Isotopic labelling-based analysis elucidates biosynthesis pathways in Saccharomyces cerevisiae for Melatonin, Serotonin and Hydroxytyrosol formation.

Yeasts can synthetise bioactive compounds such as Melatonin (MEL), Serotonin (SER) and Hydroxytyrosol (HT). Deciphering the mechanisms involved in their formation can lead to exploit this fact to increase the bioactive potential of fermented beverages. Quantitative analysis using labelled compounds, 15-N2 l-tryptophan and 13-C tyrosine, allowed tracking the formation of the above-mentioned bioactive compounds during the alcoholic fermentation of synthetic must by two different Saccharomyces cerevisiae strains. Labelled and unlabelled MEL, SER and HT were undoubtedly identified and quantified by High Resolution Mass Spectrometry (HRMS). Our results prove that there are at least two pathways involved in MEL biosynthesis by yeast. One starts with tryptophan as precursor being known for the vertebrates' pathway. Additionally, MEL is produced from SER which in turn is consistent with the plants' biosynthesis pathway. Concerning HT, it can be formed both from labelled tyrosine and from intermediates of the Erlich pathway.

Comparative assessment of software for non-targeted data analysis in the study of volatile fingerprint changes during storage of a strawberry beverage.

Five free software packages were compared to assess their utility for the non-targeted study of changes in the volatile profile during the storage of a novel strawberry beverage. AMDIS coupled to Gavin software turned out to be easy to use, required the minimum handling for subsequent data treatment and its results were the most similar to those obtained by manual integration. However, AMDIS coupled to SpectConnect software provided more information for the study of volatile profile changes during the storage of strawberry beverage. During storage, volatile profile changed producing the differentiation among the strawberry beverage stored at different temperatures, and this difference increases as time passes; these results were also supported by PCA. As expected, it seems that cold temperature is the best way of preservation for this product during long time storage. Variable Importance in the Projection (VIP) and correlation scores pointed out four volatile compounds as potential markers for shelf-life of our strawberry beverage: 2-phenylethyl acetate, decanoic acid, γ-decalactone and furfural.

Melatonin and derived l-tryptophan metabolites produced during alcoholic fermentation by different wine yeast strains.

Melatonin is a neurohormone involved in the regulation of circadian rhythms in humans. Evidence has recently been found of its occurrence in wines and its role in the winemaking process. The yeast Saccharomyces cerevisiae is consequently thought to be important in Melatonin synthesis, but limited data and reference texts are available on this synthetic pathway. This paper aims to elucidate whether the synthetic pathway of Melatonin in Saccharomyces and non-Saccharomyces strains involves these intermediates. To this end, seven commercial strains comprising Saccharomyces cerevisiae (Red Fruit, ES488, Lalvin QA23, Uvaferm BC, and Lalvin ICV GRE) and non-Saccharomyces (Torulaspora delbrueckii and Metschnikowia pulcherrima) were monitored, under controlled fermentation conditions, in synthetic must, for seven days. Samples were analysed using a UHPLC-HRMS system (Qexactive). Five out of the seven strains formed Melatonin during the fermentation process: three S. cerevisiae strains and the two non-Saccharomyces. Additionally, other compounds derived from l-tryptophan occurred during fermentation.

Impact of gluconic fermentation of strawberry using acetic acid bacteria on amino acids and biogenic amines profile.

This paper studies the amino acid profile of beverages obtained through the fermentation of strawberry purée by a surface culture using three strains belonging to different acetic acid bacteria species (one of Gluconobacter japonicus, one of Gluconobacter oxydans and one of Acetobacter malorum). An HPLC-UV method involving diethyl ethoxymethylenemalonate (DEEMM) was adapted and validated. From the entire set of 21 amino acids, multiple linear regressions showed that glutamine, alanine, arginine, tryptophan, GABA and proline were significantly related to the fermentation process. Furthermore, linear discriminant analysis classified 100% of the samples correctly in accordance with the microorganism involved. G. japonicus consumed glucose most quickly and achieved the greatest decrease in amino acid concentration. None of the 8 biogenic amines were detected in the final products, which could serve as a safety guarantee for these strawberry gluconic fermentation beverages, in this regard.

Alcoholic fermentation induces melatonin synthesis in orange juice.

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-QqQ-MS/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.

A survey of biogenic amines in vinegars.

