Gallic acid and Folin-Ciocalteu reagent were purchased from Merck (Darmstadt, Germany). Absolute ethanol, disodium phosphate, and ethylenediaminetetraacetic acid (EDTA) were supplied by VWR Chemicals, Avantor (Solon, OH, USA). Quercetin was obtained from Sigma-Aldrich, Merck (St. Louis, MO, USA), while dipotassium peroxydisulfate came from Biochem (Cosne-Cours-sur-Loire, France). Ferrozine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1 % antibiotic/antifungal solution were purchased from Thermo Scientific, Thermo Fisher Scientific (Kandel, Germany). Monosodium phosphate was supplied by J. T. Baker (Deventer, the Netherlands), and dimethyl sulfoxide (DMSO) and sodium chloride were obtained from Fisher Scientific (Loughborough, UK). The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) came from Acros Organics (Geel, Belgium), while sodium carbonate was purchased from Atom Scientific (Manchester, UK). The 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was obtained from TCI (Zwijndrecht, Belgium). Trypsin, phosphate-buffered saline (PBS) and minimum essential medium (MEM) were supplied by Corning (Manassas, VA, USA), and foetal bovine serum (FBS) was obtained from Biochrom KG (Berlin, Germany). Finally, the ALT enzyme assay kit was purchased from Randox Laboratories (Crumlin, Ireland).

Portuguese craft beer imperial stout (IS-N) (Porto, Portugal) was chosen because of its potential antioxidant and hepatoprotective activity demonstrated in our previous study (15), and also because of the availability of its malts. IS-N beer is made with Carafa Special III, Caramunich III, Carapils and Pilsner malts (selected malts) provided by the brewery that produces the craft beer (IS-N).

The malt samples were prepared as described by Mareček et al. (16) and Wu et al. (17), with slight modifications. The malt cereals were ground in an electric mill (Taurus^®; Aromatic, Oliana, Spain) for 40 s and then sieved through a 500 μm mesh sieve. To obtain the aqueous malt extract, 25 g of powder was added to 225 mL of distilled water and placed in a water bath at 45 °C for 15 min. After cooling, the mixture was filtered through Whatman filter paper No. 1 and the filtrates obtained were stored at −80 °C until completely frozen. The extracts were then freeze-dried (LABCONCO^®; FreeZone^®, Kansas City, MO, USA) at 0.007 kPa, with a condenser surface temperature of −72 °C for 3 days and stored at −80 °C.

To obtain the ethanolic malt extract, 1 g of powder was added to 100 mL of 95 % (V/V) ethanol solution. The extraction was carried out at room temperature by magnetic stirring (985VW0CHSEUA; VWR^®, Avantor, Leuven, Belgium) at 400 rpm for 30 min. The mixture was then filtered by gravity (Whatman filter paper No. 1) and the filtrates obtained were placed in a rotary evaporator (RV8; IKA^®, Staufen, Germany) under reduced pressure (90 kPa), at 60 rpm and a controlled temperature of 40 °C (HB10; IKA^®), until complete ethanol evaporation. The extracts were freeze-dried under the same conditions described above.

The IS-N craft beer with φ(ethanol)=8.50 %, was provided by the brewery responsible for its production. Upon receipt, the sample underwent the necessary treatments prior to experimental testing, following the procedures described in our previous study (15). The contents of the bottle were initially mixed uniformly for 10 s and then subjected to gas removal by sonication (Bandelin Sonorex^®, Bandelin, Berlin, Germany) for 40 min at a frequency of 35 kHz at room temperature. The alcohol was then removed using a rotary evaporator (RV8; IKA^®) operating at 40 °C, 60 rpm and a pressure of 90 kPa for 1 h. The sample was then freeze-dried (LABCONCO^®; FreeZone^®) under a pressure of 0.007 kPa, with the condenser surface cooled to −72 °C for three days.

The total phenolic content (TPC) by the Folin-Ciocalteu method and antioxidant activity using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and ferrozine (metal chelating activity) assays in aqueous and ethanolic extracts of malts under study were determined as described in our previous study (15).

