Maturation of Moristel in Different Vineyards: Amino Acid and Aroma Composition of Mistelles and Wines with Particular Emphasis in Strecker Aldehydes

The aim of this article was to assess the influence of the harvest date on the composition of amino acids and derived aromatic compounds in grape-mistelle and wine of the Moristel variety, in different vineyards. Two vineyards were sampled in 2016 and another one in 2017. At each sampling point, grapes were collected, destemmed, crushed and divided into four aliquots. The first three were fermented, and the latter was treated with ethanol, to produce 1-week macerates containing 15% ethanol (v/v)-mistelles. Overall, 10 mistelles and 33 wines were produced. Amino acids, Strecker aldehydes and aroma compounds were analysed. Amino acid profiles are characteristic of the vineyard and level of ripeness, converging with maturation. In fermentation, major amino acids, except proline, are consumed at a relatively fixed and specific tax, while consumption of 13 amino acids is determined by the ratios of alanine, glutamic acid, serine and threonine, with γ-aminobutyric acid. After fermentation, amino acid precursors to Strecker aldehydes are maxima in unripe and overripe samples, while Strecker aldehydes are maxima in unripe wines. No direct correlations between precursor amino acids in mistelle and aromatic compounds in wine have been found. Nevertheless, must amino acid profiles could determine wine aroma composition

A two-run heart-cut multidimensional gas chromatography method using flame ionization and mass spectrometry for automated and robust determination of nearly complete wine aroma-volatile profiles

A quantitative analytical method capable of determining the concentrations of 81 aroma-relevant wine volatiles covering nine orders of magnitude was developed and validated in this study. The method is based on stir bar sorptive extraction (SBSE) of 200 μL of wine diluted with 1.8 mL NaCl brine with pH 3.5. Volatiles thermally desorbed from the stir bars were separated in two runs in a heart-cut multidimensional gas chromatographic system and quantified using either a flame ionization detector (FID) in the first dimension (27 aroma compounds) or a mass spectrometer in the second dimension (54 aroma compounds, transferred to 22 cuts). Typical limits of compound detection lay around 0.02 mg/L by FID or ranged from 0.001 to 0.30 μg/L by mass spectrometry detector, liying below the corresponding odor thresholds in all cases. Linearity, reproducibility, and recovery were considered satisfactory for most compounds, with typical R2 values of 0.989–0.999, relative standard deviation below 10 % for 37 compounds and between 10 and 20 % for 44 compounds, and recovery rates of approximately 100 % (85–109 %) for all but acetaldehyde. An analysis of 20 wine samples completed our validation of the method, showing that a single-sample preparation procedure combined with heart-cut multidimensional two-detector gas chromatography can determine wine volatile concentrations ranging from 350 mg/L of isoamyl alcohol to 3.8 ng/L of 3-isobutyl-2-methoxypyrazine

Accurate quantitative determination of the total amounts of Strecker aldehydes contained in wine. Assessment of their presence in table wines

Strecker aldehydes (SAs) are key determinants of wine shelf-life and can be present in unoxidized wines in odorless forms, such as hydroxyalkylsulfonates, imines or acetals. A robust and accurate method for the determination of total forms of SAs, based on the classical derivatization with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA) and in the selective solid phase extraction of derivatives has been optimized and validated. Matrix effects have been solved by the use of adequate internal standards and by large-enough equilibration times under anoxic conditions. Method figures of merit are highly satisfactory in terms of detection limits ( 0.997), reproducibility (5–13%) and recoveries (RSDs, between 2 and 10%, for 3-methylbutanal, 14%). The analysis of total SAs in 108 Spanish wines revealed that between 52% and 70% of unoxidized red wines and likely a similar fraction of white wines, contain levels of SAs high enough to cause oxidative aromas if bound forms of SAs cleave

Determinación de histamina y de la microbiota responsable en productos lácteos: potenciales soluciones para reducir su acumulación en quesos madurados

