Changes in phenolic acid and isoflavone contents during soybean

Subscriber access provided by Iowa State University | Library

Bioactive Constituents, Metabolites, and Functions

Changes in phenolic acids and isoflavone contents during soybean drying and storage Cristiano Dietrich Ferreira, Valmor Ziegler, Jorge Tiago Schwanz Goebel, Jessica Fernanda Hoffmann, Ivan Ricardo Carvalho, Fabio Clasen Chaves, and Mauricio de Oliveira J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06808 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

Journal of Agricultural and Food Chemistry

Changes in phenolic acid and isoflavone contents during soybean drying and storage

Cristiano Dietrich Ferreira1*, Valmor Ziegler2, Jorge Tiago Schwanz Goebel1, Jessica Fernanda Hoffmann1, Ivan Ricardo Carvalho3, Fabio Clasen Chaves1, Mauricio de Oliveira1*

1

Department of Agroindustrial Science and Technology, Federal University of Pelotas,

96010-900, Pelotas, RS, Brazil. 2

Instituto Tecnológico em Alimentos para a Saúde, Universidade do Vale do Rio dos

Sinos, 93022000, São Leopoldo, RS, Brazil 3 Department

of Science and Technology of Seeds, Federal University of Pelotas, 96010-

900, Pelotas, RS, Brazil.

* Corresponding author: Maurício de Oliveira ([email protected]); Cristiano Dietrich Ferreira ([email protected]) Tel/Fax: +555332757284

ACS Paragon Plus Environment


Page 2 of 37

1

Abstract

2

The changes in phenolic acid and isoflavone profile of soybean genotypes (Nidera 5909

3

RR and BMX Força RR) dried at different temperatures and stored for 12 months were

4

investigated. In both cultivars, there was a reduction of the germination capacity and an

5

increase of fungal incidence with the increase of drying temperature and storage time.

6

Multivariate analysis of phenolic acids allowed for the differentiation among treatments.

7

Cultivar Nidera 5909 RR, dried at 110 °C, showed an interaction with characters of

8

greater relevance for differentiation, being influenced by the increase of bound coumaric,

9

and syringic, and free-hydroxybenzoic, syringic and coumaric acids. Multivariate

10

analysis of isoflavones showed a strong affinity of the aglycone isoflavones (genistein,

11

glycitein, and daidzein) within the Nidera 5909 RR cultivar at all drying temperatures and

12

with BMX Força RR cultivar at the highest temperatures. These results indicate that the

13

release and interconversion of isoflavone malonyl-β-glucosides and β-glucosides into

14

aglycone forms are simultaneous reactions during storage.

15

Keywords: Glycine max (L.) Merril; drying temperature; molds; isoflavones aglycones;

16

seed viability.

17 18

2 ACS Paragon Plus Environment

Page 3 of 37

19


Introduction

20

Soybean (Glycine max (L) Merrill) is rich in bioactive compounds such as

21

carotenoids, tocopherols, and phenolics.1 Phenolic compounds are abundant in soybeans

22

and can be found in free form (912.91 μg/g), free conjugates (1818.14 μg/g), and bound

23

components (1285.56 μg/g). Examples of these compounds found in soybeans include

24

gallic acid, protocatechuic acid, vanillic acid, syringic acid, epicatechin, p-coumaric acid,

25

ferulic acid, rutin, isoquercitrin, quercitrin, and quercetin.2 Soy is rich in isoflavones

26

(1640.7 µg/g of malonyl-glycosides, 524.7 µg/g of β-glucosides, and 81.4 µg/g of

27

aglycones),3 which have antioxidant, anti-osteoporosis, anticarcinogenic, antimutagenic

28

and bactericidal activity.4 Isoflavones are found in aglycone forms (genistein, glycitein,

29

and daidzein) and their respective glycosylated forms (malonyl-glycosides, β-glucosides,

30

and acetyl-glycosides), totaling 12 isoforms.5

31

Soybean composition is influenced by genotype, soil fertility, altitude,

32

temperature, and postharvest processing including drying, storage, and processing

33

methods.6,7,8 Hot air drying is widely used to reduce moisture and maintain seed quality

34

during storage. However, drying at high temperatures can result in physical damage to

35

the grains, for example cracking when the soybean is dried at temperatures of 110 °C and

36

140 °C.9 Surface damage, grain moisture, temperature, and associated microorganisms

37

are the main factors that degrade seeds during storage.10 Damaged seeds present a high

38

incidence of fungal infestation, which is the main responsible factor that contributes to

39

reduced seed germination, since, they alter seed metabolism and chemical composition.11

40

Although isoflavones are resistant to thermal degradation, they are easily

41

interconverted into their isoforms under heating conditions.5 Lee and Lee6 reported that

42

oven drying at 100 °C promoted a reduction of malonyl-glycosides, and an increase of β-

43

glucoside and aglycone forms, while, roasting and explosive puffing reduced malonyl-

44

glycoside derivatives, and increased acetyl-β-glycosides and β-glycosides forms. 1 ACS Paragon Plus Environment


Page 4 of 37

45

According to Niamnuy et al.12, total soybean isoflavone content is reduced at the high

46

drying temperatures of 130 °C and 150 °C, when compared to 50 °C and 70 °C. However,

47

under high drying temperature conditions, the release of aglycone isoflavone is promoted.

48

Soybean storage under drastic conditions (84% RH / 30 °C for 9 months) promotes the

49

conversion of malonyl-β-glycoside and β-glycoside to aglycones. Under these conditions,

50

the seed reaches 18% moisture, signaling towards the synthesis of β-glycosidase

51

enzymes, which at their optimum temperature (30 °C) present a high rate of hydrolysis.13

52

The conversion of isoflavone malonyl-glucosides, acetyl-β-glucosides, and β-glucosides

53

into their aglycone forms is accelerated by heat, acid, alkaline, and enzyme hydrolysis.14

54

Isoflavone β-glucoside absorption by the intestine is poor due to its high molecular weight

55

and hydrophobicity. However, colon microbiota is capable of hydrolyzing isoflavone β-

56

glucosides into aglycones.15

57

The objective of this study was to determine the effects of high drying temperature

58

and long-term storage, using the germination and fungi incidence as parameters for

59

monitoring the profile of phenolic acids and isoflavones.

60 61

Material and methods

62

Plant material

63

Two soybean cultivars (BMX Força RR and Nidera 5909 RR) with different

64

physical and chemical characteristics (supplementary file 1) were cultivated in adjacent

65

plots in the town of Morro Redondo, Rio Grande do Sul state, Brazil, latitude 31° 32'

66

34.184" S, longitude 52° 34' 12.702" W, altitude of 108 meters, according to crop

67

management practices used in the region. Seed maturation was monitored and when it

68

reached 20% moisture, which enables mechanical threshing, plants were hand harvested

69

and mechanically threshed (model BCO 80 MAX URP) 820 RPM. Grains were pre-

70

cleaned to remove foreign matter, impurities, and damaged and malformed grains. A 2 ACS Paragon Plus Environment

Page 5 of 37


71

parcel of plants was left to dry in the field during 10 days (15 °C - minimum temperature;

72

33 °C - maximum temperature; 24 °C - average temperature) until seeds had 13.5%

73

moisture content (Field drying).

74 75

Soybean drying and storage

76

Soybean moisture after harvest was determined using an oven at 130 °C with

77

forced air circulation for 3 hours.16 Moisture content was 20.3% and 19.2% for cultivars

78

BMX Força RR and Nidera 5909 RR, respectively. Air temperature (28 °C) and relative

79

humidity (60%) were monitored using an analog psychrometer. Seeds were dried in a

80

drying oven at 30, 50, 70, 90, and 110 °C. Three samples of soybeans (1.2 kg each) were

81

placed in raffia bags. Soybeans were taken out of the oven every 20 minutes for

82

homogenization, bulk temperature measurement, and weighing. The bulk temperature

83

was determined by placing the soybeans in a thermal box coupled to a mercury

84

thermometer, and the reading was carried out after two minutes of stabilization (based on

85

pre-tests). Seeds were dried until 13.5% moisture content and taken to a chamber at 16

86

°C for seven days, in order to equalize the temperature and moisture content among

87

samples. Samples (0.9 kg) were then stored in polyethylene bags (0.2-mm-thick plastic

88

film) at 25 °C for 12 months. Samples were collected at the beginning of storage and after

89

12-month storage for phenolic acid and isoflavone profile analyses. Samples were

90

collected at 4, 8, and 12 months of storage to determine germination capacity.

