wine fermentation experiment

EXPERIMENT NO. 12

Wine fermentation, table of contents, introduction, use of a hydrometer, list of reagents and instruments, a. equipment, b. reagents.

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Yeast Fermentation and the Making of Beer and Wine

Once upon a time, many, many years ago, a man found a closed fruit jar containing a honeybee. When he drank the contents, he tasted a new, strange flavor. Suddenly his head was spinning, he laughed for no reason, and he felt powerful. He drank all the liquid in the jar. The next day he experienced an awful feeling. He had a headache, pain , an unpleasant taste in his mouth, and dizziness — he had just discovered the hangover. You might think this is just a tale, but is it? Several archaeological excavations have discovered jars containing the remains of wine that are 7,000 years old (McGovern, 2009), and it is very likely that humankind's first encounter with alcoholic beverages was by chance. How did this chance discovery lead to the development of the beer and wine industry (Figure 1), and how did scientists eventually learn about the biological mechanisms of alcohol production?

The History of Beer and Wine Production

Over the course of human history, and using a system of trial, error, and careful observation, different cultures began producing fermented beverages. Mead, or honey wine, was produced in Asia during the Vedic period (around 1700–1100 BC), and the Greeks, Celts, Saxons, and Vikings also produced this beverage. In Egypt, Babylon, Rome, and China, people produced wine from grapes and beer from malted barley. In South America, people produced chicha from grains or fruits, mainly maize; while in North America, people made octli (now known as "pulque") from agave, a type of cactus (Godoy et al. 2003).

At the time, people knew that leaving fruits and grains in covered containers for a long time produced wine and beer, but no one fully understood why the recipe worked. The process was named fermentation, from the Latin word fervere , which means "to boil." The name came from the observation that mixtures of crushed grapes kept in large vessels produced bubbles, as though they were boiling. Producing fermented beverages was tricky. If the mixture did not stand long enough, the product contained no alcohol; but if left for too long, the mixture rotted and was undrinkable. Through empirical observation, people learned that temperature and air exposure are key to the fermentation process.

Wine producers traditionally used their feet to soften and grind the grapes before leaving the mixture to stand in buckets. In so doing, they transferred microorganisms from their feet into the mixture. At the time, no one knew that the alcohol produced during fermentation was produced because of one of these microorganisms — a tiny, one-celled eukaryotic fungus that is invisible to the naked eye: yeast . It took several hundred years before quality lenses and microscopes revolutionized science and allowed researchers to observe these microorganisms.

Yeast and Fermentation

Figure 1: Fermented beverages such as wine have been produced by different human cultures for centuries. Christian Draghici/Shutterstock. All rights reserved. In the seventeenth century, a Dutch tradesman named Antoni van Leeuwenhoek developed high-quality lenses and was able to observe yeast for the first time. In his spare time Leeuwenhoek used his lenses to observe and record detailed drawings of everything he could, including very tiny objects, like protozoa, bacteria , and yeast. Leeuwenhoek discovered that yeast consist of globules floating in a fluid, but he thought they were merely the starchy particles of the grain from which the wort (liquid obtained from the brewing of whiskey and beer) was made (Huxley 1894). Later, in 1755, yeast were defined in the Dictionary of the English Language by Samuel Johnson as "the ferment put into drink to make it work; and into bread to lighten and swell it." At the time, nobody believed that yeast were alive; they were seen as just organic chemical agents required for fermentation.

In the eighteenth and nineteenth centuries, chemists worked hard to decipher the nature of alcoholic fermentation through analytical chemistry and chemical nomenclature. In 1789, the French chemist Antoine Lavoisier was working on basic theoretical questions about the transformations of substances. In his quest, he decided to use sugars for his experiments, and he gained new knowledge about their structures and chemical reactions. Using quantitative studies, he learned that sugars are composed of a mixture of hydrogen, charcoal (carbon), and oxygen.

Lavoisier was also interested in analyzing the mechanism by which sugarcane is transformed into alcohol and carbon dioxide during fermentation. He estimated the proportions of sugars and water at the beginning of the chemical reaction and compared them with the alcohol and carbon dioxide proportions obtained at the end. For the alcoholic reaction to proceed, he also added yeast paste (or "ferment," as it was called). He concluded that sugars were broken down through two chemical pathways: Two-thirds of the sugars were reduced to form alcohol, and the other third were oxidized to form carbon dioxide (the source of the bubbles observed during fermentation). Lavoisier predicted (according to his famous conservation-of-mass principle) that if it was possible to combine alcohol and carbon dioxide in the right proportions, the resulting product would be sugar. The experiment provided a clear insight into the basic chemical reactions needed to produce alcohol. However, there was one problem: Where did the yeast fit into the reaction? The chemists hypothesized that the yeast initiated alcoholic fermentation but did not take part in the reaction. They assumed that the yeast remained unchanged throughout the chemical reactions.

Yeast Are Microorganisms

In 1815 the French chemist Joseph-Louis Gay-Lussac made some interesting observations about yeast. Gay-Lussac was experimenting with a method developed by Nicolas Appert, a confectioner and cooker, for preventing perishable food from rotting. Gay-Lussac was interested in using the method to maintain grape juice wort in an unfermented state for an indefinite time. The method consisted of boiling the wort in a vessel, and then tightly closing the vessel containing the boiling fluid to avoid exposure to air. With this method, the grape juice remained unfermented for long periods as long as the vessel was kept closed. However, if yeast (ferment) was introduced into the wort after the liquid cooled, the wort would begin to ferment. There was now no doubt that yeast were indispensable for alcoholic fermentation. But what role did they play in the process?

When more powerful microscopes were developed, the nature of yeast came to be better understood. In 1835, Charles Cagniard de la Tour, a French inventor, observed that during alcoholic fermentation yeast multiply by gemmation (budding). His observation confirmed that yeast are one-celled organisms and suggested that they were closely related to the fermentation process. Around the same time, Theodor Schwann, Friedrich Kützing, and Christian Erxleben independently concluded that "the globular, or oval, corpuscles which float so thickly in the yeast [ferment] as to make it muddy" were living organisms (Barnett 1998). The recognition that yeast are living entities and not merely organic residues changed the prevailing idea that fermentation was only a chemical process. This discovery paved the way to understand the role of yeast in fermentation.

Pasteur Demonstrates the Role of Yeast in Fermentation

Pasteur performed careful experiments and demonstrated that the end products of alcoholic fermentation are more numerous and complex than those initially reported by Lavoisier. Along with alcohol and carbon dioxide, there were also significant amounts of glycerin, succinic acid, and amylic alcohol (some of these molecules were optical isomers — a characteristic of many important molecules required for life). These observations suggested that fermentation was an organic process. To confirm his hypothesis, Pasteur reproduced fermentation under experimental conditions, and his results showed that fermentation and yeast multiplication occur in parallel. He realized that fermentation is a consequence of the yeast multiplication, and the yeast have to be alive for alcohol to be produced. Pasteur published his seminal results in a preliminary paper in 1857 and in a final version in 1860, which was titled "Mémoire sur la fermentation alcoolique" (Pasteur 1857).

In 1856, a man named Bigo sought Pasteur's help because he was having problems at his distillery, which produced alcohol from sugar beetroot fermentation. The contents of his fermentation containers were embittered, and instead of alcohol he was obtaining a substance similar to sour milk. Pasteur analyzed the chemical contents of the sour substance and found that it contained a substantial amount of lactic acid instead of alcohol. When he compared the sediments from different containers under the microscope, he noticed that large amounts of yeast were visible in samples from the containers in which alcoholic fermentation had occurred. In contrast, in the polluted containers, the ones containing lactic acid, he observed "much smaller cells than the yeast." Pasteur's finding showed that there are two types of fermentation: alcoholic and lactic acid. Alcoholic fermentation occurs by the action of yeast; lactic acid fermentation, by the action of bacteria.

Isolating the Cell's Chemical Machinery

By the end of the nineteenth century, Eduard Buchner had shown that fermentation could occur in yeast extracts free of cells, making it possible to study fermentation biochemistry in vitro . He prepared cell-free extracts by carefully grinding yeast cells with a pestle and mortar. The resulting moist mixture was put through a press to obtain a "juice" to which sugar was added. Using a microscope, Buchner confirmed that there were no living yeast cells in the extract.

Upon studying the cell-free extracts, Buchner detected zymase, the active constituent of the extracts that carries out fermentation. He realized that the chemical reactions responsible for fermentation were occurring inside the yeast. Today researchers know that zymase is a collection of enzymes (proteins that promote chemical reactions). Enzymes are part of the cellular machinery, and all of the chemical reactions that occur inside cells are catalyzed and modulated by enzymes. For his discoveries, Buchner was awarded the Nobel Prize in Chemistry in 1907 (Barnett 2000; Barnett & Lichtenthaler 2001; Encyclopaedia Britannica 2010).

Around 1929, Karl Lohmann, Yellapragada Subbarao, and Cirus Friske independently discovered an essential molecule called adenosine triphosphate ( ATP ) in animal tissues. ATP is a versatile molecule used by enzymes and other proteins in many cellular processes. It is required for many chemical reactions, such as sugar degradation and fermentation (Voet & Voet 2004). In 1941, Fritz Albert Lipmann proposed that ATP was the main energy transfer molecule in the cell.

Sugar Decomposition

Glycolysis — the metabolic pathway that converts glucose (a type of sugar) into pyruvate — is the first major step of fermentation or respiration in cells. It is an ancient metabolic pathway that probably developed about 3.5 billion years ago, when no oxygen was available in the environment . Glycolysis occurs not only in microorganisms, but in every living cell (Nelson & Cox 2008).

Because of its importance, glycolysis was the first metabolic pathway resolved by biochemists. The scientists studying glycolysis faced an enormous challenge as they figured out how many chemical reactions were involved, and the order in which these reactions took place. In glycolysis, a single molecule of glucose (with six carbon atoms) is transformed into two molecules of pyruvic acid (each with three carbon atoms).

In order to understand glycolysis, scientists began by analyzing and purifying the labile component of cell-free extracts, which Buchner called zymase. They also detected a low-molecular-weight, heat-stable molecule, later called cozymase. Using chemical analyses, they learned that zymase is a complex of several enzymes; and cozymase is a mixture of ATP, ADP (adenosine diphosphate, a hydrolyzed form of ATP), metals, and coenzymes (substances that combine with proteins to make them functional), such as NAD + (nicotinamide adenine dinucleotide). Both components were required for fermentation to occur.

The complete glycolytic pathway, which involves a sequence of ten chemical reactions, was elucidated around 1940. In glycolysis, two molecules of ATP are produced for each broken molecule of glucose. During glycolysis, two reduction-oxidation (redox) reactions occur. In a redox reaction, one molecule is oxidized by losing electrons, while the other molecule is reduced by gaining those electrons. A molecule called NADH acts as the electron carrier in glycolysis, and this molecule must be reconstituted to ensure continuity of the glycolysis pathway.

The Chemical Process of Fermentation

In the absence of oxygen (anoxygenic conditions), pyruvic acid can follow two different routes, depending on the type of cell . It can be converted into ethanol (alcohol) and carbon dioxide through the alcoholic fermentation pathway, or it can be converted into lactate through the lactic acid fermentation pathway (Figure 3).

Since Pasteur's work, several types of microorganisms (including yeast and some bacteria) have been used to break down pyruvic acid to produce ethanol in beer brewing and wine making. The other by-product of fermentation, carbon dioxide, is used in bread making and the production of carbonated beverages. Other living organisms (such as humans) metabolize pyruvic acid into lactate because they lack the enzymes needed for alcohol production, and in mammals lactate is recycled into glucose by the liver (Voet & Voet 2004).

Selecting Yeast in Beer Brewing and Wine Making

Humankind has benefited from fermentation products, but from the yeast's point of view, alcohol and carbon dioxide are just waste products. As yeast continues to grow and metabolize sugar, the accumulation of alcohol becomes toxic and eventually kills the cells (Gray 1941). Most yeast strains can tolerate an alcohol concentration of 10–15% before being killed. This is why the percentage of alcohol in wines and beers is typically in this concentration range. However, like humans, different strains of yeast can tolerate different amounts of alcohol. Therefore, brewers and wine makers can select different strains of yeast to produce different alcohol contents in their fermented beverages, which range from 5 percent to 21 percent of alcohol by volume. For beverages with higher concentrations of alcohol (like liquors), the fermented products must be distilled.

Today, beer brewing and wine making are huge, enormously profitable agricultural industries. These industries developed from ancient and empirical knowledge from many different cultures around the world. Today this ancient knowledge has been combined with basic scientific knowledge and applied toward modern production processes. These industries are the result of the laborious work of hundreds of scientists who were curious about how things work.

