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Home , Acids in wine, Canaiolo, Chaptalization, Yeast in winemaking

Basic and Enology - 1 Winemaking predates recorded history. During the Cro-Magnon era, 45 thousand years ago, surely some family neglected some they had collected. When they rediscovered the grapes, they found that the grapes had broken down and had escaped. They ate and drank the cracked grapes and drank the “juice”. An hour or so later, some family members got giggly and soon they all got sleepy and took a nap. The juice had naturally fermented into . They had been introduced to a new beverage.

Winemaking has been known to be part of the diet of man since he settled in the Tigris- Euphrates basin several thousand years ago.

It is widely accepted that vinifera grapes originated in Asia Minor in an area between and south of the Black and Caspian Seas. This area of the Caucasus and northern Mesopotamia (present day Syria) is where grapes were first cultivated. Men found these wild grapes growing into the treetops. They then took cuttings and planted vines and found certain varieties to their liking. As early human groups traveled, these domesticated grapes were carried with them for planting in their new homes. Over the past ten thousand years many different empires have dominated the lands in and around the Mediterranean. After they occupied new lands, the victors planted the grapes they were familiar with at home. When they found new grapes in the occupied territories, they brought them back for planting in their homelands.

The oldest evidence of wine production is residues found in ancient Neolithic wine jars (amphora) found in the Iranian Z agros Mountains and dated to about 5,000 B.C.

M ap of the ancient N ear E ast and E gypt, showing the distribution of the modern wild grapevine in purple shading. G rape remains (primarily pips) recovered from N eolithic and L ate U ruk sites are indicated by the grape cluster symbol. T he occurrences of wine jars, which have been chemically identified as such, are indicated by the jar symbol.

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Later, the ancient Phoenicians settled on the rim of the Mediterranean and on Cyprus and Sicily and planted the vines they had used at home. The Greeks made their influence felt on the Island of Cyprus by 1,000 B.C. From about 350 B.C., the Dalmatian coast in present day Croatia was used as a Greek passage. The Greeks also settled in other parts of the Adriatic Sea and in Southern . The Romans moved into these Greek territories around 165 B.C. and brought back for cultivation hosts of and vegetables. From 830 AD until the 19th century, Croatia was dominated or controlled by the Franks, Byzantines, Hungarians, Venetians, Hapsburgs and finally the Austro-Hungarians. The Phoenicians, Greeks, and Romans, as did these newer infiltrators, always brought their grape vines with them to their new territories. They also brought newly discovered vines home with them.

Since Cro-Magnon times, we’ve learned much about how to make wine. Winemaking has four basic phases: GRAPES> > > > > > > > > > > > > STABILITY > > > > > > > AGING BIOLOGICAL MICROBILOGICAL PHYSICAL CHEMICAL 1. BIOLOGICAL. Grapes grow and ripen. At , the potential quality of the wine is set. The winemaker can try to attain that potential, but not improve it. We do as little as possible, e.g., pumping, filtering, clarifying, etc., that can take from the quality potential. Winemaking is a negative craft. We need to minimize the damage we can cause. 2. MICROBIOLOGICAL/ ENZ YMATIC. Called fermentation. ( and ) produce . Yeast enzymes convert grape into , CO2 and hundreds of other compounds. Bacterial enzymes convert malic to . 3. PHYSICAL OR CLARIFICATION. Stability is guided. Particles are settled with gravity or forced through screens. combines with and precipitates (KHT). Fining agents are added to make wine more pleasing to . 4. CHEMICAL or AGING. Ultimate quality is realized. Various components of wine combine with each other or and form new substances. When aging in wooden containers, the wood adds bouquet and flavor.

The phases overlap. Some enzymatic action happens during grape ripening. Settling occurs during fermentation. Aging begins during clarification. Clarification continues during aging. Microbiological activity can continue into the bottle.

Winemaking treatments include: character, extraction from grapes, skins and microbes, aging effects and possibly wood extracts, residual sugar, CO2, extra alcohol, acid, oxidation products and flavorings. Some will be explained later. 2

When are aged, the grape aroma decreases and the bouquet increases. Wines aged in wood, pickup wood characters. Red wines, with more tannin than whites, take longer to become less harsh. With long aging, diminishes and so does “varietal character”.

Sugar increases the wine body, masks off flavors and gives sweet tastes most Americans prefer.

Sparkling wines contain excess CO2 and young whites and reds can have a touch of spritz.

During fermentation, most yeast stops working when the alcohol content exceeds 14%. Adding alcohol, with high proof, to raise alcohol to 16 to 20% will add resistance to yeast and bacterial activity.

Flor yeast fermentation gives bouquets and heating gives Madeira nutty characters.

GRAPES The most important fruit crop grown in the world. Grapes are good for wine because: 1. High sugar content 2. Fruit 3. and other ideal substrates for yeast fermentation 4. Tannins from grape skins and seeds retard oxidation of wine 5. Grape flavors last longer than other fruit flavors 6. Grapes provide raw material for a wide range of wine types

The botanical genus Vitis has two sub-genera. 1. Euvitis (true grape) or “bunch grapes”. They grow in bunches. 2. Muscadinia Grow as separate . Euvitis has about 60 species. Almost all are important in winemaking. They originated in the northern hemisphere. Most species is V. vinifera. It is native to the area of Asia Minor, south of the Black and Caspian seas (Turkey, Iraq and Iran). The most important species indigenous to America is V. labrusca, Concord being most widely known variety.

Within V. vinifera are many varieties, like , , etc. Grape growers found they can retain the quality of the parent grape vines by rooting cuttings taken from mature vine wood. Offspring produced in this way are “clones”. As identical to parents as possible. All Vitis vines are either male or female. Early cutting propagation attempts failed since the vines were invariably female. The presence of “unproductive” male vines was essential to a productive . Sometime in the history of grape culture, a spontaneous mutation of flower type occurred, resulting in vines with flowers that could

3 fertilize themselves. The mutation occurred long ago, causing “perfect flowers” to be had. (The North American native vines do not have “perfect flowers”.)

During the last century, interest in producing new varieties by deliberately pollinating the flowers of on cultivar (varietal) with another. European botanists imported American grape cuttings to use. Unfortunately, American grapevine pests were inadvertently introduced into Europe. First, they imported powdery mildew. V. vinifera has no resistance. It swept through Europe. Next, the grape louse vastatrix. The eventual solutions were sprays and American . American rootstock was sent and Phylloxera was controlled. Some of the rootstock was infected with downy mildew and black rot, causing new problems.

COMPOSITION OF WINE Compounds Grape % Dry wine % Water 70-85 85-93 Carbohydrates () 15-25 0.1-0.3 Alcohols 0.0 7-15 Organic Acids 0.3-1.5 0.3-1.1 Phenolic compounds 0.05-0.15 0.05-0.35 Nitrogen compounds 0.03-0.17 0.01-0.09 Carbonyl compounds 0.0 0.001-0.050 Inorganic compounds 0.3-0.5 0.15-0.40

Carbohydrates Approximately 90% to 94% of soluble solids are sugars. The non-sugar portion of soluble solids consists of acids, salts, tannin, coloring material, , etc. The soluble solids in grapes and must can be estimated with refractometers or hydrometers. The units used in the U.S. is ° or °Balling. 1° Brix is 1 g per 100 g of solution. Refractometers measure refractive index, a mediums’ bending of light. The refractive index changes rapidly with temperature. Temperature corrections should be made. A 20g/ 100 ml reading at 68°F, would read about 20.45 at 56° F and 19.45 at 80° F. Since only 2 or so drops are used ion a “refract”, the sample error may be a problem. A hydrometer is a floating instrument which indicates the specific gravity of the liquid in which it floats. Temperature corrections must also be made with hydrometer readings. Most hydrometers are calibrated at 68°F. The approximate correction is + 0.03° Brix for each °F above the calibrated temperature and -0.03° Brix for each °F below the calibrated temperature. Make sure the hydrometers are clean and float freely. Generally, refractometers are used in the and hydrometers at the . Alcohol on must and wines affects readings of hydrometers and refractometers differently. A 12% alcohol in a water solution gives a -4° Brix reading with a hydrometer, but a + 4° Brix reading with a refractometer. Extracts (these are nonvolatile

4 materials in wine, which is basically everything but alcohol) contribute about + 2° Brix, so dry wines give about -2° Brix readings with a hydrometer and + 6° Brix reading with refractometer.

If a fermenting wine has a reading of 19 ° Brix at 53°F, the temperature difference vs. hydrometer calibration would be -15°F. Multiplying 15 times -0.03° Brix, would give -0.45° Brix. So the actual record should be closer to 18.5 ° Brix at 53°F.

A very common problem in the winery occur every harvest. Once wines are fermenting, the progress and soundness of each fermenter is checked several times a day. A lab tech or a winemaker will go from tank to tank taking “temperatures”. With , clipboard, hydrometer, a cylinder and sampling device, each tank of fermenting wine will be sampled in the cellar. Recording is made of each tank as to its temperature, sugar and general health, e.g., clean, high foam, sluggish, sulfide, etc. The sample of fermenting juice is taken from the tank and poured into the cylinder. Once the hydrometer is plunged into the cylinder, the foam makes reading the hydrometer nigh unto impossible. The hydrometer must be plunged up and down to get the foam to dissipate. Many times so much plunging takes place that a bit more sample is needed. I’ve had some high foam tanks where two or three extra samplings were needed until the fermenting wine was stable enough to be read. After the measurement the sample is tasted.

Fermentable sugars are 6- reducing sugars (named this because they are able to reduce the alkaline Cu+ + ion in Fehling’s solution, which is a reagent used in analytical work as a test for sugars) containing one of the following reactive groups: O O OH II II I -C-H -C-CH2OH -C-O-C- Terminal aldehydes α-hydroxy hemiacetal The main sugars in grapes are and (reducing sugars). At maturity, the ratio (glucose:fructose) ranges from 0.7:1.0 to 1.5:1.0…normally equal. Fructose is approximately twice as sweet as glucose and 1-1/ 2 times sweeter than sucrose (table sugar). When sucrose (non-reducing) is added to juice to raise the sugar (), it is hydrolyzed ( is the interaction between a salt and water) to two simple sugars, glucose and fructose, aided by acids in the juice or enzymes in the yeast.

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Dry wine contains less than 0.1% reducing sugar (often non-fermentable sugars like ). Detectible sweetness varies with grapes and tasters. In low alcohol whites, threshold is about 0.4% and in some reds it’s > 1.5%.

The riper the grapes, the more non-sugar solids (extracts) remain after fermentation, including organic acids, minerals, , , etc. Extract in wines range from 0.7% to > 3.0%. Average white wines are about 2% and red about 2.5%. Most important polysaccharides in grapes are , which consist of galacturonic acid and methyl galacturonate chains crosslinked with various sugars. Pectin ranges from 0.02 to 0.06%, but normally is around 0.15%. Pectins are higher in V. labrusca than V. vinifera. Gums and mucilages made of complex chains of sugars are also present. Pectin-splitting enzymes are found in grape skins. Sometimes winemakers will add commercial pectic enzymes. Pectins and gums exist in as negatively charged colloidal particles…tending to form clarification resistant hazes. Very tight filtration can remove these particles. This tight filtration may, however, strip the wine. Grape skins are covered with a thin wax like layer known as cutin (called bloom). It consists mostly of oleanolic acid and caryophyllin (C10H16O) 3, which is a triterpene also found in clove oil.

Alcohols In 1810, Gay Lussac discovered the general chemical equation for the breakdown of sugar into and :

C6H12O6 (glucose & fructose) 2CH3CH2OH (ethanol) + 2CO2 (carbon dioxide) A chemist by the name of Louis Pasteur discovered that when a combination of specific salts were incorporated into the process of fermentation, the reaction proceeds at a faster rate; this combination of salts became known as Pasteur’s salts. Pasteur’s salts solution consists of 2.0 g potassium , .2g phosphate, .2g , and 10g in 860 mL of water.

Theoretical alcohol yields are 51.1% by weight and 59.0% by volume. The alcohol yield is lower if fermentation is not complete and if temperature is too high. In those cases, more energy is used by yeast for growth rather than fermentation and the more vigorous loss of CO2 pulls EtOH with it. 6

Rules-of-thumb: Whites °Brix X 0.59 = % alcohol by volume Reds °Brix X 0.54 = % alcohol by volume

Yeast or HAc, in the presence of O2, can metabolize EtOH, especially in open fermenters. With high surface/ volume ratio in fermenters, lot of EtOH is lost. Usually 7% to 14% of EtOH is formed, but 18% has been reached. Alcohol influences wine fragrance, taste and body. A 4% aqueous solution can taste as “sweet” as a 2% solution of glucose. Alcohol can enhance the apparent sweetness of sugar solutions. Alcohol, especially in red wines, can hide tannin bitterness. At alcohol below 10%, red wines can taste thin and bitter. Tannin increases the threshold of sweetness detection and reduces apparent sweetness. Alcohol > 14% can also be “hot”. An alcohol-water solution has a greater viscosity and more body than water alone.

Other alcohols found in wine, besides EtOH, generally come from splitting of pectins by enzymes and not from fermentation. or methyl alcohol (CH3OH), usually is less than 200 ppm (1 ppm is 1/ 10,000 of 1%) and has little flavor. Some longer chain alcohol are formed during fermentation and give wines complex bouquets; isopropyl alcohol or fusel oils (these are alcohols with more than 2 ), isobutyl alcohol (50-200 ppm in wine) and amyl alcohol (in distillation, problem called “tails”). 35% of higher alcohols arise from carbohydrates and the balance from amino acids. 2-phenylethanol smells like and is prevalent in the muscadine variety of southeast U.S.

2-phenylethanol Some wine alcohols are polyols or polyalcohols, having more than one –OH (hydroxyl) group per molecule; e.g., which is sweet and viscous. OH I HCH I HCOH I HCH I OH Glycerol

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Normal has 0.5-1.5% (wt) glycerol and tasting thresholds are approximately 1.5% and wines average 0.7%. Fermentation temperature, yeast, pH, initial °Brix and aeration all have an effect on glycerol production. Large additions of SO2 to juice prior to fermentation increases glycerol production. Red wines usually have higher levels of glycerol than white wines because of higher fermentation temperatures. But, generally it is too low in concentration to be noticed in taste or viscosity.

2,3-butanediol is another sweet tasting polyol. Generally, about 500 ppm in wine. Sorbitol, a sugar alcohol, is very low in concentration in wine. Mannitol, produced by lactic acid bacteria, is present in bacterial spoilage. Normally appears with high temperature stuck . can also cause increases in Mannitol. The Discovery of Evaporative Convection In 1855, James Thompson wrote a letter to the Royal Society in Great Britain titled, “On Certain Curious Motions Observable at the Surfaces of Wine and Other Alcoholic Liquids”. What Thompson had seen in his glass was evaporative convection driven by unbalanced surface tension. In a partially filled wine glass, a film of wine wetting the inside surface of the glass will writhe, shrink into droplets, and run down in what appears to be “legs”. This display is caused by a surface tension engine.

Surface tension are the forces acting in the surface of a liquid which govern such phenomena as wetting solids by liquids, capillary rise of liquids in tubes and wicks and curvature of a free liquid surface as in a titration burette or mercury thermometer.

Water in a tube. Mercury in a thermometer. The surface tension is not the same in different liquids. It is about three times greater in water than alcohol. At a wine surface, the alcohol in the wine evaporates more quickly than the water. The concentration of water is higher at the point where the surface wine touches the glass. Because of that, the surface tension of the wine right at the surface exceeds that of the bulk wine beneath. As the wine evaporates, it also removes heat from the surface region, thereby reducing the surface temperature below that of the bulk of the wine. For virtually all liquids, the surface tension rises as the temperature falls. Evaporation, by reducing the surface temperature, thus raises the surface tension.

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Any liquid whose surface layer has a tension higher than the value of the bulk is potentially unstable, because the potential energy of the surface is not at the minimum. The evaporating liquid is unstable with respect to surface forces, and tends to exhibit surface tension driven natural convection in seeking to rearrange intself into a more stable configuration. Wine will, because of surface tension, climb the inside of the glass. As it does, more alcohol wil evaporate, the temperature will fall and the surface tension will rise, and the wine will climb…until gravity dominates and the wine falls in legs. Organic acids Contain a double bonded (O= ) atom and a single bonded hydroxyl (OH-) group, attached to the same carbon (C-). This is a carboxyl group. H-O-C= O or -COOH I

Acids give wine their characteristic tastes, clean and lightly tart. Other wine components moderate the acid and give balance in alcohol, sugar, minerals, tannins, etc. The most important are below.

Tartaric Acid Lactic Acid C2H2(OH)2(COOH)2 COOHCH2CH(OH)COOH CH2CHOHCOOH CH2COOH Tartaric acid is normally > 50% of the total acidity. It is the strongest acid. It is not respired during grape ripening, as is malic acid.

Reminder: Respiration releases the energy required to activate the chemical changes that occur in the vine. Sugar and other compounds are transformed into simpler substances. The equation for respiration is:

C6H12O6 + 6 O2 6CO2 + 6 H2O + energy Sugar oxygen carbon water dioxide This reaction is the reverse of the photosynthesis reaction. In photosynthesis, energy is stored; in respiration, energy is released. Respiration is an oxidation process activated by enzymes.

Grapes grown in warmer regions usually have higher tartaric: malic ratios. Tartaric, and it K-salts largely control wine pH (effective acidity), which affects color, bacterial resistance and taste. As K moves into grape berries, tartaric acid is partly converted from free acid to salts, largely Potassium bitartrate (KHT). More than half is in that form at harvest. Since KHT is less soluble in EtOH than H2O, during fermentation, some precipitates. 9

Wine Crystals: Wine Diamonds

On occasion, when a bottle of wine is opened, you will notice a crystalline deposit that looks like a cluster of coarse salt at the bottom of the bottle or possibly glistening at the end of the . The clear diamond-like crystals found in wine are potassium bitartrate in crystalline form. This is basically the same stuff as the cream of tartar in your kitchen pantry.

The principal acids found in grapes, and hence wine, are tartaric and malic acids. These acids are produced by the grape as it develops. Potassium also exists naturally in grapes. Some of the potassium and tartaric acid form KHT. When the wine is under chilly conditions, more of the tartaric acid and potassium bind together. At lower temperatures, the KHT is less soluble. The marriage produces potassium bitartrate (KHT) crystals: wine diamonds. They are completely harmless and quite natural. In Europe these wine diamonds are accepted as a sign that the wine is a natural one. To many these diamonds are even appreciated. Americans, however, are used to wine being without the crystals.

The winery, in order to fulfill the consumers’ expectations, forces a pre-crystallization at the winery. This process is called “cold stabilization”. It beats the crap out of the wine. The wine is chilled to just above freezing; generally 23º F. It is held at this temperature for about two weeks. During that time, the KHT is formed in the wine. In order to remove all the KHT, the wine is filtered at this cold temperature. The KHT, and host of other things, are removed from the wine at this cold temperature. During this handling, the wine also becomes saturated with oxygen. That’s a no-no. After the cold filtration, the wine is then run through a heat exchanger to bring it up to cellar temperature. But the wine must now be sparged with nitrogen gas to remove the oxygen, and again, a few other aromatic compounds. Any winemaker who has ever tasted any wine before and after “cold stabilization” wants to cry. If the winemaker is patient, this same removal of KHT will occur over a longer period of time at cellar temperature. Most red wines left in barrels for 12 months or more are not subjected to this abuse. Some do not want any of their wines to become “cold stabilized” in a hurried fashion. They educate their market. Bravo!

So, let’s marvel at these little wine diamonds. If you’re lucky to find them, raise your glass and enjoy the wine with the assurance that this wine has been handled as naturally and gently as possible and allowed to attain the highest quality.

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At high temperature in the vineyard, malic acid is respired. In warm climates it may comprise 10% to 40% of total acid, but up to 70% in cool climates. The malic acid may later be reduced to even weaker lactic acid by (MLF). Lactic acid is a minor byproduct of fermentation and is generally < 0.1% in wine. After MLF, it can be up to 0.6%. It has a mild sour taste. It is also found in buttermilk, sour cream and cheese. Sometimes it can smell like spoiled or as a result of undesirable ML bacteria. is also in very small amounts. It is added by some winemakers to acidulate wine. It is not as tart, nor “clean” as tartaric acid, but it does not create the precipitation in the bottle risk that KHT can offer. It is also used to complex with (Fe) to prevent Fe- phosphate haze in wine. This is very rare with use of stainless steel in wineries, now. , a minor grape acid, increases during fermentation at 1/ 100the the rate of alcohol increase in wine to about 0.1%. Acidity and pH The two most commonly measured aspects of juice and wine acidity are pH and titratable acidity (TA).

The TA has no known effect on chemical or reactions or microbial activity and is basically important only for sensory perception of finished wines. It is thought this is because of the partial titration of the acidity in wine by the saliva in the mouth. Saliva is slightly basic, containing mostly bicarbonate ions. The presence of wine, causes the saliva flow somewhat in proportion to the quantity of neutralization required and this is generally correlated with TA. Individuals may be low and high salivators and the flow varies.

The pH, on the other hand is an indication of the extent to which the acid mixture has been neutralized during grape maturation and acidity adjustment. It is not correlated with the amount of acid present but is more influenced by the acids strength.