For TPC determination, 250 μL of the sample (1 mg/mL), distilled water (blank) or standard gallic acid solutions (5–100 μg/mL) were mixed with 2.5 mL of 0.2 M Folin-Ciocalteu reagent and incubated for 5 min at room temperature in the dark. Subsequently, 2 mL of sodium carbonate solution (75 g/L) were added, and the final volume was adjusted to 5 mL with distilled water. After 1 h of incubation under the same conditions, the absorbance was measured at 760 nm in a UV-Vis spectrophotometer (model UV-1600PC; VWR, Avantor, Leuven). The total concentration of phenolic compounds was determined by comparison with the standard curve of gallic acid, and the results were expressed in milligrams of gallic acid equivalents (GAE) per gram of sample.

For ABTS assay, 2.45 mM of dipotassium peroxydisulfate solution was added to 7.0 mM ABTS and the reaction mixture was kept in the dark for 16 h at room temperature to form ABTS radical. The obtained solution was diluted in PBS until it reached an absorbance of 0.70±0.05 at 734 nm. Next, 0.3 mL of the sample (malt extracts from IS-N craft beer), Trolox standard (1–1000 μg/mL) or distilled water (blank) were added to 2.7 mL of the ABTS⁺˙ solution. After 30 min of incubation at room temperature in the dark, the absorbance was measured at 734 nm in a UV-Vis spectrophotometer (model UV-1600PC; VWR, Avantor, Leuven). Antioxidant activity was expressed as average inhibitory concentration IC₅₀ (μg/mL) and the percentage of inhibition was calculated according to the established formula:

In the ferrozine assay, 50 μL of sample (beer or positive control, EDTA) were added to a 0.15 mM Fe(II) sulfate solution and left to stand for 5 min, protected from light. Next, 50 μL of 0.5 mM Fe(II) cyanide were added, the mixture was shaken vigorously and left for 10 min at room temperature protected from light. The absorbance was measured at 562 nm using a microplate reader (Multiskan FC; Thermo Scientific, Thermo Fisher Scientific, Woodlands, Singapore). Metal chelating activity was expressed as IC₅₀ (μg/mL), and the percentage inhibition was calculated according to the following formula:

The antioxidant potential using the 2,2-diphenyl-1-picrylhydrazyl assay (DPPH) was determined as described by Silva et al. (18), with slight modifications. Briefly, 19.4 μL of sample (1, 5, 10, 25, 50, 100, 250 and 500 μg/mL of malt extracts, craft beer (IS-N), or quercetin as positive control) was added to 175 μL of light-protected DPPH radical (100 μM). The absorbance was measured in a microplate reader (Multiskan FC; Thermo Scientific, Thermo Fisher Scientific) at a wavelength of 520 nm. The readings were repeated every minute for 1 hour. The ability to neutralise the DPPH radical was expressed as IC₅₀ (μg/mL) and the percentage of inhibition was calculated using the following formula:

HepG2 cells were maintained as described by Oliveira et al. (19). The cells were cultured in 25 cm³ flasks in MEM medium supplemented with 10 % (V/V) FBS and 1 % (V/V) antibiotics (ampicillin and streptomycin) and incubated at 37 °C with 5 % CO₂ (AL01-01-100; Advantage-Lab^®, Schilde, Belgium). The culture medium was changed every 2 days, and subcultured when 60/80 % confluence was reached, with the addition of 0.25 % trypsin-EDTA.

To study the cytotoxicity of craft beer, aqueous malt extracts and malt extracts prepared with 95 % (V/V) ethanol, the methods described by Carvalho et al. (20) and Viegas et al. (21) were used, with slight modifications. Briefly, HepG2 cells were incubated in 96-well plates (VWR^®, Avantor, Radnor, PA, USA) at a density of 2.0·10⁵ cell/well, 48 h before incubation with the samples. The cells were then treated with different concentrations of the samples (1−500 μg/mL) for 24 and 48 h (100 µL final volume/well), and cytotoxicity was estimated using the MTT assay. After the incubation period, 10 µL of MTT solution (5 g/mL) were added to each well and left for 1 h in an atmosphere of 5 % CO₂ at 37 °C (AL01-01-100; Advantage-Lab^®). The medium was then removed and the formed formazan crystals were dissolved in a φ(DMSO,ethanol)=50 % solution. Absorbance was measured at 570 nm using a microplate reader (Multiskan FC; Thermo Scientific, Thermo Fisher Scientific, Woodlands). The results were expressed as a percentage of cell viability relative to the control (cells without extract) using the following equation:

Alanine aminotransferase (ALT) enzyme activity was measured using the method described by González et al. (22), with assay kits obtained from Randox Laboratories. HepG2 cells were treated with aqueous and ethanolic extracts of the malts, and with craft beer (IS-N) in aqueous, and ethanolic (φ=8.5 %) solvents. Concentrations were selected based on cell viability results, choosing, for each sample, the concentration that induced the highest and lowest cell viability. After incubation for 24 and 48 h, the supernatant was removed from the wells, and enzyme activities were determined immediately. Results were expressed in international units per litre (IU/L).

Statistical analysis was carried out using GraphPad Prism^® v. 8.0 software (23). The results of the assays were analysed in triplicate and expressed as mean±standard deviation. The TPC and antioxidant potential of the samples were compared using a one-way analysis of variance (ANOVA) with Tukey's multiple comparison test for pairwise comparisons. The correlation between the mean TPC and the antioxidant assays was determined by the correlation coefficient (r), calculated by Pearson's product-moment correlation.

For the cell viability assay, the one-way ANOVA with Dunnett's multiple comparison test was used to compare each value with the control (cells without extract). The metabolic activity of the aqueous and ethanolic extracts of each malt and beer sample at the same concentration was compared using Sidak's multiple comparison test (one-way ANOVA). The same test was used to compare the levels of liver enzymes produced by incubating the cells with two concentrations of the same extract. For all assays, statistical differences were considered significant when p<0.05.

Malt is the main source of phenolic compounds in beer (75–80 %) (18). Phenolic compounds are secondary metabolites containing at least one aromatic ring linked to hydroxyl groups or other structural elements. These compounds represent a group of substances with different chemical structures, differing in their resistance to free radicals and metal chelation, as well as other reactions that occur in beer or living cells (5). Recognised as potent antioxidants, phenolic compounds play critical roles in the sensory properties, colour and colloidal stability of beer, and also contribute to its antioxidant activity (24, 25). Table 1 shows the results of the TPC determined in the four malts. The TPC values of craft beer (IS-N) were determined in our previous study (TPC as w(GAE)=(8.3±0.2) mg/g) (15).

In this study, the TPC, expressed as GAE, of malts varied considerably between the extracts analysed ((7.1±0.5)–(28.2±0.5) mg/g). The ethanolic extract of Caramunich III had the highest value ((28.2±0.5) mg/g) and was statistically superior to the others (p<0.05), followed by the aqueous extract of the same malt. On the other hand, the ethanolic extract of Carapils had the lowest TPC ((7.1±0.5) mg/g), significantly lower than the others (p<0.05). These results indicate that the amount of TPC changes the malt colour, showing that darker malts such as Caramunich III and Carafa III have a higher mass fraction of phenolic compounds than lighter malts such as Pilsner and Carapils.

At the European level, the colour of malts and beer is expressed in units of the European Brewery Convention (EBC). Pilsner malt (2.5 and 4 EBC, data from Sr Cervejeiro Online Brewery Store) is the lightest of the malts analysed and is classified as a base malt, used mainly to provide fermentable sugars because of its high enzymatic activity (18, 26, 27). Meanwhile, Carapils (10−20 EBC), Caramunich III (140−160 EBC) and Carafa III (1250−1400 EBC, for all three malts data available from Sr Cervejeiro Online Brewery Store) are considered specialty malts (27). Due to the heat treatment they undergo, a loss of enzymatic activity is observed and for this reason these malts are traditionally used in smaller quantities (usually around 5 %) than base malts (26). Their main function is to contribute to specific sensory characteristics of the beer, such as colour, aroma and flavour (18, 26).

