Alcoholic fermentation induces melatonin synthesis in orange juice

J. Pineal Res. 2014; 56:31–38 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular, Biological, Physiological and Clinical Aspects of Melatonin Doi:10.1111/jpi.12093 Journal of Pineal Research Alcoholic fermentation induces melatonin synthesis in orange juice Abstract: Melatonin (N-acetyl-5-methoxytryptamine) is a molecule implicated in multiple biological functions. Its level decreases with age, and the intake of foods rich in melatonin has been considered an exogenous source of this important agent. Orange is a natural source of melatonin. Melatonin synthesis occurs during alcoholic fermentation of grapes, malt and pomegranate. The amino acid tryptophan is the precursor of all 5-methoxytryptamines. Indeed, melatonin appears in a shorter time in wines when tryptophan is added before fermentation. The aim of the study was to measure melatonin content during alcoholic fermentation of orange juice and to evaluate the role of the precursor tryptophan. Identification and quantification of melatonin during the alcoholic fermentation of orange juice was carried out by UHPLC-QqQMS/MS. Melatonin significantly increased throughout fermentation from day 0 (3.15 ng/mL) until day 15 (21.80 ng/mL) reaching larger amounts with respect to other foods. Melatonin isomer was also analysed, but its content remained stable ranging from 11.59 to 14.18 ng/mL. The enhancement of melatonin occurred mainly in the soluble fraction. Tryptophan levels significantly dropped from 13.80 mg/L (day 0) up to 3.19 mg/L (day 15) during fermentation. Melatonin was inversely and significantly correlated with tryptophan (r = 0.907). Therefore, the enhancement in melatonin could be due to both the occurrence of tryptophan and the new synthesis by yeast. In summary, the enhancement of melatonin in novel fermented orange beverage would improve the health benefits of orange juice by increasing this bioactive compound.
ndez-Pachón1, M. S. Ferna S. Medina2, G. Herrero-Martı́n1,
1,3, I. Cerrillo1, G. Berna B. Escudero-López1, F. Ferreres2, F. Martı́n1,3, M. C. Garcı́a-Parrilla4 and A. Gil-Izquierdo2 1 Área de Nutrición y Bromatologı́a, Departamento de Biologı́a Molecular e Ingenierı́a Bioquı́mica, Universidad Pablo de Olavide, Sevilla, Spain; 2Departamento de Ciencia y Tecnologı́a de Alimentos, CEBASCSIC, Murcia, Spain; 3CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Universidad Pablo de Olavide, Sevilla, Spain; 4Área de Nutrición y Bromatologı́a, Departamento de Nutrición y Bromatologı́a, Toxicologı́a y Medicina Legal, Universidad de Sevilla, Sevilla, Spain Key words: alcoholic fermentation, food analysis, melatonin, orange juice, tryptophan, UHPLC-MS/MS Address reprint requests to Marı́a-Soledad
ndez-Pachón, Área de Nutrición y Ferna Bromatologı́a, Departamento de Biologı́a Molecular e Ingenierı́a Bioquı́mica, Universidad Pablo de Olavide, Carretera de Utrera Km 1, E-41013, Sevilla, Spain. E-mail: msferpac@upo.es Received July 24, 2013; Accepted September 6, 2013. Introduction In the last two decades, numerous epidemiological and intervention studies have associated the consumption of fruits and vegetables with a lower risk of diseases such as cardiovascular disease, cancer and ageing-related disorders [1–3]. Citrus fruits have been recognized to be rich sources of bioactive compounds, and orange juice is known for its ascorbic acid, carotenoid and flavonoid content [4]. Furthermore, an inverse correlation between moderate alcohol consumption and the occurrence of coronary heart disease has been established [5–7]. The health effects of dietary bioactive compounds and moderate alcohol consumption could be used as novel commercial beverage of low alcoholic graduation prepared from orange juice by controlled alcoholic fermentation. Indeed, it is remarkable that beverage developed by alcoholic fermentation of orange juice continues with increased effort; this has led to the development of fruit wines rich in bioactive compounds [8–10]. Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine synthesized in the pineal gland and many other organs of animals from L-tryptophan metabolism via sero- tonin [11]. This indoleamine has been implicated in several biological processes including the modulation of circadian rhythm, immune responses, free radical-scavenging activity, activation of antioxidant enzymes, reproductive activity and bone metabolism and improves glucose tolerance, insulin action and lipid profile being beneficial for diabetes, obesity or dyslipidemia [12–19]. Melatonin is also present as metabolite in plants and edible products such as tomatoes, strawberries, grapes, cherries, olive oil, nuts and cereals [20–25]. Given that the melatonin is absorbed when melatonin-containing foods are eaten [21, 26], the intake of these foods could prevent the reduction in melatonin plasma concentration, which occurs with age [27]. The orange fruit is a natural source of melatonin. Sae-Teaw and colleagues [28] analysed the melatonin concentration in orange extracts and found that it contained 150 pg/g. Johns and colleagues [29] described the melatonin content of orange to be 150 pg/g. The beneficial effects of melatonin of orange juice consumption have been shown as it increased plasma antioxidant capacity after its intake [28]. Moreover, melatonin synthesis by yeast during alcoholic fermentation in white and red wines [30–33], beer [34] and 31 Fern
andez-Pachón et al. Table 1. Quality parameters of orange juice before and after fermentation Days of fermentation TA (g citric acid/L) pH Total glucids (g/L) TPI (mg/L) Alcohol (% v/v) 8.48  0.02 8.85  0.02 3.48  0.20 3.43  0.20 78.2  5.64 53.7  4.65 793  0.50 722  12.7 0 0.87  0.01 0 15 Values are expressed as mean  DS of four independent alcoholic fermentation processes analysed in triplicate. TA, titratable acidity; TPI, total polyphenols index. pomegranate wine [35] production has been observed. Also, it has been observed that yeasts synthesize melatonin isomers (ISO) during alcoholic fermentation involved in the winemaking process [36, 37]. These ISO seem to share also similar biological functions. Mor and colleagues [38] studied the antioxidant activity of several melatonin isomers. According to the results of Mor and colleagues [38], Gómez and colleagues indicated that the isomer which they had identified in wine was even more potent than melatonin itself [37]. The amino acid tryptophan is the precursor of all 5-methoxytryptamines (or indoleamines), including melatonin, and the biosynthetic pathway is via serotonin (5-hydroxy-tryptamine) in the case of yeasts, plants and mammals [11, 39, 40]. Taking into account the formation of melatonin by yeasts during alcoholic fermentation in grape and pomegranate wines, it was of interest to evaluate whether this indoleamine is also synthesized during the elaboration process of the new beverage of the fermented orange juice. Thus, the aim of the present study was to determine, for the first time, whether melatonin and ISO are synthesized during the alcoholic fermentation process of orange juice. Likewise, the relation between levels of tryptophan, melatonin and ISO during the process was also evaluated. The possible enhancement of melatonin and ISO in the new fermented orange drink could improve the health benefits of orange juice by increasing its bioactive compounds. Materials and methods Chemicals and reagents N-acetyl-5-methoxytryptamine (melatonin) standard was purchased from Fluka (Neu-Ulm, Germany). All LC-MSgrade solvents (acetonitrile and methanol) were obtained from J. T. Baker (Phillipsburg, NJ, USA). Formic acid, ammonium acetate, sodium hydroxide and dimethyl sulfoxide (DMSO) were purchased from Panreac Qu
ımica S.A. (Barcelona, Spain). Boric acid was from Probus (Badalona, Spain). Calcium disodium EDTA, Bis-Tris reagent and tryptophan standard for derivatization were obtained from Sigma-Aldrich (Madrid, Spain). The 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate (AQC) reagent was purchased from Chemos GmbH (Regenstauf, Germany). Solutions were prepared by diluting with Milli-Q water produced using an Elixâ3 Millipore water purification system coupled to a Milli-Q module (model Adventage 10) (Molsheim, France). All reagents were of analytical grade. Alcoholic fermentation procedure The company ‘Grupo Hesperides Biotech S.L.’ carried out the controlled alcoholic fermentation of commercial pas32 teurized juice made from Citrus sinensis L. var. Navel late (Huelva, Spain) (patent: WO2012/066176A120120524). The criteria for selecting this orange juice were the compositional homogeneity, microbiological stability and organoleptic quality (data not shown). These aspects are necessary for adequate development of the fermentation process and consumer acceptance of final product. The fermentation was carried out in two parallel PVC tanks (5 L) at 20°C for 15 days in repose. The yeast strain