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Article

Variations in Isoflavone During Soybean Maturation and Their Thermal Process-Dependent Conversion

by
Ji Hye Kim
,
Jae-Hee Kim
and
Seok Hyun Eom
*
Graduate School of Green-Bio Science, College of Life Sciences, Kyung Hee University, Yongin 17104, Republic of Korea
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(9), 2155; https://doi.org/10.3390/agronomy15092155
Submission received: 21 August 2025 / Revised: 3 September 2025 / Accepted: 8 September 2025 / Published: 9 September 2025
(This article belongs to the Special Issue Quality and Safety of Crops and Crop-Based Foods)
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Abstract

Immature soybean seeds, after steaming or boiling, are widely consumed in Northeast Asia. However, changes in isoflavones and antioxidant activities during processing at different seed stages remain underexplored. In this study, soybean seeds at four maturity stages (R5–R8) were analyzed for 12 isoflavones and evaluated after steaming. Total isoflavone content increased from R6 to R7 and remained stable to R8, presenting a 10-fold increase in R7 than in R6. Levels of malonyl derivatives, such as malonylgenistin, malonyldaidzin, and malonylglycitin, consistently decreased with longer steam treatment at all seed stages. In contrast, β-glycoside forms, such as genistin and daidzin, increased after steaming, with notably high content at R7. Additionally, ABTS radical scavenging activity and total phenolic content showed strong positive correlations with total and major isoflavones, whereas DPPH radical scavenging activity showed no correlation with maturity stage or steam treatment. These findings indicate that isoflavone stability and conversion are strongly affected by seed maturation and that the R7 stage offers a favorable balance for high isoflavone and antioxidant intake in soybean seeds.

1. Introduction

Soybean (Glycine max) is a major source of oil and protein as well as a rich source of bioactive compounds, especially isoflavones [1]. Isoflavones, structurally similar to phytoestrogens, exhibit antioxidant, anti-inflammatory, and anticancer activities, making them valuable components of functional foods, cosmetics, and pharmaceuticals [2]. In Asia, including the Republic of Korea and Japan, soybeans harvested at various maturity stages are widely consumed following thermal processing, such as in edamame, gochujang, and miso [3,4].
Soybeans are typically boiled, steamed, or roasted to improve palatability, reduce antinutritional factors, and enhance digestibility [5,6,7]. These treatments also alter isoflavone content and composition. In soybean seeds, isoflavones occur as malonylglycosides, acetylglycosides, β-glycosides, and aglycones and can undergo thermal degradation or structural conversion during heating [8,9]. Malonyl isoflavones, the dominant forms in raw seeds, are thermally unstable and readily convert to more heat-stable forms, such as β-glucosides or aglycones, during heat treatment [10]. However, the degradation or conversion varies depending on the internal and external environmental changes, such as processing method, heating duration, cultivar, and seed maturity [11,12,13].
Previous studies have highlighted the importance of seed maturity for determining isoflavone conversion during thermal processing [7]. The findings revealed that immature and mature soybeans show distinct patterns of isoflavone degradation or conversion when subjected to heating (wet or dry). For example, malonylglycosides underwent deconjugation under wet heating than dry heating, while acetylglycosides appeared in mature seeds, regardless of moisture conditions. In other studies [14,15,16], they have also emphasized various responses in isoflavone profiles under different thermal conditions. These results highlight that seed maturity affects isoflavone transformation in the seeds.
Although the importance of seed maturity in isoflavone stability during thermal processing was previously explored, their study focused on comparing immature and mature soybeans. A comprehensive investigation based on defined seed maturity and their specific responses to steaming has not yet been conducted. Moreover, soybean seeds at various stages of maturity exhibit distinct physiological characteristics, including water content, chlorophyll levels, and enzymatic activity [7]. These factors influence heat transfer during processing, affecting isoflavone conversion.
Isoflavones are the primary secondary metabolites responsible for soybean antioxidant activity [17], and their bioactivity and bioavailability depend on chemical structure. Although malonylglycosides are the most abundant isoflavones in soybeans, they exhibit low absorption in the human body [18]. Conversely, aglycone and β-glycosides show higher bioavailability and stronger antioxidant activity [19]. Aglycones are absorbed more rapidly, bind estrogen receptors more effectively, and provide stronger antioxidant defense [18,20]. To optimize their functional potential in food applications, understanding isoflavones’ structural transformations during thermal processing is essential.
Therefore, this study investigated isoflavone accumulation patterns during soybean seed maturation and evaluated steaming effects on isoflavone conversion and antioxidant activity across four maturity stages.

