Occurrence of melatonin and indolic compounds derived from L-tryptophan yeast metabolism in fermented wort and commercial beers

1 This is the peer reviewed version of the article accepted for publication in Food Chemistry 1 Journal Volume 331, 2020, 127192, which has been published in final form at 2 https://doi.org/10.1016/j.foodchem.2020.127192 3 4 Occurrence of melatonin and indolic compounds derived from L-tryptophan yeast 5 metabolism in fermented wort and commercial beers 6 Edwin Fernández-Cruz1, Fernando Carrasco-Galán1, Ana B. Cerezo-López1, Eva 7 Valero3, Morcillo-Parra, M.A.2, M. Jesus Torija2, Ana M. Troncoso1, M. Carmen 8 García-Parrilla1*. 9 1Departamento de Nutrición y Bromatología, Toxicología y Medicina Legal. Facultad 10 de Farmacia. Universidad de Sevilla. C/ Profesor García González 2, 41012, Sevilla, 11 Spain 12 2Departament de Bioquimica i Biotecnologia. Facultat d’enologia. Universitat Rovira i 13 Virgili. C/ Marcel·lí Domingo 1. Campus Sescelades 43007, Tarragona, Spain 14 3 Departamento de Biología Molecular e Ingeniería Bioquímica, Universidad Pablo de 15 Olavide, Ctra. Utrera, Km 1, Sevilla 41013, Spain 16 *Corresponding author: M.C. Garcia-Parrilla 17 E-mail address: [email protected] 18 19 20 21 22 23 2 24 25 Highlights: 26 • PTFE filters were the most convenient to diminish losses of tryptophan 27 metabolites 28 • Melatonin is stable in beer matrix for 30 days at -20ºC 29 • 5-HTRP, NA5-HT, 3-IAA and L-TRP EE described for the first time in 30 commercial beers 31 • NA5-HT and 3-IAA produced during the alcoholic fermentation of wort 32 3 Abstract 33 Melatonin and serotonin are bioactive compounds present in foods and beverages and 34 related to neuroprotection and anti-angiogenesis, among other activities. They have been 35 described in wines and the role of yeast in their formation is clear. Thus, this study 36 evaluates the content of these bioactives and other related indolic compounds in beer. For 37 this purpose, commercial beers were analyzed by a validated UHPLC-HRMS method and 38 sample treatment optimized due to the low concentrations expected. Moreover, a wort 39 was fermented with different commercial beer yeast (Abbaye, Diamond, SafAle, 40 SafLager) in order to monitor the formation of these bioactives during the elaboration 41 process. 42 Results show that indolic compounds such as N-acetylserotonin and 3-indoleacetic acid 43 are produced during the alcoholic fermentation of wort. Moreover, the occurrence of four 44 indolic compounds (5-hydroxytryptophan, N-acetylserotonin, 3-indoleacetic acid, L- 45 tryptophan ethyl ester) in commercial beers is reported for the first time. 46 Keywords: melatonin, 3-indoleacetic acid, beer, bioactive, HRMS, fermentation, 47 Saccharomyces cerevisiae 48 4 1. INTRODUCTION 49 Beer is one of the most consumed alcoholic beverages in the European Union (EU 28), 50 being its consumption of 359,112 miles of hL in 2016. Spain is the third country in beer 51 production (38,634 miles of hL), being its consumption per capita of 46 L per year (The 52 Brewers of Europe, 2017). 53 Beer contains alcohol, amino acids, carbohydrates, vitamins and also bioactive 54 compounds such as polyphenols and melanoidins, mostly from hops and malt (González- 55 San José, Rodríguez, & Valls-Bellés, 2016). The amino acid L-tryptophan (L-TRP) is 56 considered as a non-preferred nitrogen source for yeast (Beltran, Novo, Rozès, Mas, & 57 Guillamón, 2004), therefore its presence in wort and beer usually goes unnoticed since it 58 is not crucial for the fermentation step. Nevertheless, L-TRP is the precursor of some 59 bioactive compounds such as