References and Recommended Reading

Barnett, J. A. A history of research on yeast 1: Work by chemists and biologists, 1789–1850. Yeast 14 , 1439–1451 (1998)

Barnett, J. A. A history of research on yeast 2: Louis Pasteur and his contemporaries, 1850–1880. Yeast 16 , 755–771 (2000)

Barnett, J. A. & Lichtenthaler, F. W. A history of research on yeast 3: Emil Fischer, Eduard Buchner and their contemporaries, 1880–1900. Yeast 18 , 363–388 (2001)

Encyclopaedia Britannica's Guide to the Nobel Prizes (2010)

Godoy, A., Herrera, T. & Ulloa, M. Más allá del pulque y el tepache: Las bebidas alcohólicas no destiladas indígenas de México. Mexico: UNAM, Instituto de Investigaciones Antropológicas, 2003

Gray, W. D. Studies on the alcohol tolerance of yeasts . Journal of Bacteriology 42 , 561–574 (1941)

Huxley, T. H. Popular Lectures and Addresses II . Chapter IV, Yeast (1871). Macmillan, 1894

Jacobs, J. Ethanol from sugar: What are the prospects for US sugar crops? Rural Cooperatives 73 (5) (2006)

McGovern, P. E. Uncorking the Past: The Quest for Wine, Beer, and Other Alcoholic Beverages. Berkeley: University of California Press, 2009

Nelson, D. L. & Cox, M. M. Lehninger Principles of Biochemistry , 5th ed. New York: Freeman, 2008

Pasteur, L. Mémoire sur la fermentation alcoolique .Comptes Rendus Séances de l'Academie des Sciences 45 , 913–916, 1032–1036 (1857)

Pasteur, L. Studies on Fermentation . London: Macmillan, 1876

Voet, D. & Voet, J. Biochemistry. Vol. 1, Biomolecules, Mechanisms of Enzyme Action, and Metabolism , 3rd ed. New York: Wiley, 2004

Classic papers:

Meyerhof, O. & Junowicz-Kocholaty, R. The equilibria of isomerase and aldolase, and the problem of the phosphorylation of glyceraldehyde phosphate . Journal of Biological Chemistry 149 , 71–92 (1943)

Meyerhof, O. The origin of the reaction of harden and young in cell-free alcoholic fermentation . Journal of Biological Chemistry 157 , 105–120 (1945)

Meyerhof, O. & Oesper, P. The mechanism of the oxidative reaction in fermentation . Journal of Biological Chemistry 170 , 1–22 (1947)

Pasteur, L. Mèmoire sur la fermentation appeleé lactique . Annales de Chimie et de Physique 3e. sér. 52 , 404–418 (1858)

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Winemaking at Home

By William C. Hurst, Extension Food Scientist

Legal Obligations

Ingredients.

• Winemaking Process

Wine Recipes

Winemaking supplies.

Wine can be made from any material capable of growing yeast. This process of active yeast growth on foodstuff is called alcoholic fermentation. The yeast feeds on the fruit sugar and converts it into ethyl alcohol and carbon dioxide gas. This process gradually depletes the sugar content as the percentage of alcohol increases. The distinct flavor and aroma of an individual wine comes from small quantities of other chemical substances produced during fermentation. In addition, the flavor and aroma are further enhanced by proper aging of the wine.

Fruits, berries and grapes readily support fermentation to produce wine. For this reason, they are the best sources to use in making wine. Many people, especially those having home fruit orchards, wild or cultivated grapes and berries growing on their property, have taken up home winemaking as a hobby. They have found it to be a challenging and rewarding diversion from the daily life routine.

This making of good quality wine involves more than just following granny’s recipes. Winemaking is a science and as such requires close attention to detail. Fermentation must be carefully controlled to avoid spoilage which renders the wine unpalatable. Cleanliness is essential in all phases of winemaking. If utensils are not thoroughly cleaned and sterilized, they may contaminate the wine. Ingredients, particularly the fruit, must be of high quality, free from decay and external contaminates.

The purpose in writing this bulletin is to provide essential material and detailed instructions for successfully making wine at home. The information described here is designed for beginners who do not know where to begin and for those experienced amateurs who frequently run into difficulties and disappointments.

IMPORTANT NOTE: Under federal law the head of a family is allowed to produce, for home consumption only, up to 200 gallons of wine annually without payment of tax. However, before starting to make beer one must file, in duplicate, copies of Form 1541 with the Internal Revenue Service Regional office. The address of the Southeast Regional office is:

Assistant Regional Commissioner Internal Revenue Service Alcohol and Tobacco Tax Division Federal Office Building 275 Peachtree Street Atlanta, Georgia 30303

The equipment required for making wine depends a great deal upon the quantity to be processed during a given period of time. On the average, the home wine maker will only produce from one to five gallons at a time. A list of equipment and other supplies needed to make wine at home follows:

A rolling pin, or food grinder, or food chopper is used to crush the fruit. Choppers with quarter-inch plates are ideal for crushing fruit without splitting the seeds.

A primary fermentor is a large (8 gallon) open container in which the ingredients are mixed and initial fermentation takes place. Food grade polyethylene containers are best to use because they are easy to clean and do not affect the taste of the wine. Glass or ceramic pots and stainless steel containers are heavy and difficult to handle. All other materials, especially aluminum and copper, should be avoided due to their toxicity. Wooden containers are acceptable but are difficult to keep clean.

A secondary fermentor is a smaller (1, 3 or 5 gallon) container having and opening only large enough to securely place a fermentation lock. Several sizes may be needed for wine making. These containers are made of heavy glass or high-density polyethylene. Fermenting juice is transferred to these containers one to seven days after the start of fermentation.

A fermentation lock is a device attached to a rubber stopper which is designed to fit into the secondary fermentor. It is important for the device to be secured so carbon dioxide gas can escape from the fermentor without letting in any air. Air contact can spoil the wine by oxidation or can contaminate the wine with acetic bacteria. These bacteria convert the alcohol in the fermenting juice to vinegar. Several commercially made locks are available or one can be made with a one-hole stopper, glass tubing, and a length of rubber or plastic tubing as follows: Insert the piece of glass tubing in the stopper. Place both in the small opening of the fermentor. Attach one end of the hose to the exposed glass tubing and submerge the other end in a mason jar filled with cold water.

A hydrometer is a simple and inexpensive instrument which takes the guesswork out of winemaking. It is used to measure the amount of sugar in the fruit juice prior to fermentation. It also tells when fermentation is completed. A hydrometer is essential for consistent results in home winemaking. Its use is further explained in this publication. The hydrometer and cylinder are purchased as a set from a scientific supply house.

A siphoning unit , 3/8-inch diameter rubber hose or plastic tubing which is five to six feet long, is commonly used to transfer wine from one container to another (racking), and finally into the bottles. The best flow rate is achieved when the upper container is about 18-inches above the lower container.

Campden tablets or potassium metabisulfite are used as a source of sulfur dioxide to check the growth of wild yeast and vinegar bacteria in the fermenting juice or finished wine. These agents also protect the color and flavor of the wine.

A wooden hand corker , or mallet, and size no. 9 cork are used in corking standard wine bottles. The corks must be sterilized before insertion. Submerge the corks in a solution containing two quarts hot water and ½ level teaspoon potassium metabisulfite or 4 Campden tablets for 15 minutes.

Rinse and soak them in fresh tap water an additional 15 minutes. Rinse and allow excess water to drain off, then drive the softened corks into filled bottles with a mallet or corker allowing a 3/8-inch headspace. After corking, store the bottles in an upright position for one week to allow time for the corks to harden. Then turn the bottles on their side to keep the corks moist and store the bottles at 60º F.

Standard wine bottles with screw tops or standard size champagne bottles with plastic corks are good for holding wine. These can be obtained at restaurants. It is best to use plastic bottles that are tinted since light affects the color of some wines. If tinted bottles are not available, use clear glass, but store them in a dark place.

Clean and sterilize used wine bottles with hot water and a weak solution of bleach (1 tablespoon bleach to a gallon of water). Screw caps and plastic corks are sterilized using potassium metabisulfite.

A pressing bag or straining cloth is needed to separate the fruit pulp solids from the liquid. A burlap or well-sewn canvas bag, 12-inches wide by 14-inches deep, is best for straining. However, having nothing better, cheesecloth or a diaper can be stretched over a “food grade” polyethylene bucket or stainless steel mixing bowl. Clothespins can be used to hold the straining cloth in place when the fruit pulp and juice are poured into the container. Remember to wash and sterilize straining bags and cloth before use.

Miscellaneous items which will be needed include: a large saucepan, a long-handled plastic spoon, a measuring cup and a set of measuring spoons, masking tape or pieces of rope, a funnel, extra cheesecloth, also gummed labels, a marking pen, a long-handled brush for cleaning, and bathroom or platform type scales for weighing juice.

The maturity and condition of the fruit being used for fermentation is very important in winemaking. Green fruit is generally high in acid and low in sugar content. Fruit of this type gives a tartness to wine, lacking a true fruity flavor. Fully ripe fruit is at its lowest acid and highest sugar content, and this is when fruit is at its best for winemaking, for it leaves a pleasant fruit flavor to the finished wine. Sound, firm fruit will contain most of the supplements necessary to complete the fermentation process. Over-ripe or bruised fruit has begun to mold and decay even though it may not be noticeable. If fruit of this type is used, a disagreeable flavor will result.

Flavor and taste of the finished wine is largely determined by the type of yeast used in fermentation. For best results, use only a true wine yeast such as Montrachet No. 522, which is available from wine supply houses. An acceptable second choice is Baker’s yeast. Yeasts are living organisms and as such require a balanced diet for proper development. The muscadine grape, when properly grown, is one of the few fruits containing all the necessary food supplements for yeast growth. Most other fruits are lacking in some part of the yeast requirements, even some wine grapes. A balanced yeast food known as nutrient must be added with the yeast at the start of fermentation. Yeast nutrient is commercially available at wine supply houses. Good substitutes for this nutrient include a pinch of ammonium phosphate (commercial fertilizer) or a small handful of minced raisins per gallon of crushed fruit.

Fruits vary a great deal in their acid content or tartness. Lack of acid gives wine an insipid taste while too much acid gives a sharp tang and can affect fermentation. Correct wine acidity brings out the full flavor of the fruit. Acid strength in the juice should be from one-half to three-fourths of one percent. Grapes are high in tartaric acid and usually the finished grape wine has this desired acidity. Some, such as apple wine, are low in acidity and require the addition of citric acid or some lemon juice to the fruit juice before fermentation begins to improve the flavor. Add approximately one tablespoon lemon juice per gallon strained juice or pulp.

Tannin gives wine its bite or astringency to overcome the greatest fault in wine, that of insipidness or flatness. Pears and grapes contain sufficient tannin in the skins and stems for winemaking, however, many other fruits including all white wines are lacking in tannin. To give wine its proper astringency, tannin must be added to these wines. Powdered tannin can be purchased commercially at wine supply houses with directions for its use. If tannin is not readily available, add several tablespoons of strong tea to the fermenting crushed fruit to improve the astringency of the finished wine.

The amount of sugar used controls the sweetness of the wine and at the same time is directly responsible for its alcohol content. Sugar, when fermented, gives one-half its amount of alcohol by volume. Thus, when completely fermented, a fruit having 22 percent sugar (fruit sugar plus added sugar) will give a wine with 11 percent alcohol. Wine containing less than 10 percent alcohol content will be thin and sour and will not keep. Under normal circumstances most yeasts have difficulty completing fermentation at sugar levels above 24 percent. Hence any sugar content in excel of 24 percent is going to remain in the finished product, resulting in sweet wine.

Sugar used in winemaking can be either ordinary household sugar or corn sugar also known as dextrose. Dextrose can be purchased at wine supply houses and is said to produce smoother wine when large amounts of sugar are needed. Sugar is best added to the raw juice in the form of sugar syrup, prepared by dissolving two cups sugar in one cup boiling water. One cup of sugar syrup is then equivalent to a 50 percent sugar solution. Adding several cups of sugar syrup every few days until the total sugar requirement has been met is the best method of keeping a good strong fermentation in progress.

Ordinarily, a wine of good alcoholic strength will clear itself if allowed to stand. But it sometimes happens, after several rackings, that a wine apparently good in other respects refuses to clear up properly. These wines may be artificially clarified using powdered kitchen gelatin. Measure out one level teaspoon gelatin for every five gallons wine. Add the gelatin to about a quart of drawn wine and dissolve by warming to 100º F while mixing well. Return this quart to the original bulk of wine and stir for 10 minutes, seal and let stand until wine clears. Since the gelatin settles tannin, along with other impurities, it is necessary to add tannin (amount equal to that of gelatin used) back to the wine.