2. Materials and Methods

2.1. Chemicals

All solvents used were of HPLC grade. Standards of 12 individual isoflavones were obtained from LC Laboratories (Woburn, MA, USA), Nacalai Tesque (Kyoto, Japan), and GenDEPOT (Katy, TX, USA), as described by Lim et al. (2021) [21]. Ascorbic acid (Daejung Chemical & Metal Co., Siheung, Republic of Korea) and gallic acid (Daejung Chemical & Metal Co., Siheung, Republic of Korea) were used as a standard compound in the antioxidant activity and total phenolic content (TPC) assays.

2.2. Plant Materials

Soybean seeds (Glycine max L. cv. Pungwon) used in this study were obtained from Pulmuone Food Co. (Chungbuk, Republic of Korea), as described by Qu et al. [7]. Seeds were soaked in distilled water for 8 h and then sown in 72-hole plug trays (2 × 2 × 4.5 cm per cell, width × length × depth; Asia Seed Co., Ltd. Seoul, Republic of Korea) filled with horticultural soil (Baroker, Seoulbio Co., Eumseong, Republic of Korea). After the first true leaves matured, seedlings were transplanted into pots and grown under natural sunlight at Kyung Hee University.
Pods were harvested at four maturity stages (Figure 1A): R5, immature green seed; R6, fully developed green seed; R7, beginning of seed maturation; R8, fully matured yellow seed. Seed maturity was verified by measuring fresh weight, water content, and soluble solid content (SSC) (Figure 1B–D). From R5 to R7, fresh weight and SSC increased significantly, followed by a decrease at R8. Water content gradually decreased with maturity. These morphological and physicochemical indicators guided seed harvesting for subsequent steam treatments.

2.3. Thermal Processing

Harvested soybean pods were placed on gauze-lined steamers over boiling water and steamed for 5, 10, 20, or 40 min. After steaming, seeds and pods were separated, and the seeds were first stored at −80 °C and subsequently freeze-dried using a freeze dryer (Ilshin Biobase Co., Seoul, Republic of Korea). Dried samples were ground using a commercial mixer and analyzed for isoflavone and flavonoid content as well as antioxidant activity and TPC.

2.4. Measurement of Physiochemical Characteristics

Seed weight was measured before and after thermal processing. For each maturity stage, dry weight was determined from 20 individual steam-treated seeds after freeze-drying. Water content was calculated following Qu et al. [7]. For total chlorophyll (TChl) determination, 15 mg of freeze-dried sample was extracted with 1 mL of 80% acetone in darkness with shaking at 15 °C for 4 h. The extract was filtered through a 0.45-μm syringe filter (Futecs Co., Ltd., Daejeon, Republic of Korea), and filtrate absorbance was measured at 645 and 663 nm using an S-4100 spectrophotometer (Scincon Co., Seoul, Republic of Korea). TChl content (mg/g dry weight) was calculated using the following equation [22]:
TChl = [(20.2 × OD645) + (8.02 × OD663)] × v/w,
where v is the extract volume (1 mL) and w is the sample weight (15 mg). For the SSC and pH measurement, 50 mg of freeze-dried sample was extracted with 1 mL of distilled water in a shaking incubator at 25 °C for 1 h and the extract was filtered through the syringe filter. After that, SSC was measured using a digital refractometer (PAL-1, Atago, Tokyo, Japan) placing 0.2 mL of extract on the refractometer’s surface at 25  ±  1 °C. The pH value was obtained using a pH meter (Hanna HI1131, HANNA Instruments, Woonsocket, RI, USA).

2.5. HPLC Analysis of Soybean Isoflavones

Isoflavone analysis followed Qu et al. [7] with minor modifications. Freeze-dried soybean powder (20 mg) was suspended in 1 mL of 58% acetonitrile and extracted overnight at 27 °C in a shaking incubator. The extract was then centrifuged at 12,000 rpm for 5 min, filtered through a 0.45-μm syringe filter, and injected into a HPLC system (Waters 2695 Alliance; Waters Inc., Milford, MA, USA). Chromatographic separation was performed using a Prontosil 120-5-C18 SH column (5.0 μm, 200 × 4.6 mm; Bischoff, Leonberg, Germany), with a mobile phase consisting of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). The gradient elution program was as follows: 0–35 min, 16%–25% B; 35–40 min, 25%–50% B; 40–47 min, 50%–65% B; 47–50 min, 65%–16% B; 50–52 min, 16% B. The flow rate and injection volume were 0.8 mL/min and 10 μL, respectively. Detection was conducted at 254 nm using a photodiode array detector (Waters 996 PDA detector; Waters Inc., MIlford, MA, USA). Isoflavones were identified and quantified using retention times and standard calibration curves.

