Land and Food Systems, Faculty of
DSpace
UBCV
Castellanos López, Elia Cristina
Master of Science - MSc
University of British Columbia
The interactions between geographical and biotic factors and the winemaking process influences the composition and sensorial characteristics of wine. The yeast carrying out the fermentation play a key role in shaping the aroma profile of the resulting wine. Commercially available strains of Saccharomyces cerevisiae (S. cerevisiae) help improve reproducibility and predictability of wine by establishing well-controlled fermentations that maintain wine quality. However, indigenous S. cerevisiae strains may be better acclimated to the conditions of specific wine producing regions and may enhance their typical sensory properties and characteristic profiles. The objectives of my research were to assess the oenological characteristics of six indigenous S. cerevisiae strains in lab-scale fermentations and to carry out pilot-scale fermentations in a winery with two strains and evaluate the aromatic composition of the finished wines. Six indigenous S. cerevisiae strains isolated from spontaneous fermentations in Okanagan Crush Pad (OCP) winery were selected for this research. Sterile Pinot Noir and Pinot Gris grape juice was used to perform 250mL lab-scale fermentations carried out at 25°C and 20°C, respectively. Primary metabolites were quantified by high performance liquid chromatography coupled to a refractive index detector (HPLC-RID), and volatile compounds were quantified by solid phase microextraction coupled to gas chromatography and mass spectrometry (SPME-GC/MS). After evaluation of their metabolite production and fermentation performance, the OCP-125 and OCP-088 strains were selected for winery pilot scale fermentations. Fermentations were carried out in triplicate 250 L stainless steel barrels, at OCP winery in Pinot Gris and Pinot Noir juice during the 2022 grape harvest. After fermentation was complete, major metabolites (organic acids, sugars, and ethanol) were quantified using HPLC-RID. Residual sugars were identified in both wines - mainly fructose, ranging between 16 g/L and 20 g/L. The alcohol by volume of the finished wine ranged between 10.82 and 11.58 %. Volatile aroma compounds were quantified by SPME-GC/MS and wines produced with OCP indigenous strains had significantly higher concentrations of ethyl decanoate, a fatty acid ester with a fruity aroma, than commercial wines. Wines produced with OCP-125 and OCP-088 strains had a similar aroma composition, producing an off-dry wine with a desirable aroma bouquet.
Suitability of indigenous Saccharomyces cerevisiae strains isolated from spontaneous fermentations as wine fermentation starter cultures by Elia Cristina Castellanos López BSc., Universidad de Guadalajara, Mexico 2019 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSDOCTORAL STUDIES (Food Science) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2024 © Elia Cristina Castellanos López, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Suitability of indigenous Saccharomyces cerevisiae strains isolated from spontaneous fermentations as wine fermentation starter cultures submitted by Elia Cristina Castellanos López in partial fulfilment of the requirements for the degree of Master of Science in Food Science Examining Committee: Vivien Measday, Professor, Food Science, UBC Supervisor Simone Castellarin, Professor, Plant Science, UBC Supervisory Committee Member Wesley Zandberg, Professor, Chemistry, UBC-Okanagan Supervisory Committee Member Ben Monpetit, Professor, Viticulture and Enology, University of California Davis Additional Examiner iii Abstract The interactions between geographical and biotic factors and the winemaking process, influences the composition and sensorial characteristics of wine. The yeast carrying out the fermentation play a key role in shaping the aroma profile of the resulting wine. Commercially available strains of Saccharomyces cerevisiae (S. cerevisiae) help improve reproducibility and predictability of wine by establishing well-controlled fermentations that maintain wine quality. However, indigenous S. cerevisiae strains may be better acclimated to the conditions of specific wine producing regions and may enhance their typical sensory properties and characteristic profiles. The objectives of my research were to assess the oenological characteristics of six indigenous S. cerevisiae strains in lab-scale fermentations and to carry out pilot-scale fermentations in a winery with two strains and evaluate the aromatic composition of the finished wines. Six indigenous S. cerevisiae strains isolated from spontaneous fermentations in Okanagan Crush Pad (OCP) winery were selected for this research. Sterile Pinot Noir and Pinot Gris grape juice was used to perform 250mL lab-scale fermentations carried out at 25°C and 20°C, respectively. Primary metabolites were quantified by high performance liquid chromatography coupled to a refractive index detector (HPLC-RID), and volatile compounds were quantified by solid phase microextraction coupled to gas chromatography and mass spectrometry (SPME-GC/MS). After evaluation of their metabolite production and fermentation performance, the OCP-125 and OCP-088 strains were selected for winery pilot scale fermentations. Fermentations were carried out in triplicate 250 L stainless steel barrels, at OCP winery in Pinot Gris and Pinot Noir juice during the 2022 grape harvest. After fermentation was complete, major metabolites (organic acids, sugars, and ethanol) were quantified using HPLC-RID. Residual sugars were identified in both wines - mainly fructose, ranging between 16 g/L and 20 g/L. The alcohol by volume of the finished wine ranged between 10.82 and 11.58 %. Volatile aroma compounds were quantified by SPME-GC/MS and wines produced with OCP indigenous strains had significantly higher concentrations of ethyl decanoate, a fatty acid ester with a fruity aroma, than commercial wines. Wines produced with OCP-125 and OCP-088 strains had a similar aroma composition, producing an off-dry wine with a desirable aroma bouquet. iv Lay summary Wineries around the world rely on commercial starter cultures that are genetically similar. Unlike commercial strains, indigenous yeasts are well-suited to local conditions, potentially elevating the sensory profile of the region's wines. Yeast (S. cerevisiae) strains were isolated from Okanagan Crush Pad (OCP) vineyards and winery, these strains belong to the Pacific wine regions and are genetically distinct from commercial strains. We assessed six indigenous OCP S. cerevisiae strains in lab-scale fermentations and selected OCP-088 and OCP-125 strains for pilot-scale winery fermentations. These two strains fermented 250L tanks of Pinot Gris and Pinot Noir juice at OCP winery in the 2022 harvest. Our findings highlight the potential of indigenous yeast strains to produce fruity aromas and contribute to the development of a distinct British Columbian wine starter culture. This research paves the way for the creation of region-specific wine starter cultures, ensuring the reproducibility and quality of our local wines. v Preface Experimental design was made under the guidance of my supervisor, Dr. Vivien Measday. I performed all the experiments described in Chapters 2 and 3. I worked with Yuritzel Moreno to establish a protocol for the HPLC analyses in the UBC Faculty of Land and Food Systems (LFS) Analytical Core Facility. Once the protocol was established, I performed all the HPLC runs and data analysis for the samples described in Chapters 2 and 3. Gas chromatography analysis was performed under the supervision of Dr. Joana Pico Carbajo, at the LFS Analytical Core Facility. I was responsible for all of the sample preparation as well as chromatogram integration and data analysis. Pilot scale fermentations, presented in Chapter 3, were performed under the supervision of winemaker Matt Dumayne, at Okanagan Crush Pad (OCP) winery, Summerland, BC. vi Table of contents Abstract ..............................................................................................................................................iii Lay summary ..................................................................................................................................... iv Preface ................................................................................................................................................ v Table of contents ............................................................................................................................... vi List of tables ..................................................................................................................................... viii List of figures ..................................................................................................................................... xi List of abbreviations ....................................................................................................................... xiv Acknowledgements .......................................................................................................................... xvi Dedication ....................................................................................................................................... xvii 1. Introduction ............................................................................................................................... 1 1.1 Winemaking and terroir ............................................................................................................ 1 1.2 Wine Fermentation ................................................................................................................... 1 1.2.1 Grape Processing ................................................................................................................ 1 1.2.2. Spontaneous Fermentation............................................................................................... 3 1.2.3 Inoculated Fermentation ................................................................................................... 4 1.3 Saccharomyces cerevisiae ......................................................................................................... 4 1.3.1. S. cerevisiae role in industrial fermentations. ................................................................... 4 1.3.2. Indigenous S. cerevisiae strains ......................................................................................... 5 1.4 Chemical composition of wine and wine yeast metabolism ..................................................... 6 1.4.1 Higher Alcohols. ................................................................................................................. 7 1.4.2 Terpenes and Terpenoids. .................................................................................................. 8 1.4.3 Esters .................................................................................................................................. 8 1.4.4 Fatty Acids .......................................................................................................................... 9 1.5 Desirable oenological-aroma traits for Pinot Noir and Pinot Gris wines ................................ 10 1.6 Thesis objective and hypothesis ............................................................................................. 12 2. Evaluation of indigenous S. cerevisiae strains in laboratory scale Pinot Noir and Pinot Gris fermentations .......................................................................................................................... 13 2.2 Materials and methods ........................................................................................................... 14 2.2.1 Yeast strains ..................................................................................................................... 14 2.2.2 Laboratory scale fermentations ....................................................................................... 14 2.2.3 Wine characterization ...................................................................................................... 15 2.2.4 Statistical Analysis ............................................................................................................ 20 vii 2.3 Results ..................................................................................................................................... 20 2.3.1 Fermentation performance .............................................................................................. 20 2.3.2 Primary metabolite production ........................................................................................ 23 2.3.3 Volatile aroma composition in Pinot Gris and Pinot Noir wines ...................................... 29 2.3 Discussion ................................................................................................................................ 41 2.3.1 Fermentation performance .............................................................................................. 41 2.3.2 Volatile metabolite production ........................................................................................ 43 3. Evaluation of two indigenous S. cerevisiae strains in pilot scale fermentations ................ 46 3.1 Synopsis ................................................................................................................................... 46 3.2 Materials and methods ........................................................................................................... 47 3.2.1 Yeast strains ..................................................................................................................... 47 3.2.2 Pilot scale fermentations.................................................................................................. 47 3.2.3 Wine metabolite analysis ................................................................................................. 48 3.2.4 Statistical analysis ............................................................................................................. 48 3.3 Results ..................................................................................................................................... 48 3.3.1 Primary metabolites ......................................................................................................... 48 3.3.2 Secondary metabolites ..................................................................................................... 51 3.4 Discussion ................................................................................................................................ 69 4. Conclusions and future directions ............................................................................................. 72 4.1 Conclusion ............................................................................................................................... 72 4.2 Future directions ..................................................................................................................... 73 References ........................................................................................................................................ 74 Appendix .......................................................................................................................................... 80 viii List of tables Table 1.1 Aroma compounds forming the vinous aroma (Ferreira, 2010) ....................................... 10 Table 2.1 Retention times (RT) of primary metabolites analyzed using HPLC-RID. ...................... 15 Table 2.2 Volatile compounds analyzed with SPME-GC/MS. ........................................................ 16 Table 2.3 Total weight loss in Pinot Gris fermentations. Results show mean (3n) ± SD. Data was analyzed using a one-way ANOVA, which showed no significant difference between strains (p ≥ 0.05) .................................................................................................................................................. 21 Table 2.4 Total weight loss in Pinot Noir fermentations. Results show mean (3n) ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. ................................ 23 Table 2.5 Concentration of primary metabolites identified in Pinot Gris laboratory scale wines, fermented by 7 S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate by HPLC, results show mean ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. ................................................................................................................................. 26 Table 2.6 Concentration of primary metabolites identified in Pinot Noir laboratory scale wines, fermented by 7 S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate, results show mean ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. ......................................................................................................................................... 29 Table 2.7 Concentration (µg/L) of volatile aroma compounds quantified by SPME-GC/MS in Pinot Gris laboratory scale wines, fermented by 7 S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate and the mean concentration [(µg/L) ± SD] of each compound is shown. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that the strains are significantly different at p≤ 0.05 for each compound. ........................... 35 Table 2.8 Concentration (µg/L) of volatile aroma compounds quantified by SPME-GC/MS in Pinot Noir laboratory scale wines, fermented by 7 S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate, and the mean concentration [(µg/L) ± SD] of each compound is shown. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that the strains are significantly different at p≤ 0.05 for each compound. ........................... 38 Table 3.1 Initial parameters of OCP pilot scale fermentation grape must ........................................ 49 Table 3.2 Concentration of primary metabolites identified in OCP-088 and OCP-125 Pinot Gris and Vin Gris/Pinot Noir pilot scale fresh wine (3n per strain). Samples were analysed in triplicate, ix results show mean ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. ......................................................................................................................................... 50 Table 3.3 Concentration of primary metabolites identified in Pinot Gris and Vin Gris pilot scale bottled wines, fermented by the OCP-088 and OCP-125 S. cerevisiae strains (3n per strain). The analysis was carried out 3 weeks after bottling. Samples were analysed in triplicate, results show mean ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. ...... 51 Table 3.4 Concentration (µg/L) of VOC quantified by SPME-GC/MS in Pinot Gris pilot scale wines, fermented by 2 S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate, results show mean (µg/L) ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. ................................................................................................................... 58 Table 3.5 Concentration (µg/L) of VOCs quantified by SPME-GC/MS in Piot Noir pilot scale wines, fermented by 2 S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate, results show mean (µg/L) ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. ................................................................................................................... 62 Table 3.6 OAVs for Pinot Gris pilot-scale wines ............................................................................. 66 Table 3.7 OAVs for Pinot Noir pilot-scale wines............................................................................. 67 Table A.1 Concentration (µg/L) of volatile aroma compounds quantified by SPME-GC/MS in Pinot Gris laboratory scale fermentations, fermented by 7 S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate, results show mean (µg/L) ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. ...................................................................... 80 Table A.2 Concentration (µg/L) of volatile aroma compounds quantified by SPME-GC/MS in Pinot Noir laboratory scale fermentations, fermented by 7 S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate, results show mean (µg/L) ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. ...................................................................... 86 Table A.3 Concentration (µg/L) of volatile aroma compounds quantified by SPME-GC/MS in Pinot Gris pilot-scale wines, fermented by two S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate, results show mean (µg/L) ± SD. Data was analyzed using a one-way ANOVA x followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. ........................................................................................... 93 Table A.4 Concentration (µg/L) of volatile aroma compounds quantified by SPME-GC/MS in Pinot Noir pilot-scale wines, fermented by two S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate, results show mean (µg/L) ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. ........................................................................................... 98 Table A.5 Calculated values for LOD (µg/L) and LOQ (µg/L) of lab-scale and pilot-scale fermentations. .................................................................................................................................. 105 Table A.6 Concentration (µg/L) of volatile aroma compounds quantified by SPME-GC/MS in Pinot Noir and Pinot Gris juice, in vintage 2019 and 2022. Samples were analysed in triplicate, results show mean (µg/L) ± SD. ................................................................................................................. 110 xi List of figures Figure 1.1 Winemaking process. ........................................................................................................ 2 Figure 1.2 Color extraction during maceration process for wine making with red grape varietals. .. 3 Figure 1.3 Chemical structures of fusel alcohols commonly found in wine. ..................................... 7 Figure 1.4 Fusel alcohol formation via Ehrlich pathway. .................................................................. 8 Figure 1.5 Terpene and terpenoid chemical structure. A. Common terpene structure, B. Simple terpenoid structure. .............................................................................................................................. 8 Figure 1.6 Chemical structure of acetate esters (A) and ethyl esters (B) commonly found in wine. . 9 Figure 2.1 Cumulative weight loss of Pinot Gris 20°C fermentations. Fermentation progression was measured by weight loss every 24 hours. Fermentations were carried out in triplicate for each of the strains. Results show the average weight loss for each timepoint, standard deviation (SD) data not shown for presentation purposes *Indicates the commercial M2 control strain. .............................. 21 Figure 2.2 Cumulative weight loss of Pinot Noir 25°C fermentations. Fermentation progression was measured by weight loss. Fermentations were carried out in triplicate for each of the strains Results show the average weight loss for each timepoint, SD data not shown for presentation purposes. *Indicates commercial M2 control. ................................................................................................... 22 Figure 2.3 Concentration of glucose and fructose identified in Pinot Gris laboratory scale wines, fermented by the M2 commercial wine strain and 6 OCP indigenous strains (3n per strain). Samples were analysed by HPLC in triplicate; error bars indicate SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. ...................................................................... 24 Figure 2.4 Concentration of ethanol in Pinot Gris laboratory scale wines, fermented by the M2 commercial wine strain and 6 OCP indigenous strains (3n per strain). Samples were analysed in triplicate by HPLC; error bars indicate SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. ................................................................................................................... 25 Figure 2.5 Concentration of glucose and fructose in Pinot Noir laboratory scale wines, fermented by seven S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate by HPLC; error bars indicate SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. ......................................................................................................................................... 27 xii Figure 2.6 Concentration of ethanol in Pinot Noir laboratory scale wines, fermented by seven S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate by HPLC; error bars indicate SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. ...... 28 Figure 2.7 PCA plots showing A) Distribution of S. cerevisiae strains and their contribution to variation in aroma compound production from Pinot Gris lab fermentations. B) Biplot showing aroma compounds and distribution of strains in Pinot Gris lab-scale fermentation wines. .............. 