1

MSc IN FOOD BIOTECHNOLOGY

UNIVERSITY OF PATRAS
DEPARTMENT OF CHEMISTRY




Nano-tubular cellulose/biopolymer composite in wine fermentation:
Volatile byproducts


GKIKA EFTHYMIA



Patras 2012





2



- /
:






2012

Acknowledgements

The present study took place in the laboratory of Food Chemistry and
Biotechnology of Chemistry Department in the University of Patras under the
supervision of the Assistant Professor M. Soupioni.
Having completed successfully this project, I would like to express my gratitude
to my supervisor Prof. M. Soupioni for her guidance and trust, and the opportunity I
was given to become familiar with several methods of analysis. I would also
particularly like to thank the PhD student John Servetas for the cooperation, support
and help during the project. Also I would like to thank the professors of the
commission that supervise my project Prof. A. Koutinas and Prof. M. Kanellaki. A
3

special thank is addressed to all the students in the laboratory (under, post graduates
and PhDs) for their help and advices.
Finally, I would like to thank my family for their psychological support which
helped me to fulfill my project.







Patras, July 2012









Abstract
A new winemaking technology with immobilized yeast cells at a tubular
cellulose/biopolymer composite is proposed with potential application in industrial
scale winemaking. The biocatalyst was prepared by immobilization of Saccharomyces
cerevisiae AXAZ-1 yeast cells on starch gel and diffusion of the yeast-gel system into
delignified sawdust (GS biocatalyst) and its suitability for fermentation of grape must
was investigated by using it in fermentations at 30
o
C and 7
o
C. The fermentation rate
and other kinetic parameters were compared with free yeast cells and immobilized
yeast cells on delignified sawdust (DC biocatalyst) at high and low temperatures. Cell
immobilization was shown by electron microscopy and by the efficiency of the
immobilized biocatalysts for alcoholic fermentation. The operational stability of the
4

biocatalyst was good and no decrease of its activity was observed, even at 7
o
C.
Fermentation times were low (only 41 days at 7
o
C), while ethanol productivities were
high compared with the other two biocatalysts, especially at low temperature. The
produced wines were analyzed for volatile byproducts by GC and it was observed that
the levels of ethyl acetate increased by the drop of the temperature, while
concentrations of higher alcohols reduced in wines. At 7
o
C the results showed that
wines produced by GS contained higher concentrations of ethyl acetate and lower
amyl alcohols concentrations than the corresponding values achieved by free cells and
DC biocatalyst, resulting in final product of improved quality.













/
, .
Saccharomyces cerevisiae
AXAZ-1 -
(GS )
30
o
C 7

C.

(DC )
.

.
5


7
o
C. (
41 7
o
C),
, .

(GC)
, .
7
o
C GS


DC ,
.








Contents


Abstract .......................................................................................................................... 3
....................................................................................................................... 3
Introduction ......................................................................... 5
1.1 Introduction .............................................................................................................. 5
1.2 Wine ......................................................................................................................... 6
1.2.1 History of the wine................................................................................................ 6
1.2.2 Chemical constituents of wine .............................................................................. 6
6

1.2.2.1 Water .................................................................................................................. 7
1.2.2.2 Alcohols ............................................................................................................. 7
1.2.2.3 Sugars ................................................................................................................. 8
1.2.2.4 Acids .................................................................................................................. 9
1.2.2.5 Phenols ............................................................................................................... 9
1.2.2.6 Aldehydes and ketones ...................................................................................... 9
1.2.2.7 Esters ................................................................................................................ 10
1.2.2.8 Nitrogen-containing compounds ...................................................................... 10
1.2.2.9 Vitamins ........................................................................................................... 10
1.2.2.10 Dissolved gases .............................................................................................. 10
1.2.2.11 Minerals ......................................................................................................... 10
1.2.3 The winemaking process..................................................................................... 10
1.2.4 Wine consumption and health ............................................................................. 12
1.3 Fermentation .......................................................................................................... 12
1.3.1 Alcoholic fermentation ....................................................................................... 12
1.3.2 Microorganisms used in alcoholic fermentation ................................................. 13
1.3.3 Volatile by-products............................................................................................ 13
1.4 Saccharomyces cerevisiae ..................................................................................... 13
1.4.1 Taxonomic hierarchy .......................................................................................... 13
1.4.2 Description and morphology............................................................................... 14
1.4.3 Life cycle ........................................................................................................... 14
1.4.4 The importance of Saccharomyces cerevisiae .................................................... 14
1.5 Immobilization ....................................................................................................... 15
1.5.1 Advantages of immobilization method ............................................................... 16
1.5.2 Immobilization techniques .................................................................................. 16
1.5.3 Prerequisites of the support materials ................................................................. 17
1.5.4 Applications of cell immobilization in food industry ......................................... 17
7

1.6 Delignification ....................................................................................................... 18
1.7 Starch gelatinization............................................................................................... 18
1.8 Aim of the project .................................................................................................. 19
Materials &Methods ...................................................................... 21
2.1 Materials and methods of analysis ........................................................................ 21
2.1.1 Laboratory equipment ........................................................................................ 21
2.1.2 Yeast strain and media ........................................................................................ 22
2.1.3 Biomass production ............................................................................................ 22
2.1.4 Grape must .......................................................................................................... 22
2.1.5 Immobilization supports ..................................................................................... 22
2.1.5.1 Preparation of Delignified Cellulosic material (DCM)-Delignification .......... 22
2.1.5.2 Starch gelatinization ........................................................................................ 22
2.1.6 Immobilization of yeast cells .............................................................................. 23
2.1.6.1 Immobilization of S.cerevisiae AXAZ-1 on DCM (DC biocatalyst) .............. 23
2.1.6.2 Preparation of the cellulose/biopolymer composite biocatalyst (GS) .............. 23
2.1.7 Fermentations at 30 oC and 7 oC ........................................................................ 23
2.2 Assays ................................................................................................................... 23
2.2.1 Kinetics of fermentations .................................................................................... 24
2.2.2 Determination of residual sugars ........................................................................ 24
2.2.3 Determination of ethanol .................................................................................... 24
2.2.4 Determination of volatile by-products ................................................................ 24
2.2.5 Determination of alcohol (% v/v) ....................................................................... 25
2.2.6 Electron microscopy ........................................................................................... 25
Results &Discussion ...................................................................... 26
3.1 Cell immobilization ............................................................................................... 26
3.2 Winemaking ........................................................................................................... 27
3.2.1 Kinetics of fermentations ................................................................................... 27
8

3.2.2 Kinetic parameters of fermentations ................................................................... 28
3.2.3 Volatile byproducts ............................................................................................ 29
Conclusions ...................................................................... 32
4. Conclusions .............................................................................................................. 32
References ..................................................................... 33
5. References ................................................................................................................ 33
5.1 English Literature .................................................................................................. 33
5.2 Greek literature ...................................................................................................... 37
5.3 Web sites ................................................................................................................ 38































9





1.1 Introduction

Wine is considered as one of the most ancient fermented beverages dated from
6000 BC and is produced by fermenting crushed grapes by the use of various types of
yeasts. According to the preferable type of wine, different varieties of grapes and
strains of yeasts are used. The procedure of wine production is called winemaking and
starts with the selection of the grapes and ends with the bottling of produced wine.
The grape juice is converted into an alcoholic beverage with the process of alcoholic
fermentation. During alcoholic fermentation sugars such as disaccharides, contained
in grapes must, interact with yeast and are turned into ethanol, CO
2
and others
volatile and non-volatile compounds.
In recent years, cell immobilization techniques have become increasingly
important and are being successfully applied in industrial processes such as the
production of alcohols (ethanol, butanol and isopropanol), organic acids (including
malic, citric, lactic and gluconic acids), enzymes (cellulase, amylase, lipase and
others) and biotransformation of steroids for hormone production, wastewater
treatment, and food applications (beer and wine). Cell immobilization in alcoholic
fermentation is a rapidly expanding research area because of its beneficial
technological and financial advantages compared to the conventional free cell system
(Tsakiris et al. 2004a). Immobilized cells increase fermentation productivity, improve
the cost of bioprocesses as they provide the opportunity of cell recovery and
recycling, they influence yeast metabolism and offer the possibility of fermenting at
low temperatures contributing to the improvement of the organoleptic characteristics
of the wine, leading to higher quality wine products (Korkoutas et al. 2004,
Mallouchos et al. 2003a). There are many techniques proposed for the immobilization
of the cells. Some of them are: entrapment within a porous matrix, encapsulation,
10

cell flocculation and immobilization on solid carrier surfaces (Korkoutas et al. 2004,
Kandylis et.al. 2008a).
A lot of support materials for the immobilization of yeast as far as the
production of wine is concerned have been studied (Colagrande et al. 1994, Divies et
al. 1994, Kourkoutas et al. 2004). These materials are either organic or inorganic and
generally they are cheap enough and abundant. Sodium alginate, Ca-alginate, -
alumina pellets (Loukatos et al. 2000), mineral kissiris (Bakoyianis et al. 1992, 1993,
Argiriou et al. 1996), gluten pellets (Bardi et al. 1997a), -carrageenan, DEAE-
cellulose (Lommi and Advenainen 1990), and much more have been used. All these
can be taken by nature and they can be either used as they are or after a slight
alteration at their porosity or their surface. Some are constructed synthetically.
Unfortunately, none of them fulfilled the food-grade properties as these materials
bring forward some disadvantages as their stability and purification are concerned.
So, some other materials have also been considered in order to achieve the desired
characteristics, such as pieces of quince, apple, raisins, watermelon (Kourkoutas et al.
2001, 2002, Tsakiris et al. 2004b, Veeranjaneya Reddy et al. 2008) or delignified
cellulose (Bardi and Koutinas 1994). These supports have successfully been used for
low-temperature winemaking resulting in wines with improved organoleptic
characteristics (taste and aroma) (Sipsas et al. 2009).
Organoleptic characteristics of wine are of great importance and the
combination of them (flavor) gives each wine its distinctive character. Flavor is a
complex combination of taste and aroma and it depends on many factors such as
chemical constitution. Aroma is the result of a combination of components produced
by yeast during fermentation. These compounds are volatile compounds, acetates and
ethyl esters, higher alcohols, fatty acids, ketones and aldehydes (Bardi et al. 1997b).
Other significant components of wines are higher alcohols such as amyl alcohols and
isobutyl alcohol which considered to be the most important.
The winemaking in low temperature gives a final product of great organoleptic
character. The fermentation in such low temperature (<5
o
C), though, calls for yeast
strains resistant at these conditions. Cryotolerant and alcohol-tolerant yeast strains
(Saccharomyces cerevisiae) are capable to develop at 6-7
o
C and have given wine of
great clarity, stable color and better quality. Wine of better quality have been
produced by yeast strains AXAZ-1, AXAZ-2, Visanto-1 which apart from their
alcohol and cryotolerant characteristics have also given wines with alcohol
11

concentration up to 10% in only 155 days (Argiriou et al. 1992, Bakoyianis et al.
1992, Bardi and Koutinas 1994, Bardi et al. 1997b). Acceleration of malolactic wine
fermentation has also been achieved by using cell immobilization (Agouridis et al.
2005).


