Growth and Aflatoxin Production by Aspergillus Parasiticus NRRL 2999 in the Presence of Potassium Benzoate Or Potassium Sorbate and at Different Initial Ph Values

820 Journal of Food Protection, Vol. 50, No. 10, Pages 820-825 (October 1987) Copyright0 International Association of Milk, Food and Environmental Sanitarians Growth and Aflatoxin Production by Aspergillus parasiticus NRRL 2999 in the Presence of Potassium Benzoate or Potassium Sorbate and at Different Initial pH Values GULAM RUSUL and ELMER H. MARTH* Downloaded from http://meridian.allenpress.com/jfp/article-pdf/50/10/820/1651075/0362-028x-50_10_820.pdf by guest on 25 September 2021 Department of Food Science and The Food Research Institute, University of Wisconsin-Madison, Madison, Wisconsin 53706 (Received for publication February 18,1987) ABSTRACT tionery, fruit and vegetable products, and casings used for fermented meat products (29). Reports indicate that Experiments were done to determine how different concentra sorbate is effective against many species of yeasts, molds tions of potassium benzoate or potassium sorbate in a glucose- and bacteria; this has been discussed in two recent re yeast extract-salts medium with an initial pH value of 3.5, 4.5 views (20,29). Bandelin (1) reported that the minimum or 5.5 affected growth and aflatoxin production by Aspergillus concentration of sorbic acid required to inhibit Alternaria parasiticus NRRL 2999. The pH of the medium, weight of solani is 0.02%, whereas 0.08% inhibited growth of mycelium and amount of aflatoxin produced were determined Penicillium citrinum and Aspergillus niger. Sauer and after 3 and 7 d of incubation. Aflatoxin was determined using reversed-phase high-performance liquid chromatography. Burroughs (27) observed that 0.5 and 1.0% K-sorbate Maximum concentrations of potassium sorbate and potassium prevented mold growth for 2 and 3 weeks, respectively, benzoate that permitted growth were 0.2% and 0.4%, respec in corn with 18% moisture, whereas similar concentra tively, in a medium with an initial pH of 5.5. When the initial tions of sorbic acid prevented mold growth for 7 and 12 pH was 4.5, the maximum concentrations of potassium sorbate weeks, respectively. Effects of K-sorbate or sorbic acid and potassium benzoate that permitted growth were 0.05% and on growth and aflatoxin production by A. parasiticus and 0.10%, respectively, but there was an extended lag phase. In A. flavus have been reported (7,8,14,22,24,30,33). creasing concentrations of potassium benzoate or potassium sor Benzoic acid and benzoates are widely used as anti- bate decreased amounts of aflatoxin B[ and Gi produced after mycotic agents to preserve beverages, fruit products, bak 3 d in a medium with initial pH values of 5.5 or 4.5. Cultures growing in the medium containing 0.1, 0.15 or 0.20% potas ery products and other foods. Most molds are inhibited sium benzoate or potassium sorbate and with an initial pH of by concentrations of 0.05 to 0.10% (9). Bandelin (1) re 5.5 were somewhat inhibited at 3 d of incubation, which was ported that 0.15% benzoic acid inhibited growth of A. characterized by a slow decrease in pH, low mycelium dry solani, whereas 0.2% benzoic acid was required to inhibit weight and small amounts of accumulated aflatoxins. After 7 A. niger and P. citrinum. Uraih et al. (31) found that d these cultures overcame the initial inhibition and produced presence of benzoic acid and Na-benzoate reduced substantial amounts of aflatoxins and mycelium. This was also amounts of aflatoxin B, and Gj produced by A. flavus, true for cultures growing in a medium with an initial pH of and also delayed onset of sporulation by the mold. They 4.5 and containing potassium benzoate or potassium sorbate. also observed that the reduction in aflatoxin production By decreasing the initial pH of the medium from 5.5 to 4.5, was proportional to the increase in amount of benzoic amounts of potassium benzoate or potassium sorbate required acid and Na-benzoate in the medium. Chipley and Uraih to achieve inhibition decreased by a factor of 10. (10), compared effects of various derivatives of benzoic acid on growth and aflatoxin production by A. parasiticus Aflatoxins are a group of secondary metabolites pro and A. flavus, and noted that 0.01% of nitrobenzoate or duced by certain strains of Aspergillus flavus. Aflatoxins p-aminobenzoate stimulated growth of A. flavus but re pose a quadruple threat to both humans and animals as duced aflatoxin release, whereas ethyl aminobenzoate re they are toxigenic, carcinogenic, mutagenic, and duced growth but stimulated aflatoxin biosynthesis. Ethyl