S ELECTIVE A CTION

Microbial inhibition by sorbate is variable and depends on species, strains, composition of substrate, pH, aw, additives present, food-processing treatments, temperature of storage, gas atmosphere, type

of packaging, and concentration of sorbate. Variations and resistance to inhibition by sorbate may lead to failures in preservation and defective food products (Sofos, 1989).

Early studies indicated that sorbate could be used as a selective agent for catalase-negative lactic acid-producing bacteria and clostridia because it was highly inhibitory against catalase- positive organisms (Phillips and Mundt, 1950; Vaughn and Emard, 1951; Emard and Vaughn, 1952; York and Vaughn, 1954, 1955). In contrast, other studies have reported either no effect or inhibition of lactics and clostridia by sorbate (Costilow et al., 1955; Hansen and Appleman, 1955; Hamdan et al., 1971). Overall, however, the inhibitory action against lactics by sorbate is less than that against yeasts, which explains the usefulness of the compound as a preservative in vegetable fermentations. Another bacterium that appears to be more resistant to inhibition by sorbate than other spore formers is Sporolactobacillus (Botha and Holzapfel, 1987). Growth of Gluconobacter oxydans in the presence of sublethal concentrations of sorbic acid before determination of the minimal inhibitory concentration (MIC) resulted in a substantial increase in the MIC within 1 hour (Eyles and Warth, 1989). In general, various species and strains of microorganisms exhibit different sensitivities to inhibition by sorbate. Varying sensitivities of bacterial species and strains to sorbate may lead to shifts in the microbial flora during storage of foods (Chung and Lee, 1981, 1982; Lahellec et al., 1981; Blocher et al., 1982; Lynch and Potter, 1982; Blocher and Busta, 1983, 1985; McMeekin et al., 1984; Kondaiah et al., 1985).

In addition to bacteria, under certain conditions some species and strains of yeasts and molds are resistant to inhibition by sorbate. Yeast strains resistant to sorbate belong to the genera Zygosac- charomyces, Saccharomyces, Torulopsis, Brettanomyces, Candida, and Triganopsis (Warth, 1977, 1985; Splittstoesser et al., 1978; Restaino et al., 1982, 1983; Bills et al., 1982; Cole et al., 1987; Lenovich et al., 1988; Mihyar et al., 1997). Of 100 yeast strains isolated from spoiled foods and beverages, most tolerated 150 ppm sorbic acid, 40% tolerated 500 ppm, and two strains of Z. bailii tolerated 800 ppm of sorbic acid (Neves et al., 1994). Resistance of yeasts to inhibition by sorbate depends on species and strains, sorbate concentration, pH, inoculum level, storage temperature, and previous exposure of the organism to low levels of sorbate (Sofos, 1989). When the yeast cells have been previously adapted to sorbate in media containing glucose or sucrose, subsequent exposure of the cells shows little effect of solute type on sorbate resistance (Lenovich et al., 1988). However, potassium sorbate suppressed growth of Z. bailii in salsa mayonnaise more than sodium benzoate (Wind and Restaino, 1995). Potassium sorbate or sodium benzoate resulted in complete inhibition of Z. rouxii in high moisture prunes (El Halouat et al., 1998).

Resistance of osmotolerant yeasts to inhibition by sorbate was acquired by preconditioning the yeast to sorbate (Bills et al., 1982). One proposed mechanism of resistance of osmotolerant yeasts has involved an inducible, energy-requiring system that transports the preservative out of the cell (Warth, 1977). Other proposed mechanisms of yeast resistance to sorbate at reduced aw have been

related to yeast cell shrinkage and decreases in membrane pore size, retarding the flow of sorbate into the cell (Restaino et al., 1983), or protection of enzyme systems from inhibition by sorbate through production of compatible solutes, such as polyols (Bills et al., 1982). Exposure of Saccha- romyces cerevisiae to sorbic acid caused strong induction of two plasma membrane proteins, one of which was identified as adenosine triphosphate (ATP)-binding cassette transporter (Pdr12), which is essential for the adaptation of yeast to growth under weak acid stress and confers weak acid resistance by mediating energy-dependent extrusion of water soluble carboxylate ions (Holyoak et al., 1996; Piper et al., 1998). Exposure of S. cerevisiae to 0.9 mM sorbic acid at pH 4.5 resulted in the increased transcription and translation (upregulation) of genes encoding 10 different proteins and the downregulation of three proteins (de Nobel et al., 2001). Functional categories of genes that are induced by sorbic acid stress included cell stress (particularly oxidative stress), transposon function, mating response, and energy generation. The induction of Hsp26, a heat shock protein of S. cerevisiae, which occurs during adaptation to sorbic acid, confers resistance to the inhibitory effects of sorbic acid (de Nobel et al., 2001).

