The Effect of Selected Winter Wheat Cultivars and the Growing Season on the Antioxidant Activity, Polyphenol Profile, and Organoleptic Assessment of Beers Produced from Them

Abstract

Wheat cultivars significantly affect the quality of grain and then malt, which are used in the production of wheat beers, determining their potential use in brewing. Nowadays, consumers are increasingly choosing products with high biological activity, even for alcoholic beverages. Furthermore, organoleptic testing is crucial, as despite their high physicochemical parameters, wheat beers may not be met with consumer acceptance. The antioxidant activity and total polyphenol content of the obtained wheat beers were determined and identified, and organoleptic analysis was performed. Wheat beers obtained from malt derived from the Elixer cultivar of wheat were characterized by the highest polyphenol profile values (3.01 mg·L⁻¹) and antioxidant activity determined by the ABTS method (0.72 mg TE·L⁻¹). The Lawina cultivar also had a high ABTS value (0.73 mg TE·L⁻¹). Total polyphenol content (89.07 mg·L⁻¹), antioxidant activity (DPPH; 1.08 mg TE·L⁻¹), antiradical activity (FRAP; 1.58 mg Fe²⁺·L⁻¹), and organoleptic profile were highest in wheat beers obtained from malt derived from the Gimantis cultivar of wheat. Based on the conducted research, it was found that beers obtained from Gimantis wheat are characterized by the best quality parameters, including antioxidant activity. The Gimantis cultivar showed the least variation in quality of wheat beers in terms of the growing season and is recommended as a wheat cultivar for the brewing industry. It is also recommended to conduct further research on beer products obtained from the analyzed winter wheat varieties, including the antioxidant potential of finished non-alcoholic products.

1. Introduction

Malt and hops are the basic raw materials from which biologically active compounds with antioxidant properties are extracted, including polyphenols, vitamins, fiber, and bitter acids found in wheat beers [1,2]. Antioxidant compounds play many roles in beers, including those responsible for flavor changes. Physical, chemical, and biological factors such as light intensity, pH changes, oxygen levels, and yeast load influence the flavor [3]. Free radicals found in the body, whose task is to damage proteins, DNA structure, or lipids, are captured by biologically active chemical compounds derived from beer. They can also chelate transition metals, limiting their availability. Antioxidants have a beneficial effect on the human body, preventing lifestyle diseases, including coronary heart disease or cancer [4]. The antioxidant compounds contained in beer increase bone density and improve the immunological properties of the human body. Although beer contains antioxidant compounds, the amount of these is low compared to that of fruits like chokeberries, blueberries, raspberries, strawberries, and blueberries. Wheat beer contains an average of 120–150 mg·L⁻¹ of antioxidant and phenolic compounds, significantly lower than that of some common vegetables, fruits, and nuts (apple 200–1000 mg·100 g⁻¹, flaxseed 1530 mg·100 g⁻¹, hazelnuts 500 mg·100 g⁻¹, etc.). However, it should not be forgotten that beers contain ethyl alcohol, which may contribute to the development of alcoholism. Despite the health benefits of beers, they should be consumed in moderation due to the ethyl alcohol they contain, or non-alcoholic beers should be consumed [5]. Technological processes that allow for oxidative stabilization, e.g., pasteurization, may negatively affect both the polyphenol content and the antioxidant activity of beers [6,7,8].

The most important group of antioxidant compounds in beers are polyphenols. The polyphenolic compounds contained in wheat beers are biologically active substances with different chemical structures, which are related to varying antioxidant and antiradical activity, as well as other reactions that occur during beer production and storage [9]. Polyphenolic compounds are responsible for the flavor impressions of the resulting beer product, and the main groups of polyphenols that can be detected in beers are cinnamic acids, hydroxybenzoic acids, flavan-3-ols, flavones, flavonols, and flavanones [5]. The proportion of polyphenolic compounds in the finished product depends on many factors. The first is the malting and drying process of green malt (Maillard reaction products). Another method is the mashing process, which can be performed in two ways: wet mashing, which causes less damage to the fruit-seed coat and husk (in the case of barley malt), resulting in a lower content of ferulic acid and phenolic compounds in the wort; and dry mashing, which depends on the particle size of the mash used [1,5]. The mashing process (temperature ranges used in the process and mashing time) influences the content of polyphenolic compounds, including cinnamic acid. During wort boiling, the content of polyphenolic compounds changes (increases) because polyphenolic compounds from hops are transferred into the wort [1,5]. Polyphenolic compounds, especially oligomers, combine with proteins to form complexes that cause sedimentation or adsorb onto yeast cells and are removed during decanting of young beer from the yeast slurry [1]. Polyphenols also influence the degree of haze in beers [10,11]. During beer storage, changes in polyphenol content occur, influenced by storage time, light exposure, and temperature range. The greatest decreases in phenolic compound content in beers occurs during the first three months of storage [5]. Phenolic compounds present in beers are responsible for the appearance (color, clarity) and sensory impressions (flavor stability) of the beer product [5]. Depending on the type of compound, they can cause perceptions of bitterness, astringency, body, or affect the sensation of fullness of flavor, which are caused by, among others, p-coumaric acid, caffeic acid, gallic acid, hydroxybenzoic acid, chlorogenic acid, and nonanal [1]. It is worth paying attention to the content of gallic acid in beers, as its concentration in the finished product is an indicator of quality due to the rate of oxidation and degradation [5]. In wheat beers, properly selected yeast strain and fermentation conditions enhance the flavor profile of the beer product.

