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Article
Phosphate fertilizer and growing environment changes the phytochemicals, oil quality, and nutritional composition of Roundup Ready GM and conventional soybean Tatiane Scilewski da Costa Zanatta, Roberta Manica-Berto, Cristiano Dietrich Ferreira, Michele Maciel Crizel Cardozo, Cesar Valmor Rombaldi, Rui Carlos Zambiazi, and Álvaro Renato Guerra Dias J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05499 • Publication Date (Web): 20 Mar 2017 Downloaded from http://pubs.acs.org on March 20, 2017
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Journal of Agricultural and Food Chemistry
Phosphate fertilizer and growing environment changes the phytochemicals, oil
1 2
quality, and nutritional composition of Roundup Ready GM and conventional
3
soybean
4 5
Tatiane Scilewski da Costa Zanattaa, Roberta Manica-Bertoa, Cristiano Dietrich
6
Ferreiraa*, Michele Maciel Crizel Cardozoa, Cesar Valmor Rombaldia, Rui Carlos
7
Zambiazib, Álvaro Renato Guerra Diasa
8 9
a
Department of Agro-industrial Science and Technology, Federal University of Pelotas,
10
96010-900, Pelotas, RS, Brazil
11
b
12
Pelotas, CEP 96010-900, CP 354, RS, Brazil
Center of Chemical, Pharmaceuticals and Food Sciences, Federal University of
13 14
* Corresponding author: Cristiano Dietrich Ferreira (
[email protected])
15
Tel/Fax: +00555332757284
16 17 18 19 20 21
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Abstract
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Phosphorus (P) intake, genotype, and growth environment in soybean cultivation can
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affect the composition of the soybean. This experiment was conducted in two locations
25
(microregions I and II) using a randomized complete block design, including
26
conventional soybean cultivars (BRS Sambaíba), genetically modified cultivars (Msoy
27
9144 Roundup Ready—RR), and varying doses of phosphorus fertilizer (0, 60, 120, and
28
240 kg/ha P2O5). Soybeans were evaluated for chemical composition, total phenols,
29
phytic acid content, individual isoflavone content, antioxidant activity, oil quality, fatty
30
acid profile, total carotenoid content, and individual tocopherol contents. Multivariate
31
analysis facilitated reduction in the number of variables with respect to soybean
32
genotype (BRS conventional Sambaíba and GM Msoy 9144 RR), dose of P2O5
33
fertilizer, and place of cultivation (microregion I and II). BRS Sambaíba had higher
34
concentrations of β-glucosides, malonylglucosides, glycitein, and genistein than Msoy
35
9144 RR, which showed a higher concentration of daidzein. The highest concentrations
36
of isoflavones and fatty acids were observed in soybeans treated with 120 and 240 kg/ha
37
P2O5, regardless of the location and cultivar.
38 39
Keywords: Glycine max L. (Merril); phosphate fertilizer; isoflavones; tocopherols;
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antioxidant scavenging
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1. Introduction
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Soybean (Glycine max L. Merril) is an important oilseed crop, with a global
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production of 308 million tons. The largest producers are the United States, Brazil,
44
Argentina, and China, totaling 84.3% of world production.1 In these countries, the
45
Roundup Ready Genetically Modified soy is highly prevalent; it constitutes 94% of
46
soybean production in the USA.
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Moreover, the soybean crop has gained prominence for its nutritional
48
characteristics, such as its high levels of bioavailable protein and the quality of its lipid
49
fraction, which is rich in unsaturated fatty acids essential for humans and high levels of
50
natural antioxidants such as carotenoids and tocopherols.2 Phytic acid constitutes 80%
51
of the existing phosphorus reserves in the plant; when present in appropriate
52
concentrations, it exerts antioxidant activity owing to its binding ability with the ions
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Zn2+, Fe2+/3+, Ca2+, Mg2+, Mn2+, and Cu2+, although excessive levels of phytic acid may
54
cause nutritional disorders.3 Soy also has high levels of isoflavones, which show some
55
antioxidant activity but are mainly recognized for their estrogen-regulating potential,
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thereby reducing the effects of menopause.4
57
Vegetables are highly influenced by environmental, genetic, and nutritional
58
conditions.5 A major genetic modification (GM) in soybean is resistance to the
59
glyphosate
60
enolpyruvoylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the shikimate
61
pathway responsible for the biosynthesis of aromatic amino acids (phenylalanine,
62
tyrosine, tryptophan), which are important in protein synthesis. These aromatic amino
63
acids are also precursors of lignin, alkaloids, flavonoids, and cinnamic acid.4,
64
previous studies, Zobiole et al.7 showed that GM soybeans had reduced levels of α-
65
linolenic acid and iron but elevated levels of oleic acid. Another study by Liang et al.8
herbicidal
molecules.
Glyphosate
blocks
the
activity
of
5-
6
In
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indicated that isoflavone content is influenced more by genetic than environmental
67
factors. Zhang et al.9 studied the composition of the oil of 13 genotypes grown in the
68
United States, Brazil, Argentina, Canada and China, and reported that the greatest
69
variation in the fatty acid profile was in oleic acid levels, which ranged from 18.38 to
70
30.62 g/100 g of oil. However, saturated fatty acids levels were more stable. In the same
71
study, the genotypes of soybeans grown in the United States and Brazil showed higher
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lutein content and lower δ-tocopherol content when compared to the genotypes grown
73
in China. Kumar et al.10 observed an interaction between the genotype and growth site
74
in the oleic, linoleic, linolenic, and stearic acids. They also reported variation in the
75
phytic acid content (27.8-45.0 mg/g) with respect to changes in temperature and soil
76
type. John et al.11 studied 27 soybean genotypes, grown in 3 regions of the United States
77
during 2 growing seasons one month apart. They reported a reduction in the
78
biosynthesis of anti-nutritional factors when soybean was grown in the first season. This
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behavior was observed in different regions and coincided with higher temperatures
80
during the pod filling stage. Low temperatures during the grain filling stage have been
81
associated with higher levels of isoflavones.12
82
Balanced fertilization is essential for the nutrition of the soybean crop, with
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phosphorus being an especially important macronutrient. Although phosphorus is not
84
present in large amounts, it participates in many metabolic pathways and physiological
85
reactions, mainly in the form of inorganic phosphate and ATP (adenosine
86
triphosphate).13 According to Yin et al.14, phosphorus acts primarily in the initial stages
87
of leaf growth and grain filling; they reported that high doses of phosphorus promote an
88
increase in protein, palmitic, oleic, and linolenic acid contents, whereas lead to a
89
decrease total lipid content. The interactions among the production factors such as
90
genotype, cultivation environment, and fertilization have been extensively studied to
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determine their effects on the quantitative variables that compose grain yields, such as
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number of pods, and number and weight of grains. However, there is little information
93
associating growing environment, phosphate fertilizer, and genotype on the chemical
94
composition, oil quality, and phytochemical composition of these beans. Optimization
95
of inputs in the production of soybean has become a standard practice worldwide, in
96
order to produce the most high quality soybeans possible on a given plot of land. In
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addition, excessive phosphorus-based fertilization damages the soil, water, and
98
organisms, leading to poorer yields in the future. Thus, the aim of this study, was to
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compare the conventional (BRS Sambaíba) and GM (Msoy 9144 RR) varieties of
100
soybean grown in two regions, with 0, 60, 120, or 240 kg/ha P2O5 fertilizer on the
101
chemical composition, contents of total phenols, phytic acid, individual isoflavones,
102
total carotenoids, individual tocopherols, as well as antioxidant activity, oil quality, and
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fatty acid profile.
