Skip to main content
NCBI home page
Scientific Reports logoLink to Scientific Reports
. 2022 Apr 1;12:5541. doi: 10.1038/s41598-022-09408-1

Metabonomics analysis of flavonoids in seeds and sprouts of two Chinese soybean cultivars

Weiwei Bi 1,2,3,✉,#, Guixing Zhao 2,#, Yutong Zhou 4, Xiaoyu Xia 2, Jinsheng Wang 2, Guangjin Wang 2, Shuwen Lu 5, Wenjin He 2, Taifei Bi 3, Jinrong Li 2
PMCID: PMC8975843  PMID: 35365712

Abstract

A popular food in China, soybean seeds and sprouts contained many biologically active substances which are beneficial to the human body, such as flavonoids. Northeast of China is the main producing area of soybean. The experimental materials came from the main soybean producing areas in Northeast China, this study compared flavonoids of two China cultivars of soybeans, Heinong52(HN52) and Heinong71(HN71). Here, we also considered the effects of germination on the chemical profile of flavonoids. Using a LC–ESI–MS/MS system, 114 differential flavonoid metabolites were identified. A total of 18 metabolites were significantly different between the two soybean varieties before germination, of which 14 were up-regulated and 4 were down-regulated. After germination, 33 significantly different metabolites were found in the two soybean sprouts, of which 19 were up-regulated and 14 were down-regulated. These experimental results revealed significant up-regulation of metabolites in soybean sprouts compared with soybean seeds, thus suggesting that soybean germination may increase content of flavonoid metabolites. There are six main pathways for the synthesis of flavonoids: isoflavonoid biosynthesis, flavonoid biosynthesis, flavone and flavonol biosynthesis, biosynthesis of secondary metabolites, and biosynthesis of phenylpropanoids. Soybean seeds lack flavone and flavanol biosynthesis and develop the capacity for this biosynthetic pathway after germination as sprouts. Isoflavonoid biosynthesis is the most abundantly utilized pathway.

Subject terms: Biochemistry, Plant sciences

Introduction

China is the first country to grow and domesticate the soybean(Glycine max(L.) Merr.), with a cultivation history of at least 4,000 years. Nowadays, soybean is gaining popularity in many countries largely as a vegetable protein and oil source, owing to its bean composition of approximately 40% protein and 20% oil1. Soybean is an important nutritional component of diets and is used in many foods, such as soybean oil, soybean sprout, paste, soymilk, and tofu2. Mostly present in plant leaves and fruits, flavonoids primarily exist as glycosides combining with sugars. An important phenolic secondary metabolite in plants, flavonoids can support plant disease resistance, and in the human body have roles such as anti-oxidation, anti-cancer, and anti-aging3,4. Certain major flavonoids, such as flavonols, flavonol glycosides, isoflavones, chalcones, anthocyanins and procyanidins have been previously isolated and identified from soybeans5,6. Additionally, recent research showed that pinto and black beans as excellent dietary sources of natural antioxidants and thus may serve as disease preventative and healthy foods7. Moreover, a recent review analyzing epidemiological studies in the last ten years and the flavonoid content of diets concluded that dietary flavonoids may prevent a range of different types of cancer8. In fact, free phenolic compounds, such as flavonols and isoflavones, have been found to have with immunomodulatory and cellular antiproliferative functions9.

The sprouting process induces a variety of biochemical changes in soybean seeds, leading to the accumulation of primary and secondary metabolites10. Some studies have found that germination and sprout formation of legumes are accompanied by a significant increase in total flavonoids content with antioxidant potential11–13. Generally, content of genistein derivatives is about two times higher than that of daidzein derivatives in raw soybeans14–16. Germination can induce isoflavone profile changes in soybeans including decreases of β-glycosides and increases of both 6ʹ-O-malonyl-β-glucosides and aglycones17–19. During germination, a 13% increase of total isoflavone content was found by Kim, et al20. One comparative study of the chemical profiles of three Egyptian cultivars of fava beans; Sakha 3, Nubaria 3, and Giza 843, focused on the effects of germination on the chemical profiles of phenolic compounds and saponins and characterized 65 metabolites based on UV spectra, accurate MS, and MS/MS data. Germination was found to dramatically increase the quantities of flavonoids and saponins to which biological activities are attributed20. A previous study has suggested that germination could enhance the content of isoflavonesin soybeans21. Accordingly, studies based on metabolomics have applied combined techniques, such as chromatographic separation (UHPLC and/or gas chromatography) and mass spectrometry, for comprehensive metabolic analysis of plant substrates22,23. Recent technological developments in the field of metabolomics, especially as a result of the widespread use of LC-high resolution MS have considerably improved accuracy and sensitivity of metabolite detection24. For example, non-target LC–MS-Orbitrap metabolomics methods were used to compare and analyze the biologically active metabolites of frozen, boiled, and canned chickpeas, lentils, and white beans25. The main purposes of this study were to: (1) compare the flavonoid contents in soybean seeds and sprouts grown under dark conditions for 3 days at 24 °C (2) analyze the metabolism of flavonoids in soybean sprouts.

