Effect of Processing on Some Quality Parameters of Flour and Bread Made from Wheat Grain Biofortified with Zn and Se

Millions of people have inadequate Se and Zn intakes, but agronomic biofortification could prevent this. This study evaluated the effect of the combined Zn and Se biofortification on the quality parameters of grain, and on the composition of minerals (Zn, Se, Mg, Ca and Fe) and their availability in bread-making wheat (Triticum aestivum L.) products, white flour, wholemeal bread and white bread were evaluated. The studied treatments were soil Zn (no Zn, and 50 kg Zn ha−1) and foliar applications (0, 10 g Se ha−1, 8 kg Zn ha−1, and 10 g Se ha−1 + 8 kg Zn ha−1) and were tested in a two-year field experiment (2017–2018, 2018–2019). The foliar combined biofortification increased the concentration of both minerals in white flour, wholemeal bread and white bread by about 33%, 24% and 51%, respectively for Zn, and 3.3-fold, 3.4-fold and 2.7-fold for Se, showing a synergistic effect on Se concentration with the Se and Zn combination. While the loss of Zn and Se during the milling process was41% and 18%, respectively, baking caused a loss of 15% and 19%, respectively, for wholemeal bread, and up to 61% and 29% for Zn and Se for white bread. Hence, although the consumption of wholemeal bread instead of white bread may enhance Zn and Se intake more than biofortification, until consumption habits change, the biofortification of wheat can help to mitigate inadequate Zn and Se intakes in the general population.


Introduction
Zinc (Zn) and selenium (Se) are essential micronutrients for many organisms, including for animals and humans. An adequate Se and Zn intake has beneficial effects in humans, such as prevention against several cancers, benefits to the immune system and growth, and some protection against the aging process and cardiovascular diseases [1][2][3][4]. However, according to the 2019 Report of the Food and Agriculture Organization [5], mineral malnutrition, or "hidden hunger" is a global problem affecting around 60% of the world's population. From that global percentage, 30% is caused by a Zn deficient intake and 15% by a Se-deficient intake [6,7]. This malnutrition, which causes poor health in a population, takes place not only in developing countries but also in developed ones.
In order to achieve an adequate intake, set to about 55-70 µg Se day −1 by Elmadfa et al. [8] and 12-15 mg Zn day −1 by the National Research Council [9], agronomic biofortification with Se and Zn has proven to be a fast, reliable, and sustainable strategy in the short term. The success of this strategy is based on different constraints such as: (1) minerals being carried out on soils with low Se and/or Zn bioavailability, such as those found in the semi-arid Mediterranean area [10]; (2) using fertilizers containing the necessary nutrients in soluble forms, such as zinc sulfate [11] or sodium selenate [12,13]; and (3) treatments being carried out on staple crops [14], such as wheat [15]. However, greater efforts should still be made to achieve the Zero Hunger goal, established by the Agenda of the United Nations by 2030.