91 92

Analyses.

93

Germination capacity

94

Germination capacity was determined in four replicates composed of 100 seeds

95

each collected randomly from each treatment, distributed in germination paper moistened

96

with distilled water (3 x the weight of the paper) and taken to a germination chamber 3 ACS Paragon Plus Environment


Page 6 of 37

97

(EletroLab, Model EL 222/4RS) at 25 °C and 80% relative humidity. The number of

98

germinated seeds was counted after 7 days of incubation. Were considered germinated

99

seeds that had radicle emission and foliar beginnings. Results were expressed as a

100

percentage of sprouted seeds.17

101 102

Fungal colonies

103

Fungal colonies were determined using the Blotter test.17 Two sheets of

104

autoclaved filter papers were placed inside sodium hypochlorite (0.07%) and alcohol

105

(70%) disinfected germination box. The sheets were soaked with 2.5 times their weight

106

with water. One hundred soybean seeds were evenly distributed over the paper. The boxes

107

were taken to an incubator at 25 °C ± 2 °C, 80% RH (relative humidity), with a

108

photoperiod of 12 hours light/12 hours dark. After 24 hours the boxes were brought to -

109

20 °C for 24 hours to prevent seed germination. The boxes were then returned to the

110

incubator where they remained for seven days. Seeds were examined under a magnifying

111

glass and microscope in order to identify fungal reproductive structures to determine

112

fungal identity at the genus level. Results were expressed as fungal colonies per 100

113

soybean seeds. Two or more fungal genera can affect the same seed.

114 115

Extraction of free phenolics

116

Soybeans were ground in a laboratory mill (Perten 3100, Perten Instruments,

117

Huddinge, Sweden) equipped with a 35-mesh sieve to obtain uniform particle size.

118

Ground soybean meal was defatted with hexane for 8 hours using a Soxhlet apparatus.

119

Defatted soybean flour (2.0 g) was extracted twice with 80% methanol at a 1:20 ratio

120

(w/v). For each extraction, the mixture was kept for 1 h at room temperature on an orbital

121

shaker (Certomat Biotech International) at 150 rpm. Samples were then centrifuged 4 ACS Paragon Plus Environment

Page 7 of 37


122

(Eppendorf 5430-R) at 7600 x g for 15 min and the supernatants obtained from each

123

extraction were combined and concentrated to dryness using a rotary evaporator

124

(Heidolph, Laborota Model 4000, Kelheim, Baviera, Germany) at 35 °C. The dried

125

extract was redissolved in 25 mL of 80% methanol.18

126 127

Extraction of bound phenolics

128

The dried residue (after solvent evaporation) from the extraction of the free

129

phenolics was dried overnight in a forced air flow oven set at 35 °C. One g of dry sample

130

was suspended in 20 mL of NaOH solution (4 M) and left under stirring for 4 h at 35 °C.

131

Sample pH was then adjusted to 2.0 (using HCl 6 M) and extracted four times with 20

132

mL of ethyl acetate. The ethyl acetate fractions were combined, concentrated in a rotary

133

evaporator, and redissolved in 10 mL of 80% methanol.19

134 135

Phenolic profile by HPLC-QToF/MS

136

Free and bound extracts were used for HPLC-ESI-QToF-MS analysis. Samples

137

were filtered through a 0.22 μm nylon membrane filter (Merck Millipore Corporation,

138

Darmstadt, Hesse, Germany). The HPLC-ESI-QToF/MS analysis was performed on a

139

Prominence UFLC system (Shimadzu, Japan) coupled to a quadrupole-time-of-flight in

140

a tandem mass spectrometer (Impact HD, Bruker Daltonics, Bremen, Germany). Phenolic

141

compounds were separated using a Luna C18 column (2.0 x 150 mm, 100 Å, particle size

142

3 μm) (Phenomenex Inc., Torrance, CA, USA). Mobile phases were 0.1% aqueous formic

143

acid (pH 2.8; solvent A) and acetonitrile (solvent B). The elution gradient was: 0–2 min,

144

10% B; 2–10 min, 10–75% B; 10-15 min, 75% B; 15–18 min 75-90% B; 18-21 min, 90%

145

B, 21-23 min, 90-10% B, 23-30 min, 10% B at a flow rate of 0.2 mL/min. Sample

146

injection volume was 10 μL. Parameters for MS analysis were set using negative

147

ionization mode with spectra acquired over a mass range from m/z 50 to 1200. Parameters 5 ACS Paragon Plus Environment


Page 8 of 37

148

were: capillary voltage, +4.0 kV; drying gas temperature, 180 °C; drying gas flow, 8.0

149

L/min; nebulizing gas pressure, 2 bar; collision RF, 150 Vpp; transfer time 70 μs, and

150

pre-pulse storage, 5 μs. Moreover, automatic MS/MS experiments were performed

151

adjusting the collision energy values as follows: m/z 100, 15 eV; m/z 500, 35 eV; m/z

152

1000, 50 eV with nitrogen as the collision gas. The MS data were processed through Data

153

Analysis 4.0 software (Bruker Daltonics, Bremen, Germany). The identity of caffeic acid,

154

p-coumaric acid, ferulic acid, gallic acid, p-hydroxybenzoic acid, syringic acid, and

155

vanillic acid was confirmed with external standards (Sigma-Aldrich). For quantitative

156

analysis, an external calibration curve for each available phenolic standard was

157

constructed (39 to 10,000 ng/mL). Results were expressed as μg/100 g dry weight (DW).

158 159

Isoflavone profile by HPLC-QToF/MS

160

Free and bound extracts were used for HPLC-ESI-QToF-MS analysis of the

161

isoflavones, performed in the same equipment previously described. A reverse phase

162

column (Shim-pack XR ODS analytical column, 2.0 mm x 75 mm x 2.2 μm particle size,

163

Shimadzu, Japan) was used for the analysis. The mobile phase was a gradient prepared

164

from 0.1% formic acid in water (component A) and methanol (component B). The

165

gradient program for the HPLC was as follows: 0–1 min, 15–15% B; 1–10 min 70% B;

166

10–12 min 70% B; 12–15 min 15% B, and the flow rate was 0.25 mL/min. Sample

167

injection volume was 10 μL and the column temperature was 35 ºC. Mass spectra in the

168

m/z range 50–1200 were obtained by using electrospray ionization in the positive mode.

169

The mass spectrometric conditions were optimized as follows: gas temperature 200 ºC,

170

drying gas flow rate 9.0 L/min, nebulizer gas pressure 2.0 bar, and capillary potentials

171

4500 V. The mass axis was calibrated using 10mM sodium formate as an internal

172

calibration solution.


Page 9 of 37


173

For quantitative analysis, a calibration curve for each isoflavone standard (µmol/mL) was

174

constructed. Results were expressed as mmol/100 g dry weight (DW), as the mean ±

175

standard deviation of four replicates.