In recent years, interest in producing malt and beer from raw materials other than spring barley has been growing. Among the wide range of alcoholic beverages, wheat beer is highly attractive to consumers. To obtain high-quality wheat malt, it is necessary to select a cultivar characterized by the appropriate raw material parameters (the most important are a total protein content of 9.5–11.5% d.m. and a grain uniformity above 85%) and to modify the malting process to achieve good physicochemical and sensory characteristics of the produced beer. Progress in wheat breeding is leading to the production of high-protein cultivars, primarily intended for flour production, which have not been used in the production of wheat malt. Compared to spring wheat, winter wheat grains are characterized by a relatively low total protein content, resulting in a malt with higher extractivity and the ability to produce a product that meets high consumer expectations. Wheat beers, regardless of their style, should be characterized by high-quality final fermentation products and sensory attributes that influence consumer perception of their appeal. The market for wheat beers and those enriched with various health-promoting ingredients, such as fruits, vegetables, and herbs, is constantly expanding and is becoming increasingly attractive among female consumers. Sensory evaluation of beers is a fundamental step in determining the quality of the produced beer. It allows not only for determining consumer desirability and acceptance but also for optimizing production processes and developing new products [12]. The overall quality of beer is primarily influenced by five sensory attributes: taste, aroma, bitterness, palatability, and carbonation.

The aim of this study was to determine the effect of selected winter wheat varieties and the growing season on the antioxidant activity, polyphenol profile, and organoleptic assessment of wheat beers produced from them.

2. Materials and Methods

2.1. Research Material and Location and Field Experiment Conditions

The research material consisted of grains of four winter wheat cultivars—Elixer, Lawina, Gimantis, and Rockefeller—from field experiments conducted in the 2020/2021, 2021/2022, and 2022/2023 growing seasons in Jelcz-Laskowice (51°21′ N; 17°35′ W), be-longing to the IUNG-PIB, Department of Herbology and Agricultural Cultivation Techniques, Wrocław. Wheat was fertilized N at a level of 60 kg N·ha⁻¹. Field experiment conditions are presented in Belcar and Gorzelany [13].

2.2. Preparation of Malts

Representative samples of raw materials were cleaned on Vögel sieves (Laborset Co., Łódź, Poland), and the grain fraction >2.5 mm was taken for testing, spread on metal plates for germination, and soaked until the grain moisture content reached 45% (sprayed twice with tap water from an atomizer at 15 ± 1 °C during the day at 12 h intervals). The sample dishes were placed in a climate cabinet with a relative humidity of 90% and a temperature of 15 ± 1 °C. After 5 days of germination, the raw material was dried in a laboratory dryer with the following operating times and then germinated: 15 h at 40 °C, 3 h at 50 °C, 3 h at 65 °C, and 2 h at 80 °C.

2.3. Wheat Beer Brewing Process

The wheat beers were brewed using the infusion method. The raw material consisted of 50% wheat malt obtained from field experiments (four analyzed winter wheat cultivars, three analyzed growing seasons) and 50% commercial barley malt purchased from the Viking Malt malthouse. The malt was placed in a ROYAL RCBM-30CK mash-boil kettle (Royal Catering, Warsaw, Poland) (assuming a process efficiency of 80%), and water was added at a rate of 3 L per kilogram of malt. Mashing was carried out for 60 min at 67 °C (with vigorous stirring for the first 30 min), after which the mashing temperature was raised to 72 °C and held for 15 min. The next step was to raise the mashing temperature to 78 °C and hold for 10 min. After obtaining a negative iodine test result, the mashing process was terminated, and the mash was then filtered and sweetened with water at 78 °C.

The sweetened wort was placed in a ROYAL RCBM-30CK mash-boil kettle and heated to 100 °C at a rate of 2 °C/1 min. The wort was boiled for 60 min. During the boil, hops were added in three doses:

0 min of boiling—3 g of Amarillo hops (9.9% α-acid content);

after 45 min of boiling—1.5 g of Amarillo hops;

after 60 min of boiling (aroma hopping)—3 g of Amarillo hops.

The hot wort was cooled. After 30 min, the wort temperature reached 20 °C. Each wort had an extract of 12.0 °P. The cooled wort was transferred to a 30 L fermentation tank and inoculated with Saccharomyces cerevisae Safale US-05 yeast (6 × 10⁹ g⁻¹), previously rehydrated according to the manufacturer’s instructions (0.58 g d.m.·L⁻¹ wort), and fermented at 21 °C for 14 days. The beer was then bottled after adding a 0.3% sucrose solution in water to achieve refermentation and carbonation. Organoleptic and physicochemical tests were performed one month after bottling.

The wheat beers were designated as follows: E (Elixer cultivar), L (Lawina cultivar), G (Gimantis cultivar), and R (Rockefeller cultivar), and 1 (first), 2 (second), and 3 (third growing season). A total of 12 wheat beers were produced.

2.4. Total Polyphenol Content and Polyphenol Profile of Wheat Beers

The total polyphenol content (mg·L⁻¹) was determined using the method described by Dvořáková et al. [14] according to EBC method 9.11 [15]. The 10 mL of degassed beer sample and 8 mL of CMC/EDTA reagent (Carboxymethyl cellulose/ethylenediaminetetraacetic acid) were transferred to a 25 mL volumetric flask and thoroughly mixed. Then 0.5 mL of ferric reagent (3.5% ammonium iron citrate) was added to the sample, which was then thoroughly homogenized. After that, 0.5 mL of ammonia reagent (ammonia–water, 1:2) was added and thoroughly mixed. Finally, the volume was made up to 25 mL with distilled water and homogenized. The absorbance at 600 nm was measured after 10 min, for reaction to take place and stabilize.

A UPLC analyzer equipped with a binary pump, column and sample manager, photodiode array detector (PDA), and tandem quadrupole mass spectrometer (TQD) (Waters, Milford, MA, USA) was used to determine the polyphenolic compounds according to the methodology described by Żurek et al. [16]. Separation was performed using the UPLC BEH C18 column (1.7 µm, 100 mm × 2.1 mm, Waters) at 50 °C, at a flow rate of 0.35 mL/min. The injection volume of the samples was 5 µL. The mobile phase consisted of water (solvent A) and 40% acetonitrile in water, v/v (solvent B). The following TQD parameters were used: capillary voltage of 3500 V; con voltage of 30 V; con gas flow 100 L/h; source temperature 120 °C; desolvation temperature 350 °C; and desolvation gas flow rate of 800 L/h. Polyphenolic identification and quantitative analyses were performed on the basis of the mass-to-charge ratio, retention time, specific PDA spectra, fragment ions, and comparison of data obtained with commercial standards and literature findings.

