Selenium Speciation in Malt, Wort, and Beer Made from Selenium

Article pubs.acs.org/JAFC

Selenium Speciation in Malt, Wort, and Beer Made from SeleniumBiofortified Two-Rowed Barley Grain Sara Rodrigo,*,† Oscar Santamaria,† Yi Chen,‡ Steve P. McGrath,‡ and Maria J. Poblaciones† †

Department of Agronomy and Forest Environment Engineering, University of Extremadura, Avenida Adolfo Suárez s/n, 06007 Badajoz, Spain ‡ Sustainable Soils and Grassland Systems, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United Kingdom ABSTRACT: Selenium (Se) biofortification of barley is a suitable strategy to increase the Se concentration in grain. In the present paper, the suitability of this Se-biofortified grain for making Se-enriched beer is analyzed. The aim of the present study was to evaluate the effect of different Se fertilizer doses (0, 10, and 20 g of Se ha−1) and forms (sodium selenate or sodium selenite) on the Se loss during the malting and brewing processes and Se speciation in grain, malt, wort, and beer. Samples were analyzed using inductively coupled plasma−mass spectrometry (ICP−MS) and high-performance liquid chromatography (HPLC)−ICP−MS for total Se and speciation. Mashing−lautering was the process with the greatest Se loss (83.8%). After malting and brewing, only 7.3% of the initial Se was retained in beer, mainly in selenite form. Even so, the fertilizer application of sodium selenate at 20 g ha−1 increased the total Se concentration almost 6-fold in the final beer in comparison to the use of grain derived from unfertilized barley. The present paper provides evidence that the use of Se-biofortified barley grain as a raw material to produce Se-enriched beer is possible, and the results are comparable to other methods in terms of efficiency. KEYWORDS: selenium, speciation, beer, brewing, HPLC−ICP−MS

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INTRODUCTION Selenium (Se) is an essential trace element for humans and animals. An adequate Se intake has beneficial effects in humans, such as prevention of several cancers, benefits in immune system,1 and some protection against the aging process and cardiovascular diseases.2 According to Elmadfa,3 the European recommended dietary allowance (RDA) of Se for humans is about 55 μg of Se day−1, slightly lower than the current Se intake recommendation of 1 μg of Se kg−1 of body weight day−1 reported by the Committee on the Medical Aspects of Food Policy (COMA).4 According to Roman Viñas et al.,5 there is an inadequate intake of Se in more than 20% of the European population. In this context, the Se intake should be increased to reach the recommended values. Several strategies to remedy Se deficiency have been tried. Because yeast (Saccharomyces cerevisiae Meyen ex E. C. Hansen) has been shown to accumulate and transform inorganic Se to organo-Se compounds,6 Se-enriched yeast supplements have been widely used.7 Another strategy to remedy the Se-deficient intake is the use of Se-enriched common dietary foodstuff obtained by means of Se biofortification of crops.8 Cereals constitute a major source of Se because they are consumed in large amounts in many human diets. Among cereals, two-rowed barley (Hordeum vulgare L. ssp. distichum) is one of the most ancient and widely distributed crops. Currently, about two-thirds of barley crops are used for animal feed, approximately one-third for malting and about 2% for human food directly. Spain produces annually more than 8 million tons of barley grain, and annual consumption is approximately 78 kg of beer per capita.9 Two-rowed barley has already demonstrated to be a suitable candidate to be included in Se biofortification programs under Mediterranean conditions.10 However, that study was focused on the Se accumulation in the grain, but very scarce © XXXX American Chemical Society

