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

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.. 85

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

i

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

ii

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)

iii

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

iv

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 L-leucine 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

v

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 co-fermentation 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

vi

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 phenylethanol and its ester 2-phenylethyl acetate (rose-like aroma) with the addition of L-leucine 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

2-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

vii

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 (methyl-1-butanol),

2-phenylethyl 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

viii

LIST OF TABLES

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

phenylalanine on day 21 99

ix

LIST OF FIGURES

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

x

LIST OF FIGURES (continued)

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 fermentation (n); EC-1118 (◊) and CBS254 (∆) in

co-fermentation: (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 (u), 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 (u), 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 (u), 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 (u), 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 (u), 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

xi

LIST OF FIGURES (continued)

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

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

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

• 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

3

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

wine-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

4

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 fermentative 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

non-• 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

5

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

6

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)

& 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’

8

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

11

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

phenylethyl 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

13

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 co-

fermentation 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

15

Scientific classification:

Kingdom: Plantae Order: Sapindales

Family: Sapindaceae

CHAPTER 2

Literature review

2.1 Nutritional status of longan juice

2.1.1 Introduction to longan fruits

2.1.1.1 Description of longan (Chomchalow, 2004)

Fig 2.1 Longan fruits

Species: Dimocarpus longan Lour (Syn Euphoria longana Lamk., Nephelium longana Cambess, Euphoria morigera Gagnes., and Euphoria scandens Winit & Kerr.)

Longan is a sub-tropical fruit in the family Sapindaceae and a prolific bearer thriving in monsoonal regions (pronounced wet and dry seasons) It is closely related to lychee having similar leaf and flower characteristics but the fruit is quite different, both

in colour (brown instead of red) and skin texture (smooth instead of bumpy) (Fig 2.1) The major difference between lychee and longan, however, is the taste of fruits While lychee has both a sweet and sour taste with a pleasing aroma, longan has only sweet with

no sour taste and very little aroma

Longan is a medium to large (10 to 20 m tall) evergreen tree with a dense canopy, brittle wood and corky bark that splits and peels The tree shape depends greatly on the cultivar varying from erect to spreading The inflorescence is large (30 to 50 cm long), multi-branched and leafless The flowers are small and yellow brown The fruits are

16

similar to those of the lychee, but smaller, smoother and yellowish-tan in colour The translucent flesh or aril is white to off-white or pinkish surrounding a large red-brown, brown, or black seed that separates easily from the flesh, which is sweet, juicy, aromatic and gives its name as “dragon eyes” It is milder in flavour and less acidic than lychee

Longan fruit is large in sugar and very nutritious It has been used as a dietary supplement in China since ancient time, and is widely applied in herb medication for benefiting the mind and spleen Longan fruit can be served fresh or processed as dried fruit, jam, and wine Like other fruits, longan has both volatile compounds and non-volatile compounds including organic acids, sugars, vitamins, amino acids

2.1.2 Volatile compounds

2.1.2.1 Volatile compounds in longan juice

Volatile compounds make a direct influence on the sensory characteristics of fresh and processed fruit products, the aroma of which is formed by a complex group of chemical substances (e.g., aldehydes, alcohols, ketones, esters, lactones, terpenes) The concentration of these volatile compounds is generally as low as ppm level and can be affected by a number of factors such as agronomy (variety, climatological conditions, ripening stage) (Douillard & Guichard, 1990; Vendramini & Trugo, 2000) and

technology (harvest, post-harvest treatments, storage and processing conditions -

Douillard et al., 1990; Lin, Rouseff, Barros & Naim, 2002)

Longan possesses a typical volatile composition (Table 2.1) Twenty eight volatile compounds of fresh longan were identified by Zhang, Zeng & Li (2008) by a

combination sampling method coupled with gas chromatography-mass spectrometry

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(GC-MS), among which five typical volatiles contributing significantly to the difference

in volatile profile characteristics of longan were ethyl acetate, β-ocimene, ethylidene-cyclohexene, allo-ocimene, 3,4-dimethyl-2,4,6-octatriene Besides, all the

1-ethyl-6-alkenes identified were interestingly C10 1-ethyl-6-alkenes with conjugated structures such as

