Food Chemistry 146 (2014) 531–537
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Antioxidant activities of Se-SPI produced from soybean as accumulation and biotransformation reactor of natural selenium Juwu Hu a,b, Qiang Zhao a, Xiang Cheng a, Cordelia Selomulya c, Chunqing Bai a, Xuemei Zhu a, Xionghui Li b, Hua Xiong a,⇑ a b c
State Key Laboratory of Food Science and Technology, Nanchang University, Jiangxi 330047, China Jiangxi Academy of Sciences, Jiangxi 330029, China Department of Chemical Engineering, Monash University, Clayton, Victoria 3800, Australia
a r t i c l e
i n f o
Article history: Received 27 March 2013 Received in revised form 5 September 2013 Accepted 16 September 2013 Available online 26 September 2013 Keywords: Selenium Soybean Selenoprotein Accumulation Biotransformation Antioxidant activity
a b s t r a c t A study to compare the uptake, translocation, and distribution of selenium (Se) in soybean planted in natural seleniferous soil in Fengcheng city of China was conducted to clarify the relationship between the Se content levels of soybean proteins and their radical scavenging activity. The data showed that the total Se content in different parts of soybean plants varied with the growth periods. The selenoprotein (Se-SPI) content increased remarkably with the increase of Se content in seleniferous soils. The Se-SPI content obtained from the region with the highest Se level was almost 18 times higher than that of the control group, while antioxidant activities were about 4-fold compared to the control, suggesting that Se played a positive role in enhancing the antioxidant activity of Se-SPI. The increase in the Se level also led to changes in amino acids composition, but with nearly no effects on the subunit composition of soybean Se-SPI. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Selenium (Se) is an essential trace element for humans, animals and some species of microorganism. It is well known primarily for its antioxidant activity, anti-inflammatory, chemopreventive, antiviral, and anticarcinogenic properties (Rayman, 2000). Inadequate intakes of Se can cause health disorders, including oxidative stress-related conditions, reduced fertility and immune functions, and an increased risk of cancers (Rayman, 2002; Whanger, 2004). It has been estimated that about one billion people globally may suffer from inadequate intake of Se. Food is the major source of Se for the general population. Therefore, fortification of Se is very important for both nutritional demand and prevention of diseases associated with Se-deficiency. The fortification of Se can be achieved by different means including addition of Se supplements to the usual diet, consumption of food naturally rich in Se, or food that has been previously enriched in Se such as vegetables fertilised with inorganic Se (Li & Wang, 2004). The fortification of Se in food could also be obtained by plants, as Se could be naturally accumulated or biotransformed in plants grown on seleniferous soils. This method may be used to fortify
⇑ Corresponding author. Tel./fax: +86 791 86634810. E-mail address: [email protected] (H. Xiong). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.087
plants with Se and thereafter help alleviate Se deficiency in humans and animals (Gomez-Galera et al., 2010). However, just like most trace elements, Se is necessary for animal and human health, but can be toxic to human and animal at higher levels (Rayman, 2000). Accordingly, Se can be beneficial to plants growth at low Se levels and be detrimental at high Se levels. Plants could take up Se due to its similarity to sulphur, and thus remove excess Se from seleniferous areas. Food produced from seleniferous soils could contain exceptionally high amounts of Se, although this type of soil is not very prevalent. In practice, selenite or selenate can be added to fertilizers, seeds can be treated with aqueous selenium, or crops sprayed with selenium salts as safe and effective means to increase the Se intake in food for both animals and humans (Aspila, 2005). However Se concentration of a particular food may vary depending on the soil in which the agricultural crop was grown and the ability of plants to take up and accumulate this element (Gupta & Gupta, 2000). Improper uses of Se fortification methods could result in high Se intake and environmental contamination through irrigation (Banuelos, Lin, Wu, & Terry, 2002). Examples of seleniferous areas include the Western USA and Hubei province in China, where Se levels may exceed 10 mg Se kg1, whereas other areas such as the Northeastern USA, Finland, and New Zealand have little or no Se in their soils (Hartikainen, 2005). Fengcheng, located in central Jiangxi province of China, is
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known as the Chinese eco-selenium Valley with high quality natural seleniferous soil resource. Statistical data from the Jiangxi Province Bureau of Quality and Technical Supervision show the distribution area of natural seleniferous soil of Fengcheng is around 524.7 km2 with Se content in the range of 0.4–0.99 lg g1, and an average of about 0.538 lg g1 (suitable Se-enriched range was 0.4–3.0 lg g1). We have analysed rice and soybean planted in this region, and found their Se contents to be 0.096 ± 0.011 lg g1 and 0.124 ± 0.009 lg g1, respectively, suggesting that they are novel natural Se-enriched food resources. More importantly, Se in the soil exists in states that are available to be absorbed and utilised easily by plants. After biotransformation by plants, Se is incorporated as selenocysteine at the active site of a wide range of proteins. At these sites, Se exerts its biological function in several selenoproteins (Brown & Arthur, 2001). The protein in soybean seeds accounts for about 40% of the dry seed weight, and is the major source of plant proteins for human and animal nutrition, which would be among the most effective plants for biofortification. However, to the best of our knowledge, the comparison of uptake, translocation, and distribution of Se, as well as functional properties of selenoproteins in soybean cultured in different regions at different Se content levels have never been reported before, especially for soybeans planted in natural seleniferous soils. The purposes of this study were to measure the Se contents and status of different parts of soybean growing in seleniferous soils and to analyse nutrient compositions and antioxidant activities of Se-enriched protein extracts. The study was designed to assist in systematically understanding the accumulation and biotransformation processes of natural Se of soybean during different periods for the development of efficient natural Se-enriched food supplements.
2.3. Extraction of different Se states in different soils The various Se states of soil were extracted sequentially as described by Zhang, Zhou, and Zhang (1997) with some modifications. Briefly, water soluble state Se (H2O–Se), acid soluble state Se, exchanged form Se, organic combination state Se, and residual state Se were extracted with ultrapure water, 3.0 mol L1 HCl, 0.1 mol L1 K2PO4 + KH2PO4 (pH 7.0), 1 mol L1 K2S2O8, and HNO3 + HF + HClO4 (v/v/v, 8:1:1), respectively. The extraction solvent to soil ratio was set at 30:1 (v/m), under ultrasonic extraction for 2.5 h. Each supernatant was obtained by centrifugation at 4800 rpm for 15 min and the residue was extracted twice, after which the supernatants were mixed. Three samples (5 ml) were taken from each sample for analysis. The determination of Se in solution was performed by an atomic fluorescence spectrophotometry (Model AI 3300, Aurora Technologies, Canada). 2.4. Preparation of soybean protein isolate Soybean protein isolate was prepared by an alkaline extraction, followed by precipitation at the isoelectric point according to Sorgentini, Wagner, and Anon (1995). The defatted soybean seed sample (100 g) was extracted with 1000 ml water adjusted at pH 8.0 with 2 mol L1 NaOH at room temperature for 3 h under continuous stirring. The supernatant was obtained by centrifugation at 4800 rpm for 15 min and the residue was extracted twice. The proteins from combined supernatants from the extractions were isoelectrically precipitated at pH 4.5 using 2 mol L1 HCl and kept at 4 °C for 1 h. The precipitate was recovered by centrifugation at 4800 rpm for 15 min, and then washed with deionized water. The protein isolates were freeze-dried and stored at 4 °C until further use. The total soybean protein content (g/100 g) in seeds was estimated by AOAC (1990), using a nitrogen conversion factor of 6.25.