176 177

Statistical analysis

178

Data obtained were submitted to the presupposition of the statistical model, where

179

the linearity and homogeneity of the residual variances were identified, as well as the

180

additivity of the model. Subsequently, the analysis of variance was performed at 5%

181

probability in order to identify the interaction between soybean genotypes x drying

182

conditions x storage periods of the seeds. When identifying the interaction, the sources of

183

variation were dismembered to the simple effects, in the same way, the non-significant

184

interactions proceeded the dismemberments to the main effects. Simple effects for the

185

qualitative factors were proceeded by Tukey at 5% probability. For the source of

186

quantitative variation, the linear regression was performed with the adjustment of the

187

highest significant degree of the polynomial at 5% probability by the t-test, where a

188

specific regression trend was obtained for each level of the qualitative factor. In order to

189

identify the multivariate trend of the measured characters and their specific affinities to

190

the treatments, it was obtained by the Biplot principal components analysis, obtained

191

through the rotated data matrix for eigenvalues and eigenvectors, the scores (representing

192

the characters) projected in the X and Y plane together with the factorial loads

193

(representing the treatments).

194 195

Results

196

Germination capacity and mold incidence as a function drying and storage

197

At the beginning of the storage, for both cultivars, low germination rates were

198

observed for seeds dried at 90 °C and 110 °C (Figure 1). During storage, the germination 7 ACS Paragon Plus Environment


Page 10 of 37

199

rate of cultivar BMX Força RR submitted to field drying remained unaltered. A trend of

200

reduction in germination capacity was observed at 4, 8 and 12-month storage for both

201

cultivars, however, the reduction observed for cultivar Nidera 5909 RR was more abrupt.

202

After a 12-month storage period, germination capacity was below 30% for all drying

203

temperatures (30, 50, 79, 90, and 110 °C) for both cultivars except for field drying for

204

cultivar BMX Força RR. Soybean regardless of the cultivar dried at the highest drying

205

temperatures (90 °C and 110 °C) had 0% germination capacity at the end of the 12-month

206

storage period.

207

Fungi of the genus Rhyzopus sp., Penicillium sp., and Aspergillus sp. were

208

identified in both cultivars, in all drying, and storage temperatures, however fungi of the

209

genus Alternaria sp. were identified only at the beginning of the storage except for those

210

dried at 110 °C (Table 1). At the beginning of the storage, total fungal colonies varied

211

from 68 (field drying) to 21 (110 °C) in cultivar BMX Força RR and from 82 colonies

212

(field drying) to 153 colonies (110 °C) in cultivar Nidera 5909 RR. Increase in drying

213

temperature promoted reductions in Alternaria sp., being eliminated at 110 °C, for both

214

cultivars. After storage, Alternaria sp. was not identified in any of the treatments, for both

215

cultivars. Drying temperature promoted minimal changes in the incidence of Aspergillus

216

sp., Penicillium sp., and Rhizopus sp. for BMX Força RR. However, for Nidera 5909 RR,

217

an increase in colony number of 35 of Aspergillus sp., 6 of Penicillium sp., and 67 of

218

Rhizopus sp. was observed for seeds dried at 110 °C when compared to field drying.

219

After storage, there were an increase in total fungal incidence at all drying

220

temperatures when compared to the beginning of storage. For cultivar Nidera 5909 RR

221

the highest fungal infestation was observed at 70 °C (225 colonies), 90 °C (240 colonies),

222

and 110 °C (232 colonies) at 12-months storage. After storage, the cultivar BMX Força

223

presented higher contamination by Aspergillus sp., while the cultivar Nidera 5909

224

showed higher contamination by Penicillium sp. and Rhizopus sp. at all drying 8 ACS Paragon Plus Environment

Page 11 of 37


225

temperatures. At the drying temperatures of 70, 90, and 110 °C, the Nidera 5909 RR

226

cultivar showed 100% of contamination by Penicillium sp. and Rhyzopus sp.

227 228

Phenolic acids profile

229

The p-coumaric, ferulic, gallic, p-hydroxybenzoic, and syringic acids were

230

identified and quantified in the free fraction, while p-coumaric, ferulic, gallic, p-

231

hydroxybenzoic, syringic, caffeic, and vanillic acids were identified and quantified in the

232

bound fraction for both cultivars, drying temperatures and storage time. Analysis of

233

variance (P < 0.05) revealed significant effects and interactions for all sources of

234

variation (soybean genotype, drying temperature, and storage period) for free and bound

235

phenolic compounds (Tables 2 and 3 and Supporting information 2, 3, 4, and 5). The

236

interaction among soybean genotype x drying temperature x storage period was

237

significant for free-syringic, free-gallic, free-coumaric, bound-vanillic, bound-gallic, and

238

bound-epicatechin contents (Supporting information 5); drying conditions x storage

239

periods for free-syringic, free-hydroxybenzoic, free-coumaric, bound-ferulic, bound-

240

epicatechin, and bound-caffeic acids content (Supporting information 4); soybean

241

genotypes x storage periods for free-syringic, free-hydroxybenzoic, free-ferulic, free-

242

coumaric, bound-syringic, bound-hydroxybenzoic, bound-epicatechin, and bound-caffeic

243

acids (Supporting information 3); soybean genotypes x drying conditions for free-

244

syringic, free-coumaric, bound-syringic, bound-vanillic, bound-hydroxybenzoic, bound-

245

gallic, bound-coumaric and bound-caffeic acids (Supporting information 2).

246

To verify a single trend for phenolic compounds, a multivariate approach was

247

applied on eight treatments (2 soybean genotype x 2 drying temperature x 2 storage time)

248

and 13 variables (phenolic acids). Results of the principal component analysis for

249

phenolics are shown in figure 2. The first principal components PCI (35.15%) and PCII

250

(32.95%) were responsible for explaining 68.10% of the total variation of the experiment. 9 ACS Paragon Plus Environment


Page 12 of 37

251

Although without the high multivariate representativeness it was possible to identify

252

specific trends of the combined treatments and the variables.

253

At the beginning of the storage, the free-gallic acid was responsible for the

254

differentiation of BMX Força RR dried in the field and at 110 °C. After storage, the free-

255

ferulic acid is strongly associated with BMX Força RR dried at 110 °C and bound-ferulic

256

acid with the BMX Força RR dried in the field. At the beginning of storage, bound-vanillic

257

acid was the compound that contributed to differentiate Nidera 5909 RR cultivar dried at

258

110 °C, followed by bound-gallic acid, bound-epicatechin, and bound-hydroxybenzoic

259

acid, which presented the lowest contribution (Figure 2 and Table 4). After storage, the

260

free-hydroxybenzoic, free-coumaric, bound-caffeic, bound-syringic and bound-coumaric

261

acids differentiated the cultivar Nidera 5909 RR dried in the field from 110 °C.

262

The distancing of the plot of the variation source and the free-gallic acid character

263

is due to the differential effects of the interaction (Supplementary file 5). The bound-

264

ferulic and free-ferulic presented increases after storage, being the highest values found

265

for the cultivar BMX Força RR (Supplementary file 3). The bound-vanillic acid showed

266

high concentration at the beginning of storage, mainly for Nidera 5909 RR cultivar dried

267

at 110 °C, however bound-epicatechin, bound-gallic acid, and bound-hydroxybenzoic

268

acid also contributed to differentiate this treatment, but with a lower contribution of the

269

characters (Supplementary file 5, and Table 4). The bound-coumaric and bound-syringic

270

acids were higher in the cultivar Nidera 5909 RR and were influenced predominantly by

271

the drying temperature of 110 °C (Supplementary file 2), while free-hydroxybenzoic and

272

bound-caffeic acid were higher after storage at Nidera 5909 RR 110 ° C (Supplementary

273

file 4). The free-coumaric and free-syringic were the compounds that presented the

274

greatest increases in absolute values (Supplementary file 5), justifying these compounds

275

to present greater power of distinction for dried Nidera 5909 RR at 110 °C.

276 10 ACS Paragon Plus Environment

Page 13 of 37

277


Isoflavone profile

278

The isoflavones were identified only in the free fraction and no isoflavones were

279

identified in the bound fraction for both soybean genotypes. The acetyl-glucoside forms

280

of isoflavones were not detected in any of studied conditions. BMX Força cultivar

281

consisted of 57.2% malonyl-glucosides, 37.6% β-glycosides, and 5.1% aglycones,

282

whereas, Nidera 5909 cultivar consisted of 65.7% malonyl-glucosides, 25.5% β-

283

glucosides, and 8.7% aglycones (supplementary file 6).

