MODULATION OF PAPAYA WINE FLAVOUR COMPOUND FORMATION BY YEASTS LEE PIN ROU NATIONAL UNIVERSITY OF SINGAPORE 2012 MODULATION OF PAPAYA WINE FLAVOUR COMPOUND FORMATION BY YEASTS LEE PIN ROU (B. Appl. Sc.(Hons.), National University of Singapore) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2012 THESIS DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety, under the supervision of Dr Liu Shao Quan, (in the Food Science and Technology research laboratory, S13-05), Chemistry Department, National University of Singapore, between January 2009 and November 2012. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. The content of the thesis has been partly published in: 1. Lee, P. R., Chong, I. S. M., Yu, B., Curran, P., & Liu, S. Q. (2012). Effects of sequentially inoculated Williopsis saturnus and Saccharomyces cerevisiae on volatile profiles of papaya wine. Food Research International, 45, 177–183. 2. Lee, P. R., Kho, S. H. C., Yu, B., Curran, P., & Liu, S. Q. (2012). Yeast ratio is a critical factor for sequential fermentation of papaya wine by Williopsis saturnus and Saccharomyces cerevisiae. Microbial Biotechnology. (In press). DOI:10.1111/1751-7915.12008. 3. Lee, P. R., Yu, B., Curran, P., & Liu, S. Q. (2011). Impact of amino acid addition on aroma compounds in papaya wine fermented with Williopsis mrakii. South African Journal of Enology and Viticulture, 32, 220–228. 4. Lee, P. R., Yu, B., Curran, P., & Liu, S. Q. (2011). Effect of fusel oil addition on volatile compounds in papaya wine fermented with Williopsis saturnus var. mrakii NCYC 2251. Food Research International, 44, 1292–1298. i 5. Lee, P. R., Li, X., Yu, B., Curran, P., & Liu, S. Q. (2011). Non-Saccharomyces yeasts and wine. In: A. S. Peeters (Ed.), Wine: Types, Production and Health (Chapter 12). Hauppauge, New York, USA: Nova Science Publishers. ISBN: 978-1-61470-804-9. 6. Lee, P. R., Ong, Y. L., Yu, B., Curran, P., & Liu, S. Q. (2010). Evolution of volatile compounds in papaya wine fermented with three Williopsis saturnus yeasts. International Journal of Food Science and Technology, 45, 2032–2041. 7. Lee, P. R., Ong, Y. L., Yu, B., Curran, P., & Liu, S. Q. (2010). Profile of volatile compounds during papaya juice fermentation by a mixed culture of Saccharomyces cerevisiae and Williopsis saturnus. Food Microbiology, 27, 853–861. 8. Lee, P. R., Yu, B., Curran, P., & Liu S. Q. (2010). Kinetics of volatile organic compounds during papaya juice fermentation by three commercial wine yeasts. Nutrition & Food Science, 40, 566–580. Lee Pin Rou Name Signature 1st November 2012 Date ii ACKNOWLEDGEMENTS First and foremost, I would like to express my deepest and sincere gratitude to my supervisor Dr Liu Shao Quan for his valuable advice, supervision, guidance and encouragements throughout the entire project. I feel honoured to have him as my supervisor and I have learned a lot from his valuable ideas, profound knowledge, and rich research experience. I am also deeply grateful for his kindness and patience throughout the four years and his countless efforts in guiding me to complete my research project and thesis. Next, I would like to extend my gratitude to Dr Yu Bin, who has provided me with meticulous guidance, and timely advice on the method development for the solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS). I would also like to sincerely thank the flavourists from Firmenich for their precious time and efforts during the wine sensory evaluations. I would like to express my warmest appreciation to my previous honours year and UROPS students, Miss Ong Yuen-Ling Irene, Miss Irene Saksono, Miss