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Bergamot and olive extracts as beer ingredients: their influence on nutraceutical and sensory properties

Bergamot and olive extracts as beer ingredients: their influence on nutraceutical and sensory... Citrus bergamia and Olea europaea L. variety Carolea are accounted as niche functional food for their high content of bio active compounds. Their extracts were used as adjunct to produce two beers with different styles, Blanche and Weiss, rich in antioxidants for a pool of consumers interested in a healthy lifestyle. The nutraceutical properties of these two beers were compared to Blanche and Weiss without any addition to verify if the beers enriched with natural extracts changed their aromaticity, flavors, and functionality. The antioxidant activity changed in the order: blanche bergamot beer > Weiss olive beer > blanche basal beer > Weiss basal beer. The phenolic profile of bergamot beer was qualitatively and quantitatively the richest in bio-compounds. Pearson’s correlation evidenced that total phenols contained in bergamot and olive beers were positively and significantly correlated with the antioxidant activities and precisely, with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and total antioxidant capacity (TAC). Correlation data evidenced that the bergamot was the beer with the greatest antioxidant activity and bioactive compound amount. This study highlighted as the addition of these natural extracts together with the right productive process improved sensorial beer properties, satisfying consumer taste while potentially increasing the beneficial effects on human health. Keywords Antioxidants · Craft beers · Flavonoids · Phenols · Sensory properties Introduction Beer is one of the oldest most popular alcoholic beverages over the world [1], typically, produced with natural ingredi- ents as barley (Hordeum vulgare), hops (Humulus lupulus L.), water, and yeast. In many cases, beers can be integrated * A. Muscolo with other cereals, sugars or natural herbal extracts called amuscolo@unirc.it adjunct. Hops are multi-functional ingredients with preserv- * Mt. Russo ative role which, release bitter compounds (humulones) [2] mariateresa.russo@unirc.it adding flavor, aroma, and bitterness to the beers. Beer is a F. Salafia drink that contains valuable nutritional compounds as car- fabio.salafia@unirc.it bohydrates, amino acids, minerals, vitamins, and polyphe- R. Di Sanzo nols. About 30% of beer polyphenols come from hops, and rosa.disanzo@unirc.it 70% come from malts [3]. The antioxidants and polyphenols S. Carabetta associated with a low alcoholic content determine the func- sonia.carabetta@unirc.it tional quality of the beers. Numerous researches evidenced that modest beer consumes can have anti-inflammatory and Dipartimento di Agraria, Mediterranea University of Reggio antioxidant properties [4, 5], with numerous human health Calabria, Via dell’Università, 25, 89124 Reggio Calabria, Italy benefits [6 –8]. Currently, there is an increased interest of consumers for craft beers (CB), which are distinctly fla- Food Chemistry, Safety and Sensoromic Laboratory (FoCuSS Lab), Dipartimento di Agraria, Mediterranean vored, have a quality value unique and sensory properties University of Reggio Calabria, Via dell’Università, 25, details [9]. Craft beers are unpasteurized, unfiltered, without 89124 Reggio Calabria, Italy Vol.:(0123456789) 1 3 2068 European Food Research and Technology (2022) 248:2067–2077 the addition of nitrogen or carbon dioxide under pressure can change during the process even if the raw materials [10], but with the addition of aromatic herbs, spices, fruit, and the applied treatments are the same. Slight changes in honey, sugar, and coffee, that satisfy sensorial perception structure, or even changes in shape, can alter the antioxidant and improve their nutritional and functional values increas- activity of a compound. As it is known, beer shelf life and ing the number of bio-compounds that can have beneficial flavor are partially influenced by its antioxidant status [13]. impacts on one's health [11]. The goal of this manuscript was To stabilize the productive process, we used 20 hectolitre to develop beers using natural extracts from fruits accounted Tanker EVO 2000 BBC Inox technological plant that allows as niche functional food, bergamot (Citrus bergamia) and to optimize production capacity while respecting quality. Olive (Olea europaea L. variety Carolea), whose beneficial The new fully automatic brewhouse wisely combines tech- properties depend on the specific geographic areas in which nological research and the flexibility of the artisan process. they grow (Grecanic costal area of Calabria Region, south Automation, in fact, allows the replication of tested recipes, Italy). Bergamot contains flavonoids and other beneficial optimizing production times and energy, allowing the brewer components that contribute to its antioxidant, anti-inflam- to give maximum expression to enhance the raw materials. matory capacity and its ability to reduce cholesterol. Olives Every detail has been carefully studied to minimize contact have important nutritional values depending on richness with oxygen from the malt grinding phase to the transfer to in monounsaturated fat, fiber and vitamin E together with the fermentation tank. The whole system is also designed to the presence of several phytochemicals, mostly phenolic be washed and sanitized automatically. Brewing plant has compounds that, acting as scavengers of reactive oxygen also cold chain system important to maintain the products species, have the capability to protect from oxidative dam- during the stabilization of the beer. In the brewery, there age [12]. Extracts of Bergamot and Olive, rich in bioactive is an automatic isobaric and under nitrogen bottling plant compounds, have been used as adjunct for personalizing a (Isobaric, Gai) to reduce at the minimum of the risks of beer style and for producing functional beers, moderately beer oxidation. To produce the blanche, malt, barley, and alcoholic and rich in antioxidants, for a pool of consumers oats were mixed in a well-established proportion, the hops interested in a healthy lifestyle. In this manuscript, the qual- were Styrian Golding and Czech Saaz. Extract of bergamot ity of the craft beers enriched with bergamot or olive extract juice after terpene elimination was added to the blanche. was compared to craft beers of the same typology (basal). Weiss was prepared using malt and barley and Hallertau Aroma beer fingerprints were also assessed to verify if the Hersbrucker as hop. The extract from olive fruit of Carolea addition of extracts affected the aromaticity and flavor of cultivar was added. The setting of the production process, the basal beers. the concentration of the ingredients used, and their mix ratio are covered by industrial secret. The fermentation batch was repeated three times for each typology of beer. Materials and methods Sample preparation Chemicals and reagents The present work analyzed and compared the chemical and Acetonitrile (ACN) HPLC grade, and formic acid were pur- sensory properties of two craft Beers produced in Calabria: chased from Sigma-Aldrich (Milan, Italy), Ultra-pure water Heraclea, Blanche with the addition of bergamot juice was obtained using a Milli-Q system (Millipore, Milan, extract, not filtered and not pasteurized; Elais, Weiss with Italy). Flavonoids standard (Coumaric acid, 4-Hydroxyben- the addition of olive extract not filtered and not pasteur - zoic acid, Caffeic acid, Ethylgallate, Ferulic acid, Kamp- ized. The two Calabrian beers have been compared to the ferol, Naringin, Protocatechuic acid, Syrengin acid, Vanillic Blanche and Weiss without any addition to verify if the beers acid, Hesperidin, Sinensetin, Neoeriocitrin, (−) Epicatechin, enriched with natural extracts changed their aromaticity, fla- Neohesperidin, Tangeretin, Chlorogenic acid and Nobiletin) vors, and functionality. was purchased from Extrasynthese (Genay Cedex, France). The analysis has been carried out in triplicate for each All the other chemicals were purchased from Sigma-Aldrich batch and for each typology of Craft beer. The samples were (Milan, Italy). degassed at 20 °C prior to testing, using magnetic stirrer until all gas has been released. Productive process Brewing is an extremely complicated process involving Beer analytical methods highly complex chemical and biochemical reactions under highly variable process conditions. Therefore, both the com- The