284

Analysis of variance for the isoflavone content is presented in table 5. There was

285

significant interaction (P < 0.05) among soybean genotype x drying temperature x storage

286

periods for isoflavone content. To show these effects the linear regressions were

287

performed (Figure 3, Supplementary file 7). For both cultivars, the isoflavones malonyl-

288

genistin, malonyl-glycitin, and malonyl-daidzin showed a tendency of reduction

289

according to the increase of drying temperature, at the beginning and after storage (Figure

290

3A, 3B and 3C, and supplementary file 7). Few changes were observed for β-genistin and

291

β-daidzin content (Figure 3D and 3F), regardless of drying temperature and storage time.

292

For β-glycitin in the cultivar Nidera 5909 RR at the beginning of the storage there was a

293

tendency to increase as the drying temperature increased (Figure 3E), however, after

294

storage, contrary behavior was observed. At the beginning of storage, increases were

295

observed for genistein and daidzein in the Nidera 5909 RR cultivar. After storage,

296

increases in genistein, glycitein and daidzein contents were observed (Figure 3G, 3H, and

297

3I).

298

The multivariate approach was performed based on 24 treatments (2 soybean

299

cultivars x 6 drying temperatures x 2 storage time) and 9 nine variables (individual

300

isoflavones) in order to identify a single trend. The results of the principal component

301

analysis for isoflavones are shown in figure 4. The first principal components PCI

302

(54.5%) and PCII (27.6%) were responsible for explaining 82.1% of the total variation of 11 ACS Paragon Plus Environment


Page 14 of 37

303

the experiment. Malonyl-genistin, malonyl-daidzin, and malonyl-glycitin were the

304

isoflavones responsible for the differentiation of the beginning of the storage, for both

305

cultivars, regardless of the drying temperature. The β-daidzin and β-genistin were the

306

main responsible for the differentiation of the cultivar BMX Força RR after storage.

307

Genistein, glycitein, daidzein, and β-glycitin were responsible for the differentiation of

308

Nidera 5909 RR cultivar dried at 110 °C at the beginning of storage and after storage of

309

Nidera 5909 RR for all drying conditions, but also had influence on the cultivar BMX

310

Força RR, dried at 70 °C, 90 °C and 110 °C.

311 312

Discussion

313

Germination capacity and mold incidence

314

The reduction in germination capacity with the increase of drying temperature and

315

time of storage (Figure 1) is in agreement with the results found by Hartmmann-Filho et

316

al.11 who performed soybean drying at different temperatures, followed by storage for

317

180 days in non-hermetic containers, and reported germinations of 100, 97, 88, 28, and

318

1% at 40, 50, 60, 70 and 80 °C, respectively, the reduction was intensified linearly during

319

storage. Germination capacity is a quality parameter used to evaluate seed viability and

320

directly reflects the integrity of the seed’s membranes and enzymes. The reduction in

321

germination capacity with drying process (Figure 1) is associated with the temperature

322

that the grains reach during drying, because in both cultivars the grain temperature was

323

29, 42, 54, 59, and 70 °C, respectively at air temperatures of 30, 50, 70, 90, and 110 °C

324

(data not shown). According to Stewart et al.20, the maximum seed temperature for

325

soybean drying is 40 °C, when germination and seed vigor are maintained. In addition,

326

high temperatures induce protein and oil modification occurs. An increase in drying

327

temperature above 50 °C promotes a reduction in the enzymatic activity and/or unfolding


Page 15 of 37


328

and/or irreversible denaturing of the proteins. Enzyme damage may be the primary

329

responsible for the reduction of germination capacity during storage.22

330

The highest incidence of Alternaria sp. in soybean subjected to field drying

331

conditions is due to the greater survival rate under conducive environmental conditions,

332

however, this fungus is more susceptible to degradation during drying and storage

333

processes (Table 1). These results agree with Bhattacharya and Raha22 who reported that

334

field molds are reduced gradually during storage, as they are unable to survive in moisture

335

equilibrium less than 90%, being replaced by storage fungi such as Aspergillus sp. Nidera

336

5909 RR soybean was more susceptible to drying temperatures, presenting a greater

337

reduction at the beginning and after 12-months storage (Figure 1B). Overall, cultivar

338

Nidera 5909 RR presented higher fungal infestation in all treatments when compared to

339

cultivar BMX Força RR. This behavior indicates greater cellular damage and less

340

resistance to pathogen infestation in the cultivar Nidera 5909 RR, consequently, more

341

damaged seeds facilitated the spore germination of Aspergillus sp., Penicillium sp., and

342

Rhizopus sp. present in the soybeans. The highest incidence of Rhyzopus sp., Penicillium

343

sp., and Aspergillus sp. was observed in soybean submitted to the highest drying

344

temperatures, followed by 12-month storage period, probably due to the greater surface

345

damage of the grains, which directly influence soybean germination (Figure 1). Oilseeds

346

possess high water activity which is associated with a large number of deteriorating

347

microorganisms, of which fungi are the main responsible for the rapid decrease of

348

germination capacity and seed vigor during storage.23 However, other factors also

349

influence quality, such as a reduction in lipid content, increase in free fatty acids and

350

oxidation products and production of mycotoxins.24

351 352

Phenolic acid profile



Page 16 of 37

353

The two cultivars used showed a similar phenolic acids profile (Supplementary

354

files 2, 3, 4, and 5). Xu and Chang25 who evaluated 30 soybean genotypes grown in the

355

North Dakota-Minnesota region, reported the presence of gallic, protocatechuic,

356

trihydroxybenzoic, p-hydroxybenzoic, vanillic, syringic, chlorogenic, p-coumaric +

357

syringaldehyde, m-coumaric, ferulic, sinapic, O-coumaric, and trans-cinnamic acids.

358

They also reported that the concentration of these compounds varied depending on the

359

variety, site, and year of cultivation. No studies were found evaluating the phenolic acid

360

profile of soybean seeds submitted to high drying temperatures, followed by long-term

361

storage. Ziegler et al.1 stored soybean seeds with different moisture contents and

362

temperatures and reported an increase in total phenolic content and a reduction in vanillic

363

acid content in soybean stored for 12 months at 15 and 18% moisture content and

364

temperatures above 25 °C. Phenolic acids derived from cinnamic or benzoic acids were

365

found in higher concentration in the bound fraction of soybean (Supplementary files 2, 3,

366

4, and 5), most likely because they make up the lignin present in the cell wall. Phenolic

367

acids are modified during drying and storage, due to oxidation or catalyzed by microbial

368

activity. According to Waggoner et al.26, the increase in the oxidation of organic matter

369

promotes the increase of more oxidized phenols of lignin (syringic acid and vanillic acid)

370

when compared to less oxidized aldehydes (vanillin and syringaldehyde). The increase of

371

free phenolic acids after storage in soybean dried at high temperatures (Figure 2,

372

Supplementary files 2, 3, 4, and 5) is probably due to the high incidence of Rhyzopus sp.

373

and Pennicilium sp (Table 1). According to McCue and Shetty27, the α- and β-glucosidase

374

enzymes produced by fungi of the genus Rhyzopus sp. promote the increase in free

375

phenolic content through the cleavage of carbohydrates associated with lignocellulosic

376

materials. According to Bruchert28, the degradation of syringic and vanillic acids can be

377

performed by microorganisms, especially anaerobes, through the cleavage of the ether


Page 17 of 37


378

bond between the phenolic subunits and the subsequent disruption of the aromatic ring

379

by bacteria or fungi.