2. Materials and methods
2.5. Purification of high Se-containing soybean protein isolate
2.1. Soils and soybean materials
The lyophilized C region soybean protein isolate was dissolved in 0.05 mol L1 Tris–HCl (pH 8.0). The solution (1.0 mL) was passed through a Q Sepharose Fast Flow anion exchange column (1.0 25 cm) (Sigma–Aldrich Co., St. Louis, MO), which had been equilibrated with 0.05 mol L1 Tris–HCl (pH 8.0) buffer. Anion exchange chromatography was performed using 0.8 mol L1 NaCl and 0.05 mol L1 Tris–HCl (pH 8.0) buffer as a mobile phase with a flow rate of 1.0 mL min1. All protein purification steps were done at 4 °C, and proteins eluted from the columns were monitored at the measuring absorbance at 280 nm. Meanwhile, the Se content of each peak was measured by atomic fluorescence spectrophotometry. The peak with the highest Se content was collected, lyophilized, and stored at 15 °C until further use. The attained high Se-containing protein was termed Se-soybean protein isolate (Se-SPI).
Collection of soils with different Se contents. Soils were obtained from three different regions in Fengcheng, Jiangxi, China. They were from Hehu village (A region), Shangzhuang village (B region), and Dong village (C region). The Se content in the soils is in the order of A region < B region < C region, which were confirmed by our own measurements in this work. The trial was carried out on 100 m2 plots in triplicate. Five subplots were selected at the four corners (25 25 cm) and the centre of the plot (1 1 m). Soil samples were collected from the surface soil (0–25 cm) with a shovel in each subplot, then mixed and milled. Finally a subsample (about 2 kg) was taken for use (Pérez-Sirvent, Martínez-Sánchez, GarcíaLorenzo, Molina, & Tudela, 2009).
2.2. Preparation of soybean
2.6. Determination of Se content
This trial was carried out on 50 m2 plots in triplicate for each treatment. The soybean seed species (Jiangxi 90-77-2, harvested on July 10th, 2010) used in the three regions was purchased from Jiangxi Academy of Agricultural Science, as the most widespread cultured species in the studied region. After the soybean seedlings were cultured, samples of soybean in C region were obtained every 15 days to measure the total Se content in the soybean’s roots, stems, leaves, and seeds at different growing periods. The determination of Se in solution was performed by an atomic fluorescence spectrophotometry (Model AI 3300, Aurora Technologies, Canada).
Three samples (0.2 g, or 10 mL) for testing were taken from prepared samples above for analysis. The test samples were digested with 10 mL of mixture of HNO3 and HClO4 (v/v, 9:1) at 150 °C for 2.5 h. After cooling, 5 mL of 6 mol L1 HCl was added to the digested samples to reduce Se6+–Se4+. When cooled, the digested solution was diluted with ultrapure water to 25 mL. Then 10 mL of the solution was transferred to a reaction vessel, with 2 mL of 6 mol L1 HCl and 1 mL of 10% (w/w) K3Fe(CN)3 added. The control undergoing the same procedure was used to provide a blank value. The determination of Se in solution was performed by an atomic fluorescence spectrophotometry (Model AI 3300, Aurora
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Technologies, Canada). The measuring conditions were conducted according to Wu, Jin, Shi, and Bi (2007) with some modifications.
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Hydroxyl radical scavenging effect ð%Þ ¼ ½ðA1 A2 Þ=A1 A0 100%
ð2Þ
2.7. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) SDS–PAGE was run according to the method of Laemmli (1970) at 4% stacking gel and 15% separating gel using a Bio-Rad Mini PROTEANÒ 3 system (Bio-Rad Laboratories, Hercules, CA, USA), at a constant current setting of 250 mA for approximately 1.5 h. Protein bands were stained with 0.125% Coomassie Brilliant Blue R-250 and destained with 25% ethanol and 10% acetic acid. The protein bands were estimated using pre-stained molecular weight standards (Sigma Chemical Co., St. Louis, MO), ranging from 10 to 170 kDa. 2.8. Amino acid analysis Amino acid compositions were determined by an HPLC system for automatic amino acid analysis (L-8800, Hitachi, Japan) after protein samples were hydrolysed with 6 M HCl at 110 °C for 24 h in a sealed tube, according to the method reported by Gehrke, Wall, Absheer, Kaiser, and Zumwalt (1985). The methionine and cysteine contents were determined after performic acid oxidation. The tryptophan content was determined after alkaline hydrolysis. The amino acid compositions were reported as g/ 100 g protein. 2.9. Determination of superoxide radical scavenging effect The superoxide radical-scavenging activities of different Se content soybean protein isolates and the Se-SPI were examined using a pyrogallol autoxidation system (Marklund & Marklund, 1974) with some modification. A reaction solution containing 80 lL of pyrogallol (10 mmol L1) and 4.5 mL of Tris/HCl/EDTA buffer (50 mmol L1, pH 8.0) was prepared. Then 0.5 mL of samples (0–1 mg mL1) was added. After incubation at room temperature for 30 min, the absorbance of the supernatant was determined at 365 nm by an UV spectrophotometry (TU1901, Purkinje General Instrument Co., Ltd., Beijing, China). The trolox (0–1 mg mL1) was used for the comparison group. The scavenging effect was calculated according to the following equation:
Scavenging effect ð%Þ ¼ ½1 ðA1 A2 Þ=A0 100%
Fig. 1. Se states (A) and total Se contents (B) of soil, soybean and soybean protein in different regions.
ð1Þ
where A0 is the absorbance of the control (without sample), A1 is the absorbance in the presence of the sample, and A2 is the absorbance of the sample without pyrogallol. 2.10. Determination of hydroxyl radical scavenging effect The hydroxyl radical scavenging assay was modified on the basis of the method described by Li, Jiang, Zhang, Mu, and Liu (2008). Different Se-SPI samples (50 lL, at a final concentration of 0–1 mg mL1 in 0.1 mol L1 sodium phosphate buffer, pH 7.4) were first added to a 96-well microplate, and then followed by the addition of 50 lL of 3 mmol mL1, 10-phenanthroline (in phosphate buffer), and 50 lL of 3 mmol mL1 FeSO4 (in water). To initiate the reaction, 50 lL of 0.01% aqueous H2O2 was added, and the reaction mixture was covered and incubated at 37 °C for 1 h with shaking. The absorbance was measured at 536 nm with a spectrophotometer, while solutions without protein sample and H2O2 were used as blank and control, respectively. Trolox (0– 1 mg mL1) was used for comparison as well. The hydroxyl radical scavenging activity was assessed according to the following equation:
Fig. 2. Se contents of leaf, stem, root and soybean in different growing periods (15 d: bud and seeding stage; 30 d: bud differentiation stage; 45 d: flowering and pods formation stage; 60–75 d: pod filling stage; 90 d: maturation stage).