104 105
2. Materials and methods
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2.1. Reagents
107
Folin-Ciocalteu reagent, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)
108
(ABTS), 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), fatty acids
109
(palmitic, estearic, oleic, linoleic, and linolenic), were all obtained from Sigma-Aldrich
110
Co., USA. The reagents used in the spectrophotometric analysis and chromatography
111
were HPLC grade and, for the remaining analyses, we used reagents analytical standard.
112 113
2.2. Characterization of soil and environment
114
This experiment was conducted in 2011–2012 in commercial areas referred to as
115
micoregion I (Pé de Galinha, 7°52′48″S, 46°00′00 W, 333 m altitude), and microregion
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II (Serra do Penitente, 8°40′29″S, 46°00′34″W, and 524 m altitude), both in the state of
117
Maranhão, Brazil.
118
For each micro-region, the experiment was conducted in a randomized complete
119
block design arranged in a factorial scheme with four replications. Factor A
120
corresponded to the genotype (BRS Sambaíba and Msoy 9144 RR), and factor B
121
corresponded to phosphorus doses (0, 60, 120, or 240 kg/ha P2O5) in the planting furrow
122
in the form of triple superphosphate.
123
Medium cycle genotypes (135 days) with similar yields were used. Each
124
experimental unit comprised seven rows of 7.0 m length, spaced 0.5 m apart, with 11
125
seeds per meter. During the development of the soybean crop, technical management
126
strategies used in the region were adopted.
127
The crop was harvested manually when 95% of the pods had the typical color of a
128
ripe pod. The usable area of each plot was 15 m2 after excluding 0.5 m from the ends of
129
each plot. After collection, immature and damaged crop plants were excluded, and the
130
remainder, useable plants were stored in an Ultrafreezer (-18 °C) until analysis.
131 132
2.3. Analysis
133
2.3.1. Chemical composition
134
The moisture content was determined using a drying oven set at 105 ± 3 °C with
135
natural air circulation for 24 h, according to the method described in American Society
136
of Agricultural Engineers.15 The lipid content was determined by extracting with
137
petroleum ether for over 8 h using a Soxhlet apparatus according to method 30-20 of the
138
Approved Methods of the American Association of Cereal Chemists16. Nitrogen was
139
determined by the Kjeldahl method and the protein content was obtained using the
140
conversion factor 6.25 according to method 46-1316. The ash content was determined by
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heating in a muffle furnace at 600 °C for 6 h according to method 08-0116. Crude fiber
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determination by acid digestion was performed as described by Angelucci et al.17 The
143
carbohydrate content was determined by difference. The results were expressed as %
144
(dry weight basis).
145 146
2.3.2. Total phenolic compounds and antioxidant activity by ABTS radical
147
Phenolics were extracted according to the method described by Dueñas et al.18
148
with some modifications. Soybean flour (2.0 g) was extracted twice with 80% methanol
149
at a ratio of 1:20 (w/v). For each extraction, the mixture was kept on a mechanical
150
shaker (Certomat Biotech International) for 1 h at 150 x g at 25°C. After centrifuging
151
(Eppendorf 5430-R) at 7600 rpm for 15 min, the supernatants obtained from each
152
extraction were combined and concentrated to dryness using a rotary evaporator
153
(Heidolph, Laborota Model 4000, Kelheim, Baviera, Germany) at 35 °C. The dried
154
methanol extract was dissolved in 25 mL of 50% methanol and used as a crude extract
155
for total free phenolic.
156
The total amount of phenolic compounds in the crude extract were quantified
157
according to the Folin-Ciocalteau method described by Zieliński and Kozłowska,19 with
158
some modifications. Extracts (100 µL) were added to 400 µL of distilled water.
159
Thereafter, 250 µL of Folin-Ciocalteau reagent (1 M) were added. After 8 min of
160
stabilization, 1250 µL of a 7% sodium carbonate solution (w/v) was added. After
161
reacting for 120 min, the absorbance of the mixture was measured at 725 nm (Jenway
162
Spectrophotometer, 6705 UV/Vis, Stone, Staffordshire, U.K.). Gallic acid was used as a
163
calibration standard. The results were expressed as mg of gallic acid equivalents
164
(GAE)/100 g of soybean (dry weight basis).
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The antioxidant activity was measured using the method described by Re et al.20
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An aliquot of 10 mL of stock solution ABTS radical (7 mM) and 175 µL of stock
167
solution potassium persulfate (140 mM) were mixed and placed in the dark for 16
168
hours. Before the analysis, the ABTS•+ solution (2.45 mM) was diluted with ethanol:
169
water solution (45:55, V/V) until an absorbance of 0.700 ± 0.02 at 734 nm. For color
170
reaction, in Falcon tubes were added 100 µL of the phenolic extract and 3900 µL of
171
ABTS•+ solution (0.700 ± 0.02). This mixture was allowed to stand at room
172
temperature for 6 min, and the absorbance was immediately recorded at 734 nm using a
173
UV spectrophotometer. The ABTS free radical scavenging activities in the crude
174
methanol extracts were expressed as µmol of trolox equivalent (TE)/100 g of soybean
175
(dry weight basis).
176 177
2.3.3. Phytic acid
178
Phytic acid content was determined using the method described by Haug and
179
Lantzsch.21 Soybean flour (0.01 g) was extracted with 1500 µL of 0.2 M HCl for 30 min
180
at 25 °C and centrifuged at 17,200 × g for 15 min (Eppendorf Centrifuge 5430R).
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Supernatant (500 µL) was added to a test tube, and 1000 µL of ferric solution (0.1g of
182
FeCl3 in 1000 mL of H20) was added. The test tube was then covered with a stopper and
183
incubated in a boiling water bath (100 °C) for 30 min. After cooling to room
184
temperature, and centrifugation at 17,200 × g for 15 min. In 500 µL of supernatant were
185
added to 750 µL of 2′,2-bipyridine solution (1% v/v). The absorbance was immediately
186
measured at 519 nm. The results were expressed as mg of phytic acid equivalent/100 g
187
(dry weight basis).
188 189
2.3.4. Isoflavones
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The samples were extracted in triplicate with 80% aqueous methanol (20:1 v/w)
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under agitation for 2 h at 4 °C, according to the method described by Genovese and
192
Lajolo.22 The homogenates were filtered through Whatman No. 06 filter paper and
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concentrated until methanol elimination on a rotatory evaporator (Rotavapor RE 120,
194
Büchi, Flawil, Sweden) at ≤40 °C. The volume of the extracts was adjusted to 5 mL
195
with HPLC grade methanol, and aliquots were filtered through a 0.22 µm PTFE filter
196
unit (polytetrafluoroethylene, Millipore Ltd., Bedford, MA, USA) for HPLC injection.
197
Isoflavone separation and quantitation was performed with a C18 Synergy 4 µm Fusion
198
RP (25 cm × 4.6 mm id) column (Phenomenex, Torrance, CA, USA) and a Hewlett
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Packard 1100 system equipped with autosampler, diode array detector, and
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ChemStation software (Agilent Technologies, Palo Alto, CA, USA). Elution solvents
201
were as follows: A, water: acetonitrile: acetic acid (95:5:0.1) and B, acetonitrile: acetic
202
acid (99.9:0.1). The solvent gradient was the same used by Genovese and Lajolo,22 at a
203
flow rate of 1 mL/min. Eluates were monitored at 255 and 320 nm and samples were
204
injected in duplicate. Identification was made based on the spectra and retention time in
205
comparison to known standards, and quantification was based on external calibration.