Samples Acquisition and Processing (Milling and Baking)
The wheat grain used in the present study for milling and baking to produce white flour and wholemeal bread and white bread was obtained from a previous field experiment where combined biofortification with Zn and Se had been performed [21]. The experiment had been conducted during two consecutive growing seasons, 2017-2018 and 2018-2019, in Badajoz, southern Spain (38 • 540 N, 6 • 44 W; 186 m above sea level), on Xerofluvents soil under rainfed Mediterranean conditions. The years during the experiment were climatically different. While 2017-2018 was similar to that of the average year in terms of precipitation (477 vs. 456 mm), with a rainy March; 2018-2019 was much drier (<300 mm), with a significant rainfall shortage in February and March. The soil characteristics of the study area are described in detail in Reynolds-Marzal et al. [21]. Briefly, the soil texture was a clay loam, with an acid pH (6.4 ± 0.2; mean ± standard error), low organic matter (1.31 ± 0.09%), and a medium content of total N (0.12 ± 0.007%), extractable P (4.9 ± 0.05 g P kg −1 ) and K (321 ± 8 mg kg −1 ). The previous field experiment had followed a split-plot design with four replications randomly distributed and a plot size of 15 m 2 (3 × 5 m). The treatments for the main plot (soil Zn application) and subplot (foliar application) factors are shown in Table 1. More detailed information about the cropping and treatments can be found in Reynolds-Marzal et al. [21]. The soil Zn application, which was only performed at the beginning of the first study year, increased the available Zn concentration in soil by up to more than 1.00 mg kg −1 , and no significant decrease was observed throughout the experimental period [21]. The grain, harvested in early July in both study years, was properly preserved until processing. By milling the harvested grain finely (<0.45 mm) with a corundum mill (WolfgangMOC, Munich, Germany), wholemeal flour was obtained. White flour (refined) was obtained using a Laboratory Mill CD 1 (Chopin, France). Baking was carried out in our lab according to the official methodology of the American Association of Cereal Chemists (AACC) No.10-09.01 [33]. From each flour sample, 50 g was weighed, and 30 mL of distilled water was added, and the mixture was slowly kneaded for 10 min until a uniform dough was obtained. This dough was baked at a temperature of 180 • C for 35 min in an Imetec Zero-Glu baker. The flour samples of two replicates had to be homogeneously mixed in pairs in order to obtain an adequate amount of each treatment for baking.

Sample Analysis
Before any measurement, all the samples were oven-dried at 70 • C to a constant weight. Consequently, all the results were given on a dry weight basis in order to compare the results. In the wholemeal flour samples, total N content was determined using the Dumas combustion method (Leco FP-428 analyzer), and protein was determined by multiplying the total N by 5.7 as a conversion factor. Dry gluten content was determined using the Glutomatic system (Perten Instruments, Hägersten, Sweden). When the flour was refined, the white flour yield (expressed in %) was determined. From the white flour, wholemeal bread and white bread samples, extractable Ca, Mg, Fe, and Zn were obtained by using DTPA (diethylenetriaminepentaacetic acid) and were measured by inductively coupled plasma optical emission spectroscopy (ICP-MS) (Agilent 7500ce, Agilent Technologies, Palo Alto, CA, USA) after a digestion with ultra-pure concentrated nitric acid and 30% w/v hydrogen peroxide using a closed-vessel microwave digestion protocol (Mars X, CEM Corp, Matthews, NC), as described in Reynolds-Marzal et al. [21]. Extractable Se was obtained by using KH 2 PO 4 (0.016 mM, pH 4.8) at a ratio of 10 g of the dry-weight sample to 30 mL KH 2 PO 4 w/v [34] and then determined by ICP-MS as described above. For quality assurance in each batch of samples, blanks and an internal control were used as reference material, with recovery of 96% and 91%, respectively, for Zn and Se. All the results are reported on a dry weight basis.
The potential mineral bioavailability in the samples was estimated by obtaining the relation between phytates, which reduced mineral absorption by the gut, especially for metal ions, and the Zn, Ca, Fe, or Mg molar ratio. Phytates were estimated as phytic acid, which was obtained according to the procedure described by Haug and Lantzsch [35], which measures the Fe in the supernatant after the precipitation of ferric phytate. Zinc bioavailability was also estimated through the total daily absorbed Zn (TAZ) according to the following equation (Equation (1)) proposed by Miller et al. [36]: where TDZ is total daily dietary zinc (mmol d −1 ) and TDP is the total daily dietary phytate (mmol d −1 ), considering the average consumption in Spain of 189 g wheat flour d −1 [37] and 125 g bread d −1 in adult couples [38]. The parameters A max , K R , K p in the model, were estimated as 0.13, 0.10, and 1.2 mmol d −1 , respectively.