Sorbic Acid and Sorbates 57

DEGRADATION

Animals and certain microorganisms can metabolize sorbate, under certain conditions, as a fatty acid through β-oxidation. When sorbate levels are high, there is also evidence of some ω-oxidation (Deuel et al., 1954b; Lück, 1980). Like caproic and butyric acids, under normal conditions of alimentation, sorbate is completely oxidized to carbon dioxide and water. Because it is metabolized like other fatty acids, sorbate yields 6.6 kcal/g, of which 50% is biologically usable.

Some mold strains can grow and metabolize sorbate under certain conditions as detected in cheeses and fruit products (Melnick et al., 1954b; Sofos, 1989). Mold strains of the genus Penicil- lium isolated from cheese treated with sorbate were able to grow and metabolize high (0.18% to 1.20%) sorbate levels (Marth et al., 1966; Bullerman, 1977; Finol et al., 1982). It should be noted that 0.1% sorbate is usually sufficient to inhibit sensitive molds (Liewen and Marth, 1985a,b). It appears that selection may occur in sorbate-treated cheeses for certain molds tolerant to the compound (Schroeder and Bullerman, 1985). Products of sorbate metabolism by molds include 1,3-pentadiene, which is a volatile compound formed through a decarboxylation reaction and has a kerosene-like, plastic paint, or hydrocarbon-like odor (Marth et al., 1966; Liewen and Marth, 1985a–c). Other strains of molds that may degrade sorbate belong to the genera Aspergillus, Fusarium, Mucor, and Geotrichum (Sofos, 1989). It appears, however, that there is no apparent relationship between sorbate resistance and the toxigenic properties of molds (Tsai et al., 1988). In general, although many molds are sensitive to inhibition by sorbate, certain strains are resistant and can metabolize the compound, using it as a carbon source. Degradation of sorbate by molds depends on species and strains, prior exposure to subinhibitory levels of sorbate, level of inoculum, amount of sorbate present, and type of substrate (Sofos, 1989).

In addition to certain molds, some bacterial strains may also degrade sorbate under appropriate conditions. This metabolism is mostly associated with lactic acid-producing bacterial strains present as high inocula in sublethal concentrations of sorbate (Crowell and Guymon, 1975; Horwood et al., 1981; Liewen and Marth, 1985a). Degradation of sorbate by lactic acid bacteria has been associated with geranium-type off-odors in wines and fermented vegetables, caused by ethyl sorbate, 4- hexenoic acid, 1-ethoxyhexa-2,4-diene, and 2-ethoxyhexa-3,5-diene (Edinger and Splittstoesser, 1986a,b; Sofos, 1989). In general, a geranium-like odor is usually associated with wines treated with sorbate and contaminated with high microbial loads.

INTERACTIONS

The antimicrobial activity of sorbates is influenced by compositional, processing, and environmental factors, such as concentration, other ingredients, pH, aw, temperature, gas atmosphere, packaging,

microbial flora, inoculum size, and other additives (Sofos and Busta, 1981; Sofos, 1989; Steels et al., 2000). These factors can act synergistically or be antagonistic and either enhance or negate the antimicrobial activity of sorbate (Sofos, 1989, 1992).

The MIC of sorbic acid increases with the size of the inoculum; large inocula at high cell concentrations therefore require considerably higher concentrations of the inhibitor to prevent growth than do dilute cell suspensions. A study found a pronounced positive inoculum effect of Z. bailii resistance to sorbic acid activity, which was not an artifact caused by insufficient growth time, dehydration of cultures, substantial metabolism of sorbic acid, or binding of sorbic acid to dead cells, but an inoculum effect that may be caused by the diversity of the cells in inocula or initial contamination as regards to sorbic acid resistance (Steels et al., 2000). The study showed that the resistance of Z. bailii to sorbic acid was largely the result of the presence of a small fraction of resistant cells and was not heritable or the result of the existence of a mixed culture (Steels et al., 2000). Increasing the concentration of sorbic acid from 200 to 1000 mg/L in apple cider decreased the D50°C value of E. coli O157:H7 from 36 to 5.2 min, about a 7-fold increase in lethality

of apple juice from 18 to 5.2 minutes, whereas benzoic acid reduced it to 0.64 min (Splittstoesser et al., 1996).