Saccharomycetaceae var. Pichia kluyveri was isolated from the natural microbiota present in the orange fruit and used for inoculation of the fermentation. The selected yeast strain ferments only reducing sugars, allowing low alcohol content in the final product (0.8–1.2% v/v). Before sample collection, fermentation liquid was agitated and mixed by using magnetic agitators to promote sample homogenization. Samples were collected every 2 days (day 0 [original orange juice], day 1, day 3, day 5, day 7, day 9, day 11, day 13 and day 15) and immediately stored at 20°C until analysis. Table 1 provides the quality parameters: titratable acidity (TA), pH, total sugars and alcohol [41], and total polyphenols index (TPI), determined by Folin-Ciocalteu method [42]. Melatonin analysis Samples were processed according to Rodrı́guez-Naranjo and colleagues [30, 31] with minor modifications. Briefly, a volume (10 mL) of each sample was centrifuged at 4500 g for 10 min, and both the supernatant and pellet were separated. The pellet was then extracted with DMSO (500 lL). Samples were evaporated to dryness under vacuum. The residues were resuspended in methanol/water (1:1, v/v), up to a concentration of 3:1 (v/v) and treated with mild ultrasonic for 10 min at room temperature. The reconstituted extracts were filtered through a 0.45-lm nylon membrane (Simplepure; Membrane Solutions, Spring View Lane plano, TX, USA) before the analysis. Melatonin determination and quantification was analysed using a UHPLC-QqQ-MS/MS (UPCL-1290 Series and a 6460 QqQ-MS/MS; Agilent Technologies, Waldbronn, Germany) with an Acquity BEH C18 column (2.1 9 150 mm; 1.7 lm; Waters, Milford, MA, USA). Chromatographic separation was achieved using a binary gradient consisting of (A) water and (B) methanol as LC-grade solvents both containing 0.1% formic acid (v/v). The flow rate was 0.30 mL/min using a linear gradient (t; %B): (0.00; 40), (1.50; 40), (1.51; 90), (3.50; 90), (3.51; 40). The volume injection was 20 lL. Multiple reaction monitoring (MRM) in positive mode was carried out. The MS data obtained were compared with the authentic Melatonin in fermented orange juice markers to produce a final melatonin identification and investigate the ISO present in samples. Melatonin quantification was based on the 233/216 MRM transition [30, 31, 35]. This MRM transition was selected because of its specificity and its better signal-to-noise ratio. Melatonin standard external calibration was used to quantify melatonin and its ISO in samples. Chromatographic profile is shown in Fig. 1. Nitrogen was used as the collision gas for the fragmentation by collision-induced dissociation of the compounds at the collision cell of the triple quadrupole mass spectrometer. Mass spectrometer parameters were set as follows: drying gas flow: 8 L/min; sheath gas flow: 12 L/min; sheath gas temperature: 350°C; nebulizer pressure: 30 psi; capillary voltage: 4000 V and nozzle voltage: 1000 V. MassHunter software version B 04.00 was used for MS control and data gathering, and MassHunter soft- (A) (B) (C) Fig. 1. Chromatographic profile recorded using UHPLC-MS/MS with multiple reaction monitoring (MRM) mode for the 233/216 transition of (A) melatonin (MEL) standard (0.312 lM), (B) supernatant of orange juice (isomer (ISO) and MEL) and (C) supernatant of fermented orange juice (day 15) (ISO and MEL). ware version B 03.01 was used for data processing, peak integration and linear regression. Tryptophan analysis Samples were centrifuged at 10,000 g during 10 min (model EBA 21, Hettich Zentrifugen, Tuttlingen, Germany), and the supernatant was transferred to a limited-volume vial. Tryptophan content was determined as previously described by Salazar and colleagues [43]. An aliquot of 500 lL of the supernatant was mixed with 12.5 lL of extraction buffer [MeOH/water (50% v/v)] and vortexed for 30 s on ice followed by incubation on ice for 5 min and sonicated in an ultrasound bath for 1 min. The homogenates were then centrifuged (centrifuge 5804 R, Hamburg, Germany) for 10 min at 17,900 g at 4°C. The supernatant was transferred to a limited-volume vial. Extracts were immediately derivatized. Derivatization of tryptophan was carried out following a method described previously by Salazar and colleagues [43] and Nagumo and colleagues [44]. Briefly, 350 lL of borate derivatization buffer (0.2 M sodium borate, pH 8.8 with 5 mM calcium disodium EDTA), 50 lL of tryptophan standard or sample and 100 lL of