The TA measures the “acid” population, the pH the “acid” strength. A wine’s total acidity only gives the sum of the free acids without taking their strength into account. The actual acidity, or concentration of hydrogen ions (H+ ), represented by the pH, relates not only to the quantity but also to the strength of the acids. There are strong and weak acids. The acid strength is represented by their dissociation constant, i.e., the proportion in which they liberate H+ . In direct value, the concentration of H+ ions in wines ranges from 0.001 to 0.0001 g/ l. A more convenient way of expressing these numbers is by using a logarithmic value. The pH is exactly the co-logarithm of the concentration of H+ ions.

This discussion will only consider the major compounds influencing acidity in grapes, malic and tartaric acids and their potassium salts. Other quantitatively less important organic acids, such as acetic, succinic, citric and lactic acids will not be discussed. Plus, 11 concentrations of minerals, such as sodium or calcium, and other cations will be passed over. Tartaric and malic acids are quantitatively the most important organic acids in grapes and wine. Tartrate is synthesized in the grape from carbohydrates, while malate is directly formed from CO2 in the tissue of young berries. Both acids are enzymatically degraded by the vine during ripening, but malic acid is metabolized more readily to generate energy and synthesize carbohydrates. The variety specific ratio of tartaric to malic acid increases in the final stages of maturity, especially in warmer regions and . We have discussed this in the past. Changes in acidity level, as they reflect grape , may be of value. Malate is consumed as an energy source during . Figure 4 shows how tartrate levels generally remain level during veraison, while Malate levels decrease.

During grape dehydration, tartrate levels may increase a bit.

Start with definitions… Organic acids in grapes. Chemical compounds are held together in a stable form as a result of a negatively charged anion bound to positively charged cations. Figure 1 shows that an is composed of a carbon group (the anion) and two hydrogen cations (H+ ). In a dried form, the molecule is stable, and tartaric acid, for example, is a crystalline powder. Inside a grape berry, the acid is partly “ionized” or “dissociated”. That is the ion and cation float freely in the juice. The degree to which this separation takes place is called “ionization”. The degree varies with the type of acid and the pH.

If one or both of the hydrogen cations are exchanged with a , such as K or Na, the molecule is called a “salt”. Potassium bitartrate has great importance to winemakers. This is the K salt of tartaric acid, known as cream of tartar. 12

Acidity measurements…Total acidity. This is a measure of the combined quantity of organic acids and their salts in a grape juice sample (Figure 1). Total acidity, as shown in Figure 2, accounts for all combinations of anions (tartrate, malate, etc.) and their corresponding cations (hydrogen or potassium). Because of the required sophisticated analytical techniques, wineries generally do not measure total acidity.

Acidity measurements…Titratable acidity (T.A.). This measures the total available amount of H+ cations in solution. It is easily determined by titrating a grape juice sample with alkaline sodium hydroxide until a certain pH is reached (pH 8.2 is used in the U.S.). T.A.’s in juice range from 5 to 16 g/ l, expressed as their tartaric acid equivalent. This titration only accounts for the free organic acids and one-half of their salts, depending on the extent of exchange. See Figure 3.

Extent of exchange. The percentage to which hydrogen has been replaced by potassium is the “extent of exchange”. Generally, about one quarter to one-third of the hydrogen ions are exchanged with potassium. This portion will not show up when measuring T.A. Figure 4 is an example of a titration curve. It goes from pH 3.4 to pH 8.2. We can see that some of the acid was already neutralized by the potassium present in the grapes. How this exchange takes place will be later explained.

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Acidity measurement…pH. pH is a measure of the free H+ ions in solution. pH is inversely related to the logarithm of the concentration of free hydrogen ions. (H+ ) This means the lower the pH, the more H+ are present and the more acidic the sample. This value depends mainly of three factors: 1. The amount of tartaric acid that has its hydrogen ions exchanged against potassium. See figure 5. The more K+ has replaced H+ , the higher the pH. Less H+ ions.

2. The ratio of tartaric to malic acid. Both acids have two hydrogen cations, tartaric is the stronger acid because it ionizes more freely at a lower pH. Thus, a higher ratio of tartaric to malic acid in juice will result in lower pH. 3. The relative ionization of malic and tartaric acids. Acidity measurement…Potassium (K+ ). Potassium is a mineral essential to plant growth. Levels of potassium in California grapes range from 560 to 2,785 mg/ l (average 1,850 mg/ l). Grape skins can carry up to 9,000 mg/ l in the outer layers. The skin, while contributing only 10% of the berry weight, contains up to 40% of the berry’s potassium. (Remember extent of exchange. White press , with more juice drawn from the skins and hence higher potassium levels, have lower T.A.’s than free run juice. Also mechanically harvested grapes, because of skin degradation and red wines because of prolonged , also have lower T.A.’s.) Acidity, pH and potassium play important interchanging and interfacing role s in juice and wine physical, chemical and microbiological stability. How important is T.A.? The two standard measures of juice or wine acidity are T.A. and pH. The T.A. has a greater impact on the way a juice or wine tastes than does pH. Perceptions of

14 tartness are dependent on T.A. As its T.A. goes up, a wine tastes tarter, but the microbes in the wine are unaffected by T.A. See Figure 6.

Aside: Perceived sourness in wine decreases with higher levels of residual sugar and alcohol. But, with increased levels of phenolics and related astringency and bitterness, sourness is enhanced. And how about pH? Looking at a wines microbial and chemical stability, the wines pH is its most important factor. The pH tells us how much of the added to a juice or wine is present as “molecular” or “free” SO2. This form of SO2 is the one that kills unwanted bacteria and yeast. The proportion of SO2 present as Free SO2 diminishes exponentially as pH increase toward 4.0. See Figure 7. This tells the winemaker that higher levels are needed to attain the desired FSO2 .

Aside: Why is pH so important? The lower the wine pH, the more effective is bentonite at removing haze-forming proteins. (Bentonite is a fining agent used for removal of unstable proteins.) Also, the wines color is more stable due to accelerated polymerization reactions versus oxidation reactions. Oxidative browning is slowed due to presence of free SO2 which scavenges oxidation products such as hydrogen peroxide. Wine are fairly insensitive to pH. But bacteria are tightly restricted in their growth and fermentation abilities by lower pH, even when SO2 is absent. Many California winemakers take pride in keeping wine above 3.60, feeling the wines have more flavor. Acidification has traditionally been recommended to produce balanced and microbiologically stable wines, but not to make wines in a “French” style. Remember, it is not the lowering of pH that influences perceived tartness, but increase in T.A. It is technically possible, but not legally permissible to reduce pH with inorganic acids, like

15 sulfuric acid (H2SO4). This provides higher amounts of free SO2, but does not significantly increase the T.A. or perceived tartness. Influence of Potassium (K+ ). In the vine roots, enzymes evenly exchange hydrogen cations (H+ ) with soil Potassium (K+ ). This exchange takes place increasingly after véraison. A transport system is instituted that brings nutrients up into the vine and fruit, and it appears that these nutrients are partly created when tartaric acid is turned into potassium bitartrate. Most plants perform the same way. The problem exists in heavily farmed areas where soil pH can decrease and related problems. Enzymatic transport of hydrogen from the berries and vine causes the grape juice left behind to become more neutral. See Figure 5 on the H+ :K+ transport.

Together with organic acids in the berry, this exchange determines the actual pH measured ion the must and resulting wine. A related problem with potassium exists. This is generally only faced by producers of sparkling wines. When grapes are harvested early, the pH may be < 3.0. Studies have shown that if the pH is this low and the potassium in the must is < 1,000 mg/ l, a sluggish or stuck fermentation may occur. Yeast requires the potassium to maintain a neutral pH within the cell. Viticultural influences. The fertilization practices in the vineyard and the potassium content of the soil only have an indirect weight on K+ concentration in the grapes and pH in the juice. Since nutrient uptake mechanisms are very complex and pH is not solely dependent on K+ concentration, an elimination of potassium fertilization will not automatically lead juices with higher acids. The vine health and fermentation performance, however, may both be negatively influenced. Once the exchange enzymes in the roots are saturated with K+ , fertilization and/ or soil potassium content have no impact on potassium accumulation in the vine. Actually, the rootstock itself has the greatest influence on potassium uptake. It has been shown that vines planted on specific can have twice as much bloom petiole potassium as vines planted ion their own roots. Also, areas infected with phylloxera or nematodes will have reduced potassium uptake activity in the root zone and soil moisture will also influence potassium uptake. Competition in the vineyard can help limit potassium uptake. Things like vine placement, spacing and crop level are important. A region where these things are widely controlled is

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Cognac in . To produce a wine that will be distilled, SO2 cannot be used. To make sure that the wines are microbiologically stable, low pH wines are needed. Also, much of Europe has very close spacing in their vineyards, generally a meter by a meter. In these cases, the close roots compete with each other for available potassium. Further, with more vines per hectare (2.47 acres per hectare), the available potassium is distributed over more grape cluster. While potassium deficiencies in grape vines can cause troubles in the vine and with yeast fermentation, many area yields low potassium and hence low pH grapes. Chalky soils, as exist in and Cognac, have low levels of potassium and high pH. These soils exhibit less exchange and therefore low pH, high T.A. juices. Warm climates give rise to rapid malic acid respiration. This causes a decrease in both Titratable Acidity and Total Acidity. Under warm conditions, with K+ being the same or higher than in cooler climates, the extent of exchange increases since there is less acid present. Since most of the acid reduction results from malic respiration, and also since tartaric is the stronger acid, the pH may change little even though the ratio between the acids changes. If there is a significant potassium exchange, the pH may rise. Or, to complicate the picture, the pH may fall if there is a faster production of malic and tartaric acids in the grape than mineral uptake. This latter condition occurs after rainfall or irrigation just after véraison. Berry size can fluctuate significantly over a short period. During ripening, a thermal expansion or water stress-related shrinkage can take place. Also, at night a water influx may occur resulting in expansion. Figure 8 shows the impact of berry expansion on pH and T.A.

Small increase in berry diameter lead to dramatic increases in berry volume and therefore lower total acid concentration and lower T.A. However, pH is determined by the ratio of tartaric to malic acid, as well as the extent of exchange of their hydrogen ions with potassium. Three pH and T.A. combinations are generally expected: 1. Over ripe fruit or hot climates lead to high pH and low T.A. due to acid respiration

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2. Early-harvested grapes or cool climates can give low pH and high T.A. grapes due to high levels of unexchanged organic acids. 3. In very long growing seasons in cool climates, one might expect both high T.A. and high pH due to large extent of exchange of accumulated acids with potassium. What about the winemaker? Delivering the same grapes, with identical T.A. and pH to several different winemakers will normally result in several different wines with different T.A. and pH. Various reasons cause these differences. 1. During fermentation, yeast utilizes most of the amino acids offered by the grapes. It also produces new organic acids, mostly succinic. The new organic acids are produced partly at the expense of the tarter malic acid, which the yeast utilizes. The CO2 produced during fermentation is an acid, also. The CO2 retained in solution, can noticeably increase T.A. 2. If malolactic fermentation (MLF) takes place, the conversion of L-malic to L-lactic acid and CO2 leads to a decrease in acidity. Since all the L-malic acid is used during MLF, it is important to know the percentage of acidity is due to malic acid before MLF occurs. MLF can be induced and also, if desired, prevented from happening. (Compounds that have the same molecular formula, but are different compounds as to how they react are called isomers. The reaction difference is because the identical atoms have different molecular structure. Isomers that differ from one another only in the way the atoms are oriented in space are called stereoisomers. Some isomers, because of their different chemical structure, will cause plane-polarized light to rotate in a right or left direction. These are dextr0rotatory and levorotatory substances. The symbols + and -, or d and l are used to indicate rotation.

L-malic acid D-malic acid

Stereochemistry has shown that DL-Malic acid has both. Only L-Malic acid occurs in grapes. The addition of DL-Malic to juice could have the L-Malic converted to L-Lactic acid in MLF. The D-Malic would remain. 3. Potassium bitartrate (KHT) is less soluble in higher levels of alcohol and at lower temperatures. Precipitation of KHT will usually occur during and after fermentation. The loss of KHT will reduce T.A. Any change in pH will depend on the starting pH. If the starting pH is less than 3.8, precipitation of KHT will cause the pH to fall. If the starting pH is greater than 3.8, the pH will rise. If the starting pH is 3.8, it will remain the same. (Note, depending on the alcohol level, the pivotal pH can be 3.6.) Acidification by addition of tartaric will cause a proportional amount of supersaturated KHT to fall out of solution, thereby increasing the T.A. and lowering the pH.

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After an acid addition or deacidification, most winemaker cannot easily predict or calculate the pH or T.A. because of these complex and often incomplete interactions. Too many other details on the acid profile are needed. In the real world, winemakers have several options. With the must or juice they can acidulate with tartaric acid or use hydrogen/ potassium cation exchange. Deacidification can happen with addition of potassium carbonate or anion exchange. The exchange columns are not very gentle on the juices.

The figures and much of the text was taken from “Acidity, pH, and potassium for Grapegrowers” by Christian Butzke and Roger Boulton in Practical Winery & Vineyard, September/October 1997.

Most acids in wine are nonvolatile and odorless. Acetic acid (HAc) is volatile. It is the main acid in . Wine exposed to 02 when in the presence of certain bacteria, form HAc and (EtAc) EtAc is an of HAc and EtOH. HAc gives much of the vinegar smell. EtAc smells more like nail polish remover. Both are measured as Volatile Acid (V.A.). When V.A. is > 0.1%, most consumers detect a vinegar smell. The amount of V.A. is usually determined by distillation and titration and is usually measured in grams of acetic acid per 100 ml.

CH3COOH CH3COOC2H5

Acetic Acid Ethyl Acetate

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Basic Winemaking and Enology - 2 COMPOSITION OF WINE Compounds Grape Must % Dry wine % Water 70-85 85-93 Carbohydrates (sugars) 15-25 0.1-0.3 Alcohols 0.0 7-15 Organic Acids 0.3-1.5 0.3-1.1 Phenolic compounds 0.05-0.15 0.05-0.35 Nitrogen compounds 0.03-0.17 0.01-0.09 Carbonyl compounds 0.0 0.001-0.050 Inorganic compounds 0.3-0.5 0.15-0.40

Phenolic Compounds Phenols and related compounds can affect the appearance, taste, fragrance, mouth-feel and antimicrobial properties of wine. They come primarily from grapes and stems, are produced by yeast metabolism, or are extracted from wood cooperage. This group includes the red pigments, the brown-forming substrates, the astringent flavors, and the bitter substances of grapes. The maximum average total phenolic content in: Red grapes is 5,500 ppm White grapes is 4,000 ppm Stems is 2,000 ppm ¼ to 1/ 3 of phenols are in skins, most of the rest is in the seeds.

Technically, phenols are cyclic compounds possessing one or more hydroxyl groups (OH-) attached to an aromatic ring. Some are readily oxidized and precipitate.

Two distinct phenol groups occur in grapes and wine; flavonoids and nonflavonoids. Both flavonoid and nonflavonoid polymers are termed tannins because of their ability to tan leather.

There are also compounds that do not possess one or more hydroxyl groups on the phenyl ring. These are not strictly phenols, but will be included in this discussion.

Examples are:

Gallic Acid Vanillin C7H6O5 C8H8O3 A non-flavonoid. Not volatile. Astringent. A non-flavonoid. Volatile. Vanilla aroma.

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Phenolic and Related Substances in Grapes and Wine General Type Example Major Source Flavonoids Flavonols Quercetin grapes Kaempferol grapes Myricetin grapes

Anthocyanins Cyanin grapes Delphinin grapes Petunin grapes Peonin grapes Malvin grapes

Flavan-3-ols Catechin grapes Epicatechin grapes Gallocatechin grapes Procyanidins grapes Condensed tannins grapes Nonflavonoids Benzoic acid Benzoic acid grapes & Vanillic acid oak Gallic acid grapes & oak Protocatechuic grapes & oak Hydrolyzable tannins grapes

Benzaldehyde Benzaldehyde grape, oak & yeast Vanillin oak Syringaldehyde oak

Cinnamic acid p-Coumaric acid grapes & oak Ferulic acid grapes & oak Chlorogenic acid grapes Caffeic acid grapes

Cinnamaldehyde Coniferaldehyde oak Sinapaldehyde oak

Tyrosol Tyrosol yeast Table from Ron S. Jackson, Wine Science Second Edition.

Flavonoids are characterized as molecules possessing two phenols joined by a pyran (oxygen-containing) carbon-ring structure. The most common in wines are flavonols, catechins (flavon-3-ols), and, in red wines, . Small amounts of free a Leucoanthocyanins (flavan-3,4-diols) also exist.

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Flavonoids may be free, polymerized to other flavonoids, sugars, or nonflavonoids, or be a combination of these. Those esterified (creation of fragrant class of compounds formed by rxn of an acid and alcohol) to sugars and nonflavonoids are called glycosides and acyl derivatives.

Polymerization of catechins and leucocyanidins produce procyanidins. Procyanidin polymers possess molecular weights ranging between 1,000 and 4,000 and have a distinctive -binding property. The tannin polymers are all astringent, bitter and precipitate with proteins (fining agents and natural). Seeds are the main source of these tannins.

The flavonoids (phenolpropanoids) make up 85% of the total phenol content (> 1,000 mg/ l) of red wines. In white wines flavonoids are less than 20% of the total phenol content (< 50 mg/ l). They come from seeds and skins, mainly. The skins have all the anthocyanins of the grape. Flavonoids are any of a group of aromatic compounds that includes many common pigments (as the anthocyanins and flavones); and non-flavonoids is a general term for phenolic compounds in wine not possessing the features of flavonoid molecules. The flavonoids are plant pigments, creating a rainbow of colors. In addition, many flavonoids function as and protect plants from damaging free radicals. They are also given credit for reduced heart disease in humans.

Flavonoid extraction during production depends on many factors. Besides what might come from wood cooperage, the upper flavonoid content depends on the amount in the fruit. This content varies with variety, climate condition and fruit maturity. Traditional fermentation, with cold maceration, pump-overs and extended maceration, extract more phenolic compounds than . Phenol removal also changes with pH, SO2 content, EtOH level, and fermentation temperature and length. Due to the multiple factors, no other major wine component shows greater variation. Furthermore, phenolic content of a wine changes more than anything else during aging.

Nonflavonoids are structurally simpler than flavonoids, but have a more diverse origin. In wines not aged in wood, the primary nonflavonoids are derived from hydroxycinnamic (most numerous and variable) and hydroxybenzoic acids. They are easily extracted from grapes during crushing. The hydroxycinnamic derived compounds occur esterified to sugars, alcohols and organic acids (mainly Tartaric). The Tartaric acid , in the presence of pectin esterases (enzymes) are broken down to their monomers (a low molecular weight carbon compound that combine to form polymers). One of these, o-diphenol caftaric acid plays an important role in oxidative browning and phenolic polymerization in must. In small amounts, one of the oxidative derivatives of caftaric acid and coutaric acids provide much of the straw yellow-gold color in some white wines.

The non-flavonoid phenols provide no flavor, but some fragrance. They can be volatile (tyrosol, syringaldehyde or vanillin) or nonvolatile (gallic acids). These come largely from grape juice but also from wood and action of mold, bacteria and yeast. They are present in juice and formed during fermentation and hence, their level is less sensitive to processing conditions than other phenols. Volatile phenols are usually below odor thresholds (1-50 ppm), but may contribute to odor through additive or synergistic effects. A number of hydrolyzable tannins come from wood. These are extractable phenols with taste thresholds about 7-15 ppm.

Phenolic acids are broken into hydroxycinnamates and the hydroxybenzoates. The important hydroxycinnamates in grapes are derivatives of caffeic, p-coumaric and ferulic acids. They occur mainly

3 in the free running juice and are about the same in white and red wines. Their actual form in the grape is as esters on tartaric acid – caftaric, coutaric and fertaric acids. The hydroxycinnamates are important in giving white wines a desirable gold color. The hydroxybenzoates are products of mold action in wines. The most important is gallic acid. Ordinarily low in juice, gallic acid increases with contact.

Wines matured in oak possess high levels of hydroxybenzoic acid derivatives. After a series of interactions, polymers of ellagic acid are formed. Esters of these acids may enhance red by forming copigments with anthocyanins. They are also active consumers of oxygen in wines aged in small wooden cooperage.

Anthocyanin pigments. In flowers and fruits, pigments exist as brilliant red and blue colors and are significant pigments in red wines. In grapes, anthocyanins predominantly exist as glucosides. Glucosides are formed through bonding of flavonoid components, called anthocyanidins, with glucose. The sugar component increases the chemical stability and water solubility of the anthocyanidins. Further complexings can take place. Young reds have 100-500 ppm total anthocyanin content.

Grape anthocyanins are divided into five classes; cyanins, delphinins, malvins, peonins and petunins. Amounts among classes vary widely with variety and growing conditions. The proportion among anthocyanins influences hue and color stability.