30 Figure 2.8 PCA plots showing A) Distribution of S. cerevisiae strains and their contribution to variation of aroma compound production in Pinot Noir lab fermentations. B) Biplot showing aroma compounds and distribution of strains in Pinot Noir lab-scale fermentation wines. ......................... 31 Figure 2.9 Total concentration of alcohols, carboxylic acids, esters, phenols and terpenoids in Pinot Gris wines fermented by seven S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate; error bars indicate SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each group. ........................................................................................................................................ 33 Figure 2.10 Total concentration of alcohols, carboxylic acids, esters, phenols and terpenoids in Pinot Noir wines fermented by seven S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate; error bars indicate standard deviation. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test.. Different letters indicate that strains are significantly different at p≤ 0.05 for each group. .............................................................................. 34 Figure 3.1 Fermentation kinetics of S. cerevisiae strains “OCP-125” and “OCP-088” in Pinot Noir (A) and Pinot Gris (B) must. Fermentation was monitored by measuring °Brix ± SD (n = 3). The arrow in (B) indicates re-inoculation of OCP-088 on fermentation day 7. ....................................... 49 Figure 3.2 Log-scale Heatmap of VOCs quantified in Pinot Gris pilot-scale wines. Data shows mean values (3n) of the fresh wines (OCP-125 and OCP-088), the bottled wines (Bottled-OCP-125 and Bottled-OCP-088) and the Pinot Gris juice, 2022 harvest. ........................................................ 53 Figure 3.3 Log-scale Heatmap of VOCs quantified in Pinot Noir pilot-scale wines. Data shows mean values (3n) of the fresh wines (OCP-125 and OCP-088), the bottled wines (Bottled-OCP-125 and Bottled-OCP-088) and the Pinot Noir juice, 2022 harvest. ........................................................ 54 Figure 3.4 Pinot Gris wine composition of VOCs chemical groups in the fresh wines (OCP-125 and OCP-088) and the bottled wines (Bottled-OCP-125 and Bottled-OCP-088) .................................... 55 Figure 3.5 Pinot Noir wine composition of VOCs chemical groups in the fresh wines (OCP-125 and OCP-088) and the bottled wines (Bottled-OCP-125 and Bottled-OCP-088) ............................. 56 xiii Figure 3.6 PCA biplot showing VOCs and distribution of strains in Pinot Gris pilot-scale winery fermentations. Fresh wines: OCP-125, OCP-088; bottled wines: B-OCP-088, B-OCP-125 and commercial wines: PG-KF and PG-SB (A) and individual distribution of strains (B). .................... 57 Figure 3.7 PCA biplot showing VOCs and distribution of strains in Pinot Noir pilot-scale wines. Fresh wines: OCP-125, OCP-088; bottled wines: B-OCP-088, B-OCP-125 and commercial wine: Freeform-VG (A) and individual distribution of strains (B). ............................................................ 61 Figure 3.8 Estimated aroma sensory profile of the volatile compounds. OAV of volatile compounds calculated as the log of the ratio of compound concentration to odor threshold for Pinot Gris (A), Pinot Noir (B) pilot-scale fermentation wines and PG-SB, PG-KG (C) and Freeform-VG (D) commercial wines. ............................................................................................................................. 65 Figure A.1 Distribution of chemical composition of VOCs groups in Pinot Gris laboratory scale fermentations. .................................................................................................................................. 103 Figure A.2 Distribution of chemical composition of VOCs groups in Pinot Noir laboratory scale fermentations. .................................................................................................................................. 104 xiv List of abbreviations ABV Alcohol by volume ADY Active dry yeast ANOVA Analysis of variance ATP Adenosine triphosphate BC British Columbia CAR Carboxen CO2 Carbon dioxide ddH2O Distilled-deionized water DVB Divinylbenzene D3 Three deuterium atoms GC Gas chromatography HPLC High performance liquid chromatography HSD Honest significant difference KF King Family Vineyard KI Kovats index LC Liquid chromatography LFS Land and Food systems LOD Limit of detection LOQ Limit of quantification MS Mass spectrometry NaCl Sodium chloride NAD+ Nicotinamide adenine dinucleotide NIST National Institute of Standards and Technology OAV Odor activity value OCP Okanagan Crush Pad xv OD Optical density ODT Odor detection threshold PCA Principal component analysis PDMS Polydimethylsiloxane PG Pinot Gris PN Pinot Noir RID Refractive index detector Rpm Revolutions per minute RT Retention time S. cerevisiae Saccharomyces cerevisiae SB Switchback vineyard SD Standard Deviation SIM Selected ion monitoring SO2 Sulphur dioxide SPME Solid phase microextraction Std Standard T Target ion UBC University of British Columbia VG Vin Gris YAN Yeast assimilable nitrogen YPD Yeast extract-peptone-dextrose VOC Volatile aroma compound v/v Volume by volume xvi Acknowledgements First and foremost, I want to express my gratitude to my supervisor, Dr. Vivien Measday, who supported me since day one and have always showed faith in my abilities. Not only did she shared her knowledge and experience, but also spent a lot of her time helping me to revise and edit my thesis, she also funded me for the duration of the program, without her continuous support this wouldn’t have been possible. Secondly, I want to thank my committee members, Dr. Wesley Zandberg and Dr. Simone Castellarin, for their advice and input during this time. Special thanks to Dr. Joana Pico-Carbajo, who spent hours with me, guiding me and teaching me, thank you for always giving me encouragement. I would also like to thank Okanagan Crush Pad winery, Christine Coletta and Matt Dumayne, for their constant support and guidance during my time at the winery. To Mitacs Accelerate and the Investment Agriculture Foundation of BC, for providing funding to carry out this research. My lab colleagues, thanks to each of you for being such great team players, especially Yuritzel Moreno, who showed me since the beginning how to navigate UBC and the lab and Jackson Moore, who took a big load of work to make sure my experiments were going well. Finally, I would like to thank my family and friends. My parents, who even from far away were always cheering for me, my husband, for his unconditional support through these years, my friends from home, for being there every step of the way, and my Vancouver friends, who helped me build a home away from home. xvii DEDICATION To my parents, From the warmth of our home in Mexico to the frosty winds of Canada, your encouragement has been my constant companion. Thank you for teaching me how to fly. 1 1. Introduction 1.1 Winemaking and terroir Winemaking, the transformation of grapes into wine by yeast, is a complex process that has been practiced for centuries. From grape harvesting and pressing to fermentation, aging and bottling, winemakers use a combination of traditional and modern techniques to achieve desired flavour and aroma profiles. The unique style and characteristics of wine are shaped by various factors, including the geographical location of the vineyard, local climate, soil composition, grape variety, vineyard management practices and the winemaking process (Zang et al., 2022). These regionally distinct wine characteristics, commonly known as terroir, hold significant importance in the world of winemaking (Bokulich et al., 2016). Terroir encompasses all environmental factors and interactions at a specific geographical site, resulting in the development of regional-specific microbial strains and grape metabolites (Cheng et al., 2020;Knight et al., 2015). The resulting interactions of these factors, significantly impacts the flavour, aroma and overall quality of the wines produced. 1.2 Wine Fermentation 1.2.1 Grape Processing The winemaking process follows different steps that can vary according to the desired style of wine (Figure 1.1). Wine production begins with the grape harvest, where the ideal range of sugar content, measured in °Brix (1g of sucrose in 100g of solution), varies depending on the grape variety. For white grapes, the optimum range is typically between 20-24°Brix, while red grapes are expected to have higher sugar levels, ranging from 22-26°Brix. Once harvested, the grapes undergo sorting and destemming, which are often accomplished using equipment such as a destemmer crusher (Joshi et al., 2022). 2 For white wines, the next step involves pressing the grapes, which can be done using various methods such as a pneumatic press or a basket press (Figure 1.1). The resulting juice, often referred to as ‘must’, is then separated from the grape skins and clarified through processes such as cold settling, filtration, or centrifugation. In contrast, red wines require a different approach - the juice remains in contact with the grape skins to extract color, a process known as maceration. The duration of maceration varies depending on the desired style of the red wine (Figure 1.2) For instance, rosé wines undergo a short maceration period of 2-20 hours to achieve a lighter color compared to red wines. The juice is then separated from the skins, and fermentation proceeds similarly to white wine production. For red wines, maceration can last from 2-4 days before fermentation, where the must is chilled to 10-15°C to prevent microbial activity, Alternatively, maceration can continue until fermentation is complete (10-35 days). In some cases, extended maceration is preferred, which can last from 3 days to over a month after fermentation. Figure 1.1. Winemaking process. 3 Figure 1.2 Color extraction during maceration process for wine making with red grape varietals. After the initial treatment, the juice is transferred to fermentation tanks or barrels, where alcoholic fermentation occurs. Wine fermentation is either accomplished naturally (without yeast inoculation), or by adding a selected commercial wine yeast strain into the must. The fermentation temperature for white wines typically ranges from 10°C to 18°C, whereas for red wines it ranges from 20°C to 30°C. Winemakers typically add sulphur dioxide (SO2) as an antimicrobial and antioxidant to the juice and finished wine. SO2 helps control the growth of wild yeast and bacteria and minimizes oxidation. It is added during juice settling and at the end of fermentation to prevent browning due to oxidation. Proper levels of SO2, and specifically for extended maceration, inert gas blanketing, are recommended to prevent oxidation and microbial spoilage during this process (Mills et al., 2008; Erten et al., 2006; Joshi et al., 2022; Morata et al., 2018) 1.2.2. Spontaneous Fermentation For centuries, winemakers have relied on natural, also called spontaneous, fermentation to complete alcoholic fermentation of grape juice, which can result in a more complex and unique sensory profile when compared to wine made by inoculated fermentation with a single yeast strain. Spontaneous fermentation relies on the microbes naturally present on the grapes and in the winery. Typically, non-Saccharomyces yeasts, which include various species of Candida, Pichia, Hanseniaspora, Debranomyces, Klockera and Torulaspora, initiate the fermentation process. However, as the ethanol levels increase, non-Saccharomyces species gradually decline, allowing more ethanol tolerant yeast, such as Saccharomyces cerevisiae (S. cerevisiae), to take over and 4 complete the fermentation of sugars to ethanol (Erten et al., 2006). In modern-day winemaking, winemakers use natural fermentations to employ indigenous yeasts present in the vineyard and winery environments that introduce specific regional attributes into the wine. The fermentative activity carried out by indigenous yeasts imparts distinctive aromatic notes to the final product, connecting a typical wine to its specific terroir (Lappa et al., 2020). For this reason, modern-day winemakers have a growing interest in low-intervention winemaking practices, such as spontaneous fermentation, and the use of indigenous yeasts, as they seek to create unique attributes that differentiate their products and showcase the distinct character of their wine regions (Zabukovec et al., 2020; Díaz et al., 2013; Capece et al., 2010). While this approach enhances the desired regional characteristics, it also carries the risk of a slow/sluggish or stuck fermentation (Zabukovec et al., 2020). Consequently, the outcome can vary unpredictably from year to year, making it challenging to control and reproduce the fermentation (Díaz et al., 2013). 1.2.3 Inoculated Fermentation Inoculated fermentation is initiated by the addition of a commercial yeast strain, typically S. cerevisiae, to the grape must. S. cerevisiae plays a crucial role in winemaking by converting sugars in grape must, which are equimolar glucose and fructose, into ethanol and carbon dioxide (Mouret et al., 2021). Commercial S. cerevisiae strain inoculation allows for rapid fermentations that complete in a timely manner, providing winemakers more control of the fermentation process and improving wine quality. However, the utilization of commercial strains, restricts the participation of the microbial population on the grapes in the fermentation and reduces the complexity of the resulting wine (Cheng et al., 2020; Barrajón et al., 2011). The availability of a wide range of yeast strains on the market provides winemakers with the means to ensure fermentation control and produce quality wines. Commercial yeasts strains have become a common choice in winemaking, particularly strains of S. cerevisiae, due to their ability to achieve reproducible and controlled fermentations, while minimizing the risk of wine spoilage (Blanco et al., 2013; Tristezza et al., 2014). Nevertheless, relying on commercial strains may lead to wine standardization and mask the distinctive qualities and aromas contributed by the indigenous strains (Mendes et al., 2022). 1.3 Saccharomyces cerevisiae 1.3.1. S. cerevisiae role in industrial fermentations. S. cerevisiae has been extensively studied due to its significance in industrial fermentations, particularly in the production of fermented alcoholic beverages such as wine, beer, and sake. S. cerevisiae fermentative behavior and technological characteristics have been well documented, showing it to be a major contributor to the complexity and sensory quality of these alcoholic 5 beverage products. This yeast species demonstrates superior adaptability to winemaking conditions, exhibiting fast growth and high biomass productivity (Maicas, 2020; Zabukovec et al., 2020). S. cerevisiae rapidly converts sugars to ethanol and, due to its high ethanol tolerance, can outcompete other yeast and bacterial species. The exceptional fermentative capacity of S. cerevisiae and its ability to tolerate various conditions, such as high osmotic stress, low pH, high ethanol levels, and nutrient starvation, enable it to dominate the fermentation process (Molinet et al., 2020). Winemaking strains of S. cerevisiae also exhibit high tolerance to the most common antimicrobial and antioxidant additive used in winemaking, SO2 (Buglass, 2022). S. cerevisiae is not only responsible for converting sugar into alcohol and CO2 but also plays an equally important role in the formation of secondary metabolites, contributing to the conversion of grape aroma precursors into volatile wine aromas. For example, S. cerevisiae possesses the ability to produce and secrete enzymes, such as β-glucosidases, that contribute to the release of aromatic compounds bound to sugar molecules, known as glycosides, releasing the aromatic compound, and volatilizing it, which allows for sensory perception (Maicas et al., 2016; Moreno-Arribas et al., 2009). Other constituents of the grape must, such as fatty acids and amino acids are metabolized by S. cerevisiae into higher alcohols, esters, and glycerol which also contribute to wine aroma and mouthfeel (Moreno-Arribas et al. 2009). 1.3.2. Indigenous S. cerevisiae strains The use of selected indigenous yeasts isolated from grapes in a specific wine region represents a valuable alternative to spontaneous fermentation, allowing for the optimization of the unique attributes of the vineyard. Employing local, indigenous, selected strains of S. cerevisiae as starter cultures is advantageous since these yeasts are acclimated to the specific conditions of the wine producing region. In contrast, the use of commercial yeast strains can lead to the gradual replacement of local microflora resulting in a reduction in microbial biodiversity. (Nikolaou et al., 2006 ; Tristezza et al., 2014). Indigenous yeast strains can be isolated from the grape juice or fermenting must. The selection of starter cultures is made by assessing the potential of a yeast strain to efficiently transform the sugars present in the must into alcohol without negative by-products. Other characteristics such as resistance to SO2, growth at high and low temperatures and ethanol tolerance should also be considered. The evaluation is typically carried out in lab-scale fermentations first in synthetic grape juice media, followed by fermentations in grape juice. Other enological properties, such as fermentation by-products, and production of unwanted sulfuric compounds, are also considered afterwards, depending on the desired wine style. The main goal is to have a strain that not only completes the sugar fermentation but also positively impacts the overall profile of the final product (Lopes et al., 2007; Rainieri et al., 2000) 6 A number of studies have shown oenological properties of interest by indigenous S. cerevisiae strains isolates. For example, (Maicas, 2020) showed that in Grignolino, a neutral Italian red grape variety, an indigenous strain highlighted a rose aroma, positively impacting the aromatic profile of the wine. Furthermore, Nikolaou et al., 2006, carried out fermentations with six isolated S. cerevisiae strains and their results clearly showed a difference between strains in aroma compound production, which leads to difference in sensory perception. Similarly, Suzzi et al., 2012, showed that some autochthonous (indigenous) yeast strains produced more alcohols and others more fruity esters. In comparison, the commercial strain that was used in the same study, did not preserve the typical characteristics of their wine region, even though it produced a good wine. The sensory analysis revealed that the best wines were those fermented by an indigenous starter culture (Suzzi et al., 2012). Finding a balance between utilizing reliable industrial yeasts and exploring the potential of indigenous yeasts is a topic of ongoing interest in the winemaking and wine research community. By incorporating the best of both approaches, winemakers can aim to produce wines that combine quality, consistency, and the uniqueness of their regional origins. 1.4 Chemical composition of wine and wine yeast metabolism Wine yeast metabolism is very important in the winemaking process as it directly influences the chemical composition and the organoleptic characteristics of the final product. The aromas that form the wine’s bouquet can be categorized into two groups: primary aromas, originating from the grape varietal and often referred to as varietal aromas, and secondary aromas, which are those shaped by the fermentation and winemaking process. The chemical composition of grapes greatly influences the formation of aroma compounds by S. cerevisiae, which includes the release of aromatic compounds from non-volatile precursor molecules. The genetic profile of different S. cerevisiae strains also plays a significant role in producing a variety of metabolites that contribute specific aromas to the wine (Mendes et al., 2022) Yeast strains perform different metabolic processes during fermentation. Primary metabolism is essential for growth, cell division, and survival, producing metabolites such as ethanol, glycerol, acetaldehyde, and acetic acid. The production of ethanol by yeast aids the extraction of grape constituent into wine (Joshi et al., 2022). Ethanol also contributes significantly to wine body and mouthfeel and facilitates the release of volatile aromas, including esters, phenols and terpenoids, to the olfactory system, contributing indirectly to wine aroma perception. Typically, ethanol levels produced during wine fermentation are between 6 and 16% alcohol by volume (ABV), with 11-14% ABV being the most common. Another notable by-product is glycerol, the third most abundant 7 compound produced during fermentation, which is present in most wines at levels of 2-8 g/L and contributes to mouthfeel (Buglass, 2022). In addition to primary metabolites, many secondary metabolites are produced during the fermentation process that contribute to the flavour and aroma of the finished wine. Secondary metabolism is nonessential for growth. Different metabolic routes, such as the Ehrlich pathway, the ethyl-ester synthesis pathway, and the mevalonate pathway, produce small molecules, such as fusel alcohols, esters, carbonyls, fatty acids, thiols and terpenoids. These secondary metabolites contribute to the organoleptic properties of the wines, including its aroma flavour and mouthfeel (Hirst et al., 2016; Erten et al., 2006). The production of secondary metabolites involves a variety of factors, including the nature and concentration of the compounds present in the juice, the yeast strain used for the fermentation process and its ability to transform them, and the conditions used during winemaking (Nikolaou et al., 2006; Hirst et al., 2016; Orlic et al., 2007). 1.4.1 Higher Alcohols. In addition to ethanol, wines contain a variety of other alcohols that contribute to the sweetness and aroma of the final product. These alcohols, known as higher alcohols or fusel alcohols (Figure 1.3), are the most abundant volatile compounds formed during fermentation and involve amino acid metabolism. The formation of higher alcohols is dependent on the yeast species involved and occurs through both catabolic and anabolic pathways. The conversion of amino acids through deamination and decarboxylation leads to the production of higher alcohols such as isoamyl alcohol. However, the quantity of higher alcohols present in wine must be carefully regulated. Concentrations up to 400 mg/L can enhance the aroma, but excessive amounts can negatively impact the quality of the wine (Joshi et al., 2022; Larnbrechts et al., 2000.) Figure 1.3 Chemical structures of fusel alcohols commonly found in wine. Fusel alcohols are formed through the Ehrlich pathway (Figure 1.4). The first step involves a transamination reaction which converts the amino acid into an a-keto acid. Subsequently, various pyruvate decarboxylase enzymes catalyze the conversion of the α-keto acid into an aldehyde. In the last step, an alcohol dehydrogenase is responsible for reducing the aldehyde to a fusel alcohol (Hirst et al., 2016). 8 Figure 1.4 Fusel alcohol formation via Ehrlich pathway. 1.4.2 Terpenes and Terpenoids. Terpenes contribute to the floral and fruit aromas found in wines. Many of the primary aromas in wine (varietal aromas) are in the form of higher terpene alcohols, such as citronellol, linalool, and geraniol. Terpenoids, derived from terpenes (Figure 1.5), are responsible for producing aromatic characteristics described as rose, geranium, and floral (Joshi et al., 2022) Figure 1.5 Terpene and terpenoid chemical structure. A. Common terpene structure, B. Simple terpenoid structure. Terpenes and terpenoids are present, in grapes, both free and as glycosides (bound by sugar molecules). During fermentation, yeast glycosidase enzymes play a crucial role in the release and volatilization of terpenoids. The enzymatic release process can be a one or two-step process, depending on the sugar bond attached to the precursor glucoside. In the case of disaccharide glycosides, the initial step involves the liberation of the terminal sugar, followed by the β-glucosidase enzyme, which cleaves the remaining glycosidic bond to release the terpenoid compound. Terpenoid production has traditionally been associated with the hydrolysis of glycosidic linkages. However, S. cerevisiae can produce monoterpenes through the mevalonic acid pathway (Joshi et al., 2022;Maicas et al., 2016;Carrau et al., 2008) 1.4.3 Esters Esters are essential components of wine quality and contribute to the fruity flavor of wine. These compounds are formed through esterification, a chemical reaction that combines acids with alcohols. The synthesis of esters in wine occurs primarily through the action of alcohol acyl-coA 9 transferase enzymes on acyl-coA and alcohols. Ester production is largely attributed to yeast metabolism. During the fermentation process, two types of esters are generated: acetate esters and ethyl esters (Figure 1.6). The production of acetate esters is regulated by the expression of two alcohol acetyl transferases, and their formation depends on the availability of fusel alcohols. Acetate esters contribute to diverse odors such as rose, banana and sweet fruit (phenylethanol acetate, isoamyl acetate and ethyl acetate respectively). On the other hand, ethyl esters, such as ethyl butanoate, ethyl octanoate and ethyl decanoate, are composed of ethanol and fatty acids. The fatty acids are activated by coenzyme A and undergo esterification in conjunction with ATP, ethanol, and other enzymes (Joshi et al., 2022; Katarína et al., 2014; Hirst et al., 2016) Figure 1.6 Chemical structure of acetate esters (A) and ethyl esters (B) commonly found in wine. 1.4.4 Fatty Acids Long-chain fatty acids play a role in wine chemistry because they are produced as by-products during the synthesis of saturated fatty acids by yeasts. In wine, various fatty acids are present in small quantities. Short-chain fatty acids, including acetic, propanoic, and butanoic acids, are formed as by-products of the fermentation process. Among these, acetic acid is the predominant volatile acidic compound in wines, typically accounting for over 90% of volatile acidity. The optimal concentration of acetic acid in wine ranges from 0.2 to 0.7 g/L. Other fatty acids, such as hexanoic acid, octanoic acid and butanoic acid can also have an impact in the aroma perception of wine, these acids can be responsible for imparting off-flavor aromas (sour, cheese, soapy) to the final product (Hirst et al., 2016; Mendes et al., 2022). Carbonyl compounds have an important role in the flavour profile of wine. These aldehydes contribute to a variety of aromas, from tree-fruit to citrusy notes. Acetaldehyde is the predominant compound in wines, accounting for over 90% of the total aldehyde content. In white wines, acetaldehyde serves as indicator of oxidation due to chemical oxidation of ethanol. The conditions 10 that promote higher-alcohol production also favor the formation of small quantities of aldehydes (Larnbrechts et al., 2000). 