2.6. Measurements of Antioxidant Activity and Total Phenolics

For the analysis of antioxidant activity and total phenolics (TPC), 15 mg of ground sample was extracted with 1 mL of 80% methanol in a shaking incubator for overnight at 25  ±  1 °C. The extract was centrifuged at 12,000× g for 5 min at 25 °C and the supernatant was stored at 4 °C until further analysis. To access the antioxidant activity, both the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging ability were used. The TPC was performed using the Folin–Ciocalteu method. These procedures were conducted following the protocol of Kim et al. [23]. The results were expressed as mg of vitamin C equivalent (VCE)/g dry weight for antioxidant activity, and mg of gallic acid equivalent (GAE)/g dry weight for TPC.

2.7. Statistical Analysis

All treatments were conducted in triplicate. Statistical differences between treatments were analyzed using Tukey’s HSD test at p < 0.05 (SAS software Enterprise Guide version 7.1; SAS Institute Inc., Cary, NC, USA). Pearson correlation coefficients were calculated for correlation analyses.

3. Results

3.1. Physicochemical Properties of Soybean Seeds During Maturation and Steaming

Figure 2 and Table 1 show the morphological and physicochemical properties, respectively, of soybean seeds harvested at four maturity stages (R5–R8) after steaming. As seeds matured, size increased to a maximum at R7, followed by a slight decrease at R8 (full maturity; Figure 2). This trend was consistent with results for dry weight and SSC, which peaked at R7 and declined at R8 (Table 1). In contrast, water content and TChl content steadily decreased with maturity, alongside visible color changes from green (R5) to yellow (R8) (Figure 2 and Table 1). Slightly acidic pH was observed at R5, with pH increasing to a peak at R7 before declining at R8. Steaming did not significantly alter water content or dry weight compared with untreated controls at any treatment duration. However, seed color lightened, especially at R5 and R6, due to chlorophyll degradation, which intensified with prolonged steaming (Figure 2 and Table 1). Additionally, SSC decreased with prolonged steaming, whereas pH showed no clear steaming duration–related pattern.

3.2. Total Isoflavone Contents of Soybean Seeds During Maturation and Steaming

As shown in Figure 3 and Supplementary Figure S1, 12 individual isoflavones were quantified using HPLC using standard curves (Supplementary Figure S2), and total isoflavone content was calculated as their sum. In early seed maturity stages, isoflavone levels were low, with 0.37 and 0.59 μg/g dry weight (d.w.) observed at R5 and R6, respectively. Content increased approximately 10-fold with maturity, showing a significant rise from R6 and R7. In steamed seeds, total isoflavone content exhibited stage-specific thermal responses (Figure 4). At R5, R6, and R8, prolonged steaming caused gradual content decreases, whereas a distinct pattern emerged at R7, where total isoflavone levels increased with steaming duration, peaking at 40 min. To identify which compounds maintained high total isoflavone levels at R7 after extended steaming, all 12 isoflavones were analyzed in detail.

3.3. Conversion of 12 Individual Types During Maturation and Steaming of Soybean Seeds

Figure 5 shows changes in the levels of 12 isoflavones in soybean seeds at different maturity stages after up to 40 min of steaming. Among these, malonylgenistin (MGNI) was most abundant and consistently decreased across all stages with prolonged steaming. After 40 min, MGNI levels decreased by approximately 60% in R5 and R6 seeds, around 40% in R7 seeds, and almost 50% in R8 seeds compared with untreated controls (0 min). In contrast, acetylgenistin (AGNI) was not detected at any maturity stage or under any steam treatment. Genistin (GNI) was absent in R5 seeds under all conditions but increased in later maturity stages with longer steaming durations, rising over 3-fold after 40 min of processing compared with the control. These findings suggest that MGNI mainly degrades to the β-glycoside GNI, rather than to AGNI or genistein, during steaming.
Levels of malonyldaidzin (MDZI), the second most abundant isoflavone, also decreased steadily at all maturity stages with longer steaming durations (Figure 5); however, the reduction was slower in R7. As MDZI levels declined, its likely degradation products, acetyl daidzin (ADZI) and daidzin (DZI), were undetected at R5 and R6 under all treatments but present at R7 and R8, where levels increased with steaming duration, peaking at 40 min. Levels of daidzein (DZE), the aglycone form of DZI, generally declined with prolonged steaming, unlike its acetyl and β-glycoside forms; DZE was undetectable at R5 and R6, and appeared only at low levels in R7 and R8 seeds. These results suggest that the malonyl form converts to acetyl and β-glycoside but not aglycone forms.
Levels of malonylglycitin (MGLI) decreased with longer steaming durations, exhibiting stage-specific responses (Figure 5). In R5 seeds, MGLI content declined slightly, with no significant differences among treatment durations. In R6 and R7 seeds, MGLI levels remained stable up to 20 min but declined significantly after 40 min of steaming. In R8 seeds, a content reduction of >80% occurred at 20 min compared with the untreated control (0 min). Acetylglycitin (AGLI) was absent in R5 and R6 seeds under all conditions. In contrast, AGLI levels decreased steadily in R7 and R8 seeds with steaming duration, with a more pronounced decline observed at R8. Levels of the β-glycoside glycitin (GLI) showed a similar pattern to MGLI, i.e., an initial increase followed by a decline with longer steaming periods. The aglycone glycitein (GLE) was absent at R5 and R6 but appeared at R7 and R8, where levels rose steadily with longer steaming durations. These findings indicate that GLI derivatives undergo structural transformation during heat processing, with malonyl and acetyl groups degraded and converted primarily into the aglycone GLE rather than into β-glycosides. This conversion was more pronounced in later seed maturity stages, especially R8.

