Isoflavones of the Soybean Components and the ... - ACS Publications

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Isoflavones of the Soybean Components and the Effect of Germination Time in the Cotyledons and Embryonic Axis Alécio Quinhone Júnior† and Elza Iouko Ida*,† †

Departamento de Ciência e Tecnologia de Alimentos, Programa de Pós Graduaçaõ em Ciência de Alimentos, Universidade Estadual de Londrina, Londrina, Paraná 86057-970, Brazil ABSTRACT: The aim of this study was to evaluate the content of different forms of isoflavones of BRS 284 soybean components and the effect of germination time in the cotyledons, radicle, and hypocotyl. Seeds were germinated until 168 h at 35 °C and collected each 24 h. The isoflavone content was determined by ultraperformance liquid chromatography, and the data were subjected to regression analysis. In cotyledons, germination time had a quadratic effect on daidzin and genistin contents and a linear effect on malonyldaidzin and malonylgenistin contents. In radicles, germination time had a quadratic effect on daidzin, glycitin, malonylgenistin, and malonylglycitin contents in addition to a linear effect on malonyldaidzin content. In hypocotyls, germination time showed a cubic effect on daidzin and genistin contents, a quadratic effect on malonyldaidzin, malonylgenistin, and malonylglycitin contents, and a linear effect on genistein content; glycitin, daidzein and glycitein were detected in a few germination times. KEYWORDS: soybean sprouts, hypocotyl, aglycone, radicle, seedling

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INTRODUCTION

Soybean germination can be used as a processing method to improve the sensorial quality6 and nutritional value10,18 of soybean seeds because it reduces the amount of undesirable substances, such as phytic acid,19,20 oligosaccharides,17,21 and trypsin inhibitors, in addition to lipoxygenase activity.10 Germination can also promote a significant increase of vitamins,22 phytosterols, tocopherols,23 isoflavones,24,25 and isoflavone aglycones.10,14,17,23 In addition, the germination process is simple and inexpensive, and soybean sprouts have been investigated for several decades due to the high content and quality of protein and essential amino acids.6 The germination time may exert distinct effects depending on the soybean sprout components or isoflavone forms. In soybean cotyledons germinated for 72 h, the content of β-glucosides does not vary significantly, but the malonylglucoside content increases throughout the germination time. In radicles, however, a decrease in malonylglucoside and β-glucosides content occurs.13 In contrast, in cotyledons and hypocotyls germinated for 120 h, a decrease in malonylglucoside and β-glucoside contents in addition to an increase in aglycone content have been reported.14 However, the biotransformation of the various forms of isoflavones in soy components as a function of germination time has not been fully explored, and a few existing studies did not follow the entire process of germination considering the different forms of isoflavones in the various components of the germinated soybeans. The objective of this study was to evaluate the content of different forms of isoflavones of BRS 284 soybean components and the effect of germination time up to 168 h on the content of these isoflavones in cotyledons and the embryonic axis.

Soybean (Glycine max (L.) Merrill) and its derivatives have received more attention in recent years due to their potential beneficial effects on human health, such as protective effects against bone loss in postmenopausal women,1 prevention of cardiovascular diseases,2 relief of menopausal symptoms,3 and protective effects against breast cancer4 and prostate cancer.5 The main soy isoflavones are divided into aglycones (daidzein, genistein, and glycitein), β-glucosides (daidzin, genistin, and glycitin), malonylglucosides (6″-O-malonyldaidzin, 6″-O-malonylgenistin, and 6″-O-malonylglycitin), and acetylglucosides (6″O-acetyldaidzin, 6″-O-acetylgenistin, and 6″-O-acetylglycitin).6 During seed development of soybean, aglycones are synthesized by the phenylpropanoid metabolic pathway and stored in vacuoles as β-glucosides and malonylglucosides.7,8 However, aglycones and acetylglucosides are absent or in low concentrations.9,10 Isoflavone contents in soybean seeds display a broad range of variation because their synthesis and accumulation are affected by multiple environmental and genetic factors.11,12 Isoflavones may vary between soybean components. Total isoflavone content in the embryonic axis can be 5.0−25.0-fold higher than that in the cotyledons, whereas the seed coat is substantially free of isoflavones.8,13,14 During the course of soybean domestication, China gradually transformed soybean seeds in various forms of foods that are versatile, tasty, and digestible, such as tofu, sauce, paste, extract, and soybean sprouts.15 Soybean sprouts, which are produced after 6−7 days of germination, are an important vegetable source, are rich in nutrients, are available all year, and have been consumed for thousands of years in countries, such as Korea, China, and Japan.16 However, interest in healthy and functional foods has increased the demand for soybean sprouts in the West.17 © 2014 American Chemical Society

Received: Revised: Accepted: Published: 8452

March 7, 2014 July 28, 2014 July 29, 2014 July 29, 2014 dx.doi.org/10.1021/jf502927m | J. Agric. Food Chem. 2014, 62, 8452−8459

Journal of Agricultural and Food Chemistry

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Figure 1. Components of soybean seeds germinated by 168 h.

Figure 2. Typical UPLC chromatogram of isoflavones in a standard solution.

