Protein stabilisation of New Zealand Sauvignon Blanc

Abstract

Identifying alternatives to bentonite fining and/or reducing bentonite use to achieve protein stability has long been a goal of the wine industry. Unstable proteins in white and rosé wines have been demonstrated to be pathogenesis-related proteins which originate from the grape, survive vinification and remain soluble in finished wine. This thesis contributes information about protein in and stabilisation of New Zealand wines. The aims of this study were first to develop methods for protein quantification and characterisaton, and then further to investigate viticultural and oenological influences on wine protein stabilisation. This study was carried out on Sauvignon blanc from the Marlborough region in consecutive vintages, 2007 and 2008. Three methods, Coomassie Brilliant Blue (CBB) assay, lithium dodecyl sulphate polyacrylamide gel electrophoresis (LDS-PAGE) and sodium dodecyl sulphate capillary gel electrophoresis (SDS-CGE), were utilised to investigate effects of vintage, vineyard site, pruning, pH adjustment, timing of bentonite addition, stabilisation test parameters and type of adsorbent. Wine protein recovery by acetone precipitation was more effective than that by ultrafiltration (10 kDa molecular weight cut-off); the latter gave a 21% loss of total protein by SDS-CGE. Proteins with molecular weight between 19 and 33 kDa were the predominant fractions determined by both LDS-PAGE (70%) and SDS-CGE (98%), but the 64 kDa fraction observed by LDS-PAGE (21%) was not detectable in SDS-CGE. However, the SDS-CGE provided better resolution than LDS-PAGE with 8 fractions (9.6, 19, 21 22, 24.6, 26.5, 27.6 and 32 kDa) detected. For a range of wines (n = 18) surveyed results indicated that the CBB assay resulted in wine protein concentrations (average 113.3 mg L⁻¹; CV = 16%) about 42% higher compared to LDS-PAGE (r² = 0.80; CV = 8%) and about 4.2 times lower compared to SDS-CGE (r² = 0.62; CV = 16%). The rapid and simple CBB assay coupled with the finding that a narrow range of protein content (10 to 25 mg L⁻¹) remained in most stabilised wines (n = 102) could be a good method to predict bentonite requirement. Among 5 vineyard sites in Marlborough, one consistently showed the lowest protein concentration in juice and wine, lowest haze formation and lowest bentonite requirement regardless of pruning treatment and vintage, whereas other vineyards varied when pruning treatments and/or vintages were compared. Two juice protein peaks at 22 and 28 kDa in SDS-CGE appeared to be related to two main wine protein fractions at 22 and 26 kDa, respectively. The 28 kDa fraction was reduced and became heterogeneous after fermentation, while the 22 kDa fraction remained unaffected. Bentonite requirement determined by a standard hot/cold test (80°C for 6 hours followed by 4°C overnight) was correlated with total and individual protein concentrations and haze level; there was a good correlation of bentonite requirement with the 26 and 32 kDa fractions (r² = 0.78; p < 0.001), but less good with protein haze, total protein concentration and the predominant 22 kDa fraction (r² < 0.50; p < 0.05). The presence or absence of bentonite during fermentation seemingly did not affect fermentation kinetics to below 0 °Brix regardless of pH, although lower juice pH (2.80 and 3.00) tended to result in sluggish fermentation. The presence of bentonite in the ferment improved the rate of completion of fermentation for the slower fermentation (pH 2.80). Adjusting the pH of juice and/or wine modified the bentonite requirement by two mechanisms: reduced wine pH improves protein adsorption efficiency by bentonite fining, and decreased juice pH results in a lower wine protein content. Bentonite addition during fermentation was the most efficient in terms of protein removal but fining after fermentation resulted in the lowest overall dosage. The molecular weight profile of proteins from wines produced at microvinification and commercial scales were identical, although different fermentation scales slightly affected wine protein content. Protein content and molecular weight profiles in stabilised wines were affected by the original juice pH with more complex patterns from high pH juice. Heat treatment at 90 °C for 1 hour produced the most protein haze and was the most sensitive in haze reduction in response to incremental bentonite fining compared to 80 °C for 2, 6 and 15 hours treatments. There seemed to be a coincident point at approximately 3 nephelometric turbidity units which gave the same predicted bentonite requirement for all heat tests. Na bentonite was the most efficient adsorbent in protein removal, followed by NaCa bentonite, polymeric resin and cation-exchange resin. Protein adsorption isotherms from the Langmuir model indicated that the adsorption capacity for Na bentonite was about 2 and 3 times higher compared to that for NaCa bentonite and polymeric resin, respectively. Na bentonite showed slightly higher binding affinity (Langmuir constant) than NaCa bentonite, but much greater when compared to polymeric resin. The polymeric resin was favorable for use in a regenerable continuous process but suffered from recovery inefficiency. Some tendency for selective removal by bentonite was noted between wine protein fractions based on molecular weight examined by SDS-CGE. However, this was not affected by wine pH (2.80 and 3.85) or bentonite type (Na or NaCa). Based on pH effects and lees volume considerations, NaCa bentonite is suitable for low pH wines and high bentonite requirement, whereas Na bentonite is suitable for high pH wines or low bentonite requirement.