Vegetable sprouts enriched with iron: Effects on yield, ROS generation and antioxidative system

Scientia Horticulturae 203 (2016) 110–117

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Vegetable sprouts enriched with iron: Effects on yield, ROS generation and antioxidative system ´ Arkadiusz Przybysz ∗ , Mariola Wrochna, Monika Małecka-Przybysz, Helena Gawronska, ´ Stanisław W. Gawronski Laboratory of Basic Research in Horticulture, Faculty of Horticulture, Biotechnology and Landscape Architecture, Warsaw University of Life Sciences−SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland

a r t i c l e

i n f o

Article history: Received 27 June 2015 Received in revised form 10 March 2016 Accepted 15 March 2016 Keywords: Antioxidative system Crucifers Fe enrichment Legumes Ros Sprouts

a b s t r a c t Iron(Fe) deficiency is a widespread nutritional disorder affecting human health and interest is therefore growing in producing plants enriched with this element as a dietary source of Fe. Owing to high nutritional value and improved bioaccessibility of essential elements, sprouts are promising targets for enrichment. In this study an attempt was made to evaluate the effects of enriching vegetable sprouts with Fe on: (i) the concentration of Fe and other ions, (ii) biomass accumulation, (iii) the levels of ROS and (iv) the activity/content of the constituent elements of enzymatic and non-enzymatic anti-oxidative systems. The germination of sprouts in the presence of Fe generated a considerable increase in the concentration of this element, especially in alfalfa (2.3-fold increase). Sprouts enriched with Fe were not depleted of other valuable ions, but their biomass accumulation generally slightly decreased, which was particularly pronounced in the sprouts of crucifers. A higher Fe concentration promoted generation of ROS and simultaneously increased activity of antioxidative enzymes (APX, CAT and GR), and greater contents of phenolic compounds and ascorbic acid. These new findings demonstrate that in the elaboration of enrichment technology, attention must be paid to the possibly elevated levels of ROS in the food products obtained. Of the sprouts tested, radish and alfalfa grown in the presence of Fe in concentrations 12 and 24 mg/L proved to be the most suitable for enrichment with Fe as they accumulated considerable amounts of Fe at still acceptable levels of accumulated biomass and relatively low generation of ROS. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Iron(Fe) deficiency is one of the most widespread nutritional disorders affecting human health and has been recognised as causing 2.4% of the total global burden of diseases (Rodgers et al., 2004). WHO estimates that around two billion individuals worldwide are anaemic (de Andrade Cairo et al., 2014). In Europe, Fe deficiency is thought to affect large proportions of the population, particularly children and menstruating or pregnant women (Hercberg et al., 2001). Fe deficiency is common due to both insufficient intake and its low bioavailability for recipients (Clemens, 2014). Inadequate intake of Fe is a result of diets dominated by cereals, which are less dense in micronutrients than other food products such as vegetables, red meat and eggs (Clemens, 2014; de Andrade Cairo et al.,

∗ Corresponding author. E-mail address: arkadiusz [email protected] (A. Przybysz). http://dx.doi.org/10.1016/j.scienta.2016.03.017 0304-4238/© 2016 Elsevier B.V. All rights reserved.

2014). This associates Fe deficiency with poverty and implies difficulties in achieving sufficient intake through dietary diversification, which might be too costly for a large part of the population. Pharmaceutical preparations used as supplements of Fe are often taken in a spontaneous and uncontrolled way, which in certain conditions may cause overdosing and increase the risk of infectious diseases (Jeruszka-Bielak et al., 2011). Therefore, an alternative could be to produce plants enriched with Fe. According to many authors, enrichment with Fe is the most practical, sustainable and costeffective long-term solution for controlling Fe deficiency (Baltussen et al., 2004; Zimmermann and Hurrell, 2007). Highly promising targets for enrichment are sprouts, which are quick and cheap to produce and entirely independent of external conditions. Sprouts constitute a growing market segment in developed countries. Their consumption has become increasingly popular among people seeking healthy diets (Ebert, 2012). Edible sprouts, e.g. from crucifers vegetables and legumes, are sources of valuable natural substances such as vitamins, amino acids, fibre, trace elements and various antioxidants (Martinez-Villaluenga