1.2 Wine

The term wine refers to the alcoholic beverage made of fermented juice of
grapes [1]. Wine can be also made from other fruits and are usually named after the
fruit from which they are produced e.g., elderberry wine, dandelion wine, apple wine
etc. The English word wine comes from the Proto-Germanic winam, which is derived
from the Latin word vinum. The Latins borrowed this term from the ancient Greek
term oinos.
In winemaking are usually used fresh grapes, which are the ripe fruits of the
vine. Over ripened or the slightly raisined fruits can be also used, provided they are
receptive to the must production. The great variety of grapes used in winemaking
derived from the species V.Vinifera (genus Vitis). The 5,000 varieties of this species
differ in color, size and shape of the berries, in the composition of the juice and
flavor, ripening time and resistance to disease.
One of the most important factors related to the types of wines which gives the
unique character to the wine is the quality of raw material. This depends on the
variety, climate, soil, cultivation methods and the topography of the vineyard. The
quality of wine depends on the different winemaking techniques followed by local
winemakers. Usually, the effort to increase the yield per hectare results in the
degradation of wine quality, while selecting the winemaking technique leads to
specific products (Ribreau-Gayon et al. 2006).
Both the wine and the grape are biological environments within which take
place many chemical and biological changes during the vinification, the maintenance
and the aging. The intervention to the above process is designed to control conditions
in order to facilitate the useful and obstruct the harmful changes.

12

1.2.1 History of the wine
The vineyard has an archeological record dating back many millions of years.
Before the ice age there were vines even in present-day Polar Regions and especially
in Iceland and northern Europe. The glaciers have significantly limited the natural
distribution and forced the geographic isolation of many varieties, some of which
evolved in different species. After the end of the ice age, the vine was evolved in
areas with more favorable climate, such as the Caucasia (Turkey, northern Iraq,
Azerbaijan and Georgia) and Mesopotamia. It is also generally believed the
domestication of the wine grape (Vitis vinifera) occurred in the same area (Zohary and
Hopf 2000).
Most researchers believe that winemaking was evolved in southern Caucasia, in
the present-day of Georgia million years ago (5000-7000 B.C.) as there is evidence of
grapes kernel in Neolithic potteries from Georgia that suggests that contemporaneous
wine production was dispersed throughout the region. Older examples of fermented
beverages have been discovered (McGovern et al. 2004), but they appear to have been
produced from rice, honey, and fruit (hawthorn and/or grape). Also, researchers at
Hajji Firuz Tepe, in the northern Zagros Mountains of Iran discovered wine residues
in potteries by identifying traces of tartaric acid by spectroscopic methods (McGovern
et al. 1996, Garnier et al. 2003). Another evidence of intentional winemaking appears
in the representations of wine presses from the region of Egypt about 5000 years ago.
Recent studies have discovered the existence of amphorae in the tomb of King
Tutankhamen (3150 B.C.) in which were detected traces of both white and red wine
(Guasch-Jane et al. 2006).
In Greece the viticulture began in 4,000 BC and there is evidence, found on
artifacts, that it was known to the Minoan and Mycenaean civilizations. The oldest
winery in the world is maintained until today in Crete and includes a stone wine press.
Ancient Greeks considered that wine was a gift from the gods and worshiped
Dionysus, a creature with the mind of man and the instincts of a beast, as god of wine.
Festivals honoring Dionysus were held during winter months and were celebrated by
performing arts and wine drinking. Vineyards, grapes and wine drinking festivities
were painted on hundreds of ancient Greek artifacts of clay, marble and metal.
Often, the ancient Greeks added herbs and spices to wine to mask spoilage. From
texts of Theophrastus seems that in Greece it was known the beneficial effect of aging
on the quality. Greeks stored and transported wines in airtight, ceramic vessels called
13

amphorae. The amphorae had various shapes with two handles, and they were used to
signify the city that produced and traded the particular wine. The storage in amphorae
had its benefits because it allowed them to store wine for long periods thus creating
brilliant aged vintage wines [2].
During the 17
th
century, the wine began to take on its modern expression. About
this time in Western Europe, the use of sulfur in barrel treatment became fairly
common, which greatly increased the likelihood of producing better quality wines and
extending their aging potential. In the mid- 1600s in England, the production of strong
glass bottles was occurred. With mechanization, glass bottles became the standard
container for both wine maturation and transport. The production of bottles and the
introduction of cork as a bottle closure provided conditions favorable for the
production of modern wine. The cork stayed wet in this position, so the wine
remained isolated from oxygen and had the opportunity to develop a smooth
character. The perfection of wine distillation significantly contributed to the
production of better quality wines. Distilled spirits were added to the fermenting juice
to prematurely stop fermentation. As a consequence, grape sugars were retained,
along with the extraction of sufficient pigments, to produce a sweet, dark-red wine.
Although alcohol distillation was first developed by the Arabs, the adoption of the
technique in medieval Europe was slow. Thus, fortified wines are of relatively recent
origin.
In the mid 19
th
century, Pasteur mentioned the central importance of
microorganisms (yeasts and bacteria) to fermentation and used heat to destroy
undesirable microorganisms in wine. This discovery set in motion a chain of events
that has produced the incredible range of wines that typify modern commerce
(Jackson 2008).

Fig. 1: Wine in ancient years
14


1.2.2 Chemical constituents of wine
The understanding of the chemical nature of wine has started since the late
1960s. This knowledge is beginning to guide vineyard and winery practice toward the
production of better-quality wine. The great increase in the number of compounds
found in wine is due to the developments in techniques such as gas chromatography
(GC), thin-layer chromatography (TLC), high-performance liquid chromatography
(HPLC), infrared spectroscopy, solid-phase microextraction (SPME) and nuclear
magnetic resonance (NMR) spectroscopy. Especially valuable has been the
combination of gas chromatography with mass spectrometry (Hayasaka et al. 2005).
The vast majority of chemicals found in wine are the metabolic by-products of yeast
activity during fermentation.
Wine consists of two primary ingredients, water and ethanol. However, the
basic flavor of wine depends on an additional 20 or more compounds and an even
larger number of compounds contribute to the differences that distinguish one varietal
wine from another.

Fig. 2: Chemical composition of wine
1.2.2.1 Water
The water is derived from grape juice and forms about 85% of wine. As the
predominant chemical constituent of grapes and wine, water plays a critical role in
15

establishing the basic characteristics of wine and especially the basic flow
characteristics of wine. Even the occurrence of tears in a glass of wine is partially
dependent on the properties of water. In addition, the high specific heat of water slows
the warming of wine in a glass. Water is also an essential component in many of the
chemical reactions involved in grape growth, juice fermentation, and wine aging
(Jackson 2008).
1.2.2.2 Alcohols
Alcohols are organic compounds containing one or more hydroxyl groups
(OH). Simple alcohols contain a single hydroxyl group, whereas diols and polyols
contain two or more hydroxyl groups, respectively. They constitute approximately 5-
15% of wine and are responsible for its sensory properties. They are formed by
conversion of sugars during yeast fermentation.

Ethanol
Ethanol is the most important alcohol in wine. Although small quantities are
produced in grape cells, the main source of ethanol in wine is yeast fermentation and
it is considered the principal organic by-product of fermentation. Ethanol
concentration in wine is above 1415% but higher levels can be reached by addition
of sugar during fermentation. The achievement of the desirable ethanol levels is the
result of controlling of many factors such as sugar content, fermentation temperature,
and yeast strain.


Fig. 3: Ethanol molecule

Ethanol is crucial to the aging and sensory properties of wine. During
fermentation, the increasing alcohol content progressively limits the growth of
microorganisms and suppresses microbes that might produce off-odors. Ethanol acts
as an important cosolvent, and along with water, plays a critical role in extracting
16

constituents from grapes. As a result, ethanol is particularly important in solubilizing
non-polar aromatics and affects their volatility. Ethanol also influences the types and
amounts of aromatic compounds produced as it affects the metabolic activity of
yeasts. Furthermore, it acts as an essential reactant in the formation of several
important volatile compounds.
Ethanol has multiple effects on taste by directly enhancing sweetness through its
own sweet taste, indirectly modifying the perception of acidity, making acidic wines
less sour and at high concentrations, it produces a burning sensation. Ethanol can also
increase the intensity of bitterness, while decreasing the astringency of tannins. It also
helps to dissolve volatile compounds produced during fermentation and those formed
during maturation in wood cooperage, reducing the escape of aromatic compounds
with carbon dioxide during fermentation. However, alcohol concentrations below 7%
contribute to the release of many aromatic compounds, affecting the aromatic
distinctiveness of a wine (Guth 1998).
Ethanol plays several roles in the aging of wine. Along with other alcohols, it
slowly reacts with organic acids to produce esters. Its concentration also influences
the stability of esters. In addition, ethanol reacts with aldehydes to produce acetals
(Jackson 2008).

Methanol
Methanol is not a major constituent in wines and has no direct sensory effect as
its concentration levels in wine are about 0.1-0.2 g/liter. Of the over 160 esters found
in wine, few are associated with methanol. Methanol is metabolized to formaldehyde
and formic acid which are both toxic to the central nervous system and especially the
optic nerve, causing blindness. For this reason, methanol must never accumulate to
toxic levels under legitimate winemaking procedures.


Fig. 4: Methanol molecule
17

The methanol content of fermented beverages is associated with the pectin
content of the substrate as the amount of methanol found in wine is primarily
generated from the enzymatic breakdown of pectins. After degradation, methyl groups
associated with pectin are released as methanol. Wine generally has the lowest
methanol content of any fermented beverage as grapes are low in pectin. The
methanol content can be increased by adding pectolytic enzymes to juice or wine to
aid clarification or by adding distilled spirits to the wine.

Higher alcohols
Higher or fusel alcohols are alcohols with more than two carbon atoms. They
may be present in healthy grapes, but in very small amounts. However, most higher
alcohols found in wine are the by-products of yeast fermentation and they commonly
account for about 50% of the aromatic constituents of wine, excluding ethanol.
The most important higher alcohols are the straight-chain alcohols: 1-propanol,
2-methyl-1-propanol (isobutyl alcohol), 2-methyl-1-butanol, and 3-methyl-1-butanol
(isoamyl alcohol). 2-Phenylethanol (phenethyl alcohol) is the most important phenol-
derived higher alcohol.


Fig. 5: Principle higher alcohols found in wine: A. 3-methyl butanol (isoamyl
alcohol), B. 2-methyl butanol (active amyl alcohol), C. 2-methyl propanol (isobutyl
alcohol), D. 1-propanol (n-propyl alcohol), E. phenethyl alcohol, F. tryptophol, G.
tyrosol
Most straight-chain higher alcohols have a strong pungent smell. At low
concentrations (~0.3 g/liter or less), they have little effect to the odor of the wine but
18

at higher levels, their influence is increased and give wine some of its distinctive
aromatic character.
The formation of higher alcohols during fermentation is markedly influenced by
many factors. Synthesis is favored by the presence of oxygen, high fermentation
temperatures, and the presence of suspended material in the fermenting juice. On the
other hand, the presence of sulfur dioxide and low fermentation temperatures suppress
production. Another factor that influences the production of higher alcohols is the
yeasts strain used in fermentation. Yeasts vary considerably in their efficiency to
produce higher alcohols.

Polyols and sugar alcohols
Glycerol is the most important wine polyol. In dry wine, glycerol is commonly
the most abundant compound, after water and ethanol. It is often higher in red
(~10mg/liter) than white (7mg/liter) wines. Glycerol has a slight sweet taste but it is
unlikely to be noticeable in a sweet wine, and plays a minor role in dry wines.
Variety, maturity, and health all affect the amount of glycerol present in grapes.
During fermentation, yeast strain, temperature, sulfur dioxide, and pH level can
influence the synthesis of glycerol.
Sugar alcohols, such as alditol, arabitol, erythritol, mannitol, and sorbitol, are
commonly found in small amounts in wine. Higher concentrations usually are the
result of fungal infection in the vineyard or bacterial growth in the wine. Sugar
alcohols can be oxidized by some acetic acid bacteria to the respective sugars.



