Ammonium Bicarbonate Inhibition of Mycotoxigenic Fungi and Spoilage Yeasts

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1 324 Journal of Food Protection, Vol. 53, No. 4, Pages (April 1990) Copyright International Association of Milk, Food and Environmental Sanitarians Ammonium Bicarbonate Inhibition of Mycotoxigenic Fungi and Spoilage Yeasts DAVID A. DEPASQUALE, ANWAAR EL-NABARAWY, JOSEPH D. ROSEN and THOMAS J. MONTVILLE* Department of Food Science, New Jersey Agricultural Experiment Station, Cook College, Rutgers-the State University, New Brunswick, NJ 08903, U.S.A. (Received for publication August 7, 1989) ABSTRACT Sodium bicarbonate inhibits growth and aflatoxin production by Aspergillus parasiticus. This survey determined that other mycotoxigenic fungi were also sensitive to bicarbonates. Sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, ammonium sulfate, and sodium chloride were added to buffered or unbuffered potato-dextrose agar to determine the bicarbonate effect on growth and morphology of six mycotoxigenic fungi. Three nonmycotoxigenic fungi and four yeast species were also examined. Ammonium bicarbonate at 0.11M completely inhibited the growth of Fusarium tricinctum NRRL 13442, F. tricinctum NRRL 13426, F. graminearum NRRL 5883, F. sporotrichioides NRRL 3249, Penicillium griseofulvum NRRL 989, Aspergillus ochraceus NRRL 3174, A. flavus NRRL 1957, A. niger, and P. notatum. Sodium chloride and ph elevated through the use of ampso-naoh, capso-naoh, or glycine-naoh buffer did not display an inhibitory effect on the filamentous fungi examined. Buffered ammonium sulfate treatments (ph approximately 9.0) completely inhibited all of the mycotoxigenic fungi, but at ph 5.6, ammonium sulfate treatments were not inhibitory. Sodium bicarbonate (0.11M) was effective only against P. griseofulvum, A. flavus NRRL 1957, A. niger, and P. notatum, causing viability reductions of 5.6, 3.7, 4.9, and 2.9 log cycles, respectively. Potassium bicarbonate was generally as inhibitory as the sodium salt. In contrast, elevated ph, alone, appeared to account for the >6 log reduction observed for the yeasts Lipomyces starkeyi, Geotrichum candidum, Kluyveromyces marxianus, and Debaryomyces hansenii. Mycotoxigenic fungi are widespread in nature and have been implicated in many crop-related diseases. The major mycotoxin-producing fungi are members of the genera Fusarium, Aspergillus, and Penicillium (3). Humans and domestic animals throughout the world consume crops invaded by mycotoxigenic species of Fusarium. Fusarium mycotoxins, including the trichothecenes, zearalenone, moniliformin, and fusarin C, are structurally related, biologically active compounds (9). Joffe (11) reported that severe outbreaks of alimentary toxic aleukia were caused by the ingestion of cereal grains 'Permanent address: Department of Poultry Disease, Faculty of Veterinary Medicine, Cairo University, El-Giza, Egypt. contaminated with Fusarium sporotrichioides. These 1944 outbreaks resulted in 10% of the Orenburg (USSR) district's population becoming infected. The consumption of Fusarium-mfested grains is also responsible for numerous swine disease (4,5,15,25). Hemorrhagic symptoms in cattle and oral lesions in chickens may result from the ingestion of grains contaminated with Fusarium sporotrichioides (10). Aspergillus mycotoxins have been intensely studied since the discovery of the aflatoxins nearly three decades ago. Aflatoxins are produced by toxigenic Aspergillus flavus and A. parasiticus. Acute and chronic toxicities, resulting from aflatoxin exposure, are well established in a variety of animal species (19). Other Aspergillus species can be toxigenic. Aspergillus ochraceus, for example, produces the potent nephrotoxin, ochratoxin A. A. ochraceus are widely distributed in corn, barley, and wheat (20). In animals, ingestion of grains contaminated with ochratoxin A results in tubular kidney necrosis and liver degeneration (18). In humans, a kidney disease, Balkan endemic nephropathy, is believed to be caused by ochratoxin A ingestion (12). Many toxic metabolites have been isolated from foods and feeds contaminated with Penicillium. Patulin, a carcinogenic and mutagenic compound, has been isolated in juice made from apples infested by Penicillium (6). Additional Penicillium-produced mycotoxins include penicillic acid, citrinin, and the rubratoxins (6,14). Mycotoxin production is commonly prevented by inhibiting the growth of molds on feed stuffs through the use of antifungal agents such as sorbates, propionates, and benzoates (23), butylated hydroxyanisole (1), or by storage under modified atmospheres (8,13,14,29). The cost or technical sophistication of these methods make them unsuitable for use at the farm level or in developing areas of the world. We have previously shown that a safe inexpensive GRAS (generally recognized as safe) compound, sodium bicarbonate, is lethal to Aspergillus parasiticus in microbiological media (16) and reduces aflatoxin production when monocultures of A. parasiticus are grown on autoclaved corn (17). In an effort to determine whether bicarbonate treatment is effective against a wide range of fungi, we exam-

