Effect of pH, Acidulant, Time, and Temperature on the Growth and Survival of Listeria monocytogenes

Abstract

The effect of different acids, pH, incubation time, and incubation temperature on the growth and survival of four strains of Listeria monocytogenes in tryptic soy broth was compared. Hydrochloric acid (HC1), acetic acid (AA), lactic acid (LA), malic acid (MA), and citric acid (CA) were used to acidify tryptic soy broth to pH values 4.4, 4.6, 4.8, 5.0, and 5.2 pH. Incubation times were 1, 3, 7, 14, and 28 d at 10, 25, and 35°C. The inhibition of L. monocytogenes in the presence of high acidity appears to be a function of acid and incubation temperature. Based on equal pH values, the antimicrobial activity is AA > LA > CA > MA > HC1 at all incubation times and temperatures. When based on equal molar concentration, the activity appeared to be CA > MA > LA > AA > HC1 at 35 and 25°C, and MA > CA > AA > LA > HC1 at 10°C. Greatest antimicrobial activity occurred at 35°C. Greatest survival occurred at 10°C and greatest growth occurred at 25°C. Final pH of the medium was as low as 3.8 in HC1 at 28 d. All strains grew well at pH values lower than the minimum previously reported (5.5-5.6). The importance of L. monocytogenes as an agent of foodborne disease has become increasingly apparent (1,6). Acidity and/or pH is an important preservative factor in many foods, either alone or in combination with other microbial barriers, such as refrigerated storage. Refrigerated storage, however, is no guarantee of protection against growth of L. monocytogenes since it can grow at refrigeration temperatures (5,10). L. monocytogenes has been reported to be unable to survive and/or grow poorly in low pH environments with 5.5-5.6 being the critical cutoff point for growth (8,9,13). However, recent reports have indicated growth of L. monocytogenes at pH 5.0 in unclarified cabbage juice adjusted with lactic acid (4), pH 5.2 in cottage cheese (12), and pH 4.39 in trypticase soy, glucose, yeast extract broth, adjusted with HC1, at 30°C (7). The purpose of this study was to extend the information on the relative effects of pH, acidulant and temperature on growth of L. monocytogenes. MATERIALS AND METHODS Test organisms Four strains of L. monocytogenes (H4 and H8, environmental isolates, and 4B and 31 C, dairy isolates) were obtained from Mrs. Jenny Scott, National Food Processors Association (Eastern Research Laboratory), Washington, DC. The environmental isolates were serotype la and the dairy isolates serotype 4b. The L. monocytogenes cultures were maintained on tryptic soy agar (TSA; Difco) slants incubated for 24 h at 35°C, and held at 5°C. They were grown in tryptic soy broth (TSB; Difco) 24 h at 35°C before use. Preparation of acid solutions and test broths One molar stock solutions of acetic (AA), lactic (LA), hydrochloric (HCL), malic (MA), and citric acids (CA) were prepared. Appropriate amounts of the stock acid solutions were added to 250 ml TSB to give the desired pH as measured with a pH meter and combination pH electrode. The acidified TSB (TSBA) was sterilized by autoclaving at 121°C for 15 min. After cooling, an aliquot was taken and the pH rechecked. Molarity of the different acids at different pH levels in test broth was also calculated (14). Effect of various acids For each acid and pH tested, 9.9 ml samples of TSBA were aseptically placed in sterile test tubes (16 x 150 mm). A volume of 0.1 ml of a 24 h test culture was inoculated into each tube and the tubes incubated at 10, 25, and 35°C. Tubes were checked for visible growth after 1, 2, 7, 14, and 28 d. The pH was measured in all tubes after 28 d. Cell numbers were determined by plating on TSA plus 0.6% yeast extract (Difco), after making serial dilutions of a 0.1 ml sample of broth in 9.9 ml of 0.1 % peptone diluent. Plates showing between 30 and 300 colonies after incubation at 35°C for 72 h were counted and the numbers converted to logs. Data are presented as the log of the count reduction. RESULTS AND DISCUSSION Table 1 shows the effect of time, temperature, acidu-lant, and pH on growth of strains of L. monocytogenes.