β-ocimene, β-myrcene, 3-carene, etc., among which three monoterpenic isomers

(β-ocimene, allo-ocimene and 3,4-dimethyl-2,4,6-octatriene) constituted a high contribution

proportion Meanwhile, Lapsongphol and Mahayothee (2007) found that major volatile compounds detected in fresh longans were cis-ocimene, β -ocimene, ethyl acetate and ethanol, also based on HS-SPME-GCMS to investigate the impact of drying temperature

on volatile compounds of dried longans Three major constituents (ethanol, ethyl acetate and cis-ocimene) have also been identified by Susawaengsup and coworkers (2005) using the same method for analyzing the volatile components of five fresh longan cultivars including Biew Kiew, Chompoo, Edor, E-haew and Kahlok

A previously remarkable study by Chang et al (1998) characterized a total of 102 different volatile compounds by vacuum distillation and dichloromethane extraction followed by GC and GC-MS Depending on the functional groups, these compounds can

be grouped into 9 classes: hydrocarbons, alcohols, acids, phenols, ketones, aldehydes, furans, esters, and miscellaneous compounds Among those, ocimene has been identified

as the predominant and characteristic volatile compound in the raw fruit whereas other volatile compounds contribute to the flowery and sweet odor of the fruit

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2.1.2.2 Analysis of volatile compounds in fruit juice

Determination of volatile compounds in a sample generally includes two steps which are sampling and instrumental analysis Several sampling methods have been used for the analysis of fruit volatiles, among which there are some well-known conventional methods such as purge and trap (P&T) (Liu, Zhou, Xie & Zhu, 1999), simultaneous distillation extraction (SDE) (Torres, Talens, Carot, Chiralt & Escriche, 2007) or steam distillation (SD) (Siani, Garrido, Monteiro, Carvalho & Ramos, 2004) However, these traditional sampling means always require long extraction time, large amounts of solvents and multiple steps In addition, unstable fruit volatiles may be thermally decomposed and degraded during thermal extraction or distillation.As a result, although being simple and straightforward procedures, they are still applied extensively for fragrance and aroma characterization, either alone or combined with other sampling-preparation procedures

(Augusto, Lopes & Zini, 2003)

Recently, a method called as supercritical fluid extraction (SFE) has been used to sample fruit volatiles, especially non-polar volatiles (Morales, Berry, McIntyre &

Aparicio, 1998) Nevertheless, the lack of suitable solvents for polar analytes and the high cost of the SFE analysis limit its wide application

Solid phase micro-extraction (SPME), developed by Pawliszyn and co-workers

(Arthur & Pawliszyn, 1990), is a simple and solvent-saving sampling method, and has been widely used in environmental, biological, pharmaceutical, field analyses and

fragrance and aroma studies The benefit brought by SPME is its ability to address the need for concentrating the analytes in the headspace The selectivity of the extraction of target analytes in the gaseous phase can be significantly altered through the use of

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different liquid phases on the fiber, therefore resulting in various types of fiber coatings For examples, the polydimethylsiloxane/ divinylbenzene (PDMS/DVB) fiber is

composed of a mixed coating containing PDMS, a liquid phase that favors the absorption

of non-polar analytes, and DVB, a porous solid that favors the adsorption of the more polar analytes; the carboxen (CAR) fiber is composed of a porous carbon that is best for the extraction of small molecules (C2-C6 analytes) (Supelco website) Although the extraction capability of HS-SPME is limited, it has been considered as a good choice for sample preparation in trace fragrance and aroma analyses Besides, it should be noted that the extraction quality of the fiber depends on the temperature of the fiber as it increases, the partition coefficient will decrease (Zhang & Pawliszyn, 1995)

preservative in fruit drinks and juices because the pH imparted by natural and added acids

is not sufficient to ensure long-term microbial stability The content of organic acids in fruit juices not only influences their flavour but also their stability, nutrition, colour, acceptability and storage quality Therefore, it is important to be able to precisely

determine food acids present for quality control purposes, as well as for meeting various laws and regulations and for labeling purposes Organic acids account for a significant fraction of not only musts but also wines and vinegars Their origins are diverse, the most important being biosynthesis by the vine, metabolic pathways related to sugar

fermentation, malolactic fermentation and ethanol oxidation (in the case of acetic acid in vinegars)

2.1.3.2 Sugars

Chang et al (1998) determined free sugar content in Euphoria longana longan

fruit flesh and dried longan by high performance liquid chromatography It was found that longan fruit flesh contains total sugar at 67.35 g/100 g longan fruit flesh based on dry