Fig. 6: A. Sorbitol, B. Erythritol, C. D- Mannitol, D. Glycerol
19

1.2.2.3 Sugars
Sugars are carbohydrates with several hydroxyl groups and an aldehyde or
ketone group. Simple sugars bond either together to form polymers such as pectins,
gums, starches, and cellulose, or with other secondary metabolites such as lactones
and anthocyanidins, to form glycosides.
The principal sugars in grapes are glucose and fructose and they often occur in
roughly equal proportions. Sugars other than glucose and fructose, such as sucrose,
occur, but in relatively insignificant amounts. Sucrose is enzymatically split into
glucose and fructose during fermentation. Grape sugar content varies depending on
the species, variety, maturity, and health of the fruit. Some species, such as V.vinifera,
reach a sugar concentration of 20%, whereas some other species rarely reach this
level.


Fig. 7: Glucose, fructose and sucrose molecules

Grape sugar content is critical to the growth and metabolism of the yeast. One
of the primary wine yeast, Saccharomyces cerevisiae, derives most of its metabolic
energy from glucose and fructose. Unfermented sugars are called residual sugars. In
dry wines, the residual sugar content consists primarily of pentose sugars, such as
arabinose and xylose, and small amounts of unfermented glucose and fructose. What
is more, sugars can also be synthesized and released by yeast cells.
The residual sugar content of dry wine is generally less than 1.5 g/liter. At this
concentration, the perception of sweetness is undetectable and the nutritive value of
these sugars is usually insufficient to constitute a threat to the microbial stability of
wine. At higher concentrations, though, residual sugars increasingly pose a microbial
20

hazard. When the residual sugar content rises above 0.2%, the sweetness starts to
become detectable. Although residual sugars are of great importance to the sweetness
of wine, fermentable sugars in grapes are essential for fermentation. Sugars are
metabolized to ethanol, higher alcohols, fatty acid esters, and aldehydes, which give
different wines much of their aromatic character.

1.2.2.4 Acids
Acids are compounds that are characterized by the ionization and release of
hydrogen ions (H
+
) in water. For the majority of table wines, the acceptable total
acidity ranges between 5.5-8.5 mg/liter. Acidity in wine is divided into volatile and
fixed acidity. Volatile acidity refers to acids that can be readily removed by steam
distillation, whereas fixed acidity includes those that are poorly volatile. Total acidity
is the combination of both categories and can be expressed in terms of tartaric, lactic,
sulfuric, or acetic acid equivalents.
Acids are very important for the characteristics of wines as they produce a
refreshing or sour taste depending on their concentration and they usually reduce
wines sweetness. What is more, their releasing during crushing is probably
instrumental in the initiation of acid hydrolysis of nonvolatile precursors in the fruit
(Winterhalter et al. 1990). The low pH, maintained by acids, plays an important role
in color stability of red wines as anthocyanins lose their red color when pH rises, and
has a beneficial antimicrobial effect as most bacteria do not grow at low pH values.
Furthermore, acids help the precipitation of pectins and proteins and the solubilization
of copper and iron that can induce haziness the finished wine.

Acetic acid
During fermentation, small amounts of acetic acid are produced by yeasts.
Acetic acid at levels lower than 300 mg/liter in wine can be a desirable flavorant, but
at levels greater than 300 mg/liter, it gives wine a sour taste and taints its fragrance.
High levels of acetic acid are usually associated with contamination of grapes, juice,
or wine with acetic acid bacteria.


Malic acid
21

Malic acid may constitute about half the total acidity of grapes and wine. Low
concentrations of malic acid give wine a flat taste and make it susceprible to microbial
spoilage, whereas high levels of malic acid give wine a sour taste.

Lactic acid
During fermentation, a small amount of lactic acid is produced by yeast.
However, lactic acid usually comes from metabolic activity of bacteria (lactic acid
bacteria) from the process of malolactic fermentation. During this process the harsher-
tasting malic acid is converted to the smoother-tasting lactic acid.

Tartaric acid
Tartaric acid is the other major grape acid, along with malic acid. It is
metabolized by few microorganisms and thus, it is usually the preferred acid added to
increase the acidity of high pH wines.

1.2.2.5 Phenols
Phenols are a large and complex group of cyclic benzene compounds,
possessing one or more hydroxyl groups and they are of primary importance to the
characteristics and quality wine and especially red wines, as they affect the
appearance, taste, aroma, and antimicrobial properties of wine. They are primarily
derived from grape and only trace amounts are derived from yeast metabolism.
The major phenolics found in wine are either members of the flavonoids or
nonflavonoids. Flavonoids are characterized by a C
6
-C
3
-C
6
skeleton, consisting of two
phenolic rings joined by a central pyran (oxygen-containing) ring. The most common
flavonoids in wine are flavonols, catechins, and anthocyanins (red wines). Flavonoids
may exist free or in polymers with other flavonoids, sugars, nonflavonoids, or a
combination of these. At grapes, they have an antimicrobial function against
pathogens, and insect pests. Nonflavonoids possess a C
6
-C
3
skeleton and are
structurally simpler than flavonoids. Nonflavonoids are derivatives of
hydroxycinnamic and hydroxybenzoic acids. The most numerous and variable are
hydroxycinnamic acid derivatives and they occur as esters with tartaric acid, but may
also be associated with sugars, various alcohols, or other organic acids. The most
common nonflavonoid in grapes, caftaric acid is one of the primary substrates for
22

polyphenol oxidase and plays an important role in oxidative browning of must. Both
flavonoid and nonflavonoid polymers, generically termed tannins and affect in many
ways the odor, the color and the taste of the wine (Jackson 2008).

1.2.2.6 Aldehydes and ketones
Aldehydes are carbonyl compounds with a terminal carbonyl functional group
(C=O). Ketones are also carbonyl compounds with the carbonyl group located on an
internal carbon.
Some aldehydes very important in the generation of wine aroma, such as
hexanals and hexenals, are produce by grapes. However, most aldehydes found in
wine are produced during fermentation. Acetaldehyde is the major wine aldehyde. It
is one of the early metabolic by- product of fermentation and it is considered as a very
important compound in stabilizing the color of red wines. Other aldehydes,
occasionally having a sensory impact on wine, are furfural and 5-(hydroxymethyl)-2-
furaldehyde.
Some ketones, such as norisoprenoid ketones and -damascenone are found in
grapes and they play appear to be significant in the aroma of several red grape
varieties. Many other ketones are produced during fermentation, but few appear to
have sensory significance. The major exception is diacetyl, which at low
concentrations (<5 mg/liter) gives a buttery, nutty, or toasty flavor. However, at
slightly above its sensory threshold, diacetyl may begin to donate a caramel-like
attribute (Rogerson et al., 2001). Another ketone, acetoin has a sugary, butter-like
character and is of great sensory importance to table wines, in which it occurs at low
concentrations.

1.2.2.7 Esters
Esters are formed as condensation products between the carboxyl group of an
organic acid and the hydroxyl group of an alcohol or phenol. Over 160 specific esters
have been identified in wine, but their significance in wine aroma is negligible as they
are found only in trace amounts. However, some esters, such as acetate esters, derived
from acetic acid and fusel alcohols, and ethyl esters, formed between ethanol and fatty
acids, occur at or above their sensory thresholds. Esters are synthesized in grapes, but
23

in amounts that are sensorially insignificant. Some other esters found in wines are
produced by yeasts (Jackson 2008).

1.2.2.8 Nitrogen-containing compounds
Many nitrogen-containing compounds are found in grapes and wine, including
inorganic forms such as ammonia and nitrates, and diverse organic forms, including
amines, amino acids, pyrazines, nitrogen bases, proteins, and nucleic acids. Complex
organic nitrogen compounds (pyrimidines, proteins, and nucleic acids) are essential
for the growth and metabolism of grape and yeast cells, but are seldom involved
directly in the sensory attributes of wine.

1.2.2.9 Vitamins
Vitamins contain a series of diverse chemicals involved in the regulation of
cellular activity. They are found in small quantities in grape, juice, and wine, and their
concentration is generally decreased during fermentation and aging. Vitamin levels in
wine are inadequate to be of major significance in human nutrition, but they usually
are sufficient for microbial growth.

1.2.2.10 Dissolved gases
Wines contain varying amounts of several gases such as CO
2
, O
2
, and SO
2
. All
except nitrogen can have significant effects on the sensory properties of wine.

1.2.2.11 Minerals
Many mineral elements, such as lead, copper, iron, calcium, chlorine, sodium,
potassium, sulfur, and aluminum are found in grapes and wine. At normally occurring
levels, many minerals are important cofactors in vitamins and enzymes. However,
heavy metals such as lead, mercury, cadmium, and selenium are potentially toxic, and
their occurrence in wine at above trace amounts usually indicates contamination. At
higher than normal levels, minerals such as iron and copper also can be undesirable as
they catalyze oxidative reactions, modify taste characteristics, or induce haziness.
24



1.2.3 The winemaking process
Winemaking has been around for thousands of years. In its basic form,
winemaking is a natural process that requires very little human intervention. There are
five basic steps to making wine: harvesting, destemming and crushing/pressing,
fermentation, clarification and stabilization, and bottling. The steps for making white
wine and red wine are essentially the same, with some differences.

Harvesting
Harvesting is the picking of the grapes and in many ways the first step in wine
production. Grapes are either harvested mechanically or by hand. As the grapes ripen
the concentration of sugars and aroma compounds rises, and the concentration of
acids falls. The aim at harvest is to pick the grapes at their optimum composition. The
time of harvesting is determined by a combination of science and old-fashioned
tasting tests for the level of sugar, pH of the grapes, acid (Titratable Acidity as
expressed by tartaric acid equivalents) and flavor compounds. Other considerations
include phenological ripeness, and tannin development. When optimum levels are
reached, the grapes are harvested [3].

Destemming and crushing/pressing
Destemming is the process of separating stems from the grapes. Depending on
the winemaking procedure, this process may be undertaken before crushing with the
purpose of lowering the development of tannins and vegetal flavors in the resulting
wine. Destemming is not compulsory; white wines are often fermented with the stems
and leaves, but these are always removed in red wines because they contain tannins
and they leave a vegetable taste on the wine.
After the destemming (in some cases these steps are done simultaneously)
comes the crushing/pressing. This part of wine production consists in extracting the
juice from the berries by squeezing them gently until they release their juices. In red
wines, the berries are crushed and the skins are left with the must (the initial grape
juice) so they absorb the tannins for their rich deep red color. The skins are taken out
25

after fermentation. In the case of white wines, the grapes are squeezes, not crushed, to
limit the absorption of tannins which would color the wine.