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et al., 2010; Vidal-Valverde et al., 2002). Sprouting improves the nutritional value of seeds, thanks among other things to their higher vitamin content, better quality of protein and enhanced digestion (Lintschinger et al., 2000). Therefore, sprouts are excellent examples of ‘functional food’, defined as lowering the risk of various diseases and/or exerting health-promoting effects (Pa´sko et al., 2009). Up to now sprouts have been enriched with several elements, e.g. Se and Zn (Sugihara et al., 2004; Zou et al., 2014). In the case of ´ Fe, successful enrichment has been obtained for soybean (Zielinska´ and Siger, 2012), rice Dawidziak et al., 2012; Zielinska-Dawidziak ´ (Wei et al., 2013), wheat (Zielinska-Dawidziak et al., 2014b), radish, broccoli and alfalfa (Park et al., 2014). However, the success of enrichment cannot be evaluated solely on the level of accumulated Fe. Fe bioavailability and the effects of enrichment with this element on the yield and quality of obtained food products also have to be taken into consideration. Bioavailability of Fe is already ´ well understood (Clemens, 2014; Hurrell and Egli, 2010; ZielinskaDawidziak et al., 2012). There are two main types of dietary Fe: nonheme Fe, which is present in both plant and animal tissues, and heme Fe, which comes from animal products only. Absorption of nonheme Fe is usually much lower and is determined by many factors: Fe promoters (ascorbic acid, amino acids, prebiotic carbohydrates, beta-carotene, muscle tissue) enhance Fe absorption, whereas Fe inhibitors (phytate, polyphenols, calcium, proteins) limit Fe absorption (Clemens, 2014; Hurrell and Egli, 2010). Fortunately, the metabolic changes that occur during germination increase the bioaccessibility of essential nutrients, as the reduction in non-nutritional components such as phytic acid releases Ca, Zn and Fe from bounded forms (Ahmadzadeh and Prakas, 2007; Greiner and Konietzny, 2006). In certain situations Fe enriched sprouts can be a source of toxic substances in human diet. It has been shown that production of sprouts enriched with Fe requires the use of solutions with a high ´ chemical purity to prevent accumulation of toxic metals (ZielinskaDawidziak et al., 2014a). Free Fe, although essential for metabolism, when is in excess is very redox-active and harmful to cells as it can convert anion superoxide and hydrogen peroxide into hydroxyl radicals via Haber-Weiss and Fenton reactions. Hence, it results in an initiate formation of ROS (Reactive Oxygen Species) (Sharma and Dietz, 2009) and consequently most probably these compounds appear in human diets. Furthermore some antioxidants, such as phenolic compounds, decrease Fe bioavailability and this duality also has to be taken into account when the enrichment strategy is being developed (Clemens, 2014; Hurrell and Egli, 2010). To the best of the authors’ knowledge, works published so far have not studied sprouts enriched with Fe as a potential dietary source of elevated concentrations of ROS. In this work an attempt was made to evaluate the effects of the enrichment of selected sprouts with Fe on: (i) the concentration of Fe and other ions, (ii) biomass accumulation, (iii) the level of ROS and (iv) the activity/content of the constituent elements of enzymatic and non-enzymatic anti-oxidative systems.

2. Material and methods 2.1. Plant material and growing conditions For this study sprouts from crucifer vegetables: broccoli (Brassica oleracea var. botrytis italica) and radish (Raphanus sativus var. redicula), and legumes: alfalfa (Medicago sativa L.) and mung bean (Vigna radiate L.) were chosen. Sprouts of these species are popular and accepted by customers on markets on global scale. Seeds were ˙ purchased from PNOS Ozarów Mazowiecki (Polish seeds producer) and sown on plastic trays (dimensions 27 × 17 × 3 cm) lined with

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filter paper and moistened with distilled water (control) or Fe in concentrations of 6, 12, 24 and 36 mg/L. Fe was used in the form of C10 H13 FeN2 O8 (Ferric EDTA). Ferric EDTA improves Fe solubility and thus bioavailability for plants, especially in conditions of higher pH. Sprouts were grown under standard production conditions in a growing chamber (Sanyo MLR-350H, Japan) at 25/18 ◦ C, 12/12 h day/night, irradiance of 250–280 ␮mol/m2 /s PAR (photosynthetic active radiation) and relative humidity 85% for 7 days. During the sprouts’ growth, solutions were supplemented as required. After one week, the sprouts were harvested and rinsed twice in tap water and then in distilled water to remove all ions and substances embedded on their surface. Some of them were immediately used for analysis (levels of ROS, biomass accumulation) and the remaining were frozen in liquid nitrogen and stored at −80 ◦ C until farther analysis (activity of antioxidative enzymes, content of phenolic compounds and total ascorbate, ions concentration).