Chong Siew-May Irene, Miss Kho Hui-Chern Stephanie, Mr Saputra Anthony and Mr Toh Mingzhan, for their help and commitment in conducting some fermentation experiments in my research project. I would also like to take this opportunity to thank my fellow postgraduates, Mr Li Xiao, Ms Sun Jingcan, Ms Cheong Mun Wai, Ms Chan Li Jie and Ms Chen Dai, for their advice, technical assistance and moral support rendered throughout the project. I am also grateful for all the technical assistance provided by the staff and laboratory technicians of the Food Science and Technology Programme. Last but not least, I would like to thank my family members and my husband for their unwavering support and encouragement in my life. iii PUBLICATIONS LIST List of publications derived from this thesis 1. Lee, P. R., Chong, I. S. M., Yu, B., Curran, P., & Liu, S. Q. (2013). Effect of precursors on volatile compounds in papaya wine fermented by mixed yeasts. Food Technology and Biotechnology, 51, 92–100. 2. Lee, P. R., Chong, I. S. M., Yu, B., Curran, P., & Liu, S. Q. (2012). Effects of sequentially inoculated Williopsis saturnus and Saccharomyces cerevisiae on volatile profiles of papaya wine. Food Research International, 45, 177–183. 3. Lee, P. R., Kho, S. H. C., Yu, B., Curran, P., & Liu, S. Q. (2012). Yeast ratio is a critical factor for sequential fermentation of papaya wine by Williopsis saturnus and Saccharomyces cerevisiae. Microbial Biotechnology. (In press). DOI:10.1111/1751-7915.12008. 4. Chan, L. J., Lee, P. R., Li, X., Chen, D., Liu, S. Q., & Trinh, T. T. T. (2012). Tropical fruit wine: an untapped opportunity. In D. Cabel (Ed.), Food & Beverage Asia. Dec/Jan 2011/2012 (pp. 48–51). Singapore: Pablo Publishing Pte Ltd. ISSN: 2010-2364. 5. Lee, P. R., Yu, B., Curran, P., & Liu, S. Q. (2011). Impact of amino acid addition on aroma compounds in papaya wine fermented with Williopsis mrakii. South African Journal of Enology and Viticulture, 32, 220–228. 6. Lee, P. R., Yu, B., Curran, P., & Liu, S. Q. (2011). Effect of fusel oil addition on volatile compounds in papaya wine fermented with Williopsis saturnus var. mrakii NCYC 2251. Food Research International, 44, 1292–1298. 7. Lee, P. R., Li, X., Yu, B., Curran, P., & Liu, S. Q. (2011). Non-Saccharomyces yeasts and wine. In: A. S. Peeters (Ed.), Wine: Types, Production and Health (Chapter 12). Hauppauge, New York, USA: Nova Science Publishers. ISBN: 978-1-61470-804-9. (In press) iv 8. Lee, P. R., Ong, Y. L., Yu, B., Curran, P., & Liu, S. Q. (2010). Evolution of volatile compounds in papaya wine fermented with three Williopsis saturnus yeasts. International Journal of Food Science and Technology, 45, 2032–2041. 9. Lee, P. R., Ong, Y. L., Yu, B., Curran, P., & Liu, S. Q. (2010). Profile of volatile compounds during papaya juice fermentation by a mixed culture of Saccharomyces cerevisiae and Williopsis saturnus. Food Microbiology, 27, 853–861. 10. Lee, P. R., Yu, B., Curran, P., & Liu S. Q. (2010). Kinetics of volatile organic compounds during papaya juice fermentation by three commercial wine yeasts. Nutrition & Food Science, 40, 566–580. List of related publications 1. Lee, P. R., Toh, M., Yu, B., Curran, P., & Liu, S. Q. (2013). Manipulation of volatile compound transformation in durian wine by nitrogen supplementation. International Journal of Food Science and Technology, 48, 650–662. 2. Lee, P. R., Boo, C. X., & Liu, S. Q. (2013). Fermentation of coconut water by probiotic Lactobacillus acidophilus and Lactobacillus casei. Annals of Microbiology. (In press). DOI: 10.1007/s13213-013-0607-z. 3. Lee, P. R., Tan, R. M., Yu, B., Curran, P., & Liu, S. Q. (2012). Sugars, organic acids, and phenolic acids of exotic seasonable tropical fruits. Nutrition & Food Science. (In press). 