Proteins in White Wines: Their Interaction With Tannins And Aroma Compounds

The presence of protein in white wines represents a major problem for the wine industry mainly due to the fact that proteins generate haze in the bottled white wines. Protein instability, which results in wine haze formation, is due to some grape PR-Proteins that thanks to their intrinsic resistance survive the vinification process, pass into the wine where cause the appearance of undesirable haze and deposits, leading to rejection by consumers. Protein hazing of white wines is considered to be a three-step process, involving protein denaturation followed by aggregation into colloidal particles able to scatter the visible light and make the wine turbid. Because of the complexity and the variability of the wine matrix, the factors and mechanisms involved in this process are still largely unknown. Commonly, winemakers prevent haze formation by removing the proteins through the use of bentonite. However, this treatment causes loss of wine and, being unspecific, also the removal of some aroma compounds. It has been calculated that the total cost deriving from bentonite treatments corresponds to a worldwide total amount of 1 billion dollars per year. Therefore, basic and applied research is still needed to solve the problem of protein haze formation in white wines. Firstly, the present thesis faces the problem of the impairment of aroma due to bentonite fining. In particular, the study arises from a previous investigation which suggested the existence of an interaction between proteins and aroma compounds. In this context, the interaction of the main wine protein VVTL1 with some fatty acid ethyl esters (FAEE), which are important fermentative aroma compounds has been investigated. Due to the difficulty to determine this interaction at the molecular level, Synchrotron Radiation Circular Dichroism (SRCD) has been used to study the secondary structure of the wine protein as affected by the interactions with FAEE having different chain lengths. Subsequently, the research continued with the investigation of the role played by tannins in the phenomena leading to protein instability of white wines. To this purpose, the effects of several polyphenols (deriving from wine and not) on the stability of VVTL1 has been investigated using SRCD. In parallel, the capability of tannins to react with the proteins over time in bottled wine has been evaluated by Dynamic Light Scattering (DLS) studies in a model wine system. In addition, the thermal stability of two purified proteins, which are representative of the major classes of proteins in white wine (i.e. a class IV Chitinase and the VVTL1), has been investigated by Differential Scanning Calorimetry (DSC) in the presence of the tannins purified from wine at different times after bottling. Finally, the last part of the research focuses on the possibility to produce good quantities of grape proteins in pure form starting from the in vitro culture of berry pulp tissues. These proteins can be used for molecular and functional characterisation. In particular, with this technique it is possible to label the proteins by cultivating the cellular tissues in the presence of N15 which allows the study of their fine structure and interactions by spectroscopic methods

The Secondary Structure of a Major Wine Protein is Modified upon Interaction with Polyphenols

Polyphenols are an important constituent of wines and they are largely studied due to their antioxidant properties and for their effects on wine quality and stability, which is also related to their capacity to bind to proteins. The effects of some selected polyphenols, including procyanidins B1 and B2, tannic acid, quercetin, and rutin, as well as those of a total white wine procyanidin extract on the conformational properties of the major wine protein VVTL1 (Vitis vinifera Thaumatin-Like-1) were investigated by Synchrotron Radiation Circular Dichroism (SRCD). Results showed that VVTL1 interacts with polyphenols as demonstrated by the changes in the secondary (far-UV) and tertiary (near-UV) structures, which were differently affected by different polyphenols. Additionally, polyphenols modified the two melting temperatures (TM) that were found for VVTL1 (32.2 °C and 53.9 °C for the protein alone). The circular dichroism (CD) spectra in the near-UV region revealed an involvement of the aromatic side-chains of the protein in the interaction with phenolics. The data demonstrate the existence of an interaction between polyphenols and VVTL1, which results in modification of its thermal and UV denaturation pattern. This information can be useful in understanding the behavior of wine proteins in presence of polyphenols, thus giving new insights on the phenomena that are involved in wine stability

Identification and relationship of the autochthonous ‘Romé’ and ‘Rome Tinto’ grapevine cultivars

The ‘Romé’ variety is considered an Andalusian (southern region in Spain) autochthonous black grape cultivar. However, several white and black grapevine accessions are known by this name, according to Vitis International Variety Catalogue. The aim of the present work was to clarify the identity of the ‘Romé’ and ‘Rome Tinto’ as black grapevine cultivar. Eight accessions known as ‘Romé’ and two as ‘Rome Tinto’ were analyzed using 30 OIV descriptors and 22 SSR loci. The morphologic and genetic analysis showed that all accessions studied presented the same genotype and phenotype and grouped with South Spanish cultivars. This study helps to clarify the confusion over the identity of ‘Romé’ grapevine cultivar, and provides a solid basis to develop a germplasm collection to protect grapevine diversity and to recover cultivars that may be in danger of extinction

Mining and validating grape (Vitis L.) ESTs to develop EST-SSR markers for genotyping and mapping