melatonin (MEL), serotonin (5-HT) and 3-indoleacetic acid 60 (3-IAA) (Rodriguez-Naranjo, Gil-Izquierdo, Troncoso, Cantos, & García-Parrilla, 2011; 61 Mihaljević Žulj, Tomaz, Maslov Bandić, Puhelek, Jagatić Korenika, & Jeromel, 2015) 62 reported in fermented beverages as wine. Since yeast metabolism is essential for the 63 synthesis of above mentioned bioactive compounds in wine, it seems reasonable to 64 evaluate their presence in beer as well as to evaluate their production during the alcoholic 65 fermentation of wort. 66 The analysis of MEL in foods and beverages has been the focus of a number of studies. 67 Thus, it has been quantified by means of ELISA in grapes (Iriti, Rossoni, & Faoro, 2006), 68 and beers (Garcia-Moreno, Calvo, & Maldonado, 2013). However, ELISA shows a huge 69 variability when tested in complex matrices different from biological fluids (Rodriguez- 70 Naranjo et al., 2011). Recent advances in the analytical field mostly consist on the use of 71 ultra-high-performance liquid chromatography coupled to High Resolution Mass 72 Spectrometer (UHPLC/HRMS) has become the preferred technique for the analysis of 73 5 bioactive compounds in food. By these means it is possible to identify and quantify 74 metabolites unequivocally at trace levels (1-100 ng/mL), permitting the simultaneous 75 analysis of a number of different compounds involved in the synthetic pathways or at 76 least chemically related. Recently, our research group has used it to determine L-TRP 77 derived compounds related with MEL metabolism such as 5-hydroxytryptophan (5- 78 HTRP), N-acetylserotonin (NA5-HT), 5-HT and also others coming from different 79 pathways such as tryptamine (TRY) and L-tryptophan ethyl ester (L-TRP EE) in synthetic 80 grape must (SM) (Fernández-Cruz, Álvarez-Fernández, Valero, Troncoso, & García- 81 Parrilla, 2016). Kocadağlı, Yilmaz and Gökmen (2014) reported the presence of MEL in 82 beer at 94.5 ng/mL using HPLC/MS-MS. In any case, the main analytical challenging 83 issues include the low concentration of MEL found in foods, sometimes near to the limits 84 of detection, its amphipathic characteristics and the high reactivity of the molecule. 85 Beer presents in its composition some compounds from the starch sources such as dextrins 86 (González-San José et al., 2016) that could negatively affect the analytical determination 87 of bioactive compounds such as MEL and 5-HT which are expected in ng/mL. Sample 88 pre-treatment is an important step to avoid the presence of interfering substances in the 89 UHPLC/HRMS analysis. For this purpose, solid phase extraction (SPE) is the most 90 suitable technique since it is widely used for complex matrices as beer, due to its low 91 solvent consumption, sample clean-up, quickness and simplicity (Boyaci et al., 2015). 92 Besides, a prior filtration step is recommended to avoid clogging in the SPE cartridges 93 before loading samples (Boyaci et al., 2015). Nylon (NY), polytetrafluoroethylene 94 (PTFE) and cellulose acetate (CA) filters are used in the analysis of MEL in grape skin 95 and wine, and in metabolomic studies related with yeast (Iriti et al., 2006; Stege, Sombra, 96 Messina, Martinez, & Silva, 2010; Rodriguez-Naranjo et al., 2011; Smart, Aggio, Van 97 Houtte, & Villas-Bôas, 2010). Since filters can retain small compounds, their suitability 98 6 evaluation for the analysis of L-TRP metabolites in beer matrix is worthy for 99 methodology development. However, in order to quantify the expected trace levels, beer 100 samples have to be concentrated as seen in other food matrices such as wine (Rodriguez- 101 Naranjo et al., 2011). 