Extraction of Se species in buckwheat sprouts grown from seeds soaked in various Se solutions

Food Chemistry 123 (2010) 941–948

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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Extraction of Se species in buckwheat sprouts grown from seeds soaked in various Se solutions Petra Cuderman a, Ljerka Ozˇbolt a, Ivan Kreft b, Vekoslava Stibilj a,* a b

Jozˇef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1111 Ljubljana, Slovenia

a r t i c l e

i n f o

Article history: Received 31 January 2009 Received in revised form 14 April 2010 Accepted 26 April 2010

Keywords: Buckwheat sprouts Se species HPLC-UV-HG-AFS HPLC-ICP-MS

a b s t r a c t The transformation of selenium (Se) in buckwheat sprouts grown from seeds soaked in various Se solutions (Se-methionine (10 mg Se L 1), selenate or selenite (5, 10, 20 mg Se L 1)) was investigated. The extraction procedure was optimised by (a) using optimal extraction media (water, phosphate buffer, 0.1, 0.2, 0.3 M HCl, the enzyme protease alone or in combination with cellulase, amylase or lipase), and by (b) optimising the ratio between sample and enzyme. For Se speciation analysis extracts with the highest percentage of soluble Se were analysed, and additionally the stability of the extracts was investigated. The results showed that uptake of Se by sprouts was dependent on the form and concentration of Se in the solution used for soaking. Optimal extraction efficiencies were obtained by hydrolysis with 0.3 M HCl and protease. Selenate (23.7–29.7% from Se(VI) sprouts and in trace amounts from Se(IV) and SeMet sprouts), Se-methionine (2.4–7.9%) and selenite (traces) were detected in all supernatants, regardless of soaking solution. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Se is an essential nutrient for animals and humans, and forms the active centre of Se-containing enzymes (selenoenzymes), such as glutathione peroxidase, thioredoxin reductase, and iodothyronine deiodinase. As Se plays an important role in human nutrition, it is important that the diet is sufficiently supplied with it. The Se content of food varies depending on the Se content of the soil where the animal was raised or the plant was grown: organ meats and seafood, 0.4–1.5 lg g 1; muscle meats, 0.1–0.4 lg g 1; cereals and grains, less than 0.1 to greater than 0.8 lg g 1; dairy products, less than 0.1–0.3 lg g 1; and fruits and vegetables, less than 0.1 lg g 1 (WHO, 1987). The Se supply in almost all European countries is below the recommended daily intake. In these countries, Se fortification of foods and the use of Se supplements are quite popular to compensate for the low Se intake from diet (Lintschinger, Fuchs, Moser, Kuehnelt, & Goessler, 2000). Moreover, field treatment with Se or direct supplementation of food and fodder is not enough. The chemical form in which Se is present in the food used is of principal importance to improve the Se nutrition of livestock and people. Nowadays, common buckwheat is becoming an increasingly important alternative crop in Europe. It is used as food (grain, sprouts) or herb (plant or leaves for herbal tea) (Smrkolj, Stibilj, Kreft, & Germ, 2006). Since the nutritional characteristics of this * Corresponding author. Tel.: +386 1 588 5352; fax: +386 1 588 5346. E-mail address: [email protected] (V. Stibilj). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.04.063

plant, like the high content of proteins and essential amino acids, suggest that Se is preserved as selenoamino acid derivatives, in particular, selenomethionine in proteins, similar to selenized yeast. Buckwheat sprouts are prized for their subtle, nutty flavour and high nutritional value. They are widely available and consumed in Japan and Korea. Therefore buckwheat is expected and known to be (Lintschinger et al., 2000) a good nutritional source of Se. Smrkolj et al. (2006) reported that SeMet is one of the main selenocompounds in Se-enriched buckwheat seeds. In addition, Kitaguchi, Ogra, Iwashita, and Suzuki (2008) confirmed that the main Se species present in Se-enriched seeds of buckwheat, treated with barium selenate, was SeMet. Lintschinger et al. (2000) treated sunflower (Helianthus annuus), wheat (Triticum aestivum) and alfalfa (Medicago sativa) seeds with selenate solution. The sunflower sprouts obtained were resistant and had the highest uptake rates, but almost 100% of the Se was extracted with water and found to be nonmetabolized selenate. The metabolism of selenate by wheat, alfalfa was inversely related to the uptake rates. At low Se enrichment <20% of the total Se content within the sprouts remained as inorganic Se, indicating a high metabolic rate (Lintschinger et al., 2000). Comparable results were reported by Chan, Afton, and Caruso (2010). In the selenite-enriched soybean root and leaf, inorganic Se utilised over 90% of the peak areas, and only a small portion of Se was converted to organoselenium compounds. In contrary, SeMet and SeCys2 were the predominant Se compounds found in the bean (Chan et al., 2010). Sprouts of several edible plants (10 families and 28 species) were cultivated hydroponically in a high Se environment (10 lg mL 1 of Se as selenite), and the