information is found regarding the effect of the biofortification in the final Se concentration in beer after the manufacturing process. At this regard, only a study carried out in Australia by Gibson et al.11 has been found to deal with the use of Seenriched barley grain by crop biofortification to obtain Seenriched beer. However, in that study, the fertilizer application rates were much higher than those recommended in the literature for barley10 and only total Se by inductively coupled plasma−mass spectrometry (ICP−MS) was analyzed, without analyzing the chemical forms of this Se. Other authors12,13 proposed a different approach to increase the Se concentration in beer and other beverages, such as white wine. This procedure consisted of adding sodium selenite during the beer-making process directly to the fermentation medium to use the capacity of yeast to accumulate and biotransform Se. Finally, Gibson et al.11 also proposed the Se biofortification of the grain during the malting process. In that case, the inorganic form of Se (especially sodium selenate) was added to the water used in the germination stage of the grain. To obtain precise information about the Se benefit, it is important to know not only the amount of Se in beer but also the chemical forms in which Se is presented. According to Thiry et al.,14 the organic Se forms, such as SeMet (selenomethionine), are the most efficient in the long term to prevent Se deficiency. Conversely, inorganic Se forms give a more rapid response to an acute shortage of Se, but they have a higher risk of becoming toxic in the long term. Several authors15,16 have found differences in Se bioavailability in Received: February 18, 2014 Revised: May 28, 2014 Accepted: May 28, 2014

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dx.doi.org/10.1021/jf500793t | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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Table 1. Percentage (Mean ± Standard Error) of Total ICP Selenium Loss, Related to the Total Se Remaining from the Previous Process, in Each Partial Process (Malting, Mashing−Lautering, and Fermentation−Maturation) and in the Whole Malting−Brewing Process, in Relation to the Dose and Form of Se in the Fertilizera treatment −1

0 g ha 10 g of Se 20 g of Se 10 g of Se 20 g of Se % average

ha−1 ha−1 ha−1 ha−1

selenate selenate selenite selenite

malting 1.8 11.9 5.5 3.3 2.9 5.1

± ± ± ± ± ±

2.5 9.8 7.7 4.6 3.2 2.0 C

mashing−lautering 76.8 84.5 84.6 85.8 87.2 83.8

± ± ± ± ± ±

11.6 0.6 2.1 2.4 0.3 8.8 A

fermentation−maturation 54.3 58.0 55.9 45.4 58.3 54.4

± ± ± ± ± ±

18.0 8.2 2.4 8.7 5.0 3.0 B

whole brewing 88.6 94.2 93.6 92.4 94.8

± ± ± ± ±

4.2 1.5 1.1 0.6 0.6

a

Letters indicate significant (p < 0.05) differences in the Se losses between the different brewing processes according to ANOVA and Fisher’s LSD test. For each process, the one-way ANOVA on the effect of the treatment on the Se loss showed no significant effect (p > 0.05). a further 24 h. Roots were manually removed to obtain clean malt that was milled in a corundum mill (WolfgangMOC, Germany). Brewing consisted of the following main processes: mashing, lautering, fermentation, and maturation. The mashing process was performed in 2 L of distilled water per sample for 1 h at 54 °C, followed by 1 h at 67 °C. For lautering, wort was separated from spent grains with a laboratory sieve and then boiled for 90 min. After boiling, wort was quickly cooled and filtered. For fermentation, 2 g of dried S. cerevisae yeast (SaftBrew T-58, Fermentis, Lesaffre Group, France) was added to wort. After a 9 day fermentation period, yeast was removed from the container bottom, and the resulting beer was left to mature for 20 days. Aliquots of grain, malt, wort, and beer were taken at the end of each process to evaluate the Se losses during malting, mashing− lautering, and fermentation−maturation. Measurement of Total Se with ICP−MS and Se Speciation with High-Performance Liquid Chromatography (HPLC)−ICP− MS and Data Presentation. For each sample of each fraction (grain, malt, wort, and beer), total Se was determined by ICP−MS (Agilent 7500ce, Agilent Technologies, Palo Alto, CA) operating in the hydrogen gas mode. This analytical method was developed by the Elemental and Molecular Analysis Service of the University of Extremadura (Spain). Previously 1 g per sample was digested with ultrapure concentrated nitric acid (2 mL) and 30% (w/v) hydrogen peroxide (2 mL) using a closed-vessel microwave digestion protocol (Mars X, CEM Corp., Matthews, NC) and diluted to 25 mL with ultrapurified water.20 Sample vessels were thoroughly washed with acid before use. For quality assurance, a blank and a standard (tomato leaf material, NIST 1573a) were included in each batch of samples. All of the results were reported on a dry weight basis. Speciation was carried out by HPLC−ICP−MS following proteolytic digestion with protease and lipase21 using an Agilent 1050 HPLC pump and a Hamilton PRPX100 250 × 4 mm column (Cole-Parmer, London, U.K.). It was performed following the methodology proposed by Li et al.22 This method was slightly modified in the present study using 40 mM NH4NO3 instead of 50 mM NH4NO3 in the mobile phase, because it was found to be more effective. ICP−MS monitored m/z 78 and 80, with hydrogen flow into the octopole reaction cell of 4 mL min−1. Peak areas for quoted species were measured, percentage value for a particular peak calculated relative to the total area under the chromatogram, and compared to the total Se content. Percentages of each fraction, selenomethionine (SeMet), selenite, selenate, oxidized selenomethionine (SeMetO), and others (unknown fraction), were calculated in each step of the brewing process. Three replicates from every fraction (grain, malt, wort, and beer) of the 10 g ha−1 sodium selenate treatment were analyzed to assess technical repeatability. Statistical Analysis. In each manufacturing process (malting, mashing−lautering, and fermentation−maturation) as well as in the whole malting and brewing process, the effect of the fertilizer treatment (0, sodium selenate at a dose of 10 and 20 g ha−1, and sodium selenite at a dose of 10 and 20 g ha−1) and the manufacturing process (malting, mashing−lautering, and fermentation−maturation) on the percentage losses of total Se (determined by ICP−MS) was statistically analyzed by two-way analysis of variance (ANOVA) and multiple comparison tests [Fisher’s least significant difference (LSD)

