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Losses of essential mineral nutrients by polishing of rice differ among genotypes due to contrasting grain hardness and mineral distribution
Journal of Cereal Science 56 (2012) 307e315
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Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Losses of essential mineral nutrients by polishing of rice differ among genotypes due to contrasting grain hardness and mineral distribution Thomas H. Hansen a, Enzo Lombi b, Melissa Fitzgerald c, Kristian H. Laursen a, Jens Frydenvang a, Søren Husted a, Chanthakhone Boualaphanh c, d, Adoracion Resurreccion c, Daryl L. Howard e, Martin D. de Jonge e, David Paterson e, Jan K. Schjoerring a, * a
Plant and Soil Science Section, Department of Plant and Environmental Sciences, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark Centre for Environmental Risk Assessment and Remediation, University of South Australia, Building X, Mawson Lakes Campus, Mawson Lakes, South Australia SA-5095, Australia c Grain Quality and Nutrition Centre, International Rice Research Institute, DAPO 7777 Metro Manila, Philippines d Rice and Cash Crop Research Institute, NAFRI, Vientiane, Lao Democratic People’s Republic e Australian Synchrotron, 800 Blackburn Road, Clayton, Victoria 3168, Australia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 November 2011 Received in revised form 18 April 2012 Accepted 9 July 2012
The effect of different polishing techniques on loss of mineral elements from rice grains was quantified using a panel of indica and tropical japonica genotypes, previously classified as differing in ease of polishing. Gradients in mineral elements across the bran-endosperm interface were quantified using microscaled precision abrasive polishing in combination with inductively coupled plasma mass spectrometry and synchrotron X-ray fluorescence microscopy. Frictional polishing, similar to that of commercial mills, i.e. 8e10% loss of grain weight, reduced the concentration of Fe, Mg, P, K and Mn by 60e80% in all genotypes. Following gentler polishing (3e5% weight loss), genotypes classified as difficult to polish showed smaller decreases in Fe, Mg, P, K and Mn compared to genotypes classified as easy to polish. The concentration of other elements, e.g. Zn, S, Ca, Cu, Mo and Cd, showed comparable reductions (<30%) irrespective of polishing technique or ease of polishing. The different patterns of polishing losses of minerals reflected their distribution within the grain. Five-fold differences in the reduction of Zn concentration during polishing were observed for different genotypes which started with similar Zn concentrations in the unpolished grain, thus showing clear potential for selecting genotypes with reduced polishing losses of Zn. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: ICP-MS and synchrotron X-ray fluorescence microscopy Iron and zinc Polishing Rice grain
1. Introduction The micronutrients iron (Fe) and zinc (Zn) are often lacking in the human diet and their accompanying deficiencies cause major health problems (Tanumihardjo et al., 2008). Worldwide, Fe and Zn deficiencies are estimated to afflict more than 2 billion people, mostly women and children in developing countries. The major cause of Fe and Zn deficiency is low intake due to diets consisting mostly of a few staple foods with low micronutrient density, e.g. polished rice and other cereals. The consequences in terms of malnutrition and health are devastating and can result in stunted growth, disease, and increased mortality. Besides Fe and Zn, the dietary intake of other mineral elements, particularly selenium (Se), iodine (I), calcium (Ca) and magnesium (Mg) may be * Corresponding author. Tel.: þ45 35333495. E-mail address: [email protected] (J.K. Schjoerring). 0733-5210/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jcs.2012.07.002
inadequate in both developed and developing countries (White and Broadley, 2009). The most widely recognized strategies for reducing micronutrient malnutrition in human populations consist of supplementation with pharmaceutical preparations, food fortification or dietary diversification. These solutions are often impractical for economic and/or social reasons. Therefore enrichment of cereal food staples through plant breeding or genetic engineering is seen as a potentially more sustainable and less expensive strategy (Ortiz-Monasterio et al., 2007). Iron and Zn concentrations in different wheat, maize and rice genotypes can vary several-fold (Gomez-Becerra et al., 2010; Lee et al., 2011; Shi et al., 2010; Simic et al., 2009). Similarly, genotypic variations exist in the concentration of Se, Ca and Mg in vegetable products which can be exploited in breeding programmes (White and Broadley, 2009). Improved intake of mineral elements by rice consumers may be achieved by minimizing the substantial losses that result from
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polishing. In this process, the bran (i.e. the germ/embryo and the aleurone/pericarp layers), which constitutes 8e10% of the rough rice kernel mass, is removed from the brown rice, thereby resulting in white rice grains. The outer grain layers are much denser in minerals than the inner parts, causing a substantial decline in the concentration of mineral elements in the polished grain. Nevertheless, polishing is a preferred step in postharvest production of rice because (i) most consumers prefer the taste and texture of the white rice and (ii) the high oil content of the bran increases risks of rancidity, thereby reducing the shelf life. In addition, polishing is carried out because it decreases the cooking time, which is an important property for poor rice consumers (Fitzgerald et al., 2009). The intensity of polishing depends on the type of mill and the duration of the treatment (Graves et al., 2009; Roy et al., 2011). The relationship between polishing time and loss of material is nonlinear due to increasing adhesion of the bran layers from outer to inner layers, while the different endosperm fractions seem to have comparable hardness (Lamberts et al., 2007). Furthermore, differences in shape and size of rice grains also contribute to the quantity of material lost during polishing (Liang et al., 2008). The loss of mineral elements associated with polishing of rice is not directly related to the loss of grain mass because elements have a heterogeneous distribution within the grain (Lombi et al., 2009). The effects of polishing on losses of macro- and microelements have been studied in rice grain subjected to different polishing rates (Bryant et al., 2005; Itani et al., 2002). These studies generally show decreased concentrations of macronutrients moving from the outer edge to the centre of the grain, as is also the case for micronutrients, although with some marked differences among elements (Lamberts et al., 2007; Ogiyama et al., 2008). Polishing characteristics are also important in order to reveal the distribution of mineral elements in relation to other grain factors affecting their bio-availability such as phytic acid (Liang et al., 2008). The current interest in developing new biofortified rice varieties suggests a need for further information on genotypic differences in polishing losses and how these relate to the distribution of mineral elements in the grain. For instance, the distribution of Zn in white rice did not differ between genotypes with different grain shapes (Liang et al., 2008), while losses of Se differed between genotypes and could be correlated to the degree of polishing (Liu et al., 2009). The aim of the present work was to relate polishing losses of mineral elements to their gradient across the bran-endosperm interface in a panel of indica and tropical japonica rice genotypes, previously classified as differing in ease of polishing. For this purpose a micro-scaled precision polishing procedure was developed. As support for the micro-scaled polishing experiment, a first assessment of mineral losses in the different genotypes was obtained by polishing in a commercial frictional mill and a gentler abrasive mill type. The micro-scaled polishing procedure allowed full-quantitative measurements by ICP-MS of the distribution of mineral elements across the outer grain layers. The element distribution for whole grains was obtained by synchrotron X-ray fluorescence microscopy. This technique was here used for the first time to quantify the distribution of elements in rice grains and validate these results against ICP-MS data. 2. Materials and methods 2.1. Plant material A panel of 12 advanced breeding lines of rice (Oryza sativa L.) was selected by the International Rice Research Institute (IRRI) based on differences in ease of polishing as determined by the length of time required to remove the bran layers completely from
the grain using gentle abrasion. The International Rice Information System (IRIS) unique identifier (IRIS-ID) for these lines is shown in Supplementary Table S1 together with information about grain properties. The IRIS-ID will be used in all reference to the breeding lines. The selected lines comprised two groups of which group 1 consisted of the genotypes that were difficult to polish and group 2 of those that were easy to polish (Supplementary Table S1). The panel also included a traditional variety from the Philippines, Perurutong. This was included because of its high fiber content, high nutritional value, high content of the antioxidant anthocyanin, relatively high content of minerals including iron, and its black pericarp, which is difficult to remove completely. The 13 genotypes were grown in the dry season of 2007 at IRRI’s experimental station in the Philippines. A total of 150 kg N ha1 was applied in three equal applications at transplanting, at mid-tillering and at panicle initiation. P and K, each at 30 kg ha1, were applied at transplanting. Insects, weeds and diseases were managed as per IRRI’s standard protocols. Water was drained at late grain-filling. Grain was harvested at maturity, and dried to 14% moisture before further processing. The unpolished seeds of the 13 rice genotypes showed a considerable span in concentration of mineral elements (Supplementary Table S2). For Fe and Zn, the difference between the genotypes with highest and lowest concentration in the unpolished grain was 65e80%. These differences were not (P > 0.05) related to the polishing ease of the grain. Six genotypes were subjected to analysis in a micro-polishing experiment, comparing genotypic differences in element losses over time. This panel consisted of three genotypes from the first panel (Perurutong and IRIS-146-14597 from Group 1, and IRIS-66445376 from Group 2) as well as three Laotian varieties, viz. Hom Nang Nouan (HNN), Kai Noi Leung (KNN) and Thasano 1 (TSN1). One of the varieties, TSN1, was also investigated in detail using synchrotron XFM. HNN and KNL are traditional, low yielding, but highly priced varieties, while TSN1 is a high yielding, improved variety released in 1993. TSN1 responds well to N application and is a popular variety with good polishing quality and acceptable eating quality (Fitzgerald et al., 2011). The Laotian rice genotypes were grown under similar conditions at an experimental field station in the Lao People’s Democratic Republic (Lao PDR) and received a total application of 60 kg N ha1, split on equal dressings at transplanting and mid-tillering. 