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Table 1 Total amino acid composition of soybean protein from different regions. Amino acid (%)
A region
B region
C region
Threonine Valine Isoleucine Leucine Lysine Tryptophan Methionine Phenylalanine Histidine Total essential amino acid Aspartic acid Tyrosine Serine Glutamic acid Glycine Arginine Cysteine Proline Alanine
3.352 ± 0.168 3.152 ± 0.189 3.285 ± 0.131 6.327 ± 0.253 5.567 ± 0.223 1.406 ± 0.070 0.523 ± 0.209c 4.445 ± 0.178 1.980 ± 0.138 30.037 9.432 ± 0.189 7.777 ± 0.155 4.703 ± 0.329 15.739 ± 0.315 3.270 ± 0.229 5.490 ± 0.329 0.682 ± 0.034c 7.827 ± 0.391 3.409 ± 0.102
3.410 ± 0.238 3.248 ± 0.292 3.245 ± 0.389 6.375 ± 0.573 5.784 ± 0.347 1.466 ± 0.043 0.207 ± 0.015b 4.425 ± 0.354 2.177 ± 0.109 30.130 9.577 ± 0.479 7.731 ± 0.463 4.886 ± 0.147 16.163 ± 0.808 3.427 ± 0.171 5.723 ± 0.515 0.316 ± 0.037b 8.345 ± 0.500 3.477 ± 0.278
3.503 ± 0.211 3.292 ± 0.230 3.196 ± 0.127 6.394 ± 0.256 5.652 ± 0.283 1.513 ± 0.106 0.112 ± 0.003a 4.413 ± 0.309 2.196 ± 0.154 30.159 9.614 ± 0.577 7.712 ± 0.536 4.914 ± 0.196 16.325 ± 0.653 3.412 ± 0.204 5.812 ± 0.232 0.102 ± 0.012a 8.496 ± 0.595 3.553 ± 0.247
Total unessential amino acid Total soybean protein content (g/100 g)
57.647 41.93 ± 2.10a
59.329 44.71 ± 1.79a,b
59.838 46.64 ± 2.33b
Each value is expressed as mean ± standard deviation of triplicate tests with at least three measurements. Means within the same row with different letters are significantly different (p < 0.05), according to Duncan’s multiple-range test.
2.11. Statistical analysis The analysis of the variance was performed with the Statistical Analysis System software 8.2 (SAS, USA). Differences among means were evaluated using Duncan’s multiple range tests. The significance was established at p < 0.05. 3. Results and discussion 3.1. Distribution of Se content in soil, soybean protein, and soybean organ
Fig. 3. (A) SDS–PAGE analysis, band 1–3: soybean protein from A region, B region and C region. M: markers, Group molecular weights I: 70.0–100.0 kDa, II: 45.0– 55.0 kDa, III: 35.0–40.0 kDa, IV: 15.0–25.0 kDa; (B) Se content of different soybean protein subunits from different regions.
where A1, A2, A0 are the absorbance of the control, samples and blank, respectively.
The biological utilisation of Se depends on the amount of Se in environmental media, as well as the existence and transformation of Se states (Hartikainen, 2005). The Se-combined states were classified as available state, potential available state and useless state (Sharmasarkar & Vance, 1995). Available state Se mainly refers to water-soluble Se, including selenite and selenate that could be easily absorbed and utilised by plants. Their contents are often regarded as the key parameter to evaluate if the soil has sufficient effective or utilisable Se. Potential available Se mainly consists of acid soluble state Se, exchanged state Se, and organic combination state Se, as direct sources of the available Se, while residual state Se could not be utilised (Sharmasarkar & Vance, 1995). In the three collected soils, water-soluble state Se content in the C region was the highest, approximately 10 times than that of in A region (Fig. 1A). The potential available state Se of the total Se content were 30.4%, 25.2%, and 41.7% for A, B, and C region, respectively (Fig. 1A). The result implied that the total Se in C region could be utilised by plants more effectively. The total Se contents of soybean and soybean protein from C region were also much higher than those of B and A regions (Fig. 1B). Hence, Se contents of soybean and soybean protein could be determined from contents of water-soluble state Se and potential available state Se in the soil. Soybean growing process included bud and seeding stage, bud differentiation stage, flowering and pods formation stage, pod filling stage and maturation stage. Fig. 2 showed that the order of total content of Se was root > leaf > stem in bud and seeding
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stage, bud differentiation stage, and flowering and pods formation stage. The order of total Se content of different organs was root > leaf > soybean > steam in pod filling stage, while Se distribution in different organs was root > soybean > leaf > stem in the mature period (harvest). The Se contents of root in flowering and pods formation stage, and pod filling stage were lower than those in the bud and seeding stage. There were significant differences between mechanisms involved in uptaking and transporting of selenate, selenite and organic Se compounds like selenomethionine (Se-Met) (Terry, Zayed, De Souza, & Tarun, 2000). Selenite absorption is a passive absorption that requires little energy (Abrams, Shennan, Zasoski, & Burau, 1990), while selenate absorption is an active transportation that could be inhibited by respiratory depressant (azides or dinitrophenol) or low temperature. Organic selenium, such as seleno methionine, would also be absorbed by plant roots by active transportation (Sors, Ellis, & Salt, 2005). Compared to the selenite and organic Se, selenate was much easier to be transferred and
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absorbed from roots, and then accumulated in leaves. Most selenite was in the root and would be converted into organic Se quickly, especially in the form of seleno-amino acids (Zayed, Lytle, & Terry, 1998). Although the initial stage of Se being absorbed and converted into selenocysteine is the same in Se accumulator plants (such as soybean) and Se non-accumulator plants (such as grain), the subsequent steps of their metabolic pathways are different. In Se accumulator plants, selenocysteine was metabolized through formation of leno-amino acids (Brown & Shrift, 2008), including seleno-methyl-selenocysteine, c-glutamyl-seleno-methyl-selenocysteine, and selenocystathione (Terry et al., 2000). However, in Se non-accumulator plants, Se combined with selenocysteine would act as zymoprotein when getting into proteins (Terry et al., 2000). Thus, due to the difference of metabolic pathways, Se accumulator plants could withstand high concentration Se substance, while Se non-accumulator plants are more susceptible to Se poisoning (Sors et al., 2005).
Fig. 4. (A) Elution diagram from an anion exchange column of high Se-containing protein from C region; (B) SDS–PAGE analysis of peak 4 from anion exchange chromatography. Left lane, molecular weight markers; Right lane, peak 4 after anion exchange chromatography.
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Se distribution and concentration in different organs of plants varied with different kinds of plants. In Se accumulator plants, such as soybean, Se is accumulated in leaves first, and then in seed (fruit) parts. But in Se non-accumulator plants, such as grain, the content of Se accumulation in the roots is often the same as those in seeds or grain (Terry et al., 2000). A study of 17 different vegetables showed that the Se contents of edible parts were lower than those of non-edible parts (Hamilton & Beath, 1963). As shown in Fig. 2, the Se contents in roots and stems were higher than those in soybean fruit during the whole soybean growing period, while the Se contents in roots and stems decreased in the soybean at the mature period. Se could be volatilised as dimethyl-diselenide via oxidation and subsequent methylation of ethylselenocysteine. Moreover, the volatilisation of dimethyldiselenide was observed in the Se hyper-accumulator Asparagus racemosus, known to accumulate large quantities of Se-methylselenocysteine (Sors et al., 2005).
Peak 4 exhibited the highest Se content of 0.256 lg g1, which was also markedly higher than those of crude proteins (0.164 lg g1, C region). Therefore, this fraction was targeted for further purification. SDS–PAGE analysis showed that peak 4 contained a single band (Fig. 4B), indicating a novel high Se-containing soybean protein was attained, namely Se-SPI with a molecular weight of about 18.0 kDa. The Se content of Se-SPI was significantly higher and the composition of amino acids was possibly different from other proteins corresponding to the other three major protein peaks. Liu, Zhao, Chen, Gu, and Bu (2012) had reported that selenium-containing proteins from rice have strong antioxidant activities. Although the chemical form of the Se-SPI still remained unknown, in the following section the improvement of antioxidant activity related to the Se content was investigated.