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performed in a germination chamber (model Mangelsdorf, J. Prolab São José dos Pinhais, PR, BR) with natural light at 35 °C (±1 °C) and relative humidity of 100% for 24, 48, 72, 96, 120, 144, and 168 h, as described by Yoshiara et al.27 The effect of germination time on the isoflavone content in radicles and hypocotyls was considered only after 48 h of germination when it was possible to observe these components and separate them manually. Germinated seeds were collected at each germination time, and the cotyledons, hypocotyls, and radicles (Figure 1) were separated manually and in sufficient quantity for analysis. Epicotyls and seed coats of germinated seeds were discarded. Thereafter, the components were identified, freeze-dried, ground (model A-11, Ika), placed in polyethylene bags, and stored at −22 °C for analysis. The components of ungerminated seeds were used for comparison. Analytical Procedures. Moisture content of the lyophilized samples was determined in a drying oven at 105 °C. The percentage of each structural part of the soybean seed was determined in triplicate using 100 g of seeds. Cotyledons, the embryonic axis, and seed coats were manually separated and weighed. The content of the different forms of isoflavones was also analyzed in these components. Prior to quantification of isoflavones, the lyophilized and milled samples were defatted with hexane at 1:10 (w/v) for 1 h at room temperature by continuous and rotary agitation followed by vacuum filtration. The isoflavone extraction was performed in triplicate with 0.1 g of defatted sample with 2 mL of extraction solution containing ultrapure water, acetone, and ethanol (1:1:1, v/v/v) as described by Yoshiara et al.28 The quantification of all isoflavones forms was performed simultaneously, in triplicate, using a UPLC system as described by Handa et al.29 The different forms of isoflavones and the sum of all forms of isoflavones were expressed as micromoles of the form

MATERIALS AND METHODS

Materials and Reagents. Soybean seeds of the BRS 284 cultivar, a conventional cultivar, grown during the 2011/2012 season were provided by a specialist company in the region and were used for this study. According to the Empresa Brasileira de Pesquisa Agropecuária,26 this cultivar is used for industrial processing, and it has an average size of 14.6 g per 100 seeds and contains 38.7% protein and 20.4% lipids; because of these characteristics, BRS 284 was used for this study. All reagents used were of analytical grade and from different sources. Acetylglucosides and malonylglucosides were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and the β-glucosides and aglycones were obtained from Sigma−Aldrich Co. (St. Louis, MO, USA) and were used as isoflavone standards. All standard solutions were filtered through a 0.22 μm syringe filter (Millex-GV, Millipore, Billerica, MA, USA) prior to injection into the ultraperformance liquid chromatography (UPLC) instrument. Experimental Design of the Germination Process. Soybean seeds were previously selected to eliminate damaged seeds, stained seeds, or any foreign material. Fifty soybean seeds were placed on two pieces of germination paper (37.5 cm × 28.2 cm; Germitest) previously wetted with distilled water and supported by a perforated plate. The seeds were then covered with another piece of paper of which the bottom edge was folded, and the papers were curled longitudinally to form rolls. To evaluate the effect of soybean germination time on the different forms of isoflavones in cotyledons and the embryonic axis, a randomized complete block design with three replications was used. For each block and germination time, 15 paper rolls were used. Germination was 8453

dx.doi.org/10.1021/jf502927m | J. Agric. Food Chem. 2014, 62, 8452−8459


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Table 1. Isoflavone Content (μmol g−1) in Seeds, Cotyledons, and the Embryonic Axis of BRS 284 Soybeansa isoflavone glucoside daidzin genistin glycitin malonylglucoside malonyldaidzin malnoylgenistin malonylglycitin aglycone daidzein genistein glycitein total isoflavone

seed

cotyledon

embryonic axis

2.273 ± 0.249a 0.618 ± 0.030a 1.376 ± 0.192a 0.279 ± 0.031a 6.381 ± 0.315a 1.530 ± 0.124a 4.132 ± 0.088a 0.718 ± 0.109a 0.104 ± 0.004a nd 0.104 ± 0.004a nd 8.757 ± 0.566a

2.056 ± 0.067a 0.464 ± 0.019a 1.592 ± 0.048a nd 5.401 ± 0.185a 1.134 ± 0.034a 4.267 ± 0.152a nd 0.109 ± 0.002a nd 0.109 ± 0.002a nd 7.565 ± 0.254a

15.819 ± 1.284b 5.059 ± 0.340b 2.459 ± 0.288b 8.301 ± 0.656b 37.701 ± 2.550b 12.715 ± 0.961b 6.405 ± 0.372b 18.581 ± 1.217b 0.324 ± 0.029b 0.223 ± 0.022 0.101 ± 0.008a nd 53.844 ± 3.863b

Mean ± standard deviation; nd: not detected; value with different letter in the same row is significantly different at 5% level according to Tukey’s test. a

Figure 3. (a) β-glucoside, (b) malonylglucoside, (c) aglycone, and (d) total isoflavone contents in cotyledons of germinated BRS 284 soybeans. of isoflavone per gram of sample on a dry and defatted basis. A typical UPLC chromatogram of isoflavones in the standard is show in Figure 2. Experimental Design and Statistical Analysis. The results of the content of different forms of isoflavones in soybean seeds and their components were subjected to analysis of variance (ANOVA), and the means were compared by Tukey’s test (p < 0.05). Regression analyses between germination time and content of different forms of isoflavones were also performed using all the experimental data with Statistica 10.0 software (StatSoft Inc., Tulsa, OK, USA).

