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Composition of mineral elements and bioactive compounds in tartary buckwheat and wheat sprouts as affected by natural mineral-rich water
Accepted Manuscript Composition of mineral elements and bioactive compounds in Tartary buckwheat and wheat sprouts as affected by natural mineral-rich water Paula Pongrac, Mateja Potisek, Anna Fraś, Matevž Likar, Bojan Budič, Kinga Myszka, Danuta Boros, Marijan Nečemer, Mitja Kelemen, Primož Vavpetič, Primož Pelicon, Katarina Vogel-Mikuš, Marjana Regvar, Ivan Kreft PII:
S0733-5210(16)30015-7
DOI:
10.1016/j.jcs.2016.02.002
Reference:
YJCRS 2082
To appear in:
Journal of Cereal Science
Received Date: 4 November 2014 Revised Date:
5 February 2016
Accepted Date: 7 February 2016
Please cite this article as: Pongrac, P., Potisek, M., Fraś, A., Likar, M., Budič, B., Myszka, K., Boros, D., Nečemer, M., Kelemen, M., Vavpetič, P., Pelicon, P., Vogel-Mikuš, K., Regvar, M., Kreft, I., Composition of mineral elements and bioactive compounds in Tartary buckwheat and wheat sprouts as affected by natural mineral-rich water, Journal of Cereal Science (2016), doi: 10.1016/j.jcs.2016.02.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Composition of mineral elements and bioactive compounds in Tartary buckwheat
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and wheat sprouts as affected by natural mineral-rich water
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Paula Pongraca,*,1, Mateja Potiseka, Anna Fraśb, Matevž Likara, Bojan Budičc, Kinga
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Myszkab, Danuta Borosb, Marijan Nečemerd, Mitja Kelemend, Primož Vavpetičd, Primož
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Pelicond, Katarina Vogel-Mikuša,d, Marjana Regvara, Ivan Krefte
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a
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SI-1000 Ljubljana, Slovenia
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Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111,
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Institute, National Research Institute, Radzikow, 05-870 Blonie, Poland
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Laboratory of Quality Evaluation of Plant Materials, Plant Breeding and Acclimatization
National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia Jožef Stefan Institute, Jamova 37, SI-1000 Ljubljana, Slovenia
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Slovenian Forestry Institute, Večna pot 2, SI-1000 Ljubljana, Slovenia
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[email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected] [email protected]; [email protected]
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*Corresponding author 1 Present address: The James Hutton Institute, Errol Road, Invergowrie, Dundee, UK, DD2 5DA Tel: +44 (0) 1382568855 Fax: +44 (0) 8449285429 Email: [email protected]
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Keywords:
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Dietary fibre; Fagopyrum tataricum; Flavonoids; Nutritional composition; Triticum
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aestivum
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ACCEPTED MANUSCRIPT Abstract
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The aim was to determine how the nutritional composition of Tartary buckwheat
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(Fagopyrum tataricum Gaertn.) and wheat (Triticum aestivum L.) sprouts is affected by
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the mineral composition of different waters used during their cultivation. We used tap
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water (TW) and two mineral-rich waters (MRWs), namely moderately mineral-rich water
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(MMRW) and extremely mineral-rich water (EMRW) originating from springs that
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contain naturally present mineral elements. Grain germination was not negatively
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affected by MRWs, however EMRW impeded radicle growth, and consequently
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prevented sprout development. In comparison to cultivation in TW, cultivation in MMRW
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resulted in higher Na, Mg, K and Mn concentrations in both sprouts. There were no
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water-related effects on distribution of mineral elements within plant species, however
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there were differences in Ca distribution. In Tartary buckwheat Ca was located in inter-
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vasculature mesophyll, presumably as oxalate crystals. In wheat Ca predominated in
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epidermis. Only in Tartary buckwheat cultivation in MMRW resulted in less dietary fibre
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and catechin and more quercetin. By capturing compositional profiles of mineral
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elements and bioactive compounds in Tartary buckwheat and wheat sprouts we
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identified the potential for selective enhancement of MMRW. We suggest further work
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using different spring MRWs to identify optimal conditions for cultivation of different
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sprouts.
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1 Introduction
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Mineral element malnutrition has negative influence on human health (Stein et al.
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2010). It is caused by a long-term diet with low concentrations and/ or bioavailability of
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mineral elements. To increase the concentrations and/or bioavailability of mineral
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elements in diet, agronomic and/or genetic approaches can be used. In addition,
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appropriate food processing includingthermal processing, mechanical processing,
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soaking, fermentation, and germination can also be applied (e.g., Nelson et al. 2013).
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Germinated seeds and sprouts are a particularly rich source of bioavailable
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mineral elements, along with other bioactive compounds (Nelson et al., 2013).
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Numerous sprouts with various nutritional qualities are commercially available. Wheat
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(Triticum aestivum L.) sprouts are an excellent source of mineral elements (Plaza et al.,
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2003; Kulkarni et al., 2006), antioxidants (Alvarez-Jubete et al., 2009) and dietary fibre
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(Koehler et al., 2007). However, germination changes the mineral element content of
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wheat (Plaza et al., 2003) and significantly increases the soluble dietary fibre content
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(Koehler et al., 2007). Other nutrient and phytochemical changes that take place after
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germination of wheat grain have been reviewed recently by Nelson et al. (2013). In
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contrast to wheat, Tartary buckwheat (Fagopyrum tataricum Gaertn.) is one of the less
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commonly sprouted grains and as such, is less studied. However Tartary buckwheat
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shows favorable nutritional compositions, with high antioxidative potential and is a rich
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source of rutin (Kim et al., 2008). There is some evidence that the mineral element
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composition of both wheat and common buckwheat sprouts and of Tartary buckwheat
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sprouts can be improved with the use of artificially produced mineral-rich water (MRW)
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Abbreviations used: EMRW, extremely mineral-rich water; I-NSPs, insoluble non-starch polysaccharides; LAPs, lignin and associated polyphenols; MRW, mineral-rich water; MMRW, moderately mineral-rich water; NSPs, non-starch polysaccharides; S-NSPs, soluble non-starch polysaccharides; TDF, total dietary fibre; TW, tap water.
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antioxidant activity was observed in Tartary buckwheat sprouts cultivated in this
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artificially produced MRW, without any accompanying changes to the content of rutin,
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quercetin or crude fibre (Hsu et al., 2008). However, the use of spring MRWs, i.e. salty
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waters that contain naturally present mineral elements has not yet been tested in
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sprout production and cultivation. Therefore, the aim of the study was to test chosen
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spring MRWs in cultivation of wheat and Tartary buckwheat sprouts and to evaluate
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how these naturally present mineral elements will influence their mineral element and
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bioactive compound compositions.
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In this study we germinated and cultivated sprouts in tap water (TW) and two
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spring MRWs (moderately MRW, MMRW and extremely MRW, EMRW) which were
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chosen based on their mineral element concentrations. We also assessed the mineral
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element and bioactive compound compositions of the initial grains to quantify the
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factors of change in the mineral element concentration that take place at the sprouting
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stage of each plant species. To assess whether water regime can influence the
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nutritional content of sprouts we measured the following nutritional composition
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parameters of the sprouts: the concentration and localisation of mineral elements, the
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amount of total dietary fibre content (i.e., comprising total and individual soluble and
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insoluble, non-starch polysaccharides and lignin with associated polyphenols) and
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flavonoids (i.e., rutin, quercetin, and catechin).
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2 Materials and methods
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2.1 Plant material, experimental set-up and sample preparation
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We used the Tartary buckwheat cultivar ‘Wëllkar’, which originates from Luxemburg
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and was recently reintroduced to Slovenia (Mlin Rangus, Dolenje Vrhpolje at 4
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Šentjernej, Slovenia). The grain used in this study came from the second Slovenian cropping (in 2012). For wheat, we used the winter wheat cultivar ‘Ficko’ as a
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representative Slovenian wheat cultivar (provided by Agricultural Institute of Slovenia).
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For both grains, the mature, air dried grain was kept in paper bags in the dark at room
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temperature. For grain analysis, we homogenised whole grains in liquid nitrogen, using
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a pestle and mortar (Pongrac et al., 2013). For sprout production, 10 g of grains (~490
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of Tartary buckwheat; ~135 of wheat) were placed in an automatic sprouter
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(EasyGreen® MicroFarm System, EasyGreen Factory Inc., Nevada, USA) where they
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were watered by misting every 3 h during the day (five times), and twice during the
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night (with a 4-h and 5-h gap) with the same quantity (i.e., 2.0 L day-1) of either TW or
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with one of the chosen spring MRWs. Spring water Radenska Classic (Radenska d. d.,
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Radenci, Slovenia) was used as MMRW and Donat Mg® (Droga Kolinska, Adriatic
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Grupa, Slovenia) as EMRW (Table 1; data provided from the producers), which were
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selected based on appropriate mineral element composition. These MRWs were not
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fortified or changed in any way before bottling or before their use in our experiments.