380 381

Isoflavone profile as a function drying and storage

382

The isoflavone profile found in this study are in agreement with Xu and Chang25

383

that evaluated the isoflavones profile of 30 soybean genotypes from the North Dakota-

384

Minnesota region. They reported higher concentrations of the isoflavones malonyl-

385

glucosides and β-glucosides, respectively, with 75% and 20%, whereas acetyl-glucosides

386

and aglycones are found in lower concentrations. The isoflavone profile varies according

387

to genotype and environmental conditions (temperature, fertilization, and place of

388

cultivation) 6, and about 80% to 90% of the isoflavones are found in soy cotyledon.29

389

These results are in agreement with Hou and Chang13, who reported that the total

390

isoflavones are slightly altered by thermal processes such as cooking and frying.

391

However, the conversion of malonyl-glucosides forms to acetyl-β-glucosides and β-

392

glucosides is favored under thermal treatment conditions. No studies were found with

393

soybean drying and storage evaluating the isoflavones profile. Drying at 110 °C reduced

394

the germination for both cultivars to 0% (Figure 1), indicating that the metabolic activity

395

of soybean was permanently compromised, which implies that changes in isoflavones do

396

not arise from seed metabolism. Probably, the synthesis or release of these metabolites is

397

due to the action of enzymes produced by microorganisms, mainly fungi that were present

398

in high concentrations in dry grains at high temperatures (Table 1). According to McCue

399

and Shetty, the α- and β-glucosidase enzymes, α-amylase, and β -glucuronidases

400

synthesized by fungi are involved in the remobilization or degradation of structural

401

components, releasing phenolic compounds.

402

At the beginning of storage, glycosylated forms of genistein (Malonyl and β-

403

glucosides) were the predominant isoflavones (Figure 3 and 4, and supplementary file 6). 15 ACS Paragon Plus Environment


Page 18 of 37

404

These results are in agreement with Niamnuy et al.14, who reported that genistein and its

405

glycosylated forms are the predominant isoflavones in soybean. In both cultivars, the

406

highest reductions of malonyl-genistin (absolute values) were observed when soybean

407

was submitted to high drying temperatures. Similar results were reported by Lee and Lee6

408

who observed a faster degradation of malonyl-genistin when compared to malonyl-

409

daidzin, and consequently, genistin was formed more rapidly than daidzin during oven

410

drying. According to Niamnuy et al.14, the highest conversion rate occurs from malonyl-

411

glucosides to β-glucosides, followed by malonyl-glucosides to aglycones and malonyl-

412

glucosides to acetyl-β-glucosides. Interconversions may occur from malonyl-glucosides

413

to acetyl-β-glucosides (by decarboxylation), from malonyl- and acetyl-glucosides to β-

414

glucosides (by de-esterification), and malonyl-, acetyl- and β-glucosides to aglycones (by

415

hydrolysis), all being accelerated by thermal, acidic, alkaline, and enzymatic hydrolysis.12

416

Damage caused by the drying process was observed during storage and directly

417

influenced the isoflavone profile. At the beginning of the storage, important reductions in

418

germination capacity were observed at 90 °C and 110 °C (Figure 1), however, during the

419

storage, germination reductions occurred in most treatments, indicating a higher level of

420

cellular damage, favoring the establishment of fungi (Table 1). Fungal contamination is

421

the main deteriorating factor in stored soybeans, since during feeding they synthesize, α-

422

and β-glucosidases, thereby favoring the release of aglycone isoflavones. These results

423

agree with Hou and Chang13 studying the storage of soybeans in 84% RH / 30 °C for 9

424

months. They reported reductions in the isoflavones malonyl-, acetyl- and β-glucosides

425

contents and increase in isoflavones aglycone contents upon 9 months of storage. Similar

426

behavior was observed by Ziegler et al.8 evaluating protein concentrate extracted from

427

soybeans stored at 12% and 15% of moisture content at different temperatures for 12

428

months. They reported increases of 151.6%, 133.4%, and 1721.7%, respectively, for

429

daidzein, genistein and glycitein under conditions of 15% moisture and 32 °C storage 16 ACS Paragon Plus Environment

Page 19 of 37


430

temperature. The α- and β-glucosidase enzymes produced by Rhyzopus sp. promote the

431

increase in free phenolics through the cleavage of carbohydrates associated with

432

lignocellulosic materials.27 The principal components analysis allows to associate

433

treatments and variables (Figure 4), clearly identifying that isoflavone aglycones

434

(genistein, glycitein, and daidzein) are responsible for the separation of the most drastic

435

treatments. At higher temperatures, mainly, 90 and 110 °C, soybean has a reduction of

436

germination capacity (Table 1), indicating compromised enzymatic activities, with little

437

resistance to moldy activity, and explains the high conversion rate of glycosylated

438

isoflavones to aglycones. Isoflavones aglycones are more reactive when compared to their

439

glycosylated forms and better utilized by the human organism,30 however, there are still

440

no precise data on the application and purification of isoflavones extracted from seeds

441

with a high fungal infestation, as well as their application in products. The increase in the

442

total isoflavone content after storage in dry seeds at higher temperatures, mainly in the

443

cultivar Nidera 5909 RR, was higher due to the increase in genistein, daidzein, but mainly

444

glycitein. The increase in glycitein was higher than the interconversion rate of malonyl-

445

glycitin, which indicates that the seeds may present some insoluble isoflavones, which

446

remained bound, and did not allow for their quantification by the currently used methods.

447

However, the action of fungi probably promoted the release of glycitein, facilitating its

448

quantification in the soluble phase. This hypothesis will be tested in an upcoming study.

449

Regardless of soybean cultivar, the increase in drying temperature promoted a

450

reduction in germination capacity and an increase in fungal incidence during the storage.

451

The multivariate analysis allowed different treatments (soybean genotypes, drying

452

conditions and storage period for phenolics and isoflavones, which showed to be closely

453

related to fungal viability and contamination (Figure 1 and Table 1). The Nidera 5909 RR

454

cultivar showed more susceptibility to deterioration at elevated temperatures and longer

455

storage periods when compared to BMX Força RR cultivar. Nidera 5909 RR cultivar 17 ACS Paragon Plus Environment


Page 20 of 37

456

presented increments of free-syringic, free-coumaric, and free-hydroxybenzoic, and

457

reduction in bound-vanillic at high drying condition and 12-months storage (Figure 2 and

458

Supplementary file 5). The highest drying temperature and 12-months of storage time,

459

promoted the conversion of malonyl-glucosides and β-glucosides into their bioactive

460

aglycones forms. The higher concentration of aglycone isoflavones in soybeans is

461

desirable since they have higher health benefits when compared to their glycosylated

462

forms, however, further studies are still needed to identify the best processing to avoid

463

the degradation of these isoflavones and microbiological contamination. Monitoring of

464

germination and fungal incidence provides an excellent parameter for the control of

465

metabolic profile quality for different genotypes.

466 467

Acknowledgments

468

The authors would like to thanks to FAPERGS (Fundação de Amparo à Pesquisa do

469

Estado do Rio Grande do Sul), CNPq (Conselho Nacional de Desenvolvimento Científico

470

e Tecnológico), SDECT-RS (Secretaria do Desenvolvimento Econômico, Ciência e

471

Tecnologia do Estado do Rio Grande do Sul) and Polo de Inovação Tecnológica em

472

Alimentos da Região Sul (Polo de Alimentos).

473 474

Supporting Information. S1. Characteristics of BMX Força RR and Nidera 5909 RR

475

Cultivars Evaluated in the Field Drying Conditions; S2. Interaction Between Soybean

476

Genotypes x Drying Conditions for Phenolic Acids (μg/100 g DW); S3. Interaction

477

Between Soybean Genotypes x Storage Periods for Phenolic Acids (μg/100 g DW); S4.

478

Interaction Between Drying Conditions x Storage Periods for Phenolic Acids (μg/100 g

479

DW); S5. Interaction Among Soybean Genotypes x Drying Conditions x Storage Periods

480

for Phenolic Acids (μg/100 g DW); S6. Interaction Among Soybean Genotypes x Drying


Page 21 of 37


481

Conditions x Storage Periods for Isoflavones (mmol/100 g DW); S7. Regression

482

Equations for Isoflavones as a Function of Temperature, Genotype, and Storage.

