Comparative study of the effects of selenium nanoparticles and selenite on selenium content and nutrient quality in soybean sprouts
Article Category: Original Article
Published Online: Dec 31, 2022
Page range: 223 - 234
Received: Jan 21, 2022
Accepted: Oct 25, 2022
DOI: https://doi.org/10.2478/fhort-2022-0017
Keywords
© 2022 Shen Rao et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
Soybean (
Because vegetables can take up and transform Se, they are ideal Se carriers for daily dietary Se supplementation. Due to the chemical similarities of Se and sulphur, plants generally take up and assimilate it through the sulphur metabolism system (Lima et al., 2018). Se can be converted into seleno-amino acids, such as selenocysteine (SeCys) and selenomethionine (SeMet) (Lima et al., 2018). Methylated SeCys (MeSeCys) and methylated SeMet (MeSeMet) may be formed from SeCys and SeMet, respectively (Chen et al., 2019; Kolbert et al., 2019). These organic Se forms have bioactive properties that are helpful to human health, including anti-cancer (Radomska et al., 2021) and inflammatory regulation (Hariharan and Dharmaraj, 2020). As a result, they are crucial nutritional molecules for plants. Soybeans have a high capability for Se accumulation (Chan et al., 2010). SeMet, SeCys, MeSeCys and SeCys2 are among the Se compounds identified in soybean, while SeMet is the most abundant (Lu et al., 2018; Deng et al., 2022). Exogenous Se treatments can also boost antioxidant defences and raise salt tolerance in soybean plants, according to a recent study (Rahman et al., 2021). Nonetheless, the majority of these findings came from soybean seeds or plants, few studies focussed on the effects of exogenous Se on soybean sprouts.
Studies have demonstrated that exogenous Se treatments exert influences on the nutrient of crops. Selenate treatments reduce overall glucosinolate levels while increasing flavonoid content in broccoli florets (Rao et al., 2021a). By contrast, Se yeast increased overall glucosinolate levels while decreasing flavonoid content (Gui et al., 2021). Se yeast and selenite both had opposing effects on vitamin C levels in broccoli florets, with the former increasing while the latter decreasing (Gui et al., 2021). Similarly, selenite has been found to reduce the amount of vitamin C in broccoli sprouts (Tian et al., 2016). These findings suggest that various Se sources and phases of development have varied effects on crop nutrient molecules.
Selenium nanoparticles (SeNPs) are a kind of elemental Se with bioactivity for plants. Bioactive SeNPs generally are produced by some Se-resistant microorganisms, such as
To reveal the differences between SeNPs and selenite in cultivating Se-enriched soybean sprouts, different concentrations of SeNPs and selenite were employed to treat hydroponically cultivated soybean sprouts in this study. Total Se content, Se speciation and several important nutritional indicators of soybean sprouts were compared. The findings of this study will contribute to a better understanding of the role of exogenous Se in the production of Se-enriched soybean sprouts.
Soybean seeds (cultivar: ‘Dongnong690’) were purchased from Wuhan Fengyuan Seed Co. Ltd. (Wuhan, China). Sodium selenite was bought from Sigma-Aldrich (Shanghai) Trading Co. Ltd. (Shanghai, China). Bioactive SeNPs (suspension with a Se concentration of 5 mg · mL−1) with a particle size of 100–600 nm were obtained from the Institute of Agricultural Economics and Technology, Hubei Academy of Agricultural Sciences (Wuhan, China). Soybean seeds were soaked in deionised water for 8 h. Sixty plump seeds were selected, put on a plastic mesh plate (mesh size: 0.3 cm length × 0.3 cm width) and then germinated in a plastic hydroponic box (34 cm length × 25 cm width × 4.5 cm depth). The plastic hydroponic box was added with 1 L of deionised water. Deionised water was replaced with SeNPs or selenite solutions after the plantule reached a length of 1 cm. A single factor experiment was carried out, and the test variable was Se concentration. The concentrations of the SeNPs or selenite solutions were set as follows: 10 μM, 20 μM, 40 μM, 80 μM and 100 μM. Deionised water was used as control. The solutions with SeNPs or selenite were renewed every 2 days. The plants were placed in an illumination incubator for growth with the following growth parameters: 24 °C and 16 h of illumination during the day, 20 °C and 8 h of darkness during the night, 75% of relative humidity and 300 μmol · m−2 · s−1 of optical density. Each treatment was set with three biological replications. Soybean sprouts were collected, weighed and separated into roots and shoots after 7 days of two exogenous Se treatments. Fresh samples were frozen in liquid nitrogen and stored at −80 °C. Samples for Se detection were dried at 60 °C, crushed and kept in an airtight container.