102 Thus, the aims of this work are: (i) to optimize sample treatment in order to improve the 103 analysis of bioactive indolic compounds in beers, (ii) to study different conditions of 104 temperature and concentration during storage time in order to set the most suitable storage 105 conditions for beer samples, (iii) to unveil the production of indolic compounds during 106 alcoholic fermentation in wort, and (iv) to study the occurrence of different L-TRP 107 derived compounds in commercial beers, widely consumed in Spain, using a validated 108 UHPLC/HRMS method. 109 2. MATERIAL AND METHODS 110 2.1 Beer samples 111 Beer brands were selected as being representative of beer consumption in Spain. The 112 Brand Foodprint 2018 study (Kantar Worldpanel, 2018 113 https://www.kantarworldpanel.com) about the main food brands sold was used to extract 114 the most consumed beers in Spain by autonomous region, including alcohol-free beers. 115 As a result, 19 different beer brands were purchased from local supermarkets in the glass- 116 bottle format. More details of each beer sample are described in Table 1. Most beers were 117 Lager type, with the exception of Guinness (Stout type), and had an alcoholic content 118 between 0.0 and 7.2º. 119 2.2 Beer Sample treatment 120 Commercial beers were degassed for 30 minutes in an UB-1488 ultrasonic bath 121 (J.P.Selecta, Barcelona, Spain). All samples were filtered before the solid phase 122 7 extraction (SPE) procedure. SPE was performed in C18 Bond Elut SPE cartridge 123 (VARIAN, Agilent) which has been widely used for sample cleaning to study MEL in 124 fermented beverages (Rodriguez-Naranjo et al., 2011) and indolic compounds in SM 125 (Fernández-Cruz, Álvarez-Fernández, Valero, Troncoso, & García-Parrilla, 2017) and 126 wines of different grape varieties (Fernández-Cruz, Cerezo, Cantos-Villar, Troncoso, & 127 García-Parrilla, 2019a). 128 All cartridges were conditioned with 2 mL of methanol and after that with 2 mL of milliQ 129 water. Then, 2 mL of samples were loaded. Cartridges were subsequently washed with 2 130 ml of a 10% methanol:water solution. The indolic compounds under study were eluted 131 with 1 mL of methanol in dark brown eppendorfs and dried in a vacuum concentrator 132 (HyperVACLITE, GYOZEN, Korea) at 30 ºC, 2000 rpm. Pellets were subsequently 133 rediluted in dark HPLC vials with 500 µL solution of 10% methanol:water with formic 134 acid (0.1 %) prior to UHPLC/HRMS analysis. 135 2.3. Reagents 136 Standards of 9 indolic compounds including 3-IAA, 5-HTRP, 5-HT, L-TRP, L-TRP EE, 137 MEL, NA5-HT, TRY and tryptophol (TOL) were supplied by Sigma Aldrich (Barcelona, 138 Spain). Merck (Darmstadt, Germany) provided methanol of LC/MS grade. Prolabo ® 139 (Obregon, Mexico) supplied formic acid for LC/MS with at 99% purity. 140 2.4. Filtration optimization 141 In order to elucidate the effect that filtration caused on the concentrations of indolic 142 compounds, most usual filters were tested such as nylon and polytetrafluoroethylene. 143 Additionally, cellulose acetate filters were also included since they are used to perform 144 the quenching step for extracellular matrices fermented with yeast (Smart et al., 2010). 145 For the sake of comparison, three different stock solutions (LOQ, 1.5 x LOQ and 3x LOQ) 146 8 of the 9 indolic compounds were prepared. Values based on the LOQ concentrations 147 (Table 1 of supplementary material) previously described by Fernández-Cruz et al. 2016. 