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Three independent experiments were established at room temperature (21°C)
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and away from direct sun at a light intensity of 10 µmoles m-2 s-1. The germination (on
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5th day) and pH of the solution (using litmus indication paper; Macherey-Nagel,
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Macherey-Nagel GmbH & Co., Düren, Germany) were evaluated. We harvested the
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sprouts eight days after sowing the grains. For buckwheat sprouts this is the optimal
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time for the simple removal of non-edible husks (Kim et al., 2004). The sprouts (roots
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and shoots of Tartary buckwheat sprouts, shoots of wheat sprouts) were rinsed with tap
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water, blotted and weighed (fresh weight). Up to ten individuals were used for cell-type
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specific localisation analysis, while the rest were freeze-dried for 5 days at 0.240 mbar
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and -30 °C (Alpha Christ 2-4). The freeze-dried mat erial was weighed (dry weight),
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homogenised in liquid nitrogen using a pestle and mortar and kept at -20 °C in air-tight
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containers until chemical analyses. The water content was calculated as the difference
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between the fresh weight and the dry weight.
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For cell-type specific mineral element localisation analysis cotyledons of Tartary buckwheat and wheat sprouts were frozen in liquid propane, sectioned at -25ºC in
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cryotome to 50 µm thick sections and freeze dried for three days (Klančnik et al., 2014).
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2.2 Chemical analyses
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2.2.1 Mineral elements
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Concentrations of Na, Mg, P, S, K, Ca, Mn, Fe, Ni, Cu, Zn and Mo were analysed in the
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TW and in the grain and sprout plant material, using inductively coupled plasma-mass
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spectrometry and inductively coupled plasma-optical emission spectroscopy, as
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previously described (Pongrac et al., 2013). The Cl concentration in the TW and plant
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materials was determined by X-ray fluorescence spectrometry, as described by
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Nečemer et al. (2008). For water analysis, total-reflection X-ray fluorescence
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spectrometry was used, while the plant materials were analysed by standard energy-
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dispersive X-ray fluorescence spectrometry, using the Fe-55 radioisotope as the
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excitation source.
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The spatial distribution of Na, Mg, P, S, Cl, K, Ca, Fe and Zn in freeze dried
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cotyledon sections was analysed with micro-proton induced X-ray emission (micro-
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PIXE) as described previously (Pongrac et al., 2013; Klančnik et al., 2014).
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2.2.2 Total dietary fibre
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Total dietary fibre (TDF) was determined in the powdered plant material using an
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enzymatic-chemical method, the ‘Uppsala method’ (Theander et al., 1994). The TDF
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represents the sum of the non-starch polysaccharides (NSPs) and lignin with
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associated polyphenols (LAPs). The NSPs were determined according to Englyst and 6
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insoluble fractions, referred to as S-NSPs and I-NSPs, respectively. The samples (100
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mg), after enzymatic hydrolysis of starch, were centrifuged and split into soluble
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(ethanol precipitates from supernatant) and insoluble (remaining pellet) fractions. Each
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of these fractions was hydrolysed to monosaccharides in 1M sulphuric acid (100 °C, 2
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h). The released monosaccharides were converted to the volatile alditol acetate
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derivatives and quantified on capillary quartz column Rtx-225 (0.53 mm × 30 m) using
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Autosystem XL gas chromatography (Autosystem XL, Perkin Elmer), equipped with an
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autosampler, splitter injection port and flame ionization detector. The carrier gas was
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He. The content of NSPs was calculated as the sum of eight released
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monosaccharides (rhamnose, fucose, ribose, arabinose, xylose, mannose, galactose
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and glucose) of both fractions. The LAPs were determined gravimetrically (Theander
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and Westerlund, 1986). The percentage content of LAPs was calculated on the basis of
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the loss in weight for the incineration of the dried insoluble material, and is expressed
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on a dry matter basis.
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2.2.3 Flavonoids
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For the extraction of the flavonoids rutin, quercetin and catechin, 2 mL methanol:water
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(60:40, v/v) was added to 100 mg powdered plant material at room temperature for 40
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min. After centrifugation at 10000× g for 10 min, the supernatant was filtered (0.22 µm;
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Millipore). We quantified the amount of rutin and quercetin in 50 µL of the filtrate using
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the same HPLC system and method as described in Watanabe and Ito (2002). (+)-
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Catechin was determined in 50 µL of the filtrate using HPLC (López-Serrano and Ros
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Barceló, 1999), with a 4.6 mm ID × 25 cm Waters Spherisorb, ODC-II, C18 column,
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and using a Waters 2690 HPLC system and a Waters 996 photodiode array detector.
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All of the data were processed with the Waters Millenium 2010 LC version 2.10
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software.
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2.3 Statistical analysis
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We tested for differences in mineral element concentrations, dietary fibre and flavonoid
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compositions for grains and sprouts grown in TW vs. MRW for each species separately
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using analysis of variance and Tukey HSD post-hoc tests to separate treatment water
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effects, with significance assessed as p <0.05. We used Levene’s test to test for
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equality of variances and the Shapiro-Wilk W test to test for normality of data. When the
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data did not satisfy the analysis of variance assumptions we used non-parametric
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Kruskal-Wallis one-way analysis of variance by ranks, and for pairwise comparisons,
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Mann-Whitney U post-hoc tests, to a significance level of p <0.05.
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The factors of change in each mineral element concentrations in sprouts was calculated by dividing the average concentration in sprouts of each species and at each
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treatment in comparison to initial grain mineral element concentrations. The micro-PIXE
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spectra were analysed and numerical matrices (pixel-by-pixel concentration matrices)
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were generated by GeoPIXE II software package. These numerical matrices were used
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to generate quantitative distribution maps and co-localisation maps using “RGB
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correlator” plug-in within PyMCA (http://pymca.sourceforge.net/) (Klančnik et al., 2014).
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3 Results and discussion
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3.1 Germination and sprout development
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During germination and seedling development mineral elements are released from their
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storage compounds in grains to be available for the growing embryo. Simultaneously
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the loss of dry matter (mostly of non-fibrous carbohydrates) due to respiration is an
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important factor in concentrating the mineral elements and bioactive compound 8
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of mineral elements through developing roots also takes place. Thus the mineral
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elements available at this point have an important role in the mineral element nutrition
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of the seedling. In our experiments we studied three different waters: tap water (TW)
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and two natural mineral-rich waters (MRWs), moderately mineral-rich water (MMRW)
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and extremely mineral-rich water (EMRW) to determine the effects of naturally present
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mineral elements in the chosen MRWs on the mineral element and bioactive compound
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composition of sprouts compared to TW. Mineral element compositions of waters used
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are given in Table 1. We chose two different plant species: a representative
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monocotyledonous (wheat) and dicotyledonous plant (Tartary buckwheat), to determine
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whether there are differences in their responses to the same cultivation conditions.
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Grain germination was not significantly affected by the MRW treatments (Table 1). By contrast, sprouts were more sensitive to the composition of MRWs. In particular,
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EMRW inhibited radicle and cotyledon development (results not shown) of all of the
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sprouts, making further analyses impossible. This observation indicated that not all
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spring MRWs could be directly used for the production of sprouts. This is likely due to
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the high concentrations of particular mineral elements leading to toxicity, imbalances
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and interactions of mineral elements (e.g., problematically low Ca to Mg ratio in EMRW
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leading to Ca deficiency and in extreme cases to death of roots tips) as well as other
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factors, including pH. Monitoring the pH of the waters revealed considerable increases
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compared to initial water pH (Table 1) with EMRW having pH on average 10.7 (range,
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9-12), in comparison to the MMRW, where the pH was on average 9.05 (range, 7-10).
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Under TW growing conditions, the pH was on average 6.4 (range, 6.0-6.5). Alkaline
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(pH 9.5-10.0) salt stress has previously been shown to be more inhibitory in terms of
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germination of wheat grain than neutral (pH 6.6-6.9) salt stress (Lin et al., 2012). Since
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the experiments were designed to resemble conditions consumers could reproduce at
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MMRW, sprouts of both plant species developed as comparably well as those
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cultivated in TW (both fresh and dry biomass; Table 2). These observations indicate
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that it was not only pH but also the mineral element composition of waters that defined
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the development of the sprouts studied here. Because sprouts in EMRW did not
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survive, the remaining analyses were performed only on sprouts cultivated in TW and
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MMRW.
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3.2 Mineral element composition of the sprouts
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When grains were sprouted in TW, the concentrations of almost all mineral elements
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were higher in sprouts of both species than in the grain (Table 2). There were
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differences between the two plant species, with Tartary buckwheat having higher
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concentrations of Mg, P, Ca, Fe, Ni, Cu, Zn and Mo and lower concentrations of S, Cl,
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K and Mn than wheat sprouts. Other studies have generally also reported an increase
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in the mineral elements in sprouts when compared to grain, although species and
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cultivation-specific effects can occur (Plaza et al., 2003; Lee et al., 2006; Kulkarni et al.,
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2006; Hübner et al., 2010; Nelson et al., 2013). Because of the marked difference
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between species shown here, it would be worthwhile to screen various plant species
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and genotypes cultivated under the same conditions to determine the variability of
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mineral element compositions and to identify species with exceptional compositions.