Statistical Analysis
For each year separately (2017-2018 and 2018-2019), the effect of the soil Zn application (0SZn and 50SZn), foliar application (0F, 8FZn, 10FSe, and 8FZn + 10FSe) and their interaction on each parameter evaluated in the grain, i.e., wholemeal flour (crude protein, dry gluten and white flour yield), white flour, wholemeal bread and white bread (concentration of Zn, Se and the other minerals studied (Ca, Fe, Mg, Se and Zn), phytic acid, phytate/mineral molar ratios and daily absorbed Zn, TAZ) were analyzed by mixed models through split-plot ANOVAs. Significant differences in the means were compared using Fisher's protected least significant difference (LSD) test at p ≤ 0.05. Simple linear regressions and Pearson's correlation coefficient were used to examine the relationship between the concentration of Zn and Se in grain (wholemeal flour) an their equivalents in white flour and wholemeal bread and white bread. All analyses were performed with the Statistix v. 8.10 package (Analytical Software, Tallahassee, FL, USA).

Effect of Zn and Se Biofortification on the Zn and Se Concentrations and Zn bioavailability in White Flour, Wholemeal Bread and White Bread
In the three fractions considered (white flour, wholemeal bread and white bread) both Zn's concentration and its bioavailability, measured as the molar phytate/Zn ratio and the daily absorbed Zn (TAZ), were significantly affected by the foliar application in both study years (Table 2). In the wholemeal bread, the Zn concentration was also affected by the soil Zn application, but only in 2018-2019 (Table 2). On average, the foliar treatments containing Zn increased the Zn concentration in the three fractions (white flour, wholemeal bread and white bread) by almost 34%, 36% and almost 34%, respectively in 2017-2018, and by 29%, more than 14% and almost 52%, respectively in 2018-2019 (Table 2). In the case of the wholemeal bread in 2018-2019, the application of 50 kg Zn ha −1 to the soil resulted in Zn concentration values in this fraction of 36.9 ± 1.1 mg kg −1 on average versus values of 30.1 ± 1.6 mg kg −1 on average in the Zn unfertilized soils. The foliar Se application did not produce any effect on the concentration of Zn in any of the three fractions considered or in any of the study years ( Table 2).
The phytic acid was affected by the main effect of foliar application and by its interaction with soil Zn application, but only in 2018-2019, when the wholemeal bread was the fraction considered (Table 2), although differences among treatments were quite small. The molar phytate/Zn ratio was significantly lower in the treatments containing Zn in the three fractions (white flour, wholemeal bread and white bread) in both study years  Table 2). The increase in the daily absorbed Zn (TAZ) obtained when Zn was supplied via foliar application was, on average, 31%, 32% and 42% for white flour, wholemeal bread and white bread, respectively in 2017-2018, and 24%, 14% and 50%, respectively, in 2018-2019 (Table 2). In this case, soil Zn application did not affect Zn bioavailability (Table 2). Table 2. Effect of the foliar treatment on the concentration of Zn and Se, phytic acid, molar phytate:Zn ratio and TAZ (daily absorbed zinc) in each fraction considered (white flour, wholemeal bread and white bread) in the two study years. Values are expressed as mean value ± standard error (n = 4 for flour and n = 2 for bread). Levels of significance (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001) for main effects (Zn soil application and foliar application) and interactions according to ANOVAs are also shown.

Degrees of freedom
Different letters for each response variable, fraction and year indicate significant differences (p ≤ 0.05) according to LSD test.
Regarding Se, its concentration in the three fractions (white flour, wholemeal bread and white bread) was only affected by the main effect of foliar application, except for wholemeal bread in 2018-2019 ( Table 2). The foliar treatments containing only Se increased the Se concentration by 2.2-fold, 3.3-fold and 2.2-fold on average in white flour, wholemeal bread and white bread, respectively, in comparison with the non-fertilized controls. However, interestingly in the case of the white flour in 2017-2018 and white bread in 2018-2019, when Zn was also supplied via foliar application in combination with Se, these increases were significantly higher, reaching values of 3.3-fold on average for white flour and 2.7-fold for white bread (Table 2). For the three fractions, the Se concentrations obtained were generally much higher in 2017-2018 than in 2018-2019 (Table 2).