Materials and methods

Samples preparation

According to our previous detection of isoflavones in 12 main soybean varieties grown in Heilongjiang, China, two varieties with high isoflavone content were selected for experimentation26. The two soybean cultivars Heinong52 (HN52), and Heinong71(HN71) used in this experiment were cultivated at the Harbin Soybean Research Institute (Modern Agriculture Demonstration Zone of Heilongjiang Academy of Agricultural Sciences, Minzhu Township, Daowai District, Harbin; 126.65°E, 45.78°N), Heilongjiang Academy of Agricultural Sciences, Harbin, China(statement as follow). Soybean seeds were harvested from three replicates of each cultivar for a cropping year and stored at room temperature until analysis of flavonoids compounds. Briefly, high-quality soybean grains were selected, rinsed with clean water, soaked in distilled water at 24 °C for 4 h, spread in a single layer on a tray, covered with gauze, and placed in an artificial climate box (HPG-1600H, Harbin Donglian Electronic Technology Development Co., Ltd. Harbin, China) at about 24 °C and 80% humidity to germinate for 3 days in the dark. The growth chamber was set up to spray water for 3 min every 1 h. After germination, approximately 20 g FW(fresh weight) of soybean sprouts (HNS52,HNS71) per cultivar with three replicates every sampling day were randomly collected from containers, and freeze-dried at − 50 °C for 20 h. The freeze-dried sprouts and seeds were crushed using a mixer mill (MM 400, Retsch) with a zirconia bead for 1.5 min at 30 Hz. 100 mg powder was weighed and extracted overnight at 4 °C with 1.0 mL 70% aqueous methanol. Following centrifugation at 10,000 g for 10 min, the extracts were absorbed (CNWBOND Carbon-GCB SPE Cartridge, 250 mg, 3 mL; ANPEL, Shanghai, China, www.anpel.com.cn/cnw) and filtrated (SCAA-104,0.22 μm pore size; ANPEL, Shanghai, China, http://www.anpel.com.cn/) LC–MS analysis(contained “2.2 HPLC Conditions” and “2.3 ESI-Q TRAP-MS/MS”) .

HPLC conditions

The sample extracts were analyzed using a LC–ESI–MS/MS system (HPLC, Shim-pack UFLC SHIMADZU CBM30A system, www.shimadzu.com.cn/; MS, Applied Biosystems 4500 Q TRAP, www.appliedbiosystems.com.cn/). We used the following steps to analyze, HPLC: column, Waters ACQUITY UPLC HSS T3 C18 (1.8 µm, 2.1 mm*100 mm); solvent system, water (0.04% acetic acid): acetonitrile (0.04% acetic acid); gradient program,100:0 V/V at 0 min, 5:95 V/V at 11.0 min, 5:95 V/V at 12.0 min, 95:5 V/V at 12.1 min, 95:5 V/V at 15.0 min; flow rate, 0.40 mL/min; temperature, 40 °C; injection volume: 5μL. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS27.

LIT and triple quadrupole (QQQ) scans were acquired using a triple quadrupole-linear ion trap mass spectrometer (Q TRAP), API 4500 Q TRAP LC/MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in a positive ion mode and controlled by Analyst 1.6.3 software (AB Sciex). The parameters of ESI source operation were as follows: ion source, turbo spray; source temperature 550 °C; ion spray voltage (IS)5500 V; ion source gas I (GSI), gas II(GSII), curtain gas (CUR) were set at 55, 60, and 25.0 psi, respectively; the collision gas(CAD) was high. Instrument tuning and mass calibration were performed with 10 and 100 μmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scans were acquired as MRM experiments with collision gas (nitrogen) set to 5 psi. DP and CE for individual MRM transitions was done with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period28–30. Similarly, Valente et al. calculated the metabolic profile and antioxidant activity of 7 European broad bean seed varieties31 and their pods (by-products) using HPLC–DAD-MS/MS-based methods and found the existence of 105 phenolic compounds, alkaloids, jasmonic acids, and organic acids32.

Statistics analysis

For all experiments, three independent assays were carried out. One-way analysis of variance was used to study the differences between means, with a significant level at P < 0.05. Data analysis was carried out with SPSS (Windows version 12.0, SPSS Inc., Chicago,IL, USA). All data are presented as mean ± standard error of means.

Results and discussion

Principal component analysis

Principal component analysis (PCA) was performed on metabolic data to visualize the biological variability of soybean seeds and sprouts. These results indicated that the dominating source of variance was the differential flavonoid content in the two soybean varieties across all germination stages (PC1). From Fig. 1A, the PC1 of 4 groups and mix was 58.6%. The PC1 of two soybean seeds was 77.25%. The PC1 of two soybean seeds and sprouts were74.46% and 81.16% respectively. The PC1 of all samples was greater than 50%. These results indicated that the grouping of samples is reasonable. Through PCA of samples (including quality control samples MIX), in order to have a preliminary understanding of the overall metabolic differences between samples in each group and the degree of variability between samples within the group. The PCA results show the trend of metabolome separation between groups, suggesting whether there is a difference in metabolome between sample groups. The X axis represents the first principal component, and the Y axis represents the second principal component. Grouping principal component analysis: before doing difference analysis, first perform principal component analysis on the grouped samples for difference comparison to observe the degree of variability between the difference groups and the samples within the group.

Figure 1.

Figure 1

Open in a new tab

PCA score chart of mass spectrum data of each group of samples and quality control samples. Mix: quality control samples, (A): all samples of PCA. (B): Two soybean seeds of PCA; (C):Soybean and sprouts of PCA(HN52 and HNS52); (D): Soybean and sprouts of PCA(HN71 and HNS71).

Differential metabolic analysis of flavonoids

From Table 1, a total of 114 flavonoid metabolites were detected in the two soybean varieties and their sprouts. These consisted of 41 isoflavones, 26 flavonoids, 14 flavonols, 10chalcones, 6 dihydroflavones, 5 tannins, 4 dihydroflavonols, 3 carbonosides, 2 flavonoids, 1 flavanol, 1 proanthocyanidin, and 1anthocyanin. These results indicate that the flavonoids in soybeans and sprouts are mainly composed of isoflavones (35.96%). 13 substances detected in this study were the same as those detected in Egyptian cultivars of chickpea in a previous study30. These were C13H16O10 (6-O-Galloyl-glucose), C13H16O10(3-O-Galloyl-glucose),C13H16O10 (2-O-Galloyl-glucose), C27H30O16(Kaempferol-3-O-neohesperidoside), C27H30O16 (5,7-Dihydroxy-4-methoxyflavone-3-O-xylose-(1–6)-glucose), C21H22O11 (Aromadendrin-7-O-glucoside), C27H30O16 (Quercetin-3-O-robinobioside), C27H30O17 (Quercetin-3-O-sophoroside (Baimaside)), C33H40O21 (Kaempferol-3-O-sophoroside -7-O- glucoside) , C27H32O15 (Eriodictyol-7-O-Rutinoside (Eriocitrin)), C27H30O16 (Quercetin-3-O-rutinoside (Rutin)), C28H32O17 (2'-Hydoxy, 5-methoxy Genistein-4',7- O-diglucoside), C27H30O15 (Eriodictyol-7-O-Rutinoside (Eriocitrin)), respectively. This indicated that these flavonoids may be universal in beans.

