Determination of Concentration of Prenylated Flavonoids and Analysis of Physicochemical Parameters of Beers Available on the Polish Market

Abstract

A total of 35 commercially available beers (of various beer styles) produced in Poland were analysed in this study to assess the concentration of xanthohumol and isoxanthohumol, prenylated flavonoids originating from the hops, which are known to possess multiple health-benefitting properties. High-performance liquid chromatography coupled with a UV/VIS DAD detector was utilised to identify and quantify hop flavonoids. Additionally, physicochemical parameters, such as wort extract content, extract content, alcohol content, and degree of attenuation, were analysed in all the samples. The xanthohumol content of the Polish beers varied the most from the analysed flavonoids and ranged from 0.029 to 2.459 mg per L of the beer. The concentration of the isoxanthohumol was, on average, higher and ranged from 0.621 to 2.510 mg per L.

1. Introduction

Beer is a popular beverage acquired through the alcoholic fermentation of wort, which is produced from malt and hops [1]. These ingredients are the source of most of the bioactive components in the finished product [2]. In recent years, many studies have focused on strategies to increase the content of phenolic compounds in beer, such as, for example, the production of beers with dark malts or the addition of fruit or fruit juices [1,3,4,5]. However, compounds with very interesting, health-benefiting properties do not necessarily need to be introduced through the addition of novel ingredients because hops are an excellent source of a particularly interesting group of compounds, namely prenylated flavonoids [6,7].

The main prenylated flavonoids originating from hops, and that are present in beer, are xanthohumol (XN) and isoxanthohumol (IXN) [8]. These compounds received considerable attention in recent years because numerous beneficial effects associated with their consumption have been reported [9]. Among them, XN has attracted the greatest interest, as it exhibits high antioxidant potential, and multiple studies have demonstrated its potential role in the alleviation of several non-communicable diseases affecting the world’s population, such as diabetes, cancer, stroke, cardiovascular disease, hyperlipidaemia, and obesity [10,11,12,13,14,15,16]. Research data have also shown that XN possesses antibacterial and antiviral properties, which could eventually be used against many common pathogens, such as herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), human immunodeficiency virus (HIV), or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [17,18].

A crucial aspect of XN is that beer is almost the sole source of this compound for humans [19]. Furthermore, XN is the most abundant prenylated flavonoid in the hop cones, accounting for approximately 0.1–1% of their dry weight [20,21,22]. However, there are significant challenges associated with the production of beer with a high concentration of XN, as this compound tends to isomerise into a less active form, IXN, during boiling of the wort, which is a critical step in the brewing process [1,23]. Additionally, XN is lost at several stages of beer production, including centrifugation in the whirlpool, filtration after wort cooling, and adsorption onto cold-precipitated proteins during beer fermentation, and it is also removed with the yeast in the process of beer filtration [24].

Nevertheless, it should be noted that several approaches for increasing the concentration of XN and other prenylated flavonoids in finished beer have been identified [25]. One technology that can be used to increase XN content is the dry-hopping procedure, in which hop cones or hop pellets are added to the beer after the completion of primary yeast fermentation. This technique is primarily used for certain beer styles (a general term referring to beers brewed using specific methods), such as India Pale Ale (IPA) and related styles, including Pale Ale, American IPA, West Coast IPA, and New England IPA [26]. During brewing of IPAs and similar beers, a large part of XN does not isomerize, as the beer is not heated during dry-hopping, during which a large portion of hops is typically added [27]. Furthermore, the presence of ethanol in beer enhances the extraction of XN from hops because XN and other prenylated flavonoids are considerably more soluble in organic solvents such as ethanol than in water [28]. Another way of increasing the concentration of XN in beer is the production of dark beers, using chocolate or caramel malts, as previous studies indicated that XN may form chemical complexes with Maillard reaction products [29,30]. However, these findings have not been definitively proven, and the influence of these findings has not yet been conclusively confirmed; interactions between XN and Maillard reaction products remain hypothetical.

In this study, beers of different types (commonly referred to as “beer styles”) were analysed to assess the range of XN and IXN concentrations in beers available on the Polish market.

The aim of the study was to determine whether the different beer styles could possess higher concentrations of hop prenylated flavonoids, which can potentially direct consumer preference for choosing particular beer styles characterised by higher prenylated flavonoid concentration. The concentrations of xanthohumol and isoxanthohumol were analysed in 35 commercially available beers produced in Poland. Additionally, basic physicochemical parameters, including extract content, alcohol content, degree of attenuation, and the original wort extract, were determined.

2. Materials and Methods

2.1. Materials

2.1.1. Research Material (Beer Samples)

The research material studied and described in this manuscript consisted of 35 commercially available beers sold on the Polish market, representing a variety of beer styles. Eight samples were of the American Indian Pale Ale style (AIPA); nine samples were of the Pale Ale style (PALE); three samples were of the Indian Pale Ale style (IPA); two samples were of the stout style (ST), three samples were of the porter style (POR); four samples were of the lager style (LAG); four samples were of the Pilsener style (PIL); and two samples were of the witbier style (WIT). All of the beers were manufactured by different breweries. Beers were stored at 4 °C, in darkness (a refrigerated cellar).