Free anthocyanins disappear as pigments condense and precipitate. The wines can also be discolored by SO2 and increased pH. The major anthocyanin in V. vinifera is malvidin. With one glucose unit attached it is called malvidin glucoside. Grape hybrids have two glucose units are diglucosides (a good ID for vinifera vs. hybrids). Other flavonoid phenol exist; anthocyanogens, catechins, flavonols (including light-yellow anthoxanthin pigments) and flavones. Flavonoids (mostly in skins) provide flavor in red wine and are usually present at 5 to 10 times threshold level. Because of low or no skin contact, they are essentially absent in white wines. This might give a reason for maceration. Catechins (a major anthocyanin) are bitter, but not astringent. In white wines they can be 100-200 ppm and 1,000 ppm in reds. The intermediate and higher tannins (polymers of catechins) are astringent, but less bitter. Tannin (can tan hides to leather) are divided into hydrolyzable (esters of gallic or ellagic acid) and non- hydrolyzable. They combine with protein and can be “fined” with gelatin, egg white or other protein fining agent. During fining, the insoluble tannin/ protein complex settles, carrying other particles too. Other bitter particles (like gallic acid) can be fined out. Flavonols and flavones are very minor in white wines, but can be 20-100 ppm in reds. Leucoanthocyanins (mostly in Reds) provide astringency and aid in fining. Pigments and tannins, though not very soluble in cold water are more so in hot water and alcohol solutions. Early in red fermentation, tannins are precipitated by grape protein or yeast and at this stage the major phenolic compounds are pigments. Once the proteins are gone, tannin enters and remains in the wine. Cold maceration will be discussed later.

Copigmentation is a very old winemaking method that only recently been widely studied. The question is how to uncover the complex relationship between the color we see in a glass of wine and its red pigments, the anthocyanins. The fact that the amounts of perceived color and measureable pigments in young red wines do not directly add up, has not been previously understood. Now we know that grapes contain varying amounts of invisible compounds in their skins, copigments or cofactors, that can make the red pigments

4 appear darker than they would by themselves. These compounds can also make the anthocyanins soluble in wine and hence more stable. It is now known that copigmentation can account for between 30% and 50% of the color in young red wines and it is primarily influenced by the levels of the noncolored cofactors.

Copigmentation is the enhancement of color of natural plant pigments by presence of non-colored components. It results from the “stacking” or aggregation of pigment molecules with molecules in solution. This involves the anthocyanin glucosides and certain phenolic acids, flavonoids, and other groups. The potential color enhancement is fixed for a given pigment-cofactor pair and the observed color in solution depends on the concentration of pigments and other factors.

This phenomenon not only causes an increase in depth of “redness” in the wine but also a shift in the hue from a red to a blue dominance. It has been thought it was polymeric phenols, aka tannins, in wines that kept the pigments in solution and that by extracting more tannin from skins and seeds more color could be extracted and retained in the wine. Various forms of maceration are employed. It is now known that the level of pigment that a wine can actually hold is preset by the quantity of cofactors in the grape and the amount of copigmentation that develops. Further, the maximum color is typically observed by the fourth to seventh day, depending on variety. Many studies have shown that additional contact time between skins and wine cannot provide additional content of color.

While some tannins are found in the skins, much higher levels are found in the seeds. Over half of these tannins are extracted during a normal 5-day red wine fermentation. The tannins continue to be extracted during extended maceration. This is long after the extraction of phenols and tannins from skins has ceased. These seed tannins are typically bitter. They play no role in color enhancement.

There are some weak cofactors in the juice of most grapes. When stronger cofactors are present, the weak ones are pushed aside. More interestingly, there are some white grapes with stronger cofactor in their juice than some red varieties. It is not surprising that some white skin grapes have been added to certain red grapes prior to fermentation. This has been happening for centuries in , the Rhone and Burgundy. As wines age, the pigments are converted into red and brown polymers due to oxidation and condensation reactions. Copigmentation is thought to favor the amount of red polymer that can be formed by protecting pigments against competing browning reactions during aging.

Several monomeric phenols exhibit coarseness in the form of astringency or bitterness. It used to be thought that only polymers of phenols (i.e., tannins) had these properties. It is now clear monomers are also involved. It is possible that there is a hiding of these astringent and bitter phenols in the copigments stacks. The “co-fermentation” of white skins with red grapes may also lead to changes in mouthfeel that are positive. This is quite apart from any color enhancement.

There appears to be a minimum amount of anthocyanin before copigmentation is detectable. Most red wines are expected to be in the concentration range of significant copigmentation while most white and blush wines are not. That is why the blue and purple tones are absent in blush wines, yet red wines made from the same variety often display this trait.

Color enhancement with copigmentation has been found to be between two and ten times that expected from pigment alone with typical values being four to six times.

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The role of cofactors and copigmentation during skin contact and fermentation of wines can be seen as twofold. First, the binding of free anthocyanins into copigmented forms enables more pigment to be retained, resulting in higher total anthocyanin content in the wine. Second, the copigmented anthocyanins provide much more color than they would have if they were in the free form. Wines made from grapes low in cofactors will not be able to form much copigmentation and will have low pigment contents. This seems to be the case for and and is why sometimes poorly colored wines result fro darkly colored grapes. Such wines are generally cherry-red when young and show no signs of purpleness. Other wines, from grapes richer in cofactors, will form more copigmentation, capture more pigment, and have deeper color with blue and purple tones characteristic of significant copigmentation.

Evidence suggests an equilibrium based on adsorption-desorption is established between the concentrations in the wine and the cellular concentration in the skin tissue, with significant reductions as the EtOH content increases. The role of copigmentation is to shift pigments out of the free anthocyanin pool of the adsorption equilibrium, causing more pigment to move from skin into the wine. The extent to which this occurs now seems to be limited by the concentration of certain cofactors in the skins at harvest and their solubility under wine conditions.

The existence of an extraction equilibrium is easily demonstrated by taking skins taken from red grapes after the maximum pigment concentration has been attained and putting them into a white juice. While the pigments are not able to move into the wine from which they were taken, where an equilibrium concentration has been reached, they will readily move from the skin tissue into the new juice until a new equilibrium is established.

Cofermentation of white and red grapes has been traditionally practiced in parts of Europe. In France, white grapes are added to red fermenters in Burgundy and the Rhone. In Chianti a six year study in the 1980’s showed the best color enhancement of one-year-old Sangiovese wines occurred when and comprised 5% to 15% of the grape mix and a further 10% was from . The Sangiovese is a red grape and the other three white.

A common challenge for winemakers is to understand the color exhibited by different wines and that displayed when they are blended. This is especially true when they are young wines in which most of the color is accounted for by the free and copigmented anthocyanins. New equilibrium may be found.

It is also known that the extraction of pigments from grape skins either prior to, during, or following fermentation is not complete. There are still 30% to 40% of anthocyanins remaining in the crushed skins. We have a long way to go. Much of the Copigmentation discussion was taken from work done by Roger Boulton at UC Davis.

The Role of SO2 in extraction of pigments is important. Cold maceration is a process used more for Pinot noir than any other red variety. It involves holding crushed grapes at temperatures below 10 °C (50 °F) for up to a week prior to fermentation. Levels of SO2 of from 0 to 150 mg/ l are added. The cooling and SO2 addition prevents fermentation from starting during the maceration period. The main reason for the cool temperature is to help control enzyme and microbial activity. Oxidative enzyme activity and microbiological effects would be expected to have a negative influence on quality. The SO2 helps in this regard too, but it is added to act as an extraction agent to break down grape cell walls and bind to anthocyanins and other phenols. The ability of SO2 to extract anthocyanins has been known for some time.

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It should also be noted that the amount of in wine after fermentation is directly proportional to the amount of SO2 added prior to fermentation. Even if acetaldehyde is bound, it can have a long term effect on wine quality and stability. As fermentation progresses in the must after it is warmed and inoculated, much of the SO2 bound to anthocyanins may be released to bind preferentially with acetaldehyde. This frees up the bound anthocyanin ions. Therefore, the bleaching effect of SO2 must be balanced with its extractive properties, and possible longer effects of acetaldehyde levels should be considered when choosing the cold maceration method. A good balance appears to be a moderate addition of 50 mg/ l of SO2 as it provides greater extraction compared to no SO2 is added, but gives more color due to a lower bleaching effect when compared with prefermentation addition of 100 mg/ l of SO2. This sulphur addition is adequate to control free acetaldehyde during fermentation and it does not lead to high total acetaldehyde production levels. Yeasts produce acetaldehyde in response to SO2 addition.

Nitrogen compounds. Nitrogen has a chief importance in stimulating yeast and bacterial growth. It occurs in (NH3), nitrates (NO3), amino acids, peptides, proteins and . The average total nitrogen content of grape must is about 600 ppm. Examples:

(NH4)2HPO4

Diammonium phosphate (DAP) Proline ()

Ammonia appears in must and wine largely as ammonium ion NH4+ . In must it averages 125 ppm and ranges from 5-175 ppm. The N content of soils and its form have great influence on must NH4+ content. Yeast use up much of the N during fermentation. NH3 content in wines is generally near 10 ppm. Some grapes lack N. DAP or urea are used to increase N. Some evidence shows N increases can improve quality. However, added NH3 can increase certain amino acids, especially histidine which may be undesirable.

Amino acids, especially proline which averages 500 ppm, are present in must and wine. Yeast produces amino acid from the ammonium and sugar in the must. The natural amino acids in must stimulate yeast growth and increase the fermentation rate. During yeast growth, many amino acids decrease. Some are needed for development of lactic acid bacteria and hence MLF. ML and other lactic acid bacteria form physiologically active in wine (tyramine causes increases in blood pressure and does the opposite when consumed.) Some people can get stuffy noses or other allergic reactions from red wines and it’s often blamed on histamine.

Proteins, complex chains of amino acids, are found in grapes and some remain in wine. Proteins can be around 400 ppm in wine. They precipitate during aging. The precipitation is increased at certain pH values and can be caused by blending resulting in a pH shift. Protein haze shows up in white and pink wines. This is called a heat unstable wine. The haze results from a complex of protein-phenolic compounds. Proteins can be reduced by adding bentonite, heating grape must or adding small amounts of tannin to the possibly heat unstable wines.

Other nitrogen compounds, such as nitrates and nitrites, a variety of amines, vitamins, nucleotides and peptides (short chains of 2-4 amino acids) are found in very small amounts and are of little importance. 7

Determining the Nitrogen Content of Must. Yeast Assimilable Nitrogen (YAN) is a combination of organic or alpha amino nitrogen (NOPA) and inorganic nitrogen or ammonia. Both of these are measured in must. Low levels of YAN have been associated with sluggish fermentation and production of off characters, especially sulfides. Sluggish and stuck fermentations, coupled with serious sulfide formation, have become increasingly common and are often associated with deficiencies of yeast assimilable nitrogen in the must. However, excessive concentrations of certain nitrogen compounds have been associated with elevated levels of ethyl carbamate and other fermentation problems. Knowledge of nitrogen status is essential for effective fermentation management. Nitrogen compounds are essential macronutrients for yeast, and are required for cell growth, multiplication, and yeast activity. Analysis of only alpha amino nitrogen or only ammonia nitrogen does not provide an accurate indication of total nitrogen status for a given must. Ammonia is the preferred form of nitrogen for yeast nutrition. Wineries routinely supplement nitrogen deficient musts with at the start of fermentation to provide adequate nitrogen levels. Additional ammonia analysis and adjustments during fermentation may also be beneficial in minimizing the risk of stuck fermentations and sulfide formation. Ammonia results are expressed as mg NH3 per liter. These values may be expressed as NOPA equivalents by multiplying NH3 results by 0.82. It is a measurement of primary amino acids usable by yeast. NOPA does not include proline, which is not utilized by yeast, or ammonia. NOPA results are expressed as mg nitrogen per liter. The YAN guidelines were discussed in production earlier in the term.

Carbonyl Compounds. Aldehydes and are formed during fermentation under oxidizing conditions. Aldehydes have a double bonded oxygen atom and hydrogen atom attached to a C. Few are significant, but many are in wine. Ketones are less active but chemically similar to aldehydes. They contain doubly bonded oxygen atoms attached to a C. O O II Aldehyde I II I Ketone __ C __ H __ C __ C__ C__ I I Two of the aldehydes in wine are: H O I II Acetaldehyde CH3CHO H __ C __ C __ H I H

Hydroxymethyl furfural C6H6O3

Acetaldehyde is an intermediate product in alcohol production, but is mostly reduced to EtOH. In table wine at 50 ppm, it is an unwanted oxidation. In “oxidized” wines, like Sherry, a concentration of > 300 ppm is desirable. In water, acetaldehyde can be detected at 1.5 ppm. In wine, with SO2, the threshold is around 100 ppm. In moderate concentrations, it has a “nutty” bouquet.

Acetaldehyde is partly responsible for “bottle sickness”. This is an aroma found in recently bottled wines. It tends to make the wine smell dull. Usually disappears in a few weeks. This dulling effect can happen at

8 other times. Poor cellar practices can show high O2 pickup and creation of acetaldehyde. SO2 dissolved in H2O reacts with acetaldehyde to form a nonvolatile bisulfite complex. This removes the acetaldehyde smell and gives a fresher fragrance. This reaction is reversible. If the SO2 is depleted by volatilization or oxidation, the acetaldehyde smell can return. Some bisulfite reactions occur during fermentation and a high level of SO2 traps acetaldehyde and prevents its reduction to EtOH. The result is more acetaldehyde at the end of fermentation than is created in lower SO2 fermentations.

Acetaldehyde reacts with pigments in red wines. Its level in red wines is lower than whites or pinks. The reaction can intensify colors, especially violet.

Hydroxymethyl furfural is an aldehyde formed by dehydration of fructose, possible when wine is baked or grape juice is concentrated at too high a temperature. Some musts are heated and this is a good indicator. It has a caramel like odor. Up to 300 ppm is found in some , Madeira and other baked wines. It is also found in wines made partly from raisins like Malaga. Occasionally some “hang time” reds have a touch of caramel on the nose.

Diacetyl and , ketones found in wine with too much oxidation, are also formed by MLF. During normal fermentation, acetoin is 25-100 ppm and later decreases. Normally, is 0.2 ppm. Above 0.9 ppm it can smell like sour milk. Diacetyl is found in heated . Some MLF wines also have it. Look for buttery nose on wines.

C4H6O2

Inorganic compounds come from the soil. Organic compounds from living things.

Inorganic compounds represent about 10% of the sugar-free extract in wine. (Extract is the non-volatile dissolved components of juice or wine; includes but is not limited to sugar; contributes to estimates of density or specific gravity.) They consist of + charged metal atoms or ammonium group (cations) and a – charged element or group (anions). Common table salt is an inorganic compound consisting of sodium cation (Na+ ) and anion (Cl-).

What’s in wine? Average Wine Chemical Concentration Cations Formula (ppm) Potassium K+ 1,000 Sodium Na+ 80 Calcium Ca+ + 50 Magnesium Mg+ + 100 Iron-Ferris Fe+ + 2 Iron-Ferric Fe+ + + 2 –Cuprous Cu+ 0.15 Copper-Cupric Cu+ + 0.15

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Average Wine Chemical Concentration Anions Formula (ppm) Chloride Cl- 60 - - - Phosphate PO4 300 - - Sulfate SO4 700

Potassium (K+ ) is the most important cation found in things that grow. The levels of K depend on grape variety, soil, climate, time of harvest and a few other minor criteria. During alcohol fermentation a large amount of K precipitates (ppt) as Potassium bitartrate (KHT). The cooler fermentation and storage temperature causes white wines to have lower K levels than reds. Sodium (Na+ ) is common in soil. It is not taken up by plants as much as K is. Natural Na level in wine is + about 35 ppm. Certain wine additives, like NaSO4, increase Na levels as does ion exchange prior to bottling. Ion exchange replaces Na for K to ppt KHT. + + Calcium (Ca ) is common in soil and in wine. Sometimes Ca forms Calcium tartrate (CaC4H4O6) or Calcium oxalate (CaC2O4). Both ppt. If Calcium sulfate (CaSO4), or gypsum, is added to increase acidity or Calcium carbonate (CaCO3), basically chalk, is added to decrease acidity, the Ca increases. Magnesium (Mg+ + ) is generally not important. It can slightly influence tartrate stability and acid taste in wine. Iron (Fe+ + ) and (Fe+ + + ) content is usually 1 to 2 ppm, unless there is contact with iron in equipment or storage. Fe (mostly in steel) from equipment (pumps, filter, and fittings) used to be quite common, now it is rare in the U.S. During the primary fermentation, 25% to 80% of Fe is removed by yeast. If Fe> 7 to 10 ppm in wine, cloudiness may appear, or increased oxidation may occur. In wines kept away from air, 80% to 95% is Ferrous (Fe+ + ), but the Ferric (Fe+ + + ) state increases as wine is aerated. The ppt of ferric phosphate (FePO4) is seen as a cloudiness known as “white casse”. The addition of Citric Acid will form a soluble + + + Ferric citrate (FeC6H5O7) compound, eliminating the white casse. Fe can react with polyphenolic compounds and ppt as blue-black film, known as “blue casse” in red wines. Rare. Copper (Cu+ ) (Cu+ + ) is normally 0.1 to 0.3 ppm in must and wine. It can also be picked up from brass or copper equipment or fittings. In the vineyard, mixture is used to control downy mildew on native American vines. It is not a problem on the west coast. Bordeaux mixture (in French, bouillie delaise) is a fungicide spray made of copper sulfate and slaked (CaO). It gives French and German vineyards their characteristic blue-green tinge in summertime. Treatments with this fungicide can increase Cu. Wine with mercaptans and is sometimes treated with copper sulfate (CuSO4). This also increases Cu levels. Copper cloudiness can occur in wines with Cu+ + as low as 0.2 ppm. This is caused by a protein-copper complex. The main anions in wine are tartrate, malate, lactate, acetate, nitrate, chloride, phosphate and sulfate. normally come from the soil and yeast nutrients added to the must. Small amounts of are present in normal grape musts. During fermentation, some yeast reduces sulfate to bisulfite or hydrogen sulfide. - - - SO4 → HSO3 → H2S It is important to use low H2S producing yeast strains. Increasing sulfate may cause slight bitterness in the wine.

Odorous compounds Odor results from volatility. Aroma comes from grapes and bouquet from fermentation and aging. Together they form the wine fragrance or “nose”. Many compounds are at very low

10 concentration, even below threshold, but additive or synergistic effects can let even trace components influence fragrance. Volatile compounds include alcohols, organic acids and there esters, phenolic compounds and carbonyl compounds. The main compound in wine, water, is odorless. When we smell a wine, ethyl alcohol (EtOH) represents 90% of all the molecules entering the nose. High levels of EtOH mask other fragrances, but fortunately EtOH does not have a very strong odor and other things can be detected. Fruity smells are associated with esters. Esters are reaction products of organic acids and alcohols. Even aqueous solutions of organic acids and alcohols can react very slowly to form esters. Two important esters are:

From to vinegary. Foxy grapes character.

Ethyl acetate CH3COOC2H5 Methyl anthranilate H2NC6H4CO2CH3

Most esters come from grapes or are produced by yeast enzymes during fermentation. When yeast stops growing, most ester formation stops. Ester formation is favored when suspended solids are removed before fermentation (by settling or centrifuge), use of good yeast strains, maximum yeast cell growth and high fermentation temperatures. The problem with high temperature fermentation is that the increased amount of esters produced is lost more rapidly by volatilization. A fermentation of 54 °F to 59 °F gives most esters in the final wine. Varieties of Vitis labrusca contain 0.1 to 1 ppm methyl anthranilate. This gives a “foxy” aroma.

Muscat and to a lesser extent , contain alcohols called monoterpene alcohols. Linalool, or linalyl alcohol, the major in , rises in concentration in the grapes after veraison. It also smells very much like the cereal Fruit Loops. C10H18O or (CH3)2C= CH (CH2)2C(CH3)(OH)CH= CH2

Cabernet sauvignon contains nitrogen compounds (pyrazines) also found in bell peppers. After prolonged bottle aging much of the character is gone.

Gases. Several gases dissolve in wine to some extent; O2, CO2, N2, H2S and SO2. The reduction/ oxidation () potential in wine or must depends mainly on the amount of dissolved oxygen in the wine. When O2 is present, components of wine or must can be oxidized; when absent, they can be reduced. Such reactions significantly affect aging and quality potential.

Oxidation occurs when an atom loses one or more electron. Its positive valence increases. Some other atom must take up the lost electron(s) and reduce in valence. This is called reduction. Oxidation is always accompanied by reduction. Each reaction by itself is called a "half-reaction", simply because we need two (2) half-reactions to form a whole reaction. In notating Redox reactions, chemists typically write out the electrons explicitly: Cu (s) ----> Cu2+ + 2 e-

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This half-reaction says that we have solid copper (with no charge) being oxidized (losing electrons) to form a copper ion with a plus-2 charge. Notice that we have a "balance" between both sides of the reaction. We have one (1) copper atom on both sides, and the charges balance as well. The symbol "e-" represents a free electron with a negative charge that can now go out and reduce some other species, such as in the half-reaction:

2 Ag+ (aq) + 2 e------> 2 Ag (s)

Here, a silver ion (silver with a positive charge) is being reduced through the addition of two (2) electrons to form solid silver. The abbreviations "aq" and "s" mean aqueous or solid, respectively. We can now combine the two (2) half-reactions to form a Redox equation:

We can also discuss the individual components of these reactions as follows. If a chemical causes another substance to be oxidized, we call it the oxidizing agent. In the equation above, Ag+ is the oxidizing agent, because it causes Cu(s) to lose electrons. Oxidants get reduced in the process by a reducing agent. Cu (s) is, naturally, the reducing agent in this case, as it causes Ag+ to gain electrons.

