冷藏过程中原料乳脂质代谢变化的靶向脂质组学分析

Targeted lipidomic analysis of the changes in lipid metabolism of raw milk during refrigeration

为探究原料乳在4℃冷藏过程中的脂质变化,从而指导乳及乳制品的后期加工,该研究采用超高效液相色谱-三重四级杆复合线性离子阱质谱技术分别对冷藏第0、2、3、4、6天的原料乳脂质进行绝对定性定量分析。结果表明,原料乳共检出20种脂质亚类、880种脂质分子,其中甘油三脂、磷脂酰乙醇胺、磷脂酰胆碱亚类含量最高;以P值<0.05,变量重要性投影(variable importance projection,VIP)>1为标准共筛选出420种显著性差异脂质代谢物,以P值<0.01,VIP>1为标准共筛选出98种极显著性差异脂质代谢物。整个冷藏过程中,极显著性差异脂质分子含量呈下降趋势,其中第3天与第4天对比组(D3 vs D4)脂质变化最为明显,冷藏3~4 d是原料乳脂质变化的关键阶段。对D3vs D4组的极显著差异脂质进行京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)通路分析,10种脂质分子被注释到6条代谢途径中,其中磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰肌醇、磷脂酰基丝氨酸被注释到甘油磷脂代谢及神经酰胺、鞘磷脂被注释到鞘脂代谢是该阶段的主要代谢途径。研究结果为探明原料乳冷藏过程中的脂质变化、划定脂肪水解的关键期提供理论依据,进而为后期乳制品的加工提供数据参考。

Milk and dairy products are often used as one of the most important sources of nutrients in recent years.Among them,the raw milk is required to be refrigerated at 4℃for several days before processing.However,the nutrients can vary greatly in the raw milk with the refrigeration period.Especially,a series of biochemical reactions can still occur in lipids,thus resulting in the deterioration of the quality of raw milk.This study aimed to investigate the lipid changes in the raw milk during refrigeration at 4℃.The targeted lipidomic analysis of raw milk was performed on the 0th,2nd,3rd,4th,and 6th days of refrigeration using UHPLC-QTRAP-MS/MS.The results showed that a total of 20 lipid subclasses and 880 lipid molecules were detected in the raw milk.Among them,TG(triglycerides),PE(phosphatidylethanolamine),and PC(phosphatidylcholine)were the most abundant subclasses.Furthermore,the content of most lipid molecules fluctuated repeatedly and then gradually decreased during the periods of refrigeration.A few lipids showed complex changes with no clear pattern during refrigeration.PCA analysis revealed that there was a difference in the lipid composition among the five groups of samples during refrigeration.A total of 420 significantly different lipid metabolites were obtained at P<0.05,and VIP>1.Namely,141,36,227,and 16 significantly different lipid species were identified for the four pairs of raw milk samples(D0 vs D2,D2 vs D3,D3 vs D4,and D4 vs D6,respectively).Among them,the comparative groups D0 vs D2 and D3 vs D4 shared the more significantly different lipid species,where the subclasses of these lipids were richer.Furthermore,a total of 98 highly significant different lipids were screened by P<0.01,VIP>1,where 71 and 27 highly significant different lipids were screened in each of the comparison groups D0 vs D2 and D3 vs D4,corresponding to 2 and 9 subclasses,respectively.There were no highly significant different lipids in the comparison groups D2 vs D3 and D4 vs D6.Subsequently,the lipid molecules upregulated or down-regulated in the D0 vs D2 and D3 vs D4 groups were screened by FC>1.5 or FC<0.67,indicating outstanding trends.The raw milk was mainly decomposed by TG(triglyceride)at the initial stage of refrigeration(0-2 d),due to the low-temperature environment and the presence of antibacterial substances in the milk.There were fewer microorganisms in the raw milk.Endogenous enzymes were attributed to the down-regulation of TG in the raw milk,such as lipoprotein lipase.Also,a variety of functional lipids were down-regulated in the middle stage of refrigeration(3-4 d),such as PE(phosphatidylethanolamine),PC(phosphatidylcholine),Cer(ceramide),resulting in a dramatic reduction of raw milk quality.At the same time,the microorganisms grew rapidly as well.The dominant bacteria in raw milk(such as Pseudomonas and Flavobacterium)produced the lipase and protease,and then decomposed the milk fat globules,finally releasing the lipids and endogenous enzymes in them.Additionally,the resulting lipolytic enzymes interacted with the endogenous enzymes,leading to the downregulation of functional lipids at this stage.The content of highly significant different lipid molecules showed an overall decreasing trend over the refrigeration,with the most outstanding lipid changes in the comparison group D3 vs D4.Therefore,3-4 d of refrigeration was a critical stage for the lipid changes in the raw milk.Meanwhile,KEGG pathway analysis of the highly significant different lipids in the group D3 vs D4 showed that 10 lipid molecules were annotated to six metabolic pathways.Among them,PC(phosphatidylcholine),PE(phosphatidylethanolamine),PI(phosphatidylinositol),and PS(phosphatidylserine)were annotated to glycerophospholipid metabolism.Cer(ceramide)and SM(sphingomyelin)were annotated to sphingolipid metabolism.Both of them were attributed to the main metabolic pathways for 3-4 d refrigeration.