methods for the analysis of the beers, and precisely alcohol, bitterness, color, pH, foam, haze, and shelf life are positions and the concentrations of the reducing substances 1 3 European Food Research and Technology (2022) 248:2067–2077 2069 described in Analytica EBC by European Brewery Conven- absorbance measured at 620 nm. There was a linear rela- tion [14, 15]. tionship between the absorbance and the amount of sugar Beer pH was measured with pH-meter. The determination present in the sample. This method determines both reduc- of alcohol content was performed by measuring the density ing and non-reducing sugars because of the presence of the of distillate from the degassed beer sample, which distil- strongly oxidizing sulfuric acid. Like the other methods, it is late is assumed to contain all the alcohol in the sample and non-stoichiometric, and therefore, it is necessary to prepare nothing else except water. The alcohol content of beer is a calibration curve using known glucose concentrations. For expressed in percentage (% v/v). ascorbic acid determinations, the method reported in Mus- colo et al. [18] was used. Color determination Anti‑oxidant activity assays Color of the degassed beer was measured at 430 nm by a UV–Vis spectrophotometer according to the EBC method The method reported in Muscolo et al. [18] was used to [14]. Color was expressed in EBC units and calculated determine the 2,2′-diphenyl-1-picrylhydrazyl radical according to the formula: C = A430·f·25 where C gives the (DPPH∙) scavenging assay. DPPH∙ concentration in the color (EBC), f is the dilution factor, and A430 is the absorb- cuvette has been chosen to give absorbance values of ~ 1.0. ance at 430 nm. The reaction mixtures were composed of: 10 μL of each extract, 700 μL DPPH∙ and 95% ethanol brought to 1.0 mL. Turbidity and bitterness assay The change in absorbance of the violet solution was meas- ured at 517 nm after 30 min of incubation at 37 °C. DPPH Beer turbidity was carried on by nephelometric method. The activity was expressed as μM of Trolox (T) using a calibra- amount of turbidity is expressed in EBC units. The amount tion curve (1.0–50 μM T). of turbidity was measured in NTU units and was expressed Total antioxidant capacity (TAC), as the oxygen radical in EBC unit. Bitterness was measured using a spectropho- absorbance capacity (ORAC) assay, which measures anti- tometer (UV–Vis) at 275 nm [14]. oxidant inhibition of peroxyl radical-induced oxidations and represents a measure of total antioxidant capacity, has been Anti‑oxidant compounds’ determination determined as reported in Papalia et al. [19]. Total phenolic content (TP) Polyphenols’ profile determination The method described by Singleton and Rossi [16] per- The beer samples were degassed by magnetic stirring formed the TP analysis. The absorbance was measured at (500 rpm) for 8 h and subsequently subjected to filtration 725 nm. Quantification was carried out based on the stand- through a 0.45 μm regenerated cell membrane filter (Aisino ard curve of tannic acid, and the concentration of TP was Corporation). expressed as tannic acid (TA) milligram per L of extract. The polyphenolic profile of the beer samples was assessed by Ultra-High-Performance Liquid Chromatography system. Total flavonoids’ content (TF) The equipment consisted of a Photo-Diode Array detector (RP-UHPLC-DAD, Shimadzu, Milan, Italy), equipped with TF was determined by the colorimetric method described a column oven (CTO-20AC), an autosampler (SIL-30AC), by Chang et  al. [17] with modifications. The reaction an in-line degasser (DGU-20A5R), a communication mod- between flavonoids and aluminum chloride forming the ule (CBM-20A), two parallel flow pumps with double piston 3+ flavonoid–Al complex was determined at 510 nm. The (LC-30AD), and photodiode array detector (SPD-M30A). results were expressed as milligram of quercetin (QE) per Chromatographic separation was carried out with a Kine- L of extract. tex C18 50 mm × 3 mm × 1.7 μm d.p. column (Phenomenex), in addition, a Kinetex C18 guard column was used (Phe- Total carbohydrates and ascorbic acid detection nomenex). The analyses were conducted using the follow- ing optimized chromatographic conditions: water with 0.1% Total carbohydrates were detected using the anthrone formic acid (mobile phase A), acetonitrile with 0.1% formic method with minor modifications [18]. Sugars react with acid (mobile phase B), flow 0.6 mL/min, and oven tempera- the anthrone reagent under acidic conditions to yield a ture 40 °C. blue–green color. The samples were mixed with sulfuric Analysis was performed in gradient elution as follows: 1% acid and the anthrone reagent and then boiled until the B for 5 min, then a gradient 15 min from 1% to 30% of B and reaction was completed. The solution was then cooled the 7.5 min of higher gradient from 30% to 65% of B, and finally 1 3 2070 European Food Research and Technology (2022) 248:2067–2077 washing and reconditioning of the system, with a separation It allows to pre-screen the chemical compounds and to give time of the analytes considered of about 28 min. sensory features from the Heracles chromatograms. The parameters for the photodiode array detector were: To obtain maximum sensor response, the operating spectrum resolution 256, split width 8 nm, cell tempera- parameters were optimized as previously reported in Mus- ture, 40 °C, and sampling rate 40 Hz. Data acquisition was colo et al. [21] with modifications as follows: 1 mL of each obtained in the range 190–400 nm and the chromatograms sample diluted with water (1:5) was placed in a 10 mL glass were acquired at the maximum absorbance of the com- vial, sealed with magnetic plugs. The vials were placed in pounds of interest. the Heracles autosampler (Odor Scanner HS 100, Gerstel, Mülheim, Germany) for headspace generation leaving to Sensory analysis equilibrate for approximately 20 min at 40 °C. Gas accu- mulated in the headspace of the sample was used for the The ultra-rapid gas chromatographic analysis (UFGC) by analysis. Syringe pierced the silicone septum of the mag- odor analyzer called Heracles II (mod. Heracles II, Alpha netic plug and for each sample, approximately, sampled MOS, Toulouse, France) was used for the analysis, using a 1 mL the headspace delivered at 125 µL/s by the autosam- detector system containing two short different polarity col- pler to the injector at 270 °C. The 1 mL headspace aliquot umns (MXT-5 a polar and MXT-1701 slightly polar) con- was, before the chromatographic separation, adsorbed on a nected to 2 flame ionization detectors (FID) for a global fin- TENAX absorbent trap maintained at 40 °C for 30 s, while gerprint and a data acquisition and processing system (Alpha the carrier gas (H ) flowed (flow rate: 1 mL/min) through MOS proprietary software (Alpha Soft). it to concentrate the analytes and to remove excess air and It consisted of an Odorscanner headspace autosampler moisture. Desorption was obtained by increasing the tem- (mod. HS 100, CTC Analytics, Zwingen, Switzerland), to perature of the trap up to 240 °C in 30 s and the sample was automate sampling and injection, a detector system contain- injected. The thermal program started at 40 °C (held for ing two short different polarity metal columns working in 18 s) and increased up to 250 °C at 3 °C/s and held for 30 s. parallel: a non-polar column (MXT-5: 5% diphenyl, 95% The total separation time was 118 s. methylpolysiloxane) and a slightly polar column (MXT- 1701: 14% cyanopropylphenyl, 86% methylpolysiloxane), Statistical analysis length of 10 m, diameter of 180 μm (Restek)., connected to two flame ionization detectors (FID1 and FID2) for a global Analysis of variance was carried out for all the data sets. fingerprint and a data acquisition and processing system One-way ANOVA with Tukey's Honestly. Powerful Statis- AlphaSoft 12.4 software (Alpha MOS proprietary software). tical Analysis and Graphics Software for Windows 7 was Two chromatograms are obtained at the same time, allow- used for all the statistical analyses. Effects were significant ing a well-defined identification of chemical compounds. at p ≤ 0.05. The integrated solid adsorbent trap thermo-regulated by Pel- tier cooler (0–260 °C) achieves an efficient pre-concentration of light volatiles and shows a great sensitivity (in the pg Results and discussion range). With fast column heating rates (up to 600 °C/min), results are delivered within seconds and the analysis cycle The main factors characterizing beer are alcohol content, time is from 5 to 9 min. color