Sodium Benzoate Inhibits Growth of or Inactivates Listeria monocytogenes

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1 525 Journal of Food Protection, Vol. 51, No. 7, Pages (July 1988) Copyright International Association of Milk, Food and Environmental Sanitarians Sodium Benzoate Inhibits Growth of or Inactivates Listeria monocytogenes MOUSTAFA A. EL-SHENAWY and ELMER H. MARTH* Department of Food Science and The Food Research Institute, University of Wisconsin-Madison, Madison, Wisconsin 5370 (Received for publication February 26, 1988) ABSTRACT The ability of Listeria monocytogenes to grow or survive was determined using tryptose broth at ph 5.6 or 5.0, supplemented with 0, , or 0.3% sodium benzoate, and incubated at 4,13,21 or 35 C. The bacterium grew in benzoatefree controls under all conditions except at 4 C and ph 5.0. At ph 5.6 and 4 C, after 60 d, L. monocytogenes (initial population ca /ml) was inactivated by 0.2, 0.25 and 0.3% sodium benzoate. Other concentrations of benzoate permitted slight growth during the first 36 d of incubation followed by a decrease in populations of the pathogen. At ph 5.0 and 4 C, from 0.15 to 0.3% benzoate completely inactivated the pathogen in 24 to 30 d, whereas the other concentrations caused a gradual decrease in the population during the 66-d incubation period. At 13 C and ph 5.6, L. monocytogenes grew (more at lower than higher concentrations of benzoate) in the presence of all concentrations of benzoate except 0.25 or 0.3%, which prohibited growth throughout a 264-h incubation period. Reducing the ph to 5.0 minimized growth at the two low concentrations of benzoate and caused slight decreases in population at the other concentrations of benzoate. At 21 and 35 C and ph 5.6, appreciable growth of L. monocytogenes occurred in the presence of 0.2% or less sodium benzoate, whereas higher concentrations were inhibitory, permitting little if any growth by the pathogen. Reducing the ph to 5.0 allowed limited growth of the pathogen at 21 and 35 C when the medium contained 0.05 or 0.1% sodium benzoate. Higher concentrations caused either complete inhibition or inhibition plus partial or complete inactivation of the pathogen during incubations of 117 h at 21<>C or 78 h at 35 C. Benzoic acid has long been used as an antimicrobial additive for foods. The sodium salt is preferred because of the low aqueous solubility of the free acid. In use, the salt is converted to the acid, the active form (14). Sodium benzoate is generally considered to be most active against yeasts and bacteria, and less active against molds (7). Levels of usage up to 0.2 and 0.3% are common, and 1.25% is the maximum permitted for liquid egg yolk in some countries (7). Benzoic acid is used primarily in foods and beverages with ph values at or below 4.0 to 4.5 (11). This preservative causes no deleterious effects in humans when consumed in small amounts (7). It occurs naturally in cranberries, prunes, cinnamon and cloves (7, 27). Recognition of Listeria monocytogenes as an agent of foodborne disease has increased in the last few years. The pathogen'can cause abortion in pregnant women as well as meningitis in newborn infants and immunocompromised adults (17,26,31). Also, this bacterium is pathogenic for animals and can cause abortion (33) and mastitis (15). L. monocytogenes can be transmitted from infected animals to humans (16,21,24,25) and also can be transmitted to humans through consumption of some foods of animal origin. Recent outbreaks of listeriosis have led to the implication of a variety of foods (1,13,19,30), especially dairy products, as a source of this pathogen (2,3,5,13,23). During 1985 listeriosis claimed the lives of at least ca. 100 people (1). Nearly all of the victims were newborn infants whose mothers had eaten a certain variety of soft Mexican-style cheese produced in California (28). L. monocytogenes is found throughout the environment (5). It's psychrotrophic nature has been firmly documented (10,18,22). It not only survives but also grows at temperatures as low as 3 C in tryptose phosphate broth (20), 4 C in milk (10,28) and 0 C in sterile meat after d (22). The inhibitory effect of sodium benzoate on some bacteria that cause foodborne illness or food spoilage has been studied by using different laboratory media as well as food products under various conditions (6,8,9,29). For sodium benzoate to be effective the ph of the substrate must be low since the undissociated acid is believed to be responsible for its antimicrobial activity. Currently, virtually no information is available on the response of L. monocytogenes to commonly used food preservatives; consequently this work was done to determine the effect of different concentrations of sodium benzoate at two ph values and four temperatures, on growth and survival of L. monocytogenes. MATERIALS AND METHODS Organism We chose L. monocytogenes strain V7 (serotype I) because it was originally isolated from food (milk). The stock culture, obtained from the Food Microbiology Laboratory Culture Collection, Department of Food Science, University of Wisconsin- Madison, was maintained through bimonthly transfers on tryptose

2 526 EL-SHENAWY AND MARTH agar (Difco Laboratories, Detroit, MI) and storage at 4 C. To prepare for an experiment, inocula from stock cultures transferred to tryptose broth (TB) (Difco) which was incubated for 24 h at 35 C under normal atmospheric conditions. Subsequently, to insure an active culture, two transfers of the cultures to new tubes of TB were made followed by incubation as just described. From the last transfer 1 ml was added to 99 ml of TB, this was repeated and 1 ml from the last bottle was inoculated into test samples so that the initial CFU/ml would be between 2.5 x 10 2 to 2 X Medium Tryptose broth, before sterilizing, was adjusted to ph 4.7 to 5.0 when an experiment was to be done at 5.0 and from 5.3 to 5.6 when an experiment was to be done at ph 5.6. The indicated values were used because addition of preservative to the broth was accompanied by an increase in ph of the medium. After sterilizing the medium and adding the preservative, the final ph was fixed exactly using 0.1 N HC1 or 0.1 N NaOH (if required) and a ph meter (model 601A Digital Ionalyzer, Orion Research Incorporated, Cambridge, MA). The NCI and NaOH solutions were sterilized by filtration. Tryptose broth was prepared in bottles in quantities of 96, 96.5, 97, 97.5, 98 and 98.5 ml to give a final quantity of 100 ml after adding the preservative and inoculum. All trials were done in duplicate. -EJ- T 40 Days Ch 0.255? -* ? Figure 1. Behavior of L. monocytogenes in the presence of various concentrations of sodium benzoate at ph 5.6 and 4 C. Preparation of preservative Concentrations of sodium benzoate used in this work were 0.05, 0.1, 0.15, 0.2, 0.25 and 0.3%. We chose these concentrations because they are in the permissible range of usage for this preservative. Sodium benzoate was prepared as a stock solution of 10%, which was sterilized by filtration. Quantities of 0.5, 1, 1.5, 2, 2.5 or 3 ml of the preservative were added to broth after autoclaving and before final adjustment of the ph. Counting techniques Cultures were agitated before sampling. Duplicate 1-ml portions from each sample were then appropriately diluted (if necessary) in freshly made saline (0.85%) solution. This was followed by duplicate surface plating of 0.1 ml of specific dilutions on tryptose agar. Sampling intervals were adjusted to observe the lag, logarithmic and stationary phases of growth in the control and treatments, when growth occurred. Plates were incubated under normal atmospheric conditions for 48 h at 35 C. Colonies were counted with the aid of a Darkfield Quebec Colony Counter (American Optical corp., Buffalo, NY). Growth or inactivation curves were then constructed for each temperature and ph. Generation time Generation time was calculated with the following formula: Generation time = l/log 10 (P2)-Log 10 (Pl)/ 0.301OVT,). Where, T ] = time of PL T 2 = time of P2, PI and P2 CFU/ml at T, CFU/ml at T,. Determination of sodium benzoate To determine if the amount of sodium benzoate changed in