1.5 Desirable oenological-aroma traits for Pinot Noir and Pinot Gris wines Aroma components, including varietal aromas from grapes and fermentative aromas developed during fermentation, play a crucial role in determining the quality of wine. The complexity of wine can vary depending on numerous factors, such as wine type, grape variety, starter culture and fermentation process, (Ruiz et al., 2019; Costantini et al., 2021). The composition of the base wine aroma, referred to as vinous aroma, consists of a specific mixture of approximately 27 compounds present in most wines (Table 1.1) However, the composition of this base aroma can differ due to factors such as must sugar concentration, yeast strain used for fermentation, and the level of anaerobiosis (Ferreira, 2010) Wine style plays a very important role in the wine industry, influencing consumer preferences. (Zang et al., 2022) Table 1.1 Aroma compounds forming the vinous aroma (Ferreira, 2010) Chemical group Compound Alcohols Ethanol Isobutanol Hexanol Isoamyl alcohol β-Phenylethanol Methionol Acids Acetic acid Butyric acid Hexanoic acid Octanoic acid Decanoic acid Isobutyric acid 2-Methyl butyric acid Isovaleric acid Ethyl esters Ethyl acetate Ethyl butyrate Ethyl hexanoate 11 Chemical group Compound Ethyl esters Ethyl octanoate Ethyl decanoate Ethyl isobutyrate Ethyl 2-methyl butyrate Ethyl isovalerate Isobutyl acetate Isoamyl acetate β-Phenylethyl acetate Others Diacetyl Acetaldehyde The desired aroma characteristics for red wines and wine wines differ significantly due to variations in grape varietal and the winemaking processes. Red wines are typically known for a more complex and intense bouquet, showing a wide range of fruity, floral, and spicy notes. On the other hand, white wines are known for their fresh aromas, typically showing fruity, floral, herbal, and mineral notes. When comparing the aroma profiles of Pinot Noir and Pinot Gris there are notable distinction; Pinot Noir often exhibits red fruit aromas such as cherry, strawberry and cherry, additionally, it can show other floral and earthy notes. In comparison, Pinot Gris is known for fresh and vibrant fruit aromas, including citrus notes, such as lemon and grapefruit, as well as stone fruits, like peach and pear. Often floral and mineral notes can be identified in Pinot Gris wines as well. Pinot Noir wines have been characterized to have a complex mixture of diverse compounds such as monoterpenoids (linalool and geraniol), esters (ethyl butanoate, ethyl hexanoate, ethyl octanoate, hexyl acetate, and ethyl acetate), higher alcohols (3-methylbutanol, and 2-methylpropanol), fatty acids (butanoic acid, hexanoic acid, and octanoic acid) and aldehydes (benzaldehyde). The most abundant component of Pinot Noir wines consists of higher alcohols (Joshi et al., 2022;Longo et al., 2021). For Pinot Gris wines, esters, including isoamyl acetate, phenylethyl ester and ethyl heptanoate, are one of the most important compounds because they are responsible for the fruity aromas of white wines. Nonetheless, other compounds such as, 3-methyl-butanol, 3-hexen-1-ol, hexanoic acid, have been identified at higher concentrations in this white wine (Grigorica et al., 2017). 12 1.6 Thesis objective and hypothesis Okanagan Crush Pad (OCP) winery is a low intervention winery that is focused on producing small lot “terroir expressive wines”. This research is part of a collaboration with OCP, as part of a long-term partnership, with the objective of testing the potential of indigenous S. cerevisiae strains, that have been isolated from spontaneous fermentations at OCP, as starter cultures for wine fermentations. OCP aims to develop their own indigenous yeast strains as winery starter cultures, ensuring consistency, preventing spoilage, and highlighting the unique terroir of Okanagan wines. The primary objective of this research is to assess the suitability of indigenous S. cerevisiae strains, derived from OCP vineyards and winery environment, as wine fermentation starter cultures. To achieve this goal, the study will address two specific objectives: 1) Evaluate six indigenous S. cerevisiae strains on a laboratory scale to determine their proficiency in conducting wine fermentations. 2) Conduct pilot-scale fermentations with two indigenous strains selected from objective 1 within a winery setting to assess the oenological characteristics and volatile aroma composition of the resulting wine. This evaluation will help determine the potential of indigenous S. cerevisiae strains as wine fermentation starter cultures. 13 2. Evaluation of indigenous S. cerevisiae strains in laboratory scale Pinot Noir and Pinot Gris fermentations 2.1 Synopsis Vineyards and wineries in every wine region are home to a variety of indigenous microorganisms, including yeast, which, along with the unique soil type, climate, and terrain, encompasses the concept of terroir. The Okanagan Valley is one of the largest wine producing regions in Canada. This valley stretches from the border with United States to more than 250 km north. The wine region is divided in eleven sub-regions, each with specific soil and climate conditions (https://winebc.com/discover-bc-wine-country/okanagan-valley/ accessed 2023-08-02). Okanagan Crush Pad (OCP) winery is located in Summerland, BC. Along with organic practices in their vineyards, they rely on spontaneous wine fermentations in an effort to highlight the Okanagan Valley terroir of their wines. One of the main disadvantages of spontaneous fermentations, when compared to inoculated fermentations with commercial strains, is the risk of having stuck fermentations, as well as inconsistent wine profiles from one vintage to the next. It is well known that yeast strains play a key role in wine quality, impacting aroma and flavor profiles as well as mouthfeel. Native Saccharomyces cerevisiae (S. cerevisiae) may have better performance during wine fermentation since they are well adapted to the conditions of the wine region. To select an indigenous S. cerevisiae strain as wine fermentation starter culture, a series of steps must be followed. To begin with, the S. cerevisiae strain must be isolated, and a genomic analysis carried out to identify the strain as genetically distinct from commercial strains (Marr et al., 2023). Afterwards, small scale fermentations must be performed in order to evaluate their technological characteristics, such as ethanol production and fermentation vigour, and their qualitative characteristics, such as fermentation by-products (Rainieri et al., 2000). In this study six indigenous S. cerevisiae strains that were isolated from OCP spontaneous fermentations were tested in lab-scale fermentations, to evaluate their potential as wine fermentation starter cultures (Marr et al., 2023). Firstly, we assessed their ability to carry-out the fermentation process. Secondly, we quantified primary and secondary metabolites, to compare their performance to a commercial wine strain. To carry out the fermentations, Pinot Gris and Pinot Noir grapes, were chosen, as they are some of the main grape varietals for white and red wine production in the Okanagan Valley ( https://winebc.com/our-wines/wine-varietals accessed 07/01/2024). 14 2.2 Materials and methods 2.2.1 Yeast strains Six strains of indigenous S. cerevisiae (OCP-041, OCP-080, OCP-088, OCP-090, OCP-095, OCP-125), isolated from spontaneous OCP grape fermentations were evaluated (Marr et al., 2023). A commercial strain “M2” (Lallemand Oenology, ENOFERM M2) was used as a reference control. Stock S. cerevisiae strains were stored at - 80° C in 20% v/v glycerol and grown on YPD agar for 72 h at 25° C prior to liquid culture preparation. 2.2.2 Laboratory scale fermentations Pinot Noir and Pinot Gris juice was provided by OCP winery (Summerland, BC), from the 2019 harvest and stored at -20°C until needed. Pinot Noir juice was whole cluster pressed, receiving the same treatment as Pinot Gris, similar to rose style grape processing, and the fermentation was carried out without skins. Both Pinot Gris and Pinot Noir juice were prefiltered using a 2.0 µm glass fiber prefilter (AP2004700, Merck Millipore Ltd., USA). Free SO2 was measured using the aeration-oxidation method (OIV-MA-AS323-04) and calculated to have an initial SO2 concentration of 10 ppm. Potassium metabisulfite (439008, Fisher Scientific) was added to the juice to maintain 10 ppm of SO2. The yeast assimilable nitrogen (YAN) content was measured by the formol titration method (Gump et al., 2003) and adjusted to 150 mg/L using Fermaid K™ (Bosagrape, Burnaby, BC). The adjusted must was sterile filtered using 0.22 µm vacuum-driven top-up filter (Millipore, USA), under sterile conditions. Each OCP and the M2 strain was inoculated to a test tube containing YPD liquid media (10 g/L of yeast extract, 20 g/L of bacteriological peptone, 20 g/L of dextrose) and incubated overnight at 25°C. The OD600 was measured using a Thermo Scientific™ BioMate™ 160 UV-Visible spectrophotometer. The OD600 readings were used to calculate the volume required to inoculate each strain at an OD600 of 0.2 in 130 mL of YPD liquid media. The strains were incubated in the YPD media at 25°C, 180 rpm for ≈ 2.5 h (until the OD600 was between 0.6 and 0.8). The OD600 was measured again to calculate the volume required to inoculate each fermentation flask at an OD600 of 0.1. The calculated amount was transferred to a 15 mL Falcon tube and centrifuged for 5 min, 2360.71g, 4°C. The excess of liquid was removed, and the yeast pellet was gently added to each flask, containing 250 mL of Pinot Gris or Pinot Noir juice, respectively, in sterile conditions. Fermentation flasks were sealed with rubber bungs and an airlock containing 4mL of ddH2O. The single-strain fermentations were carried out in triplicate (3n) in both Pinot Gris and Pinot Noir must. 15 The initial weight of the fermentation flask with juice and inoculated yeast was registered, and the fermentation was monitored by weight loss every 24 hours. Pinot Gris and Pinot Noir fermentations were conducted at 20°C and 25°C, respectively. A negative control of uninoculated Pinot Gris and Pinot Noir juice was included to confirm the absence of contaminating microbes. 2.2.3 Wine characterization 2.2.3.1 Primary metabolites Once no fermentation activity was recorded, an aliquot of 50 mL was centrifuged for HPLC analysis, supernatant was stored in 15 mL Falcon tubes at 4°C prior to the analysis. Primary wine metabolites (citric acid, tartaric acid, malic acid, acetic acid, glucose, fructose, glycerol, and ethanol) were measured by high performance liquid chromatography (HPLC) with a refractive index detector (RID) using an Agilent 1260 Infinity II LC system (Agilent Technologies, California, USA). A 1.5mL volume of wine was filtered using 0.22 µm nylon filters (VWR, USA) into 2mL HPLC vials. An ion exchange column NUCLEOGEL ION 300 OA (Macherey-Nagel, Düren, Germany) was used to elute the metabolites, with 5mM H2SO4 as the mobile phase. The column and RID temperatures were set to 71°C, and 55°C, respectively. 20 µL of each sample was injected into the system at a 0.5 mL/min flow rate for a total run time of 35 min. A six-point calibration curve using high-purity (<98%) compounds from Sigma-Aldrich (Table 2.1) was prepared to confirm metabolite retention times (RT). A calibration standard was injected at the beginning, middle and end of each run to monitor instrument performance. Table 2.1 Retention times (RT) of primary metabolites analyzed using HPLC-RID. Compound RT1 Calibration range Citric acid 10.41 0-1 g/L Tartaric acid 11.12 0-7.5 g/L Glucose 12.15 0-40 g/L Malic acid 12.80 0-7.5 g/L Fructose 13.18 0-40 g/L Acetic acid 19.54 0-1.25 % v/v Glycerol 17.70 0-2.5 % w/v Ethanol 26.80 0-15 % v/v 1RT range (min) ± 0.5 min 16 2.2.3.2 Secondary metabolites From each fermentation flask, a volume of 50 mL of fermented must was stored at -80°C for solid-phase microextraction followed by gas chromatography-mass spectrometry (SPME-GC/MS) analyses. Free volatiles were analyzed following the SPME-GC/MS protocol and instrumental conditions reported in Pico et al. 2022, using a 7890A GC coupled to a 597C single quadrupole MS detector, equipped with a GC Sampler 80 (SPME autosampler) and Chemstation software (version E.02.02.1431) (Agilent Technologies, California, USA). Samples were prepared by mixing 1.5 g of NaCl and 5 mL of fermented must in a 20 mL headspace vial and each biological replicate was injected in triplicate. D3-Ethyl butyrate and D3-linalool (CDN ISOTOPES, Canada) were added as internal standards to achieve a final concentration of 2 ppm into each vial. Wine samples were preincubated for 5 min at 45°C with an agitation speed of 250 rpm, followed by exposure to a 1 cm and 50/30 µm DVB/CAR/PDMS SPME fiber (Sigma Aldrich, Ontario, Canada) for 30 min at 250 rpm. Volatile compounds were desorbed for 3 min in the injector at 250°C. Volatile compounds were separated by a polar DB Wax column (30 m length x 0.25 mm inner diameter x 0. 25 μm film) (J&W Scientific, California, USA). The GC was operated under programmed temperature conditions: from 35°C (4.36 min) to 65°C (3 min) at 1.5°C/min, up to 138°C (0 min) at 3°C/min, and up to 23°C (5 min) at 60°C/min for a total run time of 58.23 min. The interface, ion source, and quadrupole temperatures were 250°C, 230°C, and 150°C, respectively. Electron impact ionization was performed with an energy of 70 eV and the quadrupole was simultaneously operated in SCAN (including a mass range of 35–500 m/z) and selected ion monitoring (SIM) mode. Table 2.2 Volatile compounds analyzed with SPME-GC/MS. Volatile compound Identification RT1 Experimental KI2 Literature KI T3 Q1, Q24 Alcohols 1-Propanol Std/NIST/KI5 4.42 1050 1051 59 42, 60 2-Methyl-1-butanol Std/NIST/KI 5.02 1105 1158 57 56, 41 2-Methyl-1-propanol Std/NIST/KI 6.81 1111 1110 43 42, 41 3-Methyl-1-butanol Std/NIST/KI 11.35 1064 1190 70 41, 55 17 Volatile compound Identification RT1 Experimental KI2 Literature KI T3 Q1, Q24 Alcohols 2-Hexanol Std/NIST/KI 21.05 1219 1217 45 69, 87 2-Heptanol Std/NIST/KI 19.08 1318 1318 45 55, 83 1-Hexanol Std/NIST/KI 21.06 1344 1344 56 55, 43 3-E-Hexen-1-ol Std/NIST/KI 21.44 1349 NA 41 67, 82 3-Z-Hexen-1-ol Std/NIST/KI 22.76 1366 1366 41 67, 57 2-E-Hexen-1-ol Std/NIST/KI 24.62 1391 NA 57 41, 67 2-Z-Hexen-1-ol Std/NIST/KI 25.32 1403 NA 57 67, 41 1-Octen-3-ol Std/NIST/KI 28.85 1448 1448 57 72, 43 1-Heptanol Std/NIST/KI 29.21 1453 1453 70 55, 56 2,3-Butanediol Std/NIST/KI 31.75 1508 1512 45 43, 57 1-Octanol Std/NIST/KI 35.71 1553 1553 56 55, 41 1-Nonanol Std/NIST/KI 40.85 1661 1661 56 55, 70 2-Ethyl-1-hexanol NIST/KI 30.52 1469 1470 57 41, 55 Hotrienol NIST/KI 37.25 1585 1585 71 82, 43 Aldehydes Hexanal Std/NIST/KI 5.28 1070 1070 56 44, 41 2-(E)-Hexenal Std/NIST/KI 10.78 1173 NA 41 69, 55 Octanal Std/NIST/KI 15.50 1263 1263 43 41, 45 Nonanal Std/NIST/KI 22.79 1367 1367 41 57, 67 Furfural Std/NIST/KI 27.10 1425 1425 96 95, 39 2-E-Nonenal Std/NIST/KI 32.90 1500 NA 70 55, 41 2,4-Nonadienal, (E,E)- Std/NIST/KI 41.17 1669 1670 81 41, 67 2,4-E,E-Hexadienal Std/NIST/KI 22.44 1362 1365 81 96, 67 Carboxylic acids Hexanoic acid Std/NIST/KI 47.47 1831 1831 73 60, 87 Nonanoic acid Std/NIST/KI 52.89 1999 2123 60 73, Decanoic acid Std/NIST/KI 53.49 1998 2227 60 73, Esters Methyl-2-methyl butanoate Std/NIST/KI 3.68 1034 NA 88 57, 41 18 Volatile compound Identification RT1 Experimental KI2 Literature KI T3 Q1, Q24 Esters Ethyl butanoate Std/NIST/KI 4.19 1045 1045 71 88, 43 Ethyl-2-methyl butanoate Std/NIST/KI 4.61 1055 NA 57 102, 85 Ethyl isovalerate Std/NIST/KI 5.02 1064 1064 88 85, 57 Isoamyl acetate Std/NIST/KI 6.43 1057 1101 43 70, 55 Isoamyl propionate Std/NIST/KI 9.96 1172 1176 57 70, 29 Hexyl acetate Std/NIST/KI 14.83 1233 1235 43 56, 61 Ethyl heptanoate Std/NIST/KI 19.08 1306 1310 88 45, 113 Ethyl dodecanoate Std/NIST/KI 47.47 1838 1837/1839 88 101, 43 Ethyl hexanoate NIST/KI 11.16 1201 1197.9/1209 88 99, 43 Ethyl octanoate NIST/KI 26.12 1413 1412 88 101, 127 Ethyl decanoate NIST/KI 38.80 1614 1615 88 101,43 2-Phenethyl acetate Std/NIST/KI 45.54 1779 1771 104 43, 91 Ketones 2-Heptanone Std/NIST/KI 9.29 1150 1151 43 58, 71 6-Methyl-5-hepten-2-one Std/NIST/KI 18.66 1312 1314 43 108, 69 2-Nonanone Std/NIST/KI 22.58 1364 1366 58 43, 81 2-Undecanone Std/NIST/KI 37.01 1578 1578 58 43, 71 Phenols Benzothiazole Std/NIST/KI 49.55 1890 1912 135 108 Benzaldehyde Std/NIST/KI 51.50 1472 1473 106 105,77 Benzyl alcohol Std/NIST/KI 47.72 1839 1839 79 108, 107 Phenylethyl alcohol Std/NIST/KI 48.94 1873 1873 91 92, 122 Guaiacol Std/NIST/KI 46.96 1817 1816 109 124, 81 Eugenol Std/NIST/KI 53.03 1998 2116 164 149, 77 Phenylacetaldehyde Std/NIST/KI 37.66 1590 1592 91 92,120 Terpenoids α-(+)-Pinene Std/NIST/KI 3.84 1038 NA 93 91, 92 19 Volatile compound Identification RT1 Experimental KI2 Literature KI T3 Q1, Q24 Terpenoids β-(+)-Pinene Std/NIST/KI 5.82 1082 NA 93 69, 91 Sabinene Std/NIST/KI 6.43 1105 1105 93 91, 77 –α -(-)-Phellandrene Std/NIST/KI 8.20 1132 NA 93 91, 77 Myrcene Std/NIST/KI 8.58 1139 1139 93 69, 41 α -Terpinene Std/NIST/KI 8.86 1143 NA 121 93, 136 1,4-Cineole Std/NIST/KI 9.10 1147 1164 111 43, 71 S-Limonene Std/NIST/KI 9.77 1157 1183 68 93, 67 β -phellandrene Std/NIST/KI 10.15 1163 1163 93 91, 77 Eucalyptol Std/NIST/KI 10.44 1168 1168 43 81, 108 Γ-Terpinene Std/NIST/KI 12.40 1218 1218 93 91, 136 Ocimene (isomer 2) Std/NIST/KI 13.26 1231 NA 93 91, 79 Cymene Std/NIST/KI 13.73 1237 1237 119 134, 91 (+)-Rose oxide Std/NIST/KI 19.70 1326 1331 139 69, 83 (-)-Rose oxide Std/NIST/KI 20.53 1337 1341 139 69, 83 Limonene oxide Std/NIST/KI 25.59 1406 1408 43 67, 41 Linalool oxide Std/NIST/KI 26.82 1422 1425 59 94, 111 Linalool oxide (isomer2) Std/NIST/KI 29.21 1453 1453 59 94, 111 Linalool Std/NIST/KI 35.15 1542 1542 71 93, 55 Caryophyllene Std/NIST/KI 35.58 1551 1550 133 93, 91 α-Humulene Std/NIST/KI 39.05 1620 1620 93 121, 80 Neral Std/NIST/KI 40.15 1645 1645 69 41, 84 α-terpineol Std/NIST/KI 41.33 1672 NA 59 93, 121 (+) Valencene Std/NIST/KI 41.32 1672 1715 161 204, 105 Y-terpineol Std/NIST/KI 41.48 1676 1683 93 121, 136 Geranial Std/NIST/KI 42.40 1697 1718 69 41, 84 Citronellol Std/NIST/KI 44.72 1757 1757 69 41, 81 β-Damascenone Std/NIST/KI 45.44 1776 1777 177 69, 41 Farnesene Std/NIST/KI 45.44 1776 1775 41 93, 55 Nerol Std/NIST/KI 45.69 1779 1780 69 41, 93 20 Volatile compound Identification RT1 Experimental KI2 Literature KI T3 Q1, Q24 Terpenoids α-Ionone Std/NIST/KI 46.76 1811 NA 121 93, 136 Geraniol Std/NIST/KI 47.57 1834 1834 69 41, 93 β-Ionene Std/NIST/KI 49.86 1899 NA 177 43, 135 Farnesol (isomer1) Std/NIST/KI 53.95 1997 2318 69 41, 81 Farnesol (isomer 2) Std/NIST/KI 54.10 1997 NA 69 41, 91 Terpinolene NIST/KI 12.23 1216 1251 121 93, 136 1Retention time (min) (RT), 2Kovats index (KI) ± 5 KI, 3Target ion (T), 4Qualifier ions (Q1, Q2), 5Identification with Standard (Std), National Institute of Standards and Technology (NIST) MS library and KI. 2.2.4 Statistical Analysis Primary metabolite data was analysed using a one-way ANOVA, followed by a Tukey’s post hoc test (The jamovi project (Version 2.3)). Results were considered significant at p ≤ 0.05. Principal component analysis (PCA) (RStudio 2021) was used to observe the distribution of strains based on their aromatic composition. 2.3 Results 2.3.1 Fermentation performance Each of the six OCP indigenous strains was first analyzed to determine if they could complete a fermentation based on cumulative weight loss over time (Figure 2.1). Weight is used as indicator of fermentative activity due to the CO2 being released as a by-product of the conversion of glucose and fructose to ethanol. The fermentation was considered complete when the 24-hour interval weight loss was ≤ 0.05g for 3 consecutive days. With the aim to imitate the winery conditions, as suggested by OCP winemaker Matt Dumayne, a trial run of Pinot Gris fermentations at 15°C was performed (data not shown). The result of this trial run was that many of the S. cerevisiae strains were unable to consume 50% of the sugars after 60 days of fermentation. Therefore, higher temperatures (20°C for Pinot Gris and 25°C for Pinot Noir) were selected as the fermentation temperatures for this experiment. The fermentation was considered to have started when weight loss was recorded for the first time and when the bubbling was first observed in the 250 mL fermentation flasks. OCP-125 was the first yeast strain to begin the fermentation process with the Pinot Gris juice, which occurred 3 days after inoculation (Figure 2.1). OCP-095, OCP-090, OCP-088, OCP-080 and M2 strains began fermenting 21 4 days after inoculation whereas OCP-041 took 6 days to start fermenting (Figure 2.1). The fermentation activity of OCP-041 stopped after 52-54 days of fermentation, followed by OCP-095 and OCP-080 at 58-61 days. OCP-125, OCP-088, OCP-090 and the commercial control M2 stopped fermenting after 61-65 days. However, there was no statistically significant difference between strains when comparing the cumulative weight loss at the end of fermentation. Figure 2.1. Cumulative weight loss of Pinot Gris 20°C fermentations. Fermentation progression was measured by weight loss every 24 hours. Fermentations were carried out in triplicate for each of the strains. Results show the average weight loss for each timepoint, standard deviation (SD) data not shown for presentation purposes *Indicates the commercial M2 control strain. Table 2.3 Total weight loss in Pinot Gris fermentations. Results show mean (3n) ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test, which showed no significant difference between strains (p ≥ 0.05) Strain Weight loss (g) OCP-095 23.64±0.58 OCP-080 24.56±1.07 OCP-125 25.45±1.41 OCP-041 23.71±1.02 OCP-090 27.00±3.47 OCP-088 26.01±2.64 22 Strain Weight loss (g) M2 26.72±2.93 The Pinot Noir fermentations were carried out at 25°C. All seven strains showed fermentation activity 24 hours after inoculation (Figure 2.2). OCP-095 exhibited a cumulative weight loss of 18.30±0.34 g by the end of fermentation. This value was found to be statistically lower (p≤ 0.05) than all other strains tested. The commercial control, M2, demonstrated the highest cumulative weight loss, totaling 25.92±1.41 g. This result was also statistically higher (p≤ 0.05) than all six indigenous OCP strains examined. On the other hand, among the indigenous strains, OCP-041 (20.67±0.42 g), OCP-080 (20.97±0.40 g), OCP-090 (21.92±0.43 g), and OCP-088 (21.23±0.27 g) displayed significant similarity in their total cumulative weight loss. OCP-125 (22.47±0.17 g) was found to be statistically different from OCP-041, OCP-095, and M2 but exhibited similarity with the other three indigenous strains (Table 2.4). Figure 2.2 Cumulative weight loss of Pinot Noir 25°C fermentations. Fermentation progression was measured by weight loss. Fermentations were carried out in triplicate for each of the strains Results show the average weight loss for each timepoint, SD data not shown for presentation purposes. *Indicates commercial M2 control. 