bitterness, and variety and intensity of flavors. These Heracles Analyzer provides a unique signature for each characteristics are standardized and allow a uniform deter- sample examined, through a chemical and/or olfactory fin- mination of the overall qualities of any beer. Data showed gerprint, quantifying specific molecules in complex matri- that all the beers had the same alcohol content. Conversely, ces [20]. The response of each detector is converted into a significant differences were observed in color intensity, chemical fingerprint by powerful software. For the calcu- expressed as EBC, that is also a measure of beer turbidity lation of Kovat’s indices and the identification of volatile and in IBU that is not a sensorial perceived beer parameter organic compounds, the alkane C6–C16 standard solution of bitterness, but it is rather the expression of the amount of was used. Collected data were analyzed using Kovats Reten- iso-alpha acids presents, that not only plays an essential role tion Index (RI) values, and specific compounds were identi- in enhancing foam stability, but, as reported by Ano et al. fied by AroChemBase library of chemical compounds with [22], has a role in the suppression of neuro-inflammations name, formula, CAS number, molecular weight, Kovats and improvement of cognitive functions appearing useful retention Index, sensory attributes, and related bibliography, for the prevention of dementia. Among the analyzed beers, was used for confirming identification. The AroChembase the highest EBC value was observed for the Weiss, and (Alpha MOS, Toulouse, France) is an add-on module that the lowest one for Heraclea (bergamot extract) and Elais can be used within the Heracles AlphaSoft 12.4 software. (olive extract). These data evidenced a less turbidity of both 1 3 European Food Research and Technology (2022) 248:2067–2077 2071 Heraclea and Elais with respect to the basal Blanche and a pH of 4.6 and a protein content of 0.6 g/100 mL (Tables 1, Weiss beers. The bitterness was the highest in the beers with 2). The protein beer values should be close to zero, because the addition of the fruit extracts. The parameters of bergamot proteins, binding to the polysaccharides, form insoluble and olive beers were within the range of Blanche and Weiss complexes which cause turbidity, compromising bever- category, respectively (Table 1). The pH average values of age stability [24]. Data obtained evidenced a less clarity the analyzed beers were in the normal range of category of basal Weiss and Blanche beers related to their protein to which they belong (between 3.8 and 4.7). pH is a really content. All the beers analyzed did not contain cholesterol important parameter, not only because it conditions the beer and fibers (Table  2). Total carbohydrates were a bit lower flavor and taste, influencing its quality, but also because it in the basal Weiss and Blanche than in Heraclea and Elais works as a preservative creating an adverse environment for (Table  2), but in any case, lower than 3.3–4.4 g/100 mL many pathogenic and food-spoilage microorganisms [23]. range recommended in the literature [25]. The ash content The addition of bergamot and olive extracts to Heraclea and ranged from 0.15% to 0.21% (Table 2). Data are in agree- Elais decreased the pH values, in respect to the Blanche ment with those reported by Alcázar et al. [26], who found and Weiss basal beers, respectively (Table 1). Heraclea and the total ash values ranging from 0.061% to 0.158%. Vita- Elais had the same pH (4.2) and the same protein content min C was significantly elevated in Heraclea and Elais, (0.2 g/100 mL) (Table 2), and basal Weiss and Blanche had 20-fold greater than basal Blanche and Weiss. Vitamin C is a potent water-soluble antioxidant in humans, protecting tissues, lipids, and proteins from oxidative damages, and it Table 1 Organoleptic properties of color (EBC), bitterness (IBU) is also a key regulator of immune function, cellular growth, alcohol by volume (%) of Heraclea (blanche with bergamot juice and differentiation [27]. The DPPH and TAC activities were extract), basal Blanche, Elais (Weiss with olive extract), and basal Weiss noticeable the highest in Heraclea and Elais beers as con- sequence of the greatest amount of antioxidant compounds ID pH EBC IBU Alcohol present (Table 2). As expected, the lipid value was close to a c b Heraclea 4.2 ± 0.2 7.5 ± 0.6 15 ± 1.0 5% zero for all the beers, and this is relevant for the quality of b b d Blanche 4.6 ± 0.1 11 ± 0.8 11 ± 0.5 5% beer, because the lipids act negatively on the formation of a c a Elais 4.2 ± 0.1 8 ± 0.4 17 ± 0.5 5% foam breaking the protein network [1]. The moisture val- b a c Weiss 4.6 ± 0.2 17 ± 0.7 13 ± 0.7 5% ues agreed with those recommended by Taylor [28], who stated that the minimum moisture percentage in beer has to Data are the mean of three independent experiments ± standard errors be 90% (Table 2). The energy content (Kcal/100 mL) was *Different letters, in the same row, indicate significant differences at p ≤ 0.05 lesser than the maximum limit allowed for commercial beers Table 2 Nutritional values of Heraclea Basal Blanche Elais Basal Weiss different craft beers Cholesterol nd nd nd nd Fibre nd nd nd Nd a a a a Ash 0.15 ± 0.06 0.18 ± 0.03 0.14 ± 0.04 0.19 ± 0.07 a a a a Moisture % 93.85 ± 2 92.74 ± 1.7 93.12 ± 1.8 92.13 ± 2 b a b a Protein 0.2 ± 0.06 0.6 ± 0.04 0.2 ± 0.05 0.6 ± 0.05 a b b b Carbohydrates 3.14 ± 0.3 2.36 ± 0.3 2.78 ± 0.2 2.77 ± 0.1 b d a c Phenols 468.49 ± 11 356.89 ± 10 530.14 ± 15 435.03 ± 16 b d a c Flavonoids 295.89 100.24 326.78 192.33 a c a b Flavonoids/protein 0.071 0.020 0.083 0.042 a c b c Vitamin C 30 ± 1.43 1.5 ± 0.36 20 ± 1.76 1.6 ± 0.25 b d a c DPPH 48.75 ± 1.36 37.21 ± 1.03 52.71 ± 1.46 42.44 ± 1.63 a d a c TAC 289 ± 2.41 199.12 ± 4.05 296 ± 3.34 247.90 ± 2.37 Lipids nd nd nd Nd b b a b Energy 12.56 ± 1.3 11.44 ± 1.5 15.82 ± 1.5 12.88 ± 1.2 Heraclea (blanche beer with bergamot extract), basal Blanche, Elais (Weiss beer with olive extract), and −1 basal Weiss. Protein (mg/100  mL), Carbohydrates (g/100  mL), Phenols (mg GAE L ), flavonoids (mg −1 −1 −1 Quercetin L ), Vitamin C (mg/100  mL ), 2,2-diphenyl-1-picrylhydrazyl (DPPH mg Trolox L ), total −1 antioxidant capacity (TAC mg Trolox L ), and energy (Kcal/100 mL) Data are the mean of three independent experiments ± standard errors *Different letters, in the same row, indicate significant differences at p ≤ 0.05 1 3 2072 European Food Research and Technology (2022) 248:2067–2077 (35 kcal/100 mL) according to Decree No. 6.871, of June the inhibition of hydroxy-methylglutaryl-coenzyme A reduc- 4, 2009 [29]. Regarding bio-compounds, total polyphenols tase (HMG-CoA reductase) [37], which in turn caused the were the highest in Elais, followed by Heraclea (Table 2). decrease of fatty acids and cholesterol in human blood with Flavonoids had the same trend of polyphenols. The ratio hypo-cholesterolemic action [38, 39]. of flavonoids/proteins was significantly the highest in the Comparing basal Blanche with Heraclea raised the beers prepared with bergamot and olive extracts, evidencing absence of the chromatographic peaks 1, 2, 3, 4, and 5 a great binding interaction of some flavonoids with carrier (Fig. 1) and a very low total quantity of the quantified phe- proteins (Table 2) that can facilitate the absorption of flavo - nols 11.44  ng/µL (Blanche) and 43.53  ng/µL (Heraclea) noids at intestinal level [30, 31]. (Table 3). The chromatograms of the phenolic profiles of the ana- Elais had a more complex phenolic profile (Fig.  1) than lyzed beers evidenced compounds typical of beers (Fig. 1). the basal Weiss, and this difference was due to the addition The phenolic profile of Heraclea was qualitatively and of the olive extract rich in bio-compounds. Elais contained quantitatively the richest in bio-compounds than the other more total flavonoids and phenolic acids (7.36 ng/µL) than beers. Peaks 1, 2, and 3 (Fig. 1) correspond to neoerioc- the beer belonging to its category (2.10 ng/µL). The differ - itrin, naringin, and neohesperidin, respectively, molecules ences could be principally related to the doubled amount of present in bergamot fruits in a very high quantity (Table 3). syringic acid and ethylgallate as emerges from Fig. 1 (chro- These bio-compounds possess various biological activities matographic peaks 6 and 7) and Table 3. Considering the such as antidiabetic, antiatherogenic, antidepressant, immu- data obtained, Heraclea and Elais beers had a qualitative and nomodulatory, antitumor, anti-inflammatory, DNA