The antimicrobial action of sorbate is pH dependent and increases as the pH of the substrate decreases, approaching its dissociation constant (pKa = 4.76) (Cowles, 1941; Hoffman et al., 1944;

Rahn and Conn, 1944; Lück, 1976, 1980; Sofos and Busta, 1981; Cerruti et al., 1990). Although activity is greater at low pH values, sorbates have the advantage of being effective at pH values as high as 6.5 (Bell et al., 1959, Lück, 1976; Sofos et al., 1979a; Sofos and Busta, 1980, 1981); however, certain studies have indicated antimicrobial activity by sorbate at pH values as high as 7.0 (Raevuori, 1976; Chung and Lee, 1982; Statham and McMeekin, 1988). In contrast, the maximum pH for antimicrobial activity by most other common food preservatives is lower — for example, 5.0 to 5.5 and 4.0 to 4.5 for propionate and benzoate, respectively (Sofos and Busta, 1981). The increased activity of sorbates at pH values higher than 5.5 is advantageous because it allows for their use in foods of higher pH values in which preservatives, such as parabens, might not be effective owing to their increased solubility in fat. In certain instances, sorbates can partially or totally replace benzoate even in foods of lower pH to avoid possible off-flavors caused by the higher benzoate levels needed for inhibition and to extend the range of microbial groups inhibited compared to benzoate or propionate used singly (Melnick et al., 1954a; Gooding et al., 1955; Sofos and Busta, 1981, 1982, 1993).

The increased antimicrobial activity of sorbate at lower pH values has been attributed to the increased amount of undissociated acid present, which is believed to be the effective antimicrobial form (Lück, 1980; Sofos and Busta, 1981; Lund et al., 1987; Sofos, 1989; Skirdal and Eklund, 1993). This popular theory has been questioned, however (Sofos, 1989). Studies have indicated that the dissociated sorbic acid also had antimicrobial activity, but it was 10 to 600 times less inhibitory than the dissociated acid (Eklund, 1983; Statham and McMeekin, 1988). In environments of pH higher than 6.0, however, more than 50% of the inhibition was the result of dissociated sorbic acid (Eklund, 1983). Nevertheless, it is believed that both undissociated and dissociated sorbic acids have antimicrobial activity (Skirdal and Eklund, 1993). Use of artificial saltwater prevented dissociation of the 1% sorbic acid, which exhibited favorable antimicrobial properties against Vibrio vulnificus, supporting previous evidence that sorbic acid is effective in its undisso- ciated form (Sun and Oliver, 1994). Sorbic acid (0.1%) enhanced destruction of E. coli O157:H7 cells at pH 3.4 but not at pH 6.4 (Liu et al., 1997). Increased amounts of fat in a product reduce the concentration of sorbate in the water phase, where it is needed for microbial control (Oka, 1960). Other food ingredients (e.g., salt and sugars) also reduce the concentration of sorbate in the aqueous phase (Gooding et al., 1955; Liewen and Marth, 1985a). Sugar and salt, however, act synergistically to enhance the antimicrobial activity of sorbate (Costilow et al., 1955, 1956, 1957; Sheneman and Costilow, 1955; Acott et al., 1976; Robach and Stateler, 1980; Beuchat, 1981c). In general, solutes should increase the inhibitory activity of sorbate by reducing the aw of the substrate

(Sofos, 1989; Cerruti et al., 1990). Sucrose, glucose, and sodium chloride, however, have reduced the synergistic effect of sorbate and heat on thermal inactivation of microorganisms (Beuchat, 1981a–c; Cerruti et al., 1988). Sodium chloride (1.25 and 2.5%) reduced the inhibition of Clostrid- ium botulinum by sorbate in a nutrient broth (Wagner and Busta, 1984, 1985a,b). Preconditioning of Saccharomyces rouxii cells in 60% sucrose + 0.1% sorbate rendered the cells more sensitive to inhibition by sorbate than preconditioning in 0% sucrose + 0.1% sorbate (Bills et al., 1982). Lowering the aw enhanced the resistance of the same organism to increasing concentrations of

sorbate (Restaino et al., 1981, 1983). Not only are certain strains of microorganisms resistant to inhibition by sorbate, but increased levels of microbial contamination reduce antimicrobial activity. Thus, sorbates should be used to preserve foods processed using good manufacturing practices, not as a substitute for appropriate sanitation and hygienic practices.

