和吸收器官，同时还具有免疫、内分泌以及新陈代谢的功能。肠道是鱼类最大的免疫器官，鱼类肠道健康对于鱼体的整体健康起到非常重要的作用。肠道健康下降，不仅导致机体健康下降，还降低营养物质消化吸收率，大量未消化营养物质排入水体，引起水环境污染，水质变差，进而降低鱼体健康水平，形成疾病频繁暴发。
近年来，随着我国水产养殖产量的逐年增加，水产饲料的产量远不能满足市场的需求。目前，我国水产饲料的市场空间在1000万吨以上。在提高水产饲料产量的同时还必需考虑如何降低饲料的成本，以获得最大的经济效益。蛋白质是饲料中的主要营养物质，也是决定饲料成本的关键因素。水产养殖动物大多数都要求在饲料中有较高的蛋白质含量，同时还特别依赖动物性蛋白源鱼粉，这就大大提高了水产养殖动物饲料的成本。尤其是鱼粉在世界范围内将永远是一种越来越短缺的资源。因此，人们试图利用价格比较低廉的植物蛋白部分或全部替代动物蛋白，以此配制水产养殖动物的饲料，达到降低成本、提高经济效益的目的。然而大量研究表明，在肉食性鱼饲料中植物蛋白源仅能少量替代鱼粉，植物蛋白源替代鱼粉后会引发鱼类肠道炎症，具体表现为肠道绒毛高度降低、肠道绒毛粘连、固有层及黏膜下层炎性细胞的浸润、上皮细胞核上吸收空泡减少等。而植物蛋白源中的抗营养因子，如大豆皂甙是引起鱼类肠炎的主要因素。
因植物蛋白源替代鱼粉已作为水产饲料生产中降低饲料成本的主要途径，因此其带来的鱼类肠炎问题不容忽视也不可避免。因此需要选择鱼类专用的饲料肠道炎症修复剂，确定修复剂的使用剂量，对植物蛋白源引起的肠道炎症进行修复，从而提高鱼类肠道健康水平，维持鱼类养殖业的健康可持续发展。
发明内容
本发明的目的是提供一种大菱鲆肠炎病的营养性修复剂，能有效的修复由于抗营养因子及植物蛋白源导致的大菱鲆肠炎，从而弥补现有技术的不足。
本发明提供一种大菱鲆肠炎病的营养性修复剂，包含有谷氨酰胺、精氨酸和苏氨酸；
其中谷氨酰胺、精氨酸和苏氨酸质量百分比的组成为：谷氨酰胺40％-60％，精氨酸20％-30％，苏氨酸20％-30％。
所述的营养性修复剂在制备大菱鲆饲料中的应用；在检测到抗营养因子及植物蛋白源引起的大菱鲆肠道发生炎症后，使用该营养性修复剂，时间为4周-8周。
一种使用该营养性修复剂的饲料配方为：鱼粉54％-58％、小麦粉26％-30％、鱼油1.5％-2.5％、大豆卵磷脂1.5％-2.5％、维生素和矿物质混合物2％-3％、氯化胆碱0.4％-0.6％、丙酸钙0.8％-1.2％、乙氧基喹啉0.04％-0.06％、微晶纤维素4.8％-4.9％；营养性修复剂使用添加量为2％-4％。
本发明再一个方面提供一种含有豆粕的大菱鲆饲料及与本发明营养性修复剂的配合使用方式。该饲料中使用豆粕来部分替代鱼粉。其一种具体配方为：鱼粉38％-42％、豆粕28％-32％、小麦粉17％-19％、鱼油5.5％-6％、大豆卵磷脂1.8％-2.2％、维生素矿物质混合物2％-3％、磷酸二氢钙0.4％-0.6％、氯化胆碱0.4％-0.5％、丙酸钙0.08％-0.12％、乙氧基喹啉0.04％-0.06％。在投喂该大菱鲆饲料之后使用本发明营养性修复剂4周-8周，使用添加量剂量为2％-3％，能够显著减轻大菱鲆的肠道炎性症状，修复肠道。