206
The 12 isoflavone standards were from LC Laboratories (Woburn, MA, USA).
207
Calibration was performed by injecting the standards three times at five different
208
concentrations (R2 ≥ 0.999). The results were expressed as mg of individual
209
isoflavones/100 g (dry weight basis).
210 211
2.4. Oil analysis
212
2.4.1. Oil extraction
213
The oil was extracted from soybean flour with petroleum ether for over 8 h using
214
a Soxhlet apparatus. The solvent was removed using a rotary evaporator (Heidolph,
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Laborota Model 4000, Kelheim, Baviera, Germany) under a vacuum at 35 °C. The oil
216
was collected in vials and stored at −18 °C.
217 218
2.4.2. Quality of oil
219
The lipid acidity was determined according to American Oil Chemists' Society
220
(AOCS) method Ca 5a-40,23 and expressed as a percentage of oleic acid. The peroxide
221
content was determined according to AOCS method Cd 8-53,23 and expressed as
222
milliequivalents of active oxygen/Kg of oil. The iodine index was determined using the
223
AOCS method Cd 1-25,23 and expressed as % of absorbed iodine.
224 225
2.4.3. Total carotenoids
226
The total carotenoid content was determined according to the methodology
227
described by Rodriguez-Amaya.24 Briefly, 2.5 g of oil, previously filtered, was added to
228
a 10 mL volumetric flask. The volume was completed with a 3:1 (v/v) isooctane:
229
ethanol mixture. The volumetric flasks were covered with aluminum foil to avoid the
230
degradation of carotenoids due to light exposure, and the absorbance was immediately
231
read at 450 nm. The results were expressed as mg of β-carotene/100 g of oil.
232 233
2.4.4. Fatty acids profile
234
A gas chromatograph (GC-14B, Shimadzu, Kyoto, Japan) with a flame
235
ionization detector (FID) and a fused silica capillary column measuring 30 m × 0.25
236
mm × 0.25 µm DB-225 (50% cyanopropyl methyl and 50% methyl phenyl silicone,
237
J&W Scientific, Folsom, CA, USA) was used. The injector and detector were both
238
maintained at 250 °C. Nitrogen, at a rate of 1.0 mL/min, was used as the carrier gas.
239
The fatty acids were derivatized according to methodology proposed by Hartman and
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Lago.27 A 100 mg sample of oil was weighed in a test tube, added to 500 µL of 100
241
µmol/L of KOH in methanol; and the samples left in a water bath (Quimes, Dubnoff,
242
England) at 60 °C for 90 min. After reaching room temperature, 1500 µL of 1000
243
µmol/L of H2SO4 was added and again the samples were placed in the water bath for 90
244
min. After cooling, 2000 µL of n-hexane were added and the samples agitated by vortex
245
for 30 s. The hexane phase was partially transferred to a 1500 µL flask, from which 1.5
246
µL was taken and injected into the gas chromatograph with a 1:50 split ratio. The initial
247
column temperature of 100 °C was maintained for 0.5 min and then increased to 150 °C
248
at a rate of 8 °C/min. After 0.5 min at 150 °C, the temperature was increased to 180 °C
249
at a rate of 1.5 °C/min. The column was held at 180 °C for 5 min and was increased to a
250
final temperature of 220 °C at a rate of 2 °C/min. The temperature was maintained for 6
251
min, for a total analysis time of 58 min. The identification of free fatty acids was
252
performed according to the retention time of the chromatographic patterns (myristic,
253
palmitic, oleic, linoleic, linolenic acids, all obtained from Sigma-Aldrich Co., USA).
254
The Class-GC10 software (Shimadzu, Kyoto, Japan) was used to acquire and process
255
the GC data.
256 257
2.4.5. α, δ and γ-tocoferols
258
The determination of the levels of alpha (α), gamma (γ) and delta (δ) tocopherols
259
was adapted from the second methodology of Pestana et al.25 and described by Ziegler et
260
al.26 The results were expressed in mg of tocopherol (α, γ, or δ)/100 g of oil.
261 262
2.5. Multivariate analyses
263
The results were expressed as means and standard errors. Comparison of the
264
treatment factors was performed using a multivariate analysis such as the principal
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265
component analysis (PCA). For the PCA, the variables were extracted from a
266
correlation matrix. Thus, the information contained in the original variables was
267
included in a smaller number of underlying variables called principal components
268
(PCs). The criteria for exclusion of a variable was recommended by Jolliffe28 and PCs
269
were selected, which included 70–90% of the total variance. After selecting the number
270
of PCs, the respective eigenvalues were obtained using corresponding eigenvectors. The
271
results were plotted graphically using a Biplot model from the scores and loads of the
272
selected main components.
273 274
3. Results
275
Table 1 shows the chemical and physical characteristics of the soil. Figure 1
276
shows the climate data (minimum temperature, maximum temperature, and
277
precipitation) obtained from the meteorological station of the National Institute of
278
Meteorology (Inmet, 2012). The results obtained in the analysis generated 29 dependent
279
variables (Table 1 and Table 2). To reduce this number of descriptive factors, the
280
dependent variables were submitted to Principal Component Analysis (PCA), but the
281
variability was maintained. To explain the distribution of the groups, a smaller number
282
of PCs was required. These were based on the amount of high and/or medium
283
correlations between the dependent variables and depended on the population being
284
studied.27 Therefore, according to the rule established by Jolliffe,28 only the first two
285
PCs were used for analysis by envisaging 70% variation.
286
In microregion I, the first two PCs explained 71.5% of the total variation. PC1 and
287
PC2 were responsible for 45.5% and 26%, respectively, which allowed the plot of the
288
scores and the factorial charge of the components for each cultivar and different doses
289
of phosphorus (Figure 2A). The formation of different groups showed the differences
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between conventional soy and GM soy. The eigenvectors corresponding to PC1 were
291
the result of the loading of the original variables in this component and represented a
292
measure of the relative importance of each variable. In PC1, β-glucosides (-0.20), C16:0
293
(0.28), C18:1 (-0.27), C18:2 (0.27), C18:3 (0.25), saturated fatty acids (0.27),
294
unsaturated fatty acids (-0.27), δ-tocopherol (-0.23), and α-tocopherol (0.25) contributed
295
to this differentiation. In PC2, malonylglucosides (-0.23), daidzein (0.32), glycitein (-
296
0.30), genistein (-0.25), ABTS (0.28), and lipid acidity (0.29) also contributed to this
297
differentiation. The highest levels of β-glucosides, malonylglucosides, glycitein, and
298
genistein were observed in conventional soy, whereas daidzein, ABTS, and lipid acidity
299
were observed at highest levels in GM soy (Table 2 and Figure 2A).
300
In the analysis of conventional soybean grown in microregion I, the separation was
301
observed at doses 0 and 120 kg/ha P2O5, forming a group, and the variables responsible
302
for differentiation were acetylglucosides, aglycones, glycitein, genistein, and C16:0.
