Antioxidant Capacity, Phenolic and Organoleptic Profiles of Beers Maturated with Bilberries

Abstract

Beer is probably one of the oldest alcoholic beverages, and regarding consumption, is third place after water and tea. Due to the consumer’s pursuit of novel tastes and aromas, craft brewers are trying to meet the consumer’s need, so brewing beer with a fruit addition is the new twist. Fruit incorporation into the brewing process leads to alterations in the sensory properties and chemical composition of beers, but most of the articles published on this topic are mainly concentrated on beers with fruits different from bilberries. The data on bilberry beers are still scarce. Therefore, our projects are based in this field to investigate beer production with bilberries. In our previous research, we found interesting changes in the protein profile of bilberry beers with different original extracts. Therefore, the aim of this study was to investigate the influence of the original extract of beer maturated with bilberries on the phenolic profile (determined by HPLC analysis), the antioxidant capacity (measured by the ABTS, DPPH, FRAP, and CUPRAC methods), and sensory characteristics. The reported data can contribute to the development and production of bilberry beers with high antioxidant capacity and pleasant sensory properties.

1. Introduction

Fruit addition in beer is well known in beer production in Belgium. The oldest beer style, lambic, can be produced plain or with the addition of different fruits. According to Nardini and Garaguso [1], whole fruits like cherries and raspberries are traditionally used in Belgium to produce lambic beers, and usually cherries are added in a 150–400 g/L dose. Nowadays, there has been a noticeable trend towards increased fruit incorporation into the brewing process and this can be carried out in lots of beer styles, not only sours. A fruit addition can make beer look really colorful, depending on the type of fruit used, and fresher, because of the acids in the fruit. On the other hand, fruits can add new compounds, e.g., specific polyphenols, and this way can increase the concentration of bioactive compounds. Black forest fruits, such as cherries, bilberries, raspberries, and strawberries, are commonly used [1,2,3]. Mileva et al. [4] also worked with bilberries and according to them, when they added 100 g/L of bilberries during maturation, a 1.3- to 1.4-fold increase in polyphenols and 1.2- to 1.4-fold increase in flavonoids was observed. Our unpublished research revealed that the concentration of phenolic compounds increased with raising the bilberry dose from 70 to 170 g/L, and beers with the maximum bilberry content possessed better sensory characteristics.

On another hand, it is important to investigate how these fruit beers will be accepted on the market. The fruits will increase the cost, but all these new looks, tastes, and aromas will satisfy the consumer’s novel needs and, according to Qing Yang et al., 2021 [5], female consumers especially will be really pleased. Also, the consumers may be tired of drinking IPAs, so this new twist will take fruit beers one step further than hoppy beers, and the benefits from hops phenolics can be substituted with those of fruit phenolics, which can bring the same or even bigger antioxidant activities depending on the beer style and recipe [6].

There are numerous studies on the phenolic compounds in beer. Most of them de-scribe ferulic and p-coumaric acids as the most abundant phenolic acids in beer. Vanillic, gallic, and sinapic acids have also been reported as being important for beer [7]. The majority of phenolic acids in beer are present in a bound form as glycosides, esters, and bound complexes [7,8]. The phenolic composition of beer is determined by the raw materials and the brewing process [7].

Some researchers report a strong correlation between phenolic compounds and antioxidant capacity of beers [1,7,8]. However, none of them consider the influence of technological parameters on the individual phenolic compounds and the antioxidant activity of beers, especially bilberry beers. The antioxidant capacity of different beers was studied by many researchers [1,2,3,4,9,10,11,12,13,14]. Most of them investigated beers from local market without fruits [9,10,11,12,13,14]. Some papers provide an information about fruit beers [1,2,3,4], but only Mileva et al. [4] investigated bilberry beer and determined its antioxidant activity using just DPPH methods. The application of only one method to determine the antioxidant capacity is not enough because the antioxidants in foods and drinks may act via different mechanisms, not only as free radical scavengers.

Bilberries possess a wealth of phenolic compounds, showcasing notable antioxidant properties and offering extensive advantages for human well-being, as well as a promising avenue for enhancing beer profiles. Moreover, bilberries contain flavonoids and phenolic acids, chlorogenic acid being predominant among the relatively few phenolic acids. Flavonoids such as catechin, epicatechin, quercetin, myricetin, rutin, and other flavonoid glycosides have been identified in bilberries (Vaccinium myrtillus L.) [15,16].

According to our previous research and analysis, interesting changes occurred in the sensory characteristics and chemical composition of beer and yeast metabolites when blueberries, bilberries, cherries, cherry pomace, and juice were added during the main fermentation and maturation [17,18,19,20,21].

We investigated how the addition of cherry juice and pomace during different stages of beer fermentation influenced the basic beer parameters, the content of yeast metabolites (ethanol, aldehydes, higher alcohols, esters, and vicinal diketones), sensory characteristics, phenolic compounds, and antioxidant capacity of the beers produced [20,21]. The addition of cherry juice and pomace led to the appearance of fruity notes and an increase in the phenolic compounds content and the AOA of the beer samples. The increase was much more significant and the sensory characteristics were better when the pomace was added. Also, the results showed that the higher ester concentration in beers with pomace made them preferable to the tasting panels than beers with cherry juice [20,21].

We also studied the effect of adding blueberry at the beginning of maturation on yeast metabolites [17]. The results showed that blueberries affected positively esters and vicinal diketones formation for all the beers. Blueberry addition had no significant effect on higher alcohols and aldehyde formation.

Another investigation was performed for the changes in some phenolic compounds and protein profiles of the beers with a bilberry addition during fermentation, as well as the influence of these changes on the body in the mouthfeel and the head retention of the resulting beers [18,19]. It was found that independently of the original extract, the bilberry addition led to a significant reduction in the protein concentration and the number of protein fractions. Beers with bilberry additions contained between 74% and 94% less protein than the control beers. Despite the significant changes in the protein profiles, the beers with bilberry had very good mouthfeel, body, and head retention [19].