2.1.2. Chemical Reagents and Standards

Chemical reagents used in this study were: methanol (99%), phosphoric (V) acid (85%), formic acid (99%), acetonitrile (99.5%), and diatomaceous earth acquired from Chempur company (Piekary Śląskie, Poland). Standards used in this study (xanthohumol and isoxanthohumol) were prepared in-house according to previously validated methods [31,32,33].

2.2. Methods

2.2.1. Preparation of the Beer Samples for Extraction of the Prenylated Flavonoids

Sixty mL of each beer sample, before the extraction process, was degassed by sonication in the ultrasonic bath XUB5 (Grant Instruments, Shepreth, UK) for 15 min. Beer was then centrifuged in the centrifuge tubes (volume 150 mL) in the MPW-351R (MPW Medical Instruments, Warsaw, Poland) centrifuge (5000 rpm, 10 min). Fifty mL of the samples were transferred to a 100 mL volumetric flask, and 0.1 mL of phosphoric (V) acid (85% purity) was added. The flask was then filled to volume with distilled water.

2.2.2. Extraction of the Beer Prenylated Flavonoids

The high hydrophobic analytes from beer, such as isoflavonoids and prenylflavonoids, were extracted using a solid-phase extraction (SPE) column Strata C18-E (Phenomenex, Torrance, CA, USA), with a sorbent mass of 500 mg and column volume of six mL (80 mm height and 11 mm internal diameter), according to the methodology previously described by Jurkova et al. [34]). SPE was performed using a solid-phase extraction system (SPE system), which was constructed from twelve previously mentioned Strata C18-columns assembled with a vacuum manifold AHO-6023 (Phenomenex, Torrance, CA, USA) and a vacuum pump MZ 2C NT (Cleaver Scientific, Rugby, UK). Four solvents were used to perform the extraction of beer prenylated flavonoids:

• Solvent 1—Methanol (99%).
• Solvent 2—Water: phosphoric (V) acid (100: 0.2 v/v).
• Solvent 3—Methanol: water: phosphoric (V) acid (10: 80: 0.2 v/v).
• Solvent 4—Methanol: phosphoric (V) acid (100: 0.2 v/v).

The underpressure of the SPE system was set at the level of 7–8 kPa. Sorbent in the columns was conditioned by eluting ten mL of Solvent 1 and ten mL of Solvent 2. Solvents were added to the columns by dripping a volume of the solvents drop by drop, with a ratio of around 100 drops per minute. Just after the conditioning, acidified beer (Section 2.2.1.) was poured onto the columns. Ten mL of Solvent 3 was added after the beer was filtered to reduce polar impurities from the samples. The addition of the beer and Solvents 1–3 was kept at the same rate throughout all the analyses. After the Solvent 3 was eluted from the column, the SPE system was left with a working vacuum pump for an additional ten min; however, the valve of the vacuum system was opened to ‘dry out’ the columns. After ten min, the valve was closed, and the underpressure of the SPE system was set at the level of 7–8 kPa. Needles were attached to the ends of the columns of the SPE system, and five mL of Solvent 4 was added to the columns to elute the analytes. Analytes were collected into 2.5 mL tubes, which were then filled with Solvent 4 to a final volume of 2.5 mL. Solvent 4 with the analytes was then filtered through the 0.45 µm syringe filter and analysed by means of HPLC. Extraction of the prenylated flavonoids was performed thrice for each of the analysed beers.

2.2.3. Identification and Quantitation of Xanthohumol and Isoxanthohumol

XN and IXN in beers were identified and quantified by means of high-performance liquid chromatography (HPLC) using a Waters 2690 chromatograph (Waters, Milford, MA, USA) equipped with a Photodiode Array Detector Waters 996 (Water Corporation, Milford, MA, USA). Analysis was performed using reversed phase column Kinetex 5u XB-C18 100A (Phenomenex, Torrance, CA, USA) (250 mm × 4.6 mm), which was thermostated at 28 °C. Samples were thermostated at 12 °C. Eluents used for the HPLC analysis were: eluent A—1% v/v formic acid in H₂O, and eluent B—1% v/v formic acid in acetonitrile. The injection volume was equal to ten µL. The chromatographic program was as follows:

• 65% eluent A/35% eluent B for 10 min;
• Reduction in eluent A from 65% to 10% over 8 min;
• 10% eluent A/90% eluent B for 4 min;
• Increase in eluent A to 65% during 1 min;
• 65% eluent A/35% eluent B for 5 min.

Total program time was 26 min, pressure was 2600 psi (at 65% v/v eluent A), and flow rate was equal to 1.5 mL per min. R² for the calibration curve of XN was 0.9994, while for IXN it was 0.9988. Each of the beers was analysed in triplicate (one analysis per beer extract).

2.2.4. Preparation of the Beer Samples for the Analysis of the Physicochemical Parameters

Two hundred mL of the beer sample, before the extraction process, was poured into 500 mL glass flasks and degassed by shaking on a laboratory shaker for 30 min. Diatomaceous earth was added to the degassed beer, which was then filtered through the paper filter (Macherey-Nagel, Düren, Germany) MN 614 ¼; 320 mm diameter).