3.4. Antioxidant Activities and Total Phenolic Contents

Figure 6 shows the antioxidant activities and TPC of soybean seeds at different maturity stages before and after steaming. Notably, DPPH radical scavenging activity remained constant across all maturity stages and steaming treatments, with no significant differences observed (Figure 6A). In contrast, ABTS radical scavenging activity and TPC increased with seed maturity in unsteamed seeds, peaking at the R8 stage (Figure 6(B-1,C-1)). After steaming, ABTS activity and TPC increased in R5 seeds up to 20 min of steaming before declining as treatment duration increased. In R6 and R7 seeds, no significant changes occurred with steaming. However, in R8 seeds, both ABTS activity and TPC tended to decrease as steaming duration increased (Figure 6(B-2,C-2)).

4. Discussion

This study investigated the effects of steaming on isoflavone composition and antioxidant activity in soybean seeds harvested at four well-defined maturity stages (R5–R8). Through this approach, we identified distinct isoflavone thermal stability at the R7 stage. These findings emphasized the importance of seed maturity in determining the thermal behavior of isoflavones.
Although certain maturity stages, such as R5 and R8, are not commonly used in commercial processing, our data suggest their potential applicability in industrial products, considering their thermal responses. For instance, soybeans at the R5 stage have a tender texture and mild flavor and are well suited for minimally processed products such as edamame. On the other hand, R8 stage soybeans, which are traditionally used in fermented products like miso and gochujang, can benefit from optimized thermal processing strategies to preserve or enhance isoflavone content. Therefore, our findings provide practical guidance for tailoring processing conditions based on seed maturity to improve the functional quality of soybean-based foods.
Our results revealed that total and individual isoflavone levels, antioxidant activity, and TPC were significantly altered by maturity and steaming duration. The total isoflavone accumulation pattern across stages aligned with previous studies [16,24,25]. Although the peak accumulation stage may vary by cultivar, R7 seeds contained isoflavone levels >3-fold higher than those in earlier-stage seeds.
Physicochemical traits, such as dry weight, water content, chlorophyll content, and pH, were strongly associated with isoflavone accumulation across maturity stages (Figure 7). Total isoflavones correlated positively with dry weight (r = 0.84, p < 0.0001), suggesting that as seeds matured and biomass accumulated, isoflavone biosynthesis or retention increased. Additionally, pH positively correlated with total isoflavone content (r < 0.82, p < 0.0001), indicating that more mature seeds with higher isoflavone levels had lower acidity. Conversely, water content and TChl content were negatively correlated with isoflavone levels (r = –0.75 and –0.63, respectively), implying that maturation processes involving water loss and chlorophyll decline favor isoflavone accumulation. Although SSC increased with seed maturity, it showed no significant correlation with isoflavone accumulation, likely because sugars degrade readily during heating, regardless of higher initial concentrations. These results confirm that seed physicochemical properties reliably indicate maturity stage and are closely linked to isoflavone composition and thermal behavior.
Isoflavone profiles in steamed seeds exhibited structure-dependent thermal responses [26]. Malonyl isoflavones, which showed a consistent decrease in content with increasing steaming duration across all maturity stages, were thermally labile [27,28,29]. Their structures make them susceptible to decarboxylation or de-esterification during heating [30,31,32]. In particular, MGNI loss coincided with accumulation of the β-glycoside form GNI, without formation of the acetylated form AGNI, suggesting direct MGNI-to-GNI conversion during steaming. Yue et al. [33] reported that GNI has a half-life of around 144–169 min at 100 °C, far longer than the 40 min maximum steaming conducted in this study, making severe GNI degradation unlikely. Indeed, the observed GNI increase with extended steaming supports the notion that the MGNI deconjugation rate outpaced the GNI thermal degradation rate, resulting in net accumulation.
