SO2 -Първа част

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SO2 -Първа част

Мнениеот vinoirakia » Нед Апр 25, 2010 9:51 am

Sulphur Dioxide

1. Introduction
2. SO2 Production by Yeast
3. Sodium and Potassium Salts
4. Forms and Functions of Sulphur Dioxide in Wine
5. SO2 Binding
6. The Properties of SO2
7. Free SO2 and pH


1. Introduction
Sulphur dioxide, often abbreviated to sulphite or SO2, has been used in winemaking since Roman times. It is used extensively in modern winemaking, predominantly for its suppression of yeast and bacterial action, and its anti-oxidant properties. It is possible to make wine successfully without using sulphites, but this allows less control, and results in reduced ageability, consistency and biological stability.

This article outlines the properties, forms, and uses of sulphur dioxide. Attention is given to the SO2 in general, the different forms of SO2 that exist in wine, and the issues of pH, temperature, SO2 binding, oxidative protection, SO2 removal, practical aspects of addition, hyperoxidation, SO2 testing, storage, stock solutions, sulphur wicks and Campden tablets.

In terms of the practical information presented here, the article is aimed at home winemakers. However, both theoretical and general practical aspects are presented. Winemakers who do not wish to concern themselves with the more theoretical side to SO2 use can simply skip these sections.



2. SO2 Production by Yeast
Sulphur dioxide is a natural by-product of yeast during fermentation [Zang and Franzen, 1966]. Usually less than 30 mg/l is formed. Zang and Franzen [1967] observed levels of 7-128 mg/l in 20 German wines, while Wьrdig and Schlotter [1967] reported 13-114 mg/l produced in 20 ferments. Heinzel et al. [1976] found levels ranging 6-296 mg/l, and levels ranging 3.2-640 mg/l under aerobic conditions. Levels over 100 mg/l have also been reported by Rankine and Pocock [1969], Eschenbroch [1974], Dott et al. [1976], and Suzzi et al. [1985]. The amount produced depends on various factors, including the yeast strain and the wine environment [Rankine, 1968; Eschenbruch, 1974; Romano and Suzzi, 1993; Wьrdig and Schlotter, 1967; Dittrich and Staudenmayer, 1968; Rankine and Pocock, 1969]. The production of SO2 by yeast tends to be higher in musts with a low level of suspended solids [Liu and Gallander, 1982]. Eschenbroch and Bonish [1976] found that pH had an influence on the SO2 production of some strains, but not others.


The disadvantages of using SO2 tend to be limited to its excessive use. For example, high concentrations of SO2 have an offensive odour and taste, and the formation of H2S and mercaptans under extended yeast lees ageing can increase at higher SO2 concentrations. It should be noted that a small percentage of the population is allergic to SO2, and vulnerable individuals may experience asthmatic attacks when exposed to very low levels (~1 mg) of SO2. This is, however, uncommon. A number of people believe they suffer from an SO2 allergy when this is in fact not the case. To ascertain if an individual is allergic, simply have them eat a packaged dried fruit that has had SO2 added as a preservative - the SO2 levels in these almost always exceed that which would be found in wine.



3. Sodium and Potassium Salts
Two salt forms of sulphite are generally used in winemaking: potassium metabisulphite (K2S2O5) and sodium metabisulphite (Na2S2O5).

The molecular weight of sodium metabisulphite is 190.2 and that of potassium metabisulphite is 222.4, whereas that of sulphur dioxide (SO2) is 64.1. The salts dissociate giving two moles of SO2 for each mole of the salt. Thus, the SO2 content of sodium metabisulphite is 2 x 64.1/190.2 = 67.4% and that of potassium metabisulphite is 2 x 64.1/222.4 = 57.6%.

Winemakers generally prefer to use the potassium form for sulphite additions, since this increases the level of potassium in the wine which may later help to precipitate tartrates when cold stabilising. Others claim that the sodium form can contribute a `salty' flavour to wine.

4. Forms and Functions of Sulphur Dioxide in Wine
4.1. Dissociation of Forms
Potassium metabisulphite dissociates in water to potassium ions (K+) and singly ionised bisulphite, (HSO3)-. (Sodium metabisulphite dissociates in the same way.)

Metabisulphite dissociates in the following way to form these fractions:


K2S2O5 + H2O ===> 2K+ + 2(HSO3)-


Sulphur dioxide is a bifunctional acid, and dissociates into three fractions. The quantity of each of these fractions depends on the thermodynamic constants and the pH. The dissociation is almost instantaneous.

The three fractions are molecular SO2 (SO2), sulphite (SO32-), and bisulphite (HSO3-). Dissociation of the various fractions is almost immediate.