另一种具体的含有玉米蛋白粉的大菱鲆饲料及与本发明营养性修复剂的配合使用方式。该饲料中使用玉米蛋白粉来替代部分鱼粉。其一种具体配方为：鱼粉35％-45％、玉米蛋白粉25％-30％、小麦粉20％-24％、鱼油5.5％-6.5％、大豆卵磷脂1.8％-2.2％、维生素矿物质混合物2％-3％、磷酸二氢钙0.4％-0.6％、氯化胆碱0.4％-0.5％、丙酸钙0.08％-0.12％、乙氧基喹啉0.04％-0.06％、微晶纤维素1％-2％。在投喂该大菱鲆饲料之后使用本发明营养性修复剂4周-8周，使用添加量剂量为1.5％-2.5％，能够显著减轻大菱鲆的肠炎症状，修复肠道。
本发明的大菱鲆营养性肠炎修复剂在投喂含有引起大菱鲆肠炎的抗营养因子以及植物蛋白源的饲料后使用，能够显著减轻大菱鲆肠炎的发生、修复肠道。包括显著提高肠道绒毛高度、降低肠道绒毛粘连、降低固有层及黏膜下层炎性细胞的浸润、增加上皮细胞核上吸收空泡。而且相比采用使用抗生素等药物治疗，本发明的大菱鲆营养性肠炎修复剂更加安全、可靠。
附图说明
图1：分值为1的鱼类肠道绒毛H&E染色结构图；
图2：分值为10的鱼类肠道绒毛H&E染色结构图；
图3：分值为1的鱼类肠道绒毛固有层H&E染色结构图；
图4：分值为10的鱼类肠道绒毛固有层H&E染色结构图；
图5：分值为1的鱼类肠道绒毛粘膜下层H&E染色结构图；
图6：分值为10的鱼类肠道绒毛粘膜下层H&E染色结构图。
具体实施方式
下面结合附图对本发明进行详细的描述。
实施例1大菱鲆肠炎病营养性修复剂组分的筛选
申请人在研究中发现，如果只是简单的将已报到的肠道保护剂应用于使用植物蛋白取代鱼粉的大菱鲆饲料中，其修复肠炎的效果并不理想。因此以能够为肠道细胞提供能量、提高肠道黏膜修复能力为原则，筛选获得了本发明的营养性修复剂组分。
以鱼粉为主要蛋白源，小麦粉为主要糖源，鱼油为主要的脂肪源，配制大菱鲆全鱼粉饲料，作为基础饲料。将谷氨酰胺(G)、精氨酸(A)以及苏氨酸(T)3种氨基酸进行组合，添加到基础饲料中，设计7种修复剂的实验饲料，分别简写为G、A、T、G+A、G+T、A+T和G+A+T，3种氨基酸的添加量为1％。另外在基础饲料中添加1.0％的大豆皂甙作为引起大菱鲆肠炎的饲料，具体见表1。养殖设施采用300L玻璃钢桶，每桶放养30尾大菱鲆鱼苗，起始重量8.5克左右。每个桶作为一个重复，每种饲料设三个重复。共设计8个实验处理，8个实验处理的大菱鲆先投喂大豆皂甙组饲料4周，再投喂基础饲料及7种实验饲料8周。以投喂基础饲料的大菱鲆作为对照组。
养殖过程中流水养殖，充气。在为期12周的养殖试验中，海水的溶氧在7.8-8.2mg/L，pH 7.5-8.0，盐度28-30，温度14-16℃。每天投喂2次，分别在07:00、19:00，饱食投喂。
表1：实验饲料成分表
饲养试验结束后，每个实验用桶随机抽取20尾鱼，解剖获得鱼体肠道样品，人工去除脂肪组织，将肠道样品于PBS溶液中清洗以后，取1厘米左右的组织块用10％福尔马林溶液中固定24h后，转移到70％的酒精溶液中，放置于4℃冰箱中保存。肠道组织石蜡包埋切片采用常规方法进行。经H&E染色、封片后，于显微镜下进行观察。观察指标包括肠道绒毛高度、肠道绒毛粘连水平、固有层宽度、固有层细胞(白细胞浸润、结缔组织增生)、粘膜下层宽度、粘膜下层细胞(白细胞浸润、结缔组织增生)、上皮细胞细胞核上空泡、上皮细胞细胞核位置。对每个指标进行打分，分值范围1-10。打分说明见表2。打分前将样品顺序打乱，打分过程通过盲评的方式。
表2：肠道组织结构评价分数体系
实验结果采用SPSS 13.0软件进行分析。数据采用多因素方差分析，P<0.05时认为差异显著。