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chemical species of Sein these Se-enriched sprouts were identified by Sugihara et al. (2004). They reported that by performing speciation analysis of the HCl extract, SeMeSeCys was found to be the main Se species in all sprouts. In addition SeMet, Se(IV), c-glutamyl-Se-methylselenocysteine and an unknown Se compound were detected in several high Se sprouts. Recently, the speciation of Se-containing molecules, such as selenoproteins and selenometabolits, has been primarily carried out by means of extraction procedures, mainly using the non-specific enzyme protease, followed by separation and detection by a hyphenated technique, namely high performance liquid chromatography (HPLC) in conjunction with inductively coupled plasma mass spectroscopy (ICP-MS), hydride generation atomic absorption spectrometry (HG-AAS) or hydride generation atomic fluorescence spectrometry (HG-AFS) (Dumont, Vanhaecke, & Cornelis, 2006; Mazej, Falnoga, Veber, & Stibilj, 2006; Polatajko, Jakubowski, & Szpunar, 2006). The present study investigates the metabolism of Se in buckwheat sprouts from seeds soaked in various Se solutions in order to investigate the uptake of Se and to identify the Se species present in the sprouts. Therefore, the extraction procedure was optimised by (a) using optimal extraction media (water, phosphate buffer, hydrochloric acid, the non-specific enzyme protease alone or in combination with the specific enzymes cellulase, amylase or lipase), and by (b) optimising the ratio between sample and enzyme. In addition, extract stability was studied. The accuracy of the results obtained by HPLC-UV-HG-AFS was checked by HPLC-ICPMS. 2. Materials and methods 2.1. Reagents and standards The following chemicals were used: 96% H2SO4 (Merck, Suprapur), 65% HNO3 (Merck, Suprapur), 30% HCl (Merck, Suprapur), 36% HCl (Merck, p.a.), 30% H2O2 (Merck, p.a.), V2O5 (Merck, p.a.), NaOH (Merck, puriss p.a.) and NaBH4 (Fluka, Purum p.a.), (NH4)2HPO4 (Fluka Chemie, puriss p.a.), pyridine (Fluka Chemie, puriss p.a.), diammoniumhydrogen citrate (Fluka Chemie, puriss p.a.), citric acid (Fluka Chemie, puriss p.a.), MeOH (Primar, Fisher Scientific UK, trace analysis grade), protease XIV from Streptomyces griseus (type XIV: bacterial, 4.4 units/mg solid; Sigma P-5147), aamylase from porcine pancreas (type VI-B, 19.6 units/mg solid; Sigma A-3176), cellulase from Aspergillus niger (0.57 units/mg solid; Sigma C-1184), lipase from porcine pancreas (type II, 30– 90 units/mg protein, Sigma L-3126). For preparation of Se solutions for seed soaking and Se analysis, Na2SeO3 (Se(IV), Sigma–Aldrich, >98%), Na2SeO4 (Se(VI), Sigma–Aldrich, SigmaUltra), selenomethionine (SeMet, Fluka Chemie, >99%), selenocystine (SeCys2, Fluka Chemie, >98%) and selenomethylselenocysteine (SeMeSeCys, Fluka Chemie, >98%) were used. Stock solutions of Se species containing about 1 mg Se g 1 in water were prepared and kept at 4 °C. For preparation of solutions and sample treatment, ultra-pure water (Milli Q, Millipore Corporation, Bedford, MA, USA) was used. For Se speciation analysis standards were prepared at concentrations of approximately 100 ng Se per g for each species in supernatants of the control group of buckwheat sprouts, to check for the possible different retention times of Se species caused by matrix interactions in the measurement system. 2.2. Germination and sample preparation Common buckwheat (Fagopyrum esculentum) seeds, cultivar Darja, were bought in a Seedman’s Shop in Ljubljana, Slovenia. About 500 mL (346 ± 30 g) of seeds (average seed mass was