Since wine is acidic, hydrogen ions are present (H+) and the bisulphite (HSO3-) can then transform into sulphur dioxide:

HSO3- + H+ <===> H2O + SO2

singly ionized bisulphite + hydrogen ion water + unionized (molecular) sulphur dioxide

Additionally,
HSO3- + H2O <===> H+ + SO32-
singly ionized bisulphite + water hydrogen ion + doubly ionized sulphite
Thus, the relationships of the forms of SO2 in wine are shown completely by:
H2O + SO2 <===> H+ + (HSO3)- <===> 2H+ + SO32-
water + molecular sulphur dioxide hydrogen ion + bisulphite hydrogen ion + sulphite

The amount of each free SO2 that is in each fraction (bisulphite, sulphite, and molecular) is determined by the pH. Figure 1 shows the distribution of the different species for various pH values.

4.2. Functions of the Different Forms
4.2.1. Bisulphite (HSO3-)
Bisulphite is the predominant form of free SO2 at wine and juice pHs.
It causes the inactivation of polyphenol oxidase (PPO) enzymes and the binding and/or reduction of brown quinones in juice. (PPO enzymes are the enzymatic catalysts which cause oxidative browning of juice.) For more information, see section 11.1. Bisulphite is a successful anthocyanin (the predominant colouring matter in red fruits) extractive, yet it also bleaches colour and slows anthocyanin polymerisation reactions with other phenols. Bisulphite possesses a low, and largely insignificant, antiseptic affect on yeasts (roughly 20 times less active than SO2 in wines with reducing sugars). It is odourless, but has a salty, bitter taste.


4.2.2. Molecular (or active) SO2
Molecular SO2 exists as either a gas or as single molecules in juice and wine. It is the most important form of SO2 in wine. It is responsible for antimicrobial activity [Rahn and Conn, 1944; Rhem, 1964; Macris and Markakis, 1974; Beech et al., 1979; King et al., 1981]. Rehm and Wittman [1962] found the antibacterial activity of molecular SO2 to be 500 times greater than bisulphite (HSO3-). It also possesses antioxidant activity (see Section 11, "SO2 and Oxidation"). It is volatile and is responsible for the odour and sulphurous taste of SO2.


4.2.3. Sulphite (SO32-)
At typical wine pHs the quantity of this form of sulphur dioxide is minute and its reaction with oxygen is very slow. It is the only form which reacts with oxygen directly. It is odourless and tasteless at normal concentrations.



5. SO2 Binding
5.1. General
A portion of the SO2 added to wine will become bound with compounds in the wine. This portion is called "bound" (or "combined" or "fixed") SO2. The remainder is called "free" SO2 (FSO2). "Total" SO2 (TSO2) is the sum of free and bound SO2. Figure 2 represents this graphically.

It is the bisulphite form of SO2 which binds with other compounds. The bound SO2 compounds are therefore often termed "bisulphite addition products" and are sometimes referred to as "hydroxy-sulphonates". Unstable SO2-bound products may provide a reserve that feeds free SO2 when it subsides through oxidation or vaporisation. However, it should be noted that the degree of this re-partitioning is dependent on the binding kinetics of individual SO2-bound products and may not be practically significant for most wines.

The SO2 which becomes bound is no longer available as free SO2. Since it is free SO2 that exhibits antimicrobial activity and oxidative protection, it is important to consider the amount of SO2 that will become bound.

Bound SO2 does not possess antiyeast activity, but the fractions of bound SO2 which are bound to acetaldehyde and pyruvic acid exhibit antibacterial action (this action is 5-10 times weaker than that of free SO2, though it is often present at 5-10 times the concentration of free SO2).


5.2. Compounds that bind
Binding compounds include carbonyl compounds, ketonic acids, sugars, quinones, anthocyanins, and others [Burroughs and Sparks, 1973a, 1973b, 1973c]. These will be dealt with individually below.


5.2.1. Carbonyl binding
SO2 combines with the carbonyl groups of aldehydes [Hennig and Burkhardt, 1960a, 1960b]. Carbonyl compounds represent the predominant compounds which bind to SO2. By far the most significant carbonyl compound involved in SO2 binding is acetaldehyde.

Acetaldehyde (CH3CHO), also called ethanal, is a natural intermediate product during fermentation. It is oxidised ethanol and is the compound which gives sherry its characteristic (oxidised) aroma. The main factors influencing acetaldehyde concentrations are the yeast strain, the juice thiamine content, and the amount of SO2 added to the must. The increased presence of free SO2 in the wine during fermentation also increases its production [Lafourcade, 1955]. This is undesirable, since the aroma of acetaldehyde is not favourable (at least at significant levels) in wine. Additionally, any addition of SO2 to a fermenting wine will immediately become bound with acetaldehyde, leaving it ineffective for its intended purpose. It is therefore best to avoid making SO2 additions during fermentation.

Bisulphite binds with the carbonyl oxygen atoms of acetaldehyde readily, making acetaldehyde the compound which binds most quantitatively with SO2. For example, Ough [1959] found that 12 out of 17 wines had 100% of their BSO2 bound to acetaldehyde. Though Rankine [1966] examined wines in which not all bound SO2 could be accounted for by acetaldehyde binding. Kerp [1904a, 1904b, 1904c] noted that SO2 is bound almost entirely by acetaldehyde, except in heavily sulphited wines in which the excess SO2 was bound by sugars. Analysing data from 8 wines from Peynaud and Lafourcade [1952], shows that 100% of all acetaldehyde was bound to SO2. The analysis of data from 16 Californian wines [Ough, 1959], however, suggests that most of the wines in that study have 60-100% of their acetaldehyde bound to SO2.

