Integrated metabolomic and microbiomic analysis of the effects of dietary Candida utilis replacement of fishmeal on the growth, physiology, and intestinal microbiota of southern catfish (Silurus meridionalis)

Liver samples of southern catfish were sent to Majorbio Bio-pharm Technology Co., Ltd. for untargeted metabolomic sequencing referring to previous study [27]. Grinding beads were added to the liver samples, and metabolites were extracted via low-temperature ultrasound using an extraction solution (methanol:water = 4:1, v/v) containing four internal standards (including L-2-chlorophenylalanine at 0.02 mg/mL). The samples were then incubated at -20℃ for 30 min, centrifuged at 13,000 g for 15 min at 4℃, and the supernatant was transferred to injection vials with inner liners for instrumental analysis. Quality control (QC) samples were prepared by mixing equal volumes of metabolites from all samples, and one QC sample was inserted every 5–10 samples during instrumental analysis to evaluate the repeatability of the entire analytical process. LC-MS analysis was performed on a Thermo Fisher Scientific ultra-high-performance liquid chromatography-tandem Fourier transform mass spectrometry (UHPLC-Orbitrap Exploris 240) system. After instrumental analysis, the raw LC-MS data were imported into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) for baseline filtering, peak detection, integration, retention time correction, and peak alignment. Finally, a data matrix including retention time, mass-to-charge ratio (m/z), and peak intensity was obtained. Meanwhile, LC-MS spectral information was matched against public metabolomic databases (HMDB: http://www.hmdb.ca/; Metlin: https://metlin.scripps.edu/) and Majorbio’s in-house database to acquire metabolite information.

本研究参照已有文献[27]的方法，将南方鲶的肝脏样本送至上海美吉生物医药科技有限公司（Majorbio Bio-pharm Technology Co., Ltd.）进行非靶向代谢组学测序（untargeted metabolomic sequencing）。向肝脏样本中加入研磨珠，采用含4种内标（包括浓度为0.02 mg/mL的L-2-氯苯丙氨酸）的提取液（甲醇:水=4:1，体积比），通过低温超声法提取代谢物。随后将样本置于-20℃下孵育30分钟，再于4℃、13000 g条件下离心15分钟，取上清液转移至带有内衬管的进样小瓶中，以待仪器分析。质量控制（Quality Control, QC）样本通过混合所有样本的等量代谢物制备得到；在仪器分析过程中，每5~10个样本插入1个QC样本，以评估整个分析流程的重复性。采用赛默飞世尔科技（Thermo Fisher Scientific）的超高效液相色谱-串联傅里叶变换质谱（UHPLC-Orbitrap Exploris 240）系统进行液相色谱-质谱联用（Liquid Chromatography-Mass Spectrometry, LC-MS）分析。仪器分析完成后，将原始LC-MS数据导入代谢组学分析软件Progenesis QI（沃特世公司，美国米尔福德）进行基线过滤、峰识别、积分、保留时间校正及峰对齐操作。最终得到包含保留时间、质荷比（mass-to-charge ratio, m/z）及峰强度的数据矩阵。同时，将LC-MS谱图信息与公共代谢组学数据库（HMDB：http://www.hmdb.ca/；Metlin：https://metlin.scripps.edu/）及美吉生物的内部数据库进行匹配，以获取代谢物相关信息。