DYNAMICS CHANGES OF VOLATILE COMPOUNDS DURING LONGAN JUICE FERMENTATION WITH SINGLE AND MIXED CULTURES OF YEASTS TRINH THI THANH TAM (B Eng.) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2011 ACKNOWLEDGEMENTS I would like to express my sincere gratitude to: • Professor LIU Shao Quan for his enthusiastic instruction, his precious time, constant support and patience during two years of my master thesis project • My family members for their spiritual support • Dr YU Bin for his guidance on using GCMS machine at Firmenich Company • Ms Lee Chooi Lan, Ms Lew Huey Lee, Ms Jiang Xiaohui and Mr Abdul Rahman bin Mohd Noor for their technical support • All FST postgraduates and friends for their encouragement and understanding throughout the project i ACHIEVEMENTS ACCEPTED MANUSCRIPT FOR JOURNAL PUBLICATION Thi-Thanh-Tam Trinh, Bin Yu, Phillip Curran & Shao-Quan Liu Effect of L-isoleucine and L-phenylalanine addition on aroma compound formation during longan juice fermentation by a co-culture of Saccharomyces cerevisiae and Williopsis saturnus South African Journal of Enology and Viticulture Submitted in Feb 2010 and accepted in Jun 2010 Thi-Thanh-Tam Trinh, Bin Yu, Phillip Curran & Shao-Quan Liu Growth and fermentation kinetics of mixed cultures of Saccharomyces cerevisiae var bayanus and Williopsis saturnus var saturnus at different ratios in longan juice International Journal of Food Science and Technology Submitted in Jun 2010 and accepted in Sep 2010 SUBMITTED MANUSCRIPTS FOR JOURNAL PUBLICATION Thi-Thanh-Tam Trinh, Bin Yu, Phillip Curran & Shao-Quan Liu Dynamics of volatile compounds during longan juice fermentation by three yeasts from the genus Williopsis Acta Alimentaria Submitted in Nov 2010 (under review) Thi-Thanh-Tam Trinh, Bin Yu, Phillip Curran & Shao-Quan Liu Enhanced formation of targeted aroma compounds during longan juice fermentation by Williopsis saturnus var saturnus CBS254 with the addition of selected amino acids Applied Microbiology and Biotechnology Submitted in Jul 2010 (under review) ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS i ACHIEVEMENTS ii TABLE OF CONTENTS iii SUMMARY vi LIST OF TABLES viii LIST OF FIGURES ix CHAPTER Introduction 1.1 Background 1.1.1 An overview of wine-making 1.1.2 The role of yeasts in wine fermentation 1.1.3 Wine flavours: characterization, formation and quality 1.2 Aims and objectives 11 1.3 Overview of the thesis structure 12 CHAPTER 15 Literature review 15 2.1 Nutritional status of longan juice 15 2.1.1 Introduction to longan fruits 15 2.1.2 Volatile compounds 16 2.1.3 Non-volatile compounds 22 2.2 Fermentation of longan juice 25 2.2.1 Fruit wines and their prospects 25 2.2.2 Yeast strains for longan wine fermentation 26 2.2.3 Fermentation conditions 29 CHAPTER 33 Materials and methods 33 3.1 Materials: fruits and chemicals 33 iii TABLE OF CONTENTS (continued) Page 3.2 Yeasts and culture media 34 3.3 Methods 34 3.3.1 Preparation of sterile longan juice for fermentation 34 3.3.2 Fermentation 36 3.3.3 Longan wine analysis and yeast enumeration 37 3.3.4 Analysis of volatile compounds in longan wine 37 3.3.5 Statistical analysis 38 CHAPTER 40 Results and discussion 40 Dynamics of volatile compounds during longan juice fermentation by three yeasts from the genus Williopsis 40 4.1 Volatile compounds in longan juice 40 4.2 Yeast growth, sugar consumption and pH changes during longan juice fermentation 41 4.3 Dynamic changes in volatile compounds during longan juice fermentation 43 CHAPTER 53 Results and discussion 53 Enhanced formation of targeted aroma compounds during longan juice fermentation by Williopsis saturnus var saturnus CBS254 with the addition of Lleucine and L-phenylalanine 53 5.1 Yeast growth, total soluble solids and pH changes during longan juice fermentation 53 5.2 Kinetic changes in volatile compounds during longan juice fermentation 55 5.3 Volatile compounds in longan wine at the end of fermentation with and without the added leucine and phenylalanine 63 CHAPTER 66 Results and discussion 66 iv TABLE OF CONTENTS (continued) Page Growth and fermentation