The aim of this study was to investigate the changes in individual phenolic acids and flavonoids, the antioxidant capacity, as well as the sensory characteristics of beers with different original extracts maturated with bilberries. The novel information received from this study will reveal the impact of brewing processes on the sensory characteristics, the phenolic compounds, and the antioxidant capacity, as well as the relationships between them, and will be helpful for the choice of better technological regimes for bilberry beer production.

2. Materials and Methods

2.1. Raw Materials

Pilsner malt (Weyermann, Bamberg, Germany), Perle and Cascade hops (Bulhops, Rakitovo, Bulgaria), and dry yeast Saccharomyces pastorianus Saflager W 34/70 (Fermentis, Lille, France) were utilized. Bilberries (Vaccinium myrtillus L.) were procured frozen from Bulfruct Ltd., Kostenets, Bulgaria, and stored in a freezer until use. The bilberry parameters were as follows: total dissolved solids—16.49% (w/w); Glucose—2.11 g/100 g; Fructose—2.02 g/100 g; Sucrose—0.03 g/100 g.

2.2. Beer Production

Three variants of beers without bilberries were produced, with an original extract of 12 °P, 14 °P, and 16 °P (control beers), along with three variants of beers maturated with bilberries, also with an original extract of 12 °P, 14 °P, and 16 °P.

The Pilsner malt was milled using a Corona hand mill and mixed with preheated water in various malt–water ratios (1:4.6 to 1:3.9), to achieve worts with desirable original extracts. An infusion mashing technique was employed under the following conditions: 20 min at 60 °C, 20 min at 65 °C, 25 min at 72 °C, and 1 min at 78 °C [19,20]. After mashing, the sweet wort was lautered, the spent grains were sparged, and the entire wort was boiled for approximately 60–90 min to achieve original extracts of 12 °P, 14 °P, and 16 °P. Bitter hops (20 g Perle—9% α-bitter acids) were added 10 min after the boiling started, and aromatic hops (17.14 g Cascade—7% α-bitter acids) were added 7 min before the boiling ended. The total α-bitter acid concentration was 60 mg/L. After the hot trub removal, the wort was cooled to 14 °C. All processes were conducted using a Home Brew 50 all-in-one 50 L brewing system (TM INOX, Plovdiv, Bulgaria). The aerated wort was placed in a stainless steel cylindroconical fermenter (TM INOX, Bulgaria), and yeast was added as per the manufacturer’s instructions. Fermentation was carried out at 14 °C and monitored hydrometrically. The “green beer” was transferred to small stainless-steel fermenters at 60% fermentation completion. The fermenter had previously been flushed with CO₂ and pasteurized bilberries (167 g/L) were added before slowly transferring the “green beer” via a hosepipe that reached the bottom of the small fermenter. After the transfer, a pressure of 0.5 bar was reached in the fermenter using CO₂. The maturation lasted for 14 days at 14 °C, followed by lagering for 5 days at 2 °C, both under pressure. All variants were duplicated. After lagering, the beer was bottled using a “beer gun” (Blichmann Engineering, Lafayette, IN, USA).

2.3. Beer Analysis

2.3.1. Sample Preparation

Beer Preparation

When the beer was bottled, the bilberries were separated from the finished beers. The required amount of beer was set aside for physicochemical analyses, while the remaining portion was stored at 2 °C until the day of the sensory evaluation. The beer for physicochemical analysis was filtered through filter paper Macherey-Nagel MN 619 14 Ø 320 (Düren, Germany) and frozen. It was defrosted immediately before the specific analysis. After defrosting, the beer was treated with methanol to precipitate proteins, left to stand for 30 min, and then filtered through filter paper. If necessary, additional dilutions with deionized water were performed.

Bilberry Preparation

To determine the phenolic content, individual phenolic compounds, and antioxidant capacity of the bilberries, 10 g of defrosted and mashed fruit was mixed with 100 mL of acidified methanol (0.1% v/v HCl). The mixture was extracted for 24 h at 4 °C in the dark. After filtration, the filtrate was topped up to 100 mL with acidified methanol.

2.3.2. HPLC Analysis of Phenolic Compounds

HPLC was conducted following the method outlined by Denev et al. [22], employing an Agilent 1220 HPLC system (Agilent Technology, Santa Clara, CA, USA) equipped with a binary pump and UV-Vis detector (Agilent Technology, USA). Separation was achieved using an Agilent TC-C₁₈ column (5 μm, 4.6 mm × 250 mm) at 25 °C, with detection at a wavelength of 280 nm. The mobile phases comprised 0.5% acetic acid (A) and 100% acetonitrile (B) at a flow rate of 0.8 mL/min. The gradient elution commenced with 14% B, linearly increasing to 25% B between the 6th and 30th minutes, then reaching 50% B at the 40th minute. Individual compounds were identified by comparing their retention times with those of standard solutions including gallic acid, 3,4-dihydroxy benzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, ellagic acid, catechin, epicatechin, rutin, naringin, myricetin, quercetin, naringenin, and kaempferol. All standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) and were with HPLC grade purity. The results were quantified using standard curves constructed on the basis of peak areas of individual compound standard solutions and expressed as mg/L sample.

2.3.3. Determination of Total Phenolic Compounds (TPCs) Using Folin–Ciocalteu Reagent (FCR)

The TPCs were assessed using a FC reagent following the methodology outlined by Nedyalkov et al. [20]. Briefly, 1 mL of sample prepared according to Section 2.3.1 was combined with 4 mL of freshly prepared FCR solution (1:10 v/v with water), followed by the addition of 5 mL of sodium carbonate (7.5%, w/v). After incubation at room temperature for 60 min, the absorbance of the mixture was measured at 765 nm against a blank prepared with distilled water. The results were quantified as mg of gallic acid equivalents (GAE)/L.