The Winemaking Process

Step 1 — cleaning.

Clean all winemaking equipment and bottles thoroughly before starting an operation. Don’t use soap or detergents as their residues can impart an off-taste to the wine. Use hot water and a long-handled stiff brush to clean primary and secondary fermentors and bottles. To remove the industrial film off polyethylene, fill the container with hot water and add a pound of baking soda; then let set several hours before rinsing. After cleaning, swirl a weak bleach solution (1/4 cup bleach per gallon of water) in the containers to disinfect them. Use hot water to rinse through the siphon hose.

Step 2 – Preparing the Fruit

To begin, remove stems and unripe fruit. Trim or remove all rotten fruit. Rinse the fruit with cool water and allow to drain in a colander. Crush the fruit in order to free the juice for fermentation. Soft fruits such as berries and grapes can best be handled by spreading a thin layer in the bottom of a large flat sauce pan and crushing them with a rolling pin or potato masher. Much better results are obtained with solid fruits, such as apples, when they are crushed in a food chopper.

NOTE: If white wine is desired, the juice is immediately separated from the fruit skins and pulp using a pressing bag or straining cloth and transferred to the secondary fermentor. For red wine, transfer the entire contents (juice, pulp, skins) to the primary fermentor.

Step 3 – Determining Sugar Content

The sugar content present in the juice prior to fermentation must be measured in order to know the exact amount of sugar needed to increase the level to 22 percent. To measure sugar content with a hydrometer, fill the glass cylinder with a sample of strained juice and immerse the heavy end of the hydrometer in it. Put the jar on a level surface and let the hydrometer ride in the juice until it becomes perfectly still. When it comes to rest, away from the sides of the cylinder, read the value at the surface of the liquid (not the part of the liquid which has climbed up the side of the instrument). Record this figure immediately so it will not be forgotten! The temperature of the juice should be about 75º F when the reading is taken.

Step 4 – Sterilizing Fruit

Dissolve either four Campden tablets or one-half teaspoon potassium metabisulfate in a pint of strained juice. Add this back to either the primary fermentor containing crushed fruit or to the secondary fermentor containing strained juice. Mix thoroughly, cover the container with double layered cheesecloth, secure in place with a rope, and wait four hours before proceeding to Step 5.

Step 5 – Adding of Nutrients

For fruits, other than grapes, add sufficient tannin (tea), citric acid (lemon juice), yeast nutrient (raisins) and sugar syrup to either the crushed fruit or strained juice as called for in the recipe.

Step 6 – Activating Wine Yeast

Activate a package of wine yeast (or Baker’s yeast as the second choice) by dissolving it in a cup of strained juice. Add this to the crushed fruit or strained juice and mix well.

Step 7 – Extracting Color for Red Wine

Cover the primary fermentor containing the crushed fruit with double layered cheesecloth, secure in place with a rope, and store in a warm place (60-70º F) such as a cellar or basement. Fermentation should begin within 24 hours. By the second day, the pulp should be in strong ferment. The solids in the pulp will float to the surface to form a cap over the fermenting juice.

At least twice a day this cap should be stirred thoroughly into the juice, always replacing the cover. It is most important that the fermenting pulp and juice be protected from small vinegar flies and other insects by covering during this period. Allow the fruit pulp to ferment from three to seven days in order to extract the desired color from the skins.

Step 8 – Straining Fruit Pulp

Using a pressing bag or several layers of cheesecloth, squeeze or strain the solids from the fermenting juice into a large saucepan. It is best to strain off the clear liquid first by holding back the surface solids, then gather the remaining solids in the bag or cloth and squeeze dry by slowly applying pressure. Do not squeeze hard enough to force pulp through the cloth and into the juice. Transfer the contents to the secondary fermentor using a plastic funnel to avoid spillage.

Step 9 – Determining Sugar Needed

The amount of sugar required to raise the sugar level of the juice to 22 percent can be calculated by a formula called the Pearson Square. It is diagramed below.

An example of how to use this formula for making five gallons of wine is as follows:

A sample of strained peach juice, drawn from a bushel of freshly crushed peaches was found to contain 10 percent sugar (value read at liquid’s surface when tested with a hydrometer). After primary fermentation to extract color, 35 pounds of fermenting juice – determined by the difference between fermentor weight and fermentor weight + juice weight – were separated from the skins, seeds, and pulp. How much sugar (cups sugar syrup) should be added to the 35 pounds of juice to increase the initial sugar level of the juice from 10 to 22 percent?

• Place in the upper left-hand corner or a square the percent of sugar in the syrup. Syrup prepared at a rate of two cups sugar to one cup water contains 50 percent sugar.
• In the lower left-hand corner place the percent of sugar (10) in the strained juice.
• In the center of the square place the percent of sugar level (22) desired.
• Now subtract the center figure from the figure in the upper left-hand corner, and place the remainder in the lower right-hand corner (50 – 22 = 28).

Subtract the figure in the lower left-hand corner from the figure in the center and place the remainder in the upper right-hand corner (22-10 = 12).

This last figure (12) is the number of cups of a 50 percent sugar syrup which must be added to 28 pounds of juice to increase the sugar level from 10 to 22 percent. But in this example, there are 35 pounds of juice. Therefore, to determine the number of cups of syrup required, simply divide the total pounds of juice (35) by the number in the lower right-hand corner of the square (28) and multiply by the number in the upper right-hand corner (12). First, divide 35 by 28 (= 1.25), the multiply the quotient by 12 (1.25 x 12 = 15). The result (15) will be the number of cups of sugar syrup which must be added to 35 pounds of juice to increase the sugar level from 10 to 22 percent.

Step 10 – Fermenting Red Wine

Dissolve the sugar required in water at a rate of two cups sugar to one cup water. Sterilize by brining to a rolling boil for a few minutes before setting aside to cool to room temperature. Add several cups of the sugar syrup to the fermenting juice and swirl gently to distribute. Don’t add the entire sugar syrup solution to the fermenting juice because it may cause excessive bubble formation, resulting in overflow. Adding all the syrup may also stun or kill the yeast producing an inferior wine of low alcohol strength.

Next, seal the fermentor with a snug-fitting fermentation lock and set this container in a place having a constant temperature of between 60-70º F. Within a day or two, carbon dioxide bubbles will be seen steadily emerging from the fermentation lock, an indication that active fermentation has begun. Fermentation must be vigorous so that the heavier than air carbon dioxide gas produced will lie on top of the juice like a blanket to stop the yeast from obtaining oxygen from the air. Carefully remove the lock and add two cups sugar syrup, then quickly and securely replace the lock back in place on the fermentor. Gently swirl the container to dissolve the syrup. Repeat this process every four to five days or until the solution is entirely used. Fermentation will proceed about a month.

Step 11 – Fermenting White Wine

If fruit juice only had been used, there should be no solids to strain out and the liquid would have been placed into the secondary fermentor. While the strained juice is being sterilized, dissolve the sugar required in water, sterilize and cool as before. Follow the same recommendations for adding sugar as were stated for making red wine. Store the container at a temperature of between 60-70º F until fermentation almost ceases.

Step 12 – Racking the Wine

During the process of fermentation, a whitish, fine sediment, comprised mainly of dead yeast cells, is deposited on the bottom of the secondary fermentor. Racking is the process designed to separate the wine from this sediment and is performed by siphoning the wine from one container to another when fermentation has practically come to a stop. Using a chair or table, place the secondary fermentor 18 inches above the tope of a cleaned and sterilized five-gallon fermentor located on the floor. Insert a rubber hose approximately three-eights-inch in diameter and about five feet long into the secondary fermentor. Siphon the wine from this upper container into the lower one taking care not to stir up the sediment.

During this first racking, allow the wine to aerate slightly by falling from the middle of the container to the bottom. This oxygen is utilized by the remaining yeasts in the wine to complete the fermentation process. Add one-eighth-teaspoon potassium metabisulfite or one Campden tablet for every three gallons of siphoned wine to prevent spoilage. Replace the fermentation lock and wait a month before the next racking in order for fermentation to run to completion.

Some wines continue a very slow fermentation for several months and remain cloudy. They may require racking three or four times before they become sparkling clear and are ready for bottling. In each case, racking should be done without aeration, that is, the siphoning hose should be submerged below the liquid in the lower container to keep air out of the wine.

Step 13 – Clarifying the Wine – Optional

The chilling (30-40º F) of grape wine, after racking, prompts the natural clarification of this wine. Unwanted matter and suspended solids including potassium bitartrate, are precipitated as crystals in the bottom of the container. Other wines can be treated with gelatin if they will not clear under any circumstances. The gelatin combines with tannin present in the mix to form a milky colored solution. This will in turn settle slowly, dragging down any suspended materials to the bottom of the fermentor. It will take about a month for the wine to clarify, after which it is siphoned from the sediment. The disadvantage of using this treatment is that tannin, which gives wine its unique taste, is removed. It is necessary then to mix tannin back into the wine using approximately the weight of gelatin needed to initiate clarification.

Step 14 – Bottling the Wine

When fermentation has completely stopped, the wine should be sparkling clear and ready to bottle. Wine should be siphoned without aeration into bottles for aging. This limits the chance of off-flavor and off-color development due to oxidation.

Also, dissolve one-eighth-teaspoon potassium metabisulfite or one Campden tablet for every three gallons of wine in the fermentor prior to siphoning into bottles to prevent bacterial spoilage. An inexpensive hand corker, which is driven by a mallet, can be used to drive corks into wine bottles. In addition, a small corking machine operated by levers can be used to compress corks into the bottles. If screw-tops or plastic tops are used, cap the bottles loosely the first day of filling to allow residual gases to escape. Next day, tighten them in place.

Once wine has been bottled, each bottle should be labeled giving information as to its sweetness, flavor, clarity, etc. Also, a file or notebook should be kept on winemaking procedures so fine adjustments can be made from one year’s production to another in order to produce wines exactly as you would like to have them.

Step 15 – Storing the Wine

Wines age best at an even 60º F temperature. A clean, dry basement shielded from light is an ideal place for wine storage. Wine should be stored on its side to keep the cork moist and to prevent the entrance of spoilage bacteria. During aging, subtle changes in the flavor of wine take place contributing to each wine’s characteristic bouquet and appeal. Sedimentation may take place with aging. If sediment does occur, carefully decant the clear wine to a clean bottle before serving.

Wine can be consumed anytime after bottling; however, increased aging will mellow and bring out the unique flavors of the wine. For superior clarity and flavor, wine must be aged about a year before consumption.

A wine recipe can be nothing more than a basic guide to making wine. This is due to the variables that occur in the composition of a specific fruit when grown under varying climatic, soil, and seasonal conditions. Generally, hot, dry weather will produce more sugar in a fruit, while cool, damp weather tends to increase the acidity. In addition, the degree of fruit maturity affects its sugar-acid ration, riper fruit being higher in sugar content and green fruit being more acidic. These are some of the factors that must be considered in formulating an acceptable wine recipe.

Some selected fruit recipes which will make both dry and sweet wines follow. For consistent results in making good quality dry wines, a hydrometer should be used to measure critical sugar needs; much more sugar than the critical amount will produce a sweet wine. To prevent bacterial spoilage, wines must have 11 percent alcohol content. If a sweet wine is desired, a simple method for determining sweetness is to taste the wine when fermentation stops; then add enough sugar syrup to make the wine as sweet as desired prior to racking.

To purchase winemaking supplies, use your favorite Internet search engine, such as www.google.com , to locate a supplier near you or contact your local Extension office.

Trade and brand names are used only for information. The Georgia Cooperative Extension, The University of Georgia College of Agricultural and Environmental Sciences does not guarantee nor warrant the standard of any product mentioned, neither does it imply approval of any product to the exclusion of others which may also be suitable.

Beadle, L.P., Making Fine Wines and Liquers at Home . 1 st Ed., The NoonDay Press, New York, 1972.

Carrol, D.E., “Making Muscadine Table Wine” , Department of Food Science, North Carolina State University, Raleigh, NC, 1972.

Flora, L.F., “Home Winemaking with Muscadine Grapes” , Department of Food Science, Georgia Experiment Station, Experiment, Georgia, 1976.

Slater, L.G., The Secrets of Making Wine From Fruits and Berries . 1 st Ed., Terry Publishing Company, Lilliways, Washington, 1965.

Appreciation is expressed to Bill Rosser for his assistance in developing photographs. The author is also grateful to Dr. James A. Christian, Head, Extension Food Science Department and Dr. C. J. B. Smit, Chairman, Food Science Division for their valuable comments in the review of this publication.