Effect of Zn and Se Biofortification on Quality Parameters and Mineral Status in White Flour, Wholemeal Bread and White Bread
Regarding the quality parameters of the grain studied, crude protein was not significantly influenced (p value > 0.05) by any of the factors considered in any of the study years. On average, crude protein values were 13.1 ± 0.2% in 2017-2018, and 16.0 ± 0.1% in 2018-2019. The dry gluten percentage was significantly affected by the main effect of foliar application (degrees of freedom (df)=3; p-value = 0.002) and by its interaction with soil Zn application (df = 3; p-value=0.044) in 2017-2018, and by foliar application (df = 3; p-value = 0.029) in 2018-2019. The foliar treatments containing Se were found to decrease the dry gluten content in the wheat grain (Figure 1a), although this especially happened when soil Zn was also applied and in the study year with a higher rainfall, 2017-2018 ( Figure 1a). In that year, the dry gluten values were almost 20% lower than those in 2018-2019, on average. The white flour yield was also significantly influenced by the main effect of foliar application (df = 3; p-value = 0.014), and by its interaction with soil Zn application (df = 3; p-value = 0.034), but only in 2017-2018. In this case, the foliar treatments containing Zn, especially when soil Zn was also applied, tended to decrease the white flour yield (Figure 1b When the molar ratio between phytates and the concentration of each mineral was analyzed, the pattern observed for the influence of the factors considered was exactly the same in the case of Mg and Ca for white flour, and quite similar in the case of Fe (Table  3). Thus, biofortification caused a general increase in this molar ratio in white flour, especially when either Zn or Se was applied alone. Such an increase presented more limited  (Table 3). In general terms, biofortification when either Zn or Se was applied alone tended to decrease the concentration of Mg and Ca in white flour, especially in 2017-2018 (Table 3). The concentration of Fe significantly varied in response to the S × F interaction in 2017-2018, but without a clear pattern. In any case, the differences in mineral status due to biofortification were not relevant (Table 3). Table 3. Concentration of Mg, Ca and Fe, and their molar phytate:mineral ratios in white flour, wholemeal bread and white bread, expressed as mean value ± standard error (n = 4 flour and = 2 for bread), as affected by the foliar application (F) and/or by its interaction with soil Zn application. Levels of significance (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001) for main effects (Zn soil application and foliar application) and interactions according to ANOVAs are also shown. When the molar ratio between phytates and the concentration of each mineral was analyzed, the pattern observed for the influence of the factors considered was exactly the same in the case of Mg and Ca for white flour, and quite similar in the case of Fe (Table 3). Thus, biofortification caused a general increase in this molar ratio in white flour, especially when either Zn or Se was applied alone. Such an increase presented more limited values when both micronutrients were applied in combination (Table 3). In the case of the molar phytate/Mg and phytate/Fe ratios in 2018-2019, there was a significant effect of either foliar application or theS × F interaction on their values in wholemeal bread and white bread (Table 3). However, differences in the molar ratios between phytates and any of the minerals studied had limited importance.

Zn and Se Losses Due to Preparation Processes: From Grain to Bread
Two processes were considered: milling to obtain white flour and baking using either, wholemeal flour or white flour to obtain wholemeal bread or white bread, respectively.
The correlations between the concentration of either Zn or Se in grain (wholemeal flour) and that in white flour, wholemeal bread or white bread were significant in all cases ( Figure 2). The Pearson s correlation coefficients (R) were higher in general terms for Se than for Zn, ranging from 0.72 to 0.91 for Zn, and from 0.96 to 0.98 for Se. Observation of the ratios between the concentration of either Zn or Se in each product (white flour, wholemeal bread and white bread) and that of the grain suggested a loss of Zn and Se during the milling process (to obtain white flour) of approximately 41% and 18%, respectively. The baking process using wholemeal flour caused a loss of approximately 15% and 19% of Zn and Se, respectively, but the loss increased to 61% and 29% for Zn and Se, respectively, when white flour was used instead (Figure 2).

