Kinetic changes of volatile compounds during longan juice fermentation with single and mixed cultures of yeasts

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 1 ...................................................................................................................... 1
Introduction ....................................................................................................................... 1
1.1. Background .............................................................................................................. 1
1.1.1. An overview of wine-making............................................................................ 1
1.1.2. The role of yeasts in wine fermentation ............................................................ 5
1.1.3. Wine flavours: characterization, formation and quality .................................... 6
1.2. Aims and objectives ............................................................................................... 11
1.3. Overview of the thesis structure ............................................................................. 12
CHAPTER 2 .................................................................................................................... 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 3 .................................................................................................................... 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 4 .................................................................................................................... 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 5 .................................................................................................................... 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 6 .................................................................................................................... 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 7 .................................................................................................................... 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 8 .................................................................................................................... 99

Conclusions, recommendations and future works ..................................................... 100
8.1. Conclusions .......................................................................................................... 100
8.2. Recommendations and future works .................................................................... 101
BIBLIOGRAPHY ......................................................................................................... 102
APPENDICES ............................................................................................................... 113
1. Spread plating method for yeast enumeration ......................................................... 113
2. 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= 1 : 100 and 1 : 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 1 : 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 1 : 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 ..................... 9
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 1 : 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 ..................................................................... 3

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


LIST OF FIGURES (continued)
Description

Page

Fig. 5.4 Kinetics of alcohols 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 (■). ....................... 60
Fig. 5.5 Kinetics of acids 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 (■). ....................... 61
Fig. 5.6 Kinetics of aldehydes 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 (■). ....................... 62
Fig. 6.1 Kinetics of yeast growth, Brix and pH changes during longan juice fermentation
by Saccharomyces cerevisiae var. bayanus EC-1118 (♦), Williopsis saturnus var.
saturnus CBS254 (▲) and co-fermentation ( ); EC-1118 (◊) and CBS254 (∆) in cofermentation: (A) EC-1118 : CBS254 = 1 : 100 cfu mL-1; (B) EC-1118 : CBS254 = 1 :
1000 cfu mL-1. ................................................................................................................... 68
Fig. 6.2 Kinetics of acetate esters during longan juice fermentation by S. cerevisiae var.
bayanus EC-1118 ( ), W. saturnus var. saturnus CBS254 (▲) and co-fermentation (■):
(A) EC-1118 : CBS254 = 1 : 100 cfu mL-1; (B) EC-1118 : CBS254 = 1 : 1000 cfu mL-1.
........................................................................................................................................... 70
Fig. 6.3 Kinetics of ethyl esters during longan juice fermentation by S. cerevisiae var.
bayanus EC-1118 ( ), W. saturnus var. saturnus CBS254 (▲) and co-fermentation (■):
(A) EC-1118 : CBS254 = 1 : 100 cfu mL-1; (B) EC-1118 : CBS254 = 1 : 1000 cfu mL-1.
........................................................................................................................................... 73

Fig. 6.4 Kinetics of alcohols during longan juice fermentation by S. cerevisiae var.
bayanus EC-1118 ( ), W. saturnus var. saturnus CBS254 (▲) and co-fermentation (■):
(A) EC-1118 : CBS254 = 1 : 100 cfu mL-1; (B) EC-1118 : CBS254 = 1 : 1000 cfu mL-1.
........................................................................................................................................... 76
Fig. 6.5 Kinetics of fatty acids during longan juice fermentation by S. cerevisiae var.
bayanus EC-1118 ( ), W. saturnus var. saturnus CBS254 (▲) and co-fermentation (■):
(A) EC-1118 : CBS254 = 1 : 100 cfu mL-1; (B) EC-1118 : CBS254 = 1 : 1000 cfu mL-1.
........................................................................................................................................... 78
Fig. 6.6 Kinetics of acetaldehyde during longan juice fermentation by S. cerevisiae var.
bayanus EC-1118 ( ), W. saturnus var. saturnus CBS254 (▲) and co-fermentation (■):
(A) EC-1118 : CBS254 = 1 : 100 cfu mL-1; (B) EC-1118 : CBS254 = 1 : 1000 cfu mL-1.
........................................................................................................................................... 79