2 BICARBONATE INHIBITION OF FUNGI 325 ined the influence of sodium, potassium, and ammonium bicarbonate against six mycotoxigenic fungi, three nonmycotoxigenic fungi, and four species of yeast. We report here that ammonium bicarbonate effectively inhibited the growth of all organisms tested. MATERIALS A METHODS Cultures and inoculum generation Penicillium griseofulvum NRRL 989, Aspergillus ochraceus NRRL 3174, A.flavus NRRL 1957 (nonaflatoxigenic), Fusarium sporotrichioides NRRL 3249, Fusarium graminearum NRRL 5883, Fusarium tricinctum NRRL 13426, and Fusarium tricinctum NRRL were obtained as lyophilized preparations from Northern Regional Research Center, U.S. Department of Agriculture, Peoria, Illinois. These preparations were dissolved in sterile distilled water, streaked onto potato dextrose agar (PDA; Difco Laboratories, Detroit, MI), and incubated for 7 d at C. Single colonies were then isolated, inoculated on PDA slants, incubated as above, and stored at 4 C for use as stock cultures. Natural isolates of Aspergillus niger and Penicillium notatum from the Department of Food Science, Rutgers University, were similarly maintained. Fungal spores were generated by culturing each species on 100 ml of PDA in a 500 ml Erlenmeyer flask incubated at 25 C for 7 d. The conidia were recovered by washing the PDA surface with sterile 0.85% saline, containing 0.01% Tween 80 (Sigma Chemical Co., St. Louis, MO) as a spore dispersal agent. The resulting spore suspensions were filtered through 4 layers of sterile cheesecloth to remove any hyphal fragments. The filtered spores were then centrifuged at 12,000 g and washed three times with sterile saline, centrifuging prior to each wash. The final spore preparations were resuspended in the appropriate volume of sterile saline to yield a direct microscopic count of approximately 10 8 spores/ml. Cultures of Lipomyces starkeyi, Geotrichum candidum, Kluyveromyces marxianus var marxianus, and Debaryomyces hansenii were obtained from the Department of Microbiology and Biochemistry, Rutgers University. These were maintained on Yeast Potato Dextrose agar (YPD, Difco). Inocula for the experiments were obtained by growing the yeasts in YPD broth for 5 d at 25 C to a final density of about 10 6 cells per ml as determined by direct microscopic count. Media PDA was the basal medium for fungal inhibition while YPD was used for the yeast. In both cases, the media were adjusted to ph 5.5 with 1 N NaOH before sterilization at 121 C for 15 min. Sodium bicarbonate (SB), ammonium bicarbonate (AB, Church and Dwight Co., Inc., Princeton, NJ), and potassium bicarbonate (KB, Sigma) were aseptically added to the tempered (45 C) media at final concentrations of 0.11 M or 0.22 M. Controls for the effect of the ammonium ion and sodium ion were prepared by adding ammonium sulfate (AS) or sodium chloride (Sigma) so that final media concentrations equalled the [ammonium] or [sodium] found in sodium bicarbonate and ammonium bicarbonate, respectively. The media's ph values were monitored using a flat membrane combination electrode (Fisher Scientific, Springfield, NJ) connected to a Chemcadet ph meter (Cole Parmer, Chicago, IL) standardized against buffers at ph 4.00 and Three buffered PDA systems, with alkalinity similar to that caused by the bicarbonate, were also prepared as follows: Ampso-NaOH buffered PDA contained 0.2M ampso (3-[l,l- Dimethyl-2-hydroxyethel)amino]-2-hydroxypropane sulfonic acid) (Sigma) and 0.2M NaOH adjusted to 100ml with distilled water so that when mixed with 100 ml of double-strength PDA a final ph of 9.2 was obtained. Capso-NaOH buffered PDA was prepared in a similar manner except that 0.2 M capso (3-[Cyclohexylamino]-2-hydroxyl-l-propanesulfonic acid) (Sigma) replaced ampso. Glycine-NaOH buffered PDA was prepared using 0.2 M glycine instead of either the capso or ampso. Preliminary experiments, not described here, demonstrated that glycine did not effect fungal growth. Additional treatments for each buffered PDA system were composed to include 0.11 M ammonium bicarbonate, sodium bicarbonate, or ammonium sulfate. Unmodified PDA controls were used to assess 100% viability for each organism. Bicarbonate lethality studies To quantitatively determine the effect of the test compounds on fungal growth, conventional plate counts were performed (in duplicate) as detailed below. The spore inocula were serially