nano-tubular cellulosebiopolymer composite in wine fermentation volatile byproducts

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    MSc IN FOOD BIOTECHNOLOGY

    UNIVERSITY OF PATRAS

    DEPARTMENT OF CHEMISTRY

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

    GKIKA EFTHYMIA

    Patras 2012

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    - /

    :

    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

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    biocatalyst was good and no decrease of its activity was observed, even at 7 oC.

    Fermentation times were low (only 41 days at 7 oC), 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 oC 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 cerevisiaeAXAZ-1 -

    (GS )

    30 oC 7C.

    (DC )

    .

    .

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    7 oC. (

    41 7 oC),

    , .

    (GC)

    , .

    7 oC 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

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    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

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    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

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    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

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    Introduction

    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 andstrains 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 increasinglyimportant 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,

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    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 (

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    1.2.1 History of the wineThe 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 appearsin 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

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    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

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    1.2.2 Chemical constituents of wineThe 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 yeastactivity 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 WaterThe 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

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    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

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    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

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    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

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    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 AcidsAcids 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 acidDuring 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

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    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 PhenolsPhenols 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-C3-C6 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). Flavonoidsmay 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-C3 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 mostcommon nonflavonoid in grapes, caftaric acid is one of the primary substrates for

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    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 ketonesAldehydes 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 (

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    in amounts that are sensorially insignificant. Some other esters found in wines are

    produced by yeasts (Jackson 2008).

    1.2.2.8 Nitrogen-containing compoundsMany 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 VitaminsVitamins 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 gasesWines 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 MineralsMany 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.

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    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

    Harvesti ng 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 arereached, 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 stemsand 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

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    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, whichmakes 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

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    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 stabilizationAfter 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 whichthe 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

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    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 healthWine 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 andsocial 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.

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    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 fermentationAlcoholic fermentation is the biochemical conversion of exozes (C 6H12O6) to

    ethanol and CO 2 with simultaneous energy release, and it is conducted by yeastsunder anaerobic conditions.

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    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 fermentationAlcoholic 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.

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    The most common wine yeast is Saccharomyces cerevisiae. This yeast ferment s

    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-productsThe 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-CoAester. 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 derivedfrom deamination of amino acids resulting from the breakdown of the yeast

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    and grape must proteins. Their production depends on the microorganism

    strain and the temperature.

    1.4 Sacchar omyces cerevisiaeSaccharomyces 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 asexualreproduction.

    1.4.1 Taxonomic hierarchy

    Kingdom: Fungi

    Phylum: Ascomycota

    Subphylum: Saccharomycotina

    Class: SaccharomycetesOrder: Saccharomycetales

    Family: Saccharomycetacae

    Subfamily: Saccharomyetoideae

    Genus: Saccharomyces

    Species : S. cerevisiae

    1.4.2 Description and morphologySaccharomyces 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

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    the large diameter and 1 to 7 m at the small diameter. Mean cell volumes are 29 or

    55 m3 for a haploid or a diploid cell, respectively (Pons et al. 1993).

    Fig. 11: S.cerevisiae by electronic microscope

    1.4.3 Life cycleS. 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 specificlocation 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).

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

    1.4.4 The importance of Sacchar omyces cerevisiaeSaccharomyces 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 beused for the isolation and identification of different strains (Romano et al. 2003).

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    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).

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    1.5.1 Advantages of immobilization methodThe 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 withsimplified process designs.

    Reduction of maturation times for some products.

    1.5.2 Immobilization techniquesDepending 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

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    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 freemovement 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

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    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.

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

    1.5.3 Prerequisites of the support materialsThe 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.

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    The immobilization method and the support should be easy, cheap and

    simple to scale-up.

    1.5.4 Applications of cell immobilization in food industryImmobilized 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.

    http://www.wisegeek.com/what-are-polymers.htmhttp://www.wisegeek.com/what-is-lignin.htmhttp://www.wisegeek.com/what-is-lignin.htmhttp://www.wisegeek.com/what-are-polymers.htm
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    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 4C1 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

    http://en.wikipedia.org/wiki/Carbohydratehttp://en.wikipedia.org/wiki/Staple_foodhttp://en.wikipedia.org/wiki/Potatohttp://en.wikipedia.org/wiki/Wheathttp://en.wikipedia.org/wiki/Ricehttp://en.wikipedia.org/wiki/Ricehttp://en.wikipedia.org/wiki/Wheathttp://en.wikipedia.org/wiki/Potatohttp://en.wikipedia.org/wiki/Staple_foodhttp://en.wikipedia.org/wiki/Carbohydrate
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    Materials Methods

    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

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    Electronic microscope: JEOL model JSM-6300 Glass cylinders Conical flasks oBe 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 mediaSaccharomyces 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)2SO 4, 0,1% w/v KH 2PO 4, 0,5% w/v MgSO 4.7H 2O, 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 productionThe 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, thecells produced were transferred in 2L culture medium, stirring for one day at 30C

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    The delignified cellulosic material (DCM) was then dried for 48 hours, within

    chamber, at 30 oC. 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 oC to over 70 oC. 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.

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    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)2SO 4, 0,1% w/v KH 2PO 4, 0,5% w/v MgSO 4.7H 2O,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 oC.

    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 30o

    C. The next day, the dry biocatalyst (DC) was packed within air-fight vessel covered with aluminum foil and

    stored at 4 oC 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

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    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 oC 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 oC 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 oC and 7 oCThe prepared biocatalysts were separately used for the fermentation. Two

    different fermentations were carried out at 30 oC and 7 oC respectively. For each

    fermentation 900mL of sterilized must (12 oBe) 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 30o

    C to ferment. At the same time, 3 alike flasks were

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    prepared as above, with the difference that the flasks were placed inside refrigerator at

    7 oC to ferment.

    Fermentations were monitored by measuring the oBe density and stopped whenoBe density reached 0 oBe.

    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 oBe 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 sugarsThe residual sugars in wine samples were determined with the HPLC method.

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    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).

    http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/lc.htmhttp://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/lc.htm
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    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

    http://en.wikipedia.org/wiki/Chromatographyhttp://en.wikipedia.org/wiki/Analytic_chemistryhttp://en.wikipedia.org/wiki/Separation_processhttp://en.wikipedia.org/wiki/Vaporisedhttp://en.wikipedia.org/wiki/Chemical_decompositionhttp://en.wikipedia.org/wiki/Chemical_decompositionhttp://en.wikipedia.org/wiki/Vaporisedhttp://en.wikipedia.org/wiki/Separation_processhttp://en.wikipedia.org/wiki/Analytic_chemistryhttp://en.wikipedia.org/wiki/Chromatography
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    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-O2 (pressure 0,6 and 0,2 Kg/cm2

    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).

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    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 th