Status and Revision History In Review on Feb 04, 2009 Published on Feb 25, 2010 Published with Full Review on Jan 18, 2013

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Picking the grape is just the beginning of making great wine Image adapted from: Karsten Wurth, CC0

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The chemistry of wine: Part 2 Fermentation

When it comes to wine, grapes are just the beginning.

The chemistry doesn’t stop once the grape is picked . In fact it’s just getting started.

The way in which the grape is crushed and pressed can release different compounds which affect the final flavour of the wine. Crushing breaks the grape apart and allows the juice-containing pulp and seeds to mingle with the skin and stems. Since the seeds and stems contain the more bitter-tasting components, de-stemming grapes prior to crushing, or ensuring gentle crushing to leave the seeds intact, can help to stop some of the bitter flavours developing in the wine.

If creating a white wine, the grape juice is usually pressed away from the skins and seeds. For red wines, the juice remains with the skins, since it’s the skins that contain the colour pigment.

The next step is fermentation.

Winemakers have two options here. They can wait for the naturally occurring yeasts already present on the grapes and winery equipment to begin fermentation, a process that is called spontaneous or ‘wild’ fermentation, or they can add commercial yeasts to the mix. The latter are popular because they begin the fermentation more quickly and help winemakers control the style and quality of the wine they produce. The most common commercial yeast used is Saccharomyces cerevisiae , which is also used in bread making and brewing.

Once yeasts have been added, they get to work converting the available sugars in the juice into alcohol and carbon dioxide, with at least 12 core enzymes involved in this process.

Fermentation is the key biochemical process in which the sugars are converted to alcohol, and literally hundreds of different, complex chemical compounds are also formed. These compounds create the seemingly endless variety of flavours or ‘notes’ that you read about on the label, including chocolate, pepper, butter, spice, smoke, citrus … even bubble-gum.

These flavours don’t mean that the wine actually contains chocolate or pepper or butter, such additions are generally not made or permitted. But, it may share some of the same molecules, or similar molecular arrangements, to those scents you’re familiar with, and which your nose and brain can categorise. These compounds can come from the grapes, the yeast, or even the wooden barrels used for ageing.

For example, fruity notes can come from esters produced by yeast, while the buttery flavour used to describe many chardonnay wines comes from a compound called diacetyl—a typical by-product of the microbial activity in winemaking. Floral notes in muscat or riesling are due to monoterpenes, and the small fruit or violet aromas found in some pinot noir come from norisoprenoids. Vanilla flavours can be the result of wines being aged in oak, as chemicals from the wood are transferred to the wine. To learn more about how the chemistry of wine continues to evolve through ageing and storing, read the next article in our series .

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Advances in wine fermentation.

1. Wine Fermentation: New Approaches for a Traditional Process

1.1. saccharomyces cerevisiae is not the unique microorganism in wine fermentation, 1.2. genetic modification of yeasts and vines, 1.3. micro-oxygenation, 1.4. low-alcohol wine production, 1.5. other types of fermentation in winemaking, 1.5.1. malolactic fermentation, 1.5.2. bottle fermentation, 1.5.3. carbonic maceration, 1.6. vessels, 1.7. turning wine waste into fuel, conflicts of interest.

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Share and Cite

Maicas, S. Advances in Wine Fermentation. Fermentation 2021 , 7 , 187. https://doi.org/10.3390/fermentation7030187

Maicas S. Advances in Wine Fermentation. Fermentation . 2021; 7(3):187. https://doi.org/10.3390/fermentation7030187

Maicas, Sergi. 2021. "Advances in Wine Fermentation" Fermentation 7, no. 3: 187. https://doi.org/10.3390/fermentation7030187

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How Does Wine Fermentation Work ?

by Chris Russell, Contributor

Fermentation is the process by which grape “must” (a fancy winemaking term for unfermented grapes or juice) transforms into wine. During fermentation, yeast—our microbiological friends—convert grape sugars into alcohol. There’s a lot more than just alcohol production going on, though. Fermentation drives complex chemical reactions that affect the flavor, aroma, and even color of the finished wine.

Not Just Alcohol

At its simplest, fermentation is often described as the conversion of one molecule of glucose into two molecules each of ethanol (or ethyl alcohol) and carbon dioxide: C 6 H 12 O 6 → 2C 2 H 5 OH + 2CO 2 . While that’s arguably the most important result, yeast are complex organisms that perform a wide array of biochemical processes in a fermenting wine.

Some of the compounds produced or affected by fermentation include:

• Esters : Esters are aromatic compounds that contribute delicate fruity, citrusy, or floral aromas to a young wine. They exist in must as precursors that are bound to sugar molecules. As the yeast consume the sugar, the esters are liberated and become volatile.
• Tannins : Present naturally in grape skins and seeds , tannins are antioxidant polyphenols that give wines dryness, astringency, and mouthfeel. The alcohol produced during fermentation enhances tannin extraction, while fermentation byproducts react with tannins, altering their structure, and, in turn, their perceived levels of astringency and bitterness.
• Acetaldehyde : Created by yeast in the penultimate step on the pathway to ethanol, low levels of acetaldehyde can enhance fruity aromas in wine. In high concentrations, acetaldehyde may yield unwanted bruised apple–like aromas and flavors. Acetaldehyde’s ability to catalyze tannin polymerization plays an instrumental role in the stabilization of red wine structure and mouthfeel.
• Anthocyanins : This highly reactive family of compounds in red grape skins gives red wine its color and antioxidative properties. These compounds polymerize in the presence of acetaldehyde to form a vast array of stable color components.
• Sulfites : As with ethanol, yeast produce sulfites during fermentation to fend off competition from other microorganisms. These natural sulfites can somewhat protect the wine from microbial spoilage and premature oxidation, but their levels are typically bolstered by winemakers after fermentation.
• Amino acids : Unfermented grape juice, or must, is rich in nitrogen-containing amino acids. Yeast consume most of these amino acids during fermentation, using the nitrogen to construct proteins and amino acids necessary to live and reproduce. Amino acids are the most important family of compounds in yeast nutrition and health.

From Many to One

The sweet, nutrient-rich must is an ideal medium for growing diverse species of yeast during the fermentation process. Naturally present yeast may include the familiar Saccharomyces , found in bread and beer, as well as more exotic genera such as Candida , Kloeckera , and Hansenula . As a result, the beginning of fermentation involves a lot of biodiversity, with many different types of yeast competing for resources. If allowed to ferment, each type of yeast leaves behind its own particular signature of flavor and aroma compounds.

Since not all yeasts are suitable for making wine, many wineries employ sulfites to suppress the activity of wild yeasts before fermentation, followed by inoculation with a commercially developed, cultured strain of Saccharomyces yeast. While this usually yields a predictable fermentation dominated by one particular variety of yeast, it doesn’t leave a lot of room for the natural microbiological diversity of the vineyard to shine through.

At 2Hawk, we prefer to take a gentler approach, encouraging the growth of desirable yeast found in our vineyard. These naturally occurring yeasts contribute a subtle, nuanced complexity to the finished wine that reflects the unique character of the Rogue Valley and our vineyard in particular.

As fermentation progresses, some species of yeast begin to rapidly convert the natural sugars present in the must into carbon dioxide and ethanol, or ethyl alcohol. Produced as a defense mechanism, few species of yeast can tolerate even moderate levels of ethanol. At around 4–5% ABV, or alcohol by volume, many of the yeast species present at the start of fermentation—like the Candida mentioned earlier—die off. As ethanol levels continue to rise, one strain— Saccharomyces— emerges as the victor of this fierce competition and embarks on fermenting the wine to dryness.

Influencing Fermentation

Various types of yeast make their own individual contributions to a fermenting wine, but fermentation is influenced by other factors as well.

Sugar Content

The higher the initial sugar content of the must, the more alcohol will be present in the finished wine—if allowed to ferment to dryness. More alcohol means a tougher job for the yeast, making monitoring yeast health important: unhealthy, stressed yeast are more likely to produce undesirable flavor and aroma compounds.

Fermentation Temperature

Fermentation temperature is also crucial. Lower temperatures preserve fragile, volatile aromatics in the wine, retaining a more “fruity” character. If temperatures are too low, however, yeast work slowly—or they may have difficulty fermenting all of the sugar. Higher temperatures allow for better grape skin tannin extraction but tend to drive off fruity flavors and aromas. If temperatures are too high, yeast may work at a frantic pace, producing undesirable flavors and aromas along the way. At the extreme upper end of the temperature range, yeast can even generate so much heat they die, which can impart an unpleasant “cooked” character to wines.

Finding the right fermentation temperature is all about balance. Red wines are typically fermented at the warmer end of the spectrum—between 70 and 85 degrees Fahrenheit—to take advantage of better tannin extraction. White wines are more often fermented at the cooler end of the spectrum, around 45 to 60 degrees Fahrenheit. Because fermentation is an exothermic process (i.e., it generates heat), winemakers must often take steps to actively control fermentation temperatures, particularly in large batch fermentations of several tons. At 2Hawk, we tend toward small batches, allowing for better passive cooling and reduced needs for active temperature control systems.

Fermentation Vessel

In addition to the fermentation vessel’s material, the shape and size of the vessel can be an influential consideration. Smaller fermentation vessels have more surface area for a given mass, meaning they can more easily shed excess heat from fermentation. Vessels with a rounder shape aid convection currents in the fermenting wine, facilitating fermentation by keeping temperatures uniform and yeast in suspension.

After Fermentation

Most of the time, fermentation is complete when yeast have consumed all the sugar they can, usually meaning the wine is fermented to dryness. Most of the yeast will die at this stage, gradually settling to the bottom of the fermentation vessel where they become known as “lees.” Depending on the wine style and winemaker’s preferences, the wine may be allowed to rest on the lees for some time, or it may be “racked”—transferred—to another vessel to begin the aging process without the yeast lees. The presence of yeast lees can have a profound effect on the aroma, flavor, and texture of the wine as it ages.

We hope you enjoyed getting an inside look at this complex but essential aspect of the winemaking process. For a more general overview of winemaking, including what happens before and after fermentation, take a look at our earlier blog post, How is Wine Made? Can we answer any other questions you have about making or tasting wine? Please let us know! We’d be happy to help.

Meanwhile, if you’d like to learn more about Rogue Valley wines, here are a few ways:

• Read our Fall 2018 Harvest Wrap-Up to see why we’re excited about our 2018 vintage!
• Visit the tasting room to sample our current wines.
• Follow us on Facebook and Instagram to keep up with the latest happenings.

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I began the fermentation process at a 70* temp. Then the temperature dropped to the 40’s. I left the batch undisturbed as per the kit instructions. However, I did check for expansion. There was hardly any. Today is the 11th day but for the last 3 or 4 days I noticed it was bubbling tiny bubbles and needs “burping” regularly. Should I let it work for a couple more days? The temperature is still very cold. 40* range.

Most wine yeast will work very very slowly, if at all, in that temperature range. You’ll probably want to start by checking the sugar content of the must, either using a hydrometer or just by taste. See if it’s obviously sweet or far from your expected finishing gravity. If so, you’ll want to move the fermenter somewhere warmer, at least 60-65F, give the fermenter a gentle swirl to coax yeast back into suspension, and let it work for at least a few more days. Make sure you have a means to release pressure from the fermenter aside from manual burping, as a vigorous fermentation can easily cause sealed containers to explode.

If there’s no sugar left, the bubbles you’re seeing could be the result of malolactic fermentation, which produces smaller, finer bubbles than alcoholic fermentation. You will want to either let it complete, or stabilize the wine according to the kit’s instructions.

If you have any additional questions, please feel free to run them by Kiley, our Winemaker. I’ll pass along his contact info via email. Happy fermenting, and cheers!

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The Wine Making Process Step by Step

• by Anna Maria
• October 24, 2020 May 3, 2021

How is wine made?

Below is a very basic explanation of the wine making process step by step. There’s a lot more to it but this is all you need to know.

Humans have been making wine for over 8,000 years . Without any scientific understanding, ancient people believed that wine mysteriously transformed from grape juice as a gift from the gods.  Trial and error was the core way to help wine evolve into more delicious stuff throughout the years. It is as simple as picking grapes off the vine, smooshing them into juice, and waiting for the magic to happen. While the basic concept remains the same, it wasn’t until about 150 years ago that Louis Pasteur discovered how alcoholic fermentation occurred. He ran experiments that determined that it was a live yeast bacteria that converts sugar into alcohol. This was the breakthrough moment that changed the wine industry as we know it.