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The results of sprout cultivation in MMRW compared to TW are not
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straightforward, with sometimes markedly different effects between the two species
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(Table 2, Online resource Table 1). In both species the concentrations of Na, Mg and
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K in MMRW were higher than when cultivated in TW, while concentrations of Cl were
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significantly higher in Tartary buckwheat sprouts, but did not differ between TW and
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MMRW cultivated wheat sprouts (Table 2). These differences reflect higher Na, Mg, Cl 10
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Zn were lower in MMRW than TW cultivated sprouts of both species, and in wheat
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sprouts Fe and Cu concentrations were also lower than in TW (Table 2). The observed
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lower concentration of Ca may be a result of the formation of Ca precipitates in the form
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of Ca carbonate in MMRW but not in TW, while lower Zn concentrations are a result of
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lower Zn concentrations in the MMRW (Table 1). In addition, lower concentrations of
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Zn in sprouts could result from antagonistic effects of Ca on Zn uptake in plants (Baker,
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1978 and references therein). Although the interaction between Ca and Zn was
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observed already few decades ago, the mode of action remains obscure. Likewise, it is
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difficult to explain the lower concentrations of Fe and Cu in wheat sprouts cultivated in
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MMRW compared with TW. It is possible this results from negative interactions
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between Fe and Cu and other mineral elements present in high concentrations in the
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solution. In general, studying the interactions of mineral elements in plants requires
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experiments that are designed to carefully to exclude unplanned effects. Determining
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the extent of these interactions was beyond the aims of this study and we cannot draw
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any strong conclusions on the mechanisms governing these outcomes.
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than in TW result in re-distribution of minerals at the tissue level, we performed
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quantitative localisation analysis, which display tissue-specific localisation of mineral
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elements in Tartary buckwheat and wheat sprouts (Online resource Figure 1A, B).
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There was no apparent difference in the localisation of mineral elements in TW-
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cultivated and MMRW-cultivated sprouts, except in the concentration ranges indicating
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no re-distribution of mineral elements as a result of increased external mineral
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elements occurred. However, there was a striking difference in Ca distribution between
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the two species. Co-localisation images of Ca, Mg and S (in these images Ca is
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illustrated withred, Mg with green and S with blue colour) were generated for easier
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Tartary buckwheat sprouts Ca was located in inter-vasculature mesophyll (Fig. 3 A, B)
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while in wheat sprouts Ca was located in epidermis (Fig. 3 C, D). The difference in the
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concentrations and the spatial distribution of Ca in the two species studied is in line
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with generally accepted difference between dicotyledonous and monocotyledonous
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plants (e.g., see White and Broadley, 2003; Conn and Giliham, 2010). However,
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different Ca distributions have been demonstrated also for various monocotyledonous
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plants, with circa-vasculature and/or epidermal distributions reported (Klančnik et al.,
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2014). Because of these differences it is not possible to make generalisations for
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monocotyledonous plants. Due to confined distribution of Ca in Tartary buckwheat we
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assume that these Ca hot-spots are Ca-oxalate crystals. However, the direct
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assignment of Ca hotspots in Tartary buckwheat sprouts to Ca-oxalate crystals is
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hindered by low resolution of the distribution maps (resolution was 2.3 µm). Despite
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this, there is indirect evidence to support our assumption. In particular i) the diameter of
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the hot-spots is 8-25 µm, which is a typical diameter of Ca-oxalate crystals, ii) their
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spherical shape resembles shapes of Ca-oxalate crystals, iii) plants in the same family
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and order as Tartary buckwheat (Polygonaceae, Caryophyllales) are known to form Ca-
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oxalate crystals, and iv) high oxalate concentrations are reported in buckwheat
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cotyledons (Shen et al,. 2004). The formation of Ca-oxalate in plants is linked to high-
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capacity regulation and protection against herbivory (White and Broadley, 2003 and
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references therein). Since oxalate is believed to negatively interfere with Ca absorption
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during animal digestion, resolving the relationship between the Ca location in plant
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tissue and nutrient bioaccessibility will help identify plant features leading to maximal
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Ca absorption by humans and in this way influence Ca malnutrition (Yang et al., 2012).
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3.3 Dietary fibre of the sprouts 12
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heart disease, stroke, hypertension, diabetes, obesity, and certain gastrointestinal
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diseases in humans (Anderson et al., 2009). Plant cell walls constitute the major part of
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dietary fibre (McDougall et al., 1996). Carboxyl groups on pectins and glucuronoxylans,
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both essential part of plant cell walls, are known to bind cations, especially Ca, but also
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Mg and other divalent cations. Ca bound as Ca-pectate is essential for strengthening
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cell walls and plant tissues (White and Broadley, 2003). In addition, Mn and Cu are
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important as activators of various enzymes including those involved in the synthesis of
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lignin (Broadley et al., 2012). This prompted us to evaluate dietary fibre changes taking
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place in sprout development (TW-cultivated sprouts) and in sprouts cultivated in
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MMRW. In sprouts of both plants species, we measured increases in TDF in
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comparison to the grain (Fig. 1). In TW-cultivated Tartary buckwheat sprouts, this
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increase was primarily from enhanced LAPs synthesis, whereas in wheat, both the l-
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NSPs and LAPs were significantly increased. This was also demonstrated by Koehler
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et al. (2007) who showed that in wheat sprouts TDF increased after germination. This
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increase was mostly due to an increase in soluble dietary fibre and a decrease in
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insoluble dietary fibre (see Koehler et al., 2007). However, this generalisation is not
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consistent across species. For example, in oat and barley grains germinated at 20°C,
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an increase in insoluble dietary fibre observed in 6-day post germination seedlings was
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accompanied by a decrease in soluble dietary fibre resulting in increased TDF in oats
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(Avena sativa L.) and similar levels TDF in grains and seedlings of barley (Hordeum
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vulgare L.; Hübner et al., 2010). These observations indicate species-specific changes
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in TDF and its constituents during germination and sprout development. However, as
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the data on TDF are presented on a dry weight basis, the water content of each stage
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should also be taken into account. Sprouts contain on average of 90% water, while in
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grain, this is 7% (8.45% ±0.27% in Tartary buckwheat grain, and 6.07% ±0.07% in
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wheat grain), which indicates that TDFs calculated on a fresh weight basis will be much
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lower in the sprouts than the grain, or that a considerable amount of raw sprouts will
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needed to be consumed to reach the high dietary fibre intake achieved from grain or
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whole grain flour consumption. The composition of I-NSPs and S-NSPs changed considerably between grain
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and sprouts of Tartary buckwheat and wheat (Fig. 2, for exact data and statistics see
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Online resource Table 2). Tartary buckwheat grains and sprouts contained all eight
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individual monomers of polysaccharides, while in the wheat sprouts fucose was not
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found in the S-NSPs fraction in either of the water treatments. In the TW-cultivated
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Tartary buckwheat the most prominent changes between grain and sprouts was an
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increase in the galactose and a decrease in the glucose contents in both fractions (Fig.
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2A, C). Glucose has previously been reported to be the main sugar in 7-day-old
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sprouts of common buckwheat (Kim et al., 2004). In wheat sprouts, all constituents of
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the I-NSP fraction increased compared to the grain, with galactose contributing most to
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this increase. In contrast, in the S-NSP fraction rhamnose, mannose and galactose of
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the wheat sprouts increased, while the rest decreased (Fig. 2B, D). This again
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suggests species-specific changes in the compositions of the TDF.
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We found a lower TDF content in Tartary buckwheat sprouts cultivated in the
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MMRW than in the TW, primarily due to the lower LAPs. Through enzyme activation
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processes, lignin synthesis is linked to Mn and Cu (Broadley et al., 2012). However,
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assuming a linear relationship, increased Mn and unchanged Cu concentrations in our
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experiment cannot explain the observed decrease in LAPs. Saline stress in the form of
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increasing concentrations of NaCl (up to 100 mM) increased total phenolic content in
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common buckwheat sprouts, presumably through the activation of enzymes involved in
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phenylpropanoid pathway resulting in the accumulation of phenolic compounds (Lim et
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al., 2012). In our experiments we cannot uncover any single factor as clearly
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that the conditions in MMRW did not represent saline stress to the plants as similar
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increases in LAPs was not observed and that other factors or their combinations are
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responsible for the decrease in LAPs. In addition, significantly less S-NSP was seen in
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MMRW cultivated Tartary buckwheat sprouts compared to TW cultivated sprouts with
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arabinose, mannose and galactose contributing most to this decrease (Fig. 2, Online
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resource Table 2). Within I-NSP, fucose, ribose, xylose, galactose and glucose were
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significantly lower in MMRW-cultivated Tartary buckwheat sprouts compared with TW-
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cultivated sprouts (Fig. 2, Online resource Table 2). In wheat sprouts we found no
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differences in LAPs between TW and MMRW treatments (Fig. 1), accompanied by no
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differences in the composition of I-NSPs and S-NSPs between TW and MMRW (Fig. 2,
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Online resource Table 2). We conclude that these effects are species or genotype
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specific (see also Lim et al., 2012). Other plant species and cultivars should be tested
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to determine the level of variation in TDF when cultivating sprouts in MMRW or any
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other spring MRWs.
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3.4 Flavonoids in the sprouts
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The flavonoids in buckwheat grain have been repeatedly connected with anti-oxidative,
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anti-inflammatory, and anti-hypertensive effects (e.g. Alvarez-Jubete et al., 2009).
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Rutin, quercetin and catechin were only detected in Tartary buckwheat grain and
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sprouts and not at all in wheat grains or sprouts, since wheat does not contain any of
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these compounds. Tartary buckwheat sprouts contained higher rutin and catechin
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concentrations and lower concentrations of quercetin than the grain (Table 2). Other
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reports on Tartary buckwheat sprouts have shown higher concentrations of rutin (on
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average 22100 mg kg-1; Kim et al., 2008), but lower concentrations of quercetin (on
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average 100 mg kg-1; Kim et al., 2008) and catechin (594 mg kg-1 in Lee et al., 2006).