2.2.5. Analysis of the Beer Basic Physicochemical Parameters

Physicochemical parameters of beer, such as ethyl alcohol content, extract content, beer density, wort extract content, and beer final attenuation was assessed using an Anton Paar DMA 4500M oscillating densitometer connected with Anton Paar Alcolyzer Beer ME (Anton Paar, Graz, Austria). Three analyses were performed for each beer type.

2.3. Data Analysis

Results of the analysis of beers’ physicochemical parameters, as well as data concerning the concentration of XN and IXN in the tested samples, were statistically analysed in the Statistica 12.5 software with one-way analysis of variance (α = 0.05) and the Tukey post hoc test (α < 0.05). Results are shown as means with a standard deviation.

3. Results

In this study, 35 commercially available beers from the Polish market were analysed. Acquired beers were characterised as beverages of different beer styles. Performed analyses involved the analysis of the beers’ physicochemical parameters and concentration of prenylated flavonoids (XN and IXN).

3.1. Concentration of Prenylated Flavonoids in the Beers Available on the Polish Market

The concentrations of XN and IXN in the beers analysed in this study are presented in

Table 1. Concentration of prenylated flavonoids (XN, IXN) in analysed beers.

No 1	Beer Sample	XN Content	IXN Content
		(mg/L)	(mg/L)
1	AIPA1	0.143 ± 0.020 e	2.234 ± 0.134 a
2	AIPA2	0.167 ± 0.034 e	2.154 ± 0.176 ab
3	AIPA3	0.777 ± 0.045 b	1.490 ± 0.129 cd
4	AIPA4	0.159 ± 0.028 e	1.932 ± 0.129 b
5	AIPA5	0.150 ± 0.027 e	1.898 ± 0.096 b
6	AIPA6	0.135 ± 0.024 e	2.147 ± 0.152 ab
7	AIPA7	0.442 ± 0.052 d	2.510 ± 0.182 a
8	AIPA8	0.186 ± 0.024 e	2.093 ± 0.052 b
9	PALE1	0.105 ± 0.014 f	1.400 ± 0.079 d
10	PALE2	0.125 ± 0.027 ef	1.128 ± 0.097 e
11	PALE3	0.069 ± 0.018 fg	1.338 ± 0.087 d
12	PALE4	0.074 ± 0.012 fg	1.310 ± 0.088 d
13	PALE5	0.626 ± 0.063 c	1.057 ± 0.112 f
14	PALE6	0.431 ± 0.035 d	0.610 ± 0.063 g
15	PALE7	0.151 ± 0.026 e	1.604 ± 0.202 c
16	PALE8	0.135 ± 0.025 ef	1.793 ± 0.161 bc
17	PALE9	0.114 ± 0.023 f	1.269 ± 0.096 de
18	IPA1	2.459 ± 0.198 a	1.623 ± 0.112 c
19	IPA2	0.204 ± 0.046 e	1.374 ± 0.092 d
20	IPA3	0.039 ± 0.005 g	1.119 ± 0.091 e
21	ST1	0.541 ± 0.067 cd	1.575 ± 0.118 c
22	ST2	0.102 ± 0.036 f	1.278 ± 0.084 de
23	POR1	0.581 ± 0.072 c	0.649 ± 0.061 g
24	POR2	0.563 ± 0.041 cd	0.621 ± 0.056 g
25	POR3	0.108 ± 0.034 f	0.973 ± 0.072 f
26	LAG1	0.029 ± 0.018 g	1.096 ± 0.064 f
27	LAG2	0.078 ± 0.013 fg	2.159 ± 0.105 b
28	LAG3	0.166 ± 0.016 e	0.232 ± 0.047 h
29	LAG4	0.040 ± 0.014 g	0.243 ± 0.038 h
30	PIL1	0.051 ± 0.017 g	0.508 ± 0.078 g
31	PIL2	0.454 ± 0.043 d	1.248 ± 0.136 de
32	PIL3	0.084 ± 0.023 fg	1.364 ± 0.042 d
33	PIL4	0.040 ± 0.012 g	2.106 ± 0.156 ab
34	WIT1	0.467 ± 0.036 d	2.380 ± 0.174 a
35	WIT2	0.437 ± 0.042 d	1.750 ± 0.157 bc

¹ Data are shown as mean ± standard deviation (n = 3). Letters in the column (a, b, …, h) denote homogenous groups (Tukey test, α = 0.05). Red colour indicates homogenous groups with the lowest average result, and green colour indicates homogenous groups with the highest average result.

. Representative chromatograms showing the XN and IXN peaks are provided in the Supplementary Data (Figures S1 and S2). The highest XN concentration was detected in the IPA1 sample (2.459 mg/L), which was markedly higher than that observed in all other samples. Other samples, characterised by relatively high XN concentrations, included AIPA3 (0.777 mg per L) and PALE5 (0.626 mg/L), as well as POR1 and POR2 (0.581 and 0.563 mg/L). The lowest XN concentration was detected in lager-style beer LAG1 (0.029 mg/L).