4. Lee, P. R., Saputra, A., Yu, B., Curran, P., & Liu, S. Q. (2012). Biotransformation of durian pulp by mono- and mixed-cultures of Saccharomyces cerevisiae and Williopsis saturnus. LWT-Food Science and Technology, 46, 84–90. v 5. Tan, A. W. J., Lee, P. R., Seow, Y. X., Ong, P. K. C., & Liu, S. Q. (2012). Volatile sulphur compounds and pathways of L-methionine catabolism in Williopsis yeasts. Applied Microbiology and Biotechnology, 95, 1011–1020. 6. Lee, P. R., Saputra, A., Yu, B., Curran, P., & Liu, S. Q. (2012). Effects of Saccharomyces cerevisiae and Williopsis saturnus on the volatile and aromatic profiles of grape wine. Food Biotechnology, 26, 307–325. vi TABLE OF CONTENTS Page THESIS DECLARATION . i ACKNOWLEDGEMENTS . iii PUBLICATIONS LIST iv SUMMARY xiii LIST OF TABLES .xv LIST OF FIGURES xviii LIST OF ABBREVIATIONS .xxvi CHAPTER Introduction 1.1 Basic knowledge of wine and winemaking . 1.2 History and trends of tropical fruit wine fermentation 1.3 Objectives of project CHAPTER Literature review 2.1 Nutritional information of papaya fruit 2.1.1 General information of papaya 2.1.2 Non-volatile composition of papaya 2.1.2.1 Nutritional composition of papaya 2.1.2.2 pH and organic acid composition of papaya .10 2.1.2.3 Sugar composition of papaya .11 2.1.2.4 Amino acid and phenolic acid composition of papaya 11 2.1.3 Volatile composition of papaya 13 2.2 Wine fermentation 16 2.2.1 Biochemistry of alcoholic fermentation 16 2.2.2 Volatile compounds produced during wine fermentation 18 2.2.2.1 Analysis of volatile compounds in wine .21 vii 2.2.3 Yeast strains and evolution of inoculation strategies in wine fermentation 22 2.2.4 Fermentation conditions affecting yeast growth and evolution 25 CHAPTER Materials and Methods 3.1 Materials, yeast strains and culture media .30 3.2 Preparation and pretreatment of papaya juice 31 3.3 Preparation of starter cultures and fermentation conditions .32 3.4 Analytical determinations and yeast enumeration 34 3.5 Volatile compound analysis 36 3.6 Sensory analysis .38 3.7 Statistical analysis .38 CHAPTER Dynamics of volatile compounds during papaya juice fermentation by three commercial wine yeasts 4.1 Introduction 40 4.2 Results and discussion 41 4.2.1 Fermentation profiles of three commercial wine yeasts .41 4.2.2 Volatile profiles of papaya juice .45 4.2.3 Dynamic changes of volatile compounds during papaya juice fermentation .47 4.2.4 Principal component analysis .56 4.3 Conclusions 57 CHAPTER Evolution of volatile compounds in papaya wine fermented with three Williopsis saturnus yeasts 5.1 Introduction 58 5.2 Results and discussion 59 viii Appendix G Supplementary figures and tables for Chapter (Effects of sequentially inoculated Williopsis saturnus var. mrakii NCYC2251 and Saccharomyces cerevisiae var. bayanus R2 on volatile profiles of papaya wine) 12 10 o Brix (%) 0 12 15 18 21 12 15 18 21 Time (days) pH 0 Time (days) Fig. G1. Brix and pH changes during mixed and sequential fermentations of S. cerevisiae var. bayanus R2 and W. saturnus var. mrakii NCYC2251 in papaya wine. Mixed-culture (); positive sequential (▲); negative sequential (■). Positive and negative sequential fermentations are defined as in Fig. 9.1. (Error bars = standard deviation). 