Basic winemaking chemistry

INPUTS PROCESSING OUTPUT
Grapes
Yeast
Sulphur dioxide
Other additives
Fermentation
Post fermentation
Alcohol
Acids
Polyphenolics
Other outputs
Glossary

Inputs: Grapes

Juice

Juice contains:
  • water (approx. 77%);
  • sugars ( approx. 22%) - glucose and fructose in approximately equal proportions;

[N.B. If juice is in excess of 14 Bé (i.e. about 14% (v/v) alcohol), the juice is unlikely to ferment to dryness as most yeasts cannot tolerate more than 14% (v/v) alcohol.]

Grape sugar content determines the maximum combined ethanol and residual sugar level of wine.

  • pectins (approx. 0.1%), which affect pulp consistency, juice viscosity, draining, pressing, juice clarification and wine filterability;
  • nitrogen compounds (approx. 0.1%), including amino acids and ammonium salts (which act as nitrogen sources for yeast and bacteria);
  • traces of alcohols (ethanol and glycerol);
  • acids - tartaric (5g. L-1), malic 5g. L-1, and small amounts of citric acid (Note that acid levels may fall in warm or hot conditions);
  • traces of volatiles (flavour compounds).

Seeds and stems.

There are usually two seeds per grape. Seeds and stems, along with skins, provide the phenolic material to wine which largely determines the wine style and quality. This material is partially extracted by prolonged contact of the skin and seeds with the juice during fermentation. Seeds and stems provide flavour (in this case, astringency and bitterness), whereas skins provide colour.

Skins

Physical damage (e.g. birds, mechanical harvesting, Botrytis) should be minimised to avoid microorganism growth. Such growth is favoured by warmth and proceeds with time. Skins provide a protective layer that prevents contact of microorganisms with the juice. Skins, along with seeds and stems, provide the phenolic material to wine which largely determines the wine style and quality, as well as inorganic constituents, such as the K, Ca, Mg, Na cations. (See acid as an additive to input).

Oxidase enzymes

Presence of oxidase enzymes in certain varieties of grapes may enable a chemical reaction to take place which in turn may cause oxidation to take place.

Fungal infections

Presence of fungal infections in grapes may enable a chemical reaction to take place which may cause oxidation.

Inputs: Yeast

A microorganism, such as yeast, is required to promote alcoholic fermentation. Rapid inoculation with a a strongly growing yeast culture will reduce the risk of contamination by wild yeasts.

Yeast cells are particularly well adapted to produce energy by fermentation. A wide range of yeasts types occur naturally, but the strongly fermentative yeasts such as Saccharomyces cerevisiae) which are the ones required for winemaking are rarely abundant in nature. The more abundant yeasts are the weakly fermentative types, such as Kloeckera, Hanseniaspora, and Candida, and these are often responsible for the early stages of fermentation, sometimes resulting in off flavours at this fermentation stage. However, S. cerevisiae, although not preventing the growth of weak fermenters, is ultimately the one that dominates wine fermentation. (Note that there are many different strains of S. cerevisiae, not all of which are suitable for winemaking). As pH decreases below 3.5, growth of S. cerevisiae will decrease.

The yeast strain to be used depends on:

  • SO2 tolerance,
  • fermentation temperature (Saccharomyces yeasts tend to be more tolerant of low temperature than bacteria),
  • alcohol tolerance (but all yeast action may be inhibited by fortification of wine to about 18% ethanol),
  • rate of settling after fermentation, and
  • flavour effects.

Undesirable effects of yeast. Can cause:

  • haziness and gas production, if wine contains residual sugar.
  • off flavours, if either
    • wine contains residual sugar, or
    • wine is exposed to air during fermentation.
  • release of sulphur dioxide if there is insufficient nitrogen in the must.
  • the release of acetaldehyde, a very strong SO2 binding agent. Consequently, yeast activity will eventually convert all free SO2 to a bound form.

Fungicides that are sprayed in the vineyard to control vine pathogens will leave residues on the grape that can inhibit the growth of wine yeasts.

Oxidative yeasts are quite unsuitable for winemaking and are generally easily controlled by SO2 addition, as are the weakly fermentative yeasts.

Inputs: Sulphur dioxide (SO2)

Sulphur dioxide will inhibit oxidation and minimise the risk of contamination by acetic acid bacteria. Sulphur dioxide does not affect yeast growth in juice as much as it does in wine.

Sulphur dioxide may be present in either free (FSO2) or bound (BSO2) form. FSO2 may be either molecular (SO2) or ionic (called bisulfite HSO3- and sulfite HSO32- ions). It is only the molecular form that is antimicrobial (and particularly antibacterial) or volatile. However, all three free forms are considered to have antioxidant activity.

At juice/wine pH, the bisulfite form predominates, making up more than 90% of the total. As the pH increases, the molecular form decreases in abundance. Molecular SO2 drops from 10% at pH=2.8 to 1% at pH=3.8.

An equilibrium exists between FSO2 (free) and BSO2 (bound). All suplhur dioxide initially added is free. So if sulphur dioxide is added to wine already containing suphur dioxide, the proportion of free will initially rise then gradually fall as some of it becomes bound. As more FSO2 is bound, the capacity of binding agents (e.g. acetaldehyde, anthocyanins) is used up, so further additions of sulphur dioxide will result in a greater proportion existing in the free form. Conversely, if the equilibrium is removed by some of it acting as an antioxidant, there will be less sulphur dioxide in total and, although some BSO2 will become free in an attempt to restore equilibrium, a lesser proportion will exist in the free form. However, if the sulphur dioxide is removed as a result of an increase in concentration of the binding agent, the balance itself is changed and FSO2 is not released.

