Comparison of resistance of fungal spores to gamma and electron beam radiation

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International Journal of Food Microbiology 26 (1995) 269-277

Comparison of resistance of fungal spores to gamma and electron beam radiation G. Blank Department

*, D. Corrigan

of Food Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada Received 8 March 1994; accepted 20 September 1994

Abstract The irradiation sensitivity of fungal spores to either gamma or electron beam irradiation was evaluated in distilled water. The D,, (the dose required to reduce the initial population by 90%) gamma values ranged from 0.236 to 0.416 kGy and from 0.209 to 0.319 kGy for Penicillium and Aspergirius species, respectively. The D,, electron beam values ranged from 0.194 to 0.341 and from 0.198 to 0.243 kGy for Penicillium and Aspergillus species, respectively. Of the aspergilli species evaluated, only half exhibited significantly (P < 0.05) greater sensitivity to the electron beam treatment compared to gamma irradiation. Four of the six penicillia species evaluated also exhibited significantly (P < 0.05) higher sensitivities to electron beam treatment. Keywords:

Resistance;

Fungi; Gamma

radiation;

Electron

beam radiation

1. Introdluction Exposing eliminating

food to radiation treatment delays spoilage and improves safety by or reducing pathogenic microorganisms. The effectiveness of the

treatmem is dependent on several factors including the composition of the food, the number and type of microorganisms and the dose (Diehl, 1990). Food preservation using radiation is principally achieved using a gamma source such as 6oCo or electrons. generated by high-energy electron beam accelerators. Electron beams and gamma rays differ greatly in their ability to penetrate matter. Generally gamma rays exhibit higher penetration into food compared to electron beams (Cleland and Pageau, 1985). In foods where surface decontamination and/or

* Corresponding author. Tel.: + 1 (204) 474-8742. Fax: + 1 (204) 261-1488. 0168-1605/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0168-1605(94)00129-4

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.I. Food Microbiology 26 (1995) 269-277

disinfestation is desired, for example, in the case of cereals containing either molds or insects, the use of electron beam radiation may be preferred. This preference is largely based on economics due in part to the greater efficiency of the irradiator (Diehl, 1990; CAST, 1989). The susceptibility of microorganisms and/or their spores to gamma radiation has been well established (Thayer, 1993; Saleh et al., 1988). In contrast, information pertaining to the resistance of food microorganisms exposed to accelerated electrons is relatively sparse. Therefore the aim of this investigation was to evaluate the resistance of microorganisms to electron beam radiation and compare results to those obtained by gamma radiation. Spores of commonly occurring fungi which were part of an on going study with respect to grain decontamination, were employed as test microorganisms.

2. Materials and methods 2.1. Fungal cultures and maintenance The fungal organisms used in this investigation included: Aspergillus echinulatus ATCC 1021, Aspergillus niger ATCC 52172, Curvularia geniculata ATCC 11153 and Penicillium roqueforti ATCC 10110. These organisms were obtained from the America Type Culture Collection, Rockville, MD. Aspergillus ochraceus NRRL 3174 and Aspergillus versicolor NRRL 573 were obtained from National Regional Research Laboratories (Agriculture Research Service, USDA, Peoria, IL). Aspergillus fimigatus (832), Aspergillus glaucus (838), Penicillium aurantiogriseum (3298), Penicillium granulactum (526), Penicillium verrucosum (798) and Penicillium viridicatum (1117) were obtained from the Plant Pathology Laboratory, Manitoba Agriculture, Winnipeg, MB. Altemaria altemata and Cladosporium cladosporiodes isolates were obtained from Agriculture Canada, Winnipeg, MB. Penicillium cyclopium isolated locally from grain was confirmed by the USDA Agriculture

Research Service, Peoria, IL. All cultures were grown on potato dextrose agar (PDA, Difco Laboratories, Detroit, MI) slants at room temperature for 14 days and maintained at 4°C. Cultures were transferred to fresh PDA slants every 30 days. 2.2. Conidiospore preparation and harvest Conidiospores from maintenance cultures were inoculated onto fresh PDA slants and incubated at 25°C for 14 days in the dark. Resultant conidia crops were harvested in the following manner: sterile distilled water (5 ml) was added to culture slants and the conidia were gently dislodged using a sterile glass rod. The conidia suspensions were collected in sterile screw-cap test tubes (16 X 100 mm) containing 15 ml of sterile distilled water and twice filtered using sterile Pasteur pipettes (4.62 mm) containing loosely packed glass wool. The procedure was used to remove mycelial fragments and conidial clumps (Saleh et al., 1988). The

