Metabolomic Profiling and Antioxidant Capacity Changes in Longzi Black Barley During Germination

Abstract

Longzi black barley (Hordeum vulgare L. var. nudum, LBB), a highland barley variant with superior nutritional properties, has gained increasing attention for its health-promoting benefits. However, the metabolic changes during its germination process remain poorly understood. This study investigated the metabolic changes and antioxidant capacity during LBB germination. The results revealed significant dynamic changes in total flavonoid and total phenolic contents during germination, with the total flavonoids significantly decreasing by 32.59% initially (0-12 h, from 2.64 to 1.78 mg/g) and then slightly rebounding by 15.34% at 72 h, while the total phenolics decreased by 36.35% in the early stages (0-12 h, from 6.52 to 4.15 μmol/g) and increased markedly by 44.73% in the later stages (60-72 h, reaching 6.13 μmol/g) of germination. A metabolomic analysis identified 1015 metabolites, primarily including flavonoids, phenolic acids, amino acids and their derivatives, and alkaloids. During germination, the total flavonoid content continuously decreased by 24.24%, the phenolic acids showed no significant change, the amino acids and their derivatives increased significantly by 3.63-fold, and the alkaloid content increased slightly by 1.30-fold in the early stages (0-12 h) and significantly by 3.39-fold in the later stages (12-60 h). The study revealed the metabolic changes during the germination of LBB, providing scientific evidence for the further utilization of its nutritional value.

Keywords: antioxidant capacity; bioactive substances; germination; highland barley; metabolomic analysis.

Figure 1

Changes in physiological active substances and antioxidant capacity: (A) total flavonoid content; (B) total phenol content; (C) DPPH radical scavenging capacity; (D) ABTS radical scavenging activity; (E) FRAP assay. Different letters indicate significant differences between time points (p < 0.05). Data are presented as the mean ± SD (n = 3).

Figure 2

Metabolomic profiling of LBB during germination: (A) distribution of identified metabolites by chemical classification; (B) A principal component analysis (PCA) score plot of metabolites in LBB samples at different germination stages (LBB-0, LBB-12, and LBB-60, with three biological replicates each); (C) changes in total abundance of different metabolite classes during germination; (D) numbers of up- and down-regulated metabolites in pairwise comparisons between germination stages (fold change ≥2 or ≤0.5, VIP ≥ 1).

Figure 3

An analysis of flavonoid metabolites during LBB germination: (A) changes in abundance of major flavonoid compounds during germination (different letters indicate significant differences at p < 0.05); (B) metabolic pathway of flavonoid biosynthesis; (C) metabolic pathway of anthocyanin biosynthesis; (D) metabolic pathway of flavone and flavonol biosynthesis. The colored bars indicate fold changes (FC) between different germination stages (LBB-12 vs. LBB-0, LBB-60 vs. LBB-0, and LBB-60 vs. LBB-12).

Figure 4

An analysis of phenolic acid metabolites during LBB germination: (A) changes in abundance of major phenolic acid compounds during germination (different letters indicate significant differences at p < 0.05); (B) metabolic pathway of phenylpropanoid biosynthesis. The colored bars indicate fold changes (FC) between different germination stages (LBB-12 vs. LBB-0, LBB-60 vs. LBB-0, and LBB-60 vs. LBB-12). Compounds in red dashed boxes represent key intermediates in the pathway.

Figure 5

Changes in abundance of amino acids and their derivatives during LBB germination. Different letters above the bars or violin plots indicate significant differences between germination stages (p < 0.05). The MS signal intensity reflects the relative abundance of each compound at different germination time points (LBB-0, LBB-12, and LBB-60). Violin plots show the distribution of biological replicates.

Figure 6

Changes in abundance of alkaloid metabolites during LBB germination. The relative abundance of total alkaloids and individual alkaloid compounds is shown as the MS signal intensity at different germination stages (LBB-0, LBB-12, and LBB-60). Different letters indicate significant differences between germination stages (p < 0.05). The violin plots represent the distribution of biological replicates for each compound.