protec- quantitative profile of flavonoids superior to the examined tive, hypolipidaemic, antioxidant, peroxisome proliferator- beers of the respective category. activated receptors (PPARs) [32], and memory improver Pearson’s correlation coefficient evidenced that total phe- [30, 33, 34]. Compounds 4 and 5 (Fig.  1), identified as nols contained in Heraclea and Elais were positively and Melitidin and Brutieridin [35, 36], are phenols present only significantly correlated with DPPH (r = 0.829 and 0.866, in Bergamot. These molecules have a similar structure to respectively) and TAC (r = 0.737 and 0.662, respectively), statins (drugs that decrease fatty acids and cholesterol in the while only the Weiss positively correlated with (DPPH blood), and numerous studies reported their involvement in r = 0.549) even if at minor extent (Fig.  2A); Among the Fig. 1 Chromatogram profiles of beers: Heraclea (blanche with bergamot juice extract, blue), basal Blanche (green), Elais (Weiss with olive extract, red), and basal Weiss (black) 1 3 European Food Research and Technology (2022) 248:2067–2077 2073 Table 3 Polyphenolic content Compounds Heraclea SD Blanche SD Elais SD Weiss SD (ng/µL) of different craft beers Phenolic acids b* c a  Coumaric acid 0.060 ± 0.001 0.044 ± 0.001 0.086 ± 0.003 < LOD – a b a  4-Hydroxybenzoic acid 0.516 ± 0.023 0.507 ± 0.010 0.542 ± 0.012 < LOD – c a b d  Caffeic acid 0.175 ± 0.027 0.258 ± 0.012 0.222 ± 0.010 0.086 ± 0.005 b c a  Chlorogenic acid 0.461 ± 0.019 0.266 ± 0.002 0.705 ± 0.078 < LOD _ b a c  Ferulic acid 0.359 ± 0.018 1.766 ± 0.076 0.052 ± 0.002 < LOD – a b c  Protocatechuic acid 0.434 ± 0.018 0.332 ± 0.012 0.227 ± 0.014 < LOD c d a b  Syringic acid 0.185 ± 0.001 0.107 ± 0.001 1.218 ± 0.002 0.637 ± 0.016 b a  Vanillic acid 1.020 ± 0.380 < LOD – 1.238 ± 0.077 < LOD – a b  Ethyl gallate < LOD – < LOD – 2.791 ± 0.083 1.378 ± 0.048 Flavonoids a b  Hesperidin 4.895 ± 0.074 0.973 ± 0.160 < LOD – < LOD – b a  Sinensetin 0.052 ± 0.030 0.092 ± 0.004 < LOD – < LOD –  Neoeriocitrin 4.025 ± 0.031 < LOD – < LOD – < LOD –  (−) Epicatechin 1.768 ± 0.033 < LOD – < LOD – < LOD – ± 0.136 < LOD – < LOD – < LOD –  Neohesperidin 18.041 a c b  Tangeretin 0.068 ± 0.005 0.021 ± 0.001 0.046 ± 0.003 < LOD – b a  Kampferol 0.113 ± 0.001 < LOD – 0.229 ± 0.001 < LOD –  Naringin 9.328 ± 0.216 < LOD – < LOD – < LOD –  Nobiletin 0.243 ± 0.015 – – – – < LOD –  Melitidin 0.633 ± 0.013 < LOD < LOD < LOD < LOD < LOD –  Butieridin 1.122 ± 0.016 < LOD < LOD < LOD < LOD < LOD –  Total 43.53 4.37 7.36 2.10 – Heraclea (Italian blanche with bergamot juice extract), basal Blanche, Elais (Weiss with olive extract), and basal Weiss Data are the mean of three independent experiments ± standard errors *Different letters, in the same row, indicate significant differences at p ≤ 0.05 single phenolic acids, we observed different correlations diabetes, high blood pressure, preeclampsia, atherosclerosis, depending on the amounts of single compounds. The best acute renal failure, Alzheimer’s, and Parkinson’s. Bergamot correlation with both antioxidant activities was observed for and olive extracts for their high polyphenol and flavonoid Heraclea followed by Elais. The less bioactive single phe- content have been tested in the numerous experimental and nolic acids appeared to be vanillic and chlorogenic acids and clinical studies, which demonstrated beneficial effects on ethyl gallate (Fig. 2A). Total flavonoids contained in Hera- human health and in particular showed their involvement in clea and Elais positively correlated only with TAC (r = 0.933 the inhibition of proliferation of many kinds of cancer cell and 0.866, respectively) (Fig. 2B). Among the single flavo- lines including melanoma, colon, breast, squamous, leuke- noids relived, melitidin, kaempferol, neoesperidin, hesperi- mia, lung, prostate, colorectal, and hepatomas and neurode- din, and neoriocitrin, contained in Heraclea correlated both generative disease [40–44]. with DPPH and TAC (Fig. 2B). The correlation data evi- Aroma fingerprints (Fig.  3), extrapolated from the chro- denced a diversity of action of the single compounds belong- matographic profiles of beers and odor maps run with Her - ing to the different classes. Considering these data, Heraclea acles system [45], showed significant differences between was the beer with the greatest antioxidant activity and the Elais and Heraclea, compared to the basal beers. Elais and greatest amount of bioactive and functional compounds fol- Heraclea had the greatest aromatic richness, due to the addi- lowed in ranking by Elais, basal Blanche, and basal Weiss. tion of bergamot and olive extracts which, in turn, increased The chemical structure of an antioxidant determines its the aromatic characteristics of the beer style, adding more intrinsic reactivity versus free radicals and other ROSs, intense aromatic nuances. These were mainly attributable influencing the antioxidant activity. Thus, the measure of to carbonyl compounds, in particular butanal in Elais and antioxidant activity/capacity of food in general is essential hexanal in Heraclea, that provided green, grassy, and pea- for studying the efficiency of food antioxidants in prevent- like flavors. Carbonyl compounds are normally low in beer, ing and treating the diseases related to oxidative stress like because yeasts, during the fermentation process, have the 1 3 2074 European Food Research and Technology (2022) 248:2067–2077 DPPH TAC Heraclea BlancheElais WeissHeracleaBlanche ElaisWeiss Coumaric acid -0,98 1-0,98 0-0,84 0,327 0,929 0 4-Hydroxybenzoic a1 c -0,82 0,327 00,924 -0,81-0,140 Caffeic acid 0,982 -0,57 0,327 -0,65 0,836 -0,96-0,14 0,653 Chlorogenic acid 0,277 -0,81 0,866 00,924 -0,82-0,760 Ferulic acid1 -0,87 -0,65 00,924 0,177 0,786 Protocatechuic acid 0,982 0,5 0,866 00,98 -0,65-0,76 Syringic acid 0,997 0,397 0,866 0,693 0,953 -0,74 0,756-0,69 Vanillic acid -0,33 0-0,910 0,058 00,3 0 Ethyl gallate0 0 0,933 0,37800 -0,94-1 Total phenols 0,829 0,155 0,866 0,549 0,737 -0,29 0,66 2-1 DPPH TAC Heraclea BlancheElais WeissHeraclea BlancheElais Weiss Hesperidin 0,822-0,33 00 0,543-10 0 Sinensetin 0,327 -0,8700 0,663 -0,76 00 Neoeriocitrin0,912 000 0,687 00 0 (-)Epicatechin -0,33 00 00,05800 0 Neohesperidin0,933000 0,72600 0 Tangeretin-0,98 0,5-0,5 0-0,84 -0,65 0,327 0 Kampferol0,866 0-0,980 0,6101 0 Naringin 0,132000 0,500 0 Nobiletin0,904 000 0,67300 0 Melitidin0,993000 0,87500 0 Butieridin -0,87000 -0,9200 0 Flavonoids -0,99 -0,33-0,5 -0,870,933 -0,98 0,866-1 Fig. 2 Pearson’s correlations (r) between phenols (a), flavonoids (b), (yellow-boxed dots p < 0.05, green-boxed dots p < 0.01). The red dots and antioxidant activities. The boxed dots show the significant cor - indicate negative correlation relations between values, and the color shows the level of correlation ability to completely remove aldehydes by reduction to alco- ethanol, and the acid group is a medium-chain fatty acid. hol counterparts. Their presence in the aromatic profile of Ethyl octanoate, in particular, gives the sour apple aroma. Heraclea and Elais was therefore attributable to the added Esters are volatile compounds impacting greatly aroma- extracts [46]. ticity of beer and conferring, if in moderate quantities, a Among the sensory markers, the presence of the two fruity-flowery aroma. esters, butyl acetate and ethyl octanoate, connotes the In Heraclea, in addition to the aforementioned two esters, aroma of Elais. The first compound is an acetate ester, 2-phenylethanol and 2-methyl-1-propanol alcohols were the second is an ethyl ester in which the alcohol group is detected. This high content in alcohol compounds (also 1 3 European Food Research and Technology (2022) 248:2067–2077 2075 Fig. 3 Odor maps fingerprints of beers: Heraclea (blanche with ber - a E MXT-5-FID1 + E MXT-1701-FID2; b EC MXT-5-FID1 + EC gamot juice extract), basal Blanche, Elais (Weiss with olive extract), MXT-1701-FID2; c H MXT-5-FID1; d H MXT-1701-FID2 + HC and basal Weiss, with Heracles system equipped with two parallel MXT-5-FID1 + HC MXT-1701-FID2 non-polar column (MXT-5) and slightly polar column (MXT-1701): 1 3 2076 European Food Research and Technology (2022) 248:2067–2077 included in the article's Creative Commons licence and your intended known as fusel alcohols) could lead to a pungent smell and use is not permitted by statutory regulation or exceeds the permitted taste. use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. 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Bergamot and olive extracts as beer ingredients: their influence on nutraceutical and sensory properties