seed coat. These results were similar to those reported by Yuan et al.14 However, Ribeiro et al.13 reported that BRS 213 soybeans have 86.8% cotyledons, 3.2% embryonic axis, and 10.0% seed coat. According to Phommalth et al.,25 the proportion of soybean components depends on its size, so smaller seed sizes result in a greater percentage being the embryonic axis. BRS 284 soybeans (Table 1) had 8.757 ± 0.566 μmol of total isoflavones per gram of soybean on a dry basis. The isoflavone content of soybean seeds can vary according to biotic and abiotic factors, such as cultivar, harvest year,30,31 sowing date, growing location,9 pathogen load in soybean growing season,32 and elicitors.33

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RESULTS AND DISCUSSION Content of Different Forms of Isoflavones in Soybean Seed Components. The BRS 284 soybean seeds were composed of 89.9% cotyledons, 2.4% embryonic axis, and 7.7% 8454



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Figure 4. (a) β-glucoside, (b) malonylglucoside, (c) aglycone, and (d) total isoflavone contents in radicles of germinated BRS 284 soybeans.

Effect of Soybean Germination Time on Different Forms of Isoflavones in Cotyledons. Regression analysis of the effect of germination time on daidzin (R2 = 0.80, p < 0.01) and genistin (R2 = 0.78, p < 0.01) contents in germinated soybean cotyledons indicated that the quadratic model showed a good fit to the experimental data (Figure 3a). However, glycitin was not detected in germinated cotyledons. In cotyledons germinated up to 72 h, the daidzin and genistin contents decreased by 1.5- and 4.2-fold followed by an increase of 1.7- and 2.5-fold between 72 and 168 h, respectively. This initial decrease in daidzin and genistin contents may be associated with the translocation of these isoflavones from cotyledons to radicles.7 The daidzin content in cotyledons germinated for 168 h was similar to ungerminated cotyledons, but the concentration of genistin was reduced by 1.7-fold (Figure 3a). In soybean cotyledons, the amount of β-glucosides decreases until 120 h of germination.14 However, Ribeiro et al.13 described that the germination time of 72 h of BRS 213 soybean cotyledons does not influence the profile of the genistin and daidzin isoflavones. The nondetection of glycitin in soybean cotyledons throughout the germination process has also been observed by Kudou et al.8 in ungerminated soybean cotyledons and by Ribeiro et al.13 in ungerminated soybean cotyledons and soybean cotyledons germinated for 72 h. Regression analysis of the effect of germination time on malonyldaidzin (R2 = 0.91, p < 0.01) and malonylgenistin (R2 = 0.92, p < 0.01) contents in germinated soybean cotyledons indicated that the linear model showed a good fit to the experimental data (Figure 3b). However, malonylglycitin was not detected in germinated cotyledons. Between 0 and 168 h of germination, the malonyldaidzin and malonylgenistin contents

The concentration of the various forms of isoflavones in BRS 284 soybean (Table 1) varied according to its components (p < 0.05). The total isoflavone content in the embryonic axis was 7.1fold higher than that in cotyledons. These results were similar to those described by Yuan et al.14 However, Silva et al.34 reported that the total isoflavone content in the embryonic axis is 6.5− 15.5-fold higher than in cotyledons. In addition, Berger et al.31 observed that the total isoflavone content in cotyledons is dependent on environmental conditions and that the embryonic axis genotype has greater influence. Isoflavone forms have not been found in seed coats of BRS 284 soybeans as observed.8,34 Thus, considering the percentage of each component in the soybean, 84.2% and 15.8% of the total isoflavones are present in cotyledons and the embryonic axis, respectively. Similar results have been observed by Tsukamoto et al.9 However, 51.8% and 48.2% of total isoflavone content is located in cotyledons and the embryonic axis, respectively, in BRS 213 soybeans.13 These differences in the concentration and distribution of isoflavones among soybean components may be attributed to differences in cultivars, growing conditions,30,31 seed size,35 and other factors. In BRS 284 soybeans, cotyledons and the embryonic axis showed similar proportions of β-glucosides, malonylglucosides, and aglycones isoflavones (Table 1). In relation to total isoflavones, the seeds, cotyledons, and embryonic axis showed 26.0%, 27.2%, and 29.4% β-glucosides, 72.9%, 71.4%, and 70.0% malonylglucosides, and 1.2%, 1.4%, and 0.6% aglycones, respectively. In soybean, the daidzein, genistein, and glycitein isoflavones are synthesized via the phenylpropanoid pathway and are stored in the vacuole as malonylglucosides and β-glucosides.7,8 8455



