微胶囊饲料的研制及对日本对虾仔稚幼体消化生理影响研究
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摘要
海水仔稚幼体赖以生存的生物活饵由于营养不全面、生产成本高、易携带病原菌、供给不稳定等缺点与不足,成为制约海水养殖产业化发展的瓶颈。微粒饲料不仅能有效弥补生物活饵的缺点与不足,而且质量和产量均可有效保证,更适合工厂化育苗生产。
本论文的主要研究内容如下:
1.采用喷雾干燥法、复合凝聚法、锐孔凝固浴法、流化床法制备微胶囊饲料,对微胶囊的物理性能进行分析比较。喷雾干燥法采用的壁材分别为明胶、明胶和麦芽糊精复合物,大部分微胶囊饲料粒径小于178μm,加工过程包膜维生素C的保留率仅为20.51%。壁材明胶、明胶和麦芽糊精复合物两种微胶囊的脂类包埋率分别为26.59%、18.07%,氮保留率分别为36.03%、24.93%。微观形态显示喷雾干燥法有较好的包埋效果,以明胶为壁材的微胶囊表面有褶皱,而以明胶和麦芽糊精为壁材的微胶囊表面有孔洞。复合凝聚法制备的壁材明胶和阿拉伯胶复合物的微胶囊出现粘连,微观形态没有明显的微胶囊效果,脂类包埋率和氮保留率分别为26.37%、27.47%,不适合于制备芯材组成复杂的微胶囊饲料。锐孔凝固浴法制备的微胶囊饲料粒径较大,表面粗糙凹陷,且有大量的裂缝,缺乏工业生产设备,难以大量制备微胶囊饲料。流化床法先将基础饲料通过挤出-滚圆法制成微丸,后于流化床底喷包衣制备成壁材乙基纤维素的微胶囊饲料,饲料形态规则,粒径合适,表面为均匀光滑一致的包衣膜,包衣效果良好,微胶囊饲料的包含率为97.2±1.7%,脂类包埋率为63.2±3.7%。在35.0‰的NaCl溶液中250-420μm的微胶囊饲料浸泡20 min、40 min、60 min氮保留率分别为73.6±2.6%、65.8±3.5%、53.7±4.2%。
2.分别以明胶和乙基纤维素作为包衣壁材,流化床方法制备微胶囊饲料,饲料表面的微观形态为连续、致密的包衣膜,包衣完整,包衣效果明显。加工过程维生素C的保留率为88.2%。微胶囊饲料的粒径合适、沉降速度慢,分散性能好。壁材明胶微胶囊饲料、壁材乙基纤维素微胶囊饲料的产率分别为97.3%、98.2%;包含率分别为92.2%、95.8%;脂类包埋率分别为76.8%、85.3%;浸入35.0‰的NaCl溶液1 h溶出的游离氨基酸总量分别占游离氨基酸总量的87.14%、82.82%;pH值分别为5.98、6.24。壁材乙基纤维素微胶囊饲料在不同体系(pH、温度)的缓释性能优于壁材明胶微胶囊饲料。
3.将微胶囊饲料饲喂10日龄的日本对虾糠虾Ⅱ期幼体20天,养殖试验分为四组:对照组饲喂50%虾片+50%卤虫;试验组Ⅰ饲喂50%壁材明胶的微胶囊饲料+25%虾片+25%卤虫;试验组Ⅱ饲喂100%壁材明胶的微胶囊饲料;试验组Ⅲ饲喂100%壁材乙基纤维素微胶囊饲料。各试验组幼体均能摄食饲料,试验组Ⅲ幼体有着最佳的生长效果,体重增长313.4%,而对照组体重增长仅为213.4%。与对照组相比,微胶囊饲料组(试验组Ⅱ、试验组Ⅲ)的日本对虾幼体在体重、全长和成活率方面均有显著性提高。试验组Ⅱ与试验组Ⅲ相比,日本对虾幼体的体重和成活率差异不显著,但全长差异显著。微胶囊饲料组的幼体与对照组相比较,蛋白酶活力、淀粉酶活力有显著性差异,但是碱性磷酸酶活力没有显著性差异。试验组Ⅱ和试验组Ⅲ相比,淀粉酶活力和碱性磷酸酶活力差异不显著,但胰蛋白酶活力差异显著。本研究中壁材明胶、乙基纤维素对日本对虾幼体消化酶(胰蛋白酶、淀粉酶、碱性磷酸酶)的活力没有显著性的影响,说明了幼体的消化酶活力主要受饲料中的营养成分调控。
4.组织学切片观察各试验组日本对虾幼体的肠道和肝胰脏结构,中肠管壁组织由内向外依次分成四层结构,分别是纹状缘、粘膜层、粘膜下层、肌层,肝胰脏主要结构有分泌细胞、吸收细胞和消化腺,相比较,未见异常,不同壁材的微胶囊饲料对消化道的结构无明显影响,能满足幼体消化系统的正常发育。各试验组的日本对虾肠道上皮细胞未见异常。日本对虾幼体肠道吸收营养成分的重要方式是胞饮作用。对各试验组日本对虾幼体中肠的营养状态微观结构发现,营养成分从少到多,依次为对照组、试验组Ⅰ、试验组Ⅱ、试验组Ⅲ,证实微胶囊饲料在消化道中有着明显的缓释作用,壁材乙基纤维素微胶囊比壁材明胶微胶囊缓释作用更强。
5.在蛋白含量相同的前提下,改变芯材蛋白源(酪蛋白和鱼粉)的比例,以了解幼体对蛋白源的可选择性,分别对饲料和幼体的氨基酸进行测定,对幼体的生长和消化酶活力分析发现,饲料配方中酪蛋白含量的增加降低了幼体的生长性状和消化酶活力。饲料配方中鱼粉含量的增加要优于酪蛋白含量的增加。在粗脂肪相同的前提下,以鱼油取代饲料配方中的裂壶藻粉,分别对饲料和幼体的脂肪酸组成进行分析比较,日本对虾幼体的脂肪酸18:2n-6的含量能反映饲料的脂肪酸18:2n-6含量差异。鱼油替代裂腹藻粉,能促进幼体的生长和消化酶活力的提高。