23 Table 2.4 Total weight loss in Pinot Noir fermentations. Results show mean (3n) ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. Strain Weight loss (g) OCP-095 18.30±0.34a OCP-080 20.97±0.40bc OCP-125 22.47±0.17b OCP-041 20.67±0.42c OCP-090 21.92±0.43bc OCP-088 21.23±0.27bc M2 25.93±1.4d 2.3.2 Primary metabolite production 2.3.2a Pinot Gris The initial sugar content of the Pinot Gris must was determined to be 116.54±1.45 g/L of glucose and 150.81±4.35 g/L of fructose. Before inoculation, the Brix content of the must was measured using a digital refractometer (SPER SCIENTIFIC, AZ, USA). While the typical °Brix range for white wine varietals falls between 20 and 24°Brix, this Pinot Gris must registered 25.3 °Brix, indicating an above-average sugar concentration. The wines fermented with OCP-090, OCP-125, OCP-088, OCP-095 and OCP-080 showed no significative difference (p ≤ 0.05) in residual glucose and fructose content in comparison to the commercial control M2 (Figure 2.3). However, the wines fermented with OCP-041 had significantly higher quantities of glucose and fructose after fermentation, with 16.04±2.83 g/L and 57.65±3.01 g/L respectively (Figure 2.3). 24 Figure 2.3 Concentration of glucose and fructose identified in Pinot Gris laboratory scale wines, fermented by the M2 commercial wine strain and 6 OCP indigenous strains (3n per strain). Samples were analysed by HPLC in triplicate; error bars indicate SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. Similarly, ethanol production of OCP-041 (10.88±0.35% v/v) was significantly lower than M2 (12.08±0.05% v/v) and OCP-090 (11.99±0.22% v/v). OCP-080, OCP-088, OCP-095 and OCP-125 showed no significant difference in ethanol production when compared to each other, OCP-090 or M2 (Figure 2.4). 25 Figure 2.4 Concentration of ethanol in Pinot Gris laboratory scale wines, fermented by the M2 commercial wine strain and 6 OCP indigenous strains (3n per strain). Samples were analysed in triplicate by HPLC; error bars indicate SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. The organic acids quantified (citric acid, tartaric acid, malic acid, and acetic acid) in the Pinot Gris fermentations did not show major differences among strains for tartaric and malic acid (Table 2.5). However, OCP-041 fermentations contained a higher content of acetic acid (0.09±0.01 % v/v), and a lower concentration of citric acid (0.57±0.01 g/L) compared to all other strains (Table 2.5). Glycerol concentration, on the other hand, was significantly higher in OCP-041 and OCP-080 Pinot Gris fermentations (Table 2.5). 26 Table 2.5 Concentration of primary metabolites identified in Pinot Gris laboratory scale wines, fermented by 7 S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate by HPLC, results show mean ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. Citric acid (g/L) Tartaric acid (g/L) Malic acid (g/L) Acetic acid (%V/V) Glycerol (%V/V) OCP-041 0.57±0.01a 2.01±0.10a 1.61±0.09a 0.09±0.01a 0.56±0.01b OCP-090 0.68±0.03bc 1.88±0.06a 1.80±0.03a 0.05±0.01b 0.46±0.02a OCP-095 0.68±0.04bc 2.01±0.06a 1.80±0.29a 0.07±0.01b 0.45±0.01a OCP-125 0.66±0.04c 2.04±0.15a 1.72±0.11a 0.05±0.01b 0.48±0.02a OCP-080 0.67±0.02c 1.77±0.10a 1.67±0.05a 0.05±0.01b 0.51±0.03b OCP-088 0.68±0.06bc 1.78±0.16a 1.64±0.15a 0.06±0.00b 0.50±0.01a M2 0.75±0.04b 1.99±0.21a 1.81±0.06a 0.06±0.00b 0.45±0.02a 2.3.2b Pinot Noir The Pinot Noir must had an initial sugar concentration of 22.5 °Brix, falling within the typical range for red wine varietals, which is 22-26 °Brix. The sugar content was 127.29±3.50 g/L of fructose and 104.62±1.83 g/L of glucose. The grapes were processed as Pinot Gris, with no skin contact, however since the whole cluster was pressed the must resulted in a light pink color reminiscent of a rosé wine style. The wines inoculated with the commercial M2 control strain had the lowest levels of residual sugar, with 0.11±1.00 g/L of glucose and 2.55±1.13 g/L of fructose (Figure 2.5). The levels of residual glucose in the OCP-125 and OCP-090 Pinot Noir fermentations was not statistically different from the commercial control M2, with 1.94±0.75 g/L and 2.72±1.47 g/L respectively (Figure 2.5). Except for the M2 strain, these two OCP strains also had the lowest levels of residual fructose with 21.89±4.31 g/L for OCP-125 and 28.58±5.41 g/L for OCP-090 (Figure 2.5). For OCP-080, OCP-088, OCP-041 and OCP-095 strains both residual glucose and fructose levels were statistically similar and higher than the M2 strain (Figure 2.5). Therefore, only the M2 strain was capable of fermenting all the sugars in the Pinot Noir juice. 27 Figure 2.5 Concentration of glucose and fructose in Pinot Noir laboratory scale wines, fermented by seven S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate by HPLC; error bars indicate SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤0.05 for each compound. As expected, wines fermented with the commercial M2 control strain had the highest ethanol percentage with 12.77±0.03% v /v (Figure 2.6). OCP-090 and OCP-125 had the highest ethanol production of the 6 indigenous strains, with final concentrations of 11.22±0.40% v/v and 11.48±0.01% v/v, respectively. 28 Figure 2.6. Concentration of ethanol in Pinot Noir laboratory scale wines, fermented by seven S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate by HPLC; error bars indicate SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. Similar to the Pinot Gris fermentations, OCP-041 Pinot Noir fermentations contained the greatest amount of glycerol (Table 2.6). The concentrations of organic acids in Pinot Noir wines had small, but significant, differences between strains. For example, wines fermented with M2 had slightly higher malic acid concentrations (3.55±0.40 g/L), whereas OCP-041 malic acid levels were significantly lower (2.83±0.06 g/L) (Table 2.6). Acetic acid level showed similar trends to glycerol, where OCP-041 and OCP-095 fermentations had the highest concentrations (0.11±0.00 and 0.10±0.00% v/v, respectively) while M2 and OCP-125 had the lowest concentrations (0.06±0.02 and 0.07±0.00% v/v, respectively). 29 Table 2.6 Concentration of primary metabolites identified in Pinot Noir laboratory scale wines, fermented by 7 S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate, results show mean ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. Citric acid (g/L) Tartaric acid (g/L) Malic acid (g/L) Acetic acid (%v/v) Glycerol (%v/v) OCP-041 1.03±0.01a 2.95±0.32a 2.83±0.06a 0.11±0.00a 0.67±0.00a OCP-090 1.01±0.01ab 2.90±0.43a 2.97±0.04ab 0.07±0.00cd 0.53±0.00bc OCP-095 1.00±0.01b 2.80±0.20a 2.84±0.09a 0.10±0.00ab 0.54±0.00bc OCP-125 1.03±0.01a 2.50±0.04a 3.30±0.13ab 0.07±0.00cd 0.54±0.00bc OCP-080 1.04±0.00a 2.74±0.11a 3.13±0.27ab 0.09±0.00bd 0.59±0.01c OCP-088 1.02±0.00a 2.77±0.02a 3.15±0.23ab 0.09±0.01bd 0.58±0.01c M2 1.02±0.01ab 2.55±0.34a 3.55±0.40b 0.06±0.02c 0.46±0.07b 2.3.3 Volatile aroma composition in Pinot Gris and Pinot Noir wines A Principal Component Analysis (PCA) plot was used to visualize how OCP indigenous strain fermentations differed in volatile aroma composition from the commercial M2 control strain fermentation. For Pinot Gris fermentations, the PC1 axis, which explains 41.5% of variation in the data, shows a pronounced separation between the commercial control M2, and the indigenous OCP strains (Figure 2.7). The separation on the PC2 axis, which explains 23.5% of the variation, also separates the OCP strains, with the OCP-041 strain showing more variation in comparison to the other five OCP strains. 30 Figure 2.7 PCA plots showing A) Distribution of S. cerevisiae strains and their contribution to variation in aroma compound production from Pinot Gris lab fermentations. B) Biplot showing aroma compounds and distribution of strains in Pinot Gris lab-scale fermentation wines. For the PCA plot of volatile aroma compounds detected in the Pinot Noir fermentations, the PC1 axis explains 43.7% of variation in the data, which is similar to the Pinot Gris PCA plot data (Figures 2.7 and 2.8). The separation between the commercial M2 control strain and the indigenous OCP strains in the Pinot Noir PCA plot is also very clear (Figure 2.8). The PC2 axis, which explains 18.5% of the variation, separates the OCP strains. In this case, OCP-080 and OCP-095 strains have more variation in their volatile aromas compared to the other OCP strains. A) B) 31 Figure 2.8. PCA plots showing A) Distribution of S. cerevisiae strains and their contribution to variation of aroma compound production in Pinot Noir lab fermentations. B) Biplot showing aroma compounds and distribution of strains in Pinot Noir lab-scale fermentation wines. We created a list of 87 volatile compounds (Table 2.2) based on Pico et al., 2023 and Ferreira, 2010. The presence or absence of these 87 volatile compounds in the fermentations were assessed using SPME-GC/MS. The compounds that were identified above the limit of quantification in the Pinot Gris lab fermentations are presented in Table 2.7. (For a full table, see Table A.1). Major differences between the M2 and OCP strains can be observed for a few volatile compounds. For instance, the level of 1-propanol (alcohol aroma) is almost 2-fold higher in the M2 A) B) 32 versus the OCP fermentations whereas ethyl-butanoate (pineapple aroma) concentrations are 10-fold higher in OCP versus the M2 fermentations (Table 2.7). The total levels of alcohols, carboxylic acids, esters, phenols and terpenoids were calculated for each Pinot Gris fermentation. For each group, the concentrations of the following compounds were included: • Higher alcohols: 1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-heptanol, 1-hexanol, 3-E-hexen-1-ol, 1-octanol and 2-ethyl-1-hexanol. • Carboxylic acids: hexanoic acid, decanoic acid and nonanoic acid. • Esters: ethyl butanoate, isoamyl acetate, hexyl acetate, ethyl dodecanoate, ethyl hexanoate, ethyl octanoate, ethyl decanoate and 2-phenethyl acetate. • Phenols: benzothiazole, benzyl alcohol, phenylethyl alcohol, eugenol and phenylacetaldehyde. • Terpenoids: γ-terpinene, ocimene, linalool, α-terpineol, geranial, citronelol, geraniol and terpinolene. When looking at the totals for each group of volatile compounds in the Pinot Gris fermentations, we can observe the differences between M2 and OCP-041 in particular, which are statistically different in the concentration of total phenols, terpenoids and esters (Figure 2.9). In terms of alcohols, only OCP-125 and OCP-088 have a statistical difference with OCP-125 fermentations containing the highest quantity of total alcohols (114,860.98±14,745.72 mg/L) and OCP-088 fermentations containing the lowest number of total alcohols (76,944.56±99.24 mg/L). The total levels of carboxylic acids are similar for all of the 7 different strains. For the Pinot Noir fermentations, there was not a significant difference in one specific compound between M2 and all six OCP strains (Table 2.8). There were however some compounds with differences between M2 and individual OCP strains. To better observe the differences, as mentioned above for Pinot Gris, the total concentrations of the five main groups of volatile compounds were calculated (Figure 2.10). Significant differences in esters and alcohols concentrations were observed between M2 and the OCP-095 strain. The concentration of esters was notably higher in M2 fermentations (6681.89±1762.57 µg/L) compared to OCP-095 (3652.56±893.31 µg/L). Similarly, for higher alcohols, M2 fermentations exhibited a significantly higher concentration at 154,270.73±25,541.02 µg/L compared to OCP-095 (107,611.98±11,188.52 µg/L). 33 Figure 2.9. Total concentration of alcohols, carboxylic acids, esters, phenols and terpenoids in Pinot Gris wines fermented by seven S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate; error bars indicate SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each group. 34 Figure 2.10 Total concentration of alcohols, carboxylic acids, esters, phenols and terpenoids in Pinot Noir wines fermented by seven S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate; error bars indicate standard deviation. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each group.35 Table 2.7 Concentration (µg/L) of volatile aroma compounds quantified by SPME-GC/MS in Pinot Gris laboratory scale wines, fermented by 7 S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate and the mean concentration [(µg/L) ± SD] of each compound is shown. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that the strains are significantly different at p≤ 0.05 for each compound. OCP-125 OCP-088 OCP-080 OCP-090 OCP-095 OCP-041 M2 1-Propanol 311.67±33.58bc 270.90±41.09bc 385.81±23.65c 342.45±37.48bc 299.08±23.13bc 250.65±42.65b 646.12±93.51a 2-Methyl-1-butanol 55.47±8.48 60.08±13.23 56.84±2.69 60.87±8.44 46.25±5.14 57.24±8.44 49.00±5.91 3-Methyl-1-butanol* 113783.04±14801.27a 75833.11±9855.03b 107777.73±18099.48ab 109061.63±15397.42ab 83990.40±10160.93ab 93769.33±5826.74ab 90525.40±4485.09ab 2-Heptanol 7.81±0.06 7.88±0.04 7.76±0.02 7.82±0.01 7.78±0.04 10.68±0.41a 7.77±0.04 1-Hexanol 670.68±100.17 731.25±63.07 666.60±63.54 679.31±34.83 859.37±58.37 776.92±149.84 634.16±46.98 3-E-hexen-1-ol 20.5±2.35ab 20.36±1.75ab 20.47±1.11ab 21.33±0.94ab 24.14±0.59b 22.18±2.96ab 15.78±2.77a 1-Octanol 0.36±0.01 0.39±0.03 0.40±0.02 0.36±0.01 0.40±0.01 0.40±0.04 0.35±0.01 2-Ethyl-1-hexanol 11.44±1.16 20.60±6.92a 10.62±7.36 9.12±2.78 11.49±1.46 11.30±2.47 7.32±3.81 Hexanoic acid 51.08±11.98 47.25±6.13 52.28±12.02 40.55±4.91 36.14±1.98 34.01±6.15 44.53±0.69 Nonanoic acid* 152.65±27.40 141.35±8.10 182.42±35.47 137.53±25.52 165.85±36.83 127.11±15.03 110.80±13.96 Decanoic acid* 136.47±27.22 120.85±14.76 125.20±16.89 132.37±11.59 117.34±29.48 123.20±41.69 105.93±21.11 36 OCP-125 OCP-088 OCP-080 OCP-090 OCP-095 OCP-041 M2 Ethyl butanoate 103.61±8.92a 140.6±8.59b 138.27±18.43b 124.45±5.18ab 115.37±2.77ab 102.31±3.30a 11.87±1.08c Isoamyl acetate 1661.23±199.54b 992.06±163.17a 1566.09±260.35ab 1442.78±336.99ab 1188.37±53.79ab 976.85±119.40b 1217.97±165.01ab Hexyl acetate 24.27±5.13a 25.14±2.77a 33.66±4.56ab 42.05±2.96b 37.67±3.11ab 26.70±8.12a 45.78±8.21b Ethyl dodecanoate 36.48±5.50bcd 32.31±3.90abc 31.09±4.95ab 44.80±3.21d 16.97±1.65e 16.95±2.17ae 43.48±4.24cd Ethyl hexanoate* 177.34±43.30ab 214.99±30.68ab 218.95±5.14ab 201.88±34.46a 165.47±7.51a 124.65±26.93b 224.52±32.00a Ethyl octanoate* 1161.17±76.80ab 812.49±158.56 ab 951.50±90.46 ab 1031.44±251.52 ab 951.17±122.76 ab 748.95±320.47a 1523.73±476.06b Ethyl decanoate* 1039.64±29.43a 724.60±99.72ab 786.51±21.68ab 970.83±249.40ab 696.06±139.05ab 572.62±173.66b 1098.80±227.26a 2-Phenethyl acetate 110.83±1.85c 47.68±4.73b 73.47±6.81a 84.69±0.18a 74.78±4.97a 74.05±6.49ab 48.24±9.09b 2-Undecanone 1.95±0.01ab 1.98±0.02ab 1.94a 1.96±0.02a 2.51±0.27b 2.16±0.11ab 1.91±1.01ab Benzothiazole 252.60±64.52 193.21±14.08 216.77±24.28 230.25±17.27 260.19±61.29 160.91±11.29 191.33±51.14 Benzyl alcohol 35.49±1.76 35.03±1.30 35.46±0.78 34.17±1.21 33.48±0.28 34.47±1.60 34.85±0.55 Phenylethyl alcohol 1358.59±234.39a 793.35±116.33bc 876.97±145.66abc 1227.62±129.93a 969.98±4.03ac 916.98±141.92abc 572.33±41.57b Eugenol 4.20±0.02ab 4.16±0.02ab 4.22±0.03a 4.20±0.02a 4.12±0.01b 4.14±0.03ab 4.14±0.95ab 37 OCP-125 OCP-088 OCP-080 OCP-090 OCP-095 OCP-041 M2 Phenylacetaldehyde 10.57±0.24a 4.53±0.09 5.35±0.47 6.18±0.71 6.55±0.53 5.52±0.23 4.62±0.06 γ-Terpinene 5.56±0.17 5.33±0.04 5.42±0.04 5.36±0.04 5.33±0.03 5.38±0.02 5.29±0.06 Ocimene 1.02±0.14 a 0.37±0.14 0.28±0.02 0.44±0.07 0.58±0.03 0.36±0.20 0.00 Linalool 5.99±0.17 5.81±2.01 6.96±1.98 5.32±1.54 6.70±2.12 4.08±2.16 1.49±0.11 α-Terpineol 34.81±0.77ab 36.89±3.39b 32.42±1.48a 32.46±0.82a 42.37±2.06c 65.84±7.46e 52.01±2.80d γ-Terpineol 1.57±0.02ab 1.46±0.05ab 1.43±0.03a 1.67±0.11b 1.45±0.11ab 1.39±0.06ab 1.48±0.63ab Geranial 33.42±0.08 33.29±0.03 33.27±0.05 33.27±0.03 33.32±0.03 NDa 33.24±0.03 Citronellol 7.79±0.01 7.76±0.01 7.75±0.01 7.80±0.01 7.76±0.01 7.78±0.03 7.72±0.03 Geraniol 20.10±1.72 18.38±0.29 19.08±0.36 18.92±1.36 18.66±0.24 18.48±0.24 40.71±38.65 Terpinolene* 5.03±0.08b 4.98±0.03ab 4.99±0.03ab 4.99±0.05ab 4.97±0.02a 4.95±0.01a 4.95±0.04a *Semi-quantified compounds 38 Table 2.8 Concentration (µg/L) of volatile aroma compounds quantified by SPME-GC/MS in Pinot Noir laboratory scale wines, fermented by 7 S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate, and the mean concentration [(µg/L) ± SD] of each compound is shown. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that the strains are significantly different at p≤ 0.05 for each compound. OCP125 OCP088 OCP080 OCP090 OCP095 OCP041 M2 3-Methyl-1-butanol* 154652.7±22062.5a 131353.8±5504.14ab 128904.2±547.53ab 143945.8±13814.62ab 106851.6±10594.44b 128159.8±15618.04ab 153420±25496.10a 1-Hexanol 924.57±37.51abc 839.11±20.60ab 890.14±55.01abc 871.15±128.55ab 1042.17±6.76c 979.13±2.77bc 806.78±26.52a 3-E-hexen-1-ol 22.48±3.71 26.30±1.96 26.09±2.48 23.51±3.72 24.32±4.49 24.24±2.13 23.31±4.15 2-E-hexen-1-ol 7.74±0.34 20.30±6.75a 7.87±1.12 6.54±2.40 6.10±1.14 7.36±2.01 4.30±1.79 1-Octanol 2.58±0.43ab 2.43±0.09ab 3.09±0.22a 2.21±0.67ab 2.53±0.58ab 2.90±0.70a 1.56±0.25b 1-Nonanol 13.62±1.32c 6.88±1.47a 8.48±0.66b 11.31±3.05bc 8.21±0.93ab 10.86±1.73bc 6.74±0.65a 2-Ethyl-1-hexanol* 22.50±1.63 17.10±6.98 14.05±6.14 23.36±8.11 24.12±6.55 20.14±8.31 12.60±5.40 Hexanoic acid 32.1±6.29 31.60±2.62 53.80±5.32 37.10±4.31 39.20±9.81 35.42±10.42 46.28±17.72 Nonanoic acid* 165.84±36.17 206.09±11.25 247.81±40.40 201.46±6.67 192.70±70.36 203.76±42.51 186.52±41.44 Decanoic acid* 150.47±28.22 186.46±23.34 210.05±40.37 155.86±15.61 128.74±17.05 149.74±23.45 141.99±10.89 Ethyl butanoate 105.75±10.97ab 85.28±6.55a 93.63±6.35a 105.47ab 72.78±3.44a 114.69±8.80ab 141.89±7.21b Hexyl acetate 51.98±5.45a 27.78±6.77b 50.21±1.30a 38.40±1.91ab 28.30±7.09b 54.89±0.95a 41.24±4.61ab 39 OCP125 OCP088 OCP080 OCP090 OCP095 OCP041 M2 Isoamyl acetate 1270.78±166.68a 892.10±9.81ab 1133.71±36.51ab 1124.11±262.07a 549.059±129.67b 941.79±118.72ab 1319.00±1570.09a Ethyl dodecanoate 46.58±3.12ab 40.67±7.93bc 37.72±2.51bc 40.88bc 33.24±0.81bc 30.47±1.59c 58.01±8.11a Ethyl hexanoate* 265.55±60.94 216.93±25.67 255.36±41.43 251.98±18.27 225.62±46.04 257.91±22.01 306.11±17.70 Ethyl octanoate* 2370.51±641.81 1669.95±216.33 2295.91±269.44 2276.67±526.33 1630.54±447.70 1786.45±433.91 2519.82±791.88 Ethyl decanoate* 1521.88±569.43 1615.46±298.44 1603.33±192.91 1586.02±219.50 1006.41±388.14 1185.00±178.80 2194.36±699.60 2-Phenethyl acetate 95.9±15.56ab 106.9±5.74a 94.2±10.92ab 74.40±2.32ab 64.73±2.03b 87.8±13.00ab 90.84±5.37ab Benzothiazole 246.18±17.50ac 373.60±80.06ab 387.72±58.54ab 327.60±48.48ab 417.25±34.79b 333.99±53.05ab 172.16±47.17c Benzyl alcohol 44.85±2.85 45.06±1.12 47.74±1.54 44.20±0.99 43.54±5.03 45.97±1.20 48.62±2.55 Phenylethyl alcohol 5003.12±116.39a 4373.12±167.03ab 4664.87±167.74a 4406.49±428.00ab 4184.60±627.29ab 3563.78±249.40b 4350.72±507.29ab Eugenol 5.85±0.02 5.88±0.02 5.82±0.04 5.86±0.06 5.86±0.04 5.82±0.05 5.94±0.06 Phenylacetaldehyde 9.34±0.84 9.72±1.02 10.32±1.49 9.56±2.71 13.76±0.30a 10.58±0.83 9.12±0.2 γ-Terpinene 5.72±0.08 5.57±0.07 5.60±0.13 5.64±0.11 5.60±0.12 5.53±0.02 6.10±0.41 Ocimene 0.64±0.13 0.25±0.14 0.15±0.06 0.23 0.47±0.06 0.27±0.11 0.65±0.22 40 OCP125 OCP088 OCP080 OCP090 OCP095 OCP041 M2 Linalool 5.47±0.80a 1.02±0.01b 4.53±1.12a <LOQb 8.38±0.71c 4.05±1.00a 5.98±0.23a α-Terpineol 32.81±0.58b 18.93±8.20ab 50.44±4.54c 15.30±5.47a 20.74±6.09ab 16.43±1.80a 22.63±5.88ab γ-Terpineol 1.10±0.08 1.03±0.11 1.11±0.08 1.14±0.03 1.27±0.08 1.10±0.02 1.25±0.16 Geranial 134.17±7.50 133.65±0.22 130.86±3.29 135.60±12.12 134.75±3.93 132.07±11.38 127.16±2.75 Citronellol 10.84±0.87 9.94±0.39 10.66±0.50 10.54±0.30 9.88±0.12 11.38±1.97 10.88±0.47 Geraniol 19.45±0.42ab 19.27±0.95ab 19.01±0.50ab 18.24±0.43a 19.19±0.73ab 18.76±0.91ab 20.60±0.84b Terpinolene* 5.47±0.04 5.36±0.03 5.42±0.06 5.36±0.04 5.36±0.02 5.37±0.05 5.43±0.09 *Semi-quantified compounds 41 2.3 Discussion The main objective of this chapter was to evaluate the performance of six OCP indigenous strains isolated from spontaneous OCP fermentations and to determine their ability to carry out alcoholic fermentation. The strains were isolated either from Pinot Gris grapes picked from the vineyard and taken directly back to the lab for small-scale spontaneous fermentation (OCP-041, OCP-090, OCP095), or from a larger-scale Pinot Gris winery spontaneous fermentation (OCP-080, OCP-088, OCP-125) (Marr et al., 2023). When these OCP strains were isolated from spontaneous fermentations, there were other strains present in those fermentations and therefore the performance of each individual strain in a fermentation was unknown. 2.3.1 Fermentation performance The initial sugar concentration in the Pinot Gris juice for the 2019 vintage was above average (25.3°Brix). Neither OCP indigenous strains nor the commercial control M2 were able to complete the fermentation to dryness (<5 g/L of residual sugars) (Figure 2.3). On the other hand, in Pinot Noir must, with initial 22.5°Brix, M2 was able to ferment to dryness - this, however, was not the case for any of the OCP indigenous strains (Figure 2.5). It is well known that S. cerevisiae is glucophilic, which means that it will consume glucose before fructose during the fermentation process. Sugar utilization is dependent on the yeast genetic background as well as external conditions (Guillaume et al., 2007). The ability of the yeast to break down sugars increases with nitrogen availability in the media which supports other studies demonstrating that fructose utilization is stimulated by nitrogen supplementation (Berthels et al., 2004 ; Mendes-Ferreira et al., 2004). Both Pinot Gris and Pinot Noir fermentations were carried out with a minimal nitrogen supplementation (up to 150 ppm) (Lallemand Wine Group, 2015), which, along with fermentation temperature, can have an impact on residual sugars for the indigenous strains. Zabukovec et al., 2020 reported that fermentations with 200 ppm of YAN have poor alcoholic fermentation kinetics due to a low nutritional status. Mendes-Ferreira et al., found that the minimum YAN requirement for a dry wine production was 267 ppm, demonstrating that lower levels might be good to support growth but will lead to stuck fermentations, which also observed during laboratory fermentations. Besides nutrient availability, which is crucial for the yeast to carry its metabolic activities, the fermentation performance of S. cerevisiae is influenced by other factors, including fermentation temperature, which can impact fermentation efficiency and time. S. cerevisiae optimal fermentation temperature has been reported in a range of 28-33°C because its ability to perform at higher temperatures provides an advantage when they have to compete with other species during the 42 fermentation process (Valentine et al., 2019; Lip et al., 2020; Alonso-del-Real et al., 2017). When below the optimal temperature, S. cerevisiae alters its metabolism which impacts production of secondary metabolites in wines (Du et al., 2022). For alcoholic beverages, with the aim of preserving volatile aromas during fermentation, the temperatures selected by wineries are typically suboptimal (~15-25°C) to prevent volatilization of aroma compounds. The low fermentation temperatures not only make the fermentation time longer, but also increases the risk of stuck fermentations (Lip et al., 2020). In this study, the fermentation temperature selected for Pinot Gris juice was 20°C and 25°C was selected for Pinot Noir juice. We observed that the fermentation time of Pinot Gris was double the time it took the yeast in Pinot Noir must to complete fermentation (Figure 2.1 and 2.2). Not only did the duration of fermentation benefit from a higher fermentation temperature, but also the sugar consumption improved for all seven S. cerevisiae strains in the Pinot Noir juice, although this could also be due to the lower initial sugar concentration in the Pinot Noir juice (Figure 2.3 and 2.4). According to Lallemand, the company that manufactures the commercial control strain, Enoferm M2 (Lallemand, USA), M2 has a temperature range of 13-30°C. We can clearly note how with a higher temperature, more sugar was consumed – the percentage of sugar consumed by the M2 strain in the Pinot Gris fermentation was 81% while >98% of sugars was consumed in the Pinot Noir fermentation. Nonetheless, is important to keep in mind that other intrinsic factors not considered in this study, such as the micronutrient composition of the juice and dissolved O2 might have offered Pinot Noir fermentations an advantage, on top of a more optimal fermentation temperature. Interestingly, for both the Pinot Noir and Pinot Gris fermentations, the OCP-041 strain produced more glycerol than the other indigenous OCP strains and the M2 commercial strain (Table 2.5 and 2.6). Our data suggests that the carbon flux of strain OCP-041 has shifted towards glycerol production, which could be advantageous to explore as an alternative for producing wines with a lower ethanol content. Glycerol contributes to the mouthfeel and adds to the complexity of the wine (Costantini et al., 2021; Goold et al., 2017). Furthermore, the OCP-041 strain also produced a significantly higher amount of acetic acid in both Pinot Gris and Pinot Noir fermentations (Table 2.5 and 2.6). The production of glycerol and acetic acid have a direct relationship, particularly when the initial sugar concentration is high. When producing glycerol, the yeast can produce too much NAD+ and disrupt the redox balance in the cell (Chidi et al., 2018). Utilization of the NAD+ by producing acetic acid from acetaldehyde can restore the redox balance (Chidi et al., 2018). These observations lead us to hypothesize that the OCP-041 strain might perform better in must with lower sugar concentrations, producing less acetic acid and more ethanol. 