Phosphate Fertilizer and Growing Environment Change the

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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

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quality, and nutritional composition of Roundup Ready GM and conventional

3

soybean

4 5

Tatiane Scilewski da Costa Zanattaa, Roberta Manica-Bertoa, Cristiano Dietrich

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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,

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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

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* Corresponding author: Cristiano Dietrich Ferreira ([email protected])

15

Tel/Fax: +00555332757284

16 17 18 19 20 21

1 ACS Paragon Plus Environment


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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

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(microregions I and II) using a randomized complete block design, including

26

conventional soybean cultivars (BRS Sambaíba), genetically modified cultivars (Msoy

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9144 Roundup Ready—RR), and varying doses of phosphorus fertilizer (0, 60, 120, and

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240 kg/ha P2O5). Soybeans were evaluated for chemical composition, total phenols,

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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

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genotype (BRS conventional Sambaíba and GM Msoy 9144 RR), dose of P2O5

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fertilizer, and place of cultivation (microregion I and II). BRS Sambaíba had higher

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concentrations of β-glucosides, malonylglucosides, glycitein, and genistein than Msoy

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9144 RR, which showed a higher concentration of daidzein. The highest concentrations

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of isoflavones and fatty acids were observed in soybeans treated with 120 and 240 kg/ha

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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,

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Argentina, and China, totaling 84.3% of world production.1 In these countries, the

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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

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characteristics, such as its high levels of bioavailable protein and the quality of its lipid

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fraction, which is rich in unsaturated fatty acids essential for humans and high levels of

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natural antioxidants such as carotenoids and tocopherols.2 Phytic acid constitutes 80%

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of the existing phosphorus reserves in the plant; when present in appropriate

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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

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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

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Vegetables are highly influenced by environmental, genetic, and nutritional

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conditions.5 A major genetic modification (GM) in soybean is resistance to the

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glyphosate

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enolpyruvoylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the shikimate

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pathway responsible for the biosynthesis of aromatic amino acids (phenylalanine,

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tyrosine, tryptophan), which are important in protein synthesis. These aromatic amino

63

acids are also precursors of lignin, alkaloids, flavonoids, and cinnamic acid.4,

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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

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Glyphosate

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indicated that isoflavone content is influenced more by genetic than environmental

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factors. Zhang et al.9 studied the composition of the oil of 13 genotypes grown in the

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United States, Brazil, Argentina, Canada and China, and reported that the greatest

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variation in the fatty acid profile was in oleic acid levels, which ranged from 18.38 to

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30.62 g/100 g of oil. However, saturated fatty acids levels were more stable. In the same

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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

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in China. Kumar et al.10 observed an interaction between the genotype and growth site

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in the oleic, linoleic, linolenic, and stearic acids. They also reported variation in the

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phytic acid content (27.8-45.0 mg/g) with respect to changes in temperature and soil

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type. John et al.11 studied 27 soybean genotypes, grown in 3 regions of the United States

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during 2 growing seasons one month apart. They reported a reduction in the

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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

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during the pod filling stage. Low temperatures during the grain filling stage have been

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associated with higher levels of isoflavones.12

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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

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present in large amounts, it participates in many metabolic pathways and physiological

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reactions, mainly in the form of inorganic phosphate and ATP (adenosine

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triphosphate).13 According to Yin et al.14, phosphorus acts primarily in the initial stages

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of leaf growth and grain filling; they reported that high doses of phosphorus promote an

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increase in protein, palmitic, oleic, and linolenic acid contents, whereas lead to a

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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

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associating growing environment, phosphate fertilizer, and genotype on the chemical

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composition, oil quality, and phytochemical composition of these beans. Optimization

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of inputs in the production of soybean has become a standard practice worldwide, in

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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

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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

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soybean grown in two regions, with 0, 60, 120, or 240 kg/ha P2O5 fertilizer on the

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chemical composition, contents of total phenols, phytic acid, individual isoflavones,

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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

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Folin-Ciocalteu reagent, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)

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(ABTS), 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), fatty acids

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(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

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This experiment was conducted in 2011–2012 in commercial areas referred to as

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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

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Maranhão, Brazil.