reconstituted AQC (10 mM AQC in acetonitrile) [45, 46] was added in a 2-mL propylene vial. The vial was vortexed for several seconds, and then was left to rest for 1 min at room temperature. Thereafter, the vial was heated in a heating block for 10 min at 55°C. After this step, it was removed from the heating block, and the sample was injected in a UHPLCtriple quadrupole mass spectrometer (UHPLC-MS/MS). Analysis of tryptophan was accomplished by reversed phase by means of UHPLC-MS/MS technology, as reported by Salazar and colleagues [43] and Nagumo and colleagues [44] with slight modifications. Briefly, chromatographic separation was carried out on an AccQ Tag Ultra BEH column (2.1 9 100 mm; 1.7 lm; Waters, Dublin, Ireland). The applied solvent system for gradient separation consisted of two types of eluents: the mobile phase A consisted of 50 mL of an aqueous solution (acetonitrile, formic acid and acetate ammonium in water (5 mM), 10:6:84, v/v/v) diluted with 950 mL of Mili-Q water, and the mobile phase B was a mixture of acetonitrile and formic acid (99.9:0.1, v/v). 20 lL of the derivatized tryptophan standard or sample was injected onto the column and eluted at a flow rate of 0.5 mL/min according to the gradient profile as follows: 99.9% A at 0–0.5 min, 90.9% A at 5.7 min, 78.8% A at 7.7 min, 40.4% A at 8–10 min, 10% A at 10.01–12.00 min and 99.9% A at 12.01– 14.00 min. The acquisition time was 12 min for standard or sample. Concentrated tryptophan was prepared by dissolving in Bis-Tris buffer pH 6.5. Calibration standard was generated by diluting the stock solution at 1, 0.5, 0.25, 0.12, 0.06 and 0.03 mM. Identification of tryptophan was achieved using a UHPLC system coupled with a 6460 tandem mass spectrometer (Agilent Technologies). Data acquisition and processing were performed using the MassHunter software version B.04.00 from Agilent Technologies. The MS analysis was applied in the MRM mode, which was performed using the positive ionization mode. The working conditions for the MS parameters were 33 Fern
andez-Pachón et al. extraction and NMR identification in addition to ion trap mass spectrometer [31]. Table 2 reports the content of melatonin and ISO in the soluble and pellet fractions in orange juice during fermentation, and Fig. 2 shows the evolution of total melatonin and ISO contents (sum of supernatant and pellet content). Melatonin content increased progressively during the fermentation process in the soluble fraction of orange juice. From day 7, melatonin content (8.82 ng/mL) was significantly higher than that of day 0 (2.49 ng/mL). Moreover, the highest melatonin content (day 15: 20.0 ng/ mL) was observed at the end of fermentation (Table 2). A major portion of the melatonin content was present in the supernatant, but small amounts were identified in the pellet fraction. Additionally, melatonin concentration underwent a significant increase in the pellet after alcoholic fermentation from day 0 (0.66 ng/mL) to day 7 (1.45 ng/mL). Thereafter, the content increased until day 15 (1.85 ng/mL), but this evolution was not significant. The results indicate that the increase in melatonin content along orange juice alcoholic fermentation is mainly due to the increase in the soluble fraction. Total melatonin content (sum of supernatant and pellet content) during fermentation is presented in Fig. 2A. It is observed that total melatonin underwent a marked 7-fold increase from day 0 (3.15 ng/mL) until maximal value at day 15 (21.9 ng/mL) (P < 0.001). The significant difference was also found after day 7 (P < 0.05). Several studies of melatonin levels have been based on fresh fruits (grapes, cherries and tomatoes) [22, 48, 49]. In recent years, the enhancement of melatonin after fermentation processes has been studied. The rise in melatonin in wines is due to its synthesis during alcoholic fermentation by Saccharomyces cerevisiae [30–32, 35, 50]. Accordingly, an enrichment of melatonin content during the production of beer by yeast contribution was also observed [34]. Melatonin level in fermented orange juice (21.9 ng/mL) is higher than that found for fresh fruits. Johns and colleagues [29] measured melatonin concentrations in pineapple (302 pg/g), banana (8.9 pg/g), mango (699 pg/g) and papaya (241 pg/g). Fermented orange juice reached similar values to other fermented products. Melatonin content varied depending on wine variety from 5.1 to 130 ng/mL optimized for this device, and they were set as follows: gas flow: 