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Abstract

Citrus bergamia and Olea europaea L. variety Carolea are accounted as niche functional food for their high content of bio active compounds. Their extracts were used as adjunct to produce two beers with different styles, Blanche and Weiss, rich in antioxidants for a pool of consumers interested in a healthy lifestyle. The nutraceutical properties of these two beers were compared to Blanche and Weiss without any addition to verify if the beers enriched with natural extracts changed their aromaticity, flavors, and functionality. The antioxidant activity changed in the order: blanche bergamot beer > Weiss olive beer > blanche basal beer > Weiss basal beer. The phenolic profile of bergamot beer was qualitatively and quantitatively the richest in bio-compounds. Pearson’s correlation evidenced that total phenols contained in bergamot and olive beers were positively and significantly correlated with the antioxidant activities and precisely, with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and total antioxidant capacity (TAC). Correlation data evidenced that the bergamot was the beer with the greatest antioxidant activity and bioactive compound amount. This study highlighted as the addition of these natural extracts together with the right productive process improved sensorial beer properties, satisfying consumer taste while potentially increasing the beneficial effects on human health. Keywords Antioxidants · Craft beers · Flavonoids · Phenols · Sensory properties Introduction Beer is one of the oldest most popular alcoholic beverages over the world [1], typically, produced with natural ingredi- ents as barley (Hordeum vulgare), hops (Humulus lupulus L.), water, and yeast. In many cases, beers can be integrated * A. Muscolo with other cereals, sugars or natural herbal extracts called amuscolo@unirc.it adjunct. Hops are multi-functional ingredients with preserv- * Mt. Russo ative role which, release bitter compounds (humulones) [2] mariateresa.russo@unirc.it adding flavor, aroma, and bitterness to the beers. Beer is a F. Salafia drink that contains valuable nutritional compounds as car- fabio.salafia@unirc.it bohydrates, amino acids, minerals, vitamins, and polyphe- R. Di Sanzo nols. About 30% of beer polyphenols come from hops, and rosa.disanzo@unirc.it 70% come from malts [3]. The antioxidants and polyphenols S. Carabetta associated with a low alcoholic content determine the func- sonia.carabetta@unirc.it tional quality of the beers. Numerous researches evidenced that modest beer consumes can have anti-inflammatory and Dipartimento di Agraria, Mediterranea University of Reggio antioxidant properties [4, 5], with numerous human health Calabria, Via dell’Università, 25, 89124 Reggio Calabria, Italy benefits [6 –8]. Currently, there is an increased interest of consumers for craft beers (CB), which are distinctly fla- Food Chemistry, Safety and Sensoromic Laboratory (FoCuSS Lab), Dipartimento di Agraria, Mediterranean vored, have a quality value unique and sensory properties University of Reggio Calabria, Via dell’Università, 25, details [9]. Craft beers are unpasteurized, unfiltered, without 89124 Reggio Calabria, Italy Vol.:(0123456789) 1 3 2068 European Food Research and Technology (2022) 248:2067–2077 the addition of nitrogen or carbon dioxide under pressure can change during the process even if the raw materials [10], but with the addition of aromatic herbs, spices, fruit, and the applied treatments are the same. Slight changes in honey, sugar, and coffee, that satisfy sensorial perception structure, or even changes in shape, can alter the antioxidant and improve their nutritional and functional values increas- activity of a compound. As it is known, beer shelf life and ing the number of bio-compounds that can have beneficial flavor are partially influenced by its antioxidant status [13]. impacts on one's health [11]. The goal of this manuscript was To stabilize the productive process, we used 20 hectolitre to develop beers using natural extracts from fruits accounted Tanker EVO 2000 BBC Inox technological plant that allows as niche functional food, bergamot (Citrus bergamia) and to optimize production capacity while respecting quality. Olive (Olea europaea L. variety Carolea), whose beneficial The new fully automatic brewhouse wisely combines tech- properties depend on the specific geographic areas in which nological research and the flexibility of the artisan process. they grow (Grecanic costal area of Calabria Region, south Automation, in fact, allows the replication of tested recipes, Italy). Bergamot contains flavonoids and other beneficial optimizing production times and energy, allowing the brewer components that contribute to its antioxidant, anti-inflam- to give maximum expression to enhance the raw materials. matory capacity and its ability to reduce cholesterol. Olives Every detail has been carefully studied to minimize contact have important nutritional values depending on richness with oxygen from the malt grinding phase to the transfer to in monounsaturated fat, fiber and vitamin E together with the fermentation tank. The whole system is also designed to the presence of several phytochemicals, mostly phenolic be washed and sanitized automatically. Brewing plant has compounds that, acting as scavengers of reactive oxygen also cold chain system important to maintain the products species, have the capability to protect from oxidative dam- during the stabilization of the beer. In the brewery, there age [12]. Extracts of Bergamot and Olive, rich in bioactive is an automatic isobaric and under nitrogen bottling plant compounds, have been used as adjunct for personalizing a (Isobaric, Gai) to reduce at the minimum of the risks of beer style and for producing functional beers, moderately beer oxidation. To produce the blanche, malt, barley, and alcoholic and rich in antioxidants, for a pool of consumers oats were mixed in a well-established proportion, the hops interested in a healthy lifestyle. In this manuscript, the qual- were Styrian Golding and Czech Saaz. Extract of bergamot ity of the craft beers enriched with bergamot or olive extract juice after terpene elimination was added to the blanche. was compared to craft beers of the same typology (basal). Weiss was prepared using malt and barley and Hallertau Aroma beer fingerprints were also assessed to verify if the Hersbrucker as hop. The extract from olive fruit of Carolea addition of extracts affected the aromaticity and flavor of cultivar was added. The setting of the production process, the basal beers. the concentration of the ingredients used, and their mix ratio are covered by industrial secret. The fermentation batch was repeated three times for each typology of beer. Materials and methods Sample preparation Chemicals and reagents The present work analyzed and compared the chemical and Acetonitrile (ACN) HPLC grade, and formic acid were pur- sensory properties of two craft Beers produced in Calabria: chased from Sigma-Aldrich (Milan, Italy), Ultra-pure water Heraclea, Blanche with the addition of bergamot juice was obtained using a Milli-Q system (Millipore, Milan, extract, not filtered and not pasteurized; Elais, Weiss with Italy). Flavonoids standard (Coumaric acid, 4-Hydroxyben- the addition of olive extract not filtered and not pasteur - zoic acid, Caffeic acid, Ethylgallate, Ferulic acid, Kamp- ized. The two Calabrian beers have been compared to the ferol, Naringin, Protocatechuic acid, Syrengin acid, Vanillic Blanche and Weiss without any addition to verify if the beers acid, Hesperidin, Sinensetin, Neoeriocitrin, (−) Epicatechin, enriched with natural extracts changed their aromaticity, fla- Neohesperidin, Tangeretin, Chlorogenic acid and Nobiletin) vors, and functionality. was purchased from Extrasynthese (Genay Cedex, France). The analysis has been carried out in triplicate for each All the other chemicals were purchased from Sigma-Aldrich batch and for each typology of Craft beer. The samples were (Milan, Italy). degassed at 20 °C prior to testing, using magnetic stirrer until all gas has been released. Productive process Brewing is an extremely complicated process involving Beer analytical methods highly complex chemical and biochemical reactions under highly variable process conditions. Therefore, both the com- The methods for the analysis of the beers, and precisely alcohol, bitterness, color, pH, foam, haze, and shelf life are positions and the concentrations of