148 The stock solutions were filtered with the different membrane materials and were dried 149 in a vacuum concentrator (HyperVACLITE, GYOZEN, Korea) at 30º C, 2000 rpm. 150 Pellets were subsequently rediluted in dark HPLC vials with 500 µL solution of 10% 151 methanol:water with formic acid (0.1 %) prior to UHPLC/HRMS analysis (Fernández- 152 Cruz et al., 2016) Stock solutions at LOQ, 1.5x LOQ and 3x LOQ values no filtrated were 153 also analysed as control. Results are expressed as a rate between concentration of samples 154 after use of different filter materials (PTFE, nylon and cellulose acetate) and samples with 155 no filtration step. 156 2.5. Stability of samples and storage conditions 157 A standard pilsner beer was used to monitor likely alterations on indolic compounds 158 concentration along storage of samples at different temperatures. Previously, beer was 159 degassed 30 minutes by stirring to remove the carbon dioxide. Beer samples were 160 enriched with 200 ng/mL of stock solutions. Then, 1 mL of each solution was placed in 161 dark vials. This cluster was prepared in triplicate in order to storage beer matrix at three 162 temperatures: 4 ºC, -20 ºC and -80 ºC. Sampling (1 mL) was performed at the initial point, 163 and then after 7, 15 and 30 days of storage period. 164 2.6. Wort alcoholic fermentation 165 The wort was prepared by mixing a liophylized commercial blonde barley malt extract (8 166 EBC, La tienda del cervecero, La Palma, Cartagena, Murcia) with sterile water up to a 167 density of 1050 g/cm3. Subsequently, wort was brought to boil for 5 minutes and cooled 168 down in order to add hop up to reach 12 IBUs (isomerized hop extract 6%, La tienda del 169 cervecero, La Palma, Cartagena, Murcia). Four fermentation experiments were conducted 170 9 as follows: 750 mL of wort was placed 1L-bottles with an initial concentration of 106 171 cel/mL of the different Saccharomyces cerevisiae yeast Abbaye and Diamond were 172 purchased by Lallemand (Bayern, Germany); SafLager and SafAle were supplied by 173 Fermentis (Marcq-en-Baroeul, France). Each yeast was provided as active dried yeast 174 (ADY), being rehydrated before inoculation, following the manufacturer’s instructions. 175 Fermentations were carried out at 22ºC for ale type (Abbaye, SafAle) and at 16ºC for 176 lager type yeast (Diamond, SafLager). An initial sample of the inoculum was taken before 177 inoculation, and then, sampling was performed daily for seven days until the end of 178 alcoholic fermentation. A volume enough to reach 2x109 cells was taken from bottles and 179 centrifuged at room temperature for 5 minutes at 7600 rpm to separate supernatant from 180 cell pellet. The former was stored at -80º C until UHPLC/HRMS analysis. 181 2.7. Quenching and intracellular metabolites extraction 182 Each cell pellet was submitted to a quenching procedure according to Álvarez-Fernández, 183 Fernández-Cruz, Valero, Troncoso and García-Parrilla (2019). Briefly, the resuspended 184 pellet, in milliQ water, was mixed with a pre-cooled quenching solution (-23º C) 185 (glycerol:saline solution, 3:2, v/v) in a 1:4 proportion. Then, cells were centrifuged at 186 36036 g for 20 minutes at -20º C and the resulting supernatant was discarded. The pellets 187 were mixed with a cold-washing solution (glycerol:saline solution, 1:1, v/v) and 188 centrifuged again with the same conditions previously described. Eventually, pellets were 189 stored at -80º C before intracellular metabolites extraction. 190 Intracellular extraction was performed based on previous works (Álvarez-Fernández et 191 al., 2019). The procedure consisted on mixing pellet with a cold extraction solution (-30º 192 C) (methanol:milliQ water, 1:1, v/v) and centrifuged twice at 36086 g for 20 minutes at - 193 20º C, preserving the supernatant. Resulting extracts were stored at -80º C until SPE 194 procedure and UHPLC/HRMS analysis. 