Wine is saturated at 8 ppm O2. This is dissolved oxygen. During yeast growth, the yeast uses all the oxygen. After fermentation, dissolved CO2 protects wine from excessive oxidation, through the first couple of . After that, O2 is absorbed and reactions occur. The O2 level is influenced by the amount of SO2, phenol (like tannins), ascorbic acid, iron, copper and other substances. Oxygen dissolves in must at the start of fermentation and accelerates yeast growth and hence fermentation rate. During red wine storage and processing, some oxygen gets in the wine during rackings, toppings, filtering and bottling. Wine can pickup over the course of its cellar stay over 1,000 ppm O2. If oxygen is introduced slowly over a prolonged period, it will be fine. After the slow oxidation and compound polymerization, oxygen should be kept away. For white and pink wines, oxygen should always be minimized.

Micro oxygenation Most young red wines contain a large amount of red colored anthocyanins. However, by their nature anthocyanins are a pretty unstable lot. They tend to want to react with other things and fall out of the wine, making it lose its color. Anthocyanins also readily react with SO2. When this happens the anthocyanin is effectively bleached. Here's where oxygen comes in. In the presence of oxygen, anthocyanins readily chemically combine with tannin molecules. These complexes are also colored but unlike free anthocyanins they are very stable and are resistant to bleaching. In the presence of dissolved oxygen the smaller tannin molecules themselves also join together (polymerization) to form long chain tannins. These are softer, more supple and far less bitter than the small tannins from which they were built. So if air is important to the polymerization of tannins, where does it come from? In a natural approach used by winemakers for centuries, red wine has been exposed to air during winery operations. Pumping it up and over the fermenting juice, and by from barrel to barrel are winemaking steps which as an aside help oxygenate the wine. In addition, the wine barrel naturally allows in air through the gaps in the staves, 12 and through the bung-hole when the winemaker opens up the barrel to top it up with wine after some is lost through evaporation.

This uncomplicated approach has one draw-back. Its effects are slow, unpredictable and un-measurable. Micro-oxygenation or Micro-ox is a process whereby a high tech machine accurately doses a wine with oxygen in the form of micro-bubbles over a relatively short period of time. The principal behind the technology is that the dose rate of the oxygen dialed up by the operator of the micro-ox machine is the same or less than the rate at which the wine uses it up in undertaking all the polymerization reactions. It is usually applied prior to malolactic fermentation (MLF) when the tannins and anthocyanins are more receptive to reacting with each other, and the sulphur dioxide levels are at their lowest.

Micro-oxygenation is a relatively new technique. The idea is simple. If maturation of wine in wood is the result of a leisurely infusion of oxygen, then why not bubble a very slow stream of oxygen into wine in a stainless steel tank. Throw in some bags of the oak chips of your choice and the expensive barreling operation can be eliminated. The dose of oxygen proposed is very small. Depending on the wine, anywhere from 1 and 10 mL 02/ L wine/ month is added.

The starting point depends on volatile sulfides, anthocyanin concentration, tannin concentration (how big is the wine?), end use of the wine and time line for the wine.

Post-MLF micro oxygenation attempts to impose control over how much oxygen a wine will be exposed to. One mL / L/ month is approximately what a new 225-liter barrel can deliver to a wine, including topping and racking. While oxygenation can be applied to wine as a rate more than 10 mL/ L/ month, a higher rate has a potential for generating compounds which react faster with SO2 than the phenolic compounds.

Not all wines should be targeted for micro oxygenation. First, the presence of sulfides must be considered. The rate of oxygenation may be set higher for a brief period to help eliminate these compounds. But, this could lead to problems. Second, the rate of oxygenation depends on how much color and tannin are in the wine. The bigger the wine, the higher the rate a winemaker might use. In contrast, the lower in color and tannin, the lower the rate of oxygenation. Third, what is the final market for the wine and when will it be needed for blending or bottling?

Experience has taught winemakers that a particular lot of wine requires, say, eight months for proper flavoring and maturation. Therefore, if the winemaker has the correct amount of oak in the tank and will micro oxygenate this tank at 1 mL/ L/ month, the wine will develop over eight months approximately the same as the wine would develop a barrel.

Micro oxygenation can help improve wines, especially when used in combination with toasted oak: 1. Stabilizes color. 2. Allows control of O2 supplied to the wine. 3. Builds middle body. 4. Minimizes vegetal character. 5. Puts a wine into balance. 6. Corrects slight sulfide problems. 7. Reduces dependence on barrels for flavor and aging.

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Many winemakers are searching for successful methods to minimize vegetal aromas and flavor characters in their wines. The combined use of toasted oak and micro oxygenation does appear to minimize vegetal characters.

Vegetal characters appear to be due to the combination of three primary components: 1) Isobutyl methoxy pyrazine and related compounds (e.g. bell pepper aromas), 2) cis-3-hexenol and related compounds (e.g. bright green, leafy aromas), and 3) Sulfides, methyl mercaptan and related compounds (e.g. asparagus aromas).

Micro oxygenation appears to affect two of the three components (sulfides and hexenols) through oxidation to minimize their contribution to vegetal characters. It is difficult to foresee a dramatic drop in the pyrazine component due to the components' stability and extremely low aroma threshold (in the low ppt).

Toasted oak used both in the fermentor and the tank for flavoring and aging appears to provide aromas and flavors to help mask vegetal characters. Toasted oak also provides compounds which will crosslink tannins, just as micro oxygenation will provide which will crosslink tannins.

The combination of these two sources of cross linkers should push tannins to form different structures. It may be possible that these crosslinked tannins will form a different source of compounds to interact with primary flavor compounds for that wine. Stronger or weaker interaction with the compounds will change perceived aromas and flavors of the wine, potentially explaining why we see less vegetal character in wines treated with toasted oak and controlled oxygenation.

The toasted oak is shown setup in this tank below. The control box is mounted at the top of the tank.

A Control Box makes data entry easy and displays the oxygen supply status at all times. From the Control Box, a flexible hose runs to an oxygen diffuser inside the tank. The diffuser is generally a micro-porous ceramic rod that generates extremely fine bubbles and ensures their rapid diffusion throughout the wine, while a protective steel cylinder surrounding the rod offers resistance to knocks..

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Carbon dioxide, CO2, is formed during fermentation. Saturated concentrations can be 0.1% to 0.3% at ambient conditions. MLF also produces CO2. CO2 in wine combines with water to form a sour taste caused by formation of carbonic acid. A “spritz” can be detected in the mouth at 0.05% to 0.06%. In whites, there is an increase in “freshness” of aroma at 0.06% to 0.12%. In , CO2 is dissolved and may complex loosely with protein or other compounds and hence is released more slowly when the cork is popped. Autolyzing yeast cells are probably the source of the compounds that complex with CO2 and sparkling wine when stored in contact with yeast (sur lie) for prolonged periods show finer bubbles. In production, CO2 “blankets” are used in tanks before filling with wine. Caution must be exercised to not make the still wine spritzy.

Nitrogen (78% of air) is more soluble in wine than oxygen, but much less than CO2. . N2 gas is used for stripping O2 by sparging wine.

Hydrogen sulfide (H2S) is produced by yeast during the reducing conditions of fermentation. Some yeasts, such as Montrachet, are higher H2S producers tan others. Late sulfuring in vineyards (after véraison) can contribute to H2S production. It is suggested that yeast starved for nitrogen containing nutrients can exude an extracellular enzyme that breaks down -containing proteins and amino acids and forms H2S. 1 to 2 ppb is detectable. Racking and aerating early can remove H2S. After several weeks it can form less volatile mercaptans and later oxidize to . These latter forms can be difficult to remove.

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Basic Winemaking and Enology – 3 Chemical Additives. Most additives to wine, such as fining agents, are removed before bottling. The only additives remaining in the wine are antioxidants and preservatives; SO2, ascorbic acid, sorbic acid and fumaric acid. Chemical structure is: SO2 K2S2O5 CH3CH:CHCH:CHCOOH Sulfur Potassium Sorbic Dioxide Meta-bisulfite (KMS) Acid

SO2 is a normal constituent in wine. Yeast metabolism has been shown to produce 12 to 65 mg/ l. Burning elemental sulfur has been used for vessel fumigation since Roman times.

Up to 60 mg/ l can be picked by wine from SO2 in fumigated barrel. This was the primary method of SO2 addition to wine until the 20th Century.

KMS reacts with grape acids to release SO2. Large wineries add SO2 gas, smaller use KMS or Sodium bisulfite (NaHSO3).

SO2 serves many roles. There are four reasons why people have used sulfur to protect wine for hundreds of years; anti- oxidant, anti-browning enzyme, anti- microbial and aldehyde binding activities.

Bisulfite salts rapidly ionize under the acidic conditions of must or wine, releasing gaseous . - 2- + SO2 + H2O H2SO3 HSO3 + H+ SO3 + 2H In wine, sulfur dioxide can exist in a variety of free and bound states. A very small % exists as free SO2 2- gas (about 2% of total sulfur dioxide). A small fraction also exists as free sulfate ions (SO3 ). Most of the - free ionic sulfur dioxide exists as bisulfite ions (HSO3 ) and the reminder of free sulfur dioxide exists as undissociated sulfurous acid (H2SO3). This is highly dependent on pH as well as on the concentration of binding compounds.

SO2 binds with carbonyl compounds (aldehydes, aldose sugars, pyruvic acids, etc.), unsaturated aliphatic (straight chains, no rings) compounds, proteins and other compounds and is oxidized by oxygen. The

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binding of SO 2 greatly reduces the active (free) concentration of SO 2. In bound form it is less toxic and 2- less effective as an anti-oxidant. In oxidized form, as sulfate, SO4 , it’s inert.

The portion of SO2 not bound or fixed is the “free sulfur dioxide”, the FSO2. The combination of the FSO2 and the bound SO2 is the “total sulfur dioxide”, the TSO2. Analysis is normally done for F/ TSO2. The TSO2 permitted in the US in wine is 350 ppm. In Europe, only 160 ppm of TSO2 is permitted for red wines and 210 ppm for whites and roses.

The lower the alcohol and TA and higher the pH, more SO2 is required. It can reduce oxidative aromas, improve color in red wines and retard bacterial growth.

SO2 is an in inhibiting enzyme-catalyzed oxidative discoloration and nonenzymatic browning during fermentation, aging and storage. With aeration during fermentation the acetaldehyde increases during the end of fermentation. It is important to keep the SO2 levels low and maintain anaerobic fermentation. As stated before, 50 ppm SO2 added prior to fermentation results in approximately double the level of acetaldehyde vs. that no SO2 addition fermented wines.

Wines produced by oxidation of phenolic substrate materials at the juice stage are rendered more oxidatively stable than wine made with SO2 protection. During white grape processing, SO 2 free juice browns rapidly. It looks like coffee with lots of cream added. SO2 added to juice retains straw and green tints. Following completion of cold settling of the SO2 free juice, the juice changed color to straw and green tints as the brown pigments settle out in the juice .

2- During fermentation, some SO2 is oxidized to sulfate (SO3 ) (perhaps by enzymes). Active fermentation soon binds the FSO2 because acetaldehyde is an intermediate in ethanol production. SO2 does stimulate the production of acetaldehyde during fermentation. Acetaldehyde is the main “binding partner” for FSO2 (50% to 80% of all FSO2), with the sugars being next in line. After fermentation enough SO2 should be added to combine completely with any residual acetaldehyde.

It is best to try to keep FSO2 at 20 ppm in wine of pH 3.2. That is certainly true when clean and complete fermentations have occurred. At bottling, FSO2 at 30 ppm is safe. SO2 kills many bacteria like . Acetobacter can oxidize EtOH to HAc. Also, 25 ppm FSO2 can inhibit MLF. The antiseptic qualities come from the small portion of dissolved SO2 gas. SO2 has wide-spectrum antimicrobial activity. About 1.5 mg/ l is generally sufficient to inhibit most spoilage yeast and bacteria.

At pH 2.8, 20% of the FSO2 is in this dissolved gas stage, while at pH 3.8, the concentration drops to 1%. This is one reason why high pH wines have a tendency to spoil. Yeasts are sensitive to SO2, especially in the presence of alcohol. High FSO2 levels can interfere with good secondary fermentations of sparkling wine. Many studies have shown that delaying SO2 addition until after fermentation reduces total phenolics and produces wines with better color as compared to addition of SO2 at crush.

Pigments of red wines loosely bind SO2. This makes FSO2 analysis difficult in red wines. SO2 does help dissolve red pigments. Red wine with SO2 retains color better than those without SO2. But, excess SO2 can bleach pigments.

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SO2 has minor effect in retarding natural yeast growth. It has been shown that SO2 of 100 ppm does not necessarily prevent growth of indigenous non- species, especially in red wines. Yeast follow a successor pattern of growth. In the first few days, species of Kloeckera, Hanseniaspora, and Hansenula frequently grow. They are soon dominated by a Saccharomyces and fermentations are completed by S. cerevisiae. It is known that S. cerevisiae is the most sensitive to SO2.

Not only is sulfur dioxide the most important antimicrobial and antioxidant additive in wine, it is also the primary sterilant of winery equipment. Care must be taken when used because of its corrosive nature. Sulfur dioxide can solubilize metal ion from unprotected surfaces.

Sulfur dioxide may also react with oak and form lignosulfurous acid. It has been proposed that, after decomposition, lignosulfurous acid may release hydrogen sulfide that reacts with pyrazines in wood. This could form must-smelling thiopyrazines. This could get in the wine.

At FSO2 levels between 15 to 40 mg/ l, most people can detect a distinctive burnt-match odor. This can be missed due to habituation. Healthy individuals can consume up to 400 mg/ l of TSO2 per day. Although SO2 can precipitate asthma attacks in sensitive individuals, most wine does not have enough FSO2 to induce an attack. For a small portion of asthmatics, all forms of sulfur dioxide are potentially allergenic. Sulfur dioxide absorbed by the blood from the digestive tract can be translocated to the lung. An asthma attack may occur.

Few winemakers use ascorbic acid ( C) and erythorbic acids. Both are anti-oxidants. When air contacts wine, certain phenolic compounds are converted to very strong oxidizing agents, which can oxidize EtOH to acetaldehyde and other unwanted compounds. SO2 itself does not react fast enough to prevent these rxns. Ascorbic acid does increase the ability of SO2 to absorb oxygen and is most effective when added just before racking or filtering. It is generally not needed

Sorbic acid has been used to preserve sweet wines and prevent second fermentation from taking place in the bottle. Usually 150 to 200 ppm is effective. 1,000 ppm is legal in the U.S. With bottle age, these wines can smell like butter, oxidized fat or geraniums when lactic acid bacteria metabolize sorbic acid. Sorbic acids, or sorbates, have been widely used in inexpensive wines.

Fumaric acid at 500 ppm can prevent MLF. It is not totally successful and can produce gassiness and off odors in bottled reds. Some winemakers use it.

Clarifying agents. Fining consists of adding to a wine a clarifying product capable of coagulating and making large particles which precipitate in the form of floccules that carry down the particles of cloudiness and clarify the wine, improve the color, flavor and stability.

The agents are grouped according to natural categories: 1. Earth; bentonite, kaolin 2. Proteins: gelatin, isinglass, casein, albumen, ox blood 3. Polysaccharides: agars 4. Carbons 5. Synthetic polymers: PVPP, nylon 6. Kieselsol (silicon dioxide)

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7. Others, including metal chelators, enzymes, etc.

Many fining agents contain an electrical charge. If this charge is the opposite of the particles in suspension, then neutralization and absorption may occur.

(Adsorption is a process that occurs when a gas or liquid solute accumulates on the surface of a solid or, more rarely, a liquid (adsorbent), forming a molecular or atomic film (the adsorbate). It is different from absorption, in which a substance diffuses into a liquid or solid to form a solution. The term sorption encompasses both processes, while desorption is the reverse process.) In a fining operation, small particles of suspended solids are induced to coalesce so that they form larger particles which, because of their density relative to that of the wine or juice, settle from solution. In most cases, the fining agent adsorbs suspended material and exerts some clarifying action by virtue of formation of particles of high density, thus increasing filterability.

The effectiveness of fining is dependent upon the agent, the method of preparation and addition, the quantity employed, the pH, the metal content, the temperature, the age of the wine, and previous treatments. Fining is a surface action performed by the agent (adsorption); therefore, the method of hydration and addition of the agent is of extreme importance.

Four common methods of adding fining agents are: 1) uniformly and slowly through a 'Y' on the suction side of a positive displacement pump while transferring or mixing; 2) uniformly and slowly through an 'in line' proportioning pump; 3) uniformly and slowly through a 'T' into a Guth-type tank mixer; or 4) added slowly in slurry form to a barrel using a dowel to stir in a figure-8 motion through the bung hole.

The main clarifying agents are generally positively charged proteins; their coagulation is carried under the influence of the tannins and sometimes solely by the wines acidity. Negatively charged earth products are also used.

Years back, things like milk, egg-white and ox-blood were most widely used. Now, the most commonly used agents are gelatin, albumens and casein, as well as bentonite.

When a solution of a fining agent (say gelatin) is mixed with a white wine, after a few minutes cloudiness is seen to appear which slowly becomes denser. The clouds become floccules, coagulate and slowly sink, leaving the wine more clear. In red wines, after the addition of a clarifying agent, the appearance of cloudiness is immediate and floccules begin to form in a few minutes. They grow rapidly and appear more and more colored; they form a meshwork that shrinks and falls to the bottom of the tank. Their first fall still leaves the wine cloudy with little floccules. The floccules continue to slowly form and settle.

This progresses until, over time, precipitation and clarification is achieved, and after a few days, the wine becomes clear. Clarification carries down the particles of suspension, which might or might not settle on their own over a very long time. It also fixes the colloidal coloring matter and carries away the tannins,

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which are more or less polymerized and cause astringency. Two stages of fining occur; the rxn of the agent and the ppt of the flocculate.

Reaction of tannin and agents. A colloidal rxn, not chemical, between the tannins and agents occur. The levels of fining agents to add vary. Trial taste tests in the lab must be done. The polyphenols of acidic wines (with low pH) are easily precipitated. The lowering of the temperature of the fined wine increases the agent’s ability to react with and precipitate tannins.

The fining mechanism is illustrated below:

The proteins used in fining are colloids with positive charges.

Colloids are special types of liquid mixture or suspension in which the particles of suspended liquid or solid are present in very finely divided but not molecular or dissolved form. Unlike ordinary suspensions, colloids do not exhibit the phenomenon of settling because of their exceedingly high ratio of surface area to volume.

The tannins and particles creating turbidity are negatively charged. When brought together, attraction, then flocculation and finally settling occurs. The tannin “denatures” the protein. It transforms them from lyophilic colloids (in stable state of suspension) into lyophobic colloids (no affinity for the medium they’re in), which are coagulated by the salts of wine. It must be remembered that fining can only function in the presence of mineral matter; Ca, Mg, K salts and ferric salts.

Role of salts in fining. The action of salts has been known since ancient times when NaCl was added directly to wine to increase clarity. Ferric (Fe+ + + ) salts are the most important. If gelatin is used to fine white wines lacking in ferric iron, flocculation is delayed or impeded. By aerating wine, ferric salts are

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formed from ferrous (Fe+ + ) salts. Clarification can be improved. Fining succeeds better on aerated wines. That is why it often recommended to fine after racking a wine.

Effect of temperature. Protein fining is poor at high temperatures. Fining in winter at 10 °C (50 °F) is considerably faster in flocculation and settling than in summer at 25 °C (77 °F).

Overfining. It is important that protein agents added to wine are completely coagulated. They must not remain in solution in the wine. The clarity of overfined wines is not stable. They can later become cloudy with temperature change or blending or contact with wood or cork tannin. Red wines are generally never overfined. Overfining whites with proteins, especially gelatin, can be common. Red wines can sometimes throw brown deposits that cover the whole inside of a bottle. Some of the protein-tannin rxn products don’t instantly ppt. Further polymerization with more tannins or pigments takes place and causes this ppt in the bottle.

To detect overfining, in the lab add to your wine sample 0.5 g of commercial tannin per liter. After 24 hours, cloudiness will appear in an overfined wine.

Fining tests. Before any cellar fining takes place it should always be tested in the lab. Each clarifying agent reacts differently depending on the wine style, composition, colloidal structure and suspended particles nature. Each wine coagulates differently. Fining tests can be done in the lab in 375-ml bottles. Various levels of a given agent can be tested against a control sample of the wine. Remember that mixing in a 375-ml bottle is easier and more thorough than in a barrel or large tank. Besides tasting the fining results, the sample bottles will also speed up the flocculation and ppt., clarity obtained by each agent and compactness of lees.

Bentonite. A clay flocculable in wine is empowered with high adsorbent and stabilizing properties. It is a natural mineral substance of the clay family, a hydrated aluminum silicate. Bentonite clay can swell considerably and fix as much as 10 times its weight in water, which allows slurries to be prepared. The negative charges its particles possess afford it potent adsorbent power of the positively charged protein molecules. For use with wine, sodium bentonite activated with sodium carbonate is generally used. It is called an alkaline rxn. The best known bentonite comes from Wyoming. The bentonite properties are defined by the colloidal structure, not by the chemical makeup.

It is estimated that a gram of bentonite in an aqueous suspension presents a surface area of about 750 m2. Say you have a 3,000 gallon tank of Chardonnay. Your lab tests showed that 2-1/ 2 #/ m of Bentonite would make it heat stable. That’s 1,135 grams/m or 3,405 grams of Bentonite for the whole tank. That bentonite has about 630 acres of surface area upon which to absorb protein.