x


LIST OF FIGURES (continued)
Description

Page

Fig. 7.1 Kinetics of yeast growth (as cell count and OD600 nm), Brix and pH changes
during longan juice co-fermentation by S. cerevisiae var. bayanus EC-1118 and W.
saturnus var. saturnus CBS254 (ratio of 1 : 1000 cfu mL-1, respectively) with and without
added amino acids. Longan juice without added amino acid (control) (♦) longan juice
with added L-isoleucine (▲), longan juice with added L-phenylalanine (■); EC-1118
(open nodes), CBS254 (filled nodes). ............................................................................... 87
Fig. 7.2 Kinetics of acetate esters during longan juice co-fermentation by S. cerevisiae
var. bayanus EC-1118 and W. saturnus var. saturnus CBS254 (ratio of 1 : 1000 cfu mL-1,
respectively). Longan juice without added amino acid (control) (♦), longan juice with

added L-isoleucine (▲), longan juice with added L-phenylalanine (■). .......................... 89
Fig. 7.3 Kinetics of ethyl esters during longan juice co-fermentation by S. cerevisiae var.
bayanus EC-1118 and W. saturnus var. saturnus CBS254 (ratio of 1 : 1000 cfu mL-1,
respectively). Longan juice without added amino acid (control) (♦), longan juice with
added L-isoleucine (▲), longan juice with added L-phenylalanine (■). .......................... 91
Fig. 7.4 Kinetics of alcohols during longan juice co-fermentation by S. cerevisiae var.
bayanus EC-1118 and W. saturnus var. saturnus CBS254 (ratio of 1 : 1000 cfu mL-1,
respectively). Longan juice without added amino acid (control) (♦), longan juice with
added L-isoleucine (▲), longan juice with added L-phenylalanine (■). .......................... 92
Fig. 7.5 Kinetics of organic acids during longan juice co-fermentation by S. cerevisiae
var. bayanus EC-1118 and W. saturnus var. saturnus CBS254 (ratio of 1 : 1000 cfu mL-1,
respectively). Longan juice without added amino acid (control) (♦), longan juice with
added L-isoleucine (▲), longan juice with added L-phenylalanine (■). .......................... 94
Fig. 7.6 Kinetics of aldehydes during longan juice co-fermentation by S. cerevisiae var.
bayanus EC-1118 and W. saturnus var. saturnus CBS254 (ratio of 1 : 1000 cfu mL-1,
respectively). Longan juice without added amino acid (control) (♦), longan juice with
added L-isoleucine (▲), longan juice with added L-phenylalanine (■). .......................... 95
Fig A.1 Metabolism pathway of leucine by yeasts ......................................................... 114
Fig A.2 Metabolism pathway of isoleucine by yeasts .................................................... 115
Fig A.3 Metabolism pathway of valine by yeasts ........................................................... 115
Fig A.4 Metabolism pathway of phenylalanine by yeasts .............................................. 116

xi


CHAPTER 1
Introduction

1.1. Background
1.1.1. An overview of wine-making

Wine is one of the oldest fermented alcoholic beverages in the world, which is
defined as the product of fermenting grape juice with yeasts. The term “wine” is also
used loosely to refer to alcoholic beverages where other fruits and even grains are used as
fermentation substrates, giving them so-called names as “fruit wine” or “rice wine”. The
potential of fruit wine is increasingly attracting consumers’ interests; however, its flavour
is relatively inferior and needs to be improved.
The process of grape wine-making generally includes the following steps:
•

Harvesting

Harvesting, the first step in wine production, is an action of picking the grapes in
vineyards, either by hand or mechanical means. The appropriate time to do harvesting is
decided by wine-makers based on sugar level (in °Brix unit), titrable acidity (expressed
by tartaric acid equivalents) and pH of the grapes. Other indicators can be considered
including phenological ripeness, berry flavour, tannin development (through seed colour
and taste).
•

Destemming

Destemming, an optionally undertaken process for the sake of reducing tannin
development and vegetal flavours in the final wine due to 2-methoxy-3-isopropylpyrazine

1


(which has an aroma reminiscent of green bell peppers being extracted, Hashizume &
Samuta, 1997), is the removing of stems from the grapes.
•