Las aminas biógenas (ABs) son compuestos generados por la descarboxilación de su aminoácido precursor. Entre las ABs más importantes se encuentran la histamina, la tiramina, la cadaverina y la putrescina. Su ingesta a elevadas concentraciones por la población en general o su consumo a bajas concentraciones en personas sensibles puede suponer un problema importante para la salud pública. Es por ello que es necesario emplear métodos sensibles para la detección de estos metabolitos en alimentos. Los productos lácteos, y en especial los quesos madurados, son uno de los alimentos habitualmente implicados en intoxicaciones por histamina, generada por microorganismos con actividad descarboxilasa, lo que alumbra la necesidad de identificar estos microorganismos.Los objetivos de esta Tesis Doctoral fueron evaluar la concentración de histamina en productos lácteos tras la puesta a punto de una técnica de análisis de ABs sensible; identificar los microorganismos responsables de la presencia de histamina en quesos comerciales; estudiar el patrón de distribución de la AB principal, la histamina, en diferentes áreas de quesos comerciales y evaluar su relación con la presencia de microorganismos productores y las características físicas y químicas y; ensayar posibles soluciones microbianas y enzimáticas que permitan reducir su acumulación en quesos de larga maduración.Inicialmente, se desarrolló un método para la determinación de cuatro ABs (histamina, tiramina, putrescina y cadaverina) en diferentes productos lácteos (leche, yogur y kéfir) basado en cromatografía líquida de alta eficacia de fase reversa (RP-HPLC) acoplado a un sistema de fluorescencia. Las ABs se extrajeron selectivamente mediante extracción en fase sólida (SPE) y posteriormente se derivatizaron con 6-aminoquinolil-N-hidroxisuccinimidilo carbamato (AQC). Se obtuvo un método con una elevada sensibilidad, con límites de detección de entre 0,12 y 0,2 mg/L. Este método fue aplicado en 37 muestras de leche, 23 de yogur, y 14 de kéfir dónde se pudo comprobar la variabilidad de ABs entre las muestras. Si bien la mayoría de las muestras de leche no superaron el límite de detección (LD), la frecuencia de aparición y la concentración de estos compuestos fue mayor en yogur y kéfir alcanzando valores de hasta 79 mg/kg de ABs totales en muestras de kéfir.Es conocido que los quesos madurados pueden contener cantidades elevadas de histamina. La microbiota presente en el queso es la responsable por la descarboxilación de su aminoácido precursor, la histidina, debido a la acción de la histidina descarboxilasa. Para conocer el origen de la histamina se llevó a cabo la identificación de los microorganismos responsables de su formación en quesos. En primer lugar, se analizó la concentración de histamina de 39 tipos diferentes de quesos comerciales, para después identificar los microorganismos responsables de su aparición. Como resultado, una tercera parte de los quesos analizados contenían más de 200 mg/kg de histamina y dos de ellos superaron los 500 mg/kg histamina. Se obtuvieron aislados microbianos de los cinco quesos con las concentraciones más altas de histamina y se analizó el material genético total de los quesos, con el fin de verificar la presencia del gen de la histidina descarboxilasa (hdc). Tras comprobar la presencia del gen hdc en dichas muestras, se amplificó la secuencia de nucleótidos del gen hdc, y se sometió a secuenciación Sanger para identificar el microorganismo responsable de la formación de histamina en las muestras. En cuatro de los cinco quesos seleccionados, el gen hdc correspondió al microorganismo Lentilactobacillus parabuchneri, referido como principal productor de histamina en quesos, mientras que en uno de los quesos se identificó a Tetragenococcus halophilus, microorganismo que presenta el gen hdc ubicado en un plásmido inestable.Una vez conocidos los microorganismos productores de histamina en quesos comerciales, se