teratogenic (16). Aflatoxin-producing molds can grow benzoate (0.02%) and methyl benzoate (0.02%) com over a range of environmental conditions (18,23), on a pletely inhibited aflatoxin biosynthesis and reduced variety of agricultural and food commodities (6,17) and growth of A. flavus by 78% and 61%, respectively. in the presence of certain concentrations of various chem Masimango et al. (22) reported that at 1% benzoic acid icals and preservatives (25). and Na-benzoate caused 23.6 and 23.2% inhibition, re Potassium sorbate (K-sorbate) has been used as an anti spectively, of aflatoxin production by A. flavus. They microbial agent in such foods as dairy, bakery, confec also observed that 0.5 and 0.1% of benzoic acid or Na- JOURNAL OF FOOD PROTECTION, VOL. 50, OCTOBER 1987 SORBATE AND BENZOATE AFFECT TOXIGENIC ASPERGILLUS 821 benzoate had no effect on growth and aflatoxin produc two layers of cheese cloth directly into the 125-ml separatory tion by A. flavus. According to Beuchat (3) presence of funnel. Five milliliters of distilled water was added to the 50 ppm Na-benzoate in the heating menstruum caused a cheese cloth to wash off residual medium. Thirty five milliliters significant decrease in decimal reduction times of conidia of chloroform (AR, Baker) was used instead of 50 ml. Analysis of A. flavus and Penicillium puberulum. Also, K-sorbate of variance of data was done using the Minitab statistical pack age on an IBM personal computer. was more effective in inhibiting colony formation than was Na-benzoate. RESULTS AND DISCUSSION The antimicrobial effectiveness of benzoate and sorbate is pH-dependent and resides in the undissociated pH of media containing K-benzoate or K-sorbate and molecule of the acid (2,4,5,19). This study was con having an initial pH or 4.5 or 5.5 ducted, first, to determine the effects of various pH After 3 d of incubation (Table 1), there was a decrease values plus different concentrations of K-sorbate and in pH in all cultures (initial pH 5.5) growing in the ab potassium benzoate (K-benzoate) on growth and aflatoxin sence or presence of K-benzoate or K-sorbate except for production by A. parasiticus in a glucose-yeast extract- those growing in the presence of 0.2% K-sorbate which Downloaded from http://meridian.allenpress.com/jfp/article-pdf/50/10/820/1651075/0362-028x-50_10_820.pdf by guest on 25 September 2021 salts medium, and, second, to compare the effectiveness showed only a slight increase in pH. Cultures with an of the two antifungal agents in controlling mold growth initial pH of 4.5 (Table 2) also exhibited a decrease in and toxin production. pH, but this was affected by concentrations of K-ben zoate or K-sorbate. The slight increase in pH exhibited MATERIALS AND METHODS by cultures growing in the presence of 0.20% K-sorbate likely resulted from minimal mycelia growth. A preliminary experiment was done to determine concentra tions of K-benzoate (Pfizer) or K-sorbate (Pfizer) that allowed growth of A. parasiticus in a medium with initial pH values of 3.5, 4.5 or 5.5. At pH 5.5, the maximum concentration of K-benzoate that allowed growth was 0.10% and that of K-sor TABLE 1. The pH values of cultures of A. parasiticus contain bate was 0.05%, whereas at pH 3.5 growth occurred only in ing various concentrations of potassium benzoate or potassium the medium containing 0.025% K-benzoate or K-sorbate. sorbate and incubated at 28°C; initial pH of medium was 5.5. Hence, concentrations of K-benzoate or K-sorbate used in later Incubation (d) experiments were 0.0, 0.025, 0.05, 0.10, 0.15 and 0.20% in a medium with an initial pH value of 5.5. When the initial Preservative 3 7 pH of the medium was 4.5, concentrations of K-benzoate used benzoate (%) were 0.0, 0.025. 0.05 and 0.10% and those of K-sorbate were 0 2.54 5.60 0.0, 0.025 and 0.05%. When the initial pH of the medium was 0.025 2.56 5.69 3.5, 0.025% or K-benzoate or K-sorbate was used. 0.05 2.59 5.80 A glucose-yeast extract-salts medium described by Yousef 0.10 2.93 5.02 and Marth (33) was used for preliminary and other experiments. 0.15 3.36 5.44 A. parasiticus NRRL 2999 was obtained from the Northern 0.20 4.28 5.14 Regional Research Center, U.S.D.A., Peoria, IL. The mold sorbate (%) was grown on slants of Mycological agar that were incubated 0 2.54 5.60 at 28°C. After 7 d, spores were harvested by adding sterile dis 0.025 2.46 5.61 tilled water and glass beads to cultures; the glass beads helped 0.05 2.48 5.67 to dislodge spores from the mycelium when shaken. The spore 0.10 3.25 5.26 suspension was pooled in a sterile 125-ml Erlenmeyer flask.

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