2.3.4. Assessment of Antioxidant Capacity (AOC) in Beers

• DPPH (2,2′-Diphenyl-1-picrylhydrazyl) Radical Scavenging Activity

The AOC was measured using a modification of the DPPH method as described in Nedyalkov et al. [20]. A total of 0.25 mL of sample prepared according to Section 2.3.1 was added to 2.25 mL of the 0.06 mM methanol solution of DPPH. The absorbance at 517 nm against a blank prepared with distilled water was read after being left for 30 min in the dark. The percentage inhibition was calculated against a control sample, prepared with methanol, and compared to a Trolox standard curve. The results were expressed in µmol Trolox Equivalent (TE)/L.

• FRAP (Ferric Reducing Ability of Plasma) Method

The FRAP analysis was performed according to the method described by Benzie and Strain [23], with some modifications. Briefly, the FRAP reagent was prepared by mixing 300 mM sodium acetate and glacial acetic acid buffer (pH 3.6), 20 mM ferric chloride hexahydrate, and 10 mM 4,6-tripryridyl-s-triazine (TPTZ) made up in 40 mM HCl in a 10:1:1 ratio. The FRAP assay was performed by adding 0.15 mL of the sample prepared according to Section 2.3.1 to 2.85 mL of the reagent and incubating for 4 min in the dark. Readings were taken at 593 nm against a blank prepared with methanol. The antioxidant capacity was determined using a calibration curve made with Trolox. The results were expressed in µmol TE/L.

• ABTS (2,2′-Azinobis-(3-ethylbenzothiazoline-6-sulfonate)) Radical Cation Scavenging Activity

The ability of beer to scavenge the ABTS+ free radical was investigated using a modified methodology previously reported by Iqbel et al. [24]. Equal amounts of 2.45 mM potassium persulfate and 7 mM ABTS were reacted for 12–16 h in the dark. The solution was then diluted with methanol in a 1:30 ratio to an absorbance of 1.1 ± 0.1 at 734 nm to form the test reagent. Reaction mixtures containing 0.15 mL of sample prepared according to Section 2.3.1 and 2.85 mL of reagent were incubated in the dark for 30 min. The absorbance was taken at 734 nm against methanol. The percentage inhibition was calculated against a control sample, prepared with methanol, and compared to a Trolox standard curve. The results were expressed in µmol TE/L.

• CUPRAC (Cupric Reducing Antioxidant Capacity) Method

The CUPRAC assay was performed as described in Apak et al. [25]. A total of 0.5 mL of the sample prepared according to Section 2.3.1 was mixed with 1 mL each of ammonium acetate buffer (pH 7), 7.5 mM neocuproine in ethanol, and 10 mM copper (II) chloride dihydrate solution. In the end, 0.6 mL of distilled water was added and the mixture was left for 30 min at room temperature. The absorbance was recorded at 450 nm against a blank prepared with distilled water. The standard curve was established using Trolox. The results were expressed in µmol TE/L.

2.3.5. Sensory Evaluation

A trained panel consisting of five members conducted the sensory evaluation of the beverages. The evaluation followed the descriptive method (13.10) outlined in the EBC guidelines [26].

2.4. Statistical Analysis

The results presented in this study show the average values derived from the 2-fold repetition of the experimental variants and a minimum of three analytical measurements for each repetition. The coefficients of variation expressing the percentage relationship between the standard deviations and the mean values were all found to be below 5%. Mean calculations were performed using Microsoft Excel™ at a confidence level of 95%. Correlation coefficients were determined using Excel’s built-in functions. Additionally, one-way ANOVA and Scheffe’s multiple range test, as detailed by Donchev et al. [27], were applied with the significance level set at p < 0.05.

3. Results and Discussion

The beers produced for the experiment were with original extracts of 12 °P, 14 °P, and 16 °P. The values of alcohol content and the real extract of all beers, with and without bilberry, were normal for these original extracts and the attenuation of the lager type yeast that was used. The results were published in our previous article [19]. Based on our earlier studies and observations, notable variations were identified in the sensory attributes, chemical profile of beer, and yeast-derived metabolites when ingredients such as blueberries, bilberries, cherries, cherry pomace, and juice were introduced during the primary fermentation and maturation processes [17,18,19,20,21].

3.1. Phenolic Profile of the Beers

3.1.1. Total Phenolic Compounds

Phenolic compounds are known to play an important role in beer stability and sensory characteristics [7,10,28]. Also, they possess antioxidant activity and may be a major factor in assuring the antioxidant potential of the diet [10]. The concentration of the total phenolic compounds determined by the FC method in the bilberry beers was between 1.8 and 2 times higher than that in the control beers. Its values ranged between 481 ± 12 and 502 ± 4 mg/L in the variants without bilberries and between 893 ± 13 and 957 ± 32 mg/L in the beers with a bilberry addition (

Table 1. Total phenolic compounds in bilberries and beers.

	Total Phenolic Compounds
Bilberries, mg GAE/100 g	365 ± 9 a
Control Beers, mg GAE/L
Original extract 12 °P	502 ± 4 b
Original extract 14 °P	491 ± 9 b
Original extract 16 °P	481 ± 12 b
Beers with bilberries, mg GAE/L
Original extract 12 °P	893 ± 13 c
Original extract 14 °P	957 ± 32 d
Original extract 16 °P	939 ± 7 d

Different letters indicate significant statistical differences (p < 0.05).