Interactions of sorbate with heat may affect the rate and extent of microbial destruction during heating, as well as dormancy and recovery of heated microorganisms (Sofos, 1989). Sorbate may enhance heat activation and destruction of spores, and it may inhibit the repair and growth of

Sorbic Acid and Sorbates 59

thermally injured organisms (Beuchat, 1980, 1981a–d, 1982; Lusher et al., 1984; Banks et al., 1988; Lopez et al., 1996; Oloyede et al., 1994; Splittstoesser et al., 1995), but the effect of sorbate on thermal inactivation and recovery of injured microorganisms is variable among species and strains. Low concentrations of sorbic and fumaric acids in the heating medium had little effect on the heat resistance of Eurotium herbariorum, a true aerophilic mold involved in the spoilage of grape preserves (Splittstoesser et al., 1989). Concentrations of sorbate as high as 0.1% had little effect on the thermal resistance of ascospores of Neosartorya fischeri, but growth of surviving spores that had been exposed to high temperatures was greatly inhibited by sorbate concentrations as low as 0.007% (Splittstoesser and Churey, 1989). Another report also indicated that potassium sorbate inhibited the heat-resistant N. fischeri (Nielsen et al., 1989). Sorbate may also eliminate the pro- tective effect of sucrose against the thermal inactivation of yeasts and molds (Sofos, 1989). Sorbic and benzoic acids affected the thermotolerance and heat shock response of S. cerevisiae depending on pH (Cheng and Piper, 1994; Sofos, 2000). At low pH, sorbate inhibited induction of thermo- tolerance by sublethal heat shock, but at pH 5.5 it acted as a powerful inducer of thermotolerance in the absence of sublethal heat treatment. Sorbic acid was found to induce thermotolerance without inducing the heat shock response through accumulation of trehalose in S. cerevisiae (Cheng et al., 1999).

It is of particular interest that 0.1% potassium sorbate did not have any significant effects on the heat resistance of Bacillus stearothermophilus spores in distilled water, and as such would therefore be important only in improving microbiological stability through inhibitory effects on germination and/or outgrowth of heat-damaged spores (López et al., 1996). Tolerance of Z. rouxii to sorbate was enhanced in cells preconditioned to elevated sorbate concentrations, especially in media containing 0.1% sorbate. Cells grown in sucrose-supplemented media tolerated higher concentrations of sorbate than did those grown in glucose-supplemented media. Heat resistance increased substantially when cells were grown in media containing sorbate, and particularly in the presence of sucrose as opposed to glucose. The finding that plasmolysis increased in cells grown in sucrose- as opposed to glucose-containing media provides evidence that cell morphology influ- ences heat resistance (Golden and Beuchat, 1992).

Inhibition of microbial growth by sorbate is more effective as storage temperature decreases, indicating that the compound should be more useful as a preservative in refrigerated foods (Pederson et al., 1961; Park et al., 1970; Park and Marth, 1972; Robach, 1980; Roberts et al., 1982; Roland and Beuchat, 1984; Roland et al., 1984; McMeekin et al., 1984; Tuncan and Martin, 1985). Com- binations of acidification and treatment with sorbate may enhance the storage stability of fruit juices even at temperatures higher than normal refrigeration (5°C) (Alli and Kermasha, 1989). Reductions of 5 to 10 log-units of E. coli O157:H7 or Salmonella typhimurium DT104 in apple cider were achieved through freeze-thaw treatments in the presence of sorbic acid (Uljas and Ingham, 1999). The antimicrobial activity of sorbate is usually enhanced under vacuum or modified gas atmosphere storage conditions, as indicated with several food items, including meat (Wagner et al., 1982; Myers et al., 1983; McMeekin et al., 1984) and fish products (Bremmer and Statham, 1983; Statham et al., 1985). Combinations of carbon dioxide and sorbate have also been reported as effective inhibitors of microbial growth (Danzinger et al., 1973; Elliot and Gray, 1981; Elliot et al., 1982, 1985; Gray et al., 1984). Sorbic acid (0.2% to 0.4%), however, did not enhance the bactericidal activity of cellulose-based edible films against Salmonella Montevideo (Zhuang et al., 1996).

Although food acids may reduce the water solubility of sorbate, they can enhance its antimicro- bial activity by increasing the concentration of undissociated sorbic acid. In addition, the specific anion itself may contribute antimicrobial activity (Juven, 1976; Huhtanen and Feinberg, 1980; Huhtanen et al., 1981, 1983). Specific effects, however, vary with substrates, microorganisms, and types of acids (Restaino et al., 1981, 1982; Sofos, 1989).