2.5. Antioxidant Activity of Wheat Beers

2.5.1. DPPH Assay

The antiradical activity of wheat beers was determined using the DPPH radical according to the method of He et al. [3]. For this purpose, a solution of DPPH (2,2-diphenyl-1-picrylhydrazyl) in ethanol was prepared at a concentration of 0.05 mM·L⁻¹. Of the prepared solution, 7.8 mL was taken into a test tube and 0.2 mL of diluted (2×) beer was added and incubated in the dark for 60 min at 37 °C, and then the absorbance was measured at a wavelength of λ = 517 nm using a V-5000 UV-Vis spectrophotometer (Shanghai Metash Instruments Co., Ltd., Shanghai, China). The blank contained distilled water instead of beer. The results were expressed as Trolox equivalent (mM TE·L⁻¹).

2.5.2. ABTS Test

The antiradical activity of wheat beers was determined using the ABTS cation radical according to the method of Re et al. [17]. A solution of ABTS (2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) at a concentration of 7 mM·L⁻¹ and a solution of potassium persulfate at a concentration of 2.45 mM·L⁻¹ were prepared. The prepared solutions were combined in a 1:0.5 ratio and stored for 12–16 h in the dark to form the ABTS cation. The ABTS⁺ solution was diluted with distilled water to an absorbance of 0.700 ± 0.02 at a wavelength of λ = 734 nm. A total of 3 mL of the diluted ABTS⁺ solution and 0.3 mL of the tested beer were added to the test tube, and after 6 min the absorbance was read at a wavelength of λ = 734 nm using a V-5000 UV-Vis spectrophotometer (Shanghai Metash Instruments Co., Ltd., Shanghai, China). The results were corrected for the dilution and expressed as Trolox equivalent (mM TE·L⁻¹).

2.5.3. FRAP Assay

The reducing capacity of wheat beers was determined using the FRAP reagent according to the method of Benzie and Strain [18] and He et al. [3]. A 10 mM·L⁻¹ TPTZ (2,4,6-tripyridyl-s-triazine) solution, a 20 mM·L⁻¹ FeCl₃·6H₂O solution, an acetate buffer at pH = 3.6, and a 40 mM·L⁻¹ HCl solution were prepared, followed by the FRAP reagent, obtained by mixing 25 mL of acetate buffer, 2.5 mL of TPTZ solution in HCl, and 2.5 mL of FeCl₃·6H₂O. Subsequently, 6 mL of FRAP solution and 0.2 mL of the tested beer were placed in a test tube and incubated at 37 °C for 10 min, and then the absorbance was measured at λ = 593 nm using a V-5000 UV-Vis spectrophotometer (Shanghai Metash Instruments Co., Ltd., Shanghai, China). The blank contained distilled water instead of beer. FRAP test results were expressed as mM Fe²⁺ ·L⁻¹.

All chemicals used in this study were of high purity (analytical grade). Chemicals were sourced from Poch (Gliwice, Poland), Chempur (Piekary Śląskie, Poland), and Sigma-Aldrich (Saint Louis, MO, USA).

2.6. Organoleptic Analysis

Organoleptic analysis was performed by a team of 11 trained people (5 women and 6 men, aged 30–40 years) in accordance with the Polish Standard PN-A-79093-1:2000 [19] with our own modifications. Beer samples were served after cooling to 10 °C, coded in random order, in transparent 250 mL plastic cups. Water was administered to rinse the mouth between each assessment. An organoleptic profile was used to evaluate the taste and aroma of the tested beers, defining qualitative characteristics (malty, fruity, sweet, grainy, intense, full-bodied, fresh, phenolic, bitter, and sour) according to the EBC 13.12 method [20].

2.7. Statistical Analysis

All described analyses were performed in triplicate. Statistica 13.3 was used for statistical analysis. The effect of wheat variety and growing season on the quality parameters of wheat beers was examined using multivariate analysis of variance (Tukey’s HSD test) at a significance level of α = 0.05.

3. Results and Discussion

3.1. Total Polyphenol Content and Polyphenol Profile of Wheat Beers

The average content of total polyphenols in wheat beers obtained from the analyzed wheat malts during the study years was 73.9 mg·L⁻¹ and was dependent on the cultivar used and the growing season (Figure 1). Wheat beers obtained from malt obtained from winter wheat of the Rockefeller cultivar were characterized by the lowest average content of total polyphenols (58.77 mg·L⁻¹), while significantly the highest content of total polyphenols was possessed by beers obtained from the malt obtained from wheat of the Gimantis cultivar (89.07 mg·L⁻¹); With an increase in the analyzed parameter of 34.01%. The average content of total polyphenols in the obtained wheat beers varied between the individual growing seasons. The highest value was obtained in the third year of the study (78.9 mg·L⁻¹) and was on average 18.88% higher compared to the second year of the study, while the results obtained in the first and third years of the study did not differ.

In the study by Belcar et al. [21] for wheat beers produced in the American Wheat Beer style from malt derived from wheat grain of the Elixer, Rockefeller, and Gimantis cultivars (pilot field experiment, 2019/2020 season), the total polyphenol content was 210 mg·L⁻¹, 211 mg·L⁻¹, and 187 mg·L⁻¹, respectively. In wheat beers, the total polyphenol content ranged from 125 to 142 mg·L⁻¹ [22]; 123.6 mg·L⁻¹ (beer brewed with 50% German wheat malt), 102.8 mg·L⁻¹ (beer brewed with American wheat malt) and from 86.9 to 89.2 mg·L⁻¹ (wheat beers brewed with Korean wheat malt; [23]). In the study by Mascia et al. [24], German commercial wheat beer had a total polyphenol content of 139 mg·L⁻¹, while Czech wheat beer had a total polyphenol content of 148 mg·L⁻¹. The lower content of total polyphenols obtained in this study limits the health-promoting properties of the obtained beer product and, moreover, limits the degree of interaction between polyphenols and proteins (especially with the optimal content of this component in grain and wheat malt [13]), which results in limited haze, which is characteristic for wheat beers and unfavorable in the case of barley beers. Polyphenols are chemical compounds with diverse structures, thanks to which they possess both antiradical and antioxidant properties associated with biologically active reactions occurring during beer production and storage [9]. The proportion of wheat malt in the raw material positively affects the total polyphenol content compared to wheat beers produced with the addition of unmalted grain [25]. Malting and drying the grain are two processes that significantly affect the total polyphenol content in the finished product, and thus, they are transferred to wheat beer during subsequent technological processes (mashing, boiling, fermentation). Gradually increasing the temperature of the air drying of the malt increases the intensity of transformations and the formation of new compounds, including Maillard reaction products. The temperature of the drying process (its gradual increase) causes isomerization of the produced polyphenolic compounds, which is important in the sensory analysis of the finished product, as it influences the perception of flavor (including flavanol polymers), astringency, bitterness, body, and overall flavor. Polyphenols also influence the degree of haze in beers [10,11]. The mashing process of wheat and barley malt (the length and temperature range of the process) and the fineness of the mashed material also influence the content of polyphenolic compounds in the finished beer product [1].