samples during the long incubation at 4 C, amounts of sodium benzoate in various samples were measured at the beginning and end of the experiment following the method of the AOAC (4). -G- Figure 2. Behavior ofl. monocytogenes in the presence of various concentrations of sodium benzoate at ph 5.0 and 4 C. RESULTS Behavior of L. monocytogenes at 4 C At 4 C and ph 5.6 growth of L. monocytogenes began after approximately 6 d of incubation and reached the maximum population after approximately 30 d (Fig. 1). Presence of 0.05% or more of sodium benzoate in the medium either inhibited growth or partially or completely inactivated inoculated cells. Complete inactivation, as determined by our methods, occurred after 60 d when concentrations of 0.2, 0.25 or 0.3% sodium benzoate were used. When we used 0.15% sodium benzoate, populations increased slightly during the first 36 d of incubation, but then

3 BENZOATE INHIBITS LISTERIA 527 " 8-7 " E Z> O 6 - R - -J O " 4 - -H ? D ? 0.205? -* ? 20 i i i I I ? 0.155? 0.205? A i * D ? -* ? Figure 3. Behavior ofl. monocytogenes in the presence of various concentrations of sodium benzoate at ph 5.6 and 13 C. Figure 5. Behavior ofl. monocytogenes in the presence of various concentrations of sodium benzoate at ph 5.6 and 21"C. decreased. This also occurred when the medium contained 0.1 or 0.05% sodium benzoate. The generation time for L. monocytogenes in the absence of sodium benzoate was 1.13 d. Addition of 0.05 or 0.1% sodium benzoate to the medium increased the generation time to 9.03 d. Ultimately there was a decrease in the population even at these low concentrations of the preservative. When the ph was decreased to 5.0 (Fig. 2), there was no growth of L. monocytogenes in the control until 50 d; after this period the population began to decrease. Addition of 0.1 or 0.05% sodium benzoate caused populations to decrease until the end of the experiment. The D-values resulting from concentrations of 0.3, 0.25, 0.2 and 0.15% ranged between d, whereas they were 36 d with 0.1 and 42 d with 0.05% sodium benzoate. During the incubation at 4 C sodium benzoate concentrations in cultures remained approximately constant. -Q ? 0.155? 0.205? -D- 0.25S? -* ? 300 Figure 4. Behavior ofl. monocytogenes in the presence of various concentrations of sodium benzoate at ph 5.0 and 13"C. Behavior of L. monocytogenes at 13"C L. monocytogenes started to grow in the control broth after about 12 h at 13 C and ph 5.6. After 4-5 d ( h) it had reached its maximum population, with a generation time of 4.5 h (Fig. 3). Addition of 0.05 or 0.1% sodium benzoate'to the medium delayed the onset of growth to about 48 h; generation times during the growth phase were approximately 13 and 21 h, respectively. Addition of 0.15 or 0.2% sodium benzoate permitted a slight increase in population after approximately 6 d but after 9 d the populations decreased to initial numbers. Addition of 0.25 and 0.3% benzoate permitted no growth, but there was some fluctuation in numbers during the test period. At ph 5.0 and 13 C (Fig. 4), the pathogen started to grow in the control broth after 12 h and entered the log phase after 24 h. A maximum population of ca cells/ml was attained after 7 d (168 h); the generation time during the log phase was about 8 h. Addition of 0.05% sodium benzoate to the medium did not completely prevent growth of L. monocytogenes; however, eventually the population returned to or below the initial count. Addition of 0.1 to 0.3% sodium benzoate completely inhibited growth, and caused some inactivation of cells during the extended incubation period. Behavior ofl. monocytogenes at 21"C At ph 5.6 and 21 C (Fig. 5), L. monocytogenes started growing in control broth after 6 h of incubation and reached its maximum population in ca. 45 h; the generation time during the log phase was 1.8 h. Addition of 0.05% sodium benzoate increased the lag phase to 18 h; after this the bacterium grew with a generation time of about 2 h. Addition of 0.1 or 0.15% benzoate also permitted growth but increased generation times to ca. 5.1 and 9 h, respectively.