2.2. Polishing methods Rough frictional polishing of similar intensity to that of commercial mills, i.e. 8e10% loss of grain weight, was compared with gentle abrasive polishing (3e5% weight loss). Both methods were applied on dehulled grain (Satake Rice Machine, Tokyo, Japan) originating from the panel of 13 genotypes differing in ease of polishing (Supplementary Table S1). The rough polishing was carried out by use of a standard laboratory frictional mill (Grainman 60-230-60-2AT, Grain Machinery Mfg. Corp., Miami, FL) for 30 s. For the more gentle abrasive polishing, a commercial paintshaker (Skandex SO400, Fast and Fluid Management, The Netherlands) adapted to polish small samples of rice was used. The mixing chamber of the paintshaker was fitted with boxes designed to hold 49 perspex capsules. Each capsule contained 5 g of brown rice and 10 g fused aluminium oxide. Polishing was achieved by shaking the boxes in the paintshaker for 1 h, whereupon the polished white grains were separated from the abradant and bran, and brushed to remove any adhering particles of bran. In order to further investigate genotypic differences in element losses over time, brown rice from a panel of six genotypes was polished abrasively using the microscaled method described by Hansen et al. (2009). This method made it possible to distinguish
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between the influence of the embryo and the pericarp-aleurone layers on the element content in the rice grains. Each sample consisted of approximately 10 rice grains, corresponding to 200e250 mg dry mass, where the embryo had been removed with the tip of a scalpel. The rice grains were abraded in small centrifuge tubes with 200 mg of acid washed quartz sand (Fluka 84878, 40e150 mesh SiO2). The polishing process was performed by high speed shaking in a ball mill (MM301, Retsch GmbH, Düsseldorf, Germany) fitted with 2 racks of 24 samples. The mill was operated at 30 Hz for nine selected time periods of 20e450 s. Before analysis, the remains of the grain were in all polishing experiments washed rapidly three times with Milli-Q water (18.2 U; Milli-Q Plus, Millipore Corporation, MA. USA) in order to remove surface dust, and then dried. Subsequent ICP-MS analysis of the rinsing water verified that the rinsing procedure did not cause leaching of mineral elements. 2.3. Sample digestion A method was developed to ensure complete digestion of brown (unpolished) rice (Hansen et al., 2009). The digestion was performed in closed vessels in a microwave oven (Multiwave 3000, software version 1.24, Anton Paar GmbH, Graz, Austria). Samples were micro-waved (1400 W) for 36 min (after 10 min ramping) at 210 C at a maximum pressure of 40 bar in 100 ml closed tubes. The digestion medium consisted of 5 ml 70% ultrapure HNO3 (J.T. Baker Instra-Analyzed Reagent, NJ, USA) and 5 ml 15% H2O2 (30% ExtraPure, Riedel de Häen, Selze, Germany). After digestion, samples were diluted to 50 ml with Milli-Q water (18.2 U; Milli-Q Plus, Millipore Corporation, MA, USA). Element concentrations in the digests of the intact brown rice grains were identical to those obtained by analysis of flour from the same batch of brown rice, verifying that complete digestion of the unpolished grains had been achieved. 2.4. Multi-element analysis Multi-element analysis of samples was performed using an ICPMS (Agilent 7500ce, Agilent Technologies, UK) equipped with a PFA micro-flow nebulizer. ICP-MS data were processed using the Plasma Chromatographic Software v. B-03-07 (Agilent Technologies, UK). Before full-quantitative analysis, samples were diluted to match the acid concentration of calibration standards (3.5% v/v HNO3). Sample uptake was maintained at approximately 0.1 ml min1 by a PFA micro-flow nebulizer (Laursen et al., 2009). Erbium (166Er) was used as internal standard (10 mg l1) to enable correction for drift and plasma instability. Thirteen-pointcalibration was performed using a commercially available standard solution (P/N 4400e132565, CPI International, Amsterdam, The Netherlands). Validation was performed by including certified reference materials (CRMs). Durum wheat NIST 8436 (US Department of Commerce, National Institute of Standards and Technology, Gaithersburgh, MD, USA) was chosen as the matrix corresponds to rice grain and the certification includes essential plant nutrients as well as elements important for human health such as Cd, Se and Fe. Data were rejected if the accuracy was <90% of the certified value of 7 CRMs or if standard deviation exceeded 10%. LOD was determined as three times the standard deviation of 7 blanks and only data above limit of detection (LOD) was accepted. 2.5. Imaging of element distribution A thin (70 mm thick) longitudinal section of the middle part of the grain was imaged on the X-ray fluorescence microscopy beamline at the Australian Synchrotron (Paterson et al., 2011). The
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sectioning process did not require any embedding and was performed as described by Lombi et al. (2009). The use of thin sections is necessary to overcome issues related to the penetration of the Xray beam. Using this technique, the distribution of the micronutrients Fe, Zn, Cu and Mn was imaged. The laterally-resolved element maps were collected using an undulator beamline equipped with a Si(111) monochromator and KirkpatrickeBaez mirrors focussing the beam to a spot size of approximately 3 2 mm2 (horizontal vertical). An incident beam energy of 10 keV was used to excite X-ray fluorescence which was collected using a 96element prototype Maia detector system (Kirkham et al., 2010). The detector was placed perpendicular to the incident beam path and was used to collect the full spectra fluorescence signal from the sample. The samples were analysed continuously in the horizontal direction (‘on the fly’) with steps of 1.25 mm in both directions. The pixel transit time was approximately 0.6 msec. The full XRF spectra were then analysed using GeoPIXE (Ryan, 2000). This software uses Dynamic Analysis to subtract background and resolve overlapping peaks to generate element maps of concentration. 2.6. Data analysis Statistical software (SAS Institute, USA) was used for data analysis by ANOVA using the general linear model (GLM). Chemometric data analysis was performed with the Unscrambler software package version 9.1 (Camo Process A/S, Oslo, Norway) as principal component analysis (PCA). Data was auto-scaled and validated using full cross-validation. Matlab version R2010 (Mathworks, Massachusetts, USA) was used for mathematical analysis of the relationship between mineral element concentration as a function of polishing time. The analysis was based on iterative error minimization of two straight lines, one for the initial steep decrease, and one for the slower decrease at extended polishing times, representing the outer grain layers and endosperm, respectively. With an inclusion criterion of R2 values >0.90, the intercept of the two straight lines was assumed to represent the transition point between outer grain layers and endosperm. In cases where two straight lines could not be established, a single linear regression analysis of mineral element concentration versus polishing time was fitted. 3. Results 3.1. Losses of nutrients as affected by polisher type and grain hardness The two polishing techniques, i.e. frictional polishing in an industrial type pressure mill or abrasive polishing in a laboratory shearing-type mill, had different impacts on the quantity of material removed (Table 1). Abrasive polishing only caused 3e5% weight loss compared to 9% weight loss during frictional polishing. This was accompanied by greater reductions in Fe, Mg, P, K and Mn concentrations upon frictional polishing as compared with abrasive polishing, while Zn, S, Ca, Cu, Mo and Cd concentrations were similar for the two polishing techniques (Table 1). Differences in the polishing ease of the grains had no influence on the concentration of mineral elements following frictional polishing, grains classified as difficult to polish showing the same decrease in concentration as genotypes classified as easy to polish (Table 1). Chemometric data analysis (Fig. 1) clearly allowed discrimination of the two polishing techniques, but was not able to separate grains differing in ease of polishing. Especially Mn, K, P, Fe, Mg and S were important for discrimination of the polishing techniques as evident from the distant loading plot location of S and the group of
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Table 1 Percentage decrease in concentration of mineral elements caused by polishing of brown rice in an industrial pressure mill (friction polishing) or in a laboratory shearing-type mill in which rice grain were shaken with aluminium oxide (abrasive polishing). The two techniques resulted in losses of about 9% of grain dry matter by friction polishing and 3e5% during abrasive polishing. Data are mean values for 13 different genotypes from the International Rice Research Institute, 9 classified as difficult to polish and 4 as easy to polish (Table S1). Values within same element followed by different letters differ significantly (P ¼ 0.05). Element
Type of polishing Friction
Abrasive
Polishing ability Easy
Difficult
Easy
Difficult
Decrease in element concentration, % Mg P S K Ca Mn Fe Cu Zn Se Mo Cd
86A 70A 7A 80A 38A 65A 82A 11A 34A 5A 8A 4A
90A 72A 3A 76A 34A 64A 80A 13A 29A 9A 1A 18A
60B 49B 2B 61B 24A 50B 47B 11A 30A 51B 11A 8A
51C 32C 4A 45C 5B 40C 38B 18A 27A 52B 4A 3A
Mn, K, P, Fe and Mn (Fig. 1B) in accordance with the two groups found in the score plot (Fig. 1A). Removal of bran by frictional polishing caused the genotypes to cluster more closely i.e. reduced some of the variation among genotypes (Fig. 1A). After abrasive polishing, grains classified as easy to polish grouped more closely together than genotypes difficult to polish, while the opposite was the case upon frictional polishing (Fig. 1A). 3.2. Polishing losses in relation to nutrient distribution across the bran-endosperm interface A step-wise micro-polishing procedure was performed to link the distribution across the pericarpealeuroneeendosperm interface of the different genotypes with the duration of the polishing. The initial step consisted of manual removal of the embryo, while in the succeeding steps, the de-embryoed brown grain was polished for specific time intervals in order to gradually abrade the aleurone layer and analyse the resulting concentration in the residual part of
