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Since this discovery the findings of chemists, biochemists and microbiologists have led to controlled conditions in winemaking, producing more varied and higher quality wines. Handbook of Enology Volume 1: The Microbiology of Wine and Vinifications uniquely combines scientific knowledge with the description of day-to-day work in the first stages of winemaking, from grape-picking to the end of the fermentation processes.

It discusses the scientific basics and technological problems of wine-making and the resulting consequences for the practitioner, providing an authoritative and complete reference manual for both the winemaker and the student. This text will be invaluable to winemakers, students of enology or vinification and chemists interested in winemaking. Buy it as a reference book and you won't be disappointed. Visit Seller's Storefront. List this Seller's Books.

Payment Methods accepted by seller. Stock Image. New Condition: nuovo Hardcover. Save for Later. Informazioni bibliografiche Titolo: Handbook of Enology. Preface to the First Edition. Preface to the Second Edition. Conditions of Yeast Development. Lactic Acid Bacteria. This line of reasoning lead to the description of the antioxidant related chemical properties of this compound in the same chapter as well as an explanation of adjuvants to sulfur dioxide: sorbic acid antiseptic and ascorbic acid antioxidant. In addition, the on lees aging of white wines and the resulting chemical transformations cannot be separated from vinification and are therefore also covered in Volume 1.

Finally, our understanding of phenolic compounds in red wine is based on complex chemistry. All aspects related to the nature of the. Preface to the First Edition corresponding substances, their properties and their evolution during grape maturation, vinification and aging are therefore covered in Volume 2. These works only discuss the principles of equipment used for various enological operations and their effect on product quality.

For example, temperature control systems, destemmers, crushers and presses as well as filters, inverse osmosis machines and ion exchangers are not described in detail. Bottling is not addressed at all. An in-depth description of enological equipment would merit a detailed work dedicated to the subject. Wine tasting, another essential role of the winemaker, is not addressed in these works. Many related publications are, however, readily available.

Finally, wine analysis is an essential tool that a winemaker should master. It is, however, not covered in these works except in a few particular. The authors thank the following people who have contributed to the creation of this work: J. Casas Lucas, Chapter 14, Sherry; A. Brugirard, Chapter 14, Sweet wines; J. Maujean, Chapter 14, Champagne; C. Poupot for the preparation of material in Chapters 1, 2 and 13; Miss F.

LuyeTanet for her help with typing. They also thank Madame B. Masclef in particular for her important part in the typing, preparation and revision of the final manuscript. Preface to the Second Edition The two-volume Enology Handbook was published simultaneously in Spanish, French, and Italian in and has been reprinted several times. The Handbook has apparently been popular with students as an educational reference book, as well as with winemakers, as a source of practical solutions to their specific technical problems and scientific explanations of the phenomena involved. It was felt appropriate at this stage to prepare an updated, reviewed, corrected version, including the latest enological knowledge, to reflect the many new research findings in this very active field.

The outline and design of both volumes remain the same. Some chapters have changed relatively little as the authors decided there had not been any significant new developments, while others have been modified much more extensively, either to clarify and improve the text, or, more usually, to include new research findings and their practical applications.

Entirely new sections have been inserted in some chapters. We have made every effort to maintain the same approach as we did in the first edition, reflecting the ethos of enology research in Bordeaux. We use indisputable scientific evidence in microbiology, biochemistry, and chemistry to explain the details of mechanisms involved in grape ripening, fermentations and other winemaking operations, aging, and stabilization. The aim is to help winemakers achieve greater control over the various stages in winemaking and choose the solution best suited to each situation. Quite remarkably, this scientific approach, most intensively applied in making the finest wines, has resulted in an enhanced capacity to bring out the full quality and character of.

Scientific winemaking has not resulted in standardization or leveling of quality. On the contrary, by making it possible to correct defects and eliminate technical imperfections, it has revealed the specific qualities of the grapes harvested in different vineyards, directly related to the variety and terroir, more than ever before. Interest in wine in recent decades has gone beyond considerations of mere quality and taken on a truly cultural dimension.

This has led some people to promote the use of a variety of techniques that do not necessarily represent significant progress in winemaking. Some of these are simply modified forms of processes that have been known for many years. Others do not have a sufficiently reliable scientific interpretation, nor are their applications clearly defined. In this Handbook, we have only included rigorously tested techniques, clearly specifying the optimum conditions for their utilization. As in the previous edition, we deliberately omitted three significant aspects of enology: wine analysis, tasting, and winery engineering.

In view of their importance, these topics will each be covered in separate publications. Casas Lucas for the paragraph on Sherry Section Introduction The cell wall The plasmic membrane The cytoplasm and its organelles The nucleus Reproduction and the yeast biological cycle The killer phenomenon Classification of yeast species Identification of wine yeast strains Ecology of grape and wine yeasts. Yet the role of yeasts in alcoholic fermentation, particularly in the transformation of grapes into wine, was only clearly established in the middle of the nineteenth century. The ancients explained the boiling during fermentation from the Latin fervere, to boil as a reaction between substances.

In , a Dutch cloth merchant, Antonie van Leeuwenhoek, first observed yeasts in beer wort using a microscope that he designed and produced. He did not, however, establish a relationship between these corpuscles and alcoholic fermentation. It was not until the end of the eighteenth century that Lavoisier began the chemical study of alcoholic fermentation. According to Fabroni, this material, comparable to the gluten in flour, was located in special utricles, particularly on grapes and wheat, and alcoholic fermentation occurred when it came into contact with sugar in the must.

