Analysis of Quality Changes in Mulberry Fruit at Three Developmental Stages Using Targeted Metabolomics Based on LC‒MS

Mulberry fruits are popular in many countries because of their good taste, high nutritional value and medicinal properties. Studying the metabolic profile during fruit development can enhance our comprehension of the changes in fruit quality. In this study, a widely targeted metabolomic approach utilizing liquid chromatography‒mass spectrometry (LC‒MS) was employed to investigate the widespread metabolic changes during fruit development and ripening in mulberry fruits and combined with multivariate statistical methods to screen for significant changed metabolites (SCMs) at different developmental stages. A total of 981 metabolites in 12 categories were detected in mulberry fruit at three developmental stages (i.e., the fruit expansion stage in which mulberry fruits are green (MG), the fruit color change stage in which they are red (MR) and the fruit ripening stage in which they are purple (MP)). There were significant changes in the metabolic profile at the color change and ripening stages compared to the fruit expansion stage. Flavonoids primarily increased during fruit ripening and accumulated significantly during the mature stage. Large amounts of phenolic acids, lipids, and organic acids accumulated significantly during fruit expansion stage and decreased significantly during ripening. However, sugars accumulated significantly during ripening and were found to be associated with the sweetness of mulberry fruit. Assessment of the levels of different sugar and acid components during fruit development revealed that fructose and glucose were the main sugars, and citric and malic acids were the main organic acids. Meanwhile, the sugar-acid ratio increased significantly, which in turn improved the flavor quality of mulberry fruit. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the SCMs were mainly enriched in metabolic pathways such as galactose metabolism, biosynthesis of secondary metabolites and flavonoid biosynthesis. The reliability of the metabolomics data was demonstrated through the validation of six genes encoding key enzymes using quantitative real-time PCR (qRT‒PCR). The results of this study provide valuable insights into the rational utilization of mulberry fruit resources and the efficient development of functional fruit products.