Crushing

Crushing is the process of gently squeezing the berries and breaking the skins to
start the liberation of berry contents. In most cases, destemming and crushing are carried
out at the same time by tramping barefoot or using mechanical crushers.
•

Pressing

Pressing is the applying of pressure to grapes or pomace (solid remains of grapes)
in order to separate juice from grapes and grape skins. This action aims at increasing the
yield of total juice volume from grapes, thus not always being necessary if free-run juice
after crushing is obtained at considerable amount.
•

Primary fermentation (1-2 weeks)

Biochemically, this step is defined as a pathway in which NADH (or other
reduced electron acceptors such as NADPH) generated by oxidation reactions in the
pathway) is re-oxidised by metabolites produced by the pathway. In microbiological
view, fermentation broadly refers to the many metabolic processes that occur during its
course, by which micro-organism obtain energy (in the form of ATP), usually from sugar
metabolism (Fig. 1.1). The main product expected from primary fermentation is ethanol.
Prior to fermentation, grape juice is sterilized by the addition of preservatives such as
sulfur dioxide, potassium sorbate etc. or other sterilization methods. The advantages of
sulfur dioxide are not only an anti-microbial agent but also an antioxidant; however, the
allowed dose of sulfur dioxide in the resultant wine is legally regulated. Without

2

sterilization, wines can easily suffer from bacterial spoilage despite the hygiene of winemaking practice.

Fig. 1.1 Yeast alcohol fermentation pathway
•

Cold and heat stabilization

Cold stabilization is used in winemaking to remove tartrate crystals
(generally potassium bitartrate) which look like clear sand grains in wine. These crystals
are formed by dropping the temperature of wine to close to freezing for 1-2 weeks after
fermentation. The separation from the wine then takes place with the crystals sticking to
the sides of the holding vessel and being left behind when the wine is drained from it.
In heat stabilization, unstable proteins are precipitated to prevent them from doing
so in the bottled wine and removed by filtration with the use of bentonite for absorption.
•

Secondary fermentation and bulk aging (3-6 months)

In this step, the fermentation continues but very slowly, in which grape proteins
are broken down, the remaining yeast cells and fine particles from grapes are allowed to
3


settle, potassium bitartrate also precipitate. This process turns originally cloudy wine into
clear wine which is then racked to remove the lees.
The aging process refers to the stabilizing of wine by doing the secondary
fermentation in either stainless steel vessels or oak barrels or glass carboys, depending on
the winemakers’ goals. Different container materials and aging duration will lead final

wine to different tastes and flavours.
•

Malolactic fermentation (Davis, Wibowo, Eschenbruch, Lee, & Fleet, 1985)

This optional fermentation is implemented by malolactic bacteria through nonfermentative pathways by which malic acid is metabolized to produce lactic acid and
carbon dioxide. The resultant wine is softer in taste and has greater complexity.
•

Blending and fining

Blending can be made in order to achieve the wine of desired taste by mixing
wines produced from different batches or different grapes.
Fining agents can also be used during wine-making to remove tannins thus
decreasing astringency and microscopic particles that could cloud the wines. The type of
fining agents used vary with wine products and batches, including gelatin, micronized
potassium casseinate, egg whites, etc. which are derived from animal or fish products,
others being non-animal based fining agents such as bentonite, diatomaceous
earth, cellulose pads, paper filters and membrane filters etc. These fining agents clarify
wine by reacting with wine components to form sediments which are removed by
filtration prior to bottling.
•

Filtration

4


Filtration is performed to result in clarification and microbial stabilization for
wine as large particles may affect the visual appearance of wine while the presence of

remaining microbes may continue re-fermenting or cause wine spoilage. In this process,
both large particles and microbes are removed, depending on the extent of clarification,
by using different pore size filters.
•

Bottling

Wine before bottling can be added with a certain amount of sulfite to preserve the
quality that may be damaged by oxygen and prevent unwanted fermentation that may
cause bacterial spoilage or continuous malolactic fermentation in the bottle. After
bottling, a traditional cork or other alternative wine closures such as synthetic corks,
screw-caps, which help reduce cork taint, are sealed on the bottles followed by an added
capsule to the top of the bottles.