kinetics of mixed cultures of Saccharomyces cerevisiae var bayanus and Williopsis saturnus var saturnus at different ratios in longan juice66 6.1 Yeast growth, total soluble solids and pH changes during longan juice fermentation 66 6.2 Kinetic changes in volatile compounds during longan juice co-fermentation 69 6.3 Comparison of volatile compounds in longan wine at the end of co-fermentation 80 CHAPTER 83 Results and discussion 85 Effect of L-isoleucine and L-phenylalanine addition on aroma compound formation during longan juice fermentation by co-culture of Saccharomyces cerevisiae and Williopsis saturnus 85 7.1 Yeast growth, changes in total soluble solids and pH during longan juice cofermentation 85 7.2 Kinetic changes in volatile compounds during longan juice co-fermentation 88 7.3 Volatile compounds in longan wine at the end of co-fermentation with and without added isoleucine and phenylalanine 96 CHAPTER 99 Conclusions, recommendations and future works 100 8.1 Conclusions 100 8.2 Recommendations and future works 101 BIBLIOGRAPHY 102 APPENDICES 113 Spread plating method for yeast enumeration 113 Metabolism pathways of some amino acids 114 v SUMMARY Three yeasts from the genus Williopsis (W saturnus var mraki NCYC500, W saturnus var saturnus CBS254 and W californica NCYC2590) were examined for their ability to ferment longan juice and to enhance formation of longan wine aroma compounds The three yeasts varied with their ability to produce and utilize volatiles W saturnus CBS254 was the best producer of ethyl acetate, isobutyl acetate, isoamyl acetate and 2-phenethyl acetate, whereas W californica NCYC2590 was the highest producer of butyl acetate W saturnus CBS254 was subsequently chosen to investigate the impact of two amino acids (L-leucine and L-phenylalanine) on the volatile profiles of longan wine with a view to enhancing longan wine aroma The results revealed the ability of this yeast to enhance isoamyl alcohol and its ester isoamyl acetate (banana-like aroma), and 2phenylethanol and its ester 2-phenylethyl acetate (rose-like aroma) with the addition of Lleucine and L-phenylalanine, respectively The increased production of the targeted acetate esters appeared to be at the expense of other acetate esters, whereas the effects on the biotransformation of other volatiles were minimal Next, co-fermentation of longan juice by mixed cultures of Saccharomyces cerevisiae var bayanus EC-1118 and Williopsis saturnus var saturnus CBS254 at two inoculation ratios (EC-1118 : CBS254= : 100 and : 1000 cfu mL-1) were performed to ascertain their impact on longan wine aroma compound formation The results showed improved aroma compound profiles in the longan wines fermented with mixed yeasts in comparison with the longan wines fermented with single yeasts in terms of increased production of acetate esters, fatty acid ethyl esters, alcohols and organic acids The impact of co-fermentation on longan wine aroma formation was affected by the ratio of S vi cerevisiae EC-1118 to W saturnus CBS254 with : 100 cfu mL-1 being more effective This research suggests that the inoculation ratio of mixed yeasts may be used as an effective means of manipulating longan wine aroma Again, the addition of L-isoleucine and L-phenylalanine on the volatile profiles of longan wine fermented by this co-culture at a ratio of : 1000 cfu mL-1 with the aim of enhancing longan wine aroma led to significantly higher concentrations of active amyl alcohol (2-methyl-1-butanol), 2phenylethyl alcohol and their corresponding acetate esters, respectively These findings suggest that yeasts from the genus Williopsis could be exploited for longan wine aroma enhancement either singly or in co-inoculation with Saccharomyces Furthermore, the added amino acids play an important role in enhancing targeted aroma compounds in longan wine Therefore, the combination(s) of a specific amino acid(s) and yeast can be employed as a valuable tool to modulate longan wine aroma vii LIST OF TABLES Description Page Table 1.1 A summary of the major volatile compounds reported in wine: molecular