The reaction for the binding of acetaldehyde and SO2 is:


CH3-CHO + HSO3- <==> CH3-CHOH-SO3-

and is independent of temperature (within normal ranges). Each milligram of acetaldehyde will bind with 1.45 milligrams of SO2. (The acetaldehyde-bisulphite compound is more correctly described as acetaldehyde-alpha-hydroxy sulphonate.)

Typical acetaldehyde concentrations in a newly fermented table wine are less than 75 mg/l. The typical concentration in table wines ranges from 20 to 400 mg/l [Rankine, 1995]. The sensory threshold has been reported as 100-125 mg/l [Berg et al., 1955].

Following fermentation, SO2 might be added in sufficient concentrations for acetaldehyde to be completely bound [supported by Kielhцfer, 1963; Blouin, 1963 and 1966]. In such cases, a typical newly fermented table wine will bind a maximum of 110 mg/l SO2, though this figure may range from 30 to 580 mg/l. These figures will vary widely from wine to wine. Additionally, the binding is quantitatively less, and the rate of binding slower, the lower the pH. Hennig [1943], however, believed that some acetaldehyde was beneficial for the development of wine bouquet.

Since SO2 will so readily bind with acetaldehyde, thus leaving it ineffective as an antimicrobial and antioxidative agent, it is suggested that acetaldehyde concentrations be kept to the minimum possible. This can be achieved by a low sulphiting of the must, low pH, low fermentation temperature, and minimal exposure to air.


5.2.2. Ketonic acid binding
Ketonic acids (alpha-ketoglutaric acid, pyruvic acid, glutaric acid, keto-2-gluconic acid, diketo-2,5-gluconic acid, galacturonic acid) also bind to SO2. Increased ketonic acid levels may be the result of nutritional deficiency, especially a lack of vitamins due to mold infected fruit [Burroughs and Sparks, 1973b] or musts which have experienced ion exchange. Different yeast strains also produce different levels of alpha-ketonic acids [Farris et al., 1982 & 1983]

Pyruvic acid forms during fermentation. It has also been shown to decrease slowly once having formed, and the addition of thiamine has been shown to reduce its formation [Peynaud and Lafon-Lafourcade, 1966; Delfini et al., 1980].

The average concentrations of pyruvic acid and alpha-ketoglutaric acid in wine range 10-500 mg/l and 2-350 mg/l, respectively [Usseglio-Tomasset, 1989]. Typical ranges are 0-100 mg/l and 15-40 mg/l, respectively [Rankine, 1966].


5.2.3. Sugars binding
Sugars (arabinose, mannose, galactose, glucose, keto-5-fructose, xylsosone) bind to SO2. Polysaccharides also bind with SO2.

Binding to glucose is significant in juice and approximately 50% of added SO2 may become bound (at addition levels of 50-100 mg/l). Glucose has a low binding rate (0.8 mg SO2 per gram glucose in the presence of 100 mg/l free SO2). According to Braverman [1963], fructose does not form an addition product with the bisulphite form of SO2. In any case, SO2 binding with fructose is low, and is certainly less than that with glucose. Arabinose binds more readily, though its concentrations in wine are usually low. Saccharose does not bind to a significant level.

Data plotted in Figure 3 show the percentage of added SO2 which has become bound in concentrated orange, lemon and grapefruit fruit juices. (Data from Tressler et al. [1980].) As is expected, juices with a higher sugar content bind with more SO2 due to their increased sugar levels.
5.2.4. Dicarbonyl group molecule binding
Dicarbonyl groups (glyoxal, methylglyoxal, hydroxypropanedial); gluconic and galacturonic acid, glyoxylic acid, oxaloacetic acid, glycolic aldehyde, glyceric aldehyde, dihydroxyacetone, acetoine, diacetyl, 5-(hydroxymethyl) furfural) bind with SO2.


5.2.5. Other binding
Bisulphite also binds to yeast, bacteria, and other protein and cellular particulates. This means SO2 additions are more effective in clarified juice. Binding can also occur with oxidation products of phenols and ascorbic acid. Phenolic compounds (proanthocyanic tannins, and particularly caffeic and p-coumaric acid) also bind reversibly with SO2 [Hennig and Burkhardt, 1960a, 1960b]. The binding is temperature and pH dependent. SO2 binds specifically with the four position carbons of monomeric anthocyanins of reds resulting in colour bleaching. With time, red wine pigments bind with non-SO2 compounds in wine and become less easily bound to SO2.


5.3. Binding kinetics
Binding is not instant. Whilst it is fastest within the first 24 hours of SO2 addition, it takes days before full binding is complete. Four to five days are typically required before binding ceases, during which time a slow decrease in free SO2 occurs, accompanied by a corresponding increase in bound SO2. After this time, equilibrium is realised and any further decreases are due to oxidation or vaporisation.