195 16 (Figure 4E), in days 1-3. SafLager was the strain that synthesized the highest MEL 342 concentration (0.08 ng/mL). Formerly, Sprenger, Hardeland, Fuhrberg, and Han (1999) 343 reported intracellular MEL in S.cerevisiae. However, in a recent study developed by our 344 group with both Saccharomyces and non-Saccharomyces strains MEL was only 345 quantified in the intracellular compartment of non-Saccharomyces strains along the whole 346 alcoholic fermentation, with values between 0.13-0.30 ng/mL (Fernandez-Cruz, et al., 347 2019b). From these data, we can confirm that MEL is synthesised by brewing yeast 348 between days 2 and 3 in agreement with wine yeast. 349 3-IAA and TOL are considered the most usual L-TRP metabolites derived from the 350 Ehrlich pathway of alcoholic fermentation. As far as we are concerned, this is the first 351 time that 3-IAA is determined in wort being present from the initial wort (Figure 3F). It 352 is significantly enriched at days 2-3 (23-34 ng/mL) and diminishes its concentration at 353 the end of alcoholic fermentation (day 7) (9.4-46.6 %) for Diamond, SafLager and 354 Abbaye strains, although the final concentrations are always higher than the initial ones. 355 Only SafAle maintained constant the 3-IAA concentration. In natural grape must 356 fermentations, 3-IAA was synthesized at similar concentrations (10-75 ng/mL) 357 (Fernández-Cruz et al., 2019a; Mihaljević Žulj et al., 2015). However, in SM it was 358 quantified in a larger extent (100-500 ng/mL) (Fernández-Cruz et al., 2017). This suggest 359 that S.cerevisiae is able to produce 3-IAA in substrates with different composition (grape 360 must vs wort), although selected strain is also determinant for the 3-IAA synthesis. 361 Nevertheless, this compound is consider as a precursor of 2-aminoacetophenone, an off 362 flavour in wine (Simat, Hoenicke, Gessner, & Christoph, 2004) but its role in yeast is not 363 clear. Its decrease during the alcoholic fermentation may be caused by the bond of 3-IAA 364 to amino acids or peptides that yeasts use for their metabolism after a convenient 365 intracellular hydrolysis (Simat et al., 2004). In the intracellular medium, 3-IAA was 366 17 reported in all the strains from the inoculum (Figure 4F), with no changes along alcoholic 367 fermentation with the exception of the lager type yeast, which increased the uptake of 3- 368 IAA at the end (1.27 ng/mL). Previously, when different yeast strains such us S. 369 cerevisiae (P24, QA23) and non-Saccharomyces (H. uvarum, T. delbrueckii) where used, 370 changes in 3-IAA uptake at the end of alcoholic fermentation were only significant in two 371 strains (QA23, T. delbrueckii) (Fernandez-Cruz et al., 2019b). This data reinforce the 372 importance of strains selection to produce 3-IAA. 373 Concerning TOL, it was by far the most abundant metabolite derived from the L-TRP 374 metabolism along the alcoholic fermentation (Figure 3G). It was not present in the initial 375 wort, but all yeast strains produced it at day 2 (794-1731 ng/mL), except Diamond (at day 376 3), with no changes until the end of alcoholic fermentation. This compound was studied 377 with more extent in natural grape must fermented with S. cerevisiae Aroma White, 378 reaching similar concentrations (Fernández-Cruz et al., 2019a) and also in SM, where 10- 379 folds higher values were reached with different yeast strains (Fernández-Cruz et al., 380 2017). In the intracellular medium, TOL was quantified in the four yeast strains (Figure 381 4G). Formerly, TOL had been described in the intracellular medium of different 382 Saccharomyces and non-Saccharomyces strains in a wide range (14-7615 ng/mL) 383 (Fernandez-Cruz et al., 2019b). This compound has proved to be a quorum sensing 384 molecule and a kinetic mediator during alcoholic fermentation (Valera et al., 2019). 