This paper reports the determination of biogenic amines by high-performance liquid chromatography (HPLC) and fluorescence detection after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in balsamic, apple, and red, white, and Sherry wine vinegars. A solid-phase extraction (SPE) with mixed-mode resins method was used before analysis. The method was successfully validated obtaining adequate values of selectivity, response linearity, precision, accuracy, and low detection and quantification limits. The total content of biogenic amines in vinegars ranged from 23.35 to 1445.2 µg/L, being lower than those reported in wines. Putrescine was the amine that showed the highest concentrations in most samples. Methylamine and phenylethylamine were not determined in any vinegar. Balsamic and "Pedro Ximénez" Sherry vinegars reached the highest amounts of biogenic amines, while apple, white and Sherry wine vinegars had the lowest concentrations. Principal component analysis using the biogenic amines as variables, allowed to separate the different kind of vinegars, excepting red vinegars.

Intake of alcohol-free red wine modulates antioxidant enzyme activities in a human intervention study.

Wine intake affects the antioxidant enzyme activities that contribute to the overall antioxidant properties of wine. The purpose of this study is to evaluate whether alcohol-free wine has any effect on antioxidant enzymes. The study was a randomized cross-over human intervention. A low phenolic diet (LPD) was designed to prevent interference from polyphenols in other food sources. In the first period, the volunteers ate only this low phenolic diet; in the second, they ate this diet and also drank 300 mL of alcohol-free wine. The enzymes under study were: superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. The activities of glutathione reductase, superoxide dismutase and catalase decreased during the LPD period and increased in the LPD+dealcoholized wine period. On the third day of intervention, significant changes were observed in glutathione reductase and superoxide dismutase activity for both intervention periods under study. Catalase activity changed significantly on the seventh day of intervention. Antioxidant enzymes modulated their activity more easily than the endogenous antioxidants, which did not undergo any changes. Our results show for the first time that the increase in the activity of the antioxidant enzymes is not due to the alcohol content in wine but to the polyphenolic composition. Therefore, alcohol-free wine could be an excellent source of antioxidants to protect people suffering from oxidative stress (cancer, diabetes, alzheimer, etc.) who should not consume alcohol.

Determination of the melatonin content of different varieties of tomatoes (Lycopersicon esculentum) and strawberries (Fragariaananassa).

Melatonin has recently been detected in various plants and foods. However, data regarding the food composition of melatonin are too scarce to evaluate dietary intake. This paper aims to identify melatonin unequivocally using LC-MS in a wide set of varieties of tomatoes (Lycopersicon esculentum) and strawberry (Fragariaananassa). Furthermore, a validated LC fluorescence was developed. This is the first time melatonin has been identified in Bond, Borsalina, Catalina, Gordal, Lucinda, Marbone, Myriade, Pitenza, Santonio, Perlino, Platero, and RAF varieties of tomatoes, as well as in strawberry (Fragaria ananassa): Camarosa, Candonga, Festival, and Primoris. Melatonin concentration was shown to vary greatly depending on the tomato varieties and harvests (2009, 2010), ranging from 4.11ng/g to 114.52ng/g fresh weight. However, the four varieties of strawberries collected during the two harvests showed greater similarity in melatonin (1.38-11.26ng/g fresh weight).

Antioxidant activity of phenolic compounds: from in vitro results to in vivo evidence.

Over last decade an increasing interest for antioxidants in foods has arisen. The healthy properties of antioxidants related to the prevention of degenerative diseases are the main cause of this boom. An antioxidant prevents the oxidation process, the initial step of development of degenerative diseases, cancer and many others. Literature encompasses analytical methodology development to assess antioxidant properties of foods and beverages. The screening of antioxidant activity of foodstuffs is the subject of a large number of articles. Special interest has been addressed to wine, tea and chocolate. However, the crucial key in the prevention of disease is the action these antioxidants exert after their consumption. Studies involving human subjects are scarce due to the requirements of availability of volunteers and conditions to test are limited. This review summarizes data related to in vitro antioxidant activity of foods, emphasizing the main role of phenolic compounds. A critical comparison is realized between the biological significance of these values and the biological significance of in vivo measurements. In addition, the Plasma Antioxidant Capacity is evaluated and selected as biomarker for in vivo antioxidant status of human organism. In a second part, data collected from different intervention studies performed up to date are compiled and discussed. This review summarized data related to in vitro antioxidant activity of foods, emphasizing the main role of phenolic compounds. A critical comparison is realized between the biological significance of these values and the biological significance of in vivo measurements. In addition, the Plasma Antioxidant Capacity is evaluated and selected as biomarker for in vivo antioxidant status of human organism. In a second part, data collected from different intervention studies performed up to date are compiled and discussed. The original contribution of this work is to compile data of Plasma Antioxidant Capacity after dietetic intervention studies taking into account the portion of food ingested. In addition, we calculated the antioxidant compounds content (phenolic content, ascorbic acid, vitamin E and carotenoids) contained in each food ingested to evaluate better their impact in Plasma Antioxidant Capacity. Intervention studies are grouped by the length of intervention and type of food ingested. Results reported in literature reveal that the increment in Plasma Antioxidant Capacity largely depends on analytical method used.