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to TW (Table 2) in line with previous studies of rutin in Tartary buckwheat sprouts
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cultivated in artificially produced MRWs and deionized water (Hsu et al., 2008). We
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observed increased quercetin concentration and decreased catechin concentrations
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(Table 2), indicating that by changing mineral element composition of solutions
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secondary metabolism of the sprouts can be changed. Other studies have reported no
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differences in quercetin concentrations in Tartary buckwheat grown in artificially
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produced MRWs and deionized water (Hsu et al., 2008). We were unable to find any
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reports on catehin concentration changes in Tartary buckwheat sprouts as affected by
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MRWs. However, other bioactive compounds have been shown to increase in
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buckwheat sprouts upon introducing mineral elements to deionized water. These
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include increased antioxidant activities and carotenoid concentrations (Hsu et al., 2008;
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Lim et al., 2012). Although the mechanisms behind these changes require detailed
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studies the result clearly suggest that nutritional composition of sprouts can be modified
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to achieve selective enhancement of bioactive compound composition in sprouts.
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4 Conclusions
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In the present study, we identified changes in mineral element and bioactive compound
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compositions of Tartary buckwheat and wheat sprouts as a result of cultivation in
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waters with different mineral element compositions. Differences in mineral element and
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bioactive compound compositions seen in the grains of the species studied cannot fully
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explain the extent of the differences seen in sprouts of the two species, rather species-
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specific mechanisms of mineral uptake play a significant role. EMRW had negative
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effects on the development of sprouts, which was seen as inhibition of radicle growth
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presumably due to mineral element concentrations and imbalances and/ or high pH.
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MMRW resulted in increased concentrations of Na, Mg, K and Mn and decreased
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sprouts. The spatial distribution of mineral elements did not change in response to
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treatments. Cell-type specific accumulation of Ca in the inter-vascular mesophyll was
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observed in Tartary buckwheat sprouts, apparently as Ca oxalate crystals, and in
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epidermis of wheat sprouts. While no effects on the TDF and its composition in the
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wheat sprouts were seen, decreased TDF and catechin was accompanied by
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increased quercetin in the Tartary buckwheat sprouts cultivated in MMRW, indicating
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that the cultivation tested interferes with the secondary metabolism in these sprouts.
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Taken together these results suggest the potential to selectively enhance mineral
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element and bioactive compositions in sprouts. Additional plant species or genotypes
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should be tested in different natural MRWs to determine optimal conditions and species
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specific characteristics to allow recommendations for specific mineral elements and
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bioactive compound composition improvements.
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Acknowledgements
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This study was supported by the P1-0212 Programme, by the Z4-4113 Project of the
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Slovenian Research Agency and by the COST Action FA0905. A. Rangus is
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acknowledged for providing Tartary buckwheat grain, D. Koron for providing wheat
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grain, Radenska d. d., Radenci, Slovenia for Radenska Classic and Droga Kolinska,
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Atlantic Grupa for Donat Mg®. We thank Prof. Linley Jesson and Dr. Tim George for
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reading the original manuscript.
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Fig. 1 Total dietary fibre (TDF) in Tartary buckwheat (A) and wheat (B) grain, and in the
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sprouts cultivated in tap water (TW) and in moderately mineral-rich water (MMRW). I-
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NSP, insoluble non-starch polysaccharides; S-NSP, soluble non-starch
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polysaccharides; LAPs, lignin and associated polyphenols. Different letters above and
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within columns indicate statistically significant differences. Data are means from 3
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independent replicates
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Fig. 2 The relative distributions of the individual monomers of polysaccharides in the
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insoluble non-starch polysaccharides (I-NSP; A, C) and the soluble non-starch
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polysaccharides (S-NSP; B, D) fractions of the total dietary fibre in the Tartary
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buckwheat and wheat grain, and in the sprouts cultivated in tap water (TW) and in
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moderately mineral-rich water (MMRW). Data are means from 3 independent replicated
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Fig. 3 Co-localisation image of Ca (red), Mg (green) and S (blue) in representative
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samples of Tartary buckwheat (A, B) and wheat (C, D) sprouts cultivated in tap water
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and in moderately mineral-rich water (MMRW)
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MMRWb
EMRWb
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Mineral elements (mg L-1) Na 4.4 440 1600 Mg 17.4 95 1060 P 0.01 0.093c <1.0c S 3.71 97d 2200d Cl 2.36 44 62 K 1.00 70 15 Ca 72.0 220 340 Mn 0.004 0.27 0.15 Fe 0.11 <0.10 <0.10 Cu 0.01 <1.0 0.007 Zn 2.47 <10 0.022 HCO3n.m. 2000 7800 pH 7.2 5.6 6 Germination (%) Tartary buckwheat 89±3 87±3 83±3.4 Wheat 85±4 83±3 77±4 a , measured with inductively coupled plasma-mass spectrometry and inductively coupled plasma-optical emission spectroscopy, except Cl, which was measured with total-reflection X-ray spectrometry; b, data provided by the producers; c, measured as HPO4; d, measured as SO4; n.m. not measured.
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Table 2 Fresh (FW; g) and dry weight (DW; g) and concentration of mineral elements, rutin, quercetin and catechin (mg kg-1) in
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grains and sprouts cultivated in tap water (TW) and moderately mineral-rich water (MMRW)
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Tartary buckwheat Wheat grain TW sprouts MMRW sprouts grain TW sprouts MMRW sprouts 0.039 ± 0.006 FW 0.020 ± 0.004 24.1 ± 3.4 21.1 ± 1.9 15.0 ± 1.7 14.3 ± 0.71 0.037 ±- 0.006 DW 0.016 ± 0.004 2.3 ± 0.1 2.1 ±0.04 1.4 ± 0.12 1.6 ± 0.16 Na 5.47 ± 0.48 a* 103 ± 27.1 a 2960 ± 599 b 9.8 ± 005 A* 112 ± 5.46 A 3610 ± 253 B Mg 2100 ± 281 a 5980 ± 433 b 9170 ± 437 c 1200 ± 34.9 A 1260 ± 55.4 A 1730 ± 44.8 B P 3960 ± 56.0 a 7390 ± 201 b 7580 ± 141 b 3420 ± 64.6 A 6360 ± 111 B 6080 ± 226 B S 1220 ± 271 a 2540 ± 57.6 b 2520 ± 94.9 b 1560 ± 394 A* 2800 ± 969 B 2590 ± 583 B a <100 832 ± 363 x 1162 ± 296 y 575 ± 16.3 A 3820 ± 796 B 5810 ± 1167 B Cl K 5120 ± 112 a 8050 ± 518 b 16900 ± 1528 c 4120 ± 55.8 A 11700 ± 869 B 23600 ± 563 C Ca 579 ± 31.8 a* 5900 ± 825 b 2830 ± 1026 a 327 ± 14.6 A* 2070 ± 22.6 C 1280 ± 394 B 23.3 ± 3.09 b 35.7 ± 0.9 C 21.1 ± 0.75 A 26.8 ± 0.13 B Mn 11.0 ± 0.23 a* 14.9 ± 1.1 a Fe 54.3 ± 14.6 a 98.9 ±14.9 b 78.1 ± 13.3 ab 31.7 ± 0.4 A 59.1 ± 4.43 C 47.5 ± 1.35 B Ni 0.40 ± 0.02 a 1.07 ± 0.50 ab 1.37 ± 0.36 b 0.22 ± 0.02 A* 0.21 ± 0.11 A 0.17 ± 0.01 A 2.48 ± 0.02 A* 5.83 ± 0.42 C 4.46 ± 0.17 B Cu 4.5 ± 0.02 a 10.1 ±1.80 b 8.07 ± 1.30 b Zn 23.1 ± 0.47 a* 213 ± 37 b 49.3 ± 5.94 a 21.6 ± 0.55 A 71.9 ± 9.58 B 33.8 ± 1.22 A Mo 0.90 ± 0.07 a 1.30 ± 0.13 b 1.30 ± 0.1 b 0.41 ± 0.06 B 0.30 ± 0.01 AB 0.29 ± 0.05 A rutin 914 ± 101 a 10000 ± 608 b 9210 ± 1850 b nd nd nd quercetin 1270 ± 231 b 687 ± 95 a 1130 ± 329 b nd nd nd catechin 4510 ± 453 a 8360 ± 658 c 7160 ± 480 b nd nd nd Different letters next to the values indicate statistically significant differences (lower case letters for Tartary buckwheat and upper case letters for wheat). Results presented are means ± standard deviation from 3 independent replicates; nd, not detected; a, measured with energy-dispersive X-ray fluorescence; *, nonparametric tests performed.