0.026 ± 0.001 g) were soaked in 500 mL of MilliQ water, or a solution of sodium selenate (5, 10, 20 mg Se L 1), or of sodium selenite (5, 10, 20 mg Se L 1), or of SeMet (10 mg Se L 1). Seeds were soaked for 4 h, than were separated from the solution, weighed to obtain the mass of water and Se absorbed, and distributed equally in plastic bowls, which were covered with filter paper. During germination seeds were treated with tap water (Se content below detection limit), as needed. Buckwheat sprouts whose seeds were soaked in (i) Se(VI) solution, were grown in April for 22 days, when the average day temperature was 13 °C (13 h of daylight) and the average night temperature was 8 °C. Buckwheat sprouts, whose seeds were soaked in (ii) Se(IV) solution were treated in May for 8 days. The average day temperature was 25 °C (15 h of daylight) and the average night temperature was 18 °C. Seeds soaked in (iii) solutions of SeMet or Se(VI) or Se(IV) at a concentration 10 mg Se L 1 were treated in July for 11 days. Sprouts were treated at 22 °C in a bright air-conditioned room (16 h of daylight). For every treatment a control group (seeds soaked in water) was included. Sprout sampling (harvest) was done when the buckwheat sprouts developed two extended cotyledon leafs. The whole sprouts were taken, including the cotyledons and roots. Six replicates of a hundred randomly chosen sprouts were weighed to obtain the average sprout mass. Buckwheat sprouts whose seeds were soaked in Se(VI), Se(IV) and SeMet solution are named Se(VI), Se(IV) and SeMet sprouts in the following text. For analysis, buckwheat sprouts were lyophilised at 50 °C and 0.050 mbar (CHRIST ALPHA 1–4, LOC-1, freeze-dryer), milled and homogenised in a planetary micro mill (FRITSCH, Pulverisette 7, Idar-Oberstein, Germany; speed 6, time 8 min with additional 2 min at speed 7). Finally, samples were sieved through a 0.25 mm nylon sieve. 2.3. Determination of total Se concentration Digestion was carried out on 0.2 g of homogenised sample. This was weighed in a Teflon tube and mineralisation performed using HNO3 (1.5 mL) and H2SO4 (0.5 mL) by heating the closed tube in an aluminium block, kept at 80 °C overnight and then for 1 h at 130 °C. After cooling, 2 mL of hydrogen peroxide was added and the tubes were heated for 15 min at 115 °C. This step was repeated. After the solution had cooled to room temperature, 0.1 mL V2O5 in H2SO4 was added and the tube reheated at 115 °C until the solution became blue in colour. To reduce selenate to selenite 2.5 mL of HCl was added to the solution and heated at 100 °C for 10 min. Samples were diluted with Milli Q water. Sensitive detection was achieved by HG-AFS with the chemical and instrumental operating conditions according to Smrkolj and Stibilj (2004). Working standard solutions of Se(IV) were prepared daily by dilution of a stock standard solution with a solution containing appropriate amounts of H2SO4 and HCl to obtain the same acid media as in the samples. To check the accuracy and precision of the method a standard reference material representing a similar matrix (NIST SRM 1570a, Trace Elements in Spinach Leaves) was analysed simultaneously. 2.4. Extraction and speciation Extraction of the sprouts was performed in triplicate by adding separately (a) 12 g of water, 25 mM phosphate buffer (pH 7.5), HCl (0.1, 0.2, 0.3 M), a solution of 25 mM phosphate buffer (pH 7.5) containing protease (1, 10, 50, 90, 150 mg) separately or in combination with a-amylase (150 mg) or cellulase (150 mg) or lipase (150 mg) to 900 mg of dry sample, or (b) the sample in 25 mM phosphate buffer (pH 7.5) was frozen in liquid nitrogen and unfrozen in hot water three times. Additionally, 150 mg protease was added. In all cases the duration of incubation was 24 h at 37 °C. After the extraction procedure extracts were centrifuged at

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11,000 rpm for 60 min at 4 °C (5804R, Eppendorf). The supernatant was filtered through 0.45 and 0.22 lm Millex GV filters (Millipore Corporation) and used for Se speciation analysis by HPLC-ICP-MS or HPLC-UV-HG-AFS. Supernatants and sediments were stored at 20 °C until analysis for total Se by HG-AFS was carried out. Sediments were digested and analysed for Se content as described above (determination of total Se concentration). Supernatants were digested in HNO3. The procedure was adapted from Stibilj, Mazej, and Falnoga (2003). To 0.5 g of supernatant 1 mL of conc. HNO3 was added and heated for 30 min at 80 °C, then for 15 min at 160 °C. H2O2 (0.5 mL) was added three times and the solution evaporated to 0.5–0.6 g of sample. About 0.5 mL of conc. HCl was added for reduction of Se(VI) to Se(IV), which was determined by HG-AFS. Working standard solutions of Se(IV) were prepared daily by dilution of a stock standard with 0.5 M HCl. The concentrations of extracted Se species were determined using an ion exchange HPLC system (Varian ProStar 210) coupled directly to an UV-HG-AFS (PS Analytical) or HPLC system (Agilent 1100, Waldbronn, Germany) coupled to an ICP-MS (Agilent 7500ce, Tokyo, Japan). The ICP-MS detection system was used for control of the results obtained by UV-HG-AFS since a lower detection limit could be achieved and since there is no appropriate certified reference material available for Se species. The Se species (SeCys2, SeMeSeCys, Se(IV), Se(VI), SeMet) were separated on an Hamilton PRP-X 100 anion-exchange column (4.1 mm  250 mm  10 lm) using an aqueous mobile phase containing 40 mM phosphate solution (pH 6) for detection by UV-HG-AFS or 3 and 10 mM citric acid (gradient elution) in 2% methanol (v/v) (pH 4.8) for detection by ICP-MS. Since SeCys2 comes with the void volume of the anion-exchange column, a Zorbax 300-SCX cation exchange column (4.6 mm  250 mm  5 lm) was used with an aqueous mobile phase containing 3 mM pyridine solution (pH 2.1) with addition of methanol (2%) for the ICP-MS system. The operating conditions were described in detail elsewhere (Cuderman, Kreft, Germ, Kovacˇevicˇ, & Stibilj, 2008; Mazej et al., 2006).