Levels of MDZI also decreased steadily with longer steaming durations, whereas its presumed degradation products, ADZI and DZI, showed time-dependent increases at R7 and R8. This suggests that MDZI was efficiently converted into acetyl and β-glycoside forms with prolonged heating [34], especially at later maturity stages. Conversely, the aglycone DZE exhibited a continuous reduction in content and was nearly undetectable after 40 min of steaming, indicating that conjugated isoflavone breakdown does not necessarily yield aglycones.
For glycitin derivatives, both MGLI and AGLI levels remained stable during the early stages of steam treatment but declined as steaming progressed, with maturity stage–specific patterns observed. Their reduction coincided with an increase in levels of the β-glycoside form GLI, particularly up to R7. However, at R8, GLE accumulation surpassed that of GLI. These results suggest that MGLI and AGLI were first converted to GLI through thermal deglycosylation, followed by further degradation into the aglycone form GLE. Based on this sequential pathway (malonyl → acetyl → β-glycoside → aglycone), GLI isoflavones show greater thermolability compared with DZE and GNE derivatives, as previously reported by Yue et al. [33].
Notably, isoflavone accumulation and degradation patterns varied markedly with seed maturity. Malonyl isoflavones are known to degrade more rapidly in immature seeds [7], and thermal processing induces maturity-specific isoflavone profiles [35]. These results align with our finding that total isoflavone levels remained high at R7, even after prolonged steaming. Four major isoflavones, namely MGNI, MDZI, GNI, and DZI, dominated total isoflavone content, as supported by their strong correlations (Figure 7). MGNI and MDZI levels decreased with steaming duration, although the reduction was less pronounced at R7, possibly due to the greater thermal stability of malonyl isoflavones or altered conversion pathways at the R7 stage. Physicochemical factors, such as pH, may also have affected isoflavone retention in R7 seeds. In contrast, GNI and DZI levels consistently increased with steaming duration, with the largest increases observed at R7, suggesting efficient malonyl-to-β-glycoside conversion without excessive degradation, promoting net isoflavone accumulation. Therefore, the R7 stage appears to offer optimal conditions for isoflavone retention and conversion during thermal processing, making it a promising maturity stage for functional soy-based food production.
Isoflavones are key contributors to soybean antioxidant activity [36]. Thus, the significant change in isoflavone profiles observed with steaming in this study likely affected soybean antioxidant properties. However, the DPPH radical scavenging activity did not vary significantly across maturity stages or steaming treatments. As shown in Figure 7, the activity showed no correlation with physicochemical traits, total and individual isoflavone, and TPC. This exhibited that isoflavone may have relatively low reactivity toward to DPPH radical. According to Ma and Huang (2014) [37], isoflavones showed no or low correlations with DPPH radical scavenging activity, which may be due to their structural characteristics, as isoflavones are presumed to be more effective in hydrogen atom transfer than in single electron transfer mechanisms. Conversely, ABTS radical scavenging activity was closely linked to total isoflavone content and TPC. Prior studies have shown that soybean antioxidant activity depends largely on isoflavones and phenolics [38,39]. Our correlation analysis (Figure 7) confirmed the isoflavone–antioxidant activity relationship, with ABTS radical scavenging activity and TPC exhibiting strong positive correlations with most isoflavones, unlike DPPH. DZI, MDZI, GNI, and MGNI correlated most strongly with ABTS radical scavenging activity, highlighting their important contributions to soybean antioxidant potential. Although aglycones often show higher antioxidant activity relative to that of conjugated isoflavones (sugar binding can reduce activity by ~50%) [1,38], our results show that structure alone is not decisive. MDZI and MGLI, despite low intrinsic activity, significantly influenced antioxidant activity owing to their relative abundance. Overall, these findings indicate that isoflavone structure and concentration shape antioxidant effects and that selecting suitable maturity stages and heat treatments can optimize soybean-based functional foods.

