Zeitschrift ffir

LebensmittelUntersuchung und-Forschung

Z. Lebensm.Unters.-Forsch.166,280--283 (1978)

@J. F. BergmannVerlag1978

Evaluating the Inhibitory Action of Honey on Fungal Growth, Sporulation, and Aflatoxin Production Ten Eyck T. Wellford 1, Thomas Eadie 2 and Gerald C. Llewellyn 1 Department of Biology,VirginiaCommonwealthUniversity,Richmond,Va. 23284,U.S.A. 2 VirginiaDivisionof ConsolidatedLaboratoryServices,Richmond,Va. 23219U.S.A.

Abschiitzung der Hemmwirkung von Honig auf Pilzwachstum, Sporulierung und Aflatoxin-Produktion Zusammenfassung. Unbehandelter Honig wurde mit toxigenen St~immen yon Aspergillus flavus NRRL 5862 und Aspergillus parasiticus NRRL 2999 beimpft. Der Pilz wuchs und sporulierte in mit verschiedenen Wasser-Mengen verdiinntem Honig, aber keine der Kulturen produzierte nachweisbare Mengen yon Aratoxin. Wachstum und nachfolgende Sporulation konnte nut in Medien mit nicht mehr als 60% Honig entdeckt werden. Medien mit 40% Honig zeigten innerhalb yon 2 Tagen Wachstum und Sporulation. Selbst in 10% w~i6riger HoniglSsung produzierte keine der Aspergillus-Arten Toxine. Diese Ergebnisse erh~irten unsere frtiheren Beobachtungen, dab reiner Honig das Pilzwachstum hemmt und dab sogar verdiinnte Honig-LSsungen f~ihig sind, die Toxin-Produktion zu hemmen. Die yon AOAC empfohlenen Arbeitsanweisungen fiir Extraktion und Diinnschichtchromatographie waren auch bei der Honig-Analyse auf Aflatoxin erfolgreich. Summary. Unprocessed honey was inoculated with toxigenic strains of Aspergillusflavus NRRL 5862 and A. parasiticus NRRL 2999. The fungi grew and sporufated in varying amounts of honey diluted with water, but none of the cultures produced detectable levels of aflatoxin. Growth and subsequent sporulation were seen only in media containing up to and including 60% of honey. Media having 40% of honey showed growth and sporulation by day two. Neither species of Aspergillus produced toxins even in 10% honey. These results confirm our earlier observations that pure honey inhibited fungal growth and now even diluted honey seems capable of inhibiting toxin production or possibly neutralizing it. The general procedures reOffprintrequeststo: T, E. T. Wellford(Addresssee above)

commended by the AOAC for extraction and thin layer chromatography were applied successfully in analyzing the honey substrate for aflatoxin.

Early twentieth century studies report the invasion of the bee hive [1, 2], and more recently the bee itself [3] by fungi including Aspergillus flavus. In the 1960's it was found that some strains of A.flavus and A. parasiticus secrete toxic and carcinogenic metabolites [4, 5]. Almost all animals seem to be sensitive to these metabolities, aflatoxins [6]. These mycotoxins are thought to be of particular importance as potential contaminants in some foods and feeds, such as storage grains and peanuts [7]. Studies with insects indicate that this group also responds to aflatoxins [8]. Previous studies in our laboratory showed that ariatoxin B 1 (AFB1), the most potent and carcinogenic of the group [9], caused increased levels of lethality in worker bees [10]. Other studies by Hilldrup et al. [11] and Gunst et al. [12] have reported that aflatoxigenic fungi can grow, sporulate, and produce toxin on most bee-related substrates. Hilldrup et al. [11] reported that only honey apparently failed to support fungal growth. To further clarify and confirm this reference to honey, the present investigation was undertaken to evaluate growth, development of mycelia, and aflatoxin occurrence in various concentrations of raw honey artificially inoculated with two aflatoxigenic fungi. In addition, an evaluation of the application of established analytical procedures for aflatoxin analysis were undertaken with honey samples.

Materials and Methods One 454g sampleof uncookedand unblendedhoney(Orange,Pure, Grade A; ShermanFoods,Inc., Bronx,N.Y.) was the singlesource ofmediumusedin thesestudies.

T. E. T. Wellford et al.: Inhibitory Action of Honey

281

Table 1. Mycelial Growth and Sporulation of Aspergilli on Honey

the addition of the extracting solvent, chloroform and water 90/10

(v/v). Honey in Media %

A.flavus NRRL 5862

A. parasiticus NRRL 2999

Growth

Growth

Sporulation

100

.

.

.

.

.

.

. --

Sporulation

80

.

