Gamma Irradiation-Recycling of Aspergillus Flavus and its Effect on Radiation Resistance and Toxin Production

Gamma Irradiation-Recycling of Aspergillus flavus and its Effect on Radiation Resistance and Toxin Production A. T. M. Faizur-Rahman 1 and E. S. Idziak Department of Microbiology and School of Food Science Macdonald Campus of McGill University Macdonald College, Quebec HOA ICO 1

Present address: Radiobiology Division Atomic Energy Centre Dacca, Bangladesh.

Abstract The effect of gamma irradiation-growth recycling on radiation resistance and toxin production by Aspergillus flavus NRRL 2999 in various natural fruit and vegetable media was studied. Radiation cycling of spores of A. fiavus nine times at radiation doses equivalent to 0 x, I x, and 2 X D IO levels did not result in spore crops with increased radiation resistance. Also there was, in general, no increase in total toxin production, on the contrary, toxin production decreased in most instances.

Resume Ce rapport concerne I'effet du recyclage accumulatif de ['irradiation gamma sur la resistance aux radiations et sur la pr?duction de toxine~ de Aspergillus f1avus NRR6 2999 dans divers milieux a base de frUIts et legumes naturels. Le recyclage de spores de A. flavus neuf fois it des doses de radiation equivalentes it 0 x, I X, and 2 X D w n'a pas augmente la resistance des spores it la radiation. De plus, dans I'ensemble, la production totale de toxines n'a pas augmente, ayant meme diminue au contraire dans la plupart des cas.

Introduction Repeated irradiation of various bacteria with gamma energy has resulted in increased radiation resistance, altered biochemical and morphological characteristics, and changes in virulence and toxicity (Davies and Sinskey, 1973, Previte et al., 1970, Epps and Idziak, 1970, Choudbury and Hossain, 1967). The results of these studies did not indicate any increased health hazard. In instances of low-dose irradiation processing systems (radurizationradicidation), fungi were observed to survive and subsequently proliferate under favourable conditions. Further, fungi, especially in tropical countries, are responsible for a large amount of food spoilage and, in some instances, toxicosis (Shank et al., 1972, Wogan, 1972, Detroy et al., 1971). Irradiation treatment of a variety of foods in developing countries offers a means of food preservation where Western methods of storage are not readily available. Other workers have shown that single doses of 60CO gamma irradiation of spores of Aspergillus flavus and A. ochraceus resulted in cultures capable of producing notably more toxin than control cultures (Applegate and Chipley, 1975, 1974, 1973). No recycling experiments comparable to those done with bacterial systems have been performed. The purpose of this study was therefore to ascertain whether a mycotoxin producing fungus subjected to. repeated doses of gamma energy became more radiatlOn resistant and/or produced increased levels of toxin.

Materials and Methods A·flavus NRRL 2999, received from Dr. M. van Wal-

beek, I-iealth Protection Branch, Ottawa, was grown on poCan. Inst. Food Sci. Technol. J. Vol. 10. No. I. 1977

tato dextrose agar (Difco), spores were harvested and single spores cultures established using appropriate dilutions. These cultures were tested for toxin production in a medium containing 2% yeast extract and 15% sucrose (YES, Davis et al., 1966). Spores from the toxigenic cultures were preserved on sterilized silica gel (Grivell and Jackson, 1969) and stored at 4°C (stock cultures). Various fruit and vegetable juices were used as media in the irradiation-growth recycling experiments. Lemon, orange, tomato and grape juices, collected by hand squeezing, were filtered through four layers of fine mesh nylon. Extracts of apple, banana and carrot were obtained by boiling slices in two volumes of distilled water (15 min.), cooling, blending, and finally filtering as above. For measuring toxin production, the media were sterilized at 121 °C for 15 min. For recycling experiments and in determining radiation resistance, the media were steamed three times for 30 min. at WO°C with a 24 hr. period in between. The final pH of the lemon, grape, apple, orange, tomato, banana, and carrot media were 2.7,3.1,3.3,3.7,4.1,5.0 and 6.0 respectively. Spores from stock cultures were allowed to germinate and eventually sporulate in the different natural media. Spores were harvested and resuspended in fresh media for subsequent radiation recycling as shown in Figure 1. STOCK SPORES