Se(IV) solution corresponded to those soaked in water (treated at the same time). The percentage dry weight in Se(VI) sprouts relative to the control group was lower by 12% at 5 mg Se(VI) L 1, 16% at 10 mg Se(VI) L 1 and 11% at 20 mg Se(VI) L 1 (Table 1). The percentage dry weight for Se(IV) sprouts was lower by 7% at 5 mg Se(IV) L 1, 8% at 10 mg Se(IV) L 1 and 17% at 20 mg Se(IV) L 1 relative to the control group. Comparing buckwheat sprouts whose seeds were soaked in different Se solutions at the same concentration, the dry weights were as follows: Se(VI) > Se(IV) > SeMet. Ximenez-Embun, Alonso, Madrid-Albarran, and Camara (2004) observed the same effect, a reduction in biomass yield, when lupines and sunflower were exposed to 5 mg L 1 of sodium selenate or sodium selenite. In contrary, they reported that Indian mustard showed no differences in biomass production when it was grown in the presence of sodium selenite, while a reduction of 20% in biomass yield was observed when sodium selenate was added to the medium (Ximenez-Embun et al., 2004). Interestingly, a 9% increase was obtained for Se(VI) sprouts against the control group. For Se(IV) sprouts 2% and in case of SeMet sprouts 7% lower dry weight was determined relative to the control group. Average masses of buckwheat sprouts were practically not dependent on the Se(VI) and Se(IV) concentration and were comparable with the control group (Table 1). If we compare the results (Table 1) obtained for Se(IV) and Se(VI) sprouts under different conditions (temperature, daylight, see Experimental part), we can see that these conditions influence the mass and dry matter of sprouts. In addition, even though higher plants do not require Se, there are increasing indications that Se may also have beneficial biological functions in higher plants. Se at low concentrations acts as an antioxidant and can stimulate plant growth, whereas at higher concentrations it acts as a pro-oxidant reducing the yield (Hertikainen, Xue, & Piironen, 2000; Xue, Hertikainen, & Piironen, 2001).

3. Results and discussion

The Se content was raised by increasing Se concentration in the soaking solution (Table 1). The dependence between Se(VI) concentration and Se content obtained is almost linear. Lintschinger et al. (2000) treated seeds of clover and wheat with Se(VI) solution (seeds were soaked for 12 h) and in the comparable concentration range, 0.78–20 mg Se(VI) L 1, they also observed a linear response between the Se content obtained in sprouts and the added Se concentration. Buckwheat sprouts treated with 5 and 10 mg Se(IV) L 1 took up a comparable amount of Se, while sprouts treated with 20 mg Se(IV) L 1 took up twice as much. When we compared the

3.1. Agricultural data (height and dry weight and mass of Se-enriched buckwheat sprouts) During the plant growth no toxic signs, like leaf necrosis, drying or plant death were noticed. No differences were observed in the height of Se(VI), Se(IV) and SeMet sprouts for different concentration ranges and environmental (temperature, light) conditions. Dry weight was a little lower when seeds were soaked in Se(VI) and

Table 1 Dry weight percentage, average mass per sprout (g). Se content (lg g

a b c d

Se content (lg g

0 5 10 20

12.13 ± 1.07 10.72 ± 0.76 10.18 ± 1.23 10.84 ± 0.79

0.097 ± 0.006 0.100 ± 0.005 0.093 ± 0.009 0.097 ± 0.003

0.050 ± 0.001 4.03 ± 0.11 9.67 ± 0.11 15.15 ± 0.32

58 ± 6 62 ± 9 53 ± 3

Water Se(IV)

0 5 10 20

7.47 ± 0.58 6.96 ± 1.45 6.90 ± 0.26 6.21 ± 0.52

0.114 ± 0.004 0.117 ± 0.003 0.095 ± 0.002 0.118 ± 0.001

0.070 ± 0.002 2.39 ± 0.22 2.59 ± 0.11 4.67 ± 0.32

27 ± 7 12 ± 1 12 ± 2

Water Se(VI) Se(IV) SeMet

0 10 10 10

12.09 ± 0.68 13.12 ± 0.30 11.88 ± 0.52 11.29 ± 0.41

0.105 ± 0.007 0.117 ± 0.003 0.126 ± 0.005 0.125 ± 0.001

0.078 ± 0.004 7.08 ± 0.18 1.87 ± 0.02 3.24 ± 0.06

73 ± 2 19 ± 1 31 ± 1

Concentration (mg Se L

(i)

Water Se(VI)

(iii)

) and percentage of Se uptake in buckwheat sprout (Results are given as average ± standard deviation). Average mass of sprout (g)b

Soaking solution

(ii)

1

Dry mass (%)a

Experiment

Four replicates. Six replicates. Three replicates. Results are given on a dry matter basis.

1

3.2. Se in buckwheat sprouts

)

1 c,d

)

Se uptake (%)

P. Cuderman et al. / Food Chemistry 123 (2010) 941–948

3.3. Optimisation of the extraction of Se species from Se-enriched buckwheat sprouts Firstly, optimisation of the procedure for extraction of Se from sprouts was done. In the optimisation procedure buckwheat sprouts grown from seeds soaked in 10 mg Se as Se(IV), Se(VI) and SeMet L 1 were used. The soluble Se content is given as a percentage of total Se in the sample (Fig. 1, Table 2). In the first part water-soluble Se was extracted from samples by either water (Milli Q), 25 mM phosphate buffer (pH 7.5) or hydrochloric acid (0.1, 0.2, 0.3 M). Using water media, the extraction efficiency was between 9% and 11% for Se(VI) sprouts and 4–12% for Se(IV) sprouts. Comparable extraction efficiencies were obtained using phosphate buffer as the extraction media, namely 8–13% for Se(VI) and 4–10% for Se(IV) treated sprouts. The same extrac-