Growing grapes for wine making

Winemaking starts in the vineyards. The wine is only as good as the grapes that go in it. Viticulture is fancy farming and it’s the fancy name for growing grapes. Grapes are a vine that grow uncontrollably. It is the job of the farmer to control the vines. The farmer tames the vines by creating support systems and training the vines to adhere to a shape. The proper word for this is trellising. It’s not so different than trellising a vine against a lattice fence. You’re guiding the vine on where to go and supporting it along the way.

There are many different ways and just as many reasons to trellis and train a vineyard. They vary depending on the climate, the unique needs of the grape variety, and the needs/abilities of the farmer. Trellising a vineyard should make it easier to care for the vineyard for everything from weed control, preventing disease, and ease of harvesting. 

Grape vines produce fruit once a year. The winemaker determines when the grapes are ripe, usually when sugar levels and acidity are in balance. In the northern hemisphere harvest season is between late July to late October. In the southern hemisphere, grapes harvest is from January to April. During harvest, harvesters cut the grapes from the vine, put them in baskets, and transport them to the winery. 

Sorting grapes

Grapes can be sorted in the vineyard or at the winery. Sorting in the vineyard means that only healthy clean grapes are picked. The harvesters discard rotten grapes or leaves them on the vine. This usually occurs at estate wineries that make wines from their own vineyards. Wineries that buy fruit from farmers will need to sort their grapes on a sorting table to make sure only healthy grapes go into the wine. 

Crushing/Destemming

Fermentation can only begin if the juice inside the grape interacts with yeast. To get the grape juice out, the grapes go into a crusher to break the berries open. The destemmer separates the berries from the stems. The stems impart harsh bitter tannins in the wine and we don’t want that.

The difference between white, red, and rose fermentation

At this step in the wine making process, white wine and red wine diverge on different paths. White grapes jump ahead to the press. Red grapes go through maceration. Red grapes that have very little contact with the grape skins make pink colored wines.

The next 2 wine making steps are happening simultaneously. 

Maceration is how wine gets its color. For red wine, skins are in contact with the juice throughout the fermentation process. The juice inside of all grape berries is clear. It is the skins that give wine its color. As the grape juice begins to ferment, alcohol and heat rise in the solution. Alcohol and heat extract pigment out of the skins and into the liquid. The longer the skins stay in contact with the juice, the darker the color will be. Red grapes that will become rose wine only macerate for up to 24 hours. Whereas, red grapes that will become red wine can macerate for up to a month.  

Wine Fermentation Process

Fermentation is the process in which the sugar in grapes converts to alcohol. This happens when the yeast begins to eat the sugar. Yeast is everywhere. Fermentation can begin with wild yeast found in the environment but usually winemakers will add reliable, predictable commercial yeast. Alcohol, carbon dioxide, and heat are the byproducts of this fermentation. This process can take anywhere from 10 to 21 days. It is important to control the temperature of the must/wine during this time. If the wine is too cold yeast will go into hibernation. If the wine is too hot, the yeasts will die. 

Pressing separates the skins and seeds from the juice. With white grapes, pressing occurs before fermentation. With red wine pressing occurs after the juice has already become wine. White juice is pressed whereas red wine is pressed. Pressing, whether done in a basket press or pneumonic press, gets the last bit of juice/wine out of the skins. Then the juice/wine is pumped into vats. The skins and seeds discarded. Some wineries use the skins to make grape distillates, fertilizer for the vineyards, or feed for animals. 

Dead yeast and other grape guts will fall to the bottom of the vats. The wine above the guts transfers to a different vat. They discard the sludge at the bottom. This is one way to clarify the wine. Wine can be clarified by bentonite clay, egg whites, or fish bones, too. Don’t fret on this. I’ll write about it soon.

Blending wine

The winemaker blends the wine whether it is a single variety wine or a blend of multiple grapes. Each vineyard site, each vat, and each barrel is different even if the grape variety is the same. The winemaker will carefully select how the wine will come together. There’s a lot of chemistry involved to make a balanced wine.  

Ageing wine

Unoaked white wines and roses need about 4 months to be ready for consumption. Reds take longer and can take up to 2 years to be market-ready. Oaked wines will spend time in the barrels to take on oak flavors and have small transfers of oxygen through the wood to aid development. 

Even after racking, some small particles will remain. Filtering the wines makes the wine clear and free of particles. To some extent, all wines pass through a filter. Some winemakers choose to minimize filtration to preserve as much character as possible. 

Last step is the bottling line. Pretty self explanatory. The machine fills the bottles, seals them with a cork or screwcap, seals them with a foil, and adds a label. Depending on the size of the winery, some of these steps like labeling will be done by hand. 

Okay so this isn’t technically part of the wine making process step by step, but as a sommelier, I feel like this is the most important. Cheers, friends!

Let me know in the comments if you have any questions about the winemaking process.

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1 thought on “The Wine Making Process Step by Step”

Thanks. You really simplified this. I feel like there is so much to learn about wine that it is hard to remember it all. Having it broken down like this makes it easier.

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• v.11(12); 2010 Dec

Fermenting knowledge: the history of winemaking, science and yeast research

Paul j chambers.

1 Australian Wine Research Institute, PO Box 197, Glen Osmond, Adelaide, SA 5064, Australia

Isak S Pretorius

In the second article of the ‘Food and Science' series, Paul Chambers and Isak Pretorius explain the central role of yeast in wine making and how biotechnology can contribute to improving the quality of wine.

Wine has been with us since the dawn of civilization and has followed humans and agriculture along diverse migration paths ( Fig 1 ). Serendipity presumably played a part in its genesis more than 7,000 years ago: damaged grapes spontaneously fermented in harvesting vessels; curious farmers tasted the resultant alcoholic beverage; the curious farmers liked what they tasted and enjoyed its effects; said farmers preferred fermented grape juice to the unfermented fruit. The fate of the grape was sealed.

One might argue that the most important test tube in the birth and growth of the modern life sciences is the fermenter…

A generalized scheme of the spread of Vitis vinifera noble varieties of grapevine and winemaking from their centre of origin in Asia Minor to other parts of the world.

One might argue that the seeds of science and technology, particularly biotechnology, were also sown at this time. Empirical observations of natural events and processes were harnessed in repeat ‘experiments'—which is to say, vintages—and improvements were made by trialling modifications to practices, retaining those that were beneficial and discarding failures, with the results communicated down through the generations. At that time, there was no EMBO reports or alternative means by which to facilitate horizontal dissemination of information, but the principle of development— sans peer review—is clear: experimentation and invention lead to progress—technological and otherwise—and new knowledge is shared and built upon.

Of course, early inventions and innovations in grape and wine production were based on little or no knowledge of the biology of grapevines or the microbes that drive fermentation. In fact, it would be several thousand years before it was even known that microscopic organisms exist: using a primitive microscope, Antonie van Leeuwenhoek observed cells for the first time in 1680 ( Fig 2 ).

Selected milestones that mark the path of research in microbiology and yeast biology that have affected, directly or indirectly, wine science and winemaking.

Scientific knowledge grows at an exponential rate, and nowhere is this more evident than in the historical milestones of chemistry and biology that have shaped our understanding of the biology of the microorganisms that drive fermentation ( Fig 2 ). This progress has been adorned with some of the most significant names in the chemical and biological sciences, including van Leeuwenhoek, Lavoisier, Gay-Lussac, Pasteur, Buchner and Koch. One might argue that the most important test tube in the birth and growth of the modern life sciences is the fermenter, and the most important model organism has been the yeast Saccharomyces cerevisiae —commonly known as baking, brewing or wine yeast. As readers might know, this is exemplified in the origin of the word enzyme—‘en' meaning within and ‘zyme' meaning leaven. Yeast has been integral to pioneering work in microbiology and biochemistry, particularly in the fields of metabolism and enzymology ( Barnett, 1998 , 2000 ; Barnett & Lichtenthaler, 2001 ).

Throughout the early decades of the twentienth century the place for S. cerevisiae in fundamental research was affirmed, and there are several good reasons for this. Our close relationship with this yeast in food and beverage production over millennia tells us that it is safe to work with; as confirmed by its ‘Generally Recognised as Safe' designation by the US Food and Drug Administration. In addition, it is inexpensive, easy to grow and can be stored for long periods in suspended animation. Perhaps the most important thing is that it has accessible genetics that can be followed through sexual and asexual cycles ( Barnett, 2007 ).

The 1970s set the stage for another explosion of knowledge, sparked by the advent of gene technology and driven by a convergence of genetics, biochemistry, cell biology, microbiology, physical and analytical chemistry, as well as computing brought together under the banner of molecular biology ( Fig 3 ). Yeast molecular biology was established when Gerald Fink's group in the USA demonstrated that yeast could be transformed with foreign DNA ( Hinnen et al, 1978 ). In the same year, Jean Beggs in the UK developed a shuttle vector between Escherichia coli and S. cerevisiae that enabled cloning in yeast ( Beggs, 1978 ). The research community now had a eukaryotic host that was amenable to genetic engineering, benefiting both fundamental research and offering the potential of precise engineering of novel strains for industrial applications. It was the first host cell for industrial-scale production of a recombinant vaccine against hepatitis B and a recombinant food-grade enzyme, chymosin, which is used in cheese processing ( Pretorius et al, 2003 ).

Selected milestones that mark the path of research in genetics and molecular biology that have affected, directly or indirectly, wine science and winemaking.

Ever since, S. cerevisiae has been one of the most important model organisms in molecular biology and emerging fields; breakthroughs and technological advances in molecular, systems, and now synthetic biology rarely happen without S. cerevisiae figuring somewhere prominently in the story ( Fig 3 ). The international yeast science community has been particularly progressive and proactive in establishing large collaborative projects and building resources that are available to the scientific community. S. cerevisiae was the first eukaryote to have its genome sequenced ( Goffeau et al, 1996 ), a feat that was achieved through an international effort that involved 600 scientists, which paved the way for the first chip-based gene array experiments ( Schena et al, 1995 ). It was the first organism to be used to build a systematic collection of bar-coded gene deletion mutants ( Winzeler et al, 1999 ; Giaever et al, 2002 ), in which there are deletion strains for most of the open-reading frames in the S. cerevisiae genome. This has enabled high-throughput functional-genomic experiments, and anyone seeking information on just about any aspect of S. cerevisiae biology has access to the amazing community resource: the Saccharomyces Genome Database (SGD; http://www.yeastgenome.org/).

All of this is important to wine research; our winemaking workhorse is centre stage in thousands of research projects worldwide, so we know more about this humble eukaryote than any other organism on the planet. It is therefore unsurprising that wine research has benefited enormously from the privileged place that S. cerevisiae occupies in life sciences research. This is particularly evident in the impact that advances in molecular biology and related fields have had on winemaking.

In the hands of molecular biologists, S. cerevisiae is the most tractable of organisms; it is amenable to almost any modification that modern biology can throw at a cell. This makes it an ideal host for generating variants with improved and even exotic phenotypes that will benefit winemaking. The following gives some examples of current research and directions in this field.

In modern winemaking, fermentations are driven largely by single-strain inoculations; pure cultures of selected strains of S. cerevisiae are added to grape must as soon as possible after crushing. This ensures greater control of vinification, leads to more predictable outcomes and decreases the risk of spoilage by other microorganisms. There are many—probably hundreds of—different yeast strains available, and the winemaker's choice can substantially effect the quality of the wine ( Lambrechts & Pretorius, 2000 ; Swiegers et al, 2005 ).

One of the reasons for the yeast-induced variation in wine quality is that, during fermentation, S. cerevisiae produces an abundance of aroma-active secondary metabolites and releases many aroma compounds from inactive precursors in grape juice, which greatly affect the sensory properties of the wine ( Swiegers & Pretorius, 2007 ). Thus, any genetic variation in wine yeast that affects the production or release of sensorially important molecules will affect wine quality. In this context it has been demonstrated, for example, that different commercial yeast strains generate wines with very different profiles of volatile thiols ( Swiegers et al, 2009 ). These thiols—which are present in grape juice as non-volatile cysteinylated precursors ( Tominaga et al, 1998 )—are often described as ‘passionfruit', ‘tropical fruits' and ‘citrus' by tasters, flavours that are particularly important in wine varieties such as Sauvignon Blanc ( Dubourdieu et al, 2006 ).

Molecular biology and its tools are crucial to our understanding of the genetic and molecular bases of yeast-driven volatile thiol release from non-volatile precursors in grape juice. Howell et al (2005) have used bioinformatic tools and the SGD to identify candidate S. cerevisiae carbon–sulphur lyase genes that might be involved in the release of volatile thiols from cysteinylated precursors during fermentation. The researchers used targeted gene deletion to remove these candidate carbon–sulphur lyases from the wine and laboratory yeast strains, and they identified four genes that potentially contribute to the release of these important aroma molecules.