245 Butyric acid Isobutyric acid 80 G C -F ID p e a k a r e a (x 0 0 ) G C - FID p e a k a re a (x1 000 0) 3000 60 2000 40 1000 20 0 12 15 18 21 18 21 15 18 21 15 18 21 9- Decenoic acid 750 500 250 12 15 18 21 T ime (days) Decanoic acid 6000 4000 2000 12 Time (days) Dodecanoic acid 1500 G C -F ID p ea k ar ea (x 0 0 ) 8000 GC-FID peak area (x10000) 15 1000 G C -FI D p ea k a rea (x 0 0 ) G C-F ID p ea k a rea (x 0 0 ) Hexanoic acid 700 600 500 400 300 200 100 12 T ime (days) T ime (days) 1200 900 600 300 0 12 T ime (days) 15 18 21 12 Time (days) Fig. G2. Changes of fatty acids during mixed and sequential fermentations of S. cerevisiae var. bayanus R2 and W. saturnus var. mrakii NCYC2251 in papaya wine. Mixed-culture (); positive sequential (▲); negative sequential (■). Positive and negative sequential fermentations are defined as in Fig. 9.1. (Error bars = standard deviation). 246 Ethanol 400000 Active amyl alcohol 900 G C- FI D p eak area (x 0 0 ) G C-FID p eak ar ea (x 10 00 ) 300000 200000 100000 600 300 12 15 18 21 Time (days) 12 15 18 1-Propanol 200 GC-FID peak area (x10000) T ime (days) 150 100 50 0 12 15 18 21 T ime (days) Fig. G3. Changes of alcohols during mixed and sequential fermentations of S. cerevisiae var. bayanus R2 and W. saturnus var. mrakii NCYC2251 in papaya wine. Mixed-culture (); positive sequential (▲); negative sequential (■). Positive and negative sequential fermentations are defined as in Fig. 9.1. (Error bars = standard deviation). 247 21 Methyl acetate Ethyl acetate 30000 1200 GC-FID p eak area (x 10 00 ) GC-FID peak area (x10000) 1400 1000 800 600 400 20000 10000 200 0 12 15 18 21 Propyl acetate 400 300 200 12 15 T ime (days) 18 21 12 15 18 21 15 18 21 T ime (days) Isobutyl acetate Active amyl acetate 1800 GC-FID peak area (x10 000) 4500 GC-FID peak area (x 00 00 ) 21 60 30 18 90 100 15 Butyl acetate 120 GC-FID peak area (x10000) GC-FID peak area (x100 00) 500 12 Time (days) T ime (days) 3600 2700 1800 1200 600 900 0 12 15 18 21 T ime (days) 12 Benzyl acetate 400 GC-FID p eak area (x 0 0 ) T ime (days) 300 200 100 0 12 15 18 21 T ime (days) Fig. G4. Changes of acetate esters during mixed and sequential fermentations of S. cerevisiae var. bayanus R2 and W. saturnus var. mrakii NCYC2251 in papaya wine. Mixed-culture (); positive sequential (▲); negative sequential (■). Positive and negative sequential fermentations are defined as in Fig. 9.1. (Error bars = standard deviation). 248 Ethyl butyrate Ethyl hexanoate 1500 200 G C-FID p eak area (x 10 0 0) GC-FID p eak area (x 0 0 ) 250 150 100 50 1200 900 600 300 12 15 18 21 Ethyl 9-decenoate 6000 15 18 21 3000 15 18 21 18 21 6000 4000 2000 0 12 15 T ime (days) 18 21 Ethyl tetradecanoate 250 200 150 100 50 12 T ime (days) Ethyl 9-hexadecenoate 400 GC-FID peak area (x100 00) GC-FID p eak area (x1 000 0) 12 Ethyl dodecanoate 8000 GC-FID peak area (x10000) GC-FID peak area (x10000) 9000 T ime (days) T ime (days) 300 200 100 T ime (days) 12 15 21 12 15 T ime (days) Ethyl hexadecanoate 400 GC-FID peak area (x10000) 18 300 200 100 0 12 15 18 21 T ime (days) Fig. G5. Changes of ethyl esters during mixed and sequential fermentations of S. cerevisiae var. bayanus R2 and W. saturnus var. mrakii NCYC2251 in papaya wine. Mixed-culture (); positive sequential (▲); negative sequential (■). Positive and negative sequential fermentations are defined as in Fig. 9.1. (Error bars = standard deviation). 249 Methyl octanoate Methyl dodecanoate 200 GC-FID peak area (x10000) GC-FID peak area (x10000) 300 200 100 150 100 50 0 12 15 18 21 T ime (days) 2-Methylbutyl hexanoate 50 15 18 21 25 15 18 21 18 21 100 50 12 15 18 21 Time (days) 12 T ime (days) Isobutyl decanoate Isoamyl decanoate 600 GC-FID p eak area (x 1000 0) 160 G C-FID peak area (x 10 00 ) 12 Isobutyl octanoate 150 GC-FID peak area (x10000) GC-FID peak area (x 10 00 ) 75 T ime (days) 120 80 40 500 400 300 200 100 12 15 18 21 Time (days) 12 15 Propyl decanoate 80 GC-FID peak area (x10000) Time (days) 60 40 20 0 12 15 18 21 T ime (days) Fig. G6. Changes of methyl and other esters during mixed and sequential fermentations of S. cerevisiae var. bayanus R2 and W. saturnus var. mrakii NCYC2251 in papaya wine. Mixed-culture (); positive sequential (▲); negative sequential (■). Positive and negative sequential fermentations are defined as in Fig. 9.1. (Error bars = standard deviation). 