1. Introduction
Vitamins and minerals are a group of compounds essential for the proper functioning of the body, requiring a balanced and proper diet in order to ensure adequate concentrations in the body [
1]. Studies show that in recent decades a diet rich in fruit, vegetables, meats, and whole grains that provide the body with the necessary vitamins and minerals for the proper function has been gradually replaced by a diet poor in micronutrients consisting mainly of processed meats, fats, processed grains and sugars [
2]. In Romania, according to the National Study on the Prevalence of Diabetes, Prediabetes, Overweight, Obesity, Dyslipidemia, Hyperuricemia and Chronic Kidney Disease (PREDATORR), 31.4% of adults aged 20–79 suffer from obesity and 34.6% are overweight [
3]. According to international studies, there is great concern because obesity is associated with an increased risk of metabolic, cardiovascular, and renal diseases, with childhood obesity increasing tenfold worldwide in the last four decades [
4,
5].
The daily requirement of vitamin and mineral intake depends on several parameters, such as gender, ethnicity, medication, drug and alcohol consumption, age, physical activity, and certain pathologies [
5,
6]. Deficiencies in vitamins and minerals resulting from inadequate dietary intake have been linked to neurological, cardiovascular, renal, and hormonal disorders, incurring additional costs to healthcare systems, and thus emerging as an important public health issue [
7,
8,
9].
Cereals, comprising staples such as wheat, rye, oat, and soybeans, represent a foundational component of global diets, and serve as primary sources of essential vitamins and minerals. Rich in carbohydrates, fiber, and plant-based proteins, cereals contribute significantly to daily nutritional intake [
10]. The productivity, quality, and composition of cereals are intrinsically linked to numerous factors, including the geographical position of crops—encompassing specific relief and climate considerations [
11]; soil type and its natural composition, including mineral content and enzymology [
12]; practices related to soil management, encompassing crop-specific agricultural tasks, crop rotation, irrigation, classical and modern fertilization treatments [
13,
14,
15,
16], and short/long-term soil monitoring [
11].
These grains also house a diverse array of vital micronutrients, including various B-group vitamins, iron, zinc, magnesium, potassium, and phenolic compounds [
17]. Phytic acid plays a crucial role, influencing the levels of minerals in cereals and the bioavailability of these minerals in the human body by forming insoluble salts (phytates) with magnesium, calcium, and iron, hindering their absorption [
18].
Antioxidant compounds in cereals, predominantly phenolic compounds, play a vital role in combating various ailments, including cancer, diabetes, and cardiovascular diseases. Wheat bran phenolic compounds exhibit proven antimicrobial properties against several pathogens [
19] and anti-inflammatory action in liver conditions by inhibiting cytokine production [
20].
The dynamic of vitamin and mineral levels in cereals undergoes a significant transformation before and after processing. The processing of cereals, such as heat treatment, milling, and other technological steps, can have a profound impact on the nutritional composition of the final product. Studies investigating these changes have revealed that, despite efforts to optimize nutrient content, cereals, particularly after heat processing, may exhibit alterations in vitamin and mineral levels. Heat treatments, such as baking or cooking, can lead to a reduction in the concentrations of certain vitamins and minerals, potentially affecting their bioavailability [
21,
22]. The enhancement of vitamin and mineral levels in bread has been achieved by incorporating rice flour, increasing the quantities of zinc, iron, potassium, phosphorus, and niacin by 10% [
23]. Improving the nutritional quality of bread remains a priority, with studies exploring the impact of processes such as boiling, irradiation, decortication, fermentation, germination, and storage on vitamin and mineral content [
24].
Considering that vitamins and minerals are essential for the optimal functioning of the entire body and that cereals are at the bottom of the food pyramid, being the most consumed food, the aim of this study was to determine the contents and levels of several vitamins and minerals before and after temperature processing in cereals harvested from a certain area in Western Romania. The importance of the study lies in the fact that cereals, and therefore bread, are some of the most consumed food products worldwide, and play an important part in human nutrition. Thus, the contents of minerals and vitamins and the dynamics of their concentration are crucial in order to achieve a balanced diet and nutritional state.
3. Materials and Methods
3.1. Cereal Selection
The studied cereals were selected based on the types of cereals cultivated in the area of Săcuieni town, Bihor County, Romania, situated at 47.35° latitude and 22.1° longitude. The soil in the experimental area is slightly acidic, with a pH between 5.95 and 6.40, low humus concentration, a low total nitrogen content of 0.0075, and low and very low mobile phosphorus and potassium contents, of 12 and 60 ppm, respectively.