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• Successful sprout development depended on the mineral element (ME) composition of waters
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• ME concentration but not distribution was affected by ME composition of water • Total dietary fibre was affected by ME composition of waters only in Tartary buckwheat sprouts
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• Quercetin and catechin, but not rutin concentration depended on ME composition
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of waters
S0733-5210(16)30015-7
DOI:
10.1016/j.jcs.2016.02.002
Reference:
YJCRS 2082
To appear in:
Journal of Cereal Science
Received Date: 4 November 2014 Revised Date:
5 February 2016
Accepted Date: 7 February 2016
Please cite this article as: Pongrac, P., Potisek, M., Fraś, A., Likar, M., Budič, B., Myszka, K., Boros, D., Nečemer, M., Kelemen, M., Vavpetič, P., Pelicon, P., Vogel-Mikuš, K., Regvar, M., Kreft, I., Composition of mineral elements and bioactive compounds in Tartary buckwheat and wheat sprouts as affected by natural mineral-rich water, Journal of Cereal Science (2016), doi: 10.1016/j.jcs.2016.02.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Composition of mineral elements and bioactive compounds in Tartary buckwheat
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and wheat sprouts as affected by natural mineral-rich water
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Paula Pongraca,*,1, Mateja Potiseka, Anna Fraśb, Matevž Likara, Bojan Budičc, Kinga
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Myszkab, Danuta Borosb, Marijan Nečemerd, Mitja Kelemend, Primož Vavpetičd, Primož
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Pelicond, Katarina Vogel-Mikuša,d, Marjana Regvara, Ivan Krefte
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a
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SI-1000 Ljubljana, Slovenia
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Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111,
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b
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Institute, National Research Institute, Radzikow, 05-870 Blonie, Poland
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c
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d
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e
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Laboratory of Quality Evaluation of Plant Materials, Plant Breeding and Acclimatization
National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia Jožef Stefan Institute, Jamova 37, SI-1000 Ljubljana, Slovenia
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Slovenian Forestry Institute, Večna pot 2, SI-1000 Ljubljana, Slovenia
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[email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected] [email protected]; [email protected]
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*Corresponding author 1 Present address: The James Hutton Institute, Errol Road, Invergowrie, Dundee, UK, DD2 5DA Tel: +44 (0) 1382568855 Fax: +44 (0) 8449285429 Email: [email protected]
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Keywords:
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Dietary fibre; Fagopyrum tataricum; Flavonoids; Nutritional composition; Triticum
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aestivum
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1
ACCEPTED MANUSCRIPT Abstract
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The aim was to determine how the nutritional composition of Tartary buckwheat
34
(Fagopyrum tataricum Gaertn.) and wheat (Triticum aestivum L.) sprouts is affected by
35
the mineral composition of different waters used during their cultivation. We used tap
36
water (TW) and two mineral-rich waters (MRWs), namely moderately mineral-rich water
37
(MMRW) and extremely mineral-rich water (EMRW) originating from springs that
38
contain naturally present mineral elements. Grain germination was not negatively
39
affected by MRWs, however EMRW impeded radicle growth, and consequently
40
prevented sprout development. In comparison to cultivation in TW, cultivation in MMRW
41
resulted in higher Na, Mg, K and Mn concentrations in both sprouts. There were no
42
water-related effects on distribution of mineral elements within plant species, however
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there were differences in Ca distribution. In Tartary buckwheat Ca was located in inter-
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vasculature mesophyll, presumably as oxalate crystals. In wheat Ca predominated in
45
epidermis. Only in Tartary buckwheat cultivation in MMRW resulted in less dietary fibre
46
and catechin and more quercetin. By capturing compositional profiles of mineral
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elements and bioactive compounds in Tartary buckwheat and wheat sprouts we
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identified the potential for selective enhancement of MMRW. We suggest further work
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using different spring MRWs to identify optimal conditions for cultivation of different
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sprouts.
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1 Introduction
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Mineral element malnutrition has negative influence on human health (Stein et al.
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2010). It is caused by a long-term diet with low concentrations and/ or bioavailability of
54
mineral elements. To increase the concentrations and/or bioavailability of mineral
55
elements in diet, agronomic and/or genetic approaches can be used. In addition,
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appropriate food processing includingthermal processing, mechanical processing,
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soaking, fermentation, and germination can also be applied (e.g., Nelson et al. 2013).
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Germinated seeds and sprouts are a particularly rich source of bioavailable
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mineral elements, along with other bioactive compounds (Nelson et al., 2013).
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Numerous sprouts with various nutritional qualities are commercially available. Wheat
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(Triticum aestivum L.) sprouts are an excellent source of mineral elements (Plaza et al.,
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2003; Kulkarni et al., 2006), antioxidants (Alvarez-Jubete et al., 2009) and dietary fibre
63
(Koehler et al., 2007). However, germination changes the mineral element content of
64
wheat (Plaza et al., 2003) and significantly increases the soluble dietary fibre content
65
(Koehler et al., 2007). Other nutrient and phytochemical changes that take place after
66
germination of wheat grain have been reviewed recently by Nelson et al. (2013). In
67
contrast to wheat, Tartary buckwheat (Fagopyrum tataricum Gaertn.) is one of the less
68
commonly sprouted grains and as such, is less studied. However Tartary buckwheat
69
shows favorable nutritional compositions, with high antioxidative potential and is a rich
70
source of rutin (Kim et al., 2008). There is some evidence that the mineral element
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composition of both wheat and common buckwheat sprouts and of Tartary buckwheat
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sprouts can be improved with the use of artificially produced mineral-rich water (MRW)
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1
Abbreviations used: EMRW, extremely mineral-rich water; I-NSPs, insoluble non-starch polysaccharides; LAPs, lignin and associated polyphenols; MRW, mineral-rich water; MMRW, moderately mineral-rich water; NSPs, non-starch polysaccharides; S-NSPs, soluble non-starch polysaccharides; TDF, total dietary fibre; TW, tap water.
3
ACCEPTED MANUSCRIPT for sprout cultivation (Lintschinger et al., 1997, Hsu et al., 2008). In addition, higher
74
antioxidant activity was observed in Tartary buckwheat sprouts cultivated in this
75
artificially produced MRW, without any accompanying changes to the content of rutin,
76
quercetin or crude fibre (Hsu et al., 2008). However, the use of spring MRWs, i.e. salty
77
waters that contain naturally present mineral elements has not yet been tested in
78
sprout production and cultivation. Therefore, the aim of the study was to test chosen
79
spring MRWs in cultivation of wheat and Tartary buckwheat sprouts and to evaluate
80
how these naturally present mineral elements will influence their mineral element and
81
bioactive compound compositions.
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In this study we germinated and cultivated sprouts in tap water (TW) and two
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spring MRWs (moderately MRW, MMRW and extremely MRW, EMRW) which were
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chosen based on their mineral element concentrations. We also assessed the mineral
85
element and bioactive compound compositions of the initial grains to quantify the
86
factors of change in the mineral element concentration that take place at the sprouting
87
stage of each plant species. To assess whether water regime can influence the
88
nutritional content of sprouts we measured the following nutritional composition
89
parameters of the sprouts: the concentration and localisation of mineral elements, the
90
amount of total dietary fibre content (i.e., comprising total and individual soluble and
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insoluble, non-starch polysaccharides and lignin with associated polyphenols) and
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flavonoids (i.e., rutin, quercetin, and catechin).
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2 Materials and methods
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2.1 Plant material, experimental set-up and sample preparation
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We used the Tartary buckwheat cultivar ‘Wëllkar’, which originates from Luxemburg
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and was recently reintroduced to Slovenia (Mlin Rangus, Dolenje Vrhpolje at 4
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Šentjernej, Slovenia). The grain used in this study came from the second Slovenian cropping (in 2012). For wheat, we used the winter wheat cultivar ‘Ficko’ as a
101
representative Slovenian wheat cultivar (provided by Agricultural Institute of Slovenia).
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For both grains, the mature, air dried grain was kept in paper bags in the dark at room
103
temperature. For grain analysis, we homogenised whole grains in liquid nitrogen, using
104
a pestle and mortar (Pongrac et al., 2013). For sprout production, 10 g of grains (~490
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of Tartary buckwheat; ~135 of wheat) were placed in an automatic sprouter
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(EasyGreen® MicroFarm System, EasyGreen Factory Inc., Nevada, USA) where they
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were watered by misting every 3 h during the day (five times), and twice during the
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night (with a 4-h and 5-h gap) with the same quantity (i.e., 2.0 L day-1) of either TW or
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with one of the chosen spring MRWs. Spring water Radenska Classic (Radenska d. d.,
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Radenci, Slovenia) was used as MMRW and Donat Mg® (Droga Kolinska, Adriatic
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Grupa, Slovenia) as EMRW (Table 1; data provided from the producers), which were
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selected based on appropriate mineral element composition. These MRWs were not
113
fortified or changed in any way before bottling or before their use in our experiments.
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Three independent experiments were established at room temperature (21°C)
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and away from direct sun at a light intensity of 10 µmoles m-2 s-1. The germination (on
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5th day) and pH of the solution (using litmus indication paper; Macherey-Nagel,
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Macherey-Nagel GmbH & Co., Düren, Germany) were evaluated. We harvested the
118
sprouts eight days after sowing the grains. For buckwheat sprouts this is the optimal
119
time for the simple removal of non-edible husks (Kim et al., 2004). The sprouts (roots
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and shoots of Tartary buckwheat sprouts, shoots of wheat sprouts) were rinsed with tap
121
water, blotted and weighed (fresh weight). Up to ten individuals were used for cell-type
122
specific localisation analysis, while the rest were freeze-dried for 5 days at 0.240 mbar
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and -30 °C (Alpha Christ 2-4). The freeze-dried mat erial was weighed (dry weight),
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homogenised in liquid nitrogen using a pestle and mortar and kept at -20 °C in air-tight
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containers until chemical analyses. The water content was calculated as the difference
126
between the fresh weight and the dry weight.