5. Conclusions

This study demonstrated that soybean seed maturity and steam-processing duration significantly influence isoflavone composition and antioxidant activity. Moreover, our study provided a clear criterion for assessing seed maturity through physicochemical characteristics as well as isoflavone content in R5 to R8 seeds. Total isoflavone content increased with seed maturity and, specifically, remained stable even after prolonged steaming at the R7 stage. Among 12 tested isoflavones, malonyl derivatives, such as MGNI and MDZI, were thermally degraded across all maturity stages, whereas β-glycoside forms, such as GNI and DZI, showed increased levels, particularly at R7, indicating a favorable thermal conversion pathway at this stage. The distinct response of R7 seeds, marked by limited degradation of heat-labile forms and efficient transformation into stable glucosides, helped preserve total isoflavone content. Physicochemical traits, such as dry weight, water content, and pH, were strongly correlated with isoflavone levels; total isoflavone content was positively correlated with dry weight and pH but negatively correlated with water and chlorophyll content, underscoring the role of physiological maturation in determining thermal responses. Furthermore, ABTS radical scavenging activity and TPC exhibited strong positive correlations with isoflavone content, whereas DPPH activity showed no correlation. Collectively, these findings identify R7 as the optimal harvest stage for producing steamed soybean-based functional foods, exemplified by the superior isoflavone stability and retention of R7 seeds.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15092155/s1, Figure S1: Isoflavone profiles of soybean seeds harvested at four developmental stages (R5 to R8), comparing untreated (non-steaming) and steamed (40 min) samples; Figure S2: Standard curves of 12 individual isoflavones.

Author Contributions

Conceptualization, S.H.E.; methodology, J.H.K., J.-H.K. and S.H.E.; software, J.H.K.; validation, J.H.K. and S.H.E.; formal analysis, J.H.K.; investigation, J.H.K. and J.-H.K.; resources, S.H.E.; data curation, J.H.K. and J.-H.K.; writing—original draft preparation, J.H.K.; writing—review and editing, S.H.E.; visualization, J.H.K. and J.-H.K.; supervision, S.H.E.; project administration, S.H.E.; funding acquisition, J.H.K., J.-H.K. and S.H.E. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the BK21 FOUR program of Graduate School, Kyung Hee University (GS-1-JO-NON-20240353).

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
SSCSoluble solid content
TPCTotal phenolic content
ADZIAcetyl daidzin
AGLIAcetyl glycitin
AGNIAcetyl genistin
DZEDaidzein
GLEGlycitein
GNEGenistein
MDZIMalonyl daidzin
MGLIMalonyl glycitin
MGNIMalonyl genistin
TITotal isoflavone
TIATotal isoflavone aglycone
TIAGTotal isoflavone acetly-glycosides
TIGTotal isoflavone glycosides
TIMGTotal isoflavone malonyl-glycosides