303
The doses of 60 and 240 kg/ha P2O5 formed a second group, and the total carotenoids,
304
δ-tocopherol, and C18:0 were responsible for the separation. For GM soy, the variables
305
responsible for differentiating the dose 0 kg/ha P2O5 from others were β-glucosides,
306
malonylglucosides, daidzein, genistein, and iodine. All of these variables showed the
307
lowest levels at this dose, and consequently, lower antioxidant activity. In the 0 kg/ha
308
P2O5 dose, the high fiber content was the main differentiating factor, whereas in the 120
309
kg/ha P2O5 dose, the highest protein content among the soybean varieties was the
310
differentiating factor. The doses of 60 and 240 kg/ha of P2O5 presented higher
311
concentrations of carbohydrate, δ-tocopherol, and antioxidant capacity when compared
312
to other doses (Table 2 and Figure 2A).
313
The first two principal components in microregion II accounted for 71.7% of the
314
total variation, wherein PC1 and PC2 were responsible for 46% and 25.7%, respectively
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315
(Figure 2B). The cultivars showed the same trends observed in microregion I, in that
316
there was differentiation between the conventional soybean and GM soybean, based on
317
the formation of different groups as seen in Figure 2B. Eigenvectors corresponding to
318
PC1 were total phenols (0.29), C18:1 (0.29), saturated fatty acids (-0.29), and
319
unsaturated fatty acids (0:29); vectors corresponding to PC2 were phytic acid (-0.28), β-
320
glucosides (0.25), malonylglucosides (0.31), daidzein (-0.34), glycitein (0.37), genistein
321
(0.31), and γ-tocopherol (0.33), all of which contributed to the differentiation of
322
cultivars. Conventional soybean was characterized by a high content of β-glucoside,
323
malonylglucoside, glycitein, genistein, and γ-tocopherol. However, a higher content of
324
both phytic acid and daidzein was found in GM soy (Table 3 and Figure 2B).
325
There was no group formation related to the dose of phosphorus for either cultivar.
326
For conventional soy, the greatest levels of total phenols, genistein, and peroxide were
327
observed at a dose of 120 kg/ha. At a dose of 240 kg/ha P2O5, the highest levels of total
328
carotenoids, malonylglucosides, and δ-tocopherol were observed. In contrast, for GM
329
soy, a 120 kg/ha P2O5 dose led to the highest levels of total phenols, phytic acid,
330
malonylglucosides, and C18:1, and the 240 kg/ha P2O5 dose led to the highest levels of
331
β-glucosides, daidzein, genistein, C18:0, C18:2, and C18:3 (Table 3 and Figure 2B).
332
Analysis of the two microregions showed differences in soybean composition
333
(Figure 2C). The two first PCs accounted for 75% of the total variance, wherein PC1
334
and PC2 were responsible for 47% and 28%, respectively. The variables that determined
335
the separation of the groups with their eigenvectors were lipids (-0.23), total carotenoids
336
(-0.21), phytic acid (0.24), lipid acidity (0.24), and γ-tocopherol (-0.26) for PC1, and
337
protein (0.34), carbohydrate (-0.33), peroxide index (0.21), and δ-tocopherol (-0.35) for
338
PC2. The highest protein, lipid, carotenoid, peroxide index, and γ-tocopherol levels
339
were observed in microregion II (Tables 2 and 3, and Figure 2C), whereas microregion I
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340
had higher levels of phytic acid and δ-tocopherol. This study showed that a dose of 240
341
kg/ha P2O5 could possibly increase the δ-tocopherol content in a GM soybean, resulting
342
in an increased antioxidant capacity.
343 344
4. Discussion
345
Similar variation in the isoflavone content of soybeans is found in the literature,
346
mainly influenced by the genotype and environment.30–33 Xu and Chang30 studied 30
347
soybean genotypes grown in North Dakota and Minnesota and reported variations in
348
total isoflavone content ranging between 118 and 286 mg/100 g soybean, of which 69%
349
were attributable to genistein and its conjugated forms. Chung et al.31 studied 9
350
American soy genotypes in Virginia, and reported total isoflavone content ranging from
351
250 to 320 mg/100 g soybean, with 75–84% comrpising malonyl genistin. The analysis
352
of individual isoflavones in soy from Korea and China showed that the Chinese
353
genotypes have higher concentrations of isoflavones than Korean genotypes.32 Brazilian
354
genotypes showed significantly lower concentrations of isoflavones than both American
355
and Korean genotypes, according to a study by Genovese et al.33 These researchers
356
reported levels of total isoflavone content between 57 and 188 mg/100 g soybean
357
among 13 Brazilian genotypes. The variation in isoflavone values in cultivars among
358
the various studies reflects not only genetic traits and growing conditions but also
359
variations in methods for determination of isoflavones.4
360
The proportions of metabolites, particularly isoflavones, differ even within a
361
single genotype when grown in different locations (microregions). Compared to
362
microregion I, microregion II had a higher altitude and higher rainfall between February
363
and April, with the maximum temperatures recorded during the experimental period and
364
lower minimum temperatures in February (Figure 1). The year of cultivation has greater
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365
influence on the isoflavone content than the location or genotype.34 Low temperatures
366
during the pod filling stage favors the accumulation of isoflavones.30,35 García-Villalba
367
et al.6 evaluated the metabolic profile consisting of 40 compounds of conventional and
368
transgenic soybean under the same conditions; they demonstrated that among the
369
compounds evaluated, they observed only the absence of 4-hydroxy-L-threonine in
370
transgenic soybean compared to conventional soybean. This result is in agreement with
371
that obtained in the present study, where the same compounds were identified in both
372
genotypes and sites, but with varying concentrations.
373
The composition of the lipid fraction in the present study is consistent with that
374
found by other authors. Ziegler et al.,26 reported a variation of 22.9 to 23.7 mg of β-
375
carotene/100 g of oil, 36.1 to 37.3 mg δ-tocopherol/100 g of oil, and 65.1 to 68.0 mg γ-
376
tocopherol/100 g in freshly harvested soybean. Yang et al.36 reported a fatty acid profile
377
of 10.5% of C16:0, 24.4% of C18:1, 53.1% of C18:2, and 7.5% of C18:3. In both
378
regions the saturated fatty acids, unsaturated fatty acids, and γ-tocopherol were
379
responsible for the separation of regions and genotypes. These results are consistent
380
with those reported in the existing literature, emphasizing that the composition of
381
saturated and unsaturated fatty acids varies for both conventional and transgenic
382
cultivars in different locations.37, 38 Increasing the amount of vitamin E (tocopherol) in
383
economically important oilseed crops such as soybean has been the focus of numerous
384
studies, leading to the higher nutritional value of these crops. GM soybeans
385
overexpressed the γ gene-tocopherol methyltransferase, which resulted in a 41-fold
386
increase in α-tocopherol content compared to the wild-type soybean.39 A positive
387
correlation between tocopherols and polyunsaturated fatty acids was observed by Rani
388
et al.40; they reported that the same allele responsible for the conversion of oleic acid
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389
(C18: 1) to linoleic acid (C18: 2) governed the expression of the omega-6 desaturase
390
gene, influencing tocopherol levels, especially γ-tocopherol.
391
The main variables responsible for the differentiation between genotype and place
392
cultivation identified in our experiment were isoflavones, fatty acid profiles, and
393
tocopherols. Our results were similar to those of Tsukamoto et al.41, in who evaluated
394
four soybean genotypes grown at different planting times and at two locations. They
395
reported similar behavior in the acid profile of fatty acids and isoflavones depending on
396
the location and date of planting. The reduction in isoflavones, linoleic and linolenic
397
acid content with the increase in oleic acid content has been reported at high
398
temperatures during the seed filling stage, but little interference has been reported as a
399
function of fertilization.