250 O-Tolylaldehyde 2,4-Dimethyl benzaldehyde 300 400 GC-FID peak area (x10000) GC-FID peak area (x10000) 500 300 200 200 100 100 12 15 18 21 T ime (days) Benzaldehyde 12 15 18 21 18 21 18 21 3-Hydroxy-2-butanone 200 300 150 GC-FID peak area (x10000) GC-FID peak area (x10000) 400 T ime (days) 200 100 100 50 12 15 18 21 T ime (days) β-Damascenone 80 12 15 T ime (days) Benzyl isothiocyanate 700 GC-FID p eak area (x 1000 0) GC-FID peak area (x10000) 600 60 40 20 500 400 300 200 100 12 15 18 21 Time (days) 12 15 2,4-Di-tert-butylphenol 210 GC-FID peak area (x10000) Time (days) 140 70 0 12 T ime (days) 15 18 21 Fig. G7. Changes of miscellaneous volatiles during mixed and sequential fermentations of S. cerevisiae var. bayanus R2 and W. saturnus var. mrakii NCYC2251 in papaya wine. Mixed-culture (); positive sequential (▲); negative sequential (■). Positive and negative sequential fermentations are defined as in Fig. 9.1. (Error bars = standard deviation). 251 Table G1. Modified frequency (MF%) value of sensory descriptors for the papaya wines (day 21) fermented by mixed and sequential fermentations of S. cerevisiae var. bayanus R2 and W. saturnus var. mrakii NCYC2251 in papaya wine. Sensory descriptors Mixed culture Positive sequentiala Negative sequentialb Acidic 60.00 56.57 60.00 Alcoholic 87.18 69.28 77.46 Buttery 66.33 50.60 69.28 Cocoa 52.15 40.00 52.92 Fruity 74.83 95.92 77.46 Fusel 80.00 72.11 78.74 Sweet 82.46 77.46 69.28 Yeasty 84.85 81.24 80.00 a Inoculation of S. cerevisiae after days’ fermentation with W. saturnus. b Inoculation of W. saturnus after days’ fermentation with S. cerevisiae. Table G2. Sensory parameters of papaya wines (day 21) fermented by mixed and sequential fermentations of S. cerevisiae var. bayanus R2 and W. saturnus var. mrakii NCYC2251 in papaya wine. Sensory descriptors Mixed culture Positive sequentialc Negative sequentiald Acidic 1.80 ± 0.84a 1.60 ± 1.34a 1.80 ± 0.84a a b Alcoholic 3.80 ± 0.45 2.40 ± 0.89 3.00 ± 0.71ab Buttery 2.20 ± 1.10a 1.60 ± 0.89a 2.40 ± 0.89a a a Cocoa 1.70 ± 1.30 1.00 ± 0.71 1.40 ± 0.55a Fruity 2.80 ± 0.45a 4.60 ± 0.89b 3.00 ± 0.00a Fusel 3.20 ± 0.84a 2.60 ± 0.89a 3.10 ± 0.74a a a Sweet 3.40 ± 0.89 3.00 ± 0.00 2.40 ± 0.55a Yeasty 3.60 ± 0.55a 3.30 ± 0.97a 3.20 ± 0.84a a,b Statistical analysis at 95% confidence level with same letters indicating no significant difference. c Inoculation of S. cerevisiae after days’ fermentation with W. saturnus. d Inoculation of W. saturnus after days’ fermentation with S. cerevisiae. 252 Appendix H Supplementary figures for Chapter 10 (Yeast ratio is a critical factor for sequential fermentation of papaya wine by Williopsis saturnus var. mrakii NCYC2251 and Saccharomyces cerevisiae var. bayanus R2) Isobutyric acid Butyric acid 6000 200 GC-FID peak area (x10000) GC-FID peak area (x10000) 250 150 100 50 4000 2000 0 12 15 18 Hexanoic acid 1000 GC-FID peak area (x10000) GC-FID peak area (x10000) 12 15 18 12 15 18 15 18 Decanoic acid 4000 800 600 400 200 3000 2000 1000 0 12 15 18 Benzoic acid 50 T ime (days) T ime (days) Dodecanoic acid 500 GC-FID peak area (x10000) 40 GC-FID peak area (x10000) T ime (days) T ime (days) 30 20 10 400 300 200 100 12 15 18 T ime (days) 12 Tetradecanoic acid 60 50 (x10000) GC-FID peak area T ime (days) 40 30 20 10 0 12 15 18 T ime (days) Fig. H1. Changes of fatty acids during papaya wine sequential fermentation inoculated with different ratios of W. saturnus var. mrakii NCYC2251 and S. cerevisiae var. bayanus R2. 