Fermentation
After the extraction of the must, comes the fermentation, which is the most
important part of the process of winemaking. The main purpose of fermentation is to
turn the must sugars into ethyl alcohol, and this takes place thanks to the yeasts.
Yeasts are normally already present on the grapes. The alcoholic fermentation can be
done with this natural yeast, but since this can give unpredictable results depending on
the exact types of yeast that are present, cultured yeast is often added to the must.
Fermentation is done in huge tanks and it takes place in four steps:
Lag phase: the yeasts become acclimatized to the conditions of the must,
high sugar concentration, low pH, temperature and sulfur dioxide.
Exponential phase: the yeasts are already acclimatized to their
surroundings and start multiplying in an exponential growth until they reach their
maximum density of population. The yeasts consume the high sugar levels and the
sugar concentration starts to descend quickly.
Stationary phase: The yeast has reached its maximum capacity, which
makes it stay stationary and fermentation continues at a steady pace. The heat
released by the fermentation keeps the wine at a stable temperature.
Death phase: During this phase the lack of sugar and high alcohol
concentration starts killing the yeasts, and the pace of the fermentation slows.
Fermentation is affected by several factors. The most important factor is
temperature. Fermentation can only take place between 5C and 38C. The
fermentation of white wines takes place at a lower temperature, between 8C and
14C; and red wines ferment between 25C and 30C. The fresh and aromatic flavors
are best achieved with lower temperatures. Other factors that affect fermentation are
the levels of sugar, the acidity levels, the presence of micro nutrients like vitamins and
even the airing of the barrel [3].
During or after the alcoholic fermentation, a secondary fermentation, malolactic
fermentation can also take place, during which the tart-tasting malic acid naturally
present in grape must is converted to softer-tasting lactic acid by specific strains of
26

bacteria (lactobacter) [4]. This fermentation is often initiated by inoculation with
desired bacteria. Malolactic fermentation tends to create a fuller mouthfeel in the
wines.
Clarification and stabilization
After the fermentation process, comes the clarification, stabilization and
filtration. These phases are essential as they prepare the wine for its consumption by
cleaning it and removing and solid particles or sediments left over, and prevent from
spoilage of wine inside the bottle.
Clarification is the process of removing any suspended particles left behind after
the pressing and fermentation. The suspended particles are normally leftover pieces of
the skin, pulp or seeds of the berries, as well as colloids (microscopic particles which
the human eye can't see without help) like gums, pectins, proteins, tartrates, active
yeast or bacteria. Clarification is very important as it gives a clear wine with bright
color and makes it more appealing to the consumer. Clarification during wine making
can be done in different ways: by racking or siphoning the wine from one tank or
barrel to the next. Another way of clarifying the wine is by filtering it through
progressively finer filters until the wine appears bright and clear.
Clarification is the first part of stabilization, as it removes the solid particles
which cause cloudiness in the wine. Cold stabilization consists in exposing the wine
to freezing temperatures to encourage the tartrates to crystallize and precipitate out of
the wine, preventing this to happen in the bottle. The crystallization of tartrates
produces small crystals and although they're harmless they can be undesirable for
customers.
If the wine contains residual sugar, it can continue to ferment in the bottle. This
produces carbonic gas which will make the wine sparkly or gassy when it's opened.
Further fermentation in the bottles is avoided by sterile filtration and bottling, to
ensure no active yeasts remain. Another way is to inject the wine with sulfur dioxide
and sorbic acid to inhibit the growth of yeast [3].


Bottling
The final stage of the wine making process involves the bottling of wine.
During bottling, a final dose of sulfite is added to preserve the wine and prevent
27

unwanted fermentation in the bottle. Once the wine is bottled, the opening is sealed
with a cork or synthetic corks. Further aging can be done in bottle.

Fig. 8: Flow diagram of winemaking (Jackson 2008)
1.2.4 Wine consumption and health
Wine is produced from natural raw materials and contains valuable components.
The alcohol contained in large amounts, glycerin and sugar principally provide the
nutritional value of wine. But the other compounds, such as various vitamins B
1

(thiamine), B
2
(riboflavin), B
6
(pyridoxine), B
12
(cobalamin), biotin (H), or folic acid,
ascorbic acid, choline, inorganic and organic salts, polyphenols and minerals, play an
important role in proper functioning of the human body (Soufleros, 1997).
Until the 1900s, wine was used in the treatment for several human diseases and
acted as an important solvent for medications. Since the 1990s, there has been
evidence about the health benefits of moderate wine consumption. One of the more
widely documented benefits relates to cardiovascular disease (Mukamal et al. 2006).
Studies have shown that moderate wine consumption contributes to a healthful
balance of low- and high-density lipoprotein in the plasma (Kinsella et al. 1993,
Soleas et al. 1997). Wine can also reduce the undesirable influences of stress, enhance
sociability, lower rates of clinical depression, and improve self-esteem. Other studies
have shown that wine consumption protects human against cancer as wine contains
catechins (flavonoid compounds) that act as antioxidants, preventing cell damage
from free radicals (Van De Wiel et al. 2001, Cordova et al. 2005). Finally, moderate
consumption of wine can help prevent diseases such as Parkinson's and Alzheimers
syndrome (Paganini-Hill 2001, Cupples et al. 2000, Luchsingera et al. 2004).
However, high alcohol consumption is associated with a number of health and
social problems such as impaired vision, violent behavior, psychological problems,
and car accidents. Uncontrolled consumption increases the risk of heart and liver
disease, circulatory problems, ulcers, cancer and irreversible brain damage.
28


Fig. 9: Wine consumption and health benefits




1.3 Fermentation

It has been established that the term "fermentation" is used to describe the
biochemical breakdown of carbohydrates within anaerobic conditions. According to
the science of biochemistry, fermentation is defined as the chemical change that
leads to the breakdown of carbohydrates under anaerobic conditions. However,
fermentation can also be carried out under aerobic conditions (Tadege et al. 1999).
There are many benefits and applications of fermentation for modern human.
The biotransformation of carbohydrate raw materials using yeast leads to:
Production of alcoholic beverages (wine, beer, spirits)
Production of alcohol for alcoholic beverages and pharmaceutical use
Production of bakery yeast
Production of SCP(single cell protein) for animal feed
Production of vinegar with the additional use of acetic acid bacteria
Preservation of fermented food produced by the alcoholic fermentation. The
ethanol and acetic acid are inhibitors of the growth of pathogenic
microorganisms (Caplice & Fitzgerald 1999)

1.3.1 Alcoholic fermentation
Alcoholic fermentation is the biochemical conversion of exozes (C
6
H
12
O
6
) to
ethanol and CO
2
with simultaneous energy release, and it is conducted by yeasts
under anaerobic conditions.
29

The alcoholic fermentation takes place via the glycolytic pathway (known as
Embden-Meyerhof-Parnas pathway). This includes all the reactions that allow living
cells to convert glucose (or fructose) to pyruvate, with simultaneous release of energy
in the form of ATP (Stryer 1997). During the series of reactions that take place,
electrons are transferred to NAD
+
reducing it to NADH. Subsequently, the
decarboxylation of pyruvate leads to formation of acetaldehyde, which is reduced to
ethanol by electron transfer from NADH. During this process the NADH is re-
oxidized to NAD
+
.

Fig. 10: Ethanol production via glycolysis pathway

Theoretically, 1 g of sugar produces 0.51 g ethanol and 0.49 g CO
2
. However, in
practice 0.46g ethanol and 0.44 g CO
2
is produced by 1 g of sugar (Bekatorou 2001a).

1.3.2 Microorganisms used in alcoholic fermentation
Alcoholic fermentation for the production of alcoholic beverages is carried out
by yeasts and usually by strains of S.cerevisiae. It is one of the most important species
for food industry as it is able to survive and be active in high pressure environment,
low temperatures and pH values. The strain selection depends on the flavor of the
final product that has to be achieved. Additionally, many common moulds of the
genera Aspergillus, Fusarium and Mucor are also known for their abilities for alcohol
production even though they are strict aerobes, requiring oxygen for their growth.
30

The most common wine yeast is Saccharomyces cerevisiae. This yeast ferments
glucose, sucrose and raffinose and metabolize glucose, sucrose, raffinose, maltose and
ethanol. However, it cannot ferment or utilize pentoses (such as arabinose) which are
usually present in small amount in wines as residual sugars (Fugelsang et al. 2010). In
addition to S. cerevisiae, other species within the Saccharomyces genus of the
indigenous grape flora that are involved in winemaking include S. ellipsoideus, S.
pastorianus, S. bayanus, S. apiculatus, S. uvarum, S. rosei, S. elegans, S. oviformis, S.
italicus, S. chevalieri, S. carlsbergensis etc. Other yeasts involved in spontaneous
alcoholic fermentation is Klockera apiculata, Klockera corticis, Hanseniaspora
guilliermonti, Hanseniaspora osmophila (Granchi et al. 2002), yeasts of the genera
Candida, Brettanomyces, Cryptococcus, Rhodotorula, Torulopsis,
Schizoblastosporion etc. (Koutinas & Pefanis 1994, Jackson 2008). Apart from the
above yeasts in grape must are found lactic bacteria belonging to the Lactobacillus,
Pedicoccus, Leuconostoc and Oenococcus genera.

1.3.3 Volatile by-products
The volatile by-products of alcoholic fermentation play an important role in the
formation of the final product flavor and their concentration depends on the yeast
strain, temperature, pH and grape must composition. The major volatiles are the
following:
Methanol: Methanol is produced by the enzymatic hydrolysis of poly-
galactouronic chain (pectin) methyl esterified carboxyl groups using pectin
methyl esterase enzyme (PME).
Ethyl acetate: There are two potential routes for ester formation: the reaction
between an alcohol (such as ethanol) or higher alcohols with a fatty acyl-CoA
ester. Ethyl acetate is the major ester in wine.
Acetaldehyde: Acetaldehyde is the major aldehyde to consider due to its
importance as an intermediate in the formation of ethanol and acetic acid.
Higher alcohols: The higher alcohols (propanol-1, isobutanol, amyl alcohols
and in specific 2-methyl-1-butanol, 3-methyl-1-butanol) contribute to the
overall wine flavor character and can be synthesized by the catabolic process
of the reduction of the corresponding aldehydes, which are usually derived
from deamination of amino acids resulting from the breakdown of the yeast
31

and grape must proteins. Their production depends on the microorganism
strain and the temperature.

1.4 Saccharomyces cerevisiae
Saccharomyces cerevisiae is undoubtedly the most important yeast species. In
various forms, it may function as the wine yeast, brewers yeast, distillers yeast, or
bakers yeast. All strains of S.cerevisiae can grow aerobically on glucose, maltose,
and trehalose but fail to grow on lactose and cellobiose [5]. However, they are not
capable to utilize nitrate as a source of nitrogen and they do not ferment pentoses.
They are able break down their food through both aerobic respiration and anaerobic
fermentation. What is more, they have the ability to have both sexual and asexual
reproduction.

1.4.1 Taxonomic hierarchy
Kingdom: Fungi
Phylum: Ascomycota
Subphylum: Saccharomycotina
Class: Saccharomycetes
Order: Saccharomycetales
Family: Saccharomycetacae
Subfamily: Saccharomyetoideae
Genus: Saccharomyces
Species: S. cerevisiae

1.4.2 Description and morphology
Saccharomyces cerevisiae is a single-celled Eukaryotic budding yeast belonging
to the Ascomycetes, a highly diverse group of fungi.
S. cerevisiae cells are generally ellipsoidal in shape and they typically display
an ovoid morphology. However, the size and shape of the yeast are affected by
growth rate, mutation, and environmental conditions (composition, temperature,
pressure, presence of oxidant agents, etc.). The cell size is ranging from 5 to 10 m at
32

the large diameter and 1 to 7 m at the small diameter. Mean cell volumes are 29 or
55 m
3
for a haploid or a diploid cell, respectively (Pons et al. 1993).