Grape expressed sequence tags (ESTs) are a new resource for developing simple sequence repeat (SSR) functional markers for genotyping and genetic mapping. An integrated pipeline including several computational tools for SSR identification and functional annotation was developed to identify 6,447 EST-SSR sequences from a total collection of 215,609 grape ESTs retrieved from NCBI. The 6,447 EST-SSRs were further reduced to 1,701 non-redundant sequences via clustering analysis, and 1,037 of them were successfully designed with primer pairs flanking the SSR motifs. From them, 150 pairs of primers were randomly selected for PCR amplification, polymorphism and heterozygosity analysis in V. vinifera cvs. Riesling and Cabernet Sauvignon, and V. rotundifolia (muscadine grape) cvs. Summit and Noble, and 145 pairs of these primers yielded PCR products. Pairwise comparisons of loci between the parents Riesling and Cabernet Sauvignon showed that 72 were homozygous in both cultivars, while 70 loci were heterozygous in at least one cultivar of the two. Muscadine parents Noble and Summit had 90 homozygous SSR loci in both parents and contained 50 heterozygous loci in at least one of the two. These EST-SSR functional markers are a useful addition for grape genotyping and genome mapping

Proteins in White Wines: Their Interaction With Tannins And Aroma Compounds

The presence of protein in white wines represents a major problem for the wine industry mainly due to the fact that proteins generate haze in the bottled white wines. Protein instability, which results in wine haze formation, is due to some grape PR-Proteins that thanks to their intrinsic resistance survive the vinification process, pass into the wine where cause the appearance of undesirable haze and deposits, leading to rejection by consumers. Protein hazing of white wines is considered to be a three-step process, involving protein denaturation followed by aggregation into colloidal particles able to scatter the visible light and make the wine turbid. Because of the complexity and the variability of the wine matrix, the factors and mechanisms involved in this process are still largely unknown. Commonly, winemakers prevent haze formation by removing the proteins through the use of bentonite. However, this treatment causes loss of wine and, being unspecific, also the removal of some aroma compounds. It has been calculated that the total cost deriving from bentonite treatments corresponds to a worldwide total amount of 1 billion dollars per year. Therefore, basic and applied research is still needed to solve the problem of protein haze formation in white wines. Firstly, the present thesis faces the problem of the impairment of aroma due to bentonite fining. In particular, the study arises from a previous investigation which suggested the existence of an interaction between proteins and aroma compounds. In this context, the interaction of the main wine protein VVTL1 with some fatty acid ethyl esters (FAEE), which are important fermentative aroma compounds has been investigated. Due to the difficulty to determine this interaction at the molecular level, Synchrotron Radiation Circular Dichroism (SRCD) has been used to study the secondary structure of the wine protein as affected by the interactions with FAEE having different chain lengths. Subsequently, the research continued with the investigation of the role played by tannins in the phenomena leading to protein instability of white wines. To this purpose, the effects of several polyphenols (deriving from wine and not) on the stability of VVTL1 has been investigated using SRCD. In parallel, the capability of tannins to react with the proteins over time in bottled wine has been evaluated by Dynamic Light Scattering (DLS) studies in a model wine system. In addition, the thermal stability of two purified proteins, which are representative of the major classes of proteins in white wine (i.e. a class IV Chitinase and the VVTL1), has been investigated by Differential Scanning Calorimetry (DSC) in the presence of the tannins purified from wine at different times after bottling. Finally, the last part of the research focuses on the possibility to produce good quantities of grape proteins in pure form starting from the in vitro culture of berry pulp tissues. These proteins can be used for molecular and functional characterisation. In particular, with this technique it is possible to label the proteins by cultivating the cellular tissues in the presence of N15 which allows the study of their fine structure and interactions by spectroscopic methods.La presenza di proteine nei vini bianchi rappresenta un problema di grande importanza per l’industria del vino, principalmente dovuto alla formazione di torbidità nei vini bianchi in bottiglia. L’instabilità proteica, nonché formazione di torbidità, è associata ad alcune proteine di difesa della pianta che per mezzo della loro intrinseca resistenza e stabilità sopravvivono al processo di vinificazione, passando nel vino dove causano la comparsa dell’indesiderata torbidità e di depositi in bottiglia, la quale non incontra le aspettative del consumatore e viene quindi scartata. La torbidità proteica nei vini bianchi è considerata come un processo a tre stadi, che coinvolge la denaturazione delle proteine seguita dall’aggregazione in particelle colloidali capaci di disperdere la luce visibile e far divenire il vino torbido. Data la complessità e la variabilità della matrice vino, i fattori e meccanismi coinvolti in questo fenomeno sono ancora largamente sconosciuti. Normalmente gli enologi prevengono la formazione di torbidità rimuovendo le proteine attraverso l’uso della bentonite. Tuttavia questo trattamento causa la perdita di vino, ed essendo aspecifico, causa anche la rimozione di alcuni composti aromatici. È stato calcolato che il costo totale derivante dall’uso di bentonite corrispondi a 1 miliardo di dollari l’anno. Pertanto è ancora necessaria della ricerca di base e applicata per risolvere il problema della formazione di torbidità proteica nei vini bianchi. La prima parte di questa tesi tratta il tema dell’impoverimento aromatico causato dal trattamento con bentonite. In particolare questo studio prende ispirazione da un precedente lavoro, il quale suggerisce l’esistenza di un’interazione tra le proteine e i composti aromatici. In questo contesto è stata studiata l’interazione della principale proteina dei vini bianchi, la VVTL1, e alcuni esteri etilici degli acidi grassi, i quali sono importanti aromi fermentativi del vino. Data la difficoltà di determinare a livello molecolare questo tipo d’interazione è stata sfruttata la luce di sincrotrone applicata al dicroismo circolare (SRCD) per studiare l’effetto di alcuni esteri etilici di acidi grassi a media catena sulla struttura secondaria della proteina del vino. In seguito, la ricerca è proseguita con lo studio mirato a comprendere l’esatto ruolo dei tannini nel fenomeno che conduce all’instabilità proteica dei vini bianchi. A questo scopo, sono stati studiati tramite sincrotrone gli effetti di diversi polifenoli (derivati o no dal vino) sulla stabilità della VVTL1. In parallelo la capacità nel tempo dei tannini di reagire con le proteine in bottiglia è stata valutata svolgendo degli studi di diffusione dinamica della luce (DLS) in vino modello. Con l’ausilio della Calorimetria differenziale a scansione (DSC), è stato inoltre studiata, la stabilità termica di due tra le più rappresentative proteine del vino (la VVTL1 e la chitinasi classe IV) in presenza di tannini purificati a tempi diversi di evoluzione da vino imbottigliato. Infine, l’ultima parte della ricerca si focalizza nella possibilità di produrre proteine dell’uva in quantità accettabili in forma pura partendo dalla coltivazione in vitro di tessuti di polpa della bacca. Queste proteine possono essere utilizzate per la caratterizzazione strutturale e funzionale. In particolare, con questa tecnica si rende possibile la marcatura delle proteine facendo crescere i tessuti cellulari d’uva in presenza di N15, il quale consentirebbe, per mezzo di metodi spettroscopici, lo studio in dettaglio della loro struttura e delle loro interazioni