Pigments of young red wines (anthocyanins) bind sulphur dioxide quite readily. The pigment is decolourised in the process, so good wine colour requires avoidance of high sulphur dioxide levels. For this reason, only traces (1-4 ppm) of FSO2 usually exist in such wines. Such low levels cannot inhibit yeast growth in wine (as opposed to juice) if residual sugar is available. High levels of FSO2 are required to stop yeast growth and leave residual sugar in the wine. Such high levels would decolourise red wines. Therefore red wines are made dry.

Benefits of sulphur dioxide include:

  • FSO2 (both molecular and ionic) gives rise to the three types of antioxidant activity:
    • reduces oxidative enzyme activity by inhibiting the action of the enzymes. This is the only process of sulphur dioxide that demonstrates a direct action to reduce oxidation.
    • FSO2 reduces hydrogen peroxide, an oxidising agent.
    • FSO2 decolourises brown phenolic oxidation products and prevents phenolic oxidation products from undergoing further reaction (i.e. FSO2 stabilises, but not prevents, the oxidation products).
  • free molecular SO2 (10% of the total free SO2 depending on pH) inhibits microorganism growth generally and bacterial growth particularly, although BSO2 may exhibit weak activity against such bacteria. However, the interaction between FSO2 and anthocyanins dramatically reduces the ability of sulphur dioxide to inhibit bacterial growth in red wines.

Disadvantages

  • high concentrations of FSO2 have a sharp, irritating odour.
  • FSO2 may inhibit growth of yeast and lactic acid bacteria Thus SO2 is not added after fermentation until MLF is completed.
  • high concentration of FSO2 and BSO2 may exceed legal limits.
  • decolourises red wine pigment.

Note that the analytical methods (both the aspiration-oxidation and the idometric titration methods) used to determine the quantity of sulphur dioxide cause BSO2 to be released and measured as part of the FSO2, thereby grossly overestimating the free form, and underestimating the bound form.

Sulphur dioxide
(up to 10%) FREE (90% or more) BOUND TO:

Molecular

Ionic

Aldehydes and keytones Grape pigments
SO2 HSO3 - SO3 2- Carbonyl compounds Phenolic compounds, especially anthocyanins
Inhibits microorganism growth, esp. bacterial growth. (Bisulphite ion) (Sulphite ion) BAPs (bisulphite addition products)
Demonstrates anti-oxidant activity Slightly inhibits microorganism growth, mainly in white wines.

Inputs: Other additives

DAP.

Diammonium phosphate, containing nitrogen compounds, is added as a yeast nutrient. Nitrogen compounds, including amino acids and ammonium salts, act as nutrients for yeast and bacteria. Sometimes low nutrient levels, particularly nitrogen levels, can lead to slow or stuck fermentations or the production of H2S. DAP is normally added to provide the necessary nitrogen nutrients.

Nitrogen in the must generates sulphides containing amino acids which are required as yeast food. Normally the amount of sulphides generated is sufficient. However, if cellular levels of amino acids become too low, enzyme systems will generate sulphides in excess of that needed. The excess reacts with the fermentation producing hydrogen sulphide, H2S. The addition of DAP to the ferment is designed ensure that the cellular levels of amino acids never get too low. Existing H2S can be removed in red wines by aeration, and in all wines by copper fining. However, to remove the cause of further H2S production, the nitrogen deficiency must be overcome.

Acid

Tartaric acid may be added to counter the neutralising effect of cations. This will increase total acidity and decrease pH. Tartaric acid is the acid of choice owing to its stability (i.e. non-volatility) and acid strength.

Pectolytic enzymes

May be added during crushing to facilitate rapid separation of juice from skins. Pectin is a long-chain gum-like binding material which tends to keep the cells together in the juice after draining and pressing. To obtain clarified juice, the pectin needs to be broken down by pectin-decomposing enzymes, or pectinases, allowing the grape flesh and cell debris to settle.

Tannin

Tannin may be added to wine where the grapes are thought to contain insufficient tannin.

Processing: Fermentation

Fermentation is an anaerobic process that produces energy (as opposed to respiration, an aerobic process that produces energy). Alcoholic fermentation is the anaerobic conversion of grape sugars (glucose and fructose - both C6H12O6) to ethyl alcohol (ethanol) by microscopic, unicellular fungi called yeasts.

C6H12O6 » 2 C2H5OH + 2 CO2 (+ heat)

The process occurs within the yeast cell. Sugars migrate into the yeast cell and are used as a nutrient, converting to ethanol and CO2 which leave the cell.

A key difference between alcoholic fermentation and alcoholic respiration is that in the former, the end products (ethanol and CO2 ) both have the same oxidation state as the starting sugars, whereas in the latter, the end products (water and CO2 ) have a higher oxidation state. In order to obtain the balance of the low oxidised ethanol with the more highly oxidised CO2 , the yeast takes a component of the sugar metabolism that under aerobic conditions would be oxidised, and converts it to acetaldehyde and then proceeds to reduce the acetaldehyde (and any other suitable substrates) to ethanol. Thus it is said that wine is a product of processes that occurs under reducing conditions.

