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Sorbate concentrations used in foods (<0.3%) usually inhibit microorganisms, whereas higher amounts may result in death. Consequently, when the sorbate hurdle is reduced or removed, the surviving microorganisms may resume growth and spoil the food. Sorbate inhibits cell growth and multiplication as well as germination and outgrowth of spore-forming bacteria, but the exact mechanisms of antimicrobial activity are not well defined (Freese et al., 1973; Freese and Levin, 1978; Sofos et al., 1986; Sofos, 1989).

Several studies have indicated that sorbate inhibits bacterial spore germination (Sofos et al., 1979d, 1986; Smoot and Pierson, 1981; Seward et al., 1982; Blocher and Busta, 1985). Inhibition has involved various species of bacteria in laboratory culture media and in foods and has been influenced by species, strains, pH, and sorbate concentration (Sofos, 1989). Published data have suggested that sorbate acts as a competitive and reversible inhibitor of amino acid-induced germi- nation (Smoot and Pierson, 1981). Another study reported that sorbate inhibited spores triggered to germinate or after germinant binding (Blocher and Busta, 1985). It was also postulated that sorbate probably inhibits a postgerminant binding step in the process of germination, and it was thus concluded that it inhibits spore commitment to germination, and not triggering of germination. Inhibition of the not well-defined connecting reactions of spore germination may be taking place through the interaction of sorbate with spore membranes and through increases in their fluidity (Blocher and Busta, 1985; Sofos et al., 1986; Sofos, 1989).

Several mechanisms of inhibition of metabolic function by sorbate have been proposed, and it may be possible that several of them may be functional under various conditions, including types and species of microorganisms, type of substrate, environmental conditions, and the type of food processing. Under certain conditions sorbates have changed the morphology and appearance of microbial cells (Statham and McMeekin, 1988; Ronning and Frank, 1989; Sofos, 1989). Such changes have been observed in yeast cells as dense phosphoprotein granules, irregular nuclei, increased numbers and variable sizes of mitochondria, and vacuoles. Cells of C. botulinum were long, with bulbous formation and defective division (Seward et al., 1982; Wagner and Busta, 1985a,b). Sorbate-treated cells of C. sporogenes were usually filamentous and nonseptate but with distorted shapes characterized by numerous bends and bulges. Septation, when present, resulted in minicells, and the inner cell wall appeared to be thickened; the outer cell wall was absent in many areas (Ronning and Frank, 1989). Treatment of Alteromonas putrefaciens with sorbate at pH 7.0 increased cell hydrophobicity and cell wall lysis on exposure to lysozyme, which could be overcome to some extent by addition of magnesium ions. There was also evidence of other membrane damage in sorbate-treated cells (Statham and McMeekin, 1988). Although the significance of such alter- ations is unknown, they may be the result of the incorporation of sorbate into specific cell structures and alteration of biosynthetic processes in the cell (Sofos et al., 1986).

Other proposed mechanisms of inhibition of microbial growth by sorbate include alterations in the morphology, integrity, and function of cell membranes and inhibition of transport functions and metabolic activity (Sofos, 1989). Death of microorganisms exposed to high concentrations of preservatives, such as sorbate, has been attributed to generation of holes in the cell membrane

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(Freese and Levin, 1978). Sorbate has decreased the assimilation of carbon from several substrates, including glucose, acetate, succinate, pyruvate, lactate, oxaloacetate, α-ketoglutarate, ethanol, and acetaldehyde (York and Vaughn, 1964; Harada et al., 1968; Sofos and Busta, 1981). Inhibition of cell metabolism by sorbate in these studies may have been the result of inhibition of enzymes, nutrient uptake, or various transport systems.

Sorbate inhibits the activity of several enzyme systems, which may lead to disruption of vital processes involved in transport functions, cell metabolism, growth, and replication. Enzymes inhibited by sorbate include alcohol dehydrogenase, fumarase, enolase, aspartase, catalase, malate dehydrogenase, α-ketoglutarate dehydrogenase, succinic dehydrogenase, and ficin (Melnick et al., 1954b; Whitaker, 1959; Azukas et al., 1961; Martoadiprawito and Whitaker, 1963; York and Vaughn, 1964; Troller, 1965). Some reports, however, have indicated no inhibition of enzymatic activity by sorbate (Sofos, 1989).

