Apartado D: El Rendimiento Académico

The growth-inhibiting or lethal effects of sulfurous acid are most intense when the acid is in the un-ionized form (Hailer, 1911). It has also been noted that bacteria are much more sensitive to sulfur dioxide than are yeasts and molds. Schelhorn (1951) observed that bisulfites had lower activity than sulfur dioxide against yeast, and the sulfites had none.

The bound forms of sulfur generally have reduced antimicrobial activity (Rhem, 1964; Schro-eter, 1966). Cruess (1912) estimated that, in grape juice, the bound forms of sulfurous acid had about 1/30 the antimicrobial effectiveness of the free form. Although sulfonates have decreased antimicrobial activity, several have been found to inhibit yeast respiration (Rhem, 1964). The order of decreasing antimicrobial activity of the sulfonates was pyruvate > benzaldehyde > arabinose >

ketoglutarate > acetone > acetaldehyde > glucose > fructose (Rhem, 1964).

Another reaction of significance is that between bisulfite and disulfide bonds:

R₁-S-S-R₂ + HSO₃^–→ R1SH + R₂-S-SO₃^– This reaction can cause conformational changes in enzymes.

Thiamine pyrophosphate, a required cofactor for many enzymatic reactions, can be destroyed by the action of bisulfite (Williams et al., 1935). Excess sulfur dioxide added to grape juice can deplete thiamine and inhibit fermentation (Ournac, 1969).

One type of activity of sulfite against the yeast cell is its reaction with cellular adenosine triphosphate (ATP) (Schimz and Holzer, 1977, 1979; Schimz, 1980) and/or its blocking of the cystine disulfide linkages. Hinze et al. (1981) also found reduced ATP activity by addition of SO₂ to lactic acid bacteria. Anacleto and van Uden (1982) suggested that the antimicrobial effects of SO₂ occurred at the surface of the cell. They proposed two receptor sites. One site was directly related to the death process. The other modulated the entropy of activation of the process. The cytoplasmic membrane has a high affinity for reaction with SO₂. Beech and Thomas (1985) give an excellent review of the many antimicrobial actions possible with SO₂. Among the activities discussed are blockage of transport, inhibition of glycolysis, nutrient destruction, and inhibition of general metabolism.

Lenz and Holzer (1985) showed that the depletion of thiamine pyrophosphate (TPP) in Sac-charomyces cerevisiae by SO₂ at levels used for juice and wine preservation caused the TPP-dependent enzymes to decrease in activity. Pyruvate decarboxylase and transketolase lost 42% and 87% of their activity, respectively. TPP activity losses were slower than ATP losses, but the overall losses were about the same.

When considering the antimicrobial activity of sulfur dioxide and its salts, three main groups of microbes are of interest in the acid beverages and fruits. These are as follows: (1) acetic acid–producing and lactic acid-producing bacteria, (2) fermentation and spoilage yeasts, and (3) fruit molds. Sulfites are used in other foods and pharmaceuticals, but their major use as an antimicrobial agent is in beverages and fruits.

BACTERIA

According to Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994), the genus Ace-tobacter is Gram-negative aerobic rods. The type species is A. aceti. These bacteria are able to

Sulfur Dioxide and Sulfites 149 oxidize ethanol in fermented beverages to acetic acid and, further, to carbon dioxide and water.

They are aerophilic and have pH optimum for growth of around 5.4 (Holt et al., 1994).

Dupuy (1959) postulated that the reversible reaction between sulfur dioxide and cysteine to form thiol esters along with thiamine and NAD⁺ degradation were the causes for inhibition of Acetobacter. He found that this genus also fixed a certain amount of the sulfur dioxide. Cruess (1912) found that 5.49 log Acetobacter per ml exposed to 100 and 200 mg/L of total sulfur dioxide in grape juice were reduced to 2.47 log and 0.3 log CFU/ml, respectively, after 36 hours. Rhem and Wittmann (1962) reported that 200 mg/L sulfur dioxide killed Acetobacter at pH 6.0 in a buffered solution. Dupuy and Maugenet (1963) noted that even small doses of sulfur dioxide inhibited the activity of the cells, but much larger doses were required for bactericidal action. Lafon-Lafourcade and Joyeux (1981) and Joyeux et al. (1984) stated that the amounts of SO₂ used in normal wine making are insufficient for acetic acid bacteria control. They indicated A. aceti can grow in red wine with 25 mg/L of unbound sulfur dioxide present. Watanabe and Ino (1984) and Juven and Shomen (1985) reported that up to 100 mg/L of total sulfites for grape juice, red wine, and soft drinks were required to control acetic acid bacteria.