the reducing substances 1 3 European Food Research and Technology (2022) 248:2067–2077 2069 described in Analytica EBC by European Brewery Conven- absorbance measured at 620 nm. There was a linear rela- tion [14, 15]. tionship between the absorbance and the amount of sugar Beer pH was measured with pH-meter. The determination present in the sample. This method determines both reduc- of alcohol content was performed by measuring the density ing and non-reducing sugars because of the presence of the of distillate from the degassed beer sample, which distil- strongly oxidizing sulfuric acid. Like the other methods, it is late is assumed to contain all the alcohol in the sample and non-stoichiometric, and therefore, it is necessary to prepare nothing else except water. The alcohol content of beer is a calibration curve using known glucose concentrations. For expressed in percentage (% v/v). ascorbic acid determinations, the method reported in Mus- colo et al. [18] was used. Color determination Anti‑oxidant activity assays Color of the degassed beer was measured at 430 nm by a UV–Vis spectrophotometer according to the EBC method The method reported in Muscolo et al. [18] was used to [14]. Color was expressed in EBC units and calculated determine the 2,2′-diphenyl-1-picrylhydrazyl radical according to the formula: C = A430·f·25 where C gives the (DPPH∙) scavenging assay. DPPH∙ concentration in the color (EBC), f is the dilution factor, and A430 is the absorb- cuvette has been chosen to give absorbance values of ~ 1.0. ance at 430 nm. The reaction mixtures were composed of: 10 μL of each extract, 700 μL DPPH∙ and 95% ethanol brought to 1.0 mL. Turbidity and bitterness assay The change in absorbance of the violet solution was meas- ured at 517 nm after 30 min of incubation at 37 °C. DPPH Beer turbidity was carried on by nephelometric method. The activity was expressed as μM of Trolox (T) using a calibra- amount of turbidity is expressed in EBC units. The amount tion curve (1.0–50 μM T). of turbidity was measured in NTU units and was expressed Total antioxidant capacity (TAC), as the oxygen radical in EBC unit. Bitterness was measured using a spectropho- absorbance capacity (ORAC) assay, which measures anti- tometer (UV–Vis) at 275 nm [14]. oxidant inhibition of peroxyl radical-induced oxidations and represents a measure of total antioxidant capacity, has been Anti‑oxidant compounds’ determination determined as reported in Papalia et al. [19]. Total phenolic content (TP) Polyphenols’ profile determination The method described by Singleton and Rossi [16] per- The beer samples were degassed by magnetic stirring formed the TP analysis. The absorbance was measured at (500 rpm) for 8 h and subsequently subjected to filtration 725 nm. Quantification was carried out based on the stand- through a 0.45 μm regenerated cell membrane filter (Aisino ard curve of tannic acid, and the concentration of TP was Corporation). expressed as tannic acid (TA) milligram per L of extract. The polyphenolic profile of the beer samples was assessed by Ultra-High-Performance Liquid Chromatography system. Total flavonoids’ content (TF) The equipment consisted of a Photo-Diode Array detector (RP-UHPLC-DAD, Shimadzu, Milan, Italy), equipped with TF was determined by the colorimetric method described a column oven (CTO-20AC), an autosampler (SIL-30AC), by Chang et  al. [17] with modifications. The reaction an in-line degasser (DGU-20A5R), a communication mod- between flavonoids and aluminum chloride forming the ule (CBM-20A), two parallel flow pumps with double piston 3+ flavonoid–Al complex was determined at 510 nm. The (LC-30AD), and photodiode array detector (SPD-M30A). results were expressed as milligram of quercetin (QE) per Chromatographic separation was carried out with a Kine- L of extract. tex C18 50 mm × 3 mm × 1.7 μm d.p. column (Phenomenex), in addition, a Kinetex C18 guard column was used (Phe- Total carbohydrates and ascorbic acid detection nomenex). The analyses were conducted using the follow- ing optimized chromatographic conditions: water with 0.1% Total carbohydrates were detected using the anthrone formic acid (mobile phase A), acetonitrile with 0.1% formic method with minor modifications [18]. Sugars react with acid (mobile phase B), flow 0.6 mL/min, and oven tempera- the anthrone reagent under acidic conditions to yield a ture 40 °C. blue–green color. The samples were mixed with sulfuric Analysis was performed in gradient elution as follows: 1% acid and the anthrone reagent and then boiled until the B for 5 min, then a gradient 15 min from 1% to 30% of B and reaction was completed. The solution was then cooled the 7.5 min of higher gradient from 30% to 65% of B, and finally 1 3 2070 European Food Research and Technology (2022) 248:2067–2077 washing and reconditioning of the system, with a separation It allows to pre-screen the chemical compounds and to give time of the analytes considered of about 28 min. sensory features from the Heracles chromatograms. The parameters for the photodiode array detector were: To obtain maximum sensor response, the operating spectrum resolution 256, split width 8 nm, cell tempera- parameters were optimized as previously reported in Mus- ture, 40 °C, and sampling rate 40 Hz. Data acquisition was colo et al. [21] with modifications as follows: 1 mL of each obtained in the range 190–400 nm and the chromatograms sample diluted with water (1:5) was placed in a 10 mL glass were acquired at the maximum absorbance of the com- vial, sealed with magnetic plugs. The vials were placed in pounds of interest. the Heracles autosampler (Odor Scanner HS 100, Gerstel, Mülheim, Germany) for headspace generation leaving to Sensory analysis equilibrate for approximately 20 min at 40 °C. Gas accu- mulated in the headspace of the sample was used for the The ultra-rapid gas chromatographic analysis (UFGC) by analysis. Syringe pierced the silicone septum of the mag- odor analyzer called Heracles II (mod. Heracles II, Alpha netic plug and for each sample, approximately, sampled MOS, Toulouse, France) was used for the analysis, using a 1 mL the headspace delivered at 125 µL/s by the autosam- detector system containing two short different polarity col- pler to the injector at 270 °C. The 1 mL headspace aliquot umns (MXT-5 a polar and MXT-1701 slightly polar) con- was, before the chromatographic separation, adsorbed on a nected to 2 flame ionization detectors (FID) for a global fin- TENAX absorbent trap maintained at 40 °C for 30 s, while gerprint and a data acquisition and processing system (Alpha the carrier gas (H ) flowed (flow rate: 1 mL/min) through MOS proprietary software (Alpha Soft). it to concentrate the analytes and to remove excess air and It consisted of an Odorscanner headspace autosampler moisture. Desorption was obtained by increasing the tem- (mod. HS 100, CTC Analytics, Zwingen, Switzerland), to perature of the trap up to 240 °C in 30 s and the sample was automate sampling and injection, a detector system contain- injected. The thermal program started at 40 °C (held for ing two short different polarity metal columns working in 18 s) and increased up to 250 °C at 3 °C/s and held for 30 s. parallel: a non-polar column (MXT-5: 5% diphenyl, 95% The total separation time was 118 s. methylpolysiloxane) and a slightly polar column (MXT- 1701: 14% cyanopropylphenyl, 86% methylpolysiloxane), Statistical analysis length of 10 m, diameter of 180 μm (Restek)., connected to two flame ionization detectors (FID1 and FID2) for a global Analysis of variance was carried out for all the data sets. fingerprint and a data acquisition and processing system One-way ANOVA with Tukey's Honestly. Powerful Statis- AlphaSoft 12.4 software (Alpha MOS proprietary software). tical Analysis and Graphics Software for Windows 7 was Two chromatograms are obtained at the same time, allow- used for all the statistical analyses. Effects were significant ing a well-defined identification of chemical compounds. at p ≤ 0.05. The integrated solid adsorbent trap thermo-regulated by Pel- tier cooler (0–260 °C) achieves an efficient pre-concentration of light volatiles and shows a great sensitivity (in the pg Results and discussion range). With fast column heating rates (up to 600 °C/min), results are delivered within seconds and the analysis cycle The main factors characterizing beer are alcohol content, time is from 5 to 9 min. color bitterness, and variety and intensity of flavors. These Heracles Analyzer provides a unique signature for each characteristics are standardized and allow a