If a white wine is heated, cloudiness appears when the wine cools again. The proteins have been “denatured” by heat. That transforms the lyophilic colloids into lypophobic (flocculable) colloids. The proteins then ppt. In a wine of this protein mix and pH, the protein cloudiness may naturally ppt right away or over a prolonged period. So, as lab test is needed to prevent these possibilities.

Several different lab tests exist. One method is to add bentonite slurry in the lab at various levels (C-1-2-3- 4-5 #/ m). Shake the bottle and then vacuum filter in the lab and collect filtrate in 25 ml screw capped

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culture tubes. Put the tubes in boiling water for 17 minutes. Let cool and examine with a light beam for cloudiness. When no haze appears, the needed bentonite is known.

The bentonite needs change easily so always check for heat stability just before bottling. Low pH wines need less bentonite than high pH wines. Normally, all red wines are naturally heat stable because of extra grape tannins. Whites and rosés both can give problems.

Wine proteins are derived primarily from grapes and autolyzed yeast. They consist of several (protein) fractions which appear to be the subunits of denatured grape enzymes. Their molecular weight varies from 20,000 to 40,000 Daltons (used to be called molecular weight). The polypeptides with molecular weights of less than 10,000 are mostly derived from yeast . The isoelectric point (pI) of wine protein fractions have been reported to be in the range of 2.5 - 8.7.

Wine proteins occasionally cause cloudiness or haze in white wine. Haze formation is poorly correlated with total protein content since only certain unstable protein fractions cause haze. When stabilizing a wine for protein, it is not necessary to remove all proteins, but only those fractions that are unstable and thus contribute to cloudiness.

A bentonite treatment is often used to remove unstable proteins from a wine. Bentonite is a negatively charged colloid which adsorbs positively charged protein and removes it from the wine. Proteins with the greatest positive charge are removed first.

The pH influences protein stability in two ways: 1. it affects protein solubility, and 2. it influences the charge (positive or negative) on the protein molecule.

To understand the role of pH one needs to understand the isoionic or isoelectric properties of proteins.

Protein solubility Proteins can be either positively (cation) or negatively (anion) charged based on pH conditions. When the positive and negative charges on protein are equal, the net charge is zero. The characteristic pH of a solution at which the net charge on protein is zero (positive and negative charges are equal) is defined as the isoelectric point (pH). The isoelectric point of a protein is an important property because it is at this point that the protein is least soluble, and therefore unstable. It should be noted that both below and above the isoelectric point (isoelectric pH) the protein will be soluble.

To understand the implication of pH, let us consider an example. Suppose we have a white wine with a pH of 3.30 and a protein fraction with a pH of 3.2. After blending with another wine, the pH of the new blend is changed to 3.2. Notice that the pH of the blend and the pH of the protein are now the same. Since the protein is in a pH solution similar to its pH value, it will become insoluble and thus unstable. The blend could therefore be protein unstable even though the wine was stable before blending. pH and the charge on protein

We have just noted above that protein can be positive or negative based on the pH of the solution. The important point to remember is that in a pH condition below its isoelectric point, the protein will carry net

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positive charge and behave like a cation. In a pH condition above its isoelectric point, the protein will carry a net negative charge and act as an anion.

In the example used above (wine with a pH of 3.3), the protein with an isoelectric point of 3.2 will carry a net negative charge. If this wine is fined with bentonite, the bentonite will not remove this protein fraction. This is because both the protein and bentonite carry a negative charge and we know that like charges repel. After blending, the pH will change to 3.2. The shift in pH will change the charge on the protein from negative to neutral which will make the protein insoluble because the pH is similar to the pI.

From these examples it should be clear that pH affects protein stability and that any change in pH, as often occurs after blending, can lead to the problem of insolubility. Therefore, it is a sound practice to stabilize a wine after blending, before it is bottled. Ref. Dr. Murli Dharmadhikari

Preparation of bentonite for use is done by hydration of its particles in hot water for at least several hours, and preferably for a day. The recommended slurry concentration is 6% weight/ volume. If you have at least 25,000 gallons of white and pink wine, assume the average addition will be 2 #/ m. That would take 50 lb of bentonite. Bentonite can be purchased in 50 lb sacks. If you have smaller lots you can prepare the exact amount of bentonite to be used the day before. Inspect the bentonite to ensure it is clean, dry and has no off odors. Put 100 gallons of water in a container with a low speed mixer installed. If you don’t have a mixer a low volume pump can be used for circulation of the slurry. Raise the water temperature to 120° F. (Not greater, or off odors may occur.) With the mixer turning, slowly add the bentonite powder. Mix until uniform and smooth and avoid clumping. Beginning the next day, this bentonite slurry can be used. Each gallon of slurry has about ½ gallon of bentonite. Add to tank with a mixing valve or other methods as noted before.

Gelatin is obtained by vacuum pan cooking of bones, tendons, gristle and skin. It must have little color or flavor. It has both adsorbent and flocculent powers. Gelatin has a positive charge in wine and hence precipitates after binding with negatively charged tannins. The fining power of gelatin is expressed as Bloom units (100 to 200) and viscosity (30 to 60 millipoise). The molecular weight of the fractions that are used vary from 15,000 to 140,000.

Gelatin is sold in sheets, pearls or powder. Sheet gel is the finest form. It is more expensive, is purer and readily dissolves. Gelatin dissolves in hot water, not boiling. After dissolving, cool the mixture slightly and mix the wine while slowly adding the gelatin mixture. Gelatin is good at reducing bitterness in young red wines. Levels of ½ to 2 #/ m are generally adequate. Turbid wines can generally be made clear at ¼ to ½ #/ m. Gelatin can be too harsh for older wines. It also is often used to help settle bentonite. After adding and mixing bentonite in a tank, 1/ 8 #/ m of gelatin is often added to white or wines. Before adding any fining agent, taste test series must be done in the lab.

Isinglass is removed from the air bladder of a sturgeon. It appears as practically transparent chips or cut into “vermicelli”. The latter is much easier to prepare. To prepare, take a kg (2.2 lb) of isinglass and add 100 liters of water diluted with 100 g of tartaric acid plus 40 g as KMS as a preservative. The isinglass swells rapidly and in a few hours forms a jelly. Constantly stir to keep it uniform.

After a few days, the jelly can be poured through a wire sieve. It can be smelly. It can be used at 1/16 to ¼ #/ m on all wines. The lees sometimes floats, may cling to the tank walls, clog filters and they always 8

create a large volume of lees. It’s a pain, but is very powerful and gives good brilliance. Overfining with isinglass is rare.

Egg albumen or egg white is soluble in cold water, with a touch of salt (in the U.S. we use KCl) added. Egg white contains 12% proteinaceous substances suitable for fining. Each egg white has 3 to 4 g of active agent. Generally, red wines are fined (especially in France) with between 5 and 8 egg whites per 60 gallon barrel. Fine red wines can take as much as 30#/m of egg white. Egg whites are not used on white wines.

It is the most delicate fining agent for red wines and is generally used only on the best wines. It can be prepared fresh, but is more often (in the U.S.) purchased in the frozen form. Work tags will note to “positive add 20 #/ m FEW”. When adding to the wine, avoid foam and aeration. Coagulating foam floats to the wine surface and does no fining. Beat the eggs gently to mix and then mix with about ten times their volume of the wine to be fined. This mixture is then added to the tank. Add about 1 oz of KCl to each lb of egg white to maximize fining. Remember the role of salt in fining.

Casein. Casein is a major protein found in milk. It is a positively charged macromolecule with a molecular weight of approximately 375,000. A liter of milk contains 30 g of casein. In association with sodium and potassium ions, it forms a soluble caseinate salt that easily dissolves in wine. In wine, the salt dissociates and insoluble caseinate is released. The caseinate adsorbs and removes negatively charged particles as it settles.

It is mainly used to remove oxidized color from white wine, remove off odors and aid in clarification. The agent is commonly sold as potassium caseinate in a powder form. To prepare, make a 2% (20 g/ l) solution of potassium caseinate in warm water and gently stir. Leave overnight and stir again until it is completely dissolved. The solution is good for a couple days. Each ml contains 20 mg of agent. Doses range from 50 to 250 mg/ l of potassium caseinate. When added to wine it should be well mixed. After solution preparation and then adding to wine, a drop in pH occurs, causing the casein to flocculate, ppt, adsorb and settle.

Carbon. Activated carbon adsorbents are used to decolorize and deodorize wine and spirits. It is made of small particles, with extremely high surface area, which range from 500 to 1,000 m2/ g (that’s up to ¼ acre of surface area per gram of carbon). To activate carbon, it is heated to 900 °C (1,652° F) to get proper pore alignment. It bonds with weakly polar molecules, especially those containing benzene rings or their derivatives. Phenolic compounds generally exist as ring structures, so they are removed by carbon additions.

The carbon pores are very small, so larger molecules (bigger than flavonoid dimers) are not adsorbed. Recommended levels range from 1/ 8 to 2 #/ m. The adsorption on the carbon surface is very fast, so results are noticed immediately. Activated carbon contains a great amount of oxygen in the pores and should be removed after addition. Due to its adsorption capability, carbon will also adsorb positive aroma and flavor components, reducing their concentration in the wine, so care must be taken when carbon is used. It works best on low pH wines and at higher temperatures.

The carbon type most suitable for decolorizing is marked KBB, and the deodorizing type is marked AAA. Examples: Norit's DARCO KB-B carbon is best suited for decolonization (adsorption of higher molecular weight molecules).

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Norit's DARCO S-51 is best suited for odor reduction (adsorption of smaller molecular weight molecules).

Both carbons are extremely pure, have excellent adsorptive capacities, and are routinely used in food applications.

Polyvinlypolypyrrolidone. PVPP is a resinous polymer not soluble in any solvent and leaves no residue in wines. It is sold in granular form with mean particle size of 100 µ (1 µ is 10-4 cm). PVPP is obtained by the polymerization of vinylpyrrolidone. The final product is a network of macromolecules. PVPP acts by adsorption. The amide bonds of PVPP form hydrogen bonds with the hydroxyl groups of polyphenols.

PVPP is used in: ~ The treatment of maderization and browning of white wines: Phenolic compounds play an important role in the color and taste of white wine, in particular during oxidation phenomena (phenol acids, catechins, and leucoanthocyans). ~ Mellowing red wines: Treatment with PVPP has little effect on anthocyanins and the color of wine, but leads to a considerable reduction of tannins and lowers the phenol value. ~ PVPP preferentially binds astringent tannins.

Treatment with PVPP offers the following advantages: - A noticeable reduction in the optical density of the wine. White wines are less yellow. In conjunction with carbon, it can be very effective in color reduction. - A reduction in catechins and leucoanthocyans, which are responsible for browning and binding of free SO2. - Sensorially, a reduction of bitterness and improved freshness and aroma. This specificity of action of PVPP is a complement to that obtained by treating with casein and with bentonite. It is recommended to treat the must or wine with PVPP after eliminating impurities and microorganisms in order to avoid uselessly saturating the active sites of PVPP.

The dosage for use varies depending on the gustatory effect sought, to be predetermined by prior lab trials and tasting. Dose range in white wines is 100-700 mg/ l and in red wines 100-200 mg/ l.

After doing lab tests, weigh the PVPP needed for your tank and suspend 250 g/ L of wine for 30 minutes in a small container. Use the wine to be clarified. The mixture is then added to the wine. Mix for 30 to 45 minutes by mixing or pumping over. It is not necessary to fine in order to eliminate the product after treatment, but it is useful to allow it to settle for several hours before carrying out filtration. Addition of PVPP can be done at any stage of production, from must to pre-bottling. In the United States, 21CFR173.50 states that the addition rate shall not exceed 60lbs/ 1,000 gallons.

Silica-gel. This is also sold commercially as kieselsol, baykisol and klebsol and is a milky aqueous solution of silicon dioxide. It generally is sold as a 30% (by weight) suspension of SiO2. It is available in both negatively and positively charge forms, and can therefore adsorb and remove both negatively and positively charged colloidal material. It is commonly used for removing bitter tastes from white wines. It

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does not produce large amounts of lees and when used on red wine, removes little color. The negatively charged particles interact with positively charged proteins, the same as bentonite or as tannin do.

It is a very clean fining agent. When using it on white wines, the use ranges from 0.1 to 0.25 ml/ l (30% soln.). Coagulation and separation from the wine begins in a very short time after addition. If using in conjunction with bentonite, add the bentonite first. This agent works well on cloudy botrytised wines.

Ref. Ron S. Jackson Wine Science Principles, Practices, Perception 2nd edition

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Basic Winemaking and Enology – 4

Saccharomyces cerevisiae var. ellipsoideus Microorganisms and Fermentation. Wine yeasts are saprophytes. They are fungi living on dead or decaying organic matter. "Sapro" is from "sapros" (rotten) spoken by people of Greece starting about 1000 B.C. A saprophyte is any plant that depends on other dead plant or animal tissue for a source of nutrition and metabolic energy, e.g., most fungi (molds) and a few flowering plants, such as Indian pipe and some orchids. Most saprophytes do not produce chlorophyll and therefore do not photosynthesize; they are thus dependent on the food energy they absorb from the decaying tissues, which they help to break down.

The controlled fermentation of grape juice seems to be as ancients as Man’s written records. Anton van Leeuwenhoek of Delft, the first to use the microscope in rigorous scientific studies, made his most important discovery early in his career. In 1674, he recognized the true nature of microorganisms. He began to observe bacteria and protozoa, or, as he called them his "very little animalcules", which he was able to isolate from different sources.

In 1687 he made drawings of what he titled “Crystals in Vinegar”. The ellipses under D, are thought to be yeast cells.

Even though van Leeuwenhoek seems to have seen yeast, the role of was not understood until Louis Pasteur published “Etudes sur le Vin” in 1866. At the time of Pasteur, a large portion of the wines of Europe spoiled readily. He showed the basic understanding of fermentation and proposed the methods for controlling wines soundness. Pasteur’s studies showed the microbiological basis for much of the spoiled wine at the time. His work was the foundation of present day . Soon the ecology of “native” yeast in the vineyards and winery, the use of single cell yeast starter culture in wine and production and the methods and source of malolactic fermentation were understood.

Although Pasteur showed the value of pure yeast cultures, it was not until the early 20th century that the Germans and French started isolating and using pure cultures. Most of this work was done in university laboratories. The commercial availability of yeast strains did not occur in the U.S. until the 1960’s.

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Up until this time, most American wine was in the dessert/ appetizer class and because of their high alcohol and high SO2; microbial spoilage was not a major problem. Soon, table wines become popular.

In the 1970’s wine production doubled in the U.S. The production went from 80% dessert/ appetizer to 80% table wine in the decade. Winemaking got much more complicated. The table wines most popular were not dry, but generally had a small amount of residual sugar. So the wines had lower alcohols and potentially fermentable sugar in the cellar tanks and bottle on the grocer’s shelf.

By the 1980’s the winery technologies had grown and most microbial concerns were controllable. The concept of sterilization became well advanced. Wines were made with varietal character as their calling card. At that time, wines were generally between 11% and 12.5% alcohol and pH’s ranged from 3.2 to 3.5.

In the nineties, wine styles began to change. Winemakers were allowing grapes to remain on the vines to attain “fruit-forward” wine styles and higher pH’s, new and native yeasts and bacteria were being used, new methods of maceration employed, sur lie aging was widespread, fermentation protocols varied and fermentations without SO2 were being used. Some new microbiological spoilage problems began to take hold.

Wild yeast lives in the vineyards and wineries. The most important wine yeast is var. ellipsoideus. At the beginning of ‘natural’ fermentation, the native yeast species are most active. These include yeast of the genera Kloeckera, Metschnikowia, Hansenula, Candida and Hanseniaspora. In cooler growing regions, Kloeckera apiculata seems to be the dominating yeast species on grapes, whereas in warmer regions it is Hanseniaspora uvarum. Many other strains exist.

In coastal California, Kloeckera normally represents the dominant native yeast on grapes at harvest.

Kloeckera apiculata-lemon shaped yeast

Like other non-Saccharomyces yeast, it possesses lower EtOH tolerance than Saccharomyces yeast. Even in non-inoculated fermentation, Kloeckera generally dies off when alcohol levels reach 5% to 6%. As the non-Saccharomyces yeast population fades, Saccharomyces will dominate and complete the fermentation.

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A. Growth of non-Saccharomyces yeast B. Growth of Saccharomyces yeast C. Growth of Oenococcus oeni D. Growth of spoilage bacteria and yeast Wine Microbiology K. Fugelsang, et al

Today, commercial yeasts are available in dry or liquid form. Freeze dried yeast is easiest to use. Yeasts are grown in sterile media until vigorously fermenting. For inoculation, the optimum time to transfer yeast to another fermenter is when the inoculum is at 15° Brix. An inoculum of 1% to 5% (by volume) is added to the grape must or juice. (See Yeast rehydration.pdf)

In order to have a clean and completed fermentation, the following should be considered: 1. Yeast strain history 2. Lack of competition from undesirable yeasts 3. Inoculation volume 4. Yeast nutrients 5. Presence of O2 6. Temperature 7. Additives in juice 8. Other wine components

Saccharomyces Cerevisiae cells observed at the microscope

Saccharomyces cerevisiae var. ellipsoideus

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Saccharomyces cerevisiae var. ellipsoideus

Saccharomyces cerevisiae var. ellipsoideus

After inoculation, the onset of fermentation in a batch of juice can occur in one day and is easily observed. In standard winemaking, yeast ferments sugar without O2 (anaerobic) and largely convert it into EtOH and CO2. In the presence of O2 (aerobic), yeast convert sugar to CO2 and H2O and gains much more energy for growth.

Rapid multiplication of yeast cells at the start of fermentation is possible because of dissolved O2 in the must or juice. After inoculation with yeast, enough O2 is available in the juice to allow the yeast cells to multiply from 1 to 200 million cells/cc. This is the necessary growth phase. Then, when the dissolved oxygen is depleted, yeast growth slows and conversion of sugar to alcohol becomes the main reaction.

A yeast growth curve is a graph depicting stages of population growth and decline in a closed environment: First, there is exponential or logarithmic growth until carrying capacity of environment is approached; Second, a gradual increase and then leveling off or stationary phase at the peak; and Finally, a very rapid population decline to zero or death phase as the population is poisoned by its accumulated waste (EtOH) in a closed environment. .

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Reds ferment quicker than whites because of higher fermentation temperature and oxygen additions during pumpovers. During fermentation, yeasts are influenced by temperature and must components and they also add both heat and other components to wine. Different yeast strains adapt to various temperatures. A good rule of thumb is, without heat loss, the temperature of fermenting must goes up 2.3 °F for each °Brix going down.

What conditions do yeast like and dislike? Al, Cu and Fe can all inhibit fermentation. At 65° to 75° F, most fermentation is completed in less than a week. At 40° to 50° F, most fermentation takes 2 to 6 weeks. Some yeast strains can ferment to 18% alcohol in making Sherries. Most yeast ferment glucose faster than fructose. Since fructose is sweeter, stopping fermentation before dryness gives higher fructose/ glucose ratio and a sweeter taste than a wine sweetened with sucrose or grape concentrate. Without alcohol, yeast works best when sugars are 1% to 2% and are retarded at sugars > 25%. German late harvests can have sugars of 40% to 65%. They ferment very slowly and yield alcohols of < 9%.

H2S production has been attributed to certain yeast strains, notably Montrachet. The problem is Montrachet gives incredibly aromatic, fruity wines. As mentioned, retained S from the vineyard, high Cu levels and lack of contribute to sulfide production. Bentonite added to juice prior to fermentation (reducing protein and foam) can also increase sulfides. Dirty and bentonited musts both ferment faster than settled, fined, centrifuged or filtered must. Juice solids < 0.5% (v/V)are very slow fermenters and those with > 2.5% are very fast.

Other Saccharomyces. The yeast used for sparkling wine production is usually . It gives coarse heavy sediment that settles rapidly during fermentation. This aids in later disgorging.

In Sauternes (with high sugar because of Botrytis) Saccharomyces bailii ferments fructose faster than glucose giving less sweet and not cloying wines.

Spanish flor yeasts and those of the Jura of France use S. bayanus or S. capensis. California flor yeast is S. fermentati. Flor yeast forms a film on the surface of the fermenting wine containing 12% to 16% alcohol and oxidizes alcohol to acetaldehyde and other products that give flor sherry their unique character. In table wine, this character is considered to be spoilage.

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In white wine fermentation the growth of yeast can sometimes stop at the point where ½ to 1/ 3 of the sugar still remains in the fermenting juice. The nongrowing, but viable, yeast cells complete the fermentation. Wine fermentation is a fermentation in which there are contributions from both cell growth and the resting phase activity.

Antimicrobial effects of sulfur dioxide. The addition of SO2 to juice leads to the rapid killing of natural bacteria and yeast that had been present on the grape skins. Few studies exist as to the sensitivity and rate of death of specific yeast strains. It is generally assumed that the levels required for enzyme inhibition provides significant reduction in microflora viability or growth.

Levels of 25 to 75 mg/ l of SO2 in juice can lead to 75% to 95% inhibition in phenol oxidase activity. Enzyme inhibition can aid in reducing yeast viability.

The level of SO2 sensitivity of wine yeast varies throughout the growth phase. The yeasts are more resistant as the stationary phase is approached. The existence of different survival patterns of log-phase growing cells and stationary-phase cells is apparent. The mechanisms of the yeast killing action are not clear, but a few theories are proposed.

Studies have shown that sulfur dioxide added to a solution of Saccharomyces cerevisiae was rapidly removed. It was suggested the sulfur dioxide was transported and bound to the yeast cell outer membrane with the aid of enzymes.