Promoters of yeast fermentation include high temperatures (but not above 32ºC), (but note that a high temperature will also facilitate faster oxidation and growth of microorganisms, including acetic acid bacteria), low pH and yeast nutrients (including sugar and nitrogen). Inhibitors of fermentation include alcohol levels (levels above 15% (v/v) will generally prevent fermentation), a high pH, the presence of molecular SO2 and low nutrient levels. Sometimes low nutrient levels, particularly nitrogen levels, can interfere with the completeness of fermentation.

The desirable aspects of yeast fermentation are:

  • even and complete conversion
  • pleasantly flavoured yeast bouquet
  • no undesirable off flavours (H2S, acetic acid, ethyl acetate)
  • good settling of yeast after fermentation.

Acids during fermentation

During fermentation:
  • Tartaric acid. Falls during wine making as increasing alcohol levels and low storage temperatures encourages the crystallisation of calcium bitartrate.
  • Malic acid. Can fall during fermentation due to:
    • partial degradation by fermentation yeast.
    • MLF, carried out by lactic acid bacteria in young (usually red) wines. All malic acid may be converted.

Temperature

Fermentation rate will increase with increasing temperature in the range of 10ºC to 30ºC. Reducing the temperature may retard the growth of all microorganisms (including yeast growth) especially below 4ºC. Lower temperatures also reduce the extent of formation of some undesirable aroma compounds (aldehydes and higher alcohols), whereas a temperature rise may greatly accelerate growth. The fastest rate of growth occurs at about 35ºC.

Lower temperatures (10ºC to 15ºC) support growth of non-Saccharomyces yeasts.

Time

As fermentation time increases, both desirable colour extraction and (sometimes) undesirable tannins increase. However, there is also, to some extent, an increase in flavour richness. Juice that has had a longer contact time with skins produce wines that age apparently more slowly, and improve in character with age.

Processing: Post fermentation

To minimise oxidation and microbe attack, temperature should be reduced to 0º C (white) and 10º C for red.

SO2

Addition of SO2 after primary fermentation will inhibit MLF. SO2 inhibits microorganism growth in wine far better than in juice.

Pectolytic enzymes

Added after separation to facilitate juice clarification (in white wines).

Acid

It is not usual to add tartaric acid after the potassium bitartrate stabilisation, as the tartaric acid increases the potassium bitartrate and causes instability. Citric acid may be added to reduce pH and increase total acidity.

Ion exchange

This replaces alkaline cations (K, Na, Mg, Ca) with acidic hydrogen ions. The purpose is to reduce pH without significantly increasing total acidity.

Outputs: Alcohol

Ethanol

Approx 11% of the output. Results from the fermentation of the glucose and fructose.

It is toxic, to varying degrees, to all the microorganisms, including the yeasts that produce it. Very low ethanol concentrations will inhibit moulds, but concentrations of 3-4% are required to inhibit weakly fermentative yeasts. Large concentrations (13-14%) are required to inhibit the strongly fermentative yeasts and lactic acid bacteria.

The combination of acidity and ethanol concentrations found in wine is enough to kill any microorganisms hostile to humans.

Fortification of the wine to about 18% ethanol is an effective means of inhibiting further yeast action.

Glycerol

Approx 1% of output. Produced in significant quantities in Botrytis affected wine. Said to increase sweetness and viscosity.

Outputs: Acids

Wine acids (tartaric, malic, citric, lactic, succinic [FIXED ACIDS] and acetic [VOLATILE] acids) are weak acids because less than 1% of the acid molecules provide H3O+ (hydronium ions). In strong acids, virtually all molecules provide H3O+ ions. It is the concentration of these ions (or pH) that determines the strength of an acid.

The term acid indicates the molecular species that is able to provide H3O+ ions by reaction with water. It indicates both ionised and unionised forms.

Unionised                   Ionised
H2Ta + H20       »       H3O+ + HTa -

The combination of acidity and ethanol concentrations found in wine is enough to kill any microorganisms hostile to humans. Acids in the final output include:

  • Tartaric (3g L-1 ) A lthough biologically stable, major losses occur during winemaking through precipitation of potassium bitartrate.
  • Malic (3g L-1 ) Presence is indicated by a green apple taste.
  • Lactic (3g L-1 ) Produced in small amounts (up to 0.5 g L-1) during fermentation and larger amounts from MLF.
  • Succinic (1g L-1 ) Significant quantities can be produced during alcoholic fermentation, although usually less than 1.0 g L-1 . Depends on yeast strain, juice composition and fermentation conditions.
  • Acetic (1g L-1 ) Small amounts (to 0.4 g L-1 ) produced during fermentation. Slightly larger amounts (to 0.5 g L-1 ) may end up in the wine owing to the reaction of LAB with citric acid and sugars that yeasts are unable to ferment. Higher levels may arise from undesirable bacteria acting upon reducing sugars or from the action of acetic acid bacteria. Acetic acid levels over 1.00 g L-1 are thought to detract from wine quality. Legal maximum is 1.5 g L-1 .

Outputs: Phenolics/polyphenolics

About 0.25% of the output. Phenolic compounds are the most abundant of the readily oxidised substrates in juice or wine. They are the components of the berry skin, seeds and pulp. These determine the wine style and quality. Both colourless and red phenolic compounds oxidise to brown products.

Red wines possess a much higher phenolic level than white wines. This means that there is a higher concentration of substrates that can be oxidised, but also that any given amount of oxidation will affect less of the total proportion of phenolic material.

