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Sorbic acid is active against yeasts and molds, as well as against many bacteria (Sofos, 2000).

Extensive research during the 1950s demonstrated the impressive effectiveness of sorbates against yeasts and molds and resulted in the extensive use of the compounds as fungistatic agents in many foods. Effective antimicrobial concentrations of sorbates in most foods are in the range of 0.02%

to 0.30%.

The inhibitory action of sorbate against yeasts was first documented in the 1950s in fermented vegetable products. The effectiveness of sorbates against yeasts has been documented by numerous studies (Emard and Vaughn, 1952; Ferguson and Powrie, 1957; Geminder, 1959; Pederson et al., 1961; Huang and Armstrong, 1970; El Halouat et al., 1998; Bracey et al., 1998; Piper et al., 1998).

Yeasts inhibited by sorbates include species of the genera Brettanomyces, Candida, Cryptococccus, Debaryomyces, Endomycopsis, Hansenula, Kloeckera, Pichia, Rhodotorula, Saccharomyces, Sporobolomyces, Torulaspora, Torulopsis, and Zygosaccharomyces (Sofos, 1989). In addition to their effectiveness in fermented vegetables, sorbates inhibit yeasts in fruit juices, wines, cottage cheese, dried fruits, and meat and fish products. Use of sorbates for inhibition of yeasts is especially important in low-pH and/or intermediate water activity (a_w) products, such as carbonated beverages, salad dressings, syrups, tomato products, jams, candy, jellies, and chocolate syrup (Restaino et al., 1982; Liewen and Marth, 1985a). Although, Zygosaccharomyces rouxii is considered resistant to sorbate treatments, use of a hurdle approach resulted in a synergistic effect (El Halouat and Debevere, 1996). Hurdles used in combination included a_w, CO₂ atmospheres, and sorbic acid.

Under an 80% CO₂ atmosphere, the inhibitory amounts of sorbic acid were reduced by 40% to 50%. Under this atmosphere and at a_w values in the studied range (0.80 to 0.90), potassium sorbate used at 150 ppm resulted in final counts, after 21 days, lower than the inoculum level (10³ CFU/g), whereas no growth was observed when the preservative concentration was increased to 220 ppm (El Halouat and Debevere, 1996).

Numerous studies have also documented the effectiveness of sorbates against molds (Emard and Vaughn, 1952; Deuel et al., 1954a,b; Melnick and Luckmann, 1954a,b; Melnick et al., 1954a,b;

Smith and Rollin, 1954a,b; Huang and Armstrong, 1970; Baldock et al., 1979; Kaul et al., 1979;

Kivanç, 1992; Garza et al., 1993; Skirdal and Eklund, 1993; Aly, 1996; Fan and Chen, 1999;

Matamoras-León, 1999). Mold species inhibited by sorbates belong to the genera Alternaria, Ascochyta, Ascosphaera, Aspergillus, Botrytis, Cephalosporium, Chaetomium, Cladosporium, Col-letotrichum, Cunninghamella, Curvularia, Fusarium, Geotrichum, Gliocladium, Helminthospo-rium, HeterospoHelminthospo-rium, Humicola, Monilia, Mucor, Penicillium, Phoma, Pepularia, Pestalotiopsis, Pullularia, Rhizoctonia, Rhizopus, Rosellinia, Sporotrichum, Trichoderma, Truncatella, Ulocla-dium, and others. A major application of sorbates in food products is their use for inhibition of molds in cheeses (Chichester and Tanner, 1972). Sorbates also inhibit molds in butter, sausages, fruits and juices, cakes, grains, bread, and smoked fish (Liewen and Marth, 1985a; Sofos, 1989).

Sorbates inhibit the formation of mycotoxins by various molds in culture media and in foods (Bullerman, 1983, 1984, 1985; Liewen and Marth, 1983, 1984; Roland and Beuchat, 1984; Lennox and McElory, 1984; Bhattacharya and Majumdar, 1984; Tsai et al., 1984; Tong and Draughon, 1985; Marshall and Bullerman, 1986; Gourama and Bullerman, 1988). However, neither sorbate nor propionate, applied at 10 mg/ml, were able to depress mycelial weight and aflatoxin production by Aspergillus flavus or Aspergillus parasiticus cultured in yeast extract-sucrose (YES) broth (Fan

Sorbic Acid and Sorbates 55 and Chen, 1999). In fact, under certain conditions, subinhibitory levels of sorbate may simulate the production of mycotoxins (Bullerman and Olivigni, 1974; Gareis et al., 1984; Liewen and Marth, 1985a,b; Rusul and Marth, 1987; Monnet et al., 1988; Sanchis et al., 1988). Stimulation of mycotoxin formation by low levels of sorbate depends on species and strains of molds, storage temperature, and other factors (Sofos, 1989).