The total Se of soybean sprouts was determined following the method of Rao et al. (2020). In brief, 0.2 g of samples was weighed, placed in a digestion tube and treated with 10 mL of guaranteed nitric acid and 2 mL of hydrogen peroxide. This solution combination was placed in a microwave digestion system and digested according to a set of instructions until it was clear. The clear solution was then mixed with water to make up 10 mL for total Se determination using hydride generation atomic f luorescence spectrometry (HG-AFS). Working conditions of HG-AFS (AFS8530, Beijing Haiguang Instrument, Beijing, China), were as follows: negative high voltage, 320 V; lamp current, 90 mA; atomisation temperature, 800 °C; carrier gas flow rate, 500 mL · min−1; injection volume, 1 mL.
Determination of Se speciations in soybean sprouts was performed using liquid chromatography-HG-AFS (LC-AFS530, Beijing Haiguang Instrument, Beijing, China) following the method of Rao et al. (2021b). The five Se standard compounds (SeCys2, SeMet, MeSeCys, Se4+ and Se6+) were bought from the National Institute of Metrology, China, and used to establish the standard equation. Se speciations were extracted by enzymatically hydrolysing 0.1 g of samples with proteinase K and pronase E under ultrasonication at 37 °C for 1 h. The solution was then centrifuged at 10,000 rpm for 20 min. The supernatant was filtered through a 0.2 μm filtration membrane for detection. LC-HG-AFS working conditions were as follows: mobile phase, 40 mM KH2PO4 + 20 mM KCl; pH, 6.0; flow rate, 1.0 mL · min−1, chromatographic column, Hamilton PRP-X100 (Hamilton Co., Reno, NV, USA); column temperature, 27 °C; injection volume, 150 μL; cathodic current, 80 mA; carrier gas flow rate, 600 mL · min−1 and negative high voltage, 320 V.
Chlorophyll, carotenoid, soluble sugar and soluble protein contents were determined according to the method recorded by Zhang and Li (2006). Briefly, 0.2 g of shoots of soybean sprouts were homogenised with 95% ethanol and centrifuged at 4,000 rpm for 10 min. Chlorophyll and carotenoid were detected at 665 nm, 649 nm and 470 nm by an ultraviolet-visible (UV-Vis) spectrophotometer. Soluble sugar content was determined via anthrone colorimetry with a UVVis spectrophotometer at 630 nm. Soluble protein was evaluated by coomassie brilliant blue G-250 colour rendering at 595 nm.
Vitamin C content was measured using a vitamin C colorimetric assay kit (A009-1-1, Nanjing Jiancheng Bioengineering Co. Ltd., Nanjing, China) according to the operation manual. Total isoflavone content was measured following the method described by He et al. (2013). Briefly, 0.2 g of soybean shoots was added with 4 mL of 80% ethanol, ultrasonically extracted at 60 °C for 30 min and centrifuged at 8,000 rpm for 10 min. The supernatant was collected and measured via a UV-Vis spectrophotometer at 260 nm. Genistein was used to plot the standard curve.