Discussion
In general terms, the combined foliar biofortification of Zn and Se increased both the Zn and Se concentration in the three fractions (white flour, wholemeal bread and white bread) by more than 33%, almost 24% and more than 51%, respectively, in the case of Zn, and 3.3-fold, 3.4-fold and 2.7-fold, respectively, in the case of Se between the study years on average. Thus, considering an average daily intake of 125 g bread day −1 [38], and according to the daily requirements [39], the combined foliar biofortification of Zn and Se in wheat, as proposed in the present study, might provide around 41% and 20% of the recommended daily intake, respectively, of Zn and Se in women and 33% and 16% of the

Discussion
In general terms, the combined foliar biofortification of Zn and Se increased both the Zn and Se concentration in the three fractions (white flour, wholemeal bread and white bread) by more than 33%, almost 24% and more than 51%, respectively, in the case of Zn, and 3.3-fold, 3.4-fold and 2.7-fold, respectively, in the case of Se between the study years on average. Thus, considering an average daily intake of 125 g bread day −1 [38], and according to the daily requirements [39], the combined foliar biofortification of Zn and Se in wheat, as proposed in the present study, might provide around 41% and 20% of the recommended daily intake, respectively, of Zn and Se in women and 33% and 16% of the recommended daily intake, respectively in men if wholemeal bread is consumed, but around 21% and 17% of the recommended daily intake, respectively for Zn and Se in women, and 17% and 14% of the recommended daily intake, respectively in men, if white bread is considered. All of these data support the suitability of bread-making wheat to be biofortified with a combined foliar application of Zn and Se. The increase of these micronutrients in the final product, i.e., bread, could be considered satisfactory, especially if wholemeal bread is consumed. However, the soil application of Zn did not increase (or had a limited influence on) the Zn (or Se) concentration in flour or in bread. This was an expected result as this soil application did not increase these concentrations in grain when studied by Reynolds-Marzal et al. [21].
The positive increase in the Zn and Se concentration in the different fractions studied after the combined foliar application of Zn and Se was always observed regardless of the specific weather conditions, although its magnitude was different in each study year. In the case of the year with a higher rainfall, 2017/2018, the combined foliar application of Zn and Se was more effective, as it produced the highest increases, especially for Se. As fertilizers were supplied via foliar application, the more extensive vegetative growth favored by the rainiest conditions might have increased the uptake area and the accumulation of Zn and Se in plant tissues. In the case of Zn, the fact that its transport to the grain through the phloem decreased under conditions of low water availability [40], might also explain its higher accumulation in the rainiest year. Moreover, soil Zn application produced a slight increase in Zn accumulation, but only in 2018-2019 when wholemeal bread was analyzed. Since soil moisture has been positively linked to Zn uptake through the roots [41], under the drier conditions of 2018-2019, the application of soil Zn might partly compensate for the lower uptake. This only happened when the wholemeal bread was considered, probably because it was the only fraction containing bran and germ, the parts where the Zn is accumulated more readily [42], and hence the effect of greater soil Zn uptake might be more pronounced. In any case, even in the extremely dry conditions of 2018-2019, the combined foliar application of Zn and Se increased the Zn concentration to a great extent in all the fractions considered.
The Zn bioavailability was clearly favored by the increase in Zn concentration in white flour and in both types of bread (wholemeal and white) produced by the foliar treatments containing Zn in both study years. However, after the foliar Zn application, this ratio reached values below 15 (values > 15 are associated with low bioavailability [43]) only in the white flour (in both study years) and in the wholemeal bread (in 2017-2018), but not in the white bread. Although baking has been found to increase the hydrolysis of phytates [31], in our case, phytic acid did not present lower values in bread in comparison with flour. Therefore, the important loss of Zn because of milling and baking, without a decrease in the phytate content, might explain the high ratios in white bread. Despite that, due to the increase in the Zn concentration that the Zn biofortification produced in all the fractions, this practice could be considered as positive regarding the increase in Zn bioavailability in all the situations. In this case, no differences between the application of Zn alone or in combination with Se were found. In the case of wholemeal bread, as the loss of Zn caused by milling was much less important, as discussed later, baking increased the Zn bioavailability, giving this product, the highest values of daily absorbed Zn (1.8-1.7 mg Zn d −1 on average) when Zn (alone or combined with Se) biofortification was performed. Nevertheless, in order to increase the intake of Zn in the general population through bread consumption, the most significant action might be the utilization of wholemeal bread rather than white bread, as for wholemeal bread, Zn accumulation and Zn bioavailability reached the highest values. However, currently, only 6.6% of the bread consumed in Spain is wholemeal bread [38]. Therefore, although consumption habits should be changed; Zn biofortification of wheat in the meantime might be a suitable strategy to increase the Zn content in bread and its bioavailability.