Table 1.

Flavonoids in soybeans seeds and sprouts.

Index Formula Compounds Class CAS
pme0309 C8H8O5 3-O-Methylgallic Acid Tannin 3934-84-7
pme0355 C15H10O4 Daidzein Isoflavones 486-66-8
mws0902 C15H12O4 Liquiritigenin Dihydroflavone 578-86-9
pme3217 C15H12O4 Isoliquiritigenin Chalcones 961-29-5
mws0037 C16H12O4 Formononetin (7-Hydroxy-4'-methoxyisoflavone) Isoflavones 485-72-3
mws0063 C15H10O5 Genistein* Isoflavones 446-72-0
pme3261 C15H10O5 6-Hydroxydaidzein* Isoflavones 17,817-31-1
mws0912 C15H10O5 2'-Hydroxydaidzein* Isoflavones 7678-85-5
pmp000344 C15H10O5 3',4',7-Trihydroxyflavone* Flavonoid 2150-11-0
Lmmp005125 C16H14O4 Pinostrobin Chalcone Chalcones 18956-15-5
mws4060 C16H14O4 Echinatin Chalcones 34221-41-5
pme1397 C15H11O5 +  Pelargonidin Anthocyanins 7690-51-9
mws0914 C15H12O5 Pinobanksin* Dihydroflavonol 548-82-3
pme0376 C15H12O5 Naringenin (5,7,4'-Trihydroxyflavanone)* Dihydroflavone 480-41-1
pme3250 C16H12O5 Biochanin A Isoflavones 491-80-5
pme3233 C16H12O5 Calycosin* Isoflavones 20575-57-9
mws0908 C16H12O5 Glycitein* Isoflavones 40957-83-3
mws0062 C15H10O6 Isoluteolin (Orobol)(5,7,3',4'-tetrahydroxyisoflavone) Isoflavones 480-23-9
pme0088 C15H10O6 Luteolin (5,7,3',4'-Tetrahydroxyflavone) Flavonoid 491-70-3
mws1094 C15H12O6 Dihydrokaempferol Dihydroflavonol 480-20-6
Lmdp005525 C17H14O5 Afrormosin Isoflavones 550-79-8
Lmqn003210 C17H14O5 5-Hydroxy-6,7-dimethoxyflavone Flavonoid 740-33-0
Zmhp003514 C16H12O6 6,7,8-Tetrahydroxy-5-methoxyflavone* Flavonoid –
mws0058 C16H12O6 Diosmetin (5,7,3'-Trihydroxy-4'-methoxyflavone) Flavonoid 520-34-3
pmp000001 C16H12O6 Hispidulin (5,7,4'-Trihydroxy-6-methoxyflavone)* Flavonoid 1447-88-7
pmp000804 C19H20O4 Isobavachalcone D Chalcones –
pmp000348 C20H18O4 Kanzonol D Other Flavonoids 155,233-20-8
Lmfn000604 C13H16O10 6-O-Galloyl-glucose* Tannin 13,186-19-1
Hmln000659 C13H16O10 3-O-Galloyl-glucose* Tannin –
Hmln000873 C13H16O10 2-O-Galloyl-glucose Tannin –
pmp000350 C20H16O5 Glabrone Isoflavones 60008-02-8
pmp001223 C20H18O5 Psoralenol Isoflavones 70522-30-4
pmp000352 C20H18O5 Licoflavone C Flavonoid 72357-31-4
pmp000351 C20H18O5 Eurycarpin A Isoflavones 166547-20-2
pmp000637 C20H20O5 Sophoraflavanone B Dihydroflavone 53846-50-7
Xmgp006913 C20H20O5 2,4,2',4'-tetrahydroxy-3'-prenylchalcone Chalcones –
pmn001389 C21H20O5 Gancaonin G Isoflavones 126716-34-5
pmp000355 C20H18O6 Licoflavonol Flavonols 60197-60-6
pmp000811 C20H18O6 Morachalcone C Chalcones 1304549-24-3
pmp000360 C21H22O5 3-Hydroxylicochalcone A Chalcones –
pmp000361 C21H22O5 Licochalcone D Chalcones 144506-15-0
pmp001224 C20H20O6 Brosimacutin G Chalcones 350,221-50-0
Cmsn000894 C14H18O11 7-O-Galloyl-D-sedoheptulose Tannin –
mws0055 C20H20O7 Tangeretin Flavonols 481-53-8
pmp000417 C21H20O9 Daidzein-4'-O-glucoside Isoflavones –
pme1587 C21H20O9 Daidzein-7-O-glucoside(Daidzin) Isoflavones 552-66-9
mws1597 C21H20O9 Puerarin Isoflavones 3681-99-0
pmp000384 C21H22O9 Isoliquiritin* Chalcones 5041-81-6
pmp000383 C21H22O9 Liquiritigenin-4'-O-Glucoside (Liquiritin)* Dihydroflavone 551-15-5
pmp000647 C25H28O6 Kushenol E Other Flavonoids 99,119-72-9
pme3504 C22H22O9 Formononetin-7-O-glycoside (Ononin) Isoflavones 486-62-4
HJN089 C21H20O10 Sophoricoside Isoflavones 152-95-4
pmp000413 C21H20O10 Genistein-8-C-glucoside* Isoflavones 66,026-80-0
mws1434 C21H20O10 Apigenin-6-C-glucoside (Isovitexin)* Flavonoid carbonoside 29,702-25-8
Lmlp005572 C21H20O10 Galangin-7-O-glucoside* Flavonoid –
mws0072 C21H20O10 Apigenin-5-O-glucoside* Flavonoid 28,757-27-9
mws0895 C21H20O10 Genistein-7-O-Glucoside (Genistin)* Isoflavones 529-59-9
pme1611 C21H22O10 Isohemiphloin* Flavonoid carbonoside 3682-02-8
mws1179 C21H22O10 Naringenin-7-O-glucoside (Prunin)* Dihydroflavone 529-55-5
HJN090 C21H22O10 Butin-7-O-glucoside* Flavonoid –
pmp000550 C22H22O10 Calycosin-7-O-glucoside Isoflavones 20,633-67-4