Five types of flour were included in our study, namely, whole wheat, hulled wheat, rye, soy, and oat. This has mainly been done because different cereals exhibit distinct nutritional profiles, containing varying concentrations of vitamins and minerals. The cereal samples were obtained from crops grown in 2022 in a conventional system on a sandy, medium-coarse-textured soil (psalmosol class). All grains (wheat, rye, soybean, and oats) were milled using an electric robot. The wheat was hulled using a dislocator (wheat finisher). A part of the resulting flour was analyzed as such, and the other part was used to obtain a dough with a soft consistency. The dough was prepared from 100 g flour and 60 g distilled water, and was subjected to a temperature of 220 °C for 30 min in an electric oven. The properties of the samples are shown in
Table 12.
Determinations were performed on the flour samples obtained from cereals, and these results were correlated with the bread samples obtained from the same cereals after the flour was transformed into dough and then subjected to heat processing.
The results obtained were analyzed comparatively to determine the effects of heat treatment on mineral content. Since the aim of the study was not to produce a product with a high acceptance among the population, further studies on sensory analysis, the rheological parameters of the dough, and the physicochemical and textural properties of the obtained preparation were not deemed necessary.
3.2. Chemicals and Reagents
Methanol (≥99.8%) and acetic acid (≥99.8%) used for high-performance liquid chromatography (HPLC) were purchased from VWR Chemicals (Solon, OH, USA). All other standards (Thiamine hydrochloride, Riboflavin, Niacinamide, Pyridoxine hydrochloride, Cyanocobalamin) used were from Sigma-Aldrich (Steinheim am Albuch, Germany). HNO3 65%, H2O2 30% and ICP multi-element standard solution IV, 1000 mg/L, were purchased from Merck (Darmstadt, Germany). Ultrapure water from an Ultraclear Evoqua purification system (Erlanger, Kentucky, AL, USA) was used to prepare the standard solutions and dilute the samples. All solvents were HPLC grade from VWR, and the ultra-pure water was obtained using the ULTRACLEAR UV UF EVOQUA Purification system, Pittsburg, PA, USA. We used the ACW Kit from Analitk Jena (Jena, Germany). All other standards used were from Sigma-Aldrich.
3.3. Determination of Vitamins B
The samples were further processed by extracting 0.250 g of sample with 1 mL MeOH. The samples were centrifuged using a Hettich D-78532 microcentrifuge (Tuttlingen, Germany) at 11,000 rpm for 2 min; the supernatant was filtered through a 0.45 µm cellulose filter and then analyzed by UHPLC Vanquisher H Dionex (Thermo Fisher Scientific, Dreieich, Germany) with a DAD detector for the analysis of thiamine (B1), riboflavin (B2), nicotinamide (B3), pyridoxine (B6) and cyanocobalamin (B12). The mobile phase was composed of ultra-pure H2O with 1% acetic acid and MeOH in gradient with a flow of 0.3 mL/min. The chromatographic column used was a AccucoreaQ 100 × 2.1 mm, 2.6 μm (Thermo Fisher, Waltham, MA, USA), kept at 25 °C. The injection volume was 8 µL and the detector was set at 270 nm.
3.4. Mineral Profile
The minerals (K, Ca, Mg, Fe, Cu and Zn) were determined using an inductively coupled plasma optical emission spectrometer Perkin Elmer Optima 5300DV (ICP-OES) (Waltham, MA, USA) after microwave-assisted digestion using a BerghofXpert system (Eningen, Germany). An amount of 500 mg of the sample was digested using 8 mL HNO3 65% and 2 mL H2O2 30% in a polytetrafluoroethylene digestion vessels, using a four-step digestion program (140, 170 and 190 °C—heating; 50 °C—cooling) for a total digestion time of 40 min. Afterward, the vessels were cooled down and the volume was made up with ultrapure water. Blanks were prepared in each lot of samples.
3.5. Moisture Content
The moisture contents of the flour and bread samples were determined using the AOAC’s official method 925.10 by drying at 105 °C until the resulting weight was constant [
26].