385 Therefore, TOL formation pathway from L-TRP metabolism seems to be favoured by 386 yeast. 387 Minor compounds such as TRY and L-TRP EE could also be quantified along the 388 alcoholic fermentation. TRY is a biogenic amine, seldom found in beers and formed by 389 decarboxylation of free amino acids or by amination/transamination of aldehydes and 390 ketones, by the action of microorganisms, mainly bacteria (Pradenas, Galarce-Bustos, 391 18 Henríquez-Aedo, Mundaca-Uribe, & Aranda, 2016). In our study TRY was produced by 392 yeast at the extracellular level from day 2-3 (16.5-32.3 ng/mL) (Figure 3H).). These 393 values are higher than those reported by the authors in SM (<0.5 ng/mL) after alcoholic 394 fermentation (Fernández-Cruz et al., 2017). In the intracellular content, TRY is present at 395 day 1, before the increase of extracellular TRY (Figure 4H). In fact, tryptamine in yeast 396 is formed via tryptophan decarboxylation as an intermediate step for serotonin formation 397 (Muñiz-Calvo et al., 2019). On the other hand, L-TRP EE was present along the alcoholic 398 fermentation, being produced at day 2-3 (Figure 3I). This compound is likely formed 399 thanks to the ethanol presence during the alcoholic fermentation (Arapitsas, Guella, & 400 Mattivi, 2018). Previously, the concentration of L-TRP EE has been reported with an 401 increased trend until 17.48 ng/mL in SM (Fernández-Cruz et al., 2016). However, other 402 studies show how L-TRP EE increases its concentration in the early stages of alcoholic 403 fermentation in grape must, but it disappears quickly after 3 days from the inoculation 404 day (Vigentini et al., 2015). The intracellular content of this compound is extremely low 405 (< 0.06 ng/mL) (Figure 4I). L-TRP EE has been also quantified in the intracellular 406 medium of S.cerevisiae at day 2-3 of alcoholic fermentation (0.11-0.88 ng/mL) 407 (Fernández et al., 2019b). 408 3.4. Indolic compounds content in commercial beers 409 Nineteen commercial beers were analysed by UHPLC/HRMS in order to study the 410 occurrence of different indolic compounds related with L-TRP (Table 2). To the best of 411 our knowledge, this paper reports for the first time the occurrence of 5-HTRP, NA5-HT, 412 3-IAA and L-TRP EE in different commercial beers that were unequivocally identified 413 by HRMS. 414 19 As expected, beers showed a remarkable content of L-TRP (Table 2), ranging from 415 348.08 ng/mL to 6508.83 ng/mL. Other authors quantified L-TRP in commercial beers 416 ranging 4.8-21500 ng/mL (Jia et al., 2011). 417 5-HTRP has been unequivocally quantified for the first time in commercial beers (Table 418 2); in fact, it is present in all beer samples (0.15-1.05 ng/mL). Conversely, in our alcoholic 419 fermentation experiments, 5-HTRP was quantified only in the first days. The other MEL 420 pathway intermediate, 5-HT, was also present in all beer samples (0.99-22.35 ng/mL). 421 This is in agreement with previous studies in which it was determined in different beers 422 (3.5-24.2 mg/L) by means of a derivatization process and a HPLC/DAD analysis 423 (Kirschbaum, Meier, & Brückner, 1999). However, this is the first time that it is 424 determined in commercial beers by means of UHPLC/HRMS, being a more accurate 425 technique especially for identification purposes. This fact could explain the huge 426 difference between the 5-HT found in the literature (3.5-24.2 mg/L) and the values 427 reported in this work. The presence of this compound in a common beverage like beer is 428 fairly interesting since it is able to perform a high inhibition of amyloid β-peptide 429 aggregation, thus evidencing its power as neuroprotective (Hornedo-Ortega et al., 2018). 