The content of MDA was measured using thiobarbituric acid (TBA) colorimetry following the method of Siddiqui et al. (2012). In brief, 0.5 g of shoots was ground with 5% of trichloroacetic acid and centrifuged at 3,000 rpm for 10 min. Two milliliters of the supernatant were transferred and added with 2 mL of 0.67% TBA. The solution was water bathed at 100 °C for 10 min and centrifuged at 3,000 rpm for 10 min. The supernatant was collected and detected at 450 nm, 532 nm and 600 nm. MDA content was calculated according to the formula. Glutathione (GSH) content was determined using a GSH assay kit (A006-2-1, Nanjing Jiancheng Bioengineering Co. Ltd., Nanjing, China) following the procedure recorded in the specification. Activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and ascorbate peroxidase (APX) were measured by using CAT (A007-1-1), POD (A084-3-1), SOD (A001-3-2) and APX (A123-1-1) assay kits produced by Nanjing Jiancheng Bioengineering Co. Ltd. (Nanjing, China).
Data were analysed using SPSS (version 19, SPSS Inc., Chicago, IL, USA) with one-way ANOVA followed by Duncan's new complex range test. All data were expressed as mean ± standard errors from three biological replicates. The significant difference was set at
Similar changing trends in the biomass of soybean sprouts were observed between SeNPs and selenite treatments. As shown in Figure 1, 10 μM, 20 μM and 40 μM of SeNPs and 10 μM of selenite significantly increased the fresh weight of soybean sprouts compared with the control. Notably, the fresh weight of soybean sprouts was remarkably lower in the 100 μM of SeNPs treatment than in the control. Different concentrations of selenite treatments, except for 10 μM, dramatically reduced soybean sprout biomass.
Figure 1
Biomass statistics of soybean sprouts. Different letters above the columns indicate significant differences at
The total Se concentration of soybean sprout shoots and roots was dramatically increased by both SeNPs and selenite. In the shoots, total Se concentration significantly increased following the increase of exogenous Se dosage (Figure 2A). For example, the total Se concentration in soybean shoots treated with 100 μM of SeNPs or selenite was 31-fold or 44-fold higher than that under control. However, compared with 80 M of SeNPs or selenite, 100 M of SeNPs or selenite resulted in a significant reduction in the total Se concentration of the shoots.
Figure 2
Total Se concentrations in the shoots (A) and roots (B) of soybean sprouts treated with SeNPs and selenite. Different letters above the columns indicate significant differences at
The application of exogenous Se substantially increased the total Se concentration in the roots of soybean sprouts. The highest total Se concentrations were 518 μg · g−1 DW and 587 μg · g−1 DW under 100 μM of SeNPs and 100 μM of selenite treatment, respectively (Figure 2B). The total Se concentration in the roots was considerably greater than that in the shoots under the same conditions. For instance, the total Se concentration was 425 μg · g−1 DW and 456 μg · g−1 DW in the roots treated with 100 μM of SeNPs and 100 μM of selenite, respectively, whereas the total Se concentration was 46 μg · g−1 DW and 66 μg · g−1 DW in the corresponding shoots.
Samples treated with 20 μM and 100 μM exogenous Se were utilised for Se speciation determination to demonstrate variations in Se speciation of soybean sprouts treated with SeNPs and selenite (Table 1). The five Se speciations can be well detected through LCHG-AFS (Figure S1 in Supplementary Material). In soybean sprouts treated with SeNPs, five different Se species were found. Se6+, on the other hand, was not detected under selenite treatments. In the shoots, the concentrations of SeCys2 and MeSeCys were higher under SeNPs than those under selenite, whereas the concentrations of SeMet and Se4+ were lower under SeNPs than those under selenite. In the roots, selenite treatments possessed higher concentrations of SeCys2, MeSeCys, SeMet and Se4+ than the SeNPs treatments. Interestingly, SeMet was the dominant Se speciation in the shoots, while the three organic Se forms in the roots, namely, SeCys2, MeSeCys and SeMet, were equivalent in concentration.