Live food is a key bottleneck to the culture of crustacean larvae and marine fish larvae. Live food poses many problems for the larviculture such as risk of pathogen transmission, expense of culture and problems with availability. The ultimate solution to the above problems is development of microdiet to partially or completely replace live food. Microdiets can provide a reliable and reproducible feed supply.
The mian contents of this research are as follows:
1. Different methods to prepare the microencapsulated diet. Gelatin and gelatin/maltodextrin as wall material were adopted to prepare the microencapsulated diet by spray-drying respectively. The frequency distributions of the microencapsulated diets were in normal distribution. The retention efficiency of coated Vitamin C was 20.51% during the spray drying process. SEM images showed the gelatin-walled microcapsules and gelatin/maltodextrin-walled microcapsules with a uniform film around the core. The lipid encapsulation efficiency of gelatin-walled, gelatin/maltodextrin-walled was 26.59%, 18.07% respectively , the nitrogen retention efficiency of the microencapsulate diet was 36.03%,24.93% respectively. The gelatin/gum-walled microencapsulated diet was prepared by coacervation. SEM images showed the diets were not with a uniform film and adhere each other. The lipid encapsulation efficiency and nitrogen retention efficiency of microencapsulated diets is 26.37%,27.47%. Coacervation is not adapted to prepare the microencapsulated diet. The calcium-alginate microencapsulated diet was prepared by pore-coagulation bath method. SEM images showed there are some depressions and many small cracks on the microcapsule coarse surface. The pore-coagulation bath method was not adapted to industrialized production. The basal diet as core material was made into pellets by extrusion-spheronization, then the pellets were put into the fluidized bed, adopting the mode of bottom spray coating to complete the coating process. SEM images showed that the appearance of the diet microencapsulated with ethyl cellulose with a uniform surface and a continuous film around the core. The inclusion efficiency, lipid encapsulation efficiency of the microencapsulated diet was 97.2±1.7%, 63.2±3.7% respectively. The nitrogen retention efficiency of the microencapsulated diet incubation in 35‰NaCl solution for 20 min, 40 min and 60 min was 73.6±2.6%, 65.8±3.5% and 53.7±4.2% respectively.