3.2. Effects of Se content on protein and amino acids
Se is considered to be an element possessing antioxidant activity. Its antioxidant activity was expressed by combining with GSH-Px, scavenging free radical, cutting off the chain reaction induced by the lipid peroxidation, analysing peroxide, and protecting the cell membrane (Tinggi, 2008). The profiles of different Se content proteins scavenging hydroxyl radical and super oxide radical were shown in Fig. 5. It was observed that the radical scavenging activities of different Se content proteins increased with protein
As shown in Table 1, the total essential amino acids content in the soybean protein from C region (30.159%) was close to those from B region (30.13%), but higher than that from A region (30.037%), while methionine and cysteine levels declined noticeably. In addition, the total soybean protein content from C region was much higher than that from the A region (p < 0.05), but was similar to that from B region (p > 0.05). Se could affect protein synthesis in soybean since seleno-amino acids, since selenocysteine and selenomethionine (Se-Met), could replace cysteine and methionine directly during protein synthesis process, leading to a reduction of cysteine and methionine (Brown & Shrift, 2008). As Se atomic size and ionisation could change the tertiary structure of protein, this particular effect was observed in some important protein synthesis and reaction performance (Brown & Shrift, 2008). On the contrary, in other plants such as tea and green algae (Hu, Xu, & Pang, 2003), the presence of Se could enhance organism for protein synthesis.
3.5. Antioxidant activities of different Se content proteins
3.3. Effect of Se content on the subunit composition of soybean protein The subunit compositions of total protein extracted from different Se containing soybeans were analysed by SDS–PAGE. Fig. 3A showed that Se content did not change the distribution of subunit of protein in soybean protein. The spectral band was a basic convergence uniform in different Se content soybean protein, with none of protein band disappearing or new protein band appearing. The strips in band 1 were fainter, possibly due to the lower total protein content (Table 1). Fig. 3A also indicated that the protein extracts from different Se containing soybean had a large distribution of molecular weights with ranging from 15.0 to 110.0 kDa. In order to investigate the Se content of the subunit composition, every band was divided into four protein sections. These sections were then separated from the gel and the Se content of each section was determined. As shown in Fig. 3B, proteins with molecular weights of 15.0–25.0 kDa had the highest Se content, while proteins with molecular weights of 70.0–100.0 kDa had the lowest Se content. The result showed that Se was inclined to combine with low molecular weight proteins, in agreement with Zhao et al. (2004) showing that the molecular weight of most proteins or protein subunits containing Se was less than 16 kDa. 3.4. A novel high Se-containing soybean protein After loading the protein sample (Se content, 0.164 lg g1, from C region) into an anion exchange column, the elution profile of protein and Se are presented in Fig. 4A, showing four major peaks.
Fig. 5. Scavenging activities of containing different Se content soybean protein against hydroxyl radical (A) and superoxide radical (B).
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concentrations. The Se content of Se-SPI (0.256 lg g1) was almost 18 times higher than that of the control protein (A region, 0.014 lg g1), while the antioxidant activity of Se-SPI was almost 4-fold higher compared to that of the control protein, although weaker than trolox. Thus, we could conclude that the improvement of the antioxidant activity of selenoprotein quantitatively depended on its Se content. The essential micronutrient Se occurs in the form of selenocysteine in selenoprotein exerting various effects including maintaining the cell reduction–oxidation balance. Previously, it was shown that selenomethionine was the major selenocompound in the protein (Zhang et al., 2009). As a free radical scavenger, selenoamino acids react with hydrated free radicals to form stable compounds before destroying biological macromolecules. Although the mechanism on how Se enhances the antioxidant activity of the protein is still unclear, when the protein is combined with Se, its antioxidant activity has been shown to remarkably improved. Our results were in agreement with previous findings, which showed that the antioxidant activities of green tea and phycocyanin were enhanced after Se was incorporated, and the scavenging effect was increased with Se levels increased. A possible explanation for the enhancing effect was the unique atom structure of Se (Shen et al., 2010). The ionisation energy of the 4p electron in selenium was much lower than that of the 3p electron in sulphur which made selenoamino acids much easier than sulfoamino acids became positive ion free radical. In addition, the selenoamino acid radical was more stable than that of sulfoamino acid because of the existence of the empty 4f orbit. When the sulphur in Se containing proteins was replaced by selenium or some new selenoamino acids were synthesized, stable positive ion free radicals that possess strong antioxidant activity were much easier to be formed by selenoamino acids. 4. Conclusion In summary, to the best of our knowledge, this was the first time that the process of Se accumulation by soybean produced from soils with different natural Se levels was reported. The effects of the Se content on amino acids and subunit composition of SeSPI, as well as antioxidant activities of the abstracted Se-SPI were also evaluated. The results indicated that Se was accumulated in leaves first, and then in seed parts during the growing stage. The Se level had positive effect on the content of Se-SPI, as it could change the amino acids composition of Se-SPI, but exhibited negligible effects on the subunit composition of Se-SPI. The antioxidant activity experiments indicated that Se-SPI possessed significantly higher antioxidant activity than the control protein, suggesting that Se played a positive role in enhancing the antioxidant activity of selenoprotein. The mechanism(s) involved in the antioxidant activities of protein isolated from Se-enriched soybean require further studies in vivo. Acknowledgements This research was supported in part by the National High Technology Research and Development Program of China (863 Program) (2013AA102203-5), the National Natural Science Foundation of China (31160317 and 31301436), and the Programs of State Key Laboratory of Food Science and Technology of Nanchang University (SKLF-MB-201006, SKLF-TS-201116). References Abrams, M. M., Shennan, C., Zasoski, R. J., & Burau, R. G. (1990). Selenomethionine uptake by wheat seedlings. Agronomy Journal, 82(6), 1127–1130. AOAC (1990). Official methods of analysis of the association of official analytical chemists (15th ed.). Washington, DC, USA: Association of Official Analytical Chemists.