1.1.2. The role of yeasts in wine fermentation
As wine is the product of fermenting grape juice with yeasts: no yeasts, no wine.
Hence, the role of yeasts in wine-making technology is very important, particularly in the
production of ethanol and aroma compounds which contribute to a wide variety of
characteristic flavour profiles for different wine styles. The yeasts used in wine
fermentation can be originated from the microbial communities of the grape berry and the
microbial communities of the winery environment. Over twenty yeast genera have been
identified from wines (Renouf, Claisse & Lonvaud-Funel, 2007), most of which belong
to non-Saccharomyces species, principally within the genera Hanseniaspora (anamorph
Kloeckera), Pichia, Candida, Metschnikowia, Kluyveromyces, occasionally species in

5


other genera such as Zygosaccharomyces, Saccharomycodes, Torulaspora, Dekkera and
Schizosaccharomyces may be present. These non-Saccharomyces yeasts initiate

spontaneous alcoholic fermentation of the juice, but are very soon overtaken by the
growth of Saccharomyces cerevisiae that dominates the mid to final stages of the process
(Fleet & Heard, 1993; Fleet, 2003; Maro, Ercolini & Coppola, 2007). However, the
diversity of indigenous yeasts vary with grape variety and winery environment, resulting
in reduced predictability of the fermentation such as stuck or sluggish fermentation and
inconsistency in wine quality. On the contrary, inoculated fermentation, which employs a
defined yeast culture, most popularly Saccharomyces cerevisiae, offers higher
consistency in quality through a more predictable and rapid process. Moreover, it is more
suitable for mass production and well accepted by industrial winemakers due to the
commercial availability of dried selected yeast strains that can be conveniently
reconstituted for inoculation into grape juice (Degre, 1993; Manzano, Medrala, Giusto,
Bartolmeoli, Urso & Comi, 2006). Nevertheless, the yeast strains of both Saccharomyces
and non-Saccharomyces yeasts have been reiteratively demonstrated to be important in
wine-making and the ester profile of finished products (Fleet, 2003; Miller, Wolff, Bisson
& Ebeler, 2007).

1.1.3. Wine flavours: characterization, formation and quality
The flavours of wine derive from a combination of volatile compounds inherently
present in grapes or other fruits (varietal flavours), secondary products formed during
wine fermentation (fermentative flavours) and ageing (post-fermentative flavours).

6


During fermentation, a diversity of volatile metabolites was produced, consisting of
esters, higher alcohols, acids, aldehydes, ketones, phenols and sulfur compounds.
Esters are considered as the most important flavour compounds among volatile
compounds in alcoholic beverages and the next major group after water, ethanol and fusel
alcohols (Ferreira, Fernandez, Pena, Escudero & Cacho, 1995). Although synthesized as
by-products of alcoholic fermentation at relatively low concentration, individually below

aroma threshold, they are the primary source of fruity aromas and their concentration
changes might make a dramatic impact on wine flavour (Gurbuz, Rouseff & Rouseff,
2006). Principal esters produced during alcohol fermentation comprise acetate esters (e.g.
ethyl acetate, isoamyl acetate and 2-phenylethyl acetate), ethyl esters of organic acids and
straight chain or branched-chain fatty acids (e.g. ethyl hexanoate, ethyl octanoate and
ethyl decanoate). Some esters have widespread use in the food industry, such as isoamyl
acetate for banana flavour, 2-phenylethyl acetate for rose petal flavour (Farbood, Morris
& Seitz, 1987).
In wine, esters can be formed by two ways: enzymatic synthesis during
fermentation (yeast and malolactic) and chemical esterification between alcohol and acids
at low pH during wine aging (Margalit, 1997). The enzymes involved in the catalysis of
ester synthesis such as esterase, lipase and alcohol acetyltransferase are produced by
many food microorganisms (Lilly, Bauer, Lambrechts, Swiegers, Cozzolino & Pretorius,
2006). The major esters reported in wine are summarized by Sumby, Grbin & Jiranek
(2010); however, other volatile compounds are also added in Table 1.1.
Wine flavour can be evaluated as the measuring ruler for the quality of wine since
it is one of the important sensory characteristics that strongly affect the consumers’

7


preference. Different styles of wine possess different flavour profiles which are distinct
or specific for that style only. For examples, Chardonnay white wines (originated in
France) give dominant citrus fruit flavours while Sauvignon Blanc white wines bring the
dominating flavours ranging from sour green fruits of apple, pear and gooseberry through
to tropical fruits of melon, mango and blackcurrant. Black-cherry and herbal flavours are
typical flavours of Merlot red wines, whereas soft tannins, strongly fruity (cherry,
strawberry, plum) are some notes of Pinot noir red wines.