Sorbate is known to inhibit the in vitro activity of many enzymes, especially sulfhydryl- containing enzymes (Kouassi and Shelef, 1995a,b). Inhibition of sulfhydryl enzymes by sorbate has been attributed to binding the compound with sulfhydryl groups and decreasing the number of such active sites on the enzyme. Inhibition of yeast alcohol dehydrogenase has been attributed to formation of a covalent bond with the sulfhydryl or ZnOH group of the enzyme and the α- and/or β-carbon of sorbate (Martoadiprawito and Whitaker, 1963). It was also proposed (Whitaker, 1959) that sorbate inhibits the sulfhydryl enzymes through the formation of a thiohexenoic acid derivative (CH3-CH=CH=RSCH-CH2CO2H). Inhibition of catalase by sorbate was attributed to the formation

of sorbyl peroxides through the autoxidation of sorbic acid. These peroxides then would inactivate catalase (Troller, 1965). Another postulation has indicated that sorbate may act competitively with acetate at the site of acetyl coenzyme A (CoA) formation (Wakil and Hubscher, 1960; Harada et al., 1968; Sofos, 1992). Binding and inhibition of CoA should result in inhibition of oxygen uptake and microbial growth (Sofos, 1992).

Lipophilic acid food preservatives, such as sorbate, may interfere with substrate and electron transport mechanisms. Inhibition of substrate transport into the cell by uncoupling it from the electron transport system results in cell starvation (Deak and Novak, 1970; Sheu and Freese, 1972, 1973; Sheu et al., 1972, 1975; Freese and Levin, 1978). Sorbic acid has inhibited the uptake of glucose (Yousef and Marth, 1983) and amino acids (Hunter and Segel, 1973; Tuncan and Martin, 1985), as well as the electron transport system (Anderson and Costilow, 1963; Freese et al., 1973). Inhibition of nutrient uptake may be the result of neutralization of the proton-motive force (PMF) needed for substrate uptake, inhibition of the electron transport system, inhibition of syn- thesis or depletion of ATP, inhibition of transport enzymes, and inhibition of metabolic energy utilization by the amino acid transport systems (Sofos, 1989). This effect may be occurring through incorporation of sorbic acid, or unsaturated fatty acid, into the cell membrane, where it may cause steric disorganization of active membrane transport proteins (Sofos, 1989, 1992). The highly reproducible and characteristic thermograms of S. aureus metabolism were significantly affected by sorbic acid in a concentration-dependent manner (Sayeed and Sankaran, 1991). Both the peak heat and total heat dissipation profiles were affected by 50% of the common concentration (0.2%) for use in foods. Additionally, the suggestion that inhibition of the membrane function is the primary site of action for sorbic acid is strengthened by an observation of damage to the outer cell membranes by sorbate ions at pH 7.0 in Alteromonas putrefaciens (Sayeed and Sankaran, 1991). Thus, microcalorimetric measurement of the effects of sorbic acid on S. aureus cells has clearly shown that the acid acts in its dissociated anionic form primarily on the substrate transport systems creating a starvation condition in the cells. Secondary events such as partial inhibition of the electron transport and inhibition of the activity or synthesis of catalase enzyme may have resulted in cell death (Sayeed and Sankaran, 1991). Sorbic acid could conceivably inhibit microorganisms as a weak-acid preservative, a membrane-active compound, or a specific inhibitor of metabolism (Azukas et al., 1961). There is evidence to suggest that protein denaturation or enhanced protein turnover is a further consequence of weak acid stress resulting from the expression of many proteins,

which act as molecular chaperones (de Nobel et al., 2001). Conversely, it has been shown that sorbic acid acts at high pH where weak-acid preservatives are not expected to be active (Stratford and Anslow, 1996). Sorbic acid in its MIC releases far fewer protons than other weak-acid preser- vatives. Similar degrees of inhibition by sorbic alcohol and sorbic aldehyde suggest that sorbic acid does not act as a weak-acid preservative. Specific inhibition of metabolism is also unlikely because inhibitory activity is shared by several small carbon compounds. Correlation of sorbic acid resis- tance with ethanol tolerance and the partition coefficient strongly suggest an inhibitory role for sorbic acid as a membrane-active compound (Stratford and Anslow, 1998).

Cells of Z. rouxii cultured in media supplemented with sorbate contained higher percentages of C18:1 fatty acids than cells cultured in media without sorbate (Golden et al., 1994). Because inhibition of microbial growth by sorbate has been associated with decreased levels of ATP (Harada et al., 1968; Hunter and Segal, 1973), a proposed mechanism of ATP depletion includes hydrolysis of ATP by the primary sodium/hydrogen pump in an attempt to maintain ion balance in the cell (Przybylski and Bullerman, 1980). As the hydrogen influx exceeds the pumped efflux, a shift in charge may potentially take place and lead to a decrease in the net negative intercellular charge. This could then discharge the pH gradient required for ATP formation according to the chemostatic theory of oxidative phosphorylation (Sofos, 2000). In another study using a novel method to measure pH in growing cells, little correlation was found between reduced growth on exposure to sorbic acid and reduction of intracellular pH (pHi) (Bracey et al., 1998). In fact, growth inhibition corre-