Repeated red wine consumption and changes on plasma antioxidant capacity and endogenous antioxidants (uric acid and protein thiol groups).

This study aims to ascertain the in vivo antioxidant properties of red wine by determining how it affects antioxidant biomarkers (plasma antioxidant capacity (PAC) and endogenous antioxidants such as uric acid or protein thiol groups). Antioxidant biomarkers have been assessed in eight healthy human volunteers after repeated intakes of 300 mL of red wine every day for a week. During this intervention period, volunteers followed a low phenolic diet designed to prevent the phenolic compounds in wine from interfering with the phenolics from other foods or beverages. This diet was followed throughout the week that the volunteers drank wine and for another control week when they drank water. Biomarkers were determined before the subjects taking part in the study started the intervention period with red wine (Monastrell variety) and 1, 3, and 7 days after. PAC was evaluated by the Ferric Reducing Ability of Plasma assay (FRAP), and the Oxygen Radical Absorbance Capacity assay using fluorescein (ORAC-FL). In addition, the concentrations of endogenous antioxidants such as uric acid, albumin, bilirubin, and protein thiol groups were analyzed. The FRAP method shows that PAC increased after the week of wine consumption but decreased after the week without wine consumption. The uric acid concentration did not show any changes that were significantly different from our results in acute wine intake studies. Protein thiol groups decreased significantly (p < 0.05) with the low phenolic diet, but this decrease was not statistically significant if the diet was taken with red wine (p < 0.05).

Different radical scavenging tests in virgin olive oil and their relation to the total phenol content.

Four different antioxidant tests (ABTS*+, DPPH, ORAC, beta-carotene-linoleate model system) were used to determine the free-radical scavenging activity of 39 extra virgin olive oils (EVOO) and compare the total phenol content by the Folin-Ciocalteu method. The correlation between the total phenols and antioxidant capacities measured by the four methods was very high, and highest with ABTS*+ (R2 = 0.9905). Some of these methods of measurement were applied to olive-oil samples (OO), with approximately a 50% decrease in the value of the antioxidant capacity in comparison with values found for EVOO. In conclusion, the results show that all the methods tested were suitable for determining the antioxidant capacity of olive oil. The Picual variety of extra-virgin olive oil showed high antioxidant activity.

Radical scavenging ability of polyphenolic compounds towards DPPH free radical.

Free radical scavenging activity of different polyphenolic compounds commonly present in wine has been evaluated using DPPH method. The experiments were performed with different amounts of phenols within the linear interval of response and with an excess of DPPH in all cases. In these conditions, for most of the compounds tested, the reaction was biphasic. Total stoichiometry values n confirm the implication of more than one step in the process. Flavan-3-ol compounds showed the highest values, especially procyanidins B1 (9.8) and B2 (9.1). In this family, n values coincide with the number of hydroxyl groups available. EC(50) and TEC(50) parameters have been calculated. EC(50) values are extremely diverse, being the procyanidins B1 and B2 the most potent scavenging compounds and resveratrol the less one. TEC(50) considers the rate of reaction towards the free radical. (+)-Catechin and (-)-epicatechin are the phenolic compounds that need more time to react. In contrast, caftaric and caffeic acids are the phenolic acids that react more rapidly. Antioxidant efficacy (AE) is a parameter that combines both factors. Compounds as kaempferol, with a high EC(50) value, could be considered as an antioxidant with low relevance, but instead shows the highest AE value of the phenolic compounds tested, due to its fast rate of reaction, what is of great biological importance.

Acute intake of red wine does not affect antioxidant enzymes activities in human subjects.

