Colored hulless barley (Hordeum vulgare L.) is a high-quality germplasm resource that is rich in nutrients, such as protein, β-glucan, flavonoids, amino acids, vitamins, microelements, and dietary fiber. However, a systematic evaluation of the metabolites present in colored cultivars is lacking. This study employed an untargeted metabolomics approach using ultra-high-performance liquid chromatography-tandem mass spectrometry (LC-MS) to analyze the metabolic profiles of five hulless barley cultivars with different grain colors. Six-hundred and eight metabolites in various chemical categories were detected; ABC transporters, flavonoid biosynthesis, and anthocyanin biosynthesis were found to be the most significant metabolic pathways. Principal component analysis and orthogonal partial least squares-discriminant analysis revealed significant metabolic differences among the samples, and the colored barley cultivars could be separated from white barley. Black, blue, and purple grains rich in natural antioxidants were identified as promising ingredients for the development of cereal-based functional foods. These findings showed that the nutritional function and quality of hulless barley were strongly correlated with its natural color, thereby improving our understanding of the metabolic mechanisms and functional health value associated with different seed coat colors in different hulless barley cultivars. Our results thus provide a theoretical basis for the future study of hulless barley-related products.
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