Beyond tannins, exploring the complexity of grape seed using non targeted analysis and molecular network

Abstract

Grape seeds are an important part of grape, especially in the context of red wine. They are also frequently used as markers of grape maturity and as contributors to wine quality. The aim of this study is to explore the diversity of grape seeds compounds and to partially characterize their evolution during ripening. Data for this study were collected from bunches sampled at different stages of maturity (from bunch closure to 2 weeks after harvest) in 2024. Whole grape bunch were frozen, and the seeds were then extracted from the berries. For three key sampling dates (bunch closure, end of veraison and harvest), the grape seeds were dissected into the three main layer, external layer (cuticle, epidermis, outer integument), middle layer (middle and inner integument) and endosperm. Samples were ground and extracted for 24 h in methanol, then filtered and dried under vacuum. They were the re-dissolved to the same concentration (1 mg/mL), filtered and analyzed by HPLC MS/MS. All results were used to build a molecular network using the fragmentation pattern and retention time of the molecules (1), using the software MetGem (2). MetGem database and manual interpretation of fragmentation pattern recognition were used to annotate compounds of interest within the molecular networks. Two molecular networks were built separately: one including seed, skin and pulp, to identify compound specific to seed or shared with other berry tissues, and a second one using only data from the dissected seed to locate compounds of interest in the different seed layers. Thanks to the molecular networking approach, cluster (group of related compounds) are formed on the basis of MS2 spectrum similarity, which make it easier to annotate molecules grouped together if at least one appears in a database. For example, epicatechin gallate, a well-known grape seeds compound, is grouped with epicatechin vanillate (recently described in grape seeds) and other potential epicatechin derivatives that have not previously been reported in grape seed before. This cluster found in our second molecular network also provides information on the localization on these compounds. The same process was applied to all compound detected in grape seed to obtain a comprehensive list of annotated (database hits) and putative (good fragmentation pattern and consistent molecular formula) grape seed compound.

References

Watrous, J et al. (2012). Mass spectral molecular networking of living microbial colonies. Proc. Natl. Acad. Sci. U. S. A., 109 (26), E1743–E1752.

Olivon, F et al. (2018). MetGem Software for the Generation of Molecular Networks Based on the t-SNE Algorithm. Anal. Chem., 90 (23), 13900–13908.