2. The diets microencapsulated with gelatin and ethyl cellulose for larval shrimp (Penaeus japonicus) were produced using fluidized bed coating process. The two microencapsulated diets were within a broad size range. The size of the diet microencapsulated with ethyl cellulose was smaller than that of the diet microencapsulated with gelatin. SEM images showed the appearance of the microencapsulated diets was a dense film with a superior physical quality. The retention efficiency of vitamin C was 88.2% in the coating process. Despite the less amount of wall material used, the diet microencapsulated with ethyl cellulose had a better performance with regard to production efficiency, lipid encapsulation efficiency and free amino acid retention efficiency compared with the diet microencapsulated with gelatin. The pH value of the diet microencapsulated with gelatin and ethyl cellulose was 5.98, 6.24 respectively. The diet microencapsulated with ethyl cellulose had better performance with slow release characteristic in different solution (pH, temperature) compared with the diet microencapsulated with gelatin.
3. The mysisⅡlarval shrimp (Penaeus japonicus) 10 days after hatch (DAH) were fed different diets respectively for 20 days. There were four treatments. Control: 50% shrimp flake+50% Artemia; GroupⅠ: 50% diet microencapsulated with gelatin + 25% shrimp flake +25% Artemia; GroupⅡ: 100% diet microencapsulated with gelatin; GroupⅢ: 100% diet microencapsulated with ethyl cellulose. The growth and survival of the larval shrimp confirmed the microencapsulated diets with good digestibility. There were significant differences in growth and survival, trypsin activity, and amylase activity of the larvae between the two microencapsulated diet groups and the control (P<0.05), but there was no significant difference in alkaline phosphatase activity in the larvae of each group (P>0.05). There were significant differences in total length and trypsin activity of the larvae between the two microencapsulated diet groups (P<0.05). The results showed that the larval digestive enzymes adapted to the feed composition, and the wall materials had no significant effect on the digestive enzymes activity of the larval shrimp.
4. The microscopic structure of the intestine was classified as four layers, striated border, mucosa, submucosa and muscular layer. There were many goblet cells and columnar cells in the hepatopancreas. No available information about the differentiation of the intestine and hepatopancreas of the shrimp larvae from different treatments was detected under histological analysis. A large basal nucleus, abundant mitochondria, some zymogen granules and lipid droplets in intestine epithelium were visible. The big mitochondria with large number mainly scattered at the top of the cells. The pinocytotic activity of the gut enterocytes for their intracellular digestion.The nutrient components in intermediate intestine of shrimp larvae decreased gradually in the order of GroupⅢ, GroupⅡ, GroupⅠand the control. Wall material had no significant effect on the digestive system and the microencapsulated diets with slow and controlled release characteristic in the digestive tract of the larval shrimp.
5. On the condition that the protein content is same, change the composition of amino acid in the feed formulation by changing the the content of casein and fish meal. The amino acid of the diet and the larval shrimp were compared. Increasing the content of casein and decreasing the content of fish meal in the feed formulation decreased the growth performace of the larval shrimp and digestive enzyme activity. On the condition that the fat content is same, fish oil substitute the Schizochytrium algal meal in the feed formulation. The composition and content of fatty acid of the diet and larval shrimp were compared. The fatty acid 18:2n-6 showed a dramatic proportional increase in larval tissue relative to its proportional composition in both the live and formulated diets. Fish oil substitute Schizochytrium algal meal in the feed formulation can improve growth performance and digestive enzyme activity of the larval shrimp.