2.2. Levels of ROS and activity/content of the constituent elements of enzymatic and non-enzymatic anti-oxidative systems The levels of superoxide anion-radical (O2 ◦− ) and hydroxyl radical (OH◦ ) were determined spectrometrically (Spectrometer UV–vis U2900, Hitachi, Japan) in freshly ground plant material at wavelengths of 580 and 540 nm respectively (Chaitanya and Naithani, 1994). In order to determine the activity of enzymes in the antioxidative system, sprouts were ground and subsamples of 200 mg were suspended in 100 mM potassium phosphate buffer (pH 7.8) containing Triton X-100 (0.5%), polyvinylpolypyrollidone (PVPP; 8%) and l-ascorbate (L-AA; 5 mM). The mixture was centrifuged (48000 × g, 4 ◦ C, 20 min) and the activity of the enzymes measured spectrophotometrically (Spectrometer UV/VIS U2900, Hitachi, Japan) at wavelengths of 240, 290 and 340 nm respectively for ascorbate peroxidase (APX) (Nakano and Asada, 1987; modified by Łata et al., 2005), catalase (CAT) (Beers and Sizer, 1952; modified by Łata et al., 2005) and glutathione reductase (GR) (Foyer and Halliwell, 1976, modified by Łata et al., 2005). To assess the content of phenolic compounds, the subsamples of sprouts (500 mg) were homogenised in liquid nitrogen and sonicated in an ultrasonic bath for 30 min in the presence of 5 ml of 70% EtOH. After centrifugation (24000 × g, 4 ◦ C, 10 min), the extract was poured into a new tube and the remaining materials sonicated again. The two extracts obtained were combined, sonicated for 15 min and centrifuged. Supernatant was diluted 1:5 to total 1 ml, then 100 ml of Fast Blue BB (FBBB) was added and vortexed. Subsequently 100 ml of 5% NaOH was added and samples were incubated in the dark for 60 min. The total phenolic content was measured spectrophotometrically (Spectrometer UV/VIS U2900, Hitachi, Japan) at a wavelength of 420 nm and gallic acid (GA) was used as standard (Medina, 2011). Total ascorbate (sum of the ascorbate and dehydroascorbate) was measured with HPLC system (Waters, Milford, MA, USA) after complete oxidation of ascorbate oxidase (Polle et al., 1990). Dehydroascorbate was derivitised with o-phenyloamine, and the reaction product was detected as a fluorescent compound (350/450 nm). The Symmetry C18 column (250 × 4.6 nm, 5 ␮m, Waters) was used at the following conditions: temperature of column 25 ◦ C, flow rate 1 ml/min, run time 12 min and isocratic elution with 20% MeOH plus 800 mM K2 HPO4 (pH 7.8). The results were calculated using a standard curve. The HPLC system was equipped with System Breeze with a binary solvent delivery system (1525), a degasser, an autosampler with a thermostat with the scale of 4–40 ◦ C (M 717 PLUS), a scanning fluorescence detector (M 474) and the thermostat for the column 5–85 ◦ C (Peltier).

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2.3. Determination of ion concentration The dried sprouts were ground in a laboratory mill and 0.3 g was placed in glass tubes and wet mineralised with 15 ml of concentrated nitric acid. The amounts of Fe, K, Ca, Mg, Zn and Mn were determined by atomic absorption spectrometry (Solaar M6, Thermo scientific, USA). 2.4. Biomass accumulation Fifty sprouts were randomly selected from each tray and their fresh weight was recorded. They were then oven-dried for 24 h at 105 ◦ C and then for three days at 75 ◦ C (KCW-100 drying chamber, PREMED, Poland) to obtain constant dry matter. 2.5. Statistics Data were subjected to analysis of one factorial ANOVA using Statgraphics Plus 4.1. (Statpoint Technologies Inc., Warrenton, VA, USA). The Shapiro–Wilk test was used to examine the normality of distribution, while Bartlett’s test verified the homogeneity of variances. Differences between means of combinations were evaluated by post-hoc Tukey’s Honestly Significant Difference test (HSD). Significance of means was evaluated at the P < 0.05 level. There were three biological replications (tray with sprouts). The number of replications (separate sampling) for a given parameter ranged between three and six, and is indicated in the specific tables. 3. Results 3.1. Ion concentration In most cases, enrichment with Fe led to a significant increase in the concentration of this element in all the sprouts species examined (Table 1). Of the species tested, supplementation with Fe was most efficient in alfalfa (increase of 33.6–129.9%) followed by broccoli (increase of 15.3–81.2%). In sprouts of radish and mung bean, the Fe concentration also increased, but to a lesser extent with increases in the range of 6.6–50.4% and 36.6–60.6% respectively. The highest Fe concentration after enrichment was noted in radish, followed by alfalfa, both grown in the presence of 36 mg/L Fe. The lowest concentration of Fe was recorded in mung bean. Fe concentration in control sprouts was also the highest in radish, while the lowest was in mung bean (Table 1). The Fe treatments affected the concentration of several other elements, but the recorded changes were very small and usually insignificant (Table 1). The concentration of Mg slightly decreased in radish (by 5.3–15.2%), while there was no change in all other species. Growing sprouts in the presence of Fe usually negatively affected the concentration of Ca (by 1.8–7.5%, 5.6–11.6%, 5.0–14.1% and 3.5–11.8% in broccoli, radish, alfalfa and mung bean respectively). However, when alfalfa sprouts were treated with 6 and 12 mg/L of Fe concentration of Ca increased (by 6.3–6.8%). The concentration of Zn increased in alfalfa (by 5.6–42.3%), but significantly only when the sprouts were enriched with 6 mg/L of Fe, was not changed in mung bean, in broccoli the concentration of this element slightly reduced (by 4.0–12.0%), while in radish it also decreased (by 7.4–8.8%), but only in 6 and 12 mg/L of Fe. The concentration of K and Mn barely changed after the sprouts were enriched with Fe (Table 1). 3.2. Biomass accumulation Enrichment with Fe had no effect on dynamics and the capacity of germination (data not shown). Sprouts grown in the presence of Fe developed normally, showing no symptoms of Fe toxicity

