本研究為了解枯草芽孢桿菌 E20 (Bacillus subtilis E20)調節白蝦免疫之機制，因此本實驗分為兩組，分別為不添加 B. subtilis E20的控制組及含109 CFU kg-1 B. subtilis E20的益生菌組 (E20)，並投餵白蝦八週後，採集肝胰腺並利用氫譜核磁共振對水溶性代謝物進行分析。結果顯示共有18種代謝產物被指認，其中有4種代謝產物有顯著差異 (p<0.05)，分別為副黃嘌呤、3-甲基黃嘌呤、肌酸酐及麩醯胺酸，而經由逆向高效液相層析儀及分光光度計確認後，這4種代謝產物的含量於 E20組中皆顯著高於控制組 (p<0.05)。另一方面，透過次世代定序對兩組白蝦肝胰腺進行轉錄體差異分析，發現 E20組中有許多免疫相關基因表現量顯著高於控制組，如超氧化物歧化酶、絲裂原活化激酶及其上游激酶、溶菌酶、熱休克蛋白70等。此外，E20組中的醣基化路徑相關基因表現量亦有顯著增加或相對控制組有增加趨勢，經由即時聚合酶連鎖反應確認相關基因之表現，發現醣基化路徑相關基因表現包括 Glutamine fructose-6-phosphate aminotransferase及UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase以E20組明顯高於控制組 (p<0.05)。綜合代謝物及轉錄體的分析結果可知，肝胰腺中麩醯胺酸含量增加，可使白蝦體內醣基化基質增加，進而提升白蝦體內蛋白質被醣基化的比率。其中，對免疫相當重要的熱休克蛋白70及熱休克轉錄因子，經即時聚合酶連鎖反應及西方墨點法證實，熱休克蛋白70及熱休克轉錄因子的表現量於 E20組中皆有顯著的增加 (p<0.05)。此外，消化率實驗中，E20組的白蝦對所有胺基酸的吸收皆有提升，其中麩醯胺酸則有顯著性的增加。總結來說，B. subtilis E20可能透過促進白蝦對麩醯胺酸的吸收，進而促進蛋白醣基化的表現，以增加肝胰腺中被醣基化之蛋白量，如熱休克蛋白70等，進而提升白蝦免疫力。

This study examined the mechanisms of action of Bacillus subtilis E20 in activating the immunity of shrimp via dietary administration. The white shrimp were firstly divided into two groups, one group was fed a control diet (a basal diet) and the other fed an E20-containing diet (a basal diet with 109 CFU kg-1 of B. subtilis E20 added). After the 8-week feeding regimen, the composition of hepatopancreas metabolite in shrimp were investiagted using 1H nuclear magnetic resonance (1H NMR) based metabolomic analysis. Results from the 1H NMR analysis revealed that 18 hepatopancreatic metabolites were matched and identified among treatments, of which 4 metabolites were significantly different (p<0.05). In addition, after performing the reverse-phase high-performance liquid chromatography (RP-HPLC) and spectrophotometric analyses, 4 metabolites have been confirmed including hypoxanthine, 3-methylxanthine, creatinine, and glutamine. On the other hand, results from transcriptome analysis indicated a significant increases in immune-related gene expressions in the hepatopancreas of E20-fed shrimp, including superoxidase dismutase (SOD), mitogen-activated protein kinase (MAPK), MAPK kinase 7, lysozyme, and heat shock protein 70 (HSP70). Interestingly, the real-time ploymerase chain reaction (Q-PCR) were also employed to find that glycosylation pathway-related genes, that are able to bind glutamine, in the E20 group increased significantly as compared with the control (p<0.05), including glutamine fructose-6-phosphate aminotransferase (GFPT) for transfer of amino groups on the substrate, and UDP-N-acetylglucosamine-peptide N-acetylglucosaminyl transferase. Both 1H NMR metabolomic-based and next generation sequencing (NGS) analyses demonstrated that increased glutamate in the hepatopancreas may lead to increase of the glycosylation of foodstuff, resulting in increased the rate of protein glycosylation in shrimp. Results from Q-PCR analysis also substantiated that mRNA expression of HSP70 in hepatopancreas of E20-fed shrimp was significantly higher than that of the control (p<0.05). Moreover, the digestibility experiments were conducted, showing that E20-fed shrimp used all glutaric acid then siginicantly converted into glutamate in hepatopancrease. In conclusion, B. subtilis E20 may increase the absorption of glutamine, thereby promoted the process of glycosylation, resulting in increase of the content of glycosylated HSP70 in the hepatopancreas, and then activating white shrimp immunity.

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