In , a French physicist named Charles Cagnard de La Tour proved for the first time that the yeast was a living organism. According to his findings, it was capable of multiplying and belonged to the plant kingdom; its vital activities were at the base of the fermentation of sugar-containing liquids. The German naturalist Schwann confirmed his theory and demonstrated that heat and certain chemical products were capable of stopping alcoholic fermentation. In , Meyen used this nomenclature for the first time. This vitalist or biological viewpoint of the role of yeasts in alcoholic fermentation, obvious to us today, was not readily supported.

Liebig and certain other organic chemists were convinced that chemical reactions, not living cellular activity, were responsible for the fermentation of sugar. In his famous studies on wine and beer , Louis Pasteur gave definitive credibility to the vitalist viewpoint of alcoholic fermentation. He demonstrated that the yeasts responsible for spontaneous fermentation of grape must or crushed grapes came from the surface of the grape; he isolated several races and species. He even conceived the notion that the nature of the yeast carrying out the alcoholic fermentation could influence the gustatory characteristics of wine.

He also demonstrated the effect of oxygen on the assimilation of sugar by yeasts. Louis Pasteur proved that the yeast produced secondary products such as glycerol in addition to alcohol and carbon dioxide. Since Pasteur, yeasts and alcoholic fermentation have incited a considerable amount of research, making use of progress in microbiology,. In taxonomy, scientists define yeasts as unicellular fungi that reproduce by budding and binary fission. Certain pluricellular fungi have a unicellular stage and are also grouped with yeasts.

Yeasts form a complex and heterogeneous group found in three classes of fungi, characterized by their reproduction mode: the sac fungi Ascomycetes , the club fungi Basidiomycetes , and the imperfect fungi Deuteromycetes. The yeasts found on the surface of the grape and in wine belong to Ascomycetes and Deuteromycetes. The haploid spores or ascospores of the Ascomycetes class are contained in the ascus, a type of sac made from vegetative cells. Asporiferous yeasts, incapable of sexual reproduction, are classified with the imperfect fungi. In this first chapter, the morphology, reproduction, taxonomy and ecology of grape and wine yeasts will be discussed.

Cytology is the morphological and functional study of the structural components of the cell Rose and Harrison, The yeast cell contains cellular envelopes, a cytoplasm with various organelles, and a nucleus surrounded by a membrane and enclosing the chromosomes. Figure 1. Like all plant cells, the yeast cell has two cellular envelopes: the cell wall and the membrane. The periplasmic space is the space between the cell wall and the membrane. The cytoplasm and the membrane make up the protoplasm. The term protoplast or sphaeroplast designates a cell whose cell wall has been artificially removed.

In order to take advantage of these properties, the winemaker or enologist must have a profound knowledge of these organelles. It essentially consists of polysaccharides. It is a rigid envelope, yet endowed with a certain elasticity. Its first function is to protect the cell. Protoplasts placed in pure water are immediately lysed in this manner. Cell wall elasticity can be demonstrated by placing yeasts, taken during their log phase, in a hypertonic NaCl solution. The cell wall appears thicker and is almost in contact with the membrane. The cells regain their initial form after being placed back into an isotonic medium.

On the contrary, it is a dynamic and multifunctional organelle. Its composition and functions evolve during the life of the cell, in. In addition to its protective role, the cell wall gives the cell its particular shape through its macromolecular organization. It is also the site of molecules which determine certain cellular interactions such as sexual union, flocculation, and the killer factor, which will be examined in detail later in this chapter Section 1. Finally, a number of enzymes, generally hydrolases, are connected to the cell wall or situated in the periplasmic space.

Their substrates are nutritive substances of the environment and the macromolecules of the cell wall itself, which is constantly reshaped during cellular morphogenesis. Chitin represents a minute part of its composition. The most detailed work on the yeast cell wall has been carried out on Saccharomyces cerevisiae —the principal yeast responsible for the alcoholic fermentation of grape must. It can be chemically fractionated into three categories: 1. It has very few branches. Its degree of polymerization is Under the electron microscope, this glucan appears fibrous.

It ensures the shape and the rigidity of the cell wall. It is always connected to chitin. It has very few branches, like the preceding glucan. It has an amorphous aspect under the electron microscope.

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It gives the cell wall its elasticity and acts as an anchor for the mannoproteins. It can also constitute an extraprotoplasmic reserve substance. It links the different constituents of the cell wall together. It is also a receptor site for the killer factor. They can be extracted from the whole cell or from the isolated cell wall by chemical and enzymatic methods.

Chemical methods make use of autoclaving in the presence of alkali or a citrate buffer solution at pH 7. The enzymatic method frees the mannoproteins by digesting the glucan. This method does not denature the structure of the mannoproteins as much as chemical methods. Zymolyase, obtained from the bacterium Arthrobacter luteus, is the enzymatic preparation most often used to extract the parietal mannoproteins of S.

The action of protease contaminants in the zymolyase combine, with the aforementioned activity to liberate the mannoproteins. These activities also facilitate the extraction of the cell wall mannoproteins of the S. The mannoproteins of S. Their degree of glycosylation varies. Four forms of glycosylation are described Figure 1. The mannose of the mannoproteins can constitute short, linear chains with one to five residues.