the grain. The step-wise polishing procedure was carried out for nine time intervals distributed over 450 s, finally resulting in removal of about 10% of the dry matter of the grain. Removal of the embryo led, in all genotypes, to a significantly (P ¼ 0.05) lower concentration of Fe and Zn concentration in the deembryoed grain relative to the intact grain (Fig. 2). This was the case despite the fact that the embryo only accounted for about 2% of the seed weight. Genotypic differences in the contribution of the embryo was observed for Mg and P where the concentration decreased significantly in Perurutong, IRIS-66-445376 and IRIS146-14597, while the remaining three genotypes KNL, TSN1 and HNN were only slightly affected by the removal of the embryo (Fig. 3). The concentration of S was not affected by embryo removal in any of the genotypes (Fig. 3). The Fe concentration reached a transition point after 50 2 s of polishing, marking that the outermost Fe-dense tissues had been removed (Fig. 2). A similar transition point was never reached for Zn, showing a more uniform Zn distribution across the aleuroneeendosperm interface than was the case for Fe (Fig. 2). Nevertheless, distinct genotypic differences in the rate of Zn loss during polishing were observed. The steepest decline in Zn concentration across the aleuroneeendosperm interface occurred for IRIS-146-14597 and KNL, 2-fold higher than that for HNN. Compared over the whole polishing period, IRIS-66-445376 showed the largest decline in Zn concentration (59%) relative to the unpolished grain, whereas the Zn concentration in HNN only was reduced by 14%. These large genotypic differences in Zn losses appeared despite the fact that the losses of grain dry matter were similar for the genotypes (around 15% after 450 s of polishing). For Fe, IRIS-66-445376 exhibited the steepest decline across the aleuroneeendosperm interface, 2-fold higher than that for TSN1, showing genotypic differences in the Fe gradient within the aleurone layer. The loss of grain matter was also in this case very similar (data not shown). For all genotypes, the Fe concentration of the grain parts remaining after polishing was more than 75% lower than that of the de-embryoed brown grain. At the end of polishing, KNL contained the lowest concentrations of Zn (10.2 mg g1) and Fe (0.5 mg g1), whereas Perurutong contained the highest concentrations of Fe and Zn (24 and 7 mg g1, respectively). The polishing profile of Mn was very similar to that for Fe, showing a transition point at 45 3 s (Fig. 4). The same was the case for P, Mg and K, although with slightly later transition points of about 70 3 s. By contrast, S, Cu, Se, and Cd were more evenly distributed, and resembled the profile of Zn, i.e. with the
Fig. 1. Principal component analysis of the decrease in mineral element concentration due to polishing of brown rice. Panel A is the score plot, panel B the corresponding loading plot. Principal components 1 and 2 explained 44% and 22% of the variance, respectively. (C,B) denote friction polishing and (-,,) abrasive polishing. Filled symbols are for grains empirically classified as difficult to polish, open symbols for grains classified as easy to polish. Insert in panel B shows magnification of overlapping elements in the loadings plot.
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Fig. 2. Concentration of Fe and Zn in rice grain of six genotypes as a function of polishing time. The embryo was first removed manually (time point before 0) whereupon polishing was carried out for different time intervals in a micro-scaled abrasive mill, starting at time zero. Filled circles denote iron (Fe), open circles zinc (Zn). Values are means SE (n ¼ 3). Based on iterative error minimization, two straight lines, one for the initial steep decrease and one for the slower decrease at extended polishing times, were fitted to the data. With an inclusion criterion of R2 values >0.90, the intercept of the two straight lines was assumed to represent the transition point between outer grain layers and endosperm. In cases where two straight lines could not be established, a single linear regression analysis of mineral element concentration versus polishing time was performed.
concentration much less affected by polishing, and without a clear transition point between outer grain layers and endosperm. High-definition synchrotron X-ray fluorescence was used to further refine the analysis of the distribution of Fe, Zn, Mn and Cu across the outer grain layers and within the embryo of TSN1 (Fig. 5). The highest element concentrations were found in the embryo, particularly in the scutellum, which plays a role in nutrient exchange between the embryo and endosperm. The central part of the embryo, the plumule and radicle, also exhibited high concentrations of Cu, Mn and Zn, but not Fe. The epiblast surrounding the central part of the embryo generally contained lower micronutrient concentrations, with the exception of some Zn hot spots. High Mn, Fe, Zn and Cu concentrations were recorded in the outer grain layers, composed of the pericarp (fruit coat), the tegmen (seed coat) and the aleurone layer (Fig. 5). As also observed in the stepwise micro-polishing procedure (Figs. 3 and 4), Mn and Fe were confined to the outer grain layers, while Zn and Cu concentrations decreased much less steeply towards the endosperm (Fig. 5c). Mn was localized in a very narrow band, with a peak concentration only about 20 mm below the grain surface (Fig. 5c). The Fe peak was broader than that of Mn, and attained maximum concentration about 50 mm below the grain surface. 4. Discussion Polishing is an important operation leading to the production of white rice. The degree of polishing has a significant effect on the quality and nutritional aspects of white rice, affecting properties
such as content of essential minerals (Figs. 2e4; Liang et al., 2008), phytochemicals and grain breakage (Mohapatra and Bal, 2010). Particularly in relation to Fe and Zn, it is important to develop new polishing procedures which can minimize the losses of micronutrients while preserving desired characteristics related to sensory quality and food processing. Even a very short duration (30 s) of friction polishing in the laboratory polisher used in the present work was accompanied by substantial loses of minerals such as Fe, Mg, P, K and Mn (Table 1). This type of polisher can be directly related to commercial systems and illustrates how important the polishing process is for conservation of essential micronutrients. The much gentler abrasive polishing procedures, adapted here, reduced mineral losses (Fig. 1; Table 1) and made it possible to fine-tune the polishing, reducing sample size, duration and loss of material. As abradant, aluminium oxide or quartz sand was used. Both of these materials have a high hardness but, nevertheless, their effectiveness decreased over time due to adsorption of abraded material. The ratio between grain and abradant is therefore critical for the resulting degree of polishing. In some commercially available abrasive laboratory mills, abraded material can be removed during the polishing process (Lamberts et al., 2007; Mohapatra and Bal, 2010). However, in these mill types, the polishing process cannot be micro-scaled and contamination with elements may become a problem. In the present study, small closed systems accommodating perspex capsules were used to avoid contamination problems. The loss of mineral elements associated with polishing of rice is not directly related to the loss of grain mass because elements have
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Fig. 3. Concentration of Mg, P, S and K in rice grain of 6 genotypes as a function of polishing time. The embryo was first removed manually (time point before 0) whereupon polishing was carried out for different time intervals in a micro-scaled abrasive mill, starting at time zero. Left: Filled circles Mg and open symbols P. Right: Filled circles S and open circles K. Values are means SE (n ¼ 3). Curve fitting was performed as described in Fig. 2.
a heterogeneous distribution within the grain (Lombi et al., 2009). The general pattern is that element concentrations show a steep decline across the aleuroneeendosperm interface and thereafter a more gradual decline towards the centre of the grain (Figs. 2e4; Itani et al., 2002; Lamberts et al., 2007). However, there are some marked differences between elements which have profound influence on their loss rate during polishing (Figs. 2e4; Ogiyama
et al., 2008). A relatively large proportion of Fe, Mg, P, K and Mn is associated with the outer grain layers and these elements are accordingly rapidly lost during polishing (Figs. 2e4; Table 1). In contrast, Zn, S, Ca, Cu, Mo and Cd are more uniformly distributed and only show a minor decline in concentration towards the centre of the endosperm (Figs. 2e4; see also Bryant et al., 2005 and Ogiyama et al., 2008). Due to the more uniform distribution and
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Fig. 4. Concentration of Mn, Cu, Mo and Se versus polishing time of grains of 6 rice genotypes: Perurutong (closed circles), IRIS-66-445376 (open circles), IRIS-146-14597 (closed squares), KNL (open triangles), TSN1 (closed triangles) and HNN (open squares). The embryo was first removed manually (time point before 0) whereupon polishing was carried out for different time intervals in a micro-scaled abrasive mill, starting at time zero. Values are means SE (n ¼ 3). Curve fitting was performed as described in Fig. 2.
less steep decline, polishing losses become relatively small for elements such as Zn and Cu (Table 1). Reduced polishing will thus have the largest impact on Fe preservation, less so for Zn, while Cu and Se will not be affected.
The differences in element distribution may to some extent reflect that of chelating ligands such as phytate (Liang et al., 2008; Persson et al., 2009; Wang et al., 2011) and proteins (GomezBecerra et al., 2010; Kutman et al., 2011) which also can affect the
Fig. 5. Synchrotron X-ray fluorescence microscopy of Cu, Fe, Mn and Zn in grain of the genotype TSN1. A. Light microscope image of the longitudinal grain section (dorsal side on top). B. Element concentration maps. C. Element concentration profile from selected area (white box). The data was obtained by using the transect function in GeoPIXE over an area 201 pixel wide. Red dashed lines shows the thickness of the layer of grain material that was abraded by 8e10% polishing.