9 L/min, nebulizer: 40 psi, capillary voltage: 4000 V, nozzle voltage: 1000 V, gas temperature: 325°C, sheath gas temperature: 390°C, and jetstream gas flow: 11 L/min. The MS parameters for fragmentor (ion optics; capillary exit voltage) and collision energy were optimized. The allocation of these parameters along with preferential MRM transition of the analyte generated the most abundant product ions. In this sense, the preferential MRM transition obtained for tryptophan corresponded to the AMQ moiety (171+), a result from the collision-induced cleavage at the ureide bond of AMQ adduct of amino acid [43, 47]. Statistical analysis Fermentation was performed in quadruplicate, and all analyses were in triplicate. The analysis of variance (oneway ANOVA; Duncan) was applied to establish significant differences between the means obtained during the fermentation process. A probability value of P < 0.05 was adopted as the criteria for significant differences. Pearson’s correlation coefficient (r) was used to determinate the correlation between the parameters evaluated. A P < 0.05 was adopted as the criteria for significant correlation. These analyses were performed with SPSS version 15 software (SPSS Inc., Chicago, IL, USA). Results and discussion Melatonin has been identified in orange [28, 29]. However, this is the first time that alcoholic fermentation influences melatonin formation in orange juice. Melatonin analysis during alcoholic fermentation of orange juice was carried out by UHPLC-QqQ-MS/MS as it has been validated as a selective and sensitive method for an appropriate identification and quantification of melatonin in wine samples [31]. As the authentic maker is not available to identify the current isomer detected by UHPLC-QqQ-MS/MS, tentative identification by comparing relative abundance of minority fragment ions to identify position isomer is adequate. However, the total identification of a novel compound requires the isolation by previous solid phase Table 2. Melatonin (MEL) and MEL isomer (ISO) contents of orange juice during the alcoholic fermentation process MELa (ng/mL) Days of fermentation Supernatant 0 1 3 5 7 9 11 13 15 2.49 2.38 3.31 6.67 8.82 13.7 14.9 18.8 20.0          0.03a 0.16a 0.43a 1.10ab 0.67b 1.30c 1.42cd 1.50de 2.02e ISOb (ng/mL) Pellet 0.66 0.48 0.95 0.98 1.45 1.46 1.98 1.93 1.85          0.00a 0.00a 0.24ab 0.15ab 0.10bc 0.03bc 0.13c 0.27c 0.16c Supernatant 8.28 8.52 8.58 9.20 9.71 9.83 10.3 10.4 10.5          0.73a 0.53a 0.72ab 0.66abc 0.46abc 0.50abc 0.48bc 0.30bc 0.32c Pellet 3.31 2.79 2.86 3.15 3.34 2.89 3.70 2.48 3.66          0.58a 0.51a 0.50a 0.51a 0.28a 0.46a 0.41a 0.42a 0.16a Results are expressed as mean  S.E.M. of four independent alcoholic fermentation processes analysed in triplicate. Values with different roman letters (a–e) in the same column indicate means significantly different at P < 0.05. a Melatonin was measured by UHPLC-MS/MS. b ISO amount calculated by use of MEL standard. 34 Melatonin in fermented orange juice 25 (A) *** MEL (ng/mL) 20 *** *** *** 15 * 10 5 0 0 1 3 5 7 9 11 13 15 Time (days of fermentation) 20 (B) Fig. 2. Evolution in the total content (supernatant and pellet) of melatonin (MEL) (A) and isomer (ISO) (B) of orange juice during the alcoholic fermentation process. Symbols represent mean values and error bars the S.E.M. *P < 0.05, ***P < 0.001 as compared with day 0. ISO (ng/mL) 18 16 14 12 10 8 6 0 [31] or 18 ng/mL [50]. Mena and colleagues [35] obtained the maximum melatonin content of 8.78 ng/mL during fermentation of pomegranate. These differences among fermented products can be explained by the basal concentration of melatonin before fermentation onset, growth phase and type of yeast [32], which determine the concentration of melatonin in the final product. Melatonin has up to nine known ISO (including itself) [51]; nomenclature for these has been recently proposed [36]. Possible appearance of ISO can occur during the winemaking process as single compounds or together with melatonin [31]. ISO content was also evaluated during alcoholic fermentation of orange juice. Analyses of ISO fractions separately showed that ISO content gradually increased in the soluble fraction during the orange juice fermentation process. This improvement was significant between day 0 (8.28 ng/mL) and day 11 (10.3 ng/mL). Thereafter, the increase was not significant, but at day 15 (10.5 ng/mL), the maximum value was achieved. Before fermentation, ISO concentration in