400
Some of the effects related to the place of cultivation in our experiment reflect the
401
temperature differences that occurred during seed development depending on the
402
planting date. This was observed in the present study, with the largest temperature
403
variation noted in February in the microregion II, a period in which the seed was
404
developing (Figure 1). It is known that grain crops grown at lower minimum
405
temperatures have a higher concentration of lipids and proteins.40,42 This was seen in the
406
seeds grown in microregion II (Figure 1), which showed the highest percentages of
407
these variables (Table 3).
408
In conclusion, the multivariate analysis facilitated reduction in the number of
409
variables with respect to genotype (BRS conventional Sambaíba and GM Msoy 9144
410
RR), dose of P2O5 fertilizer, and place of cultivation (microregion I and II). Isoflavones
411
showed great variation in all treatments. In both locations, the conventional soybean
412
showed a higher content of β-glycosides, malonylglycosides, glycitein, and genistein
413
than did GM soy, which was characterized by relatively high daidzein content. Higher
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414
levels of β-isoflavone, glycosides, malonylglycosides, glycitein and genistein were
415
mainly responsible for the differentiation of the doses 60, 120 and 240 kg/ha of P2O5
416
and of the soybean cultivated without fertilizers, which was also influenced by the
417
location. PCA showed no differences between P doses but showed differences between
418
the genotype and place of cultivation. This indicates that the increase in the dose of
419
fertilization does not improve the nutritional quality, oil quality, or metabolites,
420
suggesting that the rationalization of the inputs is an important tool for agriculture. The
421
difference in altitude between microregions directly influenced the higher protein, lipid,
422
total carotenoid, and γ-tocopherol concentrations found in microregion II, and the
423
higher phytic acid and δ-tocopherol content in microregion I.
424
The results of the present study are valuable for crop management, highlighting that
425
a) knowledge on the average climatic conditions of the place of cultivation facilitates
426
synchronization of the pod filling stage with the desired temperature, aiming for
427
improvement in quality; b) aids selection of the most suitable genotype for cultivation in
428
the region; and c) the increase in the dose of phosphate fertilization does not result in an
429
increase in soybean quality. Future studies are needed to elucidate the origin of the main
430
metabolic changes in this experiment resulting from variation in cultivars and fertilizers,
431
with an emphasis on the change in altitude of the place of cultivation.
432 433
Acknowledgments
434
We would like to thank CAPES (Coordenação de Aperfeiçoamento de Pessoal
435
de Nível Superior), CNPq (Conselho Nacional de Desenvolvimento Científico e
436
Tecnológico), FAPERGS (Fundação de Amparo à Pesquisa do Estado do Rio Grande
437
do Sul), SCT-RS (Secretaria da Ciência e Tecnologia do Estado do Rio Grande do Sul)
438
and Polo de Inovação Tecnológica em Alimentos da Região Sul.
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439
Supporting Information Available
440 441
Figure S1. Typical chromatogram of isoflavones by HPLC monitored at 255 nm. β-
442
glucosides (A, B, and E), malonylglucosides (C, D, and F), daidzein (G), glycitein (H),
443
and genistein (I).
444
Figure S2. Typical chromatogram of fatty acids profile by GC-FID. Palmitic (A),
445
estearic (B), oleic (C), linoleic (D), and linolenic (E).
446
Figure S3. Typical chromatogram of tocopherols by HPLC monitored at 290 nm. (A) δ-
447
tocopherol, (B) γ- tocopherol, and (C) α- tocopherol.
448 449
References
450
1. FAO. Food and Agriculture Organization of the United Nations. Food and
451
agricultural commodities production. Country rank in the world, by commodity.
452
Available from: http://faostat3.fao.org/browse/Q/QC/E. Accessed Jun 27, 2016.
453
2. Slavin, M.; Cheng, Z.; Luther, M.; Kenworthy, W.; Yu, L. Antioxidant properties and
454
phenolic, isoflavone, tocopherol and carotenoid composition of Maryland-grown
455
soybean lines with altered fatty acid profiles. Food Chem. 2009, 114, 20–27.
456
3. Kumar, V.; Rani, A.; Tindwani, C.; Jain, M. Lipoxygenase isozymes and trypsin
457
inhibitor activities in soybean as influenced by growing location. Food Chem. 2003,
458
81, 79–83.
459
4. Balisteiro, D. M.; Rombaldi, C. V.; Genovese, M. I. Protein, isoflavones, trypsin
460
inhibitory and in vitro antioxidant capacities: Comparison among conventionally and
461
organically grown soybeans. Food Res. Int. 2013, 51, 8–14.
462
5. Bøhn, T.; Cuhra, M.; Traavik, T.; Sanden, M.; Fagan, J.; Primicerio, D.
463
Compositional differences in soybeans on the market: Glyphosate accumulates in
19 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
464
Page 20 of 32
Roundup Ready GM soybeans. Food Chem. 2014, 153, 207–215.
465
6. García-Villalba, R.; León, C.; Dinelli, G.; Segura-Carretero, A.; Fernández-Gutiérrez,
466
A.; Garcia-Cañas, V.; Cifuentes, A. Comparative metabolomic study of transgenic
467
versus conventional soybean using capillary electrophoresis–time-of-flight mass
468
spectrometry. J. Chromatogr. A. 2008, 1195, 164–173.
469
7. Zobiole, L. H. S.; Bonini, E. A.; de Oliveira, R. S.; Kremer, R. J.; Ferrarese, O.
470
Glyphosate affects lignin content and amino acid production in glyphosate resistant
471
soybean. Acta Physiol. Plant. 2010, 32, 831–837.
472
8. Liang, H.-Z.; Wang, S.-F.; Wang, T.-F.; Zhang, H.-Y.; Zhao, S.-J.; Zhang, M.-C.
473
Genetic analysis of embryo, cytoplasm and maternal effects and their environment
474
interactions for isoflavone content in soybean [Glycine max (L.) Merr.]. Agr. Sci.
475
China. 2007, 6, 1051–1059.
476
9. Zhang, X.; Gao, B.; Shi, H.; Slavin, M.; Huang, H.; Whent, M.; Sheng, Y.; Yu, L.
477
Chemical composition of 13 commercial soybean samples and their antioxidant and
478
anti-inflammatory properties. J. Agric. Food Chem. 2012, 60, 10027–10034.
479
10. Kumar, V.; Rani, A.; Solanki, S.; Hussain, S. M. Influence of growing environment
480
on the biochemical composition and physical characteristics of soybean seed. J. Food
481
Compos. Anal. 2006, 19, 188–195.
482
11. John, K. M. M.; Khan, F.; Luthria, D.; Garrett, W.; Natarajan, S. Proteomic analysis
483
of anti-nutritional factors (ANF’s) in soybean seeds as affected by environmental and
484
genetic factors. Food Chem. 2017, 218, 321–329.
485
12. Kim, E.-H.; Lee, O.-K.; Kim, J. K.; Kim, S.-L.; Lee, J.; Kim, S.-H.; Chung, I.-M.
486
Isoflavones and anthocyanins analysis in soybean (Glycine max (L.) Merill) from
487
three different planting locations in Korea. Field Crop. Res. 2014, 156, 76–83.