Most yeasts produce SO2 by reducing sulfate in grape must. Acetaldehyde formed during fermentation binds bisulfite in wine, leaving little FSO2. Wine generally needs SO2 at dryness.

In fermentation, a carbon compound serves as a terminal acceptor of the electrons that are generated in the pathway in the course of converting the sugar metabolites to energy in the form of ATP. Cellular ATP is . It is an ester of adenosine that contains three phosphate groups; supplies energy for many biochemical cellular processes by undergoing enzymatic hydrolysis.

Some studies have shown that sulfur dioxide was binding to a membrane-bound compound causing uncontrolled loss of cellular ATP and hence cell viability. Cell membrane binding is suggested by some, others suggest that the molecular form of SO2 enters the yeast cell by diffusion.

Little data exists to support any of the above proposals.

The toxicity of acidic forms of sulfite solutions has been studied for some time. Work has shown that the molecular form was several hundred times more toxic than the bisulfite form. - 2- + SO2 + H2O H2SO3 HSO3 + H+ SO3 + 2H

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Relative abundance of molecular SO2, Bisulfite and Sulfites vs. pH Wine Microbiology K. Fugelsang, et al

Sulfur dioxide is a gas under normal conditions. It is very soluble in water. Approximately 40 volumes of SO2 gas are soluble for each volume of water at 20 °C (68°F) and 55 volumes of SO2 gas are soluble for each volume of water at 10 °C (50°F). The influence f temperature on solubility is important. It must be noted that higher percentages of SO2 gas solution can be created at lower temperature, but that a saturated SO2 gas solution at 50°F, it warmed to 68°F has the potential to release approximately 50 g/ l (or 15 volumes) of SO2 gas. So, be careful.

SO2 gas, when dissolved in water, is a moderately strong acid. It has pKa values of 1.77 and 7.20 while in wine or juice.

PKa: Negative log of the dissociation constant (Ka) of an acid species. Equilibrium Constants: Many chemical and biochemical reactions do not proceed to completion but instead reach an equilibrium condition at which point the forward and reverse reactions occur at the same rate such that there is no net change in product(s) concentrations. There are many factors that influence the establishment of equilibrium. The Principle of Le Chatelier states that equilibrium will shift in the direction that counteracts the applied force. For example, increasing temperature will favor the reaction that absorbs heat. There are many types of chemical and biochemical equilibria. Each type has an equilibrium constant, K, which describes numerically the relationship between the forward and reverse reactions. A large K value means the reaction is favored in the direction of that reaction while a low K value indicates that the opposite reaction is favored. Type of Reaction Equilibrium Equilibrium Equilibrium Constant Constant Expression Water + - + - Dissociation 2H2O H3O + OH Kw = [H3O ] [OH ] Kw: Ion product Constant

+ - + - Acid-Base HA H + A Ka = [H ] [A ] Kd, Ka, Kb: Acidity [HA] (dissociation) Constant

- 2- + SO2 + H2O H2SO3 HSO3 + H+ SO3 + 2H The hydrated SO2, or molecular form, is the major form below pH 1.86. 7

The molecular form seems also the most effective in reducing yeast viability. What level of molecular sulfur dioxide is needed to prevent growth of typical wine yeast and bacteria? The various studies on molecular sulfur dioxide requirements for yeast control are outlined below: Molecular SO2 Medium (mg/ l) Medium 1.3 Juice 6.6 Model wine 0.83 Medium 1.56 Wine 1.5 As reported in Principles and Practices of Winemaking

Winemakers are familiar with the concept of F/ TSO2. As sulfur dioxide is a very reactive substance, it binds with many compounds; acetaldehyde, sugars, anthocyanins and other carbonyl groups. This is the bound SO2. As stated before, the FSO2 is not bound and is the most effective part. The free and bound portions combine to give the TSO2. - + 2- + SO2 + H2O H2SO3 HSO3 + H SO3 + 2H Because of its properties, FSO2 is more significant. Remember, depending on the pH, various forms of sulfur dioxide are in a wine solution. From the studies available, the real effectiveness of sulfur dioxide as an antimicrobial and antioxidant agent is due to the molecular sulfur dioxide present in the wine. The problem is the molecular sulfur dioxide is only present in small percentages. pH of wine % of free SO2 in molecular form 3.0 6.0 3.2 4.0 3.4 2.5 3.6 1.5 3.8 1.0 4.0 0.6 A couple examples: Wine X has FSO2 of 20 ppm and pH of 3.0. The molecular SO2 level would be 6% of 20 ppm = 1.2 ppm Wine Y has FSO2 of 20 ppm and pH of 3.8. The molecular SO2 level would be 1% of 20 ppm = 0.2 ppm

What is the significance of this difference? The level of molecular sulfur dioxide needed to stop bacterial growth and prevent any oxidation is believed to be at least 0.6 ppm. So, in the examples above, Wine x would be protected but Wine Y would not, even though it has 20 ppm FSO2.The usefulness of molecular sulfur dioxide is also related to alcohol level. Understandably, the higher the alcohol, the less molecular sulfur dioxide needed. The guidelines recommended are: With 12% alcohol, a wine needs a minimum of 0.8 ppm molecular sulfur dioxide. With 14% alcohol, a wine needs a minimum of 0.6 ppm molecular sulfur dioxide.

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Estimating molecular sulfur dioxide. One molecular sulfur dioxide level will not satisfy the needs of all wines. Different levels are needed depending on the pH and alcohol. To get 0.8 ppm molecular sulfur dioxide, the following table can be used: pH FSO2 3.0 13 3.1 16 3.2 21 3.3 26 3.4 32 3.5 40 3.6 50 3.7 60 The table can be handy. The level of molecular sulfur dioxide can also be calculated with a formula. It can easily be programmed into an Excel spread sheet.

(pH - 1.83) Molecular SO2 (ppm) = FSO2 / (1 + 10 )

This formula is not alcohol dependent. With the file Free SO2 pH Distribution, FSO2 levels can be found vs. pH to attain 0.8 ppm molecular SO2.

An important note must be added. The Inter Winery Analysis Group did a recent survey of US winery lab accuracy. In 2002 they published results showing a coefficient of variation for both pH and sulfur dioxide in a laboratory proficiency program was between 19% and 39%. Any errors of this magnitude in lab analysis make all of the above of little value.

Finally, to achieve a level of 0.8 ppm molecular sulfur dioxide the following amounts of FSO2 have been widely recommended:

White wines: pH 3.0 – 3.2 10-20 ppm FSO2 pH 3.2 – 3.4 20-30 ppm FSO2 pH 3.4 – 3.5 30-50 ppm FSO2

Red wines: pH 3.4 – 3.6 10-20 ppm apparent FSO2, or 50-150 ppm TSO2

Sulfur dioxide behaves differently in red wine than white. The pigment in red wines also binds with sulfur dioxide. Therefore the FSO2 measured in red wines (apparent FSO2) is not the same as in white wine. The level of molecular sulfur dioxide is not changed, but the FSO2 amount is different.

What is suggested is, if you want to stay clear of microbial troubles, don’t make white wine at pH of 3.5 and above or red wines at pH of 3.6 and above.

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Bacteria. The antibacterial properties of sulfur dioxide are not any more straight forward. Studies have shown both the free and bound form of sulfur dioxide can inhibit bacterial growth. The pH is also very important. Wine yeasts are quite tolerant of pH. Yeast growth does not change significantly over the normal range of wine pH values, and overall fermentation characteristics are little affected by pH. On the other hand, wine bacteria do not tolerate low pH values, and wine pH strongly influences both bacterial growth rate and bacterial fermentation characteristics. This is why malolactic fermentation is not likely to occur in wines with pH values lower than 3.3. Bacterial activity is reduced in low pH wines, and many of the bacterial problems become insignificant when wine pH is low.

Some forms of bacteria have been shown to bind with acetaldehyde. The bacteria consumed some of the acetaldehyde present, releasing free sulfur dioxide that prevented further bacterial growth. Some forms of bacteria were not hampered. One study found effects on both growth and malolactic fermentation due to both free and bound sulfur dioxide at pH 4.8. Under wine conditions (pH 3.5), it was found that 10 mg/ l of acetaldehyde-bound sulfur dioxide reduced growth significantly, but not completely. At 30 mg/ l, loss of all viable cells occurred.

More importantly the primary MLF bacteria, Oenococcus oeni (Leuconostoc oenos) has been found to be especially sensitive to levels of acetaldehyde-bound sulfur dioxide in the 20 mg/ l to 60 mg/ l range. It is thought that this is due to the release of free sulfur dioxide on the consumption of acetaldehyde or its sensitivity to its bound form.

Malolactic bacteria and malic fermenting yeast. Cool region grapes are high in acidity and have high ratios of malic acid to other acids. A malolactic fermentation (MLF) converts L- malic (a diacid) to L-lactic (a monoacid) acid plus CO2, and other products. NAD- Nicotinamide-adenine dinucleotide plays a central role in yeast metabolism. NAD/ NADH: Nicotinamide adenine dinucleotide is an important coenzyme in oxidation reduction reactions. Enzymes and salts aid in the reaction. MN+ + + + L-Malic acid + NAD + CO2 + NADH + H L-lactic acid + NAD malic enzyme lactic dehydrogenase

A molecule not superimposable on its mirror image is referred to as “chiral”. Achiral is superimposable. Chiral molecules possess optical activity; they rotate the plane of plane- polarized light. Achiral do not. Some molecules rotate light clockwise (+ ) and some counterclockwise (-). They are called (+ ) d-dextrorotary and (-) l-levorotary.

After MLF, the acidity is halved. It can reduce T.A. by 1/ 3. This bacterial fermentation generally follows sugar fermentation. It increases wine complexity, but decreases fruitiness. It is done to reduce acidity but also to microbiologically stabilize the wines, even in warm regions like California. In warm regions, winemakers then must acidulate wines with tartaric, citric or d-malic acid. MLF gives reduced acidity and produces lactic ester, acetoin, and diacetyl (like melted butter) which account for aroma and taste changes. During MLF the wine gives off odors which go away or can be removed at MLF completion with racking. Few German producers use MLF since delicate white wines show “off” odor easily.

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To prevent MLF from occurring, keep the wine clean, have good levels of molecular SO2 and store in containers with untainted history. Once MLF has occurred in a wooden container, it is almost impossible to prevent future residents from doing the same. New wines can undergo MLF when kept on lees, ala sur lie, but not always. ML bacteria thrive in the presence of autolyzed yeast. The important thing is to maintain the molecular sulfur dioxide level at 0.8 ppm. It also helps if storage temperature is < 60 °F.

To encourage MLF, add the culture before the yeast fermentation is completed, at about 5 °Brix. After completion of alcohol fermentation, leave the wines (whites) sur lie, keep the temperature > 65 °F and add no SO2. The primary MLF strain is Oenococcus oeni (ML 34, PSU 1, among others).

Oenococcus oeni (Leuconostoc oenos)

They seem to grow better in red wines containing grape skins. A good culture medium is juice diluted with 4 parts water. ML culture can be added to reds at about 5 °Brix and should be at 1% to 5% (v/ v) of juice. In whites, skin contact helps ML culture. Inoculate after yeast fermentation has started when there is no FSO2, but before alcohol gets too high. If you have no MLF bacteria, it is also been possible to add 5% wine that has undergone MLF or putting wine into barrels which contained wine that had finished MLF. Be careful that the previous MLF had been clean. Tracking MLF can be easily done with paper chromatography or more costly enzymatic methods. Measuring the T.A. and pH is not a reliable tracking method. All you care to know is if all the malic acid is gone. Bacteria can produce lactic acid from carbohydrates in wine and KHT can ppt. during fermentation and reduce acidity. ML bacteria can be removed through 0.45 µ (micron) membrane filters. MLF in the bottle can produce haze, gas, acid reduction and a sauerkraut aroma. Yeast and bacteria that causes spoilage. Candida vini and C. valida are film yeasts that grow on low alcohol wines. They oxidize alcohol to CO2 in the presence of O2. To prevent, keep containers full. This is a very common problem. Candida stellata can produce a mousy odor in wine.

Candida stellata CBS 157 Dekkera/ is a spore forming yeast that produces a “horsey” or “barnyard” nose. Most winemakers consider “Brett” and its sporulating equivalent to threaten wine quality. The sensitivity of the spoilage yeast Brettanomyces to sulfur dioxide is a major concern to present

11 day winemakers. It is generally accepted to have 0.8 mg/ l of molecular sulfur dioxide to control Brettanomyces and any other spoilage organism.

Brettanomyces bruxellensis

Brettanomyces

The lactic bacteria can cause problems. They grow best without O2. These include , Leuconostoc and . They produce haze, sediment and sour “sour milk” nose.

Acetobacter aceti, A. pasteurianus and A. peroxydans are found in wine.

Electron micrograph of Acetobacter diazotrophicus cells held together by a mucilage-type material found inside the sugarcane tissue that was colonized by this bacterium

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Bacteria which turn alcohol and some sugars into acetic acid or vinegar; Acetobacter aceti.

They produce EtAc which smells like nail polish remover. They form a very thin surface film. Alcohols > 15%, pH < 3.2 and FSO2 > 100 ppm discourage acetic bacteria. wines are susceptible to spoilage. Without fermentation, there is no CO2 protection. Pomace rapidly acetifies and fruit flies (Drosophila melanogaster) carry acetic bacteria.

C2H5OH + O2 → CH3COOH + H2O Ethanol Oxygen Acetic acid Water or for more spoilage odors

C2H5OH + CH3COOH → CH3COOC2H5 + H2O Ethanol Acetic acid Ethyl acetate Water Lactobacillus trichiodes grow on fortified wines up to 21% alcohol and forms Mannitol (a hexa- alcohol, common in late harvest German wines) from fructose and also CO2 , EtOH, HAc and lactic spoilage. The bacteria form a protective coating of polysaccharides. The wine becomes so thick, a finger dipped in the wine can lift a “rope”; hence “ropieness” is used to describe the condition.

Botrytis cinerea (Gray mold)

Saccharomyces cerevisiae var. ellipsoideus

Saccharomyces cerevisiae var. ellipsoideus

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All photos were given by Isak S. Pretorius, The Research Institute

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Basic Winemaking and Enology – 5 Preventing clarity problems. Wine hazes can be caused by suspended particles, pectins, proteins, metal salts (especially Ca, Cu and Fe) and microbes (bacteria and yeast).

Grape solids generally settle with aid of gravity. Fining agents can speed the process.

Grapes contain pectins (complex carbohydrates) which, being colloidal, are very difficult to remove with fining agents or filtration. Enzymes in grapes break these down. Adding pectic enzymes can break them down and hence improve yield.

Proteins can cause haze and flocculent, especially after warm storage. The tannin in red wines combines with the proteins and causes precipitation. White and pink wines can have problems. It has been shown that it is best to add bentonite clay to remove the unstable proteins. Remember the discussion on fining in Basic Winemaking 3. Bentonite adsorbs protein instantly, so mix well when adding to wine.

Adding small amounts of grape tannin can help clarify white wine, but excessive tannin can add harshness, can darken a wine as they oxidize and form oxidizing agents in the presence of O2 and oxidize other wine components. Be very careful if you add tannins to whites to help clarify.

Cu, Ca and Fe salts cause haze. At one time haze caused by Cu and Fe was common, but with present day use of stainless steel in equipment and tubing, it is very rare. Cu haze can result from Cu additions in the vineyard. Bordeaux mixture additions are common in Europe and the East Coast of the U.S.

(Bordeaux mixture is what the French call bouillie bordelaise, is a fungicide spray made of copper sulfate and slaked lime, widely used in Europe against Oidium and mildew. It is this that so often gives French and German vineyards their blue-green tinge during summer.)

+ + CuSO4 is sometimes added to wine to remove H2S. If the Cu is not removed with Cufex of similar products, a haze may appear.

+ Ca is a problem when CaCO3 or Ca containing additives are used to reduce acidity. This is a rare occurrence in California.

Bacterial infection can also cause wine haziness. Keep your wine clean and the molecular SO2 at 0.8 ppm.

Preventing Color Problems. Prevent oxidation and use activated carbon to remove browning in whites.

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Preventing Loss of Fragrance. This problem is dependent on grape variety, care in handling and yeast selection.

Preventing off odors and flavors. Major off odors found in wine is caused by acetaldehyde, excess SO2, H2S and related S-compounds, VA, bacteria and yeast. External sources like poor cleaning practices, cooperage, filters and corks can also create problems.

As noted before, tannins in red wines are anti-oxidants and hence white wines, with lower tannin levels, are more prone to oxidize. Also, reds wines, because of their longer aging are more prone to VA.

Acetaldehyde occurs because of minor oxidation and can be removed with SO2. Before adding in the cellar, run some lab trials. Allow a couple days after adding SO2 in the lab to decide level. In some cases, problem wines can be blended and lost with others.

Some problem wine can also be saved by adding to next years fermenters. The yeast will reduce the acetaldehyde in the oxidized wine to alcohol. This doesn’t always work, so use caution.

VA can be similarly reduced by adding to new fermenters. During fermentation, EtAc will be driven off. It is possible to remove HAc with ion exchange (IX) columns, but EtAc is not removed. So, if it’s a “sweet-sour” smell, IX may not work. Blending out works and reverse osmosis can be effective, but fairly rough.

Reducing odorous sulfur compounds. SO2 can be objectionable at 20 to 50 ppm. To reduce, one can try to splash the wine in a tub to add O2. This is rarely satisfactory. You can also add small amounts slowly to fermenting wine and the CO2 will carry off some of the SO2 and the acetaldehyde produced as the intermediate in EtOH fermentation, will combine with the balance.

A more effective method is the addition of hydrogen peroxide (H2O2). The sulfur dioxide reacts with hydrogen peroxide and forms sulfate ions and water. Sulfates are basically inert. The correct amount to add will depend on the sulfite level in the wine. For a wine at 80 ppm sulfites, 1 ml of 3% hydrogen peroxide, the form sold in pharmacies, will remove the sulfites in one bottle of wine (750 ml). Some people do this before drinking wine.

Sometimes SO2 must b e reduced to encourage malolactic fermentation or secondary fermentation in a bottle. Many yeasts have trouble with 11 to 12% (v/ v) alcohol wines and FSO2 > than 20 ppm. The TTB will allow 3 ppm H2O2 addition. 3 ppm of H2O2 can oxidize 6 ppm SO2.

Hydrogen sulfide - H2S (rotten eggs).If detected, add SO2 and rack and aerate or add CuSO4. TTB allows 0.5 ppm Cu addition and only 0.2 ppm Cu remaining.

H2S + CuSO4 → CuS (insoluble)

If not removed, after two weeks, H2S could become a .

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In organic chemistry, a thiol is a compound that contains the functional group composed of a sulfur atom and a hydrogen atom (SH-). Being the sulfur analogue of an alcohol group (OH-), this functional group is referred to either as a thiol group or a sulfhydryl group.

More traditionally, are often referred to as mercaptans. The two most common in wine are: CH3CH2SH ethyl mercaptan CH3SH methyl mercaptan

Mercaptans are not volatile and will not come off with aeration. They do, however, smell “skunky”. They are later converted to disulfides. These are just as stinky and almost impossible to remove. In chemistry, a bond is a single covalent bond derived from the coupling of thiol groups. The linkage is also called an SS-bond or disulfide bridge. The overall connectivity is therefore C-S-S-C.

Disulfide bonds are usually formed from the oxidation of sulfhydryl (SH-) groups, as depicted.

Neither aeration nor Cu+ + works in removing disulfides.

Interestingly, winemakers have found disulfides are very soluble in olive oil. Mineral oil also works. Make sure the olive oil is fresh and not rancid. It can be added to the wine, mixed and skimmed. Add about 1 oz of olive oil or USP mineral oil to each gallon of wine. Fill the container completely. Avoid air contact. Mix daily for about a week. Take sample below the oil and smell and taste the wine. If OK, rack the wine. Discard stinky oil.

It is possible to treat the wine with ascorbic acid which will break the disulfide back down to sulfide and adding copper sulphate (CuSO4.5H2O) solution to remove the sulfide.

Testing before treatment is absolutely necessary because it is possible to confuse the off-odor for Brettanomyces, which has a barnyard odor and cannot be eliminated by treating it for mercaptans.

To test a possible problem wine, it is necessary to first make two stock solutions: One of copper sulphate which is done by dissolving 4.1 grams of CuSO4 in a little water and bringing the volume up to one liter with distilled water. (Use 10 ml of this solution with 90 ml of distilled water to make 100 ml total for the lab test.) One of ascorbic acid which is done by dissolving 10 grams of C6H8O6 in a little water and bringing the volume up to one liter with distilled water.

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Next, put 100 ml of the suspect wine into three glasses. Use the first glass as the control. Put 5 drops of the diluted copper sulphate solution into glass number two and stir well. Into glass number three, put 5 drops of the ascorbic acid solution, stir well and, after a few minutes, add 5 drops of the copper sulphate solution and stir well. The following table illustrates the possible results.

Glass Number 3 Glass Number2 Possibilities Ascorbic Acid/ Copper Results Copper Sulphate Sulphate

First No change in smell No change in smell Not a sulfide problem

Reduction or Second No change in smell Disulfide elimination of smell H S, mercaptan and Third Reduction of smell Elimination of smell 2 disulfide H S and/ or Fourth Elimination of smell Elimination of smell 2 mercaptan After Yair Margalit: Winery Technology & Operations.

