Studies on grape, wine and grape seed proteins and development of methods for their biochemical and functional characterization

Grape and wine proteins represent a subject of study that in recent years has received increasing attention from the international research, mainly due to the fact that important issues on wine can be clarified through the study of the nature and the properties of these macromolecules. Among the reasons of increased concern for winemakers there is the problem of haze development in bottled white wines, known as “protein casse”, due to the presence of residual amounts of insoluble proteins that can become unstable and precipitate during wine storage, causing the appearance of sediments and turbidity. These precipitates are generally the result of denaturation and subsequent aggregation of heat-unstable wine proteins deriving from grapes and belonging to the functional category of plant pathogenesis-related (PR) proteins, namely thaumatin-like proteins (TLPs) and chitinases. It has been observed that these proteins are resistant to acidic pH, proteolysis and fermentation conditions, hence they survive the winemaking process. In this thesis, firstly, the knowledge about chitinases was elaborated since recent scientific papers have described them as proteins potentially more susceptible to precipitation in white wines. In this context, several chitinase isoforms have been purified from Manzoni Bianco grape juice and their electrophoretic behaviour was characterized, deducing important functional and biochemical information on the properties of these enzymes. Subsequently, the research has focused on white wine protein aggregation by means of an innovative instrument, the Izon qNano, for polydisperse nanoparticles detection and quantitation in heat-tested samples. In detail, the role towards aggregates formation upon heating played by TLPs, chitinase, phenolics and polysaccharides, all purified from the same unfined white wine, was investigated via reconstitution experiments to better understand the contribution of each compound on haze formation. Taking into account both the high number and the big size of aggregates formed upon heating, the chitinase revealed to be easily unfolded by heat, thus making it more reactive with other wine macromolecules than TLPs. Among the latter, two isoforms showed to be more prone to form aggregates. It was then demonstrated that TLPs, being present in the starting wine at a much higher concentration than the chitinase, may contribute to the problem of wine haze, even though recent studies revealed their secondary role in haze development. Since the research in enology needs to find a precise method that allows an accurate quantification of the protein amount in wines and grape juices, in this study two colorimetric assays were compared: the Bradford method (based on the Coomassie Brilliant Blue, CBB) and the potassium dodecyl-sulphate (KDS) protein precipitation followed by the bicinchoninic acid assay (KDS/BCA). Some main factors that can potentially affect protein quantification in wine and grape juices were analysed including ethanol, polyphenols and protein glycosylation. Moreover, the response of different proteins towards CBB and BCA reagents was studied. The Bradford assay did not prove to be accurate for wine protein quantification as it was affected by the presence of interfering substances in the matrices (ethanol and polyphenols) and by the aminoacid composition of the proteins tested. On the contrary, by applying the KDS/BCA method, the matrix didn’t show any statistically significant effect on the slope of the protein calibration curve and there were less differences between the protein average responses. Furthermore, the BCA method, directly applied on the samples, was almost insensitive to the sugars present in glycoproteins and mannoproteins purified and resuspended in an aqueous medium. Finally, the storage proteins expressed in the grape seed endosperms were studied systematically, through fractional extractions, electrophoretic analyses and mass spectrometry. These proteins are expressed independently from environmental conditions and their composition has been shown to be species-specific. In particular, the most represented proteins in grape seed endosperms were isolated and identified by mass spectrometry as 11S globulin-like proteins. For the first time, an apparent 7S globulin-like protein was discovered. Finally, it was verified that the doublet of 40 kDa, subunit of the 11S globulin-like protein of 65 kDa, according to its pronounced polymorphism, could be used as “molecular marker”Le proteine dell’uva e del vino rappresentano un argomento di studio che negli ultimi anni ha ricevuto crescente attenzione da parte della ricerca internazionale, soprattutto a causa del fatto che importanti aspetti enologici possono essere chiariti attraverso lo studio della natura e