使用益生菌增加烏魚的成長表現、免疫反應以及對奴卡氏菌的抵抗能力

Abstract

水產養殖對供應人類蛋白質的重要性日益增加，因此需要追求更高的產量，造成養殖密度的提高，使魚類養殖環境惡化，並衍生出細菌性疾病的爆發，導致水產養殖業巨大的經濟損失。過去研究證實使用益生菌作為魚類飼料補充劑是增強免疫反應和提高存活率的重要策略之一。烏魚在台灣是一種相當普遍養殖的一種養殖物種，也有相當高的經濟價值。烏魚養殖中，Nocardia seriolae是一種常見的病原菌，其爆發常會造成烏魚高比例的死亡，且奴卡氏菌症常常是一種慢性的感染，病徵不明顯，不容易防治，因此本研究旨在使用益生菌來讓烏魚更健康並提高奴卡氏菌症的抗性。本實驗先以體外方式鎖定有潛力的益生菌，篩選出較能於腸胃道環境下存活、具生物安全性，並能產生消化酶和抗菌能力的益生菌。最終篩選出Lactobacillus rhamnosus、Lactobacillus reuteri和Bacillus Subtilis natto作為飼料添加劑，分別混合成含1*109 CFU/g益生菌濃度的飼料，投餵給烏魚苗吃，經過28天的投餵後，以奴卡氏菌攻毒。研究結果顯示，有餵食益生菌的組別生長表現比控制組來的好，在餵食L. reuteri和B. Subtilis natto的組別生長表現有顯著性的提升；益生菌在免疫基因的調控結果顯示，在餵食L. rhamnosus和L. reuteri的組別中的促發炎因子IL-1β和TNF-α都有顯著性的上升，而其他抗奴卡相關基因中，F3在L. rhamnosus和B. subtilis natto 的組別表現量都有顯著上升，TNFRSF25在餵食L. reuteri 和B. subtilis natto組別表現量有顯著性的上升，MMP13只有在L. rhamnosus組別有上升，結果顯示益生菌在免疫基因的調控都有不同程度的幫助，且餵食28天的免疫調控比14天來的好。餵食益生菌的組別在攻毒實驗中，存活率比沒有餵食益生菌的控制組來的高，其中L. rhamnosus顯著提升烏魚對N. seriolae抵抗能力。有鑑於此，這三株益生菌可進一步作為烏魚養殖中對抗奴卡氏菌症策略之基礎。

The product of aquaculture is more and more important in protein supply for human. The production of aquaculture is increasing leading to a high breeding density and deterioration fish farming environment. Therefore, the outbreak of bacterial diseases occurs more frequently and causes huge economic losses in the aquaculture industry. For resolving the problem, using probiotics as a fish feed supplement is an efficient strategy. Grey mullet is a common cultured species with high economic value in Taiwan. Nocardia seriolae is a pathogen of Nocardia disease causing high mortality in grey mullet. Nocardia disease often has long incubation period; thus how to prevent it is still a difficult task. The study aimed to protect grey mullet against N. seriolae by probiotic supplement. Firstly, in vitro trials were used to select the potential probiotics that had good resistance in the simulated gastrointestinal tract environment. Moreover, biosafety, secretion of digestive enzymes, and antibacterial capabilities were demonstrated. Accordingly, Lactobacillus rhamnosus, Lactobacillus reuteri and Bacillus Subtilis natto were selected for further in vivo experiment. The concentration of 1*109 CFU/g of the probiotics was mixed with the feed and fed to grey mullet fry. After 28 days of feeding, the results showed that the growth performance of the group fed with probiotics was better than that of the control group, especially the B. Subtilis natto groups. The pro-inflammatory genes, IL-1β and TNF-α, were induced in the groups fed L. rhamnosus and L. reuteri. Among the anti- N. seriolae -related genes, F3 was significantly increased in the L. rhamnosus and B. subtilis natto groups, and TNFRSF25 was significantly increased in the L. reuteri and B. subtilis natto groups. Moreover, the expression level of MMP13 was only enhanced in the L. rhamnosus group. The results revealed that probiotics were helpful in the regulation of immune genes to varying degrees, and the immune regulation of feeding for 28 days was better than that for 14 days. Finally, the survival rate was higher than in the probiotic treated groups than control group, which indicated that the probiotics could improve the resistance of grey mullet to N. seriolae. In conclusion, the present study suggested that L. rhamnosus and L. reuteri, and B. subtilis could be appropriate candidates for N. seriolae protection.