evaluó la distribución de histamina en cuatro zonas distales de una cuña (corteza periférica e interna y núcleo periférico e interior) de 12 quesos duros de larga maduración producidos con leche cruda de oveja o mezcla de leches, y se procedió a la identificación de los microorganismos que contenían el gen hdc mediante secuenciación Sanger. Se observó un patrón de distribución de histamina en las diferentes zonas acumulándose en el centro y disminuyendo en la corteza. Sin embargo, no se pudo establecer una correlación entre la distribución de la histamina y la microbiota presente, ya que la distribución de los microorganismos era similar en todas las partes del queso. En cuanto a la relación entre el análisis de las propiedades físicas y químicas de las diferentes áreas y la concentración de histamina se observó que la histamina tendía a acumularse en los lugares más salados, más húmedos y menos oxidados de la cuña.Finalmente, se evaluaron medidas basadas en el uso de la enzima Diamino Oxidasa (DAO) y de microorganismos potencialmente degradadores de histamina (Lacticaseibacillus casei 4a y 18b, Lactobacillus delbrueckii subsp. bulgaricus Colección Española de Cultivos Tipo (CECT) 4005 y Streptococcus salivarius subsp. thermophilus CECT 7207, dos cultivos iniciadores comerciales de yogur (Abiasa y CHR Hansen), y la levadura Debaryomyces hansenii), para disminuir la acumulación en quesos de este metabolito tras 100 días de maduración. Se fabricaron un total de 8 lotes de queso de leche de vaca pasteurizada a los que se les añadieron los microorganismos mencionados anteriormente o la enzima DAO, todos ellos conteniendo L. parabuchneri DSM 5987 (excepto el lote de queso de control negativo), responsable de la producción natural de histamina en el queso. Se analizaron las propiedades fisicoquímicas y sensoriales (olor), así como la concentración de histamina a lo largo de 100 días de maduración del queso. Como resultado, todas las estrategias aplicadas redujeron significativamente la concentración de histamina. Al final del periodo de maduración, se consiguió una reducción de más del 45% en los quesos preparados con D. hansenii, del 43% en aquellos preparados con las dos cepas de L. casei, del 42% en los quesos elaborados con L. bulgaricus y S. thermophilus, y del 23% en aquellos a los que se adicionó DAO. No se observaron cambios fisicoquímicos significativos (peso, pH, actividad del agua, color o textura) en los quesos como consecuencia de la adición de los cultivos microbianos o DAO. Sin embargo, la adición de microorganismos degradadores de histamina produjo quesos con un olor diferente al percibido en las muestras control, que no fue percibido como desagradable.Como conclusión, cabe destacar la puesta a punto de un método sensible para la determinación de ABs en productos lácteos que permite detectar niveles bajos de estos metabolitos. Los microorganismos identificados como productores de histamina en quesos no proceden de los cultivos iniciadores empleados en su elaboración, sino que son microorganismos contaminantes. La concentración de histamina aumenta en las zonas interiores y disminuye en las zonas exteriores del queso lo que desvela la importancia de llevar a cabo una estrategia de muestreo cuando se pretende determinar la cantidad de histamina presente en un queso. La elaboración de quesos con bajos niveles de histamina se podría lograr mediante el empleo de técnicas sensibles para la determinación de este metabolito en productos lácteos que permitiera prevenir su entrada en el mercado, la utilización de leche libre de microorganismos productores de histamina, la limpieza y desinfección de los equipos para evitar la contaminación durante la obtención de la leche y su procesado en la industria láctea con microorganismos productores de histamina, o el uso de microorganismos degradadores o DAO que pudieran degradar la histamina a lo largo de la etapa de maduración del queso.<br /