Except for the pigments of red wine (anthocyanins) they are colourless or light straw in colour. Phenolic compounds account for astringency (desirable in reds) and bitterness (undesirable in both reds and whites).

The term "phenolic compounds" includes a wide range of substances that posses the functional group first discovered in the compound phenol. Also referred to as polyphenolics. Tannin is a phenolic compound that provides both the flavour (primarily bitterness) and mouthfeel (astringency) to wine.

Phenolic compounds determine the style and quality of a wine, influencing the colour, taste and perhaps the aroma of wine.

Phenolic compounds
Flavonoids (95%) Non-flavonoids (5%)
Derived from skins (33%) and seeds, stems (67%) Derived from juice/pulp
Monomers Dimers Trimers Polymers Present in all wines, but particularly important to white wines which contain no other phenolic material.
Red pigments (provide colour) Tanins (provide flavour)
Includes anthocyanins found in outermost cellular layers of the skin Bitterness Mouthfeel (astringency)
  • Polymerisation of the above groups of flavonoids occurs over time.
  • The combination of four phenolic monomers provides the optimum astringency. (No astringency with less than four and less than optimum astringency with more than four.)
  • During ageing, polymerisation continues and leads to a decrease in astringency.
  • Red pigments (including anthocyanins) become progressively incorporated into polymeric material.
  • Vivid crimson gives way to dull brick-red colour.
  • Oxidised phenolics provide the brown colour to wine.

  • Outputs: Other

    Inorganic constituents

    Cation content (especially K), neutralises and buffers organic acids and may cause undesirably high pH values in hot dry regions (where K uptake in fruit is high). Other cations include Mg, Ca, Na.

    Anion content (e.g. phosphate, chloride and sulphate) are of little direct importance.

    Nitrogen compounds

    About 0.05% of the output. Causes haziness in white whine and may be removed by fining (with, say, bentonite). Note that nitrogen compounds, including amino acids and ammonium salts form part of the input grape juice (approx. 0.1%).

    Volatile compounds

    Some flavour components can be significant at concentrations of only a few parts per million (ppm).

    GLOSSARY OF WINEMAKING CHEMISTRY

    Acetaldehyde Aldehydes are one of the functional groups (-CHO) of hydrocarbons. Acetaldehyde is produced by the sugars during fermentation, but is readily reduced to ethanol, so only very small concentrations of aldehyde ever exist. It is also produced by the oxidation of ethanol in the must and as a by-product of phenolic compound oxidation in wine.

    Acetaldehyde (a very strong SO2 binding agent) is absent in juice and is released by yeast during fermentation. Consequently, yeast activity will eventually convert all FSO2 to a bound form. Thus, the post-fermentation level of FSO2 is virtually zero.

    SO2 binds so strongly to acetaldehyde that at first approximation the presence of free SO2 can be ignored until sufficient SO2 has been added to bind all acetaldehyde.

    Acetic acid bacteria. Strict aerobes. Widely distributed in nature. Comes from damaged fruit and Botrytis infected fruit. Acetic acid bacteria can lead to:

    • high levels of acetic acid, a condition called "volatility" (or volatile acidity).
    • when combined with ethanol, ethyl acetate (nail polish remover).
    • production of oxidised sugars by microbial action (as opposed to direct air contact). Oxidised sugars have the capacity to bind strongly to SO2 which may interfere with the normal free levels of SO2 in winemaking.

    No significant growth of acetic acid bacterium occurs below pH 3.0. The higher the pH, the more rapid the growth. The rapid initiation of fermentation will accelerate the rise of alcohol level inhibiting alcohol-intolerant microorganisms, including acetic acid bacteria.

    ethanol + acetic acid bacteria » acetic acid

    Acidity See pH and titratable acidity. The taste intensity of acids is:

    malic > tartaric > citric > lactic.

    Aerotolerant An organism that does not use molecular oxygen (O2) but is not affected by its presence.

    Anthocyanins Located in the outer cells of the grape skins. One of the objects of fermentation, at least for red wines, is to extract the anthocyanins from the skins. Anthocyanins are a phenolic compound which provides the colour in red wine. The typical level in newly fermented wine is about 0.5 g. L-1.

    Anthocyanins are a type of pigment that has both red and colourless forms that exist in equilibrium. However, a decrease in pH changes the equilibrium by increasing the proportion of anthocyanins in the red form.

    FSO2 binds to anthocyanins quite strongly, although not as strongly as to acetaldehyde. In fact, low levels of free SO2 can exist in equilibrium with anthocyanin-bound SO2. This binding decolourises the anthocyanins and the interaction dramatically reduces the ability of SO2 to inhibit bacterial growth in red wines.

    After wood maturation and MLF, anthocyanins become incorporated into polymeric phenolic material. This means that SO2 can be used during bottling with a much diminished effect on the colour of the wine.

    Ascorbic acid This is often referred to as an antioxidant. However, it can actually increase the rate of participation of dissolved oxygen in the oxidation process. Ascorbic acid prevents the dissolved oxygen reacting with juice/wine substrates as it reacts with dissolved oxygen more rapidly than the juice/wine substrates do. However, the oxidising agent hydrogen peroxide is produced in this process, so it is necessary to add SO2 with the ascorbic acid in order to scavenge the H2O2. Ascorbic acid is also used to convert quinones back to their original unoxidised form.