They are linked to the peptide chain by O-glycosyl linkages on serine and threonine residues. The glucidic part of the mannoprotein can also be a polysaccharide. It is linked to an asparagine residue of the peptide chain by an N -glycosyl linkage. The mannan linked in this manner to the asparagine includes an attachment region made up of a dozen mannose residues and a highly ramified outer chain consisting of to mannose units. Some of these side-chains possess a branch attached by a phosphodiester bond. A third type of glycosylation was described more recently. It can occur in mannoproteins, which make up the cell wall of the yeast.

The nature of the glycan— peptide point of attachment is not yet clear, but it may be an asparaginyl—glucose bond. The fourth type of glycosylation of yeast mannoproteins is the glycosyl—phosphatidyl—inositol anchor GPI. This attachment between the terminal carboxylic group of the peptide chain and a membrane phospholipid permits certain mannoproteins, which cross the cell wall, to anchor themselves in the plasmic membrane. The region of attachment is characterized by the following sequence Figure 1. A Cphospholipase specific to phosphatidyl inositol and therefore capable of realizing this cleavage.

The four types of glucosylation of parietal yeast mannoproteins Klis, Several GPI-type anchor mannoproteins have been identified in the cell wall of S. These zones are a type of raised crater easily seen on the mother cell under the electron microscope Figure 1. This chitinic scar is formed essentially to assure cell wall integrity and cell survival. Yeasts treated with D polyoxine, an antibiotic inhibiting the synthesis of chitin, are not viable; they burst after budding. The presence of lipids in the cell wall has not been clearly demonstrated. It is true that cell walls.

Scanning electron microscope photograph of proliferating S. The budding scars on the mother cells can be observed. The cell wall can also adsorb lipids from its external environment, especially the different fatty acids that activate and inhibit the fermentation Chapter 3. Chitin are connected to the cell wall or situated in the periplasmic space. This enzyme catalyzes the hydrolysis of saccharose into glucose and fructose.

Its molecular weight is Da. The periplasmic acid phosphatase is equally a mannoprotein. These enzymes are involved in the reshaping of the cell wall during the growth and budding of cells. Their activity is at a maximum during the exponential log phase of the population and diminishes notably afterwards. These endogenous enzymes are involved in the autolysis of the cell wall during the.

This ageing method will be covered in the chapter on white winemaking Chapter The inner layer is connected to a small quantity of chitin Figure 1. Its elasticity is due to the outer amorphous layer. The intermolecular structure of the mannoproteins of the outer layer hydrophobic linkages and disulfur bonds equally determines cell wall porosity and impermeability to macromolecules molecular weights less than This substance provokes the rupture of the disulfur bonds, thus destroying the intermolecular network between the mannoprotein chains. The composition of the cell wall is strongly influenced by nutritive conditions and cell age.

The proportion of glucan in the cell wall increases External medium. Cytology, Taxonomy and Ecology of Grape and Wine Yeasts with respect to the amount of sugar in the culture medium. Certain deficiencies for example, in mesoinositol also result in an increase in the proportion of glucan compared with mannoproteins. The cell walls of older cells are richer in glucans and in chitin and less furnished in mannoproteins. For this reason, they are more resistant to physical and enzymatic agents used to degrade them. Finally, the composition of the cell wall is profoundly modified by morphogenetic alterations conjugation and sporulation.

This organelle is essential to the life of the yeast. Like all biological membranes, the yeast plasmic membrane is principally made up of lipids and proteins. The plasmic membrane of S. Glucans and mannans are only present in small quantities several per cent. The lipids of the membrane are essentially phospholipids and sterols. They are amphiphilic molecules, i. The three principal phospholipids Figure 1. Phosphatidylserine PS and diphosphatidylglycerol or cardiolipin PG are less prevalent.

Free fatty acids and phosphatidic acid are frequently reported in plasmic membrane analysis. They are probably extraction artifacts caused by the activity of certain lipid degradation enzymes. The fatty acids of the membrane phospholipids contain an even number 14 to 24 of carbon atoms. The most abundant are C16 and C18 acids. They can be saturated, such as palmitic acid C16 and stearic acid C18 , or unsaturated, as with oleic.

All membrane phospholipids share a common characteristic: they possess a polar or hydrophilic part made up of a phosphorylated alcohol and a non-polar or hydrophobic part comprising two more or less parallel fatty acid chains Figure 1. In an aqueous medium, the phospholipids spontaneously form bimolecular films or a lipid bilayer because of their amphiphilic characteristic Figure 1.

The lipid bilayers are cooperative but non-covalent structures. They are maintained in place by mutually reinforced interactions: hydrophobic interactions, van der Waals attractive forces between the hydrocarbon tails, hydrostatic interactions and hydrogen bonds between the polar heads and water molecules. The examination of cross-sections of yeast plasmic membrane under the electron microscope reveals a classic lipid bilayer structure with a thickness of about 7. The membrane surface appears sculped with creases, especially during the stationary phase.

However, the physiological meaning of this anatomic character remains unknown. The plasmic membrane also has an underlying depression on the bud scar. Ergosterol is the primary sterol of the yeast plasmic membrane. In lesser quantities, 24 28 dehydroergosterol and zymosterol also exist Figure 1. Sterols are exclusively produced in the mitochondria during the yeast log phase.

