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.

Volatile profile characterisation of Chilean sparkling wines produced by traditional and Charmat methods via sequential stir bar sorptive extraction.

The volatile compositions of Charmat and traditional Chilean sparkling wines were studied for the first time. For this purpose, EG-Silicone and PDMS polymeric phases were compared and, afterwards, the most adequate was selected. The best extraction method turned out to be a sequential extraction in the headspace and by immersion using two PDMS twisters. A total of 130 compounds were determined. In traditional Chilean sparkling wines, ethyl esters were significantly higher, while acetic esters and ketones were predominant in the Charmat wines. PCA and LDA confirmed the differences in the volatile profiles between the production methods (traditional vs. Charmat).

Recent developments in the analysis of musty odour compounds in water and wine: A review.

One of the most common taints in foods is a musty or earthy odour, which is commonly associated with the activity of microorganisms. Liquid foods, particularly wine and water, can be affected by this defect due to the presence of certain aromatic organic compounds at very low concentrations (ng/L) consistent with human threshold perception levels. The volatile compounds responsible for a mouldy off-aroma include approximately 20 compounds, namely, haloanisoles, geosmin, 2-methylisoborneol, several alkyl-methoxypyrazines, 1-octen-3-ol, 1-octen-3-one, trans-octenol, 3-octanone, fenchol and fenchone. Methods for determining these very low concentrations of odour compounds must be extremely sensitive and selective with efficient preconcentration treatments. A number of extraction techniques based on LLME (liquid-liquid microextraction), SPME (solid-phase microextraction) or SBSE (stir-bar sorptive extraction) can be applied and should be selected on a case-by-case basis. Moreover, recent developments in GC instrumentation coupled to different detection systems can effectively increase the selectivity and sensitivity of the analysis of target compounds.

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.

Validation of an analytical method for the determination of ethyl carbamate in vinegars.

A solid phase extraction method (SPE) using Isolute ENV+ cartridges was validated for the determination of ethyl carbamate (EC) in different kinds of vinegars. The method proved to be quite sensitive, precise and accurate, improving the recovery and LQD of other existing methods for the same purpose. For the optimization of the method, different pH values of the sample were tested, resulting 5.5 the most adequate. Among the 14 samples analysed, only 5 of them had contents of EC above the quantification limits, ranging between 6.73 µg/L and 56.4 µg/L. The highest value was found in red wine vinegar. Taking into account the amount of vinegar consumed in a meal and the limits established for alcoholic beverages in some countries, the levels of ethyl carbamate in the vinegars tested in this work were acceptable.

Determination of amino acids in grape-derived products: a review.

The amino acids present in foods and beverages affect the quality of these products and they play an important role in enology. Amino acids are consumed by yeasts as a source of nitrogen during alcoholic fermentation and are precursors of aroma compounds. In this review various chromatographic methodologies for the determination of amino acids are described, and specific applications for the analysis of amino acid content are discussed. Amino acids usually need to be derivatized to make them more detectable. Several derivatizing reagents have been employed for the determination of amino acids in enological applications, and each has its advantages and disadvantages.

Volatile and sensory profile of organic red wines produced by different selected autochthonous and commercial Saccharomyces cerevisiae strains.

Organic wines were produced at pilot scale to select the best autochthonous and commercial yeast strains to obtain wines with high organoleptic qualities. We tested the behaviour of five S. cerevisiae yeast strains and determined their volatile composition and organoleptic characteristics by sensory analysis. A total of 51 volatile compounds were quantified in the wines produced. The concentration of most of the volatile compounds was significantly influenced depending on which yeast strain was inoculated. The differences observed in the volatile composition of the wines appear to be quantitative rather than qualitative. In general, acetals were the most abundant group of volatile compounds in all the samples studied, followed by alcohols without ethanol. The highest contents of volatile compounds were found in two of the wines produced by autochthonous yeast strains. The results obtained in the sensory analysis suggest that autochthonous yeast produced wines of higher organoleptic quality because this sample gave the highest value for the general impression attribute.

Optimization and validation of headspace sorptive extraction for the analysis of volatile compounds in wine vinegars.