or nutrient deficiency, but in most cases their biomass accumulation, expressed as both fresh weight and dry matter, decreased with increasing Fe concentration (Table 2). The enrichment with Fe reduced biomass accumulation, in some cases significantly, in sprouts of alfalfa (by 0.8–16.1% and 4.4–13.3% for fresh weight and dry matter respectively), and both species of the crucifer family: radish (by 15.3–32.4% and 2.9–15.9% for fresh weight and dry matter respectively) and broccoli (by 9.1–31.3% and 12.5–17.3% for fresh weight and dry matter respectively). Contrarily, dry matter increased in mung bean (by 5.9–8.1%), while fresh weight similarly to other species decreased, although insignificantly. In all examined species the negative effect of Fe on biomass accumulation was usually more pronounced in fresh weight than in dry matter (Table 2). 3.3. Levels of ROS and activity/content of the constituent elements of enzymatic and non-enzymatic anti-oxidative systems The enrichment of sprouts with Fe strongly altered levels of ROS and activity/content of selected elements of antioxidative system (Tables 3–5). The levels of the two examined ROS increased significantly in broccoli (by 16.0–112.3% and 29.2–204.9% respectively for O2 ◦− and OH◦ ) and radish (by 18.3–28.5% and 60.8–176.5% respectively for O2 ◦− and OH◦ ), but in radish level of O2 ◦− increased only in two higher Fe concentrations (Table 3). Production of both ROS was also greatly enhanced in mung bean (by 71.3–116.1% and 26.1–43.5% respectively for O2 ◦− and OH◦ ), but recorded changes were significant only in case of O2 ◦− . In alfalfa the level of O2 ◦− significantly increased (by 19.2–61.5%), while OH◦ generally decreased (by 11.7–29.1%). Of the tested species, the highest levels of O2 ◦− were recorded in mung bean, while the lowest were in alfalfa; in the case of OH◦ the highest and lowest levels were noted in broccoli and mung bean respectively (Table 3). Independently of the examined species and Fe concentration, enzyme activity in the antioxidative system was greater in sprouts enriched with Fe (Table 4). An increase in the activity of APX was recorded in all species and amounted to 47.8–77.1%, 5.5–93.4%, 17.1–95.2% and 17.7–43.2% in broccoli, radish, alfalfa and mung bean respectively. This increase was significant in sprouts of the species of crucifer family exposed to the most of Fe concentrations and alfalfa grown in the presence of 36 mg/Fe. The addition of Fe to the growing medium also resulted in higher GR activity, which was greater than in the controls by 56.6–191.0%, 20.0–142.0%, 8.8–59.6% and 24.3–55.0%, and CAT, which increased by 13.9–52.5%, 1.6–6.6%, 2.9–41.7% and 23.9–39.0% in broccoli, radish, alfalfa and mung bean respectively. Increase in GR activity was significant in all examined sprouts species, but they differed in Fe concentrations in which significance was recorded. CAT activity was significantly higher only in broccoli (Table 4). The enrichment with Fe usually induced the production of phenolic compounds in both crucifers and mung bean (Table 5). The content of these compounds increased to the greatest extent in broccoli (by 3.1–217.7%), followed by mung bean (by 28.6–50.0%) and radish (by 13.0–26.0%). Recorded changes were significant in mung bean (all Fe concentrations) and broccoli (36 mg/L Fe). Only in the case of alfalfa sprouts effect of Fe on phenolic compounds was less evident and not unidirectional since their content decreased in 12 and 24 mg/L Fe, but increased in the lowest and highest concentrations. When comparing species, higher content of phenolic compounds was recorded in broccoli and radish, while the lowest in alfalfa (Table 5). Growing sprouts in the presence of Fe led to a significant increase in the ascorbic acid content in alfalfa (by 46.3–155.9%) and broccoli (by 75.3–149.2%) (Table 5). Mung bean sprouts had also higher content of ascorbic acid (by 76.6–82.8%), however in case of this species effect of Fe was insignificant. The exception

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Table 1 Selected ions concentration in sprouts of broccoli, radish, alfalfa and mung bean grown in the presence of Fe ions, n = 3. Ion concentration

Fe

Mg

Ca

Zn

K

Mn

*

Fe

Broccoli

mg/L

mg/g dry matter

Control 6 12 24 36 Control 6 12 24 36 Control 6 12 24 36 Control 6 12 24 36 Control 6 12 24 36 Control 6 12 24 36

0.085 a* 0.098 ab 0.111 b 0.146 c 0.154 c 3.34 a 3.34 a 3.37 a 3.35 a 3.32 a 4.77 a 4.42 a 4.41 a 4.68 a 4.69 a 0.050 a 0.048 a 0.045 a 0.044 a 0.044 a 13.6 a 13.5 a 13.4 a 13.2 a 12.9 a 0.033 a 0.039 a 0.037 a 0.032 a 0.028 a

Radish

Alfalfa

Mung bean

0.516 a 0.550 a 0.559 a 0.639 ab 0.776 b 3.02 a 2.58 a 2.56 a 2.73 a 2.86 a 4.29 a 3.79 a 3.80 a 4.04 a 4.05 a 0.068 ab 0.063 ab 0.062 a 0.068 ab 0.070 b 15.7 a 14.6 a 14.6 a 15.3 a 15.5 a 0.021 a 0.023 a 0.023 a 0.020 a 0.021 a