10:1 ratio (♦); 1:1 ratio (▲); 1:10 ratio (■). (Error bars = standard deviation). 253 Ethanol 700000 G C-FID p eak area (x 0 0 ) 600000 G C -FID p eak area (x 0 0 ) 1-Propanol 800 600 500000 400000 400 300000 200000 200 100000 12 15 18 Time (days) Active amyl alcohol G C -FID p eak are a (x 0 0 ) G C - F ID p e a k ar e a (x 0 0 ) 12 15 18 Isobutyl alcohol 2500 1800 1200 600 2000 1500 1000 500 12 15 18 Time (days) 12 15 18 Time (days) 2-Ethyl hexanol 1-Decanol 270 G C -F ID p ea k are a (x10000) 120 G C-FID p eak area (x 0 0 ) Time (days) 80 40 180 90 0 Time (days) 12 15 18 12 15 18 Time (days) Fig. H2. Changes of alcohols during papaya wine sequential fermentation inoculated with different ratios of W. saturnus var. mrakii NCYC2251 and S. cerevisiae var. bayanus R2. 10:1 ratio (♦); 1:1 ratio (▲); 1:10 ratio (■). (Error bars = standard deviation). 254 Methyl acetate n-Butyl acetate 240 G C-FI D p eak ar ea (x 0 0 ) GC-FID p eak area (x1 00 ) 1500 1000 500 160 80 0 12 15 18 Amyl acetate 150 100 50 12 15 18 Benzyl acetate 900 GC-FID peak area (x1 0000) GC-FID p eak area (x 0 0 ) 200 Time (days) Time (days) 600 300 T ime (days) 12 15 18 12 15 18 Time (days) Fig. H3. Changes of acetate esters during papaya wine sequential fermentation inoculated with different ratios of W. saturnus var. mrakii NCYC2251 and S. cerevisiae var. bayanus R2. 10:1 ratio (♦); 1:1 ratio (▲); 1:10 ratio (■). (Error bars = standard deviation). 255 Ethyl butyrate Ethyl nonanoate 150 GC-FID peak area (x10000) GC-FID peak area (x10000) 600 400 200 100 50 0 12 15 18 Ethyl decanoate 60000 12 15 18 15 18 15 18 15 18 Time (days) T ime (days) Ethyl 9-decenoate 10000 GC-FID peak area (x10000) GC-FID p eak area (x 00 ) 8000 40000 20000 6000 4000 2000 12 15 18 Time (days) Ethyl dodecanoate 4500 12 T ime (days) Ethyl tetradecanoate 270 G C-FID p eak area (x 0 0 ) GC-FID p eak area (x 0 0 ) 3600 2700 1800 900 180 90 0 12 15 18 Time (days) Ethyl hexadecanoate 300 12 T ime (days) Ethyl 9-hexadecenoate 700 G C-FID p eak area (x1 000 0) G C-FID p eak area (x 0 0 ) 600 200 100 500 400 300 200 100 0 T ime (days) 12 15 18 12 T ime (days) Fig. H4. Changes of ethyl esters during papaya wine sequential fermentation inoculated with different ratios of W. saturnus var. mrakii NCYC2251 and S. cerevisiae var. bayanus R2. 10:1 ratio (♦); 1:1 ratio (▲); 1:10 ratio (■). (Error bars = standard deviation). 256 Methyl butyrate 1200 Methyl octanoate 700 G C -F ID p eak ar ea (x 0 0 ) GC-FID peak area (x10000) 600 800 400 500 400 300 200 100 12 15 18 Time (days) Methyl dodecanoate 12 15 18 15 18 Methyl tetradecanoate 60 G C-FID p eak area (x 0 0 ) G C -FID p ea k area (x 0 0 ) 150 Time (days) 100 50 40 20 12 15 18 Time (days) 12 Time (days) Methyl 9-hexadecenoate 60 GC-FID peak area (x100 00) 50 40 30 20 10 0 12 15 18 T ime (days) Fig. H5. Changes of methyl esters during papaya wine sequential fermentation inoculated with different ratios of W. saturnus var. mrakii NCYC2251 and S. cerevisiae var. bayanus R2. 10:1 ratio (♦); 1:1 ratio (▲); 1:10 ratio (■). (Error bars = standard deviation). 