Polyphenolic compounds in the analyzed wheat beers were identified based on characteristic spectral data: mass-to-charge ratio (m/z) and absorption maximum. Seven polyphenolic compounds were identified, the spectral properties of which are presented in

Table 1. Polyphenol profile of wheat beers obtained from malt obtained from Elixer, Lawina, Gimantis, and Rockefeller wheat grain, identified using UPLC-PDA-TQD-MS (beer E1, L1, G1, R1—first year of research; beer E2, L2, G2, R2—second year of research; beer E3, L3, G3, R3—third year of research).

	Chemical Compound [mg·L−1]	Rt [min]	MS [m/z]	MS/MS [m/z]	λmax [nm]	E1	E2	E3	L1	L2	L3	G1	G2	G3	R1	R2	R3
1	Chlorogenic acid	2.96	353	191	299sh, 327	0.08 a ± 0.00	0.15 b ± 0.01	0.09 a ± 0.00	0.10 a ± 0.00	0.10 a ± 0.01	0.09 a ± 0.01	0.07 a ± 0.01	0.10 b ± 0.00	0.09 b ± 0.01	0.08 a ± 0.01	0.13 c ± 0.00	0.10 b ± 0.00
2	Kaempferol 3-O-glucoside	3.33	447	285	264, 352	0.10 a ± 0.01	0.13 b ± 0.02	0.09 a ± 0.01	0.09 a ± 0.02	0.08 a ± 0.01	0.08 a ± 0.01	0.09 a ± 0.01	0.23 b ± 0.03	0.10 a ± 0.01	0.13 b ± 0.02	0.21 c ± 0.02	0.08 a ± 0.01
3	(+)catechin	3.48	289	141	277	0.13 a ± 0.02	0.13 a ± 0.02	0.14 a ± 0.02	0.15 a ± 0.02	0.15 a ± 0.05	0.14 a ± 0.05	0.14 a ± 0.04	0.15 a ± 0.02	0.18 b ± 0.06	0.15 b ± 0.04	0.11 a ± 0.02	0.14 b ± 0.03
4	Kaempferol rhamnoside-pentoside	3.69	563	285	264, 351	0.89 b ± 0.21	0.94 c ± 0.22	0.67 a ± 0.16	0.90 c ± 0.21	0.46 b ± 0.00	0.40 a ± 0.00	0.64 b ± 0.00	0.78 c ± 0.18	0.58 a ± 0.00	0.61 b ± 0.00	0.36 a ± 0.02	0.71 c ± 0.16
5	(+)catechin 3-O-glucoside	3.71	451	289	277	0.31 a ± 0.00	0.40 b ± 0.00	0.32 a ± 0.00	0.35 c ± 0.00	0.26 a ± 0.03	0.29 b ± 0.04	0.25 a ± 0.03	0.40 c ± 0.00	0.35 b ± 0.05	0.30 b ± 0.02	0.18 a ± 0.08	0.33 c ± 0.05
6	Kaempferol rhamnoside-pentoside-pentoside	3.85	695	563, 285	264, 350	0.24 b ± 0.03	0.28 c ± 0.02	0.22 a ± 0.02	0.22 c ± 0.01	0.18 b ± 0.02	0.15 a ± 0.01	0.24 b ± 0.02	0.22 a ± 0.03	0.22 a ± 0.02	0.24 c ± 0.01	0.10 a ± 0.00	0.19 b ± 0.02
7	Kaempferol rhamnoside-pentoside	3.91	563	285	264, 350	1.08 b ± 0.05	1.28 c ± 0.08	0.83 a ± 0.05	0.94 c ± 0.04	0.67 b ± 0.01	0.57 a ± 0.01	0.89 c ± 0.01	0.85 b ± 0.08	0.75 a ± 0.01	0.94 c ± 0.01	0.46 a ± 0.04	0.87 b ± 0.08
8	Kaempferol 3-O-rutinoside	4.91	593	285	264, 352	0.13 a ± 0.00	0.22 c ± 0.00	0.17 b ± 0.00	0.12 c ± 0.00	0.09 b ± 0.01	0.07 a ± 0.01	0.17 b ± 0.01	0.14 a ± 0.01	0.16 b ± 0.02	0.09 b ± 0.01	0.02 a ± 0.00	0.18 c ± 0.01
Sum						2.96 b ± 0.23	3.53 c ± 0.25	2.53 a ± 0.17	2.87 c ± 0.22	1.99 b ± 0.01	1.79 a ± 0.00	2.49 a ± 0.01	2.87 b ± 0.18	2.43 a ± 0.00	2.54 b ± 0.01	1.57 a ± 0.10	2.60 b ± 0.18

a–c—statistically significant differences at the confidence level of p = 0.05.

