sauerkraut and fermented milk. Ascorbic acid is another natural component of foods and, in addition, may be used artificially as an antioxidant. Organic acids ...
Z Lebensm Unters Forsch (1992) 194:124-128
Zeitschrift ffir
9 Springer-Verlag 1992
Original paper Effect of six organic acids on staphylococcal growth and enterotoxin production Ana Domeneeh, F. Javier Heruandez, Jose A. Orden, Joaquin Goyaehe, Belen Lopez, Guillermo Suarez, and Esperanza Gomez-Lucia Departamento de Patologia Animal I, Facultad de Veterinaria, Universidad Complutense, E-28040 Madrid, Spain Received July 19, 1991
Einflull sechs organiseher Siiuren auf das Waehstum von Staphylococcen und auf die Enterotoxin-Produktion Zusammenfassung. Vier Staphylococcen-Stfimmewurden bei 37 ~ fiir 24 h in Brfihe inkubiert und mit Milch-, Citronen-, Ascorbin-, Brenztrauben- und Propionsfiure gesfiuert und dabei die Uberlebensrate und die Ffihigkeit zur Enterotoxin-Produktion studiert. Die Sfiuren wurden entsprechend der Hfiufigkeit ihrer industriellen Verwendung ausgewfihlt. Periodisch wurden Proben gezogen, um die Keimzahl, pH und das Vorhandensein der Enterotoxine A, B, C und D zu bestimmen. Bei einer bestimmten Sfiure war der EinfluB auf das Wachstum und die Enterotoxin-Synthese unterschiedlich. Die am st/irksten hemmende Sfiure auf das Wachstum der Stfimme FRI100 und FRI-472 war Brenztraubensfiure, fiir den Stamm FRI-137 war es die Milchs~iure, wfihrend alle sechs Sfiuren aufden Stamm S 6 gleich wirksam waren. Milchs~iure hemmte die Enterotoxin-Synthese sehr, wiihrend der EinfluB von Essig- und Citronensfiure beinahe Null waren. Enterotoxine wurden durch pH-Werte im Sfiurebereich inaktiviert; Enterotoxin B war am stfirksten resistent gegenfiber Inaktivierung. Summary. Four Staphylococcus aureus strains were incubated at 37 ~ C for 24 h in broth progressively acidified with lactic, citric, ascorbic, acetic, pyruvic and propionic acids, and their survival rate and enterotoxin producing ability was studied. Acids were chosen based on their frequent use by the food industry. Periodically, samples were withdrawn to determine counts, pH and the presence of enterotoxins A, B, C, and D. For a given acid, the effect on growth and enterotoxin synthesis was different. The most inhibitory acid for the growth of strains FRI100 and FRI-472 was pyruvic acid, for strain FRI-137 was lactic acid, all six acids were equally effective on strain $6. Lactic acid was very inhibitory to enterotoxin synthesis, but the effect on this parameter of acetic and citric acids was almost nil. Enterotoxins were seen to be Offprint requests to: G. Suarez
inactivated at acid pH values; enterotoxin B was the most resistant to inactivation.
Introduction Staphylococcal intoxication is one of the main foodborne diseases. In Spain, foods most frequently involved include meat derivatives, dairy products, bakery products, and sauces [1]. However, it is generally considered that dairy and meat products and sauces are unfavorable substrates for the growth of microorganisms causing intoxication or food alteration because of their low pH. Studies have shown that staphylococci are able to grow at low pH levels (4.04.6) [2, 3]. Nevertheless, the effect of pH depends on the staphylococcal strain and the subsl;ance that determines the pH [4, 5]. In general, it seems that growth is less affected than enterotoxin production [4]. Most authors seem to agree that staphylococcal enterotoxins are not produced at pH values below 5.0 [2, 4, 6]. However, enterotoxins have been shown to be synthesized at low pH values [7], even in the presence of a low number of staphylococci [2, 8]. In foods, acidity may have a natural origin or be the consequence of the addition of an organic acid during the manufacture of the product. Pyruvic acid is a metabolite of lactose fermentation in certain dairy products (butter and fermented creams). Acetic and lactic acid may originate by the action of lactic bacteria in products such as sauerkraut and fermented milk. Ascorbic acid is another natural component of foods and, in addition, may be used artificially as an antioxidant. Organic acids most frequently used by the food industry as preservatives are lactic, acetic and citric acids. These acids are especially important in the acid foods involved in staphylococcal intoxication in Spain [1]. Thus, 4% acetic acid (vinegar) or citric acid (lemon juice) is added to mayonnaise dressings. Lastly, in some acid products (cakes, certain types of cheese), propionic acid is habitually added due to its antifungal properties.
125 M a n y acidified foods are left at r o o m temperature for periods of time that would allow staphylococcal growth and enterotoxin synthesis, and cause massive intoxication. The aim of the present work was to study the influence of different organic acids used by the food industry, and their ability to prevent staphylococcal growth and the synthesis, stability and inactivation of enterotoxins, as a way of preventing staphylococcal intoxication, as no such study has been undertaken systematically with six organic acids.