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nutritional quality of rice in combination with polishing procedures. It is generally assumed that a substantial part of both Zn and Fe in the cereal grain is bound to phytate (Greffeuille et al., 2011; Mills et al., 2005; Simic et al., 2009). Up to 80% of the P content of cereal grain may be present in the form of phytate and losses of Zn and Fe during polishing would therefore be expected to match that of P (Wang et al., 2011). However, this was only true for Fe, where both the initial steep losses as a function of polishing time (Figs. 2 and 3) and the total losses (Table 1) closely matched the pattern for P. In contrast, Zn was much more uniformly distributed across the aleuroneeendosperm interface closely matching that of S (Figs. 2 and 3). The coupled distribution of Fe together with P versus Zn together with S is in agreement with results from a recent speciation analysis showing that a substantial part of Fe co-eluted with P, while Zn co-eluted with S containing ligands (Persson et al., 2009). However, the concentration of Zn declined more (27e30%) upon polishing than did that of S, which was hardly affected (Table 1; Figs. 2 and 3). A substantial part of the Zn is localized in the embryo as well as in the outermost layers of the grain (Figs. 2 and 5) and further research is needed to unravel the speciation of this Zn. In addition to ligands such as phytate and protein, the non-protein amino acid nicotianamine (NA) and other low-molecular-weight ligands may play a role (Lee et al., 2011). Synchrotron XFM is a powerful technique used to visualize the distribution of elements in seeds (Lombi et al., 2009, 2011). There was good agreement between the quantitative data obtained by micro-polishing combined with ICP-MS analysis (Fig. 2) and synchrotron XFM images (Fig. 5). Both techniques showed that Mn was more confined to the surface layers of the grain than Fe, while the Zn concentration declined less steeply towards the core endosperm (Fig. 5). The Fe, Mn, Cu and Zn concentrations in the aleurone layer estimated by XFM imaging were in good agreement with ICPMS measurements of the same genotype (TSN1), showing 55 mg Fe kg1 and 35 mg Zn kg1 in the aleurone layer (Hansen et al., 2009). However, endosperm concentrations of Fe and Mn measured by synchrotron XFM were close to zero, i.e. lower than the ICP-MS derived concentrations (Fig. 4), reflecting that XFM was not sufficiently sensitive to image the low concentrations of the two elements in the endosperm. Polishing characteristics differ with grain properties such as length and width (Supplementary Table S1; Liang et al., 2008). The rice used in the present study consisted of nine japonica genotypes which have a relatively short and plump grain (length to width ratio just above 2; Supplementary Table S1) and low amylose content (12e19%). The four indica genotypes included in the study had long, slender grain (length to width ratio above 3; Supplementary Table S1) with low and intermediate amylose content (19e23%). All the japonica genotypes were empirically classified as relatively difficult to polish, while the indica genotypes were classified as easy to polish (Supplementary Table S1). However, there were no clear differences with respect to mineral losses between the two germplasm classes following the more intense frictional polishing (Table 1). In contrast, genotypic differences were observed after the relatively gentle abrasive polishing which caused a smaller reduction in the concentration of mineral elements in genotypes classified as difficult to polish compared to genotypes classified as easy to polish (Table 1). The genotypes clustered much more densely upon the more intense frictional polishing than after the more gentle abrasive polishing, reflecting less variability in endosperm concentrations of mineral elements compared to outer grain layers (Fig. 1). Distinct genotypic differences, up to 5-fold, in the reduction of Zn and Fe concentrations during polishing were observed (Fig. 2). This was the case even though the amount of dry matter lost was very similar (data not shown). A special case was the genotype Perurutong, a landrace
from the Philippines with a black pericarp, which after polishing still had 14-fold higher total Fe concentration than the genotype (KNL) which had the lowest Fe concentration. 5. Conclusions It is concluded that there is a clear potential for selecting rice genotypes with reduced losses of Zn and Fe after polishing. Further exploration of this potential needs to be combined with other quality parameters, such as cooking ability and sensory quality following polishing. A much greater proportion of Fe is lost by polishing than is the case for Zn. This reflects their distribution pattern within the grain, Fe being mainly confined to the outer grain layers while Zn is more evenly distributed. Gentle polishing will therefore have a greater effect on reducing Fe losses than is the case for Zn. The information generated in the present work illustrates that there is an interaction between grain properties and polishing techniques which needs to be taken into account in order to reduce losses of essential mineral elements, and further work can be undertaken to determine optimal industrial polishing techniques for maximizing mineral content. This information is important in relation to plant breeding, biofortification, and postharvest techniques aiming at delivering new genotypes enriched in essential mineral elements. Acknowledgements Financial support was provided by the Danish Ministry of Food, Agriculture, and Fisheries (3304-FVFP-09-B-004) and the EU-FP projects META-PHOR (FOOD-CT-2006-03622) and PHIME (FOODCT-2006-016253). Appendix A. Supplementary Material Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.jcs.2012.07.002. References Bryant, R.J., Dorsch, J.A., Peterson, K.L., Rutger, J.N., Raboy, V., 2005. Phosphorus and mineral concentrations in whole grain and milled low phytic acid (lpa) 1-1 rice. Cereal Chemistry 82, 517e522. Fitzgerald, M., Boualaphanh, C., Calingacion, M., Cuevas, R.P., Jothityangkoon, D., Sanitchon, J., 2011. Yield and quality of traditional and improved Lao varieties of rice. ScienceAsia 37, 89e97. Fitzgerald, M.A., McCouch, S.R., Hall, R.D., 2009. Not just a grain of rice: the quest for quality. Trends in Plant Science 14, 133e139. Gomez-Becerra, H.F., Erdem, H., Yazici, A., Tutus, Y., Torun, B., Ozturk, L., Cakmak, I., 2010. Grain concentrations of protein and mineral nutrients in a large collection of spelt wheat grown under different environments. Journal of Cereal Science 52, 342e349. Graves, A.M., Siebenmorgen, T.J., Saleh, M.I., 2009. A Comparative study between the McGill #2 laboratory mill and commercial milling systems. Cereal Chemistry 86, 470e476. Greffeuille, V., Kayode, A.P.P., Icard-Verniere, C., Gnimadi, M., Rochette, I., MouquetRivier, C., 2011. Changes in iron, zinc and chelating agents during traditional African processing of maize: effect of iron contamination on bioaccessibility. Food Chemistry 126, 1800e1807. Hansen, T.H., Laursen, K.H., Persson, D.P., Pedas, P., Husted, S., Schjoerring, J.K., 2009. Micro-scaled high-throughput digestion of plant tissue samples for multielemental analysis. Plant Methods 5. Itani, T., Tamaki, M., Arai, E., Horino, T., 2002. Distribution of amylose, nitrogen, and minerals in rice kernels with various characters. Journal of Agricultural and Food Chemistry 50, 5326e5332. Kirkham, R., Dunn, P.A., Kuczewski, A., et al., 2010. The Maia spectroscopy detector system engineering for integrated pulse capture, low-latency scanning and real-time processing. In: 10th International Conference on Synchrotron Radiation and Instrumentation 2009. AIP Conference Proceedings, vol. 1234, pp. 240e243. Kutman, U.B., Yildiz, B., Cakmak, I., 2011. Improved nitrogen status enhances zinc and iron concentrations both in the whole grain and the endosperm fraction of wheat. Journal of Cereal Science 53, 118e125.
T.H. Hansen et al. / Journal of Cereal Science 56 (2012) 307e315 Lamberts, L., De Bie, E., Vandeputte, G.E., Veraverbeke, W.S., Derycke, V., De Man, W., Delcour, J.A., 2007. Effect of milling on colour and nutritional properties of rice. Food Chemistry 100, 1496e1503. Laursen, K.H., Hansen, T.H., Persson, D.P., Schjoerring, J.K., Husted, S., 2009. Multielemental fingerprinting of plant tissue by semi-quantitative ICP-MS and chemometrics. Journal of Analytical Atomic Spectrometry 24, 1198e1207. Lee, S., Persson, D.P., Hansen, T.H., Husted, S., Schjoerring, J.K., Kim, Y.S., Jeon, U.S., Kim, Y.K., Kakei, Y., Masuda, H., Nishizawa, N.K., An, G., 2011. Bio-available zinc in rice seeds is increased by activation tagging of nicotianamine synthase. Plant Biotechnology Journal 9, 865e873. Liang, H., Li, Z., Tsuji, K., Nakano, K., Nout, M.R., Hamer, R.J., 2008. Milling characteristics and distribution of phytic acid and zinc in long-, medium- and shortgrain rice. Journal of Cereal Science 48, 83e91. Liu, K.L., Cao, X.H., Bai, Q.Y., Wen, H.B., Gu, Z.X., 2009. Relationships between physical properties of brown rice and degree of milling and loss of selenium. Journal of Food Engineering 94, 69e74. Lombi, E., Scheckel, K.G., Pallon, J., Carey, A.M., Zhu, Y.G., Meharg, A.A., 2009. Speciation and distribution of arsenic and localization of nutrients in rice grains. New Phytologist 184, 193e201. Lombi, E., Smith, E., Hansen, T.H., Paterson, D., de Jonge, M.D., Howard, D.L., Persson, D.P., Husted, S., Ryan, C., Schjoerring, J.K., 2011. Megapixel imaging of (micro)nutrients in mature barley grains. Journal of Experimental Botany 62, 273e282. Mills, E.N.C., Parker, M.L., Wellner, N., Toole, G., Feeney, K., Shewry, P.R., 2005. Chemical imaging: the distribution of ions and molecules in developing and mature wheat grain. Journal of Cereal Science 41, 193e201. Mohapatra, D., Bal, S., 2010. Optimization of polishing conditions for long grain basmati rice in a laboratory abrasive mill. Food and Bioprocess Technology 3, 466e472. Ogiyama, S., Tagami, K., Uchida, S., 2008. The concentration and distribution of essential elements in brown rice associated with the polishing rate: use of ICPAES and Micro-PIXE. Nuclear Instruments & Methods in Physics Research Section B-Beam Interactions with Materials and Atoms 266, 3625e3632.