实验结果如下表3所示。
表3：实验处理对大菱鲆肠道组织炎性结构变化的影响
结论：由实验结果可见，在大菱鲆肠炎发病后饲喂大菱鲆肠道炎症修复剂能够显著减轻大菱鲆的肠道炎症症状。包括显著提高肠道绒毛高度、降低肠道绒毛粘连、降低固有层及黏膜下层炎性细胞的浸润、增加上皮细胞核上吸收空泡。在饲喂的大菱鲆肠道炎症修复剂中，以G+A+T处理组，即谷氨酰胺、精氨酸及苏氨酸的组合修复效果最为显著。因此筛选此组合作为大菱鲆肠炎病修复剂的组分。
实施例2大菱鲆肠炎病营养性修复剂组分配比的筛选
以鱼粉为主要蛋白源，小麦粉为主要糖源，鱼油为主要的脂肪源，配制大菱鲆全鱼粉饲料，作为基础饲料。将谷氨酰胺(G)、精氨酸(A)及苏氨酸(T)3种氨基酸按照1:1:1、2:1:1、1:2:1和1:1:2的比例添加到基础饲料中，添加总量为3％，设计4种修复剂的实验饲料，分别简写为GAT(1:1:1)、GAT(2:1:1)、GAT(2:1:1)和GAT(1:1:2)。另外以基础饲料中添加1.0％的大豆皂甙作为引起大菱鲆肠炎的饲料，具体见表3。养殖设施采用300L玻璃钢桶，每桶放养30尾大菱鲆鱼苗，起始重量8.5克左右。每个桶作为一个重复，每种饲料设三个重复。共设计5个实验处理，5个实验处理的大菱鲆先投喂大豆皂甙组饲料4周，再投喂基础饲料及4种实验饲料8周。以投喂基础饲料的大菱鲆作为对照组。
养殖过程中流水养殖，充气。在为期12周的养殖试验中，海水的溶氧在7.8-8.2mg/L，pH 7.5-8.0，盐度28-30，温度14-16℃。每天投喂2次，分别在07:00、19:00，饱食投喂。
表4：本实施例实验饲料成分表
饲养试验结束后，每个实验用桶随机抽取20尾鱼，解剖获得鱼体肠道样品，人工去除脂肪组织，将肠道样品于PBS溶液中清洗以后，取1厘米左右的组织块用10％福尔马林溶液中固定24h后，转移到70％的酒精溶液中，放置于4℃冰箱中保存。肠道组织石蜡包埋切片采用常规方法进行。经H&E染色、封片后，于显微镜下进行观察。观察指标包括肠道绒毛高度、肠道绒毛粘连水平、固有层宽度、固有层细胞(白细胞浸润、结缔组织增生)、粘膜下层宽度、粘膜下层细胞(白细胞浸润、结缔组织增生)、上皮细胞细胞核上空泡、上皮细胞细胞核位置。对每个指标进行打分，分值范围1-10。打分说明见实施例1的表2。打分前将样品顺序打乱，打分过程通过盲评的方式。
实验结果采用SPSS 13.0软件进行分析。数据采用多因素方差分析，P<0.05时认为差异显著。
实验结果如下
表5：实验处理对大菱鲆肠道组织炎性结构变化的影响
结论：由实验结果可见，在大菱鲆肠炎发病后饲喂大菱鲆肠道炎症修复剂能够显著减轻大菱鲆的肠道炎症症状。包括显著提高肠道绒毛高度、降低肠道绒毛粘连、降低固有层及黏膜下层炎性细胞的浸润、增加上皮细胞核上吸收空泡。在饲喂的大菱鲆肠道炎症修复剂中，以GAT(2:1:1)处理组，即谷氨酰胺、精氨酸及苏氨酸的组合比例为2:1:1修复效果最佳。因此筛选此比例作为大菱鲆肠炎病修复剂的配比。
实施例3大菱鲆肠炎病修复剂对豆粕引起的肠炎的修复效果评价及浓度筛选