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Figure 1. (A) Morphology of soybean pods and seeds at four maturity stages (R5–R8). (B–D) Corresponding changes in (B) fresh weight, (C) water content, and (D) soluble solid content (SSC). Different letters indicate significant differences between each stage at p < 0.05 based on Tukey’s HSD test.
Figure 1. (A) Morphology of soybean pods and seeds at four maturity stages (R5–R8). (B–D) Corresponding changes in (B) fresh weight, (C) water content, and (D) soluble solid content (SSC). Different letters indicate significant differences between each stage at p < 0.05 based on Tukey’s HSD test.
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Figure 2. Morphological appearance of soybeans at four maturity stages (R5–R8) following 0, 5, 10, 20, and 40 min of steaming. R5, immature green seeds; R6, fully developed green seeds; R7, beginning of seed maturation; R8, fully matured yellow seeds.
Figure 2. Morphological appearance of soybeans at four maturity stages (R5–R8) following 0, 5, 10, 20, and 40 min of steaming. R5, immature green seeds; R6, fully developed green seeds; R7, beginning of seed maturation; R8, fully matured yellow seeds.
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Figure 3. (A) HPLC chromatograms of 12 isoflavones, and (B) total isoflavone content in soybean seeds at different maturity stages. 1, daidzin; 2, glycitin; 3, genistin; 4, malonyldaidzin; 5, malonylglycitin; 6, acetyldaidzin; 7, acetylglycitin; 8, malonylgenistin; 9, daidzein; 10, glycitein; 12, genistein. Uppercase letters indicate significant differences among maturity stages (p < 0.05, Tukey’s HSD test).
Figure 3. (A) HPLC chromatograms of 12 isoflavones, and (B) total isoflavone content in soybean seeds at different maturity stages. 1, daidzin; 2, glycitin; 3, genistin; 4, malonyldaidzin; 5, malonylglycitin; 6, acetyldaidzin; 7, acetylglycitin; 8, malonylgenistin; 9, daidzein; 10, glycitein; 12, genistein. Uppercase letters indicate significant differences among maturity stages (p < 0.05, Tukey’s HSD test).
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Figure 4. Total isoflavone content in soybean seeds at four maturity stages after steaming for 5, 10, 20, and 40 min. Data are means ± standard errors (n = 3). Different lowercase letters indicate significant differences among steaming durations within the same maturity stage; different uppercase letters indicate significant differences among maturity stages at the same time (p < 0.05, Tukey’s HSD test).
Figure 4. Total isoflavone content in soybean seeds at four maturity stages after steaming for 5, 10, 20, and 40 min. Data are means ± standard errors (n = 3). Different lowercase letters indicate significant differences among steaming durations within the same maturity stage; different uppercase letters indicate significant differences among maturity stages at the same time (p < 0.05, Tukey’s HSD test).
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Figure 5. Content of 12 isoflavone types in soybean seeds at four maturity stages (R5–R8) after steaming for 5, 10, 20, and 40 min. Data are means ± standard errors (n = 3). Different lowercase letters indicate significant differences among steaming durations within the same maturity stage; different uppercase letters indicate significant differences among the stages at the same time (p < 0.05, Tukey’s HSD test).
Figure 5. Content of 12 isoflavone types in soybean seeds at four maturity stages (R5–R8) after steaming for 5, 10, 20, and 40 min. Data are means ± standard errors (n = 3). Different lowercase letters indicate significant differences among steaming durations within the same maturity stage; different uppercase letters indicate significant differences among the stages at the same time (p < 0.05, Tukey’s HSD test).
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Figure 6. (A) DPPH and (B) ABTS radical scavenging activities and (C) total phenolic content in soybean seeds at four maturity stages (R5–R8). (A-1–C-1) Unsteamed seeds and (A-2–C-2) steamed seeds treated for 5, 10, 20, and 40 min. Data are means ± standard errors (n = 3). Different lowercase letters indicate significant differences among steaming durations within the same maturity stage; different uppercase letters indicate significant differences among maturity stages at the same time (p < 0.05, Tukey’s HSD test).
Figure 6. (A) DPPH and (B) ABTS radical scavenging activities and (C) total phenolic content in soybean seeds at four maturity stages (R5–R8). (A-1–C-1) Unsteamed seeds and (A-2–C-2) steamed seeds treated for 5, 10, 20, and 40 min. Data are means ± standard errors (n = 3). Different lowercase letters indicate significant differences among steaming durations within the same maturity stage; different uppercase letters indicate significant differences among maturity stages at the same time (p < 0.05, Tukey’s HSD test).
