Comparative Occurrence of Resident Fungi on Gamma Irradiated and Steam Sterilized Sorghum Grains (Sorghum bicolor L.) for Spawn Production in Ghana

Sorghum is one of the important cereals consumed by humans, animals and also used for the production of mushroom spawns in Ghana. Aim: Identification of fungi present on sorghum grains before and after pretreatment (steam and gamma radiation) principally for mushroom cultivation. Original Research Article Kortei et al.; BBJ, 7(1): 21-32, 2015; Article no.BBJ.2015.042 22 Methodology: The total number of mycoflora (Log10 CFU g -1 ) of sorghum grains and their relative frequency (percentage occurrence) associated with the raw grains and the mycoflora present after subjecting the sorghum grains to gamma radiation doses of 0, 5, 10, 15, 20, 25 and 32 kGy at a dose rate of 1.7 kGy/h from a Cobalt-60 source (SLL-515, Hungary) and moist heat at a temperature of 100120oC for 22.5 hours was evaluated. Mycological analysis was done by direct plating method on Cooke’s and Dichloran Rose Bengal Chloramphenicol (DRBC) media. Results: Nine fungal species belonging to six genera were associated with the sorghum grains. Among these fungi were Cladosporium macrocarpum, Trichoderma harzianum, Fusarium oxysporum, Rhodotorula spp., Penicillium spp., Aspergillus niger, Aspergillus fumigatus, Aspergillus ochraceous and Aspergillus flavus. Comparatively higher fungal counts of 3.27 and 3.82 Log10 CFU g -1 were recorded for non-pretreated while lower counts of 0.5 Log10 CFU g -1 were recorded for pretreated sorghum grains. Gamma radiation and moist heat significantly (P<0.05) reduced total fungal populations by an average of 2.4 and 2.1 log cycles, respectively. Rhodotorula sp. (11.5%), Penicillium sp. (34.6%), Aspergillus fumigatus (29.9%) persisted on the moist heat sterilized while only Rhodotorula sp. (100%) persisted on gamma irradiated grains. Conclusion: These data indicate possible health hazards for humans and animals upon consumption of such contaminated food grain by toxigenic moulds and also reveal the sensitivity of fungal species to gamma radiation and moist heat as a selective substrate for oyster mushroom spawn preparation.


INTRODUCTION
Sorghum (Sorghum bicolor (L.) Moench) is the fifth most important cereal after rice, wheat, maize and barley [1]. It is the staple food grain for over 750 million people who live in the semiarid tropics of Africa, Asia, and Latin America [2,3]. The sorghum crop is still a principal source of energy, protein, vitamins and minerals for millions of poor people in these regions. Besides its traditional use as food crop, sorghum has other alternative uses such as livestock and poultry feed, potable alcohol, starch, ethanol production, numerous industrial purposes [4]. Significantly among the list is its usage for mushroom spawn production.
Numerous fungi associated with sorghum grains are implicated as macro / micro organisms responsible for competition with the mycelium of the cultivated mushroom. The source of contamination is largely dependent on such factors as place of origin, physiological maturity, storage quality, grain density are of serious concern due to their fungi toxigenic potential. Again, some major effects of fungal deterioration of grains include decreased germination, discoloration, development of visible mold growth, musty or sour odors, dry matter loss and nutritional heating, caking, and the potential for production of mycotoxins in the grain. According to [5], the toxigenic moulds commonly isolated from foods or grains are Aspergillus, Penicillium and Fusarium. In storage conditions, Aspergillus and Penicillium are predominant and the Fusarium spp. is an important plant pathogen. Aflatoxins (AFB 1 , AFB 2 , AFG 1 and AFG 2 ) are mycotoxins produced by Aspergillus flavus (AFB 1 and AFB 2 producer) and A. parasiticus (AFB 1 , AFB 2 , AFG 1 and AFG 2 producer). These species are commonly recognized in grains as maize or peanuts. Aflatoxin B1 is most toxic of the group followed in decreasing toxicity by AFG 1 , AFB 2 and AFG 2 . Aflatoxins are recognized in some species as responsible for toxic signs and lesions, reduced growth, immune suppression and liver cancer [6,7]. The International Agency for Research on Cancer has classified AFB 1 as a probable human carcinogen [8].
In Ghana, Pleurotus ostreatus (Jacq. Ex. Fr) Kummer, strain EM-1, is the most cultivated mushroom [9]. The spawns of this species of mushroom has been prepared using moist heat sterilized sorghum grains. The spawn which is often the innoculum is a network of pure culture of fungal vegetative tissues interweaves a medium such as cereal grain [10,11,12]. Published works reveals that different media has been used for spawn production such as wheat [13,14,15] Gamma irradiation as a physical treatment effectively eliminates spoilage and pathogenic microorganisms in foods [18,19,20,21] and has been utilized for the reduction and elimination of pathogens in foods [22,23]. However in order to utilize irradiation as a food processing technology, it is imperative to study the radiation sensitivity of contaminating microorganisms since this provides a basis for accurate estimation of inactivation doses [24,25]. Sensitivity to irradiation varies among microbial and fungal species and is affected by the components of foods and temperature during irradiation and subsequent storage [26,27]. The D 10 -value (decimal reduction dose) is the radiation dose required to inactivate 90% of a viable bacterial population or reduce the population by a factor of 10 [28]. There is a comparatively great range of D 10 -values and therefore differences in resistance to gamma radiation by various microorganisms of public health significance. Published data [29,30] on D 10 values for Aspergillus flavus was 0.43 and 0.5 kGy in buffered saline solution and in smoked herrings, respectively.
Studies on the relative radiation-resistant fungal species by Abouzeid et al. [29] illustrated that Aspergillus and Penicillium species are relatively sensitive to ionizing radiation with a D 10 values between 0.25 and 0.65 kGy whereas other species in the genus Fusarium are more resistant requiring high but safe D 10 v alues of 0.65 to 1.5 kGy. Estimation of D 10 -values may be incorporated into risk assessments for designing processes for reduction of microbial populations in food [31]. This paper seeks to assess the mycofloral population, species diversity and compare the effect of gamma irradiation and moist heat sterilization on the mycofloral population of sorghum grains for spawn preparation.