Fig. 11: S.cerevisiae by electronic microscope

1.4.3 Life cycle
S. cerevisiae is an extremely well studied organism, with a clearly defined and
experimentally manipulable life cycle. Yeast cells can either be haploid, containing
one set of chromosomes, or diploid, containing a double set of chromosomes.
Diploids are formed when a-type haploids cells are mixed with -type haploid cells.
The life cycle of yeast involves mitotically propagating haploid forms of two distinct
mating types, and a diploid form that can either grow vegetatively or can be induced
into a meiotic developmental pathway through manipulation of the nutrient conditions
of the growth media.
Both haploid and diploid cells can replicate asexually through budding (mitotic
growth of yeast cells). During this process, growth of the cell is directed to a specific
location on the surface of the mother cell and a new cell is formed.
When diploid cells are in a hostile nutritive medium (depleted of fermentable
sugar, poor in nitrogen and very aerated) are induced to initiate meiosis and
sporulation, resulting in the formation of asci (a kind of sac with a thick cell wall).
Each one contains four haploid ascospores issued from meiotic division of the
nucleus. These haploid ascospores can germinate giving rise to two a-, and two -cell
cultures (Madigan et al. 2009).

33


Fig. 12: Life cycles of Saccharomyces cerevisiae (Kavanagh 2011)

1.4.4 The importance of Saccharomyces cerevisiae
Saccharomyces cerevisiae is one of the most important yeasts in traditional and
industrial winemaking. What is more, it is a popular "model" organism in the
laboratory because it is a unicellular eukaryote that shows many advantages such as:
It can be cultured easily
It grows rapidly
Its entire genome is known
It can be easily transformed with genes from other sources
The most important commercial use for S. cerevisiae is in food and alcoholic
beverages production. This yeast is alcohol-resistant. It is tolerant to SO
2
and
completely consumes the sugars of the grape and converts them under anaerobic
conditions mainly to alcohol (ethanol) and aromatic compounds rather than biomass.
It grows and ferments rapidly under acidic and high osmotic conditions and at low
fermentation temperatures (Jackson 2008). These features of the S. cerevisiae make it
an important microorganism in the production of alcoholic beverages and spirits.
Specific strains of the microorganism differ in their efficiency of the production of
aromatic compounds and byproducts (Ubeda & Briones, 2000). This property can be
used for the isolation and identification of different strains (Romano et al. 2003).
34

The importance of the yeast in the production of volatile compounds in wines
has been reported by many researchers (Patel & Shibamoto 2002, Fleet 2003, Ubeda
& Briones 2000, Antonelli et al. 1999). Important role in this also seems to play the
inoculation of the must with pure or mixed yeast culture, the amount of the inoculum
and the inoculation time (Fraile et al. 2000, Mateo et al. 1998).
However, the S.cerevisiae has a major disadvantage. It has limited range of use
of substrates. For example, cellulosic substrates or substrates which contain lactose
can be used only after hydrolysis. This is due to lack of necessary enzymes for the
hydrolysis.
In the present study, an alcohol-resistant, psychrophile and bottom-fermenting
S.cerevisiae strain (AXAZ-1) is used.













1.5 Immobilization

Immobilization is a technique in which cells of microorganisms are physically
confining or localizing in a certain defined region with retention of their biological
activity (Bickerstaff 1997). This gives the opportunity to increase the stability and
viability of the cells and make possible their repeated or continued use.
In the last decades the cell immobilization technique is of particular interest
because of the many advantages it offers as a method for the production of alcoholic
beverages and generally for conventional microbial fermentations. Various
immobilization methods and numerous carrier materials were tried (Walsh 2002).
35

1.5.1 Advantages of immobilization method
The use of immobilized cells offers many advantages over free cells
fermentations (Kourkoutas et al. 2004, Willaert & Nedovic 2005, Dervakos & Webb
1991):
The immobilization support may act as a protective agent against
physicochemical effects of pH and temperature resulting in prolonged activity
and stability of the biocatalyst.
Higher cell densities per unit bioreactor volume, which leads to high
volumetric productivity and shorter fermentation times.
Increased substrate uptake and yield improvement and increased tolerance to
high substrate concentration.
Feasibility of continuous processing and low temperature fermentation.
Easier product recovery through reduction of separation and filtration
requirements, thus reducing cost for equipment and energy demands.
Regeneration and reuse of the biocatalyst for extended periods in batch
operations, without removing it from the bioreactor.
Reduction of risk of microbial contamination due to high cell densities and
fermentation activity.
Lower capital costs because of the ability to use smaller bioreactors with
simplified process designs.
Reduction of maturation times for some products.
1.5.2 Immobilization techniques
Depending on the biocatalyst to be immobilized, there are many techniques to
achieve the immobilization. These are the following:
Non-specific adsorption/absorption on solid carrier surfaces
This is the most common immobilization technique. In this method, cell
immobilization on a solid carrier is carried out by physical adsorption on different
supports like wood, glass, ceramic or plastic materials (Willaert 2006), due to
electrostatic forces or by ionic and hydrogen bonding between the cell membrane and
the carrier.
This technique has many advantages. Some of them are the following. This
method causes little damage to cells, it is simple and quick. What is more, no
chemical changes occur to the support or cells leading to high viability. However, it is
36

observed a high leakage of biocatalyst from the support, increasing the risk of
contamination of the product (Bickerstaff 1997).
Covalent attachment
This technique involves covalent bond formation between the biocatalyst and
the support material. This bond is formed between the reactive groups of the
biocatalyst and the reactive groups of the surface of the support. There are several
reactive groups such as the amino group (-NH
2
), the carboxyl group (-COOH), the
hydroxyl group (-OH). The most widely used support for immobilization by covalent
bonding is silica gel. Covalent attachment to the carrier can be induced using linking
agents such as metal oxides, glutaraldehyde or aminosilanes (Verbelen et al. 2006).
Soluble enzymes bonding on insoluble supports are preferred for this method as
this type of biocatalyst offers increased chemical and physical stability to the
immobilized system, such as better ionic, thermal and acidic tolerance. Among the
advantages of this method is that it reduces the leakage of biocatalyst from the
support. However, its high cost and its complexity limit the use of this method in
industrial scale.



Entrapment
This method of immobilization based on the localization of an enzyme, or cells
or organelles etc. within the lattice of a polymer matrix or membrane. In this type of
immobilization, the cells are free in solution, but restricted in movement by the lattice
structure of a gel. The porosity of the gel lattice is controlled to ensure that the
structure is tight enough to prevent leakage of the biocatalyst but allow free
movement of nutrients and the product. Characteristic examples of this type of
immobilization are the entrapment into polysaccharide gels like alginates, collagen,
agar, chitosan and polygalacturonic acid or other polymeric matrixes like gelatin,
collagen and polyvinyl alcohol (Bickerstaff 1997, Park and Chang 2000, Kourkoutas
et al. 2004).
Although it is a widely used method in food industry and high biomass loadings
can be obtained, entrapment has several drawbacks, such as diffusion limitations of
substrates and products (the cell membrane and the layer of polymer limit the
37

diffusion), which significantly affects the rate of bioprocesses. Another disadvantage
is the chemical and physical instability of the gel and that there is no possibility for
regeneration of cells.

Encapsulation
Encapsulation is a process in which tiny particles or droplets of a micro
component (antioxidant, vitamin, natural colorant, enzyme, etc) are surrounded by
another material, which is wall material/coating/carrier or embedded in a homogenous
or heterogeneous matrix resulting in the formation of small capsules. Encapsulation
encloses enzymes etc within spherical, semipermeable membranes larger than 1000
m diameter, whereas microencapsulation encloses enzymes etc within spherical,
semipermeable membranes of 3-800 m diameter (Straathof and Adlercreutz 2000).
This method of immobilization is very accurate and has many advantages. Some
of them are that the method provides great stability and activity of the immobilized
enzymes, enzymes are immobilized without a chemical or structural modification and
there is the possibility of simultaneous immobilization of different enzymes in a
single step (co-immobilization). However, encapsulation method has also some
disadvantages such the limited diffusivity of high molecular weight substrates, there is
a possibility of occasionally inactivation of enzyme or/and leakage of the enzyme
from microencapsule (Walker and Rapley 2002, Macario et al. 2009).
Cell flocculation (aggregation)
Flocculation can be considered as an immobilization technique as the large size
of the cell aggregates makes their use in reactors possible without needing a carrier.
Cells or enzymes join to each other in order to form a large, three-dimensional
structure. This immobilization technique can be achieved either by chemical or by
physical methods. Artificial flocculating agents or cross-linkers can be used to
enhance aggregation in cell cultures that do not naturally flocculate.
Flocculation is affected by numerous parameters, such as nutrient conditions,
agitation, Ca
2+
-concentration, pH, fermentation temperature (Sampermans et al.
2005). However, this method has the disadvantage of low stability of the produced
biocatalysts and it is not widely used.
38


Fig. 13: Basic methods of cell immobilization (Kourkoutas et al. 2004)


1.5.3 Prerequisites of the support materials
The immobilized cell technology for alcoholic beverages production has certain
requirements for the support and method used (Kourkoutas et al. 2004, Willaert and
Nedovic 2005):
The surface of the carrier should provide the appropriate functional
groups to facilitate the cells adhesion.
The carrier should facilitate handling and regeneration.
High cell viability and operational stability of the biocatalyst.
The biological activity of the immobilized cells should not be limited
by the immobilization method.
The porosity of the support should be controllable to permit the free
movement of nutrients, products and gases.
The carrier should retain its chemical and biological stability so as to
be resistant to degradation by enzymes, solvents and pressure changes.
39

The immobilization method and the support should be easy, cheap and
simple to scale-up.

1.5.4 Applications of cell immobilization in food industry
Immobilized cell techniques have a wide range of applications in food
biotechnology. These techniques are not only used for food production by itself, but
also for food additives production, and for food product finishing such as aroma and
taste improvement processes.
Such techniques have been used firstly for winemaking and brewing. Many of
these techniques not aim basically to the production of wine or beer by itself, but to
carry out secondary fermentations such as malolactic fermentation of wine or to
improve the volatile byproduct composition of beer and wine. A big part of such
applications is relative to potable alcohol production, but also to fuel-alcohol
production of food industry byproducts. More than that, the usage of other food
industry byproducts, such as molasses, whey and others, has been substantially aided
by the development of immobilization, for baker's yeast production or single cell
protein and other valuable products useful as provenders. Moreover, more specific
techniques of immobilization, such as encapsulation have been used for the
production of probiotic additives for yogurt, fermented milk and other functional
foods.



1.6 Delignification

Delignification is the removal of the structural polymer lignin from plant tissues
and wood. Lignin is after cellulose, one of the most abundant organic polymers on
Earth. Its primary structure is not defined due to the heterogeneity and the complexity
of the structure.
Cellulose is a carbohydrate which is composed of multiple -D-
glycopyranose bonded with -(1-4) bonds forming a strong and stable
polymer.
40

Lignin consists of benzoic ring which joints 3C chain and other
functional groups methoxyl, hydroxyl and propane chain. Lignin is
soluble to solution of NaOH 1%.