LA EXCAVACIÓN Y SUS RESULTADOS ARQUEOLÓGICOS

Dado que la concentración de materiales en el talud del camino a que nos hemos referido anteriormente ocupaba un área bastante restringida, nos decidimos a plantear la excavación precisamente en esta zona, penetrando hacia el Norte en el talud todo lo que permitiese, hasta llegar a la vertical del escarpe del Llano del Zamborino. Sin embargo, una de las mayores dificultades que encontramos al emprender la excavación fue la existencia de ese conjunto de estratos de conglomerados rojos y arcillas, de una potencia de 9'45m, que era necesario retirar con el fin de plantear los cortes directamente sobre los niveles fosilíferos. Debido a la gran potencia de ese conjunto de estratos, la labor de desmonte supuso la pérdida de numerosos dias de trabajo de excavación, toda vez que ignorábamos si eran estériles o no, por lo cual este desmonte debió hacerse de una manera metódica y cuidada hasta que se comprobó su absoluta esterilidad

Gas chromatography-mass spectrometry strategies for the accurate and sensitive speciation of sulfur dioxide in wine

Understanding the chemistry of wine oxidation requires the accurate and sensitive quantitative determination of the most important molecular species which SO2 can form. An analytical strategy based in three independent static headspace GC–MS determinations is proposed in order to obtain information about the total, nominally free and truly free levels of SO2. Nominally free forms are directly determined after sample acidulation, total forms require the previous incubation at 100 °C, and truly free forms are determined after preconcentration of the headspace of the undisturbed sample in an alkaline solution. The two first determinations provide results equivalent to those reported by the aeration-oxidation (A-O) method, with lower limits of detection (1 mg L−1) and better repeatabilities (<4.0%). Results from the analysis of different wines revealed that levels of nominally free are systematically in excess than those of truly free SO2, which suggests that free SO2 determined by any method using previous acidulation includes at least two different species of SO2, which may have different antioxidant behavior.This work has been funded by the Spanish Ministry of Economy and Competitiveness (Project AGL2014-59840). V.C. has received a grant from the Spanish FPU program. Funding from Diputación General de Aragón (T53) and Fondo Social Europeo is acknowledged.Peer reviewe

Novel GC-MS strategies for the accurate and sensitive speciation of SO2 in wine

Trabajo presentado en la XVI Reunión Científica de la Sociedad Española de Cromatografía y Técnicas Afines (SECyTA 2016), celebrada en Sevilla del 2 al 4 de noviembre de 2016.Sulfur dioxide has been widely used in winemaking because of its antioxidant, antioxidasic and antiseptic properties. SO2 may appear in wine under different forms due to its acid‐base properties and to the more or less reversible adducts that it can form with acetaldehyde, sugars, polyphenols and other carbonyls. Speciation of this molecule is essential because its bioactive and antioxidant activities are extremely dependent on the specific chemical species. Total levels of SO2 are important because of safety and legal reasons. Bound sulfur dioxide represents a complex pool of diverse molecules from which free SO2 can be released. Such release, inevitably will have consequences on wine sensory properties, since the cleavage of SO2 adducts will release anthocyanins, and sensory relevant aldehydes [1]. Bisulfite (HSO3 ‐) is important due to its antioxidant and antioxidasic properties and molecular SO2 (SO2 plus H2SO3) is the most bioactive form and the main responsible for microbial stability. The reference method of aspiration/titration for determining free SO2 fails when levels drop below 7‐8 mg/L. Moreover, this method is based on an unspecific determination in which acid volatile compounds can produce interferences which can be important at low levels. Additionally, there are doubts about whether the free SO2 determined by the reference method is all equally active, because it has been shown that wines with similar levels of SO2 have different level of protection [2]. Three different methods based on GC‐MS have been developed for the speciation of SO2. Total forms are determined by HS‐GC‐MS of the acidified sample after incubation at 100ºC. Free and weakly‐bound SO2 is similarly determined but incubation is carried out at 40ºC. Similar results to the official method are achieved but limits of detection are much better (1 mg/L of free SO2) and the method is free from interferences. Finally, molecular SO2 is quantified also by HS‐GC‐MS analysis of an acidified fraction obtained by purging the wine with nitrogen and trapping SO2 in an aqueous alkaline solution (pH 11.5). Results have demonstrated that between 10 and 80% of the free SO2 measured by the reference method is in fact forming complexes with polyphenols which are cleaved during the analysis.N