488
13. Taiz, L.; Zeiger, E. Fisiologia Vegetal. 5 ed. Artmed: Porto Alegre, Brazil, 2013,
20 ACS Paragon Plus Environment
Page 21 of 32
Journal of Agricultural and Food Chemistry
489
918 pp.
490
14. Yin, X.; Bellaloui, N.; McClure, A. M.; Tyler, D. D.; Mengistu, A. Phosphorus
491
fertilization differentially influences fatty acids, protein, and oil in soybean. Am. J.
492
Plant. Sci. 2016, 7, 1975–1992.
493
15. ASAE. Moisture measurement-unground grain and seeds. In Standards Engineering
494
Practices Data, Saint Joseph, Michigan, USA: American Society of Agricultural
495
Engineers, 2000.
496
16. AACC. General methods. In Approved Methods of the American Association of
497
Cereal Chemists’, Method 30-20, method 46-13, and method 08-01. Saint Paul,
498
Minessota, USA: American Association of Cereal Chemists, 1995.
499
17. Angelucci, E.; Carvalho, C. R. L.; Carvalho, P. R. N.; Figueiredo, I. B.; Mantovani,
500
D. M. B.; Moraes, R. M. Manual técnico de análises de alimentos. Campinas: São
501
Paulo, Brasil: Instituto de Tecnologia de Alimentos, 1987, 52–53 pp.
502
18. Dueñas, M.; Hernández, T.; Lamparski, G.; Estrella, I.; Muñoz, R. Bioactive
503
phenolic compounds of soybean (Glycine max cv. Merril): modifications by different
504
microbiological fermentations. Pol. J. Food Nutr. Sci. 2012, 62, 241–250.
505
19. Zieliński, H.; Kozłowska, H. Antioxidant activity and total phenolics in selected
506
cereal grains and their different morphological fractions. J. Agric. Food Chem. 2000
507
48, 2008–2016.
508
20. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Riceevans, C.
509
Antioxidant activity applying an improved ABTS radical cation decolorization assay.
510
Free Radic. Biol. Med. 1999, 26, 1231–1237.
511 512 513
21. Haug, W.; Lantzsch, H. J. Sensitive method for the rapid determination of phytate in cereals and products. J. Sci. Food Agr. 1983, 34, 1423–1426. 22. Genovese, M. I.; Lajolo, F. M. Determination of isoflavones in soy product. Ciênc.
21 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
514
Page 22 of 32
Tecnol. Aliment. 2001, 21, 86–93.
515
23. AOCS. Official and tentative methods of the American Oil Chemists' Society:
516
including additions and revisions (6th ed), method Ca 5a-40, method Cd 8-53, and
517
method Cd 1-25. Champaign, IL. American Oil Chemists' Society. 2009.
518 519
24. Rodriguez-Amaya D. B. A guide to carotenoid analysis in foods. Washington, DC: ILSI Press, 1999.
520
25. Pestana, V. R.; Zambiazi, R. C.; Mendonça, C. R.; Bruscatto, M. H.; Lerma-Garcia,
521
M. J.; Ramis-Ramos, G. Quality changes and tocopherols andγ-orizanol
522
concentrations in rice bran oil during the refining process. J. Am. Oil Chem. Soc.
523
2008, 85, 1013–1019.
524
26. Ziegler, V.; Vanier, N. L.; Ferreira, C. D.; Paraginski, R. T.; Monks, J. L. F.; Elias,
525
M. C. Changes in the bioactive compounds content of soybean as a function of grain
526
moisture content and temperature during long-term storage. J. Food Sci. 2016, 81,
527
762–768.
528 529 530 531 532 533
27. Hartman, L.; Lago, R. C. A. Rapid preparation of fatty acid methyl esters from lipids. Lab. Pract. 1973, 22, 475–476. 28. Jolliffe, I.T. Principal Component Analysis, (2th ed.). New York: Springer-Verlag, 2002. 29. Manly, B. F. J. Multivariate statistical methods: a primer. London: Chapman and Hall, 2004.
534
30. Xu, B.; Chang, S. K. Characterization of phenolic substances and antioxidant
535
properties of food soybeans grown in the North Dakota-Minnesota Region. J. Agric.
536
Food Chem. 2008, 56, 9102–9113.
22 ACS Paragon Plus Environment
Page 23 of 32
Journal of Agricultural and Food Chemistry
537
31. Chung, H.; Hogan, S.; Zhang, L.; Rainey, K.; Zhou, K. Characterisation and
538
comparison of antioxidant properties and bioactive components of Virginia
539
Soybeans. J. Agric. Food Chem. 2008, 56, 11515–11519.
540
32. Lee, Y. W.; Kim, J. D.; Zheng, J.; Row, K. H. Comparisons of isoflavones from
541
Korean and Chinese soybean and processed products. Biochem. Eng. J. 2007, 36,
542
49–53.
543
33. Genovese, M. I.; Hassimotto, N. M. A.; Lajolo, F. M. Isoflavone profile and
544
antioxidant activity in Brazilian soybean varieties. Food Sci. Tech. Int. 2005, 11,
545
205–211.
546
34. Lee, S. J.; Ahn, J. K.; Kim, S. H.; Kim, J. T.; Han, S. J.; Jung. M. Y.; Chung, I. M.
547
Variation in isoflavones of soybean cultivars with location and storage duration. J.
548
Agric. Food Chem. 2003, 51, 3382-3389.
549
35. Kim, E.-H.; Lee, O.-K.; Kim, J. K.; Kim, S.-L; Lee, J.; Kim, S.-H. Chung, I.-M.
550
Isoflavones and anthocyanins analysis in soybean (Glycine max (L.) Merill) from
551
three different planting locations in Korea. Field Crop. Res. 2014, 156, 76–83.
552 553
36. Yang, T.-S., Chu, Y.-H., & Liu, T.-T. Effects of storage conditions on oxidative stability of soybean oil. J. Sci. Food Agric. 2005, 85, 1587–1595.
554
37. Milinski, M. C.; Visentainer, J. V.; Martin, C. A.; Arias, C. A. A.; Matsushita, M.;
555
Souza, N. E. Proximate composition and fatty acids profile of Brazilian conventional
556
and transgenic soybeans (Glycine max (L.) Merrill) cultivars. Electron. J. Environ.
557
Agric. Food Chem. 2007, 6, 1905–1911.
558
38. Galão, O. F.; Carrão-Panizzi, M. C.; Mandarino, J. M. G.; Santos Júnior, O. O.;
559
Maruyama, S. A.; Figueiredo, L. C.; Bonafe, E. G., Visentainer, J. V. Differences of
560
fatty acid composition in Brazilian genetic and conventional soybeans (Glycine max
561
(L.) Merrill) grown in different regions. Food Res. Int. 2014, 62, 589–594.
23 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 24 of 32
562
39. Lee, K.; Yi, B.-Y.; Kim, K.-H.; Kim, J.-B.; Suh, S.-C.; Woo, H.-J.; Shin, K.-S.;
563
Kweon, S. –J. Development of efficient transformation protocol for soybean (Glycine
564
max L.) and characterization of transgene expression after Agrobacterium-mediated
565
gene transfer. J. Korean Soc. Agric. Biotechnol. Chem. 2011, 54, 37–45.
566
40. Rani, A.; Kumar, V.; Verma, S. K.; Shakya, A. K.; Chauhan, G. S. Tocopherol
567
content and profile of soybean: Genotypic variability and correlation studies. J.