The rate of binding is dependent on the dissociation constant, K, for the individual binding reaction. The lower the value for K, the more favoured the formation of a bisulphite addition product [see Burroughs and Whiting, 1960]. As an example, acetaldehyde has a low K value compared to many other compounds in wine. The binding of SO2 to acetaldehyde is strong and rapid. For example, at a pH of 3.3, 98% of acetaldehyde was found to bind within 90 minutes of addition, and total combination was complete within 5 hours. In one study [Joslyn and Braverman, 1954], 90-95% was found to bind after just 2 minutes.

A number of K values are given in Table 1.

Table 2. Dissociation constants for SO2 binding with select compounds

Substance Source 1 Source 2 Source 3 Source 4 Source 5 Source 6
Formaldehyde 1.2 × 10-7
Acetaldehyde 2.5 × 10-6 5 × 10-4 1.5 × 10-6 1.5 × 10-6 1.5 × 10-6
alpha-ketoglutaric acid 8.8 × 10-4 8.8 × 10-4 5 × 10-4
Benzaldehyde 1 × 10-4
Acetone 3.5-4.0 × 10-3 3.8 × 10-3
Furfural 7.2 × 10-4
Chloral 3.5 × 10-2
Arabinose 3.5 × 10-2
Glucose 2.2 × 10-1
Pyruvic acid 0.3 × 10-3 1.4 × 10-4 4.0 × 10-4 4.0 × 10-4 3 × 10-4
Glucose 9 × 10-1 6.4 × 10-1 6.4 × 10-1 6.4 × 10-1
Fructose 1.5
Sucrose 5.4


Source 1 - Kolthoff and Stenger [1942] at 25 C
Source 2 - Blouin [1966] at 20 C
Source 3 - Burroughs and Sparks [1973a] and Beech et al. [1979] at pH=3
Source 4 - Burroughs and Whiting [1960] at pH 3-4, 20 C, 50 mg/l FSO2, in cider
Source 5 - Rankine [1966]
Source 6 - Usseglio-Tomasset [1989]

The value of the dissociation constant, K, increases with increasing temperature. For example, the K value for acetaldehyde increases by 5 times from 25°C (77°F) to 37.5°C (99.5°F) [Kerp, 1903; Kerp and Bauer, 1904 and 1907a,b].

It is difficult to generalise binding kinetics for all wines. Nevertheless, Blouin [1966] found that, at a free SO2 of 20 mg/l, substances with a K value less than 3 × 10-6 bound completely with SO2, whilst substances with a K value equal to or larger than 3.1 × 10-2 bound less than 1%. The presence of metals was also found to increase binding.



5.4. Relationship between total and free SO2
Molecular SO2 provides the predominant protective qualities of SO2 in wine. Since the molecular SO2 present is only a portion of the free SO2, it is important to take into consideration the fact that a fraction of SO2 additions will become bound and will no longer remain as free.

The relationship between the amount of added SO2 and the amount of SO2 remaining free is complex. It is clear, however, that it is governed by the total SO2 content of the wine and the ability of the individual wines' compounds to bind with SO2. The higher the total SO2 content, the less that a further SO2 addition will bind. The relationship between free SO2 and bound SO2 is shown in Figure 4. The rate of binding can be seen to decrease as the free SO2 concentration increases.

The exact relationship between free and bound SO2 will vary from wine to wine, but can be determined for an individual wine by making a range of SO2 additions to sample of the wine (for e.g., sample 1 has 10 mg/l SO2 added, sample 2 has 20 mg/l SO2 added, etc). The free SO2 is then measured in the samples some time later (4-5 days later is safest). An understanding of the wine's SO2 binding response at that particular point in it's life can then be determined. Such a task is, however, laborious and time consuming. Instead, many winemakers assume an empirical law of binding. This simply estimates the portion of added SO2 which will become bound on addition.


5.5. Approximate rules for SO2 binding
Many winemakers assume that about 50% of their SO2 addition to a wine becomes bound when the total SO2 is below 30-60 mg/l. After this level, added SO2 is generally considered not to bind, providing free SO2 almost exclusively. (Though some winemakers assume that thereafter some SO2 does become bound (usually about 30%).)

Peynaud notes that roughly one third (33%) of added SO2 becomes bound [Peynaud, 1984, p.271,250] under a free SO2 content of 100 mg/l.

Margalit [1996, p.268] cites the SO2 binding data of Schaeffer [1987], who found that 70-85% of SO2 became bound when added to newly fermented Gewurztraminer wine. Margalit [1990, p.26] further notes that for healthy fruit, around half of any SO2 addition will become bound when the total SO2 concentration is under 50-60 mg/l. Above 50-60 mg/l total SO2, any addition is considered to contribute to free SO2 in its entirety, i.e. no binding occurs.

