CHAPTER 1 INTRODUCTION TO WINE

TABLE OF CONTENTS TABLE OF CONTENTS i LIST OF TABLES ii LIST OF FIGURES ii CHAPTER INTRODUCTION TO WINE 1.1 History of wine 1.2 Classification of wine 1.3 Wine making process 1.3.1 Harvesting 1.3.2 Crushing and pressing 1.3.3 Fermentation 1.3.4 Clarification 1.3.5 Aging and bottling CHAPTER YEASTS IN WINEMAKING 2.1 Sacchromyces cerevisiae 2.1.1 Taxonom: Morphology and special characteristics 2.1.2 Nutrition and growth 2.1.3 Life cycle and reproduction 2.2 Spoilage yeast strains 10 CHAPTER 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 Page | i LIST OF TABLES Table Wine classification based on five different standards Table 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 Some images of yeast: S cerevisiae TBS (a&b) and S cerevisiae TNS (c&d) Figure Structure of S cerevisiae cell Figure Reproductive cycle Saccharomyces cerevisiae Page | ii CHAPTER 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 Page | 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 below briefly illustrates how wines are classified based on five different standards Table Wine classification based on five different standards Red wine According to color According to the CO2 pressure 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 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℃ 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 sugar content Light-bodied wine According to the wine body Medium-bodied Wine Full-bodied Wine Ordinary Wine lighter in color and have fewer tannins darker and have more texture on the tongue deepest color and abundant tannins 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 According to the grape harvest time Page | 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 – 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 Page | of glucose (or fructose) yields two molecules of ethanol as well as carbon dioxide The net equation for this reaction is: Hexose + ADP → Ethanol + CO2 + 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 82oC can be used to preciptate proteins that cause clouding by reacting with copper or other metals Page | 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 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 Page | CHAPTER 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 (a) (b) (c) (d) Figure Some images of yeast: S cerevisiae TBS (a&b) and S cerevisiae TNS (c&d) Page | 2.1.1.2 Typical characteristics of cell structures Figure 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 Page | 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 Page | and starches into carbon dioxide and ethanol Yeast cells contain almost all substances necessary for life, such as proteins, carbohydrates, lipids, enzymes, vitamins, amino acids, minerals Sulfur is also present in proteins, as well as coenzyme A If the lack of sulfur happens, it will damage the metabolism and synthesis of enzymes and proteins Under anaerobic conditions, sulfur is reduced to H2S Aerobic Respiration: C6H12O6 (sugar) + O2 → CO2 + H2O + energy Anaerobic Respiration: C6H12O6 (sugar) → C2H5OH (alcohol) + CO2 + energy 2.1.3 Life cycle and reproduction There are two types of yeast cells during this life cycle: haploid and diploid Each haploid cell only replicates by budding under ideal conditions If these mating types come into contact with each other, gametophytes will form and sexual reproduction will begin Furthermore, the zygote multiplies via budding, resulting in the formation of many diploid cells, which are larger than haploid cells These huge diploid cells, like haploid cells, are self-contained and replicate through budding Under the unfavorable condition, the diploid big cell turns spherical and acts as the parent cell Meiosis separates the parent cell's nucleus into haploid nuclei Each nucleus collects cytoplasm and forms spores Each ascospore is a spherical, thick-walled structure made up of ascospore septa compartments Ascospores create haploid dwarf cells when they germinate Figure Reproductive cycle Saccharomyces cerevisiae (1): Asexual reproduction (budding) (2): Sexual reproduction (3): The process of cyst formation containing spores We can show clearly from the illustrated cycle, when exposed to severe conditions, such as nutritional depletion, diploid cells can perform meiosis and create four haploid spores, each consisting of two (a) spores and two (α) spores To