568
Amer. Oil Chem. Soc. 2007, 84, 377–383.
569
41. Tsukamoto, C.; Shimada, S.; Igita, K.; Kudou, S.; Kokubun, M.; Okubo, K.;
570
Kitamura, K. Factors affecting isoflavone content in soybean seeds: Changes in
571
isoflavones, saponins, and composition of fatty acids at different temperatures during
572
seed development. J. Agric. Food Chem. 1995, 43, 1184–1192.
573
42. Li, Q.; Hu, Y.; Chen, F.; Wang, J.; Liu, Z.; Zhao, Z. Environmental controls on
574
cultivated soybean phenotypic traits across China. Agric. Ecosyst. Environ. 2014,
575
192, 12–18.
576 577 578 579 580 581 582 583 584 585 586 587 588 589
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590 591 592 593 594 595 596 597 598 599 600 601
Table 1. Physical and chemical parameters of soil from microregion I and microregion
602
II before the experiment. Microregion I
Microregion II
39 6 55
49 9 42
1.68 16.01 0.07 3.37 1.17 0.00 5,70 5.41
2.83 12.62 0.14 3.21 1.56 0.00 8,98 5.00
Physical parameters Clay (%) Silt (%) Sand (%) Chemical parameters Organic matter (%) P (mg/ dm3) K (mg/ dm3) Ca(cmolc/ dm3) Mg(cmolc/ dm3) Al (cmolc/ dm3) Cation exchange capacity (cmolc/ dm3) pH of soil 603
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Page 26 of 32
Table 2. Dependent variables evaluated in the soybean flour and oil in the conventional (BRS Sambaíba) and transgenic (Msoy 9144) cultivars with variation in phosphorus doses in microregion I. Microregion I Dependent variables
Msoy 9144 240 (a) 0(a) 60(a) 120(a) 240(a) Soybean flour 8.0±0.1 7.6±0.2 7.9±0.1 8.1±0.0 8.0±0.1 7.9±0.1 7.8±0.0 7.6±0.4 5.5±0.5 5.1±0.0 4.8±0.1 5.0±0.0 5.1±0.0 5.0±0.0 5.8±0.7 4.9±0.0 6.2±0.2 6.3±0.5 6.4±0.3 6.3±0.1 6.9±0.2 6.0±0.0 6.6±0.2 6.4±0.2 22.4±0.7 21.5±0.4 21.6±0.6 23.1±0.2 20.7±0.1 22.1±0.5 22.1±0.8 21.7±0.6 38.6±0.4 36.9±0.7 38.6±0.3 37.2±0.3 37.6±0.0 36.5±0.4 38.8±0.3 36.4±0.4 19.0±0.9 22.3±0.8 20.5±0.6 20.1±0.5 21.5±0.3 22.1±0.2 18.5±1.2 22.7±0.5 2697±124.5 2283±91.2 2350±69.9 2179±47.9 2510±83.1 2437±60.0 2574±61.7 2672±46.7 4466±13.9 4383±219.6 4662±42.0 4349±151.6 4525±426.8 4753±4.9 4779±116.5 4609±100.4 67.3±0.8 75.4±0.5 69.7±0.3 69.1±0.6 59.2±0.5 61.8±0.0 64.6±0.5 65.0±0.2 31.1±0.6 33.2±0.2 32.8±0.0 30.5±0.3 25.7±0.3 28.2±0.3 28.5±0.3 28.4±0.2 12.1±0.3 11.1±0.3 12.0±0.0 11.6±0.2 11.5±0.2 11.9±0.2 11.9±0.2 11.1±0.4 35.7±0.4 42.8±0.4 37.4±0.5 37.4±0.5 48.2±0.6 52.9±0.5 56.1±0.9 56.4±1.5 11.8±0.4 10.2±0.2 11.4±0.2 11.3±0.3 6.9±0.7 7.9±0.2 6.7±0.1 7.1±0.5 64.5±0.4 68.6±0.0 63.7±0.4 63.4±0.4 53.9±0.3 56.0±0.9 57.5±0.3 56.8±0.7 255.1±23.6 237.2±7.6 220.4±4.3 229.6±9.1 238.6±10.4 272.6±23.1 256.6±1.4 309.2±12.2 Soybean oil 0.8±0.0 0.8±0.0 0.9±0.0 0.8±0.0 1.0±0.0 1.09±0.0 1.0±0.0 1.0±0.1 0.3±0.0 0.3±0.0 0.3±0.0 0.3±0.0 0.3±0.0 0.35±0.0 0.3±0.0 0.3±0.0 8.6±0.6 9.4±0.0 9.2±0.3 9.5±0.1 9.4±0.1 9.55±0.0 9.7±0.0 9.4±0.0 12.0±0.4 10.3±0.1 11.2±0.1 10.3±0.1 13.5±1.3 10.51±0.0 12.3±1.5 10.6±0.0 2.8±0.1 3.4±0.0 2.6±0.1 3.4±0.0 3.3±0.3 3.42±0.0 3.3±0.2 3.4±0.0 20.0±3.4 25.7±0.8 24.3±0.8 26.4±0.5 9.9±8.6 26.70±0.0 18.2±8.5 26.8±0.7 58.7±2.4 54.2±0.6 55.5±0.7 53.7±0.3 65.4±6.1 53.44±0.0 59.5±6.2 53.0±0.6 0(a)
Moisture (b) Ash (b) Fiber (b) Lipid (b) Protein (b) Carbohydrate (b) Total phenolics (c) Phytic acid (d) β-glucosides (e) Malonylglucosides (e) Acetylglucosides (e) Daidzein (e) Glycitein (e) Genistein (e) ABTS (f) Lipid acidity (b) Peroxide index (g) Iodine index (b) Palmitic acid 16:0 (b) Estearic acid 18:0 (b) Oleic acid 18:1 (b) Linoleic acid 18:2 (b)
BRS Sambaiba 60(a) 120(a)
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Linolenic acid 18:3 (b) 6.4±0.4 6.2±0.1 6.2±0.0 6.0±0.0 7.2±0.6 5.94±0.0 6.6±0.6 5.9±0.0 Saturated fatty acid (b) 14.8±0.6 13.7±0.1 13.8±0.0 13.8±0.0 16.8±1.6 13.93±0.0 15.6±1.7 14.1±0.0 Unsaturated fatty acid (b) 85.1±0.6 86.2±0.1 86.1±0.0 86.2±0.0 82.7±1.8 86.07±0.0 84.3±1.7 85.9±0.0 Total carotenoids (h) 13.8±1.2 16.3±0.5 14.9±0.5 18.2±0.2 14.9±0.4 18.45±0.7 14.6±0.1 14.9±1.9 δ- tocopherol (i) 31.3±0.5 38.3±0.8 32.2±0.4 37.2±1.3 31.8±0.3 37.89±1.1 31.8±0.2 38.1±1.6 γ- tocopherol (i) 73.7±0.8 76.7±0.2 73.7±0.4 73.9±1.5 72.6±1.0 74.74±1.0 72.7±0.7 77.2±2.1 α- tocopherol (i) 0.5±0.2 0.0±0.0 1.6±0.0 0.0±0.0 2.0±0.3 0.00±0.0 2.1±0.1 0.0±0.0 (a) Kg/ha P2O5 (b) %; (c) mg of GAE/100 g; (d) mg/100 g; (e) mg/100 g; (f) mg of TE/100 g; (g) meq of O2/Kg; (h) mg of β-carotene/100 g of oil; (i) mg/100 g of oil
