A Case Study on Minerals Accumulation in Grains and Flours of Bread Wheat Fertilized with ZnSO₄ and Tecnifol Zinc ^†

Abstract

In the present day, there is an increasing demand for foods capable of fulfilling the nutritional needs of consumers, leading to a search for food products with a nutrient content able to promote a healthier lifestyle. In this study, an agronomic biofortification itinerary of Triticum aestivum L. (cv. Paiva) was conducted in an experimental field, located in Beja, Portugal, comprising foliar fertilization with ZnSO₄ and Tecnifol Zinc in three different concentrations for each fertilizer along the plant cycle. The mineral quantification (S, K, Ca, and Zn) of whole bread wheat flour and refined bread wheat flour was measured using an X-ray fluorescence analyzer (XRF analyzer), whereas the micro-energy dispersive X-ray fluorescence system (μ-EDXRF) was used to quantify the minerals within the different regions of the wheat grain (embryo, endosperm, and vascular bundle). All the minerals presented lower values in the refined flour relative to the whole bread wheat flour, in which K had higher values followed by S and finally Ca with the lower values in both types of flour. The different minerals were spread around the various regions of the grain; however, they were more concentrated in the embryo and vascular bundle. The values are similar for both fertilizers, with a slight difference regarding Zn values, namely increasing with ZnSO₄. To sum up, as the different minerals tend to accumulate in the embryo and vascular bundle, the whole bread wheat flour presents the richest option, promoting a healthier diet for consumers.

1. Introduction

The global population, in 2030, is expected to grow to between 8.5–8.6 billion; in 2050 it will reach from 9.4 to 10.1 and, at last, the population is expected to reach between 9.4 and 12.7 billion people in 2100, accentuating the cleavage between developing and developed countries [1,2]. Between 2019 and 2050, such a demographic increase will find more than half of this exponential growth of the world population in sub-Saharan African countries. Conversely, other regions such as East and Southeast Asia, Central and South Asia, Latin America and the Caribbean, and Europe and North America are projected to peak before the end of the existing century, but these values will start declining [1]. This entails an increase in food production, especially staple crops like bread wheat. Despite the predominance of such a staple crop in 2019/2020, it reached a production of about 760 million tons, in 2020/2021 increased to 776.5, and in 2021/2022 is being estimated, approximately, to reach 769.6 million tons [3]. Currently, there is an increasing demand for foods likely to meet the nutritional needs of consumers, fostering the search for food products with a nutrient content that may contribute to a healthier lifestyle. Human health will experience myriad benefits from the consumption of whole wheat as it contains phytochemicals (flavonoids, carotenoids, polyphenols, phenolic acids, and others), dietary fibers, and vitamins and minerals. These beneficial factors help reduce obesity, cancer, cardiovascular disease, and type II diabetes. What distinguishes whole wheat flour from refined wheat flour is the fact that the former keeps the outer layer of the kernel where the above-mentioned components are more concentrated [4,5]. Sulfur (S) is important for human beings as it is part of the amino acids methionine and cysteine, as also coenzymes and cofactors [6]. The microelement potassium (K) is linked with the nervous system, playing an important role in the maintenance of intracellular osmolality [7]. Calcium (Ca) is essential to mineral homeostasis, operates in the cardiac, nervous, and musculoskeletal systems, and acts as a cofactor [8]. Zinc (Zn) plays a key role at regulatory, functional, and structural levels in the human body and interacts with various proteins and enzymes [9]. This study aims to compare the chemical composition (S, K, Ca, and Zn) of whole and refined bread wheat flour and the accumulation of minerals when applying the fertilizers ZnSO₄ and Tecnifol Zinc.

2. Materials and Methods

2.1. Experimental Field

An experimental field at 38°01′52.38″ N; 7°52′53.72″ W, in Beja (Portugal) was chosen to cultivate the Triticum aestivum L. Paiva variety. Although the last days of December 2018 saw the sowing of the bread wheat field, the harvest season fell by the end of June 2019. The sowing process was implemented according to a randomized block design comprising four repetitions. This field has been divided into 48 plots (24 plots for each one of both fertilizers), each one with an area of 9.6 m² (8 m × 1.2 m), comprising 2 m between repetitions and 0.4 m between plots. An NPK fertilization and 50 kg Zn·ha⁻¹ were applied in the field beforehand. The Zn biofortification encompassed the use of ZnSO₄ and Tecnifol Zinc foliar spraying at booting, heading, and milk stages (this final stage only for Tecnifol Zinc) in late April and May 2019, with three different concentrations applied (0—control (T0), 8.1 (T1), and 18.2 (T2) kg·ha⁻¹ for ZnSO₄; and 0—control (T0), 1.3 (T1), and 2.6 (T2) kg·ha⁻¹) and 46% of urea. Considering the plant life cycle, the total rainfall accumulation was about 5.43 mm, with a daily maximum of 1.85 mm; the average maximum and minimum temperatures were 22 °C and 11 °C, respectively, (with a maximum temperature of 39 °C and a minimum of 0 °C).

