CHAPTER 1 INTRODUCTION TO WINE

Some notable events in the 19th century are the first observation of bacteria in wine Louis Pasteur, 1858, the first observation of a reduction in wine acidity Berthelot and De Fleurieu,

TABLE OF CONTENTS

TABLE OF CONTENTS i

LIST OF TABLES ii

LIST OF FIGURES ii

CHAPTER 1 INTRODUCTION TO WINE 1

1.1 History of wine 1

1.2 Classification of wine 2

1.3 Wine making process 3

1.3.1 Harvesting 3

1.3.2 Crushing and pressing 3

1.3.3 Fermentation 3

1.3.4 Clarification 4

1.3.5 Aging and bottling 5

CHAPTER 2 YEASTS IN WINEMAKING 6

2.1 Sacchromyces cerevisiae 6

2.1.1 Taxonom: Morphology and special characteristics 6

2.1.2 Nutrition and growth 8

2.1.3 Life cycle and reproduction 9

2.2 Spoilage yeast strains 10

CHAPTER 3 FACTORS THAT AFFECT THE WINE FERMENTATION 11

3.1 Temperature 11

3.2 Sugar concentration 11

3.3 pH 11

3.4 Oxygen 12

3.5 Wine concentration and carbon dioxide (CO2) 13

3.6 Starter culture 13

REFERENCES iii

LIST OF TABLES

Table 1 Wine classification based on five different standards 2 Table 2 Some common yeasts in grape, musts and wines that can be considered spoilage yeast species

in a wide range of food products 10

LIST OF FIGURES

Figure 1 Some images of yeast: S cerevisiae TBS (a&b) and S cerevisiae TNS (c&d) 6 Figure 2 Structure of S cerevisiae cell 7 Figure 3 Reproductive cycle Saccharomyces cerevisiae 9

CHAPTER 1 INTRODUCTION TO WINE

Wine has been a popular beverage of mankind for thousands of years Our natural fondness of this drink stems from the wonderful taste, its nutritious properties and not least its psychotropic (intoxicating) effects

1.1 History of wine

No one can know precisely when was wine first created? Wine is far older than recorded history and could date back over 20 million years ago as fermenting yeasts evolved together with fruit-bearing flowering plants ─ in ancient times, wine was considered a magical, spontaneous gift of nature Archaeological evidence suggests the earliest production of grape wine took place at sites in Georgia and Iran ─ from as early as 6000

BC Winemaking spread from Egypt, Phoenicia, and Greece (5000 BC – 4500 BC); and then arrived in Europe and northern Africa (1500 BC) A-thousand-year later, wine was being produced in India and China

Advances in production methods (such as vine cultivation, pottery production, and winemaking practices) peaked around 200 to 400 AD and followed by a period of 1200 to 1400-years during which progress in wine technology slowed and was generally restricted to monastic religious orders in western Europe The Romans also began to use barrels in the 3rd century AD From the 1600s, cork was used as a stopper for wine, associated with the increased use of glass bottles; thus, production of glass reaches a level high enough to see an improvement in the transporting and storing of wine

The development of wine production methods began to accelerate in the 18th century, probably because

of changes in trade relations in Europe and led to the appearance of vintage, age-worthy wines The 19th and 20th centuries were periods of great change for the wine industry, with many important discoveries and innovations Some notable events in the 19th century are the first observation of bacteria in wine (Louis Pasteur, 1858), the first observation of a reduction in wine acidity (Berthelot and De Fleurieu, 1864), the first proof that fermentation is carried out by living cells of yeast (Louis Pasteur, 1864), and Discovery of the

‘fermentation enzyme’ (Büchner, 1897) Also, in 1864, there was the first sighting of Phylloxera in France which destroyed much of the world's vineyards, outside of America’s for nearly 20 years

By the early 20th century, due to the two World Wars, the fields became the battleground for over a decade, churned up, and sowed with destruction However, scientists still worked hard and published lots of vital discoveries on wine production In the 1910s, flor yeast was introduced In the 1920s, vine improvement programs were started in some European countries A series of events occurred in the 1930s, such as Bentonite for wine clarification, Elucidation of the life cycle of Saccharomyces cerevisiae yeast, and Yeast propagation Besides that, with access to refrigeration, it has become easy for wineries to control the temperature of the fermentation process and produce high-quality wines in hot climates And in 1964, an idea of bag-in-a-box

of wine was applied

Wine has soon become a popular drink, and the wine industry worldwide has been worth billion of dollars Italy, France, Spain, the United States, and China are leading producers of wine in the 21st century