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Effect of dietary organic versus inorganic selenium in laying hens on the productivity, selenium distribution in egg and selenium content in blood, liver and kidney. Journal of Trace Elements in Medicine and Biology, 18(1), 65–68. Li, Y., Jiang, B., Zhang, T., Mu, W., & Liu, J. (2008). Antioxidant and free radicalscavenging activities of chickpea protein hydrolysate (CPH). Food Chemistry, 106(2), 444–450. Liu, K., Zhao, Y., Chen, F., Gu, Z., & Bu, G. (2012). Purification, identification, and in vitro antioxidant activities of selenium-containing proteins from selenium-enriched brown rice. European Food Research and Technology, 234, 61–68. Marklund, S., & Marklund, G. (1974). Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. European Journal of Biochemistry, 47(3), 469–474. Pérez-Sirvent, C., Martínez-Sánchez, M., García-Lorenzo, M., Molina, J., & Tudela, M. (2009). Geochemical background levels of zinc, cadmium and mercury in anthropically influenced soils located in a semi-arid zone (SE, Spain). Geoderma, 148(3–4), 307–317. Rayman, M. P. (2000). The importance of selenium to human health. The Lancet, 356(9225), 233–241. Rayman, M. P. (2002). The argument for increasing selenium intake. Proceedings of the Nutrition Society, 61, 203–215. Sharmasarkar, S., & Vance, G. F. (1995). Fractional partitioning for assessing solidphase speciation and geochemical transformations of soil selenium. Soil Science, 160(1), 43. Shen, Q., Zhang, B., Xu, R., Wang, Y., Ding, X., & Li, P. (2010). Antioxidant activity in vitro of the selenium-contained protein from the Se-enriched Bifidobacterium animalis 01. Anaerobe, 16(4), 380–386. Sorgentini, D. A., Wagner, J. R., & Anon, M. C. (1995). Effects of thermal treatment of soy protein isolate on the characteristics and structure–function relationship of soluble and insoluble fractions. Journal of Agricultural and Food Chemistry, 43(9), 2471–2479. Sors, T., Ellis, D., & Salt, D. (2005). Selenium uptake, translocation, assimilation and metabolic fate in plants. Photosynthesis Research, 86(3), 373–389. Terry, N., Zayed, A., De Souza, M., & Tarun, A. (2000). Selenium in higher plants. Annual Review of Plant Biology, 51(1), 401–432. Tinggi, U. (2008). Selenium: Its role as antioxidant in human health. Environmental Health and Preventive Medicine, 13(2), 102–108. Whanger, P. (2004). Selenium and its relationship to cancer: An update. British Journal of Nutrition, 91(1), 11–28. Wu, H., Jin, Y., Shi, Y., & Bi, S. (2007). On-line organoselenium interference removal for inorganic selenium species by flow injection coprecipitation preconcentration coupled with hydride generation atomic fluorescence spectrometry. Talanta, 71(4), 1762–1768. Zayed, A., Lytle, C. M., & Terry, N. (1998). Accumulation and volatilisation of different chemical species of selenium by plants. 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Antioxidant activities of Se-SPI produced from soybean as accumulation and biotransformation reactor of natural selenium Juwu Hu a,b, Qiang Zhao a, Xiang Cheng a, Cordelia Selomulya c, Chunqing Bai a, Xuemei Zhu a, Xionghui Li b, Hua Xiong a,⇑ a b c
State Key Laboratory of Food Science and Technology, Nanchang University, Jiangxi 330047, China Jiangxi Academy of Sciences, Jiangxi 330029, China Department of Chemical Engineering, Monash University, Clayton, Victoria 3800, Australia
a r t i c l e
i n f o
Article history: Received 27 March 2013 Received in revised form 5 September 2013 Accepted 16 September 2013 Available online 26 September 2013 Keywords: Selenium Soybean Selenoprotein Accumulation Biotransformation Antioxidant activity
a b s t r a c t A study to compare the uptake, translocation, and distribution of selenium (Se) in soybean planted in natural seleniferous soil in Fengcheng city of China was conducted to clarify the relationship between the Se content levels of soybean proteins and their radical scavenging activity. The data showed that the total Se content in different parts of soybean plants varied with the growth periods. The selenoprotein (Se-SPI) content increased remarkably with the increase of Se content in seleniferous soils. The Se-SPI content obtained from the region with the highest Se level was almost 18 times higher than that of the control group, while antioxidant activities were about 4-fold compared to the control, suggesting that Se played a positive role in enhancing the antioxidant activity of Se-SPI. The increase in the Se level also led to changes in amino acids composition, but with nearly no effects on the subunit composition of soybean Se-SPI. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Selenium (Se) is an essential trace element for humans, animals and some species of microorganism. It is well known primarily for its antioxidant activity, anti-inflammatory, chemopreventive, antiviral, and anticarcinogenic properties (Rayman, 2000). Inadequate intakes of Se can cause health disorders, including oxidative stress-related conditions, reduced fertility and immune functions, and an increased risk of cancers (Rayman, 2002; Whanger, 2004). It has been estimated that about one billion people globally may suffer from inadequate intake of Se. Food is the major source of Se for the general population. Therefore, fortification of Se is very important for both nutritional demand and prevention of diseases associated with Se-deficiency. The fortification of Se can be achieved by different means including addition of Se supplements to the usual diet, consumption of food naturally rich in Se, or food that has been previously enriched in Se such as vegetables fertilised with inorganic Se (Li & Wang, 2004). The fortification of Se in food could also be obtained by plants, as Se could be naturally accumulated or biotransformed in plants grown on seleniferous soils. This method may be used to fortify
⇑ Corresponding author. Tel./fax: +86 791 86634810. E-mail address: [email protected] (H. Xiong). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.087
plants with Se and thereafter help alleviate Se deficiency in humans and animals (Gomez-Galera et al., 2010). However, just like most trace elements, Se is necessary for animal and human health, but can be toxic to human and animal at higher levels (Rayman, 2000). Accordingly, Se can be beneficial to plants growth at low Se levels and be detrimental at high Se levels. Plants could take up Se due to its similarity to sulphur, and thus remove excess Se from seleniferous areas. Food produced from seleniferous soils could contain exceptionally high amounts of Se, although this type of soil is not very prevalent. In practice, selenite or selenate can be added to fertilizers, seeds can be treated with aqueous selenium, or crops sprayed with selenium salts as safe and effective means to increase the Se intake in food for both animals and humans (Aspila, 2005). However Se concentration of a particular food may vary depending on the soil in which the agricultural crop was grown and the ability of plants to take up and accumulate this element (Gupta & Gupta, 2000). Improper uses of Se fortification methods could result in high Se intake and environmental contamination through irrigation (Banuelos, Lin, Wu, & Terry, 2002). Examples of seleniferous areas include the Western USA and Hubei province in China, where Se levels may exceed 10 mg Se kg1, whereas other areas such as the Northeastern USA, Finland, and New Zealand have little or no Se in their soils (Hartikainen, 2005). Fengcheng, located in central Jiangxi province of China, is
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known as the Chinese eco-selenium Valley with high quality natural seleniferous soil resource. Statistical data from the Jiangxi Province Bureau of Quality and Technical Supervision show the distribution area of natural seleniferous soil of Fengcheng is around 524.7 km2 with Se content in the range of 0.4–0.99 lg g1, and an average of about 0.538 lg g1 (suitable Se-enriched range was 0.4–3.0 lg g1). We have analysed rice and soybean planted in this region, and found their Se contents to be 0.096 ± 0.011 lg g1 and 0.124 ± 0.009 lg g1, respectively, suggesting that they are novel natural Se-enriched food resources. More importantly, Se in the soil exists in states that are available to be absorbed and utilised easily by plants. After biotransformation by plants, Se is incorporated as selenocysteine at the active site of a wide range of proteins. At these sites, Se exerts its biological function in several selenoproteins (Brown & Arthur, 2001). The protein in soybean seeds accounts for about 40% of the dry seed weight, and is the major source of plant proteins for human and animal nutrition, which would be among the most effective plants for biofortification. However, to the best of our knowledge, the comparison of uptake, translocation, and distribution of Se, as well as functional properties of selenoproteins in soybean cultured in different regions at different Se content levels have never been reported before, especially for soybeans planted in natural seleniferous soils. The purposes of this study were to measure the Se contents and status of different parts of soybean growing in seleniferous soils and to analyse nutrient compositions and antioxidant activities of Se-enriched protein extracts. The study was designed to assist in systematically understanding the accumulation and biotransformation processes of natural Se of soybean during different periods for the development of efficient natural Se-enriched food supplements.