The mean XN concentration across all analysed beers was 0.298 mg/L, and substantial variation was observed among different beer styles. AIPA-style beers were characterised with an average XN concentration of 0.27 mg/L; Pale Ale-style beers possessed an average concentration of 0.2 mg of XN per L. IPA-style beers showed the highest average XN concentration (0.91 mg/L), followed by stouts (0.32 mg/L) and porters (0.45 mg/L). In contrast, markedly lower average XN concentrations were found in lager beers (0.08 mg/L) and Pilseners (0.16 mg/L). Witbier-style beers exhibited an average XN concentration comparable to that of porters (0.45 mg/L). In contrast to XN, markedly different results were obtained for IXN concentrations in the analysed beers.

Five AIPA-style beer samples exhibited the highest IXN concentrations among all tested beers, ranging from 2.093 to 2.510 mg/L, with the highest value determined in sample AIPA7. The lowest IXN concentrations were detected in the LAG3 and LAG4 samples (0.232 and 0.242 mg/L, respectively), which also exhibited low XN concentrations. The mean IXN concentration across all analysed beers was substantially higher than that of XN and amounted to 1.436 mg/L.

The average IXN concentration was 2.057 mg/L in AIPA beers, 1.279 mg/L in Pale Ales, 1.372 mg/L in IPAs, and 1.427 mg/L in stouts. The lowest average IXN concentration was observed in porters (0.714 mg/L), followed by lagers (0.933 mg/L) and Pilseners (1.307 mg/L). The highest average IXN concentration was detected in witbier-style beers (2.065 mg/L).

3.2. Physicochemical Parameters of the Beers Available on the Polish Market

Basic physicochemical parameters of the beer include alcohol content and wort extract of the wort, which are among the main pieces of information provided by producers on beer labels [35,36]. These parameters, together with other characteristics of the analysed beer samples, are presented in

Table 2. Physicochemical properties of the analysed beers.

No 1	Beer Sample	Wort Extract Content	Alcohol Content	Extract Content	Degree of Attenuation
		(% w/w)	(% v/v)	(% w/w)	(%)
1	AIPA1	16.33 ± 0.13 d	6.07 ± 0.09 e	6.60 ± 0.16 c	57.55 ± 0.88 i
2	AIPA2	16.34 ± 0.12 d	6.10 ± 0.10 e	6.57 ± 0.13 c	57.76 ± 0.96 i
3	AIPA3	16.30 ± 0.14 d	6.09 ± 0.08 e	6.54 ± 0.12 c	57.86 ± 0.81 i
4	AIPA4	16.20 ± 0.20 d	6.05 ± 0.07 e	6.51 ± 0.13 c	57.81 ± 0.81 i
5	AIPA5	16.27 ± 0.14 d	6.05 ± 0.06 e	6.58 ± 0.13 c	57.54 ± 0.92 i
6	AIPA6	16.11 ± 0.15 d	6.05 ± 0.06 e	6.41 ± 0.11 cd	58.22 ± 0.82 i
7	AIPA7	14.54 ± 0.12 f	5.76 ± 0.07 ef	5.30 ± 0.16 f	61.81 ± 1.31 g
8	AIPA8	16.23 ± 0.11 d	6.81 ± 0.06 c	5.30 ± 0.11 f	65.50 ± 0.95 ef
9	PALE1	12.86 ± 0.14 h	5.99 ± 0.08 e	3.77 ± 0.13 h	69.31 ± 0.89 c
10	PALE2	12.82 ± 0.13 h	5.65 ± 0.07 f	3.74 ± 0.12 h	69.46 ± 0.74 c
11	PALE3	12.94 ± 0.15 h	5.69 ± 0.11 f	3.80 ± 0.11 h	69.25 ± 0.85 c
12	PALE4	12.89 ± 0.13 h	5.64 ± 0.09 f	3.83 ± 0.13 h	68.90 ± 0.76 cd
13	PALE5	11.40 ± 0.12 i	4.72 ± 0.12 h	3.83 ± 0.12 h	65.09 ± 0.68 ef
14	PALE6	12.56 ± 0.16 h	4.55 ± 0.09 h	5.27 ± 0.14 f	56.46 ± 0.56 i
15	PALE7	13.24 ± 0.13 gh	5.50 ± 0.10 f	4.41 ± 0.13 g	65.17 ± 0.91 ef
16	PALE8	13.43 ± 0.12 g	5.20 ± 0.09 g	5.09 ± 0.14 f	60.47 ± 0.67 h
17	PALE9	12.51 ± 0.13 h	4.64 ± 0.07 h	5.08 ± 0.12 f	57.84 ± 0.64 i
18	IPA1	17.34 ± 0.14 c	6.46 ± 0.14 d	6.99 ± 0.16 b	57.54 ± 0.82 i
19	IPA2	16.23 ± 0.14 d	6.86 ± 0.11 c	5.21 ± 0.12 f	66.07 ± 0.92 e
20	IPA3	19.04 ± 0.22 b	8.22 ± 0.07 b	5.84 ± 0.12 e	67.24 ± 0.83 de
21	ST1	14.22 ± 0.15 f	4.13 ± 0.07 i	7.64 ± 0.13 a	44.45 ± 0.64 k
22	ST2	15.67 ± 0.19 e	5.56 ± 0.09 f	6.77 ± 0.15 bc	54.81 ± 0.61 j
23	POR1	20.95 ± 0.17 a	9.66 ± 0.08 a	5.42 ± 0.14 f	72.06 ± 1.01 b
24	POR2	15.40 ± 0.24 e	6.77 ± 0.11 c	4.52 ± 0.13 g	69.00 ± 1.39 c
25	POR3	13.66 ± 0.13 g	5.12 ± 0.08 g	5.45 ± 0.12 f	58.41 ± 0.89 i
26	LAG1	12.70 ± 0.16 h	5.40 ± 0.07 fg	4.03 ± 0.12 h	66.85 ± 0.74 de
27	LAG2	15.23 ± 0.14 ef	6.64 ± 0.12 cd	4.56 ± 0.14 g	68.41 ± 0.79 cd
28	LAG3	11.53 ± 0.15 i	5.36 ± 0.08 fg	2.91 ± 0.13 j	73.64 ± 0.78 ab
29	LAG4	11.70 ± 0.25 i	5.56 ± 0.05 f	2.76 ± 0.09 j	75.32 ± 0.99 a
30	PIL1	11.39 ± 0.13 i	5.04 ± 0.06 g	3.29 ± 0.13 i	69.61 ± 0.72 c
31	PIL2	12.71 ± 0.14 h	5.20 ± 0.05 g	4.36 ± 0.15 g	64.22 ± 0.84 f
32	PIL3	12.46 ± 0.22 h	5.06 ± 0.09 g	4.34 ± 0.13 g	63.71 ± 0.76 fg
33	PIL4	12.35 ± 0.17 h	4.99 ± 0.15 g	4.34 ± 0.14 g	63.41 ± 0.80 fg
34	WIT1	15.48 ± 0.19 e	5.71 ± 0.12 f	6.33 ± 0.15 d	57.18 ± 0.62 i
35	WIT2	16.04 ± 0.23 de	5.90 ± 0.08 e	6.59 ± 0.16 c	56.91 ± 0.64 i

