Comparing Nonsynergistic Gamma Models with Interaction Models To Predict Growth of Emetic Bacillus cereus when Using Combinations of pH and Individual Undissociated Acids as Growth-Limiting Factors

ABSTRACT A combination of multiple hurdles to limit microbial growth is frequently applied in foods to achieve an overall level of protection. Quantification of hurdle technology aims at identifying synergistic or multiplicative effects and is still being developed. The gamma hypothesis states that inhibitory environmental factors aiming at limiting microbial growth rates combine in a multiplicative manner rather than synergistically. Its validity was tested here with respect to the use of pH and various concentrations of undissociated acids, i.e., acetic, lactic, propionic, and formic acids, to control growth of Bacillus cereus in brain heart infusion broth. The key growth parameter considered was the maximum specific growth rate, μmax, as observed by determination of optical density. A variety of models from the literature describing the effects of various pH values and undissociated acid concentrations on μmax were fitted to experimental data sets and compared based on a predefined set of selection criteria, and the best models were selected. The cardinal model developed by Rosso (for pH dependency) and the model developed by Luong (for undissociated acid) were found to provide the best fit and were combined in a gamma model with good predictive performance. The introduction of synergy factors into the models was not able to improve the quality of the prediction. On the contrary, inclusion of synergy factors led to an overestimation of the growth boundary, with the inherent possibility of leading to underestimation of the risk under the conditions tested in this research.

Possible Involvement of Undissociated Acid Molecules in the Acid Response of the Chorda Tympani Nerve of the Rat

To test whether undissociated acid is capable of exciting the chorda tympani nerves in rats, we have used buffered acid solutions as taste stimuli. These solutions were prepared by adding alkali to weak acids, such as acetic acid, so that the proportion of undissociated and dissociated acids was varied whereas keeping the total acid concentration constant. When acetic acid solutions, adjusted to wide ranges of pH by NaOH, were applied to the tongue, the response magnitude of the chorda tympani nerves was not varied systematically with pH changes. However, if the sodium effect was eliminated by amiloride or replacement of cation by potassium or Tris[hydroxymethyl]aminomethane; NH2C(CH2OH)3 (Tris-base), the chorda tympani response was reduced systematically as pH increased. Similar results were obtained with citric acid and ascorbic acid. This pH-dependent change in taste nerve response to acid cannot be solely attributed to the proton gradient because the response magnitude induced by hydrogen itself, which was estimated from responses to strong acids, was much smaller than that by equi-pH acetic acid (∼85%). Thus we cannot explain the pH-dependent responses of the chorda tympani nerves to weak acids unless effects of undissociated acid molecules are postulated. It is therefore concluded that undissociated acids in weak acid solutions can be a stimulant to taste receptor cells.

Utilization and Transport of Acetic Acid in Dekkera anomala and Their Implications on the Survival of the Yeast in Acidic Environments

The yeast Dekkera anomala IGC 5153 exhibited a restricted ability to use weak acids as the only carbon and energy sources. Of the monocarboxylic, dicarboxylic, and tricarboxylic acids tested, only acetic acid was used in such a way. The cells were able to grow at acetic acid concentrations of 0.1 to 3% (vol/vol) over a pH range of 3.5 to 5.5, and the specific growth rates decreased exponentially with the increase of the undissociated acetic acid concentration in the culture medium. Transport assays carried out in cells that exhibited higher specific growth rates showed the presence of an acetate-proton symport associated with a simple diffusion component of the undissociated acetic acid, the weight of the latter increasing with the undissociated acid concentration in the culture media. The acetate carrier was shared by propionic, formic, and sorbic acids and was inducible and repressed by glucose and concentrations of undissociated acetic acid in the culture medium above 0.3% (vol/vol). In undissociated acetic acid repression conditions, the lowest values for the yeast specific growth rates were obtained, and the simple diffusion of the undissociated acid was the only mechanism involved in the acetic acid uptake by the cells. The results will be discussed in terms of the high tolerance of D. anomala to the acidic stress conditions present in wine.

Effects of Ethanol and Other Alkanols on Transport of Acetic Acid in Saccharomyces cerevisiae

ABSTRACT In glucose-grown cells of Saccharomyces cerevisiae IGC 4072, acetic acid enters only by simple diffusion of the undissociated acid. In these cells, ethanol and other alkanols enhanced the passive influx of labelled acetic acid. The influx of the acid followed first-order kinetics with a rate constant that increased exponentially with the alcohol concentration, and an exponential enhancement constant for each alkanol was estimated. The intracellular concentration of labelled acetic acid was also enhanced by alkanols, and the effect increased exponentially with alcohol concentration. Acetic acid is transported across the plasma membrane of acetic acid-, lactic acid-, and ethanol-grown cells by acetate-proton symports. We found that in these cells ethanol and butanol inhibited the transport of labelled acetic acid in a noncompetitive way; the maximum transport velocity decreased with alcohol concentration, while the affinity of the system for acetate was not significantly affected by the alcohol. Semilog plots of V max versus alcohol concentration yielded straight lines with negative slopes from which estimates of the inhibition constant for each alkanol could be obtained. The intracellular concentration of labelled acid was significantly reduced in the presence of ethanol or butanol, and the effect increased with the alcohol concentration. We postulate that the absence of an operational carrier for acetate in glucose-grown cells of S. cerevisiae, combined with the relatively high permeability of the plasma membrane for the undissociated acid and the inability of the organism to metabolize acetic acid, could be one of the reasons why this species exhibits low tolerance to acidic environments containing ethanol.

Some Physical Properties of Aqueous L-Ascorbic Acid Solutions

The density, viscosity and refractive index of aqueous L-ascorbic acid solutions, in the concentration range 0.01-1.20 M, have been measured at 25�C. The partial molal volume of the undissociated acid at infinite dilution has been found to be 105.74 cm3 mol-1. Equations which describe the density and relative viscosity of the solutions have also been given.

LOSS OF VIABILITY BY STAPHYLOCOCCUS AUREUS IN ACIDIFIED MEDIA

Survival of Staphylococcus aureus (108 cells per milliliter) after 24 hr of incubation at 37 C in Trypticase Soy broth acidified with acetic, citric, hydrochloric, lactic, and phosphoric acids was investigated. When the organism was exposed to the medium adjusted with hydrochloric acid to pH values of 5.2–3.6, 90–99.99% of the cells were inactivated. Acetic, lactic, and phosphoric acids were more active against S. aureus than was hydrochloric, whereas citric was equivalent to hydrochloric. Mixtures of lactic and hydrochloric acids inactivated more cells than did either acid alone but mixtures of other acids with hydrochloric, while superior to hydrochloric acid itself, offered no apparent advantage over use of single acids. The undissociated acid molecule was responsible for enhanced inactivation of cells by partially dissociated acids since anions of these acids had no effect on cell survival. Cells were more susceptible to inactivation by hydrogens ions at high incubation temperatures (45 C) and when the number of bacteria was low. Cells of S. aureus were most sensitive to the effects of hydrogen ions between the 12th and 24th hr during a 120-hr incubation.