2.3. Extraction of different Se states in different soils The various Se states of soil were extracted sequentially as described by Zhang, Zhou, and Zhang (1997) with some modifications. Briefly, water soluble state Se (H2O–Se), acid soluble state Se, exchanged form Se, organic combination state Se, and residual state Se were extracted with ultrapure water, 3.0 mol L1 HCl, 0.1 mol L1 K2PO4 + KH2PO4 (pH 7.0), 1 mol L1 K2S2O8, and HNO3 + HF + HClO4 (v/v/v, 8:1:1), respectively. The extraction solvent to soil ratio was set at 30:1 (v/m), under ultrasonic extraction for 2.5 h. Each supernatant was obtained by centrifugation at 4800 rpm for 15 min and the residue was extracted twice, after which the supernatants were mixed. Three samples (5 ml) were taken from each sample for analysis. The determination of Se in solution was performed by an atomic fluorescence spectrophotometry (Model AI 3300, Aurora Technologies, Canada). 2.4. Preparation of soybean protein isolate Soybean protein isolate was prepared by an alkaline extraction, followed by precipitation at the isoelectric point according to Sorgentini, Wagner, and Anon (1995). The defatted soybean seed sample (100 g) was extracted with 1000 ml water adjusted at pH 8.0 with 2 mol L1 NaOH at room temperature for 3 h under continuous stirring. The supernatant was obtained by centrifugation at 4800 rpm for 15 min and the residue was extracted twice. The proteins from combined supernatants from the extractions were isoelectrically precipitated at pH 4.5 using 2 mol L1 HCl and kept at 4 °C for 1 h. The precipitate was recovered by centrifugation at 4800 rpm for 15 min, and then washed with deionized water. The protein isolates were freeze-dried and stored at 4 °C until further use. The total soybean protein content (g/100 g) in seeds was estimated by AOAC (1990), using a nitrogen conversion factor of 6.25.
2. Materials and methods
2.5. Purification of high Se-containing soybean protein isolate
2.1. Soils and soybean materials
The lyophilized C region soybean protein isolate was dissolved in 0.05 mol L1 Tris–HCl (pH 8.0). The solution (1.0 mL) was passed through a Q Sepharose Fast Flow anion exchange column (1.0 25 cm) (Sigma–Aldrich Co., St. Louis, MO), which had been equilibrated with 0.05 mol L1 Tris–HCl (pH 8.0) buffer. Anion exchange chromatography was performed using 0.8 mol L1 NaCl and 0.05 mol L1 Tris–HCl (pH 8.0) buffer as a mobile phase with a flow rate of 1.0 mL min1. All protein purification steps were done at 4 °C, and proteins eluted from the columns were monitored at the measuring absorbance at 280 nm. Meanwhile, the Se content of each peak was measured by atomic fluorescence spectrophotometry. The peak with the highest Se content was collected, lyophilized, and stored at 15 °C until further use. The attained high Se-containing protein was termed Se-soybean protein isolate (Se-SPI).
Collection of soils with different Se contents. Soils were obtained from three different regions in Fengcheng, Jiangxi, China. They were from Hehu village (A region), Shangzhuang village (B region), and Dong village (C region). The Se content in the soils is in the order of A region < B region < C region, which were confirmed by our own measurements in this work. The trial was carried out on 100 m2 plots in triplicate. Five subplots were selected at the four corners (25 25 cm) and the centre of the plot (1 1 m). Soil samples were collected from the surface soil (0–25 cm) with a shovel in each subplot, then mixed and milled. Finally a subsample (about 2 kg) was taken for use (Pérez-Sirvent, Martínez-Sánchez, GarcíaLorenzo, Molina, & Tudela, 2009).
2.2. Preparation of soybean
2.6. Determination of Se content
This trial was carried out on 50 m2 plots in triplicate for each treatment. The soybean seed species (Jiangxi 90-77-2, harvested on July 10th, 2010) used in the three regions was purchased from Jiangxi Academy of Agricultural Science, as the most widespread cultured species in the studied region. After the soybean seedlings were cultured, samples of soybean in C region were obtained every 15 days to measure the total Se content in the soybean’s roots, stems, leaves, and seeds at different growing periods. The determination of Se in solution was performed by an atomic fluorescence spectrophotometry (Model AI 3300, Aurora Technologies, Canada).
Three samples (0.2 g, or 10 mL) for testing were taken from prepared samples above for analysis. The test samples were digested with 10 mL of mixture of HNO3 and HClO4 (v/v, 9:1) at 150 °C for 2.5 h. After cooling, 5 mL of 6 mol L1 HCl was added to the digested samples to reduce Se6+–Se4+. When cooled, the digested solution was diluted with ultrapure water to 25 mL. Then 10 mL of the solution was transferred to a reaction vessel, with 2 mL of 6 mol L1 HCl and 1 mL of 10% (w/w) K3Fe(CN)3 added. The control undergoing the same procedure was used to provide a blank value. The determination of Se in solution was performed by an atomic fluorescence spectrophotometry (Model AI 3300, Aurora
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Technologies, Canada). The measuring conditions were conducted according to Wu, Jin, Shi, and Bi (2007) with some modifications.
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Hydroxyl radical scavenging effect ð%Þ ¼ ½ðA1 A2 Þ=A1 A0 100%
ð2Þ
2.7. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) SDS–PAGE was run according to the method of Laemmli (1970) at 4% stacking gel and 15% separating gel using a Bio-Rad Mini PROTEANÒ 3 system (Bio-Rad Laboratories, Hercules, CA, USA), at a constant current setting of 250 mA for approximately 1.5 h. Protein bands were stained with 0.125% Coomassie Brilliant Blue R-250 and destained with 25% ethanol and 10% acetic acid. The protein bands were estimated using pre-stained molecular weight standards (Sigma Chemical Co., St. Louis, MO), ranging from 10 to 170 kDa. 2.8. Amino acid analysis Amino acid compositions were determined by an HPLC system for automatic amino acid analysis (L-8800, Hitachi, Japan) after protein samples were hydrolysed with 6 M HCl at 110 °C for 24 h in a sealed tube, according to the method reported by Gehrke, Wall, Absheer, Kaiser, and Zumwalt (1985). The methionine and cysteine contents were determined after performic acid oxidation. The tryptophan content was determined after alkaline hydrolysis. The amino acid compositions were reported as g/ 100 g protein. 2.9. Determination of superoxide radical scavenging effect The superoxide radical-scavenging activities of different Se content soybean protein isolates and the Se-SPI were examined using a pyrogallol autoxidation system (Marklund & Marklund, 1974) with some modification. A reaction solution containing 80 lL of pyrogallol (10 mmol L1) and 4.5 mL of Tris/HCl/EDTA buffer (50 mmol L1, pH 8.0) was prepared. Then 0.5 mL of samples (0–1 mg mL1) was added. After incubation at room temperature for 30 min, the absorbance of the supernatant was determined at 365 nm by an UV spectrophotometry (TU1901, Purkinje General Instrument Co., Ltd., Beijing, China). The trolox (0–1 mg mL1) was used for the comparison group. The scavenging effect was calculated according to the following equation:
Scavenging effect ð%Þ ¼ ½1 ðA1 A2 Þ=A0 100%
Fig. 1. Se states (A) and total Se contents (B) of soil, soybean and soybean protein in different regions.