¹ Data are shown as mean ± standard deviation (n = 3). Letters in the column (a, b, …, k) denote homogenous groups (Tukey test, α = 0.05). Red colour indicates homogenous groups with the lowest average result, and green colour indicates homogenous groups with the highest average result.

. The alcohol content of the tested beers ranged from 4.13% (v/v) to 9.66% (v/v). The lowest alcohol concentration was observed in ST1, while the highest was in POR1. The mean alcohol content across all beer samples was 5.83% (v/v).

The highest beer extract content was observed in sample ST1 (7.64% w/w), whereas the lowest was found in sample LAG4 (2.76% w/w), with an overall average of 5.14% (w/w). The original wort extract content ranged from 11.39% (w/w) in sample PIL1 to 20.95% (w/w) in sample POR1, and the mean original wort extract was 14.49% (w/w). Average alcohol and extract contents varied depending on beer style.

The average alcohol content was 6.12% (v/v) for AIPA-style beers, 5.29% (v/v) for Pale Ale beers, and 7.18% (v/v) for IPA-style beers. The lowest average alcohol content was observed in stout-style beers (4.85% v/v), while porter-style beers exhibited an average alcohol content of 7.18% (v/v). Lager beers contained, on average, 5.74% (v/v) alcohol, Pilseners contained 5.07% (v/v), and witbier-style beers contained 5.81% (v/v).

The degree of attenuation showed the greatest variability among the physicochemical parameters assessed in this study, ranging from 44.45% in sample ST1 to 75.32% in sample LAG4, with a mean value of 63%.

4. Discussion

4.1. Concentration of Prenylated Flavonoids

4.1.1. Xanthohumol

The current state of knowledge suggests that XN is the most interesting prenylated flavonoid out of the two tested in this study, and possibly the most valuable and promising compound from all the substances identified in Humulus lupulus, which possesses multiple health-benefiting properties [37]. XN is the most dominant prenylated flavonoid present in the hops and is almost exclusively introduced into the human diet through beer-brewing technology [1,2,11]. Hops and wort play a central role in beer production, not only as fundamental raw materials but also as key determinants of the chemical composition and bioactive profile of the finished, fermented product. Wort, produced by the extraction of malted cereals with water, provides fermentable sugars, amino acids, minerals, and precursor compounds that support yeast metabolism and influence beer flavour, colour, and alcohol content. The composition of wort is strongly dependent on malting conditions, grist composition, mashing regime, and wort boiling parameters, which differ substantially among manufacturers. These technological choices affect not only fermentation performance but can also influence the stability and transformation of hop-derived compounds, including prenylated flavonoids such as XN [1].