Low fermentation temperatures, anaerobic fermentations, addition of ammonium salts, and use of non-ketogenic yeast strains may all help to minimise potential addition products and hence minimise SO2 binding [Peynaud and Lafon-Lafourcade, 1966].


6. The Properties of SO2
6.1. Antioxidant
SO2 protects both must and wine from excessive oxidation.


6.2. Antienzymatic
Sulphur dioxide inhibits oxidation enzymes (enzymatic catalysts of oxidation such as tyrosinase and laccase) and destroys them with time. It inhibits the polyphenol oxidase enzyme responsible for catalysing oxidative reactions in juice. The oxidative protection of a must is sustained by this mechanism before fermentation begins. Its inclusion in must will therefore increase the amount of oxygen available to yeast in their growth phase. The use of SO2 can help to avoid oxidasic casse from rotten fruit.


6.3. Taste
Acetaldehyde is the compound which gives sherry its characteristic oxidised (or maderised) aroma. Similarly, a small amount of wine left out in a glass overnight will show an aroma dominated by acetaldehyde. A common fault due to excessive oxidation is the presence of high concentrations of acetaldehyde. SO2 will bind with acetaldehyde, essentially removing its volatile presence and resulting in a wine with a "fresher" aroma.

The addition of SO2 at crush will increase the extraction of flavonoid phenols [Singleton et al., 1980]. These compounds will contribute to bitterness and astringency. The absence of SO2 in must will increase the oxidative polymerisation [Ough and Crowell, 1987] and precipitation of phenols.

The presence of SO2 to the must may additionally cause increased extraction of phenolics during maceration.

Wines low in SO2 are believed to have softer palates, whilst high levels increase the harshness of wine. Excessive levels cause wines to have pungent, sulphurous aromas.


6.4. Fermentation
At low levels of 5-10 mg/l, SO2 delays the onset of fermentation, but later speeds up the multiplication of yeasts and their transformation of sugars [Peynaud, 1984]. Higher additions, however, result in increased fermentation delays [Yang, 1975]. This is attributed to the SO2's destruction of fungicides, which are toxic to yeast. The delay in the initiation of fermentation assists in pre-fermentation juice settling of musts. It also allows for a greater level of dissolved oxygen in the must, due to the prevention of such oxygen being used in enzymatic oxidation reactions. These higher dissolved oxygen levels provide a healthier environment for the yeast.

In musts neither inoculated with cultured yeast nor with SO2 added, wild yeast species such as the genii Kloeckera and Candida are usually present during the early stages of fermentation, but are soon dominated by the genus Saccharomyces which take a stronger hold over the fermentation. SO2 can inhibit the former strains, allowing for the latter strains to dominate fermentation from the on-set. In modern winemaking this is generally deemed more preferable. However, there are winemakers who believe that the limited influence of wild strains can enhance a wine's character.


6.5. Colour
Use of SO2 during crush/fermentation causes an increase in colour [Harvalia, 1965] through the extraction/solvency of anthocyanins and polyphenols from fruit tissues, though at normal doses the colour increase is aesthetically insignificant [Amerine and Joslyn, 1951]. (This is because the anthocyanidin-SO2 compound is more soluble in water-ethanol than anthocyanidin alone.) Wines fermented with SO2 have also been found to better retain colour [Berg and Akiyoshi, 1962]. Excessive amounts of SO2, however, cause colour bleaching, though this discolouration of red pigments is reversible.

Anthocyanin pigments bind readily with bisulphite (HSO3-). In this reaction, the coloured anthocyanin cation binds with the bisulphite anion to form colourless anthcyanin-4-bisulphite [Jurd, 1964]. This reaction has been shown to be 85% complete with the addition of 15 mg/l SO2 [Timberlake and Bridle, 1976]. However, polymeric anthocyanins are resistant to SO2 and contribute to a significant proportion of red wine colour [Burroughs, 1975].


6.6. Browning
Sulphur dioxide reduces browning by obstructing polyphenol oxidase (PPO) enzymes. These are the enzymatic catalysts which cause oxidative browning of juice. When an apple is freshly cut and the flesh begins to turn brown, for example, this is due to the activity of PPO enzymes. The reaction is as follows:


Polyphenol oxidase (PPO) activity has been shown to be reduced by more than 90% by the presence of 50 mg/l SO2 [Dubernet and Ribйreau-Gayon, 1973; Amano et al., 1979].


It seems that the bisulphite (HSO3-) form is responsible for this, and that this occurs by irreversible structural modification rather than binding inhibition [Sayavedra-Soto and Montgomery, 1986].


6.7. Antimicrobial
At low concentrations, SO2 inhibits the development of microorganisms. At high concentrations, it can destroy a proportion of the microbial population. Molecular SO2 is the form responsible for antimicrobial action [Rahn and Conn, 1944; Rhem, 1964; Macris and Markakis, 1974; Beech et al., 1979; King et al., 1981].