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For each micro-region, the experiment was conducted in a randomized complete

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block design arranged in a factorial scheme with four replications. Factor A

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corresponded to the genotype (BRS Sambaíba and Msoy 9144 RR), and factor B

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corresponded to phosphorus doses (0, 60, 120, or 240 kg/ha P2O5) in the planting furrow

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in the form of triple superphosphate.

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Medium cycle genotypes (135 days) with similar yields were used. Each

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experimental unit comprised seven rows of 7.0 m length, spaced 0.5 m apart, with 11

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seeds per meter. During the development of the soybean crop, technical management

126

strategies used in the region were adopted.

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The crop was harvested manually when 95% of the pods had the typical color of a

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ripe pod. The usable area of each plot was 15 m2 after excluding 0.5 m from the ends of

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each plot. After collection, immature and damaged crop plants were excluded, and the

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remainder, useable plants were stored in an Ultrafreezer (-18 °C) until analysis.

131 132

2.3. Analysis

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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

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petroleum ether for over 8 h using a Soxhlet apparatus according to method 30-20 of the

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Approved Methods of the American Association of Cereal Chemists16. Nitrogen was

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determined by the Kjeldahl method and the protein content was obtained using the

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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

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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

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Phenolics were extracted according to the method described by Dueñas et al.18

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with some modifications. Soybean flour (2.0 g) was extracted twice with 80% methanol

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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

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(Eppendorf 5430-R) at 7600 rpm for 15 min, the supernatants obtained from each

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extraction were combined and concentrated to dryness using a rotary evaporator

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

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methanol extract was dissolved in 25 mL of 50% methanol and used as a crude extract

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for total free phenolic.

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The total amount of phenolic compounds in the crude extract were quantified

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according to the Folin-Ciocalteau method described by Zieliński and Kozłowska,19 with

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some modifications. Extracts (100 µL) were added to 400 µL of distilled water.

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Thereafter, 250 µL of Folin-Ciocalteau reagent (1 M) were added. After 8 min of

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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

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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:

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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

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ABTS•+ solution (0.700 ± 0.02). This mixture was allowed to stand at room

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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

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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

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Phytic acid content was determined using the method described by Haug and

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Lantzsch.21 Soybean flour (0.01 g) was extracted with 1500 µL of 0.2 M HCl for 30 min

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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

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FeCl3 in 1000 mL of H20) was added. The test tube was then covered with a stopper and

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incubated in a boiling water bath (100 °C) for 30 min. After cooling to room

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temperature, and centrifugation at 17,200 × g for 15 min. In 500 µL of supernatant were

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added to 750 µL of 2′,2-bipyridine solution (1% v/v). The absorbance was immediately

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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

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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,

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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

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unit (polytetrafluoroethylene, Millipore Ltd., Bedford, MA, USA) for HPLC injection.

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Isoflavone separation and quantitation was performed with a C18 Synergy 4 µm Fusion

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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

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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

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flow rate of 1 mL/min. Eluates were monitored at 255 and 320 nm and samples were

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injected in duplicate. Identification was made based on the spectra and retention time in

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comparison to known standards, and quantification was based on external calibration.

206

The 12 isoflavone standards were from LC Laboratories (Woburn, MA, USA).

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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

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isoflavones/100 g (dry weight basis).

210 211

2.4. Oil analysis

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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

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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

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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

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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

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described by Rodriguez-Amaya.24 Briefly, 2.5 g of oil, previously filtered, was added to

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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

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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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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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(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


Page 15 of 32


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



Page 16 of 32

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


Page 17 of 32


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



Page 18 of 32

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.


Page 19 of 32


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.

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2. Slavin, M.; Cheng, Z.; Luther, M.; Kenworthy, W.; Yu, L. Antioxidant properties and

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3. Kumar, V.; Rani, A.; Tindwani, C.; Jain, M. Lipoxygenase isozymes and trypsin

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inhibitor activities in soybean as influenced by growing location. Food Chem. 2003,

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4. Balisteiro, D. M.; Rombaldi, C. V.; Genovese, M. I. Protein, isoflavones, trypsin

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6. García-Villalba, R.; León, C.; Dinelli, G.; Segura-Carretero, A.; Fernández-Gutiérrez,

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7. Zobiole, L. H. S.; Bonini, E. A.; de Oliveira, R. S.; Kremer, R. J.; Ferrarese, O.