If disulfide is not present, addition of the copper solution will help; if disulfide is present, both ascorbic acid and copper sulphate must be used.

To determine the amount of the copper solution to use, set up a series of glasses with 100 ml of wine and add 0.05 ml, 0.1 ml, 1.5 ml, etc. of the solution. Check the smell of each glass and select the first one that no longer smells. The addition of the copper solution used is the equivalent in parts per million of copper sulphate addition, thus, 0.1 ml = 0.1 ppm. To treat a 19 liter carboy of wine with 0.1 ppm requires 0.1 ppm x 19 = 1.9 ml of the stock copper solution.

Prior to adding the copper solution, add about 25 ppm of ascorbic acid, or about 0.5 grams in a 19 liter carboy. Stir in well and wait at least one day before adding the copper solution. Ascorbic acid in conjunction with copper sulphate works very well, but it is not instantaneous; it takes several days before the odor and taste disappear. Do not exceed the recommended dosage of copper sulphate or you may induce a copper haze which will be difficult to remove.

Remember, H2S is volatile. It can become a mono-mercaptans and then a poly-mercaptan, so deal with the problem as soon as it is detected. This process is not discrete: that is, while H2S is present, it is likely that mono-mercaptans are forming; and poly-mercaptans may be forming before the H2S in its volatile form disappears.

Research shows that mercaptan formation occurs within two days after the beginning of fermentation and is at its peak at about two months after which the poly-mercaptans become

4 dominant. Since most winemakers barrel-age their wines for much longer periods, if H2S has been detected and removed in the early stages, constant checking for mercaptan odors is critical since the barrel is where the mercaptans are formed, and they will continue to develop in the bottle.

Hopeless problems. Off characters produced by yeast, bacteria, mold, wood cooperage, Brettanomyces, lactic acid bacteria, etc. are very hard to solve. In most cases they must be lost in blends. Or, the wine can be turned into DM (distilling material).

Adjusting wine acidity. When an acid is dissolved in water or wine, it partially separates or dissociates into hydrogen ions (H+ ) and an anion (A-). As we’ve discussed, the concentration of the H+ affect many things. The H+ and A- immediately recombine into an undissociated acid until equilibrium is reached. The reversible equation is: HA ↔ H+ + A- Strong acids (sulfuric, hydrochloric) dissociate almost completely. Weak acids (acetic, lactic) dissociates only slightly (about 1%). The effective acidity depends on the acid concentration and dissociation tendency of H+ . This tendency is measured as pH; the negative log of H+ ion concentration in gram-atoms per liter. (A gram-atom of H+ . weighs 1 gram.) A pH meter measure this: pH = -log (H+ ) The pH of most wines ranges from 0.001 gm/ l (pH= 3.0) to 0.0001 g/ l (pH= 4.0).

Water, with no acidity, has 0.0000001 g/ l (pH= 7.0).

Strong bases (NaOH), dissociate almost completely in H2O to give a metal cation and hydroxyl anion. NaOH ↔ Na+ + OH- + The hydroxyl anion combines with the H cation in H2O and neutralizes acidity.

The other reaction product of an acid, like HCl, and a base, like NaOH, is a salt.

HCl + NaOH → H2O + NaCl

The titratable acidity (TA) in wine is measured with a titration. This process involves the measured addition of a strong base (usually NaOH) to a sample to combine with both dissociated H+ and initially undissociated (HA) hydrogen ions. When the H+ is neutralized, undissociated acids instantly dissociate to give more free H+ , and this continues until no more HA or H+ is available. A titration until a neutral point is reached (in practice about pH 8) measures all potential H+ . This gives the TA of both strong and weal acids. An example is tartaric acid (H2T) titration.

H2T + 2NaOH → 2H2O + Na2T

Though half a dozen different acids are found in wine, the TA is expressed as tartaric acid. It is also the strongest acid in wine and gives the lowest pH values for a given TA. Note: To reduce acidity in wine, do not add a strong base. Adding a little NaOH merely causes more acid dissociation and the effective acidity is not changed much. Also, by the tine NaOH is added to

5 neutralize a substantial amount of acid, so much Na2T, or other salts, will have formed that the wine will have an unpleasant salty taste and the pH will have risen dangerously high (permitting bacterial growth and giving brown colors to red wines).

What causes “sour” taste in wine? It takes about ten times as much undissociated acid as hydrogen ion to give the same degree of sourness. But because HA exceeds H+ in wine by about a hundred fold, sour is caused mainly by the undissociated acids. For this reason, sourness is more accurately predicted by TA than pH.

An acidity index has been defined after a series of ; Ia = TA (g/ l) - pH Most table wines have a TA ranging from 5 to 10 g/ l and pH is 3 to 4. The Ia can range from 7 to 1. (10 – 3 = 7 and 5 – 4 = 1). A survey of 350 commercial wines showed an average Ia for reds of 2.5 and whites 3.8. DESIRED ACIDITY IN WINE TA (g/ l) pH Dry white 6.5 to 7.6 3.2 to 3.6 Dry red 6.0 to 7.0 3.2 to 3.6 Sweet white 7.0 to 8.5 3.0 to 3.5 Ports 6.5 to 8.0 3.0 to 3.6 Sherries 5.0 to 6.0 3.4 to 3.0 Sparkling wine like table and sweet wine (Philip Jackisch Modern Winemaking) An outline of acidity adjustment methods. Must TA’s can range from 5.0 to 10.0 g/ l. During fermentation, TA’s usually fall 1 to 2 g/ l. This is mainly resulting from H2T →KHT.

Acids in wine can be changed by 1. Adjusting H+ ions present, 2. Changing anions present, or 3. Combination of both.

Method Increase Acidity Decrease Acidity Via H+ adjustment Ion Exchange (IX) IX Via A- adjustment Plastering IX Via H+ & A- adjustment Acidulation Amelioration Blend wine Cold stabilization Freeze wine Blend wine Carbonate wine Malolactic fermentation Methods of increasing acidity.

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“Plastering” is widely used in the Xérès (sair-ress) district of Spain in the making of Sherry. CaSO4, commonly called “Plaster of Paris”, is added to must. CaT precipitates and H2SO4 replaces H2T. However, CaSO4 has low solubility and may not react completely and may be a touch bitter. IX is fairly imprecise with results.

Acidulation is the preferred way to get TA and pH changes. Even this method can lead to problems. 1. Tartaric acid can lead to KHT precipitation. 2. L-malic can lead to mlf. 3. Citric acid can convert bacterially to acetic acid.

Tartaric Acid(H2T) Malic Acid (H2M) C2H2(OH)2(COOH)2 COOHCH2CH(OH)COOH

Citric Acid (H3C) Potassium bitartrate (KHT)

C6H8O7 KHC4H4O6

Kinds of Acids in Wine

ACID QUANTITY TYPE (grams/ liter)

Tartaric 1 to 5 Malic 1 to 4 Succinic 0.4 to 1 Lactic 0.1 to 0.4 Citric 0.04 to 0.7 Acetic 0.05 to 0.5

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For each 0.10 g/ 100 ml increase in TA Add (g/ l) add g/ gal add #/ m of acid 0.85 3.3 7.3 Citric 1.o 3.8 8.3 Tartaric H2T is the stronger of the two acids, but it will be reduced via KHT production.

Freezing wine and creating ice crystals remove water. This increases alcohol, body, acidity and flavor. It is not legal to do, but it happens during the cold stabilization process.

A simple lab experiment was done. A container of wine at 12% alcohol and 5.5 g/ l TA was put in a freezer for 6 hours. The resulting wine slush was placed in a sieve and drained until 62.5 % of the wine was recovered. The remaining slush was discarded. The resulting wine was 15% alcohol and had a TA of 6.3 g/ l and the soluble solids and body were increased.

Reducing acidity by amelioration. Grapes under ripe or from cold climates may be acidic. Acids may be diluted, neutralized or removed. Amelioration, adding sugar or water to wine to reduce acidity and flavor, is widely practiced in the eastern U.S. and Europe. In California, amelioration is allowed in fruit and sparkling wines, only.

The advantages in amelioration are 1. Increasing volume, 2. It’s very inexpensive, and 3. Strong “off characteristics”, like “foxy” aromas, can be removed.

The disadvantages in amelioration are 1. Loss of color, flavor and soluble solids, 2. Acidity reduction is about half as great as the water added (i.e., adding 20% water, reduces TA by about 10%) because water increases the solubility of KHT and less precipitates before and after fermentation.

Reducing the acidity by chilling. The K in grapes combines with the H2T to form KHT, which is fairly insoluble. The alcohol produced decreases the solubility even further and some KHT + + precipitates during fermentation. Since H2T has two available H ‘s and K replaces only one of these, the resulting KHT is still acidic and its precipitation during cold stabilization reduces wine acidity.

In 8 AD, Augustus Caesar exiled the Roman poet Ovid (Publius Ovidius Nasso 43 BC -17 AD) to the remote Black Sea town of Tomis (modern Constanta in Romania). Not only was Ovid isolated from his social and intellectual center, but he had to endure a climate a bit harsher than Rome. He wrote pomes to his beloved Tristia speaking of his travails. In one poem he wrote: “and the wines stand stiff, jugless but keeping the shape of their jugs, and the people don’t drink their wine – they eat pieces of it.”

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Modern temperature scales did not come about until the 17th century when Fahrenheit and Celsius proposed their scales; therefore we don’t know what temperatures Ovid endured. However, Chemists know, by Ovid’s poem, what must have occurred. The “wine” froze. But wine was generally diluted by the Romans. However, the exact words Ovid used to describe the wine were “vina” and “meri”. These words were used to describe undiluted wine. So, it can be assumed the wine was near 12% (v/ v) alcohol. So, what could the temperature be which Ovid suffered through?

Wine is a basically a mixture of water, ethyl alcohol, sugars, acids and a bunch of other stuff. The alcohol, by itself would need -115 °C (-175 °F) to become a solid. The water will become ice at 0 °C (32°F). A mixture of the components is in between. Also, all the other partners in solution (sugar, acids, etc.) make the freezing point of the wine even lower. So, if the temperature gets down to 32°F the wine will begin to get slushy. The point is as the water molecules start to freeze, the crystals leave behind a solution higher in alcohol, sugar, acids and other components. The abandoned solution is higher in concentration of all the remaining occupants. Hence, this solution has even a lower freezing point. Generally, between 21 °F to 23°F will cause the standard table wine to “freeze”.

In the cellar, to be efficient, reduce the temperature of the wine to near the freezing point (20 °F to 25 °F, depending on the alcohol). The KHT solubility is less in colder wine. Precipitation usually takes two weeks with gravity. Introducing “seed” crystals of KHT speeds precipitation. 40 µm is the best size. Add 4 g/ l or 15 g/ gal of KHT seed and mix well. Frequent and repeated mixing increases precipitation. Rack and cold filter the wine, heat to cellar temperature. Since the wine was very cold it probably became saturated with O2. You will need to N2 sparge the wine to reduce dissolved O2 to < 1.0 ppm.

Acidity is reduced in wines with appreciable H2T and K. Reduction is generally no more than 0.5 g/ L.

Cold stability tests. There are dozens of tests to measure a wines cold stability. The state of Pennsylvania test should be adequate. Hold a wine sample for 24 hours at 35 °F. Examine sample. If no , it’s cold stable.

Reducing acidity by chemical additions. Very weak bases can reduce acidity by precipitating acid anions and leaving weaker acids behind. CaCO3 removes tartrate anions and leaves carbonic acid, which dissipates into CO2 and H2O, leaves no residual acidity. For this to work, H2T must be at sufficient levels in the wine.

H2T + CaCO3 → CaT ↓ + H2CO3

H2CO3 → H2O + CO2 ↑

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Another way to reduce acidity is to add potassium bicarbonate (KHCO3), especially if TA is < 10 g/ l.

H2T + KHCO3 → KHT ↓ + H2O + CO2 ↑

For each 0.10% of acidity that must be reduced, add 0.9 g/ l of KHCO3, or 3.4 g/ gal. The wine should be chilled to help to precipitate the KHT.

Wines that cannot be deacidified with KHCO3 have both high TA and pH. This usually occurs with wines with malic acid (H2M). Malic acid can precipitate with CaCO3. H2M + CaCO3 → CaM ↓ + H2O + CO2 ↑

Because CaT is less soluble than CaM and precipitates more easily, little CaM is removed until nearly all the CaT is removed. One can get around this by treating a small portion of the target wine with large amounts of CaCO3 containing seed crystals of both CaT and CaM, thus forcing both acids to precipitate. This is a “double salt precipitation”. A proprietary mixture of ACIDEX can reduce H2T and H2M equally. This method was developed in and only used on juice, not wine. Any excess Ca precipitates with H2T. Acidity can be reduced by 0.7% and more. This is the best and safest method for reducing acidity.

Acidex® Calculations

Desired Acidity

10 g/l 9 g/l 8 g/l 7 g/l Initial Acidity Acidex® Juice Acidex® Juice Acidex® Juice Acidex® Juice grams litres grams litres grams litres grams litres

9.5 g/l * * 8 2.8 15 4.4 46 8.7

10.0 g/l * * 15 3.6 23 5.2 47 9.7

10.5 g/l 8 2.0 23 4.4 31 6.0 62 10.0

11.0 g/l 15 2.8 31 5.2 39 6.7 69 10.7

11.5 g/l 23 3.6 39 6.0 46 7.4 77 11.4

12.0 g/l 31 4.4 46 6.7 47 8.1 85 12.0

12.5 g/l 39 5.2 47 7.4 62 8.7 92 12.7

13.0 g/l 46 6.0 62 8.1 69 9.7 101 13.3

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13.5 g/l 47 6.7 69 8.7 77 10.0 108 14.0

14.0 g/l 62 7.4 77 9.7 85 10.7 117 14.5

14.5 g/l 69 8.1 85 10.0 92 11.4 124 14.7

15.0 g/l 77 8.7 92 10.7 101 11.7 * *

Note: The table is set up for 23 liters (6 gallons) and is calculated for unfermented grape juice. If you are adjusting more than 23 liters of juice divide the Acidex© and juice amounts by 23 and multiply the result by the number of liters you have. Acidex© won’t work if it’s simply dumped into the entire amount of wine. Instead you must add the indicated amount of juice to the Acidex. The procedure is as follows: 1. Determine the initial acidity of your juice. Decide the level to which you wish to reduce it and find the correct figures in the table above. 2. Carefully measure the juice sample indicated. Do not use more - it won’t work. 3. Weigh the indicated amount of Acidex© and place it in a container at least 20% larger in volume than the juice sample. This will allow for foaming. 4. Slowly stir the juice into the Acidex©. Stir for at least 10 minutes to thoroughly distribute the acid salts. You should see some active foaming. 5. Allow the mixture to settle for several hours, preferably overnight. Put it into a refrigerator if possible or, alternately, put it in the coldest place in your wine making area. 6. Filter the juice through a lint-free cloth, cheesecloth, or a wine filter. This will remove the chalky precipitate. 7. Stir the de-acidified and filtered sample back into the main portion of the juice. 8. Test and record your acidity again to ensure your reduction has had the desired effect

Example on wine. If you want to reduce 5 gallons of wine with TA of 12 g/ l to 7 g/ l, when it has 0.4% H2T, put 65 g of ACIDEX in a mixer with about a pint of wine. Add 2.75 gallons of wine slowly with stirring. Let settle several hours and rack into untreated wine. Let the wine sit for a few months to dissipate CO2. ACIDEX needs thorough mixing. The reduction of H2T is generally greater than that of H2M. Fairly expensive stuff. Check their web site.

Reducing acidity with bacterial fermentation.

Malolactic fermentation (MLF) is commonly used. Let’s start with a red grape with a TA of 11 g/l and pH of 3.0 at harvest. Assume we will have a little over 40 tons of grapes which will yield about 7,000 gallons of wine drained and pressed. One method for MLF is next described.

One to two weeks before harvest, a freeze dried or liquid ML culture is obtained. A typical culture would be an 8 oz. sample (128 oz/ gal) and it has cells count of a billion per ml. Next, get 1-1/ 2

11 gallons of apple (or grape juice). Add an equal amount of sterile water. Add the ML culture. Put it all in a 5-gallon DJ. Seal the top with a cotton ball and place the bottle in a warm place.

When the grapes are crushed, 25 ppm SO2 can be added. Before inoculating with yeast, 75 gallons of juice is drawn from the fermenter and diluted with 75 gallons of water. The apple juice culture is added. After the wine is dry and pressed, 2.5 g/gal of CaCO3 is added to reduce TA to 10 and increase pH to 3.2. To the 7,000 gallons of wine, the ML culture is added. At cellar temperature, MLF will take about three months. Final TA will depend on the starting H2M, but should end up about 7 to 7.5 g/ l. After cold stabilization, it will be lower.

The ML culture should be 1%-4% of the total wine volume. Do not add SO2 to the wine. Remember that at pH of 3.0, the level of molecular sulfur dioxide needed to stop bacterial growth and prevent any oxidation is believed to be 0.6 ppm. Only FSO2 of 13 ppm will give 0.8 ppm of molecular SO2. The addition of SO2 to the wine will stop MLF.

Lysozyme. Lysozyme is a naturally occurring enzyme isolated from egg whites which can be used in wine to control lactic acid bacteria (LAB). Lysozyme degrades the cell wall of gram- positive bacteria such as Oenococcus, Pediococcus, and Lactobacillus. Lysozyme is not effective against gram-negative bacteria like Acetobacter and has no activity against yeast.

Gram-positive bacteria are encased in a plasma membrane covered with a thick wall of peptidoglycan. Gram-negative bacteria are encased in a triple-layer. The outermost layer contains lipopolysaccharide.

Lysozyme's effectiveness depends not only on the type of bacteria, but also the number of cells present. Unlike sulfur dioxide, lysozyme is effective at higher pHs when LAB growth is favored. Lysozyme cannot completely replace sulfur dioxide, in part because it has no anti-oxidative effect. It can, however, be used to help reduce the amount of sulfur dioxide needed to achieve microbial stability over the life of both red and white wines. In general, red wines need higher doses of lysozyme due to loss of activity through polyphenol binding.

Lysozyme can be used in the following applications: Prevent growth of LAB in unsound grapes. Growth of LAB during primary fermentation can lead to sluggish or stuck fermentations. If grapes are unsound and have the potential to develop high levels of LAB, add lysozyme to discourage their growth at the juice stage.

Recommended Dosage:

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Reds and Whites 100-200 ppm (10-20 g/ hL) (0.38-0.75 g/ gal)

Delay or prevent malolactic fermentation (MLF). Add lysozyme to hinder or block the onset of MLF at the juice stage. Some yeast strains have difficulty completing alcoholic fermentation when MLF occurs simultaneously. MLF may inhibit the primary fermentation due to the bacteria's competitive utilization of fermentable sugars or due to the production of antagonistic by-products. Recommended Dosage: Delay MLF in Reds and Whites 100-200 ppm (10-20 g/ hL) (0.38-0.75 g/ gal) Prevent MLF in Reds and Whites 300-500 ppm (30-50 g/hl) (1.10-1.90 g/ gal)

Stabilize LAB populations during sluggish or stuck alcoholic fermentations. Heterofermentative LAB can form excess amounts of VA, which may become toxic to the yeast and cause a sluggish or stuck fermentation. In addition, high VA wines may need to undergo costly treatment to remove excess VA. Treat the sluggish or stuck wine with lysozyme prior to reinoculation with yeast. Recommended Dosage: Reds and Whites 250-300 ppm (25-30 g/ hL) (0.94-1.10 g/ gal)

Inhibit the onset of MLF in the bottle. Add lysozyme to a finished wine that has partially completed MLF to inhibit the onset of MLF in the bottle. Recommended Dosage: Reds and Whites 300-350 ppm (30-35 g/ hL) (1.10-1.33 g/ gal)

Lysozyme Rehydration Procedure 1. Weigh out the quantity of lysozyme to be added. 2. Add this quantity of lysozyme to approximately 5 times its weight in warm water. · 1 kg lysozyme to approximately 1.5 gal (or approximately 5.7 L water) • 1 lb lysozyme to approximately 0.75 gal (or approximately 2.8 L water) 3. Stir this mixture gently for about 1 minute. Avoid foaming! 4. Allow this mixture to “soak-up” for at least 45 minutes. 5. Repeat steps 3 and 4 until the solution has completely dissolved into a clear, colorless liquid.

(procedure from Scott Labs)

Ion Exchange. Ion exchange can be used to lower or raise acid level. Resins with ions adsorbed on them are added to wine. Ion exchange, both cation (H+ for K+ ) and combination (H+ for K+ and OH- for various anions) are used for different acid adjustments. The most common use of in exchange is with cation resins in the H+ form. It is used for increasing TA and removing K+ from juice and wine. An ion exchange resin is an insoluble matrix normally in the form of small (1-2 mm diameter) beads, usually white or yellowish, fabricated from an organic polymer substrate. The material has highly developed structure of pores on the surface of which are sites with easily trapped and released ions. The trapping of ions takes place only with simultaneous releasing of other ions; thus the

13 process is called ion exchange. There are multiple different types of ion exchange resin which are fabricated to selectively prefer one or several different types of ions. Ion exchange resins are widely used in different separation, purification, and decontamination processes. The most common examples are water softening and water purification. Most cation exchange resins deplete a wide range of nitrogenous compounds from juice in addition to K, Ca and Mg ions. Many amino acids and vitamins are exchanged at the wine pH. This could lead to nutrient deficiencies in the juice. It can sometimes be difficult to correct these deficiencies with other nutrient additions.