257 Propyl octanoate 120 80 GC-FID peak area (x10000) 90 GC-FID peak area (x10000) Propyl decanoate 100 60 30 60 40 20 0 12 15 18 T ime (days) Isobutyl hexanoate 40 30 15 18 20 10 15 18 15 18 100 50 12 15 18 T ime (days) 12 T ime (days) Isoamyl octanoate 500 Isoamyl decanoate 210 400 G C-F ID p e ak are a (x 0 0 ) G C -F ID p e ak ar ea (x 0 0 ) 12 Isobutyl decanoate 150 GC-FID peak area (x100 00) GC-FID peak area (x10000) 50 T ime (days) 300 200 100 140 70 12 15 18 12 2-Phenylethyl octanoate 80 GC-FID p eak area (x 0 00 ) T ime (days) T ime (days) 60 40 20 0 12 15 18 T ime (days) Fig. H6. Changes of miscellaneous esters during papaya wine sequential fermentation inoculated with different ratios of W. saturnus var. mrakii NCYC2251 and S. cerevisiae var. bayanus R2. 10:1 ratio (♦); 1:1 ratio (▲); 1:10 ratio (■). (Error bars = standard deviation). 258 O-Tolualdehyde Benzaldehyde 500 400 GC-FID p eak area (x 0 0 ) GC-FID p eak area (x1 0 0) 2700 1800 900 300 200 100 12 15 18 T ime (days) 4-Methyl-2-heptanone 300 12 15 18 15 18 15 18 Time (days) 6-Methyl-5-hepten-2-one 140 G C-FID p eak area (x 0 0 ) G C-F ID p eak are a (x 0 0 ) 120 200 100 100 80 60 40 20 12 15 18 β-Damascenone 150 GC-FID peak area (x10000) 12 Benzyl isothiocyanate 500 GC-FID peak area (x10 00 0) 200 T ime (days) Time (days) 100 50 400 300 200 100 0 T ime (days) 12 15 18 12 Time (days) Fig. H7. Changes of miscellaneous volatile compounds during papaya wine sequential fermentation inoculated with different ratios of W. saturnus var. mrakii NCYC2251 and S. cerevisiae var. bayanus R2. 10:1 ratio (♦); 1:1 ratio (▲); 1:10 ratio (■). (Error bars = standard deviation). 259 Table H1. Modified frequency (MF%) value of sensory descriptors among the papaya wines (day 17) fermented with sequential cultures of W. saturnus var. mrakii NCYC2251 and S. cerevisiae var. bayanus R2 at different ratios (W. saturnus: S. cerevisiae) Sensory descriptors Acidic Alcoholic Buttery Cocoa Fruity Fusel Sweet Yeasty Ratio 10:1 Ratio 1:1 Ratio 1:10 60.98 82.16 63.25 59.16 81.39 75.83 74.16 72.46 59.16 83.67 49.05 53.33 80.62 79.06 75.83 70.71 59.16 82.16 65.19 65.19 78.26 79.06 75.83 76.65 Table H2. Sensory parameters of papaya wines (day 17) fermented with sequential cultures of W. saturnus var. mrakii NCYC2251 and S. cerevisiae var. bayanus R2 at different ratios (W. saturnus: S. cerevisiae) Sensory descriptors Ratio 10:1 Ratio 1:1 Ratio 1:10 Acidic 2.13 ± 1.36a 2.00 ± 1.41a 2.00 ± 1.41a Alcoholic 3.38 ± 0.74a 3.50 ± 0.76a 3.38 ± 0.74a a a Buttery 2.00 ± 1.07 1.38 ± 0.92 2.13 ± 1.13a Cocoa 2.00 ± 1.07a 1.63 ± 1.06a 2.13 ± 0.99a Fruity 3.31 ± 0.70a 3.25 ± 0.46a 3.06 ± 1.21a Fusel 2.88 ± 0.99a 3.13 ± 1.13a 3.13 ± 0.83a a a Sweet 2.75 ± 0.89 2.88 ± 0.83 2.88 ± 0.99a Yeasty 2.63 ± 1.30a 2.50 ± 0.93a 2.94 ± 0.68a a Statistical analysis at 95% confidence level with same letters indicating no significant difference. 260 [...]... volatile profile of papaya wine 191 11.2.2 Evaluation of fermentation conditions on volatile compounds formation in papaya wine fermented by S cerevisiae and W saturnus 192 11.2.3 Effect of flavour precursors on the volatile compounds production by S cerevisiae 192 11.2.4 Increasing ethanol content in papaya wine 192 11.2.5 Investigation of the underlying mechanism of the early... fruit wine papaya wine Hypothesis Papaya with its nutrient rich content can be used for wine fermentation and the characteristics volatile production capabilities of Saccharomyces cerevisiae and Williopsis saturnus via monoculture and multistarters fermentation can modulate and improve the aroma profile of papaya wine Specific Objectives 1 To study the impact of wine yeasts on the formation of volatile... deviation).