the supernatant was higher than the melatonin content, but the augmentation in ISO during fermentation was lower. So ISO values from day 0 to day 15 underwent a 1.26% increase, while the melatonin rise was 8%. Therefore, the final concentration of melatonin was twofold higher than ISO final value in the supernatant fraction. Conversely, ISO concentration in the pellet fraction remained unchanged during alcoholic fermentation from day 0 (3.31 ng/mL) to day 15 (3.66 ng/mL). Similar to melatonin, ISO level in pellet was higher (3.31 ng/mL) than the melatonin content (0.66 ng/ mL) before fermentation, but the increment in melatonin value from day 0 to day 15 of fermentation process was 2.8%. The final content of ISO in the insoluble fraction 1 3 5 7 9 11 13 15 Time (days of fermentation) was twofold higher than melatonin content. The total ISO content (sum of supernatant and pellet fractions) remained stable during the 15 days of orange juice fermentation, with a minor and nonsignificant increase between day 0 (11.6 ng/ mL) and day 15 (14.2 ng/mL) (Fig. 2B). ISO content of fermented orange juice was similar to that reported by Rodr
ıguez-Naranjo and colleagues [31] and Gómez and colleagues [37], who reported values of ISO in different types of wines after winemaking process: 5.2–21.9 ng/mL and 15 ng/mL, respectively. Gómez and colleagues [37] observed an enhancement of ISO content during fermentation in wines. In contrast, in the current study, ISO content did not rise during fermentation. These results indicate that ISO is not synthesized during the orange juice fermentation process. This could be explained by the type of yeast used. In this regard, Tan and colleagues [36] mentioned that yeasts producing elevated levels of ISO might be able to produce higher levels of alcohol. Fermented orange juice has a low alcoholic grade. Melatonin has a number of biological functions: glucose tolerance, circadian rhythm regulation, antioxidant defence or immune system action. We hypothesized that the consumption of fermented orange juice could induce higher beneficial effects than orange juice alone, and the difference between both beverages would be caused by the higher melatonin as ISO content does not significantly change during alcoholic fermentation. Among amino acids, specially tryptophan could be a pivotal factor as it is a precursor of melatonin in yeast [30, 39]. Rodrı́guez-Naranjo and colleagues [30] and Gómez and colleagues [37] evaluated an increase in the melatonin of wines when tryptophan was added during the fermentation process. To evaluate the influence of tryptophan on 35 Fern
andez-Pachón et al. Tryptophan (mg/L) 20 16 12 * 8 * * ** ** 4 0 0 1 3 5 7 9 11 13 15 Tryptophan (mg/L) Time (days of fermentation) 16 14 12 10 8 6 4 2 0 y = –0.48x + 12.665 r = –0.907 ** 0 5 10 15 MEL (ng/mL) melatonin synthesis, tryptophan content was analysed during alcoholic fermentation of orange juice (Fig. 3). The tryptophan concentration of orange juice was 13.8 mg/L before fermentation (day 0) and significantly decreased by day 7 (6.47 mg/L). At the end of the fermentation process (day 15), tryptophan content reached a minimal value of 3.19 mg/L. Mercolini and colleagues [25] also observed a drop in tryptophan content during grape must fermentation from 117 to 82 ng/mL. There is some controversy among authors related to factors involved in the synthesis of melatonin during fermentation. Rodrı́guez-Naranjo and colleagues [32] indicated that melatonin synthesis depends on the growth phase of the yeast and the concentration of tryptophan. However, Tan and colleagues [36] pointed to melatonin production without the presence of tryptophan and observed that the production of melatonin and ISO was independent of external tryptophan during wine fermentation, suggesting that yeasts can still synthesize this indoleamine without tryptophan in the medium. In the present study, the drop of tryptophan level during fermentation could be related to the simultaneous synthesis of melatonin. In addition, the correlation between content of tryptophan and melatonin of orange juice during fermentation was studied, and the results are presented in Fig. 4. Total melatonin concentration inversely and significantly correlated with tryptophan values (r = 0.907). Taking these results into account, we hypothesize that the enhancement in melatonin content of orange juice during fermentation could be due to both the presence of its precursor, tryptophan, and the new synthesis by yeast. This study reports for the first time the influence of alcoholic fermentation on melatonin and ISO formation in 36 Fig. 3. Evolution in the tryptophan content of the orange juice during the alcoholic fermentation process. Symbols represent mean values and error bars the S.E.M. *P < 0.05, **P < 0.01 as compared with day 0. 