Identification of marker compounds for predicting browning of fresh-cut lettuce using untargeted UHPLC-HRMS metabolomics

https://doi.org/10.1016/j.postharvbio.2021.111626Get rights and content

Highlights

  • Metabolomic profiles of lettuce changed over time after cutting.

  • Twelve Metabolites associated with browning potential were identified.

  • The identified metabolites could be used as biomarkers in breeding programs.

Abstract

Enzymatic browning negatively impacts product quality and shelf-life of packaged fresh-cut lettuce. Metabolite profiles of lettuce are affected by the browning process. The purpose of this study was to identify metabolomic marker compounds to predict lettuce browning, which could be applied to discern accessions suited for commercial production and industrial breeding programs. Romaine lettuce with different browning susceptibilities were evaluated in two independent trials and growing seasons. Metabolites were analyzed using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS). Principal component analysis (PCA) was performed to visualize clusters, trends, and discriminative ion features. Seven metabolites, including quinic acid, caffeoylquinic acid, 3-hydroxytetradecanedioic, cichorioside B, 8-deacetylmatricarin-8-sulfate, dicaffeoylquinic acid and 9S,12S,13S-trihydroxy-10Z-octadecenoic acid, increased with storage time (day 0 vs. day 3) Three metabolites, including lactucopicrin-15-oxalate, tri-4-hydroxyphenylacetyl glucoside and 15-deoxylactucin-8-sulfate, decreased with storage time (day 0 vs. day 3). Two additional phenolic metabolites, dicaffeoyltartaric and caffeoyltartaric acids, were identified as potential marker compounds, whose presence on day 0 samples immediately after cutting was negatively correlated with browning development (represented by ΔHue). The identified metabolites help to elucidate the biochemical metabolism and pathways during enzymatic browning and have the potential to serve as marker compounds for predicting browning resistant accessions.

Introduction

Lettuce (Lactuca sativa L.) is one of the most important fresh vegetable crops worldwide, and the major ingredient of packaged “ready-to-eat” fresh-cut vegetable salads. However, the development of browning discoloration on the cut edges of leaf ribs often results in the loss of product shelf-life (He and Luo, 2007; Zhou et al., 2004). Browning on cut lettuce is a complex process and is often caused by the enzyme-catalyzed oxidation of phenolic compounds (Taranto et al., 2017). It is believed that phenolic metabolite composition of lettuce is related to the browning process. During cutting, tissue injury induces activation of latent polyphenol oxidase (PPO) and leads to increased levels of phenolic compounds, which are the substrates of PPO (Cantos et al., 2001; Luna et al., 2016a). This information suggests that the phenolic composition has a major effect on browning of cut surfaces after processing. An increase in phenylalanine ammonia-lyase (PAL) activity, which occurs in response to wounding is the key factor in the biosynthesis of phenolic PPO substrates (García et al., 2017). It has been suggested that lettuce storage life is related to the activity of PAL, thus, various approaches such as heat shock or inhibitor treatment have targeted PAL to slow browning development (Choi et al., 2005; Saltveit, 2000; Tomás-Barberán et al., 1997).

A variety of methods have been used to prevent or slow down enzymatic browning of lettuce. During commercial operation, browning is often minimized by controlling oxygen levels inside the packages, via the combination of initial nitrogen flush and the use of package film with limited oxygen transmission rate (Ji et al., 2005). However, this process is costly, and tiny pinholes in the packages can lead to severe browning and loss of shelf-life. Numerous studies have also been conducted on methods other than modified atmosphere packaging. Yan et al. reported ethanol treatment as an effective approach to inhibit enzymatic browning and microbial growth on lettuce stem discs (Yan et al., 2015). Acetic acid also inhibited PAL activity and the production of wound-induced phenolics (Tomás-Barberán et al., 1997). Unfortunately, effective anti-browning agents that have both inhibition activity and compatibility with commonly used sanitizers are hard to find. Furthermore, it is worth mentioning that some chemicals used in research that may effectively inhibit browning, may not meet food safety standards, while others can impart undesirable sensory effects to vegetables (Barrett and Garcia, 2002).