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daidzin and glycitin contents decreased by 17.2- and 51.2-fold after 72 h of germination, respectively. However, Ribeiro et al.13 reported that the concentration of the daidzin and glycitin isoflavones in BRS 213 soybean radicles germinated for 72 h decrease by 6.7- and 10.9-fold, respectively. The decrease in daidzin and genistin in the early stages of germination may be associated with an intense metabolic activity of seeds and conversion of these isoflavones into aglycones and subsequent exudation by the root system.7 Genistin was not detected in radicles of germinated seeds, which has also been observed by Ribeiro et al.13 in radicles of BRS 213 soybeans germinated up to 72 h. However, Kim et al.38 reported that the genistin content ranges from 0.074 to 0.437 μmol g−1 radicles in three soybean cultivars germinated for 168 h. However, the genistin content in radicles of 17 soybean cultivars germinated for 120 h in the presence of light ranges from 0.377 to 5.480 μmol g−1 radicle.24 Regression analysis of the effect of germination time on malonyldaidzin (R2 = 0.64, p < 0.01) content in germinated soybean radicles indicated that the linear model fit to the experimental data and that the quadratic model fit to the experimental data of malonylgenistin (R2 = 0.52, p < 0.01) and malonylglycitin (R2 = 0.61, p < 0.01) contents (Figure 4b). The malonyldaidzin content increased by 1.6-fold between 48 and 168 h of germination. However, the malonylgenistin and malonylglycitin contents decreased between 48 and 72 h with a less pronounced increase after 72 h of germination (Figure 4b). When compared to the embryonic axis of ungerminated seeds, the malonyldaidzin, malonylgenistin, and malonylglycitin contents in radicles germinated for 72 h decreased by 6.9-, 13.1-, and 20.2-fold, respectively. Ribeiro et al.13 also observed a reduction in malonylglucoside content in BRS 213 soybean radicles germinated for 72 h with light. According to Graham,7 the conjugates of daidzein and genistein in addition to several unidentified aromatic metabolites are selectively excreted into root and seed exudates, and the analysis of seed exudates suggests that this is a continuous but saturable event. Regression analysis of the effect of germination time on daidzein content in soybean radicles germinated indicated that the linear, quadratic, or cubic models did not fit the experimental data (p > 0.05). However, the quadratic model showed a low fit to the experimental data for the genistein concentration (R2 = 0.44, p = 0.01). These results indicated that there was little influence of germination time on the concentration of these two aglycones between 48 and 168 h. Thus, it was verified that the mean concentration of genistein and daidzein between 48 and 168 h of germination was 0.860 ± 0.069 and 0.082 ± 0.010 μmol g−1 radicle, respectively. Glycitein was not detected in radicles throughout the entire germination time. However, radicles of 17 soybean varieties germinated for 120 h with and without light have 2.9% glycitein relative to total isoflavone content.24 In relation to total isoflavone content, the percentage of aglycone in germinated soybean radicles was 14.1%. The highest percentage of isoflavone aglycones in radicles compared to cotyledons and hypocotyls can be attributed to the highest activity of the βglucosidase enzyme in radicles39 with subsequent hydrolysis and release of aglycones that participate in interactions between plants and symbiotic microorganisms as well as act as a defense agent against infection by pathogens.40 The daidzein and genistein isoflavones are the major components in soybean root extracts responsible for inducing nod genes in Bradyrhizobium japonicum, which are responsible for the formation of nitrogen-fixing root nodules.41

increased by 3.5- and 2.4-fold, respectively. According to Ribeiro et al.,13 the malonyldaidzin and malonylgenistin contents in BRS 213 soybean cotyledons germinated for 72 h are 3.3- and 2.6-fold higher than those in ungerminated cotyledons, respectively. It is noteworthy that the soybean cotyledons germinated for 168 h had 25.3% malonyldaidzin and 65.3% malonylgenistin in relation to the total isoflavone content. The malonylglycitin isoflavone was not detected in ungerminated soybean cotyledons and soybean cotyledons germinated up to 168 h. Similar results of malonyl isoflavones have been reported by Ribeiro et al.13 It was not possible to perform regression analysis on the aglycone content for germinated soybean cotyledons, since genistein was only detected in ungerminated cotyledons and cotyledons germinated for 24 h without differing in 0 and 24 h of germination (p > 0.05) presenting an average of 0.102 ± 0.010 μmol g−1 of cotyledons (Figure 3c). Additionally, daidzein and glycitein were not detected in the cotyledons during the entire germination process. According to Ribeiro et al.,13 genistein is the only isoflavone aglycone present in BRS 213 soybean cotyledons that are either ungerminated or germinated for 6 h. However, in cotyledons of 17 soybeans germinated for 120 h with and without light, three types of aglycones are detected with daidzein showing higher concentrations than genistein and glycitein.24 According to Wang and Murphy,30 these differences may be associated with the cultivar, crop year, and location of soybean cultivation. Regression analysis of the effect of germination time on total isoflavone content (R2 = 0.92, p < 0.01) in germinated soybean cotyledons indicated that the linear model showed a good fit to the experimental data (Figure 3b) with an increase of 1.4- and 2.1-fold in total isoflavone content after 72 and 168 h of germination, respectively. Similar results have been reported by Phommalth et al.,25 who described that total isoflavone contents in cotyledons of Aga3 and Pungsannamulkong soybeans germinated for 168 h at 20 °C are 1.5- and 1.7-fold higher than in ungerminated cotyledons, respectively. However, Ribeiro et al.13 observed an increase of 2.4-fold in total isoflavone content from BRS 213 soybean cotyledons germinated for 72 h. During germination, the increase in the concentration of total isoflavones can be attributed to activity and/or synthesis of novel enzymes36 that are responsible for the isoflavone biosynthesis and metabolism as well as the degradation of proteins, lipids, or carbohydrates that can promote a reduction in seed dry matter17,37 and contribute to an increase in the relative concentration of total isoflavones. Effect of Soybean Germination Time on Different Forms of Isoflavones in Radicles. The effect of germination time on the isoflavone content in radicles was considered only after 48 h of germination when it was possible to observe the radicles and manually separate them from the other components of the embryonic axis. Regression analysis of the effect of germination time on daidzin (R2 = 0.46, p = 0.01) and glycitin (R2 = 0.83, p < 0.01) contents in germinated soybean radicles indicated that the quadratic model fitted to the experimental data (Figure 4a). However, genistin was not detected in germinated radicles. The concentration of daidzin from germinated soybean radicles showed a decrease of 1.6-fold until 120 h with a new increase of 1.5-fold up to 168 h of germination, but the glycitin content showed a marked reduction of 2.9-fold between 48 and 72 h of germination and then remained constant averaging 0.145 ± 0.021 μmol g−1 between 72 and 168 h. When compared to the embryonic axis of ungerminated seeds, it was observed that the 8456