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As with phospholipids, membrane sterols are amphipathic. The hydrophilic part is made up of hydroxyl groups in C The rest of the molecule is hydrophobic, especially the flexible hydrocarbon tail. The plasmic membrane also contains numerous proteins or glycoproteins presenting a wide range of molecular weights from 10 to The available information indicates that the organization of the plasmic membrane of a yeast cell resembles the fluid mosaic model. This model, proposed for biological membranes by Singer and Nicolson , consists of two-dimensional solutions of proteins and oriented lipids.

Certain proteins are embedded in the membrane; they are called integral proteins Figure 1. They interact. The peripheral proteins are linked to the precedent by hydrogen bonds. Their location is asymmetrical, at either the inner or the outer side of the plasmic membrane. The molecules of proteins and membrane lipids, constantly in lateral movement, are capable of rapidly diffusing in the membrane. Some of the yeast membrane proteins have been studied in greater depth. These include adenosine triphosphatase ATPase , solute sugars and amino. The yeast possesses three ATPases: in the mitochondria, the vacuole, and the plasmic membrane.

The plasmic membrane ATPase is an integral protein with a molecular weight of around Da. It catalyzes the hydrolysis of ATP which furnishes the necessary energy for the active transport of solutes across the membrane. Note: an active. The cultivation of yeasts in the presence of an easily assimilated nitrogen-based nutrient such as ammonium represses this permease. The membrane composition in fatty acids and its proportion in sterols control its fluidity. The hydrocarbon chains of fatty acids of the membrane phospholipid bilayer can be in a rigid and orderly state or in a relatively disorderly and fluid state.

In the rigid state, some or all of the carbon bonds of the fatty acids are trans. In the fluid state, some of the bonds become cis. The transition from the rigid state to the fluid state takes place when the temperature rises beyond the fusion temperature. This transition temperature depends on the length of the fatty acid chains and their degree of unsaturation. The rectilinear hydrocarbon chains of the saturated fatty acids interact strongly. These interactions intensify with their length.

The transition temperature therefore increases as the fatty acid chains become longer. The double bonds of the unsaturated fatty acids are generally cis, giving a curvature to the hydrocarbon chain Figure 1. This curvature breaks the orderly.

Handbook of Enology Volume 1 The Microbiology of Wine and Vinifications 2 nd Edition

A membrane lipid bilayer. The integral proteins a are strongly associated to the non-polar region of the bilayer. The peripheral proteins b are linked to the integral proteins. Simultaneously, the hydrolysis of ATP creates an efflux of protons towards the exterior of the cell. The penetration of amino acids and sugars into the yeast activates membrane transport systems called permeases.

The general amino acid CH3 H3C. Molecular models representing the three-dimensional structure of stearic and oleic acid. The cis configuration of the double bond of oleic acid produces a curvature of the carbon chain. Like cholesterol in the cells of mammals, ergosterol is also a fundamental regulator of the membrane fluidity in yeasts.

Ergosterol is inserted in the bilayer perpendicularly to the membrane. Its hydroxyl group joins, by hydrogen bonds, with the polar head of the phospholipid and its hydrocarbon tail is inserted in the hydrophobic region of the bilayer. The membrane sterols intercalate themselves between the phospholipids.

In this manner, they inhibit the crystallization of the fatty acid chains at low temperatures. Inversely, in reducing the movement of these same chains by steric encumberment, they regulate an excess of membrane fluidity when the temperature rises. This barrier presents a certain impermeability to solutes in function of osmotic properties. Furthermore, through its system of permeases, the plasmic membrane also controls the exchanges between the cell and the medium.

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  • The functioning of these transport proteins is greatly influenced by its lipid composition, which affects membrane fluidity. In a defined environmental model, the supplementing of membrane phospholipids with unsaturated fatty acids oleic and linoleic promoted the penetration and accumulation of certain amino acids as well as the expression of the general amino acid permease GAP , Henschke and Rose, On the other hand, membrane sterols seem to have less influence on the transport of amino acids than the degree of unsaturation of the phospholipids.

    The production of unsaturated fatty acids is an oxidative process and requires the aeration of the culture medium at the beginning of alcoholic fermentation. In semi-anaerobic winemaking conditions, the amount of unsaturated fatty acids in the grape, or in the grape must, probably favor the membrane transport mechanisms of fatty acids. The transport systems of sugars across the membrane are far from being completely elucidated. There exists, however, at least two kinds of transport systems: a high affinity and a low affinity system ten times less important Bisson, The low affinity system is essential during the log phase and its activity decreases during the stationary phase.

    The high affinity system is, on the contrary, repressed by high concentrations of glucose, as in the case of grape must Salmon et al. The amount of sterols in the membrane, especially ergosterol, as well as the degree of unsaturation of the membrane phospholipids favor the penetration of glucose in the cell. This is especially true during the stationary and decline phases. This phenomenon explains the determining influence of aeration on the successful completion of alcoholic fermentation during the yeast multiplication phase.

    The presence of ethanol, in a culture medium, slows the penetration speed of arginine and glucose into the cell and limits the efflux of protons. Section 1. They are then transported by vesicles which fuse with the plasmic membrane and deposit their contents at the exterior of the membrane. Finally, certain membrane proteins act as cellular specific receptors. They permit the yeast to react to various external stimuli such as sexual hormones or changes in the concentration of external nutrients.