………… ……………………… 53 xviii Fig 4.8 Bi-plot of principal component analysis of the major volatile compounds in papaya wine fermented with three commercial wine yeasts The major volatile compounds and numbers are given in Table 4.3…………… 56 Fig 5.1 Growth of yeasts (as optical density OD 600 nm) and oBrix changes during papaya juice fermentation by three W saturnus yeasts: W saturnus var saturnus NCYC22 (),... suggest that papaya juice fermentation by pure and multistarters of yeasts can be effective in manipulating yeast succession and modulating the volatile composition and organoleptic properties of papaya wine This may be useful for winemakers in creating novel fruit wines with flavour complexity and distinct style xiv LIST OF TABLES Description Page Table 2.1 Nutritional composition of papaya …………………………………10... 2.2 Comparison of amino acid contents of papaya (mg/100 g pulp) against grape and some other tropical fruits…………… …… ………… 12 Table 2.3 List of major volatile compounds present in fully-ripened papaya fruits……………………………………………………………… … 15 Table 3.1 Composition of fusel oil…………………………………………… 31 Table 4.1 Oenological parameters of papaya wine (day 14) fermented with three commercial wine yeasts ……………………………………………... (RPA) in papaya wine (day 21) fermented by a mixed-culture of S cerevisiae var bayanus R2 and W saturnus var mrakii NCYC2251 ……………………………………………………… … 135 Table 8.3 Concentrations of selected major volatile compounds (mg/L) in papaya wine (day 21) fermented with a mixed-culture of S cerevisiae var bayanus R2 and W saturnus var mrakii NCYC2251 ……… …… 137 xvi Table 9.1 Oenological parameters of papaya wine. .. deviation) 42 Fig 4.2 Changes of fatty acids in papaya wine during fermentation by three commercial wine yeasts: S cerevisiae var bayanus EC-1118 (), S cerevisiae var bayanus R2 (▲) and S cerevisiae MERIT.ferm (■) (Error bars = standard deviation).……………… ………… 48 Fig 4.3 Changes of ethanol and isoamyl alcohol in papaya wine during fermentation by three commercial wine yeasts: S cerevisiae var bayanus... deviation).…… 49 Fig 4.4 Changes of ethyl esters in papaya wine during fermentation by three commercial wine yeasts: S cerevisiae var bayanus EC-1118 (), S cerevisiae var bayanus R2 (▲) and S cerevisiae MERIT.ferm (■) (Error bars = standard deviation).………………………………………… 50 Fig 4.5 Changes of methyl decanoate and acetate esters in papaya wine during fermentation by three commercial wine yeasts: S cerevisiae var... deviation) … 51 Fig 4.6 Changes of acetaldehyde and acetoin in papaya wine during fermentation by three commercial wine yeasts: S cerevisiae var bayanus EC-1118 (), S cerevisiae var bayanus R2 (▲) and S cerevisiae MERIT.ferm (■) (Error bars = standard deviation).……… ………….……………… 53 Fig 4.7 Changes of benzyl isothiocyanate in papaya wine during fermentation by three commercial wine yeasts: S cerevisiae var... the biotransformation of volatile and non-volatile papaya constituents with a focus on volatile compounds during fermentation with monocultures and multistarters of Saccharomyces cerevisiae and Williopsis saturnus This is in view of developing papaya wine as a new tropical fruit wine Three commercial S cerevisiae wine yeasts, namely strains EC-1118, R2 and Merit.ferm and three W saturnus yeasts: W . MODULATION OF PAPAYA WINE FLAVOUR COMPOUND FORMATION BY YEASTS LEE PIN ROU NATIONAL UNIVERSITY OF SINGAPORE 2012 MODULATION OF PAPAYA. Effects of different sequential fermentation techniques on the volatile profile of papaya wine 191 11.2.2 Evaluation of fermentation conditions on volatile compounds formation in papaya wine. knowledge of wine and winemaking 1 1.2 History and trends of tropical fruit wine fermentation 3 1.3 Objectives of project 5 CHAPTER 2 Literature review 2.1 Nutritional information of papaya