). The original extract of the beers did not significantly affect the concentration of total phenolic compounds in the beers. M. Nardini and I. Garaguso [1] also analyzed the total phenolic compounds determined by the FC method in several beers and found that the total phenolic compounds in the fruit beers were 1.5 and 2.3 times higher than those in the conventional beers without a fruit addition. For the purposes of the experiment, they used lager, ales, and lambic beers as controls, and fruit beers with cherry, orange, grape, plum, raspberry, peach, apricot, and apple; however, they did not provide any data on bilberry beers.

Mitic et al. [10] studied phenolic compounds in lager, dark, and alcohol-free beers marketed in Serbia. Similar research was conducted by Zhao et al. [9] on beers marketed in China. The results obtained on the total phenolic compounds of our control beers were in agreement with those reported by Mitic et al. (328.22–545.32 mg GAE/L [10] and Nardini and Garaguso (321–482 mg GAE/L) [1], but higher than those found by Zhao et al. (152.01–339.12 mg GAE/L) [9]. Furthermore, our data on the total phenolic compounds of beers with bilberries were higher than those on the fruit beers analyzed by Nardini and Garaguso (399–767 mg GAE/L) [1].

The total phenolic content determined using the Folin–Ciocalteu reagent could not provide information on the differences in the phenolic acids and flavonoids in the samples; therefore, it was important to determine the individual phenolic compounds.

3.1.2. Phenolic Acids

Control Beers

The results of the HPLC analysis of the phenolic acids (

Table 2. Phenolic acids and flavonoids in bilberries and beers.

Compounds	Bilberries, mg/100 g	Original Extract 12 °P		Original Extract 14 °P		Original Extract 16 °P
		Control Beer Sample, mg/dm3	Beer Sample with Bilberries, mg/dm3	Control Beer Sample, mg/dm3	Beer Sample with Bilberries, mg/dm3	Control Beer Sample, mg/dm3	Beer Sample with Bilberries, mg/dm3
Phenolic acids
Chlorogenic	57.39 ± 1.73	19.04 ± 0.54 b	130.98 ± 2.03 c	13.51 ± 0.38 d	116.86 ± 0.54 e	34.12 ± 0.22 f	143.84 ± 0.81 g
Neochlorogenic	NSP	48.96 ± 0.35 a	77.02 ± 1.85 b	41.89 ± 0.01 c	65.28 ± 0.37 d	26.32 ± 0.51 e	39.42 ± 0.92 f
Vanillic	17.90 ± 0.53	7.78 ± 0.24 b	21.60 ± 0.64 c	8.50 ± 0.01 d	18.18 ± 0.00 e	9.00 ± 0.18 f	16.69 ± 0.61 g
Caffeic	3.97 ± 0.22	2.08 ± 0.21 b	16.13 ± 0.97 c	1.95 ± 0.04 b	16.03 ± 0.79 c	ND	21.75 ± 0.57 d
3,4-dihydrobenzoic	NSP	7.67 ± 0.40 a	39.88 ± 0.72 b	7.43 ± 0.19 a	56.43 ± 0.97 c	11.17 ± 1.15 d	32.48 ± 0.54 e
Ferulic	5.28 ± 0.04	3.60 ± 0.45 b	7.73 ± 0.48 c	3.79 ± 0.06 b	7.90 ± 0.34 c	4.85 ± 0.16 d	7.71 ± 0.26 c
p-coumaric	0.83 ± 0.09	0.90 ± 0.10 b	3.02 ± 0.11 c	0.59 ± 0.00 d	3.15 ± 0.19 c	0.84 ± 0.04 b	2.95 ± 0.04 c
Gallic	NSP	28.78 ± 0.23 a	51.08 ± 0.38 b	30.73 ± 0.03 c	35.32 ± 0.92 d	33.68 ± 0.71 e	43.28 ± 0.75 f
Cinnamic	0.53 ± 0.01	0.42 ± 0.04 b	1.10 ± 0.09 c	0.45 ± 0.04 b	1.24 ± 0.04 d	0.65 ± 0.04 e	1.62 ± 0.05 f
Rosmarinic	5.96 ± 0.17	ND	3.06 ± 0.39 a	ND	2.28 ± 0.04 a	ND	2.90 ± 0.53 a
Calculated total phenolic acids	91.86 ± 2.79	119.23 ± 2.56 b	351.60 ± 7.66 c	108.84 ± 0.76 d	322.67 ± 4.20 e	120.63 ± 3.01 b	312.64 ± 5.08 e
Flavonoids
Quercetin	3.76 ± 0.04	4.47 ± 0.53 b	4.94 ± 0.53 b	3.31 ± 0.03 c	5.02 ± 0.37 b	3.63 ± 0.44 c	8.81 ± 0.58 d
Quercetin-3-glucoside	13.41 ± 0.61	3.40 ± 0.33 b	6.05 ± 0.33 c	3.71 ± 0.45 b	5.95 ± 0.47 c	4.65 ± 0.55 d	4.68 ± 0.26 d
Rutin	175.69 ± 5.69	5.94 ± 0.49 b	216.53 ± 5.92 c	3.35 ± 0.37 d	185.86 ± 4.00 e	14.72 ± 0.01 f	240.16 ± 5.02 g
Myricetin	4.80 ± 0.16	1.94 ± 0.16 b	8.00 ± 0.31 c	3.47 ± 0.51 d	8.36 ± 0.16 c	3.87 ± 0.45 d	10.62 ± 0.42 e
Kaempferol	0.46 ± 0.01	0.90 ± 0.08 b	1.51 ± 0.17 c	1.05 ± 0.06 b	2.36 ± 0.24 d	0.91 ± 0.11 b	2.35 ± 0.18 d
Catechin	138.79 ± 0.66	23.73 ± 0.44 b	101.78 ± 2.12 c	17.97 ± 0.64 d	105.39 ± 5.30 c	26.24 ± 0.57 e	106.55 ± 3.25 c
Epicatechin	47.82 ± 0.86	28.06 ± 0.45 b	34.28 ± 0.55 c	23.17 ± 0.40 d	36.52 ± 3.08 c	33.25 ± 3.07 c	28.64 ± 1.80 b
Calculated total flavonoids	384.73 ± 8.03	68.44 ± 2.48 b	373.09 ± 9.93 c	56.03 ± 2.46 d	349.46 ± 13.62 e	87.27 ± 5.20 f	401.81 ± 11.51 g
Calculated total phenolic compounds	476.59 ± 10.82	188.37 ± 5.11 b	742.69 ± 17.59 c	164.87 ± 3.22 d	672.13 ± 17.82 e	207.90 ± 8.21 f	714.45 ± 16.59 c