    If ascorbic acid is used without free SO2 the ascorbic acid can give rise to severe browning.

    Bacteria. Most bacteria cannot survive in the typical pH found in wine (pH 3.0 to pH 3.7). Two notable exceptions are acetic acid bacteria and lactic acid bacteria.

    Baumé Percentage by volume [ i.e. %(v/v) ] of solids in juice/wine. Based on a measure of salt in liquids, but the initial measurement of the juice provides an approximation of the percentage of alcohol obtainable from the fermented juice. Baumé x 1.8, gives the º Brix, which is an approximation of the g/mL of sugar in the wine.

    Botrytis. Botrytis cinerea can lead to:

    • the collapse of berry structure and integrity.
    • the release of an oxidative enzyme, laccase, into the juice.
    • secondary infections due to the exposure of the released juice to oxygen and wild microorganisms. Particularly common is the growth of acetic acid bacteria.
    • loss of water from the grape berry resulting in a concentration of sugar flavours within the berry (e.g. Sauterne style sweet wine).

    On the vine, B. cinerea may progress, with wet conditions, to vulgar rot (e.g. when affected by acetic acid bacteria) and with continued excessive production of acetic acid, to sour rot. With warm and dry conditions, the grape berries lose moisture and the sugar concentration increases to produce noble rot.

    Once fermentation begins, mould growth is limited by anaerobic conditions and the production of ethanol.

    Decarboxylation process. A process involving the loss of CO2 .

    Enzymes. Enzymes are an important group of proteins that act as biological catalysts. That is, they speed up, or slow down, the rate of chemical reactions in living organisms, such as oxidation.

    Two important oxidative enzymes are:

    • catechal oxidase, which is present in freshly crushed must. The action of catechal oxidase is readily (and truly) inhibited by SO2. It is very important to virtually stop this enzyme action during grape harvesting and juice processing where air contact is almost impossible to avoid. However, its activity decreases with time (so it does not contribute to wine oxidation).
    • laccase, which is only found in fruit infected with botrytis cinerea. Its action is not readily inhibited by SO2 and its activity hardly decreases with time. Thus, it may cause oxidation in wine. (Note that laccase is the only enzyme that can catalyse oxidation in wine).

    Formulae

    SG of Y = Density of Y /Density of H2O (no units)
    [Density of H2O= 1 g mL-1]

    Density = g mL-1 [also g cm-3 ]

    Baumé = % (v/v) [ approx potential alcohol in wine ]
    [ juice Bé = wine % (v/v) + residual sugar Bé ]

    º Brix = % (w/v) [approx sugar content in wine/juice]
    [ 1º Brix = 1% (w/v) = approx 10 g L-1 sugar ]
    [º Brix = 1.80 x Bé]

    Concentration = solute/solution [ Solution = solute + solvent ]
    = g L-1 (for solids)
    = mL L-1 (for liquids)

    Percent by volume by volume = % (v/v) [ mL / 100 mL ]
    [ e.g. Vol of alc / Vol of wine ]

    Percent by weight by volume = % (w/v) [ g / 100 mL ]

    Percent by weight by weight = % (w/w) [ g / 100 g ]
    [ e.g. SO2 / PMS = 57.6% (w/w) ]

    Parts per million = weight of solute [mg] / volume of solution [L]
    = weight of solute [ g ] / volume of solution [1000 L ]

    Weight of PMS [g] = volume of solution [ Litres] x ppm / 576

    Molarity [M] = n / L [ number of moles per litre ]

    No of moles = g / MW [mass per molecular weight ]

    Molecular weight = molar mass
    = g / mol [ physical mass per mole ]

    Flavour. Grape flavour is dependent upon trace components, such as terpenes in Muscat and methoxypyrazines in Sauvignon Blanc. Desirable flavour and intensity depends upon the wine type (e.g. table wines) and variety. In many varieties, the components responsible for flavour are not known.

    Oxidation can destroy important aroma compounds as well as produce new and unpleasant aroma compounds. However, maximisation of grape flavour assists in fighting bacterial spoilage and oxidation.

    In cool areas, where fruit ripens slowly, flavour develops while acidity is quite high and a more stronger and more distinctive flavour usually develops. In addition, low ambient temperatures reduce rates of microorganism growth and oxidation making retention of that flavour through to the final wine intrinsically easier. However, in warm or hot areas, flavour development is generally later than the decline in acidity. Consequently, desirable flavour intensity is often associated with a high pH.

    Grape flavour has to compete with flavours provided by alcoholic fermentation, MLF, wood maturation, microbial spoilage, oxidation.

    Although flavour development is closely linked to ripeness in cool climates, ripeness must be regarded as a poor indicator of flavour development. As grape flavour cannot be adjusted, but acidity can, and as sugar levels are normally acceptable, it is sensible to harvest on the basis of grape flavour and adjust the acid level.

    See also off-flavours.

    Hydrogen peroxide. A powerful oxidising agent produced during 1) the oxidation of phenolic compounds and 2) by the reaction of ascorbic acid with dissolved oxygen. It is rapidly deactivated by SO2.

    Hydrogen sulphide. Nitrogen in the must generates sulphides containing amino acids and amino acids are required as yeast food. Normally the amount of sulphides generated is sufficient. However, if cellular levels of amino acids become too low, enzyme systems will generate sulphides in excess of that needed. The excess reacts with the fermentation producing H2S. The addition of DAP to the ferment is designed ensure that the cellular levels of amino acids never get too low. Existing H2S can be removed in red wines by aeration and SO2 addition, and in all wines by copper fining. However, to remove the cause of further H2S production, nitrogen deficiency must be overcome. See also off-flavours.