ð1Þ
where A0 is the absorbance of the control (without sample), A1 is the absorbance in the presence of the sample, and A2 is the absorbance of the sample without pyrogallol. 2.10. Determination of hydroxyl radical scavenging effect The hydroxyl radical scavenging assay was modified on the basis of the method described by Li, Jiang, Zhang, Mu, and Liu (2008). Different Se-SPI samples (50 lL, at a final concentration of 0–1 mg mL1 in 0.1 mol L1 sodium phosphate buffer, pH 7.4) were first added to a 96-well microplate, and then followed by the addition of 50 lL of 3 mmol mL1, 10-phenanthroline (in phosphate buffer), and 50 lL of 3 mmol mL1 FeSO4 (in water). To initiate the reaction, 50 lL of 0.01% aqueous H2O2 was added, and the reaction mixture was covered and incubated at 37 °C for 1 h with shaking. The absorbance was measured at 536 nm with a spectrophotometer, while solutions without protein sample and H2O2 were used as blank and control, respectively. Trolox (0– 1 mg mL1) was used for comparison as well. The hydroxyl radical scavenging activity was assessed according to the following equation:
Fig. 2. Se contents of leaf, stem, root and soybean in different growing periods (15 d: bud and seeding stage; 30 d: bud differentiation stage; 45 d: flowering and pods formation stage; 60–75 d: pod filling stage; 90 d: maturation stage).
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Table 1 Total amino acid composition of soybean protein from different regions. Amino acid (%)
A region
B region
C region
Threonine Valine Isoleucine Leucine Lysine Tryptophan Methionine Phenylalanine Histidine Total essential amino acid Aspartic acid Tyrosine Serine Glutamic acid Glycine Arginine Cysteine Proline Alanine
3.352 ± 0.168 3.152 ± 0.189 3.285 ± 0.131 6.327 ± 0.253 5.567 ± 0.223 1.406 ± 0.070 0.523 ± 0.209c 4.445 ± 0.178 1.980 ± 0.138 30.037 9.432 ± 0.189 7.777 ± 0.155 4.703 ± 0.329 15.739 ± 0.315 3.270 ± 0.229 5.490 ± 0.329 0.682 ± 0.034c 7.827 ± 0.391 3.409 ± 0.102
3.410 ± 0.238 3.248 ± 0.292 3.245 ± 0.389 6.375 ± 0.573 5.784 ± 0.347 1.466 ± 0.043 0.207 ± 0.015b 4.425 ± 0.354 2.177 ± 0.109 30.130 9.577 ± 0.479 7.731 ± 0.463 4.886 ± 0.147 16.163 ± 0.808 3.427 ± 0.171 5.723 ± 0.515 0.316 ± 0.037b 8.345 ± 0.500 3.477 ± 0.278
3.503 ± 0.211 3.292 ± 0.230 3.196 ± 0.127 6.394 ± 0.256 5.652 ± 0.283 1.513 ± 0.106 0.112 ± 0.003a 4.413 ± 0.309 2.196 ± 0.154 30.159 9.614 ± 0.577 7.712 ± 0.536 4.914 ± 0.196 16.325 ± 0.653 3.412 ± 0.204 5.812 ± 0.232 0.102 ± 0.012a 8.496 ± 0.595 3.553 ± 0.247
Total unessential amino acid Total soybean protein content (g/100 g)
57.647 41.93 ± 2.10a
59.329 44.71 ± 1.79a,b
59.838 46.64 ± 2.33b
Each value is expressed as mean ± standard deviation of triplicate tests with at least three measurements. Means within the same row with different letters are significantly different (p < 0.05), according to Duncan’s multiple-range test.
2.11. Statistical analysis The analysis of the variance was performed with the Statistical Analysis System software 8.2 (SAS, USA). Differences among means were evaluated using Duncan’s multiple range tests. The significance was established at p < 0.05. 3. Results and discussion 3.1. Distribution of Se content in soil, soybean protein, and soybean organ
Fig. 3. (A) SDS–PAGE analysis, band 1–3: soybean protein from A region, B region and C region. M: markers, Group molecular weights I: 70.0–100.0 kDa, II: 45.0– 55.0 kDa, III: 35.0–40.0 kDa, IV: 15.0–25.0 kDa; (B) Se content of different soybean protein subunits from different regions.
where A1, A2, A0 are the absorbance of the control, samples and blank, respectively.
The biological utilisation of Se depends on the amount of Se in environmental media, as well as the existence and transformation of Se states (Hartikainen, 2005). The Se-combined states were classified as available state, potential available state and useless state (Sharmasarkar & Vance, 1995). Available state Se mainly refers to water-soluble Se, including selenite and selenate that could be easily absorbed and utilised by plants. Their contents are often regarded as the key parameter to evaluate if the soil has sufficient effective or utilisable Se. Potential available Se mainly consists of acid soluble state Se, exchanged state Se, and organic combination state Se, as direct sources of the available Se, while residual state Se could not be utilised (Sharmasarkar & Vance, 1995). In the three collected soils, water-soluble state Se content in the C region was the highest, approximately 10 times than that of in A region (Fig. 1A). The potential available state Se of the total Se content were 30.4%, 25.2%, and 41.7% for A, B, and C region, respectively (Fig. 1A). The result implied that the total Se in C region could be utilised by plants more effectively. The total Se contents of soybean and soybean protein from C region were also much higher than those of B and A regions (Fig. 1B). Hence, Se contents of soybean and soybean protein could be determined from contents of water-soluble state Se and potential available state Se in the soil. Soybean growing process included bud and seeding stage, bud differentiation stage, flowering and pods formation stage, pod filling stage and maturation stage. Fig. 2 showed that the order of total content of Se was root > leaf > stem in bud and seeding
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stage, bud differentiation stage, and flowering and pods formation stage. The order of total Se content of different organs was root > leaf > soybean > steam in pod filling stage, while Se distribution in different organs was root > soybean > leaf > stem in the mature period (harvest). The Se contents of root in flowering and pods formation stage, and pod filling stage were lower than those in the bud and seeding stage. There were significant differences between mechanisms involved in uptaking and transporting of selenate, selenite and organic Se compounds like selenomethionine (Se-Met) (Terry, Zayed, De Souza, & Tarun, 2000). Selenite absorption is a passive absorption that requires little energy (Abrams, Shennan, Zasoski, & Burau, 1990), while selenate absorption is an active transportation that could be inhibited by respiratory depressant (azides or dinitrophenol) or low temperature. Organic selenium, such as seleno methionine, would also be absorbed by plant roots by active transportation (Sors, Ellis, & Salt, 2005). Compared to the selenite and organic Se, selenate was much easier to be transferred and
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absorbed from roots, and then accumulated in leaves. Most selenite was in the root and would be converted into organic Se quickly, especially in the form of seleno-amino acids (Zayed, Lytle, & Terry, 1998). Although the initial stage of Se being absorbed and converted into selenocysteine is the same in Se accumulator plants (such as soybean) and Se non-accumulator plants (such as grain), the subsequent steps of their metabolic pathways are different. In Se accumulator plants, selenocysteine was metabolized through formation of leno-amino acids (Brown & Shrift, 2008), including seleno-methyl-selenocysteine, c-glutamyl-seleno-methyl-selenocysteine, and selenocystathione (Terry et al., 2000). However, in Se non-accumulator plants, Se combined with selenocysteine would act as zymoprotein when getting into proteins (Terry et al., 2000). Thus, due to the difference of metabolic pathways, Se accumulator plants could withstand high concentration Se substance, while Se non-accumulator plants are more susceptible to Se poisoning (Sors et al., 2005).
Fig. 4. (A) Elution diagram from an anion exchange column of high Se-containing protein from C region; (B) SDS–PAGE analysis of peak 4 from anion exchange chromatography. Left lane, molecular weight markers; Right lane, peak 4 after anion exchange chromatography.