以鱼粉为主要蛋白源，小麦粉为主要糖源，鱼油为主要的脂肪源，配制大菱鲆全鱼粉饲料，作为基础饲料。将谷氨酰胺(G)、精氨酸(A)及苏氨酸(T)按照2:1:1的比例进行组合，添加到基础饲料中，添加浓度为2％、3％和4％，设计3种不同浓度修复剂的实验饲料，分别简写为GAT2.0、GAT3.0和GAT4.0。另外在基础饲料中添加40％的豆粕替代鱼粉作为豆粕组的饲料，具体见表5。养殖设施采用300L玻璃钢桶，每桶放养30尾大菱鲆鱼苗，起始重量8.5克左右。每个桶作为一个重复，每种饲料设三个重复。共设计4个实验处理，4个实验处理的大菱鲆先投喂豆粕组饲料4周，再投喂基础饲料及3种实验饲料8周。以投喂基础饲料的大菱鲆作为对照组。
养殖过程中流水养殖，充气。在为期12周的养殖试验中，海水的溶氧在7.8-8.2mg/L，pH 7.5-8.0，盐度28-30，温度14-16℃。每天投喂2次，分别在07:00、19:00，饱食投喂。
表6：实验饲料成分表
饲养试验结束后，每个实验用桶随机抽取20尾鱼，解剖获得鱼体肠道样品，人工去除脂肪组织，将肠道样品于PBS溶液中清洗以后，取1厘米左右的组织块用10％福尔马林溶液中固定24h后，转移到70％的酒精溶液中，放置于4℃冰箱中保存。肠道组织石蜡包埋切片采用常规方法进行。经H&E染色、封片后，于显微镜下进行观察。观察指标包括肠道绒毛高度、肠道绒毛粘连水平、固有层宽度、固有层细胞(白细胞浸润、结缔组织增生)、粘膜下层宽度、粘膜下层细胞(白细胞浸润、结缔组织增生)、上皮细胞细胞核上空泡、上皮细胞细胞核位置。对每个指标进行打分，分值范围1-10。打分说明见实施例1的表2。打分前将样品顺序打乱，打分过程通过盲评的方式。
实验结果采用SPSS 13.0软件进行分析。数据采用多因素方差分析，P<0.05时认为差异显著。
表7：实验处理对大菱鲆肠道组织炎性结构变化的影响
结论：由实验结果可见，在大菱鲆肠炎发病后饲喂大菱鲆肠道炎症修复剂能够显著减轻由豆粕引起的大菱鲆的肠道炎症症状，修复肠道。包括显著提高肠道绒毛高度、降低肠道绒毛粘连、降低固有层及黏膜下层炎性细胞的浸润、增加上皮细胞核上吸收空泡。在饲喂的大菱鲆肠道炎症修复剂处理组中，以3.0％的添加浓度修复效果最为显著。因此在修复豆粕引起的大菱鲆肠炎中使用该修复剂的浓度为3.0％。
实施例4大菱鲆肠炎病修复剂对玉米蛋白粉引起的肠炎的修复效果评价及浓度筛选
以鱼粉为主要蛋白源，小麦粉为主要糖源，鱼油为主要的脂肪源，配制大菱鲆全鱼粉饲料，作为基础饲料。将谷氨酰胺(G)、精氨酸(A)及苏氨酸(T)按照2:1:1的比例进行组合，添加到基础饲料中，添加浓度为1.0％、2.0％和3.0％，设计3种不同浓度修复剂的实验饲料，分别简写为GAT1.0、GAT2.0和GAT3.0。另外在基础饲料中添加26％的玉米蛋白粉替代鱼粉作为玉米蛋白粉组的饲料，具体见表5。养殖设施采用300L玻璃钢桶，每桶放养30尾大菱鲆鱼苗，起始重量8.5克左右。每个桶作为一个重复，每种饲料设三个重复。共设计4个实验处理，4个实验处理的大菱鲆先投喂玉米蛋白粉组饲料4周，再投喂基础饲料及3种实验饲料8周。以投喂基础饲料的大菱鲆作为对照组。
养殖过程中流水养殖，充气。在为期12周的养殖试验中，海水的溶氧在7.8-8.2mg/L，pH 7.5-8.0，盐度28-30，温度14-16℃。每天投喂2次，分别在07:00、19:00，饱食投喂。
表8：实验饲料成分表