relation to different food sources, being higher in wheat (83%) than in garlic (78%), fish (56%), or mushrooms (5%). It is also important to distinguish between absorption, availability, and activity. While most dietary selenium is absorbed efficiently, the retention of organic forms for further bioactivity is higher than that of inorganic forms.17 The U.S. Food and Nutrition Board Report18 suggested that more than 90% of SeMet is absorbed; SeCys (selenocysteine) appears to be absorbed very well; although almost 100% of selenate is absorbed, a significant fraction is lost in the urine; and although 50% of selenite is absorbed, it shows better retention than selenate in the organism. It is already known that SeMet is the predominant form of selenium (56−83%) in the grain of many cereals, while other selenocompounds occur in smaller proportions (selenate, 12−19%; selenocysteine, 4−12%; Se-methyl-selenocysteine, 1− 4%; and others, 4−26%).19 However, to our knowledge, how Se fertilization affects such speciation in the barley grain and how the manufacturing process affects such speciation have not yet been studied. Therefore, to advance the study of the two-rowed barley as a product able to increase the available Se levels in the organism and also to test beer as a “functional food”, the aim of the present study was (1) to evaluate the effect of different Se doses and Se fertilizer forms on the total Se losses during the manufacturing processes (malting and brewing) and (2) to evaluate the influence of those Se doses and Se forms on the chemical speciation of Se accumulated in the different products derived from the manufacturing process (grain, malt, wort, and beer).

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MATERIALS AND METHODS

Samples and Malting and Brewing Process. The barley grain used for malting and brewing in the present study was obtained from a previous Se biofortification field experiment.10 The experiment had been conducted in the growing season 2011/2012, in Badajoz, southern Spain, on a Xerofluvents soil under rainfed Mediterranean conditions. The experiment was designed as a split plot arrangement with four repetitions, including two forms of Se [sodium selenate (Na2SeO4) and sodium selenite (Na2SeO3)], one per plot, and within the plot, three application doses (0, 10, and 20 g of Se ha−1 diluted in 3 L of water) were randomly distributed. The crop area for each treatment was 15 m2 (3 × 5 m). Barley grain was harvested in early June. A more detailed description of the experiment is described by Rodrigo et al.10 Malting and brewing were carried out in our lab according to the guidelines by local brewers. Five barley grain samples (600 g), one per treatment obtained by mixing the four repetitions, were taken. Malt was obtained by germinating the barley seeds (20 °C, 100% humidity) in an incubator (GER-700, RADIBER, S.A., Barcelona) until the sprout reached 2/3 of the seed size (after approximately 30 h), then airdried for 24 h at room temperature, and kilning at 55 °C in an oven for B