2.2. Mineral Quantification of Whole and Refined Flours by XRF and Wheat Grains by µ-EDXRF

To obtain the whole wheat flour we milled the grains using the Disintegrator MPD-102 Biobase Biodustry Co., Ltd. (Shandong, China) mill. Additionally, in order to obtain refined flour, the grains were milled by a Chopin CD1 mill (Group Tripette & Renaud, Asnières-sur-Seine, France).

To determine the mineral content of whole and refined wheat flours, under a helium atmosphere an X-Ray Fluorescence analyzer (XRF analyzer) (model XL3t 950 He GOLDD +) was used [10].

To determine zinc location in grain tissues collected at harvest, we used the micro-energy dispersive X-ray fluorescence system (μ-EDXRF) (M4 Tornado™, Bruker, Germany) according to [11]. We should mention that the grains were cut in half longitudinally along the crease tissue, Zn being the target of the study and not selenium.

2.3. Statistical Analyses

The statistical analyses of the data were carried out using software R (version 3.6.3). Statistical analyses included one-way and two-way ANOVA (p ≤ 0.05) to assess significant differences. Based on the results, a Tukey’s test for mean comparison was performed, considering a 95% confidence level.

3. Results

In a nutshell, when considering the different samples (whether from different minerals, types of flours, and treatments for both fertilizers) we may say that significant differences were observed (Figure 1). The macrominerals present values in which K has the higher values, followed by S, and at last, Ca. For all the minerals the refined wheat flour showed lower values than whole wheat flour, irrespective of the fertilizer applied. It is verified that the P0Sw sample is about nine-fold larger than the P0Sr sample for K. Relative to the whole wheat flour, for both fertilizers, in the macrominerals the values diminish from the left to the right in the figures, revealing that the fertilizer Tecnifol Zinc presents lower values than ZnSO₄. In other words, the greater the amount of fertilizer applied, the bigger the decrease concerning the macro element, for both fertilizers. Considering Zn, when applying both fertilizers it is observed that as the concentration of applied fertilizer increases, the value of Zn in whole flour (as well as in refined flour) also increases. Relative to the refined flour, just for ZnSO₄ fertilizer, the values of the macrominerals increase when applying higher concentrations of the fertilizer. What is more, for the same type of flour, when applying Tecnifol Zinc, the value of the macrominerals shows that the values rise between the control and the intermediate concentration of fertilizer and then drop from the intermediate concentration to the highest concentration (nevertheless, the values of the upper concentration are higher than the control).

On the whole, when compared to the other zones of the grain, there is a greater amount of the minerals S (apart from sample O), K (apart from sample F), and Zn (apart from samples F and I) in the embryo (

Table 1. Values of S, K, Ca, and Zn contents; equipment error of Triticum aestivum L. (cv. Paiva) grains when applying the fertilizers ZnSO₄ (T0—control, T1—8.1, and T2—18.2 kg·ha⁻¹) and Tecnifol Zinc (T0—control, T1—1.3, and T2—2.6 kg·ha⁻¹). The grain quantification was divided into three zones of the grain (embryo; endosperm; vascular bundle).

Fertilizer	Treatment	Zone	Code	S	K	Ca	Zn
ZnSO4	T0	Embryo	A	4814; 241	26281; 1213	3166; 158	225.4; 11.3
		Endosperm	B	1894; 94.7	1521; 76	235; 11.7	23.69; 1.18
		Vascular bundle	C	3974; 199	12863; 643	2042; 102	90.15; 4.51
	T1	Embryo	D	3959; 198	16708; 835	2293; 115	171.2; 8.56
		Endosperm	E	2173; 109	1612; 80.6	442.9; 22.1	37.23; 1.86
		Vascular bundle	F	3310; 165	17509; 875	2851; 143	228.2; 11.4
	T2	Embryo	G	5515; 276	23159; 1158	2137; 107	255.9; 12.8
		Endosperm	H	2409; 120	2341; 117	364.0; 18.2	57.71; 2.89
		Vascular bundle	I	4490; 224	17550; 878	3615; 181	450.7; 22.5
Tecnifol Zinc	T0	Embryo	J	4013; 201	17598; 880	2053; 103	154.4; 7.72
		Endosperm	K	2085; 104	1843; 92.1	292.7; 14.6	23.97; 1.20
		Vascular bundle	L	3824; 191	16228; 811	3263; 163	93.58; 4.68
	T1	Embryo	M	4247; 212	17950; 898	1774; 88.7	242.5; 12.1
		Endosperm	N	1618; 80.9	1178; 58.9	199.2; 10	26.16; 1.31
		Vascular bundle	O	5114; 256	16566; 828	2335; 117	211.2; 10.6
	T2	Embryo	P	4190; 209	26501; 1325	3566; 178	373.3; 18.7
		Endosperm	Q	2174; 109	2100; 105	295.8; 14.8	47.27; 2.36
		Vascular bundle	R	3191; 160	14235; 712	2090; 105	327.2; 16.4