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J. Food Microbiology 26 (1995) 269-277

271

suspensmns were checked microscopically for conidial purity. The conidia suspensions were diluted to 10-5-10-6 colony forming units (CFU)/ml using sterile distilled water. Conidia concentrations were confirmed using a serial dilution technique. CFU were enumerated in duplicate on PDA (25°C 5 days). 2.3. Evaluation of recovery media for irradiated spores CFU/ml) of either P. roqueforti, A. in sterile distilled water as outlined previously. Triplicate suspensions of each organisms were irradiated (0.38 kGy) using electron beam treatment and then serially diluted using Butterfield’s phosphate-buffered dilution water (PDW). The linear accelerator (Impela I-10/1; AEEL, Whiteshell Laboratories, Pinawa, MB) used in this study produced lo-MeV electrons, with a nominal total beam power of 1 kW. Dose rate at the sample position was ca. 1 kGy/s. Survivors were enumerated in duplicate using a pour plate method employing the following media, PDA-non acidified (pH 6.3, Becton Dickinson Microbiology Systems, Cockeysville, MD), PDA-acidified (0.1% tartaric acid; pH: 3.5). Czapek-Dox agar (Difco Laboratories, Detroit, MI), standard plate count agar @PC, Becton Dickinson Microbiology Systems), SPC fortified with chloramlphenicol (Sigma Chemical Corporation, St. Louis MO, 100 mg/l) and Sabouraud dextrose agar (Difco). All plates were incubated at 25°C for 5 days. The entire protocol was repeated using an irradiation dose of 0.61 kGy. Individual spore suspensions

(105-lo6

glaucus ‘or Cl. cladosporiodes were prepared

2.4. Spore irradiation and D,, determination Aque80us spore suspensions (5 ml, 105-lo6 CFU/ml) contained in sterile screw-cap test tubes (16 x 100 mm) were randomly coded for either electron or

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Fig. 1. Evaluation of recovery media for irradiated (0.38 kGy) spores. Potato dextrose agar, PDA; acidified PDA, APDA, Czapek-Dox agar, CZD; Standard plate count agar, SPC, SPC fortified with chlorampenicol, SPCC, Sabouraud dextrose agar, SBD. Bars represent the mean f S.D., n = 6.

272

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/ Int. .I. Food Microbiology 26 (1995) 269-277

gamma irradiation treatment and dosage level. Spores treated with electron beam irradiation were exposed to the following target doses: 0, 0.3, 0.6 and 1.0 kGy at 20-22°C. All irradiation trials were performed on samples which were laid horizontally on foam insulation and packed with ice. For gamma irradiation (Gammacell 220; AECL, Whiteshell Laboratories, Pinawa, MB) test tubes containing the samples were supported in an aluminum disc assembly with holes at the circumference. The assembly was packed in a 2-l beaker of crushed ice; target irradiation doses of: 0, 0.3, 0.6 and 1.0 kGy were used. The gamma ray dose rate was 12.2 kGy/h. Irradiation treatments for both gamma and electron beam were performed in duplicate. Absorbed radiation doses were determined by using radiochromic dye films (GAF, Miller and McLaughlin, 1981) enclosed in test tubes and irradiated along with the sample tubes. The absorbance of the irradiated films was measured at 600 nm and the absorbed dose calculated from a calibration curve. Following irradia-

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Fig. 2. Survivor curves for aspergilli spores following either gamma (-_) or electron beam (- - -) treatment. A. echinulatus, a; A. fumigatus, b; A. glaucus, c; A. niger, d; A. ochraceus, e; A. versicolor, f. Bars represent means f S.D., n = 4.

G. Blank, D. Corrigan / Int. J. Food Microbiology 26 (1995) 269-277

273

tion, th.e suspensions were serially diluted in PDW and pour plated in duplicate using PDA. Incubation was carried out at 25°C for 5 days. Survival curves were constructed by plotting the survivor CFU/ml versus actual radiation dose. Curves were fitted by linear regression using Figure Plotter (Fig. P Software Corporation, Durham, NC; 1990). Radiation sensitivity was expressed in terms of D,, values. A D,, value is defined as the dose required to reduce a given population by 90% of its initial value. The D,, value was determined from the reciprocal of the slope for the straight-line portion of the survival curve (L.ey, 1983). 3. Results Recovery levels for P. roqueforti spores following irradiation appeared similar for all media (Fig. 1). In the case of A. glaucus and Cl. cladosporiodes, poorest

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Fig. 3. Survivor curves for penicillia spores following either gamma (-_) treatment or electron beam (- - -) treatment. P. aurantiogtieum, a; P. cyclopium, b; P. granulactum, c; R roqueforti, d; P. verrucosum, e; P. viridicatum, f; Bars represent means& S.D., n = 4.