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Table 2. Total ICP Se (μg kg−1) and Total Se HPLC (μg kg−1) in Barley Grain, Malt, Wort, and Beer as a Function of Se Form (Sodium Selenate and Sodium Selenite) and Se Dose Applied to the Cropa Se species (%) fraction grain

malt

wort

beer

treatment 0 g ha−1 10 g of Se 20 g of Se 10 g of Se 20 g of Se 0 g ha−1 10 g of Se 20 g of Se 10 g of Se 20 g of Se 0 g ha−1 10 g of Se 20 g of Se 10 g of Se 20 g of Se 0 g ha−1 10 g of Se 20 g of Se 10 g of Se 20 g of Se









total ICP Se (μg kg−1) 111.7 880.0 1133.9 270.0 343.5 128.8 774.1 1076.2 268.4 333.8 30.7 120.2 166.3 37.9 42.7 12.4 50.7 73.0 20.6 17.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

total HPLC Se (μg kg−1)

16.3 e 25.5 b 6.4 a 5.2 d 12.9 c 14.1 d 63.8 b 88.0 a 27.7 c 23.5 c 18.2 b 5.3 a 35.8 a 2.4 b 1.8 b 2.8 c 12.0 b 11.8 a 2.0 c 2.9 c

91.2 426.1 466.5 142.9 146.1 98.6 357.7 542.0 125.4 162.0 3.9 27.7 29.3 8.9 10.2 6.6 10.5 14.4 6.9 7.1

± 26.6

± 48.7

± 1.2

± 0.9

SeMet

selenite

SeMetO

unknown

59.0 90.2 90.4 67.5 74.2 66.6 86.8 88.6 66.8 77.3 0.0 61.5 65.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0

41.0 9.8 9.6 32.5 25.8 33.4 11.2 7.6 32.5 22.2 100.0 38.5 34.8 100.0 100.0 100.0 100.0 63.0 100.0 100.0

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.6 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0 0.0 1.9 3.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 37.0 0.0 0.0

Se species are presented as a percentage of total Se HPLC. In the total ICP Se column, mean ± standard error values are presented. Letters indicate significant (p < 0.05) differences among treatments for each product fraction according to ANOVA and Fisher’s LSD test. Technical repeatability of the HPLC measurements was determined for the 10 g of Se ha−1 sodium selenate samples in each product fraction and expressed as the average ± standard error. a

test]. The same effect of the fertilizer treatment was also analyzed by one-way ANOVA and LSD test on the total Se (determined by ICP− MS) accumulated in each product fraction (grain, malt, wort, and beer) obtained during the malting and brewing processes.

highest Se accumulation in the grain (Table 2). With regard to malt, the pattern was similar, although no differences were found between the two doses of sodium selenite. In the wort, which had much lower Se concentrations than grain because of the already mentioned losses during the malting and mashing− lautering processes, fertilization with sodium selenate was the only treatment that significantly increased the Se concentration but no difference between doses. In the final beer, again the application of sodium selenate as a Se fertilizer was the only form able to increase the total Se in the manufactured product. A clear effect of the dose was observed, with the highest dose giving the highest Se concentration (Table 2). On the basis of the Se speciation analysis (one example of a malt sample is given in Figure 1), the results obtained are shown in Table 2. The technical repeatability of the procedure, which can be observed in the total Se (HPLC) column in Table 2, can be considered adequate, because the relative standard error was less than 10% (n = 3) in almost all of the cases. Only