Other interesting result, derived from the combined application, was the significant increase in the Se concentration in white flour and white bread when foliar Zn was also applied in both study years. Thus, Zn application increased the Se accumulation by 50% in white flour and 22% in white bread, on average between study years, in comparison with Se accumulation when Se was applied alone. This synergic effect between Se and Zn regarding Se accumulation has already been described for wheat [23] and other crops such as peas [10]. As proposed in such studies, Zn application may have produced an overexpression of sulfate transporters, as suggested by Na and Salt [44], thus favoring the assimilation of Se, which is produced via the sulfur assimilation pathway [12]. However, this effect was not observed when wholemeal bread was the fraction considered, indicating that foliar Zn application may mainly enhance the Se concentration in the endosperm of the grain. This might suggest that the pathways for the accumulation of Se in grain might be different in the endosperm and in the germ and bran. As low-affinity sulfate transporters (LAST) seem to be more implicated in the transport of Se by the phloem [45] and minerals seem to be mainly supplied into the endosperm by the phloem [46], it could be hypothesized that Zn application may predominantly overexpress these LAST rather than high-affinity sulfate transporters (HAST). Nevertheless, further studies specifically designed to evaluate this aspect should be performed to confirm this question.
The success of a biofortification program, in this case for Zn and Se, should be evaluated not only in terms of the increase in their concentration in the edible parts, but also in terms of how it affects other important quality parameters of the grain. In our case, crude protein and white flour yield were not negatively influenced by any of the biofortification treatments performed. This is an important issue because farmers might not be willing to implement this practice if negative effects might appear because of biofortification. Dry gluten, a plastic-elastic protein fraction responsible for dough s physical properties, was found to be decreased by the treatments containing Se, especially in 2017-2018, the study year with higher rainfall (total rainfall = 477 mm). This slight loss of quality in terms of this variable was also observed in previous studies on wheat under Mediterranean conditions, but only when Se was supplied via foliar application alone [13]. However, in that case, this only happened in the driest year (total rainfall = 338 mm), the opposite to the results obtained in the present study. Therefore, new studies including other locations and weather conditions should be further performed to elucidate this point.
The concentration of other minerals, such as Mg, Ca and Fe, was also influenced by the foliar treatment, but only in the case of white flour. In this case, although all of the treatments with either Zn or Se alone caused a slight decrease, the combined application did not reduce the concentration of Mg and Fe in this wheat product. By contrast, the concentration of Ca in white flour decreased under any foliar treatment in 2018-2019 but especially when either Zn or Se was applied alone in both study years. The effect of Zn and Se biofortification on the Ca concentration in wheat has been shown to be beneficial [23], neutral or slightly negative [21]. Therefore, a clear relationship among these three minerals cannot be established. Nevertheless, the lower Ca concentration in white flour because of biofortification was of minor importance in comparison with the loss of Ca caused by milling (Ca concentration in the most unfavorable foliar treatment reached 90% of its concentration in the controls, while Ca concentration in white flour was on average only at 43% in relation to its concentration in wholemeal flour). In any case, this lower Ca concentration in flour caused by biofortification was not transferred to the bread, thus diminishing the importance of this negative effect for the consumer.
Regarding the losses of either Zn or Se during the preparation processes from grain to bread, it should be considered that, during milling, the bran and germ are removed from the wheat kernel. Thus, white flour is mainly composed of the endosperm, which represents approximately 80-85% of the grain [47]. However, the efficiency of the milling process might determine the yield of white flour obtained [48]. In the present study, the white flour recovery was almost 62% on average. Therefore, from the removed parts (38%), almost half (15-20%), might correspond to the bran and germ, and the rest to the endosperm. In the case of Se, its loss during the milling process was ≈18%; this percentage was very similar to that of the bran and germ removed during milling, suggesting an evenly distribution of Se throughout the kernel, such as has already been found in other studies [27]. This fact agrees well with the nonessential character of Se in plants. The Se loss during milling was within the range given by other authors [27,49].