form a diploid cell, haploid cells can combine with other haploid cells of the opposite mating type (a cell can only mate with α cell, and opposite) Next, the cytoplasm of the haploid cells is fused, and the haploid cells are fertilized, resulting in a diploid zygote Then, the zygote can go through meiosis and generate an ascus, which splits into four ascospores Finally, these haploids can germinate and become haploid cells once more Page | 2.2 Spoilage yeast strains Brettanomyces bruxellensis This type of yeast is commonly found in wineries and oak barrels used for aging that can be considered as a contaminant for wine production It is because the growth of the yeast can lead to volatile phenolcontaining compounds, which give the wine an undesired odor, variously described as “barnyard”, “horse sweat”, “band-aid” A signature chemical of this yeast is 4-ethyl phenol Kloeckera apiculata Kloeckera apiculata is one of the yeasts involved in the early stages of natural fermentation It can produce high enough levels of various esters (mainly ethyl acetate and methyl butyl acetate) to cause an ester taint – a vinegar-like aroma Other types Table shows some of the most common yeasts often found in grape, musts and wines that can be considered spoilage yeast species in a wide range of food products Table Some common yeasts in grape, musts and wines that can be considered spoilage yeast species in a wide range of food products Yeast species Food product Product with 50% sugar Spoilage compounds Alcohol, esters Mould-ripened soft cheeses Fruit juices, sauces, carbonated soft drinks, salad dressings, ketchup Alcohol, esters 4-ethylphenol, Brettanomyces bruxellensis Gas production: bubbling and package expansion Refermentation and CO2 production Sweet wines Zygosaccharomyces rouxii Effect observed Bulk, barrel matured and bottled wines 4-ethylguaiacol, Acetic acid, Tetrahydropyrindines Gas production: bubbling and package expansion Off aromas, cloudiness formation in sparkling wine, mousy aroma Unpleasant mousy and medicinal taints Food products with SO2 as antiseptic Saccharomycodes Spoilage by sediment or cloudiness formation Bottle wines Wine High acetoin level Flocculent sediment Page | 10 CHAPTER FACTORS THAT AFFECT THE WINE FERMENTATION Yeast requires certain conditions to ferment In the production process, in addition to selecting yeast strains, it is also necessary to study to create the most suitable conditions to achieve high fermentation efficiency 3.1 Temperature Depending on the floating or sinking yeast, adjust the ambient temperature accordingly For floating yeast, the suitable temperature is from 20-28oC; for submerged yeast, the suitable temperature is from 5-10oC In addition, at a temperature of 28-30oC, the alcohol evaporates, making the fermentation process happen faster, and at a temperature of 50oC onwards and below 0oC, the yeast is inactive Yeast is greatly affected by temperature: too cold and they go dormant, too hot and they indulge in an orgy of fermentation that often cannot be cleaned up by conditioning High temperatures encourage the production of fusel alcohols - heavier alcohols that can have harsh solvent-like flavors 3.2 Sugar concentration All yeasts are only capable of fermenting simple sugars such as monosaccharides (glucose, fructose) and disaccharides (maltose, sucrose), except for lactose, which can only be used by Saccharomyces lactic acid yeast Yeast is completely incapable of hydrolyzing polysaccharides When the sugar concentration is greater than 30%, alcohol fermentation will be inhibited Depending on the product of the fermentation, an appropriate concentration of sugar is used In alcohol production, people use a sugar concentration of 14-20%, the fermentation process is strong and the sugar is exhausted, then it is distilled to obtain alcohol For wine, people use a sugar concentration of 16-25% and use submerged yeast, so the fermentation is slow and after fermentation, there is still some sugar in the wine, so the wine usually has a sweet taste In beer production, the sugar concentration is usually 9-12% 3.3 pH pH plays an important role in the fermentation process Yeast can grow at a pH of - but is most suitable between - 4.5 Bacteria begin to grow at pH = 4.2 and higher, below this level only yeast can grow Therefore, during the fermentation process, pH should be adjusted to less than When pH = yeasts grow