430 As far as we are concerned, this is the first time that NA5-HT is quantified in commercial 431 beer samples (Table 2). Although it is not a main compound of beer, it appears in all 432 commercial brands (0.02-0.39 ng/mL). MEL content (9.95-29.3 pg/mL) was even lower 433 than that found for NA5-HT. Previously, MEL was reported in beers at higher 434 concentration using UHPLC/HRMS (94.5 ng/mL) (Kocadağlı et al., 2014). 435 Additionally, this is the first time that 3-IAA is quantified in commercial beers (8-143 436 ng/mL) (Table 2). Its presence is noteworthy since its anti-angiogenic effect (IC50= 437 0.9704 mM) is outstanding (Cerezo, Hornedo-Ortega, Álvarez-Fernández, Troncoso, & 438 García-Parrilla, 2017). Due to the lack of available data about bioavailability of 3-IAA in 439 20 the gastrointestinal tract, if we use MEL values (19%) (Harpsøe et al., 2015) and the 440 highest 3-IAA concentration (143 ng/mL) we would need to consume 28 bottles of beers 441 to have a bioactive effect. Thus, beers should be considered as an additional source of 3- 442 IAA, but not the main one. Regarding TOL, the concentration range was higher (53-1893 443 ng/mL) than those previously reported in beers (0.2-2.5 ng/mL) (Bartolomé, Peña-Neira, 444 & Gómez-Cordovés, 2000). 445 TRY was also quantified in commercial beers (2.64-38.41 ng/mL), at low concentration 446 but in the range found in the literature for beer (n.d - 28600 ng/mL) (Pradenas et al., 2016; 447 Nalazek-Rudnicka & Wasik, 2017). Additionally, L-TRP EE, which was quantified 448 during wort alcoholic fermentation, was also described for the first time in commercial 449 beers (0.05-2.57 ng/mL). Likely, the higher level of L-TRP EE of commercial beers with 450 respect to wort alcoholic fermentation levels is in accordance with the higher ethanol 451 content in the final product (Arapitsas et al., 2018). 452 Conclusions 453 Our results show that sample treatment is an important step to further analyse bioactive 454 compounds of L-TRP metabolism using a UHPLC/HRMS technique, as they are present 455 in low concentrations. Filters of PTFE showed to be the most convenient to diminish 456 possible loss of minor bioactive compounds such as MEL, 5-HT and 3-IAA. In addition, 457 MEL is stable in beer matrix for 30 days at -20ºC. For the first time, it was described the 458 formation of L-TRP yeast metabolites such as 5-HTRP, NA5-HT, 3-IAA and L-TRP EE 459 along wort alcoholic fermentation and also in commercial beers. This fact pinpoints beer 460 as a source of compounds with demonstrated biological activity such as antioxidant, anti- 461 angiogenic and neuroprotective properties. Although concentrations found are not enough 462 to exert a bioactive level as discussed earlier, beer could contribute to the intake of these 463 compounds in the diet. 464 21 Acknowledgements 465 This work was supported by the Spanish Ministry of Economy and Competitiveness 466 [AGL2013-47300-C3-2-R, AGL2016-77505-C3-2-R]; and the Spanish Ministry of 467 Education, Culture and Sport for the FPU grant (FPU13/04820) of E.F.-C. Authors also 468 thank Professor Fernando Govantes, Professor Carlos Santos Ocaña from the CABD 469 (Centro Andaluz de Biología del Desarrollo) and M. Carballo-Álvarez from the Biology 470 Service of the Universidad de Sevilla (CITIUS) for their technical support. 