Lipidomics-Based Comparison of Molecular Compositions of Green, Yellow, and Red Bell Peppers

Abstract

Identifying and annotating the molecular composition of individual foods will improve scientific understanding of how foods impact human health and how much variation exists in the molecular composition of foods of the same species. The complexity of this task includes distinct varieties and variations in natural occurring pigments of foods. Lipidomics, a sub-field of metabolomics, has emerged as an effective tool to help decipher the molecular composition of foods. For this proof-of-principle research, we determined the lipidomic profiles of green, yellow and red bell peppers (Capsicum annuum) using liquid chromatography mass spectrometry and a novel tool for automated annotation of compounds following database searches. Among 23 samples analyzed from 6 peppers (2 green, 1 yellow, and 3 red), over 8000 lipid compounds were detected with 315 compounds (106 annotated) found in all three colors. Assessments of relationships between these compounds and pepper color, using linear mixed effects regression and false discovery rate (<0.05) statistical adjustment, revealed 11 compounds differing by color. The compound most strongly associated with color was the carotenoid, β-cryptoxanthin (p-value = 7.4 × 10^-5; FDR adjusted p-value = 0.0080). These results support lipidomics as a viable analytical technique to identify molecular compounds that can be used for unique characterization of foods.

Keywords: Capsicum annuum; bell pepper; foodomics; lipidomics; liquid chromatography mass spectrometry (LC/MS); metabolomics; β-cryptoxanthin.

Figure 1

Pepper color is the main driver of differences between pepper conditions. (A) Principal component analysis (PCA) was performed in Agilent Technologies Mass Profiler Professional Version 14.1 (MPP) using data from all bell pepper samples. Component 1, which explains 15% of the variation, is shown on the x-axis; component 2, which explains 11.2% of the variation, is shown on the y-axis; and component 3, which explains 7.3% of the variation, is shown on the z-axis. (B) Hierarchical clustering of data from 23 replicates from six individual bell peppers. The x-axis corresponds to individual compounds detected in the peppers, which are grouped by color and listed on the y-axis. Blue lines indicate less relative abundance of a compound compared to the other 8174 compounds, while red lines indicate higher relative abundance compared to the other 8174 compounds. The vertical distance between compounds provides a rough estimation of their similarity. (C) Venn diagram illustrates overlap between the 2623 compounds detected in all colors of pepper samples (center section), the 1229 compounds detected in green peppers (green circle), but not red or yellow; the 200 compounds in both green and yellow; and the 1076 compounds in both green and red pepper. Within the yellow peppers (yellow circle), 324 compounds were detected in yellow but not red or green and 810 compounds were detected in both yellow and red bell peppers. Red peppers contained 1813 compounds that were detected in red (red circle), but not green or yellow bell peppers.

Figure 2

Relative abundance of β-cryptoxanthin detected in bell pepper samples by color. Following analysis of pepper samples using untargeted LC/MS, β-cryptoxanthin levels were compared according to their relative abundance across samples. (A) Red bell pepper has significantly higher levels compared to green and yellow. *** p < 0.001; (B) To determine whether cooking had any impact, each pepper was divided, three samples were heated for 5 min, and two samples remained raw. There was no statistically significant difference between the cooked peppers compared to raw overall or within either pepper color; (C) There was a nominal statistical difference between one non-organic, Canadian red pepper compared to two organic, Mexican red peppers; (D) There was no statistical difference between the non-organic, Mexico grown green pepper vs. the organic US grown green pepper.

Figure 2

Relative abundance of β-cryptoxanthin detected in bell pepper samples by color. Following analysis of pepper samples using untargeted LC/MS, β-cryptoxanthin levels were compared according to their relative abundance across samples. (A) Red bell pepper has significantly higher levels compared to green and yellow. *** p < 0.001; (B) To determine whether cooking had any impact, each pepper was divided, three samples were heated for 5 min, and two samples remained raw. There was no statistically significant difference between the cooked peppers compared to raw overall or within either pepper color; (C) There was a nominal statistical difference between one non-organic, Canadian red pepper compared to two organic, Mexican red peppers; (D) There was no statistical difference between the non-organic, Mexico grown green pepper vs. the organic US grown green pepper.