饲养试验结束后，每个实验用桶随机抽取20尾鱼，解剖获得鱼体肠道样品，人工去除脂肪组织，将肠道样品于PBS溶液中清洗以后，取1厘米左右的组织块用10％福尔马林溶液中固定24h后，转移到70％的酒精溶液中，放置于4℃冰箱中保存。肠道组织石蜡包埋切片采用常规方法进行。经H&E染色、封片后，于显微镜下进行观察。观察指标包括肠道绒毛高度、肠道绒毛粘连水平、固有层宽度、固有层细胞(白细胞浸润、结缔组织增生)、粘膜下层宽度、粘膜下层细胞(白细胞浸润、结缔组织增生)、上皮细胞细胞核上空泡、上皮细胞细胞核位置。对每个指标进行打分，分值范围1-10。打分说明见实施例1的表2。打分前将样品顺序打乱，打分过程通过盲评的方式。
实验结果采用SPSS 13.0软件进行分析。数据采用多因素方差分析，P<0.05时认为差异显著。
实验结果如下
表9：实验处理对大菱鲆肠道组织炎性结构变化的影响
结论：由实验结果可见，在大菱鲆肠炎发病前饲喂大菱鲆肠道炎症修复剂能够显著减轻由玉米蛋白粉引起的大菱鲆肠炎的发生、修复肠道。包括显著提高肠道绒毛高度、降低肠道绒毛粘连、降低固有层及黏膜下层炎性细胞的浸润、增加上皮细胞核上吸收空泡。在饲喂的大菱鲆肠道炎症修复剂处理组中，以2.0％的添加浓度修复效果最为显著。因此在修复玉米蛋白粉引起的大菱鲆肠炎中使用该修复剂的浓度为2.0％。
Claims (9)
1.一种大菱鲆肠炎病的营养性修复剂，其特征在于，所述的包含有谷氨酰胺、精氨酸和苏氨酸；其中谷氨酰胺、精氨酸和苏氨酸质量百分比的组成如下：谷氨酰胺40％-60％，精氨酸20％-30％，苏氨酸20％-30％。
2.如权利要求1所述的营养性修复剂，其特征在于，所述的谷氨酰胺、精氨酸及苏氨酸的比例为2:1:1。
3.权利要求1或2所述的营养性修复剂在制备大菱鲆饲料中的应用。
4.一种大菱鲆饲料，其特征在于，所述的饲料中添加了权利要求1或2所述的营养性修复剂。
5.如权利要求4所述的大菱鲆饲料，其特征在于，所述的饲料配方如下：鱼粉54％-58％、小麦粉26％-30％、鱼油1.5％-2.5％、大豆卵磷脂1.5％-2.5％、维生素和矿物质混合物2％-3％、氯化胆碱0.4％-0.6％、丙酸钙0.8％-1.2％、乙氧基喹啉0.04％-0.06％、微晶纤维素4.8％-4.9％；营养性修复剂使用添加量为2％-4％。
6.一种使用豆粕来部分替代鱼粉的大菱鲆饲料，其特征在于，所述的饲料中添加了2％-3％的权利要求1或2所述的营养性修复剂。
7.如权利要求6所述的大菱鲆饲料，其特征在于，所述的饲料的配比如下：鱼粉38％-42％、豆粕28％-32％、小麦粉17％-19％、鱼油5.5％-6％、大豆卵磷脂1.8％-2.2％、维生素矿物质混合物2％-3％、磷酸二氢钙0.4％-0.6％、氯化胆碱0.4％-0.5％、丙酸钙0.08％-0.12％、乙氧基喹啉0.04％-0.06％；2％-3％的权利要求1或2所述的营养性修复剂。
8.一种使用玉米蛋白粉来部分替代鱼粉的大菱鲆饲料，其特征在于，所述的饲料中添加了1.5％-2.5％的权利要求1或2所述的营养性修复剂。
9.如权利要求6所述的大菱鲆饲料，其特征在于，所述的饲料的配比如下：鱼粉35％-45％、玉米蛋白粉25％-30％、小麦粉20％-24％、鱼油5.5％-6.5％、大豆卵磷脂1.8％-2.2％、维生素矿物质混合物2％-3％、磷酸二氢钙0.4％-0.6％、氯化胆碱0.4％-0.5％、丙酸钙0.08％-0.12％、乙氧基喹啉0.04％-0.06％、微晶纤维素1％-2％、1.5％-2.5％的权利要求1或2所述的营养性修复剂。
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