0.244 a 0.326 ab 0.340 b 0.445 c 0.561 d 2.32 a 2.37 a 2.32 a 2.31 a 2.47 a 3.83 ab 4.07 b 4.09 b 3.64 ab 3.29 a 0.071 a 0.101 b 0.087 ab 0.068 a 0.075 a 16.3 ab 16.1 ab 15.3 a 16.3 b 16.6 b 0.017 a 0.017 a 0.018 a 0.017 a 0.018 a

0.071 a 0.071 a 0.097 b 0.104 b 0.114 b 2.35 a 2.37 a 2.31 a 2.41 a 2.27 a 1.97 a 1.90 a 1.78 a 1.74 a 2.12 a 0.054 a 0.053 a 0.052 a 0.054 a 0.050 a 20.5 a 20.7 a 20.8 a 21.5 a 20.8 a 0.012 a 0.010 a 0.011 a 0.012 a 0.011 a

The difference between the means for Fe concentrations are significant when the bars are indicated by different lower case letters.

Table 2 Biomass accumulation in sprouts of broccoli, radish, alfalfa and mung bean grown in the presence of Fe ions, n = 3. Weight of 50 sprouts

Fresh weight

Dry matter

*

Fe

Broccoli

mg/L

g

Control 6 12 24 36 Control 6 12 24 36

1.21 b* 1.10 ab 0.921 ab 0.958 ab 0.831 a 0.104 a 0.091 a 0.090 a 0.086 a 0.087 a

Radish

Alfalfa

Mung bean

2.41 b 2.70 b 2.04 ab 1.64 a 1.63a 0.309 a 0.300 a 0.292 a 0.260 a 0.263 a

1.18 b 1.10 ab 1.17 b 1.13 ab 0.990 a 0.090 a 0.086 a 0.083 a 0.082 a 0.078 a

11.4 a 10.9 a 9.92 a 10.4 a 9.91 a 1.35 a 1.45 a 1.45 a 1.43 a 1.46 a

The difference between the means for Fe concentrations are significant when the bars are indicated by different lower case letters.

Table 3 Content of anion-radical (O2 ◦ − ) and hydroxyl radical (OH◦ ) in sprouts of broccoli, radish, alfalfa and mung bean grown in the presence of Fe ions, n = 6. Measured ROS

Anion-radical (O2 ◦ − )

Hydroxyl radical (OH◦ )

*

Fe

Broccoli

mg/L

relative values

Control 6 12 24 36 Control 6 12 24 36

1.06 a* 1.47 ab 1.74 bc 1.23 ab 2.25 c 0.185 a 0.330 ab 0.449 bc 0.239 a 0.564 c

Radish

Alfalfa

Mung bean

1.58 ab 1.10 a 1.35 ab 2.03 b 1.87 b 0.051 a 0.082 a 0.091 a 0.085 a 0.141 b

1.04 a 1.38 ab 1.46 ab 1.24 ab 1.68 b 0.282 a 0.218 a 0.333 a 0.249 a 0.200 a

1.65 a 2.82 b 3.33 b 3.05 b 3.56 b 0.046 a 0.066 a 0.077 a 0.058 a 0.045 a

The difference between the means for Fe concentrations are significant when the bars are indicated by different lower case letters.

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Table 4 Activity of ascorbate peroxidase (APX), glutathione reductase (GR) and catalase (CAT) in sprouts of broccoli, radish, alfalfa and mung bean grown in the presence of Fe ions, n = 3. Measured enzyme

Ascorbate peroxidase (APX)

Glutathione reductase (GR)

Catalase (CAT)

*

Fe

Broccoli

mg/L

n kat/g fresh weight

Control 6 12 24 36 Control 6 12 24 36 Control 6 12 24 36

7.51 ab* 6.61 a 12.2 bc 13.3 c 11.1 bc 0.256 a 0.401 ab 0.621 bc 0.407 ab 0.745 c 0.488 a 0.556 ab 0.577 ab 0.590 ab 0.744 b

Radish

Alfalfa

Mung bean

6.67 a 7.04 ab 10.6 bc 12.8 c 12.9 c 0.250 a 0.300 a 0.374 a 0.532 b 0.605 b 0.439 a 0.455 a 0.460 a 0.468 a 0.446 a

4.39 a 5.14 a 5.66 a 5.45 a 8.57 b 0.307 a 0.344 a 0.334 a 0.345 a 0.490 b 0.376 a 0.387 a 0.415 a 0.390 a 0.533 a

7.61 a 9.45 a 10.9 a 8.96 a 10.6 a 0.411 a 0.564 ab 0.637 b 0.511 ab 0.537 ab 0.649 a 0.804 a 0.902 a 0.837 a 0.835 a

The difference between the means for Fe concentrations are significant when the bars are indicated by different lower case letters.