. Chlorogenic acid belongs to the group of hydroxycinnamic acid derivatives, while the remaining identified compounds belonged to the group of flavonols, represented by (+)catechin and its derivative, as well as kaempferol derivatives (in the glycoside form). In beers, sugars such as rhamnose and glucose, as well as arabinose, rutinose, and xylose, are combined with kaempferol [26]. These glycosides are responsible for taste sensations, including the sensation of astringency in the mouth and, to a lesser extent, the bitterness experienced during beer consumption [27]. The basic raw materials used in beer production: hops, malt, and, to a lesser extent, cereal grains, are sources of phenolic compounds, including flavonols [9]. Flavonols are antioxidant compounds that limit cell aging in the human body, have a beneficial effect on the cardiovascular system, and inhibit the development of certain cancer cells [1,5,27].

The chlorogenic acid content in wheat beers obtained from wheat malt prepared from the Elixer winter wheat grain ranged from 0.08 to 0.15 mg·L⁻¹ during the study period (Table 1). Among the analyzed flavonols, their content varied significantly, with the highest content being found in the following compounds: kaempferol rhamnoside-pentoside (1.50–2.22 mg·L⁻¹), and (+)catechin 3-O-glucoside (0.31–0.40 mg·L⁻¹) at much lower concentrations. The total content of identified polyphenolic compounds ranged from 2.53 mg·L⁻¹ for beer obtained from the third year of study to 3.53 mg·L⁻¹ for beer obtained from the second year of study. The average content of polyphenolic compounds in wheat beers brewed with malt from winter wheat cultivar Elixer was 3.01 mg·L⁻¹ (Table 1). In the study by Belcar et al. [25] on Witbier-type beers with the addition of 25% wheat malt and with a 50% share of wheat grain of the Elixer cultivar, the total content of identified polyphenolic compounds was 6.43 and 6.53 mg·L⁻¹, respectively, including kaempferol 3-O-rutinoside at concentrations of 0.57 and 0.65 mg·L⁻¹, respectively; kaempferol pentoside-rhamnoside at concentrations of 1.39 and 1.31 mg·L⁻¹, and kaempferol 3-O-glucoside at concentrations of 0.58 and 0.64 mg·L⁻¹, respectively. In the study by Gorzelany et al. [28] on wheat beer, the total content of identified polyphenolic compounds was 3.22 mg·L⁻¹, with the main compounds detected being kaempferol-3-O-rutinoside-7-O-glucoside at a concentration of 0.92 mg·L⁻¹ and kaempferol-3-O-glucoside-7-O-glucoside at a concentration of 1.35 mg·L⁻¹. In a subsequent study by Belcar and Gorzelany [29], the total content of identified polyphenolic compounds was 3.15 mg·L⁻¹, with the main compounds detected being the aforementioned compounds at concentrations of 0.92 mg·L⁻¹ and 1.31 mg·L⁻¹, respectively.

Depending on the year of study, the content of chlorogenic acid in wheat beers obtained from wheat malt prepared from winter wheat grain of the Lawina cultivar ranged from 0.09 to 0.10 mg·L⁻¹ (Table 1). Among the analyzed flavonols, their content varied significantly, with the highest content being found in the compounds: kaempferol rhamnoside-pentoside (0.97–1.84 mg·L⁻¹), and (+)catechin 3-O-glucoside (0.26–0.35 mg·L⁻¹) at much lower concentrations. The total content of identified polyphenolic compounds ranged from 1.79 mg·L⁻¹ for beer obtained from wheat grain of the Lawina cultivar (third year of study) to 2.87 mg·L⁻¹ (first year of study). The average content of polyphenolic compounds in wheat beers brewed with malt from winter wheat of the Lawina cultivar was 2.22 mg·L⁻¹ (Table 1). The content of chlorogenic acid in wheat beers obtained from wheat malt prepared from winter wheat grain of the Gimantis cultivar during the study years ranged from 0.07 to 0.10 mg·L⁻¹ (Table 1). Among the analyzed flavonols, their content varied significantly, with the highest content being found in the following compounds: kaempferol rhamnoside-pentoside (1.29–1.63 mg·L⁻¹), and (+)catechin 3-O-glucoside (0.25–0.40 mg·L⁻¹) at much lower concentrations. The total content of identified polyphenolic compounds ranged from 2.43 mg·L⁻¹ (for beer obtained from malt in the third year of the study) to 2.87 mg·L⁻¹ for beer obtained in the second year of the study (Table 1). The average content of polyphenolic compounds identified using UPLC-PDA-TQD-MS in wheat beers brewed with malt from winter wheat of the Gimantis cultivar was 2.60 mg·L⁻¹. The content of chlorogenic acid in wheat beers obtained from wheat malt prepared from the Rockefeller winter wheat grain during the study years ranged from 0.08 to 0.13 mg·L⁻¹ (Table 1). Among the analyzed flavonols, their content varied significantly, with the highest content of kaempferol rhamnoside-pentoside compounds (0.82–1.58 mg·L⁻¹), and (+)catechin 3-O-glucoside at a much lower concentration (0.18–0.33 mg·L⁻¹). The total content of identified polyphenolic compounds ranged from 1.57 mg·L⁻¹ for beer from the second year of study to 2.60 mg·L⁻¹ for beer obtained from malt from the third year of study. The average content of polyphenolic compounds in wheat beers brewed with malt from winter wheat of the Rockefeller cultivar was 2.24 mg·L⁻¹ (Table 1).

