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. benzoate in various samples were measured at the beginning and When we used 0.15% sodium benzoate, populations in­ end of the experiment following the method of the AOAC (4). creased slightly during the first 36 d of incubation, but then

JOURNAL OF FOOD PROTECTION, VOL. 51, JULY 1988 BENZOATE INHIBITS LISTERIA 527

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Figure 3. Behavior ofL. monocytogenes in the presence of vari­ Figure 5. Behavior ofL. monocytogenes in the presence of vari­ ous concentrations of sodium benzoate at pH 5.6 and 13°C. ous concentrations of sodium benzoate at pH 5.6 and 21"C.

tion at 4°C sodium benzoate concentrations in cultures remained approximately constant.

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 (96-120 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 popu­ lations decreased to initial numbers. Addition of 0.25 and 300 0.3% benzoate permitted no growth, but there was some Hours fluctuation in numbers during the test period. 0.055? 0.155? -D- 0.25S? At pH 5.0 and 13°C (Fig. 4), the pathogen started to -Q- Control 0.105? 0.205? -*- 0.305? grow in the control broth after 12 h and entered the log phase after 24 h. A maximum population of ca. 108 cells/ml Figure 4. Behavior ofL. monocytogenes in the presence of vari­ was attained after 7 d (168 h); the generation time during ous concentrations of sodium benzoate at pH 5.0 and 13"C. the log phase was about 8 h. Addition of 0.05% sodium benzoate to the medium did not completely prevent growth decreased. This also occurred when the medium contained of L. monocytogenes; however, eventually the population 0.1 or 0.05% sodium benzoate. The generation time for L. returned to or below the initial count. Addition of 0.1 to monocytogenes in the absence of sodium benzoate was 1.13 0.3% sodium benzoate completely inhibited growth, and d. Addition of 0.05 or 0.1% sodium benzoate to the me­ caused some inactivation of cells during the extended incu­ dium increased the generation time to 9.03 d. Ultimately bation period. there was a decrease in the population even at these low concentrations of the preservative. Behavior ofL. monocytogenes at 21"C When the pH was decreased to 5.0 (Fig. 2), there was At pH 5.6 and 21°C (Fig. 5), L. monocytogenes started no growth of L. monocytogenes in the control until 50 d; growing in control broth after 6 h of incubation and reached after this period the population began to decrease. Addition its maximum population in ca. 45 h; the generation time of 0.1 or 0.05% sodium benzoate caused populations to during the log phase was 1.8 h. Addition of 0.05% sodium decrease until the end of the experiment. The D-values benzoate increased the lag phase to 18 h; after this the resulting from concentrations of 0.3, 0.25, 0.2 and 0.15% bacterium grew with a generation time of about 2 h. Addi­ ranged between 12-14 d, whereas they were 36 d with 0.1 tion of 0.1 or 0.15% benzoate also permitted growth but and 42 d with 0.05% sodium benzoate. During the incuba­ increased generation times to ca. 5.1 and 9 h, respectively.

JOURNAL OF FOOD PROTECTION, VOL. 51, JULY 1988 528 EL-SHENAWY AND MARTH

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0.0595 0.1535 -D- 0.2558 0.055? 0.1595 -D- 0.2595 -EJ- Control -0- Control 0.1095 0.2095 -*- 0.3035 0.1095 0.2 095 -*- 0.3095

Figure 6. Behavior o/L. monocytogenes in the presence of vari­ Figure 8. Behavior ofh. monocytogenes in the presence of vari­ ous concentrations of sodium benzoate at pH 5.0 and 21°C. ous concentrations of sodium benzoate at pH 5.0 and 35°C.

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. 109/ml) was achieved after ca. 27 h of incubation. Addition of 0.05% sodium benzoate de­ layed 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. So­ dium benzoate at 0.15% further delayed the onset and also reduced the rate of growth from those observed when less 0 10 benzoate was present. Presence of 0.2,0.25 or 0.3% sodium benzoate resulted in complete inhibition of growth. 0.0595 0.1595 -D- 0.2595 At pH 5.0 and 35°C, growth of L. monocytogenes in the -e- Control 0.1095 0.2095 -*- 0.3095 control broth was inhibited only minimally. Growth began after 6 h, the maximum population was achieved after 30 h Figure 7. Behavior ofL. monocytogenes in the presence of vari­ and the generation time during the log phase was 66 min ous concentrations of sodium benzoate at pH 5.6 and 35"C. (Fig. 8). Addition of 0.05% sodium benzoate retarded growth further with a generation time of ca. 6 h, and addi­ tion of 0.15% sodium benzoate allowed slight growth after Presence of 0.2% sodium benzoate allowed slight growth 54 h of incubation,which was followed by a decrease in the and a generation time of ca. 20 h. Addition of 0.25 or 0.3% population. There was no evidence of growth when 0.2, sodium benzoate virtually completely inhibited growth of 0.25 or 0.3% sodium benzoate was present in the medium. L. monocytogenes. Instead, all three of those concentrations of sodium benzo­ Reducing the pH to 5.0 at 21 °C delayed the onset of ate caused inactivation of L. monocytogenes with absence growth in the control broth to 9 h; the maximum population of viable cells, as determined by our methods, achieved in appeared after 54 h, and the generation time during the log 60 (0.3%) to 78 (0.2%) h. 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 DISCUSSION 0.1%. A concentration of 0.15% sodium benzoate permitted slight, if any, growth after about 81 h. Addition of 0.20, L. monocytogenes is a very adaptable microorganism. It 0.25 or 0.30% sodium benzoate resulted in essentially can grow over a wide range of temperatures (28). As seen complete inhibition of growth by the pathogen. from our results, temperature of incubation was important