43 These findings emphasize the complexity of wine fermentation and highlight the need to find the optimal conditions for each strain to meet specific production objectives. The six indigenous S. cerevisiae OCP strains were capable of performing fermentation, but the fermentation conditions need to be fine-tuned to allow complete sugar consumption so that the strains could be of use for industrial wine fermentations. 2.3.2 Volatile metabolite production Secondary metabolites are responsible for shaping the aroma bouquet of wine. Out of the 87 volatile compounds that we assessed, a total of 34 compounds were detected above quantification limits in Pinot Gris and Pinot Noir lab-scale fermentations. For all 7 strains, higher alcohols accounted for the most significant proportion of aromas, followed by esters, phenolic compounds, carboxylic acids and terpenoids (Figure 4.1 and 4.2). Alcohol compounds account for the largest proportion of aroma compounds identified in the wines and the aromas attributed to these compounds are “green grass”, “wine-like”, “alcohol” and “solvent”. Nonetheless, the odour detection threshold (ODT), which is the lowest concentration of a compound in which the odor becomes perceptible to the human nose, for these compounds is higher than the concentrations identified in the fermentations for most of the compounds, thereby influencing the aroma complexity of the wines without negatively affecting its profile. The only alcohol compound with a concentration is higher than its ODT is 3-methyl-1-butanol, with an “overripe banana” aroma. The indigenous OCP-125 strain had the highest concentration of 3-methyl-1-butanol (113,783.04±14,801.27 µg/L) in Pinot Gris fermentations, which is statistically similar to the other four OCP-strains and M2 but significantly different from OCP-088 (75,833.11±9,855.03 µg/L, Table 2.7). Similarly, in Pinot Noir fermentations OCP-125 had the highest concentration of 3-methyl-1-butanol (154652.7±22062.5 µg/L), but this value was significantly different only to OCP-095 fermentations (106,851.6±105,94.44 µg/L, Table 2.8). Esters, which are the second most abundant group of volatile compounds, make the greatest contribution to desirable fruity aromas. For both Pinot Noir and Pinot Gris fermentations, OCP-090, OCP-125 and M2 had a higher quantity of esters, however, the differences are only significant between M2 and OCP-041 for Pinot Gris wines, and between M2 and OCP-095 for Pinot Noir wines (Figure 2.9 and 2.10). Isoamyl acetate is one of the most important compounds known for a positive contribution to the aroma profile, particularly for white wines (Romano et al., 2019). The aroma of isoamyl acetate is often describe as banana, and wines fermented with OCP-125, had significantly higher concentrations of this compound when compared to OCP-088 and OCP-041 (Table 2.7). The levels of isoamyl acetate in OCP-090 and M2 Pinot Gris fermentations were 44 statistically similar to OCP-125, but also to the other four strains (Table 2.7). For Pinot Noir fermentations, all strains had a very similar level of isoamyl acetate production, with exception of OCP-095 that had a lower amount (Table 2.8) In both cases, isoamyl acetate concentrations were above ODT, as well as ethyl butanoate and 2-phenetyl acetate. Interestingly, ethyl butanoate, a compound that contributes to fruity “apple” and “pineapple” aromas was significantly higher in all six OCP strains compared to the M2 strain in Pinot Gris wines (Table 2.7). Similarly, 2-phenethyl acetate, known for its “rose, “honey”, “floral” aroma descriptors, was detected in higher concentrations in Pinot Gris fermentations for OCP-125, OCP-090, OCP-095 and OCP-080 strains when compared to the commercial control M2, in Pinot Gris (Table 2.7). Both ethyl decanoate and ethyl octanoate, with aromas described as “pineapple”, “pear”, “fruity” were also abundant in both Pinot Gris and Pinot Noir fermentations and their concentrations were above the ODT. In the Pinot Gris fermentations OCP-125 and the M2 commercial strains produced a significantly higher amount of ethyl decanoate than the OCP-041 strain (Table 2.7). The M2 commercial strain also produced a significantly higher amount of ethyl octanoate than OCP-041 in Pinot Gris fermentations (Table 2.7). In the Pinot Noir fermentations however, no statistical differences among strains were identified for either ethyl decanoate or ethyl octanoate (Table 2.8). Following esters, phenolic compounds were also quantified, with phenylethyl alcohol being the most abundant - this compound is often classified as fusel alcohol. Concentrations of this compound, which has an aroma descriptor of “floral”, were above the ODT for both wines. In Pinot Gris fermentations, both OCP-125 and OCP-090 had statistically significant higher levels of phenylethyl alcohol (1358.59±234.39 µg/L and 1227.62±129.93 µg /L, respectively) compared to the M2 commercial strain (572.33±41.57 µg/L) (Table 2.7). However, in the Pinot Noir fermentations, the concentration of phenylethyl alcohol for M2 (4350.72±507.29 µg/L) was similar to all OCP strains, except for OCP-041 which had a lower amount (3563.78±249.40 µg/L) (Table 2.8). Terpenoid compounds, which are often bound to a sugar molecule in the grapes, are liberated from their glycosides during the fermentation process and volatilized. Release of terpenes by glycosidase activity can occur by the yeast strain carrying out the fermentation or by adding a commercially prepared enzyme. As observed in the results, some of the terpenoid compounds that were quantified in wines, such as linalool and geranial, which impart a “flower” aroma, were not detected in the juice (Table 4.5) (Black et al., 2015). For Pinot Gris and Pinot Noir, geranial, which provides a “floral” aroma, was the only terpenoid compound above ODT. Other grape varietals like 45 Muscat, Riesling and Gewürztraminer are known to have higher abundance of these compounds (Slaghenaufi et al., 2022). Several studies have shown similar results to this work, where indigenous S. cerevisiae yeast strains isolated from spontaneous fermentations produced wines with desirable organoleptic profiles. For example, Sauvignon Blanc wines from Slovenia that were fermented with indigenous S. cerevisiae strains had higher concentrations of 1-propanol, acetaldehyde, linalool, geraniol and 2-phenylethanol than those produced with a commercial strain (Cuš et al., 2017). In another study carried out in the Patagonian wine region, an indigenous strain was selected as a wine starter culture for this Argentinian wine producing region due to the comparable and desirable characteristics of this yeast isolate compared to commercial strains (Lopes et. al., 2007). Similarly, in central-Italy, autochthonous strains had better organoleptic profiles including moderate production of fruity esters, typical of wines of this region, that the commercial strain failed to produce (Suzzi et al., 2012) The proportionally similar aroma compositions between the commercial control and the six OCP indigenous strains demonstrate the OCP isolates ability to produce wines with desirable organoleptic characteristics (Figure 4.1 and 4.2). The differences between these aroma profiles, which will help highlight profiles that better reflect the regional-specific characteristics, can be clearly distinguished when looking at the PCA for both Pinot Gris and Pinot Noir wines where the six OCP-strains are well differentiated from M2 (Figure 2.7 and 2.8), The PCA plots also demonstrate that there are differences in the volatile aroma composition among the OCP strains, which emphasizes the roll of the starter culture in shaping the organoleptic characteristics of wine. The ability of the six OCP-indigenous strains to conduct alcoholic fermentations, comparably to a commercial control, fulfils the first objective of this research. Moving forward, two strains, OCP-088 and OCP-125, were selected to perform pilot-scale fermentations within a winery environment, with the aim to further validate their fermentation performance and assess the organoleptic profile of the resulting wine. This research will add to the continuous exploration of the potential use of indigenous strains in specific wine-regions. 46 3. Evaluation of two indigenous S. cerevisiae strains in pilot scale fermentations 3.1 Synopsis The use of an indigenous yeast strain, isolated from spontaneous fermentations as a starter culture offers winemakers the opportunity to continue their terroir driven approach by combining the advantages of inoculated and spontaneous fermentations, and reducing the risk during fermentation. In this chapter, we explore the use of two indigenous OCP strains: OCP-088 and OCP-125 in pilot scale winery fermentations of Pinot Gris and Pinot Noir Gris wines. Several studies have explored the utility of indigenous S. cerevisiae in winemaking, ranging from isolating strains in spontaneous fermentations to assessing their performance in lab-scale, pilot-scale, and full-scale winery fermentations (Lopes et al., 2007; Guerrini et al., 2021; Vazquez J., et. al., 2023). Indigenous strains have demonstrated suitability for wine production, contributing to yeast biodiversity and preventing standardization in wine (Zabukovec P., et. al., 2020). Existing literature indicates that indigenous strains have proven to be fit S. cerevisiae candidates for use as starter cultures. Lopes et al. (2007) selected an indigenous strain from 32 candidates, evaluated its ability to dominate fermentation in a winery setting, which lead to the development of a regional North Patagonian starter culture. Similarly, Vazquez et. al. (2023), confirmed the use of a selected indigenous S. cerevisiae strain, isolated from a local vineyard, as a wine starter culture in Emina Rueda cellar, Spain. Other research suggests that wines produced with selected indigenous strains exhibit distinct sensory characteristics with consumer preference for indigenously fermented wines over wines produced with commercial strains (Guerrini et al., 2021 Vazquez et al., 2023). Additionally, studies, including Aponte et al., 2020 emphasize the importance of fermentation vigor, emphasizing that a starter culture adapted to a specific winery environment can enhance the intensity and efficiency of the fermentation process. These findings underscore the practical advantages and consumer appeal of incorporating indigenous S. cerevisiae strains in winemaking practices. 47 3.2 Materials and methods 3.2.1 Yeast strains To grow sufficient quantities of yeast for winery fermentations, two Okanagan Valley indigenous Saccharomyces cerevisiae strains, OCP-088 and OCP-125, were sent to Scotts Labs, USA for propagation. The strains were grown in dextrose liquid media and achieved a cell count of 4.67x1012 cells/L for OCP-088 and 2.99x1012 cells/L for OCP-125 (Scott Labs, USA). 3.2.2 Pilot scale fermentations Pilot scale fermentations were performed at OCP Winery, in Summerland, BC during the 2022 harvest under the guidance of OCP winemaker Matt Dumayne. Pinot Gris and Pinot Noir grapes were individually processed and pressed. Before inoculation of yeast, 10 ppm of SO2 was added and the Pinot Gris must was settled for a week. For the Pinot Noir juice the must was settled for one day and the grapes were processed following white winemaking practices, making it a Vin Gris wine style. Stainless steel barrels were used for fermentation, with a grape must volume of 250 L. The must was inoculated with either S. cerevisiae strain OCP-088 or OCP-125 at a concentration of 1.25x1012 cells/barrel. Fermentations were carried out in triplicate for each must (3n = Pinot Gris, 3n = Pinot Noir). The initial parameters of the must were measured using an OenoFossTM wine analyzer (FOSS Analytics, Denmark). The initial temperature at inoculation was 12.7±0.4°C for Pinot Gris must, and 15.6±0.4°C for Pinot Noir must. Twenty-four hours after yeast inoculation, 100 g of FermControl™ BIO (2B Ferm Control, Germany) was added to each barrel as a nutrient supplementation. Temperature and °Brix were monitored daily using a Density Meter DMA 35 (Anton Paar, USA). Samples for wine metabolite analysis were taken once fermentation activity was considered to have stopped (there was no further decrease of sugar concentration or increase in ethanol production). Due to limited storage space, the wine maker decided to mix each triplicate barrel sample equally into one barrel for each of the strain fermentations, and 60 ppm of SO2 was added. The remainder of the Pinot Gris and Pinot Noir fermentations were blended with other winery fermentations. The four barrels were stored at cellar temperature (16-18°C). Three months later, an additional 60 ppm of SO2 was added to each barrel. The barrels continued in storage until bottling, four months later. 48 3.2.3 Wine metabolite analysis An aliquot of 100 mL was taken from each of the barrels for metabolite analyses 28 days after inoculation for Pinot Gris wine and 24 days after inoculation for Pinot Noir wine. The wine was shipped back to the lab in 50 mL Pyrex flasks and 50 mL Falcon tubes, on ice. Once in the lab, the wine was centrifuged for 5 min at 2360.71 g, 4°C, then the liquid was then transferred into a 50 mL flask, which was stored at -80°C to perform SPME-GC/MS analysis, and to 15 mL tubes that were stored at 4°C for HPLC-RID analysis. Both HPLC-RID and SPME-GC/MS methodologies were carried out as described in section 2.2.3 of chapter II. 3.2.4 Statistical analysis A one-way ANOVA test was conducted to analyse primary metabolite data, followed by a Tukey’s post hoc test. Results were considered significant at p ≤ 0.05. Heatmaps and pie charts were generated using MATLAB (MathWorks, USA). 3.3 Results Winery fermentations were conducted at a different site that was isolated from the winery production area to prevent potential cross-contamination with commercial strains. The lack of temperature control in this location represented a challenge during the fermentation process. To counteract the impact of colder winter days, propane heaters were used to maintain a warm environment. Because the Measday lab does not have the equipment necessary for high-density cell propagation, the two starter culture strains were grown to high density at Scotts labs and a liquid culture inoculum was used. Typically, commercial strains are sold as active dry yeast (ADY) that the winemaker rehydrates prior to inoculation. However, ADY production is an expensive process that requires testing to determine if the yeast strain is viable after desiccation. As well, ADY producers do not typically produce small lots of ADY yeast, therefore we decided to use liquid starter cultures. 3.3.1 Primary metabolites The initial parameters of the Pinot Gris and Pinot Noir must are shown in Table 3.1. SO2 was added by the OCP winemaker at 10ppm to imitate regular OCP winemaking practices. 49 Table 3.1 Initial parameters of OCP pilot scale fermentation grape must Fermentation kinetics were assessed by measuring °Brix. In the case of the Vin Gris wine (Pinot Noir), both OCP-125 and OCP-088 showed similar behaviour from the beginning of the fermentation with a reduction of 14.1±0.7 °Brix in the first 5 days of fermentation. On the other hand, for Pinot Gris, OCP-088 did not have any fermentation activity after the first inoculation as seen by the high Brix levels for the initial stages. Seven days post-inoculation, a re-inoculation of OCP-088 was done (indicated by the arrow in Figure 3.1), which added 1x1012 cells/tank to each of the three Pinot Gris must barrels. After the second inoculation, the OCP-088 fermentation began to proceed in the Pinot Gris must. Figure 3.1 Fermentation kinetics of S. cerevisiae strains “OCP-125” and “OCP-088” in Pinot Gris (A) and Pinot Noir (B) must. Fermentation was monitored by measuring °Brix ± SD (n = 3). The arrow in (A) indicates re-inoculation of OCP-088 on fermentation day 7. Samples for HPLC analysis were taken at day 24 for the Pinot Noir and day 28 for Pinot Gris fermentations - these samples will be subsequently referred to as “fresh wines”. At this time, pH SO2 (mg/L) YAN (mg/L) °Brix Glucose (g/L) Fructose (g/L) Malic Acid (g/L) Pinot Gris 3.03 10 136.9 20.4 81.70 ± 2.25 105.49 ± 1.47 3.10 ± 0.35 Pinot Noir 3.12 10 106.2 21.5 90.02 ± 0.36 117.54 ± 1.82 5.67 ± 0.05 50 fermentation activity was low and the sugar decrease in 24-hour lapses was minimal. For Pinot Gris fresh wine, OCP-125 produced more ethanol (10.78±0.10 %v/v) compared to OCP-088 (10.37±0.14 %v/v) (Table 3.2). Similarly, the residual fructose of the OCP-125 Pinot Gris fresh wine was significantly lower (22.45±0.88 g/L) than the OCP-088 Pinot Gris fresh wine (28.28±2.32 g/L) (Table 3.2). For the Pinot Noir fermentations, both OCP-088 and OCP-125 strains were statistically similar in terms of sugar consumption, with residual fructose concentrations at 23.72±2.51 g/L for OCP-088 and 26.22±0.34 g/L for OCP-125 (Table 3.2). Similarly, ethanol production was statistically similar with 11.44±0.11%v/v for OCP-088 and 11.27±0.10%v/v for OCP-125 (Table 3.2). However, glycerol levels were slightly, but significantly, higher in the OCP-088 wines (0.52±0.02 % v/v) than the OCP-125 wines (0.45±0.02% v/v) (Table 3.2). We did not detect a statistically significant difference in the quantity of organic acids between the strains, in both Pinot Gris and Pinot Noir fresh wines, with exception of malic acid. The concentration of malic acid was significantly lower in OCP-088 Pinot Noir fresh wine (5.47±0.34 g/L) compared to OCP-125 (5.71±0.35 g/L), with a statistical significance of p < 0.05. Table 3.2 Concentration of primary metabolites identified in OCP-088 and OCP-125 Pinot Gris and Vin Gris/Pinot Noir pilot scale fresh wine (3n per strain). Samples were analysed in triplicate, results show mean ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. After each fermentation was deemed complete, each corresponding triplicate fermentation was blended in equal part to fill up one tank for each strain and each juice. The wines remained in Pinot Gris Pinot Noir OCP125 OCP088 OCP125 OCP088 Glucose (g/L) 1.23±0.17a 2.17±0.56a 2.08±0.22A 1.38±0.55A Fructose (g/L) 22.45±0.88a 28.28±2.32b 26.22±0.34A 23.72±2.51A Citric acid (g/L) 0.27±0.02a 0.29±0.05a 0.53±0.04A 0.57±0.09A Tartaric acid (g/L) 3.08±0.07a 3.03±0.12a 1.57±0.06A 1.55±0.10A Malic acid (g/L) 3.26±0.26a 2.95±0.25a 5.71±0.35A 5.47±0.34B Acetic acid (%v/v) 0.01±0.01a 0.02±0.01a 0.01±0.01A 0.01±0.01B Glycerol (%v/v) 0.50±0.01a 0.52±0.02a 0.45±0.02A 0.52±0.02B Ethanol (% v/v) 10.78±0.10a 10.37±0.14b 11.27±0.10A 11.44±0.11A 51 the barrels until bottling, seven months later. When measuring primary metabolites in the bottled wine, both strains showed a slight decrease on sugar content as well as an increase in ethanol percentage when compared to the fresh wines (compare Table 3.2 and Table 3.3). It is likely that when the triplicate fermentations were blended, sufficient oxygen was introduced to allow the remaining viable yeast to metabolize more sugar. Table 3.3 Concentration of primary metabolites identified in Pinot Gris and Vin Gris pilot scale bottled wines, fermented by the OCP-088 and OCP-125 S. cerevisiae strains (3n per strain). The analysis was carried out 3 weeks after bottling. Samples were analysed in triplicate, results show mean ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. 3.3.2 Secondary metabolites A heatmap was used to visualize differences in metabolite concentrations for each type of wine which demonstrated that OCP-088 and OCP-125 wines had similar profiles of volatile organic compounds (VOCs), for each of the Pinot Gris and Pinot Noir fresh wines (Figure 3.2 and 3.3). For Pinot Gris, significative differences can be observed for nonanoic acid, with a “cheese”, “fat”, “waxy” aroma descriptor, which was higher in OCP-088 versus OCP-125 fresh wines (2635.89±376.19 ug/L and 2006.47±112.81 ug/L, respectively, Table 3.4, Figure 3.2). On the other hand, isoamyl acetate, which imparts a fruity “banana, pear” aroma was higher in OCP-125 versus OCP-088 fresh wines (2436.19±279.43 ug/L and 1950.43 ±148.40 ug/L respectively, Table 3.4, Figure 3.2). In the Pinot Noir fresh wines, significant differences between strains can be observed Pinot Gris Pinot Noir OCP125 OCP088 OCP125 OCP088 Glucose (g/L) 0.50±0.01a 1.17±0.01b 1.39±0.02A 1.01±0.04B Fructose (g/L) 15.58±0.01a 20.54±0.01b 22.12±0.01A 20.69±0.29B Ethanol (% v/v) 11.18±0.04a 10.82±0.04b 11.48±0.08A 11.58±0.03A Citric acid (g/L) 0.26±0.001a 0.25±0.001b 0.80±0.001A 0.83±0.001B Tartaric acid (g/L) 4.04±0.002a 4.32±0.02b 2.63±0.003A 2.59±0.005B Malic acid (g/L) 2.95±0.01a 2.78±0.01b 5.21±0.04A 4.82±0.09B Acetic acid (%v/v) 0.03±0.01a 0.03±0.01a 0.02±0.004A 0.02±0.003A Glycerol (%v/v) 0.52±0.03a 0.53±0.01a 0.45±0.03A 0.50±0.006A 52 for 2-phenethyl acetate, with an aroma descriptor of “floral, honey”, with a greater concentration in OCP-088 compared to OCP-125 (303.01±33.94 µg/L and 235.01±47.62 µg/L, respectively) (Table 3.5, Figure 3.3). Concentrations of ethyl dodecanoate and ethyl decanoate were higher in OCP-125 (401.56±32.38 µg /L and 5626.14±291.88 µg/L, respectively), and ethyl hexanoate was higher in OCP-088 (914.18±47.67 µg /L) - these esters are responsible for the fruity aromas in wine (Table 3.5, Figure 3.3). When comparing the fresh wines with the bottled wines, there are significative differences between VOCs. For instance, the concentration of 3-methyl-1-butanol, 2-methyl-1-propanol, 1-propanol, ethyl butanoate, ethyl-2-methyl-butanoate, ethyl isovalerate and ethyl decanoate increased in bottled Pinot Gris wines (Table 3.4, Figure 3.2). On the other hand, for Pinot Noir bottled wines, there is an increase in 1-hexanol, 3-e-hexen-1-ol, 2-methyl-1-propanol, isoamyl acetate, for both OCP-strains (Table 3.5, Figure 3.3). Furthermore, the concentration of phenylethyl alcohol increased in bottled wines for OCP-125 Pinot Noir (2314±305.34 µg/L), whereas in bottled OCP-088 remained statistically similar to the fresh wine (1642.44±85.87 µg /L). Phenylacetaldehyde was detected in both cases (Pinot Gris and Pinot Noir) in the bottled samples and was not detected in fresh wines (Figure 3.2 and 3.3). 53 Figure 3.2 Log-scale Heatmap of VOCs quantified in Pinot Gris pilot-scale wines. Data shows mean values (3n) of the fresh wines (OCP-125 and OCP-088), the bottled wines (Bottled-OCP-125 and Bottled-OCP-088) and the Pinot Gris juice, 2022 harvest. 54 Figure 3.3 Log-scale Heatmap of VOCs quantified in Pinot Noir pilot-scale wines. Data shows mean values (3n) of the fresh wines (OCP-125 and OCP-088), the bottled wines (Bottled-OCP-125 and Bottled-OCP-088) and the Pinot Noir juice, 2022 harvest. 