Selenium speciation in soybean shoots and roots.
| Tissue | Treatment | Selenium speciation (μg · g−1 DW) | ||||
|---|---|---|---|---|---|---|
| SeCys2 | MeSeCys | SeMet | Se4+ | Se6+ | ||
| Shoot | 0 | nd | nd | 0.193 ± 0.021 d | 0.035 ± 0.008 c | nd |
| N20 | 0.188 ± 0.022 b | 0.185 ± 0.048 c | 2.182 ± 0.258 c | 0.071 ± 0.023 c | 0.453 ± 0.001 b | |
| N100 | 1.192 ± 0.199 a | 0.953 ± 0.097 a | 3.789 ± 0.611 b | 0.147 ± 0.025 b | 0.792 ± 0.092 a | |
| S20 | 0.093 ± 0.019 b | 0.087 ± 0.008 d | 1.857 ± 0.189 c | 0.048 ± 0.005 c | nd | |
| S100 | 0.969 ± 0.041 a | 0.696 ± 0.012 b | 4.796 ± 0.065 a | 0.248 ± 0.006 a | nd | |
| Root | 0 | nd | nd | 0.127 ± 0.015 d | 0.034 ± 0.008 e | nd |
| N20 | 2.245 ± 0.214 d | 2.292 ± 0.286 c | 3.897 ± 0.240 c | 0.888 ± 0.051 d | 2.444 ± 0.057 b | |
| N100 | 6.318 ± 0.377 b | 7.224 ± 0.336 b | 5.734 ± 0.250 b | 1.759 ± 0.066 b | 6.870 ± 0.356 a | |
| S20 | 3.613 ± 0.084 c | 2.188 ± 0.255 c | 3.601 ± 0.178 c | 1.046 ± 0.015 c | nd | |
| S100 | 11.135 ± 0.716 a | 8.698 ± 0.235 a | 14.213 ± 0.059 a | 1.970 ± 0.021 a | nd | |
Different letters behind the data indicate significant differences at
MeSeCys: methyl selenocysteine; N100, 100 μM Se nanoparticles; N20, 20 μM Se nanoparticles; nd: not detected; S100, 100 μM selenite;S20, 20 μM selenite; Se4+: selenite; Se6+: selenate; SeCys2: selenocystine; SeMet: selenomethionine.
Chlorophyll and carotenoid contents were influenced by SeNPs and selenite treatments. As demonstrated in Figure 3A, SeNPs caused a significant increase in chlorophyll content, whereas selenite treatments (except 80 μM) led to an insignificant increase in chlorophyll content. The changes in carotenoid content were similar to the changes in chlorophyll content. Carotenoid content in soybean sprouts significantly increased following the increase of SeNPs dosages (Figure 3B). Selenite treatments increased carotenoid content as well, although only at concentrations of 10 μM and 80 μM.
Figure 3
Chlorophyll (A) and carotenoid (B) contents of soybean sprouts treated with SeNPs and selenite. Different letters above the columns indicate significant differences at
Treatments with SeNPs and selenite remarkably enhanced the generation of soluble sugar and protein in soybean sprouts (Figure 4). SeNPs and selenite both significantly increased the soluble sugar content of soybean sprouts (Figure 4A). Interestingly, the soluble sugar content of soybean sprouts under 80 μM and 100 μM of selenite treatment was much greater than that of soybean sprouts under 80 μM and 100 μM of SeNPs treatment. Selenite and SeNPs also posed a similar impact on the soluble protein level of soybean sprouts. As shown in Figure 4B, the soluble protein content prominently increased with the increase of SeNPs and selenite concentration. However, 10 μM of SeNPs or selenite conspicuously reduced the soluble protein content when compared with the control.
Figure 4
Soluble sugar (A) and protein (B) contents of soybean sprouts treated with SeNPs and selenite. Different letters above the columns indicate significant differences at
Vitamin C and isoflavone are the two kinds of important nutrient compounds in soybean sprouts. As shown in Figure 5A, SeNPs and selenite remarkably boosted the vitamin C content of soybean sprouts. For instance, the vitamin C content in soybean sprouts was 0.25 mg · g−1 FW in the control group, but it was 0.65 mg · g−1 FW and 0.81 mg · g−1 FW under 100 μM of SeNPs and selenite, respectively. They were 2.6-fold and 3.4-fold, respectively, of that under the control treatment. The total isoflavone content of soybean sprouts was also altered by SeNPs and selenite (Figure 5B). The total isoflavone content of soybean sprouts treated with SeNPs was significantly higher than that of the control. Notably, 10–40 μM of selenite remarkably enhanced the total isoflavone content of soybean sprouts, but 80 μM and 100 μM of selenite showed no significant influences compared with the control.