Electron microscopy shows that while cellulose has a rather crystalline
structure, lignin is bound to cellulose irregularly, and this way the structure of the
wood tightens. Thus the removal of lignin loosens the structure of the wooden tissue
and the attack of enzymes to the cellulose becomes more facile.
When dealing with immobilized biocatalysts, delignification of cellulosic
materials, acts beneficially. The removal of lignin from a wooden tissue, forms
appropriate holes and vents, for the suitable entrapment of yeast or bacterial cells.
Thus, delignification of the cellulosic support material of the biocatalyst enhances the
active surface of the biocatalyst and its stability (Kopsahelis et al. 2007).
The carrier for cell immobilization used in this study is sawdust of wood. The
agricultural and forestry waste consist of 20-80% cellulose, 50-80% hemi- cellulose
and lignin. The wood is composed of cellulose and hemi-cellulose at 70%, 25% lignin
and 3- 10% of other organic and inorganic compounds.
1.7 Starch gelatinization

Starch is a carbohydrate consisting of two types of molecules, amylose
(normally 20-30%) and amylopectin (normally 70-80%). Both consist of polymers of
-D-glucose units in the
4
C
1
conformation. It is the most common carbohydrate in the
human diet and is contained in large amounts in such staple foods as potatoes, wheat,
corn and rice.
Starch occurs as highly organized structures, known as starch granules. Starch
has unique thermal properties and functionality that have permitted its wide use in
food products and industrial applications. When heated in water, starch undergoes a
transition process, during which the granules break down into a mixture of polymers
in solution, known as gelatinization.
The importance of starch gel as support for cell immobilization in winemaking
has been reported in many researches. Starchy supports seem to be very promising as
they derive from products abundant in nature (potato, corn, wheat, barley), easy to
handle and especially of low cost. The biocatalysts immobilized on such supports
41

retain their operational stability for a long period and in a wide range of fermentation
temperatures producing wines of fine clarity (Kandylis et al. 2008a, Kandylis 2012).














1.8 Aim of the project

The aim of the present study was to evaluate the suitability of a
cellulose/biopolymer composite biocatalyst, prepared by immobilization of
Saccharomyces cerevisiae AXAZ-1 yeast cells on the starch gel and then diffusion of
starch gel into the delignified sawdust, for production of great quality of wine. The
biocatalyst was used for dry winemaking at 30C and also for low-temperature
winemaking (7C). The effect of the immobilized biocatalyst on volatile byproducts
of the wine was studied using gas chromatography technique and the ethanol
production and the sugar content was studied using HPLC method. For comparison
reasons, wine using free Saccharomyces cerevisiae AXAZ-1 cells and with
immobilized yeast cells on a simple delignified cellulose biocatalyst were also
produced and analyzed.






42




































2.1 Materials and methods of analysis
2.1.1 Laboratory equipment
Gas chromatographer: SHIMADZU GC-8A Gas Chromatograph
High pressure liquid chromatographer (HPLC): SHIMADZU LC-9A
43

Electronic microscope: JEOL model JSM-6300
Glass cylinders
Conical flasks

o
Be hydrometers
Sartorius precision laboratory instrumentation weighing balances
Thermometers
Chamber
Freezer
Refrigerator
Centrifugal
Steam distillation apparatus
1 and 5-mL pipettes
Alcohol hydrometer
Distilled water
2.1.2 Yeast strain and media
Saccharomyces cerevisiae AXAZ-1, an alcohol-resistant and psychrotolerant
strain, is used in the present study. It was grown in liquid culture medium consisting
of 0,1% w/v (NH
4
)
2
SO
4
, 0,1% w/v KH
2
PO
4
, 0,5% w/v MgSO
4
.7H
2
O, 0,4% w/v yeast
extract, 4% w/v glucose. Then it was sterilized by autoclaving at 130C for 15 min.

Fig. 14: Conical flask containing culture medium
2.1.3 Biomass production
The mass production was done with successive cultures of the microorganism in
liquid medium containing 4% w/v glucose. Initially, 100mL of liquid medium were
inoculated by a solid culture and the incubation time was one day at 30C. Then, the
cells produced were transferred in 2L culture medium, stirring for one day at 30C
44

and two days later the microorganism was harvested by centrifugation at 6000 rpm for
10 min. The liquid superjacent was discarded and a white biomass was collected and
placed one more time for incubation. About 15gr of yeast were obtained by this
method. These steps were repeated as many times as needed to produce sufficient
amount of biomass for the accomplishment of the experiments.
The total biomass produced, was finally separated into several proportions and
packed in air-fight packages, and was stored at 4
o
C, until its use.

2.1.4 Grape must
The grape must, which was used for alcoholic fermentations, derived from
concentrated grape must (~32
o
Be), obtained from the winery Achaia Clauss (Patras,
Greece). The grape must was used after appropriate dilution with deionized water in
order to achieve the desire
o
Be density and without further addition of nutrients. The
initial
o
Be density was 12
o
Be. The must was sterilized by autoclaving at 130C for 15
min before its use.

2.1.5 Immobilization supports
For the immobilization of yeast cells, delignified cellulosic material and corn
starch gel were used as supports. Delignified cellulosic material (DCM) was prepared
from sawdust after lignin removal with sodium hydroxide solution.

2.1.5.1 Preparation of Delignified Cellulosic material (DCM)-Delignification
In a pot of 5L were placed 300g of wood sawdust and 3L of a 1% sodium
hydroxide solution. The slurry was heated for 3h at the boiling point, with continuous
stirring, for three hours, and the volume of water during the heating was held constant
by the addition of water. This mixture was then filtered using a textile filter and the
content of the textile filter was several times washed with hot water (~80
o
C). These
two steps were repeated several times and after each washing, the content of the
textile filter was pressed to remove the water. The cellulosic material considered
delignified, when the water that was coming from the washing, was clear and
colorless.
45

The delignified cellulosic material (DCM) was then dried for 48 hours, within
chamber, at 30
o
C. The final material had less than 4% moisture, and was packed
within air-fight vessel until its next use.
The protocol for the preparation of the DCM is an alteration of the protocol
suggested by Bardi & Koutinas, 1994.


Fig. 15: Dry delignified cellulosic material (DCM)

2.1.5.2 Starch gelatinization
In a beaker containing a magnetic agitator were placed 8gr of corn starch and
100mL of deionized water. The mixture was heated from 20
o
C to over 70
o
C. The
temperature was raised gradually for about 2.5h. After 2.5h the starch gel was formed.
The formed gel was immediately used as support for yeast cell immobilization.
The protocol for the corn starch gelatinization is a protocol suggested by
Kandylis et al., 2008a.

46


Fig. 16: Formation of starch gel

2.1.6 Immobilization of yeast cells
S.cerevisiae AXAZ-1 cells were immobilized on DCM and corn starch gel.
2.1.6.1 Immobilization of S.cerevisiae AXAZ-1 on DCM (DC biocatalyst)
In a conical flask of 500mL were placed 20gr of dry DCM and 230mL of
culture medium (0,1% w/v (NH
4
)
2
SO
4
, 0,1% w/v KH
2
PO
4
, 0,5% w/v MgSO
4
.7H
2
O,
0,4% w/v yeast extract, 4% w/v glucose). The mixture was sterilized by autoclaving at
130C for 15 min. After its cooling, 4.6gr of S.cerevisiae AXAZ-1 cells were added.
The mixture was agitated well and then it was allowed to ferment for 1 day, inside a
chamber at 30
o
C.
The next day the mixture was filtered using textile filter and the wet biocatalyst
was obtained. Then the wet biocatalyst was placed on glass plate of square shape and
then it was dried for 24 hours, within chamber, at 30
o
C. The next day, the dry
biocatalyst (DC) was packed within air-fight vessel covered with aluminum foil and
stored at 4
o
C until its next use.
2.1.6.2 Preparation of the cellulose/biopolymer composite biocatalyst (GS)
After the starch gelatinization, the beaker with the formed gel was placed in an
ice bath to decrease the temperature of the gel. Then 10gr of S.cerevisiae AXAZ-1
cells were mixed with the prepared gel at ~40 C during its cooling. Then the yeast-
gel system was placed, before its coagulation, on a glass plate of square shape. After
47

that, dry, sterilized DCM was added to yeast-gel system with simultaneous agitation
of the mixture, till mixtures saturation. Then the plate with the wet
cellulose/biopolymer composite biocatalyst was placed inside a chamber at 30
o
C for
24h for complete gel formation and cell immobilization.
The next day the dry biocatalyst (GS) was scratched from the plate and it was
packed, in the form of small cakes, within air-fight vessel covered with aluminum foil
and stored at 4
o
C until its next use.


Fig. 17: A. Corn starch gel with immobilized yeast cells, B. Wet
cellulose/biopolymer composite biocatalyst, C. Dry cellulose/biopolymer composite
biocatalyst, D. Dry cellulose/biopolymer composite biocatalyst in the form of small
cakes
2.1.7 Fermentations at 30
o
C and 7
o
C
The prepared biocatalysts were separately used for the fermentation. Two
different fermentations were carried out at 30
o
C and 7
o
C respectively. For each
fermentation 900mL of sterilized must (12
o
Be) were used. The must was equally
separated into 3 conical flasks of 500mL (300mL of sterilized must into each flask)
and 7gr of DC biocatalyst were added into the first flask, 7gr of GS biocatalyst into
the second flask and 1.2gr of free yeast cells into the third one. Then the flasks were
placed inside chamber at 30
o
C to ferment. At the same time, 3 alike flasks were
48

prepared as above, with the difference that the flasks were placed inside refrigerator at
7
o
C to ferment.
Fermentations were monitored by measuring the
o
Be density and stopped when
o
Be density reached 0
o
Be.


Fig. 18: A. Flask with GS biocatalyst at the end of the fermentation, B. Flask
with DC biocatalyst at the end of the fermentation, C. Flask with free yeast cells at the
end of the fermentation, D. Flasks with biocatalysts during fermentation, from left to
right, GS biocatalyst, free yeast cells, DC biocatalyst

2.2 Assays
2.2.1 Kinetics of fermentations
Kinetics of fermentations were performed by measuring
o
Be density at various
time intervals. Liquid samples of 1mL from each flask were taken during
fermentations and kept refrigerated for further analyses.
2.2.2 Determination of residual sugars
The residual sugars in wine samples were determined with the HPLC method.
49

High-performance liquid chromatography (HPLC), also known as High-
pressure Liquid Chromatography, is a form of liquid chromatography to separate
compounds that are dissolved in solution. HPLC is a popular method of analysis as it
is easy to learn and use and is not limited by the volatility or stability of the sample
compound. It is used for the separation and determination of organic and inorganic
solutes in any samples especially biological, pharmaceutical, food, environmental,
industrial, etc.
HPLC instruments consist of: a solvent reservoir, a high pressure pump, an
injection port, a column, a column oven, a detector and a recorder. The pump provides
a steady high pressure and keeps constant the flow rate of the liquid and when sample
is injected, it is carried into the column by the mobile phase and separated in its
components. Simultaneously the chromatogram is plotted and when a component is
detected, a peak is appeared.


Fig. 19: Block diagram showing the components of an HPLC instrument
The residual sugars in wine samples were determined using a Shimadzu HPLC
system with an SCR-101N stainless steel column, an LC-9A pump, a SHIMADZU
CTO-10A oven at 60 C, and an RID-6A refractive index detector. Three times
distilled and filtered water (3D) was used as mobile phase with a flow rate of 0.8
mL/min, and 1-butanol was used as internal standard. 0.25mL samples of wine and
1.25 mL of a 1% (v/v) solution of 1-butanol were diluted to 25 mL with 3D water and
filtered via a syringe filter (Phenex NY 0.20 m), and 40 L were injected directly
into the column. Residual sugar concentrations were calculated using a standard curve
prepared by 5 standard solutions with specific concentrations of fructose, glucose and
ethanol and expressed as grams of residual sugar per litre (g/L).
50



Fig. 20: Shimadzu HPLC system


2.2.3 Determination of ethanol
The concentration of ethanol in the wine samples was measured by the HPLC
method, with the same apparatus and conditions as described for residual sugars. The
calculations were done using a standard curve prepared by 5 standard solutions with
specific concentrations of fructose, glucose and ethanol and expressed in terms of %
(v/v).
The determination of ethanol enabled calculation of ethanol productivity, which
was defined as grams of ethanol per liter liquid volume per day.