274

G. Blank, D. Corrigan /ht.

J. Food Microbiology 26 (19951269-277

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recovery was obtained on acidified potato dextrose. A similar recovery profile was obtained when the irradiation dose was increased to 0.61 kGy and/or when the protocol was repeated using a gamma source (data not included). The survivor curves for Aspergillus spores are shown in Fig. 2. Overall results indicated that electron beam treatment was significantly (P < 0.05) more effective in only half of the species evaluated. In the case of the penicillia, four of the six species exhibited significantly (P < 0.05) higher sensitivity to electron beam treatment (Fig. 3 and Fig. 4) when compared to gamma irradiation. C. geniculata and A. altemaria exhibited greater sensitivity to electron beam treatment, however, over the target dose range employed (0.0 to 1.0 kGy), it appeared that CL cladosporiodes spores were equally resistant to both forms of treatment.

G. Blank, D. Corrigan / Int. J. Food Microbiology 26 (1995) 269-277

275

Table 1 Summary of D,, values for irradiated spores Microorganism

Irradiation treatment (kGy) Gamma

Electron beam

A. echinulatus A. jkmigutus A. glaucus A. niger A. ochraceus A. versicolor P. aurantiogriseurn P. cyclopium P. granulactum P. roquefiwti P. verrucosum P. viridiccttum C. genicuiaia Cl. cladosporiodes Alt. alternata

0.319 = 0.276 a 0.250 =

0.241 b 0.198 b 0,243 a

0.245 a 0.209 a 0.282 a

0.199 b 0.198 a 0.234 a

0.236 0.397 0.239 0.416 0.266 0.333 1.798

0.194 a 0.290 b

a = a a a a a

0.201 b 0.341 0.208 0.265 1.193

b b = b

NDa

ND

2.409 a

1.099 b

a D,, values were not determined.

Values in rows having the same superscript are not significantly different (P < 0.05; Student t-test).

The D,, values for all organisms were determined from the slopes of the survivor curves and are summarized in Table 1. The D,, gamma values for the Aspergillus spp. ranged from 0.245 (A. niger) to 0.319 kGy (A. echinulatus) and from 0.198 (A. ochraceus, A. fwnigatus) to 0.243 kGy (A. glaucus) for electron beam treatment. For the Penicilfium spp., the D,, gamma values ranged from 0.236 (R aurantiogriseum) to 0.416 kGy (P. roqueforfi) and from 0.194 (P. aurantiogriseum) to 0.341 kGy (P. roqueforti) for electron beam treatment. The D10 values for both C. geniculata and A. alternata were at least 3-times greater than all of the aforementioned organisms.

4. Discussion Injured microorganisms surviving process treatments including heat or irradiation quite often require exacting recovery conditions in order to undergo repair (Foegeding and Busta, 1983). In an effort to assess the post-irradiation recovery of spores jt was deemed necessary to evaluate various commonly used media. Based on these results, all media investigated with the exception of acidified PDA yielded similar results. In the case of acidified PDA it is likely that the low pH and/or acidulant interfered with spore repair. Increasing the dose or changing the radiation source did not appear to alter the recovery profile on the media examined. O’Neill et al. (1991) investigated the effect of common grain fungi to gamma irradiation and reported no significant differences in survivors based on recovery media. Munzer (1969) also reported that when Aspergiks flaws spores were

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G. Blank, D. Corrigan /ht.