Each value is the mean ± SD of triplicate determination. Different letters for the values of beer samples in the rows indicate significant statistical differences (p < 0.05). NSP—peaks not separated well enough. ND—not detected.

) showed that gallic, chlorogenic, and neochlorogenic acids predominated in the control beers. Their total share was between 65% and 73% of all the investigated phenolic acids. According to Wanenmacher et al., most studies describe ferulic and p-couma ric acids as the most abundant phenolic acids in beer [7]. Gallic and ferulic acids were the dominant phenolic compounds identified in beers on the Chinese market [9]. Moura-Nunes et al. [11] reported that the phenolic profile of Brazilian beers was distinct from that of European beers in terms of the high gallic acid and low ferulic acid content. According to Mitic et al. [10], gallic, ferulic, and protocatechuic acids were the most abundant phenolic acids in all the analyzed beers.

The concentration of p-coumaric acid in our control beers was similar to data for other beers without fruits reported by Zhao et al. [9], Mitic et al. [10], and Moura-Nunes et al. [11]; the content of caffeic acid was similar to the results of Zhao et al. [9], but a little bit higher than reported by Mitic et al. [10]. Our control beers contained more ferulic and vanillic acids than beers analyzed by Zhao et al. [9], Mitic et al. [10], and Moura-Nunes et al. [11]. The concentration of gallic acid was similar to those determined by Mitic et al. [10], but higher than mentioned by Zhao et al. [9] and Moura-Nunes et al. [11]. According to the results of Nardini and Garaguso [1], chlorogenic and neochlorogenic acids are in traces or not detected in beers without fruits. In their review article, Wanennmacher et al. [7] have summarized the information from different researchers and chlorogenic acids in beers ranging from traces to 10.96 mg/L, while the concentration of neochlorogenic acid in beers is not mentioned. The raw materials, the malting, and brewing processes influence the chemical composition of beers [29], which may explain the variation in phenolic acids determined by different researchers and our results.

According to our data, a difference was observed in the ratio between chlorogenic acid and neochlorogenic acid in the control beers with different original extracts. The share of neochlorogenic acid decreased with the increase in the original extract of the beer. Neochlorogenic acid predominated in the beers with an original extract of 12 °P and 14 °P, while chlorogenic acid predominated in the beer with 16 °P (Table 2). The original extract of the beer influenced the phenolic acid profile in the beer without bilberries, but there was no clear trend.

Bilberries

The highest amount of the phenolic acids determined in the bilberries was held by chlorogenic (62.5%) and vanillic (19.5%) acids (Table 2). According to Moze et al. [15], Su et al. [12], and Diaconeasa et al. [16], chlorogenic acid was the main phenolic acid found in blueberries (Vaccinium corymbosum L.) and bilberries (Vaccinium myrtillus L.). This agreed with our findings. The amount of the other determined phenolic acids in the bilberries was less. The peaks of neochlorogenic, gallic, and 3,4-dihydroxybenzoic acids merged into one large initial peak and could not be determined, so they were not included in the total amount calculation. Similarly to our results, Moze et al. [15] identified caffeic, ferulic, and p-coumaric acids in bilberries. Su et al. [12] reported the presence of vanillic, ferulic, caffeic, gallic, and 3,4-dihydrobenzoic acids in blueberry samples from China. It is expected that phenolic acids from bilberries will extracted in beers during beer maturation.

Beers with Bilberries

The total concentration of phenolic acids in beers with bilberries was between 2.6- and 3.0-fold more than in the control beers, but the concentration of some individual phenolic acids was many times greater. A bigger increase was observed in the amount of chlorogenic acid—between 4.2 and 8.6 times more than control beers; caffeic acid—between 7.8 and 8.2 times more; and 3,4-dihydrobenzoic acid—up to 7.6 times more. The increase in the concentration of the other phenolic acids was smaller: 3.4 to 5.3 times for coumaric acid, and 1.1 to 2.8 times for gallic, neochlorogenic, ferulic, cinnamic, and vanillic acids. However, the concentration of all analyzed phenolic acids in the beers with bilberries was bigger than those in the control beers.

Compared to the data from the literature, our beers with bilberries contained chlorogenic acid 10 to 100 times and vanillic acid 4 to 500 times more than fruit beers analyzed by Nardini and Garaguso [1]. Most of the fruit beers mentioned in Nardini and Garaguso’s article contained much less neochlorogenic, ferulic, caffeic, and p-coumaric acids than beer with bilberries investigated by us. The concentration of neochlorogenic acid in beer with plums (60.3 ± 1.24 Nardini and Garaguso) and p-coumaric acid in one of the beers with cherries (9.40 ± 0.66 Nardini and Garaguso) and in beers with grapes (4.61 ± 0.07 Nardini and Garaguso) was higher than the concentration in our bilberry beers. The content of ferulic acid in beers with bilberries and beer with grapes (7.05 ± 0.43 Nardini and Garaguso) was comparable.