very poorly, on the contrary, bacteria grow very strongly At pH = 3.8 yeast grows very strongly, almost all bacteria have not developed In order to create an appropriate pH in the yeast culture medium, it is common to add an acid to the fermentation medium that does not affect the yeast's activity People often use citric acid to adjust pH Page | 11 Depending on the product obtained, the pH of the environment is adjusted accordingly The pH of the medium is acidic, the product obtained is ethyl alcohol If the pH of the medium is weak, the products are ethyl alcohol and glycerin, and if the pH of the medium is weak, the products are ethyl alcohol, acetic acid, and glycerol 3.4 Oxygen Oxygen is an important component in the growth of yeast cell biomass However, it is the cause of product damage in subsequent stages Only a small amount of oxygen is needed in the first stage, when the fermentation broth has reached the number of yeast cells, it prevents the fermentation liquid from contacting oxygen so that the yeast can carry out the fermentation process to convert sugar into alcohol and CO The creation of biological components, including sterols, unsaturated fatty acids, and structural constituents in numerous organic molecules, requires oxygen The yeast does not require oxygen for energy synthesis under winemaking conditions, but it does require a large amount of free oxygen for effective development Because of inhibition of fatty acid and sterol biosynthesis in yeast, a decrease in oxygen availability causes a drop in biomass production and the rate of glycolysis To speed up their growth, the yeast takes advantage of any available oxygen in the wort They can adapt and grow in the absence of oxygen using other mechanisms, but oxygen allows them to so far more efficiently The yeast should complete the adaptation phase and start primary fermentation within 12 hours under normal conditions Yeast is a facultative respirator Under aerobic conditions in the presence of oxygen, the following reaction will occur: C6H12O6 +6O2 → 6H2O + 6CO2 +Q1 ➔ increase biomass Only under anaerobic conditions does it proceed to alcoholic fermentation according to the equation: C6H12O6 → 2C2H5OH + 2CO2 + Q2 Therefore, in the presence of oxygen, alcohol fermentation will be inhibited Pasteur effect: is the inhibition of alcohol fermentation in the presence of oxygen The transition from fermentation to respiration, in addition to reducing the efficiency of alcohol and carbon dioxide production, also reduces the efficiency of sugar use Therefore, the first stage of fermentation requires air into the medium to stimulate yeast growth, then oxygen is not required to create anaerobic conditions for the most efficient alcoholic fermentation Page | 12 3.5 Wine concentration and carbon dioxide (CO2) Alcohol accumulates in the fermenter and CO2 inhibits the growth and fermentability of yeast Yeast growth is slowed down when the alcohol concentration in the fermentation medium is 1%, from to 6% has an adverse effect Most yeasts ferment at an alcohol concentration of 12-14%, only a few ferments at an alcohol concentration of 17-20% If the alcohol concentration is too high, all yeast will be inhibited The alcohol tolerance of yeast is the alcohol concentration that inhibits the growth and activity of yeast after 72 hours of culture at 30oC CO2 inhibits fermentation, but the release of CO2 has a beneficial effect on fermentation 3.6 Starter culture The number of yeast cells added to the yeast juice greatly affects the fermentation process If the number of yeast cells is appropriate, then the fermentation process goes well and the recovery efficiency is high, the product quality is better If the number of yeast cells is too small, the fermentation rate is slow If the biomass of yeast cells is too much, the fermentation medium is not enough for the yeast to grow, the yeast cells will die gradually, the product produces a strange taste, and a significant amount of yeast is wasted Page | 13 REFERENCES Introduction to wine [1] Amerine, M A (2020, April 2) The wine-making process Encyclopedia Britannica https://www.britannica.com/topic/wine/The-wine-making-process [2] Andrew, W L., Gavin, S L., & David, J W (2016) Glycolysis Understanding Wine Chemistry, 22(1), 195–204 https://doi.org/10.1002/9781118730720.ch22a [3] Boulton, R B (1996) 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