55 The composition of the wines changed over time during the eight months storage followed by bottling. In the Pinot Gris wines, the concentrations of higher alcohols and phenolic compounds significantly increased (p<0.01) in the bottled wines compared to the fresh wines for both OCP-088 and OCP-125 (Figure 3.4). The carboxylic acids have the reverse effect with a significant decrease in concentration (p<0.01) in the bottled wines, compared to the finished fermentations (Figure 3.4). For Pinot Noir wines, the total concentration of higher alcohols significantly decreased (p<0.05) in the bottled OCP-088 wine compared to the fresh wine (Figure 3.5), this is mainly due to the concentrations of 3-methyl-1-butanol, unlike Pinot Gris, Pinot Noir wines had a high concentration of this compound in fresh wines, which decreased over time (Table 3.4 and 3.5). Major differences (p<0.001) were observed between fresh wines and bottled wines in phenolic compound concentrations, which increased substantially in the bottled wines for both OCP-088 and OCP-125 strains (Figure 3.5). Figure 3.4 Pinot Gris wine composition of VOCs chemical groups in the fresh wines (OCP-125 and OCP-088) and the bottled wines (Bottled-OCP-125 and Bottled-OCP-088) 56 Figure 3.5 Pinot Noir wine composition of VOCs chemical groups in the fresh wines (OCP-125 and OCP-088) and the bottled wines (Bottled-OCP-125 and Bottled-OCP-088) In order to compare the VOCs profile of the wine with commercially produced wine during the same vintage in OCP winery, we quantified the concentration of VOCs identified above quantification limits for commercial wines produced using Pinot Gris and Pinot Noir from the same 2022 harvest (Tables 3.4, 3.5). For this comparison three commercial wines were used, Pinot Gris 2022– Switchback vineyard (PG-SB), Pinot Gris 2022- King Family Vineyard, fermented with X5 Laffort yeast (PG-KF), and Freeform Vin Gris 2022 fermented in concrete tanks (Freeform VG). PG-SB and Freeform VG were naturally fermented at the winery, with the grapes coming from the same vineyard as the juice used for this experiment. To look at the variation between Pinot Gris commercial wines, the fresh OCP-088 and OCP-125 wines and the OCP-088 and OCP-125 bottled wines, a PCA analysis was performed using the concentration of VOCs (Figure 3.6). The OCP-088 and OCP-125 fresh wines clearly clustered together, as did the OCP-088 and OCP-125 bottled wines and the commercial Pinot Gris wines 57 (Figure 3.6). The differences between the commercial wines, the fresh wines and the bottled wines, produced with OCP-strains, are indicated along the X axis, which explains 53.1% of the variation. Differences between bottled and fresh OCP wines are also observed, as expected, mainly on the Y axis which explains only 26.8% of the variation (Figure 3.6). Figure 3.6 PCA biplot showing VOCs and distribution of strains in Pinot Gris pilot-scale winery fermentations. Fresh wines: OCP-125, OCP-088; bottled wines: B-OCP-088, B-OCP-125 and commercial wines: PG-KF and PG-SB (A) and individual distribution of strains (B)A) B) 58 Table 3.4. Concentration (µg/L) of VOC quantified by SPME-GC/MS in Pinot Gris pilot scale wines, fermented by 2 S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate, results show mean (µg/L) ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. OCP-125 OCP-088 B-OCP-125 B-OCP-088 PG-SB PG-KF 3-Methyl-1-butanol 14562±240.5a 14724.15±2474.50a 126705.57±3782.36b 120577±15679.94b 132964.36±14638.40b 126556.81±10971.36b 1-Hexanol 1511.82±8.88ac 1480.25±166.24ac 1690.68±75.98ac 1844.10±68.37bc 2326.50±267.78b 1599.71±101.96ac 3-E-hexen-1-ol 18.95±4.13ab 17.27±1.12a 26.20±4.05db 33.18±4.34d 22.41±1.10bc 21.16±0.72bc 3-Z-hexen-1-ol NDa NDa <LOQb <LOQb 311.53±9.92c 162.04±8.35d 2-Methyl-1-propanol ND ND 253.84±17.58b 274.6±6.12c ND ND 1-Heptanol ND ND 139.15±22.19a 191.71±13.14b 139.45±14.42a 144.18±13.14a 1-Propanol 175.96±12.97ab 149.74±36.31a 272.06±30.29c 233.91±15.16bc ND ND 2-Ethyl-1-hexanol 5.53±3.17ab 21.28±6.06b 1.12±0.14a 1.19±0.16a 16.53±2.94b 6.53±2.94b Hexanoic acid 585.53±53.41 747.15±45.66 584.20±84.39 720.52±83.51 563.86±56.99 567.41±57.47 Nonanoic acid 2006.47±112.81a 2635.89±376.19b 2180.26±199.90ab 2171.15±154.43ab 235.95±1.07c 257.82±10.61c Decanoic acid 1009.98±120.41ab 1425.61±355.07a 525.12±110.35b 840.84±248.82b 84.56±2.80c 107.93±20.46c Ethyl butanoate 141.84±11.48a 142.88±12.24a 186.39±4.53b 203.76±3.60b 291.59±20.79c 343.18±28.61d 59 OCP-125 OCP-088 B-OCP-125 B-OCP-088 PG-SB PG-KF Ethyl-2-methyl butanoate ND ND 6.99±0.80a 8.42±1.13a 8.25±1.37a 7.63±0.58a Ethyl isovalerate <LOQa <LOQa 8.88±1.39b 18.50±0.70c <LOQa 4.80±1.53d Isoamyl acetate 2436.14±279.43a 1950.43±148.40b 1084.44±80.10c 841.02±14.78d 2385.50±313.76ab 2243.1±169.81ab Hexyl acetate 174.13±6.56a 164.16±25.16a ND ND 67.46±4.52b 52.79±5.80b 2-Phenethyl acetate 375.84±2.62a 312.69±24.74a 116.34±12.46b 101.76±6.08b 157.90±5.65b 135.04±14.03b Ethyl dodecanoate 965.14±37.10a 836.92±115.80a 784.93±112.94ac 630.76±94.74abc 541.52±91.12bc 510.23±11.23bc Ethyl_hexanoate 562.94±96.16a 616.42±51.38ab 538.78±19.90a 727.46±23.97b 597.40±42.84a 501.10±60.06ab Ethyl_octanoate 4407.60±121.36a 4429.23±354.53a 5436.86±115.22a 6819.72±453.40b 5391.70±459.28a 5141.40±451.01a Ethyl_decanoate 4207.94±146.60ab 3513.65±186.65a 4680.96±569.73b 4924.99±503.44b 1847.96±256.32c 1279.28±62.90c Benzaldehyde <LOQa 0.08±0.03a 0.45±0.04a 0.80±0.05a 5.66±0.22b 20.93±6.13c Phenylacetaldehyde <LOQa <LOQa 28.11±0.15b 24.96±3.27b 16.57±0.69c 18.48±5.85c Benzyl alcohol <LOQa <LOQa 49.54±8.09b 48.18±5.22b 86.02±5.25c 31.03±3.63b Phenylethyl alcohol 2106.29±39.18a 2381.93±163.50a 1905.14±405.42a 2040.49±386.61a 39206.41±2031.004b 32190±501.55b 60 OCP-125 OCP-088 B-OCP-125 B-OCP-088 PG-SB PG-KF γ-Terpinene 2.56±0.09a 2.51±0.03a 2.61±0.28a 2.59±0.19a 6.35±0.10b 6.40±0.02b α-Terpineol 6.89±1.16ab 6.99±2.23ab 5.06±0.49b 10.01±1.60a 14.80±0.33c 7.78±0.90a γ -Terpineol 38.11±7.78a 34.25±11.31a 22.24±0.75b 14.30±6.37b 6.28±1.21c 17.96±3.55b Geranial 100.44±7.44ac 70.35±0.90bd 85.94±11.08bc 90.53±3.14ad 48.23±9.52d 46.23±8.60d Citronellol 11.31±0.88a <LOQb 11.99±0.79a 12.02±2.10a 26.33±4.81b 19.90±5.63b Terpinolene 2.40±0.01a 2.39±0.0.1a <LOQb <LOQb ND ND 61 For Pinot Noir wines, the clustering is also similar to the Pinot Gris wines, where PC1 explains 58.4% of the variation and shows the separation between the Freeform Vin Gris wine and the wines fermented with OCP-strains (Figure 3.7). PC2 which accounts for 27.7% of the variation, separates the fresh wine and the bottled wine (Figure 3.7). Figure 3.7 PCA biplot showing VOCs and distribution of strains in Pinot Noir pilot-scale wines. Fresh wines: OCP-125, OCP-088; bottled wines: B-OCP-088, B-OCP-125 and commercial wine: Freeform-VG (A) and individual distribution of strains (B) A) B) 62 Table 3.5 Concentration (µg/L) of VOCs quantified by SPME-GC/MS in Piot Noir pilot scale wines, fermented by 2 S. cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate, results show mean (µg/L) ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. PN-OCP125 PN-OCP088 BPN-OCP125 BPN-OCP088 FREEFORM-VG 3-Methyl-1-butanol 137273.97±14459.55ab 146640.39±20199.59a 111881.83±12057.26ab 102960.65±6683.84b 109780.21±14696.67ab 1-Hexanol 1229.02±115.48a 1069.79±55.58a 1845.58±186.39b 1706.46±78.60b 1804.11±107.82b 3-E-hexen-1-ol 14.53±1.49a 13.69±1.11a 30.9±4.31b 33.03±1.34b 28.52±0.40b 2-Methyl-1-propanol ND ND 363.27±24.32a 376.92±26.28a 430.20±70.96a 1-Heptanol ND ND 131.22±23.79ab 159.85±8.03a 122.83±9.27b 1-Propanol 1343.87±146.62a 1278.18±281.58a 1292.23±93.86a 1595.11±183.42a 121.68±8.85b 2-Ethyl-1-hexanol 8.60±1.99a 3.53±0.33a 1.12±0.15a 2.65±0.09a 27.11±2.99b 1-Nonanol 8.14±2.10a 6.39±0.05a ND ND ND Hexanoic acid 224.83±51.65a 241.72±13.31a 210.11±13.71a 247.93±18.55a 220.72±29.85a Nonanoic acid 729.49±77.42ab 677.67±9.10a 784.12±119.52ab 804.15±22.78b 96.49±18.32c Decanoic acid 392.59±242.41a 423.98±70.95a 388.85±86.26a 336.83±83.44a 34.42±7.71b Ethyl butanoate 197.70±11.77a 226.08±10.88a 201.09±20.18a 268.71±34.29a 458.03±13.62b Ethyl-2-methyl butanoate ND ND 3.52±0.79a 2.30±0.04a 11.64±0.72b Ethyl isovalerate <LOQb <LOQb <LOQb <LOQb 7.20±0.80a Isoamyl acetate 1742.38±189a 2276.33±176.20ab 2748.90±202.25b 3981.11±187.02c 3167.56±80.334d 63 Odour Activity Values (OAVs) play a crucial role in determining the potential contribution of specific volatile compounds to the aroma perception of a product. OAVs quantify the perceived odour intensity of these compounds. To derive OAVs, the ratios between the concentration of each aroma compound and its odor threshold were calculated. Compounds with an OAV below one enhances the complexity of the aroma bouquet without significantly affecting odor perception. On the other hand, an OAV above one indicates an active aroma (Table 3.6). Because OAVs are theoretical, values that are close to 1 are also indicated as possible odor active compounds that may take part in the aroma bouquet (Table 3.6). PN-OCP125 PN-OCP088 BPN-OCP125 BPN-OCP088 FREEFORM-VG Hexyl acetate 274.72±11.64a 275.92±5.12a 102.22±14.70b 116.87±6.31b 54.43±3.17c 2-phenethyl acetate 235.01±47.62a 303.01±33.94b 169.61±18.41a 202.37±18.19a 205.24±26.45a Ethyl dodecanoate 401.56±32.38a 306.12±31.01c 499.90±36.38ad 465.98±19.47d 64.34±12.52e Ethyl_hexanoate 736.11±38.29a 914.18±47.67b 682.10±6.40a 962.03±55.55b 503.09±36.54c Ethyl_octanoate 6257.45±159.26a 6232.67±183.51a 5033.09±599.50bc 5208.89±339.05b 4274.43±274.56c Ethyl_decanoate 5626.14±291.88a 4650.44±359.71b 4488.12±306.41b 4181.56±212.45b 1693.28±207.73c Phenylacetaldehyde <LOQc <LOQc 11.82±6.97a 14.42±3.26ab 18.32±0.20b Benzyl alcohol 51.36±1.63a 41.19±0.63b 37.3±4.87b 41.22±3.89b 39.53±3.40b Phenylethyl alcohol 1735.97±393.98ab 1631.48±130.84a 2314±305.34b 1642.44±85.87a 40770.87±2124.66c γ-Terpinene 3.03±0.01a 3.06±0.01a 3.25±0.31a 3.15±0.12a 6.45±0.32b a-Terpineol 8.81±2.68 1.52±0.04 7.46±1.37 18.62±11.85 10.33±1.78 γ-Terpineol 23.99±5.99a 20.18±6.82a 16.9±1.11a 9.45±4.31a 10.41±1.90a Geranial 70.49±2.43a 68.74±3.17a 46.50±6.97b 48.32±3.92b 48.73±1.47b Citronellol 13.10±1.14 a 13.70±0.24a 19.18±2.39b 20.23±0.64b 25.23±3.12c 64 The OAVs for both fresh and bottled wine samples were calculated, and they were grouped into four attributes: fruity, green grass, floral and rancid, according to their aroma descriptor (Table 3.6). The log10 was calculated from the sum of OAVs for each group to better estimate the aroma profile of the wines (Figure 3.8). We can observe how, for fresh Pinot Gris and Pinot Noir wines, both OCP-125 and OCP-088 have very similar aroma characteristics (Figure 3.8 A, B). The esters responsible for fruity aromas are the principal component of the aroma bouquet, followed by fusel alcohols that add some green grass notes. The green grass attribute increases for both OCP-125 and OCP-088 in Pinor Gris and Pinot Noir bottled wines. The estimated aroma profile of the OCP bottled wines, is very similar to the aroma profiles of both the Pinot Gris and Vin Gris commercial wines produced by OCP (Figure 3.8 C and D). 65 Figure 3.8 Estimated aroma sensory profile of the volatile compounds. OAV of volatile compounds calculated as the log of the ratio of compound concentration to odor threshold for Pinot Gris (A), Pinot Noir (B) pilot-scale fermentation wines and PG-SB, PG-KG (C) and Freeform-VG (D) commercial wines. 00.511.522.533.544.5FruityGreen grassFloralRancidC)PG-SB PG-KG00.511.522.533.54FruityGreen grassFloralRancidD)FREEFORM-VG00.511.522.533.54FruityGreen grassFloralRancidA)OCP-125 OCP-088B-OCP-125 B-OCP-08800.511.522.533.54FruityGreen grassFloralRancidB)OCP-125 OCP-088B-OCP-125 B-OCP-08866 Table 3.6 OAVs for Pinot Gris pilot-scale wines PG-125 PG-088 Bottled PG-125 Bottled PG-088 PG-SB PG-KG Odour descriptor1 3-Methyl-1-butanol 62.47 58.90 435.40 414.34 531.86 5062.22 Overripe banana, fusel oil 1-Hexanol 0.60 0.59 0.68 0.74 0.93 0.64 Herbaceous, green 3-E-hexen-1-ol <1 <1 <1 <1 <1 <1 Grass 2-Methyl-1-propanol - - <1 <1 - - Fusel winey 1-Heptanol - - 28.10 38.57 46.19 47.99 Grass 1-Propanol <1 <1 <1 <1 - - Alcoholic, pungent, harsh 1-Nonanol <1 <1 - - - - - Hexanoic acid <1 <1 <1 <1 <1 <1 Sweat, cheese, rancid Nonanoic acid 0.67 0.88 0.73 0.72 <1 <1 - Decanoic acid <1 <1 <1 <1 <1 <1 Rancid, fat Ethyl butanoate 141.84 142.88 186.39 203.76 291.60 343.18 Fruity Isoamyl acetate 1381.07 975.22 542.22 420.51 1523.18 1943.22 Banana, pear Hexyl acetate 87.06 82.08 - - 33.73 26.39 Fruity, sweet floral 2-Phenethyl acetate 1.50 1.25 <1 <1 <1 <1 Floral, rose, honey Ethyl hexanoate 112.36 123.28 107.76 145.49 119.48 100.22 Green apple, pineapple Ethyl octanoate 2203.80 2214.61 2718.43 3409.86 2695.85 2570.7 Fruity Ethyl decanoate 21.04 17.57 23.40 24.62 9.23 6.40 Fruity, apple 67 PG-125 PG-088 Bottled PG-125 Bottled PG-088 PG-SB PG-KG Odour descriptor1 Phenylacetaldehyde - - 7.03 6.24 4.14 4.62 - Benzyl alcohol - - <1 <1 <1 <1 Floral Phenylethyl alcohol 2.81 3.18 2.54 2.72 52.27 42.92 Rose, floral α-terpineol <1 <1 <1 <1 <1 <1 Pine Geranial 3.14 2.20 2.69 2.83 1.52 1.45 Floral Citronellol <1 - <1 <1 <1 <1 Green lemon 1 Odour descriptors were obtained from Pulcini et al., 2022 and Vilanova et al., 2010 Table 3.7 OAVs for Pinot Noir pilot-scale wines PN-125 PN-088 Bottled PN-125 Bottled PN-088 Freeform-VG Odour descriptor1 3-Methyl-1-butanol 549.10 586.56 447.52 411.84 439.12 Overripe banana, fusel oil 1-Hexanol <1 <1 0.74 0.68 0.72 Herbaceous, green 3-E-Hexen-1-ol <1 <1 <1 <1 <1 Grass 2-Methyl-1-propanol - - <1 <1 <1 Fusel winey 1-Heptanol - - 43.74 53.28 40.55 Grass 1-Propanol <1 <1 <1 <1 - Alcoholic, pungent, harsh 1-Nonanol <1 <1 - - - - Hexanoic acid <1 <1 <1 <1 <1 Sweat, cheese, rancid Nonanoic acid <1 <1 <1 <1 <1 - 68 PN-125 PN-088 Bottled PN-125 Bottled PN-088 Freeform-VG Odour descriptor1 Decanoic acid <1 <1 <1 <1 <1 Rancid, fat Ethyl butanoate 197.70 226.08 201.09 268.71 458.03 Fruity Isoamyl acetate 871.19 1138.16 1374.45 1990.56 1583.78 Banana, pear Hexyl acetate 137.36 137.96 51.11 58.44 27.21 Fruity, sweet floral 2-Phenethyl acetate 0.94 1.21 0.68 0.81 0.82 Floral, rose, honey Ethyl hexanoate 147.22 182.84 136.42 192.41 100.62 Green apple, pineapple Ethyl octanoate 3128.72 3116.34 2516.55 2604.45 2137.21 Fruity Ethyl decanoate 28.13 23.25 22.44 20.91 8.46 Fruity, apple Phenylacetaldehyde - - 9.08 3.60 4.58 - Benzyl alcohol <1 <1 <1 <1 <1 Floral Phenylethyl alcohol 2.31 2.18 7.09 2.19 54.36 Rose, floral α-Terpineol <1 <1 <1 <1 <1 Pine Geranial 2.20 2.15 1.94 1.89 1.52 Floral Citronellol <1 <1 <1 <1 <1 Green lemon 1 Odour descriptors were obtained from Pulcini et al., 2022 and Vilanova et al., 2010 69 3.4 Discussion In this chapter, the main objective was to conduct fermentations in a pilot-scale winery setting, representing a significant scale-up from the initial lab fermentations presented in Chapter 2. The purpose was to assess whether the OCP-088 and OCP-125 indigenous S. cerevisiae strains could initiate and complete fermentations under winery conditions. Due to the substantial 1000-fold increase in scale (250 L winery barrels compared to the 250 mL lab-fermentations), the OCP indigenous strains’ adaptability to a larger environment was uncertain. The fermentations were carried out in a separate OCP building to reduce chances of cross-contamination with commercial strains being used in the OCP winery for their 2022 wine production. The experiments in this chapter aimed to bridge the gap between laboratory-scale fermentations and practical winemaking applications, providing insights into the performance of OCP indigenous strains in a winery setting. As discussed in the previous chapter, temperature, along with other factors, has an impact on fermentation time by S. cerevisiae. For pilot-scale fermentations, Pinot Gris and Pinot Noir musts were inoculated at a temperature range of 13-15°C. Rapidly, the temperature increased with the fermentative activity, oscillating between 18-20°C during fermentation, and dropping to 13-15°C towards the end once fermentation activity slowed down. For the Pinot Noir/Vin Gris wines, both the OCP-125 and OCP-088 strains were capable of starting the fermentation process, even at low temperature. On the other hand, for Pinot Gris wines, the first inoculation of the OCP-088 liquid culture was not able to start the fermentation, therefore this strain had to be reinoculated (Figure 3.1B). Even so, both OCP indigenous strains were able to consume a higher percentage of glucose and fructose than during lab-scale fermentations. Their improved performance can be attributed to several factors including must composition, a 5-fold increase in the concentration of yeast strain inoculum, less initial sugar concentration and nutrient availability. Both the Pinot Gris and Pinot Noir grape must had lower Brix in the 2022 vintage compared to the 2019 vintage - 20.4° Brix (vintage 2022) vs 25.3°Brix (vintage 2019) for Pinot Gris, and 21.5°Brix (vintage 2022) vs 22.5°Brix (vintage 2019). As well the pilot-scale fermentations had nutrient supplementation 24-hours after yeast inoculation whereas the lab fermentations had no nutrient addition after inoculation. This highlights the importance of optimizing the fermentation parameters to the individual needs of the yeast strains as discussed in Chapter 2. Other than the initial adaptation to the must upon inoculation, OCP-125 and OCP-088 had similar levels of sugar consumption and primary metabolite production for both the Pinot Gris and Pinot Noir/Vin Gris fermentations (Table 3.2). 70 Similar to the lab-scale fermentations the highest quantity of VOCs for both Pinot Gris and Pinot Noir wines and for both OCP strains are higher alcohols, followed by esters, phenolic compounds, carboxylic acids and terpenoids, showing these strains abilities to produce wines with desired organoleptic profiles (Figures 3.4, 3.5). It is important to note than minimal differences in esters concentration can make a difference in aroma perception since the odor detection threshold of these compounds is very low (Parapouli et al., 2020). The theoretical OAVs calculated to estimate aroma profiles for fresh and bottled Pinot Gris and Vin Gris/Pinot Noir wines clearly show “fruity” (apple, pear, pineapple, banana) and “green grass” as the main odor attributes, which are the two most common descriptors for white wines (Figure 3.8, Table 3.6). The concentrations of carboxylic acids were below detection thresholds in both wine varietals, showing that neither of these wines have off-flavors impacting the wine expected quality (Table 3.6). Wines produced with the OCP indigenous strains had significantly higher concentrations of ethyl decanoate than the commercial wines, which adds to a “fruity”, “apple” aroma in both Pinot Gris and Vin Gris/Pinot Noir. Interestingly, the aroma profiles of the wines were described by OCP winemaker Matt Dumayne as tropical fruit, guava, fresh cut melon and slightly herbaceous notes for Pinot Noir fermented with OCP-125. For Pinot Noir fermented with OCP-088, the wine was described as earthy, leathery, and less fruity. Pinot Gris fermented with OCP-125 was described as having a ripe red fruit profile and Pinot Gris fermented with OCP-088 was described as dusty with lighter melon and lemon/lime notes. Oxidation of the wine can occur before, during and after bottling which results in changes of the organoleptic profile. Phenylacetaldehyde, described as “honey, floral” is one of the compounds found to be contributor to the oxidative “off-flavor” of wine when present at high concentrations (Mayr et al., 2015) The presence of phenylacetaldehyde in both Pinot Noir and Pinot Noir bottled wines is likely due to oxidation of phenylethyl alcohol (Figures 3.2, 3.3, Tables 3.4, 3.4). Some acetate esters tend to decrease post-fermentation and during wine storage as a consequence of chemical reactions such hydrolysis. In the study of Cordente et al., 2021, it was shown that differences in sensory analysis for wines after 3 months of bottling was very subtle, whereas a 15-month wine has a more distinct aroma. In this study, the OAV of hexyl acetate, which has an odor description of “fruity, sweet floral”, was lower in bottled wine than in fresh wine, for both Pinot Noir and Pinot Gris (Table 3.6). No other esters were found to decrease significantly for our wines. As shown in both Pinot Gris and Pinot Noir PCA plots the wines fermented with OCP-indigenous strains were clustered together, and distinct from the commercial wines produced at the 71 winery (Figures 3.6, 3.7), demonstrating that the S. cerevisiae strain plays a key role in determining the aroma composition of the wines, and can help bring out attributes that may highlight the regional terroir. Other studies have isolated S. cerevisiae strains from specific wine-making regions and have also shown these isolates capability of producing desirable organoleptic characteristics. These studies have also shown that selecting and using a region-specific strain as a starter culture can differentiate the wines produced with commercial yeast strains and spontaneous fermentations (Vázquez et al., 2023; Cuš et al., 2017; Baǧder Elmaci et al., 2014; Suzzi et al., 2012). Interestingly, one of the main differences, between the OCP-indigenous yeast and the commercial samples was phenylethyl alcohol, which was higher in the commercial samples (Table 3.4 and 3.5). Conversely, in lab-scale fermentations OCP-125 produced two-times more phenylethyl alcohol than the commercial M2 strain in Pinot Gris fermentations (Table 2.7). Phenylethyl alcohol is associated with floral-rose aromas - this phenolic alcohol is considered one of the main compounds in wine and winery practices can influence its production (Jackson, 2020; Parapouli et al., 2020)). The different processing of the Pinot Gris wine in the 250mL lab fermentations versus the 250 L pilot scale fermentations is likely why the same strain (OCP-125) produced variable levels of phenylethyl alcohol. The utilization of indigenous yeast strains, such as OCP-125 and OCP-088, has proven to be a promising approach for enhancing the fermentation process and influencing the organoleptic profile of wines. Despite challenges during the wine making process, these indigenous strains demonstrated an overall good performance in consuming a higher percentage of glucose and fructose compared to lab-scale fermentations. The adaptability of OCP-125 and OCP-088 to varying must compositions and inoculation temperatures underscores their ability to adapt to different fermentation parameters. Beyond their fermentation capabilities, these strains contribute to the unique sensory characteristics of wines, as evidenced by the aroma profiles and clustering observed in the PCA data. Considering these findings, the use of indigenous yeast as fermentation starter cultures presents a compelling avenue for winemakers seeking to enhance the distinctive qualities of their wines. 72 4. Conclusions and future directions 4.1 Conclusion The aim of this research was to assess the suitability of indigenous S. cerevisiae strains, isolated from OCP winery and vineyards, as wine fermentation starter cultures. For the first objective, in chapter two, we evaluated six indigenous OCP strains in lab-scale fermentations and primary and secondary metabolites were quantified. Despite being unable to ferment to dryness (residual sugars <5 g/L), particularly in Pinot Gris must with a high concentration of sugars, the six OCP strains were able to metabolize sugars to ethanol and produce key fermentation byproducts (Table 2.5 and 2.6) Interestingly, strain OCP-041 exhibited a propensity for glycerol production, which can potentially be explored for production of wines with reduced ethanol content. Furthermore, the PCA plots showed that the aromatic profile of indigenous OCP strains is different from the commercial control M2, in both Pinot Gris and Pinot Noir fermentations (Figure 2.7 and 2.8). The aromatic data supports the hypothesis that the selected starter culture plays an important role in shaping the aroma characteristics of a wine and that indigenous S. cerevisiae strains can produce a wine that is distinctive from commercial strains. The six indigenous strains explored in this chapter had the ability to produce wines with a desirable VOC composition (Figure A-1 and A-2), however a further exploration of their individual requirements could help improve their fermentation performance. In chapter three, two S. cerevisiae indigenous strains were selected for pilot-scale fermentations in a winery environment to assess their performance in a practical winemaking setting. Despite the considerable scale-up from lab-fermentations, both strains adapted to the larger barrel volume, and successfully performed the fermentations. Notably, both strains demonstrated similar levels of sugar consumption and primary metabolite production for both Pinot Gris and Pinot Noir fermentations (Table 3.2). The aroma profiles of wines fermented with OCP strains were distinct from those produced with commercial strains, underscoring the potential that regional isolates have to accentuate terroir (Figures 3.6 and 3.7). Taking in consideration the results, OCP-125 is a good candidate for selection as commercial starter culture as throughout the experiments it showed more consistency in sugar consumption and ethanol production, producing wines with desirable aroma characteristics, as described by the tasting performed by the winemaker. This study highlights the importance of process optimization in winemaking, and the proper selection of a strain that can perform up to the winery standards. As well the research recognizes the importance of the role of the microorganism performing the fermentation process to shape the final profile of the wine. 73 4.2 Future directions In this study, fermentations were carried out under specific conditions, with the objective of imitating as close as possible the low-intervention winemaking techniques of OCP winery. Further study on individual-strain requirements, such as optimal fermentation temperatures and nutrient requirements would help improve fermentation performance of these indigenous strains, particularly if there is an interest in using these strains for dry wine production. Alternatively, the use of two- or more- indigenous strains in sequential fermentation, or an indigenous strain followed by a commercial strain to ensure that all sugars are consumed can be explored. Both the OCP-125 and OCP-088 strains are genetically distinct from commercial S. cerevisiae strains supporting the use of these strains as region-specific starter cultures (Marr A., et.al. 2023). These two strains have proven to be capable of growing to a high cell concentration in liquid media. Growth to high cell concentrations shows their capacity to be used in industrial settings, however their fitness and viability in the ADY production process still has to be tested. Generating OCP-125 as an ADY product would be ideal for wine production due to the ability to store the yeast product over many years. Since the winemaking process is seasonal, having liquid yeast culture produced on an annual basis would not only be inefficient but also expensive. Furthermore, pilot-scale fermentations with the other four OCP indigenous strains could be explored, since each of these strains may offer advantages or might be better adapted to specific musts or winemaking conditions. This research would increase the number of commercially available Okanagan-region wine starter cultures of which there are currently none. Although during pilot-scale winery fermentations differences were found in aromatic composition when compared to commercially produced wines, another experiment that includes the same winemaking conditions with a commercial starter culture would be necessary to better observe what regional-specific attributes are introduced when using OCP indigenous strains. For the same purpose, although OAV was used to predict the aroma profile of the wines (Table 3.5 and 3.6), a descriptive sensorial analysis would help mark the differences between wines perceived by the human senses and would help establish the advantages of using indigenous OCP strains for the organoleptic profile of BC wines. 