Bound SO2 also possesses antimicrobial activity, though this is limited. The antimicrobial activity of bound SO2 depends on the compound that the SO2 is bound to [Rehm and Wittman, 1962]. For example, acetaldehyde, pyruvate, and acetone have a significant inhibition effect, whilst glucose has only a slight inhibition effect. Generally, the antimicrobial activity of bound SO2 is not significant.

Different yeast and bacteria strains have different levels of tolerance to SO2 [Cruess, 1912]. A number of studies have attempted to determine the levels required for inhibition or death for numerous strains. One study found a 1000-fold reduction in the number of viable cells of a Brettanomyces species, certain LAB species, and other wine spoilage organisms within 24 hours at a molecular SO2 concentration of 0.8 mg/l [Beech et al., 1979]. Another found total microbial inhibition in musts at 4 mg/l molecular SO2 [Delfini, 1984]. A classic study by Beech et al. [1979] assessed the SO2 required to reduce non-growing yeast and bacterial populations by 10,000 viable cells/ml over a 24 hour period in 10% ethanol buffered solutions. They found that 0.825 mg/l molecular SO2 was required for one Saccharomyces cerevisiae strain, 0.825 mg/l for a Brettanomyces strain, 1.50 mg/l for a Zygosaccharomayces bailii strain, and 4 mg/l for a Lactobacillus plantarum strain. Based on these figures it would seem that a level of 0.8 mg/l molecular SO2 is sufficient for the suppression of the majority of yeast and bacteria strains.

It should be noted, however, that strains can build up resistance to SO2. Older cultures tend to have more resistance to SO2 [Schimz, 1980; Katchmer, 1990].

Bacteria are more susceptible to SO2 than yeasts and are considered separately below.


6.8. Antiyeast
Molecular SO2, and to a lesser extent bisulphite (HSO3-), inhibit yeast. Free SO2 essentially has an antiseptic effect [Kielhofer, 1963], and the growth of Saccharomyces has been shown to be related to its concentration [Ingram, 1948].


6.8.1. Resistance adaptation
As stated above, different yeast strains are resistant to SO2 [Porchet, 1931] to varying degrees. Some may tolerate 700 mg/l free SO2 or more.

Yeast may also adapt to an SO2 environment and become resistant to SO2. Certain yeasts have been shown to permanently adapt to 10-12 times the SO2 concentration that the parent strain could tolerate [Scardovi, 1951, 1952, 1953]. Delfini [1988, 1989, 1992a] demonstrated that a variety of yeast strains (S.cerevisiae, S. ludwigii, Zygosaccharomyces baillii, and Schizosaccharomyces japonicus) could develop successive permanent (inherited) resistance to SO2 to a final level of 9.2-11.5 mg/l molecular SO2.

It is therefore important to limit SO2 additions, and to avoid adding successive doses, as this may result in increased SO2 resistance by the yeast strain.


6.8.2. Growth
Yeast growth exhibits an extended lag phase in the presence of SO2, but this is usually followed by normal growth following the end of the lag phase [Schanderl, 1959].

SO2 is more effective on yeasts in their resting/sporulating phase, since binding with aldehydes may occur latter.


6.8.3. Complete and partial inhibition
Scardovi [1951] showed that total S. cerevisiae strain cell death occurred with 4 mg/l molecular SO2 for non-resistant variants, whilst 40 mg/l was required for resistant strains. S. cerevisiae has been shown to be sensitive to 0.5-0.9 mg/l molecular SO2, with complete inhibition occurring at >0.5-1 mg/l [Beech, 1979]. In another study, approximately 30% cell death occurred at 18 mg/l molecular SO2, whilst 70% death occurred at 42 mg/l [Farkas, 1988]. SO2 can also reduce the viability of a yeast inoculum. One study found that 15-20 mg/l free SO2 reduced the population from 106 to 104 cells/ml [Lehmann, 1987]. Nevertheless, fermentation has been seen to commonly occur (evidenced by a 0.5-1% by volume alcohol production) in musts with as much as 2000 mg/l free SO2 [Delfini, 1984].

Marcis and Markakis [1974] showed that 1.3 mg/l molecular SO2 was required to eliminate viable yeast cells in a medium. Another study showed that 1.56 mg/l was required [King et al., 1981]. Minarik [1978] found that 6.4 mg/l was required in juice, while Beech et al. [1979] found that 0.825 mg/l was required in a model wine solution, and Sudraud and Chauvet [1985] suggested 1.5 mg/l be used following fermentation and 1.2 mg/l be used during storage to prevent refermentation of residual sugar.

It may be generally accepted that 4-5 mg/l molecular SO2 can cause total inhibition of S. cerevisiae.


6.8.4. Time dependence
Total yeast death is also time dependent. Yeast uptake of SO2 is rapid, and can be complete within 3 minutes [Macris and Markakis, 1974]. It may, however, require longer periods of time for SO2 to become lethal. One study [Delfini, 1981] found total inhibition occurred at 0.29 mg/l molecular SO2 for Kloeckera apiculata, 0.67 for Pichia vini, and 1.59 for Candida vini. These concentrations became lethal after 24 hours of exposure with a cell population of 106 cells/ml. Macris and Markakis [1974] found that a population reduction of 90% took 83 minutes with 0.025 mg/l molecular SO2. In spite of these findings, Uzuka and Nomura [1986] found that, at 0.80 mg/l molecular SO2, over 50% of yeast viability was lost within 30 minutes. A similar reduction corresponds to 6 hours at 0.825 mg/l in the Beech et al. [1979] study and 20 hours in the King et al. [1981] study.