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Ortiz-Monasterio, J.I., Palacios-Rojas, N., Meng, E., Pixley, K., Trethowan, R., Pena, R.J., 2007. Enhancing the mineral and vitamin content of wheat and maize through plant breeding. Journal of Cereal Science 46, 293e307. Paterson, D., de Jonge, M.D., Howard, D.L., Lewis, W., McKinlay, J., Starritt, A., Kusel, M., Ryan, C.G., Kirkham, R., Moorhead, G., Siddons, D.P., 2011. In: The Xray Fluorescence Microscopy Beamline at the Australian Synchrotron. American Institute of Physics Conference Proceedings, vol. 1365, pp. 219e222. Persson, D.P., Hansen, T.H., Laursen, K.H., Schjoerring, J.K., Husted, S., 2009. Simultaneous iron, zinc, sulfur and phosphorus speciation analysis of barley grain tissues using SEC-ICP-MS and IP-ICP-MS. Metallomics 1, 418e426. Roy, P., Orikasa, T., Okadome, H., Nakamura, N., Shiina, T., 2011. Processing conditions, rice properties, health and environment. International Journal of Environmental Research and Public Health 8, 1957e1976. Ryan, C.G., 2000. Quantitative trace element imaging using PIXE and the nuclear microprobe. International Journal of Imaging Systems and Technology 11, 219e230. Shi, R.L., Zhang, Y.Q., Chen, X.P., Sun, Q.P., Zhang, F.S., Romheld, V., Zou, C.Q., 2010. Influence of long-term nitrogen fertilization on micronutrient density in grain of winter wheat (Triticum aestivum L.). Journal of Cereal Science 51, 165e170. Simic, D., Sudar, R., Ledencan, T., Jambrovic, A., Zdunic, Z., Brkic, I., Kovacevic, V., 2009. Genetic variation of bioavailable iron and zinc in grain of a maize population. Journal of Cereal Science 50, 392e397. Tanumihardjo, S.A., Bouis, H., Hotz, C., Meenakshi, J.V., McClafferty, B., 2008. Biofortification of staple crops: an emerging strategy to combat hidden hunger. Comprehensive Reviews in Food Science and Food Safety 7, 329e334. Wang, K.M., Wu, J.G., Li, G., Zhang, D.P., Yang, Z.W., Shi, C.H., 2011. Distribution of phytic acid and mineral elements in three indica rice (Oryza sativa L.) cultivars. Journal of Cereal Science 54, 116e121. White, P.J., Broadley, M.R., 2009. Biofortification of crops with seven mineral elements often lacking in human dietseiron, zinc, copper, calcium, magnesium, selenium and iodine. New Phytologist 182, 49e84.
Contents lists available at SciVerse ScienceDirect
Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Losses of essential mineral nutrients by polishing of rice differ among genotypes due to contrasting grain hardness and mineral distribution Thomas H. Hansen a, Enzo Lombi b, Melissa Fitzgerald c, Kristian H. Laursen a, Jens Frydenvang a, Søren Husted a, Chanthakhone Boualaphanh c, d, Adoracion Resurreccion c, Daryl L. Howard e, Martin D. de Jonge e, David Paterson e, Jan K. Schjoerring a, * a
Plant and Soil Science Section, Department of Plant and Environmental Sciences, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark Centre for Environmental Risk Assessment and Remediation, University of South Australia, Building X, Mawson Lakes Campus, Mawson Lakes, South Australia SA-5095, Australia c Grain Quality and Nutrition Centre, International Rice Research Institute, DAPO 7777 Metro Manila, Philippines d Rice and Cash Crop Research Institute, NAFRI, Vientiane, Lao Democratic People’s Republic e Australian Synchrotron, 800 Blackburn Road, Clayton, Victoria 3168, Australia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 November 2011 Received in revised form 18 April 2012 Accepted 9 July 2012
The effect of different polishing techniques on loss of mineral elements from rice grains was quantified using a panel of indica and tropical japonica genotypes, previously classified as differing in ease of polishing. Gradients in mineral elements across the bran-endosperm interface were quantified using microscaled precision abrasive polishing in combination with inductively coupled plasma mass spectrometry and synchrotron X-ray fluorescence microscopy. Frictional polishing, similar to that of commercial mills, i.e. 8e10% loss of grain weight, reduced the concentration of Fe, Mg, P, K and Mn by 60e80% in all genotypes. Following gentler polishing (3e5% weight loss), genotypes classified as difficult to polish showed smaller decreases in Fe, Mg, P, K and Mn compared to genotypes classified as easy to polish. The concentration of other elements, e.g. Zn, S, Ca, Cu, Mo and Cd, showed comparable reductions (<30%) irrespective of polishing technique or ease of polishing. The different patterns of polishing losses of minerals reflected their distribution within the grain. Five-fold differences in the reduction of Zn concentration during polishing were observed for different genotypes which started with similar Zn concentrations in the unpolished grain, thus showing clear potential for selecting genotypes with reduced polishing losses of Zn. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: ICP-MS and synchrotron X-ray fluorescence microscopy Iron and zinc Polishing Rice grain
1. Introduction The micronutrients iron (Fe) and zinc (Zn) are often lacking in the human diet and their accompanying deficiencies cause major health problems (Tanumihardjo et al., 2008). Worldwide, Fe and Zn deficiencies are estimated to afflict more than 2 billion people, mostly women and children in developing countries. The major cause of Fe and Zn deficiency is low intake due to diets consisting mostly of a few staple foods with low micronutrient density, e.g. polished rice and other cereals. The consequences in terms of malnutrition and health are devastating and can result in stunted growth, disease, and increased mortality. Besides Fe and Zn, the dietary intake of other mineral elements, particularly selenium (Se), iodine (I), calcium (Ca) and magnesium (Mg) may be * Corresponding author. Tel.: þ45 35333495. E-mail address: [email protected] (J.K. Schjoerring). 0733-5210/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jcs.2012.07.002
inadequate in both developed and developing countries (White and Broadley, 2009). The most widely recognized strategies for reducing micronutrient malnutrition in human populations consist of supplementation with pharmaceutical preparations, food fortification or dietary diversification. These solutions are often impractical for economic and/or social reasons. Therefore enrichment of cereal food staples through plant breeding or genetic engineering is seen as a potentially more sustainable and less expensive strategy (Ortiz-Monasterio et al., 2007). Iron and Zn concentrations in different wheat, maize and rice genotypes can vary several-fold (Gomez-Becerra et al., 2010; Lee et al., 2011; Shi et al., 2010; Simic et al., 2009). Similarly, genotypic variations exist in the concentration of Se, Ca and Mg in vegetable products which can be exploited in breeding programmes (White and Broadley, 2009). Improved intake of mineral elements by rice consumers may be achieved by minimizing the substantial losses that result from
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polishing. In this process, the bran (i.e. the germ/embryo and the aleurone/pericarp layers), which constitutes 8e10% of the rough rice kernel mass, is removed from the brown rice, thereby resulting in white rice grains. The outer grain layers are much denser in minerals than the inner parts, causing a substantial decline in the concentration of mineral elements in the polished grain. Nevertheless, polishing is a preferred step in postharvest production of rice because (i) most consumers prefer the taste and texture of the white rice and (ii) the high oil content of the bran increases risks of rancidity, thereby reducing the shelf life. In addition, polishing is carried out because it decreases the cooking time, which is an important property for poor rice consumers (Fitzgerald et al., 2009). The intensity of polishing depends on the type of mill and the duration of the treatment (Graves et al., 2009; Roy et al., 2011). The relationship between polishing time and loss of material is nonlinear due to increasing adhesion of the bran layers from outer to inner layers, while the different endosperm fractions seem to have comparable hardness (Lamberts et al., 2007). Furthermore, differences in shape and size of rice grains also contribute to the quantity of material lost during polishing (Liang et al., 2008). The loss of mineral elements associated with polishing of rice is not directly related to the loss of grain mass because elements have a heterogeneous distribution within the grain (Lombi et al., 2009). The effects of polishing on losses of macro- and microelements have been studied in rice grain subjected to different polishing rates (Bryant et al., 2005; Itani et al., 2002). These studies generally show decreased concentrations of macronutrients moving from the outer edge to the centre of the grain, as is also the case for micronutrients, although with some marked differences among elements (Lamberts et al., 2007; Ogiyama et al., 2008). Polishing characteristics are also important in order to reveal the distribution of mineral elements in relation to other grain factors affecting their bio-availability such as phytic acid (Liang et al., 2008). The current interest in developing new biofortified rice varieties suggests a need for further information on genotypic differences in polishing losses and how these relate to the distribution of mineral elements in the grain. For instance, the distribution of Zn in white rice did not differ between genotypes with different grain shapes (Liang et al., 2008), while losses of Se differed between genotypes and could be correlated to the degree of polishing (Liu et al., 2009). The aim of the present work was to relate polishing losses of mineral elements to their gradient across the bran-endosperm interface in a panel of indica and tropical japonica rice genotypes, previously classified as differing in ease of polishing. For this purpose a micro-scaled precision polishing procedure was developed. As support for the micro-scaled polishing experiment, a first assessment of mineral losses in the different genotypes was obtained by polishing in a commercial frictional mill and a gentler abrasive mill type. The micro-scaled polishing procedure allowed full-quantitative measurements by ICP-MS of the distribution of mineral elements across the outer grain layers. The element distribution for whole grains was obtained by synchrotron X-ray fluorescence microscopy. This technique was here used for the first time to quantify the distribution of elements in rice grains and validate these results against ICP-MS data. 2. Materials and methods 2.1. Plant material A panel of 12 advanced breeding lines of rice (Oryza sativa L.) was selected by the International Rice Research Institute (IRRI) based on differences in ease of polishing as determined by the length of time required to remove the bran layers completely from