8

8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23

1
2
3
4
5
6
7

No

Acetate esters

Isobutyl acetate
1-Butyl acetate
Active amyl acetate
Isoamyl acetate
Hexyl acetate
Ethyl 2-phenylacetate
2-Phenylethyl acetate
Ethyl esters
Ethyl acetate
Ethyl lactate
Ethyl isobutanoate
Ethyl butanoate
Ethyl 3-hydroxybutanoate
Ethyl 4-hydroxybutanoate
Ethyl 2-methylbutanoate
Ethyl isovalerate
Diethyl succinate
Diethyl malate
Ethyl hexanoate
Ethyl octanoate
Ethyl vanillate
Ethyl dihydrocinnamate
Ethyl cinnamate
Ethyl decanoate

Compound name

C4H8O2
C5H10O3
C6H12O2

C6H12O2
C6H12O3
C6H12O3
C7H14O2
C7H14O2
C8H14O4
C8H14O5
C8H16O2
C10H20O2
C10H12O4
C11H14O2
C11H12O2
C12H24O2

C6H12O2
C6H12O2
C7H14O2
C7H14O2
C8H16O2
C10H12O2
C10H12O2

Molecular
formula

141-78-6
97-64-3
97-62-1
105-54-4
5405-41-4

999-10-0
7452-79-1
108-64-5
123-25-1
7554-12-3
123-66-0
106-32-1
617-05-0
2021-28-5
103-36-6
110-38-3

110-19-0
123-86-4
624-41-9
123-92-2
142-92-7
101-97-3
103-45-7

CAS
number
Trace-170[5]
Trace-170
16.5[7]
31–5520[1,2,4,5]
1-390[4]
31-394[4,5]
Trace-260[1,2,6]
5000–63500[1,4]

3050-297500[4,5,6]
10-480[1,2,5]
70-532[1,2,4,5]
50-580[5,6]
6610[6]
Trace-30[2,5]
Trace-69[1,2,4,5]
1210-61110[4,5,6]
810[6]
150-1640[1,2,4,5]
137-2610[1,2,4,5,6]
460[6]
Trace-3[2,5]
Trace-10[1,2,5]
10-696[2,4,5,6]

Fruity (at > 100 mg mL-1), solvent, balsamic
Milk, soapy, buttery, fruity
Fruity, strawberry, lemon
Floral, fruity, strawberry, sweet
Fruity-winey, green, marshmallow
Caramel
Apple, strawberry, berry, sweet, cider, anise
Sweet fruit, pineapple, lemon, anise, floral
Fruity, fermented, floral
Brown sugar, sweet
Fruity, strawberry, green apple, anise
Sweet, fruity, ripe fruit, burned, beer
Flower, fruit, sweet, vanilla
Flower

Honey, cinnamon
Oily, fruity (grape), floral

Concentration in
wine (µg L-1)

Fruity, apple
Fruity, apple
Banana, peanut
Banana, fruity
Green, herbaceous, fruit, grape
Rose, floral
Flowery, rose

Aroma characteristics

9

7500[1], 60000
50–200, 154636[3]
1, 15[1,2]
1, 20[1]
20000
0.1, 1[1], 18[2]
0.1, 3[1,2]
200000[3]
5 [1,2]
2[2], 5[1], 12
2[2]
1[1,2]

12, 200[2]

66, 1600[3]
66, 1880[3]
5
2, 30[1]
2–480
250[1], 650

Odour threshold
(µg L-1)

Table 1.1 A summary of the major volatile compounds reported in wine: molecular formula, aroma characteristics, concentration in wine
and odour thresholds