3.6. Determination of Total Phenolic Content
Then, 5 mL of distilled water, 1.5 mL of sodium carbonate solution (10%), 0.5 mL of extracted sample and 0.5 mL of Folin–Ciocalteu solution where pipette into a 15 mL centrifuge vail. After the samples were kept for 45 min at room temperature in the dark, they were measured at a wavelength of 765 nm (Spectrum BX II, Perkin Elmer, USA). The results have been expressed in gallic acid equivalent. The samples were created in triplicate [
27].
3.7. Determination of Total Antioxidant Capacity
After methanol extraction, the samples were directly injected into PHOTOCHEM (Analytik Jena, Germany) and the antioxidant capacity was measured suing the ACW kit and expressed in equivalent ascorbic acid. The samples were made in triplicate.
3.8. Determination of Vitamin D3 (Cholecalciferol), Vitamin A (Retinyl Acetate), Vitamin K (MK4, MK7)
Here, 0.250 g samples were extracted with 1 mL MeOH. The samples were centrifuged (Microcentrifuge Hettich D-78532, Germany) at 11,000 rpm for 2 min; the supernatant was filtered through a 0.45 µm cellulose filter and then analyzed by UHPLC Vanquisher H from Dionex, Thermo Fisher Scientific, Germany, with a DAD detector for the analysis of Cholecalciferol, Retinyl Acetate, MK4, MK7. The mobile phase was composed of ultrapure water (A) and methanol (B). The isocratic elution was performed at 1 mL/min in a proportion of 98% B. The chromatographic column used was a Acclaim C30 150 × 46 µm, 5 µm (Thermo Scientific, Sunnyvile, CA, USA), kept at 30 °C. The injection volume was 10 µL and the detector was set at 265 nm [
28].
3.9. Statistical Analysis
The design of experiment (DOE) aimed to compare the levels of different cations in five different cereals (P1–P5). Thus, two factors were considered: the raw factor and the baked factor. Univariated statistical analysis consisted of two-way analysis of variance (ANOVA, p = 0.05) with a post-hoc multiple pairwise sample mean comparison test, established by Dunn–Sidak, with a confidence interval of 95% (p = 0.05).
Multivariate statistical analysis considered as samples the interaction factor when raw and baked. The variables consisted of the levels of different cations in five different cereals (P1–P5).
The multivariate approach included several methods: principal component analysis (PCA), linear discriminant analysis (LDA), multivariate ANOVA MANOVA (
p = 0.05), and hierarchical cluster analysis (HCA). All these methods enable comparisons between multivariate data that consist of samples’ multivariate profiles. Each multivariate profile sequentially gathers all the parameter values for the sample. The multivariate analysis was performed and graphically designed with a custom-made application based on standardized procedures from MATLAB 2022b CWL (The MathWorks Inc., Natick, MA, USA) [
24].
4. Discussion
According to the 2020 Global Nutrition Report, one-third of the population is obese or overweight. Bread is one of the most widely consumed foods, high in carbohydrates and fats, but unfortunately low in vitamins and minerals [
5].
Wheat is one of the most widely used cereals in the diet, being an important source of vitamins and minerals and containing little fat. After wheat grain is processed into flour, its nutrient content changes through fermentation and heat treatment in the process of producing various bakery products. After the hulling of wheat, white flour is produced, which is lower in nutrients due to the refining process [
29]. A constant concern, especially in developed countries, is to improve the quality of bread and cereal foods in general so as to ensure a healthy diet [
30]. This phenomenon has been ever more evident during the past few years, when the general interest in plant-based diets has increased substantially [
31]. The consumption of whole grain products is probably lower also due to their organoleptic properties; whole grain products being associated with lower acceptability [
32]. In European countries, wheat is the staple food crop, providing up to 50% of the total energy intake, with other cereals and beans following behind [
33]. The concentration of iron and zinc in the grain is directly influenced by the minerals available in the soil, and these minerals are found in the embryo and aleurone of the wheat grain, along with the B vitamins, calcium, and magnesium. The embryo and the aleurone are largely removed during hulling, which is why these minerals are present to a lesser extent in white flour [
29,
34].
Existing research has also confirmed the decrease in vitamin and mineral contents under the thermic treatment of cereals and legumes [
35]. Thus, our findings were similar to those obtained by Barantama and Simard, who recorded a significant decrease in minerals and vitamins after processing common beans [
36]. Additionally, studies have identified that some cooking methods involving temperature do not affect the content of Fe to such great extent as the concentrations of other minerals [
37,
38], and this aspect has also been confirmed by our findings, with the lowest impact of temperature being on Fe concentration, across all samples, with oat and soybean flours still remaining above average Fe concentrations. However, our analysis yielded different results when compared to some other studies, with Hemalatha et al. not recording any difference in the bioavailability of Zn in wheat after heat treatment [
37].