Hops are primarily added to beer for bitterness, aroma, and microbiological stability; however, they are also the exclusive source of prenylated flavonoids in beer. Manufacturers apply hops at different stages of the brewing process, such as wort boiling, whirlpool hopping, or post-fermentation dry-hopping—depending on the desired sensory profile and beer style. From the perspective of xanthohumol retention, these approaches are particularly important, as thermal treatment during wort boiling promotes the isomerisation of XN into IXN, thereby reducing the concentration of the biologically more active compound [37]. As a result, brewers who rely heavily on late hopping or dry-hopping techniques, especially in beer styles such as India Pale Ales, may theoretically achieve higher XN levels compared to those employing traditional early-boil hopping regimes [38].

Differences in hop form (whole cones, pellets, or hop extracts), hop dosage, hop variety, and hopping intensity further contribute to variability in xanthohumol content among beers produced by different manufacturers [39]. Additionally, ethanol content, wort composition, and process-related losses during clarification, fermentation, and filtration influence the final concentration of hop-derived prenylated flavonoids. Consequently, manufacturers adopt distinct brewing strategies that balance technological feasibility, sensory quality, product stability, and economic considerations, which collectively result in substantial differences in xanthohumol content across beer styles and producers [40].

As mentioned before, XN is isomerised to IXN during wort boiling, which is a crucial step in beer production, and one of the main purposes of wort boiling is precisely the isomerisation of flavour-active hop-components, thereby increasing their solubility in water [1]. Some xanthohumol is lost during beer centrifugation, where a fraction of XN is removed together with hop debris and various proteins, which form the so-called “hot break” or “hot trub” [1,24]. During subsequent wort cooling, another fraction of XN is adsorbed onto cold-precipitated proteins. During beer fermentation, some XN may be adsorbed by yeast cells [24]. As most yeast is removed from the finished beer during filtration, this further reduces XN content [1]. Importantly, the interplay between beer style, alcohol content, and filtration practices can modulate the bioavailability of XN, suggesting that even small process variations may significantly affect the functional potential of the final product [41]. Based on the data obtained in this study, beers produced using larger hop addition, with the use of more hops, or with the use of hops high in XN (such as Magnum or Brewer’s Gold) should possess higher XN concentration, as it is known that XN is introduced to the beer only from the hop material (more precisely, hop pellets, which are currently the most commonly used hop form in brewing). Unfortunately, commercial producers rarely disclose information on hop dosage or hop composition. However, certain trends may be inferred from other parameters assessed in this study, as well as from the beer styles analysed. For example, beers with higher ethanol content and extended contact with hop material (e.g., via dry-hopping) facilitate greater solubilization of XN, highlighting the role of physicochemical parameters in determining prenylflavonoid levels [1].

Out of the tested beers, some were of the American India Pale Ale (AIPA) beer style or India Pale Ale (IPA) style, which are typically dry-hopped [1,42]. Many of the AIPAs and IPAs exhibited high XN concentrations. Only three tested beers had XN concentrations exceeding 0.6 mg/L, and two of these samples were of AIPA and IPA style, while the third one was a Pale Ale, which is also often dry-hopped [1,34]. Interestingly, some beers of styles not typically associated with dry-hopping or high hop additions during wort boiling, such as stouts and witbiers, also showed relatively high XN content. Two factors likely contributed to the increased XN concentrations in these samples compared to other beers in this study. Witbier-style beers and other wheat beers are typically unfiltered, and beer filtration is the process that significantly reduces the concentration of XN, primarily due to the removal of undissolved XN [34]. Similar results were noted in the study by Gribkova et al. (2023), where the unfiltered wheat beers were characterised with high XN content, despite using hops only in one dose and boiling for 60 min [43]. Stouts and porters are dark beers, produced with dark or caramel malts, which are the source of the melanoidins in the wort and finished beer [44]. Previous studies have suggested that these compounds may inhibit the isomerization of XN to IXN during the wort boiling [20,25,29,30].

The results of this study, compared with previous analyses of XN content, indicate that XN concentrations in commercially available beers are increasing. Stevens et al. (1999), in one of the first studies analysing XN content in beers by HPLC, reported that light beers (mostly lagers and Ales) had XN concentrations ranging from 0.002 to 0.24 mg/L, which are lower than most samples analysed in the present study [24]. However, the patented ‘XAN technology’ beer from the beginning of the 21st century allowed commercial production of light beers with XN concentrations above 1 mg/L and dark, unfiltered beers with XN concentrations up to 10 mg/L [45]. Analysis of the lager beers available on the Polish market by Toboła et al. (2014) showed XN concentrations ranging from 0.006 to 0.22 mg/L, which are similar to the results reported in 1998 [46]. These trends demonstrate that evolving brewing technologies, increased use of craft brewing methods, and style diversification are driving higher XN concentrations in contemporary commercial beers [47,48,49]. It is also crucial to remember that XN concentration is highly prone to degradation over time during beer storage, especially at elevated temperatures. A study by Gahr et al. (2020) has shown that over 79% of XN content can be lost after a year of storage at 22 °C, which is why adequate handling of beer by the supplier and vendors is of utmost importance [50].