HJN091 C22H20O10 Prunetin-4'-O-glucoside Isoflavones 154-36-9
pme3400 C22H22O10 Biochanin A-7-O-glucoside (Sissotrin) Isoflavones 5928-26-7
pmp000415 C22H22O10 3'-Methoxydaidzin Isoflavones 200,127-80-6
mws0894 C22H22O10 Glycitin Isoflavones 40,246-10-4
mws1172 C22H22O10 Trifolirhizin (Maackiain-3-O-glucoside) Isoflavones 6807-83-6
Lmlp003531 C21H20O11 Luteolin-3'-O-glucoside* Flavonoid 5154-41-6
pme2459 C21H20O11 Luteolin-7-O-glucoside (Cynaroside)* Flavonoid 5373-11-5
Xmyp005654 C21H20O11 Kaempferol-4'-O-glucoside* Flavonoid –
mws1361 C21H22O11 Astilbin Dihydroflavonol 29838-67-3
Lmtn002796 C21H22O11 Aromadendrin-7-O-glucoside Flavonoid 28189-90-4
Lmlp005236 C21H22O11 Dihydrokaempferol-3-O-glucoside Dihydroflavonol 1049-08-8
Zmdp004370 C23H22O10 6''-O-Acetyldaidzin Isoflavones 71385-83-6
Lmdp003994 C23H24O10 6,4'-Dimethoxyisoflavone-7-O-glucoside (Wistin) Isoflavones 19046-26-5
mws0091 C21H20O12 Quercetin-3-O-glucoside (Isoquercitrin) Flavonols 482-35-9
mws0061 C21H20O12 Quercetin-3-O-galactoside (Hyperin) Flavonols 482-36-0
pmp000191 C23H22O11 6''-O-Acetylgenistin* Isoflavones 73566-30-0
pmp000581 C23H22O11 Apigenin-7-O-(6''-acetyl)glucoside* Flavonoid 72741-92-5
pmp000192 C24H24O11 Acetylglycitin Isoflavones 134859-96-4
Lmmp003903 C23H22O12 Kaempferol-3-O-(2''-acetyl)glucoside Flavonols –
Lmmn003398 C23H22O12 Kaempferol-3-O-(6''-acetyl)glucoside Flavonols –
Lmgp004829 C23H24O12 5,7,4'-Trihydroxy-6,8-dimethoxyisoflavone-7-O-galactosides Isoflavones –
pmp000193 C24H22O12 6''-O-Malonyldaidzin Isoflavones 124590-31-4
Zmdp004305 C28H24O9 Daidzein-7-O-(2''-benzoyl)rhamnoside Flavonoid -
Zmdp005767 C25H24O12 Formononetin-7-O-(6''-Malonyl)glucoside Isoflavones -
Zmdp004112 C24H22O13 Genistein-7-O-(6''-malonyl)glucoside Isoflavones -
pmp000194 C24H22O13 6''-O-Malonylgenistin Isoflavones 51011-05-3
pmp000195 C25H24O13 6''-O-Malonylglycitin Isoflavones 137705-39-6
Lmmp003817 C24H22O14 Kaempferol-3-O-(6''-malonyl)glucoside* Flavonols –
pmp000587 C24H22O14 Luteolin-7-O-(6''-malonyl)glucoside* Flavonoid –
Lmdp004892 C24H22O14 Kaempferol-3-O-(6''-malonyl)galactoside* Flavonols –
pmb0608 C25H24O14 Chrysoeriol-7-O-(6''-malonyl)glucoside Flavonoid –
Zmdp003677 C26H28O13 Daidzein-7-O-Glucoside-4'-O-Apioside Flavonoid 108069-01-8
Wmkn002777 C27H30O13 7-Hydroxy-3''-methoxy-isoflavone-7-primeveroside Isoflavones –
Lmnp202580 C26H28O14 Apigenin-8-C-(2''-xylosyl)glucoside Flavonoid carbonoside –
pmp000411 C26H28O14 Apigenin-7-O-(2''-glucosyl)arabinoside Flavonoid –
mws0836 C30H26O12 Procyanidin B1 Proanthocyanidins 20315-25-7
pmp000414 C27H30O14 Puerarin-4'-O-glucoside Isoflavones 117047-08-2
pmp001079 C27H30O15 Luteolin-7-O-neohesperidoside (Lonicerin) Flavonoid 25694-72-8
mws1073 C27H30O15 Apigenin-6,8-di-C-glucoside Flavonoid 23666-13-9
Lmjp002867 C27H30O15 Kaempferol-3-O-neohesperidoside Flavonols 32602-81-6
Wmkp002590 C27H30O15 5,7-Dihydroxy-4-methoxyflavone-3-O-xylose-(1-6)-glucose Flavonoid –
mws1519 C27H32O15 Eriodictyol-7-O-Rutinoside (Eriocitrin) Dihydroflavone 13463-28-0
Lmmp000897 C30H26O14 Gallocatechin-Gallocatechin Flavanols –
pmn001583 C27H30O16 Quercetin-3-O-robinobioside* Flavonols 52525-35-6
Zmxp003107 C27H30O16 Luteolin-7,3'-di-O-glucoside* Flavonoid 52187-80-1
mws0059 C27H30O16 Quercetin-3-O-rutinoside (Rutin)* Flavonols 153-18-4
Lmhp003217 C28H32O16 2'-Hydoxy,5-methoxyGenistein-O-rhamnosyl-glucoside Isoflavones –
pme1540 C28H32O16 Isorhamnetin-3-O-neohesperidoside Flavonols 55033-90-4
Lmtp003677 C27H30O17 Quercetin-3-O-sophoroside (Baimaside) Flavonols 18609-17-1
Lmhp002800 C28H32O17 2'-Hydoxy,5-methoxyGenistein-4',7-O-diglucoside Isoflavones –
Hmqp003435 C29H30O17 Apigenin-7-O-(2''-O-apiosyl)(6''-Malonyl)glucoside Flavonoid –
Hmlp003185 C30H32O18 Luteolin-7-O-(6''-malonyl)glucoside-5-O-rhamnoside Flavonoid –
Lmwp003888 C33H40O21 Kaempferol-3-O-sophoroside-7-O-glucoside Flavonols –
Open in a new tab