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Characteristics of commercial beers 654 Code Beer type Country of origin Glass bottle type Alcohol content (%) AT Pilsner lager Spain Brown glass 4.6 AB Pilsner lager Spain Brown glass 5.9 AM Pilsner lager Spain Brown glass 5.0 B0 Pilsner lager Spain Brown glass 0.0 BW American lager United States Brown glass 5.0 CB Pilsner lager Denmark Green glass 5.0 CO American lager México Clear glass 4.6 CC Pilsner lager Spain Brown glass 4.8 CZ Pilsner lager Spain Brown glass 5.9 DP Flavoured Lager Belgium Clear glass 5.9 ED Pilsner lager Spain Brown glass 5.4 EG Pilsner lager Spain Brown glass 5.5 GN Irish dry stout Ireland Black glass 5.0 HK Pilsner lager The Netherlands Green glass 5.0 MA Pilsner lager Spain Brown glass 5.5 MU Irish red ale lager Irish Brown glass 5.0 PL Weissbier lager Germany Brown glass 5.5 SM Pilsner lager Spain Brown glass 5.4 VD Märzenbier lager Spain Brown glass 7.2 655 656 657 658 659 660 661 33 Table 2. Concentration (ng/mL) of L-Tryptophan (L-TRP) and its metabolites in commercial beers. 662 Beer code L-TRP 5-HTRP 5-HT NA5-HT MEL* 3-IAA TRY TOL L-TRP EE AT 1890.85 ± 20.34 0.28 ± 0.00 6.77 ± 0.10 0.06 ± 0.00 29.2 ± 1.27 82.3 ± 0.09 18.5 ± 0.07 1511 ± 24.0 0.32 ± 0.00 AB 3648.60 ± 36.70 0.20 ± 0.00 3.97 ± 0.10 0.14 ± 0.00 13.5 ± 0.00 66.0 ± 0.95 12.6 ± 0.16 293 ± 5.98 1.02 ± 0.01 AM 3510.67 ± 80.38 0.42 ± 0.02 8.38 ± 0.10 0.26 ± 0.00 23.7 ± 0.57 7.93 ± 0.03 24.2 ± 0.29 117 ± 1.05 0.73 ± 0.01 B0 4163.76 ± 66.63 0.46 ± 0.02 22.35 ± 0.32 0.36 ± 0.00 21.7 ± 0.99 11.7 ± 0.00 2.64 ± 0.06 51.7 ± 1.22 0.05 ± 0.00 BW 3832.94 ± 83.81 0.27 ± 0.01 6.01 ± 0.12 0.15 ± 0.00 15.7 ± 0.71 25.9 ± 0.62 31.1 ± 0.75 104 ± 2.44 1.42 ± 0.01 CB 3621.13 ± 25.86 0.32 ± 0.01 5.43 ± 0.23 0.28 ± 0.01 18.4 ± 2.12 20.1 ± 0.58 23.6 ± 1.26 366 ± 15.7 0.49 ± 0.03 CO 4535.70 ± 67.84 0.30 ± 0.01 1.32 ± 0.01 0.19 ± 0.00 13.5 ± 0.42 25.0 ± 0.61 9.15 ± 0.15 82.2 ± 1.44 0.44 ± 0.01 CC 6078.06 ± 36.93 0.35 ± 0.03 11.48 ± 0.22 0.29 ± 0.00 27.15 ± 0.21 66.5 ± 0.16 29.8 ± 0.05 163 ± 0.38 0.74 ± 0.00 CZ 4108.09 ± 9.42 1.05 ± 0.02 7.14 ± 0.12 0.31 ± 0.01 18.8 ± 1.13 15.4 ± 0.48 36.6 ± 0.49 149 ± 5.26 0.81 ± 0.03 DP 4432.98 ± 398.26 0.33 ± 0.03 1.63 ± 0.17 0.25 ± 0.02 25.6 ± 2.83 143.4 ± 3.11 20.5 ± 1.71 139 ± 8.43 0.49 ± 0.04 ED 3744.20 ± 161.19 0.27 ± 0.01 1.81 ± 0.06 0.11 ± 0.00 10.85 ± 0.78 35.6 ± 0.06 14.8 ± 0.58 58.8 ± 0.99 0.89 ± 0.03 EG 4559.18 ± 75.47 0.22 ± 0.00 7.15 ± 0.07 0.21 ± 0.00 16.3 ± 0.57 63.1 ± 0.22 16.9 ± 0.23 106 ± 1.09 0.47 ± 0.01 GN 4767.53 ± 31.78 0.36 ± 0.00 0.99 ± 0.01 0.22 ± 0.00 17.7 ± 0.71 7.68 ± 0.06 38.1 ± 0.19 81.2 ± 0.01 1.52 ± 0.00 HK 3938.52 ± 10.78 0.83 ± 0.00 8.85 ± 0.02 0.35 ± 0.00 21.15 ± 0.21 7.79 ± 0.01 19.3 ± 0.24 97.3 ± 1.23 0.31 ± 0.00 MA 1102.37 ± 29.52 0.17 ± 0.01 10.64 ± 0.20 0.06 ± 0.00 20.2 ± 0.99 100 ± 0.34 16.5 ± 0.64 1354 ± 22.2 0.61 ± 0.02 MU 5935.79 ± 10.56 0.73 ± 0.01 16.23 ± 0.90 0.21 ± 0.01 19.1 ± 1.84 18.1 ± 0.53 34.6 ± 1.68 182 ± 2.61 0.58 ± 0.03 PL 1624.19 ± 26.50 0.48 ± 0.02 2.09 ± 0.05 0.39 ± 0.00 10.8 ± 0.28 11.8 ± 0.20 31.9 ± 0.08 819 ± 1.58 1.48 ± 0.04 SM 348.08 ± 5.51 0.15 ± 0.01 7.60 ± 0.04 0.02 ± 0.00 27.85 ± 1.34 109 ± 0.31 25.7 ± 0.29 1893 ± 20.6 2.57 ± 0.01 VD 6534.82 ± 378.16 0.34 ± 0.03 2.35 ± 0.00 0.21 ± 0.01 9.95 ± 1.06 40.7 ± 1.24 38.4 ± 2.79 61.4 ± 3.39 2.04 ± 0.16 * Concentration expressed in pg/mL. 663 34 L-TRP: L-tryptophan; 5-HTRP: 5-hydroxytryptophan; 5-HT: serotonin; NA5-HT: N-acetylserotonin; MEL: melatonin; 3-IAA: 3-indolacetic acid; 664 TRY: tryptamine; TOL: tryptophol; L-TRP EE: L-tryptophan ethyl ester. 665 666 667