Mycofloral profile and the radiation sensitivity (D10 values) of solar dried and gamma irradiated Pleurotus ostreatus (Jacq.Ex. Fr.) Kummer fruitbodies stored in two different packaging materials

Abstract The presence of fungi in our foods poses serious health risks as some genera of fungi may produce certain mycotoxins which have carcinogenic, mutagenic, teratogenic, and immunosuppressive effect on humans and animals alike. Fruitbodies of Pleurotus ostreatus were solar dried at a moisture content of 12.5 ± 0.2% and stored in polythene and polypropylene packs, gamma irradiated at doses of 0 (control), 1, 2, 3, 4, and 5 kGy at a dose rate of 1.7 kGy/hr from a Cobalt 60 source (SLL, 515, Hungary) and stored at room temperature 28–30°C for a period of 12 months. Mycological analyses were done at intervals of 0, 3, 6, and 12 months. A total of eleven (11) fungi belonging to eight fungal genera were isolated on both Cooke's and DRBC media; Aspergillus (A. niger, A. flavus, A. fumigatus, A. tamarii), Rhizopus (R. oligosporus), Mucor (M. racemosus), Fusarium (F. oxysporum), Penicillium (Penicillium sp.), Trichoderma (T. viride), and Rhodotorula sp. were recorded. There was a significant (p < .05) reduction in initial mycofloral population by an average of 2.2 log cycles as well as in species numbers with increasing doses of radiation. Radiation sensitivity (D10 values) also ranged between 1.68–2.78 kGy. Gamma irradiation treatment is one way which can enhance food safety through the reduction in potential pathogens and has been recommended as part of a comprehensive program to enhance food safety.

Generally, foods that are not properly handled and stored are prone to microbial contamination resulting in the occurrence of certain harmful microorganisms which pose a health hazard. According to Schardl, Panaccione, and Tudzynski (2006) a plethora of fungal spores exists in the environment and these fungal spores which when dry, float through the air and find suitable conditions where they can start the growth cycle again (FSIS, USDA, 2006) and actively growing molds. Fungal contamination of food may be one of the more insidious but seldom recognized causes of diseases. Fungi produce mycotoxins which are adaptable and potent in causing some severe diseases such as cancer and furthermore, damage vital organs such as the liver, kidney, and brain (IARC, 1993). A variety of fungi (Fusarium, Claviceps, Penicillium, Trichothecium, Aspergillus, Cephalosporium, etc.) may contaminate foods and produce illness with symptoms such as vomiting, diarrhea, headaches, chills, dizziness, and blurred vision. Prevention of fungal invasion of commodities is by far the most effective method of avoiding mycotoxin problems and could be achieved by gamma irradiation.
The long standing use of gamma radiation in the preservation of fruits, vegetables, pulses, cereals, and dried products has shown encouraging results (Addo, 2008;IAEA TECDOC, 2006;Kortei, Odamtten, Obodai,Appiah, Adu-Gyamfi et al., 2015;Kortei, 2015;Odamtten, 1986;Odamtten, Appiah, & Langerak, 1986). The recommendation by the joint FAO/IAEA/WHO Expert Committee on the wholesomeness of irradiated foods gave the impetus for the acceptability of food irradiated up to an overall dose of 10 kGy and the adoption by the Codex Alimentarius Commission for irradiated foods in 1983, has contributed to a wide acceptance of food irradiation as a preservation method.
Currently, National Public Health Authorities of 26 countries have guaranteed some 140 unconditional and provisional clearance covering many different products treated with gamma radiation for human consumption (IAEA, 2004). Nevertheless, in order to utilize irradiation as a food processing technology, it is very important to study the radiation sensitivity of contaminating microorganisms since this provides a basis for accurate estimation of lethal/killing doses (Thayer, 2000).
Sensitivity to irradiation varies among bacterial and fungal species and is affected by the components of foods and temperature during irradiation and subsequent storage (Adu-Gyamfi, Appiah, & Torgby-Tetteh, 2012;Kortei, 2015;Neimira, 2007). The D 10 -value (decimal reduction dose) is the radiation dose required to inactivate 90% of a viable microbial population or reduce the population by a factor of 10 (Smith & Pillai, 2004). Estimation of D 10 -values may be incorporated into risk assessments for designing processes for reduction in microbial populations in food (Cheroutre-Vialette & Lebert, 2000).
Yeasts and fungi play a major role in the spoilage of foods in Ghana as their growth on foods can cause major quality problems. Some fungi produce potent mycotoxins which could be carcinogenic, mutagenic, teratogenic, and allergic (Tournas, 2005;Kortei, Odamtten, Obodai,Appiah, Adu-Gyamfi et al., 2015). A good packaging material should not harbor and support growth of microorganisms and should also be able to maintain the lower water activity of the mushrooms after drying during storage. The objective of this study therefore, was to evaluate the mycofloral profile of solar dried and gamma irradiated P. ostreatus fruitbodies stored in two packaging materials and determine their radiation sensitivity (D 10 values) in vivo.

| Determination of moisture content
The method prescribed by AOAC, (1995) was employed in the determination of the moisture content.