Metabolomics is a comprehensive metabolic profiling approach that enables analysis of a wide range of metabolite classes simultaneously in a non-biased manner (Cevallos-Cevallos et al., 2009; Wolfender et al., 2009). It is an important comparative tool for studying global metabolite levels of plant materials grown under different conditions (Sun et al., 2015). Metabolomics has increasingly been used to optimize selection of advantageous accessions and improve breeding materials (Zivy et al., 2015). Untargeted metabolomic strategies, based on comparing metabolite profiles from different samples using chemometric approaches, can help identify the markers which play the most relevant role in various conditions (pre- or post-harvested) (Arapitsas et al., 2014; Pérez et al., 2010). The approach could generate highly sensitive results and facilitate high-throughput data acquisition (Nagana Gowda and Djukovic, 2014). Using this approach, García et al. explored the metabolites in iceberg and romaine lettuce, as well as the metabolic changes that occurred during storage and identified the groups of chlorogenic acids and sinapaldehyde derivatives to have positive and negative correlation, respectively, with browning development (García et al., 2019, 2018, 2017, 2016).

Romaine lettuce is easily spoiled and susceptible to enzymatic browning. In addition, the browning process was reported to occur more rapidly in romaine lettuce than in other types of lettuce, such as iceberg (Martínez-Sánchez et al., 2011). Thus, numerous approaches have been developed to control romaine lettuce browning, e.g., chemical treatment, and/or physical treatment (Tomás-Barberán et al., 1997; Zhan et al., 2012). In this study, the metabolite profiles of two groups of romaine lettuce accessions harvested in different seasons with each group having varying browning susceptibilities, were examined after cutting and the compounds responsible for browning were identified (Fig. 1). Multivariate analysis was performed to visualize group clustering, trends, or discriminative ion features among the observations. The marker compounds were putatively identified by the associated accurate mass data, MS2 fragments, and published references, together with on-line databases. Hue angle is used as an important browning index to evaluate browning development (Chisari et al., 2007). Thus, correlation between marker ion intensities and browning development (represented by ΔHue) was evaluated within the selected lettuce accessions. The purpose of this study was to identify potential marker compounds that could be applied to select the most desirable accessions both for industrial commercial production and breeding programs.

Section snippets

Chemicals

Caffeoylquinic acid, dicaffeoyltartaric acid and caffeoyltartaric acid were bought from Sigma-Aldrich (St. Louis, MO, USA). Formic acid and high-performance liquid chromatography (HPLC)-grade methanol and acetonitrile were purchased from Fisher Scientific (Waltham, MA, USA). HPLC-grade water was prepared from distilled water using a Milli-Q system (Millipore Lab., Bedford, MA). All other chemicals and reagents were of analytical grade.

Lettuce accessions and tissue preparation

Over 350 accessions of romaine lettuce (Lactuca sativa var.

Browning development represented by hue angle (h°)

In this study, the browning severity of lettuce tissues was evaluated by imaging under standardized conditions and analyzing the color parameters (L*, a*, b* and h°) using an established image processing procedure (Teng et al., 2019). This method greatly improves the efficiency of evaluation and minimizes the subjectivity associated with conventional sensory evaluation. The changes in hue angle (ΔHue) between day 0 and day 3 samples were computed and used to indicate browning potential based on

Conclusion

In conclusion, twelve metabolites were identified as potential marker compounds for romaine lettuce browning. Among them, three (caffeoylquinic acid, 9S,12S,13S-trihydroxy-10Z-octadecenoic acid and caffeoyltartaric acid) agree well with previous reports (García et al., 2017), while the other nine metabolites are proposed for the first time in romaine lettuce. These include quinic acid, 3-hydroxytetradecanedioic, cichorioside B, 8-deacetylmatricarin-8-sulfate, dicaffeoylquinic acid,

Author statement

Zhihao Liu: Methodology, Data analysis, Writing - Original draft preparation.

Jianghao Sun: Data analysis, Writing - review & editing.

Zi Teng: Methodology, Data analysis, Writing - review & editing

Yaguang Luo: Funding acquisition, Project administration, Writing - review & editing.

Liangli Yu: Writing - review & editing.

Ivan Simko: Resources, Writing - review & editing.

Pei Chen: Supervision, Project administration, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This research was supported by the Agricultural Research Service of the U.S. Department of Agriculture and an Interagency Agreement with the Office of Dietary Supplements, National Institute of Health, Department of Health and Human Services. This research was partially supported by the USDA-NIFA Specialty Crop Research Initiative (Award No 2015-51181-24283. The authors wish to thank Dr. Hui Peng and Ms. Rebecca Zhao at USDA-ARS in Salinas CA for their kind assistance in lettuce growing and

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