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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 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 , molds tions of potassium benzoate or potassium sorbate in a - and ; this has been discussed in two recent re­ 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 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 (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 (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 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. 0.15 4.33 4.76 The number of spores present per milliliter was determined by 0.20 5.68 3.64 the plate count method using Mycological agar and incubation at 28°C. One hundred and forty four milliliters of glucose-yeast ex­ tract-salts medium was dispensed into each of a series of 300- ml Erlenmeyer flasks containing the desired amounts of K-ben­ TABLE 2. The pH values of cultures of A. parasiticus contain­ zoate or K-sorbate. The pH of the medium was adjusted to 3.5, ing various concentrations of potassium benzoate or potassium 4.5 or 5.5 by adding 10 N HC1 or 40% NaOH. The medium sorbate and incubated at 28"C; initial pH of medium was 4.5. was then filter-sterilized using a 0.45-|jim filter and dispensed Incubation (d) into sterile 300-ml Erlenmeyer flasks. Six milliliters of a spore Preservative 3 7 suspension containing ca. 106 conidia per milliliter was added to each flask. The medium was then dispensed aseptically into K-benzoate (%) sterile 125-ml Erlenmeyer flasks. Each flask contained 25 ml 0 2.43 of medium and ca. 106 conidia. 0.025 2.58 After 3 and 7 d of incubation, contents of two flasks per 0.05 3.85 concentration of acid were analyzed for pH, mycelium dry 0.10 4.67 weight and aflatoxins B, and Gi; methods used were as previ­ sorbate (%) ously described (26) but with the following modifications. For 0.025 2.57 aflatoxin determination, 10 ml of medium was filtered through 0.05 4.66

JOURNAL OF FOOD PROTECTION, VOL. 50, OCTOBER 1987 822 RUSUL AND MARTH

The rate of decrease in pH decreased with increasing whereas at higher concentrations the reverse was true. concentrations of K-benzoate or K-sorbate. The decrease This is evident because the mycelial weight of cultures in pH, which may have resulted from production of or­ growing in the presence of 0.025 or 0.05% K-sorbate ganic acids by the mold during growth, was proportional was significantly (p<0.05) higher than that of cultures to the amount of growth. This explains why in the pres­ growing in the presence of 0.025 or 0.05% K-benzoate ence of subinhibitory concentrations of K-benzoate and while cultures growing in the presence of 0.10, 0.15 or K-sorbate, there was only a slight decrease in pH as a 0.20% K-benzoate had significantly (p<0.05) more result of negligible mold growth. After 7 d of incubation, mycelium than those growing in similar concentrations of there was an increase in pH of all cultures growing with K-sorbate. or without K-sorbate or K-benzoate in the medium with At 3 d of incubation, cultures growing in the medium an initial pH of 5.5 except when the medium contained containing 0.0, and 0.025% K-benzoate or 0.025% K- 0.2% K-sorbate and there was a decrease in pH. Similar sorbate with an initial pH of 4.5 (Table 4) produced sig­ trends in increase in pH were observed in cultures grow­ nificantly (p<0.05) more mycelium than did cultures ing in the medium with an initial pH value of 4.5 and growing in the presence of 0.05% K-benzoate. There was 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 containing 0.0, 0.025% K-benzoate or 0.025% K-sorbate. no significant difference (p>0.05) in mycelium dry A decrease in pH also occurred in cultures growing in weight of cultures growing in the medium containing 0.0, the presence of 0.05 and 0.10% K-benzoate or 0.05% 0.025% K-benzoate or 0.025% K-sorbate. At 3 d of incu­ K-sorbate. The increase in pH after 7 d of incubation bation, there was no growth of A. parasiticus in the may have resulted from assimilation of organic acids pro­ medium containing 0.10% K-benzoate or 0.05% K-sor­ duced earlier by the mold, elevated levels of nitrogen in bate and with an initial pH of 4.5 (Table 4). the medium (11), or autolysis of fungal cells (33).