). However, Ca is predominant in the vascular bundle, excluding samples A and P. Regarding the predominance of each of the minerals in each of the zones, it appears that for the embryo and for the vascular bundle there is a lower prevalence of Zn, followed by Ca, S, and finally, K. When comparing the same concentration of the two different fertilizers, from the same zone of the grain it appears that ZnSO₄ presented higher values than Tecnifol Zinc for the four minerals for T0—embryo, T1—endosperm, T1—vascular bundle, and T2—vascular bundle and presented lower values than Tecnifol Zinc for the four minerals of T1—embryo. Moreover, ZnSO₄ revealed lower values of Tecnifol Zinc for T2—embryo (except S), T0—vascular bundle (not including S), and T0—endosperm (only for S, K, and Zn). Finally, Tecnifol Zinc showed lower values than ZnSO₄ for T2—endosperm for the minerals K, Ca, and Zn. Regarding the comparison between the different concentrations of fertilizers (but for the same fertilizer), for the same mineral in the same grain zone, a relation was found: T0 < T1 < T2 (for ZnSO₄, for the minerals S (endosperm), K (endosperm and vascular bundle), Ca (vascular bundle), and Zn (endosperm and vascular bundle); for Tecnifol Zinc, for the minerals K (embryo) and Zn); T0 < T2 < T1 (for ZnSO₄, only the mineral Ca (endosperm); for Tecnifol Zinc, for the mineral S (embryo); T1 < T0 < T2 (for ZnSO₄, for the minerals S (embryo and vascular bundle) and Zn (embryo); for Tecnifol Zinc, for the minerals S (endosperm), K (endosperm), Ca (embryo and endosperm) and Zn); T1 < T2 < T0 (only the embryo, regarding the mineral K, when applying ZnSO₄); T2 < T0 < T1 (for Tecnifol Zinc, for the mineral S (vascular bundle)); T2 < T1 < T0 (for ZnSO₄, for the mineral Ca (embryo); for Tecnifol Zinc, for the mineral Ca (vascular bundle)).

4. Discussion

The discrepancy between the amount of the same mineral for whole and refined wheat flours, regardless of the fertilizer applied, in which the whole flour presented higher values, is related to the different industrial processing of the flours. In this way, refined flour is obtained by removing the outermost zones of the grain, where the minerals are more concentrated, being in line with the data obtained by the quantification of the grains [12]. As a result, refined wheat flours are less suitable for a healthier diet. According to some studies focused on bread wheat biofortified with ZnSO₄, S, K, Ca, and Zn are preferably located in the embryo (S) and the vascular bundle (K, Ca, Zn) [13]. However, when comparing 11 different bread wheat varieties, in 7 of them Zn was more accumulated in the embryo, followed by the vascular bundle [14]. This finding agrees with the data obtained for S and Zn, which are preferably located in the embryo, and for Ca (predominant in the vascular bundle). Our data on the predominance of K, S, Ca, and Zn in the different flour and grains (despite the zone) is supported by the studies of [13]. In general, when comparing both fertilizers, Tecnifol Zinc was the least effective. Foliar spraying of both fertilizers enhanced the Zn content in the flours according to [15]. The decreasing trend of S and Ca minerals with the increasing Zn concentration agrees with the literature [16].

5. Conclusions

Considering flours, macrominerals present values in which K has the highest values, followed by S, and finally Ca. Regardless of the fertilizer applied, the refined wheat flour showed lower values than whole wheat flour, for all the minerals. Relative to the whole wheat flour, for both fertilizers, in macrominerals the values diminished as the fertilizer concentrations increased. The values of whole wheat flour proved that ZnSO₄ was the fertilizer presenting higher values when compared with Tecnifol Zinc. Zinc showed that as the concentration of the applied fertilizer increases, the value of this mineral in the flours also increases when applying both fertilizers. In general, the minerals S, K, and Zn are predominant in the embryo, nevertheless, Ca is predominant in the vascular bundle. Regarding the predominance of each mineral in each zone, and, in descending order, it appears that for the three zones there is a lower prevalence of Zn, Ca, S, and finally K (only for embryo and vascular bundle). When comparing the same concentration of the two different fertilizers, focusing on the same section of the grain, it appears that ZnSO₄ presented higher values than Tecnifol Zinc for the four minerals, by and large. To sum up, as the different minerals tend to accumulate in the embryo and vascular bundle of the grain, and whole bread wheat flour includes these zones, this flour constitutes a healthier choice for consumers.