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For cell-type specific mineral element localisation analysis cotyledons of Tartary buckwheat and wheat sprouts were frozen in liquid propane, sectioned at -25ºC in
129
cryotome to 50 µm thick sections and freeze dried for three days (Klančnik et al., 2014).
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2.2 Chemical analyses
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2.2.1 Mineral elements
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Concentrations of Na, Mg, P, S, K, Ca, Mn, Fe, Ni, Cu, Zn and Mo were analysed in the
134
TW and in the grain and sprout plant material, using inductively coupled plasma-mass
135
spectrometry and inductively coupled plasma-optical emission spectroscopy, as
136
previously described (Pongrac et al., 2013). The Cl concentration in the TW and plant
137
materials was determined by X-ray fluorescence spectrometry, as described by
138
Nečemer et al. (2008). For water analysis, total-reflection X-ray fluorescence
139
spectrometry was used, while the plant materials were analysed by standard energy-
140
dispersive X-ray fluorescence spectrometry, using the Fe-55 radioisotope as the
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excitation source.
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The spatial distribution of Na, Mg, P, S, Cl, K, Ca, Fe and Zn in freeze dried
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cotyledon sections was analysed with micro-proton induced X-ray emission (micro-
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PIXE) as described previously (Pongrac et al., 2013; Klančnik et al., 2014).
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2.2.2 Total dietary fibre
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Total dietary fibre (TDF) was determined in the powdered plant material using an
148
enzymatic-chemical method, the ‘Uppsala method’ (Theander et al., 1994). The TDF
149
represents the sum of the non-starch polysaccharides (NSPs) and lignin with
150
associated polyphenols (LAPs). The NSPs were determined according to Englyst and 6
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insoluble fractions, referred to as S-NSPs and I-NSPs, respectively. The samples (100
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mg), after enzymatic hydrolysis of starch, were centrifuged and split into soluble
154
(ethanol precipitates from supernatant) and insoluble (remaining pellet) fractions. Each
155
of these fractions was hydrolysed to monosaccharides in 1M sulphuric acid (100 °C, 2
156
h). The released monosaccharides were converted to the volatile alditol acetate
157
derivatives and quantified on capillary quartz column Rtx-225 (0.53 mm × 30 m) using
158
Autosystem XL gas chromatography (Autosystem XL, Perkin Elmer), equipped with an
159
autosampler, splitter injection port and flame ionization detector. The carrier gas was
160
He. The content of NSPs was calculated as the sum of eight released
161
monosaccharides (rhamnose, fucose, ribose, arabinose, xylose, mannose, galactose
162
and glucose) of both fractions. The LAPs were determined gravimetrically (Theander
163
and Westerlund, 1986). The percentage content of LAPs was calculated on the basis of
164
the loss in weight for the incineration of the dried insoluble material, and is expressed
165
on a dry matter basis.
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2.2.3 Flavonoids
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For the extraction of the flavonoids rutin, quercetin and catechin, 2 mL methanol:water
169
(60:40, v/v) was added to 100 mg powdered plant material at room temperature for 40
170
min. After centrifugation at 10000× g for 10 min, the supernatant was filtered (0.22 µm;
171
Millipore). We quantified the amount of rutin and quercetin in 50 µL of the filtrate using
172
the same HPLC system and method as described in Watanabe and Ito (2002). (+)-
173
Catechin was determined in 50 µL of the filtrate using HPLC (López-Serrano and Ros
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Barceló, 1999), with a 4.6 mm ID × 25 cm Waters Spherisorb, ODC-II, C18 column,
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and using a Waters 2690 HPLC system and a Waters 996 photodiode array detector.
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All of the data were processed with the Waters Millenium 2010 LC version 2.10
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software.
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2.3 Statistical analysis
180
We tested for differences in mineral element concentrations, dietary fibre and flavonoid
181
compositions for grains and sprouts grown in TW vs. MRW for each species separately
182
using analysis of variance and Tukey HSD post-hoc tests to separate treatment water
183
effects, with significance assessed as p <0.05. We used Levene’s test to test for
184
equality of variances and the Shapiro-Wilk W test to test for normality of data. When the
185
data did not satisfy the analysis of variance assumptions we used non-parametric
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Kruskal-Wallis one-way analysis of variance by ranks, and for pairwise comparisons,
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Mann-Whitney U post-hoc tests, to a significance level of p <0.05.
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The factors of change in each mineral element concentrations in sprouts was calculated by dividing the average concentration in sprouts of each species and at each
190
treatment in comparison to initial grain mineral element concentrations. The micro-PIXE
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spectra were analysed and numerical matrices (pixel-by-pixel concentration matrices)
192
were generated by GeoPIXE II software package. These numerical matrices were used
193
to generate quantitative distribution maps and co-localisation maps using “RGB
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correlator” plug-in within PyMCA (http://pymca.sourceforge.net/) (Klančnik et al., 2014).
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3 Results and discussion
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3.1 Germination and sprout development
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During germination and seedling development mineral elements are released from their
199
storage compounds in grains to be available for the growing embryo. Simultaneously
200
the loss of dry matter (mostly of non-fibrous carbohydrates) due to respiration is an
201
important factor in concentrating the mineral elements and bioactive compound 8
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203
of mineral elements through developing roots also takes place. Thus the mineral
204
elements available at this point have an important role in the mineral element nutrition
205
of the seedling. In our experiments we studied three different waters: tap water (TW)
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and two natural mineral-rich waters (MRWs), moderately mineral-rich water (MMRW)
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and extremely mineral-rich water (EMRW) to determine the effects of naturally present
208
mineral elements in the chosen MRWs on the mineral element and bioactive compound
209
composition of sprouts compared to TW. Mineral element compositions of waters used
210
are given in Table 1. We chose two different plant species: a representative
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monocotyledonous (wheat) and dicotyledonous plant (Tartary buckwheat), to determine
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whether there are differences in their responses to the same cultivation conditions.
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Grain germination was not significantly affected by the MRW treatments (Table 1). By contrast, sprouts were more sensitive to the composition of MRWs. In particular,
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EMRW inhibited radicle and cotyledon development (results not shown) of all of the
216
sprouts, making further analyses impossible. This observation indicated that not all
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spring MRWs could be directly used for the production of sprouts. This is likely due to
218
the high concentrations of particular mineral elements leading to toxicity, imbalances
219
and interactions of mineral elements (e.g., problematically low Ca to Mg ratio in EMRW
220
leading to Ca deficiency and in extreme cases to death of roots tips) as well as other
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factors, including pH. Monitoring the pH of the waters revealed considerable increases
222
compared to initial water pH (Table 1) with EMRW having pH on average 10.7 (range,
223
9-12), in comparison to the MMRW, where the pH was on average 9.05 (range, 7-10).
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Under TW growing conditions, the pH was on average 6.4 (range, 6.0-6.5). Alkaline
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(pH 9.5-10.0) salt stress has previously been shown to be more inhibitory in terms of
226
germination of wheat grain than neutral (pH 6.6-6.9) salt stress (Lin et al., 2012). Since
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the experiments were designed to resemble conditions consumers could reproduce at
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MMRW, sprouts of both plant species developed as comparably well as those
230
cultivated in TW (both fresh and dry biomass; Table 2). These observations indicate
231
that it was not only pH but also the mineral element composition of waters that defined
232
the development of the sprouts studied here. Because sprouts in EMRW did not
233
survive, the remaining analyses were performed only on sprouts cultivated in TW and
234
MMRW.
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3.2 Mineral element composition of the sprouts
237
When grains were sprouted in TW, the concentrations of almost all mineral elements
238
were higher in sprouts of both species than in the grain (Table 2). There were
239
differences between the two plant species, with Tartary buckwheat having higher
240
concentrations of Mg, P, Ca, Fe, Ni, Cu, Zn and Mo and lower concentrations of S, Cl,
241
K and Mn than wheat sprouts. Other studies have generally also reported an increase
242
in the mineral elements in sprouts when compared to grain, although species and
243
cultivation-specific effects can occur (Plaza et al., 2003; Lee et al., 2006; Kulkarni et al.,
244
2006; Hübner et al., 2010; Nelson et al., 2013). Because of the marked difference
245
between species shown here, it would be worthwhile to screen various plant species
246
and genotypes cultivated under the same conditions to determine the variability of
247
mineral element compositions and to identify species with exceptional compositions.