2.2.4 Determination of volatile by-products
The volatile compounds in wine samples were determined by means of gas
chromatography (GC).
Gas chromatography (GC) is a common type of chromatography used in
analytic chemistry for separating and analyzing compounds that can be vaporized
without decomposition. Typical uses of GC include testing the purity of a particular
51

substance, or separating the different components of a mixture (the relative amounts
of such components can also be determined).
A gas chromatograph consists of: a source of carrier gas with one or more
pressure reduction valves, an inlet (injection port) that can be heated, a column in a
thermostatic air bath, a detector suitable for vapour phase samples. The high
temperatures are needed to vaporize the solutes of interest and maintain in the gas
phase. The injection port and the detector are generally maintained at a temperature
approximately 10% above that of the column to ensure rapid volatilization of the
sample.


Fig. 21: Block diagram showing the components of a GC instrument
The volatile compounds (acetaldehyde, ethyl acetate, propanol-1, isobutanol and
amyl alcohols) were determined by means of gas chromatography on a Shimadzu GC-
8A Gas Liquid Chromatograph with stainless steel column, packed with Escarto 5905
(a mixture of Squalene 5%, Carbowax-300 90%, di-ethyl-hexyl sebacate 5% v/v), 2 m
length and stable temperature at 70C. The detector used was FID (Flame Ionization)
with temperature 210C and high purity mixture H
2
-O
2
(pressure 0,6 and 0,2 Kg/cm
2

respectively) as fuel. The carrier gas was nitrogen of high purity with pressure 4
Kg/cm
2
and the recorder SHIMADZU C-R6A Cromatopack was also used. Samples
of 4 L of wine were injected directly into the column and the concentrations of the
above compounds were determined using standard curves and expressed as grams of
volatile compounds per litre (g/L).

52


Fig. 22: Shimadzu GC system

2.2.5 Determination of alcohol (% v/v)
Alcohol was distilled and measured using a alcoholmeter.
Distillation is a method of separating mixtures based on differences in their
volatilities in a boiling liquid mixture. Distillation is based on the fact that the matter
can exist in three phases: solid, liquid and gas. As the temperature of a pure substance
is increased, it passes through these phases, making a transition at a specific
temperature from solid to liquid (melting point) and then at a higher temperature from
liquid to gas (boiling point). The process involves evaporating a liquid into a gas
phase, then condensing the gas back into a liquid and collecting the liquid in a clean
receiver. Substances that have a higher boiling point than the desired material will not
be distilled at the working temperature, and remain behind in the flask. Applied to the
preparation of alcoholic beverages, alcohol has a lower boiling point than water (and
sugar) and thus distillation can separate the alcohol from the wine. For fermented
beverages, like wine and beer, the alcohol content is expressed in percent by volume.
This method is based on removing and collecting all of the alcohol contained in
an exactly volume of wine, by distillation. This collected distillate is then diluted back
to exactly the same volume using distilled water. The alcohol concentration of this
solution is then determined by measuring its density using a special alcohol
hydrometer, usually calibrated at 20
o
C and in % v/v.
53

For the determination of alcohol we measured 200mL of our samples using a
volumetric flask and transferred it into the 500mL distilling flask. We also added a
few pieces of kissiris into the distilling flask for avoidance of foaming. We attached
the distilling flask to condenser by means of a bent tube, we passed the tap water
through the condenser and we distilled carefully (suitable heating for normal boiling).
The previously mentioned volumetric flask was used as a receiver flask of the
distillate. We continued the distillation until the two thirds (2/3) of the original sample
and after the end of the process, we transferred the distillate into a 250ml volumetric
tube, we introduced an alcoholmeter carefully and a thermometer for the measurement
of the temperature. We read on the alcoholmeter at the bottom of the meniscus of the
alcoholic liquid and we used suitable table to convert the measurement of the
alcoholmeter at 20
o
C. So, we determined the percentage alcohol in the wine or else,
the alcoholic degrees of the wine (expressed as mL alcohol / 100mL of wine or %
v
/
v
).


Fig. 23: Steam distillation apparatus

2.2.6 Electron microscopy
Pieces of the immobilized biocatalysts (DC and GS) were washed with
deionized water and dried overnight at 30 C. The samples were coated with gold in a
Balzers SCD 004 sputter coater for 3 min and examined in a JEOL model JSM-6300
scanning electron microscope.

54


Fig. 24: JSM-6300 scanning electron microscope





















55


3.1 Cell immobilization
As referred in chapter 1.5.3, the carrier used for immobilization should have
food grade purity so as the wine produced can be safely consumed. Additionally, its
surface should be suitable for cells to adhere to and the porosity has to be adequate
and uniform. The cell immobilization was confirmed by electron microscopy.
56


Fig. 25: Electron micrographs of delignified sawdust with the immobilized cells of S.
cerevisiae AXAZ-1 (DC biocatalyst).
57



Fig. 26: Electron micrographs of corn starch gel with the immobilized cells of S.
cerevisiae AXAZ-1 diffused into delignified sawdust [cellulose/biopolymer
composite biocatalyst (GS)].



The first support used in the present project (sawdust) contains the phenolic
insoluble compound lignin which offers rigidity and creates physicochemical
boundaries to microorganisms. This compound and other toxic substances were
58

removed using sodium hydroxide (NaOH) where they are soluble resulting in a dark
brown solution (saousi 2009). This procedure resulted in a gelatinous carrier with
more pores and holes that can easily accommodate the immobilization of cells (Bardi
and Koutinas 1994). Cell immobilization was shown by the electron micrographs
showing yeast cells densely and homogenously adhered to the surface of the carrier,
as a result of natural entrapment into the tubular cellulosic material of sawdust (Fig.
25).
The other support that was used in this study (tubular cellulose/biopolymer
composite) was formed by corn starch, which was used in the form of gel, diffused
into delignified sawdust. The corn starch gel was formed by gelatinization of starch
granules at high temperatures (90-95C). At this temperature, higher than its
gelatinization temperature, corn starch granules undergo irreversible swelling, and as
they cool, crystallization is favored and an elastic gel is formed (Hoover 2001, Parker
and Ring 2001). The cell immobilization was confirmed by electron microscopy
showing yeast cells attached on the surface of corn starch gel and also mixed and
entrapped in the interior of gel matrix which coats the pores and holes of the
delignified sawdust (Fig. 26).
In addition, effective immobilization was also established by the ability of the
biocatalysts to perform efficiently fermentations of grape must at 30
o
C and 7
o
C.


3.2 Winemaking
Winemaking using psychrotolerant yeast immobilized on tubular
cellulose/biopolymer composite (GS) at high and low temperature (30
o
C and 7
o
C),
compared with simple delignified biocatalyst and free yeast cells, is reported. The
suitability of the immobilized biocatalyst for winemaking was evaluated in terms of
fermentation productivity (kinetics), and organoleptic quality, with emphasis on
volatile composition. The immobilization of cells on GS, as well as the suitability of
the immobilized biocatalyst for alcoholic fermentation, was confirmed by its good
operational stability during fermentations of grape must, even at low temperature
(7
o
C).

59

3.2.1 Kinetics of fermentations
The delignified cellulosic as well as starchy materials have been used
successfully as immobilization supports at laboratory scale for batch and continuous
wine production (Bardi and Koutinas 1994, Iconomou et al. 1996, 1995, Mallouchos
et al. 2002, 2003a, Kandylis 2008a, 2012) and beer (Bardi et al. 1996a), both at
ambient and low temperatures. Their use in the production of beer and wine
dramatically reduced fermentation time and improved quality.
The fermentations using GS biocatalyst were carried out at 30C and 7C at the
same initial
o
Be density (12
o
Be). Similar fermentations were performed using free
yeast cells and immobilized yeast cells on delignified sawdust and the kinetics of
these fermentations are shown in Fig.27, 28, 29.



Fig. 27: Kinetics of fermentation at 30C using DC biocatalyst, GS biocatalyst and
free yeast cells


Fig. 28: Kinetics of fermentation at 7C using DC biocatalyst, GS biocatalyst and free
yeast cells



Fig. 29: Comparison of kinetics of fermentation at 30
o
C and 7C using DC
biocatalyst, GS biocatalyst and free yeast cells

Generally, it can be inferred that the GS biocatalyst proved to be mechanically
and chemically stable for the whole duration of fermentations, even at low
temperature. It was capable to produce wines in 2 days at 30
o
C and 41 days at 7
o
C. It
is obvious that the decrease of temperature increased the fermentation time.
For comparison reasons, fermentations of grape must at 30C and 7C were
carried out using immobilized yeast cells on delignified sawdust (DC) and free
Saccharomyces cerevisiae AXAZ-1 yeast cells. At higher temperature (30C) GS
biocatalyst had the same fermentation time with the DC biocatalyst (2 days), while
60

free yeast cells fermentation time was higher than in the case of GS biocatalyst (7
days). At 7
o
C GS biocatalyst had lower fermentation time than DC biocatalyst, which
produced wine in 48 days, and considerably lower than free yeast cells, which needed
76 days to terminate the fermentation (Fig. 29).


3.2.2 Kinetic parameters of fermentations
The kinetic parameters of these fermentations are represented at the following
table (Table 1). The fermentations at two different temperatures (30 and 7
o
C) were
carried out with initial sugar concentrations 249.95 g/L and 179.33 g/L respectively.
In general, an increase in the fermentation time was observed with the drop of
temperature from 30 to 7
o
C. This can be attributed to the fact that cells need a longer
period of adaptation in the fermentation environment at 7
o
C.







Table 1: Kinetic parameters obtained in fermentations of grape must at 30C and 7
o
C
using GS biocatalyst, DC biocatalyst and free yeast cells.
Biocatal
yst
Fermentat
ion
Temperat
ure
(
o
C)
Fermentat
ion Time
(h)
Initi
al
o
Be
Initi
al
suga
rs
(g/L)
Residu
al
sugars
(g/L)
Ethan
ol
(%v/v
)
Conversi
on (%)
Ethanol
productiv
ity
(g/L/d)
GS 30 52 12 249.
95
16.85 12.13 93.25 44.36
DC 30 48 12 249.
95
25.88 11.40 89.64 45.03
61

Free 30 168 12 249.
95
32.4 12.59 87.03 14.20

GS 7 980 12 179.
33
20.56 11.85 88.53 2.28
DC 7 1148 12 179.
33
23.79 11.60 86.73 1.90
Free 7 1826 12 179.
33
28.41 11.93 84.15 1.24

