Soybean contains nutritional bioactive compounds, including carotenoids associated with human health benefits. Carotenoids are applicable in pharmaceuticals/nutreceuticals, cosmetic, and mainly food industries. However, an efficient and accurate method for carotenoid and chlorophyll detection and quantification has not yet been developed and validated for soybean seeds. The need for a rapid and reliable analysis method has become increasingly important. Thus, this study was initiated to develop and validate a simple, rapid, and selective reversed-phase high-performance liquid chromatographic (RP-HPLC) method for the simultaneous determination of lutein, zeaxanthin, α-carotene, β–carotene, β–cryptoxanthin, and chlorophyll–a and –b in soybean flour sample (100.00 mg) extracted using ethanol-acetone (1:1) solvents at a volume of 1.50 mL. Interestingly, the effective separation technique was achieved using the mobile phases of methyl tert-butyl ether, methanol containing 10 mM ammonium acetate, and water delivered at a 0.90 mL min⁻¹ flow rate through a C₃₀YMC Carotenoid (250 × 4.6 mm I.D., S-5 µm) column coupled with a UV-VIS detector set at 450 nm. The detector response was linear from 0.05–30.00 μg mL⁻¹ with a coefficient of determination (R²) of 0.9993–0.9999. The validated method was sensitive with a detection limit (LOD) of 0.0051–0.0300 μg mL⁻¹ and 0.0155–0.0909 μg mL⁻¹ for the quantification limit (LOQ). The recovery values were from 83.12–106.58%, and the repeatability precision ranged from 1.25–4.20% and 0.15–0.81% for the method and system, respectively. The method showed adequate precision with a relative standard deviation smaller than 3.00%. This method was also found to be applicable for profiling carotenoids and chlorophylls in other legumes. In summary, this method was successfully implemented for qualitative and quantitative determination of major carotenoids and chlorophylls in soybean and other legume seeds, which are beneficial to food industry and quality breeding programs to meet human nutrition demands globally.

Soybean (Glycine max (L.) Merrill) seeds are abundant in physiologically active metabolites, including carotenoids and chlorophylls, and are used as an affordable source of functional foods that promote and maintain human health. The distribution and variation of soybean seed metabolites are influenced by plant genetic characteristics and environmental factors. Here, we investigated the effects of germplasm origin, genotype, seed coat color and maturity group (MG) on the concentration variation of carotenoid and chlorophyll components in 408 soybean germplasm accessions collected from China, Japan, the USA and Russia. The results showed that genotype, germplasm origin, seed color, and MG were significant variation sources of carotenoid and chlorophyll contents in soybean seeds. The total carotenoids showed about a 25-fold variation among the soybean germplasms, with an overall mean of 12.04 µg g⁻¹. Russian soybeans yielded 1.3-fold higher total carotenoids compared with Chinese and Japanese soybeans. Similarly, the total chlorophylls were substantially increased in Russian soybeans compared to the others. Soybeans with black seed coat color contained abundant concentrations of carotenoids, with mainly lutein (19.98 µg g⁻¹), β-carotene (0.64 µg g⁻¹) and total carotenoids (21.04 µg g⁻¹). Concentrations of lutein, total carotenoids and chlorophylls generally decreased in late MG soybeans. Overall, our results demonstrate that soybean is an excellent dietary source of carotenoids, which strongly depend on genetic factors, germplasm origin, MG and seed coat color. Thus, this study suggests that soybean breeders should consider these factors along with environmental factors in developing carotenoid-rich cultivars and related functional food resources.