the grain using gentle abrasion. The International Rice Information System (IRIS) unique identifier (IRIS-ID) for these lines is shown in Supplementary Table S1 together with information about grain properties. The IRIS-ID will be used in all reference to the breeding lines. The selected lines comprised two groups of which group 1 consisted of the genotypes that were difficult to polish and group 2 of those that were easy to polish (Supplementary Table S1). The panel also included a traditional variety from the Philippines, Perurutong. This was included because of its high fiber content, high nutritional value, high content of the antioxidant anthocyanin, relatively high content of minerals including iron, and its black pericarp, which is difficult to remove completely. The 13 genotypes were grown in the dry season of 2007 at IRRI’s experimental station in the Philippines. A total of 150 kg N ha1 was applied in three equal applications at transplanting, at mid-tillering and at panicle initiation. P and K, each at 30 kg ha1, were applied at transplanting. Insects, weeds and diseases were managed as per IRRI’s standard protocols. Water was drained at late grain-filling. Grain was harvested at maturity, and dried to 14% moisture before further processing. The unpolished seeds of the 13 rice genotypes showed a considerable span in concentration of mineral elements (Supplementary Table S2). For Fe and Zn, the difference between the genotypes with highest and lowest concentration in the unpolished grain was 65e80%. These differences were not (P > 0.05) related to the polishing ease of the grain. Six genotypes were subjected to analysis in a micro-polishing experiment, comparing genotypic differences in element losses over time. This panel consisted of three genotypes from the first panel (Perurutong and IRIS-146-14597 from Group 1, and IRIS-66445376 from Group 2) as well as three Laotian varieties, viz. Hom Nang Nouan (HNN), Kai Noi Leung (KNN) and Thasano 1 (TSN1). One of the varieties, TSN1, was also investigated in detail using synchrotron XFM. HNN and KNL are traditional, low yielding, but highly priced varieties, while TSN1 is a high yielding, improved variety released in 1993. TSN1 responds well to N application and is a popular variety with good polishing quality and acceptable eating quality (Fitzgerald et al., 2011). The Laotian rice genotypes were grown under similar conditions at an experimental field station in the Lao People’s Democratic Republic (Lao PDR) and received a total application of 60 kg N ha1, split on equal dressings at transplanting and mid-tillering. 2.2. Polishing methods Rough frictional polishing of similar intensity to that of commercial mills, i.e. 8e10% loss of grain weight, was compared with gentle abrasive polishing (3e5% weight loss). Both methods were applied on dehulled grain (Satake Rice Machine, Tokyo, Japan) originating from the panel of 13 genotypes differing in ease of polishing (Supplementary Table S1). The rough polishing was carried out by use of a standard laboratory frictional mill (Grainman 60-230-60-2AT, Grain Machinery Mfg. Corp., Miami, FL) for 30 s. For the more gentle abrasive polishing, a commercial paintshaker (Skandex SO400, Fast and Fluid Management, The Netherlands) adapted to polish small samples of rice was used. The mixing chamber of the paintshaker was fitted with boxes designed to hold 49 perspex capsules. Each capsule contained 5 g of brown rice and 10 g fused aluminium oxide. Polishing was achieved by shaking the boxes in the paintshaker for 1 h, whereupon the polished white grains were separated from the abradant and bran, and brushed to remove any adhering particles of bran. In order to further investigate genotypic differences in element losses over time, brown rice from a panel of six genotypes was polished abrasively using the microscaled method described by Hansen et al. (2009). This method made it possible to distinguish
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between the influence of the embryo and the pericarp-aleurone layers on the element content in the rice grains. Each sample consisted of approximately 10 rice grains, corresponding to 200e250 mg dry mass, where the embryo had been removed with the tip of a scalpel. The rice grains were abraded in small centrifuge tubes with 200 mg of acid washed quartz sand (Fluka 84878, 40e150 mesh SiO2). The polishing process was performed by high speed shaking in a ball mill (MM301, Retsch GmbH, Düsseldorf, Germany) fitted with 2 racks of 24 samples. The mill was operated at 30 Hz for nine selected time periods of 20e450 s. Before analysis, the remains of the grain were in all polishing experiments washed rapidly three times with Milli-Q water (18.2 U; Milli-Q Plus, Millipore Corporation, MA. USA) in order to remove surface dust, and then dried. Subsequent ICP-MS analysis of the rinsing water verified that the rinsing procedure did not cause leaching of mineral elements. 2.3. Sample digestion A method was developed to ensure complete digestion of brown (unpolished) rice (Hansen et al., 2009). The digestion was performed in closed vessels in a microwave oven (Multiwave 3000, software version 1.24, Anton Paar GmbH, Graz, Austria). Samples were micro-waved (1400 W) for 36 min (after 10 min ramping) at 210 C at a maximum pressure of 40 bar in 100 ml closed tubes. The digestion medium consisted of 5 ml 70% ultrapure HNO3 (J.T. Baker Instra-Analyzed Reagent, NJ, USA) and 5 ml 15% H2O2 (30% ExtraPure, Riedel de Häen, Selze, Germany). After digestion, samples were diluted to 50 ml with Milli-Q water (18.2 U; Milli-Q Plus, Millipore Corporation, MA, USA). Element concentrations in the digests of the intact brown rice grains were identical to those obtained by analysis of flour from the same batch of brown rice, verifying that complete digestion of the unpolished grains had been achieved. 2.4. Multi-element analysis Multi-element analysis of samples was performed using an ICPMS (Agilent 7500ce, Agilent Technologies, UK) equipped with a PFA micro-flow nebulizer. ICP-MS data were processed using the Plasma Chromatographic Software v. B-03-07 (Agilent Technologies, UK). Before full-quantitative analysis, samples were diluted to match the acid concentration of calibration standards (3.5% v/v HNO3). Sample uptake was maintained at approximately 0.1 ml min1 by a PFA micro-flow nebulizer (Laursen et al., 2009). Erbium (166Er) was used as internal standard (10 mg l1) to enable correction for drift and plasma instability. Thirteen-pointcalibration was performed using a commercially available standard solution (P/N 4400e132565, CPI International, Amsterdam, The Netherlands). Validation was performed by including certified reference materials (CRMs). Durum wheat NIST 8436 (US Department of Commerce, National Institute of Standards and Technology, Gaithersburgh, MD, USA) was chosen as the matrix corresponds to rice grain and the certification includes essential plant nutrients as well as elements important for human health such as Cd, Se and Fe. Data were rejected if the accuracy was <90% of the certified value of 7 CRMs or if standard deviation exceeded 10%. LOD was determined as three times the standard deviation of 7 blanks and only data above limit of detection (LOD) was accepted. 2.5. Imaging of element distribution A thin (70 mm thick) longitudinal section of the middle part of the grain was imaged on the X-ray fluorescence microscopy beamline at the Australian Synchrotron (Paterson et al., 2011). The
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sectioning process did not require any embedding and was performed as described by Lombi et al. (2009). The use of thin sections is necessary to overcome issues related to the penetration of the Xray beam. Using this technique, the distribution of the micronutrients Fe, Zn, Cu and Mn was imaged. The laterally-resolved element maps were collected using an undulator beamline equipped with a Si(111) monochromator and KirkpatrickeBaez mirrors focussing the beam to a spot size of approximately 3 2 mm2 (horizontal vertical). An incident beam energy of 10 keV was used to excite X-ray fluorescence which was collected using a 96element prototype Maia detector system (Kirkham et al., 2010). The detector was placed perpendicular to the incident beam path and was used to collect the full spectra fluorescence signal from the sample. The samples were analysed continuously in the horizontal direction (‘on the fly’) with steps of 1.25 mm in both directions. The pixel transit time was approximately 0.6 msec. The full XRF spectra were then analysed using GeoPIXE (Ryan, 2000). This software uses Dynamic Analysis to subtract background and resolve overlapping peaks to generate element maps of concentration. 2.6. Data analysis Statistical software (SAS Institute, USA) was used for data analysis by ANOVA using the general linear model (GLM). Chemometric data analysis was performed with the Unscrambler software package version 9.1 (Camo Process A/S, Oslo, Norway) as principal component analysis (PCA). Data was auto-scaled and validated using full cross-validation. Matlab version R2010 (Mathworks, Massachusetts, USA) was used for mathematical analysis of the relationship between mineral element concentration as a function of polishing time. The analysis was based on iterative error minimization of two straight lines, one for the initial steep decrease, and one for the slower decrease at extended polishing times, representing the outer grain layers and endosperm, respectively. With an inclusion criterion of R2 values >0.90, the intercept of the two straight lines was assumed to represent the transition point between outer grain layers and endosperm. In cases where two straight lines could not be established, a single linear regression analysis of mineral element concentration versus polishing time was fitted. 3. Results 3.1. Losses of nutrients as affected by polisher type and grain hardness The two polishing techniques, i.e. frictional polishing in an industrial type pressure mill or abrasive polishing in a laboratory shearing-type mill, had different impacts on the quantity of material removed (Table 1). Abrasive polishing only caused 3e5% weight loss compared to 9% weight loss during frictional polishing. This was accompanied by greater reductions in Fe, Mg, P, K and Mn concentrations upon frictional polishing as compared with abrasive polishing, while Zn, S, Ca, Cu, Mo and Cd concentrations were similar for the two polishing techniques (Table 1). Differences in the polishing ease of the grains had no influence on the concentration of mineral elements following frictional polishing, grains classified as difficult to polish showing the same decrease in concentration as genotypes classified as easy to polish (Table 1). Chemometric data analysis (Fig. 1) clearly allowed discrimination of the two polishing techniques, but was not able to separate grains differing in ease of polishing. Especially Mn, K, P, Fe, Mg and S were important for discrimination of the polishing techniques as evident from the distant loading plot location of S and the group of