was radish in which the content of ascorbic acid did not change or was even lower. The highest content of ascorbic acid was recorded in broccoli, while in alfalfa it was the lowest (Table 5). 4. Discussion Sprouts enriched with essential elements are deservedly receiving growing attention. However, enrichment can be justified only when in parallel with an increased accumulation of the desired element(s) there is no reduction in biomass production or nutritional value. In this study an attempt was made to enrich sprouts with Fe and to evaluate the effects of enrichment on the yield, level of ROS and activity/content of the constituent elements of enzymatic and non-enzymatic anti-oxidative systems in the food products obtained. 4.1. Enrichment with Fe and concentration of selected ions The results of this study revealed that an increasing concentration of Fe in growing medium simultaneously brought about an increase in the Fe concentration of tested sprouts. However, the effectiveness of the enrichment varied between species and turned out to be most effective in alfalfa (2.3-fold increase) followed by broccoli (1.8-fold increase), while it was less effective in mung bean (1.6-fold increase) and radish (1.5-fold increase). These results are consistent with a previous study reporting that Fe accumulation significantly varies between species/cultivars (Wei et al., 2013). Sprouting seeds from legumes in the presence of Fe is an acknowledged method of enrichment (remarkable 28-fold

increase in soybean), primarily because they contain a high con´ centration of phytoferritin (Zielinska-Dawidziak and Siger, 2012). Bains et al. (2014) showed, that also bioavailability of Fe is enhanced in legumes sprouts, mostly due to phytates reduction and increase in the content of Fe absorption enhancers with progressing germination. In the present study, the examined legume sprouts were less efficient and differed considerably with regard to their enrichment potential, which was higher in alfalfa. Sprouting seeds in solution containing Fe was not the only possible method of enrichment. According to Park et al. (2014), the concentration of Fe in alfalfa sprouts was also significantly greater (2.0-fold increase) when seeds were soaked in Fe solution during imbibition. For broccoli and radish the effect of this treatment was also positive, but not significant (Park et al., 2014). In this work, sprouts species characterised as having the greatest increase in Fe content were not the richest in this element, since the highest Fe concentrations were found in radish, both in the control and after enrichment, while the mung bean had the lowest Fe concentrations. Therefore, to achieve the highest possible concentration of Fe in sprouts, it is important to strive for both efficient enrichment and species/varieties naturally rich in this element. In the case of sprouts, the initial concentration of Fe in seeds is crucial. There is a significant variation in Fe concentrations in seeds between and within species of legumes, where it has been found to vary between 1.4–6.6 fold (White and Broadley, 2005). This corresponds well with the 3.4-fold greater concentration of Fe in alfalfa seeds recorded in this study compared to mung bean. Due to the fact that sprouts are eaten in small quantities, the Fe concentration in the sprouts has to be high in order to have

Table 5 Content of phenolic compounds and ascorbic acid in sprouts of broccoli, radish, alfalfa and mung bean grown in the presence of Fe ions, n = 3. Measured enzyme

Content of phenolic compounds

Content of ascorbic acid

Fe

Broccoli

mg/L

␮g/g GA

Control 6 12 24 36

76.8 a 79.2 a 156. ab 147. a 244. b ␮g/g fresh weight 571. a 1006. b 1001. b 1050. b 1423. c

control 6 12 24 36

Radish

Alfalfa

Mung bean

123. a 117. a 139. a 153. a 155. a

51.8 a 57.6 a 47.1 a 50.1 a 54.9 a

64.9 a 92.5 b 97.3 b 83.5 ab 91.0 b

620. a 615. a 601. a 624. a 610. a

136. a 348. c 208. b 135. a 199. ab

227. a 415. a 401. a 410. a 405. a

*The difference between the means for Fe concentrations are significant when the bars are indicated by different lower case letters.