Swiegers et al (2007) then engineered a wine yeast, VIN13, to constitutively express a carbon–sulphur lyase gene, tna A, from E. coli . Sensory analysis revealed that, compared with its non-engineered relative, this transgenic yeast, VIN13 (CSL1), had a positive impact on the release of volatile thiols from a Sauvignon Blanc grape juice. The authors commented that wine assessors preferred the VIN13 (CSL1)-derived experimental wines to the relatively neutral VIN13-derived wines.

A similar approach has been used to engineer yeasts for the enhanced production of fruity esters ( Lilly et al, 2006a ) and to increase the production of higher, fusel alcohols ( Lilly et al, 2006b )—all of which contribute to the flavour profiles of wines. Although this work is in the early stages of development, it shows the value of yeast molecular biology, and the amazing resources that come with it.

Wine alcohol content is of growing importance to the wine industry. In some wine regions, it has been increasing during recent decades ( Godden & Muhlack, 2010 ). The main reason for this increase is that grapegrowers tend to leave fruit on the vine as long as possible to increase fruity characters—which develop as berries mature—and reduce undesirable ‘green' characters. This practice, however, produces fruit with a higher sugar content, which translates to higher ethanol concentrations in the wine.

A recent review by Kutyna et al (2010) discusses several metabolic engineering strategies that have been explored to generate wine yeasts that can divert some carbon metabolism away from ethanol production, with the aim of decreasing ethanol yields during vinification. Understanding the central metabolism of yeast and the genes that drive it has been crucial to this work. Candidate genes that are likely to influence ethanol yields can be identified from a range of sources, including the SGD, and then manipulated and cloned as required. Several laboratories have targeted the glycerol-3-phosphate dehydrogenase isozymes GPD1 and GPD2 , which divert carbon from glycolysis to glycerol production ( Michnick et al, 1997 ; Remize et al, 1999 ; de Barros Lopes et al, 2000 ).

Increased expression of either of the GPD paralogues increased glycerol and decreased ethanol yields. However, increased Gpd activity also led to increased amounts of acetic acid in the fermentation product. This was probably owing to rectification—by one or more of the five aldehyde dehydrogenase isozymes—of a redox imbalance that resulted from excessive Gpd-driven oxidation of NADH. Aldehyde dehydrogenase isozymes drive the oxidation of acetaldehyde to acetic acid with concomitant reduction of coenzymes NAD + or NADP, depending on which isozyme is involved ( Navarro-Aviño et al, 1999 ). This might be good for a yeast cell struggling with an imposed redox imbalance, but an increase in acetic acid production is not good news for winemakers; excessive vinegar is not desirable in wine. This problem was alleviated by knocking out one of the five aldehyde dehydrogenase isozymes, ALD6 ( Eglinton et al, 2002 ; Cambon et al, 2006 ).

Similar approaches have targeted S. cerevisiae pyruvate decarboxylase isozymes, alcohol dehydrogenase isozymes and glycerol transporters, leading to increased glycerol yields and reduced ethanol production ( Kutyna et al, 2010 ). However, while there are probably several good candidate ‘low-ethanol' wine yeast strains sitting in various labs around the world, none have been tested in commercial-scale, industrial fermentations. This is largely because consumers are generally unaccepting of genetically modified organisms (GMOs) in foods and beverages.

Another area of ongoing research in wine yeast molecular biology is the development of strains that flocculate—that is, form clumps—at the end of fermentation. This facilitates the process of settling them out of suspension and separating them from the wine, thereby reducing the need for clarification. The timing of flocculation is crucial; it must not happen too early, as yeast in large flocs are inefficient at sugar utilization and can generate suboptimal—stuck or sluggish—fermentations ( Pretorius, 2000 ).

Generally, wine yeasts are not good at flocculation; they do not form large clumps that settle out of suspension. Many years of research using laboratory strains of S. cerevisiae led to the identification and characterization of genes that encode cell-surface glycoproteins—including lectin-like flocculins—that cause, among other things, flocculation and subsequent settling to the bottom of the fermentation vessel ( Pretorius, 2000 ).

Recent findings have identified a problem with extrapolating basic research on laboratory strains to those used in industry; yeasts domesticated for different purposes have different phenotypes. Work by Govender et al (2008) on the flocculation genes FLO1 , FLO5 and FLO11 , for example, demonstrated the potential ability of engineered ADH2 - or HSP30 -promoter/ FLO gene combinations to switch on flocculation at the end of fermentation; ADH2 and HSP30 are both upregulated in stationary-phase cells, so their promoters are suitable candidates to drive the expression of genes in later stages of wine fermentation.

The results of this work were promising, but, when they were carried over to wine yeast, the findings were rather different. There were even substantial differences between wine yeast strains, leading the authors to caution that “optimisation of the flocculation pattern of individual commercial strains will have to be based on a strain-by-strain approach” ( Govender et al, 2010 ). Nonetheless, controlled expression of FLO genes at the end of fermentation remains a plausible technique for improving the performance of wine yeast, but the strategies required to achieve a desirable outcome might be more complex than was originally thought.

While the complexity of biological systems is a cause for excitement and wonder to most biologists, it can make engineering novel strains for industrial applications trickier than molecular biology and biotechnology textbooks might suggest. For those of us working on industrial yeast strains, it might be pertinent to directly tackle the issue of complexity and use systems biology approaches to better understand the workings of yeast metabolism. This should lead to more accurate modelling of metabolic processes for better-informed manipulations, to achieve targeted, predictable outcomes.

However, molecular biologists face one important obstacle to this progress: near worldwide refusal to permit the use of GMOs in the production of foods and beverages…

S. cerevisiae has been at the forefront of ‘-omics' research. This provides us with enormous opportunities to improve understanding of wine yeast complexity, which, in turn, will inform the design of new strains for industrial applications. Increased and improved knowledge from a huge number of studies investigating strains of S. cerevisiae at the various -omic levels gives wine yeast scientists a head start in this field ( Borneman et al, 2007 ; Petranovic & Vemuri, 2009 ).

One of the most interesting developments has come from the sequencing of a wine yeast genome, and its comparison with the genomes of a laboratory strain and an opportunistic pathogenic S. cerevisiae ( Borneman et al, 2008 ). The authors found a difference of about 0.6% in sequence information between the wine yeast and the other strains. They also found, perhaps more importantly, 100 kb of additional genome sequence in the former; enough to carry at least 27 genes. Open reading frames (ORFs) in the additional sequences do not resemble anything found in other strains of S. cerevisiae , but seem to be similar to genes found in distant fungal relatives. BLAST searches have indicated that some of the genes that are specific to wine yeast are similar to those encoding cell-wall proteins. This might contribute to the greater robustness of wine yeast, compared with laboratory strains. Other genes might encode proteins associated with amino acid uptake, which is significant in the context of wine sensory attributes; amino acid metabolism is central to the production of many sensorially important volatile aroma compounds.

Novo et al (2009) published similar findings from a different wine yeast strain (EC1118) and suggested that the extra sequence was probably the result of horiziontal gene transfer. Further work using functional genetics—to determine the effects of knocking out and overexpressing the ORFs—should enable characterization of the phenotypes of these ORFs, determine their relevance in the context of winemaking and might also reveal their origins.

There have also been numerous studies describing transcriptomic, proteomic and metabolomic analyses of wine-yeast fermentations. This work is beginning to provide insights into wine-yeast fermentations, but it is still early days. It should also be noted that much of the -omics work on wine yeast has used resources and databases that are based on laboratory strains. It is now clear that there are genomic differences between wine and lab strains of S. cerevisiae , and these might affect -omics data acquisition and analysis. For example, gene-array chips based on the reference laboratory strain S288c will not include the additional ORFs found in wine strains. This does not suggest that earlier work is invalid, but that there are likely to be gaps in it.

As the various -omics fields progress, it should be possible to build systems-based mathematical models of metabolism that will facilitate the in silico design of new wine yeast strains ( Borneman et al, 2007 ). In parallel, we see the emergence of synthetic biology where, yet again, S. cerevisiae is a key player. It should not be too long before we have customised S. cerevisiae genomic components—regulatory elements to control the expression of targeted genes, or cassettes carrying genes encoding metabolic pathways to shape wine-relevant traits, for example—available ‘off the shelf' for designing, building and refining metabolic processes in our wine yeast. But are consumers ready for this brave and exciting new world?

The engineered wine yeast strains described in this paper show the potential of novel yeast strain development to improve wine quality. But molecular biologists face a major obstacle to this progress: near world-wide refusal to permit the use of GMOs in the production of foods and beverages, at least in ‘developed' countries ( Gross, 2009 ; Pretorius & Høj, 2005 ). Wine industries in most parts of the world have eschewed the use of GMOs in commercial winemaking, leaving most new-generation wine yeasts on the laboratory shelf, where they await more enlightened times.

Two genetically modified wine yeast strains have been released to market in a limited number of countries including the USA, Canada and Moldova: ML01 and 522 EC− . ML01, a transgenic wine yeast, has genes that enable it to perform malolactic fermentation (MLF), a deacidifying secondary fermentation in which malic acid—present in grape juice—is decarboxylated to lactic acid. MLF is usually performed by the lactic acid bacterium Oenococcus oeni after alcoholic fermentation. However, this bacterium is rather fastidious, being inhibited by a range of conditions that are typical of fermented grape juice—low pH, high alcohol content, poor nutrient availability and the presence of sulphur dioxide—and can become ‘stuck' or take considerable time to complete fermentation ( Davis et al, 1985 ). In addition, lacitic acid bacteria can produce a range of biogenic amines, which are associated with health risks ( Lonvaud-Funel, 2001 ).

A wine yeast that completes both primary and secondary fermentations should therefore have great potential in the wine industry. The genetically modified wine yeast ML01 carries two foreign genes—the Schizosaccharomyces pombe malate transporter gene ( mae1 ) and the O. oeni malolactic enzyme gene ( mleA )—which are both chromosomally integrated and regulated by the S. cerevisiae PGK1 promoter and terminator ( Husnik et al, 2006 ). This enables the host wine yeast to perform MLF, in parallel with alcoholic fermentation.

The researchers went to great lengths to ensure the safety of ML01. The transgenes came from microorganisms found in wine, there were no antibiotic resistance genes or vector sequences carried by the yeast and transcriptome and proteome analysis showed no important differences in gene expression profiles between the genetically modified strain and its parent. The FDA granted ‘Generally Regarded As Safe' status to ML01, but it has not been widely adopted, even in countries where it is approved for use. This is largely owing to concerns about export markets that do not tolerate GMOs. In fact, wine industries in many countries have banned the use of GMOs in wine production, in order to avoid jeopardizing their exports. ​ exports.

The genetically modified wine yeast 522 EC− was engineered to reduce the risk of ethyl carbamate production during fermentation. Ethyl carbamate, a potential carcinogen, is the product of yeast-derived urea reacting with ethanol. It is usually produced at such low levels—if at all—that it is not a cause for concern, but it sometimes can make an appearance in some wine-producing regions.

S. cerevisiae is able to degrade urea before it is secreted and release ammonia instead, thereby reducing the risk of generating ethyl carbamate. This is achieved by the action of an enzyme encoded by DUR1,2 , but this gene is repressed by nitrogen and therefore downregulated throughout much of wine fermentation. Coulon et al (2006) placed a copy of DUR1,2 behind a constitutive ( PGK1 ) S. cerevisiae promoter, which led to a reduction in ethyl carbamate yields. Interestingly, this genetically modified yeast is self or cis cloned; it carries no foreign DNA and therefore is not transgenic. Nonetheless, because it was generated by using techniques that involved the manipulation of DNA in vitro , the regulations of many countries classify it as a GMO. Again, to the best of our knowledge, this yeast is not being used in the industry. This might be because ethyl carbamate production is not a widespread problem, but it probably also reflects the influence of GMO bans and the reluctance of winemakers to risk losing market share in countries that harbour strong anti-GMO sentiment.

Who knows what bottled masterpieces await us as we sculpt novel yeast strains in the laboratory using molecular, systems and synthetic biology

Winemaking, science and technology have interwoven histories and have grown together over the millennia, benefiting from each other. Although science is an important part of an oenologist's training and scientific methods and equipment are routinely employed in the winery, winemakers are not scientists per se . They are, perhaps more appropriately regarded as artisans, with the emphasis on the ‘art'. As for many human endeavours, the Arts progress with developments in technology; think of the use of acrylic paint in the fine arts since its introduction in the 1950s, or David Hockney's use of a Polaroid camera to create photocollages. In the way that acrylic paint and photography have provided more options to artists, enabling them to broaden their horizons, yeast science and technology is adding to the winemaker's palette. Who knows what bottled masterpieces await us as we sculpt novel yeast strains in the laboratory using molecular, systems and synthetic biology. The only real obstacle that we face is consumer acceptance of GMOs; we can only hope that rationality will eventually prevail.