74 References Alonso-del-Real, J., Lairón-Peris, M., Barrio, E., & Querol, A. (2017). Effect of temperature on the prevalence of Saccharomyces non cerevisiae species against a S. cerevisiae Wine strain in wine fermentation: Competition, physiological fitness, and influence in final wine composition. Frontiers in Microbiology, 8(FEB). https://doi.org/10.3389/fmicb.2017.00150 Aponte, M., Romano, R., Villano, C., & Blaiotta, G. (2020). Dominance of S. cerevisiae commercial starter strains during greco di tufo and aglianico wine fermentations and evaluation of oenological performances of some indigenous/residential strains. Foods, 9(11). https://doi.org/10.3390/foods9111549 Baǧder Elmaci, S., Özçelik, F., Tokatli, M., & Çakir, I. (2014). Technological properties of indigenous wine yeast strains isolated from wine production regions of Turkey. Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology, 105(5), 835–847. https://doi.org/10.1007/s10482-014-0138-z Barrajón, N., Capece, A., Arévalo-Villena, M., Briones, A., & Romano, P. (2011). Co-inoculation of different Saccharomyces cerevisiae strains and influence on volatile composition of wines. Food Microbiology, 28(5), 1080–1086. https://doi.org/10.1016/j.fm.2011.02.016 Berthels, N. J., Cordero Otero, R. R., Bauer, F. F., Thevelein, J. M., & Pretorius, I. S. (2004). Discrepancy in glucose and fructose utilisation during fermentation by Saccharomyces cerevisiae wine yeast strains. FEMS Yeast Research, 4(7), 683–689. https://doi.org/10.1016/j.femsyr.2004.02.005 Black, C. A., Parker, M., Siebert, T. E., Capone, D. L., & Francis, I. L. (2015). Terpenoids and their role in wine flavour: Recent advances. Australian Journal of Grape and Wine Research, 21, 582–600. https://doi.org/10.1111/ajgw.12186 Blanco, P., Mirás-Avalos, J. M., Pereira, E., & Orriols, I. (2013). Fermentative aroma compounds and sensory profiles of godello and albariño wines as influenced by saccharomyces cerevisiae yeast strains. Journal of the Science of Food and Agriculture, 93(11), 2849–2857. https://doi.org/10.1002/jsfa.6122 Bokulich, N. A., Collins, T. S., Masarweh, C., Allen, G., Heymann, H., Ebeler, S. E., & Millsa, D. A. (2016). Associations among wine grape microbiome, metabolome, and fermentation behavior suggest microbial contribution to regional wine characteristics. MBio, 7(3). https://doi.org/10.1128/mBio.00631-16 Buglass, A. J. (2022). An introduction to viticulture, winemaking and wine: From vineyard to wine glass. Cambridge Scholars Publishing. 131-154 Capece, A., Romaniello, R., Siesto, G., Pietrafesa, R., Massari, C., Poeta, C., & Romano, P. (2010). Selection of indigenous Saccharomyces cerevisiae strains for Nero d’Avola wine and evaluation of selected starter implantation in pilot fermentation. International Journal of Food Microbiology, 144(1), 187–192. https://doi.org/10.1016/j.ijfoodmicro.2010.09.009 75 Carrau, F. M., Boido, E., & Dellacassa, E. (2008). Terpenoids in grapes and wines: Origin and micrometabolism during the vinification process. In Natural Product Communications (Vol. 3, Issue 4, pp. 577–592). Natural Product Incorporation. https://doi.org/10.1177/1934578x0800300419 Cheng, E., Martiniuk, J. T., Hamilton, J., McCarthy, G., Castellarin, S. D., & Measday, V. (2020a). Characterization of Sub-Regional Variation in Saccharomyces Populations and Grape Phenolic Composition in Pinot Noir Vineyards of a Canadian Wine Region. Frontiers in Genetics, 11. https://doi.org/10.3389/fgene.2020.00908 Cheng, E., Martiniuk, J. T., Hamilton, J., McCarthy, G., Castellarin, S. D., & Measday, V. (2020b). Characterization of Sub-Regional Variation in Saccharomyces Populations and Grape Phenolic Composition in Pinot Noir Vineyards of a Canadian Wine Region. Frontiers in Genetics, 11. https://doi.org/10.3389/fgene.2020.00908 Chidi, B. S., Bauer, F. F., & Rossouw, D. (2018). Organic Acid Metabolism and the Impact of Fermentation Practices on Wine Acidity: A Review. J. Enol. Vitic, 39(2). https://doi.org/10.21548/39-2-3164 Cordente, A. G., Nandorfy, D. E., Solomon, M., Schulkin, A., Kolouchova, R., Francis, I. L., & Schmidt, S. A. (2021). Aromatic higher alcohols in wine: Implication on aroma and palate attributes during chardonnay aging. Molecules, 26(16). https://doi.org/10.3390/molecules26164979 Costantini, A., Cravero, M. C., Panero, L., Bonello, F., Vaudano, E., Pulcini, L., & Garcia-Moruno, E. (2021). Wine fermentation performance of indigenous saccharomyces cerevisiae and saccharomyces paradoxus strains isolated in a piedmont vineyard. Beverages, 7(2). https://doi.org/10.3390/beverages7020030 Cuš, F., Zabukovec, P., & Schroers, H. J. (2017). Indigenous yeasts perform alcoholic fermentation and produce aroma compounds in wine. Czech Journal of Food Sciences, 35(4), 339–345. https://doi.org/10.17221/398/2016-CJFS Díaz, C., Molina, A. M., Nähring, J., & Fischer, R. (2013). Characterization and dynamic behavior of wild yeast during spontaneous wine fermentation in steel tanks and amphorae. BioMed Research International, 2013. https://doi.org/10.1155/2013/540465 Du, Q., Ye, D., Zang, X., Nan, H., & Liu, Y. (2022). Effect of low temperature on the shaping of yeast-derived metabolite compositions during wine fermentation. Food Research International, 162. https://doi.org/10.1016/j.foodres.2022.112016 Erten, H., Tanguler, H., Cabaroglu, T., & Canbas, A. (2006). The influence of inoculum level on fermentation and flavour compounds of white wines made From cv. Emir. Journal of the Institute of Brewing, 112(3), 232–236. https://doi.org/10.1002/j.2050-0416.2006.tb00718.x Ferreira, V. (2010). Volatile aroma compounds and wine sensory attributes. Goold, H. D., Kroukamp, H., Williams, T. C., Paulsen, I. T., Varela, C., & Pretorius, I. S. (2017). Yeast’s balancing act between ethanol and glycerol production in low-alcohol wines. In Microbial Biotechnology (Vol. 10, Issue 2, pp. 264–278). John Wiley and Sons Ltd. https://doi.org/10.1111/1751-7915.12488 76 Grigorica, L.-G., Niculaua, M., Nechita, C. B., Nistor, A.-M., & Cotea, V. V. (2017). The impact of some commercial yeast strains on aroma compounds and sensorial analysis on two white wine varieties made in PGI Dealurile Olteniei, Romania. BIO Web of Conferences, 9, 02006. https://doi.org/10.1051/bioconf/20170902006 Guerrini, S., Barbato, D., Guerrini, L., Mari, E., Buscioni, G., Mangani, S., Romboli, Y., Galli, V., Parenti, A., & Granchi, L. (2021). Selection of indigenous saccharomyces cerevisiae strains and exploitation of a pilot-plant to produce fresh yeast starter cultures in a winery. Fermentation, 7(3). https://doi.org/10.3390/fermentation7030099 Guillaume, C., Delobel, P., Sablayrolles, J. M., & Blondin, B. (2007). Molecular basis of fructose utilization by the wine yeast Saccharomyces cerevisiae: A mutated HXT3 allele enhances fructose fermentation. Applied and Environmental Microbiology, 73(8), 2432–2439. https://doi.org/10.1128/AEM.02269-06 Gump, B. H., Zoecklein, B. W., & Fugelsang, K. C. (2003). Prediction of Prefermentation Nutritional Status of Grape Juice: The Formol Method. In Food Microbiology Protocols (pp. 283–296). Humana Press. https://doi.org/10.1385/1-59259-029-2:283 Hirst, M. B., & Richter, C. L. (2016). Review of aroma formation through metabolic pathways of Saccharomyces cerevisiae in beverage fermentations. In American Journal of Enology and Viticulture (Vol. 67, Issue 4, pp. 361–370). American Society for Enology and Viticulture. https://doi.org/10.5344/ajev.2016.15098 Jackson, R. S. (2020). Chemical constituents of grapes and wine. In Wine Science (pp. 375–459). Elsevier. https://doi.org/10.1016/b978-0-12-816118-0.00006-4 Joshi, V. K., Bordiga, M., Cosme, F., Fariña, L., Jackson, R. S., Manuel Jordão, A., Lonvaud-Funel, A., & Stockley, C. (2022). Concise Encyclopedia of Science and Technology of Wine. Katarína, F., Katarína, M., Katarína, Ď., Ivan, Š., & Fedor, M. (2014). Influence of yeast strain on aromatic profile of Gewürztraminer wine. LWT, 59(1), 256–262. https://doi.org/10.1016/j.lwt.2014.05.057 Knight, S., Klaere, S., Fedrizzi, B., & Goddard, M. R. (2015). Regional microbial signatures positively correlate with differential wine phenotypes: Evidence for a microbial aspect to terroir. Scientific Reports, 5. https://doi.org/10.1038/srep14233 Lappa, I. K., Kachrimanidou, V., Pateraki, C., Koulougliotis, D., Eriotou, E., & Kopsahelis, N. (2020). Indigenous yeasts: emerging trends and challenges in winemaking. In Current Opinion in Food Science (Vol. 32, pp. 133–143). Elsevier Ltd. https://doi.org/10.1016/j.cofs.2020.04.004 Larnbrechts, M. G., & Pretorius, I. S. (2000). Yeast and its Importance to Wine Aroma-A Review. Lip, K. Y. F., García-Ríos, E., Costa, C. E., Guillamón, J. M., Domingues, L., Teixeira, J., & van Gulik, W. M. (2020). Selection and subsequent physiological characterization of industrial Saccharomyces cerevisiae strains during continuous growth at sub- and- supra optimal temperatures. Biotechnology Reports, 26. https://doi.org/10.1016/j.btre.2020.e00462 77 Longo, R., Carew, A., Sawyer, S., Kemp, B., & Kerslake, F. (2021). A review on the aroma composition of Vitis vinifera L. Pinot noir wines: origins and influencing factors. In Critical Reviews in Food Science and Nutrition (Vol. 61, Issue 10, pp. 1589–1604). Bellwether Publishing, Ltd. https://doi.org/10.1080/10408398.2020.1762535 Lopes, C. A., Rodríguez, M. E., Sangorrín, M., Querol, A., & Caballero, A. C. (2007a). Patagonian wines: Implantation of an indigenous strain of Saccharomyces cerevisiae in fermentations conducted in traditional and modern cellars. Journal of Industrial Microbiology and Biotechnology, 34(2), 139–149. https://doi.org/10.1007/s10295-006-0178-0 Lopes, C. A., Rodríguez, M. E., Sangorrín, M., Querol, A., & Caballero, A. C. (2007b). Patagonian wines: The selection of an indigenous yeast starter. Journal of Industrial Microbiology and Biotechnology, 34(8), 539–546. https://doi.org/10.1007/s10295-007-0227-3 Maicas, S. (2020). The role of yeasts in fermentation processes. In Microorganisms (Vol. 8, Issue 8, pp. 1–8). MDPI AG. https://doi.org/10.3390/microorganisms8081142 Maicas, S., & Mateo, J. J. (2016). Microbial glycosidases for wine production. Beverages, 2(3). https://doi.org/10.3390/beverages2030020 Marr, R. A., Moore, J., Formby, S., Martiniuk, J. T., Hamilton, J., Ralli, S., Konwar, K., Rajasundaram, N., Hahn, A., & Measday, V. (2023). Whole genome sequencing of Canadian Saccharomyces cerevisiae strains isolated from spontaneous wine fermentations reveals a new Pacific West Coast Wine clade . G3: Genes, Genomes, Genetics. https://doi.org/10.1093/g3journal/jkad130 Mayr, C. M., Capone, D. L., Pardon, K. H., Black, C. A., Pomeroy, D., & Francis, I. L. (2015). Quantitative Analysis by GC-MS/MS of 18 Aroma Compounds Related to Oxidative Off-Flavor in Wines. Journal of Agricultural and Food Chemistry, 63(13), 3394–3401. https://doi.org/10.1021/jf505803u Mendes, S. D. C., Arcari, S. G., Werner, S. S., Valente, P., & Ramirez-Castrillon, M. (2022). Wild Saccharomyces Produced Differential Aromas of Fermented Sauvignon Blanc Must. Fermentation, 8(4), 177. https://doi.org/10.3390/fermentation8040177 Mendes-Ferreira, A., Mendes-Faia, A., & Leão, C. (2004). Growth and fermentation patterns of Saccharomyces cerevisiae under different ammonium concentrations and its implications in winemaking industry. Journal of Applied Microbiology, 97(3), 540–545. https://doi.org/10.1111/j.1365-2672.2004.02331.x Mills, D. A., Phister, T., Neeley, E., & Johannsen, E. (2008). Wine Fermentation. Molinet, J., & Cubillos, F. A. (2020). Wild Yeast for the Future: Exploring the Use of Wild Strains for Wine and Beer Fermentation. In Frontiers in Genetics (Vol. 11). Frontiers Media S.A. https://doi.org/10.3389/fgene.2020.589350 Morata, A., González, C., Tesfaye, W., Loira, I., & Suárez-Lepe, J. A. (2018). Maceration and Fermentation: New Technologies to Increase Extraction. In Red Wine Technology (pp. 35–49). Elsevier. https://doi.org/10.1016/B978-0-12-814399-5.00003-7 78 Moreno-Arribas, V., & Polo, M. C. (2009). Wine Chemistry and Biochemistry. Mouret, J. R., Aguera, E., Perez, M., Farines, V., & Sablayrolles, J. M. (2021). Study of oenological fermentation: Which strategy and which tools? Fermentation, 7(3). https://doi.org/10.3390/FERMENTATION7030155 Nikolaou, E., Soufleros, E. H., Bouloumpasi, E., & Tzanetakis, N. (2006). Selection of indigenous Saccharomyces cerevisiae strains according to their oenological characteristics and vinification results. Food Microbiology, 23(2), 205–211. https://doi.org/10.1016/j.fm.2005.03.004 Parapouli, M., Vasileiadis, A., Afendra, A. S., & Hatziloukas, E. (2020). Saccharomyces cerevisiae and its industrial applications. In AIMS Microbiology (Vol. 6, Issue 1, pp. 1–31). AIMS Press. https://doi.org/10.3934/microbiol.2020001 Pico, J., Nozadi, K., Gerbrandt, E. M., Dossett, M., & Castellarin, S. D. (2023). Determination of bound volatiles in blueberries, raspberries, and grapes with an optimized protocol and a validated SPME-GC/MS method. Food Chemistry, 403. https://doi.org/10.1016/j.foodchem.2022.134304 Rainieri, S. (2000). Selection and improvement of wine yeast Acquatrode: System to Detect the Level of Stress / Discomfort of Aquatic Animals View project yeast genomics View project. In Article in Annals of Microbiology. https://www.researchgate.net/publication/237429676 Romano, P., Ciani, M., & Fleet, G. H. (2019). Yeasts in the production of wine. In Yeasts in the Production of Wine. Springer New York. https://doi.org/10.1007/978-1-4939-9782-4 Slaghenaufi, D., Vanzo, L., Luzzini, G., Arapitsas, P., Marangon, M., Curioni, A., Mattivi, F., Piombino, P., Moio, L., Versari, A., Ricci, A., Segade, S. R., Rolle, L., & Ugliano, M. (2022). Monoterpenoids and norisoprenoids in Italian red wines. Oeno One, 56(3), 185–193. https://doi.org/10.20870/oeno-one.2022.56.3.5387 Suzzi, G., Arfelli, G., Schirone, M., Corsetti, A., Perpetuini, G., & Tofalo, R. (2012). Effect of grape indigenous Saccharomyces cerevisiae strains on Montepulciano d’Abruzzo red wine quality. Food Research International, 46(1), 22–29. https://doi.org/10.1016/j.foodres.2011.10.046 Tristezza, M., Fantastico, L., Vetrano, C., Bleve, G., Corallo, D., Mita, G., & Grieco, F. (2014). Molecular and technological characterization of saccharomyces cerevisiae strains isolated from natural fermentation of susumaniello grape must in Apulia, Southern Italy. International Journal of Microbiology, 2014. https://doi.org/10.1155/2014/897428 Valentine, G. D. S., Walker, M. E., Gardner, J. M., Schmid, F., & Jiranek, V. (2019). Brief temperature extremes during wine fermentation: effect on yeast viability and fermentation progress. Australian Journal of Grape and Wine Research, 25(1), 62–69. https://doi.org/10.1111/ajgw.12365 van Leeuwen, C., Barbe, J. C., Darriet, P., Geffroy, O., Gomès, E., Guillaumie, S., Helwi, P., Laboyrie, J., Lytra, G., Le Menn, N., Marchand, S., Picard, M., Pons, A., Schüttler, A., & Thibon, C. (2020). Recent advancements in understanding the terroir effect on aromas 79 in grapes and wines. Oeno One, 54(4), 985–1006. https://doi.org/10.20870/OENO-ONE.2020.54.4.3983 Vázquez, J., Mislata, A. M., Vendrell, V., Moro, C., de Lamo, S., Ferrer-Gallego, R., & Andorrà, I. (2023). Enological Suitability of Indigenous Yeast Strains for ‘Verdejo’ Wine Production. Foods, 12(9). https://doi.org/10.3390/foods12091888 Zabukovec, P., Čadež, N., & Čuš, F. (2020). Isolation and identification of indigenous wine yeasts and their use in alcoholic fermentation. Food Technology and Biotechnology, 58(3), 337–347. https://doi.org/10.17113/ftb.58.03.20.6677 Zang, X., Du, Q., Qu, R., Ye, D., Lu, Y., & Liu, Y. (2022). Analysis of Volatile Aroma Compounds and Sensory Characteristics Contributing to Regional Style of Red Wines from Hexi Corridor Based on Sixteen Grape Varieties/Clones. Fermentation, 8(10). https://doi.org/10.3390/fermentation8100501 80 Appendix Table A.1. Concentration (µg/L) of volatile aroma compounds quantified by SPME-GC/MS in Pinot Gris laboratory scale fermentations, fermented by 7 Saccharomyces cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate, results show mean (µg/L) ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. OCP-125 OCP-088 OCP-080 OCP-090 OCP-095 OCP-041 M2 1-Propanol 311.67±33.58bc 270.90±41.09bc 385.81±23.65c 342.45±37.48bc 299.08±23.13bc 250.65±42.65b 646.12±93.51a 2-Methyl-1-butanol 55.47±8.48 60.08±13.23 56.84±2.69 60.87±8.44 46.25±5.14 57.24±8.44 49.00±5.91 2-Methyl-1-propanol <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 3-Methyl-1-butanol* 113783.04±14801.27a 75833.11±9855.03b 107777.73±18099.48ab 109061.63±15397.42ab 83990.40±10160.93ab 93769.33±5826.74ab 90525.40±4485.09ab 2-Hexanol ND ND ND ND ND ND ND 2-Heptanol 7.81±0.06 7.88±0.04 7.76±0.02 7.82±0.01 7.78±0.04 10.68±0.41a 7.77±0.04 1-Hexanol 670.68±100.17 731.25±63.07 666.60±63.54 679.31±34.83 859.37±58.37 776.92±149.84 634.16±46.98 3-E-Hexen-1-ol 20.5±2.35ab 20.36±1.75ab 20.47±1.11ab 21.33±0.94ab 24.14±0.59b 22.18±2.96ab 15.78±2.77a 3-Z-Hexen-1-ol <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 81 OCP-125 OCP-088 OCP-080 OCP-090 OCP-095 OCP-041 M2 2-E-Hexen-1-ol <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 2-Z-Hexen-1-ol <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 1-Octen-3-ol ND ND ND ND ND ND ND 1-Heptanol ND ND ND ND ND ND ND 2,3-Butanediol ND ND ND ND ND ND ND 1-Octanol 0.36±0.01 0.39±0.03 0.40±0.02 0.36±0.01 0.40±0.01 0.40±0.04 0.35±0.01 1-Nonanol ND ND ND ND ND ND ND 2-Ethyl-1-hexanol 11.44±1.16 20.60±6.92a 10.62±7.36 9.12±2.78 11.49±1.46 11.30±2.47 7.32±3.81 Hotrienol <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Hexanal ND ND ND ND ND ND ND 2-(E)-Hexanal ND ND ND ND ND ND ND Octanal ND ND ND ND ND ND ND Nonanal <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Furfural ND ND ND ND ND ND ND 2-E-Nonenal ND ND ND ND ND ND ND 2,4-Nonadienal, (E,E)- ND ND ND ND ND ND ND 82 OCP-125 OCP-088 OCP-080 OCP-090 OCP-095 OCP-041 M2 2,4-E,E-Hexadenial ND ND ND ND ND ND ND Hexanoic acid 51.08±11.98 47.25±6.13 52.28±12.02 40.55±4.91 36.14±1.98 34.01±6.15 44.53±0.69 Nonanoic acid* 152.65±27.40 141.35±8.10 182.42±35.47 137.53±25.52 165.85±36.83 127.11±15.03 110.80±13.96 Decanoic acid* 136.47±27.22 120.85±14.76 125.20±16.89 132.37±11.59 117.34±29.48 123.20±41.69 105.93±21.11 Methyl-2-methyl butanoate ND ND ND ND ND ND ND Ethyl butanoate 103.61±8.92a 140.6±8.59b 138.27±18.43b 124.45±5.18ab 115.37±2.77ab 102.31±3.30a 11.87±1.08c Ethyl-2-methyl butanoate ND ND ND ND ND ND ND Ethyl isovalerate <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Isoamyl acetate 1661.23±199.54b 992.06±163.17a 1566.09±260.35ab 1442.78±336.99ab 1188.37±53.79ab 976.85±119.40b 1217.97±165.01ab Isoamyl propionate <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Hexyl acetate 24.27±5.13a 25.14±2.77a 33.66±4.56ab 42.05±2.96b 37.67±3.11ab 26.70±8.12a 45.78±8.21b 83 OCP-125 OCP-088 OCP-080 OCP-090 OCP-095 OCP-041 M2 Ethyl heptanoate <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Ethyl dodecanoate 36.48±5.50bcd 32.31±3.90abc 31.09±4.95ab 44.80±3.21d 16.97±1.65e 16.95±2.17ae 43.48±4.24cd Ethyl hexanoate* 177.34±43.30ab 214.99±30.68ab 218.95±5.14ab 201.88±34.46a 165.47±7.51a 124.65±26.93b 224.52±32.00a Ethyl octanoate* 1161.17±76.80ab 812.49±158.56 ab 951.50±90.46 ab 1031.44±251.52 ab 951.17±122.76 ab 748.95±320.47a 1523.73±476.06b Ethyl decanoate* 1039.64±29.43a 724.60±99.72ab 786.51±21.68ab 970.83±249.40ab 696.06±139.05ab 572.62±173.66b 1098.80±227.26a 2-Phenethyl acetate 110.83±1.85c 47.68±4.73b 73.47±6.81a 84.69±0.18a 74.78±4.97a 74.05±6.49ab 48.24±9.09b 2-Heptanone ND ND <LOQ <LOQ <LOQ ND <LOQ 6-Methyl-5-hepten-2-one ND ND ND ND ND ND ND 2-Nonanone <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 2-Undecanone 1.95±0.01ab 1.98±0.02ab 1.94a 1.96±0.02a 2.51±0.27b 2.16±0.11ab 1.91±1.01ab Benzaldehyde <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Benzothiazole 252.60±64.52 193.21±14.08 216.77±24.28 230.25±17.27 260.19±61.29 160.91±11.29 191.33±51.14 Benzyl alcohol 35.49±1.76 35.03±1.30 35.46±0.78 34.17±1.21 33.48±0.28 34.47±1.60 34.85±0.55 84 OCP-125 OCP-088 OCP-080 OCP-090 OCP-095 OCP-041 M2 Phenylethyl alcohol 1358.59±234.39a 793.35±116.33bc 876.97±145.66abc 1227.62±129.93a 969.98±4.03ac 916.98±141.92abc 572.33±41.57b Guaicol ND ND ND ND ND ND ND Eugenol 4.20±0.02ab 4.16±0.02ab 4.22±0.03a 4.20±0.02a 4.12±0.01b 4.14±0.03ab 4.14±0.95ab Phenylacetaldehyde 10.57±0.24a 4.53±0.09 5.35±0.47 6.18±0.71 6.55±0.53 5.52±0.23 4.62±0.06 α-(+)-pinene <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ β-(+)-pinene ND ND ND ND ND ND ND Sabinene ND ND ND ND ND ND ND 2-Carene ND ND ND ND ND ND ND α-(-)-Phellandrene <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Myrcene <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ α-Terpinene ND ND ND ND ND ND ND 1,4-Cineole ND ND ND ND ND ND ND s-Limonene <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ β-Phellandrene <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Eucalyptol ND ND ND ND ND ND ND Γ-terpinene 5.56±0.17 5.33±0.04 5.42±0.04 5.36±0.04 5.33±0.03 5.38±0.02 5.29±0.06 Ocimene 1.02±0.14 a 0.37±0.14 0.28±0.02 0.44±0.07 0.58±0.03 0.36±0.20 0.00 Cymene <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 85 OCP-125 OCP-088 OCP-080 OCP-090 OCP-095 OCP-041 M2 (+)-Rose oxide <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ (-)-Rose oxide <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Limonene oxide ND ND ND ND ND ND ND Linalool oxide (isomer 1) <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Linalool oxide (isomer 2) ND ND ND ND ND ND ND Linalool 5.99±0.17 5.81±2.01 6.96±1.98 5.32±1.54 6.70±2.12 4.08±2.16 1.49±0.11 Caryophyllene ND ND ND ND ND ND ND α-Humulene ND ND ND ND ND ND ND Neral ND ND ND ND ND ND ND α-Terpineol 34.81±0.77ab 36.89±3.39b 32.42±1.48a 32.46±0.82a 42.37±2.06c 65.84±7.46e 52.01±2.80d (+) Valencene ND ND ND ND ND ND ND γ-Terpineol 1.57±0.02ab 1.46±0.05ab 1.43±0.03a 1.67±0.11b 1.45±0.11ab 1.39±0.06ab 1.48±0.63ab Geranial 33.42±0.08 33.29±0.03 33.27±0.05 33.27±0.03 33.32±0.03 NDa 33.24±0.03 Citronellol 7.79±0.01 7.76±0.01 7.75±0.01 7.80±0.01 7.76±0.01 7.78±0.03 7.72±0.03 β-Damascenone ND ND ND ND ND ND ND Farnesene ND ND ND ND ND ND ND Nerol ND ND ND ND ND ND ND 86 OCP-125 OCP-088 OCP-080 OCP-090 OCP-095 OCP-041 M2 α-ionone ND ND ND ND ND ND ND Geraniol 20.10±1.72 18.38±0.29 19.08±0.36 18.92±1.36 18.66±0.24 18.48±0.24 40.71±38.65 β-ionene ND ND ND ND ND ND ND Farnesol (isomer 1) ND ND ND ND ND ND ND Farnesol (isomer 2) ND ND ND ND ND ND ND Terpinolene* 5.03±0.08b 4.98±0.03ab 4.99±0.03ab 4.99±0.05ab 4.97±0.02a 4.95±0.01a 4.95±0.04a *Semi-quantified compounds <LOQ: Below limits of quantification. ND: Not detected or <LOD (below limits of detection) Table A.2. Concentration (µg/L) of volatile aroma compounds quantified by SPME-GC/MS in Pinot Noir laboratory scale fermentations, fermented by 7 Saccharomyces cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate, results show mean (µg/L) ± SD Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. OCP125 OCP088 OCP080 OCP090 OCP095 OCP041 M2 1-Propanol <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 2-Methyl-1-butanol <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 2-Methyl-1-propanol ND ND ND ND ND ND ND 87 OCP125 OCP088 OCP080 OCP090 OCP095 OCP041 M2 3-Methyl-1-butanol* 154652.7±22062.5a 131353.8±5504.14ab 128904.2±547.53ab 143945.8±13814.62ab 106851.6±10594.44b 128159.8±15618.04ab 153420±25496.10a 2-Hexanol ND ND ND ND ND ND ND 2-Heptanol ND ND ND ND ND ND ND 1-Hexanol 924.57±37.51abc 839.11±20.60ab 890.14±55.01abc 871.15±128.55ab 1042.17±6.76c 979.13±2.77bc 806.78±26.52a 3-E-Hexen-1-ol 22.48±3.71 26.30±1.96 26.09±2.48 23.51±3.72 24.32±4.49 24.24±2.13 23.31±4.15 3-Z-Hexen-1-ol <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 2-E-Hexen-1-ol 7.74±0.34 20.30±6.75a 7.87±1.12 6.54±2.40 6.10±1.14 7.36±2.01 4.30±1.79 2-Z-Hexen-1-ol <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 1-Octen-3-ol ND ND ND ND ND ND ND 1-Heptanol ND ND ND ND ND ND ND 2,3-Butanediol ND ND ND ND ND ND ND 1-Octanol 2.58±0.43ab 2.43±0.09ab 3.09±0.22a 2.21±0.67ab 2.53±0.58ab 2.90±0.70a 1.56±0.25b 1-Nonanol 13.62±1.32c 6.88±1.47a 8.48±0.66b 11.31±3.05bc 8.21±0.93ab 10.86±1.73bc 6.74±0.65a 2-Ethyl-1-hexanol* 22.50±1.63 17.10±6.98 14.05±6.14 23.36±8.11 24.12±6.55 20.14±8.31 12.60±5.40 Hotrienol <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Hexanal ND ND ND ND ND ND ND 2-(E)-Hexanal ND ND ND ND ND ND ND Octanal ND ND ND ND ND ND ND 88 OCP125 OCP088 OCP080 OCP090 OCP095 OCP041 M2 Nonanal <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Furfural ND ND ND ND ND ND ND 2-E-Nonenal ND