6.8.5. Yeast selective
To a certain degree, SO2 may be used as a yeast selector. At certain doses it promotes yeast selection by hindering the multiplication of non/low-alcohol producing yeasts such as apiculates, Torulopsis, and Candida more than that of elliptic yeasts [Romano and Suzzi, 1992]. Nevertheless, Heard and Fleet [1988] showed that apiculated yeasts (Kloeckera and Hanseniaspora) grew to substantial populations (106-107 cells/ml) in a few days before receding.


6.9. Antibacterial
Lactic bacteria are sensitive to free and, to a lesser extent, bound SO2 [Fornachon, 1963].

The primary antimicrobial effect of SO2 is attributable to molecular SO2, at least up to pH 5 [Scardovi, 1951, 1952; Macris and Markakis, 1974]. Though there is evidence that bound SO2 can contribute to bacterial control [Bioletti, 1912; Rhem, Wallnofer and Wittman, 1965; Lafon-Lafourcade and Peynaud, 1974; Hood, 1983] and inhibit LAB growth [Fornachon, 1963]. This is because LAB consume acetaldehyde, which subsequently releases SO2 from the bound acetaldehyde-SO2 form [Osborne et al., 2000]. Mayer et al. [1975] found Leuconostoc oenos sensitive to levels of acetaldehyde-bound SO2 levels of 20-60 mg/l. Hood [1983] showed that just 6 mg/l of acetaldehyde-bound SO2 could inhibit the growth of Leuconostoc oenos, Leuconostoc brevis, and Pediococcus pentosaceus at pH 3.4.

In one study [Delfini and Morsiani, 1992], ten strains of Leuconostoc and four of Lactobacillus were found to cease growth above 0.5 mg/l molecular SO2. A Leuconostoc population of 2 × 106 cells/ml in a buffered synthetic medium died within 22 hours after addition of 0.84 mg/l molecular SO2. However, a number of Leuconostoc and Lactobacillus strains survived SO2 additions and resumed multiplication after a 10-60 day lag phase at molecular SO2 levels under 0.8 mg/l.

Acetic acid bacteria are also sensitive to SO2. Research conducted on Acetobacter aceti, A. liquefaciens, A. hansenii, A. pasteurianus and Gluconobacter oxydans showed that some strains were more sensitive than others. Molecular SO2 levels of 0.1-0.65 mg/l were required to effectively kill strains in juice over a 4 day period, depending on the individual strain [du Toit, 2000]. The production of VA was also found to inhibit the growth of yeast (Vin 13). Some acetic acid bacteria strains were found to produce SO2 binding compounds such as gluconic, 2 ketogluconic and 2,5 diketogluconic acids. The addition of SO2 before fermentation may therefore be of increased importance, since this will inhibit acetic acid development which in turn will prevent inhibition of yeast growth.


6.10. Overview
The complexities of SO2 inhibition/death on yeast and bacteria have not been exhaustively studied. The quantity of molecular sulphur dioxide required to inhibit specific micro-organisms depends on their individual environment and history. For example, pH impacts on yeast and bacterial growth irrespective of SO2 concentration, and should therefore be considered as a separate yet related influence. In the absence of such information, values utilised for protection must be obtained from the available data.

SO2 should not be used to stop fermentation directly, since the SO2 added will immediately become bound leaving it ineffective. If SO2 is used as a yeast inhibitor, its use should be in parallel with other techniques (such as low temperature, clarification, or sorbate) and even then should only be used when a sufficient reduction in the yeast population is attained.

SO2 remains an invaluable tool for inhibiting bacteria, which might otherwise spoil wine. It appears that levels of inhibition range from 0.5 to 0.825 mg/l molecular SO2, depending on the bacteria and strain. Based on this information, maintenance of 0.825 mg/l molecular SO2 would be a safe approach to controlling bacteria. However, levels used by winemakers to control biological stability are generally achieved through levels ranging 0.5-1.5 mg/l molecular SO2.

Currently, control of biological stability is generally achieved through levels ranging 0.5-1.5 mg/l of molecular SO2. A general opinion exists that 0.8 mg/l molecular SO2 provides sufficient protection for dry wines. (Some feel that a concentration of 0.6 mg/l is suitable for red must or wine, while 0.8 mg/l is suitable for white must or wine.) Wines with residual sugar might better be protected with levels ranging 1.5-2 mg/l. However, it should be kept in mind that these levels are rule-of-thumb and different yeast and bacteria under different conditions will act differently. Winemakers should be aware of this and arrive at usage levels suitable to their individual situation.