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Figure 5. (a) β-glucoside, (b) malonylglucoside, (c) aglycone, and (d) total isoflavone contents in hypocotyls of germinated BRS 284 soybeans.

respectively, followed by an increase of 2.8- and 1.7-fold, respectively, up to 144 h with a little tendency to change between 144 and 168 h. The glycitin isoflavone was detected at 48 h with a reduction of 4.3-fold at 72 h. After this germination time, glycitin was not detected, thus excluding regression analysis for glycitin. However, the glycitin content ranges from 0.100 to 0.776 μmol g−1 hypocotyl in 17 soybean cultivars germinated for 120 h with light.24 When compared to the embryonic axis of ungerminated seeds, the daidzin, genistin, and glycitin contents decreased by 15.0-, 7.6-, and 6.8-fold, respectively, after 48 h of germination. Graham7 reported a higher concentration of daidzein and genistein conjugates in hypocotyl hooks and reported that the contents of these isoflavones undergo a programmed and dramatic decrease between 48 and 96 h of germination. Additionally, Graham found an inverse correlation between the concentration of isoflavone conjugates and expansion of the hypocotyl hook. Regression analysis of the effect of germination time on malonyldaidzin (R2 = 0.76, p < 0.01), malonylgenistin (R2 = 0.89, p < 0.01), and malonylglycitin (R2 = 0.92, p < 0.01) contents in germinated soybean hypocotyls indicated that the quadratic model showed a good fit to the experimental data (Figure 5b). The malonyldaidzin content decreased by 3.3-fold up to 96 h of germination and returned to the initial concentration after 168 h of germination. However, the malonylgenistin and malonylglycitin contents decreased by 4.6- and 5.3-fold, respectively, up to 96 h of germination with little change between 96 and 168 h. When compared to the embryonic axis of ungerminated seeds, the malonyldaidzin content decreased by 22.0- and 4.1-fold at 96 and 168 h, respectively, and the malonylgenistin and malonylglycitin contents were decreased by 7.2- and 36.7-fold

Regression analysis of the effect of germination time on total isoflavone content (R2 = 0.35, p = 0.04) in germinated soybean radicles indicated that the quadratic model showed a low fit to the experimental data (Figure 4d), thereby indicating that the germination time between 48 and 168 h had a low effect on the total isoflavone content. Thus, in this interval of germination, the average total isoflavone content was 5.778 ± 0.125 μmol g−1 radicle. The low change observed for isoflavone content in radicles may be due to high variability in the experimental data, which is intrinsic to the experiment because germination is a biological phenomenon in which several factors in addition to time can influence the isoflavone content. In the embryonic axis of ungerminated soybean seeds, the total isoflavone content was 53.844 ± 3.863 μmol g−1 sample. Thus, after 168 h of germination, the total isoflavone content decreased by 9.3-fold. A decrease of 6.3-fold was observed by Ribeiro et al.13 in BRS 213 soybean radicles germinated for 72 h. This decline may be associated with exudation of isoflavones to the external environment or metabolism of isoflavones in other constituents.7 Effect of Soybean Germination Time on Different Forms of Isoflavone in Hypocotyls. The effect of germination time on the isoflavone content in hypocotyls was considered only after 48 h of germination when it was possible to observe the hypocotyls and to manually separate them from the other components of the embryonic axis. Regression analysis of the effect of germination time on daidzin (R2 = 0.87, p < 0.01) and genistin (R2 = 0.84, p < 0.01) contents in germinated soybean hypocotyls indicated that the cubic model fit to the experimental data (Figure 5a). Thus, the daidzin and genistin contents in soybean hypocotyls decreased by 2.1- and 2.9-fold between 72 and 96 h of germination, 8457



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to carry germination until 168 h and not eliminate the cotyledons. In addition, if the main purpose is the intake of aglycones isoflavones, the consumption of radicle and hypocotyls may be sufficient as cotyledons germinated for 168 h does not contain these forms of isoflavones.

