可可果莢是可可豆加工後留下的果實外殼，常被當成農業廢棄物，若無適當處理，對環境影響極大。可可果莢具有多種生物活性，若開發為免疫刺激物提升水產養殖生物的免疫抗病力，不只解決其處理的問題，還能幫助養殖產業的發展。本研究以四種不同的方式製備可可果莢萃取物：熱水萃取新鮮果莢萃取物 (CPE)、乙醇萃取新鮮果莢果膠 (CPP)、熱水萃取烘乾果莢萃取物 (DPE) 和乙醇萃取烘乾果莢的果膠 (DPP)。以注射的方式進行南美白蝦 (Litopenaeus vannamei) 免疫反應因子、病原感染、溫度緊迫、先天免疫相關和先天免疫訊號傳導基因等試驗。在溶藻弧菌攻擊抵抗力方面，注射劑量CPE 於40 μg shrimp-1、CPP 於12　μg shrimp-1 、DPE 於20 μg shrimp-1和DPP 於6　μg shrimp-1在實驗144小時的存活率比注射生理食鹽水的控制組分別顯著提升23.3%、20.0%、20.0%和23.3%；低溫緊迫耐受性方面，同上述注射劑量，於14℃、96小時的存活率也分別顯著高於控制組23.4%、16.7%、26.6%和23.3%；與proPO系統相關的LGBP、PE、proPO I 或proPO II基因表現量均顯著提升；與免疫訊息傳遞路徑相關基因方面，CPE和DPP二者都可增強南美白蝦TLR1和TLR3基因表現，僅CPP上調其TLR2的表現量。綜合而言，在proPO系統方面，除DPE的proPO I有顯著提升以外，其他三種萃取物都是 proPO II的表現量顯著提升，亦即顆粒細胞無法同時完整表達原酚氧化酵素 (proPO) 相關基因。而總血球數 (THCs)、酚氧化酵素 (PO)活性、呼吸爆 (RBs)、吞噬作用、LGBP和PE均顯著提升，這說明，可可果莢萃取物均可促進血球的再生釋放，進而強化酚氧化酵素活性和提高呼吸爆，並通過提升原酚氧化酵素系統的活性來增強吞噬作用。

Cacao pod husk is the residues after cacao beans processing. It is often treated as agricultural waste. Without proper treatment, it puts a severe impact on the environment. Cacao pod husk has a variety of biological activities. If developed as an immunostimulant to enhance the immune effects of aquaculture organisms, not only will it solve the problem of its treatment, but also promote the development of the aquaculture industry. In this study, three different methods were used for the preparation of extracts: fresh cacao pod husk extract (CPE) by hot water extraction, fresh cacao pod husk pectin (CPP) by ethanol extraction, Dried cacao pod husk extract (DPE) by hot water extraction, and dried cacao pod husk pectin (DPP) by ethanol extraction. Experiments of Litopenaeus vannamei injected with extracts on immune response factors, pathogenic infection, temperature stress, innate immunity-related and signaling pathways-related genes were studied. The results showed the resistance against Vibrio alginolyticus attacks, the survival rates of L. vannamei injected with CPE on 40 μg shrimp-1, CPP on 12 μg shrimp-1, DPE on 20 μg shrimp-1, and DPP on 6 μg shrimp-1 at the 144 hours of the experiments were significantly higher than those of the control group injected with saline by 23.3%, 20.0%, 20.0%and 23.3%. The results of anti-hypothermal stress tests with the same groupings and dosage protocol designed as above vibrio infection also showed the survival rate at 14°C and 96 hours were also significantly higher than that of the control group by 23.4%, 16.7%, 26.6% and 23.3%. Regarding to the proPO system, LGBP, PE, proPO I or proPO II were all significantly improved; as for signaling pathways-related genes, both CPE and DPP can enhance the expression of TLR1 and TLR3 genes, but only CPP can up-regulate the expression of TLR2. In summary, L. vannamei injected with the DPE was found with significant difference in the expression of proPO I, but the other three extracts were only showed with significant difference in the expression of proPO II; that means the granulocytes might not completely express all proPO at the same time. Total haemocyte counts (THCs), PO activity, respiratory burst (RBs), phagocytosis, LGBP, PE and part of proPO were all significantly raised, which showed that cacao pod extracts can promote the generation and release of heamocytes, thereby strengthening PO activity and improving RBs; and enhance phagocytosis by increasing the activity of the proPO system.

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