60

Slight by Slight by 6 days 13 days By 36 days 50% of the surface was covered

Slight by 6 days

40

Slight by 2 days

By 36 days most of the surface was covered

Slight by -3 days. Heavy by 6 days By 36 days most of the surface was covered

20

Slight by By 2 days 2 days. 50% of surHeavy by face was 3 days covered By 36 days the whole surface was covered

Slight by Slight by 2 days. 2 days. Heavy by 3 Heavy by 6 days days By 36 days the whole surface was covered

10

Slight by Slight by 2 days. 2 days. Heavy Extremely by 6 days heavy by 3 days By 36 days the whole surface was covered

Moderate by 2 days. Heavy by 3 days

Slight by 2 clays

Slight by 2 days. Heavy by 6 days

By 36 days the whole surface was covered

Preparation of Culture Media Forty four clear, glass, two ounce, prescription phials, plugged with cotton wool were sterilized with a small amount of water. After cooling, the water was decanted and various amounts of new, sterile water and uncooked honey were added under aseptic conditions. The concentrations of honey made with the sterile water varied from 0 to 100%. Specifically, the concentrations had the following percentages of honey by volume: 100%, 80%, 60%, 40%, 20%, 10%, and 0%. The total liquid volume in the culture phials was 5 ml.

Inoculation of Media Two known toxigenic fungal species, Aspergillusflavus NRRL 5862 and Aspergillus parasiticus NRRL 2999 were used. Equal aliquots of spores from each species were inoculated separately into triplicate phials containing the above listed concentrations of honey, The spores for both species were taken from individual potato-dextroseagar slants maintained in our laboratory. Culture phials that were not inoculated were used to determine natural flora content of the honey.

Incubation, Extraction, and Quantitation Procedures All cultures were incubated at room temperature, 25 __+3° C, in subdued light for 36 days. Daily observations for fungal growth and sporulation were made. Each culture, including both natural flora, control, and inoculated samples, were attenuated after 36 days with

Twenty-five ml of extracting solvent were placed into each bottle. The bottles were capped and shaken gently for one hour. They were allowed to stand overnight and the upper layer, acqueous chloroform, was used for aflatoxin determinations. Twenty microliter samples were compared visually with reference samples spotted simultaneously utilizing the official AOAC procedure [13]. Dilutions were made as necessary and determinations were repeated in triplicate. A set of samples "spiked" with 10 ng of aflatoxin B 1 per ml of honey was also extracted to verify the procedure.

Results

Funyal Growth and Sporulation Two days following inoculation, mycelial growth and slight sporulation were observed in the 10%, 20%, and 40% honey cultures inoculated with both fungal species. It required six days for growth and 13 days for sporulation in 60% honey. A. parasiticus cultures did not sporulate in concentrations above 40%. As indicated in Table 1, 80% and 100% cultures of honey failed to support observable growth or sporulation by either species. Some bacterial growth was observed in the samples containing 50% or less natural flora concentrations of honey. No AspergiIlus-like fungi were observed in the natural flora cultures.

Toxin Production No aflatoxins (less than 2ppb) were detected in any of the inoculated, control or natural flora samples. Samples from extracts showing heavy fungal growth were concentrated further and still no toxins were detected. Discussion

The preliminary studies undertaken by Hilldrup et al. [11] reported a similiar result; no growth or production of aflatoxin following inoculation of honey. Grout [14] claimed that osmophilic yeasts were the only type of micro-organism that could live on honey, and only then if the honey has not ripened or contained greater than 19% water. Honey normally has a water content of approximately 17% as well as a variety of organic and inorganic compounds (Table 2). When attempting to interpret the results of our study, several parameters may be considered to explain the absence of toxin production in the media. First, there is the possible influence of the osmotic state of honey, due to the large amount of sugars present. Grout [14] reported that ordinary yeasts do not cause fermentation of honey because they are unable to grow in the high sugar concentration. This would be a major factor in pure honey, and could prohibit germination and mycelial growth. But, as the osmotic

282

environment decreases, in this case by the addition of water, a point is reached where there was mycelial growth (Table 1). Another factor that must be considered is pH. In this study, the pH was found to be altered by the increased addition of water. This parameter is further complicated by the fact that as these fungi grow, the pH of the media changes [-15]. The pH of the media was measured in our study and found to range from pH 5 down to pH 2, depending on the concentration of honey in the media. Lie and Marth [-15] reported growth and toxin production after 10 days in artifical media by both A. flavus and A. parasiticus in this pH range. For this reason we believe that lack of toxin production was due mostly to the high osmotic conditions found in honey, but the influence of pH cannot be disregarded. The mould itself would not grow in concentrations greater than 60% of honey. It was shown some time ago 1-16] that various pathogenic bacteria were killed when introduced into honey. This was also thought to be due to the high osmolarity and acidity of honey. Also, Grout [14] stated that spoilage of honey by bacteria is not possible due to the high acidity of honey. In the study reported herein, even after growth of mycelia and some sporulation in the diluted honey, there were no detectable levels of aflatoxin produced by these known toxigenic strains. To confirm that the fungi had not changed or become a non-producer, spores were inoculated into sweetened-coconut-flakecultures containing 20 g of substrate and i0 ml of HaO and grown for one week at room temperature. The presence of aflatoxins was confirmed in all these cultures for both species. Absence of toxin in honey could be due to several factors in addition to those mentioned above. For example, the media could have a poor nutritional balance, but this seems unlikely, due to the numerous ingredients in honey including several sugars (Table 2). Another possibility is the presence of a substance in honey that breaks down the toxin once it is formed. There is no evidence for this, but one compound that reportedly is formed in low levels in honey is hydrogen peroxide. It is an oxidizing agent produced by glucose oxidase [-17]. It has been suggested that this enzyme is added to honey by the worker bees before it is fed to the larvae, in order to prevent contamination by bacteria [14]. Buchen and Marth [-18] have shown that hydrogen peroxide can detoxify aflatoxin. There are other aflatoxin-inhibitory substances, in addition to honey. These include phytic acid [19] and hydroxy-cinnamic acid derivatives [19, 20]. Also, the several stages of fungal growth must be considered, beginning with the germination of spores, growth of mycdia, sporulation, toxin production, and