after 168 h of germination. These results showed similar tendencies to those observed by Yuan et al.,14 who found a reduction of 9.0-, 25.6-, and 125.0-fold for the malonyldaidzin, malonylgenistin, and malonylglycitin contents in soybean hypocotyls germinated for 120 h in the presence of light. Regression analysis of the effect of germination time on genistein (R2 = 0.84, p < 0.01) content in germinated soybean hypocotyls indicated that the linear model showed a good fit to the experimental data (Figure 5c). The genistein content increased by 2.0-fold between 48 and 168 h of germination. Similar trends were observed by Yuan et al.,14 who reported an increase of 9.7-fold for the genistein content in soybean hypocotyls germinated for 72 h followed by a decrease of 1.4fold between 72 and 120 h of germination. In hypocotyls, daidzein was detected only after 120 h, and glycitein was detected only after 48 h of germination. Therefore, no regression analysis was performed for these components. In hypocotyls, the daidzein content doubled between 120 and 144 h and remained constant up to 168 h with an average of 0.258 ± 0.013 μmol g−1 hypocotyl. In hypocotyls of 17 soybean cultivars germinated for 120 h, the daidzein content ranges between 0.231 and 1.019 μmol g−1 hypocotyl.24 The daidzein content in soybean hypocotyls germinated for 120 h increases by 5.1-fold compared to nongerminated hypocotyls.14 The glycitein content after 48 h of germination was 0.230 ± 0.023 μmol g−1 hypocotyl. However, in hypocotyls of 17 soybean cultivars germinated for 120 h with and without light, the glycitein concentrations range from 0.067 to 0.814 μmol g−1.24 Regression analysis of the effect of germination time on total isoflavone content (R2 = 0.84, p < 0.01) in germinated soybean hypocotyls indicated that the quadratic model showed a low fit to the experimental data (Figure 5d). The total isoflavone content decreased by 4.6-fold between 48 and 96 h of germination followed by a 2.0-fold increase between 96 and 168 h. Comparing the embryonic axis of ungerminated soybeans with soybean hypocotyls germinated for 168 h, the total isoflavone content decreased by 9.8-fold (Table 1). Similar results have been observed in the Aga3 and Pungsannamulkong cultivars germinated for 168 h.25 In conclusion, soybean BRS 284 showed 89.9% cotyledons, 2.4% embryonic axis, and 7.7% seed coat, and 84.2% and 15.8% of total isoflavones are present in cotyledons and the embryonic axis, respectively; however, the total isoflavone content in the embryonic axis was 7.1-fold higher than that in cotyledons. The germination time exerted different effects depending on the components of germinated soybeans and the different forms of isoflavones. In cotyledons germinated up to 168 h, the total isoflavone content increased 2.1-fold in relation to ungerminated cotyledons, and aglycones were not detected. In radicles, germination time exerted low influence on the daidzein and genistein content, while the malonyldaidzin increased 1.6-fold between 48 and 168 h, and in hypocotyl, the genistein content increased by 2.0-fold between 48 and 168 h of germination. Therefore, in this study, it was concluded that, in the germination process by 168 h, the total isoflavones showed higher concentration in the cotyledons and aglycones were detected only in the hypocotyl and radicle. In the present study, the effects of germination time were modeled, and it was possible to monitor the whole process of germination in relation to the content of different forms of isoflavones and different components of soybeans germinated. Our results imply that, to obtain soybean sprouts for human consumption with higher levels of total isoflavones, it is necessary

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Corresponding Author

*Phone: +55 43 3371 5971; fax: +55 43 3371 4080; e-mail: [email protected]. Funding