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weight, particularly xylose (2.94%), fructose (2.3%), glucose (44.69%), maltose (15%) and sucrose (5.37%) in its composition

2.1.3.3 Vitamins

Ascorbic acid (vitamin C) and mineral composition in longan (Dimocarpus

longan) cultivars grown in Hawaii (Kilauea, Kurtistown and Puueo) were quantified by

Wall (2006) Longan cultivars (Biew Kiew and Scri Chompoo) were found to constitute the highest contents of ascorbic acid (60.1 mg/100 g fresh weight) compared to lychee

(Litchi chinensis) and rambutan (Nephelium lappaceum) cultivars (27.6 mg/100 g and

36.4 mg/100 g, respectively, Table 2.2) Furthermore, longans were a good source of potassium (K, 324.9 mg/100 g) and copper (Cu, 0.26 mg/100 g)

Table 2.2 Ascorbic acid and mineral composition in longan cultivars grown in Hawaii

(Adapted from Wall, 2006)

a

Dietary reference intakes (DRI) are the most recent set of dietary recommendations established

standard errors of six replications per cultivar at each location

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2.1.3.4 Amino acids

The individual amino acid proportion is obtained from USDA National Nutrient

Database in Dimocarpus longan longan fruit in comparison with that found in grape and

other tropical fruits (Table 2.3) Scion, maturity degree, temperature and other factors

such as rootstock, climate, mineral nutrition, crop level, trellising system, and diseases

may affect the concentration of amino acids in grape Arginine is found to be

predominant in Vitis vinifera variety, while glutamic acid is the most dominant amino

acid in longan followed by alanine and aspartic acid

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)

(Dimocarpus longan)

Grape

(Vitis vinifera)

Mango

(Mangifera indica)

Banana (Musa acuminata Colla)

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2.2 Fermentation of longan juice

2.2.1 Fruit wines and their prospects

Fruit wine refers to alcoholic beverages made from fruits other than grapes The popularity of fruit wine, especially tropical fruit wine, is increasing in spite of the

relatively inferior flavour Tropical fruits have a variety of characteristic flavours

partially due to their growth in the tropical climate which is normally available in tropical countries such as Cambodia, Laos, Thailand, Vietnam etc Various fruits have been used for making fruit wine, for example pineapple (Panjai, Ongthip & Chomsri, 2009; Pino & Queris, 2010), longan (Yao et al., 2004), lychee (Zhang, Zeng, Chen & Yu, 2008),

papaya, mango (Ezeronye, 2004) etc They are also a large source of vitamins and

antioxidants

Fruit wine- related research has been on-going for many years, largely based on simple transfer of information from grape wine fermentation, which does not necessarily lead to the successful development of good quality fruit wines This may be due to the differences in the composition between grapes and tropical fruits As a result, tropical fruit wine flavour is often undesirable and needs to be improved

The addition of artificial flavours into fruit wine may not improve overall quality due to flavour complexity and a lack of natural characters; besides, consumers

increasingly prefer foods and beverages containing natural flavours because of perceived health and environmental issues associated with synthetic chemicals and the production thereof (Vanderhaegen, Neven, Coghe, Verstrepen, Derdelinckx & Verachtert, 2003)

There are many factors that may affect the quality of fruit wine, comprising fruit variety, the preparation and treatment of fruit musts, fermentation conditions, aging,

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bottling, enzymatic treatments, and microbial starters and their population, some of which will be discussed in more details hereafter These factors can be adjusted to develop and improve wine quality, of which, flavour is a very important attribute

2.2.2 Yeast strains for longan wine fermentation

In the past, wine fermentation was carried out spontaneously where the

indigenous microflora was involved This process consists of a succession of yeast

growth whereby non-Saccharomyces yeasts grow during the early stages, while

Saccharomyces yeasts that are responsible for alcoholic fermentation and lactic acid

bacteria that perform malolactic fermentation develop at the later stages (Fleet et al., 1993; Fleet, 2003; Heras-Vazquez, Mingorance-Cazorla, Clemente-Jimenez &

Rodriguez-Vico, 2003) Both of those fermentations contribute to wine flavour

complexity The industrial winemaking process involves fermentation by selected wine

yeast starter cultures of S cerevisiae and S bayanus with or without malolactic

fermentation by lactic acid bacteria (mainly Oenococcus oeni) to ensure consistency of

the product and ease of control but compromises the complexity of wine flavour (lack of