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Table 3. Dependent variables evaluated in the soybean flour and oil in the conventional (BRS Sambaíba) and transgenic (Msoy 9144) cultivars with variation in phosphorus doses in microregion II. Microregion II Dependent variables BRS Sambaiba Msoy 9144 (a) (a) (a) (a) (a) (a) 0 60 120 240 0 60 120(a) 240(a) Soybean flour (b) Moisture 8.0±0.0 7.7±0.8 8.2±0.1 7.7±0.1 7.8±0.1 8.1±0.2 8.0±0.1 7.9±0.2 Ash (b) 4.8±0.0 4.8±0.0 4.7±0.0 4.5±0.1 4.6±0.1 4.7±0.1 4.9±0.0 4.8±0.0 Fiber (b) 6.2±0.1 6.4±0.0 6.5±0.1 6.3±0.1 6.3±0.1 6.1±0.0 6.3±0.0 6.1±0.0 Lipid (b) 22.6±0.5 24.8±0.4 22.5±0.4 24.3±0.3 23.0±0.2 24.0±0.3 22.1±0.2 23.8±0.1 Protein (b) 38.6±0.1 38.4±0.6 38.9±0.1 38.2±0.1 39.8±0.0 38.2±0.0 38.0±0.5 38.9±0.0 Carbohydrate (b) 19.5±0.9 17.7±0.8 18.9±0.0 18.8±0.3 18.1±0.2 18.6±0.5 20.4±0.5 18.3±0.0 Total phenolics (c) 2413±60.3 2078±77.0 2464±77.3 2221±22.7 2467±49.2 2450±52.5 2580±123.8 2104±107.3 Phytic acid (d) 3125±355.9 3056±163.8 3445±185.0 3472±38.5 3802±16.9 3493±128.5 3965±186.6 3769±52.4 β-glucosides (e) 67.4±0.9 77.2±0.5 76.3±0.1 75.8±0.2 59.4±0.6 64.7±0.3 67.2±1.0 68.4±0.2 (e) Malonylglucosides 32.2±0.3 35.4±0.2 36.7±0.1 36.9±0.5 25.6±1.7 29.7±1.5 32.6±0.1 27.7±0.3 Acetylglucosides (e) 11.2±0.4 11.7±0.3 11.5±0.5 11.3±0.4 11.5±0.5 11.5±0.4 11.3±0.3 11.3±0.3 Daidzein (e) 37.7±0.6 45.1±0.2 44.3±0.6 43.0±0.4 48.0±0.9 54.2±0.1 57.1±0.5 58.0±0.2 Glycitein (e) 11.3±0.5 11.6±0.4 11.4±0.4 11.3±0.4 7.8±1.1 7.9±0.8 7.6±0.6 7.4±0.7 (e) Genistein 66.0±0.2 70.4±0.3 74.4±0.8 71.2±0.7 57.7±1.7 61.0±0.1 60.8±1.1 62.7±0.7 ABTS (f) 242.5±2.2 238.6±16.3 254.1±6.9 250.1±5.4 254.7±7.5 254.4±8.4 248.1±5.4 220.4±9.6 Soybean oil (b) Lipid acidity 0.7±0.0 0.6±0.0 0.6±0.0 0.6±0.0 0.6±0.0 0.7±0.0 0.6±0.0 0.4±0.2 Peroxide index (g) 0.2±0.1 0.6±0.0 0.8±0.0 0.6±0.0 0.7±0.0 0.5±0.1 0.7±0.1 0.4±0.0 Iodine index (b) 9.5±0.0 9.3±0.0 9.4±0.0 9.5±0.0 9.6±0.1 9.4±0.1 9.3±0.0 9.3±0.2 Palmitic acid 16:0 (b) 11.3±0.1 10.9±0.0 11.0±0.2 11.1±0.1 11.2±0.1 11.3±0.2 11.2±0.1 11.1±0.1 Estearic acid 18:0 (b) 2.7±0.0 3.8±0.2 3.1±0.3 3.2±0.2 3.0±0.2 3.0±0.2 2.7±0.0 3.5±0.0 Oleic acid 18:1 (b) 27.5±0.9 23.8±0.2 26.1±1.2 24.6±0.5 26.4±1.6 24.7±0.8 27.7±0.5 22.6±0.7 Linoleic acid 18:2 (b) 52.5±0.9 54.8±0.1 53.3±0.9 54.6±0.3 53.0±1.2 54.7±0.8 52.1±0.4 55.8±0.5 Linolenic acid 18:3 (b) 5.7±0.2 6.5±0.2 6.2±0.1 6.2±0.1 6.2±0.2 6.0±0.2 6.0±0.0 6.7±0.0
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Saturated fatty acid (b) 14.1±0.2 14.8±0.3 14.2±0.1 14.4±0.1 14.2±0.1 14.3±0.1 14.0±0.0 14.7±0.1 Unsaturated fatty acid (b) 85.8±0.2 85.1±0.3 85.7±0.1 85.5±0.1 85.7±0.1 85.6±0.1 85.9±0.0 85.2±0.1 Total carotenoids (h) 15.3±0.8 18.6±0.5 17.3±0.5 20.7±0.9 16.7±0.9 20.4±0.1 17.1±1.4 19.1±1.0 δ- tocopherol (i) 32.2±0.8 33.0±1.6 30.8±0.1 33.8±0.1 29.5±0.4 33.5±0.8 29.3±0.5 32.8±0.8 γ- tocopherol (i) 83.6±2.1 81.4±0.2 81.7±0.6 85.6±2.7 79.8±0.3 79.3±0.5 79.0±0.9 78.4±1.7 α- tocopherol (i) 5.8±0.4 1.8±1.8 5.6±1.6 0.0±0.0 4.6±0.6 0.0±0.0 3.4±0.3 0.0±0.0 (a) Kg/ha P2O5 (b) %; (c) mg of GAE/100 g; (d) mg/100 g; (e) mg/100 g; (f) mg of TE/100 g; (g) meq of O2/Kg; (h) mg of β-carotene/100 g of oil; (i) mg/100g of oil
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250
Apr/12
30
C
200 150 100
Apr/12
Mar/12
Feb/12
Jan/12
Dec/11
50 Nov/11
Precipitation (mm)
Apr/12
Mar/12
Jan/12
Feb/12
20.5
31
Mar/12
21.0
32
Feb/12
21.5
33
Jan/12
22.0
B
Dec/11
22.5
34
Nov/11
Maximum temperature (°C)
A
Dec/11
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23.0
Nov/11
Minimum temperature (°C)
627
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Figure 1. Average minimum temperature (A), average maximum temperature (B), and
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monthly rainfall (C) between November 2011 and April 2012. Microregion I (……) and
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Microregion II ( ——).
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Figure 2. Plotting scores and factor loadings of PC1 and PC2, related to dependent
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variables analyzed. The conventional soybean BRS Sambaíba and GM Msoy 9144
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exposed to different doses of phosphorus (0, 60, 120, and 240 kg/ha P2O5) are separated
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in the microregion I (A), and microregion II (B), and for separation between
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microregions, both results are plotted (C). Sat- Saturated fatty acid; Unsat- Unsaturated
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fatty acid.
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TOC Abstract Graphic
Isoflavones
Tocopherols
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