uniform deter- sample examined, through a chemical and/or olfactory fin- mination of the overall qualities of any beer. Data showed gerprint, quantifying specific molecules in complex matri- that all the beers had the same alcohol content. Conversely, ces [20]. The response of each detector is converted into a significant differences were observed in color intensity, chemical fingerprint by powerful software. For the calcu- expressed as EBC, that is also a measure of beer turbidity lation of Kovat’s indices and the identification of volatile and in IBU that is not a sensorial perceived beer parameter organic compounds, the alkane C6–C16 standard solution of bitterness, but it is rather the expression of the amount of was used. Collected data were analyzed using Kovats Reten- iso-alpha acids presents, that not only plays an essential role tion Index (RI) values, and specific compounds were identi- in enhancing foam stability, but, as reported by Ano et al. fied by AroChemBase library of chemical compounds with [22], has a role in the suppression of neuro-inflammations name, formula, CAS number, molecular weight, Kovats and improvement of cognitive functions appearing useful retention Index, sensory attributes, and related bibliography, for the prevention of dementia. Among the analyzed beers, was used for confirming identification. The AroChembase the highest EBC value was observed for the Weiss, and (Alpha MOS, Toulouse, France) is an add-on module that the lowest one for Heraclea (bergamot extract) and Elais can be used within the Heracles AlphaSoft 12.4 software. (olive extract). These data evidenced a less turbidity of both 1 3 European Food Research and Technology (2022) 248:2067–2077 2071 Heraclea and Elais with respect to the basal Blanche and a pH of 4.6 and a protein content of 0.6 g/100 mL (Tables 1, Weiss beers. The bitterness was the highest in the beers with 2). The protein beer values should be close to zero, because the addition of the fruit extracts. The parameters of bergamot proteins, binding to the polysaccharides, form insoluble and olive beers were within the range of Blanche and Weiss complexes which cause turbidity, compromising bever- category, respectively (Table 1). The pH average values of age stability [24]. Data obtained evidenced a less clarity the analyzed beers were in the normal range of category of basal Weiss and Blanche beers related to their protein to which they belong (between 3.8 and 4.7). pH is a really content. All the beers analyzed did not contain cholesterol important parameter, not only because it conditions the beer and fibers (Table  2). Total carbohydrates were a bit lower flavor and taste, influencing its quality, but also because it in the basal Weiss and Blanche than in Heraclea and Elais works as a preservative creating an adverse environment for (Table  2), but in any case, lower than 3.3–4.4 g/100 mL many pathogenic and food-spoilage microorganisms [23]. range recommended in the literature [25]. The ash content The addition of bergamot and olive extracts to Heraclea and ranged from 0.15% to 0.21% (Table 2). Data are in agree- Elais decreased the pH values, in respect to the Blanche ment with those reported by Alcázar et al. [26], who found and Weiss basal beers, respectively (Table 1). Heraclea and the total ash values ranging from 0.061% to 0.158%. Vita- Elais had the same pH (4.2) and the same protein content min C was significantly elevated in Heraclea and Elais, (0.2 g/100 mL) (Table 2), and basal Weiss and Blanche had 20-fold greater than basal Blanche and Weiss. Vitamin C is a potent water-soluble antioxidant in humans, protecting tissues, lipids, and proteins from oxidative damages, and it Table 1 Organoleptic properties of color (EBC), bitterness (IBU) is also a key regulator of immune function, cellular growth, alcohol by volume (%) of Heraclea (blanche with bergamot juice and differentiation [27]. The DPPH and TAC activities were extract), basal Blanche, Elais (Weiss with olive extract), and basal Weiss noticeable the highest in Heraclea and Elais beers as con- sequence of the greatest amount of antioxidant compounds ID pH EBC IBU Alcohol present (Table 2). As expected, the lipid value was close to a c b Heraclea 4.2 ± 0.2 7.5 ± 0.6 15 ± 1.0 5% zero for all the beers, and this is relevant for the quality of b b d Blanche 4.6 ± 0.1 11 ± 0.8 11 ± 0.5 5% beer, because the lipids act negatively on the formation of a c a Elais 4.2 ± 0.1 8 ± 0.4 17 ± 0.5 5% foam breaking the protein network [1]. The moisture val- b a c Weiss 4.6 ± 0.2 17 ± 0.7 13 ± 0.7 5% ues agreed with those recommended by Taylor [28], who stated that the minimum moisture percentage in beer has to Data are the mean of three independent experiments ± standard errors be 90% (Table 2). The energy content (Kcal/100 mL) was *Different letters, in the same row, indicate significant differences at p ≤ 0.05 lesser than the maximum limit allowed for commercial beers Table 2 Nutritional values of Heraclea Basal Blanche Elais Basal Weiss different craft beers Cholesterol nd nd nd nd Fibre nd nd nd Nd a a a a Ash 0.15 ± 0.06 0.18 ± 0.03 0.14 ± 0.04 0.19 ± 0.07 a a a a Moisture % 93.85 ± 2 92.74 ± 1.7 93.12 ± 1.8 92.13 ± 2 b a b a Protein 0.2 ± 0.06 0.6 ± 0.04 0.2 ± 0.05 0.6 ± 0.05 a b b b Carbohydrates 3.14 ± 0.3 2.36 ± 0.3 2.78 ± 0.2 2.77 ± 0.1 b d a c Phenols 468.49 ± 11 356.89 ± 10 530.14 ± 15 435.03 ± 16 b d a c Flavonoids 295.89 100.24 326.78 192.33 a c a b Flavonoids/protein 0.071 0.020 0.083 0.042 a c b c Vitamin C 30 ± 1.43 1.5 ± 0.36 20 ± 1.76 1.6 ± 0.25 b d a c DPPH 48.75 ± 1.36 37.21 ± 1.03 52.71 ± 1.46 42.44 ± 1.63 a d a c TAC 289 ± 2.41 199.12 ± 4.05 296 ± 3.34 247.90 ± 2.37 Lipids nd nd nd Nd b b a b Energy 12.56 ± 1.3 11.44 ± 1.5 15.82 ± 1.5 12.88 ± 1.2 Heraclea (blanche beer with bergamot extract), basal Blanche, Elais (Weiss beer with olive extract), and −1 basal Weiss. Protein (mg/100  mL), Carbohydrates (g/100  mL), Phenols (mg GAE L ), flavonoids (mg −1 −1 −1 Quercetin L ), Vitamin C (mg/100  mL ), 2,2-diphenyl-1-picrylhydrazyl (DPPH mg Trolox L ), total −1 antioxidant capacity (TAC mg Trolox L ), and energy (Kcal/100 mL) Data are the mean of three independent experiments ± standard errors *Different letters, in the same row, indicate significant differences at p ≤ 0.05 1 3 2072 European Food Research and Technology (2022) 248:2067–2077 (35 kcal/100 mL) according to Decree No. 6.871, of June the inhibition of hydroxy-methylglutaryl-coenzyme A reduc- 4, 2009 [29]. Regarding bio-compounds, total polyphenols tase (HMG-CoA reductase) [37], which in turn caused the were the highest in Elais, followed by Heraclea (Table 2). decrease of fatty acids and cholesterol in human blood with Flavonoids had the same trend of polyphenols. The ratio hypo-cholesterolemic action [38, 39]. of flavonoids/proteins was significantly the highest in the Comparing basal Blanche with Heraclea raised the beers prepared with bergamot and olive extracts, evidencing absence of the chromatographic peaks 1, 2, 3, 4, and 5 a great binding interaction of some flavonoids with carrier (Fig. 1) and a very low total quantity of the quantified phe- proteins (Table 2) that can facilitate the absorption of flavo - nols 11.44  ng/µL (Blanche) and 43.53  ng/µL (Heraclea) noids at intestinal level [30, 31]. (Table 3). The chromatograms of the phenolic profiles of the ana- Elais had a more complex phenolic profile (Fig.  1) than lyzed beers evidenced compounds typical of beers (Fig. 1). the basal Weiss, and this difference was due to the addition The phenolic profile of Heraclea was qualitatively and of the olive extract rich in bio-compounds. Elais contained quantitatively the richest in bio-compounds than the other more total flavonoids and phenolic acids (7.36 ng/µL) than beers. Peaks 1, 2, and 3 (Fig. 1) correspond to neoerioc- the beer belonging to its category (2.10 ng/µL). The differ - itrin, naringin, and neohesperidin, respectively, molecules ences could be principally related to the doubled amount of present in bergamot fruits in a very high quantity (Table 3). syringic acid and ethylgallate as emerges from Fig. 1 (chro- These bio-compounds possess various biological activities matographic peaks 6 and 7) and Table 3. Considering the such as antidiabetic, antiatherogenic, antidepressant, immu- data obtained, Heraclea and Elais beers had a qualitative and nomodulatory, antitumor, anti-inflammatory, DNA protec- quantitative profile of flavonoids superior to the examined tive, hypolipidaemic, antioxidant, peroxisome proliferator- beers of the respective