First insight into endolysin mediated lysis in the wine microbiota

Descripción del proyecto

Nuevas enzimas para combatir las bacterias indeseables en la vinificación

La Unión Europea es el principal productor, consumidor y exportador de vino del mundo y contribuye a la mitad de la producción mundial de esta bebida. La vinificación requiere bacterias, pero algunas son indeseables. Para controlarlas se suelen utilizar sulfitos, aunque es preferible recurrir a métodos alternativos que no empleen sustancias químicas. Las endolisinas son enzimas hidrolíticas empleadas por los virus que infectan a las bacterias (bacteriófagos) para degradar la capa de peptidoglicanos de la bacteria hospedadora. Su potencial se ha demostrado en aplicaciones médicas y veterinarias, pero apenas se ha examinado en el sector de los alimentos y bebidas. En el proyecto ENDOWINE, que cuenta con el apoyo de las acciones Marie Skłodowska-Curie, se pretende caracterizar las endolisinas de bacteriófagos que infectan a las bacterias del vino mediante el análisis «in silico», seleccionar endolisinas candidatas y estudiar su actividad en experimentos de fermentación del vino.

Objetivo

Grapes are the largest fruit crop worldwide and in 2023, 15.9 billion litres of wine was produced in the European Union alone. Many challenges exist in the process of converting grapes into wine, including those associated with the resident wine microbiota. This microbiota consists of several micro-organisms, including Oenococcus oeni and a variety of other lactic acid bacteria. Some members, such as O. oeni, contribute mainly positively to the final product. However, there are certain species that are responsible for organoleptic defects. Therefore, protection measures are in place to target spoiling bacteria, of which the use of sulphites is one of the most common tactics. As reduction of chemical preservatives is a relevant theme in recent years, the search for alternatives is ongoing. Microbial-based alternatives are increasingly attractive and meet the demands for chemical-free food products. One such promising alternative involves the use of endolysins, which are phage-encoded hydrolytic enzymes used by phages to degrade the peptidoglycan layer of the host bacterium at the end of their lytic replication cycle.

ENDOWINE will strive to exploit phage-derived endolysins as antimicrobials in wine. With rapid activity and high specificity among the many reasons, endolysins are particularly promising. Very few experiments using endolysins in food products have been performed, but their potential has already been demonstrated in the medical and veterinary setting. Furthermore, egg white lysozyme is already applied by the wine industry.

Using a multidisciplinary approach, we will first determine the diversity of endolysins in phages infecting wine LAB through in silico analysis. Candidate endolysins will be chosen, cloned, overexpressed and purified in Escherichia coli, after which their enzymatic activities and their antimicrobial spectra will be determined. Finally, their impact on wine will be examined in small and large scale fermentation experiments.