Combinations of sorbate with antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butyl hydroquinone (TBHQ), and propyl gallate (PG), had increased antimicrobial activity compared with individual components (Klindworth et al., 1979; Davidson

et al., 1981; Lahellec et al., 1981; Morad et al., 1982; Poerschke and Cunningham, 1985). Variations existed, however, with types of microorganisms, antioxidants, and substrates. These combinations offer the advantage of simultaneous inhibition of microbial growth and development of rancidity. Potassium sorbate was found more inhibitory against yeasts than hydroxycinnamic acid (Stead, 1995).

Several studies have also indicated increased antimicrobial effects when sorbate was combined with various phosphates (Ivey and Robach, 1978; Robach, 1979b; Seward et al., 1982; Nelson et al., 1983; Wagner and Busta, 1983; Sofos, 1986a; Thomas and Wagner, 1987; Mendonca et al., 1989). Combinations of sorbate with benzoate or propionate may be used to expand the range of micro- organisms inhibited with reduced concentrations of each preservative.

The synergistic effect observed when vanillin and potassium sorbate were used in combination could be considered for use to reduce the amounts needed to inhibit mold growth (Matamoras- León et al., 1999). Penicillium glabrum was considered as the most resistant Penicillium mold tested and was found to be inhibited by the combination of 500 ppm of vanillin and 300 ppm of potassium sorbate for at least 1 month at 25°C, pH 3.5, and aw 0.98; these concentrations of

chemicals represented a greater than 50% reduction of the amounts needed to suppress growth when used individually (Matamoras-León et al., 1999). In addition, mixtures of sorbate with various antibiotics have demonstrated increased antimicrobial activity (Schmidt, 1960; Amano et al., 1968; Miller and Brown, 1984; Gourama and Bullerman, 1988). Interactions and increased antimicrobial effects have also been observed in combinations of sorbates with propionate, ascorbate, certain amino acids, fatty acids, sucrose fatty acid esters, sulfur dioxide, propylene glycol, glucose oxidase, and other compounds (Ferguson and Powrie, 1957; Ough and Ingraham, 1960; Robach et al., 1981; Kabara, 1984; Kadam et al., 1985; Marshall and Bullerman, 1986; Bell and De Lacy, 1987; Tellez- Giron et al., 1988).

Numerous publications and patents describe interactions of sorbate with many other factors, as well as multifactorial interactions (Sofos, 1989; Thomas et al., 1993; Guerrrero et al., 1994; Deak and Beuchat, 1994). The microbial stability of shelf-stable banana puree was enhanced through inhibition of inoculated osmophilic and nonosmophilic yeasts, molds, Bacillus coagulans, Clostridium pasteurianum, and Clostridium butyricum by adjustment of aw to 0.97 and pH to 3.4

and addition of 250 ppm ascorbic acid, 100 ppm potassium sorbate, 400 ppm sodium bisulfite, and mild heat (Guerrero et al., 1994). The effect of temperature, pH, sodium chloride concentration, and potassium sorbate was evaluated against growth of three foodborne bacterial pathogens (Bacil- lus cereus, verocytotoxigenic Escherichia coli, and S. aureus) using gradient gel plates (Thomas et al., 1993). Sorbate was completely effective against E. coli at all temperature/pH/NaCl combi- nations and was the most effective preservative tested against B. cereus. Increase in the acidity and/or the NaCl concentration improved the effect of all the preservatives, except nitrite when used against S. aureus. At <25°C, sorbate was more effective than benzoate against S. aureus when used with higher concentrations of NaCl (Thomas et al., 1993). As indicated, Z. bailii is highly resistant to individual inhibitory factors, including sorbate. A study found that growth was not inhibited at pH 3.8, in a medium containing 0.06% potassium sorbate, at aw of 0.93, or at 10°C (Deak and

Beuchat, 1994). However, interactions between two or more of these factors resulted in marked inhibition with the inhibitory effect of potassium sorbate occurring at the highest concentration (0.06%) combined with aw values lower than 0.95 (Deak and Beuchat, 1994). The ability of

C. botulinum nonproteolytic type B to grow in the presence of sorbic acid was at least as great at 20°C as at 30°C (Lund et al., 1990). After 14 days at 30°C in the presence of 280 mg undissociated sorbic acid/L, the log probability of growth of a single vegetative cell is approximately –4. At temperatures of 12°C and below, sorbic acid resulted in marked inhibition; the log probability of growth of a single bacterium at 12°C after 60 d at pH 5.5 in the absence acid being 0 and –1 under the same conditions but in the presence of a calculated undissociated sorbic acid concentration of