category. activated receptors (PPARs) [32], and memory improver Pearson’s correlation coefficient evidenced that total phe- [30, 33, 34]. Compounds 4 and 5 (Fig.  1), identified as nols contained in Heraclea and Elais were positively and Melitidin and Brutieridin [35, 36], are phenols present only significantly correlated with DPPH (r = 0.829 and 0.866, in Bergamot. These molecules have a similar structure to respectively) and TAC (r = 0.737 and 0.662, respectively), statins (drugs that decrease fatty acids and cholesterol in the while only the Weiss positively correlated with (DPPH blood), and numerous studies reported their involvement in r = 0.549) even if at minor extent (Fig.  2A); Among the Fig. 1 Chromatogram profiles of beers: Heraclea (blanche with bergamot juice extract, blue), basal Blanche (green), Elais (Weiss with olive extract, red), and basal Weiss (black) 1 3 European Food Research and Technology (2022) 248:2067–2077 2073 Table 3 Polyphenolic content Compounds Heraclea SD Blanche SD Elais SD Weiss SD (ng/µL) of different craft beers Phenolic acids b* c a  Coumaric acid 0.060 ± 0.001 0.044 ± 0.001 0.086 ± 0.003 < LOD – a b a  4-Hydroxybenzoic acid 0.516 ± 0.023 0.507 ± 0.010 0.542 ± 0.012 < LOD – c a b d  Caffeic acid 0.175 ± 0.027 0.258 ± 0.012 0.222 ± 0.010 0.086 ± 0.005 b c a  Chlorogenic acid 0.461 ± 0.019 0.266 ± 0.002 0.705 ± 0.078 < LOD _ b a c  Ferulic acid 0.359 ± 0.018 1.766 ± 0.076 0.052 ± 0.002 < LOD – a b c  Protocatechuic acid 0.434 ± 0.018 0.332 ± 0.012 0.227 ± 0.014 < LOD c d a b  Syringic acid 0.185 ± 0.001 0.107 ± 0.001 1.218 ± 0.002 0.637 ± 0.016 b a  Vanillic acid 1.020 ± 0.380 < LOD – 1.238 ± 0.077 < LOD – a b  Ethyl gallate < LOD – < LOD – 2.791 ± 0.083 1.378 ± 0.048 Flavonoids a b  Hesperidin 4.895 ± 0.074 0.973 ± 0.160 < LOD – < LOD – b a  Sinensetin 0.052 ± 0.030 0.092 ± 0.004 < LOD – < LOD –  Neoeriocitrin 4.025 ± 0.031 < LOD – < LOD – < LOD –  (−) Epicatechin 1.768 ± 0.033 < LOD – < LOD – < LOD – ± 0.136 < LOD – < LOD – < LOD –  Neohesperidin 18.041 a c b  Tangeretin 0.068 ± 0.005 0.021 ± 0.001 0.046 ± 0.003 < LOD – b a  Kampferol 0.113 ± 0.001 < LOD – 0.229 ± 0.001 < LOD –  Naringin 9.328 ± 0.216 < LOD – < LOD – < LOD –  Nobiletin 0.243 ± 0.015 – – – – < LOD –  Melitidin 0.633 ± 0.013 < LOD < LOD < LOD < LOD < LOD –  Butieridin 1.122 ± 0.016 < LOD < LOD < LOD < LOD < LOD –  Total 43.53 4.37 7.36 2.10 – Heraclea (Italian blanche with bergamot juice extract), basal Blanche, Elais (Weiss with olive extract), and basal Weiss Data are the mean of three independent experiments ± standard errors *Different letters, in the same row, indicate significant differences at p ≤ 0.05 single phenolic acids, we observed different correlations diabetes, high blood pressure, preeclampsia, atherosclerosis, depending on the amounts of single compounds. The best acute renal failure, Alzheimer’s, and Parkinson’s. Bergamot correlation with both antioxidant activities was observed for and olive extracts for their high polyphenol and flavonoid Heraclea followed by Elais. The less bioactive single phe- content have been tested in the numerous experimental and nolic acids appeared to be vanillic and chlorogenic acids and clinical studies, which demonstrated beneficial effects on ethyl gallate (Fig. 2A). Total flavonoids contained in Hera- human health and in particular showed their involvement in clea and Elais positively correlated only with TAC (r = 0.933 the inhibition of proliferation of many kinds of cancer cell and 0.866, respectively) (Fig. 2B). Among the single flavo- lines including melanoma, colon, breast, squamous, leuke- noids relived, melitidin, kaempferol, neoesperidin, hesperi- mia, lung, prostate, colorectal, and hepatomas and neurode- din, and neoriocitrin, contained in Heraclea correlated both generative disease [40–44]. with DPPH and TAC (Fig. 2B). The correlation data evi- Aroma fingerprints (Fig.  3), extrapolated from the chro- denced a diversity of action of the single compounds belong- matographic profiles of beers and odor maps run with Her - ing to the different classes. Considering these data, Heraclea acles system [45], showed significant differences between was the beer with the greatest antioxidant activity and the Elais and Heraclea, compared to the basal beers. Elais and greatest amount of bioactive and functional compounds fol- Heraclea had the greatest aromatic richness, due to the addi- lowed in ranking by Elais, basal Blanche, and basal Weiss. tion of bergamot and olive extracts which, in turn, increased The chemical structure of an antioxidant determines its the aromatic characteristics of the beer style, adding more intrinsic reactivity versus free radicals and other ROSs, intense aromatic nuances. These were mainly attributable influencing the antioxidant activity. Thus, the measure of to carbonyl compounds, in particular butanal in Elais and antioxidant activity/capacity of food in general is essential hexanal in Heraclea, that provided green, grassy, and pea- for studying the efficiency of food antioxidants in prevent- like flavors. Carbonyl compounds are normally low in beer, ing and treating the diseases related to oxidative stress like because yeasts, during the fermentation process, have the 1 3 2074 European Food Research and Technology (2022) 248:2067–2077 DPPH TAC Heraclea BlancheElais WeissHeracleaBlanche ElaisWeiss Coumaric acid -0,98 1-0,98 0-0,84 0,327 0,929 0 4-Hydroxybenzoic a1 c -0,82 0,327 00,924 -0,81-0,140 Caffeic acid 0,982 -0,57 0,327 -0,65 0,836 -0,96-0,14 0,653 Chlorogenic acid 0,277 -0,81 0,866 00,924 -0,82-0,760 Ferulic acid1 -0,87 -0,65 00,924 0,177 0,786 Protocatechuic acid 0,982 0,5 0,866 00,98 -0,65-0,76 Syringic acid 0,997 0,397 0,866 0,693 0,953 -0,74 0,756-0,69 Vanillic acid -0,33 0-0,910 0,058 00,3 0 Ethyl gallate0 0 0,933 0,37800 -0,94-1 Total phenols 0,829 0,155 0,866 0,549 0,737 -0,29 0,66 2-1 DPPH TAC Heraclea BlancheElais WeissHeraclea BlancheElais Weiss Hesperidin 0,822-0,33 00 0,543-10 0 Sinensetin 0,327 -0,8700 0,663 -0,76 00 Neoeriocitrin0,912 000 0,687 00 0 (-)Epicatechin -0,33 00 00,05800 0 Neohesperidin0,933000 0,72600 0 Tangeretin-0,98 0,5-0,5 0-0,84 -0,65 0,327 0 Kampferol0,866 0-0,980 0,6101 0 Naringin 0,132000 0,500 0 Nobiletin0,904 000 0,67300 0 Melitidin0,993000 0,87500 0 Butieridin -0,87000 -0,9200 0 Flavonoids -0,99 -0,33-0,5 -0,870,933 -0,98 0,866-1 Fig. 2 Pearson’s correlations (r) between phenols (a), flavonoids (b), (yellow-boxed dots p < 0.05, green-boxed dots p < 0.01). The red dots and antioxidant activities. The boxed dots show the significant cor - indicate negative correlation relations between values, and the color shows the level of correlation ability to completely remove aldehydes by reduction to alco- ethanol, and the acid group is a medium-chain fatty acid. hol counterparts. Their presence in the aromatic profile of Ethyl octanoate, in particular, gives the sour apple aroma. Heraclea and Elais was therefore attributable to the added Esters are volatile compounds impacting greatly aroma- extracts [46]. ticity of beer and conferring, if in moderate quantities, a Among the sensory markers, the presence of the two fruity-flowery aroma. esters, butyl acetate and ethyl octanoate, connotes the In Heraclea, in addition to the aforementioned two esters, aroma of Elais. The first compound is an acetate ester, 2-phenylethanol and 2-methyl-1-propanol alcohols were the second is an ethyl ester in which the alcohol group is detected. This high content in alcohol compounds (also 1 3 European Food Research and Technology (2022) 248:2067–2077 2075 Fig. 3 Odor maps fingerprints of beers: Heraclea (blanche with ber - a E MXT-5-FID1 + E MXT-1701-FID2; b EC MXT-5-FID1 + EC gamot juice extract), basal Blanche, Elais (Weiss with olive extract), MXT-1701-FID2; c H MXT-5-FID1; d H MXT-1701-FID2 + HC and basal Weiss, with Heracles system equipped with two parallel MXT-5-FID1 + HC MXT-1701-FID2 non-polar column (MXT-5) and slightly polar column (MXT-1701): 1 3 2076 European Food Research and Technology (2022) 248:2067–2077 included in the article's Creative Commons licence and your intended known as fusel alcohols) could lead to a pungent smell and use is not permitted by statutory regulation or exceeds the permitted taste. use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. 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Journal

European Food Research and Technology – Springer Journals

Published: Aug 1, 2022

Keywords: Antioxidants; Craft beers; Flavonoids; Phenols; Sensory properties

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