delle proprietà di queste macromolecole. Tra i motivi di maggiore apprensione per i produttori vi è il problema della formazione di torbidità nei vini bianchi imbottigliati, noto come “casse proteica” e dovuto alla presenza di quantità residue di proteine instabili che possono divenire insolubili e precipitare, durante lo stoccaggio dei vini, causando la comparsa di sedimenti e torbidità. Tali precipitati sono generalmente il risultato della denaturazione e successiva aggregazione delle proteine instabili del vino, identificate come derivanti dall’uva e appartenenti alla categoria funzionale delle proteine legate alla patogenesi (PR proteins) della pianta, in particolare proteine taumatina-simili (TLPs) e chitinasi. E’ stato osservato che tali proteine sono resistenti a pH acidi, alla proteolisi e alle condizioni di fermentazione, risultando le più stabili al processo di vinificazione. In questa tesi, in un primo momento, è stata approfondita la conoscenza delle chitinasi che recenti pubblicazioni hanno definito come le proteine potenzialmente più suscettibili alla precipitazione nei vini bianchi. In questo ambito, sono state purificate diverse isoforme di chitinasi dal mosto Manzoni Bianco ed è stato caratterizzato il loro comportamento elettroforetico, deducendo importanti informazioni sulle proprietà funzionali e biochimiche di questi enzimi. Successivamente l’attività di ricerca si è focalizzata sullo studio dell’aggregazione proteica nei vini bianchi per mezzo di uno strumento innovativo, l’Izon qNano, in grado di individuare e quantificare nanoparticelle polidisperse in campioni testati al calore. In particolare, è stato analizzato il ruolo svolto dalle singole proteine (TLPs e chitinasi), dei polifenoli e dei polisaccaridi, tutti purificati dallo stesso vino, nella formazione di aggregati per mezzo di esperimenti di ricostituzione, con l’obiettivo di determinare il contributo di ogni componente alla formazione di torbidità, valutando le dimensioni e la concentrazione degli aggregati sviluppati nei campioni in seguito a riscaldamento. Considerando sia l’elevato numero di aggregati formati che le notevoli dimensioni di questi ultimi, la chitinasi si è dimostrata facilmente denaturabile al calore e, come conseguenza di questo fatto, più reattiva con le altre macromolecole del vino rispetto alle taumatine. Tra queste ultime, due isoforme si sono rivelate particolarmente reattive. Si è dimostrato pertanto che le TLPs, essendo tra l’altro presenti in quantità preponderante nel vino, possono contribuire al problema dell’intorbidamento anche se in misura minore rispetto alle chitinasi, nonostante studi recenti abbiamo rivelato un loro ruolo secondario nella formazione di torbidità. Poiché è fondamentale nel campo della ricerca utilizzare un metodo preciso che stimi accuratamente la concentrazione delle proteine nei vini e nei mosti, è stato effettuato uno studio che ha messo a confronto due metodi colorimetrici per la quantificazione delle proteine nel vino: il metodo basato sulla colorazione di Bradford e il saggio dell’acido bicinconinico preceduto dalla metodica di precipitazione con potassio dodecyl solfato (KDS/BCA). Lo scopo di questo lavoro è stato quello di analizzare in dettaglio alcuni fattori che potenzialmente possono interferire nella quantificazione delle proteine nel vino, come l’etanolo, i polifenoli, la glicosilazione e la natura delle singole proteine. E’ emerso che il metodo Bradford è inaffidabile nei confronti di una quantificazione proteica precisa in vino in quanto risente sia della presenza di sostanze interferenti nelle matrici (etanolo e polifenoli) che della composizione aminoacidica delle proteine utilizzate come standard. Di contro, il metodo BCA preceduto dalla precipitazione con KDS, si è dimostrato più affidabile in quanto le varie matrici non hanno influenzato la quantificazione e la differenza tra le risposte delle proteine è risultata più attenuata. Inoltre, la tecnica del BCA, applicata direttamente sui campioni, in quanto le mannoproteine non precipitano con KDS, è risultata pressoché insensibile nei confronti degli zuccheri presenti nelle mannoproteine purificate e risospese in mezzo acquoso. Infine, sono state studiate sistematicamente, tramite estrazioni frazionate, analisi elettroforetiche e spettrometria di massa le proteine di riserva espresse nell’endosperma dei vinaccioli. Queste proteine vengono espresse indipendentemente dalle condizioni ambientali e sono tipiche delle diverse varietà. In particolare, sono state isolate ed identificate attraverso la spettrometria di massa le globuline 11S maggiormente rappresentate nell’endosperma dei vinaccioli e per la prima volta una probabile globulina 7S. Si è infine verificato che il doppietto di 40 kDa, subunità della proteina 11S di 65 kDa, visto il suo spiccato polimorfismo, può essere utilizzato come “marcatore molecolare” delle diverse varietà di Vitis vinifer