Regarding Zn, due to it being essential in plant metabolism, it mainly accumulates in the bran and the embryo, whereas the endosperm is poor in this micronutrient [42]. This fact agrees well with the Zn loss found in our study after milling, which was approximately 41%, indicating a high proportion of Zn in the removed parts, such as the bran and germ. However, this result suggests a proportion of Zn in the endosperm that was higher than that obtained by other authors [29], where the Zn loss during milling was estimated to be 70%. This could be explained by the fact that Zn fertilization via foliar application has been found to increase the Zn proportion in the wheat endosperm, an effect that is more important as the application is performed at a later growth stage [50]. This is supported by the information gathered by Wang et al. [30], where Zn loss after milling was almost 45% in Zn biofortified grain and more than 60% in unfortified wheat. Therefore, according to our results, foliar Zn biofortification might mitigate the Zn loss produced during the milling of wheat grain, as it might increase the proportion of this micronutrient in the endosperm, the main part used for bread making in most countries.
The average loss of either Zn or Se due to baking was quite similar for both micronutrients (≈15% for Zn, and ≈19% for Se), when using wholemeal flour. Thus, in the case of Se, the loss due to baking was of similar importance to that lost through milling. In other studies on wheat [27], baking has been found to have a minimal effect on Se concentration, with Se losses of <5% attributed to the small sample size and the Se detection limits of the analytical method (ICP-OES). However, in studies on other food products, such as garlic [51], the Se losses resulting from baking were quite similar to those of our study, showing values of 16%. In that study, changes in the chemical Se form caused by heating were demonstrated, in particular, Se-methylSeCys and Se-Met, which might have been partially decomposed into other Se species [51]. Therefore, in our case, it might also be expected that the heat generated during baking could have also decomposed Se-amino acids into other Se-compounds. If any of these new compounds were volatile Se-compounds, such as dimethylselenide or dimethyldiselenide, such as has been proposed in other studies [52], this could explain the loss of Se after a heat processing. Nevertheless, further studies, including Se speciation, should be performed to clarify this fact. Regarding Zn, losses because of cooking have been mainly attributed to its dissolution in water or oil, which is later discarded, when the cooking method is boiling or frying, respectively [30]. However, the effect of heat alone, such as that produced during baking or toasting, does not seem to be very negative [27], although Zn values in baked food products have been found, in general, to contain less Zn that the raw material [30]. The reasons for this loss of Zn caused by heat are poorly understood, and further studies should be required to verify, for instance, if changes in the Zn chemical form might be produced by exposure to high temperatures, which could explain the losses by volatilization, as in the case of Se. When white flour was used instead, Se and Zn losses were ≈29% and ≈61%, respectively. Both percentages were quite similar to those obtained by the sum of the loss due to milling and that of baking (=37% for Se, and =56% for Zn), suggesting a simple additive effect of both processes during white bread preparation.

Conclusions
The combined biofortification of bread-making wheat with Zn and Se was quite effective in boosting their concentration in wheat-derived products, such as white flour, wholemeal bread and white bread. Zn s bioavailability was also clearly favored by the combined biofortification. The combined application was beneficial in comparison with the application of either Zn or Se alone, as a synergic effect was observed between both micronutrients, especially for the increase in the Se concentration. The rest of the quality parameters analyzed, such as crude protein, dry gluten, white flour yield and mineral status (Mg, Ca and Fe), remained unaltered or very slightly affected after biofortification. Milling was the process where the largest loss of Zn and Se was produced. The consumption of wholemeal bread instead of white bread may enhance Zn and Se intake in the general population more considerably than biofortification. Nevertheless, although consumption habits should be changed, in the meantime, the biofortification of wheat may help to mitigate deficient Zn and Se intake. Funding: This research was funded by the Regional Government of Extremadura (Spain) and by the European Regional Development Fund (ERDF), grant number IB16093.

Institutional Review Board Statement: Not applicable.
Data Availability Statement: Not applicable.