Nevertheless, different combinations of wheat flour and flour from other cereals can be used to improve bread quality. Thus, studies show an increase in the levels of K, Mg, Na, Zn, Fe and polyphenols when combined with quinoa flour [
39]. The quality of the bread was also improved by the addition of chickpea flour, which resulted in increased levels of Na, Mg, Fe and Zn, as well as phenolic compounds and flavonoids with antioxidant activity [
40]. Furthermore, better antioxidant properties were obtained when combining amaranth with corn flour, while adding millet to corn flour yielded increased levels of Ca, Fe, Zn and fibers [
41,
42]. Higher protein levels were achieved by combining wheat flour with lentil flower or
Moringa olefeira leaf powder, with the latter also increasing the levels of polyphenols and minerals, such as Ca, Zn and Fe [
43,
44].
Flour fortification is a crucial public health strategy aimed at enhancing the nutritional value of flour by adding essential micronutrients, such as vitamins and minerals. Typically, iron, folic acid, and various B vitamins, including thiamine, riboflavin, and niacin, are commonly incorporated into flour during the fortification process [
45]. Thus, in Mexico, commercial wheat flour is fortified with folic acid, Fe and Zn, increasing the antioxidant capacity [
46].
All the analyzed samples are present in the diet after heat processing. In order to limit the unfavorable influence of temperature on bioactive compounds and antioxidant phenolic acids, as well as to preserve endogenous nutrients and reduce the production of toxic reaction compounds, Tian et al. thermally processed wheat flour to produce bread and pancakes at temperatures below 100 °C [
47]. The decrease in antioxidant capacity after heat processing was also demonstrated by Cammerata et al., who conducted studies on raw and boiled pasta obtained from wheat species [
48]. Analyses of vitamin and mineral content and antioxidant capacity were also conducted by Furuichi et al. on various barley species. However, they did not determine the levels of these compounds after thermal processing in various food preparations [
49].
Considering the differences in the quantities of vitamins and minerals in cereal flour (expressed per 100 g of the sample) and various types of food products (expressed per 100 g of the sample/bread) obtained after heat processing, we compared the two categories. This highlighted the fact that the population’s diet is deficient in vitamins, minerals, and antioxidants necessary for the optimal functioning of the body. These differences are also due to the fact that 100 g of flour does not yield 100 g of bread. The products most widely consumed by the population are those obtained from hulled wheat, which is low in these components.
The observed variations in mineral content and nutrient concentrations between raw and baked samples underscore the multifaceted nature of food processing and its impact on nutritional composition. Several factors contribute to these differences, including the effects of heat treatment, moisture loss, and biochemical transformations during baking. Heat exposure can lead to the degradation or loss of certain nutrients, particularly heat-sensitive vitamins and antioxidants, thereby influencing the overall nutrient profile of the baked samples. Additionally, moisture loss results in a concentration effect, leading to higher nutrient concentrations per unit dry weight. This phenomenon was better observed when assessing the moisture content of the samples and reporting the results of the measurements in the dry weight system, as increases in the concentration of certain minerals, such as K, were recorded.
Although the moisture content differs between flour samples compared to bread samples, and the results were reported per 100 g of product, both cereal-derived flour and bread are deficient in terms of mineral and vitamin content, as well as antioxidant capacity, relative to the daily requirements of the population. This is concerning as it predisposes the population at all ages to the development of various pathologies.
Phenolic compounds are mainly found in the bran and germ, which are removed during the refining process [
50,
51]. Polyphenolic compounds have been proven beneficial in various gastrointestinal diseases, by promoting the growth of beneficial gut microbiota, as well as interacting with macromolecules and having important antioxidative properties [
49]. After exposure to temperature, the polyphenol concentration also decreases, while the biofortification of rye plants by applying potassium iodide to the soil resulted in a significant increase in hydrophilic and lipophilic antioxidants (glutathione, ascorbic acid, phenolic compounds) [
52,
53,
54].