1.2 Classification of wine

There are many ways to classify wine Table 1 below briefly illustrates how wines are classified based

on five different standards

Table 1 Wine classification based on five different standards

According to color

Red wine fermented from grapes with skin

White wine removed skins and seeds of grapes before fermentation

Rosé wine made of red grape varieties after short-term impregnation and

fermentation

According to the

CO 2 pressure

Still wine with a carbon dioxide pressure of less than 0.05 MPa at 20℃

Sparkling wine with a carbon dioxide pressure greater than or equal to 0.05

MPa at 20℃

According to the

sugar content

If the sugar in the wine is not completely converted into alcohol after the fermentation, the remaining sugar is the residual sugar According to the amount

of sugar, the still wine and sparkling wine can be divided into the following levels:

According to the

wine body

Light-bodied wine lighter in color and have fewer tannins

Medium-bodied Wine darker and have more texture on the tongue

Full-bodied Wine deepest color and abundant tannins

According to the

grape harvest time

Ordinary Wine Material: grape is picked after natural maturity come down

Late Harvest Wine

Material: grapes are naturally ripe and wait a few days (weather permitting), and when picked, the resulting wine tends to be sweeter and more flavorful

Noble Rot Wine

Material: the harvest time of grapes is delayed first, when the weather permits, the grapes are often infected with certain noble rot bacteria

Ice Wine Material: Waiting until the temperature drops to -7℃ to -8℃,

grapefruit is frozen and then harvested

1.3 Wine making process

There are five basic stages to making wine: harvesting, crushing and pressing, fermentation, clarification, aging and bottling

1.3.1 Harvesting

Winemakers usually harvest grapes from a vineyard in late summer or early autumn when grapes are riped enough After harvested, grapes are classified to cull rotten and under-ripe grapes

1.3.2 Crushing and pressing

Crushing the whole cluster is the next step Winemakers can carry out either by crushers on an industrial scale or by people in a conventional way to collect juice (called free-run juice) and the mass of crushed grapes (called must) Depending upon the desired product, the pressing process is different

If it is to make white wine, winemakers will quickly press the must to separate the juice from the skin, seed, and solid By doing that, unwanted color (which comes from grape skin) and tannins can not leach into the white wine If it is to make red wine, the must will be unpressed; alternatively, it is left in contact with its skin to Gardner color, flavor, and additional tannins during fermentation

1.3.3 Fermentation

The transformation of grape juice into wine is essentially a microbial process, usually taking ten days

to a month or more Alcoholic fermentation is the conversion of the principal grape sugars glucose and fructose to ethanol and carbon dioxide In winemaking, alcoholic fermentation involves two stages: natural fermentation and later fermentation

Natural fermentation occurs in the first 6 – 12 hours of the fermentation process, which is caused by wild yeasts that exist on grapes naturally This phenomenon contributes to the wine sensory characteristics such as flavors, odors, aromas, and texture However, it can lead to unwanted colors and a nasty taste of the

wine Additionally, the unpredictable duration of wild yeast can happen hence taking over the Saccharomyces

and constraining the desired fermentation To avoid that, wine manufacturers commonly inoculate the must,

which prevents the growth of wild yeast, and add a starter culture of commercial yeast (Saccharomyces)

There will be a lag period (time adaption) of commercial yeast strains before cell growth and fermentation under the low substrate and high oxygen exposure conditions to supply sterols and unsaturated fatty acids necessary for ethanol tolerance

Saccharomyces metabolize glucose and fructose to pyruvate via the glycolytic pathway Primarily to

recycle cofactors, pyruvate is decarboxylated to acetaldehyde, which is then reduced to ethanol One molecule

of glucose (or fructose) yields two molecules of ethanol as well as carbon dioxide The net equation for this reaction is:

Hexose + 2 ADP → 2 Ethanol + 2 CO2 + 2 ATP Considerably less ATP is generated during fermentation than during respiration, and most eukaryotes will rely on fermentation only under anaerobic conditions However, Saccharomyces will commence fermentation even under aerobic conditions if sufficient glucose is present in the media The major outcome

of glycolysis is the production of ethanol from hexose sugars, but a portion of glycolysis products are diverted

to biomass formation, yielding glycerol and acetic acid This is achieved by two regulatory phenomena: (1)

glucose repression (the genes required for growth and metabolism are repressed by high glucose

concentration, meaning that mRNA is not made; there is no transcription), and (2) glucose inactivation (the

inhibition of activity and subsequent proteolytic destruction of many of the same proteins that are regulated

by glucose repression and also catalyzed by high sugar concentration) After that, several minor metabolites that are important to flavor can be formed; thus, changes to the fermenting grape must result from the reducing environment and entrainment of volatiles in CO2 gas In a model fermentation starting with about 22-24% sugar, 95% sugar is converted into ethanol and carbon dioxide, 1% sugar is converted into cellular materials, and 4% sugar is converted to other end products

For the following reasons, winemakers commonly let oxygen infiltrate to the must to increase the biomass of the yeast before fermentation, then set the anaerobic and low-temperature conditions to maximize the fermentation yield

1.3.4 Clarification

Some wine deposits their suspended material (yeast cells, particles of skin, etc.) very quickly Removal

of this suspended material is called clarification The major procedures involved are:

• Fining: Proteins and yeast cells are adsorbed on fining agents such as bentonite, gelatin, silica,

phytate, etc

• Filtration: Removal of yeast cells and most bacterial cells by sufficiently small pore size of filters

• Centrifugation: High-speed spinning used to clarify the must

• Refrigeration: Temperature reduction prevents both yeast growth and the evolution of carbon dioxide,

which tends to keep the yeast cells suspended

• Ion exchange: If ion exchanger is charged with sodium, it will replace the potassium in potassium

acid tartate with sodium, making a more soluble tartate

• Heating: Pasteurization at 70 to 82o

C can be used to preciptate proteins that cause clouding by reacting with copper or other metals

1.3.5 Aging and bottling

Many wines improve in quality during barrel and bottle storage Such wines eventually reach their peak and begin to decline with further aging During the aging period, acidity decreases, additional clarification, and stabilization occur as undesirable substances precipitate, and the various components of the wine form complex compound affecting flavors and aromas Wine is usually aged in wooden containers made of oak, which allow oxygen to enter and vapor of water and alcohol to escape to decrease volume for the addition of more of the same wine

Before bottling, wine may require blending, filtration, and the use of antiseptics to combat microbe development The bottle shape and color are dictated by custom and cost Some white wines, subject to change when exposed to light, are preferably bottled in brown, brownish-green, or greenish-blue colored bottles After bottling, the closure is made Red wines that may be aged in the bottle for many years are closed with corks 5 centimeters

Appropriate storage conditions include an absence of light and a low temperature at about 12 to 16oC

to prevent rapid aging and deterioration by microbial factors

CHAPTER 2 YEASTS IN WINEMAKING 2.1 Sacchromyces cerevisiae

2.1.1 Taxonom: Morphology and special characteristics

2.1.1.1 Morphology

Saccharomyces cerevisiae (S cerevisiae) is a eukaryotic, unicellular microorganism and a member of

the fungus kingdom It is a dimorphic yeast that can vary between a unicellular and a filamentous growth form Some of them can show multicellular characteristics by forming pseudohyphae or false hyphae

Saccharomyces cerevisiae belongs to the kingdom of fungi (Mycophyta), class Ascomycetes, order

Endomycetes, family Saccharomycetaceae, genus Saccharomyces, and genus Cerevisiae The size of this

yeast experiences significantly with each stage of development In general, its cell is markedly larger than a bacterial cell, making up approximately 7µm in diameter and 8-12µm in length Moreover, the temperature might exert on size or volume For example, the critical diameter of a single cell was 7.94 µm at growth temperature above 18.5oC while below 18.5oC; in contrast, it exponentially increases up to 10.2 µm The

standard of vegetative cells of S cerevisiae, the most typical in appearance and most widely used

domesticated yeast are egg-shaped, elliptical, or occasionally spherical The yeast shape is not stable; however, it depends on age, variety, and external conditions For example, in a nutrient-rich culture medium, the cell has an oval shape In anaerobic conditions, the cell is usually round-shaped whereas the cell is longer

in aerobic conditions Its color is yellowish-green

Figure 1 Some images of yeast: S cerevisiae TBS (a&b) and S cerevisiae TNS (c&d)