Quantification of aroma compounds in wine vinegars is challenging due to the complexity of the matrix and the low concentrations expected. A method for the determination of volatile compounds in wine vinegars employing headspace sorptive extraction-thermal desorption-gas chromatography-mass spectrometry (HSSE-TD-GC-MS) was developed. A central composite design was used to optimize the sampling condition. The proposed method was successfully validated and low detection and quantification limits was obtained. The application of the proposed methodology allows the determination of 53 compounds in different wine vinegars (red, Sherry). Five of them have been detected in wine vinegars for the first time.

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.

[Three cases of unusual non-germ cell tumors of the testicle]. / Tumores no germinales inusuales del testículo: tres casos clínicos.

By describing 3 clinical cases of unusual testicular non germinal tumors, including an adenoma of the rete testis, an undifferenciated sex cord tumor and a mesothelioma of the tunica vaginalis, we make a literature review of the unusual testicular tumors and testicular apendix, including their incidence and management. Also and as one of our conclusions, we expose the importance of the intraoperatory biopsy in the testicular cancer surgery, because even if it is infrecuent, the presence of this rare testicular tumors, in which if they are proven to be benign, the testicular unit could be preserved and the radical orquiectomy could be avoided.

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).

Tumores no germinales inusuales del testículo: tres casos clínicos / Three cases of unusual non-germ cell tumors of the testicle

A propósito de tres casos clínicos con tumores de histología infrecuente del testículo que incluyen el adenoma de la rete testis, el tumor indiferenciado de los cordones sexuales y el mesotelioma de la túnica vaginal, se realiza una revisión bibliográfica de los tumores inusuales del testículo y sus anexos, incluyendo su incidencia y manejo. Finalmente y a manera de conclusión, se explica la importancia de la biopsia rápida en el caso de los tumores testiculares dado que, aunque infrecuente, si ésta resultara benigna, podría evitarse la orquectomía radical y realizar una cirugía conservadora

By describing 3 clinical cases of unusual testicular non germinal tumors, including an adenoma of the rete testis, an undifferenciated sex cord tumor and a mesothelioma of the tunica vaginalis, we make a literature review of the unusual testicular tumors and testicular apendix, including their incidence and management. Also and as one of our conclusions, we expose the importance of the intraoperatory biopsy in the testicular cancer surgery, because even if it is infrecuent, the presence of this rare testicular tumors, in which if they are proven to be benign, the testicular unit could be preserved and the radical orquiectomy could be avoided

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.

Analysis for chloroanisoles and chlorophenols in cork by stir bar sorptive extraction and gas chromatography-mass spectrometry.

A complete methodology for the determination of chloroanisoles and chlorophenols in cork material is proposed. The determination is accomplished by means of a previous liquid-solid extraction followed by stir bar sorptive extraction (SBSE) coupled to gas chromatography-mass spectrometry (GC-MS). Two different liquid-solid extraction experiments were conducted and eight compounds considered (2,6-dichloroanisole, 2,4-dichloroanisole, 2,4,6-trichloroanisole, 2,4,6-trichlorophenol, 2,3,4,6-tetrachloroanisole, 2,3,4,6-tetrachlorophenol, pentachloroanisole and pentachlorophenol). From the results obtained we can conclude that high volume extraction extending extraction time up to 24h is the best choice if we have to release compounds from the inner surfaces of cork stoppers. Recovery percentages ranged from 51% for pentachloroanisole to 81% for 2,4-dichloroanisole. This method allows the determination of an array of compounds involved in cork taint at very low levels from 1.2ng g(-1) for 2,4,6-tricholoroanisole to 23.03ng g(-1) for 2,3,4,6-tetrachlorophenol.

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.

Resveratrol content in wines and musts from the south of Spain.

Trans- and cis-resveratrol and their glycosides have been determined in eighty-one samples including musts, substrate wines and commercial wines (red, white and Sherry-type wines) from the south of Spain. Sample analysis was performed using a direct injection technique by HPLC with photodiode array detector. Prior to the injection, the sample was filtered using a filter selected through a complete filtration study. Climatological data can affect total resveratrol levels. Values obtained for total resveratrol content in red wines ranged from 0.038 mg/l in a dry year to 1.164 mg/l for a normal year. The pressure applied to the berries to obtain must, has been object of study in order to ascertain its influence in final resveratrol content. For the same vintage, the total resveratrol content ranged from 0.032 mg/l in a wine, obtained from the lighest pressure musts, to 0.873 mg/l for those obtained from the highest pressure musts. No resveratrol has been found in Sherry wines, being present in the substrate wines in all cases.