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Landete Iranzo, José María
Ferrer Soler, Sergi (dir.); Pardo Cubillos, Isabel (dir.) Universitat de València - MICROBIOLOGIA I ECOLOGIA |
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Aquest document és un/a tesi, creat/da en: 2005 | |
Biogenic amines are organic bases endowed with biological activity which are frequently found in fermented foods and beverages. They are produced mainly as a consequence of decarboxylation of amino acids. High concentrations of biogenic amines can cause undesirable physiological effects in sensitive humans, especially when alcohol and acetaldehyde are present
In this work, we describe a new enzymatic method that allows directly estimating histamine concentration in synthetic media, grape must or wine (white, rosé and red) without polyphenols or sugar interferences. This new enzymatic method shows a good correlation (r=0.996, p<0.001) between the histamine concentrations and absorbances in the interval 0.4 to 160 mg l-1.
The influence of grape variety, type of vinification, wine pH, malolactic fermentation, and storage in bottle on biogenic amine concentrations was also studied. Result...
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Biogenic amines are organic bases endowed with biological activity which are frequently found in fermented foods and beverages. They are produced mainly as a consequence of decarboxylation of amino acids. High concentrations of biogenic amines can cause undesirable physiological effects in sensitive humans, especially when alcohol and acetaldehyde are present
In this work, we describe a new enzymatic method that allows directly estimating histamine concentration in synthetic media, grape must or wine (white, rosé and red) without polyphenols or sugar interferences. This new enzymatic method shows a good correlation (r=0.996, p<0.001) between the histamine concentrations and absorbances in the interval 0.4 to 160 mg l-1.
The influence of grape variety, type of vinification, wine pH, malolactic fermentation, and storage in bottle on biogenic amine concentrations was also studied. Results show important differences in putrescine and histamine concentrations between regions, varieties of grape and type of wine; differences were less appreciable for the remaining biogenic amines studied. Low pH prevented biogenic amine formation. Malolactic fermentation and short storage periods in bottle (3 - 6 months) showed increases in histamine concentration, while longer periods of storage led to a general decrease in histamine. Grape varieties, different types of winemaking, pH and lactic acid bacteria may be responsible for the differences observed in the biogenic amine concentration of wines analyzed.
Strains of lactic acid bacteria were isolated in this work, and their ability to form biogenic amines was assayed in synthetic media, grape must and wine. The species exhibiting the highest frequency of histamine production is Oenococcus oeni. However, the concentration of histamine produced by this species is lower than the produced by strains belonging to species of Lactobacillus and Pediococcus. A correlation of 100% between presence of histidine decarboxylase gene and histamine production was observed. The main wine species tyramine and phenylethylamine producer was Lactobacillus brevis whereas this character was no present or infrequent in the rest of analysed wine strains.
The sequence of tyrosine decarboxylase operon contained four complete genes encoding atyrosyl-tRNA synthetase, the tyrosine decarboxylase, a probable tyrosine permease and a Na+/H+ antiporter.
The aim of this work was also to study the influence of enological factors (organic acids, sugars, ethanol, histidine, histamine, SO2, pH and temperature) on the hdc expression and on HDC activity in Lactobacillus hilgardii, Pediococcus parvulus and Oenococcus oeni. Cell extracts and whole cells were used to study the influence of different factors on histidine decarboxylase gene (hdc) expression and histidine decarboxylase enzyme (HDC) activity. Glucose, fructose, malic acid, citric acid and histamine diminished the hdc expression, while ethanol and histidine increased the histidine decarboxylase enzyme activity. Temperature and pH had effect on the activity of HDC but not on hdc expression. Tartaric acid and L-lactic acid, and SO2 had no effect on enzyme synthesis and activity. Lb hilgardii is the most active, and O. oeni the less one, lactic acid bacteria species difer in the relative enzymatic activity but all the factors affected in the same way to Lb. hilgardii, P. parvulus and O. oeni. The hdc gene expression was lowered by glucose, fructose, malic acid, and citric acid, whereas ethanol enhanced the HDC enzyme activity. This behavior was observed for all the studied microorganisms. Thus, the conditions that normally occur during malolactic fermentation and later on, could favor the histamine production. SO2 could prevent bacterial growth, but does not diminish the HDC enzyme activity.RESUMEN
Dada la influencia negativa de la presencia de aminas biógenas en el vino, por el efecto perjudicial sobre la salud humana y por la disminución de la calidad del vino y dada también la presencia de las bacterias lácticas (BL) en los mismos, en esta tesis nos planteamos determinar la importancia de las BL en la presencia de estas aminas en el vino, así como que factores podían influir en la concentración de las mismas o en la mayor o menor producción por parte de las BL.
En primer lugar, desarrollamos un método enzimático que permite analizar la concentración de histamina en vino y la producción de la misma por BL, a continuación analizamos la concentración de histamina, tiramina, feniletilamina, putrescina, cadaverina y triptamina en vinos de distintas zonas geográficas y de distintas variedades de uva, así como la influencia del pH, de la fermentación maloláctica y de los periodos de almacenamiento, además, la capacidad aminobiogénica de bacterias aisladas de algunos vinos también fue analizada. Entre otros resultados, pudimos comprobar que era durante la fermentación maloláctica cuando incrementaban los niveles de histamina, tiramina y feniletilamina, pero no de las otras aminas estudiadas, además encontramos BL capaces de producir histamina, tiramina y feniletilamina, pero no putrescina, cadaverina ni triptamina.
Ante la necesidad de desarrollar un método molecular que nos permitiera identificar la presencia del gen de la tirosina descarboxilasa (tdc) en BL de origen enológico, nos planteamos la secuenciación y caracterización molecular del operón tdc del Lactobacillus brevis 9809, encontrándonos que este operón contiene 4 genes completos que codifican para una tirosin-RNAt sintetasa, para la tirosina descarboxilasa, para una probable tirosina permeasa y para un antiporter Na+/H+.
Posteriormente, analizamos mediante métodos moleculares y bioquímicos la capacidad de producción de histamina, tiramina y feniletilamina por parte de más de 150 cepas de BL, además de correlacionar la presencia en vinos de estas aminas con la capacidad de producción de las mismas por las BL presentes en los mismos, observamos que Lactobacillus hilgardii y Pediococcus parvulus eran los responsables de los elevados niveles de histamina en vino y que la mayor parte de los Oenococcus podían producir esta amina, pero no representaban un peligro al producir bajos niveles. En cuanto a la tiramina, todos los Lactobacillus brevis y algún Lactobacillus hilgardii producían tiramina y feniletilamina atribuyéndose a estas especies la responsabilidad de los niveles de tiramina y feniletilamina.
Finalmente, estudiamos la influencia de factores físico-químicos del vino y la fase de crecimiento sobre la producción de histamina, pudimos comprobar como la glucosa y fructosa y los ácidos málico y cítrico junto con la histamina disminuían la expresión del gen de la histidina descarboxilasa y por tanto la producción de histamina, por otro lado la histidina aumentaba dicha expresión, además la presencia de ciertos niveles de etanol y pyridoxal 5-fosfato aumentaba la producción de histamina actuando sobre el enzima histidina descarboxilasa, por último observamos como la fase de crecimiento de las BL influye decisivamente en la expresión del gen de la histidina descarboxilasa.
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