Bacterial species inhibited by sorbate belong to the genera Acetobacter, Achromobacter, Acine-tobacter, Enterobacter, Aeromonas, Alcaligenes, Alteromonas, Arthrobacter, Bacillus, Campylo-bacter, Clostridium, Escherichia, Klebsiella, Lactobacillus, Micrococcus, Moraxella, Mycobacte-rium, Pediococcus, Proteus, Pseudomonas, Salmonella, Serratia, Staphylococcus, Vibrio, Yersinia, and others (El-Shenawy and Marth, 1988; Zhao et al., 1993; Kouassi and Shelef, 1995a,b; Sofos, 2000; Koodie and Dhople, 2001). Depending on pH and concentration, sorbate inhibited or inac-tivated Listeria monocytogenes in a broth substrate (El-Shenawy and Marth, 1988) and in a cold-pack cheese food (Ryser and Marth, 1988). Potassium sorbate sensitized cells of L. monocytogenes and Zygosaccharomyces bailii to inactivation by high hydrostatic pressure (Mackey et al., 1995;

Palou et al., 1997). In other studies (Kouassi and Shelef, 1995a,b), although sorbate did not affect growth of L. monocytogenes in broth, it suppressed cysteine activation of listeriolysin. It was concluded that combinations of sorbate with propionate or lactate, which inhibited growth, could extend shelf life and increase safety (Kouassi and Shelef, 1995a,b; Sofos, 2000). At the relatively high level of 1%, potassium sorbate slightly decreased L. monocytogenes presence in two commer-cial cheese brines and thus could be used as an antilisterial agent in commercommer-cial brines, but the cost effectiveness of adding high levels (1%) of substrate is questionable, and their long-term stability in the high-salt and low pH environment of brines is not well documented (Larson et al., 1999). Staphylococcus aureus producing staphylococcal thermonuclease (TNase) retained its full activity and was not inhibited even after exposure to chilling and refrigeration temperatures when sorbic acid was applied at 0.04% to 0.5% (Kumar et al., 2000). A hydroxypropyl methylcellulose (HPMC) coating significantly reduced the number of viable Salmonella Montevideo cells on the surface of tomatoes; however, the addition of sorbic acid (0.2% to 0.4%) to HPMC did not substantially enhance bactericidal activity (Zhuang et al., 1996). The combination of 0.1% potas-sium sorbate and 0.1% sodium benzoate substantially decreased Escherichia coli O157:H7 con-tamination and suppressed the growth of yeasts and molds, which could have a protective effect on E. coli O157:H7, increasing its survival, and result in spoilage of the product (Zhao et al., 1993).

Addition of 0.05% sorbic acid was found to inhibit growth of E. coli O157:H7 in apple cider (Koodie and Dhople, 2001). However, it has also been shown that potassium sorbate or sodium benzoate did not affect survival of E. coli O157:H7 during storage of apple cider (Miller and Kaspar, 1994). Certain bacterial strains are not inhibited by sorbate, however, and some may even metabolize the compound (Sofos, 1989). Overall, however, sorbates can inhibit positive and Gram-negative, catalase-positive and catalase-Gram-negative, aerobic and anaerobic, and mesophilic and psy-chrotrophic microorganisms, as well as spoilage and pathogenic bacteria. Inhibition of bacteria by sorbate appears to cause an extension of the lag phase, with a lesser influence on rate and extent of growth (Larocco and Martin, 1981; Chung and Lee, 1981, 1982; Greer, 1982; Zamora and Zaritzky, 1987a,b; Tsay and Chou, 1989). The effect of sorbate on spore-forming bacteria may be exerted on spore germination, outgrowth, and/or vegetative cell division (Sofos et al., 1979a–d, 1980a, 1986; Smoot and Pierson, 1981; Seward et al., 1982; Blocher and Busta, 1985; Lund et al., 1987). Overall, however, sorbic acid is considered as a more effective inhibitor of yeasts and molds than bacteria (Skirdal and Eklund, 1993; Sofos, 1989).

SELECTIVE ACTION

Microbial inhibition by sorbate is variable and depends on species, strains, composition of substrate, pH, a_w, 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, SacZygosac-charomyces, 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 a_w 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, a_w, 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 D_50°C value of E. coli O157:H7 from 36 to 5.2 min, about a 7-fold increase in lethality (Splittstoesser et al., 1995). Sorbic acid (0.1%) reduced the D_50°C of E. coli O157:H7 during storage

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 (pK_a = 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 a_w 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 a_w 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 thermothermo-tolerance

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 thermothermo-tolerance