本论文的主要研究内容如下:
1.采用喷雾干燥法、复合凝聚法、锐孔凝固浴法、流化床法制备微胶囊饲料,对微胶囊的物理性能进行分析比较。喷雾干燥法采用的壁材分别为明胶、明胶和麦芽糊精复合物,大部分微胶囊饲料粒径小于178μm,加工过程包膜维生素C的保留率仅为20.51%。壁材明胶、明胶和麦芽糊精复合物两种微胶囊的脂类包埋率分别为26.59%、18.07%,氮保留率分别为36.03%、24.93%。微观形态显示喷雾干燥法有较好的包埋效果,以明胶为壁材的微胶囊表面有褶皱,而以明胶和麦芽糊精为壁材的微胶囊表面有孔洞。复合凝聚法制备的壁材明胶和阿拉伯胶复合物的微胶囊出现粘连,微观形态没有明显的微胶囊效果,脂类包埋率和氮保留率分别为26.37%、27.47%,不适合于制备芯材组成复杂的微胶囊饲料。锐孔凝固浴法制备的微胶囊饲料粒径较大,表面粗糙凹陷,且有大量的裂缝,缺乏工业生产设备,难以大量制备微胶囊饲料。流化床法先将基础饲料通过挤出-滚圆法制成微丸,后于流化床底喷包衣制备成壁材乙基纤维素的微胶囊饲料,饲料形态规则,粒径合适,表面为均匀光滑一致的包衣膜,包衣效果良好,微胶囊饲料的包含率为97.2±1.7%,脂类包埋率为63.2±3.7%。在35.0‰的NaCl溶液中250-420μm的微胶囊饲料浸泡20 min、40 min、60 min氮保留率分别为73.6±2.6%、65.8±3.5%、53.7±4.2%。
2.分别以明胶和乙基纤维素作为包衣壁材,流化床方法制备微胶囊饲料,饲料表面的微观形态为连续、致密的包衣膜,包衣完整,包衣效果明显。加工过程维生素C的保留率为88.2%。微胶囊饲料的粒径合适、沉降速度慢,分散性能好。壁材明胶微胶囊饲料、壁材乙基纤维素微胶囊饲料的产率分别为97.3%、98.2%;包含率分别为92.2%、95.8%;脂类包埋率分别为76.8%、85.3%;浸入35.0‰的NaCl溶液1 h溶出的游离氨基酸总量分别占游离氨基酸总量的87.14%、82.82%;pH值分别为5.98、6.24。壁材乙基纤维素微胶囊饲料在不同体系(pH、温度)的缓释性能优于壁材明胶微胶囊饲料。
3.将微胶囊饲料饲喂10日龄的日本对虾糠虾Ⅱ期幼体20天,养殖试验分为四组:对照组饲喂50%虾片+50%卤虫;试验组Ⅰ饲喂50%壁材明胶的微胶囊饲料+25%虾片+25%卤虫;试验组Ⅱ饲喂100%壁材明胶的微胶囊饲料;试验组Ⅲ饲喂100%壁材乙基纤维素微胶囊饲料。各试验组幼体均能摄食饲料,试验组Ⅲ幼体有着最佳的生长效果,体重增长313.4%,而对照组体重增长仅为213.4%。与对照组相比,微胶囊饲料组(试验组Ⅱ、试验组Ⅲ)的日本对虾幼体在体重、全长和成活率方面均有显著性提高。试验组Ⅱ与试验组Ⅲ相比,日本对虾幼体的体重和成活率差异不显著,但全长差异显著。微胶囊饲料组的幼体与对照组相比较,蛋白酶活力、淀粉酶活力有显著性差异,但是碱性磷酸酶活力没有显著性差异。试验组Ⅱ和试验组Ⅲ相比,淀粉酶活力和碱性磷酸酶活力差异不显著,但胰蛋白酶活力差异显著。本研究中壁材明胶、乙基纤维素对日本对虾幼体消化酶(胰蛋白酶、淀粉酶、碱性磷酸酶)的活力没有显著性的影响,说明了幼体的消化酶活力主要受饲料中的营养成分调控。
4.组织学切片观察各试验组日本对虾幼体的肠道和肝胰脏结构,中肠管壁组织由内向外依次分成四层结构,分别是纹状缘、粘膜层、粘膜下层、肌层,肝胰脏主要结构有分泌细胞、吸收细胞和消化腺,相比较,未见异常,不同壁材的微胶囊饲料对消化道的结构无明显影响,能满足幼体消化系统的正常发育。各试验组的日本对虾肠道上皮细胞未见异常。日本对虾幼体肠道吸收营养成分的重要方式是胞饮作用。对各试验组日本对虾幼体中肠的营养状态微观结构发现,营养成分从少到多,依次为对照组、试验组Ⅰ、试验组Ⅱ、试验组Ⅲ,证实微胶囊饲料在消化道中有着明显的缓释作用,壁材乙基纤维素微胶囊比壁材明胶微胶囊缓释作用更强。
5.在蛋白含量相同的前提下,改变芯材蛋白源(酪蛋白和鱼粉)的比例,以了解幼体对蛋白源的可选择性,分别对饲料和幼体的氨基酸进行测定,对幼体的生长和消化酶活力分析发现,饲料配方中酪蛋白含量的增加降低了幼体的生长性状和消化酶活力。饲料配方中鱼粉含量的增加要优于酪蛋白含量的增加。在粗脂肪相同的前提下,以鱼油取代饲料配方中的裂壶藻粉,分别对饲料和幼体的脂肪酸组成进行分析比较,日本对虾幼体的脂肪酸18:2n-6的含量能反映饲料的脂肪酸18:2n-6含量差异。鱼油替代裂腹藻粉,能促进幼体的生长和消化酶活力的提高。
Live food is a key bottleneck to the culture of crustacean larvae and marine fish larvae. Live food poses many problems for the larviculture such as risk of pathogen transmission, expense of culture and problems with availability. The ultimate solution to the above problems is development of microdiet to partially or completely replace live food. Microdiets can provide a reliable and reproducible feed supply.
