1. The Suitability of L. cv. Pinot noir Mariafeld for Sparkling Wine Production in Niagara, Ontario Esther Onguta, Lisa Dowling, Belinda Kemp, Jim Willwerth, George van der Merwe 1 and Debbie Inglis Cool Climate Oenology and Viticulture Institute (CCOVI), Brock University, 500 Glenridge Avenue, St. Catharines, Ontario, L2S 3A1, Canada, (email: dinglis@brocku.ca).dinglis@brocku.ca 1 Department of Molecular & Cellular Biology (MCB), College of Biological Science (CBS), University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1, Canada Abstract Grapes grown in Niagara, Ontario, a cool climate region, are desirable for sparkling wine production due to high acid levels. Proteins, derived from grapes and yeast, contribute to the foamability in sparkling wine. Foam persistence and stability are two factors that determine the quality of sparkling wine. This research is part of a larger project that aims to investigate the role of protein in finished sparkling wine and its effect on foamability. The objective of this study was to evaluate the suitability of the high acid grape, Pinot noir Mariafeld, for quality sparkling wine production. Grapes were harvested in 2014 and divided into two treatments: a control with no bentonite addition and a treatment that received a 1.0g/L sodium bentonite addition to remove grape proteins. Prior to undergoing primary fermentation with S. cerevisiae EC1118 the protein concentration using the Bradford assay, was 42.0 ± 5.0 µg/mL for control and 21.0 ± 2.0 µg/mL for bentonite treated. Chemical analyses were carried out at each stage of winemaking. Sodium bentonite addition affected the protein concentration but did not impact fermentation kinetics or chemical composition of the juice and base wine. Base wines were divided into eight treatments for secondary fermentation in bottle where half was inoculated with S. cerevisiae EC1118 and half with S. bayanus Brock Isolate. Sodium bentonite was added along with the liqueur de tirage to select treatments to remove yeast proteins. Further research is underway to characterise proteins and determine the suitability of Mariafeld for sparkling wine production. Introduction Factors such as climate, grape variety, clone, protein concentration, yeast species, aging, CO 2, and acidity affect sparkling wine quality (Cilindre et al., 2010). Proteins positively contribute to the foamability in sparkling wine (Pocock et al., 2011). The selection of yeast species is important in sparkling wine production. Base wine parameters such as high ethanol (% v/v), low pH, and high TA may be a stressful fermentative environment for yeast (Borrull et al., 2015). An understanding of how these factors such as grape clone, protein and yeast species, affect sparkling wine production in Niagara, Ontario is key to optimizing quality. Aims: To understand the role that proteins, derived from grapes and yeast, have on the foam persistence, foam stability and overall quality in sparkling wines To determine the role different yeast strains have on final sparkling wine quality To understand how bentonite addition impacts sparkling wine quality Experimental Design Figure 1 Methodology followed for winemaking. Table 1 Breakdown of wine treatments. Figure 2 Primary fermentation kinetics for Mariafeld Pinot noir juice to base wine for both control and bentonite treated juice with S. cerevisiae EC 1118. Note: Bars represent the standard deviation of four replicates. Results Interpretation: The control/no bentonite treatment and bentonite treated juice showed similar fermentation trends and both reached dryness (<5g/L) on Day 12. Conclusion Figure 3 Protein concentrations of juice to pre-secondary fermentation in bottle determined using the Bradford assay. Note: Bars represent the standard deviation of duplicates. Star symbol indicates a significant difference (P  0.05) in protein concentration with No Bentonite Treatment and Bentonite Treatment samples at this stage of wine production. Treatment Juice Bentonite Treatment Yeast Strain Tirage Bentonite Treatment Pressure (atm) pH Titratable acidity (g/L tartaric acid) Ethanol (% v/v) Malic acid (g/L) Reducing sugar (g/L) Protein [ ] (µg/mL) T1-S. cerevisiae-5.2 ± 0.33.02 ± 0.0110.5 ± 0.0412.5 ± 0.26.0 ± 0.50.6 ± 0.017.8 ± 2.0 T2-S. bayanus-0.0 ± 0.03.01 ± 0.0010.5 ± 0.0310.6 ± 0.15.9 ± 0.326.4 ± 0.724.8 ± 1.0 T3-S. cerevisiae+5.3 ± 0.13.03 ± 0.0010.5 ± 0.0312.3 ± 0.16.4 ± 0.10.5 ± 0.018.0 ± 4.0 T4-S. bayanus+0.0 ± 0.03.02 ± 0.0110.4 ± 0.0310.5 ± 0.25.9 ± 0.326.6 ± 0.116.0 ± 3.0 T5+S. cerevisiae-5.1 ± 0.13.02 ± 0.0110.7 ± 0.0412.2 ± 0.06.6 ± 0.10.6 ± 0.021.3 ± 1.0 T6+S. bayanus-0.0 ± 0.03.00 ± 0.0010.6± 0.0310.5 ± 0.15.9 ± 0.326.8 ± 0.520.3 ± 0.0 T7+S. cerevisiae+5.2 ± 0.03.02 ± 0.0010.6 ± 0.0412.3 ± 0.16.6 ± 0.10.6 ± 0.016.0 ± 0.0 T8+S. bayanus+0.0 ± 0.03.00 ± 0.0110.5 ± 0.0310.6 ± 0.16.6 ± 0.126.9 ± 0.014.8 ± 2.0 Table 3 Chemical analysis of wine at lees aging midpoint.* * Data represents the means ± standard deviations of three bottles, collected at the lees aging midpoint, for each treatment. All parameters were evaluated in duplicates. References Borrull, A., Poblet, M., & Rozes, N. (2015). New insights into the capacity of commercial wine yeasts to grow on sparkling wine media. Factor screening for improving wine yeast selection. Food Microbiology, 48, 41-48. Cilindre, C., Liger-Belair, G., Villaume, S., Jeandet, P., & Marchal, R. (2010). Foaming prperties of various Champagne wines depending on several parameters: Grape variety, aging, protein and CO 2 content. Analytica Chimica Acta, 660, 164-170. Pocock, K.F., Salazar, F.N., & Waters, E.J. (2011). The effect of bentonite fining at different stages of white winemaking on protein stability. Australian Journal of Grape and Wine Research, 17, 280-284. Grapes that are high in acid are desirable for sparkling wine production. Mariafeld is a high acid clone of Pinot noir that may be suitable for quality sparkling wine production. S. bayanus Brock Isolate is not a suitable yeast strain for sparkling wine produced from Mariafeld Pinot noir. Factors such as high TA, low pH and % alcohol in base wine may have contributed to the lack of viable cells to undergo secondary fermentation in bottle. The sodium bentonite addition, showed a reduction in protein concentration in the initial juice and base wine. However, protein levels observed at the lees aging midpoint did not differ between the eight treatments. Future protein measurements, including after disgorging,dosage and finished sparkling wine, will be required to determine if lees aging has an impact on protein concentration. Protein analysis involving SDS-PAGE is underway to determine the protein profile of sparkling wine and the protein composition at every stage in production. Finished wine will also be subject to quality assessment where the foam stability, foam persistence and foam height will be observed. This assessment will be used to determine the impact proteins have on sparkling wine quality and provide an understanding on optimizing quality in Ontario. Funding provided by: OMAFRA - U OF G Partnership Interpretation: The four treatments (T2, T4, T6, and T8) inoculated with S. bayanus Brock Isolate did not undergo successful secondary fermentation in bottle. The ethanol (%v/v) did not increase compared to the base wine and pressure was not observed with S.bayanus Brock Isolate treatments. Ethanol (% v/v) pH Titratable Acidity (g/L tartaric acid) Control/No Bentonite Treatment 11.5 ± 0.12.98 ± 0.0412.1 ± 0.1 Bentonite Treatment 11.3 ± 0.223.03 ± 0.0112.1 ± 0.1 Table 2 Base wine parameters. * * Data represents the means ± standard deviations of four replicates. Interpretation: The bentonite treated juice has half the protein concentration of the control/no bentonite treatment prior to primary fermentation to understand the role of grape proteins on sparkling wine quality. Grape Variety – Clone Pinot noir – Mariafeld Control/No Bentonite Treatment Control/ No Bentonite Treatment Base Wine T1 T2 T3 T4 Bentonite Treatment (1.0g/L) Bentonite Base Wine T5 T6 T7 T8 Grape Protein Reduction Tirage Culture Buildup & Yeast Protein Reduction 1 st Fermentation 2 nd Fermentation °Brix : 19.1 ± 0.1 pH: 3.08 ± 0.01 Titratable Acidity (g/L tartaric acid) : 13.1 ± 0.0 °Brix : 18.9 ± 0.0 pH: 3.09 ± 0.01 Titratable Acidity (g/L tartaric acid) : 12.4 ± 0.0 Treatment Juice Bentonite Treatment Yeast Strain Tirage Bentonite Treatment T1-S. cerevisiae- T2-S. bayanus- T3-S. cerevisiae+ T4-S. bayanus+ T5+S. cerevisiae- T6+S. bayanus- T7+S. cerevisiae+ T8+S. bayanus+ *