Ion exchange takes place in columns containing a bed of resins. They operate until the resin bed has been exhausted and must be regenerated. The regeneration is very messy and the regenerating stream is very toxic. Also, if juice had been ion exchanged, the resins can get fouled with proteins and gums, making additional cleaning necessary.

Because of the toxic nature of the regenerating stream, it is getting more difficult to dispose of them.

Biological deacidification. MLF is the most common biological means of deacidifying a wine. The yeast Schizosaccharomyces pombe also effectively decarboxylates H2M. However, the yeast also likes to generate H2S and others of its ilk. Hence, it’s not recommended.

There have been reports out of Australia in the 1960’s about some S. cerevisiae strains that can degrade nearly 50% of the H2M during alcohol fermentation. This may be worth further study.

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Basic Winemaking and Enology – 6 Aging Wine. A variety of changes take place that affect wine quality. Slow oxidation and reduction reactions are the principal ones. Wood extractives can also have significant effects on color, odor and flavor of wines aged ion wood. The rate of maturation in wooden containers varies with type of wine, extent of aeration, storage container size and temperature of the wine.

The higher the wine tannin content, SO2 and other reductive substances in a wine and the lower the wine temperature, the slower the aging will be. If aging proceeds too rapidly, the wine may not develop its potential fine qualities. Oxidation is the biggest hazard. On the other hand, very slow aging will cost more money and risks the possibility of contamination.

Most white and pink wines are aged in inert containers, like stainless steel, glass-lined, epoxy, tile-lined or very large, very old wooden tanks. Normal aging time is very short, generally 2 to 6 months.

Young whites have lighter color, more grape aromas and fermentation bouquet and less wine or barrel bouquet. They taste fresh and fruity. The lightest wines reach their maximum quality after 6 months to two years in the bottle. More full bodied, barrel fermented white wines can be aged in barrels for 18 to 24 months. This is especially true of the Chardonnays of Burgundy. Heavier, sweetened whites can be improved much longer. Some Sauternes and Germany TBA’s are great after 50 years.

Pink wines can be more delicate than whites. With just six months of bottle aging their hue can go from candy red to orangish and finally orangish brown. Before you even smell the wine a negative perception can be created. When young, they have great reddish pink colors and loads of fruity aromas. On the palate they are very fresh and lively. The most gulpable category of wines.

Light reds, low in alcohol, are best drunk young. They are just slightly more ageably serious than rosés.

Many wineries hold reds in large containers for up to a year and then in barrels for one to three years before bottling. The highest priced reds are aged in barrels for two years and more. They age slowly in cellars at 50° to 60° F. It is the decision of the winemaker when barrel aging should be stopped and the wines bottled.

Aging will not improve the quality of poor quality wine. Both under aging and over aging can damage the quality of sound and excellent wines.

Aging occurs in all wines and continues until the wine is consumed. Fruity esters are formed during fermentation and aging begins as soon as fermentation is completed. It happens in all storage containers.

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With red wine, the first thing observed is color becomes less vivid. The purple and violet tints of young wines become lighter and shift in an orange and brick direction. The fruity aromas of young reds fade. The nose becomes more complex, intense, subtle and pleasing.

With white wines, they lose the simple taste of grape and begin to acquire a very slight aged- buttery flavor. This is very nice when it occurs in great wines. It can become too pronounced and be disagreeable. The color goes toward yellow then gold and on to amber and brown.

Two distinct aging phases are noticed. In maturation the wine develops a particular character and becomes clear during its tank or barrel life. While in maturation, the wine will be exposed to intermittent and moderate amounts of oxygen. It is aerated through every winemaking process; sampling, pumping, racking, filtering, fining, topping, centrifuging, etc. It is very important that the winemaker have good equipment and cellar operators. The wines cannot tolerate large amounts of oxygen. With moderate oxygen pickup the wines lose their “rawness”.

Most of maturation changes result from the formation of esters and binding of small pigment molecules with larger ones and their resulting precipitation. Recall the esters are formed by the reaction of organic acids and alcohols. This reaction process is very slow and limited in the presence of water. Most esters in wine come from the grapes or are produced by yeast enzymes during fermentation. Most ester formation stops when yeast stop growing. Also, if wine is stored in wooden cooperage, there may also be wood extractions.

Aging, as normally defined, happens in the bottle. It is here the wine attains optimum quality. During this phase, the role of oxygen no longer is a factor. In the bottle, wine ages in the absence of air.

Controlled oxidation. Both oxidation and aging are slowed by the presence of SO2. As each 4 ppm of SO2 is oxidized it removes 1 ppm of O2 from wine. Air is mostly nitrogen and has 21% O2. Oxygen is absorbed into wine through all the cellar practices mentioned above. Also, containers not tightly sealed can admit oxygen. It takes 6 to 7 ml of O2 to saturate wine at cellar temperature. As the temperature drops (especially during cold stabilization) even more O2 will go into solution.

Wine absorbs and consumes O2 very rapidly. In a partly filled container, wine can absorb and consume 50 ml/ l from air in 24 hours. Keep you tanks topped! Don’t “do it in the morning”. Oxygen can chemically combine with wine in different ways. If only a small amount of O2 is available to the wine, it can be absorbed by the metallic ions and oxidize them. These ions then oxidize other wine components, perhaps acids.

Later in the aging cycle, tannins, pigments and SO2 are oxidized. This process can take more than a year. Oxidized tannins and pigments become less soluble in wine and precipitate. You have seen these in bottles of older red wines.

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When oxygen is introduced in excess or too rapidly, metallic ions can’t act as a primary carrier and oxygen appears to combine directly with alcohols and aldehydes. First, a flat character ensues. Same as “bottle sickness”. That lasts a few weeks. In white wines, excess oxygen can give a “sherry” character. Also, some higher alcohols can be oxidized to acids, which can form esters with EtOH and other alcohols and contribute to flavor and bouquet in aged wines. Small quantities of these esters seem fruity. However, ethyl acetate (EtAc), the principal ester, is undesirable in excess and remind one of paint thinner.

Wines with pronounced grape aromas and flavors will lose both these characters as oxidation takes place. Oxidation causes pigments, in both white and red wine, to turn brown and precipitate, darkening white wines and lightening reds. Because of more tannins and pigments, red wines can take more O2 than whites.

If acetaldehydes are formed, tannins and pigments tend to combine with it to prevent a Maderized aroma or flavor. Wines from the Island of Madeira are sweet fortified wines which are heated and held for 3 to 6 months at 50° C (122° F). This gives the wines a very caramel, baked aromas.

Tannins have bitter flavor that oxidation reduces, tending to smooth or mellow the taste. The dark color of red wines hides minor browning and color changes that accompany oxidation. Putting wines in small barrels and leaving them there is associated with oxidation and is why whites are not barrel aged a long time. Full bodied and sweet wine wines can benefit with barrel aging. The exact amount of oxygen needed for aging any given wine is not known. With micro-oxidation, we know that one mL / L/ month is approximately what a new 225-liter barrel can deliver to a wine, including topping and racking. (Basic Winemaking 2) But that says nothing of the ideal needs of a given wine.

During fermentation and early rackings, wines are saturated with CO2 and generally have good levels of SO2. Because of that, O2 has a minor affect on the wine. For normal aging, it is estimated that each liter of red wine requires at least 100 ml of O2 over the life of the wine. White, pink and light red wines uses about a tenth of that amount.

A 60-gallon barrel is about 225-liters. Wines in barrels have been reported to absorb 30-40 ml of O2 in one year. Each racking can add 5-6 ml/ l. Topping, fining, pasteurization (if done) also increase oxygen pickup. Both light and heat can speed up oxidation reactions.

Containers without extracts. These are inert containers. Since stainless steel, glass-lined tanks and similar containers are normally air tight; the importance of keeping containers topped and tightly sealed must be emphasized. As a last resort, if the container can’t be filled, CO2 can be an aid. It is good practice to purge all lines and tanks with CO2 prior to passing wine through or into them.

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Aging in wood. Wines can be aged in wood via several pathways. Generally, pink wines are never ‘aged” in wood. Wineries with nothing but wooded tanks must store pink wines in wood until bottling. The extraction of wood flavors should be avoided.

White wines can be fermented in wood; barrels, puncheons, ovals and tanks. After fermentation, the tanks can be topped and the wine allowed to remain in the wood and kept sur lie. Or, the wine can be racked off the yeast lees, the exited container cleaned and the wine returned for aging. The racked wine can remain in the same state of cleanliness or it can be filtered prior to re-entering the container. The filtered wine will pickup wood extracts more readily that the racked wine.

Red wines can be sent to wood for aging directly from the fermenter and press, after one racking or after filtration. Again, the filtered wine will pickup wood extracts more readily that the direct from fermenter or racked wine.

Oak aging tends to speed up wine clarification. Tartrate precipitation is also accelerated as a result of wood aging.

Long term aging of red wines in barrels carries risks. VA must be periodically checked. To ensure good microbial and oxidative protection, make sure that, after completion of MLF, the free molecular SO2 is between 0.6 and 0.8 ppm.

Generally, people who pay high prices for wines, feel wines aged in barrels taste better than those not. It is true that young wines develop more rapidly in small volume containers than larger ones. Young wines remain cloudy longer in tanks than in barrels. The smaller barrels have more precipitation sites on barrel walls per unit volume than do larger tanks. A rough wood surface also has more precipitation sites than smooth stainless steel tanks.

The acid level in wines increases over time because of loss of water due to evaporation and extraction of acids from wood. Alcohol, even though it evaporates, also increases in wood because water diffuses through wood more readily than alcohol.

In time, color also becomes more concentrated with wood phenol complexings.

The wood of a container contributes to complexity of taste and bouquet of mature wines. Overtones of vanilla and delicately wooded flavors can be found in the bouquet of wines kept in new barrels from their very early stages. The quantity of tannin can increase significantly. This can cause bitter or astringent perceptions.

There are also differences between American (Quercus alba) and European (Q. robur and Q. sessilis) oak. We know that oak species, forest location, toasting time and temperature, the climate in which the staves are cured, the time of stave curing and the char level can all influence the flavor composition of the wood.

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European oak has more extractable aromas than and American oak has more extractable tannins than European. American oak can have a coconut-like or even resinous aroma and European oak is clove-like. The long curing of European oak prior to coopering shows large increases in vanillin. My descriptions have been American can be “lumber yardy” and European “sweet”.

Wood cooperage. The disadvantages of wood cooperage include high cost. Mark Heinemann of Demptos Napa Cooperage quotes the latest price on a 225-liter (60 gallon) barrels at: French $960 to $1,060 Hungarian $585 to $600 American $345 to $375 Remember that the US$ has lost 22% of value vs. the Euro over the last two years.

Wood cooperage demands high maintenance costs to pre-test for leaks and repairing cooperage flaws. The winemaker, lab, QC staff and cellar crew must spend much more time inspecting and working on individual barrels. The barrels are all individual entities and require individual attention. One bad barrel in 500 can easily destroy a blend. The potential problems include oxidation, microbes, off or excess wood characters, topping, record keeping, barrel signage and storage.

Inert containers do not face most of these problems. 55-gallon stainless steel drums certainly have similar record keeping, barrel signage and storage. And all containers, no matter what they of, must be individually checked before being added to a blend. But, the possibility of oxidation, microbes, off or excess wood characters, or topping requirements do not generally apply with inert containers.

It can’t be ignored that most of the worlds “great” wines have spent a portion of their pre-bottle life in wooden cooperage. Preferred sizes run from 225 to 1,875 l (60 to 500 gal). The smaller the container, the higher is the surface to volume of wine ratio. Faster aging and more wood extract occurs in smaller containers. Many different woods have been used for wine storage.

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In barrel making or coopering, an oak tree, for example, is cut into sections of the length desired for staves and the sections are split lengthwise through the center into quarters. With only inner heartwood used, barrel staves are cut from the face of each quartered section so that the rays in the wood, which extend outward from the center, are parallel to the width of the stave. Cutting them this way reduces wine diffusion through the staves and minimizes changes of stave width due to moisture.

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What type of Toast Level is desired in a barrel?

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Oak Characters

Positive Negative

Coconut Vanilla Spicy Clove Pepper Cinnamon

Cedar Green

Planky

Sawdust

Woody Pencil

Shaving

Sappy-

Resineous

Tobacco Grass Grass Dill Dill Menthol Herbaceous Menthol Thyme Laurel

Mushroom/ Truffle Shoe Box Earthy Leather Wet Dry Leaves Cardboard Tannic Tannic Astringent Bitter

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Reference is the average of all samples after 12 months.

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Preparation and maintenance of barrels. When barrels arrive, get a flashlight and inspect inside and outside of every barrel. Smell each one. Rub you hand over the outside of the barrel to check the precision of the cooper. On well made barrels, you cannot feel the joint where the staves meet. It is wise to number each barrel. The information can be branded on one head of the barrel. For example: 7001 N-M Demp This is the first barrel was purchased in 2007. It was the first numbered. The next barrel will have 7002 branded on it. The wood is from the Never (N) forest of France. A code for each forest can be assigned. The barrel was toasted to the medium (M) level prior to leaving the cooperage. The cooper usually puts their name on the barrel head. If not, put a code. Demp refers to Demptos Napa Cooperage. You can also ask them to stamp the forest and char level before shipping to you. If all these things happen, just brand 7001. It is also good to brand the number on the stave next to the bung hole. Then the winemaker, when tasting the barrel, can take notes without looking at the barrel head. Sometimes that is not easy to do.

Fill the new barrels with water. Let them soak for a day to tighten seams. Some people add a 100 ppm SO2, acidified with 0.25% citric acid to help “sterilize” the barrel. Doing this extracts oak.

Once tight, use the barrel. Don’t let it get dry. After barrels have been used, check by smell and sight to make sure no bacteria or mold infection has started. If there are no wines to refill the barrels, rinse them with cold water and add SO2. Burn ¼ sulfur stick inside the empty barrel. Seal after. On the barrel heads, in chalk, write the “date, W&S”. Make sure no sulfur drips into barrel bottom. Sulfur at the bottom of the barrel can lead to H2S production. SO2 gas can also be sprayed into the empty barrel prior to sealing.

Some people remove the tartrates and other sediments from the barrel each time emptied or every 3 to 4 years. Hot water usually works. If not, a sodium carbonate solution is possible. The hot water and NaCO3 can also extract wood compounds. The tartrates in the barrel act as sights for future tartrate and other sediment deposits.

The sulfured barrels, when reused, should be filled with water and held overnight. This will test for tightness and remove SO2. If a barrel has developed a moldy smell, soaking with a 1% Na2CO3 (soda ash) solution at 120 °F can be tried. Not always successful.

Topping up barrels. Barrels storing “finished” wine should be kept full and tightly sealed. A wine is finished when it has completed alcohol fermentation and MLF if it has been inoculated. Both alcohol and malolactic fermentation produces CO2 and tightly closed containers can lead to problems. Generally, bungs are launched from barrels after pressure buildup.

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Some of the water in the wine diffuses through the wood pores and the volume in the container goes down. As this “” occurs, a vacuum is pulled inside a tightly sealed barrel. The winemaker has two choices.

Every four months the bungs can be removed from the barrels and the empty volume topped up. This eliminates the head space in the barrel, but adds a touch of oxygen. When the bungs are pulled prior to topping, a vacuum is released. A sucking sound occurs.

Another alternative is to pump the wine in the barrel, make it full, put in the bung and rotate the barrel 30°. You want the bung to be wet and remain swollen. Then just leave the barrel in the rotated position for the duration of the barrel aging.

Wood extractives. If wines are not left in barrels too long, the aging can add great complexity to the wines. Some 5% to 10% of the total weight of dry oak consists of tannins and other oak substances that can be dissolved in wine. The phenolic components of oak are largely nonflavonoids. This is in contrast to young wines, in which flavonoids predominate. This difference allows chemical analysis to identify wood aged wines.

Vanillin, syringaldehyde and related phenolics derived from lignin are among the compounds desired from oak aging. Lignin is the major non-carbohydrate constituent of wood. It functions as the natural plastic binder for the cellulose fibers. About 0.6% to 0.7% of the total weight of dry wood is initially extracted from the barrel. Apparently during further aging, reactions of lignin with water (hydrolysis) and oxidation releases more aromatic substances.

In a new barrel, about 3.8 g of wood per liter of wine (1/ 2 oz per gal) are extracted at a penetration of 0.5 mm (1/ 50 in) in about 2 months. This is 3 to 10 greater than oak perception thresholds. In old barrels, 6 mm (1/4 in) of penetration by wine is not unusual, suggesting that under perfect conditions, a barrel could be filled and emptied about 100 times to give a detectible oak flavor to all wine.

If wine is stored in new barrels for 2 to 3 years, about 70% of the total extractives will be removed from the barrel. This is far too much and would lead to over oaking. Normal winery practices would find barrels depleted of any possible oak flavoring after about seven years. If the barrel is well coopered and sound, it can still be very satisfactory for wine aging. To also gain oak flavor during aging, several possibilities exist. 1. Shave the insides of the barrels to expose new wood. This can compromise the structural integrity of the barrel. 2. Put new heads in the barrels. This would be like buying 25% new barrels. 3. Buy a certain percentage of new barrels every year. This would add the desired oak flavor to the final blend. 4. Add InnerStaves. 5. Use oak additives. 6. Use oak extracts.

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Oak chips and shavings or granular powder can give oak flavors in about a week. Further contact can increase extractives resulting from wood lignin hydrolysis. From 15 to 30 g of oak chips per gal of wine can add positive oak flavors and astringency to red wines.

Oak extracts are made by combining oak chips with small amounts of 100-proof alcohol (vodka is OK). Once made and kept in sealed containers they will keep indefinitely.

In cooperage, the wood surface touching the wine and giving wood extracts, increases as the cube root of the volume. Therefore, by increasing the volume on a wooden container by a factor of 1,000 would only increase the wood surface area ten times. In a 60-gallon barrel there is approximately 21 ft2 of wood in contact with the wine, or about 0.35 ft2 of wood surface area for each gallon of wine.

A 130-gallon puncheon has approximately 37 ft2 of wood in contact with the wine, or about 0.28 ft2 of wood surface area for each gallon of wine. It also takes 25% more time to get the same level of extractives. A 5-gallon wooden container has 1,000 ft2 of wood surface area for each gallon of wine. So, the size of container used, as well as its age, is an important factor in deciding how to age a wine in wood.

European oak contributes more extract and tannin to wines than American oak. On average, about one-third more flavor. The oaks have different, discernable extractives.

Oak chips and extracts. The use of oak additives is rapidly expanding in the American wine industry. (Recall Winemaking Choices –White) As stated, oak chips yield extractives within a week and with further contact can increase extractives by allowing hydrolysis of wood lignins. Extracts can fill the same roles as chips.

Off odors in wine. Excess SO2 and acetaldehyde are more common off odors in white wine than reds, because red wine pigment combines with both. They also combine with each other, so they cannot coexist as off odors.

Acetaldehyde (CH3CHO) results from oxidation of EtOH. When it shows up in a wine, that normally indicates the wine is too old or has been badly handled. It often is accompanied with browning. In some wines, like sherry, it is a natural and desirable component. It can give a “nutty” flavor. If you want to isolate the odor, compare a standard white table wine with a Fino sherry. Acetaldehyde is also a minor byproduct of alcohol fermentation. Is can smell like raw pumpkin.

Sulfur dioxide (SO2) is detected at different thresholds by different people. In excess, it causes eyes to water, tingles the nose and causes sneezing. It is reminiscent of lighted match sticks. It can effect wine flavor and give a “musty” character.

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Among the off odors in red wines, high volatile acidity (VA) is most common, followed by mercaptans and disulfides and then microbiological infections.

All wines contain small amounts of acetic acid (HAc, CH3COOH) and ethyl acetate (EtAc, CH3COOC2H5). They are both volatile and contribute to wine odor. Below 0.05%, little effect of either is detected. Higher levels cause the wine to seem to some, like vinegar.

HAc is an oxidation product and EtAc is an ester of HAc and EtOH. They can combine to give wines odors like vinegar, nail polish remover or airplane glue.

Hydrogen sulfide (H2S) can give wines a rotten egg odor. Yeast can reduce certain forms of sulfur to H2S and this can be converted to mercaptans and disulfides. Traces of these can ruin the wine. Recall in Basic Winemaking 5, we talked about tests using copper sulfate and ascorbic acid to determine if perceived off odors in wine resulted from H2S, mercaptan or disulfide, or none of those. UCD’s Dr. Linda Bisson has done lots of work on sulfide occurrence in wine. She sent a note indicating they had found from commercial samples that what people thought were sulfur off- odors had no sulfur in them at all. Many of the characters were microbially created, i.e. Brett, etc.

Microbial infection can lead to a sauerkraut odor from lactic bacteria and if sorbic acid is also present a geranium bouquet. Lactobacillus bacteria cause a “mousy” nose. MLF can produce diacetyl (CH3CO)2, which in excess smells like heated or rancid butter. Earthy odors can be bacterially caused. Wines made from mildewed grapes can smell moldy.

Corks can give “corky” or moldy odors to wine. The most insidious taint compound is 2,2,6- trichloroanisole (TCA). This results from chlorinated corks.

Brettanomyces and Dekkera yeast give horse sweat, white paste, burnt beans,…, noses.

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