The mian contents of this research are as follows:
1. Different methods to prepare the microencapsulated diet. Gelatin and gelatin/maltodextrin as wall material were adopted to prepare the microencapsulated diet by spray-drying respectively. The frequency distributions of the microencapsulated diets were in normal distribution. The retention efficiency of coated Vitamin C was 20.51% during the spray drying process. SEM images showed the gelatin-walled microcapsules and gelatin/maltodextrin-walled microcapsules with a uniform film around the core. The lipid encapsulation efficiency of gelatin-walled, gelatin/maltodextrin-walled was 26.59%, 18.07% respectively , the nitrogen retention efficiency of the microencapsulate diet was 36.03%,24.93% respectively. The gelatin/gum-walled microencapsulated diet was prepared by coacervation. SEM images showed the diets were not with a uniform film and adhere each other. The lipid encapsulation efficiency and nitrogen retention efficiency of microencapsulated diets is 26.37%,27.47%. Coacervation is not adapted to prepare the microencapsulated diet. The calcium-alginate microencapsulated diet was prepared by pore-coagulation bath method. SEM images showed there are some depressions and many small cracks on the microcapsule coarse surface. The pore-coagulation bath method was not adapted to industrialized production. The basal diet as core material was made into pellets by extrusion-spheronization, then the pellets were put into the fluidized bed, adopting the mode of bottom spray coating to complete the coating process. SEM images showed that the appearance of the diet microencapsulated with ethyl cellulose with a uniform surface and a continuous film around the core. The inclusion efficiency, lipid encapsulation efficiency of the microencapsulated diet was 97.2±1.7%, 63.2±3.7% respectively. The nitrogen retention efficiency of the microencapsulated diet incubation in 35‰NaCl solution for 20 min, 40 min and 60 min was 73.6±2.6%, 65.8±3.5% and 53.7±4.2% respectively.