7. Free SO2 and pH
As mentioned previously, molecular SO2 is the principal form of sulphur dioxide responsible for anti-microbial activity. The amount of molecular free SO2 available is a function of pH. Thus, SO2 additions should be calculated with reference to pH.

Because the significant SO2 form responsible for antimicrobial action is molecular SO2, microbial growth control without measurement of free SO2 and pH is less meaningful and certainly less assured.

The relationship between pH and molecular free SO2 can be shown as follows:

[SO2] + [H2O] <===> [HSO3-] [H+]
K = [HSO3-] [H+] / [SO2]
[HSO3-] / [SO2] = K / [H+]

Now,

[H+] = 10-pH
K = 10-pKa

So,

[HSO3-] / [SO2] = 10-pKa / 10-pH
[HSO3-] / [SO2] = 10pH - pKa


Since the sulphite form (SO32-) is almost insignificant at wine and must pH, free SO2 consists of the molecular form (SO2) and the bisulphite form (HSO3-),


Free SO2 = [HSO3-] + [SO2]

So,

( Free SO2 - [SO2] ) / [SO2] = 10pH-pKa
( Free SO2 / [SO2] ) - 1 = 10pH - pKa
Free SO2 / Molecular SO2 = 10pH - pKa + 1
Free SO2 = Molecular SO2 * ( 10pH - pKa + 1 )

King et al. [1981] found the pKa's of SO2 in water to be 1.77 and 7.20. Tartar and Garretson [1941] and Schroeter [1966] reported values of 1.76 and 7.20. These values seem to have become the accepted values in water. The ionisation constants are affected by the ethanol concentration, the presence of sugars and other organic compounds and salts, and the temperature. Figure 5 shows various pK1 values for given ethanol concentrations and temperatures [data from Usseglio-Tomasset, 1989].


Usseglio-Tomasset and Bosia [1984] reported a pK1 value of 1.78 (at 19°C) (pK2 was 7.08 at 20°C) and noted that a pK1 value closer to 2.0 was more realistic in wine-like conditions. Thus, using a value of 1.81 might be more sensible.

However, the difference between using a pK1 value of 1.77 or 1.81 is minimal in terms of free SO2 values required (for any given molecular level). Thus, for most practical purposes, the importance in using an acutely accurate pK value is not high under normal winemaking conditions.

In the calculations below, a pK value of 1.81 is adopted. This is a widely used value. Thus, the equations relating molecular and free SO2 can be written as:


Molecular SO2 = Free SO2 / ( 10(pH - 1.81) + 1 )
Free SO2 = Molecular SO2 * ( 10(pH - 1.81) + 1 )


Alternatively, values obtained using the above equations are shown in Table 2 below.


Table 3. Free SO2 required for given molecular SO2 level


pH Free SO2 (mg/l) for given molecular SO2 level

0.6 mg/l 0.8 mg/l 2 mg/l
2.8 6 9 22
2.9 8 11 27
3.0 10 13 33
3.1 12 16 41
3.2 15 20 51
3.3 19 26 64
3.4 24 32 80
3.5 30 40 100
3.6 38 50 125
3.7 47 63 157
3.8 59 79 197
3.9 74 99 248
4.0 94 125 312
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vinoirakia
 
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Re: SO2 -Първа част

Мнениеот Alambik » Вто Апр 27, 2010 9:05 pm

Не се заяждам ама тази статия да не е подарък от дядоти!? Извиниме за изказа, но видях цитати от 19..."The value of the dissociation constant, K, increases with increasing temperature. For example, the K value for acetaldehyde increases by 5 times from 25°C (77°F) to 37.5°C (99.5°F) [Kerp, 1903; Kerp and Bauer, 1904 and 1907a,b].", за да сме по-точни! И разни 0.0.... мг./Л. от 1919 г., и какво ли още не! Та на тези данни и анализи биха завидяли и в днешно време! Извинявай!!! :cry:
Alambik
 
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Re: SO2 -Първа част

Мнениеот vinoirakia » Пет Апр 30, 2010 7:12 am

Извинен си.
Говорих с Международната и европейска асоциация на измервачите.
Всички анализи преди 1950 се заличават.
Теореми, като "и все пак тя се върти", "земята е кръгла" , "падна ми ябълка на главата", "изплисках ваната" също ще се махнат.
Не знам защо Пастьор, Кюри и Менделеев и други подобни са си губили времето по лабораториите.
vinoirakia
 
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Re: SO2 -Първа част

Мнениеот Alambik » Сря Май 05, 2010 10:18 pm

A, Вие в УХТ, защо не приемате отрицателните резултати за заключение и завършек на дипломна работа, а трябва студентите на всяка цена да искарат положителни проби, резултати и изводи??? :idea: Направо .... :oops: И да си дойдем на въпроса - приемали са всичко за чиста монета! Иди успорвай нещо казано от Професор да те видя!? Това, което са постановили те е постолат, но с много изключения, които ще стават все повече с напредване на методите за анализ и науките във всички посоки! :idea:
Alambik
 
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