    The activation of these membrane proteins triggers the liberation of compounds such as cyclic adenosine monophosphate cAMP in the cytoplasm. These compounds serve as secondary messengers which set off other intercellular reactions. The consequences of these cellular mechanisms in the alcoholic fermentation process merit further study. Evolution of glucose transport system activity of S. Simultaneously, the presence of ethanol increases the synthesis of membrane phospholipids and their percentage in unsaturated fatty acids especially oleic.

    Temperature and ethanol act in synergy to affect membrane ATPase activity. The amount of ethanol required to slow the proton efflux decreases as the temperature rises. However, this modification of membrane ATPase activity by ethanol may not be the origin of the decrease in plasmic membrane permeability in an alcoholic medium.

    The role of membrane ATPase in yeast resistance to ethanol has not been clearly demonstrated. The plasmic membrane also produces cell wall glucan and chitin. The mannoproteins are essentially produced in the endoplasmic reticulum. The organelles endoplasmic reticulum, Golgi apparatus, vacuole and mitochondria are isolated from the cytosol by membranes. Glycolysis and alcoholic fermentation enzymes Chapter 2 as well as trehalase an enzyme catalyzing the hydrolysis of trehalose are present. Trehalose, a reserve disaccharide, also cytoplasmic, ensures yeast viability during the dehydration and rehydration phases by maintaining membrane integrity.

    The lag phase precedes the log phase in a sugar-containing medium. It is marked by a rapid degradation of trehalose linked to an increase in trehalase activity. This activity is itself closely related to an increase in the amount of cAMP in the cytoplasm. This compound is produced by a membrane enzyme, adenylate cyclase, in response. Glycogen is the principal yeast glucidic reserve substance.

    Animal glycogen is similar in structure. When observed under the electron microscope, the yeast cytoplasm appears rich in ribosomes. These tiny granulations, made up of ribonucleic acids and proteins, are the center of protein synthesis. Joined to polysomes, several ribosomes migrate the length of the messenger RNA. They translate it simultaneously so that each one produces a complete polypeptide chain. It is linked to the cytoplasmic membrane and nuclear membrane. It is, in a way, an extension of the latter.

    Although less developed in yeasts than in exocrine cells of higher eucaryotes, the ER has the same function. It ensures the addressing of the proteins synthesized by the attached ribosomes. As a matter of fact, ribosomes can be either free in the cytosol or bound to the ER.

    The proteins synthesized by free ribosomes remain in the cytosol, as do the enzymes involved in glycolysis. Those produced in the ribosomes bound to the ER have three possible destinations: the vacuole, the plasmic membrane, and the external environment secretion. The presence of a signal sequence a particular chain of amino acids at the N -terminal extremity of the newly formed protein determines the association of the initially free ribosomes in the cytosol with the ER.

    The synthesized protein crosses the ER membrane by an active transport process called translocation. This process requires the hydrolysis of an ATP molecule.

    Having reached the inner space of the ER, the proteins undergo certain modifications including the necessary excising of the signal peptide by the signal peptidase. In many cases, they also undergo a glycosylation. The yeast glycoproteins, in particular the structural, parietal or enzymatic mannoproteins, contain glucidic side chains Section 1.

    Some of these are linked to asparagine by N -glycosidic bonds. This oligosaccharidic link is constructed in the interior of the ER by the sequential addition of activated sugars in the form of UDP derivatives to a hydrophobic, lipidic transporter called dolicholphosphate. The entire unit is transferred in one piece to an asparagine residue of the polypeptide chain. The dolicholphosphate is regenerated. The Golgi apparatus consists of a stack of membrane sacs and associated vesicles.

    It is an extension of the ER. Transfer vesicles transport the proteins issued from the ER to the sacs of the Golgi apparatus. The Golgi apparatus has a dual function. It is responsible for the glycosylation of protein, then sorts so as to direct them via specialized vesicles either into the vacuole or into the plasmic membrane. An N-terminal peptidic sequence determines the directing of proteins towards the vacuole.

    This sequence is present in the precursors of two vacuolar-orientated enzymes in the yeast: Y carboxypeptidase and A proteinase. The vesicles that transport the proteins of the plasmic membrane or the secretion granules, such as those that transport the periplasmic invertase, are still the default destinations.

    The vacuole is a spherical organelle, 0. Depending on the stage of the cellular cycle, yeasts have one or several vacuoles. Before budding, a large vacuole splits into small vesicles. Some penetrate into the bud. Others gather at the opposite extremity of the cell and fuse to form one or two large vacuoles. The vacuolar membrane or tonoplast has the same general structure fluid mosaic as the plasmic membrane but it is more elastic and its chemical composition is somewhat different. It is less rich in sterols and contains less protein and glycoprotein but more phospholipids with a higher degree of unsaturation.

    The vacuole stocks some of the cell hydrolases, in particular Y carboxypeptidase, A and B proteases, I aminopeptidase, X-propyl-dipeptidylaminopeptidase and alkaline phosphatase. In this respect, the yeast vacuole can. Vacuolar proteases play an essential role in the turn-over of cellular proteins. In addition, the A protease is indispensable in the maturation of other vacuolar hydrolases. It excises a small peptide sequence and thus converts precursor forms proenzymes into active enzymes. The vacuolar proteases also autolyze the cell after its death.

