Sodium Benzoate Inhibits Growth of Or Inactivates Listeria Monocytogenes
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525 Journal of Food Protection, Vol. 51, No. 7, Pages 525-530 (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 53706 (Received for publication February 26, 1988) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/51/7/525/1658063/0362-028x-51_7_525.pdf by guest on 28 September 2021 ABSTRACT Recognition of Listeria monocytogenes as an agent of foodborne disease has increased in the last few years. The The ability of Listeria monocytogenes to grow or survive was pathogen'can cause abortion in pregnant women as well as determined using tryptose broth at pH 5.6 or 5.0, supplemented meningitis in newborn infants and immunocompromised with 0, 0.05. 0.1, 0.15. 0.2. 0.25 or 0.3% sodium benzoate, and adults (17,26,31). Also, this bacterium is pathogenic for incubated at 4,13,21 or 35°C. The bacterium grew in benzoate- animals and can cause abortion (33) and mastitis (15). L. free 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. monocytogenes can be transmitted from infected animals to 103/ml) was inactivated by 0.2, 0.25 and 0.3% sodium benzoate. humans (16,21,24,25) and also can be transmitted to hu Other concentrations of benzoate permitted slight growth during mans through consumption of some foods of animal origin. the first 36 d of incubation followed by a decrease in populations Recent outbreaks of listeriosis have led to the implication of the pathogen. At pH 5.0 and 4°C, from 0.15 to 0.3% benzoate of a variety of foods (1,13,19,30), especially dairy products, completely inactivated the pathogen in 24 to 30 d, whereas the as a source of this pathogen (2,3,5,13,23). During 1985 other concentrations caused a gradual decrease in the population listeriosis claimed the lives of at least ca. 100 people (1). during the 66-d incubation period. At 13°C and pH 5.6, L. Nearly all of the victims were newborn infants whose monocytogenes grew (more at lower than higher concentrations of mothers had eaten a certain variety of soft Mexican-style benzoate) in the presence of all concentrations of benzoate except 0.25 or 0.3%, which prohibited growth throughout a 264-h incu cheese produced in California (28). bation period. Reducing the pH to 5.0 minimized growth at the L. monocytogenes is found throughout the environment two low concentrations of benzoate and caused slight decreases in (5). It's psychrotrophic nature has been firmly documented population at the other concentrations of benzoate. At 21 and (10,18,22). It not only survives but also grows at tempera 35°C and pH 5.6, appreciable growth of L. monocytogenes oc tures as low as 3°C in tryptose phosphate broth (20), 4°C in curred in the presence of 0.2% or less sodium benzoate, whereas milk (10,28) and 0°C in sterile meat after 16-20 d (22). higher concentrations were inhibitory, permitting little if any The inhibitory effect of sodium benzoate on some bac growth by the pathogen. Reducing the pH to 5.0 allowed limited teria that cause foodborne illness or food spoilage has been growth of the pathogen at 21 and 35°C when the medium con studied by using different laboratory media as well as food tained 0.05 or 0.1% sodium benzoate. Higher concentrations caused either complete inhibition or inhibition plus partial or products under various conditions (6,8,9,29). For sodium complete inactivation of the pathogen during incubations of 117 benzoate to be effective the pH of the substrate must be low h at 21<>C or 78 h at 35°C. 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 Benzoic acid has long been used as an antimicrobial preservatives; consequently this work was done to deter additive for foods. The sodium salt is preferred because of mine the effect of different concentrations of sodium ben the low aqueous solubility of the free acid. In use, the salt zoate at two pH values and four temperatures, on growth is converted to the acid, the active form (14). Sodium and survival of L. monocytogenes. benzoate is generally considered to be most active against yeasts and bacteria, and less active against molds (7). MATERIALS AND METHODS Levels of usage up to 0.2 and 0.3% are common, and 1.25% is the maximum permitted for liquid egg yolk in Organism some countries (7). Benzoic acid is used primarily in foods We chose L. monocytogenes strain V7 (serotype I) because it and beverages with pH values at or below 4.0 to 4.5 (11). was originally isolated from food (milk). The stock culture, ob This preservative causes no deleterious effects in humans tained from the Food Microbiology Laboratory Culture Collec when consumed in small amounts (7). It occurs naturally in tion, Department of Food Science, University of Wisconsin- cranberries, prunes, cinnamon and cloves (7, 27). Madison, was maintained through bimonthly transfers on tryptose JOURNAL OF FOOD PROTECTION, VOL. 51, JULY 1988 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 102 to 2 X 103. 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 T Downloaded from http://meridian.allenpress.com/jfp/article-pdf/51/7/525/1658063/0362-028x-51_7_525.pdf by guest on 28 September 2021 was accompanied by an increase in pH of the medium. After 40 sterilizing the medium and adding the preservative, the final pH Days 0.0555 -0- 0.1555 -Ch 0.255? was fixed exactly using 0.1 N HC1 or 0.1 N NaOH (if required) -EJ- Control and a pH meter (model 601A Digital Ionalyzer, Orion Research 0.1053 -•- 0.2058 -*- 0.305? Incorporated, Cambridge, MA). The NCI and NaOH solutions were sterilized by filtration. Tryptose broth was prepared in Figure 1. Behavior of L. monocytogenes in the presence of vari bottles in quantities of 96, 96.5, 97, 97.5, 98 and 98.5 ml to give ous concentrations of sodium benzoate at pH 5.6 and 4°C. a final quantity of 100 ml after adding the preservative and inocu lum. All trials were done in duplicate. 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 concentra tions 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 por tions from each sample were then appropriately diluted (if neces sary) 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 -G- Control 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 Figure 2. Behavior ofL. monocytogenes in the presence of vari curves were then constructed for each temperature and pH. ous concentrations of sodium benzoate at pH 5.0 and 4°C. Generation time Generation time was calculated with the following formula: RESULTS Generation time = l/Log (P2)-Log (Pl)/ 10 10 Behavior of L. monocytogenes at 4°C 0.301OVT,). Where, T ] At 4°C and pH 5.6 growth of L. monocytogenes began = time of PL T2 = time of P2, PI •• CFU/ml at T, after approximately 6 d of incubation and reached the and P2 CFU/ml at T,. 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 Determination of sodium benzoate inactivated inoculated cells. Complete inactivation, as de To determine if the amount of sodium benzoate changed in termined by our methods, occurred after 60 d when concen samples during the long incubation at 4°C, amounts of sodium trations of 0.2, 0.25 or 0.3% sodium benzoate were used.