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a significant effect on human health. For consumption purposes, sprouts are sold in 100 g packages. Based on the results of this study, for men 100 g of enriched broccoli sprouts provide 9.0–21.0% of the recommended 8 mg Fe/day according to the Recommended Daily Allowance (RDA, Institute of Medicine, 2001), mung bean 11.5–20.9%, alfalfa 23.2–55.3% and radish 76.4–156.7%. The lowest values were usually found in the control and the highest always in sprouts grown at 36 mg/L Fe. For women (18 mg Fe/day) these values amounted to 4.0–9.3%, 5.1–9.3%, 10.3–24.6% and 33.9–69.6% of RDA respectively for broccoli, mung bean, alfalfa and radish. These data demonstrate that, even if eaten in small quantities, radish and alfalfa sprouts may contribute greatly to the daily supplementation of Fe. However, the total Fe content in diets provides little information about the content of bioavailable Fe, which is influenced by the type of food and can vary 10-fold between different meals of similar Fe content (Hallberg and Hulthen, 2000). Unfortunately, the absorption of Fe from vegetarian diets is low and depends on many factors (Clemens, 2014; Hurrell and Egli, 2010). In light of the above, Fe enrichment of plant products is even more desirable and corresponds well with the recommendation of Dietary Reference Intakes to increase Fe content in vegetarians diets by 80% to compensate for its low bioavailability, which is estimated to be 10% (Hunt, 2003). According to Ahmadzadeh and Prakas (2007) the great advantage of enrichment via sprouting seeds is the fact that germination increases Fe bioavailability through the reduction of phytic acid and other antinutrients. One of the greatest problems of plant enrichment with one ion is the impaired uptake of other ions. This should not be a risk in the case of sprouts as they consume nutrients from seeds. The results of this work showed that although enrichment with Fe affected the concentration of some other ions, the levels of recorded changes were small and in most cases insignificant. Therefore, it could be concluded that the sprouts obtained in this study were not depleted of valuable elements. A slight decrease in elements concentration recorded in this study may be explained by the possible leakage of various substances from the seeds during imbibition, as has been previously demonstrated for alfalfa, broccoli and radish sprouts, in which the accumulation of Fe was negatively associated with concentrations of Ca, Mg and Mn (Park et al., 2014). 4.2. Biomass accumulation In the present study, the dynamics and capacity of germination and growth of the sprouts were not affected by Fe, but biomass accumulation generally decreased. In the species tested, the sprout weight was reduced to the greatest extent in crucifers, while in mung bean there was almost no change. It is likely that industrial production of sprouts, which depends on timely growth rates and adequate yields, would be affected by Fe enrichment. In broccoli, radish and alfalfa, the application of Fe in concentration 36 mg/L limited biomass accumulation to too great an extent for it to be considered for enrichment purposes. It has been shown that soaking seeds with Fe solutions also adversely affects the weight of alfalfa and radish sprouts, while in broccoli stimulation was recorded, in contrast to the present study (Park et al., 2014). The loss of biomass accumulation may be caused by excess Fe, causing the overproduction of ROS, especially the hydroxyl radical, which irreversibly impairs cellular structure and damages membranes, DNA and proteins (Sharma and Dietz, 2009; Nagajyoti et al., 2010; Kobayashi and Nishizawa, 2012). Also Li et al. (2012) demonstrated that the inhibition of Triticum aestivum L. seedlings growth in response to Fe exposure was possibly attributed to increased H2 O2 generation, because excessive ROS accumulation within plant cells can lead to membrane lipid oxidation, thus affecting plant growth and development. Lack of negative effects of Fe on growth of mung bean sprouts results most probably from size of seeds.

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Large mung bean seeds provide sufficient amount of nutrients for developing seedlings, thus they have taken up less Fe than other tested species and this might explain that the toxic effect of this element on biomass production was not recorded. In order to find a compromise between the efficiency of enrichment and decrease in biomass accumulation while simultaneously offering adequate application of Fe, the duration of enrichment should also carefully be determined. In this study, enrichment was carried out for seven days, which is a standard duration for the commercial sprouts production, but this period should perhaps be reduced 4.3. Levels of ROS and activity/content of the constituent elements of enzymatic and non-enzymatic anti-oxidative systems Functional food, including plants enriched with selected ions, is considered to be a source of antioxidants. Dietary antioxidants are widely believed to scavenge and/or inhibit the production of ROS in the human body (Nemzer et al., 2014). Antioxidants consumed with plants help in the management of human disease, however many pathologies may be exacerbated when plant food products become a source of ROS, since both a deficiency and excess of ions promote the generation of ROS in plants (Bazzano et al., 2002; Smith-Warner et al., 2003). In standard germination conditions, production of ROS in cells is low (Dubey, 2010) and not affected by Fe, because the level of released Fe from ferritin is limited and Fe is mobilised for growth (Briat et al., 1999). Moreover, variety of ROS, such as O2 ◦− , H2 O2 and ascorbate free radicals generated during germination, when fatty acids are broken-down, are immediately degraded (Donaldson et al., 2008). In enriched sprouts, there may be an increased content of free Fe. Excess Fe is harmful to cells since it can convert anion superoxide and hydrogen peroxide to hydroxyl radicals, and hence increase the formation of ROS (Sharma and Dietz, 2009) both in plants and consumers of them. Therefore, one of the most critical parameters characterising the sprouts enriched with Fe, just behind the concentration of this element, should be the relationship between the level of ROS and content/activity of antioxidants. The effects of Fe enrichment on ROS generation are underestimated in the literature. Lack of Fe toxicity is often validated with the absence of significant inhibition of sprout growth or a decrease in germination rate (Park et al., 2014). In this study, the production of ROS increased in all species and Fe concentrations, including those where the effect on biomass accumulation was very small. An exception was the decreased content of OH◦ in alfalfa. Although, there are no studies directly investigating the effect of Fe enrichment on ROS formation, some researchers have hypothesised that a recorded higher activity of antioxidative enzymes (Smolik et al., 2013) and greater content of non-enzymatic antioxidants, e.g. fer´ ritin, ␤-carotene and phenolic compounds (Zielinska-Dawidziak and Siger, 2012) are associated with a higher production of hydrogen peroxide in terms of stress caused by excess Fe. According to Frossard et al. (2000), the critical toxicity level of Fe in leaves is 500 mg/kg of dry weight, thus lower than that obtained in this study for radish (most concentrations) and alfalfa (36 mg/L Fe). These data demonstrate that a side-effect of sprouts enriched with Fe can be elevated levels of ROS in human diets. Future enrichment strategies must consider this as a possible risk for consumers. Greater ROS generation originating from Fe enrichment might be countered by a simultaneous increase in antioxidative activity, and thus justify the production of enriched sprouts. In the current study, enrichment of sprouts with Fe resulted in increased activity of enzymes (APX, CAT and GR) and greater contents of phenolic compounds and ascorbic acid. These results are in line with other findings (Park et al., 2014; Smolik et al., 2013; Wei et al., 2013 ´ and Siger, 2012) demonstrating that high conZielinska-Dawidziak