    Autolysis, while ageing white wine on its lees, can affect wine quality and should concern the winemaker. Vacuoles also have a second principal function: they stock metabolites before their use. In fact, they contain a quarter of the pool of the amino acids of the cell, including a lot of arginine as well as S-adenosyl methionine. In this organelle, there is also potassium, adenine, isoguanine, uric acid and polyphosphate crystals. These are involved in the fixation of basic amino acids.

    Specific permeases ensure the transport of these metabolites across the vacuolar membrane.

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    An ATPase linked to the tonoplast furnishes the necessary energy for the movement of stocked compounds against the concentration gradient. It is different from the plasmic membrane ATPase, but also produces a proton efflux. The ER, Golgi apparatus and vacuoles can be considered as different components of an internal system of membranes, called the vacuome, participating in the flux of glycoproteins to be excreted or stocked.

    The inner membrane is highly folded to form cristae. The general organization of mitochondria is the same as in higher plants and animal cells. The membranes delimit two compartments: the inner membrane space and the matrix. The mitochondria are true respiratory organelles for yeasts. In aerobiosis, the S. In anaerobiosis, these organelles degenerate, their inner surface decreases, and the cristae disappear.

    Ergosterol and unsaturated fatty acids supplemented in culture media limit the degeneration of mitochondria in anaerobiosis. Even in aerated grape must, the high sugar concentration represses the synthesis of respiratory enzymes. As a result, the mitochondria no longer function. This phenomenon, catabolic glucose repression, will be described in Chapter 2. Cardiolipin diphosphatidylglycerol , in minority in the plasmic membrane Figure 1. The fatty acids of the mitochondrial phospholipids are in C, C, C, C In aerobiosis, the unsaturated residues predominate.

    When the cells are grown in anaerobiosis, without lipid supplements, the short-chain saturated residues become predominant; cardiolipin and phosphatidylethanolamine diminish whereas the proportion of phosphatidylinositol increases. In aerobiosis, the temperature during the log phase of the cell influences the degree of unsaturation of the phospholipids- more saturated as the temperature decreases. The mitochondrial membranes also contain sterols, as well as numerous proteins and enzymes Guerin, The two membranes, inner and outer, contain enzymes involved in the synthesis of phospholipids and sterols.

    The ability to synthesize significant amounts of lipids, characteristic of yeast mitochondria, is not limited by respiratory deficient mutations or catabolic glucose repression. The outer membrane is permeable to most small metabolites coming from the cytosol since it contains porine, a 29 kDa transmembrane protein possessing a large pore.

    Porine is present in the mitochondria of all the eucaryotes as well as in the outer membrane of bacteria. Oxidative phosphorylation takes place in the inner mitochondrial membrane. The matrix, on the other hand, is the center of the reactions of the tricarboxylic acids cycle and of the oxidation of fatty acids. The majority of mitochondria proteins are coded by the genes of the nucleus and are synthesized by the free polysomes of the cytoplasm.

    The mitochondria, however, also have their own machinery for protein synthesis. In fact, each mitochondrion possesses a circular 75 kb kilobase pairs molecule of double-stranded AND and ribosomes. It contains a few dozen genes, which code in particular for the synthesis of certain pigments and respiratory enzymes, such as cytochrome b, and several sub-units of cytochrome oxidase and of the ATP synthetase complex. Some mutations affecting these genes can result in the yeast becoming resistant to certain mitochondrial specific inhibitors such as oligomycin.

    This property has been applied in the genetic marking of wine yeast strains. Some mitochondrial mutants are respiratory deficient and form small colonies on solid agar media. It has a diameter of 1—2 mm and is barely visible using a phase contrast optical microscope. It is located near the principal vacuole in non-proliferating cells.

    The nuclear envelope is made up of a double membrane attached to the ER. It contains many ephemeral pores, their locations continually changing. These pores permit the exchange of small proteins between the nucleus and the cytoplasm.

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    Contrary to what happens in higher eucaryotes, the yeast nuclear envelope is not dispersed during mitosis. In the basophilic part of the nucleus, the crescentshaped nucleolus can be seen by using a nuclearspecific staining method. As in other eucaryotes, it is responsible for the synthesis of ribosomal RNA. During cellular division, the yeast nucleus also contains rudimentary spindle threads composed of microtubules of tubulin, some discontinuous and others continuous Figure 1.

    The continuous microtubules are stretched between the two spindle pole bodies SPB. These corpuscles are permanently included in the nuclear membrane and. The yeast nucleus Williamson, The cytoplasmic microtubules depart from the spindle pole bodies towards the cytoplasm. There is little nuclear DNA in yeasts compared with higher eucaryotes—about 14 kb in a haploid strain. It has a genome almost three times larger than in Escherichia coli, but its genetic material is organized into true chromosomes. Each one contains a single molecule of linear doublestranded DNA associated with basic proteins known as histones.

    The histones form chromatin which contains repetitive units called nucleosomes. Yeast chromosomes are too small to be observed under the microscope. Pulse-field electrophoresis Carle and Olson, ; Schwartz and Cantor, permits the separation of the 16 chromosomes in S. This species has a very large chromosomic polymorphism. This characteristic has made karyotype analysis one of the principal criteria for the identification of S.