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

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19. Zieliński, H.; Kozłowska, H. Antioxidant activity and total phenolics in selected

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cereal grains and their different morphological fractions. J. Agric. Food Chem. 2000

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48, 2008–2016.

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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.



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

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moisture content and temperature during long-term storage. J. Food Sci. 2016, 81,

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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.

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

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

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Food Chem. 2008, 56, 9102–9113.


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31. Chung, H.; Hogan, S.; Zhang, L.; Rainey, K.; Zhou, K. Characterisation and

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Soybeans. J. Agric. Food Chem. 2008, 56, 11515–11519.

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Korean and Chinese soybean and processed products. Biochem. Eng. J. 2007, 36,

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34. Lee, S. J.; Ahn, J. K.; Kim, S. H.; Kim, J. T.; Han, S. J.; Jung. M. Y.; Chung, I. M.

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Variation in isoflavones of soybean cultivars with location and storage duration. J.

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Agric. Food Chem. 2003, 51, 3382-3389.

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35. Kim, E.-H.; Lee, O.-K.; Kim, J. K.; Kim, S.-L; Lee, J.; Kim, S.-H. Chung, I.-M.

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Isoflavones and anthocyanins analysis in soybean (Glycine max (L.) Merill) from

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three different planting locations in Korea. Field Crop. Res. 2014, 156, 76–83.

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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.

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37. Milinski, M. C.; Visentainer, J. V.; Martin, C. A.; Arias, C. A. A.; Matsushita, M.;

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Souza, N. E. Proximate composition and fatty acids profile of Brazilian conventional

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and transgenic soybeans (Glycine max (L.) Merrill) cultivars. Electron. J. Environ.

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Agric. Food Chem. 2007, 6, 1905–1911.

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38. Galão, O. F.; Carrão-Panizzi, M. C.; Mandarino, J. M. G.; Santos Júnior, O. O.;

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Maruyama, S. A.; Figueiredo, L. C.; Bonafe, E. G., Visentainer, J. V. Differences of

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fatty acid composition in Brazilian genetic and conventional soybeans (Glycine max

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(L.) Merrill) grown in different regions. Food Res. Int. 2014, 62, 589–594.



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39. Lee, K.; Yi, B.-Y.; Kim, K.-H.; Kim, J.-B.; Suh, S.-C.; Woo, H.-J.; Shin, K.-S.;

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Kweon, S. –J. Development of efficient transformation protocol for soybean (Glycine

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max L.) and characterization of transgene expression after Agrobacterium-mediated

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gene transfer. J. Korean Soc. Agric. Biotechnol. Chem. 2011, 54, 37–45.

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40. Rani, A.; Kumar, V.; Verma, S. K.; Shakya, A. K.; Chauhan, G. S. Tocopherol

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content and profile of soybean: Genotypic variability and correlation studies. J.

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Amer. Oil Chem. Soc. 2007, 84, 377–383.

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

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

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

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

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42. Li, Q.; Hu, Y.; Chen, F.; Wang, J.; Liu, Z.; Zhao, Z. Environmental controls on

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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


Page 25 of 32


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



604 605 606

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)

26

ACS Paragon Plus Environment

Page 27 of 32

607 608 609 610


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

611 612 613 614 615 616 617 618 619

27



620 621

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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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622 623 624


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

628

23.0

Nov/11

Minimum temperature (°C)

627

629 630 631

Figure 1. Average minimum temperature (A), average maximum temperature (B), and

632

monthly rainfall (C) between November 2011 and April 2012. Microregion I (……) and

633

Microregion II ( ——).

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636 637

Figure 2. Plotting scores and factor loadings of PC1 and PC2, related to dependent

638

variables analyzed. The conventional soybean BRS Sambaíba and GM Msoy 9144

639

exposed to different doses of phosphorus (0, 60, 120, and 240 kg/ha P2O5) are separated

640

in the microregion I (A), and microregion II (B), and for separation between

641

microregions, both results are plotted (C). Sat- Saturated fatty acid; Unsat- Unsaturated

642

fatty acid.

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TOC Abstract Graphic

Isoflavones

Tocopherols

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Phosphate Fertilizer and Growing Environment Change the

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