| Enumeration of mycoflora
The decimal serial dilution plate technique was used in estimating fungal populations. About 10 g fresh weight of sample was placed in 250 ml Erlenmeyer flask containing 100 ml sterile distilled water. The mixture was shaken at rev/min in a Gallenkamp Orbital Shaker for 30 min. Aliquot (1 ml) of the suspension was placed in sterile universal bottles (MaCartney tubes) containing 9 ml of 0.1% peptone, and was serially diluted up to 1:10 −3 . The fungal population was enumerated on modified Cooke's medium (Cooke, 1954) and Dichloran Rose Bengal Chloramphenicol (DRBC) agar incubated at 30-32°C for 5 to 7 days for species diversity.

| Characterization and Identification of fungal isolates
Fungal isolates were examined under stereo-binocular microscope (Leica 261, Germany) using the needle mounts technique. Their identification was performed according to macro and micromorphological characteristics. All the isolates were identified up to the species using keys and manuals (Barnett & Hunter, 2006;Larone, 1986;Samson, Hoekstra, & Frisvad, 1995). The percentage (%) occurrence of fungi was calculated by the formular according to Sreenivasa, Dass, and Janardhana (2010).

| Packaging materials and storage
The mushroom samples (40 g) each were packaged in either transparent polythene (19 cm x 13 cm) and or in transparent polypropylene (18.5 cm x 12.5 cm x 5 cm) pouches and stored at the prescribed temperatures for up to 12 months.

| D 10 values determination
The D 10 value is the reciprocal of the slope of the exponential part of a survival curve. This value may also be obtained from Equation (1). The data was subjected to regression analysis. The surviving fractions, log 10

| Statistical analysis
The values obtained for total fungal counts were transformed to logarithm conversions and subjected to analysis of variance (ANOVA) and means separated by Least Significant Difference (LSD) using SPSS version 9 for windows. In the non-irradiated samples there were significant differences (p < .05) between the radiation treatments (1-5 kGy). The same trend was obtained on raising the fungi on Cooke's medium. Although there were increases in fungal population during storage in polythene and polypropylene bags for 3-12 months, the population counts were still low and within the acceptable limits counts (0.86-1.8 log 10 CFU/g) especially in samples treated with radiation doses 2-5 kGy. Similar trends were recorded for spores incubated on Cooke's medium (Figure 4).

| Percentage occurrence of fungal species in the irradiated dried mushrooms
The phenology of the resident fungi in samples stored in polythene and polypropylene packs after radiation (0-5 kGy) for up to 12 months F I G U R E 1 Mycoflora population of mushroom fruitbodies stored in polypropylene packs for up to 12 months and isolated on DRBC medium at 28-32°C  persisted on the fruit bodies from an initial >30% but increased to <50% in 12 months.

| Radiation sensitivity
Results obtained are presented in Figures 13 and 14. The curves were linear and correlation ratio values R 2 obtained were positive ranging from R 2 = .79-.95 (Table 1). From these curves the D 10 values were calculated immediately after irradiation with respect to packaging material used. The D 10 values ranged between 1.68-2.78 kGy depending on the package material. (Figures 13 and 14). The effective dose for killing fungi was close to what was found for B. cereus (Kortei et al., 2014) in both packaging packs, that is, D 10 = 0.76-3.21 kGy (Table 1).

| Mycoflora population on fruit bodies
Post irradiation storage studies revealed a marginal increase in mycoflora population. Presumably, physical environmental factors such as moisture and temperature in the packs could marginally support growth of microorganisms (Food Safety, 2003).
It was also observed that there was reduction in fungal growth  (2011) showed that a dose of 2.0 kGy was not sufficient to completely reduce the main toxigenic Aspergillus spp. resident in soil samples.
Although minor changes in the fungal morphology were observed, ultra-structural changes at cell wall level and the increase in mycotoxin production ability at 2 kGy were observed.
Storage period studies revealed a general significant increase in mycofloral population on the dried mushroom fruit bodies. Kortei The differences observed between the mycoflora of mushrooms stored in the two packaging materials were not statistically significant (p > .05). Nonetheless, for food storage, polypropylene would be the most preferred since it is more robust, aesthetically good and can keep safe from declining quality.

| Radiation sensitivity curves of fungi on fruit bodies after irradiation (Cooke's and DRBC)
Radiation sensitivity (D 10 values) of fungi on dried P. ostreatus ranged between 1.68 and 2.61 kGy for samples stored in polythene and polypropylene, respectively, plated on DRBC; D 10 values of 2.60 and 2.78 kGy were obtained for same samples stored in polythene and polypropylene packs, respectively, and plated on Cooke's medium (Table 1). Statistical differences (p < .05) observed F I G U R E 1 3 Radiation sensitivity curves of total fungi on mushroom fruit bodies immediately after irradiation and raised on Cooke's medium    reduction in mycofloral population by > 2 log cycles with the application of gamma irradiation. Gamma irradiation has been proposed as an efficient process to eliminate toxigenic fungi before the initiation of mycotoxins production and has been applied to several foods for extension of shelf life.

| CONCLUSION
The use of irradiation as a complement of the good manufacturing practices (GMP) and may constitute a strategy that could be applied together with other methods to prevent and control the presence of toxicogenic fungi in stored mushrooms.