In the study by Belcar et al. [25] in Witbier-type beers, with the addition of 25% wheat malt and 50% share of wheat grain of the Lawina cultivar, the total content of identified polyphenolic compounds was 5.79 and 5.80 mg·L⁻¹, respectively, including kaempferol 3-O-rutinoid in concentrations of 0.58 and 0.55 mg·L⁻¹, respectively; kaempferol pentoside-rhamnoside was present in both beers in concentrations of 1.04 mg·L⁻¹, and kaempferol 3-O-glucoside in concentrations of 0.58 and 0.59 mg·L⁻¹, respectively; with the addition of 25% wheat malt and 50% of Gimantis wheat grain, the total content of identified polyphenolic compounds was 6.18 and 6.24 mg·L⁻¹, respectively. Among the polyphenolic compounds analyzed, the main ones were kaempferol 3-O-rutinoside at concentrations of 0.65 and 0.67 mg·L⁻¹, respectively; kaempferol pentoside-rhamnoside at concentrations of 1.12 and 1.14 mg·L⁻¹; and kaempferol 3-O-glucoside at concentrations of 0.61 and 0.64 mg·L⁻¹, respectively. And with the addition of 25% wheat malt and 50% Rockefeller wheat grain, the total content of identified polyphenolic compounds in both beers was 6.34 mg·L⁻¹. Among the analyzed polyphenolic compounds, kaempferol 3-O-rutinoid can be distinguished in concentrations of 0.63 and 0.61 mg·L⁻¹, respectively; kaempferol pentoside-rhamnoside in concentrations of 1.14 and 1.22 mg·L⁻¹, respectively; and kaempferol 3-O-glucoside in concentrations of 0.69 and 0.60 mg·L⁻¹, respectively. In another study by Belcar and Gorzelany [29], the total content of identified polyphenolic compounds in wheat beer obtained from the Lawina cultivar was 2.18 mg·L⁻¹, with the main detected compounds being kaempferol-3-O-rutinoside-7-O-glucoside at a concentration of 0.73 mg·L⁻¹ and kaempferol-3-O-glucoside-7-O-glucoside at a concentration of 0.81 mg·L⁻¹.

Among the analyzed winter wheat cultivars from which wheat malts were prepared and then brewed, the highest average content of polyphenolic compounds was found in wheat beers obtained from malt derived from the Elixer winter wheat cultivar (3.01 mg·L⁻¹). The polyphenolic content in wheat beers obtained from malt derived from the Gimantis, Rockefeller, and Lawina cultivars was significantly lower by 13.62%, 25.58%, and 26.25%, respectively. The highest average polyphenolic content was found in the beers from the first year of the study, which were 8.46% and 13.97% higher compared to the second and third years of the field study.

As reported by Radonjič et al. [5] and Almaguer et al. [30] the average kaempferol content in barley beers was 0.10–1.64 mg·L⁻¹. Mikyška et al. [9] found that storage of lager beers causes a slight decrease in kaempferol-O-glucoside (approximately 15% after six weeks of beer aging compared to fresh wort). In lager beers, the content of kaempferol-O-glycosides was 0.17–0.26 mg·L⁻¹; the content was influenced by the method of hopping the wort (four weeks of storage [9,31]). During boiling of the wort with hops, kaempferol-O-glycosides are transferred to the wort after about 30 min of boiling (depending on the hop dose) [31]. The cultivar, cultivation conditions, or country of origin influence the average kaempferol content in hop cones, which was 1.2 mg·kg⁻¹ d.m. [5,31]. Cereal raw materials (wheat and barley malt) are also a substrate of kaempferol glycosides used in the production of wheat beers. In the study by Suchowilska et al. [32], the average kaempferol content in wheat grain was 11.4 mg·kg⁻¹ d.m., while in the study by Buśko et al. [33] it was slightly lower—6.0 mg·kg⁻¹ d.m. In the study by Özcan et al. [34], the kaempferol content in barley grain was determined to be at 19.9 mg·kg⁻¹, while in malts produced from it, the content of this flavonol was slightly higher and amounted to 21.5 mg·kg⁻¹. Kaempferol, but also the high content of other flavonol derivatives, in the analyzed wheat beers is the result of various chemical reactions occurring during boiling of wort with hops: both the process of extraction of compounds contained in hops, but also the formation of glycoside derivatives by combining aglycones originating, among others, from malts with sugars present in the wort, e.g., glucose, creating O-glycoside flavonol derivatives.

3.2. Antioxidant Activity of Wheat Beers

Compounds with antioxidant activity in the finished beer product are primarily polyphenols, but also fiber, vitamins, and bitter acids [1,2]. Technological processes that prepare the finished product for long-term consumption through oxidative stabilization, such as pasteurization, can negatively impact both the polyphenol content and the antioxidant activity of the beers [6,7,8]. The use of pasteurization significantly improves the safety of beer beverages and at the same time extends their shelf life, but if we want to obtain beer with high health-promoting properties, other preservation methods should be used, e.g., microfiltration.

Higher DPPH radical activity in wheat beers indicates a reduced content of aldehydes that reduce flavor stability, such as trans-2-nonenal [3]. The average value of antioxidant activity determined by the DPPH method in the analyzed wheat beers during the study years was 0.82 mM TE·L⁻¹ (Figure 2). The wheat cultivar used and the growing season had a significant effect on the DPPH value in beers obtained from the analyzed wheat malts. The lowest average value of this parameter was obtained for wheat beers obtained from malt obtained from winter wheat of the Lawina cultivar (0.46 mM TE·L⁻¹), while the highest value was observed for beers obtained from malt obtained from wheat of the Gimantis cultivar (1.08 mM TE·L⁻¹), which represents a 57.41% increase compared to the Lawina cultivar. The average value of antioxidant activity determined by the DPPH method in the obtained wheat beers varied in the individual growing seasons; the highest value was obtained for the first year of the study (0.84 mM TE·L⁻¹) and it was 7.14% higher compared to the third year of the study, while the results obtained in the first and second year of the study were not different.

Wheat beers produced from unmalted wheat grain of the Elixer cultivar, and with the addition of malt obtained from it, were characterized by the highest antioxidant activity determined by the DPPH method [25]. In subsequent studies on wheat beers brewed using wheat malt produced from wheat grain of the Elixer and Lawina cultivars, the DPPH value was 2.27 mM TE·L⁻¹ and 2.19 mM TE·L⁻¹, respectively [29]. While wheat beers in the American Wheat Beer style, produced from malt from wheat grain of the Elixer, Rockefeller, and Gimantis cultivars, were characterized by an average antioxidant activity of 1.60 mM TE·L⁻¹, 1.17 mM TE·L⁻¹, and 1.21 mM TE·L⁻¹, respectively [21]. Wheat beers made from wheat malt derived from the Elixer cultivar of winter wheat (field experiment) had an average DPPH of 2.27 mM TE·L⁻¹ [28]; 1.04 mM TE·L⁻¹ [35], while wheat beers made from wheat malt derived from the Lawina cultivar of winter wheat (field experiment) had an average DPPH of 2.38 mM TE·L⁻¹ [36]. Significantly lower antioxidant activity (0.5 mM TE·L⁻¹) determined by the DPPH method was recorded in top-fermented wheat beer [6].