formula, aroma characteristics, concentration in wine and odour thresholds Table 2.1 Identification and quantification of volatile compounds in fresh longan in previous studies 20 Table 2.2 Ascorbic acid and mineral composition in longan cultivars grown in Hawaii (Adapted from Wall, 2006) 23 Table 2.3 Composition of amino acids (mg/100g flesh) in longan and some other tropical fruits without refuse (adopted from USDA National Nutrient Database for Standard Reference, Release 22, 2009) 24 Table 4.1 Major volatile compounds in longan juice and longan wine fermented by three Williopsis yeasts (day 14) 46 Table 4.2 Minor volatile compounds in longan juice and longan wines fermented by three Williopsis yeasts (day 14) 47 Table 5.1 Major volatile compounds in longan wine fermented by W saturnus CBS254 with added amino acids (day 14) 65 Table 6.1 Major volatile compounds in longan wine fermented by S cerevisiae EC-1118 and W saturnus CBS254 and mixed culture 83 Table 6.2 Concentrations* of selected volatile flavour compounds produced by S cerevisiae EC-1118 and W saturnus CBS254 and mixed culture at the end of fermentation 84 Table 7.1 Volatile compounds produced by a co- culture of S cerevisiae EC-1118 : W saturnus CBS254 (ratio of : 1000 cfu mL-1) in longan wine with added isoleucine and phenylalanine on day 21 99 viii LIST OF FIGURES Description Page Fig 1.1 Yeast alcohol fermentation pathway Fig 1.2 Diagram of thesis structure 14 Fig 2.1 Longan fruits 15 Fig 3.1 Diagram of longan juice fermentation 35 Fig 4.1 Kinetics of yeast growth (as yeast count), pH and Brix changes during longan juice fermentation by three Williopsis yeasts: W californica NCYC2590 (▲), W mraki NCYC500 (♦) and W saturnus CBS254 (■) 42 Fig 4.2 Kinetics of acetate esters during longan juice fermentation by three Williopsis yeasts: W californica NCYC2590 (▲), W mraki NCYC500 (♦) and W saturnus CBS254 (■) 48 Fig 4.3 Kinetics of ethyl esters during longan juice fermentation by three Williopsis yeasts: W californica NCYC2590 (▲), W mraki NCYC500 (♦) and W saturnus CBS254 (■) 49 Fig 4.4 Kinetics of alcohols during longan juice fermentation by three Williopsis yeasts: W californica NCYC2590 (▲), W mraki NCYC500 (♦) and W saturnus CBS254 (■) 50 Fig 4.5 Kinetics of fatty acids during longan juice fermentation by three Williopsis yeasts: W californica NCYC2590 (▲), W mraki NCYC500 (♦) and W saturnus CBS254 (■) 51 Fig 4.6 Kinetics of aldehydes during longan juice fermentation by three Williopsis yeasts: W californica NCYC2590 (▲), W mraki NCYC500 (♦) and W saturnus CBS254 (■) 52 Fig 5.1 Growth of Williopsis saturnus var saturnus CBS254 (as optical density at 600 nm), Brix and pH changes during longan juice fermentation with and without added amino acids Longan juice without added amino acid (control) (♦), longan juice with added L-leucine (▲), longan juice with added L-phenylalanine (■) 54 Fig 5.2 Kinetics of acetate esters during longan juice fermentation by Williopsis saturnus var saturnus CBS254 Longan juice without added amino acid (control) (♦), longan juice with added L-leucine (▲), longan juice with added L-phenylalanine (■) 56 Fig 5.3 Kinetics of ethyl esters during longan juice fermentation by Williopsis saturnus var saturnus CBS254 Longan juice without added amino acid (control) (♦), longan juice with added L-leucine (▲), longan juice with added L-phenylalanine (■) 57 ix • Comparison of simultaneous/co-fermentation versus sequential fermentation In the former co-fermentation, two yeasts are inoculated into the juice at the same time, while in the latter one, W saturnus CBS254 is firstly inoculated followed by S cerevisiae EC1118 which is then inoculated after a few days • The addition of fusel oils into longan juice may be investigated, which aims the production of aroma compounds at being proliferated • Different conditions during fermentation may be examined for the effects on the flavour compounds of resulting longan wine, such as pH, fermentation temperature, sulfite content, oxygen level (aeration of fermentation media), added assimilable nitrogen (diammonia