lated with an increase in the intracellular adenosine diphosphate (ADP)/ATP ratio as a result of increased ATP consumption by the cells. This was partly attributed to the activation of protective mechanisms, such as increased proton pumping by the membrane H+_{-ATPase, which ensured that}

pHi did not decline when cells were exposed to sorbic acid (Henriques et al., 1997). Therefore, the

available evidence suggested that the inhibitory action of sorbic acid was the result of the induction of an energetically expensive protective mechanism that compensated for any disruption of pHi

homeostasis but resulted in less available energy for normal growth. Thus, it is believed that the inhibitory action of sorbic acid is the result of excessive consumption of cellular energy that occurs as a consequence of the cell eliciting a stress response that attempts to maintain pHi homeostasis

such that the available energy for growth and cell division is drastically reduced (Bracey et al., 1998).

Evidence also suggests that this energy-demanding stress could also be the result of the induction of ATP-driven plasma membrane pumps for active extrusion of weak acids from the cell (Henriques et al., 1997). Indeed, as indicated, a pump recently identified as the Pdr12 protein is induced by sorbic acid in S. cerevisiae (Piper et al., 1998). Previous studies with bacteria, however, found no decreases in ATP in the presence of sorbic acid (Sofos, 1989, 1992, 2000). Inhibition of microbial growth by sorbate may be based on neutralization of the PMF that exists across the cell membranes by lipophilic, weak-acid preservatives such as sorbic acid and lead to starvation of cells from compounds that are transported actively by the PMF (Eklund, 1980, 1983, 1985; Ronning and Frank, 1987, 1988; Salmond et al., 1984; Sofos, 1989). In lower pH environments, the difference across the cell membrane is large, and the amount of acid entering and dissociating in the cytoplasm is higher. This accumulation of hydrogen ions inside the cell acts as an inhibitor by interfering with metabolic processes and causing a dissipation of the transmembrane proton gradient, which is one of the components of the PMF (Sofos, 2000). According to this theory, undissociated sorbic acid acts as a protonophore, which decreases the intracellular pH and dissipates the PMF of the membrane that energizes transport of compounds such as amino and keto acids. Inhibition of uptake of such components is believed to induce a stringent-type regulatory response in the cells, resulting in inhibition of growth but in maintenance of cell viability (Sofos, 2000). A stringent response involves readjustments occurring in bacteria when amino acids become limiting or their specific ratios are disturbed (Sofos, 2000).

Preincubation of S. cerevisiae yeast cells in the presence of benzoate or sorbate at an extra- cellular pH value of 6.8 elicited a set of metabolic effects on sugar metabolism, which became

Sorbic Acid and Sorbates 71

apparent after the subsequent addition of glucose. These effects can be summarized as follows: (1) reduced glucose consumption; (2) inhibition of glucose- and fructose-phosphorylating activities; (3) suppression of glucose-triggered peak of hexose monophosphates; (4) substantial reduction of glucose-triggered peak of fructose 2,6-bisphosphate; and (5) block of catabolite inactivation of fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxy-kinase, but not of cytoplasmic malate dehydrogenase (Burlini et al., 1993). On the whole, this pattern resulted in prevention of glucose-induced switch of metabolism from a gluconeogenic to a glycolytic state. Thus, unlike former assumptions, intracellular acidification is not likely to mediate the bulk of metabolic effects of benzoate and sorbate because under the described working conditions intracellular pH was kept close to neutrality (Burlini et al., 1993). Overall, it is believed that neutralization of the pH difference across the cell membrane is not the only mechanism of microbial inhibition by sorbate (Stratford and Anslow, 1998; Bracey et al., 1998; Sofos, 2000). Reasons for this conclusion include the following:

1. Although dissociation of the acid inside the cell may eliminate the pH difference across the membrane, its effect on the other component of the PMF, which is the difference in electrical potential, is smaller. However, it is believed to be adequate to energize the uptake of substances needed for cell maintenance and growth.

2. Sorbic acid has been shown to inhibit microbial growth, although less efficiently, even at pH values near neutrality; in such situations, uptake of the compound based on pH difference across the cell membrane would not be adequate to explain the observed antimicrobial activity.

3. Neutralization of the PMF is probably not involved in growth inhibition in the presence of carbohydrates because they do not depend on this force for their transport (Sofos, 2000).

Thus, the mechanisms involved in inhibition of microbial metabolism and proliferation by sorbic and other similar lipophilic acid preservatives appear to be different, depending on the type of microorganism, substrates, and environmental conditions (Sofos, 1989, 1992).