Sulfur Dioxide

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Sulfur dioxide is widely recognized in both the wine and food industries for its antioxidative and antimicrobial properties. The current legal limit for SO2 in wines in the United States is 350 mg/L, a concentration well above levels normally used by winemakers. Nevertheless, wines that contain greater than 10 mg/L must disclose this information on the label. Because SO2 is a metabolite of yeasts during fermentation (Section 1.5.2), wines will usually contain some sulfite even though additions were not made during processing.

5.2.1.1 Forms of Sulfur Dioxide

Once dissolved in water, sulfur dioxide exists in equilibrium between molecular SO2 (SO2^H2O), bisulfite (HSO3-), and sulfite (SO32-) species as illustrated below:

This equilibrium is dependent on pH, with the dominant species at wine pH (3 to 4) being the bisulfite anion (Fig. 5.1). Besides being in equilibrium with the molecular and sulfite species, bisulfite also exists in "free" and "bound" forms. Here, the molecule will react with carbonyl compounds (e.g., acetaldehyde), forming addition products or adducts such as hydroxysulfonic acids.

100 -c

90-

80

70

V)

60

"<5

0

50

H

40

30

20

10

0 1

Molecular SO2^H2O

Bisulfite HSO3-

Sulfite SO32-

Figure 5.1. values.

Relative abundance of molecular SO2, bisulfite, and sulfite at different pH

In addition to acetaldehyde, molecules present in grape juice that react with bisulfite are pyruvic acid, a-keto-glutaric acid, dihydroxyacetone, diacetyl, anthocyanin pigments, and others (Ough, 1993b).

O OH

5.2.1.2 Microbial Inhibition

It is generally believed that the molecular sulfur species is the antimicrobial form of sulfur dioxide. Because SO2^H2O does not have a charge, the molecule enters the cell and undergoes rapid pH-driven dissociation at cytoplasmic pH (generally near 6.5) to yield bisulfite and sulfite. As the intracellular concentration of molecular SO2 decreases due the internal equilibrium, more molecular SO2 enters the cell, further increasing intra-cellular concentrations.

The amount of molecular SO2 present in any wine is not normally measured directly. Rather, the concentration is calculated knowing the concentration of free SO2 and the pH of the wine based on the following formula:

Alternatively, Fig. 5.2 can be used as a guide to approximate the amount of free SO2 needed at various pH values for yielding either 0.5 or 0.8 mg/L molecular SO2.

SO2 inhibits microorganisms by various means including rupture of disulfide bridges in proteins and reaction with cofactors including NAD+ and FAD. It also reacts with ATP, and brings about deamination of cytosine to uracil increasing the likelihood of lethal mutations. Concentrations of crucial nutrients may also be reduced (Ough, 1993b; Romano and Suzzi, 1993). Specifically, SO2 can cleave the vitamin thiamin into components not metabolically useable (Fig. 5.3). Although not normally a problem during vinification, loss of thiamin can be a concern when fermenting juice that has been stored for a period of time as muté. In the United States, the maximum allowable addition of thiamin hydrochloride is 0.6 mg/L.

Allowable Sewer

Figure 5.2. Amounts of free SO, at given pH to yield either 0.5 or 0.8 mg/L molecular SO,.

Figure 5.2. Amounts of free SO, at given pH to yield either 0.5 or 0.8 mg/L molecular SO,.

CH2-SO3H

NH2 ?HVCH2CH2OH H3C

HSO3

H3C^N/ CHVCH2CH2OH

Figure 5.3. Breakdown of thiamin by SO2.

The concentration of molecular SO2 needed to prevent growth of microorganisms varies with wine/juice pH, temperature, population density and diversity, stage of growth, alcohol level, and other factors. The frequently cited addition level of 0.8 mg/L molecular SO2 was suggested by Beech et al. (1979) as the amount needed in white table wines to bring about a 104 CFU/mL reduction in 24 h in populations of several spoilage microorganisms. Differences in sensitivity to SO2 between genera of yeasts and bacteria found in wines are known to exist (Warth, 1977; 1985; Du Toit et al., 2005). For example, work by Davis et al. (1988) with lactic acid bacteria isolated from Australia red wines indicated that strains of L. oenos (O. oeni) were less tolerant to sulfur dioxide than strains of P. parvulus. Davis et al. (1988) further suggested that wines with high total SO2 concentration may be more likely to support the growth of Pediococcus than L. oenos. In contrast, Hood (1983) reported that pediococci were less tolerant to bound SO2 than lactobacilli or leuconostocs. In practice, many wine-makers attempt to maintain 0.4 to 0.6 mg/L molecular SO2 to control Brettanomyces and other spoilage microorganisms during wine aging. Although Du Toit et al. (2005) suggested that a concentration of 0.8 mg/L molecular SO2 was needed to prevent the growth of Acetobacter pasteurianus, the authors further noted that this concentration did not completely eliminate the bacterium.

Whereas some winemakers encourage non-Saccharomyces yeasts (Section 8.4), others desire to limit their growth due to synthesis of undesirable odors and flavors. Although SO2 can suppress the non-Saccharomyces populations prior to alcoholic fermentation (Constant et al., 1998; Egli et al., 1998; Henick-Kling et al., 1998; Ciani and Pepe, 2002; Cocolin and Mills, 2003), its use at the crusher may not inhibit yeast species that have greater resistance to the additive. In fact, some species of Pichia, Saccharomycodes,

Schizosaccharomyces, and Zygosaccharomyces require at least 2 mg/L molecular SO2 for inhibition (Warth, 1985), a difficult concentration to obtain given that the presence of molecular SO2 depends highly on pH (Fig. 5.1). Mechanisms of SO2 resistance differ but are related to variable rates of diffusion across cell membranes, biosynthesis of compounds that bind SO2, and varying enzyme sensitivity (Romano and Suzzi, 1993).

5.2.1.3 Addition of SO2

Sulfur dioxide can be added to musts or wines in the forms of compressed gas, potassium metabisulfite (K2S2O5), or by burning candles containing sulfur in an enclosed container such as a barrel. In the case of compressed SO2, the amount required for a 30 mg/L addition using a gas of 90% purity can be calculated:

L 1000 mg 454 g 1gallon 0.90

= 0.279 lb SO2 per1000 gallons

Most commonly, sulfur dioxide is incorporated into grape must or wine as potassium metabisulfite. Theoretically, 2 moles of SO2 can be derived from each mole of K2S2O5. Therefore, the theoretical yield of SO2 from potassium metabisulfite would be a ratio of molecular weights for each species:

K2S2O5 K2S2O5 222.32

From the above equation, 57.6% of K2S2O5 is theoretically available to form SO2. In practice, this figure is high given that K2S2O5 decomposes upon prolonged storage.

Knowing the relative proportion of SO2 in K2S2O5, the amount required for any addition can be calculated in either g/L or lb/1000 gallons. Thus, the weight of K2S2O5 required to yield a 30 mg/L SO2 addition can be calculated:

L 1000 mg 454 g 1gallon 0.576

= 0.436 lb K2S2O5 per 1000 gallons

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