ND ND ND ND ND ND 2,4-Nonadienal, (E,E)- ND ND ND ND ND ND ND 2,4-E,E-Hexadenial ND ND ND ND ND ND ND Hexanoic acid 32.1±6.29 31.60±2.62 53.80±5.32 37.10±4.31 39.20±9.81 35.42±10.42 46.28±17.72 Nonanoic acid* 165.84±36.17 206.09±11.25 247.81±40.40 201.46±6.67 192.70±70.36 203.76±42.51 186.52±41.44 Decanoic acid* 150.47±28.22 186.46±23.34 210.05±40.37 155.86±15.61 128.74±17.05 149.74±23.45 141.99±10.89 Methyl-2-methyl butanoate ND ND ND ND ND ND ND Ethyl butanoate 105.75±10.97ab 85.28±6.55a 93.63±6.35a 105.47ab 72.78±3.44a 114.69±8.80ab 141.89±7.21b Ethyl-2-methyl butanoate ND ND ND ND ND ND ND Ethyl isovalerate <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Isoamyl acetate 1270.78±166.68a 892.10±9.81ab 1133.71±36.51ab 1124.11±262.07a 549.059±129.67b 941.79±118.72ab 1319.00±1570.09a 89 OCP125 OCP088 OCP080 OCP090 OCP095 OCP041 M2 Isoamyl propionate <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Hexyl acetate 51.98±5.45a 27.78±6.77b 50.21±1.30a 38.40±1.91ab 28.30±7.09b 54.89±0.95a 41.24±4.61ab Ethyl heptanoate <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Ethyl dodecanoate 46.58±3.12ab 40.67±7.93bc 37.72±2.51bc 40.88bc 33.24±0.81bc 30.47±1.59c 58.01±8.11a Ethyl hexanoate* 265.55±60.94 216.93±25.67 255.36±41.43 251.98±18.27 225.62±46.04 257.91±22.01 306.11±17.70 Ethyl octanoate* 2370.51±641.81 1669.95±216.33 2295.91±269.44 2276.67±526.33 1630.54±447.70 1786.45±433.91 2519.82±791.88 Ethyl decanoate* 1521.88±569.43 1615.46±298.44 1603.33±192.91 1586.02±219.50 1006.41±388.14 1185.00±178.80 2194.36±699.60 2-Phenethyl acetate 95.9±15.56ab 106.9±5.74a 94.2±10.92ab 74.40±2.32ab 64.73±2.03b 87.8±13.00ab 90.84±5.37ab 2-Heptanone ND ND ND ND ND ND ND 6-Methyl-5-hepten-2-one ND ND ND ND ND ND ND 2-Nonanone <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 2-Undecanone ND ND ND ND ND ND ND Benzaldehyde ND ND ND ND ND ND ND 90 OCP125 OCP088 OCP080 OCP090 OCP095 OCP041 M2 Benzothiazole 246.18±17.50ac 373.60±80.06ab 387.72±58.54ab 327.60±48.48ab 417.25±34.79b 333.99±53.05ab 172.16±47.17c Benzyl alcohol 44.85±2.85 45.06±1.12 47.74±1.54 44.20±0.99 43.54±5.03 45.97±1.20 48.62±2.55 Phenylethyl alcohol 5003.12±116.39a 4373.12±167.03ab 4664.87±167.74a 4406.49±428.00ab 4184.60±627.29ab 3563.78±249.40b 4350.72±507.29ab Guaicol ND ND ND ND ND ND ND Eugenol 5.85±0.02 5.88±0.02 5.82±0.04 5.86±0.06 5.86±0.04 5.82±0.05 5.94±0.06 Phenylacetaldehyde 9.34±0.84 9.72±1.02 10.32±1.49 9.56±2.71 13.76±0.30a 10.58±0.83 9.12±0.2 α-(+)-pinene <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ β-(+)-pinene ND ND ND ND ND ND ND Sabinene ND ND ND ND ND ND ND 2-Carene ND ND ND ND ND ND ND α-(-)-Phellandrene <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Myrcene <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ α-Terpinene ND ND ND ND ND ND ND 1,4-Cineole ND ND ND ND ND ND ND s-Limonene <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ β-Phellandrene <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 91 OCP125 OCP088 OCP080 OCP090 OCP095 OCP041 M2 Eucalyptol ND ND ND ND ND ND ND γ-Terpinene 5.72±0.08 5.57±0.07 5.60±0.13 5.64±0.11 5.60±0.12 5.53±0.02 6.10±0.41 Ocimene (isomer 2) 0.64±0.13 0.25±0.14 0.15±0.06 0.23 0.47±0.06 0.27±0.11 0.65±0.22 Cymene <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ (+)-Rose oxide <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ (-)-Rose oxide <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Limonene oxide ND ND ND ND ND ND ND Linalool oxide (isomer 1) <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Linalool oxide (isomer 2) ND ND ND ND ND ND ND Linalool 5.47±0.80a 1.02±0.01b 4.53±1.12a <LOQb 8.38±0.71c 4.05±1.00a 5.98±0.23a Caryophyllene ND ND ND ND ND ND ND α-Humulene ND ND ND ND ND ND ND Neral ND ND ND ND ND ND ND α-Terpineol 32.81±0.58b 18.93±8.20ab 50.44±4.54c 15.30±5.47a 20.74±6.09ab 16.43±1.80a 22.63±5.88ab (+) Valencene ND ND ND ND ND ND ND γ-Terpineol 1.10±0.08 1.03±0.11 1.11±0.08 1.14±0.03 1.27±0.08 1.10±0.02 1.25±0.16 Geranial 134.17±7.50 133.65±0.22 130.86±3.29 135.60±12.12 134.75±3.93 132.07±11.38 127.16±2.75 92 OCP125 OCP088 OCP080 OCP090 OCP095 OCP041 M2 Citronellol 10.84±0.87 9.94±0.39 10.66±0.50 10.54±0.30 9.88±0.12 11.38±1.97 10.88±0.47 β-Damascenone ND ND ND ND ND ND ND Farnesene ND ND ND ND ND ND ND Nerol ND ND ND ND ND ND ND α-Ionone ND ND ND ND ND ND ND Geraniol 19.45±0.42ab 19.27±0.95ab 19.01±0.50ab 18.24±0.43a 19.19±0.73ab 18.76±0.91ab 20.60±0.84b β-Ionene ND ND ND ND ND ND ND Farnesol (isomer 1) ND ND ND ND ND ND ND Farnesol (isomer 2) ND ND ND ND ND ND ND Terpinolene* 5.47±0.04 5.36±0.03 5.42±0.06 5.36±0.04 5.36±0.02 5.37±0.05 5.43±0.09 *Semi-quantified compounds; <LOQ: Below limits of quantification; ND: Not detected or <LOD (below limits of detection). 93 Table A.3 Concentration (µg/L) of volatile aroma compounds quantified by SPME-GC/MS in Pinot Gris pilot-scale wines, fermented by two Saccharomyces cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate, results show mean (µg/L) ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. OCP-125 OCP-088 B-OCP-125 B-OCP-088 PG-SB PG-KG 2-Methyl-1-butanol ND ND ND ND ND ND 2-Methyl-1-propanol ND ND ND ND ND ND 3-Methyl-1-butanol 14562±240.5a 14724.15±2474.50a 126705.57±3782.36b 120577±15679.94b 132964.36±14638.40b 126556.81±10971.36b 2- Hexanol ND ND <LOQ <LOQ <LOQ <LOQ 2- Heptanol ND ND ND ND ND ND 1-Hexanol 1511.82±8.88ac 1480.25±166.24ac 1690.68±75.98ac 1844.10±68.37bc 2326.50±267.78b 1599.71±101.96ac 3-E-Hexen-1-ol 18.95±4.13ab 17.27±1.12a 26.20±4.05db 33.18±4.34d 22.41±1.10bc 21.16±0.72bc 3-Z-Hexen-1-ol NDa NDa <LOQb <LOQb 311.53±9.92c 162.04±8.35d 2-E-Hexen-1-ol <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 2-Z-Hexen-1-ol <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 1-Octen-3-ol ND ND ND ND ND ND 2-Methyl-1-propanol ND ND 253.84±17.58b 274.6±6.12c ND ND 1-Heptanol ND ND 139.15±22.19a 191.71±13.14b 139.45±14.42a 144.18±13.14a 2,3-Butanediol ND ND ND ND ND ND 94 OCP-125 OCP-088 B-OCP-125 B-OCP-088 PG-SB PG-KG 1-Propanol 175.96±12.97ab 149.74±36.31a 272.06±30.29c 233.91±15.16bc ND ND 2-Ethyl-1-hexanol 5.53±3.17ab 21.28±6.06b 1.12±0.14a 1.19±0.16a 16.53±2.94b 6.53±2.94b Hotrienol ND ND ND ND ND ND Hexanal ND ND ND ND ND ND 2-(E)-hexenal ND ND ND ND ND ND Octanal ND ND ND ND ND ND Nonanal ND ND ND ND ND ND Fufural ND ND ND ND ND ND 2-E-Nonenal ND ND ND ND ND ND 2-4-E,E-Nonadienal ND ND ND ND ND ND 2,4-E,E-Hexadienal ND ND ND ND ND ND Hexanoic acid 585.53±53.41 747.15±45.66 584.20±84.39 720.52±83.51 563.86±56.99 567.41±57.47 Nonanoic acid 2006.47±112.81a 2635.89±376.19b 2180.26±199.90ab 2171.15±154.43ab 235.95±1.07c 257.82±10.61c Decanoic acid 1009.98±120.41ab 1425.61±355.07a 525.12±110.35b 840.84±248.82b 84.56±2.80c 107.93±20.46c Methyl-2-methyl butanoate ND ND ND ND ND ND Ethyl butanoate 141.84±11.48a 142.88±12.24a 186.39±4.53b 203.76±3.60b 291.59±20.79c 343.18±28.61d Ethyl-2-methyl butanoate ND ND 6.99±0.80a 8.42±1.13a 8.25±1.37a 7.63±0.58a Ethyl isovalerate <LOQa <LOQa 8.88±1.39b 18.50±0.70c <LOQa 4.80±1.53d 95 OCP-125 OCP-088 B-OCP-125 B-OCP-088 PG-SB PG-KG Isoamyl acetate 2436.14±279.43a 1950.43±148.40b 1084.44±80.10c 841.02±14.78c 2385.50±313.76ab 2243.1±169.81ab Hexyl acetate 174.13±6.56a 164.16±25.16a ND ND 67.46±4.52b 52.79±5.80b Ethyl heptanoate <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 2-phenethyl acetate 375.84±2.62a 312.69±24.74a 116.34±12.46b 101.76±6.08b 57.90±5.65c 135.04±14.03b Ethyl dodecanoate 965.14±37.10a 836.92±115.80a 784.93±112.94ac 630.76±94.74abc 541.52±91.12bc 510.23±11.23bc Ethyl_hexanoate 562.94±96.16a 616.42±51.38ab 538.78±19.90a 727.46±23.97b 597.40±42.84a 501.10±60.06ab Ethyl_octanoate 4407.60±121.36a 4429.23±354.53a 5436.86±115.22a 6819.72±453.40b 5391.70±459.28a 5141.40±451.01a Ethyl_decanoate 4207.94±146.60ab 3513.65±186.65a 4680.96±569.73b 4924.99±503.44b 1847.96±256.32c 1279.28±62.90c 2-Heptanone ND ND ND ND ND ND 6-Methyl-5-hepten-2-one ND ND ND ND ND ND 2-Nonanone ND ND ND ND ND ND 2-Undecanone ND ND ND ND ND ND Benzaldehyde <LOQa 0.08±0.03a 0.45±0.04a 0.80±0.05a 5.66±0.22b 20.93±6.13c Phenylacetaldehyde <LOQa <LOQa 28.11±0.15b 24.96±3.27b 16.57±0.69c 18.48±5.85c Benzyl alcohol <LOQa <LOQa 49.54±8.09b 48.18±5.22b 86.02±5.25c 31.03±3.63b Guaicol ND ND ND ND ND ND Eugenol <LOQ ND ND ND ND ND Phenylethyl alcohol 2106.29±39.18a 2381.93±163.50a 1905.14±405.42a 2040.49±386.61a 39206.41±2031.004b 32190±501.55b α-(+)-Pinene ND ND ND ND ND ND 96 OCP-125 OCP-088 B-OCP-125 B-OCP-088 PG-SB PG-KG β-(+)-Pinene ND ND ND ND ND ND Sabinene ND ND ND ND ND ND 2-Carene ND ND ND ND ND ND α-(-)-Phellandrene <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Myrcene <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ α-Terpinene ND ND ND ND ND ND 1,4-Cineole ND ND ND ND ND ND S-Limonene <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ β-Phellandrene <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Eucalyptol ND ND ND ND ND ND γ-Terpinene 2.56±0.09a 2.51±0.03a 2.61±0.28a 2.59±0.19a 6.35±0.10b 6.40±0.02b Ocimene <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ (+)-Rose oxide <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ (-)-Rose oxide <LOQ ND <LOQ ND ND <LOQ Limonene oxide ND ND ND ND ND ND Linalool oxide (isomer 1) <LOQ <LOQ ND ND ND ND Linalool oxide (isomer 2) ND ND ND ND ND ND Linalool <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Caryophyllene ND ND ND ND ND ND 97 OCP-125 OCP-088 B-OCP-125 B-OCP-088 PG-SB PG-KG α-Humulene <LOQ ND ND ND ND ND Neral ND ND ND ND ND ND α-Terpineol 6.89±1.16ab 6.99±2.23ab 5.06±0.49b 10.01±1.60a 14.80±0.33c 7.78±0.90a (+) Valencene ND ND ND ND ND ND γ-Terpineol 38.11±7.78a 34.25±11.31a 22.24±0.75b 14.30±6.37b 6.28±1.21c 17.96±3.55b Geranial 100.44±7.44ac 70.35±0.90bd 85.94±11.08bc 90.53±3.14ad 48.23±9.52d 46.23±8.60d Citronellol 11.31±0.88a <LOQb 11.99±0.79a 12.02±2.10a 26.33±4.81b 19.90±5.63b β-Damascenone ND ND ND ND ND ND Farnesene ND ND ND ND ND ND Nerol ND ND ND ND ND ND α-Ionone ND ND ND ND ND ND Farnesol (isomer 1) ND ND ND ND ND ND Farnesol (isomer 2) ND ND ND ND ND ND Geraniol ND ND ND ND ND ND β-Ionene ND ND ND ND ND ND Terpinolene* 2.40±0.01a 2.39±0.0.1a <LOQb <LOQb ND ND *Semi-quantified compounds; <LOQ: Below limits of quantification; ND: Not detected or <LOD (below limits of detection). 98 Table A.4 Concentration (µg/L) of volatile aroma compounds quantified by SPME-GC/MS in Pinot Noir pilot-scale wines, fermented by two Saccharomyces cerevisiae yeast strains (3n per strain). Samples were analysed in triplicate, results show mean (µg/L) ± SD. Data was analyzed using a one-way ANOVA followed by a Tukey HSD post hoc test. Different letters indicate that strains are significantly different at p≤ 0.05 for each compound. PN-OCP125 PN-OCP088 BPN-OCP125 BPN-OCP088 FREEFORM VG 2-Methyl-1-butanol ND ND ND ND ND 2-Methyl-1-propanol ND ND ND ND ND 3-Methyl-1-butanol 137273.97±14459.55ab 146640.39±20199.59a 111881.83±12057.26ab 102960.65±6683.84b 109780.21±14696.67ab 2-Hexanol ND ND ND ND ND 2-Heptanol ND ND ND ND ND 1-Hexanol 1229.02±115.48a 1069.79±55.58a 1845.58±186.39b 1706.46±78.60b 1804.11±107.82b 3-E-hexen-1-ol 14.53±1.49a 13.69±1.11a 30.9±4.31b 33.03±1.34b 28.52±0.40b 3-Z-Hexen-1-ol ND ND <LOQ <LOQ <LOQ 2-E-Hexen-1-ol ND ND ND ND ND 2-Z-Hexen-1-ol ND ND ND ND ND 1-Octen-3-ol ND ND ND ND ND 2-Methyl-1-propanol ND ND 363.27±24.32a 376.92±26.28a 430.20±70.96a 1-Heptanol ND ND 131.22±23.79ab 159.85±8.03a 122.83±9.27b 1-Propanol 1343.87±146.62a 1278.18±281.58a 1292.23±93.86a 1595.11±183.42a 121.68±8.85b 2-Ethyl-1-hexanol 8.60±1.99a 3.53±0.33a 1.12±0.15a 2.65±0.09a 27.11±2.99b 2,3-Butanediol ND ND ND ND ND 99 PN-OCP125 PN-OCP088 BPN-OCP125 BPN-OCP088 FREEFORM VG 1-Octanol ND ND ND ND ND 1-Nonanol 8.14±2.10a 6.39±0.05a ND ND ND Hexanal ND ND ND ND ND 2-(E)-Hexenal ND ND ND ND ND Octanal ND ND ND ND ND Nonanal <LOQ <LOQ <LOQ <LOQ <LOQ Fufural ND ND ND ND ND 2-E-Nonenal ND ND ND ND ND 2-4-E,E-Nonadienal ND ND ND ND ND 2,4-E,E-Hexadienal ND ND ND ND ND Hexanoic acid 224.83±51.65a 241.72±13.31a 210.11±13.71a 247.93±18.55a 220.72±29.85a Nonanoic acid 729.49±77.42ab 677.67±9.10a 784.12±119.52ab 804.15±22.78b 96.49±18.32c Decanoic acid 392.59±242.41a 423.98±70.95a 388.85±86.26a 336.83±83.44a 34.42±7.71b Methyl-2-methyl butanoate ND ND ND ND ND Ethyl butanoate 197.70±11.77a 226.08±10.88a 201.09±20.18a 268.71±34.29a 458.03±13.62b Ethyl-2-methyl butanoate ND ND 3.52±0.79a 2.30±0.04a 11.64±0.72b Ethyl isovalerate <LOQb <LOQb <LOQb <LOQb 7.20±0.80a Isoamyl acetate 1742.38±189a 2276.33±176.20ab 2748.90±202.25b 3981.11±187.02c 3167.56±80.334d 100 PN-OCP125 PN-OCP088 BPN-OCP125 BPN-OCP088 FREEFORM VG Isoamyl propionate <LOQ <LOQ <LOQ <LOQ <LOQ Hexyl acetate 274.72±11.64a 275.92±5.12a 102.22±14.70b 116.87±6.31b 54.43±3.17c 2-Phenethyl acetate 235.01±47.62a 303.01±33.94b 169.61±18.41a 202.37±18.19a 205.24±26.45a Ethyl heptanoate <LOQ <LOQ <LOQ <LOQ <LOQ Ethyl dodecanoate 401.56±32.38a 306.12±31.01c 499.90±36.38ad 465.98±19.47d 64.34±12.52e Ethyl_hexanoate 736.11±38.29a 914.18±47.67b 682.10±6.40a 962.03±55.55b 503.09±36.54c Ethyl_octanoate 6257.45±159.26a 6232.67±183.51a 5033.09±599.50bc 5208.89±339.05b 4274.43±274.56c Ethyl_decanoate 5626.14±291.88a 4650.44±359.71b 4488.12±306.41b 4181.56±212.45b 1693.28±207.73c 2-Heptanone ND ND ND ND ND 6-Methyl-5-hepten-2-one ND ND ND ND ND 2-Nonanone ND ND ND ND ND 2-Undecanone ND ND ND ND ND Benzaldehyde <LOQ <LOQ <LOQ <LOQ <LOQ Benzothiazole ND ND ND ND ND Phenylacetaldehyde <LOQc <LOQc 11.82±6.97a 14.42±3.26ab 18.32±0.20b Benzyl alcohol 51.36±1.63a 41.19±0.63b 37.3±4.87b 41.22±3.89b 39.53±3.40b Phenylethyl alcohol 1735.97±393.98ab 1631.48±130.84a 2314±305.34b 1642.44±85.87a 40770.87±2124.66c Guaicol ND ND ND ND ND Eugenol ND ND ND ND ND α-(+)-Pinene <LOQ <LOQ ND ND ND 101 PN-OCP125 PN-OCP088 BPN-OCP125 BPN-OCP088 FREEFORM VG β-(+)-Pinene ND ND ND ND ND Sabinene ND ND ND ND ND 2-Carene ND ND ND ND ND β-(-)-phellandrene <LOQ <LOQ ND ND ND Myrcene <LOQ <LOQ ND ND ND α-terpinene ND ND ND ND ND 1,4-Cineole ND ND ND ND ND S-Limonene <LOQ <LOQ <LOQ <LOQ <LOQ β-Phellandrene <LOQ <LOQ <LOQ <LOQ <LOQ Eucalyptol ND ND ND ND ND Γ-terpinene ND ND ND ND ND Ocimene <LOQ <LOQ <LOQ <LOQ <LOQ Cymene <LOQ <LOQ <LOQ <LOQ <LOQ (+)-Rose oxide <LOQ <LOQ <LOQ <LOQ <LOQ (-)-Rose oxide ND ND ND ND ND Limonene oxide ND ND ND ND ND Linalool oxide (isomer 1) <LOQ <LOQ ND ND ND Linalool oxide (isomer 2) ND ND ND ND ND Linalool <LOQ <LOQ <LOQ <LOQ <LOQ 102 PN-OCP125 PN-OCP088 BPN-OCP125 BPN-OCP088 FREEFORM VG Caryophyllene ND ND ND ND ND A-humulene ND ND ND ND ND Neral ND ND ND ND ND Α-terpineol 8.81±2.68 1.52±0.04 7.46±1.37 18.62±11.85 10.33±1.78 (+) Valencene ND ND ND ND ND γ-Terpineol 23.99±5.99a 20.18±6.82a 16.9±1.11a 9.45±4.31a 10.41±1.90a Geranial 70.49±2.43a 68.74±3.17a 46.50±6.97b 48.32±3.92b 48.73±1.47b Citronellol 13.10±1.14 a 13.70±0.24a 19.18±2.39b 20.23±0.64b 25.23±3.12c β-Damascenone ND ND ND ND ND Farnesene ND ND ND ND ND Nerol ND ND ND ND ND α-Ionone ND ND ND ND ND Farnesol (isomer 1) ND ND ND ND ND Farnesol (isomer 2) ND ND ND ND ND Geraniol ND ND ND ND ND β-Ionene ND ND ND ND ND Terpinolene* <LOQ <LOQ <LOQ <LOQ <LOQ *Semi-quantified compounds; <LOQ: Below limits of quantification; ND: Not detected or <LOD (below limits of detection). 103 Figure A.1 Distribution of chemical composition of VOCs groups in Pinot Gris laboratory scale fermentations. 104 Figure A.2 Distribution of chemical composition of VOCs groups in Pinot Noir laboratory scale fermentations. 105 Table A.5 Calculated values for LOD (µg/L) and LOQ (µg/L) of lab-scale and pilot-scale fermentations. VOCs PG Lab-scale PN Lab-scale PN Pilot-scale PG Pilot-scale LOD LOQ LOD LOQ LOD LOQ LOD LOQ 1-propanol 4.364 14.401 2.336 7.710 2.235 7.375 0.526 1.737 2-methyl-1-butanol 0.865 2.853 1.418 4.679 0.622 2.054 0.401 1.324 2-methyl-1-propanol 3.940 13.003 4.203 13.870 5.952 19.641 8.440 27.850 3-methyl-1-butanol* 0.865 2.853 1.418 4.679 0.622 2.054 0.401 1.324 2-hexanol* 0.280 0.925 0.089 0.294 0.791 2.610 0.400 1.320 2-heptanol 0.010 0.031 0.014 0.045 0.010 0.032 0.009 0.028 1-hexanol 0.280 0.925 0.089 0.294 0.791 2.610 0.400 1.320 3-E-hexen-1-ol 0.113 0.373 0.196 0.646 0.085 0.281 0.114 0.376 3-Z-hexen-1-ol 0.113 0.373 0.196 0.646 0.085 0.281 0.114 0.376 2-E-hexen-1-ol 0.196 0.648 0.198 0.675 0.189 0.623 0.124 0.409 2-Z-hexen-1-ol 0.532 1.756 0.352 1.162 0.365 1.206 0.346 1.142 1-octen-3-ol 0.032 0.105 0.032 0.105 0.027 0.090 0.023 0.075 1-heptanol 0.147 0.483 0.151 0.499 0.113 0.374 0.120 0.397 2,3-butanediol 0.421 1.263 0.235 0.776 0.832 2.746 0.721 2.379 1-octanol 0.021 0.068 0.019 0.063 0.015 0.048 0.013 0.042 1-nonanol 0.014 0.047 0.016 0.053 0.012 0.041 0.011 0.035 2-ethyl-1-hexanol* 0.280 0.925 0.089 0.294 0.791 2.610 0.400 1.320 Hotrienol* 0.185 0.609 0.213 0.704 0.143 0.472 0.142 0.467 106 VOCs PG Lab-scale PN Lab-scale PN Pilot-scale PG Pilot-scale LOD LOQ LOD LOQ LOD LOQ LOD LOQ Hexanal 1.042 3.439 1.160 3.827 0.870 2.871 0.737 2.433 2-(E)-hexenal 0.150 0.494 0.203 0.669 0.178 0.586 0.139 0.457 Octanal 0.469 1.547 0.373 1.230 0.307 1.013 0.276 0.912 Nonanal 0.152 0.502 0.176 0.581 0.143 0.472 0.142 0.467 Fufural 42.181 139.199 40.369 133.216 38.407 126.744 37.126 122.515 2-E-nonenal 0.039 0.130 0.048 0.158 0.038 0.127 0.034 0.114 2-4-E,E-nonadienal 0.003 0.011 0.004 0.014 0.003 0.011 0.003 0.009 2,4-E,E-hexadienal 0.993 3.276 1.293 4.268 1.063 3.509 0.936 3.088 Hexanoic acid 0.006 0.021 0.006 0.021 0.015 0.051 0.213 0.702 Nonanoic_acid 0.201 0.664 0.157 0.519 0.133 0.438 0.007 0.024 Decanoic_acid 0.029 0.096 0.050 0.164 0.007 0.023 0.014 0.047 Methyl-2-methyl butanoate 0.133 0.440 0.140 0.461 0.116 0.383 0.110 0.362 Ethyl_butanoate 0.105 0.345 0.127 0.420 0.091 0.301 0.083 0.275 Ethyl-2-methyl butanoate 0.046 0.151 0.046 0.151 0.043 0.143 0.039 0.130 Ethyl_isovalerate 0.123 0.404 0.130 0.430 0.110 0.363 0.100 0.329 Isoamyl acetate 4.315 14.238 8.283 27.334 0.595 1.962 2.562 8.453 Isoamyl propionate 0.007 0.024 0.008 0.025 0.006 0.021 0.006 0.018 Hexyl acetate 0.012 0.038 0.014 0.046 0.014 0.047 0.012 0.040 Ethyl_heptanoate 0.001 0.004 0.002 0.008 0.002 0.008 0.002 0.006 107 VOCs PG Lab-scale PN Lab-scale PN Pilot-scale PG Pilot-scale LOD LOQ LOD LOQ LOD LOQ LOD LOQ Ethyl_dodecanoate 0.017 0.057 0.019 0.061 0.031 0.104 2.421 7.988 Ethyl_hexanoate* 0.001 0.004 0.002 0.008 0.002 0.008 0.002 0.006 Ethyl_octanoate* 0.001 0.004 0.002 0.008 0.002 0.008 0.002 0.006 Ethyl_decanoate* 0.001 0.004 0.002 0.008 0.002 0.008 0.002 0.006 2-phenylethyl_acetate 0.000 0.002 0.001 0.002 0.000 0.002 0.000 0.001 2-heptanone 0.003 0.011 0.004 0.012 0.003 0.010 0.003 0.008 6-methyl-5-hepten-2-one 1.389 4.584 1.999 6.597 1.161 3.831 1.260 4.159 2-nonanone 0.003 0.009 0.002 0.008 0.003 0.008 0.002 0.007 2-undecanone 0.000 0.001 0.000 0.001 0.000 0.001 0.000 0.001 Benzylaldehyde 0.558 1.842 0.529 1.744 0.450 1.483 0.427 1.410 Benzyl_alcohol 0.565 1.864 0.668 2.206 0.599 1.975 0.617 2.036 Phenylethyl_alcohol 1.737 5.732 13.749 45.371 1.938 6.397 1.417 4.676 Benzothiazole 0.018 0.060 0.025 0.082 0.019 0.063 0.018 0.059 Eugenol 0.074 0.244 0.120 0.395 0.102 0.338 0.086 0.283 Guaiacol 0.007 0.023 0.009 0.028 0.008 0.028 0.007 0.022 Phenylacetaldehyde 0.402 1.327 0.496 1.636 0.397 1.310 0.314 1.036 α-(-)-phellandrene 0.000 0.001 0.001 0.003 0.000 0.002 0.001 0.002 β-(+)-pinene 0.005 0.018 0.016 0.051 0.008 0.027 0.012 0.040 Sabinene 0.005 0.017 0.006 0.021 0.005 0.017 0.005 0.017 108 VOCs PG Lab-scale PN Lab-scale PN Pilot-scale PG Pilot-scale LOD LOQ LOD LOQ LOD LOQ LOD LOQ 2-carene 0.006 0.021 0.006 0.019 0.006 0.019 0.006 0.019 Myrcene 0.000 0.001 0.000 0.002 0.000 0.001 0.000 0.001 α-Terpinene 0.000 0.001 0.000 0.001 0.000 0.001 0.000 0.001 1,4-cineole 0.002 0.008 0.003 0.009 0.002 0.008 0.002 0.006 S-limonene 0.000 0.001 0.000 0.001 0.000 0.001 0.000 0.001 B-phellandrene 0.011 0.037 0.016 0.051 0.007 0.023 0.009 0.028 Eucalyptol 0.021 0.071 0.018 0.059 0.014 0.047 0.013 0.045 γ-Terpinene 0.000 0.001 0.000 0.002 0.000 0.001 0.000 0.001 Ocimene (isomer 2) 0.002 0.005 0.002 0.006 0.001 0.005 0.001 0.005 Cymeme 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 (+)-Rose_oxide(isomer_1) 0.001 0.004 0.001 0.004 0.001 0.004 0.001 0.004 (+)-Rose_oxide(isomer_2) 0.000 0.001 0.001 0.002 0.000 0.001 0.000 0.001 Limonene oxide 0.035 0.116 0.002 0.007 0.019 0.063 0.064 0.211 Linalool_oxide(isomer1) 0.835 2.755 0.016 0.053 2.920 9.637 0.196 0.646 Linalool_oxide(isomer2) 0.229 0.755 0.164 0.541 0.214 0.708 0.151 0.499 Linalool 0.185 0.609 0.213 0.704 0.143 0.472 0.142 0.467 Caryophyllene 0.000 0.001 0.001 0.002 0.001 0.002 0.001 0.004 α-Humelene 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.001 Neral 0.016 0.052 0.015 0.051 0.008 0.028 0.115 0.380 109 VOCs PG Lab-scale PN Lab-scale PN Pilot-scale PG Pilot-scale LOD LOQ LOD LOQ LOD LOQ LOD LOQ α-Terpineol 0.007 0.023 0.009 0.030 0.008 0.027 0.007 0.024 γ-Terpineol 0.068 0.223 0.046 0.151 0.038 0.124 0.021 0.069 Geranial 0.407 1.343 1.525 5.034 0.424 1.400 0.711 2.346 (+)-Citronellol 0.109 0.359 0.147 0.486 0.087 0.288 0.083 0.272 β-Damascenone 0.008 0.028 0.009 0.028 0.007 0.025 0.007 0.022 Farnesene 0.029 0.094 0.032 0.106 0.029 0.096 0.029 0.094 Nerol 0.053 0.174 0.083 0.274 0.055 0.181 0.056 0.185 α-ionone 0.001 0.004 0.016 0.053 0.001 0.004 0.001 0.004 Geraniol 0.013 0.042 0.015 0.048 0.016 0.053 0.013 0.044 β-ionene 0.006 0.018 0.007 0.023 0.006 0.018 0.006 0.018 Farnesol(isomer1) 0.019 0.061 0.027 0.089 0.026 0.085 0.009 0.028 Farnesol(isomer2) 0.228 0.751 0.531 1.753 0.661 2.182 0.443 1.460 Terpinolene* 0.000 0.001 0.000 0.002 0.000 0.001 0.000 0.001 *Semi-quantified compounds 110 Table A.6 Concentration (µg/L) of volatile aroma compounds quantified by SPME-GC/MS in Pinot Noir and Pinot Gris juice, in vintage 2019 and 2022. Samples were analysed in triplicate, results show mean (µg/L) ± SD. Pinot Gris 2019 Pinot Noir 2019 Pinot Gris 2022 Pinot Noir 2022 1-propanol ND ND ND ND 2-methyl-1-butanol ND 21.23±0.73 ND ND 2-methyl-1-propanol ND ND ND ND 3-methyl-1-butanol 79.02±13.18 61.07±13.71 92.33±22.13 259.91±23.92 6-methyl-5-hepten-2-one ND ND ND ND 1-hexanol <LOQ <LOQ <LOQ <LOQ 2-nonanone ND ND ND ND Nonanal 0.60±0.01 <LOQ 3.29±0.12 0.32±0.03 2-E-hexen-1-ol ND ND ND ND Furfural ND ND ND ND 1-octen-3-ol ND ND ND ND Decanal 0.86±0.02 1.23±0.33 0.90±0.08 0.99±0.11 2-6-E-Z-nonadienal ND ND ND ND Neral ND ND ND ND 2-Ethyl-1-hexanol ND ND ND ND Hotrienol ND ND ND ND 3-E-hexen-1-ol 3591.36±238.94 2679.57±240.96 5245.04±418.26 3754.21±806.13 Hexanal ND ND ND ND 111 Pinot Gris 2019 Pinot Noir 2019 Pinot Gris 2022 Pinot Noir 2022 2-(E)-hexenal ND ND ND ND Hexyl acetate <LOQ <LOQ <LOQ <LOQ 3-Z-hexen-1-ol ND ND ND ND Linalool oxide (isomer1) ND ND ND ND Benzaldehyde ND ND ND ND 2,3-butanediol ND ND ND ND Caryophyllene ND 59.27±3.16 ND ND 1-nonanol ND ND ND ND Hexanoic acid ND ND ND ND Nonanoic acid ND ND ND ND Decanoic acid ND ND ND ND Methyl-2-methyl butanoate 22.84±0.03 20.56±0.01 ND ND Ethyl butanoate ND ND 4.43±0.16 31.88±1.16 Ethyl-2-methyl butanoate 0.44±0.01 0.44±0.02 ND ND Ethyl isovalerate ND ND ND ND Isoamyl acetate <LOQ <LOQ ND ND Isoamyl propionate ND ND ND ND Cymene <LOQ <LOQ <LOQ <LOQ 2-heptanol ND ND ND ND Ethyl dodecanoate ND ND ND ND 112 Pinot Gris 2019 Pinot Noir 2019 Pinot Gris 2022 Pinot Noir 2022 Ethyl_hexanoate <LOQ <LOQ ND ND Ethyl_octanoate ND ND ND ND Ethyl_decanoate <LOQ <LOQ <LOQ <LOQ 2-heptanone ND ND ND ND Octanal <LOQ <LOQ 5.16±1.92 6.97±0.49 2,4-e,e-hexadienal 24.88±1.42 21.24±1.99 19.06±3.40 46.99±10.73 1-octanol ND ND ND ND Linalool oxide (isomer2) 11.71±0.90 6.73±0.90 11.82±3.04 18.75±1.14 α-ionone ND ND ND ND Benzyl alcohol 11.75±3.04 12.30±1.53 35.08±8.80 39.55±12.21 Phenylethyl alcohol 354.94±129.28 400.13±150.70 347.93±65.58 312.91±42.44 Benzothiazole ND ND ND ND Eugenol 12.99±4.43 17.48±3.88 ND ND 2-undecanone ND ND ND ND Farnesene 11.98±1.92 <LOQ ND ND (-)-Rose oxide (isomer 2) ND <LOQ <LOQ <LOQ α-(+)-pinene ND ND ND ND β-(+)-pinene ND ND ND ND Sabinene ND ND ND ND 2-carene ND ND ND ND 113 Pinot Gris 2019 Pinot Noir 2019 Pinot Gris 2022 Pinot Noir 2022 α-(-)-phellandrene <LOQ <LOQ <LOQ <LOQ Myrcene <LOQ <LOQ <LOQ <LOQ α-terpinene ND ND ND ND 1,4-cineole ND ND ND ND S-limonene <LOQ <LOQ <LOQ <LOQ β-phellandrene 4.12±0.02 20.85±3.40 <LOQ <LOQ Eucalyptol ND ND ND ND γ-Terpinene 13.19±2.30 24.36±0.84 10.69±3.21 13.78±0.66 2-hexanol ND ND ND ND Ocimene (isomer 2) ND ND ND ND Ethyl heptanoate ND <LOQ ND ND (+)-Rose oxide (isomer 1) ND ND ND ND 2-z-hexen-1-ol 3566.53±320.78 2183.40±200.06 ND ND Limonene oxide ND ND ND ND 1-heptanol ND ND ND ND 2-E-nonenal ND ND ND ND Linalool ND ND ND ND Phenylacetaldehyde ND ND ND ND α-Humulene ND ND ND ND 2,4-nonadienal, (e,e)- 13.53±0.88 14.07±4.50 13.24±1.20 14.28±2.32 114 Pinot Gris 2019 Pinot Noir 2019 Pinot Gris 2022 Pinot Noir 2022 α-terpineol ND ND ND ND (+) Valencene 4.44±0.25 6.79±1.33 ND ND Y-terpineol ND ND ND ND Geranial ND ND ND ND Citronellol 6.08±2.08 5.75±1.08 4.48±0.64 3.94±0.93 β-damascenone ND ND ND ND 2-phenethyl acetate ND ND ND ND Nerol ND ND ND ND Guaiacol ND ND ND ND Geraniol 52.60±0.86 63.17±12.40 21.47±3.86 61.97±9.27 β-ionene ND ND ND ND Farnesol (isomer1) ND ND ND ND Farnesol (isomer 2) ND ND ND ND Terpinolene 22.46±8.14 22.46±0.30 11.05±4.30 35.40±0.55
Thesis/Dissertation
eng
Food Science
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Measday, Vivien
Suitability of indigenous Saccharomyces cerevisiae strains isolated from spontaneous fermentations as wine fermentation starter cultures
Text