60 50 40 30 20 10 0 wa ph te os r ph at e bu ffe r 0. 1M HC l 0. 2M HC l 0. 3M HC l pr ot ea N2 se + p pr r o ot te ea as se e + am pr yla ot se ea se pr + ot lip ea as se e + ce llu la se

ability of seeds to take up Se from soaking solutions containing different Se species at the same concentration, the following order was obtained: Se(VI) > SeMet > Se(IV). On analysing Se(VI) soaking solutions no Se(VI) transformations or losses were observed. Zayed, Lytle, and Terry (1998) studied uptake of Se(VI), Se(IV) and SeMet in rice, sugarbeet, broccoli and Indian mustard sprouts. Se was added to the nutrient solution after germination into the soil. Special attention should be devoted to this kind of treatment, since to date the mechanism of Se species added to the soil and consequently their influences on the environment remains unknown. The highest Se content was observed in rice, broccoli and Indian mustard sprouts treated with Se(VI) and sugarbeet sprouts treated with SeMet. The lowest Se level was determined in sprouts treated with Se(IV). The same order was also observed by De Souza et al. (1998) who treated Indian mustard with Se(VI), SeMet and Se(IV). As can be observed, the uptake of Se depends on the chemical form of the selenium added. For the plant species tested, the Se(VI) addition lead to a higher accumulation rate than Se(IV). The former anion is actively transported through the root membrane using the same channels and mechanisms as sulphate. On the other hand, there is no evidence for Se(IV) uptake by membrane transporters. The mechanism of Se(IV) uptake is not fully understood, but it is assumed to be passive. Plants can also take up organic forms of Se, such as SeMet, actively (Terry, Zayed, de Souza, & Tarun, 2000). Buckwheat seeds (average seed mass 0.026 g) soaked in water for 4 h absorbed on average 14.7 mg of water. Supposing that there is no difference in water uptake by seeds of the solution or water, then the theoretical uptake of Se (14.7 mg of water multiplied by the Se concentration in the soaking solution) was estimated as 74 ng Se for seed soaked in 5 mg Se L 1, 147 ng Se for seed soaked in 10 mg Se L 1, and 294 ng Se for seed soaked in 20 mg Se L 1. From the ratio between the measured mass of Se in the sprout (Se concentration in the sprout multiplied by the average sprout mass and dry weight) and the theoretical one in the seed, we calculated the percentage of Se uptake in buckwheat sprouts (Table 1). The order obtained was as follows: Se(VI) > SeMet > Se(IV). In the case of sprouts treated with SeMet and Se(IV), the uptake rate was about two to four times lower than for Se(VI) treated ones. Terry et al. have described that this is due to the use by selenate of the same membrane transporters than sulphate and therefore, the incorporation rate is much higher although not the metabolism (Terry et al., 2000). However, some Se, uptaken by the imbibing seed, remained in the husk, and was not used by the developing sprout. Some Se remained as well in the filtering paper and on the surface of the plastic bowl. As there are differences in Se uptake among Se species, it is possible that sprouts were less able to uptake more toxic forms, and they remained in higher proportion in the solution, imbibed by the other part of seed (husk) and in the filtering paper.

soluble Se (%)

944

Se(VI) sprouts

Se(IV) sprouts

SeMet sprouts

Fig. 1. Soluble Se (%) obtained after using different extraction media for 10 mg L Se(VI), Se(IV) and SeMet sprouts.

1

tion efficiency was obtained for SeMet treated sprouts, 10 ± 2% for both extraction media. Using hydrochloric acid, the extraction efficiency for Se(IV) stayed comparable, 9–13%, for SeMet it was twice as high, 22 ± 3%, regardless of the acid concentration used. A three to four times higher efficiency was achieved for Se(VI) sprouts; 32–35% with 0.1 M HCl; 36–43% with 0.2 M HCl and 39– 44% with 0.3 M HCl. To enhance the extraction efficiency, enzymatic hydrolysis was applied. Firstly, the non-specific enzyme protease XIV was employed. Efforts to increase the extraction efficiency included using different ratios between enzyme and sample, by breaking the cells with liquid nitrogen prior to enzymatic hydrolysis with protease XIV and by combining the non-specific enzyme with a specific one, like cellulase or amylase or lipase. When the ratio between enzyme and sample was 1:6, the extraction efficiency for Se(VI) and SeMet sprouts remained comparable to the ones obtained after acid hydrolysis of 38 ± 2% and 20 ± 2%. In the case of Se(IV) a twice higher result was obtained, 22 ± 3% (Table 2). The extraction efficiency remained the same on increasing the ratio to 1:10 and 1:18. When the ratio between enzyme and sample was 1:90 and 1:900, the results obtained were comparable with those using water media or phosphate buffer. Therefore, the lower enzyme – sample ratio (1:6) was taken in further experiments. Further, by breaking cells with liquid nitrogen before enzymatic hydrolysis with protease XIV, we succeeded in increasing the extraction efficiency only for Se(VI), to 52 ± 2%. For Se(IV) and SeMet the extraction efficiency stayed the same (approximately 20%) as for hydrolysis without liquid nitrogen. Since we were not satisfied with such low efficiencies, we tried the combination of several specific enzymes, like cellulase or amylase or lipase with protease XIV. The ratio between the two enzymes and sample was 1:1:6. In supernatants of buckwheat sprouts treated with Se(VI) the efficiency was the same using protease XIV as with amylase. Protease XIV in combination with lipase gave an around 10% higher result, but on the other hand, when combining protease XIV and cellulase an about 34% lower result was achieved than in combination with lipase. For enzymatic extracts of Se(IV) and SeMet sprouts the efficiency stayed the same, regardless of the enzyme combination used (Fig. 1). Independent of the short growing period, from 8 to 22 days, a great part of Se remained insoluble. Since we were not able to increase the extraction efficiency with a combination of different enzymes, we decided to use protease XIV as the optimal enzyme for the extraction of Se species from Se-enriched buckwheat sprouts. In further analysis, 0.9 g of sample was treated with 0.15 g of protease XIV, dissolved in 12 g of 25 mM phosphate buffer (pH 7.5). After every extraction procedure the non-soluble part was also analysed for Se content, to perform mass balance calculations.