Mycelium dry weight TABLE 3. Dry weight of mycelium (g/25 ml of medium) pro­ duced by A. parasiticus in cultures containing various concen­ The maximum concentration of K-sorbate that permit­ trations of potassium benzoate or potassium sorbate and incu­ ted growth of A. parasiticus was 0.20 and 0.05%, and bated at 28°C; initial pH of medium was 5.5. that of K-benzoate was 0.4 and 0.10%, when the medium Incubation 1 had an initial pH, respectively, of 5.5 or 4.5. At an in­ :d) itial pH of 3.5, there was no growth of A. parasiticus Preservative 3 7 at 3 d in the presence of 0.025% K-benzoate or K-sorbate ^-benzoate (%) (data not shown). Results in Table 3 demonstrate that in ( 0.76 0.79 most instances increasing concentrations of K-sorbate or 0.025 0.70 0.73 K-benzoate decreased mycelial growth in a medium with 0.05 0.70 0.73 an initial pH of 5.5. 0.10 0.61 0.79 0.15 0.55 0.95 There was no significant difference (p>0.05) in myce­ 0.20 0.49 0.81 lial dry weight of cultures growing in the medium con­ ^-sorbate (%) taining 0.0, 0.025, and 0.05% K-sorbate but there was 0 0.76 0.79 a significant difference (p<0.05) in mycelial dry weight 0.025 0.75 0.77 at 3 d of incubation between these cultures and those 0.05 0.75 0.78 growing in the presence of 0.10, 0.15 and 0.20% K-sor­ 0.10 0.59 0.77 bate in the medium with an initial pH of 5.5. There also 0.15 0.29 0.82 was a significant difference (p<0.05) in mycelial dry 0.20 0.03 0.82 weight among cultures growing in the presence of 0.10, 0.15 and 0.20% K-sorbate. At 3 d of incubation there was a significant difference TABLE 4. Dry weight of mycelium (g/25 ml of medium) pro­ (p<0.05) in mycelial dry weight among cultures growing duced by A. parasiticus in cultures containing various concen­ in the absence of K-benzoate and those growing in the trations of potassium benzoate or potassium sorbate and incu­ presence of the chemical in a medium with an initial pH bated at 28°C; initial pH of medium was 4.5. of 5.5. There was no significant difference (p>0.05) in Incubation (d) mycelia weight of cultures growing in the presence of Preservative 3 7 0.025 and 0.05% K-benzoate but there was a significant K-benzoate (%) difference (p<0.05) between these cultures and those 0 0.62 0.68 growing in the presence of 0.10, 0.15 and 0.20% K-ben­ 0.025 0.55 0.78 zoate. There was a significant difference (p<0.05) in 0.05 0.26 0.75 mycelial weight among cultures growing in the presence 0.10 NGa 0.75 of 0.10, 0.15 and 0.20% K-benzoate in a medium with K-sorbate (%) an initial pH of 5.5. 0.025 0.48 0.76 Results in Table 3 also suggest that at lower concentra­ 0.05 NG 0.77 tions K-benzoate was more inhibitory than K-sorbate, aNG = No growth