483 484

Author contributions

485

Cristiano Dietrich Ferreira: General responsible for the execution and writing of the

486

manuscript; Valmor Ziegler: Participated in the experimental design and execution of the

487

analyzes; Jorge Tiago Schwanz Goebel; He participated in experimental planning,

488

cultivation, and cultural dealings in the field; Jessica Fernanda Hoffmann: She has high

489

knowledge in chromatography and mass spectrophotometry and helped in the LC-MS

490

analysis and data treatment; Ivan Ricardo Carvalho: Assisted in the correction and

491

alteration of the statistical material, as attendance to that requested by the reviewers.

492

Fabio Clasen Chaves: Coordinator of the mass spectrometry laboratory and assisted in

493

the correction of the manuscript; Maurício de Oliveira: Responsible for the research line

494

of drying and storage, participated in the structuring of the study, interpretation of the

495

results and revision of the manuscript.

496 497

References

498

1. Ziegler, V.; Vanier, N. L.; Ferreira, C. D.; Paraginski, R. T.; Monks, J. L. F.; Elias, M.

499

C. Changes in the bioactive compounds content of soybean as a function of grain moisture

500

content and temperature during long-term storage. J. Food Sci. 2016, 81(3), 762–768.

501

2. Wang, Y.-K.; Zhang, X.; Chen, G.-L.; Yu, J.; Yang, L.-Q.; Gao, Y.-Q. Antioxidant

502

property and their free, soluble conjugate and insoluble-bound phenolic contents in

503

selected beans. J. Funct. Foods, 2016, 24, 359–372.

504

3. Lima, F. S.; Kurozawa, L. E.; Ida, E. I. The effects of soybean soaking on grain

505

properties and isoflavones loss. LWT - Food Sci. Technol, 2014, 59, 1–9.



Page 22 of 37

506

4. Wang, Q.; Ge, X.; Tian, X.; Zhang, Y.; Zhang, J.; Zhang, P. Soy isoflavone: The

507

multipurpose phytochemical (Review). Biomed. Rep. 2013, 1, 697–701.

508

5. Cho, K. M.; Ha, T. J.; Lee, Y. B.; Seo, W. D.; Kim, J. Y.; Ryu, H. W.; Jeon, S. H.;

509

Kang, Y. M.; Lee, J. H. Soluble phenolics and antioxidant properties of soybean (Glycine

510

max L.) cultivars with varying seed coat colours. J. Funct. Foods, 2013, 5, 1065–1076.

511

6. Lee, S.; Lee, J. Effects of oven-drying, roasting, and explosive puffing process on

512

isoflavone distributions in soybeans. Food Chem. 2009, 112(2), 316–320.

513

7. Zannata, T. S.; Manica-Berto, R.; Ferreira, C. D.; Cardozo, M. C.; Rombaldi, C. V.;

514

Zambiazi, R. C.; Dias, A. R. G. Phosphate fertilizer and growing environment changes

515

the phytochemicals, oil quality, and nutritional composition of Roundup Ready GM and

516

conventional soybean. J. Agric. Food Chem. 2017, 65(13), 2661–2669.

517

8. Ziegler, V.; Ferreira, C. D.; Hoffman, J. F.; Oliveira, M.; Elias, M. C. Effects of

518

moisture and temperature during grain storage on the functional properties and isoflavone

519

profile of soy protein concentrate. Food Chem. 2018, 242, 37–44.

520

9. Soponronnarit, S.; Swasdisevi, T.; Wetchacama, S. Fluidised bed drying of soybeans.

521

J. Stored Prod. Res. 2001, 37, 133–151.

522

10. Fleurat-Lessard, F. Integrated management of the risks of stored grain spoilage by

523

seedborne fungi and contamination by storage mould mycotoxins - An update. J. Stored

524

Prod. Res. 2017, 71, 22–40.

525

11. Hartmann-Filho, C. P.; Goneli, A. L. D.; Masetto, T. E.; Martins, E. A. S.; Oba, G. C.

526

The effect of drying temperatures and storage of seeds on the growth of soybean

527

seedlings. J. Seed Sci. 2016, 38(4) 287–295.

528

12. Niamnuy, C., Nachaisin, M., Laohavanich, J., Devahastin, S. Evaluation of bioactive

529

compounds and bioactivities of soybean dried by different methods and conditions. Food

530

Chem. 2011, 129, 899–906.


Page 23 of 37


531

13. Hou, H. J.; Chang, K. C. Interconversions of isoflavones in soybeans as affected by

532

storage. J. Food Sci. 2002, 67(6), 2083–2089.

533

14. Niamnuy, C.; Nachaisin, M.; Poomsa-Ad, N.; Devahastin, S. Kinetic modelling of

534

drying and conversion/degradation of isoflavones during infrared drying of soybean.

535

Food Chem. 2012. 133, 946–952.

536

15. Yeom, S.-J.; Kim, B.-N.; Kim, Y.-S.; Oh, D.-K. Hydrolysis of isoflavone glycosides

537

by a thermostable β-glucosidase from Pyrococcus furiosus. J. Agric. Food Chem. 2012,

538

60, 1535–1541.

539

16. ASAE. Moisture measurement-unground grain and seeds. In Standards Engineering

540

Practices Data, Saint Joseph, Michigan, USA: American Society of Agricultural

541

Engineers, 2000.

542

17. BRASIL. Regras para análise de sementes. Ministério da Agricultura Pecuária e

543

Abastecimento. Secretaria de Defesa Agropecuária, Brasília: SNDA/DNDV/CLAV, 399.

544

(2009).

545

18. Dueñas, M.; Hernández, T.; Lamparski, G.; Estrella, I.; Muñoz, R. Bioactive phenolic

546

compounds of soybean (Glycine max cv. Merril): modifications by different

547

microbiological fermentations. Pol. J. Food Nutr. Sci. 2012, 62, 241–250.

548

19. Zilic, S.; Mogol, B. A.; Akıllıoglu, G.; Serpen, A.; Delic, N.; Gökmen, V. Effects of

549

extrusion, infrared and microwave processing on Maillard reaction products and phenolic

550

compounds in soybean. J. Sci. Food Agric. 2014, 94, 45–51.

551

20. Stewart, O. J.; Raghavan, G. S. V.; Orsat, V.; Golden, K. D. The effect of drying on

552

unsaturated fatty acids and trypsin inhibitor activity in soybean. Process Biochem. 2003,

553

39, 483–489.

554

21. Ahern, T. J.; Klibanov, A. M. Analysis of processes causing thermal inactivation of

555

enzymes. Method. Biochem. Anal. 1988, 33, 91–127. 21 ACS Paragon Plus Environment


Page 24 of 37

556

22. Bhattacharya, K.; Raha, S. Deteriorative changes of maize, groundnut and soybean

557

seeds by fungi in storage. Mycopathologia 2002, 155, 135–141.

558

23. Nagaraja, O., Krishnappa, M. Seed borne mycoflora of niger (Guizotia abyssinica

559

cass,) and its effect on germination. Phytopathology 2009, 62(4), 513–517.

560

24. Saxena, N.; Rani, S. K. S.; Deepika, M. Biodeterioration of Soybean (Glycine max

561

L.) seeds during storage by Fungi. Int. J. Curr. Microbiol. Appl. Sci. 2015, 4(6), 1118–

562

1126.

563

25. Xu, B.; Chang, S. K. C. Characterization of phenolic substances and antioxidant

564

properties of food soybeans grown in the North Dakota-Minnesota region. J. Agric. Food

565

Chem, 2008. 56(19), 9102–9113.

566

26. Waggoner, D. C.; Wozniak, A. S.; Cory, R. M.; Hatcher, P.G. The role of reactive

567

oxygen species in the degradation of lignin derived dissolved organic matter. Geochim.