diluted in sterile 0.1% peptone and plated on each medium to give effective inocula levels ranging from 10 1 to 10 8 spores per plate, except for A. flavus, A. niger, and P. notatum, where the upper level was The plates were incubated at 25 C, examined for growth every other day, and counted after 14 d. Colony counts were then multiplied by the dilution factor and results expressed as cfu/ml of original spore suspension. Although the amount of time required for colony formation was affected by treatment, all colonies within a given treatment developed within a few days of each other, no additional colonies were formed on extended incubation, and there were no spreaders. RESULTS Low levels (0.11 M) of ammonium bicarbonate completely inhibited the growth of both Fusarium tricinctum strains, F. graminearum, F. sporotrichioides, Penicillium griseofulvum, Aspergillus ochraceus, A. flavus, A. niger, and P. notatum (Table 1). No additional colonies were formed when incubation was extended to 28 d. The viability of fungi plated on media formulated in glycine- NaOH buffer (ph 9.6) or with ammonium sulfate (ph 5.6) was generally similar to the control (untreated) media. F. sporotrichioides and the F. tricinctum strains had slight (typically less than one log) reductions at elevated ph and in the presence of ammonium sulfate. Sodium bicarbonate (0.11 M) exhibited substantial inhibition toward the growth of P. griseofulvum, A.flavus, A. niger, and P. notatum. Doubling the sodium bicarbonate concentration afforded an additional 1.3 log reduction for P. griseofulvum and completely inhibited the growth of A. flavus and P. notatum (data not shown). Potassium bicarbonate, when used, was as inhibitory as the sodium salt. Sodium chloride at 0.11 M was not inhibitory. Additional experiments examined the growth of P. griseofulvum, A. ochraceus, F. graminearum, and F. sporotrichioides in buffered (ampso-naoh, capso-naoh or glycine-naoh) PDA. Results for all three buffers were similar. A typical profile of fungal growth as a function of treatment (untreated PDA, buffered PDA, buffer+sb, buffer

3 326 MONTVILLE, DePASQUALE, EL-NABARAWY, A ROSEN TABLE 1. Influence of 0.11 M ammonium bicarbonate (AB), sodium bicarbonate (SB), potassium bicarbonate (KB); equimolar (see text) ammonium sulfate (AS) or sodium chloride; and elevated ph on viability of fungi (expressed as ability to form colonies) after 14 d at 25 C. Fungal viability (expressed as log cfu/ml of the original organism P. griseofulvum A. ochraceus F. graminearum F. sporotrichioides F. tricinctum (133442) F. tricinctum (13426) A. flavus A niger P. notatum "100% viability. b Not Done. control 8 (ph 5.5) SB (ph 9.4) inocula) on media containing: KB (ph 9.4) AB (ph 8.9) glynaoh (ph 9.6) b NaCI (ph 5.4) AS (ph 5.6) AB, and buffer+as) is demonstrated in Fig. 1. Ampso- NaOH buffered PDA (ph 8.8) data (Fig. 1) show that buffer addition to PDA (ph 5.3) did not lower the viability of any fungus tested. Buffer plus 0.11 M SB (ph 9.2) inhibited only the Penicillium sp. (>3 log reduction compared to untreated PDA). The addition of AB to buffered PDA (ph 9.0) totally suppressed the growth of all fungi. Buffered PDA plus AS (ph. 8.7) was as inhibitory as the buffer+ab treatment. Ampso-NaOH results are representative of all buffering systems employed. Fungi colonies surviving in the presence of sodium bicarbonate typically developed with altered morphologies and pigmentations. A. ochraceus colonies grown on untreated PDA, for example, were large and circular with filamentous margin. The raised surface was golden yellow and granular. The reverse color was brownish orange. Colonies grown in the presence of sodium bicarbonate 5» 4 an m, $4. 1^1 m M Q P. griseofulvum eg A. octwaceus EJ t. graminearum F. sporotrichioides PDA AMPSO AMPSO+SB AMPSO+AB AMPSO+AS Figure 1. Growth of mycotoxigenic fungi, expressed as log cful ml, in PDA alone (ph 5.3), Ampso-NaOH buffered PDA (Ampso, ph 8.8), and Ampso-bujfered PDA containing sodium bicarbonate (Ampso+SB, ph 9.2), ammonium bicarbonate (Ampso+AB, ph 9.0) or ammonium sulfate (Ampso+AS, ph 8.7). were smaller, rhizoid, and a pale tan color. Centrally-located filaments, extending from the agar surface, were observed in SB-resistant colonies. The reverse color was creamy to white. P. griseofulvum control colonies were large, olive green, with white margins and velvety. Conversely, colonies developing on media containing bicarbonate were smaller, granular and white. Filamentous centers were again apparent. F. graminearum colonies also changed color and produced filamentous tufts when grown in the presence of sodium or potassium bicarbonate. F. tricinctum colonies were normally purple to dark red and velvety. In the presence of sodium and potassium bicarbonate, the resulting