This work was partially funded by Fundaçaõ Araucária de ́ e Tecnológico do Paraná/Conselho Desenvolvimento Cientifico ́ Nacional de Desenvolvimento Cientifico e Tecnológico-CNPq (PRONEX Program). A.Q.Jr. would like to thank CNPq for a graduate scholarship, and E.I.I. is a CNPq Research Fellow. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Atteritano, M.; Mazzaferro, S.; Frisina, A.; Cannata, M. L.; Bitto, A.; D’anna, R.; Squadrito, F.; Macrì, I.; Frisina, N.; Buemi, M. Genistein effects on quantitative ultrasound parameters and bone mineral density in osteopenic postmenopausal women. Osteoporosis Int. 2009, 20 (11), 1947−1954. (2) Chan, Y.-H.; Lau, K.-K.; Yiu, K.-H.; Li, S.-W.; Chan, H.-T.; Tam, S.; Shu, X.-O.; Lau, C.-P.; Tse, H.-F. Isoflavone intake in persons at high risk of cardiovascular events: Implications for vascular endothelial function and the carotid atherosclerotic burden. Am. J. Clin. Nutr. 2007, 86, 938−945. (3) Williamson-Hughes, P. S.; Flickinger, B. D.; Messina, M. J.; Empie, M. W. Isoflavone supplements containing predominantly genistein reduce hot flash symptoms: A critical review of published studies. Menopause 2006, 13, 831−839. (4) Wada, K.; Nakamura, K.; Tamai, Y.; Tsuji, M.; Kawachi, T.; Hori, A.; Takeyama, N.; Tanabashi, S.; Matsushita, S.; Tokimitsu, N.; Nagata, C. Soy isoflavone intake and breast cancer risk in Japan: From the Takayama study. Int. J. Cancer. 2013, 133, 952−960. (5) Dong, X.; Xu, W.; Sikes, R. A.; Wu, C. Combination of low dose of genistein and daidzein has synergistic preventive effects on isogenic human prostate cancer cells when compared with individual soy isoflavone. Food Chem. 2013, 141, 1923−1933. (6) Liu, K. Soybeans as Functional Foods and Ingredients; AOCS Press: Champaign, IL, 2004; p 331. (7) Graham, T. L. Flavonoid and isoflavonoid distribution in developing soybean seedling tissues and in seed and root exudates. Plant Physiol. 1991, 95, 594−560. (8) Kudou, S.; Fleury, Y.; Welti, D.; Magnolato, D.; Uchida, T.; Kitamura, K.; Okubo, K. Malonyl isoflavone glycosides in soybean seeds (Glycine max Merrill). Agric. Biol. Chem. 1991, 55, 2227−2233. (9) Tsukamoto, C.; Shimada, S.; Igita, K.; Kudou, S.; Kokubun, M.; Okubo, K.; Kitamura, K. Factors affecting isoflavone content in soybean seeds: Changes in isoflavones, saponins, and composition of fatty acids at different temperatures during seed development. J. Agric. Food Chem. 1995, 43, 1184−1192. (10) Paucar-Menacho, L. M.; Berhow, M. A.; Mandarino, J. M. G.; Chang, Y. K.; Mejia, E. G. D. Effect of time and temperature on bioactive compounds in germinated Brazilian soybean cultivar BRS 258. Food Res. Int. 2010, 43, 1856−1865. (11) Hoeck, J. A.; Fehr, W. R.; Murphy, P. A.; Welke, G. A. Influence of genotype and environment on isoflavone contents of soybean. Crop Sci. 2000, 40 (1), 48−51. (12) Mebrahtu, T.; Mohamed, A.; Wang, C. Y.; Andebrhan, T. Analysis of isoflavone contents in vegetable soybeans. Plant Foods Hum. Nutr. 2004, 59, 55−61.

8458



Article

(13) Ribeiro, M. L. L.; Mandarino, J. M. G.; Carrão-Panizzi, M. C.; Oliveira, M. C. N.; Campo, C. B. H.; Nepomuceno, A. L.; Ida, E. I. βGlucosidase activity and isoflavone content in germinated soy bean radicles and cotyledons. J. Food Biochem. 2006, 30, 453−465. (14) Yuan, J.-P.; Liu, Y.-B.; Peng, J.; Wang, J.-H.; Liu, X. Changes of isoflavone profile in the hypocotyls and cotyledons of soybeans during dry heating and germination. J. Agric. Food Chem. 2009, 57, 9002−9010. (15) Liu, K. Food use of whole soybeans. In Soybeans Chemistry, Production, Processing, and Utilization; Lawrence, A. J., White, P. J., Galloway, R., Eds.; AOCS Press: Urbana, IL, 2008; pp 441−482. (16) Lee, J. D.; Hwang, Y. H.; Cho, H. Y.; Kim, D. U.; Chung, M. G. Comparison of characteristics related with soybean sprouts between Glycine max and G. soja. Korean J. Crop Sci. 2002, 47, 189−198. (17) Kim, W.-J.; Lee, H.-Y.; Won, M. H.; Yoo, S.-H. Germination effect of soybean on its contents of isoflavones and oligosaccharides. Food Sci. Biotechnol. 2005, 14, 498−502. (18) Kim, S.-L.; Lee, J.-E.; Kwon, Y.-U.; Kim, W.-H.; Jung, G.-H.; Kim, D.-W.; Lee, C.-K.; Lee, Y.-Y.; Kim, M.-J.; Kim, Y.-H.; Hwang, T.-Y.; Chung, I.-M. Introduction and nutritional evaluation of germinated soy germ. Food Chem. 2013, 136, 491−500. (19) Ribeiro, M. L. L.; Ida, E. I.; Oliveira, M. C. N. Efeito da germinaçaõ ́ de soja cv. BR-13 e Paraná sobre ácido fitico, fósforo total e inibidores de tripsina. Pesqui. Agropecu. Bras. 1999, 34, 31−36. (20) Ramadan, E. A. Effect of processing and cooking methods on the chemical composition, sugars and phytic acid of soybeans. Food Public Health 2012, 2, 11−15. (21) Martín-Cabrejas, M. A.; Díaz, M. F.; Aguilera, Y.; Benítez, V.; Mollá, E.; Esteban, R. M. Influence of germination on the soluble carbohydrates and dietary fibre fractions in non-conventional legumes. Food Chem. 2008, 107, 1045−1052. (22) Ahmad, S.; Pathak, D. K. Nutritional changes in soybean during germination. J. Food Sci. Technol. 2000, 37, 665−666. (23) Shi, H.; Nam, P.; Ma, A. Comprehensive profiling of isoflavones, phytosterols, tocopherols, minerals, crude protein, lipid, and sugar during soybean (Glycine max) germination. J. Agric. Food Chem. 2010, 58, 4970−4976. (24) Lee, S. J.; Ahn, J. K.; Khanh, T. D.; Chun, S. C.; Kim, S. L.; Ro, H. M.; Song, H. K.; Chung, I. M. Comparison of isoflavone concentrations in soybean (Glycine max (L.) Merrill) sprouts grown under two different light conditions. J. Agric. Food Chem. 2007, 55, 9415−9421. (25) Phommalth, S.; Jeong, Y.-S.; Kim, Y.-H.; Hwang, Y.-H. Isoflavone composition within each structural part of soybean seeds and sprouts. J. Crop Sci. Biotechnol. 2008, 11, 57−62. (26) Embrapa. Cultivares de Soja: Regiões sul e Central do Brasil 2010/ 2011, 1a ed.; Embrapa Soja: Londrina, Puerto Rico, 2010; p 62. (27) Yoshiara, L. Y.; Madeira, T. B.; Ribeiro, M. L. L.; Mandarino, J. M. G.; Carrão-Panizzi, M. C.; Ida, E. I. β-Glucosidase activity of soybean (Glycine max) embryonic axis germinated in the presence or absence of light. J. Food Biochem. 2012, 36, 699−705. (28) Yoshiara, L. Y.; Madeira, T. B.; Delaroza, F.; Silva, J. B.; Ida, E. I. Optimization of soy isoflavone extraction with different solvents using the simplex-centroid mixture design. Int. J. Food Sci. Nutr. 2012, 63, 978−986. (29) Handa, C. L.; Couto, U. R.; Vicensoti, A. H.; Georgetti, S. R.; Ida, E. I. Optimisation of soy flour fermentation parameters to produce βglucosidase for bioconversion into aglycones. Food Chem. 2014, 152, 56−65. (30) Wang, H.; Murphy, P. A. Isoflavone composition of American and Japanese soybeans in Iowa: Effects of variety, crop year, and location. J. Agric. Food Chem. 1994, 42, 1674−1677. (31) Berger, M.; Rasolohery, C. A.; Cazalis, R.; Daydé, J. Isoflavone accumulation kinetics in soybean seed cotyledons and hypocotyls: Distinct pathways and genetic controls. Crop Sci. 2008, 48, 700−708. (32) Wegulo, S. N.; Yang, X.-B.; Martinson, C. A.; Murphy, P. A. Effects of wounding and inoculation with Sclerotinia sclerotiorumon isoflavone concentrations in soybean. Can. J. Plant Sci. 2005, 85, 749− 760. (33) Zhang, B.; Hettiarachchy, N.; Chen, P.; Horax, R.; Cornelious, B.; Zhu, D. Influence of the application of three different elicitors on