2. The diets microencapsulated with gelatin and ethyl cellulose for larval shrimp (Penaeus japonicus) were produced using fluidized bed coating process. The two microencapsulated diets were within a broad size range. The size of the diet microencapsulated with ethyl cellulose was smaller than that of the diet microencapsulated with gelatin. SEM images showed the appearance of the microencapsulated diets was a dense film with a superior physical quality. The retention efficiency of vitamin C was 88.2% in the coating process. Despite the less amount of wall material used, the diet microencapsulated with ethyl cellulose had a better performance with regard to production efficiency, lipid encapsulation efficiency and free amino acid retention efficiency compared with the diet microencapsulated with gelatin. The pH value of the diet microencapsulated with gelatin and ethyl cellulose was 5.98, 6.24 respectively. The diet microencapsulated with ethyl cellulose had better performance with slow release characteristic in different solution (pH, temperature) compared with the diet microencapsulated with gelatin.
3. The mysisⅡlarval shrimp (Penaeus japonicus) 10 days after hatch (DAH) were fed different diets respectively for 20 days. There were four treatments. Control: 50% shrimp flake+50% Artemia; GroupⅠ: 50% diet microencapsulated with gelatin + 25% shrimp flake +25% Artemia; GroupⅡ: 100% diet microencapsulated with gelatin; GroupⅢ: 100% diet microencapsulated with ethyl cellulose. The growth and survival of the larval shrimp confirmed the microencapsulated diets with good digestibility. There were significant differences in growth and survival, trypsin activity, and amylase activity of the larvae between the two microencapsulated diet groups and the control (P<0.05), but there was no significant difference in alkaline phosphatase activity in the larvae of each group (P>0.05). There were significant differences in total length and trypsin activity of the larvae between the two microencapsulated diet groups (P<0.05). The results showed that the larval digestive enzymes adapted to the feed composition, and the wall materials had no significant effect on the digestive enzymes activity of the larval shrimp.
4. The microscopic structure of the intestine was classified as four layers, striated border, mucosa, submucosa and muscular layer. There were many goblet cells and columnar cells in the hepatopancreas. No available information about the differentiation of the intestine and hepatopancreas of the shrimp larvae from different treatments was detected under histological analysis. A large basal nucleus, abundant mitochondria, some zymogen granules and lipid droplets in intestine epithelium were visible. The big mitochondria with large number mainly scattered at the top of the cells. The pinocytotic activity of the gut enterocytes for their intracellular digestion.The nutrient components in intermediate intestine of shrimp larvae decreased gradually in the order of GroupⅢ, GroupⅡ, GroupⅠand the control. Wall material had no significant effect on the digestive system and the microencapsulated diets with slow and controlled release characteristic in the digestive tract of the larval shrimp.
5. On the condition that the protein content is same, change the composition of amino acid in the feed formulation by changing the the content of casein and fish meal. The amino acid of the diet and the larval shrimp were compared. Increasing the content of casein and decreasing the content of fish meal in the feed formulation decreased the growth performace of the larval shrimp and digestive enzyme activity. On the condition that the fat content is same, fish oil substitute the Schizochytrium algal meal in the feed formulation. The composition and content of fatty acid of the diet and larval shrimp were compared. The fatty acid 18:2n-6 showed a dramatic proportional increase in larval tissue relative to its proportional composition in both the live and formulated diets. Fish oil substitute Schizochytrium algal meal in the feed formulation can improve growth performance and digestive enzyme activity of the larval shrimp.
引文
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