JOURNAL OF FOOD PROTECTION, VOL. 50, OCTOBER 1987 SORBATE AND BENZOATE AFFECT TOXIGENIC ASPERGILLUS 823

At 7 d of incubation, A. parasiticus growing in the Production of qflatoxin medium containing 0.5 and 0.10% K-benzoate or 0.5% Data in Tables 5 and 6 suggest that biosynthesis and K-sorbate and with an initial pH value of 4.5 overcame accumulation of aflatoxin were influenced most by con­ this initial inhibition and produced substantial amounts of centration of K-benzoate or K-sorbate, initial pH of the mycelium. This was also true for cultures growing in the medium and extent of mycelial growth. At 3 d of incuba­ medium containing 0.10, 0.15 or 0.20% K-benzoate or tion, cultures growing in the presence of increasing con­ K-sorbate and with an initial pH of 5.5, which had a centrations of K-benzoate or K-sorbate produced decreas­ significant (p<0.05) increase in mycelium dry weight at ing amounts of aflatoxin Bi and aflatoxin Gj in the 7 d of incubation. Amounts of mycelium produced by medium with an initial pH of 5.5 or 4.5. There was no these cultures were almost equivalent to amounts pro­ significant difference (p>0.05) in amounts of aflatoxin duced by cultures growing in the presence of 0.0, 0.025, Gj produced by cultures growing in the medium contain­ 0.05% K-benzoate or K-sorbate. Data in Table 3 suggest ing 0.0 or 0.025% K-benzoate or K-sorbate and with an that 0.15% K-benzoate may have stimulated growth of initial pH of 5.5. Similarly, there was no significant dif­

A. parasiticus since maximum mycelium dry weight was ference (p>0.05) in amounts of aflatoxin Bj produced 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 produced by this culture. There was no significant by cultures growing in the presence of 0.0, 0.025 and (p>0.05) increase in mycelium dry weight of cultures 0.05% K-benzoate or K-sorbate in the medium with an growing in the presence of 0.0, 0.025 or 0.05% K-ben­ initial pH of 5.5. There was a significant difference (p< zoate and K-sorbate in a medium with an initial pH of 0.05) in amount of aflatoxin Gi produced by cultures 5.5. At 7 d of incubation, sporulation occurred in control growing in the presence of 0.0 or 0.025% compared to cultures, but there was only slight sporulation in cultures those growing in the presence of 0.05, 0.10, 0.15 or growing in the presence of 0.025 and 0.05% K-benzoate. 0.20% K-benzoate or K-sorbate.

TABLE 5. Accumulation of aflatoxin B, and Gt (\i,glml of medium) at 28°C in a medium with various concentrations of potassium benzoate or potassium sorbate; initial pH of medium was 5.5. 3d 7 d Preservative K-benzoate (%) 0 27.6 401.6 25.5 591.5 0.025 21.0 346.5 17.9 471.3 0.05 22.2 291.5 19.1 520.0 0.10 19.3 171.8 16.6 407.9 0.15 13.0 137.0 19.1 518.4 0.20 10.1 84.4 25.5 572.0 K-sorbate (%) 0 27.6 401.6 25.5 591.5 0.025 26.4 381.4 15.3 492.4 0.05 27.6 319.0 21.0 494.0 0.10 14.2 139.2 19.1 391.6 0.15 6.8 46.1 21.4 407.9 a 0.20 ND ND 22.7 367.3 aND = None detected.

TABLE 6. Accumulation of aflatoxin B, and G, (\Lglml of medium) at 28°C in a medium with various concentrations of potassium benzoate or potassium sorbate, initial pH of medium was 4.5. 3d 7 d Preservative K-benzoate (%) 0 19.3 393.3 15.2 637.8 0.025 15.9 263.1 16.7 578.9 0.05 4.0 31.5 13.8 565.2 0.10 NDa ND 26.1 502.4 K-sorbate (%) 0.025 14.0 214.8 13.8 663.3 0.05 ND ND 16.7 626.0 aND: None detected.