Compounds in beer that reduce oxidized lipid peroxidation products are biologically active compounds determined using the FRAP method [3]. Ditrych et al. [6] also observed that higher polyphenol content in beers positively affected the antioxidant potential of beers determined using the DPPH and FRAP tests. The average value of antioxidant activity determined by the FRAP method in the analyzed wheat beers during the study years was 1.51 mM Fe²⁺·L⁻¹ (Figure 3). The winter wheat cultivar used and the growing season had a significant impact on the FRAP value in beers obtained from the analyzed wheat malts. The lowest mean value of the analyzed parameter was obtained for the Rockefeller winter wheat cultivar (1.44 Fe²⁺·L⁻¹), while the highest among the analyzed wheat beers was obtained for the Gimantis malt (1.58 Fe²⁺·L⁻¹). The mean value of antioxidant activity determined by the FRAP method in the obtained wheat beers varied between growing seasons; the highest value was obtained on the second year of the study (1.56 mM Fe²⁺·L⁻¹) and was 5.13% higher compared to the first and third years of the study, while the results obtained in the first and third years of the study were not different.

Wheat beers produced in the American Wheat Beer style with malt from wheat grain of the Elixer, Rockefeller, and Gimantis cultivars were characterized by FRAP values of 2.34 mM Fe²⁺·L⁻¹, 2.55 mM Fe²⁺·L⁻¹, and 1.83 mM Fe²⁺·L⁻¹, respectively [25]. In subsequent studies on wheat beers from wheat grain of the Elixer and Lawina cultivars, FRAP was 2.79 mM Fe²⁺·L⁻¹ and 2.53 mM Fe²⁺·L⁻¹, respectively [29], while for wheat beers brewed with wheat malt obtained from winter wheat grain of the Elixer cultivar—2.19 mM Fe²⁺·L⁻¹ [28]; 0.86 mM Fe²⁺·L⁻¹ [35], and additionally for wheat beers brewed from winter wheat grain of the Lawina cultivar—2.42 mM Fe²⁺·L⁻¹ [36]. According to the authors He et al. [3], the reducing capacity of unpasteurized wheat beers ranged from 1.28 to 1.71 mM Fe²⁺·L⁻¹, while during storage, a decrease in the analyzed parameter was observed depending on the storage temperature (the highest decrease was observed at 5 °C, and the lowest at 20 °C). In the study by Ditrych et al. [6], top-fermented wheat beers had a reducing capacity of 0.70 to 0.80 mM Fe²⁺·L⁻¹.

The average ABTS value in the analyzed wheat beers during the study years was 0.69 mM TE·L⁻¹ (Figure 4). The ABTS value of beers produced from the analyzed wheat malts was influenced by the cultivar and the growing season. The highest ABTS value was found in wheat beers obtained from malts from the Elixer and Lawina wheat cultivars (0.72 mM TE·L⁻¹ and 0.73 mM TE·L⁻¹), while the significantly lowest value was obtained from the Rockefeller cultivar (0.61 mM TE·L⁻¹). The mean value of antioxidant activity determined by the ABTS method in the obtained wheat beers varied in the individual growing seasons; the highest value was obtained in the third year of the study (0.76 mM TE·L⁻¹) and was 21.05% higher than in the first year and 7.89% higher than in the second year. The average ABTS value in the second year of the study was 0.70 mM TE·L⁻¹ and was 14.29% higher than the average value obtained in the first year of the study.

In the study by Belcar and Gorzelany [29], wheat beers brewed with wheat malt produced from wheat grain of the Elixer and Lawina cultivars (field experiment) were characterized by an average antioxidant activity, determined by the ABTS method, of 1.81 mM TE·L⁻¹ and 1.97 mM TE·L⁻¹, respectively. Wheat beers obtained in the American Wheat Beer style were characterized by ABTS at the level of 2.94 mM TE·L⁻¹, 2.18 mM TE·L⁻¹, and 2.06 mM TE·L⁻¹, respectively [21]. In subsequent studies this parameter in wheat beers obtained from malt obtained from winter wheat grain of the Elixer cultivar was 1.81 mM TE·L⁻¹ [28]; 1.01 mM TE·L⁻¹ [35], and from the Lawina cultivar—0.92 mM TE·L⁻¹ [36].

3.3. Organoleptic Analysis of Wheat Beers

The organoleptic characteristics of the obtained wheat beers determine the specific beer style and shape the attractiveness and acceptance of a given beer among consumers. At the same time, they provide an opportunity to assess the use of the tested wheat cultivars in beer production. The organoleptic profile of wheat beers obtained from the four analyzed cultivars and from three years of field experiments conducted by an 11-person team of evaluators is presented in Figure 5, Figure 6, Figure 7 and Figure 8.

The organoleptic profile of the studied wheat beers varied slightly; wheat beers made with malt produced from the Elixer wheat grain (Figure 5) were characterized by malty notes caused by, among others, maltol and furaneol [37], a fresh, intense, and slightly fruity flavor, which is characteristic of wheat beers, but also with a slight sour aftertaste. Differences between the organoleptic profile results of wheat beers made with malt from different years of research were most noticeable for the attribute of full flavor; the highest value was obtained for wheat beer produced from malt obtained from the first year of the study. The beer products obtained in subsequent years of the study were characterized by an increasingly lower sensation of fullness of flavor. The perception of acidity in the beers (confirmed by the results of physicochemical analysis) led the evaluators to suggest that these beers might not be met with consumer acceptance. This was likely due to an inappropriate recipe selection or irregularities resulting from the fermentation process. Interactions occurring during fermentation and maturation of beer between esters, sulfur compounds, carbonyl and phenolic compounds, alcohols, and organic acids have a significant impact on the flavor of the resulting beer [37]. Beers characterized by distinct fruity notes, a sweet aftertaste, and a pleasant aroma are more preferred and desired by consumers compared to traditional beers [38,39]. In several studies [21,25,28,29,36], wheat beers brewed with wheat malt produced from wheat grain of the Elixer cultivar were characterized by intense cereal and malt notes and a fresh taste with a slightly bitter aftertaste.