The purpose of this work was to test if the acute intake of red wine has an effect on the activity of antioxidant enzymes. Eight healthy, non-alcoholic, non-smoking human volunteers took part in the study. Ethical approval for the study was obtained from the Ethical Research Committee of the University Hospital Virgen Macarena from Seville. Each subject fasted 12-14 hours before the experiment started. Volunteers were asked to consume 300 mL of red wine in 5 minutes. Venous blood sample was obtained by antecubital venipuncture, with heparin vacutainer. Blood extraction was performed before wine ingestion (baseline value) and 30, 55, and 120 minutes after wine intake. Blood samples were immediately centrifuged at 12,000 x g for 3 minutes, avoiding unnecessary exposure to light. Antioxidant enzymes under study were: superoxide dismutase in erythrocytes, glutathione peroxidase in whole blood, and glutathione reductase in plasma. Determinations were performed spectrophotometrically with commercial available enzymatic kits. No statistically significant changes were observed on the activities of the enzymes superoxide dismutase, glutathione peroxidase, and glutathione reductase assayed at any of the times after wine intake. The intake of red wine did not modify the short-term activity of antioxidant enzymes.

Antioxidant capacity of plasma after red wine intake in human volunteers.

Plasma antioxidant capacity (AC) has been assessed in eight healthy human volunteers after wine intake. Analytical methods used for evaluating AC included the ferric reducing ability of plasma (FRAP) and oxygen radical absorbance capacity using two different fluorescent probes, beta-phycoerythrin (ORAC-PE) and fluorescein (ORAC-FL). In addition, the concentrations of endogenous antioxidants such as uric acid, albumin, and bilirubin were determined. The suitability of analytical methods was evaluated with two different biological matrixes: plasma and serum. Plasma AC was determined before ingestion of 300 mL of red wine (baseline value) and 30, 55, and 120 min afterward. Maximum average increase in AC values was reached at 55 min. Plasma AC determined by ORAC-PE at time zero was significantly correlated with albumin concentration. Plasma AC determined with FRAP at time zero is well correlated with uric acid. Moreover, a good linear correlation was found between uric acid concentration and AC determined by FRAP in each volunteer. The maximum concentration of uric acid occurred after 55 min. Uric acid increase accounts for a nonnegligible part in FRAP values and must be evaluated when using this method for assessing AC in plasma.

The antioxidant activity of wines determined by the ABTS(+) method: influence of sample dilution and time.

The free radical scavenging activity of 42 Spanish commercial wines was determined using the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS(+)). The ABTS(+) radical was generated enzymatically using a horseradish peroxidase and hydrogen peroxide. The presence of wine phenolic compounds caused the absorbance of the radical to decay at 414nm. The measurement conditions were optimised. The total phenolic content of wines ranged from 1262 to 2389mgl(-1) for red wines and 70 to 407mgl(-1) for white wines, expressed as gallic acid equivalents. The phenolic content of Sherry wines was similar to that of white wines. Optimum dilutions for white and Sherry wines were set up as a function of their total phenolic content (for total phenol index, TPI<300mg gallic acid per liter, dilution 2.5:10 to 5:10; for TPI>300mg gallic acid per liter, dilution 1:10 to 3:10). Red wines absorb at the wavelength of measurement and dilutions between 0.35:10 and 0.1:10 are advisable. Reaction kinetics were also monitored and the antioxidant activity, expressed as Trolox Equivalent Antioxidant Capacity (TEAC), was determined at 2 and 15min of reaction. The mean values for TEAC(2min) were 5.01+/-1.40mM for red wines, 0.46+/-0.32mM for white wines and 0.26+/-0.19mM for Sherry wines. At 15min, mean values were 6.93+/-2.41mM for red wines, 0.67+/-0.47mM for white wines and 0.26+/-0.19mM for Sherry wines. The correlation coefficients were better at 2min (r=0.9012) than at 15min (r=0.8462) when compared with TPI. Hence, TEAC(2min) seems to be a more appropriate measure.

Spectrophotometric determination of total procyanidins in wine vinegars.

A widely-used method for the spectrophotometric determination of procyanidins in wines has been adapted to wine vinegar samples. Reagent concentrations have been established and the analytical method tested for possible matrix effects. The recovery of catechin was approximately 98% and the limit of reliable measurement was 0.48 mg l(-1). The within-day and between-day precisions were evaluated and according to the two-tailed F-test the precisions were statistically equivalent. Application to wine vinegars obtained by traditional and quick acetification methods showed differences in concentration between the two groups.