The inhibitory effects of potassium sorbate on the bioenergetics, phenylalanine uptake, protein synthesis, and certain aspects of cell regulation were examined in putrefactive anaerobe 3679. Undissociated sorbic acid appeared to act as a protonophore by lowering the intracellular pH and dissipating the proton motive force of the membrane. Sorbate inhibited the uptake of phenylalanine, decreased the rate of protein synthesis, and altered patterns of phosphorylated nucleotide accumulation, resulting in increased intracellular concentrations of GTP, ppGpp, and an unidentified compound (possibly pppGpp). The addition of a noninhibitory amount of tetracycline released the inhibition of growth by sorbate. Based on these results, we concluded that the inhibition of putrefactive anaerobe 3679 by sorbate resulted from a stringent-type regulatory response induced by the protonophoric activity of sorbic acid.

Putrefactive anaerobe 3679 (Clostridium sporogenes), a gram-positive bacterium, was examined by light and electron microscopy during normal growth and in a medium containing sorbate (50 mM, pH 6.5), hydrochloric acid (pH of medium adjusted from 7 to 5 with HCl), or nitrite (1 mM, pH 7). During the early exponential growth phase, untreated cells were filamentous and nonseptate, but became septate later and divided when the culture entered the stationary phase. Untreated short and filamentous cells had a double-layered cell wall. Sorbate-treated cells were usually filamentous and nonseptate, but with distorted shapes characterized by numerous bends and bulges. Septation, when present, resulted in minicells. The inner cell wall appeared to be thickened and the outer wall was absent in many areas. Acid-treated cells were similar to sorbate-treated cells but contained septa. Considerable cellular debris was present in the suspension. Nitrite-treated cells were also filamentous, bent, and bulged but the cell wall appeared normal. Considerable cellular debris was also present in suspensions of nitrite-treated cells. Changes in morphology are discussed in relation to possible mechanisms of cell growth regulation and the inhibitory action of sorbate, acid, and nitrite.

The effects of some common curing ingredients (sucrose, sodium chloride, and nitrite) on sorbate-induced inhibition were tested in putrefactive anaerobe (PA) 3679, a proteolytic species of Clostridium. A noninhibitory concentration (1%) of sucrose was not synergistic with the inhibitory action of potassium sorbate (30 mM; pH 6). The effect of sodium chloride depended upon the concentration used; a combination of 3% NaCl and sorbate (30 mM; pH 6) was more inhibitory than either compound alone but 1% NaCl reduced the inhibitory action of sorbate (30 mM; pH 6). Combining a noninhibitory concentration of nitrite (100 pM) and sorbate (30 mM; pH 6) was bactericidal to PA 3679. Nitrite raised the intracellular pH (pHi) and increased the protonmotive force (PMF) of untreated and sorbate-treated cells. Growth of PA 3679 was stimulated by the addition of a noninhibitory level of undissociated sorbic acid (5.5 mM; pH 7) or 0.1% glucose (5.5 mM) to a nutrient-deficient medium (0.25% trypticase [BBL]) but growth initiated more rapidly when sorbic acid was added than when the medium was supplemented with glucose. Results of this study are discussed in relation to the possible mechanisms of preservative action and suggest that certain recent changes in methods of food processing and packaging may increase the possibility of growth of clostridial organisms, including Clostridium botulinum.