20 25 Fig. 4. Correlation between total melatonin (MEL) content (ng/mL) and tryptophan concentration (mg/L) present in fermented orange juice. Pearson’s correlation coefficient (r) is significant at the ** 0.01 level. orange juice. According to our results, fermentation of orange juice induces a gradual synthesis of melatonin with a rise from day 7 to day 15 of alcoholic fermentation. When comparing melatonin amounts in fermented orange juice with the content in other fruits, it is suggested that this beverage is a promising source of this bioactive compound. However, ISO content remains stable during the process. Moreover, to optimize the conditions for melatonin synthesis, it is important to know the factors involved in this process. In this study, the enhancement of the melatonin content may have been dependent on tryptophan as this precursor decreases during orange juice fermentation. Fermented orange juice could be a new functional food, and its consumption could exert a potentially positive effect on health. Subsequent intervention studies are necessary to evaluate the health effects of this novel orange beverage and to verify whether any benefits observed may be, at least in part, due to melatonin and its isomers, possibly acting synergistically with other bioactive compounds present in the fruit such as flavanones and carotenoids. Acknowledgements The authors are grateful to the company ‘Grupo Hesp
erides Biotech S.L.’ for providing the samples of fermented orange juice. The authors also gratefully acknowledge the support of Junta de Andalucı́a through the Project P09-AGR4814M and of National funding agencies through the Projects AGL2011-23690, CSD007-0063 (CONSOLIDER-INGENIO 2010 ‘Fun-C-Food’) and CSIC 201170E041. The authors are also grateful to the Fundación S
eneca – CARM ‘Group of Excellence in Research’ 04486/GERM/06 and the Ibero-American Programme for Science, Technology and Development Melatonin in fermented orange juice (CYTED) – Action 112RT0460 CORNUCOPIA. The Research Project grant of BEL is supported by Junta de Andalucı́a. References 1. GEORGE TW, PATERSON E, WAROONPHAN S et al. Effects of chronic consumption of fruit and vegetable puree-based drinks on vasodilation, plasma oxidative stability and antioxidant status. J Hum Nutr Diet 2012; 25:477–487. 2. SLAVIN JL, LLOYD B. Health benefits of fruits and vegetables. Adv Nutr 2012; 3:506–516. 3. LEENDERS M, SLUIJS I, ROS MM et al. Fruit and vegetable consumption and mortality: European prospective investigation into cancer and nutrition. Am J Epidemiol 2013; 178:590–602. 4. BARROS HR, FERREIRA TA, GENOVESE MI. Antioxidant capacity and mineral content of pulp and peel from commercial cultivars of citrus from Brazil. Food Chem 2012; 134:1892– 1898. 5. FU W, CONKLIN BS, LIN PH et al. Red wine prevents homocysteine-induced endothelial dysfunction in porcine coronary arteries. J Surg Res 2003; 115:82–91. 6. ARRANZ S, CHIVA-BLANCH G, VALDERAS-MARTINEZ P et al. Wine, beer, alcohol and polyphenols on cardiovascular disease and cancer. Nutrients 2012; 4:759–781. 7. SINGH KJ, COHEN BE, NA B et al. Alcohol consumption and 5-year change in left atrial volume among patients with coronary heart disease: results from the Heart and Soul study. J Card Fail 2013; 19:183–189. 8. KLOPOTEK Y, OTTO K, BOHM V. Processing strawberries to different products alters contents of vitamin C, total phenolics, total anthocyanins, and antioxidant capacity. J Agric Food Chem 2005; 53:5640–5646. 9. YOO KM, AL-FARSI M, LEE H et al. Antiproliferative effects of cherry juice and wine in Chinese hamster lung fibroblast cells and their phenolic constituents and antioxidant activities. Food Chem 2010; 123:734–740. 10. JAGTAP UB, WAGHMARE SR, LOKHANDE VH et al. Preparation and evaluation of antioxidant capacity of Jackfruit (Artocarpus heterophyllus Lam.) wine and its protective role against radiation induced DNA damage. Ind Crops Prod 2011; 34:1595–1601. 11. CHATTORAJ A, LIU T, ZHANG LS et al. Melatonin formation in mammals: in vivo perspectives. Rev Endocr Metab Disord 2009; 10:237–243.
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