Science & Society Series on Food and Science

This article is part of the EMBO reports Science & Society series on ‘food and science' to highlight the role of natural and social sciences in understanding our relationship with food. We hope that the series serves a delightful menu of interesting articles for our readers.

Acknowledgments

Research at the Australian Wine Research Institute (AWRI) is financially supported by Australia's grapegrowers and winemakers through their investment body, the Grape and Wine Research Corporation, with matching funding from the Australian Government. Systems biology research at the AWRI uses resources provided as part of the National Collaborative Research Infrastructure Strategy (NCRIS), an initiative of the Australian Government, in addition to funds from the South Australian State Government. AWRI's collaborating partners within this NCRIS-funded initiative—which is overseen by Bioplatforms Australia—are Genomics Australia, Proteomics Australia, Metabolomics Australia (of which the Microbial Metabolomics unit is housed at the AWRI) and Bioinformatics Australia.The AWRI is part of the Wine Innovation Cluster in Adelaide.

The authors declare that they have no conflict of interest.

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Wine Making

Wine is an undistilled alcoholic beverage made from fermented fruit juice. Meanwhile, fermentation refers to the catabolic and anaerobic process of transforming sugar into carbon dioxide and ethanol with the help of bacteria, preferably in the dark. For this experiment, it aims to create wine in a laboratory by learning the process on how to produce and by appreciating the role of each material in the product. The experiment started with the preparation of each ingredient, specifically, fruit juice, active yeast, and refined sugar, and the measurement of their required weights. Next, 50 mL of fruit juice was warmed on a hot plate and then refined sugar was dissolved into the heated juice. After that, the juice heated again to a temperature of 70°C. Active yeast was added and mixed with the hot juice. The mixture was then combined with the remaining fruit juice and its density was determined using a hydrometer or pycnometer. Then, the mixture was transferred in a bottle with a cotton wad or balloon as a cover and was left to ferment in a dark room for a week. After a week, the wine was decanted and filter before being placed in a packaging bottle. The same process was repeated for the remaining wine formulations. The results showed that the wine made from the W1 formulation was much paler than from W2 and W3. It was also observed that the specific gravity of all the wines was greater before fermentation occurred. From the ingredients, yeast acted as the fermenting agent which converted the sugar compounds from the fruit juice and from the refined sugar into alcohol. Also, sugar was used to balance sweetness and to produce more alcohol in the wine since the sugars in the fruit juice is not sufficient to reach the desired ethanol content for wines.

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Wine is an alcoholic beverage made from fermented grapes or other fruits. Many tropical fruits such as mango, jackfruit, litchi, banana and cashew apple have been shown to be suitable for fermentation, mainly because of their appropriate taste, flavour, availability, high sugar and water content and overall chemical composition (Muniz et al. 2008). The natural chemical balance of these fruits lets them ferment without the addition of sugars, acids, enzymes, water, or other nutrients. Yeast consumes the sugars in the fruits and converts them into alcohol. Study was conducted to produce red wine without using any sugar and making use of the kitchen yeast strain i.e saccharomyces cereisiae and was tested for the different physical and chemical characteristics of the wine such as acidity ,sugar content and other quantitative and qualitative tests. The wine was produced in simple lab conditions and using simple lab utensils and instruments so this techniques may very well reduce the overall cost of wine production. The production and tests were conducted in small scale but this technique can be converted to large scale with few changes.

Dr. P. Saranraj

Fruit is an essential part of your diet using essential part of vitamin and minerals that contribute to overall strength for your health. Fruit wines are undistilled alcoholic beverages usually made from grapes or other fruits such as peaches, plums or apricots, banana, elderberry, or black current which are nutritive, more tasty, and mild stimulants. These fruits undergo a period of fermentation and aging. They usually have an alcohol content ranging between 5 and 13%. Wines made from fruits are often named after the fruits. No other drinks, except water and milk, have earned such universal acceptance and esteem throughout the ages as has wine. Wine is a food with a flavor like fresh fruit which could be stored and transported under the existing conditions. Being fruit-based fermented and undistilled product, wine contains most of the nutrients present in the original fruit juice. The nutritive value of wine is increased due to the release of amino acids and other nutrients from yeast during fermentation. Fruit wines contain 8–11% alcohol and 2–3% sugar with energy value ranging between 70 and 90 kcal per 100 ml. The present explained about the fermentation of wine and its quality analysis. In this present review, we discussed about fermentation, history of fermentation, Saccharomyces cerevisiae and alcoholic fermentation, fermentation of fruit juice into wine, classification of wine, factors influencing fermentation and wine quality, and Indian wine market.

George Vierra

Wine is an alcoholic beverage made from fermented grapes or other fruits. The natural chemical balance of grapes lets them ferment without the addition of sugars, acids, enzymes, water, or other nutrients. Yeast consumes the sugars in the grapes and converts them into alcohol. Different varieties of grapes and strains of yeasts produce different types of wine such as red wine ,white wine, sparkling wine, rose wine etc. Study was conducted to produce red wine without using any sugar and making use of the kitchen yeast strain i.e Saccharomyces cereisiae and was tested for the different physical and chemical characteristics of the wine such as acidity ,sugar content and other quantitative and qualitative tests. The wine was produced in simple lab conditions and using simple lab utensils and instruments so this techniques reduces the overall cost of wine production. The production and tests were conducted in small scale but this technique can be converted to large scale with few changes.

PRODUCTION OF WINE FROM THE FERMENTATION OF ORANGE JUICE BY Saccharomyces cerevisiae

Oladimeji ishaq Hassan

The fermentation of orange juice by Saccharomyces cerevisiae isolated from palm wine was carried out. The fermentation was done in two phases; the aerobic phase which lasted for 5 days and the anaerobic phase which lasted for 9 days. Some physicochemical parameters were monitored during the aerobic and anaerobic phase of fermentation. These were pH, titratable acidity, specific gravity, sugar content and alcohol concentration. The result of the physicochemical analysis showed that during aerobic fermentation there was a decline in the pH from 3.6 to 2.4; an increase in titratbable acidity from 9.10 to 16.8g/l. During anaerobic phase the pH increased from 3.0 to 4.3 while the titratable acidity decreased from 16.4 to 9.7g/l. The yeast counts increased from 5.5 ×106 to 6.9×106 cells/ml; alcohol content increased from 0 to 5.4% during aerobic fermentation, while during anaerobic there was a drop in yeast counts from 7.1×106cells/ml to 6.3×106 cells/ml; and the alcohol content increased from 5.8 to 9.6%. The sugar content and specific gravity in the wine dropped throughout during aerobic and anaerobic fermentation with sugar content dropping from initial value of 50.80 mg/ml to 2.87 mg/ml, while the Specific gravity dropped from 1.040 to 0.980osp.gr. On the 9th day of the anaerobic phase the fermentation was terminated by opening the fermentation tank, the wine was then clarified, racked and bottled.

Suna Ertunç

Summary: The goal of this study was to examine the effects of operating parameters on ethanol concentration (ethanol) in apple wine production process. Examined parameters were temperature (T), pH and sulphurdioxide concentration (SO2). Experiments were planned and executed according to a full two-level factorial experimental design method. The studied levels were 18°C and 25°C for temperature, 3 and 4 for pH and 50 and 150 ppm for SO2. Ethanol concentration of apple wine for each set of experiments was determined by GC/MS. Experimental data were analyzed by using both graphical and quantitative Exploratory Data Analysis (EDA) Techniques. The main effect of each factor on sugar consumption rate (SCR) was also examined. The results show that the effect of examined operating parameters on ethanol was negative. High temperature level caused faster fermentation rate than the one caused by low temperature. Low level of pH and high level of SO2 inhibited the activities of both harmful mic...

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Thi Hường Trần

This application note presents a simple and rapid method for the simultaneous determination of Saccharose, Glucose, Fructose, Ethanol and Glycerol. The method is designed for the quality and process control of wine. All five substances are baseline separated and no hydrolysis of Saccharose was observed. An extra sample preparation is not necessary, just a dilution. This method works with water as a low cost eluent.

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Science in School

Analysing wine at school teach article.

Author(s): Thomas Wendt

​European countries produce more than half of the world’s wine – and drink a lot of it too! These hands-on activities for schools reveal the science behind the perfect wine.

The age at which it is legal to drink alcohol varies from country to country, but most teachers would agree that drinking wine during chemistry lessons is inappropriate (and potentially dangerous!). However, producing and analysing wine at school can be fun and educational. These activities, developed at the science centre Experimenta w1 , invite students aged 15-18 to become vintners for a day, using analytical techniques to explore the changes that take place during the wine-making process.

Wine is produced by fermenting grape juice (which has particularly high levels of sugar) using specialised yeast cells. The sugar is converted into ethanol and carbon dioxide under anaerobic conditions:

C 6 H 12 O 6 + 2 ADP + 2 Pi = 2 C 2 H 5 OH + 2 CO 2 + 2 ATP

Experimenta

Experimenta w1  in Heilbronn is the largest informal learning and interactive science centre in southern Germany. In addition to the interactive exhibitions and science garden, Experimenta offers more than 30 laboratory-based programmes for school classes and individual pupils, from kindergarten level up to upper secondary school. These programmes address technology and all life sciences as well as providing teacher training.

The three main factors that determine the quality of the final product are sweetness, alcohol content and acid content. Using standard methods of a commercial wine laboratory, these three activities for the school laboratory explore how the quality of the starting grape juice and the must (fermenting grape juice) affect the final product. Each activity takes approximately 20-30 minutes.

• In activity 1, students can determine the sugar content of grape juice using refractometry. Activity 1a (see below) offers an alternative, based on density measurement.
• The exact determination of the alcohol content in commercial wines is performed by distilling the ethanol, then measur ing the viscosity of the distillate using sophisticated apparatus. In activity 2, students can use the equipment used by hobby winemakers – a vinometer – to measure the alcohol content of the must and wine.
• A well balanced wine needs a certain amount of fruit acid; the total acid content is a very important measure, because it directly affects the flavour. In activity 3, the acid content is determined by pH titration.

Four further activities can be downloaded w2 :

• Activity 1a: in an alternative to activity 1, students determine the sugar content of grape juice using density measurement instead of refractometry.
• Measuring levels of carbon dioxide, one of the products of the fermentation, is a useful way to monitor the progress of the reaction. In activity 4, students quantify CO2 levels over the course of the reaction by literally shaking the gas out of solution.
• In activity 5, students use light transmittance to investigate the difference that fining (the addition of substances that clarify the wine by precipitation) makes to the cloudiness of the finished product.
• In activity 6, students examine fermenting yeast under the microscope.

To supply the must used in these experiments, you will need to set up a simple grape juice fermentation at least one day in advance, using red grape juice (e.g. from the supermarket). You will also need some basic chemistry laboratory equipment, plus a vinometer for measuring alcohol content, a pycnometer (also known as a specific gravity bottle) and a refractometer. You can download a description of how to set up the fermentation w2 .

Determining sugar content

The sweetness of the wine is determined by the amount of sugar remaining after fermentation, together with the total acidity of the wine. A dry wine has up to 9 g/l sugar and an acidity level that is at least 2 g/l lower than the sugar content. A medium-dry wine has a sugar content of 9-18 g/l and an acidity level that should be no more than 10 g/l lower than the sugar content. A sweet wine has 18-45 g/l of sugar. To ensure the correct balance of sugar, acidity and alcohol in the final wine, it is important to determine the starting sugar concentration; if necessary, limited amounts of sugar can be added before fermentation.

The increased density of the must (compared to water) is mainly due to fermentable sugar. Density measurements or refractometry can be used to measure the sugar content, which in Germany is expressed as the must weight and measured in Oechsle (°Oe). In the English-speaking world, the sugar content is expressed in Brix (°Bx), which represents the concentration of dissolved sugar, in weight percent (wt%).

The must weight is calculated by:

must weight = (density – 1) x 1000

Where must weight is measured in °Oe and density in g/l.

As a rough estimate, 1°Oe corresponds to 2.37 g/l sugar (i.e. about 0.237 °Bx). Therefore, the sugar concentration can be estimated as:

sugar concentration = must weight x 2.37

Where sugar concentration is measured in g/l.