4 528 EL-SHENAWY AND MARTH Z> LL O 05 O 120 -EJ D * D * Figure 6. Behavior o/l. monocytogenes in the presence of various concentrations of sodium benzoate at ph 5.0 and 21 C. Figure 8. Behavior ofh. monocytogenes in the presence of various concentrations of sodium benzoate at ph 5.0 and 35 C e D * Figure 7. Behavior ofl. monocytogenes in the presence of various concentrations of sodium benzoate at ph 5.6 and 35"C. Presence of 0.2% sodium benzoate allowed slight growth and a generation time of ca. 20 h. Addition of 0.25 or 0.3% sodium benzoate virtually completely inhibited growth of L. monocytogenes. Reducing the ph to 5.0 at 21 C delayed the onset of growth in the control broth to 9 h; the maximum population appeared after 54 h, and the generation time during the log phase was about 2.1 h (Fig. 6). Presence of 0.05 or 0.1% sodium benzoate allowed growth after approximately 45 h, with generation times of about 6.8 h for 0.05% and 9 h for 0.1%. A concentration of 0.15% sodium benzoate permitted slight, if any, growth after about 81 h. Addition of 0.20, 0.25 or 0.30% sodium benzoate resulted in essentially complete inhibition of growth by the pathogen. Behavior of Listeria monocytogenes at 35"C Figure 7 gives the data obtained at ph 5.6 and 35 C. Under these conditions growth of L. monocytogenes in the control was rapid with a lag phase of about 3 h, and with a generation time of about 49 min during the log phase. The maximum population (ca /ml) was achieved after ca. 27 h of incubation. Addition of 0.05% sodium benzoate delayed the onset of growth until about 9 h and the generation time during the log phase was about 77 min. Presence of 0.1% benzoate also delayed the onset of growth by about 9 h, but the generation time was extended to 135 min. Sodium benzoate at 0.15% further delayed the onset and also reduced the rate of growth from those observed when less benzoate was present. Presence of 0.2,0.25 or 0.3% sodium benzoate resulted in complete inhibition of growth. At ph 5.0 and 35 C, growth of L. monocytogenes in the control broth was inhibited only minimally. Growth began after 6 h, the maximum population was achieved after 30 h and the generation time during the log phase was 66 min (Fig. 8). Addition of 0.05% sodium benzoate retarded growth further with a generation time of ca. 6 h, and addition of 0.15% sodium benzoate allowed slight growth after 54 h of incubation,which was followed by a decrease in the population. There was no evidence of growth when 0.2, 0.25 or 0.3% sodium benzoate was present in the medium. Instead, all three of those concentrations of sodium benzoate caused inactivation of L. monocytogenes with absence of viable cells, as determined by our methods, achieved in 60 (0.3%) to 78 (0.2%) h. DISCUSSION L. monocytogenes is a very adaptable microorganism. It can grow over a wide range of temperatures (28). As seen from our results, temperature of incubation was important

5 BENZOATE INHIBITS LISTERIA 529 in determining the efficacy of sodium benzoate against this microorganism. The ph of the medium also contributed to the total environment which enhanced the effectiveness of sodium benzoate. For example, at ph 5.6 in the absence of sodium benzoate the generation time decreased from 1.13 d to 49 min as the temperature was increased from 4 to 35 C. When the ph value was decreased to 5.0, the effect was greater and ranged from complete inhibition (and hence no generation time) at 4 C to 66 min at 35 C. Complete inhibition of growth of L. monocytogenes at 4 C and ph 5.0 was confirmed by work of Shahamat et al. (32) when they inoculated the bacterium into trypticase soy broth (TSB), and adjusted the ph to 5.0 using HC1. The same workers grew the microorganisms at 4, 22 and 37 C in a basal medium (TSB) and in a basal medium plus different concentrations of salts at various ph values and different concentrations of sodium nitrite. They found that the bacteriostatic action of sodium nitrite was greatest at ph 5.0 and at 4 C; also, they suggested that the reduction in population depended on the nature of the medium, ph and storage