JOURNAL OF FOOD PROTECTION, VOL. 50, OCTOBER 1987 824 RUSUL AND MARTH

Cultures growing in the medium containing 0.15 and if the chemical is absent from the medium. There was 0.20% K-benzoate or 0.15% K-sorbate and with an initial no significant (p>0.05) change after 7 d of incubation pH value of 5.5 produced significantly (p<0.05) less af- in amount of aflatoxin Bj present in the medium contain­ latoxin Bj than those growing in the presence of 0.0, ing 0.0, 0.025, 0.05 or 0.10% K-benzoate and with an 0.025 and 0.05% K-benzoate or K-sorbate in the medium initial pH of 5.5. with an initial pH value of 5.5. Amounts of aflatoxin Cultures in the medium containing 0.025% K-benzoate G] produced decreased significantly (p<0.05) when con­ or K-sorbate and with an initial pH of 3.5 failed to grow centrations of K-benzoate or K-sorbate increased in incre­ at 3 d of incubation but overcame this inhibition during ments from 0.05 to 0.20%. This was also true for afla­ the 3-to-7-d incubation interval. Amounts of mycelium toxin Bj. No aflatoxin Bj and G, were detected in the and aflatoxins produced were significantly (p<0.05) less medium containing 0.20% K-sorbate. than those produced by cultures growing at similar con­ At 3 d of incubation there was a significant difference centrations of K-benzoate or K-sorbate in the medium (p<0.05) in amounts of aflatoxin B, and G, produced with an initial pH of 5.5 or 4.5 (data not shown). Trends by cultures growing in the medium containing 0.0, 0.025 in inhibition and the concentration of K-sorbate required 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 and 0.05% K-benzoate or 0.025% K-sorbate and with an to inhibit growth of A. parasiticus are in general agree­ initial pH value of 4.5. Increasing the concentrations of ment with those reported by others (8,24,33). K-benzoate or K-sorbate significantly (p<0.05) decreased According to Lueck (21), only 15% undissociated sor- amounts of aflatoxin Bj and Gi produced by cultures bic acid or 5% undissociated benzoic acid is present at growing at pH of 4.5. No aflatoxin B, or Gi was de­ pH 5.5, whereas at pH 4.5 the values are 65 and 33%, tected at 3 d of incubation in the medium containing respectively. This explains why a higher concentration of 0.1% K-benzoate and 0.05% K-sorbate and with an in­ K-sorbate or K-benzoate was required to inhibit growth itial pH value of 4.5. of A. parasiticus at pH 5.5 than at pH 4.5. Gould et At 7 d of incubation, there was a significant (p<0.05) al. (15), citing work of other investigators, concluded increase in amount of aflatoxin G] produced by cultures that weak lipophilic acids such as sorbate, benzoate and growing at all concentrations of K-benzoate or K-sorbate propionate, may owe part of their effectiveness to their in the medium with an initial pH of 5.5 or 4.5. Cultures , in the undissociated form, in the cell mem­ growing in the medium containing 0.1, 0.15 and 0.20% brane and to the consequent action as 'proton ionphores.' K-sorbate or K-benzoate with an initial pH of 5.5, were In effect, lipophilic acids increase the proton flux across initially slightly or completely