2.1.1.2 Typical characteristics of cell structures

Figure 2 Structure of S cerevisiae cell

As a eukaryote, S cerevisiae contains membrane-bound organelles Compared to the structure of

bacteria, yeast involves prokaryotes and eukaryotes Along with the evolution of the nucleus and the mechanism of nuclear division(membranous nuclei, chromosomes, filamentous cell division, etc.), many bodies appear in eukaryotes but are not found in prokaryotes Yeast cells have a complex structure and finished products In the cell, there are components – corpuscles, which can be divided into intracellular or cell organelles and intracellular hosts or inclusions

A typical S cerevisiae cell would be composed of: a cell wall; plasma membrane; cytosol; nucleus;

endoplasmic reticulum; vacuole; Golgi apparatus; mitochondrion; and peroxisome

Cell envelope

The chemical composition of the cell envelope includes protein-polysaccharide complexes, a phosphate group and lipids The cell is about 25 nm thick and makes up about 25% of the cell mass In the polysaccharide part, glucan (mainly) and mannan were found Surrounding the yeast cell is a dense, soft, elastic membrane that can shape and protect the cell against external influences and toxins The yeast cell shell carries electricity It also has the effect of keeping intracellular osmotic pressure, regulating nutrients that are low-molecular-weight compounds and mineral salts through small pores into the cell

Cytoplasm

In the area between the cell wall and the cytoplasmic membrane, we find a series of enzymes, mainly hydrolytic enzymes such as B – fructofuranozidase (invertase) and acid phosphatase Some of these are enzymes bound to the cell wall Among the enzymes mentioned above, invertase is a mannoprotein in nature Mannan in the enzyme accounts for up to 50% and plays an important role in stabilizing the enzyme molecule

Plasma membrane

It is surrounded by a very thin membrane, not larger than 0.1 nm thick The membrane is a very bright border around the cytoplasm The cytoplasmic membrane has four functions: acting as an osmotic barrier, regulating nutrients from the environment into the cell, and vice versa for metabolic products out of the cell, performing biosynthesis Synthesizes some cell components (cell envelope components) where certain enzymes and cell organelles (such as ribosomes) are located

Mitochondrion

For small granules or rods, filaments, shape changes during culture, rod, single strand, or chained The

S cerevisiae cells maintained at various glucose concentrations have distinct mitochondrial morphologies

Interestingly, the mitochondria in cells grown on 0.5 percent glucose have a shape comparable to mitochondria in respiring cells Due to the formation of acetic acid, the mitochondria of cells growing at higher glucose concentrations (2 and 4%) became fractured during growth while, in the environment with low glucose concentration, by contrast, yeast cells have up to 100-200 mitochondria The structure of mitochondria changes when yeasts transition from aerobic to anaerobic conditions in the absence of lipids, the mitochondria are very simple, consisting of two membranes, but without folds However, the addition of lipids will cause folds

Nucleus

Immutable components, eukaryotes contain DNA and RNA Nuclei size is not uniform among yeast strains and even within the same strain Other organelles: vacuoles, ribosomes, Golgi endoplasmic reticulum, etc., which have the same structure as plant cells

2.1.2 Nutrition and growth

Like many fungal species, Saccharomyces cerevisiae exists in a variety of strains A heterotroph is a

term used to describe an organism that feeds on another one The chemical composition of yeast cells includes water, organic compounds, and ash which oxidize chemical bonds such as sugars, fats, and protein to

transform their energy sources S cerevisiae can ferment glucose, galactose, maltose, sucrose, raffinose, and

simple dextrin, but not lactose, mannitol, nitrate, or starch It grows optimally at 33-35oC in environments containing 10%- 30% glucose Particularly, the minimum temperature is 4oC in 10% glucose and 13oC in 50% glucose, while maximum temperature makes up approximately 38-39oC These cells can also use almost amino acids, small peptides, and nitrogen bases as their nitrogen source Above all, galactose and fructose

are known as the most efficient sugar fermenters in S cerevisiae cells There are two types of respiration: aerobic and anaerobic Some strains of Saccharomyces cerevisiae, e.g., are unable to grow anaerobically on sucrose and trehalose In contrast, through aerobic and anaerobic respiration, S cerevisiae cells convert sugars