INTRODUCCIÓN

Because the quantity of undissociated acid decreases with increasing pH (Table 2.3), the use of benzoic acid or sodium benzoate as a food preservative has been limited to those products that are acid in nature. Currently, these compounds are used primarily as antimycotic agents, and most yeasts and fungi are inhibited by 0.05% to 0.1% of the undissociated acid. Food-poisoning and spore-forming bacteria are generally inhibited by 0.01% to 0.02% undissociated acid, but many spoilage bacteria are much more resistant. Therefore, benzoic acid cannot be relied on to effectively preserve foods capable of supporting bacterial growth (Chichester and Tanner, 1972; Baird-Parker, 1980). MICs for some of the bacteria, yeasts, and fungi involved in food poisoning and food spoilage are given in Tables 2.5 and 2.6. Several factors interact to determine the MIC, including pH, temperature, genus and species of the microorganism in question, composition of the growth medium, prior exposure to the preservative, and environment from which the microorganism was originally isolated.

The sensitivity of 42 yeast cultures to sorbic and benzoic acids, potassium sorbate, and sodium benzoate was determined (Manganelli and Casolari, 1983). The scattering of MIC values was lower with potassium sorbate and benzoic acid and higher with sorbic acid and sodium benzoate. Addition of benzoate to chemostat cultures of S. cerevisiae decreased the biomass and increased the specific oxygen uptake rate of cells (Verduyn et al., 1992).

The effects of several preservatives and antimicrobial agents on aflatoxin B₁ production by A.

flavus have been reported (Bauer et al., 1981). In liquid media, all treated cultures produced measurable levels of toxin 3 to 7 days later than controls. In benzoic acid-supplemented cultures, aflatoxin B₁ production was higher than in controls. The authors concluded that subinhibitory concentrations of these compounds may stimulate toxin production in some cases.

Sodium benzoate was found to control both growth and aflatoxin production by Aspergillus parasiticus in liquid media (El-Gazzar and Marth, 1987). Increasing the concentration of sodium benzoate increased the percentage of inhibition at the end of incubation (10 days). The average accumulation of mycelial dry weight was greater in the presence of benzoate than in its absence, however, with the greatest increase occurring when the medium contained 0.3% sodium benzoate.

Different concentrations of benzoic acid were tested to determine the effective levels capable of reducing the mycelial growth of six Fusarium and eight Penicillium species by 50% (Thompson, 1997). In general, Fusarium species were more sensitive to benzoic acid (210 to 420 µg/ml) than were Penicillium species (250 to 3000 µg/ml).

TABLE 2.5

Antimicrobial Spectrum of Benzoic Acid against Selected Bacteria, Yeasts, and Fungi

a Minimum inhibitory concentration in µg/ml (ppm).

Source: Adapted from Chipley (1983, 1993); Davidson and Juneja (1990); Russell (1991);

and Steels et al. (1999).

Sodium Benzoate and Benzoic Acid 19

Growth and aflatoxin production by toxigenic strains of Aspergillus were partially or completely inhibited by the undissociated form of six organic acid preservatives, including benzoic (Rusul and Marth, 1988). Salts, such as sodium and potassium chlorides and sodium nitrate, enhanced aflatoxin production when present at low levels but became inhibitory at higher levels.

Neosartorya fischeri is one of the most frequently isolated heat-resistant fungi causing spoilage of fruit juices and other heat-processed fruit-based products (Nielsen et al., 1989). Growth of this fungus was accompanied by production of fumitremorgin mycotoxins. Fungal growth was reduced by lowering the pH of laboratory media from 7.0 to 2.5; selected organic acids promoted growth and toxin production when added to the media. Small amounts (75 mg/L) of potassium sorbate or sodium benzoate completely inhibited germination of ascospores and subsequent outgrowth.

Both fungistatic and fungicidal properties have been attributed to benzoic acid, according to the results of a study involving several strains of Trichophyton and Microsporum (Pelayo, 1979).

Sodium benzoate has been suggested as an inhibitor of cellulose-decomposing bacteria and fungi (Sauer, 1977).

Under appropriate conditions, bacteriostatic and bactericidal properties of benzoic acid can also be demonstrated. Beuchat (1980) reported that sodium benzoate (300 µg/ml) inhibited the growth of Vibrio parahaemolyticus in laboratory media and enhanced the rate of thermal inactivation of this organism at slightly higher concentrations. Ten generally regarded as safe (GRAS) substances, including benzoic acid, were tested against both the opaque and translucent morphotypes of Vibrio vulnificus (Sun and Oliver, 1995). Eight of these had a lethal effect on both morphotypes of this bacterium.

In a series of studies involving L. monocytogenes (El-Shenawy and Marth, 1988; Yousef et al., 1989), it was found that benzoic acid at concentrations of approximately 1000 to 3000 ppm had strong bacteriostatic, but relatively modest bactericidal, activities against cells in a liquid minimal medium. Incubation of cells in minimal media caused injury that depended on the temperature of incubation but not on the presence of benzoic acid. The authors questioned the suitability of benzoic acid alone to control this pathogen in foods. This organism was isolated from milk, and its survival

TABLE 2.6

Minimum Inhibitory Concentration of Benzoic Acid for Food Spoilage Yeasts

Isolate^a MIC (ppm)

Kluveromyces fragilis Kloeckera apiculata Pichia ohmeri Hansenula anomala Saccharomyces cerevisiae Zygosaccharomyces rouxii Zygosaccharomyces bisporus Candida krusei

Saccharomycodes ludwigii Schizosaccharomyces pombe Zygosaccharomyces bailii

173 188 200 223 170–450 242–330 200–350 440 500–600 500–567 600–1300

a A total of 23 isolates were tested. Most were isolated from spoiled foods that had contained preservative. Isolates were grown at 25°C in yeast extract medium containing 5% glucose (pH 3.5) without addition of benzoic acid.

Source: Adapted from Warth (1989c).

and growth were determined in media supplemented with organic acids and sodium benzoate (El-Shenawy and Marth, 1989). In general, inactivation or inhibition of growth occurred in inoculated media when a lower incubation temperature (13°C) and pH (5.0) were used and required lower levels of sodium benzoate. Combining glycerol monolaurate with benzoic acid gave greater inhi-bition of L. monocytogenes than when each preservative was tested alone (Oh and Marshall, 1994).

The effects of temperature, pH, sodium chloride, and three preservatives on the growth of three foodborne bacterial pathogens were studied using gradient gel plates (Thomas et al., 1993). Potas-sium sorbate was completely effective against Vero cytotoxigenic E. coli at all temperature/pH/NaCl combinations. It was also the most effective against B. cereus. At <25°C, sorbate was more effective than sodium benzoate against S. aureus when used with higher concentrations of NaCl. At 35°C, benzoate was the most effective preservative against S. aureus, especially when used at pH <6.

Sodium nitrite was the least effective preservative tested. Increasing the acidity and/or NaCl generally improved the effect of all the preservatives.

Potassium sorbate and sodium benzoate did not have any significant effects on the heat resis-tance of four strains of B. stearothermophilus spores (Lopez et al., 1996). However, when these preservatives were added to recovery media, they were very effective inhibitory agents for heat-damaged spores (Lopez et al., 1998). Effectiveness increased as the pH of the recovery media decreased to 5.0.

An interesting method to determine concentrations of antimicrobial agents that provide infinite microbial stability has been developed (Marwan and Nagel, 1986b). This method was based on finding the relative effectiveness of an inhibitor, like benzoic acid, at various concentrations. The relative effectiveness values of benzoic acid were established for S. bayanus and Hansenula species.

A plot of the inhibitor concentration versus the reciprocal of relative effectiveness was linear (Figure 2.3). The x-axis intercept was the concentration of the inhibitor that gave infinite microbial inhibition. The infinite inhibition concentrations for S. bayanus and Hansenula species were 330 and 180 ppm benzoic acid, respectively, when these organisms were grown in Trypticase^® soy broth. Infinite inhibition concentrations could be affected by the growth medium. The growth patterns of S. bayanus in the presence of different concentrations of benzoic acid are shown in Figure 2.4. A typical dose-response effect may be observed.

INFLUENCE OF OTHER CHEMICALS AND PHYSICAL