    Ions Loss of a positively charged hydrogen ion leaves a negatively charged ion. Tartaric and malic acid give rise to the hydrogen tartrate ion (bitartrate ion) and hydrogen malate ion; and hydrogen malate ions in turn give rise to tartrate and malate ions.

    H2Ta » HTa - » Ta 2-

    H2Ma » HMa - » Ma2-

    Ka The acid dissociation constant. An acid (HA) dissociates into the hydronium ion (H3O+) and the salt of the acid (A-).

    HA + H2O « » H3O+ +A-

    The stronger the acid the more fully it dissociates and the greater is the value of the acid dissociation constant. It is equal to:

    [H3O+] [A-]


    [HA]

    where [ ] indicates the molarity of the hydronium ion, the salt and the acid. The negative logarithm of the acid dissociation constant is referred to as the pKa.

    Lactic acid bacteria There are three genera of LAB:

    • Oenococcus (formerly Leuconostoc). Gram-positive cocci, nonmotile, ellipsoidial to spherical, pairs & chains, heterofermentative, catalase negative, optimum temperature 22°C.
    • Lactobacillus Regular, nonsporing, Gram-positive, rods or coccobaccilli, pairs & chains, heterofermentative & homofermentative, aero-tolerant, catalase negative.
    • Pediococcus Gram-positive, cocci, nonmotile, pairs & tetrads, homofermentative, aero-tolerant, catalase negative.
    Oenococcus is the most desirable. All three may also be classified as aerotolerant, facultative anaerobes.

    The higher the pH, the more rapid the growth:

    pH 3.2 - growth of any of the LAB, including O. oeni (which provides a clean MLF) is difficult to initiate. If it does grow, it will grow slowly.

    pH 3.3 - usually required for growth of any LAB, but only O. oeni grows well. This pH is the minimum required for rapid and complete MLF.

    pH 3.4 - L. oenos grows more rapidly than Lactobacillus and Pediococcus.

    pH 3.5 - Lactobacillus and/or Pediococcus is likely to be more rapid than L. oenos.

    LAB are able to tolerate the concentrations of ethanol normally associated with wines, but reduced growth is observed as temperature increases and/or as the ethanol concentration exceeds 10%(v/v). Lactic acid bacteria are very sensitive to SO2 (including the bound form), so red wines intended for MLF are usually held without SO2 addition after alcoholic fermentation until completion of MLF. Lactic acid bacteria are slightly more tolerant of low temperatures than acetic acid bacteria.

    Malic acid Malic acid (like tartaric acid) is an important acid of the grape berry and has two acidic sites. Both contribute to titratable acidity. However, it is more easily biologically degraded than tartaric acid so the level in the grape berry is more variable than for tartaric acid. The hydrogen malate ion contributes about 50% to the titratable acidity compared to the unionised acid but only about 1.5% to the actual acidity (or pH). In cold areas, where ripening is difficult, acid levels can be very high and most of the acid can be malic acid.

    Microorganisms As soon as the protective layer of the skin is broken, contact of microorganisms (including wild yeasts) with juice occurs and growth and proliferation of cell numbers follows. For this reason, red grapes are inoculated at crushing with a strongly growing selected yeast culture of the S. cerevisiae strain (thus accelerating the rise of ethanol and inhibiting alcohol-intolerant microorganisms).

    Growth promoters include sugars, oxygen and warmth. Growth inhibitors include ethanol, pH, molecular SO2, sorbic acid and temperature.

    The three basic types of microorganisms are yeasts, bacteria and moulds.

    MLF This fermentation is carried out by lactic acid bacteria and occurs within the bacterial cell. Lactic acid bacteria (LAB) convert sugars to lactic acid. One carboxyl functional group (COOH) is lost.

    C4H6O5 + LAB » C3H6O3 + CO2
    malic                    lactic
    acid                     acid

    Although lactic acid bacteria can utilise sugars as a nutrient , they are slow growing microorganisms that cannot compete with yeasts for this nutrient. Thus, yeasts will tend to use all the available sugar during yeast fermentation. At the end of fermentation, when sugar levels are low or non-existent, lactic acid bacteria use malic acid as a nutrient. It is usual to add a cultured strain of lactic acid bacteria, the Oenococcus (formerly Leuconostoc) genus being the most desirable.

    Malolactic fermentation:

    • is unusual in white wines because of their low pH and higher FSO2 level.
    • is a decarboxylation process under the control of enzymes.
    • can occur when pH > 3.7, leading to undesirable bacteria.
    • does not require O2 i.e. it is an anaerobic process. .
    • when complete, MLF renders the wine less susceptible to subsequent bacterial activity, thus avoiding MLF in the bottle. i.e. it's more microbially stable.
    • increase pH (generally) as the pK1 of lactic acid is higher than that of malic acid.
    • increases volatile acidity by 0.1 to 0.2 g. L-1.
    • reduces titratable acidity (as lactic acid is less acidic than malic acid).
    • can contribute to increased flavour complexity.
    • inhibits grape varietal aroma.
    • adds new aromas:
      • buttery (diacetyl)
      • ethyl acetate (ethyl ester of lactic acid)

    MLF can be inhibited as follows:

    • temperature kept below 15º C.
    • SO2 added after primary fermentation.
    • low pH (less than pH 3.3).
    • high alcohol level (yeast alcoholic fermentation causes the lactic acid bacteria to decrease to very low levels, although it can rise in the later stages of fermentation).