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Table 2 Se content and its species present in buckwheat sprouts supernatant obtained after extraction with 0.3 M HCl or protease XIV, using UV-HG-AFS and/or ICP-MS (*) as detection systems. Results were corrected for the Se content in protease (Cuderman and Stibilj, 2009). Experiment

Soaking solution

Concentration

Soluble Se

Se(IV) d

lg Se g-1 sample (soluble Se%; mass balance %)

ng Se g

5 10 20 20*

1.60 ± 0.04 (40) 4.30 ± 0.11 (44) 5.93 ± 0.27 (39; 79)

40 97 76 24

mg Se L Extraction with 0.3 M HCl (i) Se(VI)

1

(1.0) (1.0) (0.5) (0.2)

Se(VI) 1

SeMeta

Xb

Yc




e

sample (% ) 1129 2708 4500 4299

(28.0) (28.0) (29.7) (28.4)

(ii)

Se(IV)

5 10 20 20*

0.24 ± 0.01 (10) 0.33 ± 0.01 (13) 0.47 ± 0.02 (10; 110)




120 155 374 228

(iii)

Se(VI) Se(IV) SeMet

10 10 10

3.32 ± 0.05 (47) 0.23 ± 0.01 (12) 0.73 ± 0.02 (23; 68)

71 (1.0)
2053 (29.0)



5* 10* 20*

1.61 ± 0.04 (40; 82) 4.07 ± 0.13 (42; 83) 5.41 ± 0.40 (36; 81)

tr tr tr

1004 (24.9) 2657 (27.5) 4000 (26.4)

143 (3.5) 230 (2.4) 439 (2.9)



123 234 375 208

Extraction with protease XIV (i) Se(VI)

(ii)

Se(IV)

5 10 20 20*

0.50 ± 0.03 (21) 0.57 ± 0.02 (22) 0.94 ± 0.06 (20; 100)


tr tr tr 47 (1.0)

130 204 371 338

(5.4) (7.9) (7.9) (7.2)

(iii)

Se(VI) Se(IV) SeMet

10* 10 10 10*

2.88 ± 0.41 (41; 67) 0.36 ± 0.06 (19; 93) 0.55 ± 0.06 (17; 66)

tr tr
1675 (23.7) tr
263 (3.7) 143 (7.6) tr 117 (3.6)

(4.6) (3.3) (5.3) (4.9)

(4.7) (5.0) (5.3) (4.5)




tr = traces. a Se as SeMet. b Unknown Se species with retention time of 9.5 min, obtained on Zorbax 300-SCX, estimated as SeMeSeCys. c Unknown Se species with retention time of 20 min, obtained on Zorbax 300-SCX, estimated as SeCys2. d Percentage of Se; sum of soluble and insoluble Se according to total Se, given as an average of at least three determinations. e Percentage of Se species according total Se.

Quantitative results were obtained for Se(IV) buckwheat sprouts, while in the case of Se(VI) and SeMet sprouts only between 65– 83% of total Se was found as soluble and non-soluble parts. The results obtained were the same, regardless of the extraction media used. This means that Se could be adsorbed on tube walls or be present in volatile form, but further studies are needed to draw conclusions. Unfortunately the mass balance is not often included in speciation studies. Nevertheless, Roberge, Borgerding, and Finley (2003) reported that the mass balance for Se-enriched broccoli is between 70–100%. They suspect that there were losses of Se due to volatilisation process. 3.4. Se species in Se-enriched buckwheat sprouts To obtain information about the Se species present in buckwheat sprouts speciation analysis using HPLC-UV-HG-AFS and HPLC-ICP-MS was done. Se species were calculated based on results obtained on anion-exchange column (Table 2). HPLC-UVHG-AFS gave detection limits of 27 ng g 1 sample for Se(IV), 66 ng g 1 sample for Se(VI), 105 ng g 1 sample for SeMet (Mazej et al., 2006), whereas detection (quantification) limits for Se species on the HPLC-ICP-MS system were 15 (51) ng g 1 sample for Se(IV), 1 (5) ng g 1 sample for Se(VI) and 10 (39) ng g 1 sample for SeMet (Cuderman & Stibilj, 2009; Cuderman et al., 2008). The repeatability within one day was between 5% and 11% for Se species at concentration 100 ng g 1 (n = 4) and was comparable with the one determined within one week, which was between 8% and 12% (n = 10), regardless of the system used. Comparing the peak areas obtained for separated Se species (100 ng of Se g 1) after column separation with peak areas obtained without a column, a column recovery was determined. For HPLC-ICP-MS was as follows: 81.2% for Se(IV), 93.5% for Se(VI) and 63.6% for SeMet

