基于超高效液相色谱-高分辨质谱法的冷冻过程中猪肉差异物质代谢组学分析

Metabolomics analysis of differential substances in pork during freezing based on ultra performance liquid chromatography-high resolution mass spectrometry

中文关键词:  冷冻猪肉  冻龄标志物  超高效液相色谱-高分辨质谱法  非靶向代谢组学

英文关键词:frozen pork  frozen age marker compounds  ultra performance liquid chromatography-high resolution mass spectrometry  untargeted metabolomics

中文摘要:

      目的 基于超高效液相色谱-高分辨质谱法(ultra performance liquid chromatography-high resolution mass spectrometry, UPLC-HRMS)建立非靶向代谢组学差异物质研究方法, 以筛查猪肉在冷冻过程中的差异性代谢物。方法 比较6种不同溶剂提取的代谢物特征离子的数量, 选择最佳的提取溶剂。应用UPLC-HRMS分析冻龄为0、2、4和6个月猪肉样品的代谢物, 并结合化学计量学和正交偏最小二乘法判别分析(orthogonal partial least squares-discriminant analysis, OPLS-DA)、主成分分析(principal component analysis, PCA)、单变量统计分析和倍数变化、火山图等统计学方法分析不同冻龄时间的样品, 筛选出差异性标志物, 利用质谱数据库定性标志物。结果 5%氨化甲醇(氨水:甲醇=5:95, V:V)为最佳的提取溶剂。OPLS-DA和PCA的结果显示, 冷冻2、4、6个月的猪肉样品与新鲜猪肉样品可以被成功区分。通过Compound Discover软件分析, 在冷冻2、4和6个月时分别鉴定出了3385、3874和4111个代谢物, 通过统计分析和数据库匹配, 在新鲜猪肉与冷冻2、4和6个月猪肉之间分别鉴定出了12、13和13种差异性化合物。其中, 烟酰胺、烟酰胺腺嘌呤二核苷酸、5-脱氧-5-甲硫腺苷、次黄嘌呤、芥酰胺、18-β-甘草次酸6种化合物为各冻龄阶段共有, 且呈现出规律性变化, 可作为判别猪肉冻龄时间的差异性标志物。结论 基于UPLC-HRMS技术的非靶向代谢组学能快速有效地筛查猪肉的冻龄标志性化合物, 本研究为猪肉在冷冻过程的代谢变化规律和质量控制提供了依据。

英文摘要:

      Objective To screen the different metabolites of pork during the freezing process, untargeted metabolomics differential substance study method was established based on ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). Methods The number of characteristic ions of metabolites extracted with 6 kinds of different solvents were compared to select the best extraction solvent. UPLC-HRMS was used to analyze the metabolites of pork samples frozen at 0, 2, 4, and 6 months, then the data of samples of different freezing ages were analyzed to screen out differential markers by chemometrics and statistical methods including orthogonal partial least squares-discriminant analysis (OPLS-DA), principal component analysis (PCA), univariate statistical analysis and fold change, and volcano analysis, and the mass spectrometry database was used for qualitative markers. Results The results showed that 5% ammoniated methanol (ammonia:methanol=5:95, V:V) was the best extraction solvent. OPLS-DA and PCA showed that samples frozen for 2, 4, and 6 months could be successfully distinguished from fresh samples. Through the analysis of Compound Discover software, 3385, 3874 and 4111 metabolites were identified after freezing for 2, 4 and 6 months, respectively. Through statistical analysis and database matching, 12, 13, and 13 differential compounds were identified between fresh meat and pork frozen for 2, 4, and 6 months. Among them, 6 kinds of compounds such as nicotinamide, nicotinamide adenine dinucleotide, 5’-s-methyl-5’-thioadenosine, hypoxanthine, erucamide, 18-β-glycyrrhetinic acid were the common metabolites to each freezing age stage, and they showed regular changes, which could be used as different markers for distinguishing the freezing age of pork. Conclusion Untargeted metabolomics based on UPLC-HRMS can quickly and effectively screen pork for frozen age marker compounds, which provides a basis for the metabolism and quality control of pork during freezing process.