J. Food Microbiology 26 (1995) 269-277

subjected to gamma irradiation, only slight differences in growth were observed among Sabourand, potato dextrose and malt extract agars. Czapek agar, however, exhibited the poorest growth. In the present study, PDA was arbitrarily chosen as the recovery medium. D,, values obtained from the dose-response curves in this study were higher in all cases with gamma irradiation as compared to electron beam treatment. Chelack et al. (1991) working with Aspergillus alutaceus var. alutaceus compared the irradiation efficiency between gamma and electron beam treatment. The authors also reported higher D,, values when gamma irradiation was used. However, significant differences in treatment efficacy between irradiation sources were exhibited by only one-half and two-thirds of the aspergilli and penicillia species, respectively. Conidiospores of Altemaria and Curvulatia did, however, exhibit significantly higher resistances to gamma treatment. Differences between the two irradiation processes with regards to microbial lethality focus mainly on efficiency. The efficiency of electron accelerators is higher than that of gamma sources because the electron beam can be directed at the product or microorganism, whereas the gamma sources emit radiation in all directions (Diehl, 1990). Thus, whereas a typical 6oCo gamma source can deliver a dose rate of ca. 12 kGy per hour, an electron beam facility can deliver several tons of kGy per second (Allen et al., 1990). The resistances CD,, values) of the penicillia and aspergilli to gamma irradiation were comparable to those reported by O’Neill et al. (1991) and Saleh et al. (1988). In some instances, however, direct comparison of D,, values could not be made because of differences in either the inocuhtm size, strain or suspending menstruum. Spores of Altemaria and Curvularia exhibited higher resistances compared to the penicillia and aspergilli, regardless of radiation source. As pointed out by Saleh et al. (1988), the presence of multicelled thickwalled macrocondia may impart radiation protection to these fungi. Cladosporium which also produces multicellular thick-walled spores (Malloch, 1981) appeared to be radiation resistant within the applied dose range. In conclusion, although the D,, values for all conidiospores were higher when gamma treatment was applied, statistical results indicated that electron beam treatment was significantly more effective in only one-half to two-thirds of the organisms evaluated. The application of electron beam treatment, although lacking the penetration of 6oCo, may have clear advantages in the areas of decontamination or deinfestation. In addition, since radionuclides are not part of the process, it may help to avail fears regarding radioactive accidents incurred during processing, storage and transportation.

References Allen, D.W., Crowson, A., Leathard, D.A. and Smith, C. (1990) The effect of ionising radiation on additives present in food contact polymers, in: Food Irradiation and the Chemist (Johnson, D.E. and Stevenson, M.H., Eds.), Royal Society of Chemistry. CAST: Council for Agricultural Science and Technology (1989) Task Force Report No. 115. June 1989. Ionizing energy, in: Food Processing and Pest Control: ii. Application.

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Chelack, W.S., Borsa, J., Marquardt, R.R. and Frohlich, A.A. (1991) Role of the competitive microbial flora in the radiation-induced enhancement of ochratoxin production by Aspergillus alutaceus var. alutaceus NRRL 3174. Appl. Environ. Microbial. 57, 2492-2496. Cleland, M.R. and Pageau, G.M. (19851 Electrons versus gamma rays -alternative sources for irradiation processes, in: Food Irradiation Processing, Proceedings of a symposium held in Washington, D.C., March 1985. Int. Atomic Energy Agency, Vienna. Diehl, J.F. (1990) Biological Effects of Ionizing Radiation in Safety of Irradiated Foods. Marcel Dekker Inc.., NY. Foegeding, P.M. and Busta, E.F. (1983) Hypochlorite injury of Clostridium botulinurn spores alters germination responses. Appl. Environ. Microbial. 45, 1360-1364. Ley, F.I. (1983) Food irradiation, in: Food Microbiology: Advances and Prospects. The Society of Applied Bacteriology Symposium Series No. 11 (Roberts, T.A. and Skinner, E.A., Eds.). Academic Press, London. Malloch, D. (19811 Moulds: Their Isolation, Cultivation, and Identification. University of Toronto Press, Toronto, pp. 64-65. Miller, A. and McLaughlin, W.L. (19811 Evaluation of radiochromic dye films and other plastic dose meters under radiation processing conditions, in: High-dose Measurements in Industrial Radiation Processing, pp. 119-138. Tech. Rep. Ser. 205, International Atomic Energy Agency, Vienna. Munzer, R. (1969) Uber einige die strahlenemfindlichkeit von schimmelpilzen beeinflussende factoren. Arch. Mikrobiol. 64, 349-356. O’Neill. Damoglou, K., A.P. and Patterson, M.F. (1991) Sensitivity of some common grain fungi to irradiation on grain and in phosphate-buffered saline. Lett. Appl. Microbial. 12, 180-183. Saleh, Y.G., Mayo, MS. and Ahearn, D.G. (19881 Resistance of some common fungi to gamma irradiation. Appl. Environ. Microbial. 54, 2134-2135. Thayer, D.W. (1993) Extending shelf life of poultry and red meat by irradiation processing. J. Food Prot. 56, 831-833.