冰温贮藏对玉露香梨果心褐变的影响及代谢组学分析

Effect of ice temperature storage on core browning of Yuluxiang pear based on metabolomics analysis

【目的】探讨冰温(-1℃)贮藏下玉露香梨果心差异代谢物及其对褐变的影响机制。【方法】利用超高效液相色谱-质谱法对Y-I-C(-1℃,贮藏240 d)和Y-S-C(0℃,贮藏240 d)处理组进行非靶向代谢组学分析,通过主成分分析、偏最小二乘判别分析和KEGG富集等多元统计,筛选组间果心差异代谢物,结合贮藏期间生理变化探讨冰温对果心褐变的影响。【结果】玉露香梨采后果心共鉴定出331种代谢产物,包括黄酮、苯丙烷类、萜类、糖及糖醇、生物碱、有机酸、脂质、氨基酸、醌类、单宁、核苷酸等类物质。相对于Y-C(采后0 d)处理,Y-S-C处理组果心共有27种差异代谢物显著下调,4种物质上调。与0℃贮藏相比,-1℃贮藏延缓了果心多酚氧化酶活性的上升及总酚、总黄酮的氧化,抑制了果心褐变。Y-I-C与Y-S-C处理组筛选出50种显著差异代谢物,Y-I-C处理组上调的26种物质主要为苯丙烷类、黄酮类、有机酸类等物质,差异代谢物富集在代谢通路、氨基酸代谢、次生代谢产物的生物合成、碳水化合物代谢等途径。【结论】-1℃冰温贮藏下邻苯二酚、熊果苷、柠檬酸等物质的上调增强了果实的抗氧化性,并通过相关途径协同影响果心氧化代谢。

【Objective】Due to the factors such as harvest maturity and storage conditions,the fruit core of Yuluxiang pear is prone to browning during long-term storage,affecting fruit quality after storage.Ice temperature storage is a storage method to store fruits under the temperature between 0℃and freezing point,which can effectively delay fruit metabolism,reduce occurrence of fruit browning,maintain the color of fruit core and fruit quality of Yuluxiang pear.Metabolomics can detect and analyze the types,quantities,and changes of metabolites to find relative relationship between metabolites and physiological and pathological changes during a specific physiological period and under specific conditions.Therefore,untargeted metabolomics with metabolic profiling analysis can be used to analyze the changes in metabolites during browning process of Yuluxiang pear,and further explore the impact of ice temperature technology on fruit core browning to provide theoretical reference for clarifying the mechanism and controlling of pear browning.【Methods】The fruits of Yuluxiang pear with soluble solid content of 11.5%-12.5%were selected as the test material using near-infrared fruit non-destructive detector.After pre-cooling,the fruits were packaged in high permeability CO2 polythene fresh keeping bag and stored at(-1.0±0.5)℃and(0.0±0.5)℃,respectively.Regular sampling was conducted to measure the core browning index,total phenols,total flavonoids and polyphenol oxidase enzyme activity at every 60 days of cold storage.Each sample was represented by three biological replicates.The ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)was used to perform untargeted metabolomics analysis on the fruit core of Yuluxiang pear after 240 days stored at-1℃and 0℃.The metabolomics samples were Y-C(0 day post harvest),Y-S-C[stored at(0.0±0.5)℃for 240 days],Y-I-C[stored at(-1.0±0.5)℃for 240 days].Using multivariate statistics such as principal component analysis(PCA),and partial least squares-discriminant analysis(PLS-DA),the kyoto encyclopedia of genes and genomes(KEGG)database,differential metabolites and metabolic pathways were screened and analyzed.Combining with the determination of physiological indexes,the effect of ice temperature on pear core browning was analyzed.【Results】The core browning occurred in cold storage at 0℃on 120th day after harvest and the browning symptoms only occurred in the fruit corn during storage period.With the increase of polyphenol oxidase activity,the total phenol content in the core of Yuluxiang pear at 0℃gradually decreased,and the total flavonoid content also showed a downward trend.The ice temperature storage at-1℃delayed the decrease in total phenolic and total flavonoid content,maintained a certain level of antioxidant capacity,inhibited the increase in polyphenol oxidase activity,and lightened core browning.The metabolomics analysis revealed that a total of 331 metabolites with different relative contents were obtained in the core tissue of Yuluxiang pear before and after storage,which were mainly classified into flavonoids(72),phenylpropanoids(56),terpenes(44),sugars and polyols(40),alkaloids(34),organic acids(15),lipids(13),amino acids and their derivatives(10),tannins(4),quinones(4),nucleotides(2)and other unclassified compounds(37).Among them,the higher relative content of metabolites were organic acids,sugars and polyols and alkaloids.In the Y-S-C vs Y-C samples,31 differentially expressed metabolites were identified based on a variable importance in the projection(VIP)>1.0 and t-test<0.05,among them,4 metabolic compounds were up-regulated and 27 metabolic compounds were down-regulated.Compared with Y-C treatment,phenylpropanoid substances such as protocatechuic acid,catechol,dendrophenol and ferulic acid were down-regulated after cold storage at 0℃,and organic acids such as trifluoroacetic acid,2-isopropylmalic acid,and methylsuccinic acid were down-regulated,sugars and polyols,and flavonoids were all down-regulated as well.These differential metabolites were mapped to 11 KEGG pathways,among these pathways,valine,leucine and isoleucine biosynthesis,pyruvate metabolism,and fructose and mannose pathways were significantly enriched.50 species differentially expressed metabolites were identified in Y-I-C vs Y-S-C samples,the numbers of up-regulated and down-regulated metabolites were 26 and 24 respectively.There were 11 phenylpropanoids,among them,catechol,arbutin,protocatechuicaldehyde,quinic acid,and ferulic acid were up-regulated in expression levels when stored at-1℃compared with 0℃.There are 7 organic acid substances,among them,malic acid,2-isopropylmalic acid,trifluoroacetic acid,and citric acid were up-regulated in expression levels when stored at-1℃compared with 0℃.Other types included sugars and polyols(8),terpenes(6),flavonoids(5),lipids(3),amino acid(2),alkaloids(2),quinone(1)and others(5).The KEGG enrichment pathway enriched 50 significantly different metabolites in 25 metabolic pathways,among them,the more concentrated pathways included metabolic pathways,amino acid metabolism,biosynthesis of other secondary metabolites,carbohydrate metabolism,as well as lipid metabolism,metabolism of cofactors and vitamins,environmental information processing,metabolism of terpenoids and polyketides,membrane transport and other pathways.【Conclusion】The ice temperature storage(-1℃)delayed the increase of polyphenol oxidase activity and the oxidation of total phenols and flavonoids,and inhibited core browning of Yuluxiang pear.50 species differentially expressed metabolites were screened out in the pear core,and 26 substances were mainly upregulated,including phenylpropanoids,flavonoids,organic acids,etc.Through KEGG metabolic pathway enrichment analysis,it was found that they significantly contributed to metabolic pathways,amino acid metabolism,and biosynthesis of other secondary metabolites.In addition,TCA cycle,glycolysis/gluconeogenesis,and fatty acid degradation were also involved.The ice temperature treatment increased the antioxidant capacity of the fruits by up-regulating substances such as catechol,arbutin,and citric acid and synergistically affected the oxidative metabolism of the fruit core through related pathways.