The GS biocatalyst retained its operational stability, even at low temperature,
and produced wines of fine clarity and contained alcohol at concentrations similar to
those of dry table wines (10.5-12.5%v/v). More specifically, at 30
o
C the ethanol
concentration (12.13%v/v) and ethanol productivity (44.36 g/L/d) were high and
residual sugar concentration was 16.85 g/L with conversion 93.25%. At 7
o
C the final
product, produced by GS biocatalyst had 11.85%v/v ethanol concentration and the
ethanol productivity was 2.28 g/L/d. The concentration of residual sugars was 20.56
g/L and the conversion 88.53%. The difference in ethanol productivity is due to the
increase of fermentation time at 7
o
C. The relatively high ethanol productivity and the
high levels of conversion indicate the efficiency of the biocatalyst for alcoholic
fermentation.
The fermentation with GS biocatalyst at low temperature resulted in
fermentation time and ethanol productivity which can be accepted by industry. The
fermentation with the same biocatalyst at 30C led to faster fermentation time than
that of traditional fermentation and ethanol productivity was about 4-fold higher than
in the case of free cells. Also, comparing the fermentation time and ethanol
productivity of GS biocatalyst and free yeast cells at 7
o
C, it is obvious that GS
biocatalyst terminated the fermentation much faster than free cells and with about 2-
fold higher ethanol productivity value. This confirms similar results that have been
published during the production of wine using immobilized cells of Saccharomyces
cerevisiae AXAZ-1 in delignified and starchy materials (Bardi et al. 1996b, Bardi and
Koutinas 1994, Kandylis et al. 2008a).
Although GS biocatalyst showed better fermentation time and higher ethanol
productivity than fermentations carried out by free yeast cells, DC biocatalyst
62

presented slightly lower fermentation time and therefore slightly better ethanol
productivity than GS biocatalyst at 30
o
C. More specifically, for DC the fermentation
time was 48 hours and the ethanol productivity was 45.03 g/L/d, while for GS was 52
hours and 44.36 g/L/d respectively. However, lower concentration of residual sugars
and thus higher conversion rate achieved by GS biocatalyst compared to DC
biocatalyst at the same temperature (Table 1). At 7
o
C GS proved to be a better
biocatalyst for winemaking than DC as it shown lower fermentation time (only 41
days) and higher ethanol concentration and ethanol productivity. In addition, lower
concentration of residual sugars was achieved by GS, resulting in higher conversion
rate (Table 1).
Comparing the kinetic parameters for each fermentation, it is obvious that GS
and DC biocatalysts achieved lower fermentation times, higher ethanol productivities
and higher conversions than the corresponding values observed in fermentations with
free yeast cells at both temperatures. Similar changes in kinetic parameters of the
alcoholic fermentation have been reported by Galazzo and Bailey (1990) and Patkova
et al. (2000) in experiments with cells immobilized in calcium alginate. However, GS
biocatalyst can be considered more efficient for low temperature winemaking. This
can be attributed to the fact that this biocatalyst combines the advantages of both
starch gel and delignified cellulosic materials as immobilization supports. Previous
studies shown that starch and DC materials behave as catalysts or promoters of the
activity of enzymes involved in the process of fermentation by reducing the activation
energy (Ea) of the fermentation (Bardi and Koutinas, 1994, Bardi et al. 1996b,
Kandylis et al. 2008a, 2008b, Iconomopoulou et al. 2000). What is more, Iconomou
et al. 1995 reported that the removal of lignin from sawdust may lead to an increase in
ethanol productivity as the lignin in sawdust might inhibit fermentation.


3.2.3 Volatile byproducts
The concentration of volatile byproduct in wines constitutes an indication of the
products quality. As GS biocatalyst proved to be suitable for wine fermentation at
both 30
o
C and 7
o
C, the final consideration was the evaluation of quality of the
fermented products. The majority of the compounds that contribute to the aroma of
63

wines are produced during alcoholic fermentation of grape must; very few are derived
from grapes. The most abundant compounds in the wine aroma are acetaldehyde,
ethyl acetate, propanol-1, isobutanol (isobutyl alcohol), and amyl alcohols (Regodn
et al. 2006). The concentrations of these compounds in the produced wines are
summarized in Table 2.

Table 2: Volatile byproducts of wines produced by fermentations of grape must at
30C and 7
o
C using GS biocatalyst, DC biocatalyst and free yeast cells.
Biocatal
yst
Fermentat
ion
Temperat
ure
(
o
C)
Acetaldeh
yde
(mg/L)
Ethyl
aceta
te
(mg/
L)
Propan
ol-1
(mg/L)
Isobut
yl
alcoho
l
(mg/L
)
Amyl
alcoh
ols
(mg/L
)
Total
alcohols
(ethanol &
methanol)(%
v/v)
Total
volatile
s
(ethano
l &
methan
ol
exclude
d)
(mg/L)
GS 30 23.045 29.24
5
10.94 21.13 21.12
5
13.3 105.485
DC 30 23.61 23.31 8.69 11.165 15.28 13 82.055
Free 30 25.53 18.32 6.08 13.87 26.35 13.4 90.15

GS 7 22.58 33.05 12.375 6.06 12.06 13.2 88.125
DC 7 22.70 27.84 12.19 4.76 14.78 12.69 81.27
Free 7 24.35 21.13 9.46 10.54 19.94 13.3 85.42
As it is obvious, fermentations carried out by GS biocatalyst resulted in
products contain acetaldehyde, ethyl acetate and higher alcohols at concentrations that
contribute to pleasant flavors (Ribrau-Gayon et al. 2000) (Table 2). More
specifically, acetaldehyde concentration ranged from 23.045 to 22.58 mg/L at 30
o
C
and 7
o
C respectively. Ethyl acetate concentration increased with the drop of
fermentation temperature from 29.245 mg/L at 30C to 33.05 mg/L at 7C. Amyl
alcohols, isobutyl alcohol, and propanol-1 were detected in levels that contribute
positively to wine aroma. The concentrations of isobutyl alcohol and amyl alcohols
64

ranged from 21.13 to 6.08 mg/L and from 21.125 to 12.06 mg/L respectively.
Propanol-1 was found in relatively low concentration from 10.94 to 12.375 mg/L.

Acetaldehyde
The acetaldehyde at low concentrations is desired as it gives a pleasant fruity
aroma, while at higher levels gives the wines a pungent irritating odor (Romano et al.
1994, Liu and Pilone 2000). The acetaldehyde content in fermented beverages usually
lies between 13-40 mg/L (Longo et al. 1992) and in some cases may reach 75 mg/L
(Koutinas and Pefanis 1994). Generally, the concentration of acetaldehyde in
produced wines was in levels similar to those found in commercial wines and below
the odor threshold value (100 mg/L).
Acetaldehyde concentrations were lower in wines produced by GS biocatalyst,
and decreased further at 7
o
C, compared to those produced by DC and free yeast cells.
Fermentations using free cells resulted in higher acetaldehyde content (25.53 at 30C
and 24.35 at 7
o
C) than concentrations achieved by immobilized biocatalysts at both
temperatures. Thus, it can be argued that the combination of low temperature
fermentation and immobilized cells produce wines with low levels of acetaldehyde.
What is more, GS seems to be more efficient biocatalyst than DC as it achieved lower
acetaldehyde concentrations than the corresponding values observed in fermentations
with DC biocatalyst. Similar results have been reported by other researchers (Yajima
and Yokotsuka 2001, Kourkoutas et al. 2005, Mallouchos et al. 2003a, 2003b).
Different results from those have been reported by Peinado et al. (2006) in wine
production with immobilized cells in capsules of Penicillium where the levels of
acetaldehyde, achieved by immobilized cells, were higher than those of free cells. The
concentration of acetaldehyde depends on the activity of alcohol dehydrogenase,
which converts it to ethanol. The activity of this enzyme appears to be greater in the
immobilized cells and depends on temperature (Romano et al. 1994).


Ethyl acetate
Ethyl acetate is the most important and abundant ester in wines (Plata et al.
2003). It is of great importance for the quality of the wine as at low concentrations
65

(<50 mg/L) may be pleasant and contributes to fragrance complexity, while above
120 mg/L, it is likely to donate a sour vinegar off-odor.
Ethyl acetate as determined in the final fermentation products using GS
biocatalyst was found in higher concentrations than those identified in the final
products obtained from fermentations using DC biocatalyst and free yeast cells
(Table 2). Immobilized cells produced higher quantities of ethyl acetate than free
cells at every temperature studied. These levels contribute positively to the wine
aroma because they are above the threshold value (12.3 mg/L) and lower than the
value which is considered to have a negative impact on aroma (Etievant, 1991).
Comparing the two immobilized biocatalysts, the wines produced with GS biocatalyst
showed the highest concentrations at each temperature [29.245 mg/L (GS) and 23.31
mg/L (DC) at 30
o
C, 33.05 mg/L (GS) and 27.84 (DC) at 7
o
C]. What is more, it was
observed an increase at the ethyl acetate levels followed by the drop of the
temperature, thus confirming the fact that improved quality of wines achieved by
fermentations at low temperatures. Correlation between ethyl acetate concentration
and fermentation temperature has also been observed in previous studies (Kourkoutas
et al. 2001, 2002, Tsakiris et al. 2004a, Mallouchos et al. 2003c, Iconomopoulou et
al. 2002, Kandylis et al. 2008a, 2008b).


Higher alcohols
Higher alcohols (propanol-1, isobutyl alcohol and amyl alcohols) are considered
as the largest group of flavor compounds in wines and among them amyl alcohols and
isobutyl alcohol have high importance (Longo et al. 1992). Low concentrations of
higher alcohols below 300mg/L have a positive contribution to the aroma of wine. At
higher levels, the contribution is negative as they increasingly overpower the
fragrance.
The concentration of propanol-1 was slightly higher in wines produced by GS
biocatalyst at both temperatures. The concentrations of amyl alcohols and isobutyl
alcohol in the wines produced by GS biocatalyst were significantly reduced as the
temperature was decreased, while for free yeast cells and DC biocatalyst a slightly
decrease of amyl alcohols and isobutyl alcohol levels was observed. Generally, their
concentrations appear to decrease with the drop of fermentation temperature for all
biocatalysts (Table 2). However, the concentration of higher alcohols in all cases
66

ranged in levels that contribute to the pleasant flavor of the wines produced. The
decrease of the amount of higher alcohols in produced wines with temperature
decrease has been reported by many studies (Erten 2002, Bakoyianis et al. 1993,
Bardi et al. 1997b, Kourkoutas et al. 2005, Tsakiris et al. 2004a, 2004b, Kandylis et
al. 2008a, 2008b).

































67











4. Conclusions
Evaluating the results, it was proved that the use of a tubular
cellulose/biopolymer composite (GS) as biocatalyst, proposed in this project, is
suitable for winemaking at both high (30
o
C) and low (7
o
C) temperatures. This
biocatalyst has many advantages. It is a cheap, abundant support of food grade purity.
These advantages make GS a promising biocatalyst for winemaking at low
fermentation temperatures. Furthermore, the use of GS biocatalyst gave the best
results in kinetic parameters and formation of major volatile products, particularly at
low temperature. More specifically, comparing the kinetic parameters, GS biocatalyst
had faster fermentation times, higher ethanol productivities, lower concentrations of
residual sugars and higher conversion rates compared to free yeast cells at both
temperatures. Regarding quality, fermentations using GS, especially at low
temperature, resulted in final products that had improved quality and fine clarity as
they contained higher levels of ethyl acetate and lower amyl alcohols concentrations
than the corresponding values achieved by free cells. Comparing the two immobilized
biocatalysts, it can be concluded that the GS biocatalyst is advantageous over DC for
low temperature winemaking, as it leads to lower fermentation times giving a product
with an improved aromatic quality with lower concentrations of higher alcohols and
higher concentrations of ethyl acetate.
68

To conclude, considering that the main difficulty in the use of immobilized
biocatalysts in industrial fermentations is the lack of a low cost and safe carrier which
would not have a negative effect on product consistency and quality, the above results
seem promising and thus industrial application of GS biocatalyst has a great potential.






























69

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5.2 Greek literature
.., . (1994). " ."
, , pp. 57-102.
. (2001a). "
." , ,
, .
78

. (1997). ". .
" .
. (2009). "
." , ,
, .


5.3 Web sites
1. http://www.britannica.com/EBchecked/topic/645269/wine
2. http://www.thegreekwine.com/history.html
3. http://www.spanish-wines.org/wine-production.html
4. http://en.wikipedia.org/wiki/Winemaking
5. http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae