Intracellular biosynthesis of melatonin and other indolic compounds in Saccharomyces and non-Saccharomyces wine yeasts

For Peer Review 1 1Intracellular biosynthesis of melatonin and other indolic compounds in 2Saccharomyces and non-Saccharomyces wine yeasts 3 4Edwin Fernández-Cruz1,†, Beatriz González2,†, Sara Muñiz-Calvo3,†, María Ángeles 5Morcillo-Parra2, Ricardo Bisquert3, Ana Maria Troncoso1, María Carmen García- 6Parrilla1, María Jesús Torija2, José Manuel Guillamón3* 7 81Departmento de Nutrición y Bromatología, Facultad de Farmacia, Universidad de Sevilla, 9Sevilla, Spain 10 2Departament de Bioquímica i Biotecnologia, Facultat d’Enologia, Universitat Rovira i 11 Virgili, Tarragona, Spain 12 3Departamento de Biotecnología de Alimentos, Instituto de Agroquímica y Tecnología de 13 Alimentos, Consejo Superior de Investigaciones Científicas (CSIC), Valencia, Spain 14 15 *Corresponding author: José M. Guillamón, [email protected] +34963900022ext: 16 2303 17 18 †These authors contributed equally to this work 19 20 21 22 23 24 25 26 27 Page 1 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 2 28 Abbreviations: 5-HT: serotonin; 5-HTRP: 5-hydroxytryptophan; L-TRP: L-tryptophan; 29 TRYP: tryptamine, N-acetyl-5-HT: N-acetyl-5-hydroxytryptamine; L-TRP EE: L- 30 tryptophan ethyl ester; TOL: tryptophol; 3IAA: 3-indoleacetic acid; MLT: melatonin 31 Abstract 32 Certain bioactive compounds that derive from tryptophan have been determined in 33 fermented foods, which suggests that the role of yeast is putative in origin. Melatonin is a 34 neurohormone in humans that plays an important role in health. The activity of other 35 compounds, such as 3-indoleacetic acid, has been recently highlighted, and interest in 36 elucidating the conditions of their production has grown. 37 However, the biosynthesis of melatonin by yeasts remains unclear to a large extent. 38 Therefore, this work was undertaken to demonstrate the unequivocally synthesis of 39 melatonin and other compounds that derive from tryptophan metabolism by yeast by 40 determining them in the intracellular compartment. By high resolution mass spectrometry 41 and a validated method, tryptophan itself, melatonin, serotonin, N-acetyl-5- 42 hydroxytryptamine and 3-indoleacetic acid, were identified in the intracellular compartment 43 of Saccharomyces and non-Saccharomyces wine yeasts. 44 Keywords: exact mass, simultaneous determination, alcoholic fermentation, bioactive, 5- 45 methoxindoles, wine yeast 46 Page 2 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 3 47 1. Introduction 48 Melatonin (MLT), a neurohormone involved in circadian rhythm regulation, has been 49 found in foods like strawberry, tomatoes [1] and cherries [2, 3], and in alcoholic beverages 50 such as beer [4] and wines [5, 6]. The last-cited authors also monitored the whole 51 winemaking process, and highlighted that melatonin was formed in the fermentation stage 52 as a product of yeast metabolism [7]. This fermentative origin was later confirmed by the 53 fermentation of other products, such as orange [8]. To date however, the synthesis pathway 54 has not been unveiled in yeast and very little is known about the relevance of this molecule 55 in its metabolism and physiology. Two independent studies have recently reported the 56 protective role of intracellular melatonin against oxidative stress and UV radiation in S. 57 cerevisiae [9, 10]. 58 The MLT synthesis pathway in vertebrates uses tryptophan (L-TRP) as a precursor, which 59 is converted into 5-hydroxytryptophan (5-HTRP), serotonin (5-HT) and N-acetyl-5- 60 hydroxytryptamine (N-acetyl-5-HT) as intermediates. Other L-TRP-derived compounds 61 with an indolic ring have been detected in wines, including L-tryptophan ethyl ester (L- 62 TRP EE) [11], tryptophol (TOL) [12] , 3-indoleacetic acid (3IAA) [13] and tryptamine 63 (TRYP) [14] . Thus in order to ascertain the yeast metabolic origin of MLT and other 64 related indolic compounds, we aimed to monitor the intracellular synthesis of all these L- 65 TRP-derived compounds and their evolution during either pulses of L-TRP to resting yeast 66 cells or the alcoholic fermentation of synthetic must. To date, only the study of Sprenger et 67 al. [15] has evidenced the presence of MLT in the intracellular compartment of the yeast S. 68 cerevisiae. Since this article was published, analytical techniques have been extensively 69 developed thanks to mass spectrometry advances that allow the unequivocal identification Page 3 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 4 70 of substances at relatively low concentrations. Fernández-Cruz et al. [16] recently 71 developed and validated an analytical method by ultra high-performance liquid 72 chromatography coupled to high-resolution mass spectrometry (UHPLC/HRMS) to 73 monitor both MLT and related indolic compounds in order to lower their detection limits, 74 and to assess their occurrence in culture medium and fermented products. In this study, we 75 used this new validated analytical method to detect all these compounds intracellularly in 76 yeasts of different species, which are all involved in wine fermentation. The intracellular 77 detection of these molecules strongly reinforced the role played by yeasts in the final 78 concentration of these bioactive molecules in fermented foods. 79 80 2. Materials and Methods 81 2.1. Yeast strains 82 Six yeast strains were used in this study. Two strains (QA23 and P24) belonged to S. 83 cerevisiae. QA23 is a commercial strain and is marketed by Lallemand S.A. (Canada). P24 84 was also provided by Lallemand S.A. (Canada), but it has no commercial name as it is still 85 going through its development stage. The other four strains are non-Saccharomyces, wild 86 isolates from the winemaking Priorat region of Spain and belong to different yeast species: 87 Hanseniaspora uvarum Hu4 (CECT 13130), Starmerella bacillaris Cz4 (syn. Candida 88 zemplinina, CECT 13129), Metschnikowia pulcherrima Mpp (CECT 13131), and 89 Torulaspora delbrueckii Tdp (CECT 13135). 90 2.2. Tryptophan pulses Page 4 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 5 91 Cells were grown overnight in YPD medium (2% (w/v) bacteriological peptone, 2% (w/v) 92 glucose, 1% (w/v) yeast extract) and washed twice with distilled water before being 93 transferred to other media. After this overnight growth, yeast strains were suspended at cell 94 densities of ~ 108 cells/mL in salt medium (50mM Na2HPO4, 0.5% NaCl, adjusted to pH 95 5.8 using citric acid) and incubated in Erlenmeyer flasks with orbital agitation (150rpm) at 96 28ºC in complete darkness for 4 h. After this incubation period in salt medium, L-TRP was 97 added at a final concentration of 1 mM. Control cells were not supplemented with any 98 nutrient. Sampling was done at 30 min after adding this amino acid [15]. After this pulse, 99 10 mL of the culture were centrifuged (10 min at 4000 rpm). The obtained pellet (~ 109 100 cells) was washed twice with distilled water and transferred to a microcentrifuge tube to be 101 stored at -80 ºC until it was analysed. 102 2.3. Fermentation conditions 103 Two strains of S. cerevisiae (QA23 and P24) and two strains of non-Saccharomyces species 104 (H. uvarum CECT13130 and T. delbrueckii CECT13135) were used to test the intracellular 105 synthesis of these indolic compounds under fermentation conditions. Fermentations were 106 carried out in synthetic must (SM) (pH 3.3), as described by Riou et al. [17] , but with 200 107 g/L of reducing sugars (100 g/L glucose + 100 g/L fructose). The assimilable nitrogen 108 source was 300 mg N/L (120 mg N/L as ammonium and 180 mg N/L in the amino acid 109 form). The proportion of the different amino acids was the same as that previously 110 described by Riou et al. [17] , except for L-TRP, whose concentration was increased 5-fold 111 to boost indolic compound synthesis, but the same content in mg N/L terms (180 mg N/L) 112 was kept. The population inoculated in SM came from an overnight culture in YPD at 30ºC 113 and the cells were rinsed twice with sterile distilled water prior to transfer to SM. These Page 5 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 6 114 fermentations were performed in triplicate in laboratory-scale fermenters using 500-mL 115 bottles filled with 400 mL of SM, which were fitted with closures that enabled carbon 116 dioxide to escape and samples to be removed at 28ºC with continuous orbital shaking at 117 100 rpm. Yeast cell growth was determined by absorbance at 600 nm and by plating 118 adequate dilutions on YPD agar by the end of fermentation. YPD plates were incubated for 119 2 days at 30ºC. Fermentation was monitored by measuring the density of the medium (g/L) 120 in a Densito 30 PX densitometer (Mettler Toledo, Switzerland). Sampling was done in the 121 lag (1 h after inoculation), log (initial, medium and final exponential phases) and stationary 122 phases (2 days after cells entered the stationary phase). The volume taken during each 123 sampling was calculated to obtain 108 cells (approximately 10 OD600 units). 124 2.4. Intracellular metabolite extraction 125 Intracellular metabolites were extracted by adapting the boiling buffered ethanol method 126 previously described by Gonzalez et al. [18] . Three mL of a solution of 75% (v/v) boiling 127 absolute ethanol containing 70 mM (final concentration) of HEPES buffer (pH 7.5) were 128 added to the cell pellet. This mixture was incubated for 3 min at 80ºC and 3 min on ice. The 129 extract was concentrated by the evaporation of the volume at 45ºC in a 5301 Concentrator 130 plus/Vacufuge® plus (Eppendorf, Spain). The final intracellular content was resuspended 131 in 1 mL of ultrapure milliQ water and centrifuged for 10 min at 5,000 g at 4ºC to remove 132 insoluble particles. The supernatant was transferred to a new tube and stored at -20º C until 133 used. 134 2.5. Sample preparation Page 6 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 7 135 Samples were extracted as previously reported by Rodriguez-Naranjo et al. [6] with the 136 following modifications. Briefly, C18 SPE cartridges (Variant, Agilent) were conditioned 137 with 2 mL of methanol and 2 mL of milliQ water. A 350-µL aliquot of the sample was 138 loaded, which was followed by a washing step with 2 mL of a 10% v/v methanol/water 139 solution. The analytes were eluted with 1 mL of methanol and then evaporated to dryness at 140 34ºC, 2,000 rpm in a vacuum concentrator (HyperVACLITE, GYOZEN, South Korea). 141 The extract was reconstituted with 116 µL of water/methanol 1:1 (v/v) and stored away 142 from light at -18ºC until analysed. 143 2.6. UHPLC/HRMS parameters 144 The UHPLC/HRMS analysis was carried out under the same conditions of a validated 145 method reported by Fernández-Cruz et al. [16] in a UHPLC Dionex Ultimate 3000 system 146 (ThermoScientific, San Jose, USA). This benchtop LC-MS/MS combines quadruple 147 precursor ion selection with high-resolution, accurate-mass (HRAM) Orbitrap detection. 148 All the devices were controlled by the Chromeleon Express Software. The column used for 149 the analysis was a ZORBAX RRHD SB-C18 (2.1 x 100 mm, 1.8 µm particle size) with the 150 corresponding guard column purchased from Agilent Technologies (Waldbronn, Germany). 151 The UHPLC system was coupled to a Thermo Scientific QexactiveTM hybrid quadrupole- 152 orbitrap mass spectrometer (Bremen, Germany). The target-MS2 mode was set to run the 153 analysis with the same parameters described by Fernández-Cruz et al. [16]. For 154 identification purposes, the Xcalibur Software (version 3.0.63) was used. In order to 155 quantitate, the TraceFinderTM Software (version 3.1) (Thermo Fisher Scientific, Waltham, 156 MA) was applied. The mass characteristics of the compounds under study are shown in Page 7 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 8 157 Table S1, and include the exact mass of the protonated ion, calculated mass, ∆mass ppm 158 ranging between 0.004 and 2.55 ppm, MS/MS fragments with the molecular formula. 159 2.7. Statistical analysis 160 Differences in the intracellular indole concentrations after the L-TRP pulses were assessed 161 by a directional Student’s t-test to compare the concentration of each indole compound 162 before and after the pulse. The 0.05 probability level was chosen as the maximum point of 163 statistical significance throughout. The STATISTICA software V.7 (StatSoft, Inc, 2004) 164 was used to perform the multivariate data analysis and the principal components analysis. 165 166 3. Results and Discussion 167 3.1. Intracellular indolic compounds after a tryptophan pulse to starved cells 168 The putative synthetic pathway of MLT in yeasts is completely unknown. In order to 169 improve our knowledge about this route, we aimed to detect the intracellular synthesis of 170 the different intermediates of this pathway (L-TRP, 5-HTRP, 5-HT, N-acetyl-5-HT and 171 MLT), and other L-TRP-derived compounds (3IAA, TRYP, L-TRP EE and TOL), in 172 different yeast strains and species, which all participate in wine fermentations. 173 In a similar experiment to that reported by Sprenger et al. [15] , we incubated yeast cells for 174 4 h in a non-proliferative medium, which were pulsed with L-TRP. After 30 min, 175 intracellular metabolites of 109 cells were extracted for their analysis by UHPLC/HRMS. 176 The absolute values of these compounds before and after the L-TRP pulses are shown in 177 Tables 1 and 2, in which they are separated by higher concentrations (expressed as ng/109 178 cells ) and lower concentrations (expressed as pg/109 cells ), respectively. In order to Page 8 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 9 179 highlight which metabolites significantly increased, we calculated the P-values for each 180 indolic compound and strain before and after the L-tryptophan pulse (Table S2). Moreover, 181 to gain an overview, these values are represented in a heat-map as relative increases after 182 this pulse (Figure 1). Hierarchical clustering divided the species into two major groups: 183 cluster 1 grouped the strains of S. cerevisiae, M. pulcherrima and H. uvarum, with the two 184 S. cerevisiae strains forming a sub-cluster. Cluster 2 comprised the S. bacillaris (Cz4) and 185 T. delbrueckii (Tdp) strains. 186 The 3IAA compound was the metabolite synthesised at higher concentrations in all the 187 strains, and Tdp and Cz4 presented the greatest accumulation. The other major synthesised 188 compound was TOL, but at a much lower concentration. Both are synthesised via the Erlich 189 pathway. While 3IAA is the higher acid that derives from L-TRP, TOL is the higher 190 alcohol [19]. The synthesis of both metabolites is important because exogenous 3IAA and 191 TOL induce morphological changes [20] and modulate the cell growth of wine yeast 192 species, which suggests a possible role in microbial interaction during wine fermentation 193 [21]. Among the metabolites detected at much lower concentrations, we found three 194 intermediates of the MEL pathway of animals and plants: 5-HTRP, 5-HT and TRYP. 195 TRYP accumulated in the S. cerevisiae, M. pulcherrima and S. bacillaris strains, but not in 196 T. delbrueckii and H. uvarum ones. However, neither N-acetyl-5-HT nor MLT, these being 197 the last products of the putative pathway, were detected in any strain. Sprenger et al. [15] 198 reported high levels of methoxyindoles within the first 30 min after a L-TRP addition to 199 starved cells. Our results indicate that, after a L-TRP pulse to starved cells, is favouring 200 Ehrlich pathway over MLT biosynthetic pathway, which is more likely part of a secondary 201 metabolism. Perhaps pulses with other intermediates of the route, such as 5-HT or other Page 9 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 16 335 20. Prusty R, Grisafi P, Fink GR (2004) The plant hormone indoleacetic acid induces 336 invasive growth in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 101:4153–7 337 21. González B, Vázquez J, Cullen PJ, et al (2018) Aromatic Amino Acid-Derived 338 Compounds Induce Morphological Changes and Modulate the Cell Growth of Wine 339 Yeast Species. Front Microbiol 9:1–16 340 22. Gardana C, Iriti M, Stuknyte M, et al (2014) “Melatonin isomer” in wine is not an 341 isomer of the melatonin but tryptophan-ethylester. J Pineal Res 57:435–441 342 23. Fernández-Cruz E, Álvarez-Fernández MA, Valero E, et al (2017) Melatonin and 343 derived L-tryptophan metabolites produced during alcoholic fermentation by 344 different wine yeast strains. Food Chem 217:431–437 345 24. Curiel JA, Morales P, Gonzalez R, Tronchoni J (2017) Different non-Saccharomyces 346 yeast species stimulate nutrient consumption in S. cerevisiae mixed cultures. Front 347 Microbiol 8:1–9 348 25. González B, Vázquez J, Morcillo-Parra MÁ, et al (2018) The production of aromatic 349 alcohols in non-Saccharomyces wine yeast is modulated by nutrient availability. 350 Food Microbiol 74:64–74 351 26. Borrull A, López-Martínez G, Poblet M, et al (2015) New insights into the toxicity 352 mechanism of octanoic and decanoic acids on Saccharomyces cerevisiae. Yeast 353 32:451–460 354 Page 16 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 17 Table S1. Mass characteristics for identification purposes. Compound RT min Exact mass [M+H]+ Calculated Mass (m/z) Molecular Formula ∆mass ppm MS/MS fragments Relative intensities 5-HT 1.26 177.1022 177.10269 C10H12N2O 2.55 149.01244(C10HN2) 100 5-HTRP 1.53 221.0921 221.09239 C11H12N2O3 1.46 175.08694(C10H11ON2) 100 188.07057(C11H10O2N) 100 L-TRP 4.08 205.0971 205.09715 C11H12N2O2 0.004 146.06015(C9H8ON) 29 TRYP 4.14 161.1073 161.10697 C10H12N2 2.20 144.08079(C10H10N) 100 N-acetyl-5- HT 5.23 219.1122 219.11201 C12H14N2O2 3.60 177.00673(C11HON2) 6.86 216.10199(C13H14O2N) 100 L-TRP EE 6.63 233.1284 233.12852 C13H16N2O2 0.30 174.09129(C11H12ON) 24 TOL 7.09 162.0913 162.09164 C10H11NO 1.87 144.08104(C10H10N) 100 3IAA 7.10 176.0706 176.07104 C10H9NO2 2.50 130.06523(C9H8N) 100 MLT 7.17 233.1284 233.12852 C13H16N2O2 0.30 174.09125(C11H12ON) 100 5-HT: serotonin; 5-HTRP: 5-hydroxytryptophan; L-TRP: L-tryptophan; TRYP: tryptamine, N-acetyl-5-HT: N-acetyl-5-hydroxytryptamine; L-TRP EE: L- tryptophan ethyl ester; TOL: tryptophol; 3IAA: 3-indoleacetic acid; MLT: melatonin Page 17 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 18 Table S2. The P-values of the comparison of the means for each indolic compound and strain before and after adding 1 mM L-tryptophan n.i= not increased 5-HTRP 3-IAA L-TRP TOL TRYPT SERO MEL NACSERO L-TRP EE QA23 0.00010357 0.00092524 1.1358E-05 0.00030773 3.7844E-05 1.0791E-05 n.i n.i n.i P24 3.2167E-16 0.00309727 7.1358E-08 2.4896E-10 3.8906E-08 0.01295624 n.i n.i n.i Mpp 5.6746E-14 2.6776E-06 5.8109E-08 7.1522E-06 6.7444E-06 1.0667E-18 n.i n.i n.i Hu4 0.072464 0.01617441 0.0139287 0.00205023 n.i n.i n.i n.i n.i Cz4 5.772E-08 0.00465218 0.00014551 0.00249176 0.09672938 0.01778494 n.i n.i n.i Tdp 0.00059832 2.463E-06 1.5788E-12 1.2007E-07 n.i 2.2925E-07 n.i n.i n.i Page 18 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 19 Table 1. Concentrations of intracellular L-tryptophan (L-TRP), 3 indoleacetic acid (3IAA) and tryptophol (TOL) in the different yeast strains before and after (+) adding 1mM L-TRP Intracellular indolic compounds [ng/109 cells] Strain L-TRP 3-IAA TOL QA23 802.88 ± 130.19 6618.00 ± 2079.58 22.52 ± 5.00 QA23 + 12747.35 ± 3942.87 40273.17 ± 19552.73 184.74 ± 80.80 P24 596.96 ± 54.39 16840.5 ± 19918.13 41.20 ± 34.85 P24 + 16038.78 ± 2921.30 48127.5 ± 9790.15 444.50 ± 24.42 Tdp 867.30 ± 1141.83 54303 ± 70915.99 37.50 ± 34.91 Tdp + 59136.41 ± 3505.64 732656.5 ± 174376.77 1152.87 ± 220.23 Hu4 48.95 ± 45.46 21539.33 ± 34401.71 21.13 ± 17.33 Hu4 + 1461.73 ± 1345.40 128911.17 ± 101127.34 142.70 ± 78.57 Mpp 51.68 ± 10.83 1941.67 ± 1020.14 40.27 ± 6.61 Mpp + 8990.70 ± 1655.16 88437.83 ± 24168.56 2360.90 ± 726.52 Cz4 383.21 ± 55.29 19006.75 ± 8540.48 22.60 ± 8.13 Cz4 + 8397.83 ± 2576.33 775384.00 ± 435367.43 218.16 ± 99.73 Results are expressed as mean ± SD of biological replicates (n=3) Page 19 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 20 Table 2. Concentrations of the intracellular indole minority compounds in the different yeast strains before and after (+) adding 1 mM L-tryptophan Intracellular indolic compounds [pg/109 cells] Strain 5-HTRP 5-HT N-acetyl-5-HT MLT TRYP L-TRP EE QA23 139.00 ± 0.00 853.00 ± 23.82 n.d. n.d. n.d. n.d. QA23 + 948.50 ± 80.27 1308.00 ± 147.47 n.d. n.d. 1304.17 ± 497.07 n.d. P24 n.d. 681.00 ± 3.11 n.d. 170.33 ± 4.24 n.d. n.d. P24 + 934.83 ± 25.22 1037.17 ± 13.24 n.d. 60.83 ± 13.44 1167.67 ± 207.29 n.d. Tdp 341.50 ± 6.36 894.00 ± 115.89 n.d. n.d. n.d. n.d. Tdp + 1711.67 ± 532.67 1700.33 ± 128.04 n.d. n.d. n.d. n.d. Hu4 n.d. 819.33 ± 4.50 n.d. n.d. n.d. n.d. Hu4 + 278.33 ± 1.41 692.67 ± 15.51 n.d. n.d. n.d. n.d. Mpp n.d. n.d. n.d. n.d. n.d. n.d. Mpp + 880.67 ± 39.90 993.83 ± 15.14 n.d. n.d. 608.50 ± 189.23 n.d. Cz4 n.d. 412.00 ± 7.55 n.d. n.d. n.d. n.d. Cz4 + 990.50 ± 110.84 1048.67 ± 99.51 n.d. n.d. 147.83 ± 23.33 n.d. Results are expressed as mean±SD of biological replicates (n=3) n.d= not detected; L-TRP = L-tryptophan; 5-HTRP = 5-hydroxytryptophan; 5-HT = serotonin, N-acetyl-5-HT = N-acetyl serotonin; MLT = melatonin; 3IAA = 3-indoleacetic acid; TRYP = tryptamine; TOL = tryptophol; L-TRP EE = L-tryptophan ethyl Page 20 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 21 Table 3. Concentration of the intracellular indolic compounds in ng/108 cells from the samples taken in the different alcoholic fermentation stages in two Saccharomyces cerevisiae strains (QA23 and P24) and in two non-Saccharomyces strains (Hu4 and Tdp). Compound Strain L-TRP 5-HTRP 5-HT N-acetyl-5-HT MLT 3IAA TRYP TOL L-TRP EE QA23 ie 24584.64 ± 8348.46 1.95 ± 0.46 1.50 ± 0.81 n.d. n.q. 2.75 ± 0.96 1.77 ± 0.48 5756.87 ± 1666.29 0.75 ± 0.28 QA23 me 5174.69 ± 1830.14 1.08 ± 0.06 0.07 ± 0.03 n.d. n.q. 1.65 ± 0.16 0.73 ± 0.08 3780.78 ± 66.53 0.11 ± 0.06 QA23 fe 3841.20 ± 266.19 1.06 ± 0.01 0.06 ± 0.04 n.d. n.q. 0.94 ± 0.19 0.93 ± 0.06 4295.66 ± 240.02 0.46 ± 0.01 QA23 ff 5936.66 ± 1309.64 1.16 ± 0.06 0.03 ± 0.01 n.d. n.q. 3.31 ± 0.27 0.64 ± 0.10 8204.25 ± 982.44 0.66 ± 0.16 P24 ie 49027.84 ± 8874.80 5.46 ± 0.79 7.03 ± 1.49 n.d. n.q. 6.98 ± 0.88 3.36 ± 0.31 8303.80 ± 527.15 0.88 ± 0.06 P24 me 1501.19 ± 198.34 1.10 ± 0.04 0.12 ± 0.08 n.d. n.q. 3.80 ± 0.79 0.77 ± 0.08 7615.75 ± 1490.55 0.04 ± 0.03 P24 fe 3379.33 ± 459.49 1.06 ± 0.02 0.31 ± 0.23 n.d. n.q. 2.57 ± 0.37 1.15 ± 0.27 4253.87 ± 561.80 0.57 ± 0.12 P24 ff 2425.52 ± 474.66 1.03 ± 0.01 0.03 ± 0.03 n.d. n.q. 2.33 ± 1.29 0.81 ± 0.16 2605.30 ± 656.04 0.27 ± 0.07 Hu4 ie 3841.78 ± 3069.12 n.q. n.d. 0.30 ± 0.20 0.93 ± 1.11 4.34 ± 2.73 n.d. 133.02 ± 48.38 1.70 ± 1.01 Hu4 me 703.13 ± 2.45 n.q. n.d. 0.14 ± 0.01 0.34 ± 0.17 0.05 ± 0.01 n.d. 13.56 ± 0.07 n.d. Hu4 fe 89.26 ± 0.63 n.q. n.d. 0.13 ± 0.00 0.56 ± 0.26 0.95 ± 0.37 n.d. 36.74 ± 0.08 0.46 ± 0.01 Hu4 ff 15.31 ± 7.37 n.q. n.d. 0.13 ± 0.01 0.28 ± 0.10 0.15 ± 0.04 n.d. 20.16 ± 0.19 1.04 ± 0.67 Tdp ie 2478.68 ± 1595.96 0.03 ± 0.01 n.d. 0.14 ± 0.00 0.17 ± 0.07 4.28 ± 1.09 n.d. 98.55 ± 16.38 n.d. Tdp me 2529.59 ± 216.84 0.02 ± 0.00 n.d. 0.14 ± 0.01 0.66 ± 0.43 3.09 ± 1.76 n.d. 295.44 ± 51.35 n.d. Tdp fe 1551.73 ± 166.32 0.02 ± 0.00 n.d. 0.13 ± 0.01 0.32 ± 0.21 7.62 ± 0.86 n.d. 654.20 ± 115.09 n.d. Tdp ff 665.05 ± 230.83 0.04 ± 0.00 n.d. 0.13 ± 0.01 0.12 ± 0.03 22.63 ± 5.44 n.d. 842.79 ± 143.23 n.d. n.d. = not detected; n.q. = not quantifiable; L-TRP = L-tryptophan; 5-HTRP = 5-hydroxytryptophan; 5-HT = serotonin, N-acetyl-5-HT = N-acetyl serotonin; MLT = melatonin; 3IAA = 3-indoleacetic acid; TRYP = tryptamine; TOL = tryptophol; L-TRP EE = L-tryptophan ethyl ester; ie = initial exponential; me = middle exponential; fe = final exponential, ff =finalized fermentation. Biological replicates are indicated as mean±SD of biological replicates (n=3) . Page 21 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review 22 Figure 1. Effect of tryptophan addition for 30 min on the intracellular production of the different indolic compounds in Saccharomyces cerevisiae (QA23 and P24) and non-Saccharomyces (Mpp, Hu4, Cz4, Tdp) starved cells. Figure 2. Progression of the detected indole compounds across the different alcoholic fermentation stages for the studied yeast strains (A). The major compounds tryptophan (L-TRP) and tryptophol (TOL) were masked for better visualisation (B). ie = initial exponential; me = middle exponential; fexp = final exponential, fp =final point Figure 3. The statistical principal components analysis (PCA) of the effect of the different growth stages on the production of indolic compounds in Saccharomyces and non-Saccharomyces strains. Component 1 reflects 62.06% total variance (it negatively correlates with L-TRP, TRYP, 5-HTRP and TOL, and positively with N-acetyl-5-HT and MLT). Component 2 reflects 13.84% total variance (and positively correlates with 3-IAA and negatively with L-TRP-EE) and is arranged on two dimensions according to Components 1 and 2. The samples of Saccharomyces are grouped in the yellow ellipse, while the non-Saccharomyces are grouped in the blue ellipse, which reflect differences in indolic profiles at the species level. Page 22 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Fig 1: Effect of tryptophan addition during 30 min on intracellular production of the different indolic compounds in Saccharomyces cerevisiae (QA23 and P24) and non-Saccharomyces (Mpp, Hu4, Cz4, Tdp) starved cells. Page 23 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Fig 2: Progression of the detected indole compounds across the different stages of alcoholic fermentation for studied yeast strains (A). Major compounds tryptophan (L-TRP) and tryptophol (TOL) were masked for better visualization (B). ie = initial exponential; me = middle exponential; fexp = final exponential, fp =final point Indolic compounds (%) QA23 P24 Hu4 Tdp QA23 P24 Hu4 Tdp QA23 P24 Hu4 Tdp QA23 P24 Hu4 Tdp ie me fexp fp 100 90 80 70 60 50 40 30 20 10 0 A L-TRP EE TOL TRYPT 3IAA MLT N-acetyl-5-HT 5-HT 5-HTRP L-TRP Indolic compounds (%) QA23 P24 Hu4 Tdp QA23 P24 Hu4 Tdp QA23 P24 Hu4 Tdp QA23 P24 Hu4 Tdp ie me fexp fp 100 90 80 70 60 50 40 30 20 10 0 B L-TRP EE TRYPT 3IAA MLT N-acetyl-5-HT 5-HT 5-HTRP Page 24 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 For Peer Review Figure 3. The statistical principal components analysis (PCA) of the effect of the different growth stages on the production of indolic compounds in Saccharomyces and non-Saccharomyces strains. Component 1 reflects 62.06% total variance (it negatively correlates with L-TRP, TRYP, 5-HTRP and TOL, and positively with N-acetyl-5-HT and MLT). Component 2 reflects 13.84% total variance (and positively correlates with 3-IAA and negatively with L-TRP-EE) and is arranged on two dimensions according to Components 1 and 2. The samples of Saccharomyces are grouped in the yellow ellipse, while the non-Saccharomyces are grouped in the blue ellipse, which reflect differences in indolic profiles at the species level. . Page 25 of 25 http://mc.manuscriptcentral.com/efrt European Food Research and Technology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60