    On the other hand, MLF can be promoted by adding starter cultures or by extended contact with yeast cells which stimulates growth of lactic acid bacteria.

    Molarity. The number of moles of solute in one litre of solution i.e. mol L-1 (Note: A solution is made up of the base liquid solvent plus the added solute.) Thus, if 1 mole of HCl was added to 10 litres of H20, the result would be 0.1 M HCl .

    Mole. A mole is a quantity, just like a dozen is a quantity. It is the number of atoms in 12 g of carbon. The number of moles in an element of a given mass is equal to the mass in grams divided by the molecular weight. Thus the number of moles in 12g of carbon, which has a molecular weight of 12, is one.

    Moulds. Unlike yeast fermentation and MLF, moulds require oxygen for growth. Thus, growth promoters include oxygen and is a problem with grapes (see botritis cinerea ). However, ethanol is a growth inhibitor and thus moulds are rarely a problem in wine.

    Off-flavours/aromas. Off flavours/aromas can arise from oxidation, sulphur compounds, malolactic fermentation and volatile acidity.

    Oxidation results in the loss of desirable (fruit) flavours, then colour deterioration, development of unpleasant baggy, oily, coarse oxidation flavours. (Components in low oxidation states generally have a more pleasant aroma than their counterparts in higher oxidation states.) Oxidation can not only destroy aroma compounds, it can produce new aroma compounds, many of which are unpleasant.

    Sulphur compounds include:

    Sulphur compounds Threshold
    Hydrogen sulphide: Rotten egg gas, Devil's fart! H2S is thought to be caused by 1) residual sulphur from spraying and/or 2) nitrogen deficiency in the grapes. 50-80g L-1
    Ethyl mercaptan: Formed by the reaction of ethanol (or perhaps acetaldehyde) with H2S. Aroma: Rubber, onion. 1g L-1
    Dimethyl sulphide (DMS): Related to the way grapes are grown (minimal pruning). Can improve fruitiness and add complexity, but will eventually be become a sulphur smell (stewed, plummy). 25-60g L-1

    Malolactic fermentation results in a small (0.1 to 0.2 g L-1) increase in volatile acidity.

    Volatile acidity can result in vinegear and ethyle acetate aromas.

    Oxidation. Oxidation is a measure of the degree of oxidation of a compound. It results in an increase in the oxidised state. Reduction is the opposite of oxidation. It results in a reduction of the oxidised state.

    Oxidation can occur by a purely chemical process or one involving the action of oxidase enzymes, that is an enzyme that has the function of acting as an oxidation catalyst.

    Enzyme-catalysed oxidation is usually more important in juice, where enzyme activity is high and exposure to oxygen is relatively short. Activity will increase with temperature, but can be eliminated by very high temperatures.

    The purely chemical process is more common in wine, where enzyme activity is low and the potential for lengthy air-contact is prolonged. The chemical process is caused by exposure and dissolution of O2 in wine/juice PLUS a chemical reaction with juice/wine components, including phenolic compounds. The chemical reaction depends on the quantity of dissolved oxygen (do), the temperature, the pH (higher temperatures and pH, means faster oxidation), and the presence or absence of SO2. (Note, however, there is little evidence that SO2 can effectively prevent oxidation by dissolved oxygen. The oxidation of the phenolic compounds by dissolved oxygen produces a further oxidising agent, H2O2, and it is the action of this agent that the SO2 inhibits.)

    The concentration of dissolved oxygen that can be supported by juice or wine depends upon the solubility of the gas, which is in turn dependent upon gas pressure, O2 concentration in the gas and juice/wine temperature. However, in most situations, oxygen saturation is not closely approached so the reduction of oxidation rate through decreased temperature is more important than the potential increase of dissolved oxygen.

    Oxidation may arise from the contact of a substrate with either O2 (in the air) or an oxidising agent. Overall, red wines are more tolerant of oxidation than white wines, the degree depending upon the flavour and phenolic concentration and composition of the wine.

    Exposure to air may cause growth of oxidative yeasts as films on the surface, giving off-flavours and growth of acetic acid bacteria, producing volatility.

    Oxidation cannot be readily reversed, even through the use of SO2.

    pH pH is a measure of the concentration of hydronium ions, rather than the concentration of total acids (which is measured by titratable acidity) i.e. it is a measure of acid strength, not acid quantity.

    pH measures acidity and in juice and wine is typically between 3.0-3.7. Within this range, growth of only a few bacteria (primarily lactic acid and acetic acid bacteria) is possible.

    Verasion: acidity is high and pH is approx 2.6.

    Ripening: pH increases mainly due to partial neutralisation of the acids, a process in which they are converted to potassium salts. In warm areas, increase may be rapid in the latter stage of the ripening process and inadequate awareness of fruit ripeness could be disastrous, also affecting flavour.

    Ripe grapes: optimum is pH 3.0-3.4 , depending on the wine. This level will be obtained earlier in warm regions than in cool.

    Over-ripe grapes: The pH, especially in warm areas, can rise alarmingly above optimum level (to pH 4.0 or more) and before flavour development is adequate.