Metabolomic data is massive and multidimensional, so it is imperative to combine univariate and multivariate statistical analyses, to analyze the data from a multivariate perspective according to the characteristics of the data, and to accuratelymine differential metabolites. This study has three biological replicates. The method of combining fold change ≥ 2 and OPLS-DA model VIP (The VIP value represents the influence of the difference between the corresponding metabolites in the classification and discrimination of each group of samples in the model. It is generally believed that the metabolites with VIP ≥ 1 are significantly different.) ≥ 1 was used to screen for differential metabolites. Shown in Fig. 2A, there were a total of 18 significantly different metabolites between the two soybean varieties, of which 14 were up-regulated and 4 were down-regulated. After germination, the significantly different metabolites in the two soybean sprouts were 33, of which 19 were up-regulated and 14 were down-regulated (Fig. 2B). Moreover, there were a total of 27 significantly different metabolites between HN52 and HNS52 cultivars, of which 26 were up-regulated and 1 were down-regulated (Fig. 2C). Additionally, there were a total of 25 significantly different metabolites between HN71 and HNS71, of which 21 were up-regulated and 4 were down-regulated (Fig. 2D). These experimental results showed that both differential and up-regulated metabolites increased in soybean sprouts compared to soybean seeds, thus indicating that soybean germination can increase content of flavonoid metabolites. From a qualitative comparison, that the germination process clearly has an impact on the number of characterized metabolites, especially flavonoid compounds. In fact, the biological activity of the plant substrate is mainly due to these metabolites33–37.

Figure 2.

Figure 2

Open in a new tab

Volcano Map of Differential Metabolites. (A): HN52 vs HN71; (B): HNS52 vs HNS71; (C): HN52 vs HNS52; (D): HN71 vs HNS71.

Each point in the volcano map represents a metabolite. The abscissa represents the logarithm of the quantitative difference of a certain metabolite in the two samples; the ordinate represents the VIP value. The larger the absolute value of the abscissa, the greater the multiple difference in the expression amount between the two samples; the larger the ordinate value, the more significant the differential expression, and the more reliable the differentially expressed metabolites screened. The green dots in the figure represent down-regulated differentially expressed metabolites, the red dots represent up-regulated differentially expressed metabolites, and black represents detected but not significantly different metabolites.

As shown in Fig. 3, the flavonoids of the two varieties have a balanced distribution of the differential metabolite multiples, and the differential metabolites multiples decrease after germination. This indicated that the flavonoid metabolism of different varieties of soybeans while germinating was highly similar. The flavonoid metabolites in the same variety of soybean seeds and sprouts differ greatly in multiples, showing that flavonoid metabolism is especially active during soybean germination, and that germination is a way to accumulate flavonoids. During the growth process, small sprouts may be damaged by microbial and environmental pressures or nutrient deficiency. Therefore it is necessary for plants to synthesize secondary metabolites (such as flavonoid compounds) through different metabolic pathways to develop defense mechanisms38–40.

Figure 3.

Figure 3

Open in a new tab

Analysis of the fold difference of metabolites between different samples. (A): HN52 vs HN71; (B): HNS52 vs HNS71; (C): HN52 vs HNS52; (D): HN71 vs HNS71.

As shown in Fig. 4, there were total 33 differential metabolites, of which 12 identical metabolites in HN52 vs HNS52 and HN52 vs HN71. There were total 45 differential metabolites, of which 13 metabolites were shared in HN71 vs HNS71 and HNS52 vs HNS71. These results indicated that flavonoid metabolites increased in germinated soybeans. For example, Wu et al. discussed the effect of germination on the antioxidant potential and isoflavonoid content of chickpeas and observed increases in both antioxidant activity and isoflavonoids content with germination41. Additionally, Ayet et al. revealed that germination increased the content of soya saponins in lentils42,43. Moreover, Mekky et al. revealed that germination increased the quantities of flavonoids, phenolic acids and saponins in fava beans30.

Figure 4.

Figure 4

Open in a new tab

Venn diagram showing the relationships between different groups of metabolites.

Functional annotation and enrichment analysis of differential metabolites

In the homepage of the KEGG website, we directly enter the keyword "Flavonoids" and the top 4 pathways are all pathways involving flavonoid metabolites, including:flavonoid synthesis, anthocyanin synthesis, isoflavone synthesis, flavonoids and flavonols synthesis. Using the KEGG database44 to annotate and display the differential metabolites identified in these samples, 6 metabolite were significantly up-regulated in the isoflavone synthesis pathway, 1 metabolite was significantly down-regulated, and 13 metabolites did not change significantly (Fig. 5).

Figure 5.

Figure 5

Open in a new tab

Differential metabolite KEGG pathway diagram. Note: Red indicates that the metabolite content is significantly up-regulated in the experimental group, and blue indicates that the metabolite is detected but not significantly changed. Green indicates that the metabolite content is significantly down-regulated in the experimental group.