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RESULTS The two-way ANOVA performed to evaluate the effect of the fertilizer treatment and the manufacturing process on the losses of total Se resulting from the different malting and brewing processes showed a significant effect of the process [degrees of freedom (df) = 2; F statistic (F) = 284.42; p < 0.001] but not of the treatment (df = 4; F = 1.03; p = 0.426) nor the interaction (df = 8; F = 0.58; p = 0.781). For this reason, the multiple comparison test between the three different processes was carried out with the pooled data of the different treatments. The highest Se losses (obtained by ICP−MS) occurred significantly during the mashing−lautering processes, with a loss of 83.8% on average (Table 1). Although significantly lower, important Se losses were also observed during fermentation−maturation, with a loss percentage of 54.4%, in relation to the Se remaining after mashing−lautering. During the malting process, only 5.1% of Se was lost. On average, during the whole malting and brewing process, the loss of total ICP Se was 92.7%. As the ANOVA showed, similar Se loss patterns were observed in the different Se fertilization treatments used during barley cultivation (Table 1). The fertilization treatment also significantly affected the total ICP−MS Se obtained in the different product fractions (grain, malt, wort, and beer) derived from the manufacturing process (Table 2). In the grain, the fertilization treatment significantly increased the Se concentration in comparison to controls. Sodium selenate was the most effective fertilizer form in boosting Se in the grain, and the higher fertilizer dose gave the

Figure 1. Se species of a malt sample separated by anion-exchange− HPLC−ICP−MS. The chromatogram was monitored at Se m/z 78. C



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and malt after biofortification has been found to be in the SeMet form, similar to findings in the grain of bread-making wheat,24 hard wheat,25 rice,22 and chickpea.26 The highest loss of total Se was clearly produced during the mashing and lautering (a Se loss of around 85% on average). This Se loss was higher than that found in the same process in the already mentioned study by Gibson et al.11 In that study, the Se loss was dependent upon the capacity of the pilot brew used. They used 20 and 500 L pilot breweries, which produced Se losses during mashing−lautering of about 71.5 and 64%, respectively. Therefore, taken into account that there appears to be a link between beer capacity (or brewing scale) and Se loss, the lab scale (in 2 L volumes) used in the present paper might explain the higher Se loss found in our study. With regard to the Se speciation analysis during this process, most SeMet was removed from the Se fraction. The cause could be thought to be the degradation of this compound because of the high temperatures during boiling. However, Poblaciones et al.25 reported minimal SeMet losses after boiling in Se-enriched pasta. Thus, SeMet could be considered thermostable. Therefore, the most likely explanation for this loss would be that Se was strongly bound to the spent grain, and only a proportion of the most water-soluble compounds, such as selenite,22 was able to pass to the wort. However, when the initial concentration of SeMet in the malt was high (more than 300 μg kg−1), as occurred in the case of sodium selenate fertilization, not all SeMet was lost in the spent grain and approximately 1/18 and 1/25 of SeMet remained in the wort. Further experiments analyzing the Se concentration and the Se species contained in the spent grain should be performed to confirm this hypothesis. Finally, during the fermentation−maturation process, a large proportion of total Se was also lost (around 54% on average). Yeast (S. cerevisiae) has been shown to accumulate Se in high concentrations.27,28 This fact may explain the strong reduction in the total Se recorded in our study after this process removed the yeast from the fermented and matured wort. Thus, after malting and brewing, only around 7.3% (range of 5.2−11.4%) of the total Se contained in grain was maintained in the final beer. This proportion was only a little bit lower than 10.3 and 15.6% obtained by Gibson at al.11 in the 20 and 500 L pilot breweries, respectively. Therefore, the quite similar results obtained in the two studies carried out in different conditions might reflect they are highly consistent, regardless of the conditions used. The Se concentration of the control beer was in line with that obtained for commercial beer, because in the Mediterranean area, it has been reported13 to range between 9.2 and 17.2 μg kg−1. In the final beer obtained in the present study, no SeMet was found; instead, most Se was obtained in the selenite form. This lack of SeMet is a negative aspect of the use of Se-biofortified barley to obtain selenized beer because it is known that the bioavailability of Se organic forms is much higher than that of inorganic forms.17 Nevertheless, it has been shown above that a large percentage of Se (around 37%) was in unknown forms in beer produced from barley fertilized with the largest dose of sodium selenate. In addition, when the total Se obtained by ICP−MS is compared to that obtained by HPLC−ICP−MS, it can be observed that there was a great amount of Se that was not considered in the speciation analysis because of accuracy limitations in the procedure. Further studies including more advanced techniques may allow for the identification of these fractions, the unknown fraction, and that not included in the