phosphate) Optimization may be done to find out the optimized conditions where the expected flavour compound profile is achieved • Malolactic fermentation may be tested since it has been known to induce some desirable effects, for examples the deacidification, increased flavour and aroma complexity via bacterial activity and microbiological stabilization, which is conducted by lactic acid bacteria • The effects of glycosidases on the flavour production may be investigated Glycosidases are enzymes that catalyze the hydrolysis of glycosidically bound aroma compounds, which allows increasing the levels of volatiles in wine, thus possibly improving the organoleptic quality These enzymes are mainly produced by nonSaccharomyces yeasts or lactic acid bacteria A 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ã Use the pipettor 100-1000 àL to insert the sterile tip just below the surface of original yeast suspension and release the thumb plunger to draw up mL of aliquot and deliver into first tube that is then mixed by vortex machine • Deliver mL from the first tube into the second tube until 106 dilution is obtained Make sure that the sample is uniformly shaken before being delivered to the other tube Plating out 0.1 mL aliquots of the 106 dilution on sterile nutrient agar contained in the petri dishes: • Use the pipettor 20-200 µL to draw 0.1 mL of 104, 105, 106 dilution tubes onto agar plates Do not gouge the agar surface 113 • Use the sterile spreader to spread the suspension on the agar surface Incubate inverted at 25oC for 48 hours to develop the colonies Count all colonies formed and calculate the number of cfu (colony forming unit) in the original sample cfu mL-1 = cfu plate-1 × dilution factor × / aliquot Dilution factor = 104, 105, 106 Aliquot = 0.1 mL Metabolism pathways of some amino acids α-KGA Glu Alcohol + alcohol acyltransferases DH Leucine -> α-ketoisocaproate -> Isovaleryl-CoA -> Isovalerate esters BCAAT Alcohol DC α-OH-isocaproate -> Isovaleric acid (3-methylbutanoic acid) Isoamyl aldehyde (3-methylbutanal) Acetyl-CoA Isoamyl alcohol isoamyl acetate ester (3-methylbutanol) AAT (alcohol acetyltransferase) BCAAT = branched-chain amino acid transferase DH = dehydrogenase DC = decarboxylase Fig A.1 Metabolism pathway of leucine by yeasts 114 α-KGA Glu Alcohol + alcohol acyltransferases DH Isoleucine -> α-keto-β-methylvalerate -> α-Methylbutyryl-CoA -> 2-Methylbutanoate esters BCAAT Alcohol DC 2-methylbutanoic acid Amyl aldehyde (2-methylbutanal) Acetyl-CoA Amyl alcohol Amyl acetate ester (2-methylbutanol) AAT (alcohol acetyltransferase) BCAAT = branched-chain amino acid transferase DH = dehydrogenase DC = decarboxylase Fig A.2 Metabolism pathway of isoleucine by yeasts α-KGA Glu Alcohol + alcohol acyltransferases DH Valine -> α-ketoisovalerate -> Isobutyryl-CoA -> 2-Methylpropanoate esters BCAAT Alcohol DC α-OH-isovalerate -> Isobutyric acid (2-methylpropanoic acid) Isobutanal (2-methylpropanal) Acetyl-CoA Isobutyl alcohol isobutyl acetate (2-methylpropyl acetate) (2-methylpropanol) AAT (alcohol acetyltransferase) BCAAT = branched-chain amino acid transferase DH = dehydrogenase DC = decarboxylase Fig A.3 Metabolism pathway of valine by yeasts 115 Phenylalanine AAAT α-KGA Glu Phenylpyruvate Phenylacetaldehyde Benzaldehyde Benzalcohol Benzoic acid Phenyllactate Phenylacetic acid 2-Phenylethanol Acetyl-CoA AAT 2-Phenylethyl acetate AAAT = aromatic amino acid transferase AAT = alcohol acetyltransferase Fig A.4 Metabolism pathway of phenylalanine by yeasts 116 ... solids and pH changes during longan juice fermentation 66 6.2 Kinetic changes in volatile compounds during longan juice co -fermentation 69 6.3 Comparison of volatile compounds in longan. .. soluble solids and pH changes during longan juice fermentation 53 5.2 Kinetic changes in volatile compounds during longan juice fermentation 55 5.3 Volatile compounds in longan wine... Kinetic changes in volatile compounds during longan juice co -fermentation 88 7.3 Volatile compounds in longan wine at the end of co -fermentation with and without added isoleucine and phenylalanine