(Cuderman et al., 2008). Column recoveries for the same Se species obtained on HPLC-UV-HG-AFS were approximately 20% lower (Mazej et al., 2006). This could be ascribed to a lower decomposition of Se species with UV-HG-AFS in comparison with a plasma source. Regardless of the system used, the highest part of SeMet remained on the anion-exchange column. Since comparable extraction efficiency was obtained on hydrolysis with 0.3 M HCl or protease XIV, we decided to perform speciation analysis in both mentioned supernatants (Table 2). Using speciation analysis, around 30% of total Se for Se(VI) sprouts, 5– 15% of total Se for Se(IV) sprouts and 5–7% of total Se for SeMet sprouts were identified as soluble Se species. Results obtained for Se species content were corrected for the Se content in the enzyme used for the extraction, where necessary (Cuderman & Stibilj, 2009). Vogrincˇicˇ, Cuderman, Kreft, and Stibilj (2009) faced a similar problem, low solubility of this element (14%) in above-ground buckwheat parts (stems, leaves and inflorescences), using the same extraction procedure, protease XIV (Vogrincˇicˇ et al., 2009). Further, the stability of extracts used for Se speciation analysis was monitored over a 30 day period and Se species were found to be stable. Extracts of Se(VI) sprouts, obtained after hydrolysis with hydrochloric acid, contained the highest amount of Se(VI), 28–30% of total Se, independent of the Se concentration used as the soaking solution. Se(IV) was present in traces. Lintschinger et al. (2000) soaked sunflower, wheat and alfalfa seeds in Se(VI) solution. The Se content within the sprouts remained as Se(VI). Moreover, Slekovec and Goessler (2005) reported Se(VI) to be the major Se species in vegetative parts of onion, garlic, radish and cabbage, foliarly treated with 10 mg Se(VI) m 2 or 20 mg Se(VI) m 2. In Se(IV) supernatants we were not able to detect any Se species after separation on an anion-exchange column using the UV-HG-AFS system. With ICP-MS detection we were able to detect Se(VI) and

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some Se(IV) in trace amounts. On the cation exchange column an unknown Se species occurred, with a retention time of 9.5 min (Fig. 2). In contrast, Sugihara et al. (2004) reported that chemical analysis of Se in the HCl extract of buckwheat sprouts treated with Se(IV) (10 mg Se(IV) L 1) contained SeMeSeCys as the major Se species. The difference could be explained by the different mode of cultivation (Table 3). Sugihara et al. (2004) treated seeds hydroponically for 7 days while in the present paper seeds were soaked in various Se solutions only for a short period, 4 h. Therefore plant

a

0.018

AFS Response

0.016

0.014

0.012

0.01 0

500

1000

1500

2000

2500

3000

Retention Time (s)

b

0.013 X

AFS Response

0.011

0.009

0.007

0.005 0

200

400

600

800

1000

1200

1400

Retention Time (s)

Fig. 2. Chromatogram of acid sprout extracts from seeds soaked in 10 mg L 1 Se(IV) obtained after separation on Hamilton PRP-X 100 (a) and Zorbax 300-SCX (b) columns with UV-HG-AFS detection.

defence mechanisms, shown by the presence of SeMeSeCys in the sprouts (Terry et al., 2000), was probably develop due to a longer exposure period to high Se concentration. Further, by doing speciation analysis of extracts obtained after hydrolysis with the enzyme protease XIV, almost the same results were obtained for identified and unknown Se species (Table 2, Fig. 3). Se(IV), Se(VI) and unknown compounds were present in comparable concentration ranges, regardless of the extraction used (hydrochloric acid or protease). The most important Se species observed in each chromatogram was SeMet, and was present at 2–4% for SeMet, 5–8% for Se(IV) and 2–4% for Se(VI) buckwheat sprouts. SeMet was present as a low part of the total Se in buckwheat sprouts, while Kitaguchi et al. (2008) and Smrkolj et al. (2006) (Table 3) reported that the main Se species present in seeds of Se-enriched buckwheat, treated with Se(VI), was SeMet. However, different Se mechanism is followed in buckwheat seeds in comparison to sprouts, specially, due to a longer development/growing period. Enzymatic and alkali extractions were made by Kitaguchi et al. (2008) and SeMet was the only species that differed from those in alkali extracts (Se(IV), Se(VI) and SeMeSeCys). From the data reported by Kitaguchi et al. (2008) and our results it is evident that SeMet is present in proteins. From the experiment where seeds were soaked in 10 mg Se(IV), Se(VI) or SeMet L 1, we can conclude that the form of Se in soaking solution has a great influence on the Se species plant uptake (Table 2, Fig. 3) as well as different cultivation conditions (temperature, daylight) do not cause differences in the Se species present. The uptake of Se was highly dependent on the chemical form in which Se was supplied. Se(VI) was transported much more easily than selenite, or organic Se, such as SeMet (Table 2). Se(VI) uses the same transporter as sulphate in getting across plant membranes. Once inside, this ion is either toxic to or cannot efficiently utilise ATP sulphurylase and the subsequent enzymes leading to Cys and Met synthesis. The reduction of selenate to selenite has also been pointed out as the rate-limiting step in Se(VI) transformation (Terry et al., 2000). It therefore remains mainly as selenate in the various plant tissues, and it can readily move around the plant and, therefore will accumulate in above-ground biomass of the plant. Se(IV), on the other hand, uses an unidentified mechanism to enter the cells of plants, which is much less efficient compared to Se(VI) uptake. However, once inside the cells, Se(IV) is able to use the sulphur enzymes leading to Cys and Met synthesis. This may be possible because selenite is at a lower oxidation state

Table 3 Literature comparison of Se and its species content in Se-enriched buckwheat seeds and sprouts, treated under different conditions.