There are six primary metabolic pathways for differential metabolism of flavonoids; isoflavonoid biosynthesis, flavonoid biosynthesis, flavone and flavonol biosynthesis, secondary metabolite biosynthesis, and phenylpropanoid biosynthesis (Fig. 6). Unlike soybean seeds, soybean sprouts have flavone and flavonol biosynthesis. Isoflavonoid biosynthesis accounts for most of the pathways, and the proportions in different samples are 66.67% in HN52 vs HN71 (Fig. 6A), 41.67% in HNS52 vs HNS71 (Fig. 6B), 54.55% in HN52 vs HNS52 (Fig. 6C), and 40% in HN71 vs HNS71 (Fig. 6D), respectively. Another important pathway is the biosynthesis of secondary metabolites, and the proportions in different samples are 33.33% in HN52 vs HN71 (Fig. 6A, 16.67% in HNS52 vs HNS71 (Fig. 6B), 54.55% in HN52 vs HNS52 (Fig. 6C), 70% in HN71 vs HNS71 (Fig. 6D), respectively). Metabolic pathway data explained why isoflavones were higher in different metabolites.

Figure 6.

Figure 6

Open in a new tab

Differential metabolite KEGG classification chart. (A): HN52 vs HN71; (B): HNS52 vs HNS71; (C): HN52 vs HNS52; (D): HN71 vs HNS71. Note: The ordinate is the name of the KEGG metabolic pathway, and the abscissa is the number of metabolites annotated to the pathway and its proportion to the total number of metabolites annotated.

As shown in Fig. 7, that the larger size dot indicated isoflavonoid biosynthesis and biosynthesis of secondary metabolites representing the number of these metabolites enriched. The red point represented isoflavonoid biosynthesis in Fig. 7C, indicating that isoflavonoid was more significantly enriched. The redder points indicating isoflavonoid biosynthesis in Fig. 7A, showed that isoflavonoids were more significantly enriched.

Figure 7.

Figure 7

Open in a new tab

Differential metabolite KEGG enrichment map. (A): HN52 vs HN71; (B): HNS52 vs HNS71; (C): HN52 vs HNS52; (D): HN71 vs HNS71. Note: The abscissa represents the rich factor corresponding to each channel, the ordinate is the channel name, and the color of the point is p value. The more red, the more significant the enrichment. The size of the dot represents the number of different metabolites enriched.

Conclusion

In this study, two soybean varieties with high isoflavone content and their soybean sprouts in Heilongjiang Province, China were analyzed with flavonoid metabolome analysis. 114 differential metabolites were detected which contained 41 isoflavones and 26 flavonoids. These experimental results revealed that the differentially expressed and up-regulated metabolites in soybean sprouts increased compared to soybean seeds. The flavonoid metabolites in the same variety of soybean seeds and sprouts differ greatly in multiples, indicating that flavonoid metabolism is very active during soybean germination, and germination may be a way to accumulate flavonoids. Metabolic pathway data explained why isoflavones were higher than different metabolites. This study detected and analyzed the changes of flavonoid metabolites after soybean germination, providing data reference for the research of flavonoids, and providing ideas for future research on plant secondary metabolism.

Acknowledgements

This study was subsidized by Heilongjiang Province Postdoctoral Science Foundation (LBH-Z16184) and funded by Project of Harbin Science and Technology Bureau (2017RAQYJ034), College Project of Heilongjiang Academy of Agricultural Sciences (2017BZ14, 2020YYYF038) and Agricultural Science and Technology Innovation Leapfrog Project of Heilongjiang Academy of Agricultural Sciences (HNK2019CX01-14-5, HNK2019CX19-09).

Author contributions

W.B., G.Z., and Y.Z., X.X. wrote the main manuscript text and jinsheng Wang, guangjin Wang prepared Figs. 1–3, S.L., W.H. and T.B. prepared Figs. 4–7, Jintong Li prepared Table 1. All authors reviewed the manuscript.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Weiwei Bi and Guixing Zhao