in the case of malt, it was a little bit higher (around 13%). In the grain, Se was mainly accumulated as SeMet, regardless of the chemical Se form used in the crop fertilization, although SeMet concentrations were higher when sodium selenate was used (on average 90.6%) in comparison to sodium selenite (on average 70.9%) and controls (on average 59.0%). The dose had a positive effect on the SeMet concentration in grain, only when Se was applied as sodium selenite but not when Se was applied as sodium selenate. In grain, selenite was also recorded, especially when Se was applied as sodium selenite (on average 29.2%). No other Se species were detected in the grain. In the control grain, the pattern was quite similar to that observed for the lower dose of selenite fertilizer, although the percentage of selenite was a little higher in detriment of the percentage SeMet. According to the results, the malting process did not have any substantial effect on the distribution pattern of the different Se species proportions (Table 2). In this case, a very small percentage of SeMetO (a SeMet oxidation product) was observed, and several unknown Se species occurred when the sodium selenate fertilizer was used, which slightly decreased the percentage of SeMet. Important changes took place after the mashing−lautering process. In wort derived from non-Se-enriched barley and barley fertilized with sodium selenite, regardless of the dose, only the selenite form was detected. In wort from barley fertilized with sodium selenate, the proportion of SeMet was also substantially reduced but around 60% of SeMet was still recorded in these samples. This progressive removal of the SeMet fraction was completed after the fermentation− maturation processes. In almost all of the cases, Se measured in beer was in the selenite form. Only when barley was fertilized with the highest dose of sodium selenate, 37.0% of Se detected was unknown and the rest was in the selenite form.

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DISCUSSION The Se loss recorded in the present paper during the malting process (around 5% on average) was lower than the approximately 12−20% obtained by Gibson et al.11 in their study dealing with the use of barley grain biofortified with Se during malting and brewing. Those authors determined that the amount of Se lost in this process was very similar to the amount of Se recorded in the rootlets. Therefore, the removal of rootlets from malt might explain in a high percentage the Se loss during this process. If the germination step had been stopped before in our study, then the rootlets should be smaller, and consequently, they might contain a lower amount of Se, which might explain the differences between both studies. However, not all of the Se loss during this process can be explained with the removal of rootlets. The rest of the Se loss observed during the malting process could be explained by Se phytovolatilization in the germination step. Grant et al.23 found that plants transform the different Se forms during their metabolic activity. During germination and root development, part of Se contained in grain, mainly in the form of SeMet, may be transformed to selenomethylmethionine (SeMMet), which is the principal precursor of the volatile compound, dimethylselenide (DMSe). However, the slightly smaller proportion of SeMet that we found after malting might be explained in part by the transformation and volatilization hypothesis explained above but could also be a consequence of the transformation of SeMet to SeMetO, because easy oxidation of SeMet has already been demonstrated.13 Even so, in our study carried out on barley, most Se recorded in grain D



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almost 2400 μg kg−1, a concentration very close to that given by Gibson et al.11 considering the Se loss during malting. In following the methodology indicated in the present paper, it is also important to note that several Se-rich byproducts, generated during the brewing process, could be used as an important Se source. For example, spent grain, which includes approximately 25−30% of the malt weight, is an important byproduct from brewing that is commonly used for animal feed.29 Therefore, considering that most Se, especially SeMet, might be bound in the spent grain, it could become a highquality and an excellent feeding product for animals, able to remedy Se deficiencies and avoid their consequences for animal health and fertility.30 Finally, it is also important to note that grain yield and quality parameters of grain were not affected by Se fertilization.10 This is an important point, because it would be impossible to successfully implement a potential biofortification program if farmers obtained lower income gains as a consequence of Se application. In conclusion, this paper provides an alternative approach to the already described methods to obtain Se-enriched beer, in our case through the use of Se-biofortified barley grain as raw material. Although after malting and brewing, only 7.3% of the initial total Se was retained in the beer, the use of grain from barley fertilized with sodium selenate at 20 g ha−1 increased the final Se concentration in grain almost 6-fold. Selenium was mainly lost during the mashing−lautering process because most Se seemed to be bound to the spent grain, which could be used consequently to prevent Se deficiency in the animal feeding. The present paper provides evidence that the use of Sebiofortified barley grain as a raw material to produce Seenriched beer is possible, and the results are comparable to other methods in terms of efficiency. The final decision by farmers or brewers about the suitability of its use should be taken based on the importance of the positive or negative aspects of the different methods in each particular case and balancing investment risk and profitability according to the market preferences.