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The results of sprout cultivation in MMRW compared to TW are not
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straightforward, with sometimes markedly different effects between the two species
250
(Table 2, Online resource Table 1). In both species the concentrations of Na, Mg and
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K in MMRW were higher than when cultivated in TW, while concentrations of Cl were
252
significantly higher in Tartary buckwheat sprouts, but did not differ between TW and
253
MMRW cultivated wheat sprouts (Table 2). These differences reflect higher Na, Mg, Cl 10
ACCEPTED MANUSCRIPT and K concentrations in MMRW than found in the TW used. Concentrations of Ca and
255
Zn were lower in MMRW than TW cultivated sprouts of both species, and in wheat
256
sprouts Fe and Cu concentrations were also lower than in TW (Table 2). The observed
257
lower concentration of Ca may be a result of the formation of Ca precipitates in the form
258
of Ca carbonate in MMRW but not in TW, while lower Zn concentrations are a result of
259
lower Zn concentrations in the MMRW (Table 1). In addition, lower concentrations of
260
Zn in sprouts could result from antagonistic effects of Ca on Zn uptake in plants (Baker,
261
1978 and references therein). Although the interaction between Ca and Zn was
262
observed already few decades ago, the mode of action remains obscure. Likewise, it is
263
difficult to explain the lower concentrations of Fe and Cu in wheat sprouts cultivated in
264
MMRW compared with TW. It is possible this results from negative interactions
265
between Fe and Cu and other mineral elements present in high concentrations in the
266
solution. In general, studying the interactions of mineral elements in plants requires
267
experiments that are designed to carefully to exclude unplanned effects. Determining
268
the extent of these interactions was beyond the aims of this study and we cannot draw
269
any strong conclusions on the mechanisms governing these outcomes.
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than in TW result in re-distribution of minerals at the tissue level, we performed
272
quantitative localisation analysis, which display tissue-specific localisation of mineral
273
elements in Tartary buckwheat and wheat sprouts (Online resource Figure 1A, B).
274
There was no apparent difference in the localisation of mineral elements in TW-
275
cultivated and MMRW-cultivated sprouts, except in the concentration ranges indicating
276
no re-distribution of mineral elements as a result of increased external mineral
277
elements occurred. However, there was a striking difference in Ca distribution between
278
the two species. Co-localisation images of Ca, Mg and S (in these images Ca is
279
illustrated withred, Mg with green and S with blue colour) were generated for easier
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281
Tartary buckwheat sprouts Ca was located in inter-vasculature mesophyll (Fig. 3 A, B)
282
while in wheat sprouts Ca was located in epidermis (Fig. 3 C, D). The difference in the
283
concentrations and the spatial distribution of Ca in the two species studied is in line
284
with generally accepted difference between dicotyledonous and monocotyledonous
285
plants (e.g., see White and Broadley, 2003; Conn and Giliham, 2010). However,
286
different Ca distributions have been demonstrated also for various monocotyledonous
287
plants, with circa-vasculature and/or epidermal distributions reported (Klančnik et al.,
288
2014). Because of these differences it is not possible to make generalisations for
289
monocotyledonous plants. Due to confined distribution of Ca in Tartary buckwheat we
290
assume that these Ca hot-spots are Ca-oxalate crystals. However, the direct
291
assignment of Ca hotspots in Tartary buckwheat sprouts to Ca-oxalate crystals is
292
hindered by low resolution of the distribution maps (resolution was 2.3 µm). Despite
293
this, there is indirect evidence to support our assumption. In particular i) the diameter of
294
the hot-spots is 8-25 µm, which is a typical diameter of Ca-oxalate crystals, ii) their
295
spherical shape resembles shapes of Ca-oxalate crystals, iii) plants in the same family
296
and order as Tartary buckwheat (Polygonaceae, Caryophyllales) are known to form Ca-
297
oxalate crystals, and iv) high oxalate concentrations are reported in buckwheat
298
cotyledons (Shen et al,. 2004). The formation of Ca-oxalate in plants is linked to high-
299
capacity regulation and protection against herbivory (White and Broadley, 2003 and
300
references therein). Since oxalate is believed to negatively interfere with Ca absorption
301
during animal digestion, resolving the relationship between the Ca location in plant
302
tissue and nutrient bioaccessibility will help identify plant features leading to maximal
303
Ca absorption by humans and in this way influence Ca malnutrition (Yang et al., 2012).
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3.3 Dietary fibre of the sprouts 12
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307
heart disease, stroke, hypertension, diabetes, obesity, and certain gastrointestinal
308
diseases in humans (Anderson et al., 2009). Plant cell walls constitute the major part of
309
dietary fibre (McDougall et al., 1996). Carboxyl groups on pectins and glucuronoxylans,
310
both essential part of plant cell walls, are known to bind cations, especially Ca, but also
311
Mg and other divalent cations. Ca bound as Ca-pectate is essential for strengthening
312
cell walls and plant tissues (White and Broadley, 2003). In addition, Mn and Cu are
313
important as activators of various enzymes including those involved in the synthesis of
314
lignin (Broadley et al., 2012). This prompted us to evaluate dietary fibre changes taking
315
place in sprout development (TW-cultivated sprouts) and in sprouts cultivated in
316
MMRW. In sprouts of both plants species, we measured increases in TDF in
317
comparison to the grain (Fig. 1). In TW-cultivated Tartary buckwheat sprouts, this
318
increase was primarily from enhanced LAPs synthesis, whereas in wheat, both the l-
319
NSPs and LAPs were significantly increased. This was also demonstrated by Koehler
320
et al. (2007) who showed that in wheat sprouts TDF increased after germination. This
321
increase was mostly due to an increase in soluble dietary fibre and a decrease in
322
insoluble dietary fibre (see Koehler et al., 2007). However, this generalisation is not
323
consistent across species. For example, in oat and barley grains germinated at 20°C,
324
an increase in insoluble dietary fibre observed in 6-day post germination seedlings was
325
accompanied by a decrease in soluble dietary fibre resulting in increased TDF in oats
326
(Avena sativa L.) and similar levels TDF in grains and seedlings of barley (Hordeum
327
vulgare L.; Hübner et al., 2010). These observations indicate species-specific changes
328
in TDF and its constituents during germination and sprout development. However, as
329
the data on TDF are presented on a dry weight basis, the water content of each stage
330
should also be taken into account. Sprouts contain on average of 90% water, while in
331
grain, this is 7% (8.45% ±0.27% in Tartary buckwheat grain, and 6.07% ±0.07% in
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wheat grain), which indicates that TDFs calculated on a fresh weight basis will be much
333
lower in the sprouts than the grain, or that a considerable amount of raw sprouts will
334
needed to be consumed to reach the high dietary fibre intake achieved from grain or
335
whole grain flour consumption. The composition of I-NSPs and S-NSPs changed considerably between grain
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and sprouts of Tartary buckwheat and wheat (Fig. 2, for exact data and statistics see
338
Online resource Table 2). Tartary buckwheat grains and sprouts contained all eight
339
individual monomers of polysaccharides, while in the wheat sprouts fucose was not
340
found in the S-NSPs fraction in either of the water treatments. In the TW-cultivated
341
Tartary buckwheat the most prominent changes between grain and sprouts was an
342
increase in the galactose and a decrease in the glucose contents in both fractions (Fig.
343
2A, C). Glucose has previously been reported to be the main sugar in 7-day-old
344
sprouts of common buckwheat (Kim et al., 2004). In wheat sprouts, all constituents of
345
the I-NSP fraction increased compared to the grain, with galactose contributing most to
346
this increase. In contrast, in the S-NSP fraction rhamnose, mannose and galactose of
347
the wheat sprouts increased, while the rest decreased (Fig. 2B, D). This again
348
suggests species-specific changes in the compositions of the TDF.
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We found a lower TDF content in Tartary buckwheat sprouts cultivated in the
350
MMRW than in the TW, primarily due to the lower LAPs. Through enzyme activation
351
processes, lignin synthesis is linked to Mn and Cu (Broadley et al., 2012). However,
352
assuming a linear relationship, increased Mn and unchanged Cu concentrations in our
353
experiment cannot explain the observed decrease in LAPs. Saline stress in the form of
354
increasing concentrations of NaCl (up to 100 mM) increased total phenolic content in
355
common buckwheat sprouts, presumably through the activation of enzymes involved in
356
phenylpropanoid pathway resulting in the accumulation of phenolic compounds (Lim et
357
al., 2012). In our experiments we cannot uncover any single factor as clearly
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359
that the conditions in MMRW did not represent saline stress to the plants as similar
360
increases in LAPs was not observed and that other factors or their combinations are
361
responsible for the decrease in LAPs. In addition, significantly less S-NSP was seen in
362
MMRW cultivated Tartary buckwheat sprouts compared to TW cultivated sprouts with
363
arabinose, mannose and galactose contributing most to this decrease (Fig. 2, Online
364
resource Table 2). Within I-NSP, fucose, ribose, xylose, galactose and glucose were
365
significantly lower in MMRW-cultivated Tartary buckwheat sprouts compared with TW-
366
cultivated sprouts (Fig. 2, Online resource Table 2). In wheat sprouts we found no
367
differences in LAPs between TW and MMRW treatments (Fig. 1), accompanied by no
368
differences in the composition of I-NSPs and S-NSPs between TW and MMRW (Fig. 2,
369
Online resource Table 2). We conclude that these effects are species or genotype
370
specific (see also Lim et al., 2012). Other plant species and cultivars should be tested
371
to determine the level of variation in TDF when cultivating sprouts in MMRW or any
372
other spring MRWs.
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3.4 Flavonoids in the sprouts
375
The flavonoids in buckwheat grain have been repeatedly connected with anti-oxidative,
376
anti-inflammatory, and anti-hypertensive effects (e.g. Alvarez-Jubete et al., 2009).