568

Cosmochim. Ac. 2017, 208, 171–184.

569

27. McCue, P.; Shetty, K. Role of carbohydrate-cleaving enzymes in phenolic antioxidant

570

mobilization from whole soybean fermented with Rhizopus oligosporus. Food

571

Biotechnol. 2007, 17(1), 27–37.

572

28. Bruchert, V. Degradation of lignin monomers and oligomers by a consortium of

573

anaerobic

574

http://www.mbl.edu/microbialdiversity/files/2012/08/1996_bruchert.pdf. Accessed in

575

08/30/2018.

576

29. Tsukamoto, C.; Shimada, S.; Igita, K.; Kudou, S.; Kokubun, M.; Okubo, K.;

577

Kitamura, K. Factors affecting isoflavone content in soybean seeds: Changes in

578

isoflavones, saponins, and composition of fatty acids at different temperatures during

579

seed development. J. Agric. Food Chem. 1995, 43(5), 1184–1192.

580

30. Kim, M.; Lee, J.; Han, J. Deglycosylation of isoflavone C-glycosides by newly

581

isolated human intestinal bacteria. J. Sci. Food Agric. 2015, 95(9), 1925–1931.

bacteria.

Microbial

Diversity.

1996,

Retrieved

from


Page 25 of 37


582

Funding

583

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de

584

Nível Superior - Brasil (CAPES) - Finance Code 001



Page 26 of 37

585

Figure captions

586

Figure 1. Germination (%) of Soybean Submitted to Different Drying Temperatures

587

Followed by 12-Month Storage. BMX Força RR (A) and Nidera 5909 RR (B).

588

Figure 2. Biplot of Principal Component Analysis (PC1 vs. PC2)_for Phenolic Content

589

of Soybean Submitted to Different Drying Temperatures Followed by 12-Month Storage

590

. BMX Força RR_Field Drying (B_F_0) and 110 °C (B_1_0) 0 Months; BMX Força

591

RR_Field Drying (B_F_12) and 110 °C (B_1_12) 12 Months; Nidera 5909 RR_Field

592

Drying (N_F_0) and 110 °C (N_1_0) 0 Months; Nidera 5909 RR Field Drying (N_F_12)

593

and 110 °C (N_1_12). BSY, Bound-Syringic acid; BVA, Bound-Vanillic acid; BHY,

594

Bound-Hydroxybenzoic acid; BGA, Bound-Gallic acid; BFE, Bound-Ferulic acid; BEP,

595

Bound-Epicatechin, BCO, Bound-Coumaric acid; BCA, Bound-Caffeic acid; FSY, Free-

596

Syringic acid; FHY, Free-Hydroxybenzoic acid; FGA, Free-Gallic acid; FFE, Free-

597

Ferulic acid; FCO, Free-Coumaric acid

598

Figure 3. Isoflavones Content of Soybeans as a Function of Drying Temperature and

599

Storage. Malonyl-Genistin (A), Malonyl-Glycitin (B), Malonyl-Daidzin (C), β-Genistin

600

(D), β-Glycitin (E), β-Daidzin (F), Genistein (G), Glycitein (H), Daidzein (I).

601

Figure 4. Biplot of Principal Component Analysis (PC1 vs. PC2) for Isoflavones Content

602

of Soybean Submitted to Different Drying Temperatures Followed by 12-Month Storage.

603

BMX Força RR: Field Drying (B_F), 30 °C (B_30), 50 °C (B_50), 70 °C (B_70), 90 °C

604

(B_90) and 110 °C (B_1) for 0 and 12 Months; Nidera 5909 RR: Field Drying (N_F), 30

605

°C (N_30), 50 °C (N_50), 70 °C (N_70), 90 °C (N_90) and 110 °C (N_1) for 0 and 12

606

Months. MGE, Malonyl-Genistin; MGL, Malonyl-Glycitin; MDA, Malonyl-Daidzin;

607

BGE, β-Genistin; BGL, β-Glycitin; BDA, β-Daidzin; AGE, Genistein; AGL, Glycitein;

608

ADA, Daidzein.

609


Page 27 of 37


Table 1. Fungal Colonies per 100 Soybean Seeds Dried at Different Temperatures Followed by 12-Month Storage BMX Força RR Nidera 5909 RR Drying a b c d a Conditions AS RP PE AL Total AS RPb PEc ALd Total 0 Months Field 2 25 1 43 71 11 31 3 37 82 30 °C 22 22 8 10 62 3 26 39 24 92 50 °C 9 28 15 11 63 8 28 41 29 106 70 °C 22 33 4 2 61 64 1 50 6 121 90 °C 17 21 4 1 43 49 15 87 1 152 110 °C 14 7 1 0 22 46 37 70 0 153 12 Months Field 1 10 5 0 16 3 68 68 0 136 30 °C 32 14 48 0 94 6 85 85 0 176 50 °C 35 12 63 0 110 22 84 84 0 190 70 °C 33 29 51 0 113 25 100 100 0 225 90 °C 35 49 71 0 155 40 100 100 0 240 110 °C 33 35 39 0 107 32 100 100 0 232 a AS- Aspergillus sp.; b RP - Rhyzopus sp.; c PE - Penicillium sp.; d AL - Alternaria sp.



Page 28 of 37

Table 2. Analysis of Variance For the Effects of Soybean Genotype, Drying Conditions, and Storage Period on Free Phenolics Content. Source of Variation Soybean Genotypes Drying Conditions Soybean Genotypes * Drying Conditions Storage Period Soybean Genotypes *Storage Period Drying Conditions * Storage Period Soybean Genotypes* Drying Conditions *Storage Period Repetition CV (%) Residue a ns Not Significant; * Significant (P < 0.05) b

Mean Squares a GL FSY b 1 1 1 1 1 1 1 3 -

121364.3* 115836.9* 6626.9* 2637358.0* 384849.6* 198371.3* 2522.3* 862.2 5.2 403.55

FHY

FGA

FFE

FCO

20467.7* 40.3ns 6.9ns 89919.8ns 18226.2* 299.5* 27.6ns 27.6 10.3 35.9

0.1ns 0.0ns 1.2ns 11.4* 1.9ns 5.7ns 10.5* 1.4 3.0 1.81

48679.8* 15580.5* 230.6ns 3194.0* 2462.3* 36.8ns 353.1ns 724.0 13.0 395.05

1431220.5* 84635.3* 9894.7* 1475203.7* 887944.7* 88778.4* 11954.4* 11.5 2.8 71.19

FSY, Free-Syringic acid; FHY, Free-Hydroxybenzoic acid; FGA, Free-Gallic acid; FFE, Free-Ferulic acid; FCO, Free-Coumaric acid


Page 29 of 37


Table 3. Analysis of Variance For the Effects of Soybean Genotype, Drying Conditions, and Storage Period on Bound Phenolics Content Mean Square a

Source of Variation G L Soybean Genotypes

1

BSYb BVA 1765978.2 * 14731.9*

Drying Conditions

1

752948.6* 2488.7ns

Soybean Genotypes * Drying Conditions

1

STORAGE Period

1

Soybean Genotypes *Storage Period

1

466964.5* 171288.0* 5612.7* 2050110.0 23544.5 7194.0ns * * 20311.2 * 164795.4* 939.6ns

Drying Conditions * Storage Period Soybean Genotypes* Drying Conditions *Storage Period Repetition CV (%) Residue a ns Not Significant; * Significant (P < 0.05)

1

3880.8ns

1 3

23295.6ns 5438.3 11.2 10134.25

329.0ns

BHY 49282.3 * 13894.4 *

BGA

BFE

3.1ns

4209.0* 13877.8 *

4.6ns 230.6 * 75.3ns

0.1ns 12411.0 *

12.9ns 762.5ns

1053.4ns 40.3ns 108.4 134265.6* 1135.3ns * 4577.4 887.9 1.7 7.3 6.9 10.0 1765.86 739.19 17.79

5660.5* 80.6ns 2119.6 6.7 414.76

BEP 10720.1 * 21699.7 * 64.7ns 304.4ns 49211.7 * 18900.5 * 11426.9 * 428.7 10.3 726.16

BCO BCA 4537276.9 * 23.3 ns 6.9ns 835.4 132844.4* * 37922.6*

3668.0ns

417.6ns

79.7* 134.1 * 103.3 *

6909.0ns 1921.4 6.2 2341.40

2.9ns 6.7ns 5.7 8.70

4656.1ns

b

BSY, Bound-Syringic acid; BVA, Bound-Vanillic acid; BHY, Bound-Hydroxybenzoic acid; BGA, Bound-Gallic acid; BFE, Bound-Ferulic acid; BEP, Bound-Epicatechin, BCO, Bound-Coumaric acid; BCA, Bound-Caffeic acid.