Materials and methods Staphylococcus aureus strains. S. aureus strains FRI-100, $6, FRI137, and FRI-472, which produce enterotoxins A (SEA), B (SEB) plus SEA, C (SEC), and D (SED), respectively, were used. They were kindly provided by M.S. Bergdoll (Food Research Institute, Madison, Wis., USA), and were stored frozen in a cryoprotective medium (tryptone, 10 g; skim powder milk, 20 g; glycerol, 80 ml; distilled water 320 ml). Strains were thawed and grown in BHI broth (Pronadisa, Madrid, Spain) at 37~C for 18 h in a gryratory shaker (Lab-Line Instruments, Melrose Park, IL, USA) at 100 rpm. Colony homogeneity was verified on Baird-Parker agar (BP, Pronadisa) Organic acids. The organic acids used, prepared 1 mol in distilled water, were: lactic acid (Lac), citric acid (Cit), ascorbic acid (Asc), acetic acid (Ace) (all from Panreac, Barcelona, Spain), pyruvic acid (Pyr) (Merck, Darmstadt, FRG), and propionic acid (Pro) (Sigma, St. Louis, MO, USA). Experimental design. A 2-L-Erlenmeyer flask with 1 L BHI broth was connected by a silicone tube to a separatory funnel, placed 100 cm above, which contained 200 ml of one of the 1 mol organic acids. The acid flow was controlled by the stopcock and a Mohr press. Another silicone tube connected the flask to a 50-ml syringe that was used for sampling. The assembled system was autoclaved at 121~ C for 15 rain. Inoculation and incubation. One of the four S. aureus strains was aseptically inoculated into the flask to give a final concentration of 105-106 cfu/ml. The flask was incubated at 37~ C for 24 h on a gyratory shaker (100 rpm) as the organic acid flowed at a constant rate (7-10 ml/h). Sa..mples(50 ml) were taken immediately after inoculation and after 30 min, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h with the syringe, and were introduced into a 100-ml sterile plastic container for further processing. Control experiments, without addition of organic acid, were conducted with each strain. Staphylococcal counts and pH measurement. Serial decimal dilutions were done in 1% peptone water (Difco, Detroit, MI, USA), surface plated on Baird-Parker agar (BO) (Pronadisa, Spain) and Tryptic Soy agar (TSA) (Pronadisa, Spain), and incubated at 37~C for 48 h. The pH was measured by introducing the electrode (Crison, type 414, Barcelona, Spain) directly into the sample. Enterotoxin detection. All samples were examined for enterotoxin presence by the ELISA method described by Freed et al. [9]. The sample was previously concentrated to 1/10 by dialysis against polyethylene glycol 20,000 (Serva, Heidelberg, FRG) for 24 h at 4~ C. A standard curve was run in each plate diluting crude enterotoxin in BHI acidified with the acid used to pH 3.5. To minimize the possible effect of staphylococcal protein A, concentrated samples were diluted (1 : 1) in normal rabbit serum (NRS) (complement inactivated at 56~ for 30min) [10] and were assayed by the same ELISA technique. Statistical analysis. The variance and multiple range test of Newman-Keuls was obtained from the data of counts and pH value, with
the Biostatistics III programme using an Apple IIe (Cupertino, CA, USA) computer. The study was done for (a) each strain with regard to the six organic acids and (b) for each acid with regard to the four different strains. In both cases, analysis was independent for counts and pH. All studies were done in duplicate.
Results and discussion Influence o f p H on staphylococcal growth
Although the optimal p H for growth is 7.0, staphylococci m a y grow at other p H values depending on different factors such as temperature, presence of O2, etc. This ability to grow at acidic p H values allows staphylococcal contamination of acid type foods such as yogurt and other dairy products, generally considered as unfavorable for the multiplication of microorganisms and enterotoxin synthesis because of the low pH. The increasing importance of staphylococcal intoxication and the fact that in Spain the foods involved are mainly dairy products and sauces such as mayonnaise [1], led us to study the effect of these acids and p H on staphylococcal enterotoxin synthesis. To study the influence of organic acids and of pH, we incubated the samples under optimal conditions (37~ and aerobiosis); thus, the only limiting factor was the acid. Our results showed a different behaviour of the S. aureus strains with respect to p H (Fig. 1). Globally, statistical analysis showed that growth of S. aureus $6 was the m o s t inhibited, and the effect of p H was different from the other three strains used, which were not statistically different. In addition, no $6 colonies were detected after 24 h in any of the experiments, whereas strains FRI-100, FRI-137, and FRI-472 were present, even at very low p H (pH 2.4, FRI-472). Nevertheless, we do not consider this presence of colonies as representing multiplication of staphylococci in the acidified broth. Statistical analysis showed that in m o s t of the strains a different acid seemed to be the most inhibitory, while the other five exerted a similar effect (Fig. 1). After the computer statistical analysis, the m o s t inhibitory acid for strains FRI-100 and FRI-472 was Pyr, and for FRI-137 Lac. All six acids were similarly inhibitory. In our hands, this acid was never the most inhibitory. Our data show that citric acid was the least inhibitory, as staphylococci multiplied at a lower p H than those reported previously [12]. This acid also allowed staphylococcal growth at the lowest p H values, which could mean a less bactericidal effect. This is very important for those products such as home-made mayonnaise, where the microbiological safety relies on the addition of citric or acetic acid. F o r contrast, propionic acid (generally used as fungistatic in foods) rapidly limited the multiplication of staphylococci. Lactic acid is one of the organic acids more intensely studied due to its importance in dairy and fermented products. Thus, several authors [8, 13, 14] have found that the bactericidal effect on staphylococci started at a p H above those shown in this study. However, these previous studies were done in comOlex media, such as cheese
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