377
Rutin, quercetin and catechin were only detected in Tartary buckwheat grain and
378
sprouts and not at all in wheat grains or sprouts, since wheat does not contain any of
379
these compounds. Tartary buckwheat sprouts contained higher rutin and catechin
380
concentrations and lower concentrations of quercetin than the grain (Table 2). Other
381
reports on Tartary buckwheat sprouts have shown higher concentrations of rutin (on
382
average 22100 mg kg-1; Kim et al., 2008), but lower concentrations of quercetin (on
383
average 100 mg kg-1; Kim et al., 2008) and catechin (594 mg kg-1 in Lee et al., 2006).
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385
to TW (Table 2) in line with previous studies of rutin in Tartary buckwheat sprouts
386
cultivated in artificially produced MRWs and deionized water (Hsu et al., 2008). We
387
observed increased quercetin concentration and decreased catechin concentrations
388
(Table 2), indicating that by changing mineral element composition of solutions
389
secondary metabolism of the sprouts can be changed. Other studies have reported no
390
differences in quercetin concentrations in Tartary buckwheat grown in artificially
391
produced MRWs and deionized water (Hsu et al., 2008). We were unable to find any
392
reports on catehin concentration changes in Tartary buckwheat sprouts as affected by
393
MRWs. However, other bioactive compounds have been shown to increase in
394
buckwheat sprouts upon introducing mineral elements to deionized water. These
395
include increased antioxidant activities and carotenoid concentrations (Hsu et al., 2008;
396
Lim et al., 2012). Although the mechanisms behind these changes require detailed
397
studies the result clearly suggest that nutritional composition of sprouts can be modified
398
to achieve selective enhancement of bioactive compound composition in sprouts.
399
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4 Conclusions
401
In the present study, we identified changes in mineral element and bioactive compound
402
compositions of Tartary buckwheat and wheat sprouts as a result of cultivation in
403
waters with different mineral element compositions. Differences in mineral element and
404
bioactive compound compositions seen in the grains of the species studied cannot fully
405
explain the extent of the differences seen in sprouts of the two species, rather species-
406
specific mechanisms of mineral uptake play a significant role. EMRW had negative
407
effects on the development of sprouts, which was seen as inhibition of radicle growth
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presumably due to mineral element concentrations and imbalances and/ or high pH.
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MMRW resulted in increased concentrations of Na, Mg, K and Mn and decreased
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sprouts. The spatial distribution of mineral elements did not change in response to
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treatments. Cell-type specific accumulation of Ca in the inter-vascular mesophyll was
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observed in Tartary buckwheat sprouts, apparently as Ca oxalate crystals, and in
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epidermis of wheat sprouts. While no effects on the TDF and its composition in the
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wheat sprouts were seen, decreased TDF and catechin was accompanied by
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increased quercetin in the Tartary buckwheat sprouts cultivated in MMRW, indicating
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that the cultivation tested interferes with the secondary metabolism in these sprouts.
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Taken together these results suggest the potential to selectively enhance mineral
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element and bioactive compositions in sprouts. Additional plant species or genotypes
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should be tested in different natural MRWs to determine optimal conditions and species
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specific characteristics to allow recommendations for specific mineral elements and
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bioactive compound composition improvements.
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Acknowledgements
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This study was supported by the P1-0212 Programme, by the Z4-4113 Project of the
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Slovenian Research Agency and by the COST Action FA0905. A. Rangus is
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acknowledged for providing Tartary buckwheat grain, D. Koron for providing wheat
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grain, Radenska d. d., Radenci, Slovenia for Radenska Classic and Droga Kolinska,
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Atlantic Grupa for Donat Mg®. We thank Prof. Linley Jesson and Dr. Tim George for
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reading the original manuscript.
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Fig. 1 Total dietary fibre (TDF) in Tartary buckwheat (A) and wheat (B) grain, and in the
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sprouts cultivated in tap water (TW) and in moderately mineral-rich water (MMRW). I-
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NSP, insoluble non-starch polysaccharides; S-NSP, soluble non-starch
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polysaccharides; LAPs, lignin and associated polyphenols. Different letters above and
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within columns indicate statistically significant differences. Data are means from 3
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independent replicates
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Fig. 2 The relative distributions of the individual monomers of polysaccharides in the
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insoluble non-starch polysaccharides (I-NSP; A, C) and the soluble non-starch
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polysaccharides (S-NSP; B, D) fractions of the total dietary fibre in the Tartary
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buckwheat and wheat grain, and in the sprouts cultivated in tap water (TW) and in
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moderately mineral-rich water (MMRW). Data are means from 3 independent replicated
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Fig. 3 Co-localisation image of Ca (red), Mg (green) and S (blue) in representative
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samples of Tartary buckwheat (A, B) and wheat (C, D) sprouts cultivated in tap water
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and in moderately mineral-rich water (MMRW)
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EMRWb
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Mineral elements (mg L-1) Na 4.4 440 1600 Mg 17.4 95 1060 P 0.01 0.093c <1.0c S 3.71 97d 2200d Cl 2.36 44 62 K 1.00 70 15 Ca 72.0 220 340 Mn 0.004 0.27 0.15 Fe 0.11 <0.10 <0.10 Cu 0.01 <1.0 0.007 Zn 2.47 <10 0.022 HCO3n.m. 2000 7800 pH 7.2 5.6 6 Germination (%) Tartary buckwheat 89±3 87±3 83±3.4 Wheat 85±4 83±3 77±4 a , measured with inductively coupled plasma-mass spectrometry and inductively coupled plasma-optical emission spectroscopy, except Cl, which was measured with total-reflection X-ray spectrometry; b, data provided by the producers; c, measured as HPO4; d, measured as SO4; n.m. not measured.
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Table 2 Fresh (FW; g) and dry weight (DW; g) and concentration of mineral elements, rutin, quercetin and catechin (mg kg-1) in
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Tartary buckwheat Wheat grain TW sprouts MMRW sprouts grain TW sprouts MMRW sprouts 0.039 ± 0.006 FW 0.020 ± 0.004 24.1 ± 3.4 21.1 ± 1.9 15.0 ± 1.7 14.3 ± 0.71 0.037 ±- 0.006 DW 0.016 ± 0.004 2.3 ± 0.1 2.1 ±0.04 1.4 ± 0.12 1.6 ± 0.16 Na 5.47 ± 0.48 a* 103 ± 27.1 a 2960 ± 599 b 9.8 ± 005 A* 112 ± 5.46 A 3610 ± 253 B Mg 2100 ± 281 a 5980 ± 433 b 9170 ± 437 c 1200 ± 34.9 A 1260 ± 55.4 A 1730 ± 44.8 B P 3960 ± 56.0 a 7390 ± 201 b 7580 ± 141 b 3420 ± 64.6 A 6360 ± 111 B 6080 ± 226 B S 1220 ± 271 a 2540 ± 57.6 b 2520 ± 94.9 b 1560 ± 394 A* 2800 ± 969 B 2590 ± 583 B a <100 832 ± 363 x 1162 ± 296 y 575 ± 16.3 A 3820 ± 796 B 5810 ± 1167 B Cl K 5120 ± 112 a 8050 ± 518 b 16900 ± 1528 c 4120 ± 55.8 A 11700 ± 869 B 23600 ± 563 C Ca 579 ± 31.8 a* 5900 ± 825 b 2830 ± 1026 a 327 ± 14.6 A* 2070 ± 22.6 C 1280 ± 394 B 23.3 ± 3.09 b 35.7 ± 0.9 C 21.1 ± 0.75 A 26.8 ± 0.13 B Mn 11.0 ± 0.23 a* 14.9 ± 1.1 a Fe 54.3 ± 14.6 a 98.9 ±14.9 b 78.1 ± 13.3 ab 31.7 ± 0.4 A 59.1 ± 4.43 C 47.5 ± 1.35 B Ni 0.40 ± 0.02 a 1.07 ± 0.50 ab 1.37 ± 0.36 b 0.22 ± 0.02 A* 0.21 ± 0.11 A 0.17 ± 0.01 A 2.48 ± 0.02 A* 5.83 ± 0.42 C 4.46 ± 0.17 B Cu 4.5 ± 0.02 a 10.1 ±1.80 b 8.07 ± 1.30 b Zn 23.1 ± 0.47 a* 213 ± 37 b 49.3 ± 5.94 a 21.6 ± 0.55 A 71.9 ± 9.58 B 33.8 ± 1.22 A Mo 0.90 ± 0.07 a 1.30 ± 0.13 b 1.30 ± 0.1 b 0.41 ± 0.06 B 0.30 ± 0.01 AB 0.29 ± 0.05 A rutin 914 ± 101 a 10000 ± 608 b 9210 ± 1850 b nd nd nd quercetin 1270 ± 231 b 687 ± 95 a 1130 ± 329 b nd nd nd catechin 4510 ± 453 a 8360 ± 658 c 7160 ± 480 b nd nd nd Different letters next to the values indicate statistically significant differences (lower case letters for Tartary buckwheat and upper case letters for wheat). Results presented are means ± standard deviation from 3 independent replicates; nd, not detected; a, measured with energy-dispersive X-ray fluorescence; *, nonparametric tests performed.
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ACCEPTED MANUSCRIPT Highlights
• Successful sprout development depended on the mineral element (ME) composition of waters
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• ME concentration but not distribution was affected by ME composition of water • Total dietary fibre was affected by ME composition of waters only in Tartary buckwheat sprouts
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• Quercetin and catechin, but not rutin concentration depended on ME composition
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of waters