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Se distribution and concentration in different organs of plants varied with different kinds of plants. In Se accumulator plants, such as soybean, Se is accumulated in leaves first, and then in seed (fruit) parts. But in Se non-accumulator plants, such as grain, the content of Se accumulation in the roots is often the same as those in seeds or grain (Terry et al., 2000). A study of 17 different vegetables showed that the Se contents of edible parts were lower than those of non-edible parts (Hamilton & Beath, 1963). As shown in Fig. 2, the Se contents in roots and stems were higher than those in soybean fruit during the whole soybean growing period, while the Se contents in roots and stems decreased in the soybean at the mature period. Se could be volatilised as dimethyl-diselenide via oxidation and subsequent methylation of ethylselenocysteine. Moreover, the volatilisation of dimethyldiselenide was observed in the Se hyper-accumulator Asparagus racemosus, known to accumulate large quantities of Se-methylselenocysteine (Sors et al., 2005).
Peak 4 exhibited the highest Se content of 0.256 lg g1, which was also markedly higher than those of crude proteins (0.164 lg g1, C region). Therefore, this fraction was targeted for further purification. SDS–PAGE analysis showed that peak 4 contained a single band (Fig. 4B), indicating a novel high Se-containing soybean protein was attained, namely Se-SPI with a molecular weight of about 18.0 kDa. The Se content of Se-SPI was significantly higher and the composition of amino acids was possibly different from other proteins corresponding to the other three major protein peaks. Liu, Zhao, Chen, Gu, and Bu (2012) had reported that selenium-containing proteins from rice have strong antioxidant activities. Although the chemical form of the Se-SPI still remained unknown, in the following section the improvement of antioxidant activity related to the Se content was investigated.
3.2. Effects of Se content on protein and amino acids
Se is considered to be an element possessing antioxidant activity. Its antioxidant activity was expressed by combining with GSH-Px, scavenging free radical, cutting off the chain reaction induced by the lipid peroxidation, analysing peroxide, and protecting the cell membrane (Tinggi, 2008). The profiles of different Se content proteins scavenging hydroxyl radical and super oxide radical were shown in Fig. 5. It was observed that the radical scavenging activities of different Se content proteins increased with protein
As shown in Table 1, the total essential amino acids content in the soybean protein from C region (30.159%) was close to those from B region (30.13%), but higher than that from A region (30.037%), while methionine and cysteine levels declined noticeably. In addition, the total soybean protein content from C region was much higher than that from the A region (p < 0.05), but was similar to that from B region (p > 0.05). Se could affect protein synthesis in soybean since seleno-amino acids, since selenocysteine and selenomethionine (Se-Met), could replace cysteine and methionine directly during protein synthesis process, leading to a reduction of cysteine and methionine (Brown & Shrift, 2008). As Se atomic size and ionisation could change the tertiary structure of protein, this particular effect was observed in some important protein synthesis and reaction performance (Brown & Shrift, 2008). On the contrary, in other plants such as tea and green algae (Hu, Xu, & Pang, 2003), the presence of Se could enhance organism for protein synthesis.
3.5. Antioxidant activities of different Se content proteins
3.3. Effect of Se content on the subunit composition of soybean protein The subunit compositions of total protein extracted from different Se containing soybeans were analysed by SDS–PAGE. Fig. 3A showed that Se content did not change the distribution of subunit of protein in soybean protein. The spectral band was a basic convergence uniform in different Se content soybean protein, with none of protein band disappearing or new protein band appearing. The strips in band 1 were fainter, possibly due to the lower total protein content (Table 1). Fig. 3A also indicated that the protein extracts from different Se containing soybean had a large distribution of molecular weights with ranging from 15.0 to 110.0 kDa. In order to investigate the Se content of the subunit composition, every band was divided into four protein sections. These sections were then separated from the gel and the Se content of each section was determined. As shown in Fig. 3B, proteins with molecular weights of 15.0–25.0 kDa had the highest Se content, while proteins with molecular weights of 70.0–100.0 kDa had the lowest Se content. The result showed that Se was inclined to combine with low molecular weight proteins, in agreement with Zhao et al. (2004) showing that the molecular weight of most proteins or protein subunits containing Se was less than 16 kDa. 3.4. A novel high Se-containing soybean protein After loading the protein sample (Se content, 0.164 lg g1, from C region) into an anion exchange column, the elution profile of protein and Se are presented in Fig. 4A, showing four major peaks.
Fig. 5. Scavenging activities of containing different Se content soybean protein against hydroxyl radical (A) and superoxide radical (B).
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concentrations. The Se content of Se-SPI (0.256 lg g1) was almost 18 times higher than that of the control protein (A region, 0.014 lg g1), while the antioxidant activity of Se-SPI was almost 4-fold higher compared to that of the control protein, although weaker than trolox. Thus, we could conclude that the improvement of the antioxidant activity of selenoprotein quantitatively depended on its Se content. The essential micronutrient Se occurs in the form of selenocysteine in selenoprotein exerting various effects including maintaining the cell reduction–oxidation balance. Previously, it was shown that selenomethionine was the major selenocompound in the protein (Zhang et al., 2009). As a free radical scavenger, selenoamino acids react with hydrated free radicals to form stable compounds before destroying biological macromolecules. Although the mechanism on how Se enhances the antioxidant activity of the protein is still unclear, when the protein is combined with Se, its antioxidant activity has been shown to remarkably improved. Our results were in agreement with previous findings, which showed that the antioxidant activities of green tea and phycocyanin were enhanced after Se was incorporated, and the scavenging effect was increased with Se levels increased. A possible explanation for the enhancing effect was the unique atom structure of Se (Shen et al., 2010). The ionisation energy of the 4p electron in selenium was much lower than that of the 3p electron in sulphur which made selenoamino acids much easier than sulfoamino acids became positive ion free radical. In addition, the selenoamino acid radical was more stable than that of sulfoamino acid because of the existence of the empty 4f orbit. When the sulphur in Se containing proteins was replaced by selenium or some new selenoamino acids were synthesized, stable positive ion free radicals that possess strong antioxidant activity were much easier to be formed by selenoamino acids. 4. Conclusion In summary, to the best of our knowledge, this was the first time that the process of Se accumulation by soybean produced from soils with different natural Se levels was reported. The effects of the Se content on amino acids and subunit composition of SeSPI, as well as antioxidant activities of the abstracted Se-SPI were also evaluated. The results indicated that Se was accumulated in leaves first, and then in seed parts during the growing stage. The Se level had positive effect on the content of Se-SPI, as it could change the amino acids composition of Se-SPI, but exhibited negligible effects on the subunit composition of Se-SPI. The antioxidant activity experiments indicated that Se-SPI possessed significantly higher antioxidant activity than the control protein, suggesting that Se played a positive role in enhancing the antioxidant activity of selenoprotein. The mechanism(s) involved in the antioxidant activities of protein isolated from Se-enriched soybean require further studies in vivo. Acknowledgements This research was supported in part by the National High Technology Research and Development Program of China (863 Program) (2013AA102203-5), the National Natural Science Foundation of China (31160317 and 31301436), and the Programs of State Key Laboratory of Food Science and Technology of Nanchang University (SKLF-MB-201006, SKLF-TS-201116). References Abrams, M. M., Shennan, C., Zasoski, R. J., & Burau, R. G. (1990). Selenomethionine uptake by wheat seedlings. Agronomy Journal, 82(6), 1127–1130. AOAC (1990). Official methods of analysis of the association of official analytical chemists (15th ed.). Washington, DC, USA: Association of Official Analytical Chemists.
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