Characterization of chitinase isoforms from grape juice

Grape chitinases are recognized as being mainly responsible for protein haze formation in white wines. Vitis vinifera L. cv. Manzoni Bianco grape juice proteins were fractionated using anion exchange and hydrophobic interaction chromatographies. According to SDSPAGE and zymography, six protein bands with chitinolytic activity were subjected to mass spectrometry (MALDI-TOF/TOF MS), which assigned all the bands to Vitis vinifera class IV chitinases. These grape chitinase isoforms showing different electrophoretic and chromatographic behaviours are likely to be also distinct in their functionality in wine. This could be relevant to understand the involvement of single chitinase components in wine hazing and to develop specific winemaking techniques for their removal from wine

Caratterizzazione di isoforme di chitinasi da succo d\u2019uva

Le chitinasi dell\u2019uva sono considerate tra le principali responsabili della instabilit\ue0 proteica nei vini bianchi. In questo lavoro le proteine del mosto (cv. Manzoni bianco) sono state frazionate utilizzando una cromatografia a scambio anionico seguita da una cromatografia a interazione idrofobica. In base all\u2019analisi elettroforetica (SDS-PAGE) e alla rilevazione dell\u2019attivit\ue0 enzimatica su gel sono state identificate sei diverse bande proteiche con attivit\ue0 chitinolitica. Queste bande sono state sottoposte a spettrometria di massa (MALDI-TOF/TOF MS), che ha identificato tutte le frazioni come Chitinasi di classe IV di Vitis vinifera. Tali isoforme, che mostrano differenti caratteristiche elettroforetiche e cromatografiche, hanno probabilmente un diverso comportamento nel determinare gli intorbidamenti del vino. Quindi il loro studio \ue8 importante per meglio capire il coinvolgimento di ogni singola componente nella formazione di torbidit\ue0 e per sviluppare tecniche enologiche specifiche mirate alla prevenzione della casse proteica dei vini bianchi

Advantages of the KDS/BCA assay over the Bradford assay for protein quantification in wWhite wine and grape juice

The present study compared the performance of two colorimetric protein assays, the Bradford and the potassium dodecyl sulfate/bicinchoninic acid (KDS/BCA) assays, for use in wine and grape juice analysis. The Bradford assay was affected by protein type, whereas the KDS/BCA assay had lower protein-to-protein variation. Bovine serum albumin and lysozyme yielded an absorbance (595 nm) vs. protein concentration slope (dose\u2013response curve) similar to that of wine proteins. In the Bradford assay, the presence of 12% ethanol and 200 mg/L of wine polyphenols decreased the protein absorbance by 28 and 16%, respectively, whereas in the KDS/BCA assay such interference was not significant. Among 64 white wines, the correlation between protein haze potential, determined by a heat test, and protein content was better for the KDS/BCA assay. This study confirmed the superiority of the KDS/BCA assay over the Bradford assay for quantifying protein in white grape juice and wine, and it yielded better predictive value with respect to the risk of white wine protein instability

Foaming properties of potato (Solanum tuberosum) proteins: A study by the gas sparging method

The proteins used in food industry to make foams are mostly of animal origin, thus arising ethical and health concerns. for this reasons, protein of plant origin, such as those of potato, may represent an interesting alternative. In this research, the foaming behavior of potato proteins have been studied, using a commercial sample of potato proteins enriched in patatin (Laffort), intended for use as a fining agent in wine. The potato proteins, solubilized in buffers at different pH (3-7), were subjected to foaming by sparging N2, air and CO2, in the same conditions of pressure and time. The results showed a significant foam expansion (400%) in samples in which N2 and CO2 were sparged. However, some differences were observed in foam stability. In particular, foam was very stable when produced with N2 (40%), less stable with air (30%) and very unstable with CO2 (10%). Image analysis carried out on foams by a stereomicroscope and d32 (Sauter mean diameter) measurements showed significant differences related to the nature of the sparged gas and the pH. In particular, the foam obtained with N2 produced very small bubbles, with low polydispersity. Electrophoretic analyses of the proteins migrating into the foam revealed differences in protein patterns, especially in relation to the sparged gas, indicating that the nature of the dissolved gas affects the behavior of the different protein components. In conclusion, the patatin preparation here tested produces foams with stability and expansion which depend not only on the pH but also on the type of gas sparged in the liquid

Study of Combined Effect of Proteins and Bentonite Fining on the Wine Aroma Loss

The wine aroma loss as a consequence of treatments with bentonite is due to the occurrence of multiple interaction mechanisms. In addition to a direct effect of bentonite, the removal of aroma compounds bound to protein components adsorbed by the clay has been hypothesized, but never demonstrated. We studied the effect of bentonite addition on total wine aroma compounds (extracted from Muscat wine) in a model solution in the absence and presence of total and purified (Thaumatin-like proteins and chitinase) wine proteins . The results showed that in general bentonite alone has a low effect on the loss of terpenes, but removed ethyl esters and fatty acids. The presence of wine proteins in the solution treated with bentonite tended to increase the loss of esters with the longest carbon chains (from ethyl octanoate to ethyl decanoate), and this was significant when the purified proteins were used. The results here reported suggest that hydrophobicity can be one of the driving forces involved in the interaction of aromas with both bentonite and proteins

The proteins of the grape (Vitis vinifera L.) seed endosperm: Fractionation and identification of the major components

Grape (Vitis vinifera L.) seed endosperm proteins were characterized after sequential fractionation, according to a modified Osborne procedure. The salt-soluble fraction (albumins and globulins) comprised the majority (58.4%) of the total extracted protein. The protein fractions analysed by SDS-PAGE showed similar bands, indicating different solubility of the same protein components. SDS-PAGE in non-reducing and reducing conditions revealed the polypeptide composition of the protein bands. The main polypeptides, which were similar in all the grape varieties analysed, were identified by LC-MS/MS as homologous to the 11S globulin-like seed storage proteins of other plant species, while a monomeric 43 kDa protein presented high homology with the 7S globulins of legume seeds. The results provide new insights about the identity, structure and polypeptide composition of the grape seed storage proteins