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Figure 7. Pearson correlation heatmap showing physicochemical properties, 12 individual isoflavones, antioxidant activities (DPPH and ABTS radical scavenging), and total phenolic content (TPC) in soybean seeds. *, **, and *** indicate significances at p < 0.05, p < 0.01, and p < 0.001 using Pearson’s correlation analysis. D.W., dry weight; W.C., water content; TChl, total chlorophyll content; DZE, daidzein; DZI, daidzin; ADZI, acetyl-daidzin; MDZI, malonyldaidzin; GLE, glycitein; GLI, glycitin; AGLI, acetylglycitin; MGLI, malonylglycitin; GNE, genistein; GNI, genistin; AGNI, acetylgenistin; MGNI, malonylgenistin; TDin, total daidzin derivatives; TGly, total glycitin derivatives; TGni, total genistin derivatives; TIA, total isoflavone aglycones; TIG, total isoflavone glucosides; TIAG, total isoflavone acetylglucosides; TIMG, total isoflavone malonylglucosides; TI, total isoflavones.
Figure 7. Pearson correlation heatmap showing physicochemical properties, 12 individual isoflavones, antioxidant activities (DPPH and ABTS radical scavenging), and total phenolic content (TPC) in soybean seeds. *, **, and *** indicate significances at p < 0.05, p < 0.01, and p < 0.001 using Pearson’s correlation analysis. D.W., dry weight; W.C., water content; TChl, total chlorophyll content; DZE, daidzein; DZI, daidzin; ADZI, acetyl-daidzin; MDZI, malonyldaidzin; GLE, glycitein; GLI, glycitin; AGLI, acetylglycitin; MGLI, malonylglycitin; GNE, genistein; GNI, genistin; AGNI, acetylgenistin; MGNI, malonylgenistin; TDin, total daidzin derivatives; TGly, total glycitin derivatives; TGni, total genistin derivatives; TIA, total isoflavone aglycones; TIG, total isoflavone glucosides; TIAG, total isoflavone acetylglucosides; TIMG, total isoflavone malonylglucosides; TI, total isoflavones.
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Table 1. Physicochemical properties of soybean seeds at four maturity stages (R5 to R8) after steam processing for 5, 10, 20, and 40 min.
Table 1. Physicochemical properties of soybean seeds at four maturity stages (R5 to R8) after steam processing for 5, 10, 20, and 40 min.
StageSteaming Time
(min)		Dry Weight
(g/ea)		Water Content
(%)		TChl Content
(mg/g d.w.)		SSC
(Brixº)		pH
R500.02 ± 0.00aC78.25 ± 0.07aA0.15 ± 0.00aB0.70 ± 0.06aB5.83 ± 0.01dC
50.02 ± 0.00aD78.39 ± 0.29aA0.13 ± 0.00bA0.63 ± 0.03abAB6.25 ± 0.01aB
100.02 ± 0.00aD76.58 ± 0.58abA0.11 ± 0.00cB0.57 ± 0.03abB6.12 ± 0.01bC
200.02 ± 0.00aD77.38 ± 0.38abA0.12 ± 0.00cA0.53 ± 0.03abB5.96 ± 0.01cB
400.02 ± 0.00aD75.46 ± 0.27bA0.04 ± 0.00dC0.47 ± 0.03bBC5.54 ± 0.02eD
R600.08 ± 0.01aB66.84 ± 0.43aB0.18 ± 0.00aA0.90 ± 0.06aA6.54 ± 0.01bB
50.06 ± 0.00abC65.17 ± 0.40aB0.14 ± 0.01bA0.47 ± 0.07bB5.99 ± 0.03cB
100.06 ± 0.00bC67.01 ± 0.17aB0.14 ± 0.00bA0.47 ± 0.03bB6.81 ± 0.01aB
200.06 ± 0.00bC66.38 ± 0.65aB0.08 ± 0.01cB0.40 ± 0.00bC6.02 ± 0.03cB
400.05 ± 0.00bC67.51 ± 0.20aA0.05 ± 0.00dB0.40 ± 0.00bC6.51 ± 0.04bC
R700.15 ± 0.01aA52.53 ± 1.18aC0.07 ± 0.00aC1.23 ± 0.03aB7.01 ± 0.03bA
50.14 ± 0.00aA54.78 ± 0.88aC0.06 ± 0.00bB0.50 ± 0.00bB7.15 ± 0.01aA
100.14 ± 0.00aA53.50 ± 0.45aC0.06 ± 0.00bC0.53 ± 0.03bB7.15 ± 0.00aA
200.14 ± 0.01aA54.95 ± 0.46aC0.06 ± 0.00bC0.43 ± 0.03bBC7.11 ± 0.01aA
400.14 ± 0.00aA55.39 ± 0.49aB0.06 ± 0.00bA0.57 ± 0.03bB6.75 ± 0.01cB
R800.08 ± 0.00aB7.41 ± 0.29aD0.07 ± 0.00aC0.83 ± 0.03aB6.51 ± 0.02bB
50.09 ± 0.00aB7.61 ± 0.40aD0.07 ± 0.00aB0.77 ± 0.03aA7.14 ± 0.01aA
100.08 ± 0.00aB10.79 ± 1.03aD0.06 ± 0.01bC0.77 ± 0.07aA7.15 ± 0.01aA
200.09 ± 0.00aB12.25 ± 0.12aD0.06 ± 0.00bC0.83 ± 0.03aA7.13 ± 0.02aA
400.08 ± 0.00aB15.46 ± 3.07aC0.04 ± 0.00cC0.77 ± 0.03aA7.08 ± 0.03aA
Values are mean ± standard error from triplicate experiments. Lowercase letters indicate differences between processing durations within the same stage, uppercase letters indicate differences between stages within the same time.
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Kim, J.H.; Kim, J.-H.; Eom, S.H. Variations in Isoflavone During Soybean Maturation and Their Thermal Process-Dependent Conversion. Agronomy 2025, 15, 2155. https://doi.org/10.3390/agronomy15092155

AMA Style

Kim JH, Kim J-H, Eom SH. Variations in Isoflavone During Soybean Maturation and Their Thermal Process-Dependent Conversion. Agronomy. 2025; 15(9):2155. https://doi.org/10.3390/agronomy15092155

Chicago/Turabian Style

Kim, Ji Hye, Jae-Hee Kim, and Seok Hyun Eom. 2025. "Variations in Isoflavone During Soybean Maturation and Their Thermal Process-Dependent Conversion" Agronomy 15, no. 9: 2155. https://doi.org/10.3390/agronomy15092155

APA Style

Kim, J. H., Kim, J.-H., & Eom, S. H. (2025). Variations in Isoflavone During Soybean Maturation and Their Thermal Process-Dependent Conversion. Agronomy, 15(9), 2155. https://doi.org/10.3390/agronomy15092155

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