Several reports (Karova and Kircheva, 1982; Spirov et al., 1983) indicated that around 50 mg/L of free sulfite could preserve wine vinegar for about half a year. Cell growth was completely prevented. Additions of 100 mg/L bleached the color of the vinegar.

Bacteria common in acid fruits and beverages are the lactic acid-producing genera Lacto-bacillus, Leuconostoc, Pediococcus, and Oenococcus. The homolactic species found in wines are Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus leichman-nii, Lactobacillus plantarum, Pediococcus cerevisiae, and Pediococcus pentosaceus, and the het-erolactic species are Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus buchneri, Lacto-bacillus hilgardii, LactoLacto-bacillus trichodes, and Oenococcus oeni (Amerine et al., 1980).

Leuconostoc mesenteroides subspecies dextranicum can also cause ropiness in beverages through the formation of dextrans. Fornachon (1957) noted that sulfur dioxide and pH were important factors in controlling lactic bacteria in wines. Levels above 120 mg/L of total sulfur dioxide (free and bound) decreased the incidence of malolactic fermentation, with lower pH increasing the effectiveness of the sulfur dioxide. Amerine et al. (1980) reported that 30 mg/L of free sulfur dioxide is sufficient to inhibit malolactic fermentation in wine. In model solutions containing Lactobacillus arabinosus and L. casei, 320 and 651 mg/L of sulfurous acid were required, respectively, for antimicrobial action at pH 6.0 (Rhem and Wittmann, 1962).

Carr and Davies (1971) noted that relatively high amounts of free sulfurous acid were present in ciders that contained viable lactobacilli. They found that up to 1.5 mg SO₂ per liter in the undissociated form was bacteriostatic to L. plantarum. Concentrations above 1.5 mg/L were bac-tericidal. Lactobacillus trichodes (Fornachon et al., 1949) is described as causing a hairlike growth in fortified wines. This organism is particularly intolerant to sulfur dioxide. As low as 75 to 80 mg/L of total sulfur dioxide prevents growth and 100 mg/L kills this species. The bacterium, however, is very heat resistant and alcohol tolerant. Lafon-Lafourcade (1975) demonstrated the effectiveness of both free sulfur dioxide and bound sulfur dioxide in inhibiting Leuconostoc gracile in wine at pH 3.5. Addition of 30 mg/L of free sulfur dioxide killed the bacteria completely in 15 days; 20 days was required to kill the bacteria when the same amount of aldehyde–bisulfite complex was used.

Oenococcus oeni grows preferentially at lower pH but seems less tolerant to sulfur dioxide (Mayer, 1979). As little as 30 mg/L of added sulfur dioxide may be lethal to the species. Manca de Narda and Strosser de Saad (1987) investigated the tolerance of O. oeni, L. hilgardii, and P. pentosaceus to SO₂, pH, and ethanol. O. oeni was the most sensitive to SO₂.

Fermentation of grape juices with larger amounts of insoluble solids present resulted in wines that underwent malolactic fermentation sooner and more rapidly than those juices that contained fewer solids (Liu and Gallander, 1982, 1983). The juices high in insoluble solids ultimately had lower residual total sulfites. Liu and Gallander (1983) demonstrated that lower pH wines underwent

malolactic fermentation more slowly and that lower sulfur dioxide levels increased the fermentation rate of the O. oeni PSU-1 used. Ough et al. (1988) demonstrated that without sufficient SO₂ and adjusted pH, the growth of O. oeni in red table wine occurs readily. There was some additive effect when used with dimethyl dicarbonate but not enough to warrant the use with SO₂ for this purpose.

Piracci (1984) found 0.5 mg/L of molecular sulfite was sufficient to control malolactic bacteria growth.

Lafon-Lafourcade et al. (1983) determined that, in the Bordeaux area of France, O. oeni was the primary malolactic bacterium associated with wine. It survived alcoholic fermentation when others did not. It was found to tolerate up to 100 mg/L of SO₂, although after sulfite addition, a rapid decline in cell numbers occurred. The remaining cells later multiplied to significant numbers.