β-Damascenone
Benzaldehyde
Furfural

43
44
45

C13H18O
C7H6O
C5H4O2

C4H8O
C4H6O2

C2H4O

C2H4O2
C3H6O2
C4H8O2
C4H8O2
C6H12O2
C8H16O2
C10H20O2

C5H12O
C6H14O
C10H18O
C8H10O

C2H6O
C3H8O
C4H10O
C4H10O
C5H12O

Molecular
formula

23726-93-4
100-52-7
98-01-1

513-86-0

431-03-8

75-07-0

64-19-7
79-09-4
107-92-6
79-31-2
142-62-1
124-07-2
334-48-5

123-51-3
111-27-3
78-70-6
60-12-8

64-17-5
71-23-8
71-36-3
78-83-1
137-32-6

CAS
number

Pungent at high but fruity at low
concentration, freshly cut apples
Creamy, buttery
Creamy, milk, buttery (a negative above

threshold value)
Baked fruit, rape fruit
Bitter, almond
Sweet

Stinging (> 50 ppm), vinegary
Animal (goaty)
Rancid, cheesy, sweet
Fruity, pleasant, buttery, cheesy
Fatty, sweet
Cheesy, sour, sweaty
Rancid

Alcoholic
Fruity, alcoholic
Alcoholic
Green, fresh, fusel
Pungent, pleasant, earthy-musty (R),
ethereal-fruity (S)
Sweet, fusel, fruity-winey
Herbaceous, fatty, fruity, green
Lemon, fresh, citric, flowery
Rose, flowery, pollen, perfume

Aroma characteristicsa

9000
150000[3]
7000, 40000[1]
250-300, 30000[1]

2500, 8000[2]
6, 15[1], 25[2]
10000[1], 14000[2]
200000[1]
8100[2], 20000
173[2], 240, 10000[1]
2300[2], 8100, 200000[1]
420[2], 3000[1]
500[2], 3000
1000[2], 10000, 15000[1]
500[1]
150000[3]
100[2]
0.05[1]
350-3500
14100[2]

49200-457900[4]
740-13200[2,4]
1-40[2,4]
40093-153269[2]
31430-1383000[2,4]
1621[5]
434-4719[2,5]
411-2345[2,5]
1520-14740[4]
562-9767[2,5]
62-16861[2,5]
2963[5]
598-159331[2,5]

82[5]
Trace-5[2,5]
1-47[4]
2-308[2,4,5]

Odour threshold
(µg L-1)c

7510-44800[4]
294-3090[4]
2750-86900[2,4]
10280-124900[4]

Concentration in
wine (µg L-1)b

b

10

Aroma characteristics collated from: Aznar & Arroyo (2007), Clarke & Bakker (2004), Gómez-Míguez et al. (2007).
Concentration range derived from indicated sources: [1] Guth (1997); [2] Ferreira et al. (2000); [4] Aznar & Arroyo (2007); [5] Gomez-Miguez et al. (2007); [6] Rocha et al.
(2004); [7] Selli et al. (2008).
c
Odour thresholds determined in water except where specified: [1] Guth (1997) using matrix as 10% v/v aqueous ethanol; [2] Ferreira et al. (2000) using matrix as a
synthetic wine (11% v/v ethanol, 7 g L-1 glycerol, 5 g L-1 tartaric acid, pH 3.4); [3] Etiévant (1991) using matrix as 12% v/v aqueous ethanol.

a

Acetoin

Diacetyl

41
42

40

33
34
35
36
37
38
39

Isoamyl alcohol
1-Hexanol
Linalool
2-phenylethyl alcohol
Organic acids
Acetic acid
Propanoic acid
Butanoic acid
Isobutanoic acid
Hexanoic acid
Octanoic acid
Decanoic acid
Carbonyl compounds
Acetaldehyde

Alcohols
Ethanol
1-Propanol
1-Butanol
Isobutyl alcohol
Active amyl alcohol

24
25
26
27
28

29
30
31
32

Compound name

No

Table 1.1 (Continued)


1.2. Aims and objectives
The aim of this research was to investigate the effect of non-Saccharomyces
yeasts and the effect of co-inoculating non-Saccharomyces yeast and Saccharomyces
yeast at 2 different inoculation ratios (EC-1118 : CBS254 = 1:100 and 1:1000 cfu mL-1)
on aroma compounds in juice from the tropical longan fruit (Dimocarpus longan Lour.).