colonies were whitish to pink with faint pink long filaments. Ammonium and sodium bicarbonate at 0.11 M produced a >6 log reduction in all of the yeasts except L. starkeyi, where low levels of ammonium bicarbonate caused a 2.6 log reduction, and sodium bicarbonate was ineffective (Table 2). At 0.22 M, ammonium bicarbonate or 0.33 M sodium bicarbonate, growth of L. starkeyi was completely inhibited. High alkalinity, generated using glycine- NaOH buffered media (ph 9.7) caused total inhibition of all yeasts examined. DISCUSSION Ammonium bicarbonate (AB) completely inhibited the growth of all organisms examined in this study. The 0.11 M level of AB effectively reduced the viability (5 to 7 log 10 ) of both yeast and mold. Only one yeast, L. starkeyi, displayed any resistance to the 0.11 M AB. This organism was inhibited by approximately 3.5 log 1() at the 0.11 M level. Doubling the concentration of AB to 0.22 M, completely prevented the development of this yeast. Elevated ph (>9.0), characteristic of ampso-naoh, capso-naoh, or glycine-naoh buffered PDA, did not have an apparent effect on the cell viability of mycotoxigenic

4 BICARBONATE INHIBITION OF FUNGI 327 TABLE 2. Influence of ammonium h icarbonate (AB), sodium bicarbonate (SB), and elevated ph on log cfu/ml on medium containing growth of yeast. L. starkeyi G. candidum K. marxianus D. hansenii control ( P H ) buffer glynaoh (ph 9.7) <I AB 0.1M (ph 8.6) 2.8 SB 0.2M (ph 8.7) 0.1M (ph 9.3) M (ph 9.8) fungi. The similar trends observed for treatment in both buffered and unbuffered PDA and all three buffered systems strongly suggest that AB inhibition of filamentous fungi is not a simple ph effect. Conversely, high alkalinity (glycine-naoh buffered PDA) did inhibit yeast viability (Table 2). These data suggest that bicarbonate inhibition of spoilage yeasts may be exclusively ph related. High viability in the presence of sodium chloride ruled out inhibition by sodium ions, although sodium chloride at much higher levels can be inhibitory (26). Sodium bicarbonate (SB) and potassium bicarbonate (KB) were also inhibitory to many of the fungi tested. Unlike the broad spectrum antifungal action of AB, inhibition by KB and SB was species specific as both resistant and sensitive responses were observed, depending on the organism. Alkalinity, alone, did not inhibit SB and KB sensitive Aspergillus and Penicillium as these organisms grew well in glycine-naoh buffered PDA (Table 1). These data suggest that SB and KB inhibition may be caused by bicarbonate anions, in agreement with our previous findings for A. parasiticus (16). Xu and Hang (30), have reported that the bicarbonate anion inhibits machinery mold. Punja and Groan (21,22), in support of our findings, reported that carbonate and bicarbonate anions inhibit sclerotial germination of Sclerotium rolfsii. In contrast, nitrates, sulfate, and chloride had no significant effect on sclerotial germination and mycelial growth of Sclerotium rolfsii (22). The changes in pigmentation which accompanied growth in the presence of bicarbonate-survivors are significant because many intermediates in mycotoxin biosynthetic pathways are pigmented. Accumulation of specific intermediates above a metabolic lesion in the aflatoxin biosynthetic pathway is often accompanied by concurrent accumulation of pigmented compounds (2). For example, when grown in the presence of dichlorvos, A. parasiticus produce an orange pigment (31) which has been identified as versiconal acetate (24). An acetyl derivative of a versiconal type has been reported for A. flavus grown in the presence of benzoic acid (25,27), but this has not been confirmed by other investigators (28). We anticipate that these pigments will be useful in determining the mechanism of bicarbonate's antimycotoxigenic action. Mass spectrometry data from this laboratory indicate that averufin and versicolorin A are among the intermediates accumulated when A. parasiticus is grown in the presence of sodium bicarbonate (7). The mechanisms(s) by which bicarbonates induce alterations in fungal morphology and cause pigment accumulation in the other mycotoxigenic fungi are currently being examined as part of our ongoing studies on bicarbonate's antimycotic activity. Additionally, we reported the increased fungicidal activity of the ammonium salt relative to the sodium form. Comparison of fungal viability in AS (unbuffered ph 5.6), AS (buffered ph 8.8), and AB treatments supports a mechanism proposed by Punja, et. al. (22). It appears from the data presented in Table 1 and Fig. 1 that free ammonia is responsible for fungal inhibition. The NH 3 concentration is greater in alkaline AB preparations (ph 9.0), than in the slightly acidic ammonium sulfate treatments. 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