    The scientific community has nearly established the complete sequence of the chromosomic DNA of S. In the future, this detailed knowledge of the yeast genome will constitute a powerful tool, as much for understanding its molecular physiology as for selecting and improving winemaking strains. The yeast chromosomes contain relatively few repeated sequences.

    Most genes are only present. Cytology, Taxonomy and Ecology of Grape and Wine Yeasts in a single example in the haploid genome, but the ribosomal RNA genes are highly repeated about copies. The genome of S. The Ty elements code for non-infectious retrovirus particles. The latter can reinsert itself into any site of the chromosome. The random excision and insertion of Ty elements in the yeast genome can modify the genes and play an important role in strain evolution. It is a circular molecule of DNA, containing 6 kb and there are 50— copies per cell.

    Its biological function is not known, but it is a very useful tool, used by molecular biologists to construct artificial plasmids and genetically transform yeast strains. By definition, yeasts belonging to the imperfect fungi can only reproduce by vegetative multiplication. Some yeasts, such as species belonging to the genus Schizosaccharomyces, multiply by binary fission.

    M corresponds with mitosis, G1 is the period. As soon as the bud emerges, in the beginning of S, the splitting of the spindle pole bodies SPB can be observed in the nuclear membrane by electron microscopy. At the same time, the cytoplasmic microtubules orient themselves toward the emerging bud. These microtubules seem to guide numerous vesicles which appear in the budding zone and are involved in the reshaping of the cell wall.

    As the bud grows larger, discontinued nuclear microtubules begin to appear. The longest microtubules form the mitotic spindle between the two SPB. At the end of G2, the nucleus begins to push and pull apart in order to penetrate the bud. Some of the mitochondria also pass with some small vacuoles into the bud, whereas a large vacuole is formed at the other pole of the cell.

    The expansion of the latter seems to push the nucleus into the bud. During mitosis, the nucleus stretches to its maximum and the mother cell separates from the daughter cell. This separation takes place only after the construction of the separation cell wall and. The movement of chromosomes during mitosis is difficult to observe in yeasts, but a microtubule—centromere link must guide the chromosomes. In grape must, the duration of budding is approximately 1—2 hours. As a result, the population of the cells double during the yeast log phase during fermentation.

    Some transform into a kind of sac with a thick cell wall. These sacs are called asci. Each one contains four haploid ascospores issued from meiotic division of the nucleus. Grape must and wine are not propitious to yeast sporulation and, in principal, it never occurs in this medium. Yet Mortimer et al. Our researchers have often observed asci in old agar culture media stored for several weeks in the refrigerator or at ambient temperatures Figure 1.

    The natural conditions in which wild wine yeasts sporulate and the frequency of this phenomenon are not known. In the laboratory, the agar or liquid medium. Scanning electron microscope photograph of S. Asci containing ascospores can be observed. Wine yeasts, both wild and selected, do not sporulate easily, and when they do they often produce non-viable spores. Meiosis in yeasts and in higher eucaryotes Figure 1. Several hours after the transfer of diploid vegetative cells to a sporulation medium, the SPB splits during the DNA replication of the S phase.

    A dense body DB appears simultaneously in the nucleus near the nucleolus. The DB evolves into synaptonemal complexes—structures permitting the coupling and recombination of homologous chromosomes. After 8—9 hours in the sporulation medium, the two SPB separate and the spindle begins to form. This stage is called metaphase I of meiosis.

    At this stage, the chromosomes are not yet visible. Then, while the nuclear membrane remains intact, the SPB divides. At metaphase II, a second mitotic spindle stretches itself while the ascospore cell wall begins to form. Nuclear buds, cytoplasm and organelles migrate into the ascospores. At this point, edification of the cell wall is completed.

    The spindle then disappears when the division is achieved. Placed in favorable conditions, i. Sign a MATa cells produce a sexual pheromone a. This peptide made up of 12 amino acids is called sexual factor a. Sexual coupling occurs between two cells of the opposite sexual sign. Meiosis in S. II of meiosis; h end of meiosis; formation of ascospores. Diploid cells appear in the descendants of an ascospore. They are homothallic and have an HO gene which inverses sexual sign at an elevated frequency during vegetative division.

    This changeover Figure 1. Some strains, from a monosporic culture, can be maintained in a stable haploid state. Their sexual sign remains constant during many generations. They are heterothallic. Others change sexual sign. Sexual sign commutation model of haploid yeast cells in a homothallic strain Herskowitz et al.

    During the following cellular division, S produces two cells S and F2 that have become a cells. Laboratory strains that are deficient or missing the HO gene have a stable sexual sign. Heterothallism can therefore be considered the result of a mutation of the HO gene or of genes that control its functioning Herskowitz et al. Most wild and selected winemaking strains that belong to the S.

    It is also true of almost all of the strains that have been isolated in vineyards of the Bordeaux region. Moreover, recent studies carried out by Mortimer et al. We have made the same. In other words, the four spores issued from an ascus give monoparent diploids, capable of forming asci when placed in a pure culture. This generalized homozygosis for the HO character of wild winemaking strains is probably an important factor in their evolution, according to the genome renewal phenomenon proposed by Mortimer et al. Certain slow-growth or functional loss mutations of certain genes decrease strain vigor in the heterozygous state.

