Haze Formation and Application of Insect-derived Peptidases in Wine Fining



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Wine haze is a challenge for the wine industry as it causes global losses of more than 1 billion US dollars per year. During transportation and storage, non-fined bottled wines undergo temperature variations, which can result in the aggregation of thermolabile proteins such as thaumatin-like protein (TLP) and chitinase (CHI). Protein flocculation in wines generates particles large enough to disperse light and produce haze. To prevent this, bentonite clay is still widely employed as a fining agent, even though its detrimental effects on wine sensory. Since peptidases (or proteolytic enzymes) are able to degrade haze proteins, they could be alternatively applied as fining agents, although their activity is strictly dependent on the wine pH and temperature. Therefore, the present thesis is focused on understanding the chemistry behind the haze formation and the degradation of thermolabile proteins by peptidases to propose analytical and technological solutions for that challenge. By using advanced methods of mass spectrometry (MS), such as top-down proteomics (peptidomics), distinct cleavage spots along the structure of TLP or CHI were identified, and this technique was proposed as an analytical tool for the screening of peptidases. In addition, the cleavage profile performed by peptidases could be evaluated directly on purified recombinant TLP and CHI by heterologously expressing them in the yeast Komagataella phaffii. Thus, pure recombinant rTLP and rCHI were used as haze protein models and applied in combination with MS-based peptidomics for the screening of suitable peptidases for applications in wine fining. Insect-derived peptidases were considered as promising enzymes able to successfully cleave haze proteins under acidic conditions. Insects are organisms well adapted to diverse ecological niches and dietary habits and, consequently, they produce a broad variety of digestive enzymes along their gastrointestinal system. The spotted wing fly Drosophila suzukii is a natural pest of vine grapes and was considered as a potential source of proteolytic enzymes able to cleave wine haze proteins. The larval D. suzukii evolves and feeds on nutrients inside mature berries and, therefore, produces peptidases with activity at the acidic pH of grapes. Acid peptidases from D. suzukii, candidates to cleave wine haze proteins, could be here confirmed by identifying digested peptides from recombinant rTLP and rCHI via MS-based peptidomics. The combination of methods described in the present thesis can be applied for the identification and application of suitable peptidases in winemaking.




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