Wibowo et al. (1988) found significant delays in growth of O. oeni when SO₂ was added and quite a variable response between strains. Davis et al. (1988) tested 146 different strains of wild malolactic bacteria. All strains grew at pH 4.5 in beef broth and 20% tomato juice serum medium at 64 mg/L of total sulfur dioxide. O. oeni strains were less tolerant to the sulfur dioxide than Pediococcus paroulus strains or the Lactobacillus species. This work confirms that wines with high total sulfur dioxide concentrations are more likely to undergo malolactic fermentation with other than O. oeni strains with unfavorable sensory results.

Splittstoesser and Stoyla (1989) looked at five different regulatory-approved additives to deter-mine if they could suppress malolactic bacterial growth in grape juice as a replacement for sulfites.

None of the compounds alone, or in paired combinations, were completely effective.

Millet and Lonvaud-Funel (2000) reported that sulfites cause a portion of lactic acid and acetic acid bacterial populations to enter a viable but nonculturable (VBNC) state. The cells could not be cultured on nutrient agar plates but demonstrated metabolic activity through hydrolysis of fluores-cent esters and were countable using direct epifluorescence microscopy. They suggested that these microorganisms could cause spoilage in wines that were considered sterile using conventional counting techniques.

Roberts and McWeeny (1972), in their review, state that sulfur dioxide is more effective against the growth of Gram-negative rods, such as Escherichia coli and Pseudomonas, than in inhibiting Gram-positive bacteria. This is demonstrated in the use of sulfites in meats. The preservation of the color and odor of meats is improved by sulfite treatment and, although slowing or prevention of growth of surface bacteria is probably important, the main effect in meat appears to be the antioxidant properties (Roberts and McWeeney, 1972). Banks et al. (1985) reviewed the use of sulfite as an additive to control microbiological changes occurring in meat products. They noted that sulfites shifted the microflora of the meat to positive bacteria from the normal Gram-negative flora. The Gram-positive bacteria remaining grew more slowly than the Gram-Gram-negative bacteria, In addition, Salmonella and E. coli were inhibited to a greater extent by sulfites than other bacteria. Banks and Board (1982) tested several genera of Enterobacteriaceae isolated from sausage for their metabisulfite sensitivity. The microorganisms tested and the concentration of free sulfite (µg/ml) necessary to inhibit their growth at pH 7.0 were as follows: Salmonella, 15–109; E. coli, 50–195; Citrobacter freundii, 65–136; Yersinia enterocolitica, 67–98; Enterobacter agglomerans, 83–142; Serratia marcescens, 190–241; and Hafnia alvei, 200–241. Tompkin et al. (1980) found the addition of 100 mg/kg of SO₂ as sodium metabisulfite to canned pork inoculated with Clostrid-ium botulinum spores delayed cell growth. The delay was proportional to the concentration of the bisulfite addition. They also noted that the interaction of sulfites with nitrites caused a lowering of the nitrites available for nitrosamine formation.. Reddy and Mandokhot (1987) found that minced goat meat could be preserved up to 11 to 13 days if held at 7°C with 450 mg/L of sulfur dioxide added. The effect was inhibition of growth of the flora. Sensory tests showed no adverse results.

The shelf life of ground beef was effectively increased from 1.8 days at 7°C storage with no treatment to 12.6 days at 0°C with the addition of 250 mg/kg of sulfur dioxide. The packaging used was a gas-permeable wrapping that allowed oxidative conditions (von Holy et al., 1988).

Adams et al. (1987) found that vacuum packaging and a good oxygen barrier film decreased the

Sulfur Dioxide and Sulfites 151

spoilage in sulfite-treated sausage. This was because of the lack of oxygen delaying yeast growth and the production of sulfite-binding substances. Thus the free sulfite, which inhibited growth, was maintained for a longer period. Sodium sulfite addition in sausage was shown to affect biogenic amines. The concentration of tyramine and putrescine increased in the presence of sulfite, but the level of cadaverine was reduced (Bover-Cid et al., 2001). There was no effect on histamine, phenylethylamine, or tryptamine.