Page 30 of 37

Table 4. Contribution of the Characters to Differentiate Treatments for Multivariate Phenolics. Variable BCO BVA FHY FGA FCO BSY FSY BFE FFE BCA BHY BGA BEP a Sj, Variance of Variable

S.j a 10.79 9.79 9.63 8.29 8.07 7.71 7.38 7.28 7.01 6.77 6.61 6.59 4.82

Value (%) 10.70 9.71 9.56 8.22 8.01 7.64 7.32 7.22 6.95 6.72 6.56 6.54 4.78

b

BSY, Bound-Syringic acid; BVA, Bound-Vanillic acid; BHY, Bound-Hydroxybenzoic acid; BGA, Bound-Gallic acid; BFE, Bound-Ferulic acid; BEP, Bound-Epicatechin, BCO, Bound-Coumaric acid; BCA, Bound-Caffeic acid; FSY, Free-Syringic acid; FHY, Free-Hydroxybenzoic acid; FGA, Free-Gallic acid; FFE, Free-Ferulic acid; FCO, Free-Coumaric acid


Page 31 of 37


Table 5. Analysis of Variance For the Effects of Soybean Genotype, Drying Conditions, and Storage Period on Isoflavones Content Mean squares a Source of Variation GL MGE b MGL Soybean Genotypes 1 587829.6* 123.0* Drying Conditions 5 60008.3* 149.3* Soybean Genotypes * Drying Conditions 5 1969.3* 30.8* Storage period 1 560302.3* 3624.8* Soybean Genotypes *Storage Period 1 2684.9* 349.2* Drying Conditions * Storage Period 5 6643.5* 24.2* Soybean Genotypes* Drying Conditions *Storage Period 5 7903.1* 41.2* Repetition 3 440.7 0.6 n CV (%) 4.09 4.14 Residue 6447.87 0.66 a ns Not Significant; * Significant (P < 0.05)

MDA 312257.5* 10647.3* 1042.1* 243261.0* 4284.0* 3626.0* 3053.3* 82.3 3.56 21.76

BGE 115557.9* 3623.2* 120.53 82479.5* 12841.3* 921.2* 622.9* 114.2 n 6.72 58.41

BGL 75454.5* 1498.5* 667.4* 2550.2* 3262.0* 8284.8* 8650.8* 190.0 9.81 56.84

BDA 643701.2* 5437.3* 839.3* 91785.4* 42909.1* 219.0* 552.8* 14.4 n 3.97 41.81

AGE 53444.5* 27471.3* 3663.6* 259989.7* 8684.1* 8992.6* 6012.3* 42.5 5.19 13.74

AGL 3.6* 670538.9* 311006.0* 106785.9* 1438493.1* 552339.7* 223726.9* 94425.7 10.69 52.56

b MGE,

Malonyl-Genistin; MGL, Malonyl-Glycitin; MDA, Malonyl-Daidzin; BGE, β-Genistin; BGL, β-Glycitin; BDA, β-Daidzin; AGE, Genistein; AGL, Glycitein; ADA, Daidzein


ADA 38885.5* 40277.2* 4161.1* 391566.5* 1722.9* 10827.9* 11112.1* 10.3 3.1 8.20


Page 32 of 37

Table 6. Contribution of the Characters to Differentiate Treatments for Multivariate Isoflavones. Variable MGE MGL MDA BDA AGE AGL ADA BGE BGL a Sj, Variance of Variable

S.j a 57.76* 37.06 60.22 69.98 75.78 27.47 88.51 53.47 44.90

Value (%) 11.21 7.19 11.68 13.58 14.71 5.33 17.18 10.37 8.71

b MGE,

Malonyl-Genistin; MGL, Malonyl-Glycitin; MDA, Malonyl-Daidzin; BGE, β-Genistin; BGL, β-Glycitin; BDA, β-Daidzin; AGE, Genistein; AGL, Glycitein; ADA, Daidzein


Page 33 of 37


160 140

160

A

140 120

Germination (%)

Germination (%)

120

Field 30 °C 50 °C 70 °C 90 °C 110 °C

B

100 80 60 40

100 80 60 40

20

20

0

0

0

4

8

Storage period (months)

12

0

4

8

12

Storage period (months)

Figure 1.



Page 34 of 37

1.5

1.0

B_1_0

B_F_0

FFE B_1_12

PC II 32.95%

0.5

B_F_12

BFE

FGA

0.0 N_1_0 BVA

FHY

BCA

-0.5

FCO

BEP BHY

-1.0

N_1_12

FSY

BGA

BSY BCO N_F_12

-1.5 N_F_0

-2.0 -1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

PC I 35.15% Figure 2.


Page 35 of 37


50

500 400 300 200 100 0

Field

30 °C

50 °C

70 °C

90 °C

40

30

20

10

0

110 °C

500

B

Malony-daidzin (mmol/100g)

600

A

Malonyl-glycitin (mmol/100g)

Malonyl-genistin (mmol/100g)

700

Field

30 °C

50 °C

70 °C

90 °C

300 200 100

Field

110 °C

110

-daidzin (mmol/100g)

-glycitin (mmol/100g)

-genistin (mmol/100g)

210

200 150 100 50

10 50 °C

70 °C

90 °C

0

110 °C

Field

30 °C

70 °C

90 °C

200

100

0 50 °C

70 °C

Drying conditions

90 °C

110 °C

110 °C

100

Field

30 °C

50 °C

70 °C

Drying conditions 400

I

1500

1000

500

0 30 °C

90 °C

200

0

110 °C

Daidzein (mmol/100g)

300

110 °C

300

H

Glycitein (mmol/100g)

Genistein (mmol/100g)

50 °C

2000

G

90 °C

400

Drying conditions

Drying conditions 400

70 °C

F

250 310

50 °C

500

E

30 °C

30 °C

Drying conditions

300

D

Field

0

Drying conditions

Drying conditions

Field

BMX Força - 0 months BMX Força - 12 months Nidera 5909 - 0 months Nidera 5909 - 12 months

C 400

300

200

100

0

Field

30 °C

50 °C

70 °C

Drying conditions

90 °C

110 °C

Field

30 °C

50 °C

70 °C

90 °C

110 °C

Drying conditions

Figure 3.



Page 36 of 37

2.0 B_50_12

1.5

B_70_12 B_F_12

B_30_12

1.0

BDA

PC II 27.6%

0.5

B_110_12 B_90_12

BGE

B_F_0 B_50_0 MDA B_70_0 MGE B_90_0 B_30_0 B_110_0

0.0

-0.5

AGL

ADA AGE

N_110_12 N_90_12 N_70_12

BGEII N_F_12

MGL

N_30_12 N_50_12

-1.0

N_F_0

N_30_0

N_110_0

N_90_0

N_70_0

-1.5

N_50_0

-2.0 -1.5

-1.0

-0.5

0.0

0.5 PC I 54.5%

1.0

1.5

2.0

2.5

Figure 4. 34 ACS Paragon Plus Environment

Page 37 of 37


Table of Contents (TOC) Graphic


Changes in phenolic acid and isoflavone contents during soybean

Recommend Documents