Based on these results, it can be claimed that the bilberry addition enriches beers with many phenolic acids much more than the addition of other fruits.

The bilberry addition in the beer led to a change in the phenolic acid profile. There was a significant decrease in the shares of neochlorogenic and gallic acids. The shares of vanillic and ferulic acids also decreased but to a lesser degree. The share of chlorogenic, caffeic, and 3,4-dihydroxybenzoic acids increased significantly. The share of p-coumaric acid also increased to a lesser degree. The variants with bilberries contained rosmarinic acid, between 0.5% and 0.9% of the total amount of phenolic acids analyzed, while rosmarinic acid was absent in the control beers. The cinnamic acid share remained essentially unchanged, varying between 0.3% and 0.5% of the total amount of phenolic acids analyzed in all experimental beer variants.

The original beer extract did not affect the changes in concentration of individual phenolic acids in the same way. It did not influence the amount of rosmarinic, ferulic, and p-coumaric acids in the beers with different original extracts. No significant difference was observed in the concentration of these acids, while the concentration of chlorogenic, neochlorogenic, vanillic, and gallic acids varied in the beers with different original extracts (

Table 3. Antioxidant capacity of bilberries and beer samples.

Antioxidant Capacity	CUPRAC	FRAP	ABTS	DPPH
Bilberries, Trolox μmol/100 g	8150 ± 123 a	4581 ± 77 a	3346 ± 83 a	954 ± 67 a
Control beers, Trolox μmol/dm3
Original extract 12 °P	2248 ± 239 b	1154 ± 56 b	973 ± 25 b	996 ± 45 b
Original extract 14 °P	2914 ± 128 c	1123 ± 70 b	1429 ± 51 c	903 ± 96 b
Original extract 16 °P	2113 ± 129 b	881 ± 36 c◦	940 ± 79 b	897 ± 50 b
Beers with bilberries, Trolox μmol/dm3
Original extract 12 °P	7107 ± 518 d	3555 ± 245 d	2681 ± 44 d	2396 ± 80 c
Original extract 14 °P	9423 ± 435 e	4253 ± 253 e	4056 ± 312 e	2089 ± 74 d
Original extract 16 °P	8132 ± 20 f	3365 ± 58 d	2991 ± 112 f	2465 ± 77 c

Different letters in the columns indicate significant statistical differences (p < 0.05).

). The alteration trend was not the same for the mentioned acids. The concentration of neochlorogenic and vanillic acids decreased with the original extract’s growth, while the concentration of chlorogenic and gallic acids decreased when the original extract raised from 12 °P to 14 °P and increased when the original extract raised from 14 °P to 16 °P.

3.1.3. Flavonoids

Control Beers

The results of the HPLC analysis of flavonoids (Table 2) showed that epicatechin and catechin predominated in the beers without bilberries. They represented 68% to 76% of all flavonoids analyzed. According to Wannenmacher et al. [7], (+)-catechin is described as the most abundant flavan-3-ol monomer in beer. In the study of Zhao et al. [9], the beers analyzed exhibited a relatively high level of (+) catechin, while the (−) epicatechin values were much lower. The concentration of catechin and epicatechin in beers analyzed by other researchers were many times lower than our control beers. Zhao et al. [9] reported a range between 0.08 and 4.00 mg/L for catechin and 0.02–0.73 mg/L for epicatechin, while the data from Mitic et al. [10] were 0.57–1.27 mg/L and 0.08–0.43 mg/L, respectively. The beers analyzed by the mentioned researchers were purchased from a local market and based on their trademarks; they were not turbid. Brewers use PVPP to clarify and stabilize beers. In a study by McMurrough et al., cited by Wannenmacher et al. [7], 48% of total polyphenols, 78% of total flavonols, 79% of (+)-catechin, and 88% of (−)-epicatechin were removed from beer treated with 100 g/hL PVPP. This could explain the higher concentration of flavonoids in our beer samples. They were not treated with PVPP.

Bilberries

Flavonoids such as catechin, epicatechin, quercetin, myrecetin, rutin, and other flavonoid glycosides have been identified in blueberries (Vaccinium corymbosum L.) and bilberries (Vaccinium myrtillus L.) [15,16]. The same flavonoids as well as kaempferol and quercetin-3-glucoside were identified in bilberries used in this study (Table 2). Rutin and catechin dominated.

Beers with Bilberries

The bilberry addition during maturation led to a significant increase in the concentration of flavonoids (between 4.6- and 6.2-fold) and the flavonoid profile of the beers. The concentration of rutin raised much more than the average increase in total flavonoids. The beers with bilberries contained between 16 and 56 times more rutin than the control beers. Rutin was the dominant compound in the beers maturated with bilberries, followed by catechin and epicatechin. They represented 93% to 95% of the total flavonoid concentration determined.

According to Nardini and Garaguso [1], catechin and quercetin were present in all the fruit beers analyzed; myricetin was in seven of ten fruit beers, while rutin was only present in the orange and apple fruit beers. The concentration of catechin in fruit beers analyzed by Nardini and Garaguso ranged from 1.00 mg/L and 20.40 mg/L and quercetin between 0.3 mg/L and 7.10 mg/L and the highest values were detected in the beers with cherries. The data obtained by us for these two flavonoids demonstrated that the bilberry beers were much richer with rutin and catechin than the cherry beers and other fruit beers analyzed by Nardini and Garaguso. The concentrations of myricetin and quercetin in our samples with bilberries were higher, too.

The original extract of the control beers and the beers with bilberries affected the quantity of the flavonoids studied, but not in the same way. The catechin concentration was comparable in all three bilberry beers regardless of the original extract, while the rutin concentrations varied significantly. For the other flavonoids, in some cases, there was no statistically significant difference in the concentration of a specific flavonoid in two of the beers with different original extracts.