Yeast

The use of sulfur dioxide to deplete the wild yeast in grape juice is a standard practice dating back many years (Cruess, 1912). Rhem and Wittmann (1962) determined the inactivation levels of sulfurous acid for a variety of yeast genera (Table 5.4). Goto (1980) determined viable counts of various wild yeasts in grape juice in the presence of sulfur dioxide and found Torulopsis and Saccharomyces were the most tolerant, whereas Kloeckera, Pichia, Rhodotorula, and several other genera were very susceptible. Haznedari (1979) tested 30 strains of S. cerevisiae that had been characterized as “SO₂ resistant” for tolerance to high concentrations of sulfur dioxide. Six strains could grow well at 1000 mg/L, and five others were able to produce adequate amounts of ethanol.

In contrast, Carr and Davies (1971) found sulfur dioxide incorporated into growth medium (pH 3.4) at 25 mg/L was sufficient to kill a culture of S. cerevisiae (10⁵ CFU/ml) after 8 hours at 25°C.

Dott and Trüper (1978) found “killer yeasts” (those yeasts that when grown in mixed cultures cause the death of other yeasts) were high or medium producers of sulfite and were more resistant to sulfur dioxide.

Molecular sulfur dioxide is the most effective form for inhibiting yeast. The amount of molecular SO₂ (mg/L) in wine can be calculated using free SO₂ mg/L/(1 + 10^{pH - 1.81}). Sudraud and Chauvet (1985) found that to maintain yeast stability, 1.5 and 1.2 mg/L of molecular sulfur dioxide were necessary at the finish of fermentation and during aging, respectively. Ough et al. (1988) found that with yeast acclimatized to sulfur dioxide, between 2.0 and 3.0 mg/L were required. The yeast also showed increased resistance to the fungicide dimethyl dicarbonate. pH is extremely important in the effective use of sulfur dioxide. A 10-fold increase in molecular sulfur dioxide occurs between pH 4.0 and 3.0. Any rule of thumb addition, such as “maintain the free SO₂ at 20 mg/L,” for biological stability can be disastrous. In fact, with the pressure to reduce sulfur dioxide content, its use to prevent the growth of yeast in sweet table wines is seldom ever contemplated.

According to Warth (1985), resistance of yeast to sulfur dioxide ranges from 0.05 M free sulfur dioxide for Kloeckera apiculata to 2.8 mM for Z. bailii at 25°C under aerobic conditions at pH 3.5 in tryptone yeast extract medium. Saccharomycodes ludwigii was nearly as tolerant as Z. bailii.

Once the inhibition was overcome, the growth rates and cell yield were similar. Delfini (1989) TABLE 5.4

Range of Effective Antimicrobial Concentrations of Sulfurous Acid against Various Genera of Yeast

Genus Number of Species

Effective H₂SO₃ (mg/L)^a

Saccharomyces 13 0.10–20.20

Zygosaccharomyces 2 7.2–8.7

Pichia 1 0.20

Torulopsis 1 0.20

Hansenula 1 0.60

Candida 2 0.40–0.60

a Rhem and Wittmann (1962).

reported the existence of very sulfite-resistant S. cerevisiae, Z. bailii, S. ludwigii, and Schizosac-charomyces japonicus in the re-fermentation of sweet champagnes. Growth was found at the highest concentration of free sulfur dioxide used, and there was no correlation with the number of cells in the initial inoculum. In a cell recycled ethanol fermentation system, Chang et al. (1997) showed that sulfite up to 400 mg/L had no effect on S. cerevisiae despite a reduction in the bacterial counts in the system at the same concentration.

Spoilage yeast in dry wines are fairly rare. With modern filtration and sanitation technology, cell numbers are depleted to very low levels at bottling. Brettanomyces can contaminate a winery, especially wines in barrels, if proper sulfur dioxide levels are not maintained. In normal sweet table wines, the main spoilage yeast is Saccharomyces. With this genus, sulfur dioxide, even at the legal limits, does not always inhibit growth and fermentation. Minarik and Navara (1977) reported finding the spoilage yeast S. ludwigii in a low-alcohol wine. This particular species was found to be very resistant to sulfurous acid. S. ludwigii is also a noted spoiler in sweet grape juice containing high amounts of sulfur dioxide (Jakob, 1978). Sand (1980) suggested that Z. bailii was becoming a problem in juices, soft drinks, and wines because of its tolerance to SO₂, alcohol, and low pH.

Spoilage by Z. bailii in comminuted orange drink was controlled by 230 mg/L of SO₂ at pH 3.1 but not in the base material, which was at pH 3.7 (Lloyd, 1975).