    Sporulation, however, produces haploid cells containing different combinations of these heterozygotic characters. All of these spores become homozygous diploid cells with a series of genotypes because of the homozygosity of the HO character. Certain diploids which prove to be more vigorous than others will in time supplant the parents and less vigorous ones. This very Homothallisme. Genome renewal of a homozygote yeast strain for the HO gene of homothallism, having accumulated recessive mutations during vegetative multiplication Mortimer et al.

    In these, the spore viability rate is the inverse function of the heterozygosis rate for a certain number of mutations. The completely homozygous strains present the highest spore viability and vigor. In conclusion, sporulation of strains in natural conditions seems indispensable. It assures their growth and fermentation performance. With this in mind, the conservation of selected strains of active dry yeasts as yeast starters should be questioned.

    It may be necessary to regenerate them periodically to eliminate possible mutations from their genome which could diminish their vigor. The killer strains are not sensitive to their toxin but can be killed by a toxin that they do not produce. Neutral strains N do not produce a toxin but are resistant. The action of a killer strain on a sensitive strain is easy to demonstrate in the laboratory on an agar culture medium at pH 4.

    The sensitive strain is inoculated into the mass of agar before it solidifies; then the strain to be tested is inoculated in streaks on the solidified medium. If it is a killer strain, a clear zone in which the sensitive strain cannot grow encircles the inoculum streaks Figure 1. This phenomenon, the killer factor, was discovered in S. Killer yeasts have been classified into 11 groups according to the sensitivity reaction between strains as well as the nature and properties of the toxins involved.

    The killer factor is a cellular interaction model mediated by the proteinic toxin excreted. It has given rise to much fundamental research Tipper and Bostian, ; Young, Barre , , Radler and Van Vuuren and. Identification of the K2 killer phenotype in S. The presence of a halo around the two streaks of the killer strain is due to the death of the sensitive strain cultivated on the medium. Jacobs have described the technological implications of this phenomenon for wine yeasts and the fermentation process.

    They are in the same category as non-infectious mycovirus. The M genome 1. The L genome 4. There are three kinds of killer activities in S. According to Wingfield et al. The K2 strains are by far the most widespread in the S. They possess M VLPs of normal dimensions that code only for the immunity factor. They either do not produce toxins or are inactive because of mutations affecting the M-type RNA. Many chromosomic genes are involved in the maintenance and replication of L and M RNA particles as well as in the maturation and transport of the toxin produced.

    The K1 toxin is a small protein made up of two sub-units 9 and 9. It is active and stable in a very narrow pH range 4. The K2 toxin, a 16 kDa glycoprotein, produced by homothallic strains of S. It is therefore active at the pH of grape must and wine. The KRE2 gene is also involved in the fixation of toxins to the parietal receptor; the kre2 mutants are also resistant. The toxin linked to a glucan receptor is then transferred to a membrane receptor site by a mechanism needing energy.

    Cells in the log phase are, therefore, more sensitive to the killer effect than cells in the stationary phase. When the sensitive cell plasmic membrane is exposed to the toxin, it manifests serious functional alterations after a lag phase of about. These alterations include the interruption of the coupled transport of amino acids and protons, the acidification of the cellular contents, and potassium and ATP leakage.

    The cell dies in 2—3 hours after contact with the toxin because of the above damage, due to the formation of pores in the plasmic membrane. The killer effect exerts itself exclusively on yeasts and has no effect on humans and animals. In a work by Barre studying wild strains, manifested the K2 killer character, were sensitive and 95 neutral.

    In the Bordeaux region, the K2 killer character is extremely widespread. In a study carried out in and on the ecology of indigenous strains of S. Rossini et al. Some K2 killer strains were also isolated in the southern hemisphere Australia, South Africa and Brazil. On the other hand, most of the killer strains isolated in Japan presented the K1 characteristic.

    Most research on the killer character of wine yeasts concerns the species S. Little information exists on the killer effect of the alcohol-sensitive species which essentially make up grape microflora. However, some killer strains of Hanseniaspora uvarum and Pichia kluyveri have been identified by Zorg et al.

    Handbook of Enology, Vol 1 The Microbiology of Wine and Vinifications

    Cytology, Taxonomy and Ecology of Grape and Wine Yeasts Barre studied the activity and stability of the K2 killer toxin in enological conditions Figure 1. The killer toxin only manifested a pronounced activity on cells in the log phase. Cells in the stationary phase were relatively insensitive. The amount of ethanol or SO2 in the wine has It is also quickly inactivated by the presence of phenolic compounds and is easily adsorbed by bentonite. Scientific literature has reported a diversity of findings on the role of the killer factor in the competition between strains during grape must fermentation.

    This considerable discrepancy can probably be attributed to implantation and fermentation speed of the strains present. The killer phenomenon seems more important to interstrain competition when the killer strain implants itself quickly and the sensitive strain slowly. In the opposite situation, an elevated percentage of killer yeasts would be necessary to eliminate the sensitive population. In Bordeaux, we have always observed that certain sensitive strains implant themselves in red wine fermentation, despite a strong presence of killer yeasts in the wild microflora for example, M, an active dry yeast starter.

    In white winemaking, the neutral yeast VL1 and sensitive strains such as EG8, a slow-growth strain, also successfully implant themselves. The wild killer population does not appear to compete with a sensitive yeast starter and therefore is not an important cause of fermentation difficulties in real-life applications.