The variation in TPC observed between different malts and types of extract (aqueous vs ethanolic) corroborates the literature, which highlights the influence of the solvent on the extraction of phenolic compounds (9, 28). Other factors, such as the barley variety, the growing region, the use of fertilisers, and the germination and drying stages, also have an impact on the phenolic content and antioxidant activity of malts (26, 29, 30). The main phenolic compounds present in malt are (+)-catechin, protocatechuic acid, quercetin, ferulic acid and gallic acid (30).

According to the literature, the amount of phenolic compounds in malt tends to increase its colour intensity, especially up to around 450-500 EBC (31). This is partly due to the polymerisation and preservation of phenolic compounds during moderate drying regimes. In addition, products of the Maillard reaction, such as melanoidins, also contribute to the antioxidant potential and are formed in greater quantities during drying and roasting. However, in malts with a colour above 500 EBC, such as the Carafa III analysed in this study, there is a decrease in the content of phenolic compounds, which can be explained by the ability of melanoidins to retain simple phenolic compounds in their structure, as well as the possible inactivation of enzymes (such as ferulic acid esterase) responsible for their release from barley cell walls (31).

The literature also shows variations in TPC between different types of malt and barley. Zhao et al. (9) reported TPC values for dry barley, expressed as GAE, ranging from 2.17 to 2.56 mg/g among 14 varieties of Chinese malted barley (Gan4, Gan3 and Wupi1, Ken2 and Ken3, Humai8, Humai16, Gangpi1, Suyin1, Huaimai19, Linnong, Nongmai, KA4B and Gang2), extracted with 80 % acetone (V/V). In the study by Gąsior et al. (26), the wort obtained from Pilsner malt had a TPC of (192.6±8.7) mg/L. In contrast, in the study by Neto et al. (32), the aqueous extract of Pilsner malt revealed a considerably lower TPC of only (0.91±0.00) µg/mL.

In the study conducted by Šimić et al. (33), which evaluated nine varieties of barley and their respective malts (Barun, Bravo, Bingo, Premium, Vanessa, Tiffany, Maxim, Gazda and Rex), extraction with acidified methanol (V(HCl):V(methanol)=1:100) demonstrated a higher content of total phenolics and higher antioxidant activity in malts than in their corresponding barleys. The TPC values on dry mass basis for the malts ranged from (1.53±0.09) to (1.82±0.00) mg/g, while for the barleys they ranged from (1.27±0.03) to (1.67±0.09) mg/g. These results are in accordance with the study conducted by Dvořáková et al. (29), who found that most of the ten aqueous extracts from the malts studied (from barley varieties Prestige, Jersey, KM 1910, KM 2084, Malz, Merlin, Sebastian, Tolar, Bojos and Amulet) had on dry mass basis higher antioxidant activity than their respective barleys, ranging from 1.1 to 2.9 mg/g and from 0.6 to 1.5 mg/g, respectively.

In our previous study (15), IS-N beer had a TPC of extract (8.3±0.2) mg/g, which was lower than that observed in the malt extracts analysed, with the exception of the ethanolic extract from Carapils. This difference may be related to technological brewing processes, such as malting, the milling method, and the method and mode of mashing, which affect the final phenolic content of the wort and beer (26, 31). Moreover, the difference may also be related to the fact that, in beer formulation, dark (or special) malts are traditionally used in smaller quantities than the base malt (in this case, Pilsner), which has lower TPC values (26). This may explain the lower TPC values observed in beer than in the malts analysed individually.

The study carried out by Censi et al. (28) corroborates the differences in TPC between the different extracts used and the final beer. In the aqueous starter malts, TPC ranged from (11.7±1.8) to (16.568±2.412) mg/g, and in the ethanolic extracts it ranged from (28.1±1.0) to (72.1±1.9) mg/g. The beers analysed ranged from (19.0±1.1) to (35.8±0.15) mg/g.

The phenolic profile of beer is diverse, encompassing catechins and proanthocyanidins, prenylchalcones and their flavanone derivatives, as well as flavonols, hydroxybenzoic acids, hydroxycinnamic acids and stilbenes (34).

It should be noted that the Folin-Ciocalteu method, although widely used for drinks and plant extracts, is not specific to phenolic compounds and can be affected by other compounds with reducing activity (9, 25). Therefore, the possibility of overestimating the TPC results cannot be ruled out (25).

Beer and malts represent a heterogeneous matrix containing antioxidants with different mechanisms of action, which results in different contributions to their antioxidant capacity (35, 36). The antioxidant activity of the imperial stout craft beer (IS-N) under study was determined previously by our research group using different colorimetric antioxidant assays (ABTS and ferrozine assays) (15), as there is no standard method that can objectively characterise the total antioxidant capacity of the samples (37).

ABTS and DPPH assays allow the antioxidant potential of an extract to be measured by its ability to donate hydrogen atoms and electrons, neutralising the respective radicals with a consequent decrease in absorbance (38). Ferrozine assay, based on metal-chelating activity, estimates the antioxidant potential of the extract by its ability to chelate Fe(II), preventing their oxidation to Fe(III) ions, and thus inhibiting the formation of the hydroxyl radical (OH˙), the initiator of the oxidative chain (39). Some assays, such as ABTS and DPPH, are commonly used in beers and cereals due to their sensitivity, convenience and simplicity (33). The results of the assays in malts are shown in Table 2. Regarding the antioxidant activity of the craft beer (IS-N) determined by the DPPH assay, it was not possible to determine IC₅₀ values for the range of concentrations analysed.

According to the ABTS assay, the antioxidant potential of the malt extracts ranged from (17.3±0.8) µg/mL (Caramunich III aqueous extract) to (133.6±2.0) µg/mL (Caramunich III ethanolic extract). It should be noted that the aqueous extract of Caramunich III had the lowest IC₅₀ value, showing high antioxidant capacity (IC₅₀<50 µg/mL), with significant differences compared to other samples (p<0.05).

Analysis of the results confirms that the aqueous malt extracts generally showed greater antioxidant potential (from (17.3±0.8) µg/mL for Caramunich III to (57.1±1.2) µg/mL for Pilsner) than the ethanolic extract (from (38.2±0.8) µg/mL for Pilsner to (547±9) µg/mL for Carapils). This suggests that the antioxidant compounds with the greatest potential to neutralize 50 % of the initial amount of ABTS⁺ free radicals are more soluble in water than in 95 % ethanol, except for the ethanolic extract of Pilsner malt (activity of the ethanolic extract higher than the aqueous extract, p<0.05). The study conducted by Censi et al. (28) corroborates these results, as the aqueous extracts of the initial malt of the analysed beers showed a higher antioxidant potential, expressed as TE, ranging from (21.4±2.7) to (46.82±0.031) µmol/g, determined by the ABTS assay, than the ethanolic extracts with 70 % ethanol, with an antioxidant potential ranging from (41.3±2.8) to (97±19) µmol/g.

In our previous study (15), IS-N craft beer showed an IC₅₀ of (80.1±1.1) µg/mL determined by the ABTS assay, indicating moderate antioxidant activity (50<IC₅₀<100 µg/mL) (40), similar to that found in the present work for the ethanolic extracts of Carafa III and Caramunich III and the aqueous extract of Pilsner.

In the study by Gąsior et al. (26), the wort obtained from Pilsner malt inhibited (22.2±0.9) % of ABTS⁺ radicals. Also, in the study by Oliveira Neto et al. (32), (42.8±13.3) μL of aqueous extract from Pilsner malt neutralised 50 % of ABTS^+· radicals. Zhao et al. (9) found that the antioxidant potential, expressed as TE, of φ(acetone)=80 % extracts from malts from 14 Chinese varieties of barley, determined by the ABTS assay, ranged from 11.39 to 13.58 µmol/g of dry barley. Finally, Dvořáková et al. (29) demonstrated that the antioxidant capacity, expressed as GAE, of aqueous malt extracts from 10 varieties, determined on dry extract basis using the ABTS assay, ranged from 0.20 to 0.45 mg/g.