Figure 5
Vitamin C (A) and total isoflavone (B) contents of soybean sprouts treated with SeNPs and selenite. Different letters above the columns indicate significant differences at
The changes in MDA and five antioxidant indexes, namely, GSH, CAT, POD, SOD and APX, were determined to evaluate the effects of SeNPs and selenite treatments on the membrane oxidation status and antioxidant properties of soybean sprouts. Results showed that SeNPs reduced MDA content in soybean sprouts (Figure 6A). Compared with the control, 40 μM and 80 μM of SeNPs led to a significant decrease in MDA content. The MDA concentration of soybean sprouts was dramatically decreased by 20 μM selenite treatment, but the MDA content was significantly elevated by 80 μM and 100 μM selenite treatment. SeNPs and selenite increased the GSH content in soybean sprouts (Figure 6B). GSH levels were 37.8% and 38.2% higher under 100 μM of SeNPs and 80 μM of selenite, respectively, than those under control.
Figure 6
Changes in the MDA and antioxidant system of soybean sprouts treated with SeNPs and selenite. (A) MDA content; (B) GSH content; (C) CAT activity; (D) POD activity; (E) SOD activity and (F) APX activity. Different letters above the columns indicate significant differences at
In soybean sprouts, the activity of three antioxidant enzymes (CAT, POD and SOD) was measured to access how they responded to SeNPs and selenite treatment. The results indicated that 10–40 μM of SeNPs and 10–20 μM of selenite remarkably enhanced CAT activity in soybean sprouts, but 100 μM of SeNPs and 80–100 μM of selenite dramatically decreased CAT activity when compared with the control (Figure 6C). Unlike CAT, POD activity increased as exogenous Se concentrations increased, as illustrated in Figure 6D. POD activity of soybean sprouts treated with 100 μM of SeNPs and selenite increased 71.7% and 175.5%, respectively, when compared with 0 μM of Se treatment. In contrast, the SOD activity of soybean sprouts was not significantly affected by SeNPs, but 40–100 μM of selenite remarkably decreased the SOD activity compared with the control (Figure 6E). SeNPs and selenite significantly increased the activity of APX. As shown in Figure 6F, AXP activity under 100 μM of SeNPs and selenite treatment was 2.86 and 2.56-fold of that under the control, respectively.
This study compared the effects of two Se forms, namely, SeNPs and selenite, on the growth and quality of soybean sprouts. Gui et al. (2021) also reported that 0.1–1.6 mM of Se yeast and selenite significantly increased and decreased the growth of broccoli florets, respectively. Here, similar results were obtained that selenite inhibited the growth of soybean sprouts. SeNPs are inorganic Se form, but Se yeast contains kinds of Se forms (mainly as SeMet) and other chemical compounds (Gui et al., 2021), implying that they would pose different influences on crops. Noticeably, as a novel type of Se source, SeNPs showed no significant influence on the growth of soybean sprouts (Figure 1).
