Identificación taxonómica y clonal de bacterias acéticas, y estudio del efecto de la nisina frente a biofilms de bacterias enológicas stars

Abstract

The community of microorganisms involved in wine and vinegar elaborations is very diverse, complex and scarcely characterized, and precisely microorganisms are responsible for the fermentations that take place, i.e. the transformations of grape juice into wine and wine into vinegar. During fermentations microorganisms produce numerous changes in the composition, colour, and organoleptic attributes of the fermented final product that will determine its quality. Lactic acid bacteria (LAB) and acetic acid bacteria (AAB) are the two bacterial families involved in these transformations within the oenological context. The overall objectives of this thesis are: a) to perform clonal and taxonomic characterization of AAB responsible for the acetic fermentation of a variety of industrial vinegars elaborated by the submerged method, and b) to study the possibility of using the bacteriocin nisin in winemaking to achieve wine microbiological control and against the formation of undesired bacterial biofilms. In this thesis methodologies based on DNA analysis have been developed for clonal and taxonomic identification of AAB isolates from samples of wine- cider- and ethanol vinegars in full acetic fermentation. Moreover, the methodology of fluorescence microscopy has been developed for quantification of AAB in vinegar samples and LAB in wine samples. Regarding clonal identification, results showed as well that restriction analysis with SpeI of genomic DNA and pulsed field gel electrophoresis is an appropriate method for studying AAB strains, whereas the rapid method ERIC-PCR is appropriate for monitoring a previously characterized AAB strain and to determine its implantation in acetic fermentations. 43 new AAB clones from vinegar samples were identified, which became part of the bacteria collection of the University of La Rioja of indigenous strains responsible for fermentations. Two G. europaeus strains prevailed and could be excellent candidates for selection of starters for vinegar elaboration. An active extract was obtained from the LAB strain with the QPS status (qualified presumption of safety) by the EFSA Lactococcus lactis LM29. The extract inhibited the growth of 83.5% of the oenological LAB collection and contained 60 % white grape must and 200 ?g/ml of nisin equivalent. Nisin in this concentration prevented biofilm formation by LAB that presented this wine spoilage and microbial contamination feature. The amino acid metabolism of L. lactis in presence of ethanol was also studied and it was unveiled that this LAB utilizes the ADI (arginine deiminase) pathway of arginine degradation as mechanism of response to the presence of ethanol. It generated ornithine and ammonium, but it did not generate any biogenic amine (agmatine, spermidine, phenylethylamine, histamine, or putrescine) either in presence or in absence of ethanol in the culture broth. These results indicate that nisin preparations with the appropriate concentrations could inhibit undesired LAB and prevent biofilm formation, therefore they could constitute useful tools for the microbiological control of wines and could help to decrease the levels of sulphites that are currently used in winemaking.