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Table 1 Percentage decrease in concentration of mineral elements caused by polishing of brown rice in an industrial pressure mill (friction polishing) or in a laboratory shearing-type mill in which rice grain were shaken with aluminium oxide (abrasive polishing). The two techniques resulted in losses of about 9% of grain dry matter by friction polishing and 3e5% during abrasive polishing. Data are mean values for 13 different genotypes from the International Rice Research Institute, 9 classified as difficult to polish and 4 as easy to polish (Table S1). Values within same element followed by different letters differ significantly (P ¼ 0.05). Element
Type of polishing Friction
Abrasive
Polishing ability Easy
Difficult
Easy
Difficult
Decrease in element concentration, % Mg P S K Ca Mn Fe Cu Zn Se Mo Cd
86A 70A 7A 80A 38A 65A 82A 11A 34A 5A 8A 4A
90A 72A 3A 76A 34A 64A 80A 13A 29A 9A 1A 18A
60B 49B 2B 61B 24A 50B 47B 11A 30A 51B 11A 8A
51C 32C 4A 45C 5B 40C 38B 18A 27A 52B 4A 3A
Mn, K, P, Fe and Mn (Fig. 1B) in accordance with the two groups found in the score plot (Fig. 1A). Removal of bran by frictional polishing caused the genotypes to cluster more closely i.e. reduced some of the variation among genotypes (Fig. 1A). After abrasive polishing, grains classified as easy to polish grouped more closely together than genotypes difficult to polish, while the opposite was the case upon frictional polishing (Fig. 1A). 3.2. Polishing losses in relation to nutrient distribution across the bran-endosperm interface A step-wise micro-polishing procedure was performed to link the distribution across the pericarpealeuroneeendosperm interface of the different genotypes with the duration of the polishing. The initial step consisted of manual removal of the embryo, while in the succeeding steps, the de-embryoed brown grain was polished for specific time intervals in order to gradually abrade the aleurone layer and analyse the resulting concentration in the residual part of
the grain. The step-wise polishing procedure was carried out for nine time intervals distributed over 450 s, finally resulting in removal of about 10% of the dry matter of the grain. Removal of the embryo led, in all genotypes, to a significantly (P ¼ 0.05) lower concentration of Fe and Zn concentration in the deembryoed grain relative to the intact grain (Fig. 2). This was the case despite the fact that the embryo only accounted for about 2% of the seed weight. Genotypic differences in the contribution of the embryo was observed for Mg and P where the concentration decreased significantly in Perurutong, IRIS-66-445376 and IRIS146-14597, while the remaining three genotypes KNL, TSN1 and HNN were only slightly affected by the removal of the embryo (Fig. 3). The concentration of S was not affected by embryo removal in any of the genotypes (Fig. 3). The Fe concentration reached a transition point after 50 2 s of polishing, marking that the outermost Fe-dense tissues had been removed (Fig. 2). A similar transition point was never reached for Zn, showing a more uniform Zn distribution across the aleuroneeendosperm interface than was the case for Fe (Fig. 2). Nevertheless, distinct genotypic differences in the rate of Zn loss during polishing were observed. The steepest decline in Zn concentration across the aleuroneeendosperm interface occurred for IRIS-146-14597 and KNL, 2-fold higher than that for HNN. Compared over the whole polishing period, IRIS-66-445376 showed the largest decline in Zn concentration (59%) relative to the unpolished grain, whereas the Zn concentration in HNN only was reduced by 14%. These large genotypic differences in Zn losses appeared despite the fact that the losses of grain dry matter were similar for the genotypes (around 15% after 450 s of polishing). For Fe, IRIS-66-445376 exhibited the steepest decline across the aleuroneeendosperm interface, 2-fold higher than that for TSN1, showing genotypic differences in the Fe gradient within the aleurone layer. The loss of grain matter was also in this case very similar (data not shown). For all genotypes, the Fe concentration of the grain parts remaining after polishing was more than 75% lower than that of the de-embryoed brown grain. At the end of polishing, KNL contained the lowest concentrations of Zn (10.2 mg g1) and Fe (0.5 mg g1), whereas Perurutong contained the highest concentrations of Fe and Zn (24 and 7 mg g1, respectively). The polishing profile of Mn was very similar to that for Fe, showing a transition point at 45 3 s (Fig. 4). The same was the case for P, Mg and K, although with slightly later transition points of about 70 3 s. By contrast, S, Cu, Se, and Cd were more evenly distributed, and resembled the profile of Zn, i.e. with the
Fig. 1. Principal component analysis of the decrease in mineral element concentration due to polishing of brown rice. Panel A is the score plot, panel B the corresponding loading plot. Principal components 1 and 2 explained 44% and 22% of the variance, respectively. (C,B) denote friction polishing and (-,,) abrasive polishing. Filled symbols are for grains empirically classified as difficult to polish, open symbols for grains classified as easy to polish. Insert in panel B shows magnification of overlapping elements in the loadings plot.
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Fig. 2. Concentration of Fe and Zn in rice grain of six genotypes as a function of polishing time. The embryo was first removed manually (time point before 0) whereupon polishing was carried out for different time intervals in a micro-scaled abrasive mill, starting at time zero. Filled circles denote iron (Fe), open circles zinc (Zn). Values are means SE (n ¼ 3). Based on iterative error minimization, two straight lines, one for the initial steep decrease and one for the slower decrease at extended polishing times, were fitted to the data. With an inclusion criterion of R2 values >0.90, the intercept of the two straight lines was assumed to represent the transition point between outer grain layers and endosperm. In cases where two straight lines could not be established, a single linear regression analysis of mineral element concentration versus polishing time was performed.
concentration much less affected by polishing, and without a clear transition point between outer grain layers and endosperm. High-definition synchrotron X-ray fluorescence was used to further refine the analysis of the distribution of Fe, Zn, Mn and Cu across the outer grain layers and within the embryo of TSN1 (Fig. 5). The highest element concentrations were found in the embryo, particularly in the scutellum, which plays a role in nutrient exchange between the embryo and endosperm. The central part of the embryo, the plumule and radicle, also exhibited high concentrations of Cu, Mn and Zn, but not Fe. The epiblast surrounding the central part of the embryo generally contained lower micronutrient concentrations, with the exception of some Zn hot spots. High Mn, Fe, Zn and Cu concentrations were recorded in the outer grain layers, composed of the pericarp (fruit coat), the tegmen (seed coat) and the aleurone layer (Fig. 5). As also observed in the stepwise micro-polishing procedure (Figs. 3 and 4), Mn and Fe were confined to the outer grain layers, while Zn and Cu concentrations decreased much less steeply towards the endosperm (Fig. 5c). Mn was localized in a very narrow band, with a peak concentration only about 20 mm below the grain surface (Fig. 5c). The Fe peak was broader than that of Mn, and attained maximum concentration about 50 mm below the grain surface. 4. Discussion Polishing is an important operation leading to the production of white rice. The degree of polishing has a significant effect on the quality and nutritional aspects of white rice, affecting properties
such as content of essential minerals (Figs. 2e4; Liang et al., 2008), phytochemicals and grain breakage (Mohapatra and Bal, 2010). Particularly in relation to Fe and Zn, it is important to develop new polishing procedures which can minimize the losses of micronutrients while preserving desired characteristics related to sensory quality and food processing. Even a very short duration (30 s) of friction polishing in the laboratory polisher used in the present work was accompanied by substantial loses of minerals such as Fe, Mg, P, K and Mn (Table 1). This type of polisher can be directly related to commercial systems and illustrates how important the polishing process is for conservation of essential micronutrients. The much gentler abrasive polishing procedures, adapted here, reduced mineral losses (Fig. 1; Table 1) and made it possible to fine-tune the polishing, reducing sample size, duration and loss of material. As abradant, aluminium oxide or quartz sand was used. Both of these materials have a high hardness but, nevertheless, their effectiveness decreased over time due to adsorption of abraded material. The ratio between grain and abradant is therefore critical for the resulting degree of polishing. In some commercially available abrasive laboratory mills, abraded material can be removed during the polishing process (Lamberts et al., 2007; Mohapatra and Bal, 2010). However, in these mill types, the polishing process cannot be micro-scaled and contamination with elements may become a problem. In the present study, small closed systems accommodating perspex capsules were used to avoid contamination problems. The loss of mineral elements associated with polishing of rice is not directly related to the loss of grain mass because elements have
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Fig. 3. Concentration of Mg, P, S and K in rice grain of 6 genotypes as a function of polishing time. The embryo was first removed manually (time point before 0) whereupon polishing was carried out for different time intervals in a micro-scaled abrasive mill, starting at time zero. Left: Filled circles Mg and open symbols P. Right: Filled circles S and open circles K. Values are means SE (n ¼ 3). Curve fitting was performed as described in Fig. 2.