inhibited but overcame this the cell membrane, thus interfering with the proton gra­ inhibition and then produced large amounts of aflatoxin dient across the membrane and disrupting many of the G,. chemiosmotic-related functions by the cell. This was also true for cultures growing in the presence Results of this study suggest that at the higher pH of 0.05 and 0.10% K-benzoate or 0.025 or 0.05% K-sor­ value (5.5), K-sorbate was more effective in inhibiting bate in the medium with an initial pH of 4.5. This ability growth and aflatoxin production than was K-benzoate. of cultures to overcome initial inhibition and then pro­ This was also true at pH 4.5, because the amount of K- duce large amounts of aflatoxins when growing in the sorbate required was 50% less than of K-benzoate to presence of subinhibitory levels of preservatives has been achieve similar inhibition. reported by others (10,13,14,26,33). A significant (p< ACKNOWLEDGMENTS 0.05) increase in amount of aflatoxin B, was observed in cultures growing in the medium containing 0.15 or Research supported by the College of Agriculture and Life Sciences, 0.20% K-benzoate or 0, 10, 0.15 or 0.20% K-sorbate University of Wisconsin-Madison and by University Pertainian Malaysia and with an initial pH value of 5.5. There was a signifi­ through the support of Gulam Rusul. cant (p<0.05) decrease in amount of aflatoxin B] cul­ tures growing in the medium containing 0.025 or 0.05% REFERENCES K-sorbate and with an initial pH of 5.5. The decrease in amount of aflatoxin B] is probably the result of its 1. Bandelin, F. J. 1958. The effect of pH on the efficiency of various degradation by intracellular fungal enzymes that are liber­ mold inhibiting compounds. J. Am. Pharm. Assoc. Sci Ed. 47:691-694. ated following autolysis of the mycelium (12,33). Degra­ 2. Bell, T. A., J. L. Etchells, and A. F. Borg. 1959. Influence of dation of aflatoxins has been reported by others sorbic acid on the growth of certain species of bacteria, yeasts, (26,28,33,34). These investigators observed that the de­ and filamentous fungi. J. Bacteriol. 77:573-580. gradation usually occurs during the 7-to-10-d incubation 3. Beuchat, L. R. 1981. Influence of potassium sorbate and sodium interval. Doyle and Marth (12) reported that 7-d-old benzoate on heat inactivation of Aspergillus flavus, Penicillium puberulum and Geotrichum candidum. J. Food Prot. 44:450-454. mycelia can degrade aflatoxins but 9-d-old mycelia de­ 4. Beuchat, L. R. 1979. Control of yeasts and molds by using food grade maximum amounts of aflatoxins. Since we ob­ additives and physical methods, pp. 154-163. In M. E. Rhodes served degradation of aflatoxin B{ during the 3-to-7-d in­ (ed.), Food mycology. G. K. Hall and Co., Boston, MA. terval of incubation, it is possible that small concentra­ 5. Bosund, I. 1962. The action of benzoic and salicyclic acids on tions of K-sorbate can induce degradation of aflatoxin B] the metabolism of microorganisms. Adv. Food Res. 11:331-353. 6. Bullerman, L. B. 1986. Mycotoxins and food safety. Food Tech- at an earlier stage of mold growth than would happen nol. 40(5):59-66.