JOURNAL OF FOOD PROTECTION, VOL. 51, JULY 1988 BENZOATE INHIBITS LISTERIA 529 in determining the efficacy of sodium benzoate against this of the medium. Much more benzoate was required at pH microorganism. The pH of the medium also contributed to values near neutrality, e.g. pH 5 to 8, than at those in the the total environment which enhanced the effectiveness of moderately acid range of 2.5 to 4.5. sodium benzoate. For example, at pH 5.6 in the absence of Although our data provide information on the efficacy sodium benzoate the generation time decreased from 1.13 d of sodium benzoate against L. monocytogenes, they do not to 49 min as the temperature was increased from 4 to 35°C. permit prediction of concentrations required commercially When the pH value was decreased to 5.0, the effect was for preservation of all foods because there are many envi­ greater and ranged from complete inhibition (and hence no ronmental conditions which occur in different products that generation time) at 4°C to 66 min at 35°C. Complete inhi­ were not considered in these experiments. Examples in­ bition of growth of L. monocytogenes at 4°C and pH 5.0 clude water activity, atmosphere, initial microbial load, was confirmed by work of Shahamat et al. (32) when they type of microbial flora, and certain food components. All of inoculated the bacterium into trypticase soy broth (TSB), them might influence the antimicrobial activity of the pre­ and adjusted the pH to 5.0 using HC1. The same workers servative. Nevertheless, our data clearly indicate that L.

grew the microorganisms at 4, 22 and 37°C in a basal monocytogenes is susceptible to the action of benzoic acid. 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 medium (TSB) and in a basal medium plus different con­ Thus our observations can serve as a basis for choosing this centrations of salts at various pH values and different con­ preservative for evaluation when its use to control L. centrations of sodium nitrite. They found that the bacteri­ monocytogenes is appropriate in a product. ostatic action of sodium nitrite was greatest at pH 5.0 and at 4°C; also, they suggested that the reduction in population ACKNOWLEDGMENTS depended on the nature of the medium, pH and storage temperature. Our data agree with that assessment. Research supported by the College of Agricultural and Life Sciences, The influence of temperature and pH on the inhibitory University of Wisconsin-Madison; the American Mideast Educational and effect of sodium benzoate also is clear from our data. It was Training Services through a Peace Fellowship to Moustafa A. El-Sh- enawy; and by the Chr. Hansen's Laboratory, Inc., Milwaukee, WI. We evident that effectiveness of the various concentrations of thank Dr. R. L. Sellars for helpful suggestions regarding these experi­ the preservative was increased as the temperature was ments. reduced. For example, the bacterium could not grow at the minimum concentration of sodium benzoate (0.05%) used REFERENCES in this study when incubated at 4°C, but it grew slightly in the presence of 0.15% when the temperature was increased 1. Anonymous. 1985. Listeriosis outbreak associated with Mexican-style to 35°C. Also, the effectiveness of sodium benzoate was cheese - California. Morbid. Mortal. Weekly report 34:57-59. 2. Anonymous. 1986. FDA finds Listeria in Brie cheese from French- increased as the pH was reduced. For example, at pH 5.0 certified plant. Food Chem. News. February 17, 1986. and 13°C the bacterium could not grow in the minimum 3. Anonymous. 1986. Brie cheese recalls extended by General Foods, concentration (0.05%) of sodium benzoate, but there was U.S. Importer. Food Chem. News. March 3, 1986. slight growth in the presence of 0.1% when the pH was 4. A.O.A.C. 1975. Official methods of the Association of Official Ana­ increased to 5.6. El-Kest and Marth (12) treated different lytical Chemists. 12th ed. Assoc. Off. Anal. Chem., Washington, D.C. pp. 352. strains of L. monocytogenes with 1 ppm chlorine at differ­ 5. Barza, M. 1985. Listeriosis and milk. New Engl. I. Med. 312:438- ent temperatures (5, 25 and 30°C) and pH values. They 440. found the smallest number of survivors when cells were 6. Bowen, J. F., C. C. Starchan, and C. W. Davis. 1953. 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The effect of Hence, enhancement of the antimicrobial effect of sodium hydrogen-ion concentration on the toxicity of several preservatives to benzoate at refrigerator temperature is important since this microorganisms. Hilgardia 6:295-314. activity of the preservative can affect the length of time a 10. Donnelly, C. W., and E. H. Briggs. 1986. Psychrotrophic growth and product can be refrigerated safely. thermal inactivation of Listeria monocytogenes as a function of milk Cruess and Richert (8) noted that the retarding action of composition. J. Food Prot. 49:994-998, 1002. 11. El-Gazzar, F. E., and E. H. Marth. 1987. Sodium benzoate in the sodium benzoate on the rate of multiplication of Saccharo- control of growth and aflatoxin production by Aspergillus parasiticus. myces ellipsoideus was greater at pH values of 2.5 to 4.5 i. Food Prot. 50:503-509. than at 5.0 to 9.0. They also found that the concentrations 12. El-Kest, S. E., and E. H. Marth. 1988. 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