I

suspend in medium Incubate 30·C (98rmination. and sporulation)

HARVEST

SPORES

resuspend in fresh medium

Irradiate Incubate 30·C (C)erminotion a sporulation)

o

jrad-

I

•

HARVEST SPORES

Resuspend in fresh medium

I

I

i +1

I I I

I.... ...J

40 I Krod-l

80 I Krad-

1

I +I +I + I I

•

HARVEST SPORES

i

I

I I I

1_",_

HARVEST SPORES

I

I

I I

L~J

Fig. I. Protocol for irradiation-growth recycling of spores of A. flavlIs NRRL 2999.

5

The spore crops obtained after .the various recycling procedures were aHowed to germinate and sporulate in the different media supplemented with 1.5% agar. The resultant spores, resuspended in fresh liquid media were subjected to increasing doses of gamma irradiation (Gammacell 220, A.E.C.L. Canada, dose rate 5 'Krad/min.) to determine their radiation resistance. Survivors were enumerated on the prepared natural media supplemented with 1.5% agar. Five ml aliquots of the different media in 25 X 150 mm test tubes were inoculated with the spore crops obtained after the various recycling procedures and incubated for 7-14 days at 30°e. At selected times, cultures were withdrawn and extracted twice with chloroform. Aflatoxin B, and G 1 in these extracts were separated on silica gel T.L.e. plates developed in benzene-methanol-acetic acid (24: 2: I) for 2 hrs. (Stoloff et al. 1971). Fluorescent spots were detected under the V.V. Chromato-vue cabinet (Ultra-violet Products, Inc. Calif.), rf compared with those of B1 and G, standards on the same plate (Sigma Chemicals Co., St. Louis, Mo.), and intensity measured by scanning through a fluorimeter (T.L.e. Scanner, G. K. Turner Assoc., Calif.). Total aflatoxin production (B, and G,) was then calculated. The presence of aflatoxin B1 and G 1 was further demonstrated by spraying the plates with p-anisaldehyde spray reagent and heating at l30°C for 10 min. (Scott et al., 1970). Growth and reactions on various carbohydrate sources were determined using the A.P.I. 20E test system (Analytab Products Inc., N.Y.).

Results and Discussion None or very little growth of A. flavus occurred in lemon, apple, or tomato agar media. The radiation sensitivity of A. flavus spores from grape, orange, banana, and carrot agar media is similar-irradiation at 160 ~rads reduced the viable count by circa 4 log cycles (FIgure 2). This sensitivity is similar to that recorded for other strains of AspergillUS (Applegate & Chipley, 1975, 1974, 1973); thus spores of A. flavus are moderately sensitive to gamma energy. . In bacterial systems, low dose (1 X D,o Krad) radIation recycling was observed to produce cultures with increased radiation resistance after 5-6 cyclings (Previte et al., 1971). This was not demonstrated with A. flavus spores harvested from natural media after recycling nine times at 0, 40, and 80 Krad (0, I X and 2 X D,o Krad) (Table 1). No differences in gross morphology of A. flavus grown ill the natural media and on Czapek agar were noted as a result of the radiation recycling treatment. Several changes in carbohydrate utilization did, however, occur as a result of recycling the spores of A. flavus (Table 2). Spores recycled at 0 and 80 Krad in grape, orange, banana and carrot media produced an acid reactio.n in amylose whereas the Parent spore crop produced a baSIC reaction. Other minor changes in reaction were also noted in sorbose, rhammose, saccharose and melibiose. Sporulation of 40 Krad recycled cultures was delayed 12-14 days and subsequently greatly reduced in the low pH media, ego in grape and orange as compared both to the 0 Krad recycled control cultures and Parent stock cul6