“character” or “bouquet”), compared with the spontaneous fermentation process caused

by indigenous yeasts (Lambrechts et al., 2000; Romano et al., 2003) As a result, a

tendency is emerging using non-Saccharomyces yeasts in winemaking to take advantage

of their prospective role in imparting the complex organoleptic characteristics (Fleet, 2003; Plata, Millán, Mauricio & Ortega, 2003; Viana, Gil, Genovés, Vallés &

Manzanares, 2008) The co-inoculation of non-Saccharomyces yeasts with S cerevisiae

to improve the quality of wine has been suggested as a way of making use of spontaneous

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fermentation without the risk of stuck fermentation or spoilage, not only for grape wine but also other fruit wines such as pineapple wine (Jolly, Augustyn & Pretorius, 2003; Ciani, Beco & Comitini, 2006; Viana et al., 2008; Panjai et al., 2009)

Wine co-fermentation (simultaneous fermentation by two yeasts or more) has been evaluated to be able to impart better organoleptic quality than single fermentation

by pure Saccharomyces or non-Saccharomyces yeasts in a number of studies For

instance, non-Saccharomyces yeasts Hanseniaspora guilliermondii and Hanseniaspora uvarum co-inoculated with S cerevisiae have resulted in increased levels of 2-

phenylethyl acetate and isoamyl acetate in grape wine, respectively, and decreased levels

of ethyl acetate (Rojas, Gil, Pinaga & Manzanares, 2003; Moreira, Mendes, Guedes de Pinho, Hogg & Vasconcelos, 2008), which is excessively produced by apiculate yeasts

(Ciani & Picciotti, 1995; Plata et al., 2003) Other mixed cultures of non-Saccharomyces yeasts and S cerevisiae are also found to enhance desirable flavour compounds For instance, mixed cultures of Hanseniaspora osmophila and S cerevisiae give rise to 2-

phenylethyl acetate (Viana, Gil, Valles & Manzanares, 2009), while mixed cultures

containing Candida stellata or Candida cantarellii lead to higher glycerol but lower

acetic acid levels (Soden, Francis, Oakey & Henschke, 2000; Toro & Vazquez, 2002; Ciani et al., 2006) A combination of different S cerevisiae strains could also be

employed to modulate wine aroma (King, Swiegers, Travis, Francis, Bastian & Pretorius,

2008)

However, a mixed starter culture of defined yeast ratio should be introduced to

control the fermentation process, in which non-Saccharomyces yeasts should not die off early when co-existing with S cerevisiae during fermentation, thus resulting in wine with

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a wide variety of flavour components In this context, different yeast ratios have been

examined in several studies For instance, as the proportion of initial Hanseniaspora osmophila inoculum was increased from 0 to 90% versus S cerevisiae, the production of

2-phenylethyl acetate was enhanced (Viana et al., 2009) On the other hand, wines

inoculated with equal ratios of Saccharomyces and non-Saccharomyces yeasts have

similar compositions of ethanol, volatile acidity, ethyl acetate and ethyl lactate but

decreased levels of some alcohols, compared with single S cerevisiae-fermented wines

(Jolly et al., 2003; Kim, Hong & Park, 2008) Meanwhile, Rojas et al (2003) found an

increase in acetate ester but no effect on ethyl ester contents when mixed cultures of H guilliermondii and Pichia anomala with S cerevisiae at the ratio of 10 : 1 ratio were simultaneously inoculated, respectively Whereas, a 20 : 1 ratio of Torulaspora

delbrueckii and S cerevisiae co-culture improved the analytical profile of sweet wine, by producing 53% less volatile acidity and 60% less acetaldehyde than a pure culture of S

Most of Saccharomyces species have a vigorous fermentative ability while many

other yeast genera ferment sugars only weakly or not at all (Barnette, Payne & Yarrow, 1983), among which, the genus Williopsis was demonstrated to produce high levels of

esters, e.g isoamyl acetate (Iwase et al., 1995; Yilmaztekin et al., 2008, 2009) This genus was first defined in 1925 by Zender, since then, further species have been

accommodated within this genus (James, Roberts & Collins, 1998) It has been reported

that Williopsis saturnus var saturnus strains were known to be able to convert higher

alcohols into the corresponding acetate esters, e.g isoamyl alcohol into isoamyl acetate when isoamyl alcohol was added into fermentation medium (Vandamme & Soetaert,