A high concentration of Se accumulating in the plant body may hinder plant development and even be toxic (Kolbert et al., 2019). For example, 800 mg · L−1 of selenite significantly reduced the biomass of
Application with Se can differently affect the biosynthesis of chlorophyll in various plant species. For instance, leaf yellowing was observed in cucumber treated with 80 μM of selenite or 20 μM of selenate (Hawrylak-Nowak et al., 2015). Spraying or irrigating selenate showed no significant influences on chlorophyll content in lamb's lettuce (Hawrylak-Nowak et al., 2018). This study revealed that 0–100 μM of SeNPs or selenite has no reverse effects on photosynthesis of soybean. Carotenoids are important pigments that give leaves their red, orange or yellow colour and are important nutritional components for humans (Loladze et al., 2019). Here, SeNPs substantially increased the content of carotenoid in soybean shoots, thus promoting the nutrient quality of soybean sprouts. Soluble sugar is an essential component of vegetables and fruits (Wang et al., 2018; Hu et al., 2019). Several studies demonstrate that exogenous Se application causes an increase in soluble sugar content in some crops, such as selenite on broccoli (Gui et al., 2021), Se yeast on cabbage (Liao et al., 2021) and amino acid-chelated Se-enriched foliar fertiliser on grapefruits (Zhu et al., 2017). Similar to the results reported by these previous studies, the present study found that SeNPs and selenite stimulated the accumulation of soluble sugar (Figure 4A). Different plant species have various soluble protein accumulation patterns when they were treated with Se. For example, high dosages of Se applied on rice plants showed decreased soluble proteins (Mostofa et al., 2017), but treatment with selenate posed no significant influences on soluble protein level in
Plants are continuously defending themselves from oxidation. MDA is a useful metric for measuring the level of lipid peroxidation in plant membranes (Morales and Munné-Bosch, 2019). SeNPs treatment results in lower MDA levels than selenite treatment, implying that soybean sprouts have superior membrane integrity under SeNPs treatment than selenite treatment. GSH is a potent antioxidant molecule that plays a role in a wide range of environmental stressors in plants (Park et al., 2021). Both SeNPs and selenite increased the GSH levels in soybean sprouts, indicating that the two Se forms are beneficial to enhance the antioxidant activities. CAT plays an essential role in the defence reactions of plants because it can catalyse the extinction of hydrogen peroxide and protect cells (Anjum et al., 2016). Here, results of CAT activity indicate that a low dosage of SeNPs or selenite enhances the elimination of hydrogen peroxide, but a high dosage may aggravate the accumulation of peroxides in soybean sprouts. POD is another key antioxidase that participates in the scavenging of ROS, including hydrogen peroxide (Lukacova et al., 2021). Given that POD activity showed the highest in 100 μM SeNPs or selenite treatments (Figure 6D), POD may be the dominant peroxide remover in soybean sprouts treated with a high dosage of Se. SOD is a superoxide dismutase enzyme that includes multiple isozymes, including copper/zinc-SOD and iron-SOD (Saed-Moucheshi et al., 2021). Here, SeNPs showed no significant influence on SOD activity, but selenite posed a slight decrease in the SOD activity of soybean sprouts, indicating that SOD activity may be stable when soybean sprouts are exposed to Se. APX plays an important role in scavenging ROS, especially hydrogen peroxide in chloroplasts and relates to the redox reaction of vitamin C in plants (Li et al., 2022). Changes in APX activity were consistent with vitamin C content in the present study, indicating that Se treatments enhanced the antioxidant property of soybean sprouts. Taking the analysis together, we suggest that soybean sprouts can regulate ROS scavenging by adjusting the activity of some key antioxidases when they are treated with SeNPs and selenite.
This study revealed that both SeNPs and selenite significantly influenced the growth of soybean sprouts. The low dosage promoted the growth but the high dosage prohibited it. Both SeNPs and selenite remarkably increased the total Se concentration in soybean sprouts, although the former was less effective than the latter in terms of uptake efficiency. Five Se speciations, namely, SeCys2, SeMet, MeSeCys, Se4+ and Se6+, were detected in the shoots or roots of soybean sprouts treated with SeNPs. Four Se speciations were found in soybean sprouts treated with selenite, excluding Se6+. SeNPs and selenite increased the contents of chlorophyll, carotenoid, soluble sugar, soluble protein, vitamin C and isoflavones in soybean sprouts. SeNPs and selenite treatments differently affected MDA content and activities of several antioxidant enzymes. SeNPs treatments resulted in lower MDA content when compared with selenite. Both of SeNPs and selenite increased the GSH content. The modest dose of exogenous Se stimulated CAT activity, while the large dose inhibited it. SeNPs and selenite both increased the activity of POD and APX. SeNPs did not affect SOD activity; on the other hand, selenite reduced SOD activity. In conclusion, SeNPs may be a more suitable Se source than selenite for application in Se-enriched soybean sprouts.