Despite this increase, XN concentrations in commercially available beers remain too low to exert therapeutic effects, and any potential benefits would likely be mitigated by the volume of beer required for significant intake. Furthermore, the combination of alcohol content and relatively low XN levels strongly suggests that commercial beer should not be considered a reliable dietary source of XN, as the suggested XX consumption for various health benefits is often in the range of 1 mg of XN per kg of bodyweight [51]. Nevertheless, the integration of prenylated flavonoid analysis with physicochemical profiling, as performed in this study, provides critical insight into how brewing parameters and beer composition influence bioactive compound retention, offering guidance for both consumers and brewers interested in functional beer properties. These limitations might be partially addressed through dealcoholisation processes, but further studies would be required to evaluate this approach.

4.1.2. Isoxanthohumol

IXN is a prenylated flavonoid originating primarily from hops, like XN. IXN is formed as a result of XN isomerisation, which occurs when XN is subjected to high temperatures, such as during wort boiling [52]. IXN is more soluble in water (5.0 mg/L, 23 °C) than XN (1.3 mg/L, 23 °C); therefore, fermenting beer, which is an alcoholic beverage with relatively low alcohol content, has a higher capacity to solubilise IXN than XN [53].

The results of analyses performed in this study support this observation because almost all of the beers contained higher IXN concentrations than XN, with the exception of the IPA1 sample, which had a very high concentration of XN (2.459 mg per L). This pattern is consistent with prior research indicating that IXN generally accumulates in beer at levels exceeding those of XN due to both its higher solubility and the thermal isomerisation that occurs during wort boiling [54].

There are plausible explanations for high XN and for the rather low concentration in some of the samples. Hops contain 10–50 times less IXN than XN, which is the most prevalent prenylated flavonoid present in the hops [55]. As the isomerisation of XN to IXN occurs only at elevated temperatures, IXN is likely formed mainly during wort boiling and not during later stages of beer production [1,50]. Consequently, the ratio of XN to IXN in finished beer can serve as an indicator of hopping regime, boiling duration, and thermal exposure, providing insight into brewing practices and their impact on bioactive compound distribution [28]. Beers that are typically dry-hopped, such as American Pale Ales and Indian Pale Ales, are expected to have elevated XN concentrations, but not necessarily IXN, as the hops added to the fermenting beer are not heated. However, these beer styles are also usually kettle-hopped with higher amounts of hops than in the process of production of other beer styles to ensure bitter flavour [1,37]. This early hopping contributes to IXN formation in the boiling wort, and consequently, IXN concentrations may correlate not only with the amount of hops added but also with wort composition, pH, and other physicochemical parameters that influence compound stability [56].

It is further worth mentioning that analysis of IXN in beers can be as informative as analysis of XN, which, in recent studies, has received more attention than its isomerised form. IXN is also a biologically active compound, although it has lower antioxidant potential than XN. Importantly, IXN possesses important metabolic relevance, as it can be converted by the human intestinal microbiota into the potent phytoestrogen 8-prenylnaringenin [51]. This biotransformation underscores the physiological importance of IXN despite its lower direct antioxidant activity, and it highlights beer as a vehicle for precursors of bioactive metabolites. Recent research also indicates that IXN can be a substrate for production of many interesting, biologically active compounds, such as: (2S,2″S)- and (2R,2″S)-4′-hydroxy-5-methoxy-7,8-(2,2-dimethyl-3 hydroxy-2,3-dihydro-4H-pyrano)-flavanones; and (2R)-7,4′-dihydroxy-5-methoxy-8-(2,3-dihydroxy-3-methylbutyl)-flavanones, (2R)-, and (2S)-4′-hydroxy-5-methoxy-2″-(1-hydroxy-1-methylethyl)dihydrofuro[2,3-h] flavanones [52]. These compounds, although present at trace levels, may contribute to the overall bioactive profile of beer and warrant further investigation. Additionally, the relative stability of IXN compared to XN during storage and post-fermentation processing suggests that beer can act as a practical model for studying prenylated flavonoid chemistry in complex food matrices [56].

Finally, integrating IXN concentrations with physicochemical parameters, such as beer colour, alcohol content, bitterness, and polyphenol index, can reveal trends in how brewing practices influence bioactive compound profiles. For example, darker beers or those with higher initial hopping rates may exhibit higher IXN retention, while unfiltered beers preserve both XN and IXN more effectively. By combining these chemical and physicochemical analyses, researchers can better predict the functional potential of beers across different styles and production methods, providing practical insights for brewers aiming to maximise prenylated flavonoid content [42,57,58].