T.E.T. Wellford et al.: Inhibitory Action of Honey Table 2. Composition of Honey a Component

Percentage

(%)

Water 17.20 .79.59 Sugars Fructose 38.19 Glucose 31.28 1.31 Sucrose Maltose and other reducing disaccharides 7.31 Higher sugars 1.50 Acids (gluconic, citric, malic, succinic, formic, acetic, 0.57 butyric, lactic, pyroglutamic and amino acids) 0.26 Proteins Ash (minerals: potassium, sodium, calcium, magnesium, 0.17 chlorides, sulfates, phosphates, silicia, etc.) Enzymes Present Invertase (converts sucrose to dextrose and levulose Diastase (converts starch to dextrins) Glucose Oxidase (converts dextrose to gluconolactone) Catalase (decomposes hydrogen peroxide) Phosphatase (decomposes glycerophosphate) a From White, 1975 (17)

detoxification. At least one or more of the above noted factors could be interacting synergistically to inhibit any one of the above fungal stages. In conclusion, it is evident had mould and subsequent aflatoxin contamination is not anticipated as a problem associated with honey, even though bees themselves are affected by the toxins [10], and other bee substrates support fungal growth and toxin production [11]. Further delineation of the inhibitory process seems worthy of additional study and should include studies using a buffer system to maintian a constant pH in inoculated cultures. Also studies evaluating the action of specific enzymes and other products in the system should be included. Documentation of inhibitory properties relating to the action of enzymes and their products could hold potential value for application to other aflatoxigenic susceptible foods. Acknowledgements We wish to thank Mr. Glenn Hoke, Virginia Commonwealth University, for assisting in various aspects of the study. The authors appreciate the cooperation provided by Paul E. Irvin and H. F. McGowan of the Virginia Division of Consolidated Laboratory Services for mycotoxin analyses. Clerical assistance was provided by Brenda C. Wellford.

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(19o6)

T. E. T. Wellford et al. : Inhibitory Action of Honey 2. Burnside, C.E.:"Fungus diseases of the honey bees." U.S.D.A. Tech. Bull 149, (1930) 3. Foote, H. L.: Am. Bee J. 106, 126 (1966) 4. Asplin, F.D., Carnaghan, R. B.A.:Vet. Rec. 73, 1215 (1961) 5. Barnes, J. M., Butler, W. H.: Nature (Lond.) 202, 1016 (1964) 6. Wogan, G. N., Newberne, P. M.: Cancer Res. 27, 2370 (1967) 7. Diener, U.L., Davis, N.D.: Aflatoxin formation by Aspergillus flavus. Aflatoxin: Scientific Background, Control and Implications (Goldblatt, L. A. ed.). New York: Academic Press 1969 8. Lalor, J. H., Chinnici, J. P., Llewellyn, G. C.: Dev. Ind. Microbiol. 17, 443 (1976) 9. Butler, W.H.: Liver injury and aflatoxins. Mycotoxins in Foodstuffs. (Wogan, G. N. ed.) Cambridge, MA: MIT press 1965 10. Hilldrup, J. A., Llewellyn, G. C.: Va. J. Sci. 28, 63 (1977) 11. Hilldrup, J.A.L., Eadie, T., Llewellyn, G.C.: J. Assoc. Offic. Anal Chemists. 60, 96 (1977) 12. Gunst, K.W., Hilldrup, J.L., Llewellyn, G.C.: J. Apic. Res. In press (1978)

283 13. Horwitz, W., Chichilo, P., Reynolds, H.: Aflatoxins: Official Methods of Analysis. Ass. Off. Anal. Chem. Section 26014-26 019, Washington, DC 462 (1975) 14. Grout,R.A.: The Hive and the Honeybee. Hamilton, IL: Dadant and Sons PuN. 1963 15. Lie, J.L., Marth, E.H.~ J. Dairy Sci. 51, 1743 (1968) 16. Sackett, W.G.: Colo. Agric. EXp. Sta. Bull. 252, 18 (1919) 17. White, J. W. Jr.: Honey. The Hive and the Honey Bee. Hamilton, IL: Dadant and Sons Publ. 1975 18. Buchen, S.Y., Marth, E.H.: J. Food Prot. 40, 617 (1977) 19. Swaminathan, B., Koehler, P. E.: J. Food Sci. 41, 313 (1976) 20. Gupta, S.K., Venkitasubramanian, T.A.: Z. Lebensm. Unters.Forsch. 159, 107 (1975)

Received February 17, 1978