Regarding the differences in the phenolic profiles of the beers studied, the following hypothesis can be formulated. Probably, the different technological modes (brewing duration and malt/water ratio) used to obtain worts with different original extracts had this effect. In addition, the different alcohol concentrations formed after the wort fermentation with different original extracts could also have had an influence. These different parameters affected the composition and concentration of all substances in the beer and the degree of their interaction with the phenolic compounds. In a review article, Wannenmacher et al. [7] reported the influence of mash thickness, wort density, and boiling times on the ferulic acid concentration and polyphenol content in the wort.

3.2. Antioxidant Capacity of Beers

The antioxidant capacity of the beers was investigated using four methods. The values observed with the different methods increased in the following order: DPPH < ABTS < FRAP < CUPRAC (Table 3). This was in an agreement with the results reported by Taffulo et al. [13]. With a few exceptions, the original extract of the wort did not significantly influence the antioxidant capacity of the control beers determined by various methods.

The data from the ABTS assay (Table 3) showed that the antioxidant capacity of the beers with bilberries was between 2.8 and 3.2 times greater than the control beers. The greatest increase was observed in the 16 °P beer, while the smallest increase was in the 12 °P variant. The original extract of the beer significantly influenced its antioxidant capacity determined by the ABTS method. The AOC was the greatest in the variants with a 14 °P original extract, both with the control beer and the bilberry beer. The ABTS values of the control beers were in agreement with the ones observed by Tafulo et al. (632.2–1079.2 μM TE) [13], Zhao et al. (0.55–1.95 mmol TE/L) [9], and Nardini and Garaguso (1.29–2.03 mmol TE/L) [1], and were higher than those reported by Mitic et al. (0.17–0.33 mmol TE/L) [10] and Socha et al. (0.25–0.72 mmol TE/L) [14]. The antioxidant capacity of the beers with bilberries determined by the ABTS assay was similar to the results on fruit beers (1.62–3.53 mmol TE/L) reported by Nardini and Garaguso [1], and the beer with 14 °P demonstrated a higher value.

The DPPH radical scavenging activity of the beers analyzed (Table 3) was similar to the results of Tafulo et al. (608.1–2053.9 μM TE) [13]. Other researchers reported lower values: 0.23–0.67 mmol TE/L [12]; 0.26–0.65 mmol TE/L [10]; and 0.24–1.35 mmol TE/L [9]. The bilberry beers exhibited an antioxidant capacity between 2.3 and 2.7 times higher than that of the control beers as determined by the DPPH method. The greatest increase was observed in the 16 °P beer, while in those with original extracts of 12 °P and 14 °P the increase in the antioxidant capacity was comparable.

The antioxidant capacity of the beers with the bilberry addition determined using the CUPRAC method (Table 3) was between 3.2 and 3.8 times greater than that of the control beers. The largest increase was observed in the 16 °P beer, while in those with original extracts of 12 °P and 14 °P it was comparable. The original extract of the beers significantly influenced their antioxidant capacity determined using the CUPRAC method. It was the highest in the variants with an original extract of 14 °P, both in the control and the bilberry beers. Our data on the control beers were in agreement with those of Tafulo et al. (977.5–3423.0 μM TE) [13], while the values of the beers with bilberries were higher than those reported by Tafulo et al. [13].

The bilberry addition led to a 3.1- to 3.8-fold increase in the antioxidant capacity of the beers determined using the FRAP method. The smallest increase was observed in the 12 °P beer, while in those with original extracts of 14 °P and 16 °P it was comparable. The highest antioxidant capacity in the bilberry beers was observed in the 14 °P variant. In the control beers, the 16 °P variant had the lowest antioxidant capacity, while the values of the 12 °P and 14 °P variants were comparable. The FRAP values obtained for the control beers and the beers with bilberries were higher than those reported by Tafulo et al. (121.6–553.4 μM TE) [13]. The results reported by Mitic et al. (22.99–831.20 mmol FE/L) [10] and Nardini and Garaguso (3.08–9.76 mM Fe₂SO₄ Equivalent) [1] were expressed in different equivalents and were difficult to compare. It was interesting to note that the bilberry beers showed a metal chelating activity higher than not only the control beers analyzed but also the data on beers reported in the literature, as determined using the FRAP and CUPRAC methods.

The observations showed that the original extracts in the beers maturated with bilberries influenced their antioxidant capacity determined by different methods. A possible explanation of the observed trend may lie in the different alcohol content and differences in the concentration, molecular weight, and chemical structure of the protein and polysaccharide substances in the beer samples with different original extracts. Probably, due to these differences in the starting matrix of the beer, the phenolic compounds extracted from the bilberries interacted to varying degrees with the proteins and polysaccharides from the beer, which was reflected in the concentration of the remaining substances and the exhibited antioxidant activity in the beer. Other studies conducted by us showed significant changes in the concentration and molecular weight of the protein fractions in bilberry beers with different original extracts [19]. A number of other researchers investigated the protein–polyphenol precipitation in beer. Zhao and Sun-Waterhaus [30] reported this in their review article.

Most often, the antioxidant capacity of the beer is related to polyphenol compounds and melanoidins, as confirmed in many articles [1,7,9,10,11,14]. Some peptides possess antioxidant properties, too. In a review article, Tang-Bin Zou et al. [31] summarized the available information about the relationship between the structure of peptides and their antioxidant activities.

We found a strong correlation both between the results obtained by different methods used to assess antioxidant capacity and with the data on total phenolic compounds. The correlation coefficients between the values of the antioxidant capacity determined by different methods and total phenolic compounds (

Table 4. Correlation coefficients between antioxidant capacity determined using different methods and total phenolic compounds.