    High pH conditions are of far more concern than low pH conditions. A low pH:

    • inhibits bacterial growth, but does not significantly inhibit fermentation,
    • may inhibit MLF.
    • increases the colour of young red wines (see anthocyanins).
    • decreases the likelihood of oxidation.
    • increases the sensation of acidity (but not as much as titratable acidity).
    • increases the effectiveness of SO2.

    It is pH, not titratable acidity, that is important to the activity of SO2. At juice/wine pH, the bisulfite form of SO2 predominates, making up more than 90% of the total of the free SO2. As the pH decreases, the molecular form markedly increases in abundance from a mere 1% at pH 3.8 to 10% at pH 2.8.

    The combination of acidity (pH) and ethanol concentrations found in wine is enough to kill any microorganisms hostile to humans.

    pH = - log 10 [ H3O+ ]

    Prolonged contact with skins, as occurs in red winemaking, will lead to an increase in pH of about 0.2pH.

    pKa The negative logarithm of Ka, the acid dissociation constant. It indicates the pH of wine in which an acid will be half neutralised, i.e. the pH at which the acid (HA) and its salt (A-) are in equilibrium. Polyprotic acids, or acids with more than one carboxyl functional group (COOH) per molecule, dissociate more than once, have more than one salt, and have more than one pKa (or more than one point of equilibrium). The number of dissociations, salts and pKa's correspond to the number of carboxyl functional groups per molecule of the acid.

    No. of
    carboxyl
    functional
    groups
    Acid pK1 pK2 pK3
    (name of salt)
    1 Lactic 3.81 na na
    1 Acetic 4.75 na na
    2 Succinic 4.16 5.61 na
    2 Tartaric 2.98
    (hydrogen tartrate)
    4.34
    (tartrate)
    na
    2 Malic 3.40
    (hydrogen malate)
    5.11
    (malate)
    na
    3 Citric 3.14 4.77 6.39

    Thus, the balance of the undissociated acid and its salt(s) depends on the pH. Note that as the pH of red wines is normally in the range of pH 3.2 to 3.4, the dissociated form of tartaric acid will predominate in red wines. (The undissociated acid form will predominate below pH 2.98.)

    Pressed juice. Higher in astringent phenolics, cations (mainly potassium) and pH than free run juice.

    Quinones Very reactive oxidised phenolics produced by the oxidation of phenolic compounds. They react readily with other wine components and, in the absence of SO2, or SO2 combined with ascorbic acid, can give rise to rapid precipitation of brown material in the wine. SO2 can decolourise the brownness and prevent further reaction but the ascorbic acid can rapidly convert the reactive oxidised phenolics back to their original unoxidised phenolic form. If ascorbic acid is used without FSO2 the ascorbic acid itself can give rise to severe browning.

    Substrate The molecule upon which an enzyme acts. Oxygen substrates are those substrates which, when exposed to either O2 (in the air) or an oxidising agent, result in oxidation occurring.

    Tartaric acid Tartaric acid (like malic acid) has two acidic sites. Both contribute to titratable acidity. The hydrogen tartrate (or bitartrate) ion contributes about 50% to the titratable acidity compared to the unionised (or undissociated) acid but only about 1.5% to the actual acidity (or pH). Tartaric acid is slightly stronger and much more biologically stable acid than either malic or citric acids. This allows wine to have a lower pH, and much greater microbial stability, than other fermented fruits.

    Titratable acidity is a measure of the concentration of acids in a particular environment (e.g. grape juice), but not a measure of the concentration of hydronium ions (or pH). That is, it is a measure of the relative quantity of acids in a solution, rather than a measure of the strength of those acids.

    Verasion: titratable acidity high (35 g L -1)

    Ripening: titratable acidity falls due to dilution of fruit and loss of malic acid due to its use as a source of energy in respiration.

    Ripe grapes: optimum is 6-12 g L -1, depending on the wine. This level will be obtained earlier in warm regions than in cool.

    Over-ripe grapes: The acidity, especially in warm areas, can readily fall to below optimum level (to 3-4 g L -1 ) and before flavour development is adequate.

    Titratable acidity is a better indicator of berry maturity behaviour than pH, and relates to acid taste better than pH.

    Volatile Acidity

    • VA is 90% acetic acid and 10% other acids (formic, proprionic, butyric) .
    • Maximum VA varies from country to country (e.g. in Australian wines the maximum is 1.5 g L-1).
    • About 0.5 g L-1 formed during fermentation. Increases when 3.2 > pH > 4.0.
    • Levels above 0.8 g L-1 may become noticeable, depending on wine style.
    • Ethyl acetate (the ester of acetic acid) is often present along with acetic acid, in the ratio of 1:5. (Note that esters are the product of the reaction between a carboxylic acid and an alcohol, and provide fruit aromas.).
    • The aroma of ethyl acetate (nail polish remover) may be detected before that of acetic as it is has lower aroma threshold in wine.
    • The legal maximum for ethyl acetate varies from country to country (e.g. there is no legal maximum in Australia for ethyl acetate).

    Level Effect
    Acetic acid 500-750 mg L-1 Perceptible aroma
    1.5 g L-1 Adds to aroma and burns back of throat
    above 1.5 g L-1 Vinegar aroma, and a sour finish.
    Ethyl acetate 50 mg L-1 Perceptible, adds fruitiness
    100 mg L-1 Distinctive aroma present; adds sourness.