Regarding the DPPH assay, the antioxidant potential of the malt extracts ranged from (152.6±9.3) μg/mL in the aqueous extract of Caramunich III to (441±9) μg/mL in the ethanolic extract of Carapils. The aqueous extract of Caramunich III was the only aqueous extract for which it was possible to determine the IC₅₀ value in the range of concentrations tested (1–500 μg/mL). However, its antioxidant capacity is considered low, given that its IC₅₀>100 μg/mL (40).

On the other hand, as previously mentioned, it was not possible to determine IC₅₀ in the IS-N craft beer, in the aqueous extract of Carafa III, or either of the Pilsner malt extracts at the concentrations tested (1–500 μg/mL), indicating low antioxidant efficacy as determined by the DPPH method.

In the study by Özcan et al. (41), methanolic extracts from malt showed greater antioxidant activity than the extracts obtained with the same solvent from barley, with inhibition percentages of (67.31±0.00) % for malt and (66.48±0.00) % for barley. These results corroborate those obtained by Šimić et al. (33), who, using acidified methanol extraction, reported DPPH inhibition ranging from (63.1±1.9) to (68.4±2.0) % for malts and from (58.2±2.2) to (65.2±1.5) % for barley.

The data obtained in this study do not corroborate the results observed by Silva et al. (18), in which the beers showed better antioxidant activity than the raw materials. In the present study, aqueous extracts of Carafa III, Caramunich III, Carapils and Pilsner had greater antioxidant activity than the beer (IS-N), in the ABTS assay. Using the DPPH assay, ethanolic extracts of Carafa III and Carapils, as well as ethanolic and aqueous extracts of Caramunich III, demonstrated greater antioxidant potential than the beer.

These differences in the results can be attributed to various biotic and abiotic factors that influence plant physiology and the production of secondary metabolites with antioxidant activity, as well as the type of solvent used for extraction, which plays a crucial role in the efficiency of phenolic compound extraction (9).

In addition, the selection of barley varieties with greater metal-chelating activity is essential for beer quality, as it contributes to the stability of its flavour. This is because transition metal ions can activate the oxygen present in the drink, promoting oxidation reactions that result in the formation of compounds responsible for undesirable flavours (9).

In the present study, using the ferrozine assay, it was not possible to determine the IC₅₀ values within the range of concentrations tested (1–500 μg/mL) for the craft beer or the malt extracts. These results indicate that the samples studied had low antioxidant capacity (IC₅₀>100 μg/mL) (40).

In the study conducted by Zhao et al. (9), metal-chelating activity values, expressed as ethylenediaminetetraacetic acid equivalents (EDTAE), in Chinese malts extracted with 80 % acetone varied between 1.15 and 2.06 μmol/g dry barley, with a weak correlation between TPC and metal-chelating capacity. In the study by Silva et al. (18), the chelating activity of metals in the aqueous extracts of the evaluated malts ranged from (12.0±0.5) to (24.8±0.6) %, with the aqueous extract of Carapils malt showing a chelating percentage of (20.5±0.7) %.

In this study, for malt extracts, there was a significant correlation only between the ABTS and DPPH antioxidant assays (p<0.05) when analysing the ethanolic extracts (Table 3), as would be expected given that these assays are based on the transfer of electrons or hydrogen atoms. Furthermore, there was a negative correlation between TPC and the ABTS assay in the aqueous extracts, as well as between TPC and the ABTS and DPPH assays in the ethanolic extracts, which confirms the fact that phenolic compounds contribute to the elimination of ABTS and DPPH radicals, i.e. antioxidant capacity.

In the study conducted by Zhao et al. (9), a weak correlation was observed between the TPC of malt extracts and metal-chelating capacity, but a significant correlation of TPC with the ABTS and DPPH assays (p<0.01), as well as a significant correlation (p<0.01) between the ABTS and DPPH assays themselves, as also observed in the present study. In turn, the study by Gąsior et al. (26) reported a strong correlation between the TPC of malt extracts and the ABTS assay (r=0.75).