There have been numerous reports on techniques to minimize the amount of sulfur dioxide used in wines (Aerny, 1986a,b; Gomes and da Silva Babo, 1985; Asvany, 1985; Galassi and Mancini, 1985; Valouyko et al., 1985; Hernandez, 1985). Recommendations include cooling the fruit before crushing, adjusting the pH downward if necessary, using yeast that produces minimum amounts of sulfur dioxide-binding components, settling and racking the juices, reducing the amount of sulfur dioxide before or during fermentation, minimizing the air around the wine, using sulfide salts before fermentation, substituting other preservatives at bottling, and eliminating oxygen from bottled wine.

Hydrogen sulfide (H₂S) was used (Ubigli et al., 1982) as an alternative for pretreatment of grape juice before fermentation. The treatment was reported to be successful for juice. Schmitt et al.

(1983), however, stated that the use of sulfide salts failed to protect wine and left negative sensory characteristics in the wine. Although other agents may act as antimicrobial agents, none seem to be capable of replacing the antioxidant property of SO₂.

Yeast can form sulfite from sulfate via sulfate permease. Some yeast strains can form rather large amounts of sulfur dioxide during juice fermentation (Table 5.5). Suzzi et al. (1985) tested 1700 Saccharomyces. They found a majority of the wines in the test to contain about 10 mg/L of total sulfur dioxide (Table 5.6). This shows that most wines will not be free from sulfite labeling requirements even if no sulfur dioxide is added. Dott et al. (1977) determined the cause for sulfite production to be sulfate permease inhibition by methionine. In high-sulfite-producing yeast, the sulfate permease was not repressed by methionine. Proper selection of the yeast strain to avoid high levels of sulfite is an obvious choice. Very little of the sulfur dioxide formed by the yeast remains in the free state.

Breweries have an interest in sulfur dioxide as an antioxidant. Klimovitz and Kindraka (1989) found that endogenous sulfur dioxide varied with starting specific gravity of the brew and the sulfate content of the water used. Vernerova et al. (1983) showed that increasing the dissolved oxygen in the wort from 1 to 2 to 8 to 9 mg/L caused production of 15% to 30% more sulfur dioxide. Strain differences caused variation in the sulfur dioxide produced from 11 to 26 mg/L.

Angelino et al. (1989), investigating the sulfur dioxide content of green beer, found no correlation with dissolved oxygen levels in wort and no correlation with ATP sulfurase or sulfite reductase.

Pearlstein (1988) found that extended aeration decreased sulfite production.

Fruit juices and preserves can be protected from microbial spoilage by sulfur dioxide addition.

Generally, because the pH is high, excess amounts are used. Even at low pH (2.6 and 3.4), Sethi and Anand (1984) had to use 692 mg/L in carrot preserves to inhibit Bacillus cereus. Ranote and Bains (1982) used 350 mg/L of sulfur dioxide to preserve Kinnow, a late-harvest orange juice.

Patel et al. (1985) used an initial 1000 mg/L of sulfur dioxide in the last stages of the manufacture

Sulfur Dioxide and Sulfites 153

TABLE 5.5

Formation of Sulfur Dioxide by Various Yeasts during Grape Juice Fermentations

Yeast Sulfur Dioxide (mg/L) Reference

Saccharomyces carlsbergensis 12 Minarik (1975)

150 Eschenbruch and Bonish (1976)

80, 100, 160, 170, 1300 Heinzel et al. (1976)

S. cerevisiae 22, 35 Minarik (1975)

20, 52, 100 Eschenbruch and Bonish (1976)

5, 150 Heinzel et al. (1976)

0, 3, 4, 5 Delfini et al. (1976) 18–23

S. pastorianus 85 Minarik (1975)

S. bayanus 0, 5, 500 Minarik (1975)

300, 500 Heinzel et al. (1976)

5.5, 35.5, 44, 56, 76 Delfini et al. (1976) 17, 18, 20 Poulard and Brelet (1978)

S. uvarum 1.5, 3, 21.5 Delfini et al. (1976)

14, 18, 21, 40 Poulard and Brelet (1978)

S. chevalieri 0 Minarik (1975)

18, 20 Poulard and Brelet (1978)

S. acidifacien 26 Minarik (1975)

S. bailii 27 Delfini et al. (1976)

S. rosei 0 Delfini et al. (1976)

S. italicus 0, 1.5 Delfini et al. (1976)

S. italicus 0, 1.5 Delfini et al. (1976)