Tocopherols are natural antioxidants that increase the stability of fat-containing foods and are well known for their health benefits. To investigate the variation in seed tocopherol composition of soybeans from different origins, 493 soybean accessions from different countries (China, USA, Japan, and Russia) belonging to 7 maturity groups (MG 0–VI) were grown in 2 locations (Beijing and Hainan Provinces of China) for 2 years (2017 and 2018). The results showed that significant differences (p < 0.001) were observed among the accessions and origins for individual and total tocopherol contents. The total tocopherol content ranged from 118.92 μg g⁻¹ to 344.02 μg g⁻¹. Accessions from the USA had the highest average concentration of γ- and total tocopherols (152.92 and 238.21 μg g⁻¹, respectively), whereas a higher level of α-tocopherol (12.82 μg g⁻¹) was observed in the Russian accessions. The maturity group of the accession significantly (p < 0.001) influenced all tocopherol components, and higher levels of α-, γ-, and total tocopherols were observed in early maturing accessions, while late-maturing accessions exhibited higher levels of δ-tocopherol. The inclination of tocopherol concentrations with various MGs provided further evidence of the significance of MG in soybean breeding for seed tocopherol components. Furthermore, the correlation between the seed tocopherol components and geographical factors revealed that α-, γ-, and total tocopherols had significant positive correlations with latitude, while δ-tocopherol showed an opposite trend. The elite accessions with high and stable tocopherol concentrations determined could be used to develop functional foods, industrial materials, and breeding lines to improve tocopherol composition in soybean seeds.

Soybean seeds are primary sources of natural tocopherols used by the food and pharmaceutical industries, owing to their beneficial impacts on human health. Selection for higher tocopherol contents in seeds along with other desirable traits is an important goal in soybean breeding. In order to identify the genomic loci and candidate genes controlling tocopherol content in soybean seeds, the bulked-segregant analysis technique was performed using a natural population of soybean consisting of 1525 accessions. We constructed the bulked-segregant analysis based on 98 soybean accessions that showed extreme phenotypic variation for the target trait, consisting of 49 accessions with extremely-high and 49 accessions with extremely-low tocopherol content. A total of 144 variant sites and 109 predicted genes related to tocopherol content were identified, in which a total of 83 genes were annotated by the gene ontology functions. Furthermore, 13 enriched terms (p < 0.05) were detected, with four of them found to be highly enriched: response to lipid, response to abscisic acid, transition metal ion transmembrane transporter activity, and double-stranded DNA binding. Especially, six candidate genes were detected at 41.8–41.9 Mb genomic hotspots on chromosome 5 based on ANNOtate VARiation analysis. Among the genes, only Glyma.05G243400 carried a non-synonymous mutation that encodes a “translation elongation factor EF1A or initiation factor IF2gamma family protein” was identified. The haplotype analysis confirmed that Glyma.05G243400 exhibited highly significant variations in terms of tocopherol content across multiple experimental locations, suggesting that it can be the key candidate gene regulating soybean seed tocopherols. The present findings provide novel gene resources related to seed tocopherols for further validation by genome editing, functional characterization, and genetic improvement targeting enhanced tocopherol composition in soybean molecular breeding.

Soybeans are grown worldwide owing to their protein, oil, and beneficial bioactive compounds. Genetic and environmental factors influence soybean seed isoflavones. In the present study, we profiled the seed isoflavones in world diverse soybean germplasm grown in two locations over two years in China. Significant differences (p < 0.001) were observed between the accessions, accession origins, seed coat colors, and maturity groups for individual and total isoflavone (TIF) content. TIF content of the soybean accessions ranged from 677.25 μg g⁻¹ to 5823.29 μg g⁻¹, representing an 8-fold difference. USA soybean accessions showed the highest mean TIF content (3263.07 μg g⁻¹), followed by Japan (2521.26 μg g⁻¹). Soybean with black seed coat showed the highest (3236.08 μg g⁻¹) TIF concentration. Furthermore, isoflavone levels were significantly higher in late-maturity groups. Correlation analysis revealed significant positive associations between individual and TIF content. Malonyldaidzin and malonylgenistin showed higher correlations with TIF content (r = 0.92 and r = 0.94, respectively). The soybean accessions identified as having high and stable TIF content can be utilized in the food and pharmaceutical industries and breeding programs to develop soybean varieties with enhanced isoflavone content.