Longan was chosen to make wine based on its large amount of sugar, significant level of
polyphenols, good source of ascorbic acid, potassium, copper and other minerals
(Rangkadilok, Worasuttayangkurn, Bennett & Satayavivad, 2005; Wall, 2006).
Furthermore, a short harvesting time and easily perishable properties of longan result in
its oversupply at the initial season but considerable waste at the end. Apart from dried
and canned longan products as solutions to the problem above, wine products from
longan juice are also prospective, especially for niche market (Yao, Liang, Liu & Wu,
2004). Meanwhile, three non-Saccharomyces yeasts from the genus Williopsis namely W.
saturnus var. mraki NCYC500, W. saturnus var. saturnus CBS254 and W. californica
NCYC2590 were chosen in this research due to its ability in producing high quantity of
esters such as isoamyl acetate (Iwase, Morikawa, Fukuda, Sasaki & Yoshitake, 1995;
Yilmaztekin, Erten & Cabaroglu, 2008, 2009) while S. cerevisiae var. bayanus EC-1118,
a Saccharomyces yeast, was used owing to its vigorous fermenting ability, commercially
used on the industrial scale to ensure consistency of the product and ease of control
(Lambrechts & Pretorius, 2000; Romano, Fiore, Paraggio, Caruso & Capece, 2003).
Initial cell count difference of 2-6 logs between non-Saccharomyces and S. cerevisiae
yeasts is well-observed in spontaneous fermentation depending on yeast species and must
(Combina, Elía, Mercado, Catania, Ganga, Martinez, 2005), thus 2 and 3 log higher

11


inoculation ratio of the former yeast were examined in this study. Furthermore, with the
purpose of enhancing aroma compound formation in fermented longan juice, the impact
of added selected amino acids on the production of targeted aroma compounds in longan
wine was examined. L-leucine, L-isoleucine, and L-phenylalanine were opted as they
were demonstrated to be precursors of isoamyl acetate, active amyl acetate and 2phenylethyl alcohol respectively (Dickinson et al., 1997; Dickinson, Harrison, Dickinson
& Hewlins, 2000; Dickinson, Salgado & Hewlins, 2003) but present at little amounts in
longan (Table 2.3). Emphasis was placed on the fermentation performance and dynamics
of volatile flavour compounds during longan juice fermentation which was expressed in

peak areas obtained by flame ionization detector of the gas chromatographic system
(abbreviated as GC-FID peak area) instead of concentrations since kinetics and relative
contribution of volatile flavour compounds were only paid attention.

1.3. Overview of the thesis structure
The contents of the thesis are covered in 7 chapters (diagrammed in Fig. 1.2) as
follows:
Chapter 1 gives a background on the science of wine-making such as the steps
involved in wine production from raw materials to bottled products, the roles of yeasts
and flavour being one of the most important attributes of wine quality.
Chapter 2 is a review of literature on the nutritional compounds of longan
(including non-volatile and volatile compounds) and the fermentation of longan juice to
produce longan wine, a prospective fruit wine.

12


Materials and methods used for this research are covered in chapter 3, which
clarifies the preparation of sterile longan juice, the propagation of yeast, the fermentation
of juice and the analysis of resultant wine.
Chapter 4 describes the kinetics of volatile compound production by three yeasts
from the genus Williopsis, thus selecting one yeast species based on its flavour compound
profile to continue further researches.
The purpose of chapter 5 is to describe ways on enhancing longan wine flavour by
adding selected amino acids in fermented longan juice due to the these compounds being
demonstrated to be flavour precursors in several previous studies.
Chapter 6 presents the experiments which employ a Williopsis species selected
from chapter 4 and a commercial Saccharomyces cerevisiae strain to carry out the cofermentation of longan juice with the aim of exploiting the positive roles of both yeasts.
Two inoculation ratios of yeasts are compared in terms of their effects on the volatile
flavour compound profile.

Again, chapter 7 describes the co-fermentation experiment with selected added
amino acids with the intention of enhancing aroma compound formation in longan wine.
Finally, drawn conclusions and future works were presented in chapter 8,
followed by bibliographical list and appendices at the end of the report.

13