soybean plants on the concentrations of several isoflavones in soybean seeds. J. Agric. Food Chem. 2006, 54, 5548−5554. (34) Silva, C. E.; Carrão-Panizzi, M. C.; Mandarino, J. M. G.; Leite, R. S.; Mônaco, A. P. A. Teores de isoflavonas em grãos inteiros e nos componentes dos grãos de diferentes cultivares de soja (Glycine max (L.) Merrill). Braz. J. Food Technol. 2012, 15, 150−156. (35) Lee, S. J.; Kim, J. J.; Moon, H. I.; Ahn, J. K.; Chun, S. C.; Jung, W. S.; Lee, O. K.; Chung, I. M. Analysis of isoflavones and phenolic compounds in Korean soybean [Glycine max (L.) Merrill] seeds of different seed weights. J. Agric. Food Chem. 2008, 56, 2751−2758. (36) Bewley, J. D.; Black, A. M. Seeds-Physiology of Development and Germination, 2a ed.; Plenum Press: New York, 1994; p 445. (37) Bordingnon, J. R.; Ida, E. I.; Oliveira, M. C.; Mandarino, J. M. Effect of germination on the protein content and on the level of specific activity of lipoxygenase-1 in seedlings of three soybean cultivars. Arch. Latinoam. Nutr. 1995, 45, 222−226. (38) Kim, E. M.; Lee, K. J.; Chee, K.-M. Comparison in isoflavone contents between soybean and soybean sprouts of various soybean cultivar. Korean J. Nutr. 2004, 37, 45−51. (39) Hsieh, M. C.; Graham, T. L. Partial purification and characterization of a soybean β-glucosidase with high specific activity towards isoflavone conjugates. Phytochemistry (Elsevier) 2001, 58, 995−1005. (40) Suzuki, H.; Takahashi, S.; Watanabe, R.; Fukushima, Y.; Fujita, N.; Noguchi, A.; Yokoyama, R.; Nishitani, K.; Nishino, T.; Nakayama, T. An isoflavone conjugate-hydrolyzing-glucosidase from the roots of soybean (Glycine max) seedlings: Purification, gene cloning, phylogenetics, and cellular localization. J. Biol. Chem. 2006, 281, 30251−30259. (41) Kosslak, R. M.; Bookland, R.; Barkei, J.; Paaren, H. E.; Appelbaum, E. R. Induction of Bradyrhizobium japonicum common nod genes by isoflavones isolated from Glycine max. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 7428−7432.

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Isoflavones of the Soybean Components and the ... - ACS Publications

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