Additionally, the vitamin content is susceptible to temperature dynamics as well, with the concentration of B-vitamins being more stable in whole wheat flour than in white flour. More precisely, B1 and B6 vitamins were more susceptible to temperature changes, and their concentrations decreased significantly after the baking process, while the B2 concentration was more stable [
55,
56]. This effect has largely been shown in conventional baking processes. Nevertheless, due to the low concentrations of vitamins even in the raw samples, measuring the concentrations in the baked samples was not deemed necessary.
The fortification of wheat flour with minerals would lead to an increase in the nutritional value of the resulting dishes, and of bread in particular. This can also be achieved by adding flour obtained from mixing wheat flour with flour from various plants that have a high mineral content. Current cereal breeding programs are mainly focused on higher yields and higher technological quality for industrial processing, neglecting health and nutritional aspects, including possible allergenic factors that may emanate from substances used to increase productivity and control pests [
57,
58,
59,
60].
Moreover, the mineral composition of cereals is closely correlated with the chemical composition of the soil. As mentioned before, the soil in the experimental area is moderately acidic and with a low K concentration, which causes a lower assimilation of minerals in plants, mainly due to increased Al toxicity and impaired root growth and stress responses [
61,
62]. The organic matter consists of low levels of humus, and the low nitrogen and phosphorus contents also correlate with slowed plant growth, being strongly influenced by farming methods, with conventional ones being less harmful to the soil and environment, compared with the modern ones [
63,
64,
65].
The low vitamin and mineral content of cereals is due to soil depletion, which is also observed in organically grown cereals where no pesticides or other chemicals are used. Diets are based mainly on processed foods from crops grown in mineral-poor soils. To increase the yield of cereals, the amount of carbohydrates is increased, and the amount of minerals and proteins is decreased. Additionally, to increase yields, but also as a result of the use of chemical fertilizers and pesticides, modern crops are harvested faster. Therefore, cereals have less time to absorb nutrients from the soil. Monoculture farming practices have also led to soil depletion, which directly affects the mineral content. Industrial development, including deforestation and exploitation, also contributes significantly to soil depletion by removing the soil layer containing the minerals needed by crops [
66,
67].
Consumer preferences for refined, nutrient-poor foods have led to an increase in non-communicable chronic diseases (diabetes, obesity, cardiovascular diseases). In Romania, white bread represents the most widely consumed food. Considering the low values compared to daily requirements for minerals and vitamins, several researchers have studied ways to increase these values, obtaining qualitatively superior bakery products in terms of these compounds. Depending on the chemical composition of the flour obtained from cereals, the dough and consequently the bread will have different characteristics (water absorption, protein and starch quality, amylase activity) [
68].
The water retention capacity in bread after baking is over 50%, depending on the types of cereals used. Since this parameter is influenced by other factors (time, temperature, and equipment used in kneading, aspects related to the fermentation process, temperature, and humidity during baking) [
69], we chose to report the results obtained after baking the dough, also per 100 g of fresh weight of the product, similar to the results obtained from the analysis of the flour from which the heat-treated dough was obtained. Taking into account the moisture retained in bread and the fact that the final results were reported per 100 g of the fresh product, the latter demonstrated a lower content of minerals compared to the flour data. These values are consistent with the results obtained by other researchers, showing a reduction in mineral levels after baking the dough. For example, in the case of Mg, the decrease is from 47.73 mg per 100 g of wheat flour to 42.16 mg per 100 g of bread, representing a decrease of 11.67% [
70].
Food safety and environmental issues are important factors of increasing concern to people in both developed and developing countries. Cereals are the most widely consumed food in the world, and therefore should be an inexpensive and rich source of vitamins and minerals to reduce the incidence of chronic non-communicable diseases faced by a large part of the population regardless of age [
71,
72].
The limiting factor of this study is the lack of a detailed analysis of the consumption of each cereal type in terms of age, education, and presence or absence of chronic nutritional conditions. In view of the results obtained, it would be advisable to conduct a more comprehensive study of a larger area of cereal, vegetable and fruit crops throughout the country that should include the determination of the vitamin and mineral content of these foods consumed by the entire population, given that a deficiency of vitamins and minerals in the diet leads to various chronic diseases in the whole body, regardless of age. Nevertheless, to our knowledge, the existing evidence of the dynamic of the concentrations of various minerals and vitamins in cereals and legumes under temperature treatment is rather limited. Thus, we consider this research as an important foundation for further studies analyzing the matter.