speciation analyses. At least, it would be very interesting to know if these fractions are in organic or inorganic forms to know accurately the bioavailability of the Se remaining in beer. In addition to the approach to making Se-enriched beer through Se-biofortified barley indicated in the present study and that by Gibson et al.,11 other authors13 have proposed to add sodium selenite directly to wort during the brewing process, expecting biotransformation of selenite to organic selenium compounds by yeast. The same methodology was also used to obtain other Se-enriched beverages, such as white wine.12 A variant of this method was also proposed by Gibson et al.11 consisting of adding the inorganic form of Se in the water used for germination during malting. A comparison between these two approaches could be of great interest for farmers and beer manufacturers to establish their effectiveness, suitability, and convenience. Technically, both approaches reported a similar Se loss derived from the fermentation− maturation process (the only common in both methodologies), for example, 45−58% in our study and 40−57% in the study by Sánchez-Martinez et al.13 In both approaches, the SeMet concentration reported in the final beer once removed yeast was either negligible or very low (1−2%). With regard to easiness and cost of the method, the brewer should choose one of the two following options, which correspond with each approach: to pay a little bit more money to farmers for Seenriched barley (the method proposed in the present paper) or to introduce an additional step during the manufacturing process to include selenite/selenate in the wort or water for malting. The second option presents a high initial investment. It is true that it could be compensated in the long term if Seenriched beer can be sold at higher prices than normal beer, but meanwhile, this investment is at risk to be lost. Conversely, the first option allows for examining the market trend year to year and, consequently, paying farmers for Se-enriched barley according to that trend. On the other hand, it is important to take into account that adding selenium during the beer-making process may be interpreted as the addition of an ingredient, and this may have a negative connotation to food health authorities and beer consumers. For farmers, the application of Se would be very cheap because they can use the conventional fertilizer applications and incorporate Se at the same time. The cost of the product is also very cheap, around 4 € ha−1 (at a dose of 20 g ha−1), easy to be compensated if beer manufacturers pay more for this Seenriched product. The main weakness of the approach proposed in the present paper is the apparently low proportion of Se (ranging between 5.2 and 11.4% of the total Se contained in grain) remaining in the final beer after malting and brewing. Even so, Se biofortification provided barley grain able to produce beer with a much higher Se concentration than that obtained from non-Se-enriched barley. Beer produced with barley fertilized with sodium selenate at a dose of 20 g ha−1, showed total ICP Se concentrations almost 6 times higher than beer produced with non-Se-fertilized barley. Gibson et al.11 considered that 1 L of beer made from malt with a initial concentration of 2.2 mg of Se kg−1 would provide a male with over 100% of his recommended daily intake of Se. In our study, the maximum Se concentration in malt was 1076 μg kg−1, obtained with a fertilizer application of sodium selenate at 20 g ha−1. However, the biofortification of barley with sodium selenate in the same area at a dose of 40 g ha−1 has produced according to Rodrigo et al.10 a Se concentration in grain of

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +34-924-289-300. Fax: +34-924-286-201. E-mail: [email protected]. Funding

Rothamsted Research receives strategic funding from the U.K. Biotechnology and Biological Sciences Research Council. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS ́ The authors thank Teodoro Garcia-White for his invaluable help with the laboratory work.

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REFERENCES

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