The fermentation of all fermentable sugar in a solution of 100 °Oe (sugar concentration 237 g/l or 23.7 °Bx) yields approximately 100 g/l ethanol (or 10 wt% alcohol). Because ethanol has a density of 0.79 g/ml, this converts to 12.67 vol% ethanol. Thus:

alcohol concentration (in % volume) = alcohol concentration (in g/l) x 0.1267

Student activity 1: determining sugar content using a refractometer

The amount of sugar in the grape juice will determine both the alcohol content and the sweetness of the finished wine. In this activity, you will use the refractive index to estimate sugar content.

Refraction is the change in direction of light when it passes from one medium to another (e.g. from air into water). The light-scattering behaviour of a solution changes as the concentration of solutes (dissolved substances) increases. A refractometer uses this principle to determine the concentration of dissolved particles in a solution. In wine, these are principally sucrose.

Most handheld refractometers give the concentration of the dissolved substance either in Brix (°Bx), a scale defined in terms of sucrose content, or in Oechsle (°Oe). A solution of 20 wt% sucrose in water is 20 °Bx. Oechsle can be converted approximately into Brix by multiplying by 0.237.

• Refractometer
• 20 wt% sucrose solution
• Grape juice
• Paper towels
• Pipette 2 drops of the sucrose solution onto the glass surface of the refractometer and close the lid.
• Take a reading through the eyepiece and enter the data in table 1.
• Using a paper towel soaked in distilled water, clean the glass surface, and then dry it.
• Repeat the measurement with grape juice.
• Calculate the missing numbers in table 1 using the equations above.

• How accurate was your result for the sucrose solution compared to the expected value?
• How reproducible were your measurements? Compare them to that of other groups.
• If you carried out activity 1a as well, how comparable were your results for the two methods (density versus refractometry)?
• A typical wine has about 12 vol% alcohol. Estimate how much sugar needs to be added to the grape juice to obtain 12 vol% alcohol.

Student activity 2: determining alcohol content

The amount of alcohol obtained by fermentation depends on the sugar content of the grape juice and the alcohol tolerance of the yeast strain: most yeast strains tolerate up to 16 % alcohol. The amount of alcohol can be measured quite accurately using a vinometer, a simple device developed for hobby winemakers. It is based on the principle that the surface tension falls as the alcohol content increases.

In this activity, you will measure the alcohol content of your must.

• Coffee filter
• Distilled water
• Filter 20 ml must through a coffee filter to remove any remaining yeast cells.
• Place a small amount of filtrate in the funnel of the vinometer (figure 2B) and wait until the capillary is full. Keep the remaining filtrate for activity 3.
• Carefully invert the vinometer onto a layer of paper towels, then observe the level of liquid while it slowly drops (figure 2C). Once it stays constant, take a reading and enter it in table 2.
• Rinse the vinometer with distilled water, then repeat the measurement using the wine.

Note: The alcohol content of the must is probably much lower than that of the wine. This may be because the fermentation process is not finished. It can also indicate that remaining sugar has increased the surface tension and is affecting the reading.

• You determined the sugar concentration of the grape juice in activity 1. Based on the available sugar in the grape juice, did you expect a higher alcohol content in the wine?
• If the fermentation continued for longer, would you expect an increased alcohol content?

Total acid content

Fruit juices can contain several different acids, including tartaric, malic, citric and oxalic acid, in differing ratios, depending on the type of fruit. The predominant acid in wine is tartaric acid, which has a pH between 3 and 4. However, due to the complex mixture of different acids and bases, proteins and salts, the total acid content of wine cannot be estimated from the pH value alone. Instead, it is determined by titration to neutrality and expressed as total equivalent amount of tartaric acid in g/l. The acid content of wine is typically 4-8.5 g/l but can be as high as 15 g/l. It must always be considered in conjunction with the amount of remaining sugar (see ‘Determining sugar content’).

Tartaric acid (molecular weight 150 g) is a diprotic acid (containing two hydrogen atoms per molecule that can dissociate in water as protons) that can be fully neutralised with sodium hydroxide. Because 1 mol NaOH neutralises 0.5 mol tartaric acid (75 g/l), 1 ml 0.1 M NaOH neutralises 7.5 mg tartaric acid.

HOOC-CH(OH)-CH(OH)-COOH + 2NaOH → Na + -OOC-CH(OH)-CH(OH)-COO – Na + + 2H 2 O

Student activity 3: Determination of acidity by titration

All wines contain a certain amount of acid. The winemaker is interested in the total acidity, caused mainly by tartaric acid. The total acidity is determined by titration with diluted sodium hydroxide.

• Magnetic stirrer
• Beaker (250 ml)
• Two measuring cylinders (10 ml, 100 ml)
• NaOH solution (0.1 M)
• 10 ml must (filtered, from activity 2)

For each sample (must or wine):

• Fill the burette with NaOH solution. Enter the starting volume in table 3.
• Measure 10 ml of your sample and place it in the 250 ml beaker. Add 100 ml distilled water.
• Start the magnetic stirrer and insert the pH electrode so that the tip is in the sample, but not touching the sides of the beaker or the magnetic stirrer flea.
• Did you observe any colour changes?
• If so, at what pH value?
• What could be the reason for a colour change?
• Calculate the amount of NaOH used and the acid concentration.

Example: We used 14 ml 0.1 M NaOH to neutralise 10 ml solution. The concentration is therefore (14 x 7.5 mg/ml x 100) = 10.5 g/l acid.

Safety note

Wear safety goggles and gloves. See also the  general safety note .

In activities 1-3, you analysed the three major factors that determine the quality of the final product: sweetness, alcohol and acid content. Now it is time to evaluate your product.

• Is the total acidity within the limits for wine production?
• Did the starting grape juice contain sufficient sugar to produce the expected alcohol content?
• How long would you expect fermentation to take before the process is finished?

Acknowledgements

The author would like to thank the wine laboratory of Pfäffle GmbH in Heilbronn, Germany, for support during the development of the activities. He would also like to thank in particular Christine Dietrich and Karsten Wiese from the teacher-training college in Heilbronn for their collaboration.

Web References

• w1 – Visit the  Experimenta website
• w2 –  Instructions on how to set up the fermentation, in addition to four further wine-related activities (Word ®  document) .  The same instructions as a PDF file .
• A basic guideline for common experiments in wine analysis:

Schmitt A (1975)  Aktuelle Weinanalytik, Ein Leitfaden für die Praxis . Germany: Heller Chemie. ISBN: 978-3-9800498-3-2

• For a comprehensive overview of topics that are relevant to the hobby winemaker, see the  Fruchtweinkeller website  (in German) and the  Fruchtwein website  (also in German)

Thomas Wendt received his PhD on structural biology from the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany, in 1998. During his postdoctoral research in the USA and back in Germany, he then focused on protein biochemical and molecular biology methods. After supervising numerous students, Thomas decided to concentrate on encouraging young people to consider a scientific career. Since 2009, he has been the educational head of the teaching laboratories at Experimenta.

Supporting materials

Instructions on how to set up the fermentation (Word document)

Instructions on how to set up the fermentation (PDF file)

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The Chemistry of Wine: Fermentation

By Maureen McKenna, Certified Sommelier, CMS

For thousands of years, wine has played a big role in many cultures, starting with the oldest-known winery in Armenia discovered in 4100 B.C. through the Greek, Roman and Egyptian empires all the way up to today. Whether it’s to raise a glass of sparkling wine in celebration or just pour one to unwind at the end of a long day, wine is often a favorite drink to fortify the mood.

Cultivation of the winemaking grape, Vitis vinefera , began in what is now Iran in the 5th millennium B.C. Today, you’ll find grapes grown in nearly every country that falls between latitudes 30 and 50 degrees north and 30 and 50 degrees south. It’s between these latitudes where you find the ideal climate for vines.

But it takes more than a good grape harvest to make wine. There are other external factors that can make or break a good yield, including the terroir (i.e. soil, climate, geography), how the grapes are harvested, the fermentation process, the maturation of the wine, and of course, the winemaker.

This article focuses on fermentation . A basic overview of the chemistry involved in that process can help with understanding this fundamental step in winemaking.

There are two basic ingredients needed to ferment the juice of grapes into wine: sugar and yeast . Like all fruit, sugar is found naturally in grapes, with the sugar level increasing as the grapes ripen on the vine; a process in the wine-making world called veraison . Ripening can take one to two months, depending on the climate. The right balance of rain and sunshine ensures good sugar levels in the grapes. When ready, the grapes are picked and crushed, leaving the juice, known as must, for fermentation.

The second ingredient needed for fermentation, yeast, consumes the sugar in the must, and as a byproduct, it releases three components: ethanol , CO 2 , and heat. The CO 2 and heat escape, and the ethanol remains.

The yeast needed for fermentation can be found naturally in the environment and on the grapes themselves. This natural yeast dies off, however, when grape juice reaches 4 to 5 percent alcohol by volume, before fermentation is complete.

In order to ferment the must completely, then, the winemaker adds an anaerobic (no oxygen needed), cultured yeast called Saccharomyces cerevisiae. Depending on the temperature at which must is fermented, the process can take one to two weeks.

Following fermentation, the winemaker will store the wine in various vessels, such as barrels or stainless-steel tanks, for example, for a period of time designated by local wine laws and based on the style of wine being made. During that period, harsh acids in the wine convert into softer, more palatable acids. (Some grape varieties might need a little help, so the winemaker will kickstart the process.) Once bottled, the wine may be stored for even longer to age before reaching your table.

Whether you fancy red, white, sparkling or fortified wine, fermentation is the chemical reaction at the heart of the process. It is a practice that has been honed over thousands of years, spreading around the world and surviving history to the modern-day wine-making that we enjoy today.

The Oxford Companion to Wine, 4th Edition. Jancis Robinson and Julia Harding

Photo credit: copyright jackf / 123rf stock photo.

• Specialized Cell Structure and Function: Cellular Respiration

Here are the facts and trivia that people are buzzing about.

Genomics and biochemistry of Saccharomyces cerevisiae wine yeast strains

• Published: 06 January 2017
• Volume 81 , pages 1650–1668, ( 2016 )

Cite this article

• M. A. Eldarov 1 ,
• S. A. Kishkovskaia 2 ,
• T. N. Tanaschuk 2 &
• A. V. Mardanov 1  

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Saccharomyces yeasts have been used for millennia for the production of beer, wine, bread, and other fermented products. Long-term “unconscious” selection and domestication led to the selection of hundreds of strains with desired production traits having significant phenotypic and genetic differences from their wild ancestors. This review summarizes the results of recent research in deciphering the genomes of wine Saccharomyces strains, the use of comparative genomics methods to study the mechanisms of yeast genome evolution under conditions of artificial selection, and the use of genomic and postgenomic approaches to identify the molecular nature of the important characteristics of commercial wine strains of Saccharomyces . Succinctly, data concerning metagenomics of microbial communities of grapes and wine and the dynamics of yeast and bacterial flora in the course of winemaking is provided. A separate section is devoted to an overview of the physiological, genetic, and biochemical features of sherry yeast strains used to produce biologically aged wines. The goal of the review is to convince the reader of the efficacy of new genomic and postgenomic technologies as tools for developing strategies for targeted selection and creation of new strains using “classical” and modern techniques for improving wine-making technology.

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Natural Yeast Strains of Saccharomyces cerevisiae that are Promising for Sherry Production

Abbreviations.

autonomously replicating sequence

copy number variation

gross chromosomal rearrangement

horizontal gene transfer; Indel, insertion/deletion polymorphism

long terminal repeat

malolactic fermentation

next-generation sequencing

open reading frame

quantitative traitloci

single-nucleotide polymorphism

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Institute of Bioengineering, Federal Research Center “Fundamentals of Biotechnology”, Russian Academy of Sciences, 119071, Moscow, Russia

M. A. Eldarov & A. V. Mardanov

All-Russian National Research Institute of Viticulture and Winemaking “Magarach”, Russian Academy of Sciences, 298600, Yalta, Republic of Crimea, Russia

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Original Russian Text © M. A. Eldarov, S. A. Kishkovskaia, T. N. Tanaschuk, A. V. Mardanov, 2016, published in Uspekhi Biologicheskoi Khimii, 2016, Vol. 56, pp. 155–196.

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Eldarov, M.A., Kishkovskaia, S.A., Tanaschuk, T.N. et al. Genomics and biochemistry of Saccharomyces cerevisiae wine yeast strains. Biochemistry Moscow 81 , 1650–1668 (2016). https://doi.org/10.1134/S0006297916130046

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Received : 29 July 2016

Revised : 19 September 2016

Published : 06 January 2017

Issue Date : December 2016

DOI : https://doi.org/10.1134/S0006297916130046

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