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centration of Fe in plants causes an increase in the activity/content of different antioxidants, not just ferritin, which is directly involved in the process of Fe detoxification. Phenolic compounds and ascorbic acid are important plant antioxidants that reduce the risk of various chronic diseases in humans (Davey et al., 2000; Rice-Evans et al., 1997), but they also affect Fe absorption (Clemens, 2014; Hurrell and Egli, 2010). All nonheme food Fe that enters the common Fe pool in the digestive tract is absorbed to the same extent, which depends on the balance between the absorption inhibitors and enhancers (Hurrell and Egli, 2010). Ascorbic acid is classified as an enhancer, but phenolic compounds are inhibitors. In this work, the content of total phenolics was analysed; however it is not just their quantity, but also their type that influences Fe absorption (Hurrell and Egli, 2010). The increment in total phenolic compounds in sprouts grown in standard conditions is explained by an increase in free phenol with alkaline hydrolysis due to dismantling of the cell wall during germination (Wei et al., 2013), but stress caused by excess of Fe probably enhances this increase. Enhanced production of phenolic compounds is the typical response of plants to high concentrations of metals. The results of this work are in agreement with other studies reporting an increase in total phenolic content during Fe fortification process in rice (Wei et al., 2013), radish, alfalfa and broccoli (Park et al., 2014). A less evident effect of excess Fe on total phenolic compounds and flavonoids individually was recorded ´ in Fe-enriched soybean sprouts (Zielinska-Dawidziak and Siger, 2012). Although the inhibitory effect of phenolic compounds on Fe absorption is well proven, some researchers consider the positive relationship between the concentrations of Fe and phenolic compounds as advantageous, mostly because of their benefits to human health (Park et al., 2014). In this study, all tested species of sprouts except radish had an increased content of ascorbic acid when were grown in the presence of Fe. Bains et al. (2014) demonstrated, that in the legumes sprouts content of ascorbic acid increases with an advancement of the germination period, simultaneously enhancing bioavailability of Fe. Ascorbate also plays a key role in the reduction of Fe from the ferric complexes (Grillet et al., 2014). According to Lynch and Cook (1980) there is a dose-dependent enhancing effect of native or added ascorbic acid on Fe absorption. However, with most vegetarian diets the enhancing effect of ascorbic acid on nonheme Fe absorption is unlikely to counteract the absence of unidentified enhancers provided by meat, poultry and fish and the probably increased consumption of inhibitors of Fe absorption (Hunt, 2003). Up to now, the positive effects of antioxidants from functional food referred to prevention or amelioration of oxidative stressrelated human diseases, but their critical role in scavenging of ROS in food products of plant origin was underestimated. This was due to the fact, that plants usually were not considered as a source of ROS in human diets. In this study selected ROS and antioxidants were measured, and therefore it is difficult to state whether the increase in antioxidative potential was sufficient to mitigate the negative effects of ROS. However, based on the results obtained, 24 mg/L Fe can be suggested as a threshold concentration for safe enrichment of broccoli, radish, alfalfa and mung bean sprouts.

5. Conclusions Results obtained in this work allow to conclude that enrichment of the sprouts with Fe in the conditions applied in this study is possible, but the results obtained varied between the species tested. The concentration of Fe in Fe-enriched radish and alfalfa sprouts was high enough to be considered as an alternative dietary source of this element. Sprouts enriched with Fe were not depleted of valuable ions, but their biomass accumulation was reduced most probably

due to the toxic effects of excessive Fe. Our results showed for the first time, that enrichment with Fe significantly increased levels of ROS with at the same time increased activity/content of the enzymatic and non-enzymatic anti-oxidative system. These new data demonstrate that sprouts enriched with Fe if cultivated and enriched in certain conditions can be a source of elevated levels of ROS in human diets. Therefore, very careful analysis of all the compounds that make up the oxidative balance in plants is necessary before a new enrichment strategy is proposed. Of the sprouts tested, radish and alfalfa grown in the presence of Fe in concentrations of 12 and 24 mg/L proved to be the most suitable for enrichment with Fe, as they accumulated considerable amounts of Fe at still acceptable levels of accumulated biomass and relatively low generation of ROS.

Conflict of interest None.

Acknowledgements These studies were financed by the Ministry of Science and Higher Education in Poland within a project accompanying ´ the COST Action 905 granted to S. W. Gawronski, # 799/ NCOST/2010/0.

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