The organoleptic profile of the studied wheat beers varied slightly; wheat beers made with malt produced from the Lawina wheat grain (Figure 6) were characterized by cereal-malty notes, a fresh and slightly fruity flavor, especially for beer L3, which is a characteristic feature of wheat beers, but also a slight sour aftertaste. Differences between the organoleptic profile results of wheat beers made with malt from different years of research were most noticeable for the attribute of flavor intensity; the highest value was obtained for wheat beer produced from malt obtained from the third year of the study. Beer products obtained in previous years of the study were characterized by a decreasing perception of flavor intensity. The perception of acidity in the beers (confirmed by the results of physicochemical analysis) led the evaluators to suggest that these beers might not be met with consumer acceptance. This was likely due to an inappropriate recipe selection or irregularities resulting from the fermentation process. In the studies by Belcar and Gorzelany [29] and Belcar et al. [21], wheat beers brewed from wheat malt produced from the Lawina variety of wheat were characterized by intense cereal and malty notes, as well as a fresh and intense flavor with a slightly sour aftertaste.

The organoleptic profile of the analyzed wheat beers varied slightly; wheat beers made with malt produced from the Gimantis wheat grain (Figure 7) were characterized by cereal-malty notes and a fresh, intense, and slightly sweet flavor, which is characteristic of wheat beers. Differences between the organoleptic profile results of wheat beers made with malt from individual years of research were most noticeable for the sour and fruity flavor attributes; the highest value was obtained for wheat beer produced from malt obtained from the third year of the study. Beer products obtained in previous years of the study were characterized by increasingly lower perception of the discussed flavor attributes. The lower perception of acidity in the beers (confirmed by the results of physicochemical analysis) led the evaluators to suggest that these beers might be met with consumer acceptance after appropriate recipe selection or improvement of fermentation conditions. In the study by Belcar et al. [21], wheat beers brewed from wheat malt produced from the Gimantis wheat grain were characterized by intense malty notes, a full flavor, and a bitter aftertaste.

The organoleptic profile of the studied wheat beers varied slightly; wheat beers made with malt produced from the Rockefeller wheat grain (Figure 8) were characterized by malty notes, a fresh, slightly intense, and fruity flavor, which is characteristic of wheat beers, but also a high perception of a sour and bitter aftertaste. Differences between the organoleptic profile results of wheat beers made with malt from different years of research were most noticeable for the fruity and intense flavor attributes; the most noticeable fruity flavor was observed in wheat beers from the third year of the study and was significantly less noticeable in beers brewed with malt from previous years of the study. The flavor intensity was most pronounced in beers brewed with malt from the second year of the study, and the third year of the study was characterized by a higher flavor intensity compared to the first year of the study. The perception of high acidity in the beers, combined with a bitter aftertaste (confirmed by the results of physicochemical analysis), could contribute to potential low consumer acceptance, likely due to poor recipe selection or irregularities resulting from the fermentation process. In the study by Belcar et al. [36] wheat beers brewed with wheat malt produced from the Rockefeller wheat grain were characterized by a bitter-sweet taste and a full flavor.

The average organoleptic profile of the wheat beers studied varied, but all the beers were characterized by malty notes and, to a lesser extent, grainy notes (especially for beers brewed with malt obtained from the Gimantis and Lawina wheat cultivars), a sense of freshness, a light intensity, and a sour aftertaste, especially for those brewed with malt obtained from the Rockefeller and Lawina wheat grains. The high acidity obtained in wheat beers will most likely be the reason for a lack of acceptance by consumers (for the Rockefeller and Lawina wheat cultivars; Figure 9). The evaluation panel determined that there is a correlation between the wheat cultivar from which the wheat malt used to brew the analyzed wheat beers was produced and the quality of the resulting beer. The most balanced organoleptic profile was observed in wheat beers produced from malt obtained from the Gimantis wheat grain, regardless of the year of testing. Fermentation process conditions and the use of appropriate yeast strains significantly influence the flavor of the produced wheat beer (especially its phenolic flavor), while the temperature conditions of the fermentation process have a lesser impact. The unpleasant aftertaste of wheat beers may be related to improperly maintaining the pH of the wort and beer, or the use of chlorinated water, which causes the formation of chemical compounds—chlorophenols—in the produced beer [1]. Succinic acid, formed during fermentation, can impart a bitter aftertaste to beer [40]. In the study by Gugino et al. [41], wheat beer brewed from commercial wheat and barley malt in a 1:1 ratio was characterized by an intense yellow color, fruity, yeasty, and honeyed aroma notes, and intense flavor and carbonation. In a study by He et al. [3], the quality of fresh, cloudy wheat beers, uncured for 18 days at various temperatures, was observed and found to be optimal for storage for 0–9 days at 5 °C. During this time, wheat beer exhibited slightly hoppy, malty, and spicy notes, with abundant, delicate beer foam and a mild, fresh, and full flavor. In a study by Mascia et al. [24], Czech commercial wheat beer was characterized by fruity and phenolic notes with a full, fresh taste, while German commercial wheat beer was characterized by significantly lower sensory qualities compared to Czech wheat beer.

4. Conclusions

The brewing process influenced the polyphenol profile and antioxidant activity of wheat beers obtained from selected winter wheat cultivars from three growing seasons. The research results for beers obtained from wheat grains from individual research seasons were slightly different, with greater differences observed for the individual wheat cultivars analyzed. Wheat beers obtained from malt derived from the Elixer cultivar of wheat had the highest polyphenol profile values and antioxidant activity determined by the ABTS method. Total polyphenol content, antioxidant activity (DPPH), and organoleptic profile were highest for wheat beers obtained from malt derived from the Gimantis cultivar of wheat. The Gimantis cultivar showed the least variation in quality of wheat beers across growing seasons and is recommended as a wheat cultivar for the brewing industry.