temperature. Our data agree with that assessment. The influence of temperature and ph on the inhibitory effect of sodium benzoate also is clear from our data. It was evident that effectiveness of the various concentrations of the preservative was increased as the temperature was reduced. For example, the bacterium could not grow at the minimum concentration of sodium benzoate (0.05%) used in this study when incubated at 4 C, but it grew slightly in the presence of 0.15% when the temperature was increased to 35 C. Also, the effectiveness of sodium benzoate was increased as the ph was reduced. For example, at ph 5.0 and 13 C the bacterium could not grow in the minimum concentration (0.05%) of sodium benzoate, but there was slight growth in the presence of 0.1% when the ph was increased to 5.6. El-Kest and Marth (12) treated different strains of L. monocytogenes with 1 ppm chlorine at different temperatures (5, 25 and 30 C) and ph values. They found the smallest number of survivors when cells were exposed to chlorine at 5 C. As seen from our data, a low temperature enhanced the effect of sodium benzoate. This is important because L. monocytogenes is a psychrotrophic bacterium which can grow at refrigerator temperatures (10,18,22). In addition to its ability to grow at refrigeration temperatures, L. monocytogenes becomes more virulent when grown at a low rather than high temperature (17). Hence, enhancement of the antimicrobial effect of sodium benzoate at refrigerator temperature is important since this activity of the preservative can affect the length of time a product can be refrigerated safely. Cruess and Richert (8) noted that the retarding action of sodium benzoate on the rate of multiplication of Saccharomyces ellipsoideus was greater at ph values of 2.5 to 4.5 than at 5.0 to 9.0. They also found that the concentrations of benzoate required to prevent growth of S. ellipsoideus, Saccharomyces cervisiae, Mucor sp., two Penicillium spp., three strains of Mycoderma yeast, a lactic acid bacterium, a vinegar bacterium, Escherichia coli, Bacillus subtilis, and Bacillus sporogenes was greatly affected by the ph value of the medium. Much more benzoate was required at ph values near neutrality, e.g. ph 5 to 8, than at those in the moderately acid range of 2.5 to 4.5. Although our data provide information on the efficacy of sodium benzoate against L. monocytogenes, they do not permit prediction of concentrations required commercially for preservation of all foods because there are many environmental conditions which occur in different products that were not considered in these experiments. Examples include water activity, atmosphere, initial microbial load, type of microbial flora, and certain food components. All of them might influence the antimicrobial activity of the preservative. Nevertheless, our data clearly indicate that L. monocytogenes is susceptible to the action of benzoic acid. Thus our observations can serve as a basis for choosing this preservative for evaluation when its use to control L. monocytogenes is appropriate in a product. ACKNOWLEDGMENTS Research supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison; the American Mideast Educational and Training Services through a Peace Fellowship to Moustafa A. El-Shenawy; and by the Chr. Hansen's Laboratory, Inc., Milwaukee, WI. We thank Dr. R. L. Sellars for helpful suggestions regarding these experiments. REFERENCES 1. Anonymous Listeriosis outbreak associated with Mexican-style cheese - California. Morbid. Mortal. Weekly report 34: Anonymous FDA finds Listeria in Brie cheese from Frenchcertified plant. Food Chem. News. February 17, Anonymous Brie cheese recalls extended by General Foods, U.S. Importer. Food Chem. News. March 3, A.O.A.C Official methods of the Association of Official Analytical Chemists. 12th ed. Assoc. Off. Anal. Chem., Washington, D.C. pp Barza, M Listeriosis and milk. New Engl. I. Med. 312: Bowen, J. F., C. C. Starchan, and C. W. Davis Effect of preservatives on fermentation spoilage in pickled cherries. 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