Organic Acids And Destruction Of Pathogens

Organic acid preservatives have widespread application for preventing food spoilage and contribute to the manufacture of safe food products [87-89]. The survival or death of pathogenic bacteria in acid and acidified foods has been investigated in a variety of products, including apple cider [68,90-93], mayonnaise, dressings and condiments [76,84,94,95], and fermented meats [96-98]. The mechanism of action of organic acids is commonly attributed to acidification of the cytoplasm of target cells, but also to intracellular accumulation of anions [99]. The protonated form of weak acid preservatives may diffuse across microbial cell membranes and then dissociate in the cell cytoplasm, releasing protons and anions because the intracellular pH must be maintained at a higher value than the external environment. Internal acid anion concentrations may correlate with the cessation of growth. Goncalves et al. [100] found that the specific growth rate of L. rhamnosus approached zero at approximately 4 molar lactate (anion), with pH values between 5.0 and 6.8. In vegetable fermentations, L. plantarum was found to tolerate a lower internal pH than other LAB and, therefore, would have lower acid anion concentrations.

Data on the relative effects of various organic acids and preservatives on the inhibition of microbial pathogens are often conflicting in the scientific literature. For example, Young and Foegeding [101] showed that with equal initial pH values in brain-heart infusion broth ranging from 4.7 to 6.0 and on an equimolar basis, the order of effectiveness in inhibiting the growth of L. monocytogenes for three weak organic acids was acetic > lactic > citric. However, when based on initial undissociated acid concentrations, the order was reversed. Ostling and Lindgren [102] determined MIC values for the inhibition of L. monocytogenes by lactic, acetic, and formic acids. They found lactic acid was the most inhibitory over a range of pH values from 4.2 to 5.4, with an MIC value of less than 4 mM (protonated acid) for aerobic growth and less than 1 mM for anaerobic growth. They used cells grown in glucose-containing nutrient broth and reported MIC values for the protonated acid as no growth for 5 days. Similar MIC values for the inhibition of growth of Listeria innocua were reported as 217 mM sodium lactate at pH 5.5, corresponding to about 5mM protonated lactic acid [103], and 4.7 mM protonated lactic acid in another study [7]. Buchanan and Edelson [104] looked at the effects of a variety of organic acids on E. coli O157:H7 at a fixed concentration of 0.5% and pH 3.0. They examined the effects of citric, malic, lactic, and acetic acids on the viability of this organism; variables included growth phase and the presence or absence of glucose in the growth medium. The ability of the cells to survive when held in an acid solution varied in a strain-dependent manner. For nine strains, lactic acid was the most effective at reducing the viable cell population, and HCl was the least effective [104]. This study clearly demonstrated that strain-to-strain variability, as well as growth conditions (induction of acid resistance by growth in the presence of glucose), must be considered in studies of the effects of weak acids and low pH on E. coli.

The effect of acetate on E. coli O157:H7 was investigated by Diez-Gonzalez and Russell [70,105]. They investigated intracellular pH, acetate anion accumulation, glucose consumption rates, and intracellular potassium concentrations. They showed that E. coli O157:H7 cells could divide in the presence of about twice as much intracellular acetate anion (80 vs. 160 mM) as E. coli K12. In cells grown at a constant pH of 5.9, E. coli O157:H7 lowered its internal pH to close to 6.0 and accumulated significantly less anion when compared to E. coli K12, which kept a constant internal pH of 7. To test the theory that acetate acted as an uncoupler (i.e., ferrying protons across the E. coli cell membrane), Diez-Gonzales and Russell [105] compared the effects of acetate and the uncoupler carbonylcyanide-m-chlorophenylhydrazone (CCCP). They found that the effects of acetate and CCCP differed, specifically in reference to intracellular ATP concentrations of E. coli O157:H7. Acetate had very little or no effect on intracellular ATP, even at concentrations greater than 200mM, while about 10mM CCCP reduced intracellular ATP concentrations by about 50%. These and similar experiments showed that acetate was having effects other than simply acting as an uncoupler on E. coli O157:H7. It was also apparent from these studies that E. coli O175:H7 and E. coli K12 regulate internal pH differently.

14.5.1 Specific Effects of Acids

A complicating factor in the study of acid inhibition of microorganisms is that protonated acids and pH (which are interdependent variables linked by the Henderson-Hasselbalch equation for common conditions) may both independently inhibit growth [106], or they may interact. Tienungoon et al. [107] modeled the probability of growth of L. monocytogenes using a logistic regression procedure with a function relating specific growth rate to temperature, water activity, pH, lactic acid, and lactate ion concentrations. They found that their equation accurately predicted conditions allowing growth using their own laboratory data, as well as examples from the literature [107]. They presented no data, however, on the growth/no growth interface for L. monocytogenes, based on protonated acid and pH; they cited a lack of independent data sets available in the literature.

To address the safety concerns of the FDA and the acidified foods industry, Breidt et al. [79] investigated the specific effects of organic acids independent of pH. This study was made possible by using gluconic acid as a noninhibitory low pH buffer. While gluconic acid has been investigated for use as an antimicrobial agent in meats [108,109], it has not proven to be as effective as acetic or lactic acid. The antimicrobial effects of gluconic acid solutions were found to be primarily due to pH rather than to specific effects of the acid itself [79]. No change in the log reduction time (D value) was observed over a 100-fold range of gluconic acid concentrations (Figure 14.2).

By using gluconic acid as a noninhibitory buffer, the inhibitory effects of pH alone were compared with the combined effects of pH and acetic acid, while holding ionic strength, temperature, and other variables constant [79]. As expected, survival of E. coli O157:H7 was reduced with the addition of acetic acid at concentrations typically found in acidified foods, and with

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