Grape juice Orange juice Bonona extract Carrot extract

~ 6 :::>

......... 0--0

-

o o

w

0:

~ CJ)

5

W

-I

->~4 lI..

o

C)3

o-I

2 20

60

DOSE

100 ( Krad)

140

Fig. 2. Survival of spores of A. jiavus NRRL 2999 suspended in different natural media and subjected to varying doses of 60CO gamma Ir· radiation.

tures in the same media. In contrast 80 Krad recycled cultures sporulated at approximately the same time as the recycled controls. . Toxin production by the Parent cultures was co~sIder­ ably lower in the high acid grape and orange medIa (pH 3.1 and 4.1) than in the banana and carrot media (pH 5.0 and 6.0) (Figure 3). Davis et al. (1966), however, observed that variation in initial pH from 3.0 to 6.4 in a 20% sucros.e, 2% yeast extract medium did not greatly influence tOXill production and growth. The differenc~ ~etw~en the t~o sets of results is probably due to the vanatIOn In the medIa used. Recycling, without irradiation, resulted in a marked decrease in toxin production in grape, orange, and carrot media' whereas that in banana medium was, during the early phase of growth, slightly higher than that found with the Parent stock culture. Irradiation recycling at 40 Krad resulted in a complete loss of toxin production in grape, orange, and carrot media and in greatly reduced levels of toxin in the banana medium. Cultures recycled at 80 Krad produced less toxin than the Parent stock cultures except when recycled in orange medium. In grape juice medium (low pH), ~he amount of toxin produced by the recycled cultures dunng 7 days was approximately 50% of that of the Parent culture. After 14 J. Ins!. Can. Sci. Technol. Aliment. Vol. 10, No. I. 1977

Table \. Radiation sensitivity of Aspergillus flavus NRRL 2999 spores obtained after radiation recycling at 0, 40, and 80 Krad. D,,, (Krad) After 5 cyclings at

After 9 cyclings at

Medium

Parent Stock

oKrad

40 Krad

80 Krad

oKrad

40 Krad

80 Krad

Grape Orange Banana Carrot

34.3 37.9 30.2 34.7

3\.0 32.2 3 \.4 30.4

30.5 27.3 27.8

28.6 26.0 29.0 33.4

38.4 35.5 33.1 37.1

40.1 29.3 3 \.2 37.2

31.3 30.2 31.9 37.8

Table 2. Alterations in carbohydrate reactions by Aspergillus flavus NRRL 2999 as a result of nine recyclings at 0, 40, and 80 Krad. II Media

Amylose

Grape

B~a31

Orange Banana Carrot

B~a"

Altered reactions on 2) Melibiose Rhamnose 6)

Saccharose

Sorbose

a~B'"

a~B4I

a~b" b~A3H'"

A~B"

b~a3l"

b~a"

Carbohydrates tested - amylose, arabinose, glucose, inositol, mannose, melibiose, rhamnose, saccharose, sorbose. 2) a slight acid; A - acid; b - slight basic; B - basic. JI. 41." Alteration in reactions as a result of recycling. 31 after recycling at 0 Krad, 41 after recycling at 40 Krad 51 after recycling at 80 Krad. 61 No change in reaction as a result of recycling. II

10

5

E

....