Buckwheat

Seeds

Treatment conditions

Analytical procedure

Se content (lg g 1)

Se species (% of total Se)

Reference

Foliar spraying (15 mg Se L 1, Na2SeO4)

Enzymatic extraction with protease; HPLC-UV-HG-AFS detection Extraction with driselase and protease or NaOH; HPLC-ICP-MS detection Extraction with HCl; HPLC-ICP-MS detection Extraction with HCl and protease; HPLC-ICP-MS detection

2.9

SeMet (88%)

Smrkolj et al. (2006)

170.4

SeMet (43%), Se(IV) (1%), Se(VI) (5%), SeMeSeCys (5%)

Kitaguchi et al. (2008)

8.5

SeMeSeCys (58%), unknown Se compound (14%) SeMet (3.6%), unknown Se compound (2.8%), Se(IV) and Se(VI) (traces)

Sugihara et al. (2004) This study

Foliar spraying (500 mg Se m 2, BaSeO4; BaSeO3)

Sprouts

Hydroponically (10 mg Se L 1, Na2SeO3; 7 days) Soaking for 4 h (10 mg Se L 1, SeMet); Growing period 11 days Soaking for 4 h (5, 10, 20 mg Se L 1, Na2SeO3); Growing period 8 days Soaking for 4 h (5, 10, 20 mg Se L 1, Na2SeO4); Growing period 22 days

3.2

2.4(5); 2.6(10); 4.7(20) 4.0(5); 9.7(10); 15.2(20)

SeMet (5.4–7.9%), unknown Se compound (4.5–5.3%), Se(IV) and Se(VI) (traces) Se(VI) (23.7–29.7%), SeMet (2.4–3.7%), Se(IV) (traces)

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a

b

3.0E+03 Se(VI)

Intensity 78Se

2.0E+03 1.5E+03 1.0E+03

SeMet

5.0E+02

SeMet

Se(VI) + Se(IV)

5.0E+03

Se(IV)

Intensity 78Se

2.5E+03

6.0E+03

4.0E+03 3.0E+03 2.0E+03

X

1.0E+03

0.0E+00

0.0E+00

0

500

1000

1500

2000

2500

3000

0

500

1000

4.0E+03

d

3.0E+04

2.0E+04

2.0E+03

Se(VI)

Intensity 78Se

3.0E+03

SeMet

1.0E+03

2000

2500

3000

2500

3000

2500

3000

Se(VI) + Se(IV)

c

1500

Retention time (s)

Intensity 78Se

Retention time (s)

SeMet 1.0E+04

Se(IV) 0.0E+00

0

500

1000

1500

2000

2500

0.0E+00

3000

0

500

1000

Retention time (s)

e

Intensity 78Se

Intensity 78Se

f

SeMet

6.0E+02

4.0E+02

2.0E+02

Se(VI)

Se(IV)

0.0E+00

0

500

1000

1500

2000

1500

2000

Retention time (s)

2500

3000

3.0E+03

SeMet

2.0E+03

Y 1.0E+03

Se(IV) + Se(VI)

0.0E+00 0

500

1000

1500 2000 Retention time (s)

Retention time (s)

Fig. 3. Chromatogram of enzymatic sprout extracts from seeds soaked in 20 mg Se(IV) L 1 (a) and (b), 20 mg Se(VI) L after separation on Hamilton PRP X-100 (a), (c), (e) and Zorbax 300-SCX (b), (d), (f) columns connected to ICP-MS.

compared to sulphate. As a result, Se(IV) will not move around the plant as readily as Se(VI) and will accumulate more in the roots, where will be converted more easily to organic forms (XimenezEmbun et al., 2004). 4. Conclusions In the present work, seeds of common buckwheat, cv. Darja, were soaked in various concentrations of sodium selenate, sodium selenite and selenomethionine. The results showed that the uptake of Se by seeds was dependent on the form and concentrations of Se in the solution used for soaking, and the order was as follows: Se(VI) > SeMet > Se(IV). Hydrolysis with water, phosphate buffer, hydrochloric acid or a combination of different enzymes was used for Se speciation analysis in the sprouts. The highest extraction efficiencies were obtained with 0.3 M HCl of 39–47% for Se(VI), 10–13% for Se(IV) and around 23% for SeMet sprouts, and by enzymatic extraction with protease XIV of 36–42% for Se(VI), 19–22%

1

(c) and (d) and 10 mg SeMet L

1

(e) and (f) obtained

for Se(IV) and around 17% for SeMet sprouts. By performing mass balance calculation, about 35–20% losses in Se(VI) and SeMet sprouts were observed. Selenomethionine, selenite and selenate were detected in the supernatants of all sprouts, regardless of the soaking solution. Further, no differences occurred in the Se species present in sprouts cultivated under different conditions such as temperature and daylight. Due to the low extraction efficiencies in all investigated samples, especially in Se(IV) and SeMet sprouts, we intend to perform speciation analysis of the non-soluble part of the sample after the extraction procedure. Further, it would be important to find if the differences in mass balance calculations for Se(VI) and SeMet sprouts correspond to Se lost by volatilisation. Acknowledgements This work was financially supported by the Slovenian Reserarch Agency through the programme P1-0143, contract 1000-05310030 and projects J7-9805 and J4-2041.

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