References

  • 1.Messina MJ. Legumes and soybeans: overview of their nutritional profiles and health effects. Am J. Clin Nutr. 1999;70:439s–450s. doi: 10.1093/ajcn/70.3.439s. [DOI] [PubMed] [Google Scholar]
  • 2.Kim EH, Chung JI, Chi HY, Kim JA, Chung IM. Analysis of phenolic compounds and isoflavones in soybean seeds (Glycine max (L.) Merill) and sprouts grown under different conditions. Eur. Food Res. Technol. 2006;222:201–208. [Google Scholar]
  • 3.Beninger CW, Hosfield GL. Antioxidant activity extracts, condensed tannin fractions, and pure flavonoids from Phaseolus vulgaris L. seed coat color genotypes. J. Agr. Food Chem. 2003;51:7879–7883. doi: 10.1021/jf0304324. [DOI] [PubMed] [Google Scholar]
  • 4.Lee CH, Yang L, Xu JZ, Yeung SYV, Huang Y, Chen ZY. Relative antioxidant activity of soybean isoflavones and their glycosides. Food Chem. 2005;90:735–741. [Google Scholar]
  • 5.Aparicio-Fernandez X, Yousef GG, Loarca-Pina G, Mejia E, Lila MA. Characterization of polyphenolics in the seed coat of black jamapa bean (Phaseolus vulgaris L.) J. Agr. Food Chem. 2005;53:4615–4622. doi: 10.1021/jf047802o. [DOI] [PubMed] [Google Scholar]
  • 6.Beninger CW, Gu LW, Prior RL, Junk DC, Vandenberg A, Bett KE. Changes in polyphenols of the seed coat during the after-darkening process in pinto beans (Phaseolus vulgaris L.) J. Agr. Food Chem. 2005;53:7777–7782. doi: 10.1021/jf050051l. [DOI] [PubMed] [Google Scholar]
  • 7.Diaz-Batalla L, Widholm JM, Fahey GC, Castano-Tostado E, Paredes-Lopez O. Chemical components with health implications in wild and cultivated Mexican common bean seeds (Phaseolus vulgaris L.) J. Agr. Food Chem. 2006;54(6):2045–2052. doi: 10.1021/jf051706l. [DOI] [PubMed] [Google Scholar]
  • 8.Rodríguez-García C, Sánchez-Quesada C, Gaforio JJ. Dietary flavonoids as cancer chemopreventive agents: an updated review of human studies. Antioxidants. 2019;8:137. doi: 10.3390/antiox8050137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Du H, Huang Y, Tang Y. Genetic and metabolic engineering of isoflavonoid biosynthesis. Appl. Microbiol. Biot. 2010;86(5):1293–1312. doi: 10.1007/s00253-010-2512-8. [DOI] [PubMed] [Google Scholar]
  • 10.Chon SU. Total polyphenols and bioactivity of seeds and sprouts in several legumes. Curr. Pharm. Design. 2013;19(34):6112–6124. doi: 10.2174/1381612811319340005. [DOI] [PubMed] [Google Scholar]
  • 11.Kim MA, Kim MJ. Isoflavone profiles and antioxidant properties in different parts of soybean sprout. J. Food Sci. 2020;85:689–695. doi: 10.1111/1750-3841.15058. [DOI] [PubMed] [Google Scholar]
  • 12.Cevallos-Casals BA, Cisneros-Zevallos L. Impact of germination on phenolic content and antioxidant activity of 13 edible seed species. Food Chem. 2010;119(4):1485–1490. [Google Scholar]
  • 13.Gharachorloo M, Ghiassi Tarzi B, Baharinia M. The effect of germination on phenolic compounds and antioxidant activity of pulses. J. AM Oil Chem. Soc. 2013;90(3):407–411. [Google Scholar]
  • 14.Ebert WA, Chang CH, Yan MR, Yang RY. Nutritional composition of mungbean and soybean sprouts compared to their adult growth stage. Food Chem. 2017;237(15):15–22. doi: 10.1016/j.foodchem.2017.05.073. [DOI] [PubMed] [Google Scholar]
  • 15.Kim TJ, Sung CH, Kim YJ, Jung BM, Kim ER, Choi WS, Yoo SH. Effects of a soaking-fermentation-drying process on the isoflavone and γ-aminobutyric acid contents of soybean. Food Sci. Biotechnol. 2007;16:83–89. [Google Scholar]
  • 16.Lee JH, Renita M, Fioritto RJ, St. Martin SK, Schwartz SJ, Vodovotz Y. Isoflavone characterization and antioxidant activity of Ohio soybean. J. Agr. Food Chem. 2004;52:2647–2651. doi: 10.1021/jf035426m. [DOI] [PubMed] [Google Scholar]
  • 17.Sakthivelu G, Akitha Devi MK, Giridhar P, Rajasekaran T, Ravishankar GA, Nikolova MT, Kosturkova GP. Isoflavone composition, phenol content, and antioxidant activity of soybean seeds from India and Bulgaria. J. Agr. Food Chem. 2008;56:2090–2095. doi: 10.1021/jf072939a. [DOI] [PubMed] [Google Scholar]
  • 18.Phommalth S, Hwang YH, Jeong YS, Kim YH. Isoflavone composition within each structural part of soybean seeds and sprouts. J. Crop. Sci. Biotechnol. 2008;11:57–62. [Google Scholar]
  • 19.Quinhone Júnior A, Ida E. Profile of the contents of different forms of soybean isoflavones and the effect of germination time on the secompounds and the physical parameters in soybean sprouts. Food Chem. 2015;166:173–178. doi: 10.1016/j.foodchem.2014.06.012. [DOI] [PubMed] [Google Scholar]
  • 20.Yuan JP, Liu YB, Peng J, Wang JH, Liu X. Changes of isoflavone profile in the hypocotyls and cotyledons of soybeans during dry heating and germination. J. Agr. Food Chem. 2009;57:9002–9010. doi: 10.1021/jf902248b. [DOI] [PubMed] [Google Scholar]
  • 21.Kim WJ, Lee HY, Won MH, Yoo SH. Germination effect of soybean on its contents of isoflavones and oligosaccharides. Food Sci. Biotechnol. 2005;14:498–502. [Google Scholar]
  • 22.Mekky RH, Thabet MM, Rodríguez-Pérez C, Yousry Elnaggar DM, Mahrous EA, Segura-Carretero A, Abdel-Sattar E. Comparative metabolite profiling and antioxidant potentials of seeds and sprouts of three Egyptian cultivars of Vicia faba L. Food Res. Int. 2020;136:35–37. doi: 10.1016/j.foodres.2020.109537. [DOI] [PubMed] [Google Scholar]
  • 23.Abu-Reidah IM, Arráez-Román D, Warad I, Fernández-Gutiérrez A, Segura-Carretero A. UHPLC/MS2-based approach for the comprehensive metabolite profiling of bean (Vicia faba L) by-products: a promising source of bioactive constituents. Food Res. Int. 2017;93:87–96. doi: 10.1016/j.foodres.2017.01.014. [DOI] [PubMed] [Google Scholar]