---

GRAPE

ORANGE

20

10

~

7

z C(

...J IL

14

10 BANANA

)(

0

.... 50

C(

25

~ ~ ~

--

50

10

10

~

14 DAYS

0 K,od

••

40 K,od

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7

I

14

CARROT

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- •• ---•

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;1\

PARENT

7

••• •

100

~ * 7

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DAYS

80 K,od

Fig. 3. Affiatoxin (B, - G,) production by A. flavus NRRL 2999 subjected to nine irradiation-growth recyclIng at 0, 40, and 80 Krad in 4 different natural media. "Detectable amount of toxin produced. Can.lnst. Food Sci. Techno!. J. Vol. 10. No. I. 1977

7

days, however, the amount of residual toxin in both culture systems was at the same low level (Figure 3). In orange juice medium, although toxin production by the radiation cycled cultures after 7 and 14 days was several fold higher than that produced by the Parent culture, nevertheless the amount was either equal to (')r considerably lower than the level of toxin produced in the other media by the Parent cultures. In carrot and banana media, the 80 Krad recycled cultures produced only barely detectable amounts of toxin during all stages of growth. These results may be compared with those of other authors who observed that the surviving spores after a single irradiation dose of from 10 to 300 Krad were capable of producing notably increased levels of mycotoxin10 to 60% greater than that produced by control non-irradiated cultures (Applegate and Chipley, 1975, 1974, 1973). In our experiments the increased toxin production was observed only with spores recycled at 80 Krad in orange juice. In conclusion, radiation recycling of spores of A. flavus nine times at a radiation dose equivalent to 1 X and 2 X their 0 10 values did not result in spore crops with increased radiation resistance. In addition, radiation recycling did not, in general, result in cultures exhibiting notably increased toxin production. On the contrary, total toxin production decreased in most instances as a result of irradiation-growth recycling.

8

Acknowledgements: Financial support from Canadian International Development Agency was greatly appreciated.

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fungi of stored rice. Food Irradiation (France) 8:

30~33.

Davies. R. and A T. Sinskey. 1973. Radiation resistant mutants of Salmonella typhimurium LT. 2: development and characteristics. J. Bacteriol. 113: 133~144. Davis, H. D., U. L. Diener and D. W. Eldridge. 1966. Production of Aflatoxins B, and G, by As· pergillus flavus in a semi-synthetic medium. Appl. Microbiol. 14: 378-380. .. Detroy, R. W., E. B. Lillehoj, and A. Ciegler. 1971. Aflatoxins and related compounds In Mlcrobialloxins Vol. VI, eds. Ciegler, A., S. Kadis, and S. J. Ail, Academic Press, N.Y. pp. 19-25. Epps, N. A. and E. S. Idziak. 1970. Radiation treatment of foods. Public health significance of irradiation-recycled Salmonella. Appl. Microbiol. 19: 338-344. Grivell, A. R. and J. F. Jackson. 1969. Microbial culture preservation with silica gel. 1. Gen. Microbiol. 50: 423-425. Previte. T. J., Y. Chang, W. Scrutchfield, and H. M. EI-Bisi. 1971. Effects of radiation pasteurization on Salmonella II: Influence of repeated radiation growth cycles in virulence and resistance to radiation and antibiotics. Can. 1. Microbiol. 17: 105-110. Scott, P. M., J. W. Lawrence, and M. van Walbeek. 1970. Detection of mycotoxins by thin-layer chromatography. Application to screening of fungal extracts. App!. Microbiol. 20: 839-842. Stoloff, L., S. Nesheim, L. Yin, J. V. Rodricks, M. Stack, and A. D. Campbell. 1971. A multimycotoxin detection method for aftatoxins, ochratoxins, zearalenone, sterigmatocystin and patulin. J. Assoc. Off. Anal. Chern. 54: 91-97. Shank, R. C, G. N. Wogan, T. B. Gibson and A. Nondasuta. 1972. Dietary aflatoxin' and human liver cancer. II. Aftatoxins in market foods and foodstuffs of Thailand and Hong Kong. Fd. Cosmet. Toxicol. 10: 61-69. Wogan, G. N. 1972. Assessment of exposure to aflatoxins in Proc. of host environment interaction in the etiology of cancer in man. Primostin, Yugoslavia, pp. 237·241. Received June 29, 1976.

J. Inst. Can. Sci. Technol. Aliment. Vol. 10, No. I, 1971