Straightforward strategy for quantifying rotundone in wine at ng L-1 level using solid-phase extraction and gas chromatography-quadrupole mass spectrometry. Occurrence in different varieties of spicy wines

This paper presents a straightforward methodology to quantify rotundone in wine at ng L level. This compound, responsible for the black pepper aromatic note, may have sensorial relevance in some wines due to its low odor threshold, estimated at only 16 ng L in red wines. The proposed strategy is based on solid phase extraction and analysis by GC-MS. The detection limit value was 0.6 ng L, which is more than one order of magnitude below its odor threshold in wine. Matrix effects have not been found and a synthetic wine calibration was proposed. The precision of the method was evaluated in reproducibility terms, obtaining a very acceptable value (RSD 4%). The optimized and validated strategy was applied to quantify this molecule in thirty wines belonging to different varieties as Graciano, Maturana tinta, Schioppettino, Shiraz, Duras and Gamay. Two of these wines exhibited levels higher than 100 ng L.We are very grateful to our colleges in the Australian Grape and Wine Industry for supplying us with the standards of rotundone and for valuable discussions about the methodologies used. We would especially like to thank Tracey Siebert. We thank Olivier Geffroy (Institut Francais de la Vigne et du Vin, France) for supplying some of the wines analyzed in this work. LAAE acknowledges the continuous support of Diputación Gral. Aragón (T53) and Fondo Social Europeo.Peer Reviewe

Accurate quantitative determination of the total amounts of Strecker aldehydes contained in wine. Assessment of their presence in table wines

Strecker aldehydes (SAs) are key determinants of wine shelf-life and can be present in unoxidized wines in odorless forms, such as hydroxyalkylsulfonates, imines or acetals. A robust and accurate method for the determination of total forms of SAs, based on the classical derivatization with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA) and in the selective solid phase extraction of derivatives has been optimized and validated. Matrix effects have been solved by the use of adequate internal standards and by large-enough equilibration times under anoxic conditions. Method figures of merit are highly satisfactory in terms of detection limits ( 0.997), reproducibility (5–13%) and recoveries (RSDs, between 2 and 10%, for 3-methylbutanal, 14%). The analysis of total SAs in 108 Spanish wines revealed that between 52% and 70% of unoxidized red wines and likely a similar fraction of white wines, contain levels of SAs high enough to cause oxidative aromas if bound forms of SAs cleave.Funded by the Spanish Ministry of Economy and Compet- itiveness (MINECO) (project AGL2017-87373-C3). LAAE acknowledges the continuous support of Gobierno de Arag ́on (T29) and European Social Fund. M.B. would like to acknowledge the Spanish National Research Agency (AEI) and the Ministry of Science, Innovation, and Universities (MICIU) for her “Juan de la Cierva-Incorporaci ́on” post- doctoral grant IJC2018-037830-I. A.M.A.C. acknowledges the Depart- ment of Science, University and Knowledge Society from DGA his predoctoral grant (2020 call)

Combination of SPE and fluorescent detection of AQC-derivatives for the determination at sub-mg/L levels of biogenic amines in dairy products

Biogenic amines (BAs) are compounds generated by decarboxylation of their amino acid precursors. Their intake, even at low concentrations, can lead to several types of health problems in sensitive individuals. As they can be easily formed in fermented dairy products, their quantitative determination is very relevant. In the present paper, a method for the quantitative determination of four biogenic amines in different dairy products has been developed, validated and applied to 37 samples of milk, 23 of yogurt, and 14 of kefir. Amines were selectively extracted using solid phase extraction, subsequently derivatizatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and further determined by High Performance Liquid Chromatography with fluorescence detection. The method’s sensitivity was highly satisfactory, with limits of detection lower than 0.2 mg/L. Optimal linearity and repeatability were also achieved. BAs were not detected in most of the milk samples, but they were found frequently at high levels in yogurt and kefir samples, reaching values of up to 79 mg/kg total BAs in kefir samples. Levels measured should not be a cause for concern for the population at large, but should be known by BAs-sensitive individuals