4.2. Physicochemical Parameters

All the basic physicochemical parameters of the analysed beers, such as alcohol content, extract content, degree of attenuation, and wort extract content, varied substantially. These parameters are key indicators of beer composition and brewing efficiency and can indirectly influence the solubility, stability, and retention of prenylated flavonoids like XN and IXN [1,14]. Usually, in beer production technology, the bitterness of the hops must be balanced with the sweet taste of the sugars present in the beer; otherwise, the beer would be unpalatable [59,60,61]. This balance reflects the underlying interactions between malt-derived sugars and hop-derived compounds, which also affect the chemical environment for flavonoid solubilization and retention. For instance, higher sugar concentrations may enhance the solubilization of hydrophobic flavonoids through co-solvent effects, while excessive residual sugar may reduce bioactive perception by dilution or binding [62]. This relation also works in the other way—beers should not be too sweet (i.e., contain too much residual sugars), and without the addition of bitter compounds from hops, the resulting beverage would be overly sweet and cloying for most of the consumers [59,63,64,65]. From a chemical perspective, the interplay between sugar concentration, ethanol production, and hop-derived compounds creates a matrix that modulates prenylated flavonoid behaviour, affecting both XN and IXN extraction and stability during fermentation and maturation [1,66]. This is why, in beer-brewing technology, there are a few general principles that can describe the relationship of beer bitterness, beer alcohol content, and beer extract content. Beers with low alcohol content are perceived as more bitter than beers with high alcohol content (assuming the same amount of bittering substances). Beers with a high concentration of residual sugars (lower degree of attenuation) tend to feel less bitter than ‘dry’ beers (with a high degree of attenuation) [1]. These physicochemical properties also interact with XN and IXN retention. For example, higher ethanol content resulting from greater attenuation can enhance XN and IXN solubility, while higher residual extract can stabilise these flavonoids through complex formation or reduced precipitation [66]. Alcohol content alone, however, does not necessarily strongly influence XN and IXN content of the finished beer, as the hops are traditionally added to the non-alcoholic wort [1,22]. Nevertheless, alcohol can play a synergistic role when hops are added post-fermentation (dry-hopping), as ethanol in the fermenting beer matrix facilitates the dissolution of prenylated flavonoids. This highlights the importance of considering both production technology and physicochemical environment in understanding XN and IXN profiles [67]. However, this situation changes drastically with a beer that is produced by using a dry-hopping technique, as the hops are added to the fermented beer [37]. In these cases, high extract content combined with a high degree of attenuation (which results in higher alcohol content) may enhance XN and IXN concentrations in the final product, as the hydrophobic prenylated flavonoids have more time and favourable conditions to dissolve in the alcoholic medium [37,42,43]. However, from the analysis of only the finished product, without the knowledge of the amount of hops used, whether the beer was dry-hopped, and knowledge of the XN content of the hops, it is nearly impossible to draw definitive conclusions about basic physicochemical parameters and their influence on the XN and IXN content in the beverage. Despite this limitation, correlations between higher ethanol levels, higher degrees of attenuation, and increased XN/IXN concentrations observed in some beer styles suggest that these routine physicochemical measurements can serve as indirect predictors of prenylated flavonoid content [28]. Moreover, integrating these parameters with beer style and process information provides a more holistic understanding of how brewing practices affect bioactive compound retention, which can inform both research and practical applications in functional beer development. Finally, these findings indicate that beer physicochemical parameters not only guide sensory perception but also influence the bioactive potential of the beverage [68]. Future work should focus on systematically linking XN and IXN concentrations with alcohol content, extract, and attenuation across multiple beer styles and brewing technologies, allowing predictive modelling of flavonoid retention and more targeted functional beer production.

5. Limitations

The material studies were limited to beverages that could be purchased in Poland in the years 2020–2024. The properties of beer, as a material that is produced from seasonal crops, such as barley or hops, might be prone to various fluctuations, especially connected with the weather and places where the crops were grown. It is also necessary to note that breweries in Poland (and, generally, all around the world) do not have to disclose the year and country of origin of the components which were used to produce that particular batch of beverage, and therefore, analyses in other years, even using beers of the same brands as shown in this study, might show significantly different results.

6. Conclusions

The results of this study demonstrate that the content of hop-derived prenylated flavonoids in commercially available Polish beers is highly heterogeneous and cannot be predicted solely on the basis of beer style classification. Although beers belonging to hop-forward styles, particularly American India Pale Ales and India Pale Ales, frequently exhibited elevated concentrations of xanthohumol and isoxanthohumol, this tendency was not consistent across all samples. Notably, several beers representing styles traditionally considered less hop-intensive, such as stouts, porters, and witbiers, contained comparable or even higher levels of xanthohumol than selected IPA- or AIPA-style beers.

Isoxanthohumol was identified as the dominant prenylated flavonoid in the majority of the analysed beers, which is consistent with its formation during wort boiling as a result of xanthohumol isomerization. The presence of beers with unusually high xanthohumol concentrations suggests that technological factors, including hop dosage, hopping regime, timing of hop addition, and the use of dry-hopping, may play a more decisive role in determining final prenylated flavonoid content than beer style designation itself.

From a consumer perspective, these findings indicate that beer style cannot be treated as a reliable indicator of elevated xanthohumol intake. From a technological and research standpoint, the results highlight the need for more detailed reporting of hopping strategies and raw material composition in studies investigating bioactive hop compounds. Further investigations linking specific brewing practices with prenylated flavonoid transfer and stability may support the development of beers with intentionally enhanced xanthohumol content.