Proteins in white wines: their interaction with tannins and aroma compounds

Proteins in white wines have several potential effects which can be related to their interactions with other wine components. In particular the interaction with the low quantity of tannins present in white wine should be involved in protein haze formation, as suggested previously. Moreover it is possible that wine proteins are involved in binding aroma compounds, as it is known for several proteins in other food systems. The knowledge on these aspects is however very scant, mainly regarding the forces involved in the interactions as affected by the fine molecular structure of the interacting compounds. This PhD thesis research project is aimed to study in depth these aspects, which can help to improve the quality of white wines

Proteins in white wines: their interaction with tannins and aroma compounds - Oral Presentation

This presentation concern only a part of the PhD project. In particular the interactions between white wine tannins and proteins. The effects of tannins purified eight months after bottling from full-filled bottle and half-filled bottle on proteins were studied via reconstitution experiments in model wine. Contrarily to that purified few days after bottling, the tannins obtained after eight months show interaction with proteins already at room temperature, especially the ones from half-filled bottles. These data suggest an evolution of tannins reactivity in bottle, highlighting a fundamental role of oxygen. The study allowed clarifying the mechanisms of protein precipitation in bottled white wines

UTILIZZO DEL CHITOSANO ANIMALE NELLA CHIARIFICA DEI VINI E DEI MOSTI

PROVE DI CHIARIFICA SU SCALA DI LABORATORIO DI MOSTO E DUE VINI BIANCHI E ROSSI. CONFRONTO DEL CHITOSANO ANIMALE CON ALCUNI PRODOTTI REPERIBILI IN COMMERCI

Spectroscopy reveals that ethyl esters interact with proteins in wine

Impairment of wine aroma after vinification is frequently associated to bentonite treatments and this can be the result of protein removal, as recently demonstrated for ethyl esters. To evaluate the existence of an interaction between wine proteins and ethyl esters, the effects induced by these fermentative aroma compounds on the secondary structure and stability of VVTL1, a Thaumatinlike protein purified from wine, was analyzed by Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. The secondary structure of wine VVTL1 was not strongly affected by the presence of selected ethyl esters. In contrast, VVTL1 stability was slightly increased by the addition of ethyl-octanoate, - decanoate and -dodecanoate, but decreased by ethyl-hexanoate. This indicates the existence of an interaction between VVTL1 and at least some aroma compounds produced during fermentation. The data suggest that proteins removal from wine by bentonite can result in indirect removal of at least some aroma compounds associated with th