	CUPRAC	DPPH	FRAP	FC	TPhC (Calculated from HPC)
ABTS	0.986	0.852	0.972	0.946	0.892
CUPRAC	-	0.926	0.988	0.986	0.951
DPPH	-	-	0.933	0.974	0.992
FRAP	-	-	-	0.986	0.962
FC	-	-	-	-	0.988

) were above 0.85. It is known that polyphenols may act as antioxidants via different mechanisms: free radical inactivation by hydrogen atom transfer (HAT), or single electron transfer (SET) reactions, or by chelating transition metal ions [7]. The high correlation coefficients indicated that the phenolic compounds in the beers analyzed showed a good free radical scavenging activity and metal reducing and chelating ability. The beer samples that were rich in phenolic antioxidants had higher quality, more stable sensory properties, such as flavor and aroma, foam stability, and a longer shelf life when compared to beer with lower antioxidant activity [10].

The antioxidant activity of phenolic compounds depends on their chemical structure, the number and position of OH groups, whether the phenolic acids are cinnamic derivatives or benzoic derivatives, electron delocalization in the aromatic nucleus, or the presence of esterification or glycosylation [32,33]. It could be hypothesized that the significant increase in the antioxidant capacity of the beers with bilberries compared to the control beers was due to multiple magnification of the phenolic compounds determined, such as catechin, rutin, chlorogenic acid, and caffeic acid. According to Mitic et al. [10], caffeic, vanillic, sinapic, and ferulic acids are the major contributors to the antioxidant activity of beer. Catechin, rutin, and chlorogenic acid also possess high antioxidant activity [33]. Therefore, we found correlation coefficients between the antioxidant capacity determined using different methods and individual phenolic acids and flavonoids (

Table 5. Correlation coefficients between the antioxidant capacity determined using different methods and individual phenolic acids and flavonoids.

	ABTS	CUPRAC	DPPH	FRAP	FC
Chlorogenic	0.858	0.929	0.990	0.929	0.974
Neochlorogenic	0.603	0.596	0.602	0.702	0.617
Vanillic	0.846	0.895	0.946	0.936	0.936
Caffeic	0.839	0.926	0.980	0.902	0.968
3,4-dihydrobenzoic	0.965	0.953	0.845	0.973	0.932
Ferulic	0.902	0.947	0.952	0.950	0.971
p-coumaric	0.923	0.968	0.975	0.982	0.993
Gallic	0.519	0.628	0.843	0.672	0.729
Cinnamic	0.835	0.903	0.928	0.860	0.928
Rozmarinic	0.699	0.814	0.992	0.829	0.910
Quercetin	0.538	0.643	0.751	0.578	0.696
Quercerin-3-glucoside	0.775	0.780	0.750	0.829	0.792
Rutin	0.866	0.936	0.997	0.936	0.979
Myricetin	0.863	0.925	0.947	0.887	0.945
Kaempferol	0.943	0.955	0.850	0.902	0.924
Catechin	0.919	0.969	0.983	0.974	0.996
Epicatechin	0.539	0.529	0.474	0.597	0.545

). Based on these coefficients, it could be asserted that changes in the antioxidant capacity of the beers maturated with bilberries compared to the control beers were most strongly influenced by changes in the concentration of the following phenolic acids: chlorogenic, vanillic, ferulic, and p-coumaric, as well as the flavonoids rutin, myricetin, kaempferol, and catechin. Changes in the concentrations of cinnamic, caffeic, and, to some extent, 3,4-dihydroxybenzoic acid also showed a strong correlation with the antioxidant capacity data obtained using the different methods.

3.3. Sensory Evaluation

The organoleptic characteristics of the bilberry beers differed significantly from those of the control beers (Figure 1).

The sweet malty aroma in the control beers was replaced by a fresh and fruity aroma in the beers maturated with bilberries. The same tendency was also observed in the flavor. Similar results were achieved by Zapataa et al. when quince fruit was added to beer. The control sample exhibited more caramel notes; however, when the fruit was added, these notes were replaced by floral, fruity, and pome fruit ones [34].

The trained panelists gave a major note that the bilberry beers still possessed the beer taste, even if they look like a Radler with forest fruits (Figure 1a). It is shown in Figure 1 that the bilberry beer with original gravity 14 °P possessed the best fruit and fresh aroma among the bilberry beers. The body of the control beer with 16 °P, as expected, was the most full one. It was also expected to have reduction in body and head retention in the beer samples with bilberry addition because of the reaction between proteins and the bilberry phenolic compounds [19], but there was no significant reduction (Figure 1a). The best fruit taste and balance in the flavor and body were found in the 14 °P beer sample maturated with bilberry. The highest score for the aftertaste was also given in the 14 °P sample. It possessed the best fruit aftertaste, longest length in the aftertaste, and the most successful balance between aroma and taste.

4. Conclusions

This study provides new information about changes in the antioxidant capacity, phenolic profile, and the sensory properties of beers with different original extracts maturated with bilberries. To the best of our knowledge, the obtained data address a gap in understanding the impact of technological parameters on the characteristics of bilberry beers, with a specific focus on this aspect, rather than on any health-related considerations.

Maturing beer with bilberries introduced numerous changes in its properties. Antioxidant capacity, as well as phenolic acid and flavonoid concentrations, significantly increased. The original extract of the beers influenced all examined parameters, but in different ways. The enhanced antioxidant activity was deemed advantageous for improving beer stability. Further experiments are necessary to deeply investigate this relation.

The bilberry addition positively influenced the taste and aroma of the beers. The 14 °P variant was estimated as the beer with the most pleasant sensory characteristics among the bilberry beers investigated. The findings from this study can support the development and production of innovative bilberry-infused beers. Moreover, they hold the potential for optimizing brewing processes to achieve bilberry beers with superior antioxidant properties and sensory characteristics in the future.