a heterogeneous distribution within the grain (Lombi et al., 2009). The general pattern is that element concentrations show a steep decline across the aleuroneeendosperm interface and thereafter a more gradual decline towards the centre of the grain (Figs. 2e4; Itani et al., 2002; Lamberts et al., 2007). However, there are some marked differences between elements which have profound influence on their loss rate during polishing (Figs. 2e4; Ogiyama
et al., 2008). A relatively large proportion of Fe, Mg, P, K and Mn is associated with the outer grain layers and these elements are accordingly rapidly lost during polishing (Figs. 2e4; Table 1). In contrast, Zn, S, Ca, Cu, Mo and Cd are more uniformly distributed and only show a minor decline in concentration towards the centre of the endosperm (Figs. 2e4; see also Bryant et al., 2005 and Ogiyama et al., 2008). Due to the more uniform distribution and
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Fig. 4. Concentration of Mn, Cu, Mo and Se versus polishing time of grains of 6 rice genotypes: Perurutong (closed circles), IRIS-66-445376 (open circles), IRIS-146-14597 (closed squares), KNL (open triangles), TSN1 (closed triangles) and HNN (open squares). The embryo was first removed manually (time point before 0) whereupon polishing was carried out for different time intervals in a micro-scaled abrasive mill, starting at time zero. Values are means SE (n ¼ 3). Curve fitting was performed as described in Fig. 2.
less steep decline, polishing losses become relatively small for elements such as Zn and Cu (Table 1). Reduced polishing will thus have the largest impact on Fe preservation, less so for Zn, while Cu and Se will not be affected.
The differences in element distribution may to some extent reflect that of chelating ligands such as phytate (Liang et al., 2008; Persson et al., 2009; Wang et al., 2011) and proteins (GomezBecerra et al., 2010; Kutman et al., 2011) which also can affect the
Fig. 5. Synchrotron X-ray fluorescence microscopy of Cu, Fe, Mn and Zn in grain of the genotype TSN1. A. Light microscope image of the longitudinal grain section (dorsal side on top). B. Element concentration maps. C. Element concentration profile from selected area (white box). The data was obtained by using the transect function in GeoPIXE over an area 201 pixel wide. Red dashed lines shows the thickness of the layer of grain material that was abraded by 8e10% polishing.
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nutritional quality of rice in combination with polishing procedures. It is generally assumed that a substantial part of both Zn and Fe in the cereal grain is bound to phytate (Greffeuille et al., 2011; Mills et al., 2005; Simic et al., 2009). Up to 80% of the P content of cereal grain may be present in the form of phytate and losses of Zn and Fe during polishing would therefore be expected to match that of P (Wang et al., 2011). However, this was only true for Fe, where both the initial steep losses as a function of polishing time (Figs. 2 and 3) and the total losses (Table 1) closely matched the pattern for P. In contrast, Zn was much more uniformly distributed across the aleuroneeendosperm interface closely matching that of S (Figs. 2 and 3). The coupled distribution of Fe together with P versus Zn together with S is in agreement with results from a recent speciation analysis showing that a substantial part of Fe co-eluted with P, while Zn co-eluted with S containing ligands (Persson et al., 2009). However, the concentration of Zn declined more (27e30%) upon polishing than did that of S, which was hardly affected (Table 1; Figs. 2 and 3). A substantial part of the Zn is localized in the embryo as well as in the outermost layers of the grain (Figs. 2 and 5) and further research is needed to unravel the speciation of this Zn. In addition to ligands such as phytate and protein, the non-protein amino acid nicotianamine (NA) and other low-molecular-weight ligands may play a role (Lee et al., 2011). Synchrotron XFM is a powerful technique used to visualize the distribution of elements in seeds (Lombi et al., 2009, 2011). There was good agreement between the quantitative data obtained by micro-polishing combined with ICP-MS analysis (Fig. 2) and synchrotron XFM images (Fig. 5). Both techniques showed that Mn was more confined to the surface layers of the grain than Fe, while the Zn concentration declined less steeply towards the core endosperm (Fig. 5). The Fe, Mn, Cu and Zn concentrations in the aleurone layer estimated by XFM imaging were in good agreement with ICPMS measurements of the same genotype (TSN1), showing 55 mg Fe kg1 and 35 mg Zn kg1 in the aleurone layer (Hansen et al., 2009). However, endosperm concentrations of Fe and Mn measured by synchrotron XFM were close to zero, i.e. lower than the ICP-MS derived concentrations (Fig. 4), reflecting that XFM was not sufficiently sensitive to image the low concentrations of the two elements in the endosperm. Polishing characteristics differ with grain properties such as length and width (Supplementary Table S1; Liang et al., 2008). The rice used in the present study consisted of nine japonica genotypes which have a relatively short and plump grain (length to width ratio just above 2; Supplementary Table S1) and low amylose content (12e19%). The four indica genotypes included in the study had long, slender grain (length to width ratio above 3; Supplementary Table S1) with low and intermediate amylose content (19e23%). All the japonica genotypes were empirically classified as relatively difficult to polish, while the indica genotypes were classified as easy to polish (Supplementary Table S1). However, there were no clear differences with respect to mineral losses between the two germplasm classes following the more intense frictional polishing (Table 1). In contrast, genotypic differences were observed after the relatively gentle abrasive polishing which caused a smaller reduction in the concentration of mineral elements in genotypes classified as difficult to polish compared to genotypes classified as easy to polish (Table 1). The genotypes clustered much more densely upon the more intense frictional polishing than after the more gentle abrasive polishing, reflecting less variability in endosperm concentrations of mineral elements compared to outer grain layers (Fig. 1). Distinct genotypic differences, up to 5-fold, in the reduction of Zn and Fe concentrations during polishing were observed (Fig. 2). This was the case even though the amount of dry matter lost was very similar (data not shown). A special case was the genotype Perurutong, a landrace
from the Philippines with a black pericarp, which after polishing still had 14-fold higher total Fe concentration than the genotype (KNL) which had the lowest Fe concentration. 5. Conclusions It is concluded that there is a clear potential for selecting rice genotypes with reduced losses of Zn and Fe after polishing. Further exploration of this potential needs to be combined with other quality parameters, such as cooking ability and sensory quality following polishing. A much greater proportion of Fe is lost by polishing than is the case for Zn. This reflects their distribution pattern within the grain, Fe being mainly confined to the outer grain layers while Zn is more evenly distributed. Gentle polishing will therefore have a greater effect on reducing Fe losses than is the case for Zn. The information generated in the present work illustrates that there is an interaction between grain properties and polishing techniques which needs to be taken into account in order to reduce losses of essential mineral elements, and further work can be undertaken to determine optimal industrial polishing techniques for maximizing mineral content. This information is important in relation to plant breeding, biofortification, and postharvest techniques aiming at delivering new genotypes enriched in essential mineral elements. Acknowledgements Financial support was provided by the Danish Ministry of Food, Agriculture, and Fisheries (3304-FVFP-09-B-004) and the EU-FP projects META-PHOR (FOOD-CT-2006-03622) and PHIME (FOODCT-2006-016253). Appendix A. Supplementary Material Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.jcs.2012.07.002. References Bryant, R.J., Dorsch, J.A., Peterson, K.L., Rutger, J.N., Raboy, V., 2005. Phosphorus and mineral concentrations in whole grain and milled low phytic acid (lpa) 1-1 rice. Cereal Chemistry 82, 517e522. Fitzgerald, M., Boualaphanh, C., Calingacion, M., Cuevas, R.P., Jothityangkoon, D., Sanitchon, J., 2011. Yield and quality of traditional and improved Lao varieties of rice. ScienceAsia 37, 89e97. Fitzgerald, M.A., McCouch, S.R., Hall, R.D., 2009. Not just a grain of rice: the quest for quality. Trends in Plant Science 14, 133e139. Gomez-Becerra, H.F., Erdem, H., Yazici, A., Tutus, Y., Torun, B., Ozturk, L., Cakmak, I., 2010. Grain concentrations of protein and mineral nutrients in a large collection of spelt wheat grown under different environments. Journal of Cereal Science 52, 342e349. Graves, A.M., Siebenmorgen, T.J., Saleh, M.I., 2009. A Comparative study between the McGill #2 laboratory mill and commercial milling systems. Cereal Chemistry 86, 470e476. Greffeuille, V., Kayode, A.P.P., Icard-Verniere, C., Gnimadi, M., Rochette, I., MouquetRivier, C., 2011. Changes in iron, zinc and chelating agents during traditional African processing of maize: effect of iron contamination on bioaccessibility. Food Chemistry 126, 1800e1807. Hansen, T.H., Laursen, K.H., Persson, D.P., Pedas, P., Husted, S., Schjoerring, J.K., 2009. Micro-scaled high-throughput digestion of plant tissue samples for multielemental analysis. Plant Methods 5. Itani, T., Tamaki, M., Arai, E., Horino, T., 2002. Distribution of amylose, nitrogen, and minerals in rice kernels with various characters. Journal of Agricultural and Food Chemistry 50, 5326e5332. Kirkham, R., Dunn, P.A., Kuczewski, A., et al., 2010. The Maia spectroscopy detector system engineering for integrated pulse capture, low-latency scanning and real-time processing. In: 10th International Conference on Synchrotron Radiation and Instrumentation 2009. AIP Conference Proceedings, vol. 1234, pp. 240e243. Kutman, U.B., Yildiz, B., Cakmak, I., 2011. Improved nitrogen status enhances zinc and iron concentrations both in the whole grain and the endosperm fraction of wheat. Journal of Cereal Science 53, 118e125.
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