  • 24.Ammar S, Contreras MDM, Belguith-Hadrich O, Bouaziz M, Segura-Carretero A. New insights into the qualitative phenolic profile of Ficus carica L. fruits and leaves from Tunisia using ultra-high-performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry and their antioxidant activity. RSC Adv. 2015;5(26):20035–20050. [Google Scholar]
  • 25.Alseekh S, Perez de Souza L, Benina M, Fernie AR. The style and substance of plant flavonoid decoration; towards defining both structure and function. Phytochem. 2020;174:112347. doi: 10.1016/j.phytochem.2020.112347. [DOI] [PubMed] [Google Scholar]
  • 26.Bi WW, Zhao GX, Xia XY, Wang GJ, Bian KX, Zhang FY. Comprehensive analysis on soybean sprout characteristics of main soybean varieties in Heilongjiang Province. Soybean Sci. 2021;40(02):209–216. [Google Scholar]
  • 27.Torres-Fuentes C, Contreras MDM, Recio I, Alaiz M, Vioque J. Identification and characterization of antioxidant peptides from chickpea protein hydrolysates. Food Chem. 2015;180:194–202. doi: 10.1016/j.foodchem.2015.02.046. [DOI] [PubMed] [Google Scholar]
  • 28.Mohseni-Mehran S, Golshani B. Simultaneous determination of levodopa and carbidopa from fava bean, green peas and green beans by high performance liquid gas chromatography. J. Clin. Diagn. Res. JCDR. 2013;7(6):1004. doi: 10.7860/JCDR/2013/5415.3072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Abu-Reidah IM, Contreras MDM, Arráez-Román D, Fernández-Gutiérrez A, Segura-Carretero A. UHPLC-ESI-QTOF-MS-based metabolic profiling of Vicia faba L. (Fabaceae) seeds as a key strategy for characterization in foodomics. Electrophoresis. 2014;35(11):1571–1581. doi: 10.1002/elps.201300646. [DOI] [PubMed] [Google Scholar]
  • 30.Mekky RH, Abdel-Sattar E, Segura-Carretero A, Contreras MDM. Phenolic compounds from sesame cake and antioxidant activity: a new insight for Agri-Food residues’ significance for sustainable development. Foods. 2019;8(10):432. doi: 10.3390/foods8100432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Valente IM, Cabrita ARJ, Malushi N, Oliveira HM, Papa L, Rodrigues JA, Fonseca AJM, Maia MRG. Unravelling the phytonutrients and antioxidant properties of European Vicia faba L. seeds. Food Res. Int. 2019;116:888–896. doi: 10.1016/j.foodres.2018.09.025. [DOI] [PubMed] [Google Scholar]
  • 32.Mekky RH, Contreras MDM, El-Gindi MR, Abdel-Monem AR, Abdel-Sattar E, Segura-Carretero A. Profiling of phenolic and other compounds from Egyptian cultivars of chickpea (Cicer arietinum L.) and antioxidant activity: a comparative study. RSC Adv. 2015;5(23):17751–17767. [Google Scholar]
  • 33.Rodríguez-Pérez C, Zengin G, Segura-Carretero A, Lobine D, Mahomoodally MF. Chemical fingerprint and bioactivity evaluation of Globularia orientalis L. and Globularia trichosantha Fisch. C. A. Mey. using non-targeted HPLC-ESI-QTOF-MS approach. Phytochem. Anal. 2019;30(2):237–252. doi: 10.1002/pca.2809. [DOI] [PubMed] [Google Scholar]
  • 34.Abouzed TK, Contreras MDM, Sadek KM, Shukry MH, Abdelhady D, Gouda WM, Abdo W, Nasr NE, Mekky RH, Segura-Carretero A, Kahilo KA-A, Abdel-Sattar E. Red onion scales ameliorated streptozotocin-induced diabetes and diabetic nephropathy in Wistar rats in relation to their metabolite fingerprint. Diabetes Res. Clin. Pract. 2018;140:253–264. doi: 10.1016/j.diabres.2018.03.042. [DOI] [PubMed] [Google Scholar]
  • 35.Gurfinkel DM, Rao AV. Soyasaponins: The relationship between chemical structure and colon anticarcinogenic activity. Nutr. Cancer. 2003;47(1):24–33. doi: 10.1207/s15327914nc4701_3. [DOI] [PubMed] [Google Scholar]
  • 36.Hwang E-S. Antioxidant and quinone reductase activity of soyasaponins in hepa1c1c7 mouse hepatocarcinoma cells. Prev. Nutr. Food Sci. 2017;22(4):300. doi: 10.3746/pnf.2017.22.4.300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Megías C, Cortés-Giraldo I, Girón-Calle J, Alaiz M, Vioque J. Characterization of Vicia (Fabaceae) seed water extracts with potential immunomodulatory and cell antiproliferative activities. J. Food Biochem. 2018;42(5):e12578. [Google Scholar]
  • 38.Fabiola AG-O, Martín-Martínez ES, María EV, Yolanda R-A, José De JB, Rosalva ME. Profile analysis and correlation across phenolic compounds, isoflavones and antioxidant capacity during germination of soybeans (Glycine max L.). CyTA. J. Food. 2017;15(4):516–524. [Google Scholar]
  • 39.Dueñas M, Hernández T, Estrella I, Fernández D. Germination as a process to increase the polyphenol content and antioxidant activity of lupin seeds (Lupinus angustifolius L.) Food Chem. 2009;117:599–607. [Google Scholar]
  • 40.Michalak A. Phenolic compounds and their antioxidant activity in plants growing under heavy metal stress. Pol. J. Environ. Stud. 2006;15:523–530. [Google Scholar]
  • 41.Wu Z, Song L, Feng S, Liu Y, He G, Yioe Y, Liu SQ, Huang D. Germination dramatically increases isoflavonoid content and diversity in Chickpea (Cicer arietinum L.) seeds. J. Agr. Food Chem. 2012;60(35):8606–8615. doi: 10.1021/jf3021514. [DOI] [PubMed] [Google Scholar]
  • 42.Valente IM, Maia MRG, Malushi N, Oliveira HM, Papa L, Rodrigues JA, Fonseca AJM, Cabrita ARJ. Profiling of phenolic compounds and antioxidant properties of European varieties and cultivars of Vicia faba L. pods. Phytochemistry. 2018;152:223–229. doi: 10.1016/j.phytochem.2018.05.011. [DOI] [PubMed] [Google Scholar]
  • 43.Ayet G, Burbano C, Cuadrado C, Pedrosa M, Robredo L, Muzquiz M, De la Cuadra C, Castano A, Osagie A. Effect of germination, under different environmental conditions, on saponins, phytic acid and tannins in lentils (Lens culinaris) J. Sci. Food Agr. 1997;74(2):273–279. [Google Scholar]
  • 44.Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28(1):27–30. doi: 10.1093/nar/28.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Scientific Reports are provided here courtesy of Nature Publishing Group

RESOURCES