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7. Bullerman, L. B. 1983. Effects of potassium sorbate on growth 21. Lueck, E. 1977. Antimicrobial food additives. Springer-Verlag, and aflatoxin production by A. parasiticus and A. flavus. J. Food Berlin. Prot. 46:940-942. 22. Masimango, N., J. L. Ramaut, and J. Ramacle. 1979. Contribution 8. Bullerman, L. B. 1979. Effects of potassium sorbate on mycotoxin a 1'etude du role des additifs chimiques dans la lutte contre 1'af- production and growth of Aspergillus parasiticus, Penicillium com­ latoxine. Rev. Ferment. Ind. Aliment. 33(4): 116-123. mune and Penicillium patulum in broth cultures. Presented at the 23. Northolt, M. D., and L. B. Bullerman. 1982. Prevention of mold 39th Annual Meeting of the Institute of Food Technologists, St. growth and toxin production through control of environmental con­ Louis, MO, June 10-13. ditions. J. Food Prot. 45:519-526. 9. Chipley, J. R. 1983. and benzoic acid. pp. 11- 24. Przybylski, K. S., and L. B. Bullerman. 1980. Influence of sorbic 35. In A. L. Branen and P. M. Davidson (eds.), Antimicrobials acid on viability and ATP content of conidia of Aspergillus in foods. Marcel Dekker, Inc., New York. parasiticus. J. Food Sci. 45:375-376,385. 10. Chipley, J. R., and N. Uraih. 1980. Inhibition of Aspergillus 25. Ray, L., and L. B. Bullerman. 1982. Preventing growth of poten­ growth and aflatoxin release by derivatives of benzoic acid. Appl. tially toxic molds using antifungal agents. J. Food Prot. 45:953- Environ. Microbiol. 40:352-357. 963. 11. Ciegler, A., R. E. Peterson, A. A. Lagoda, and H. H. Hall. 1966. 26. Rusul, G., F. E. El-Gazzar, and E. H. Marth. 1986. Growth of Aflatoxin production and degradation by A. flavus in 20-liter fer- and aflatoxin production by Aspergillus parasiticus in a medium mentor. Appl. Microbiol. 14:826-833. containing potassium chloride or a mixture of potassium chloride 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 12. Doyle, M. P., and E. H. Marth. 1978. Aflatoxin is degraded by and sodium chloride. J. Food Prot. 49:880-885. fragmented and intact mycelia of Aspergillus parasiticus grown in 27. Sauer, D. B., and R. Burroughs. 1974. Efficacy of various chemi­ 5 to 18 days with and without agitation. J. Food Prot. 41:549-555. cals as grain mold inhibitors. Trans. Am. Soc. Agr. Eng. 17:557- 13. Draughon, F. A., and J. C. Ayres. 1981. Inhibition of aflatoxin 559. production by selected insecticides. Appl. Environ. Microbiol. 28. Shih, C. N., and E. H. Marth. 1972. Production of aflatoxin in 41:972-976. a medium fortified with sodium chloride. J. Dairy Sci. 55:1415- 14. Garies, M., J. Bauer, A. von Montgelas, and B. Gedek. 1984. 1419. Stimulation of aflatoxin B, and T-2 toxin production by sorbic 29. Sofos, J. N., and F. F. Busta. 1981. Antimicrobial activity of sor­ acid. Appl. Environ. Microbiol. 47:416-418. bate. J. Food Prot. 44:614-647. 15. Gould, C. W., M. H. Brown, and B. C. Fletcher. 1983. 30. Udagawa, S., M. Kobatake, and H. Kurata. 1977. Reestimation Mechanisms of action of procedures, pp. 67-95. of the preservation effectiveness of potassium sorbate as a food ad­ In T. A. Roberts and F. A. Skinner (eds.), Food microbiology: ditive in jams and marmalade. Bull, of the Nat'l Inst, of Hygenic advances and prospects. Academic Press, London U.K. Sciences (Eisei Shikenjo Hokoku) 95:88-92. [In Food Sci and 16. Heathcote, J. G. 1984. Aflatoxins and related toxins, pp. 89-130. Technol. Abstr. 1979, 11(3):105]. In V. Betina (ed.), Mycotoxins-production, isolation, separation 31. Uraih, N., T. R. Cassity, and J. R. Chipley. 1977. Partial charac­ and purification. Elsevier Science Publishers B. V., Amsterdam, terization of mode of action of benzoic acid on aflatoxin biosyn­ Netherlands. thesis. Can. J. Microbiol. 23:1580-1584. 17. Hesseltine, C. W., O. L. Shotwell, J. J. Ellis, and R. D. 32. Uraih, N., and J. R. Chipley. 1976. Effect of various acids and Stubblefield. 1966. Aflatoxin formation by Aspergillus flavus. Bac­ salts on growth and aflatoxin production by Aspergillus flavus terid. Rev. 30:795-805. NRRL3145. Microbios 17:51-59. 18. Holmquist, G. U., H. W. Walker, and H. M. Stahr. 1983. Influ­ 33. Yousef, A. E., and E. H. Marth. 1981. Growth and synthesis of ence of temperature, pH, water activity and antifungal agents on aflatoxin by Aspergillus parasiticus in the presence of sorbic acid. growth of Aspergillus flavus and A. parasiticus. J. Food Sci. J. Food Prot. 44:736-741. 48:778-782. 34. Yousef, A. E., S. M. El-Gendy, and E. H. Marth. 1980. Growth 19. Jay, J. M. 1986. Modern food microbiology, 3rd ed. Van Nostrand and biosynthesis of aflatoxin by Aspergillus parasiticus in cultures Co., New York. pp. 259-296. containing nisin. Z. Lebensm. Unters. Forsch. 171:341-343. 20. Liewen, M. B., and E. H. Marth. 1985. Growth and inhibition of microorganisms in the presence of sorbic acid: A review. J. Food Prot. 48:364-75.

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