Collection of Samples
Sorghum samples (approximately 1000 g) were collected from Madina market, Accra, Ghana in 2013. Samples were brought to the laboratory in sterile plastic bags and kept at 4ºC. All the samples were subjected to mycological analysis.

Moist Heat Sterilization
Grains were steeped in water overnight for about 12 hours. About 265 g of grains were packed into bottles and then transferred into transparent heat resistant polypropylene bags (24 cm x 38 cm) and then plugged with cotton wool and covered with plain sheets. The sheets were held in place with rubber bands. The grains were sterilized in an autoclave (Priorclave, Model PS/LAC/EH150, England) at 121ºC for 1h.

Irradiation
Sorghum grains were soaked overnight and packaged as described above and then irradiated at doses 0, 5, 10, 15, 20, 25 and 32 kGy at a dose rate of 1.7 kGy per hour in air from a cobalt 60 source (SLL 515, Hungary). Doses were confirmed using the ethanol-chlorobenzene (ECB) dosimetry system at the Radiation Technology Centre of the Ghana Atomic Energy Commission, Accra, Ghana.

Determination of pH
According to AOAC [32].

Determination of Moisture Content
According to AOAC [32]

Enumeration of Mycoflora
The 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 140 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 [33] and Dichloran Rose Bengal Chloramphenicol (DRBC) agar incubated at 30-32ºC for 5 to 7 days for species diversity.

Characterization and Identification of Fungal Isolates
Fungal isolates were examined under stereobinocular microscope (Leica 261, Germany)

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 (N/N 0 ) of microorganisms, was calculated and used as relative changes of their actual viable cell counts. The D 10 values were calculated by plotting log 10 (N/N 0 ) against dose (D) according to the equation Where No is the initial viable count; N is the viable count after irradiation with dose D; D is the radiation dose [38,27]. The linear correlation coefficient (r 2 ) and the regression equations were also calculated.

Statistical Analysis
The values obtained for total fungal counts were transformed to logarithm conversions and subjected to analysis of variance (ANOVA) using SPSS (Chicago, IL) version 9 for windows.

RESULTS AND DISCUSSION
Results of the influence of gamma radiation and moist heat sterilization (steam) on the relative abundance and total microbial population on the surface of the sorghum grains indicated some significant (P<0.05) difference. Both methods of pretreatments were effective in reducing the microbial load. The non-pretreated (control) sample harbored comparatively higher fungal counts of 3.27 and 3.82 log 10 CFU/g enumerated from the Cooke's and DRBC growth media, respectively, (Fig. 1). Gamma radiation dose of 5kGy was able to reduce the mycofloral population by 1. Steam sterilization was also effective in reducing the total fungal population by 1.6 and 2.6 log cycles, respectively, for the two growth media. The effectiveness of steam sterilization in reducing the total fungal population was comparable to doses 10 kGy and 15 kGy. Doses beyond 15 kGy further reduced the total fungal populations to range 0.9-0.5 log 10 CFU/g on Cooke's medium. However on DRBC, steam sterilization reduced total fungal population to 1.23 log 10 CFU/g which corresponds to an intermediary of 10 and 15 kGy. Statistically, there were no significant (P>0.05) differences recorded between steam sterilization and gamma radiation doses of 10, 15, 20, 25 and 32 kGy (Fig. 1).
Results obtained corroborate results reported by Rico et al.
[40] who observed a 1-to-2 log cycle reduction in initial microbial count of 10 6 CFU/g with steam, while gamma irradiation at 10 kGy resulted in a 5-log cycle reduction in same initial microbial count as they investigated the comparative effect of steaming and irradiation on the physicochemical and microbiological properties of dried red pepper (Capsicum annum L.). In a similar work, Al-Bachir and Al-Dawi [41] reported a 1-to-2 log cycle reduction in total aerobic plate count with steam while a 4-log cycle reduction was recorded with 10 kGy dose of gamma radiation as they compared the effect of gamma irradiation and heating on the microbiological properties of licorice (Glycyrrhiza glabra L.) root powders.
Radiation sensitivity of fungi isolated from sorghum grains cultured on Cooke's and DRBC growth media were 7.9±kGy and 6.4±kGy respectively ( Fig. 2 (Fig. 4). Generally on both growth media, fungal populations and species number decreased as    Table 2. Surveys conducted worldwide also revealed that, A. flavus and A. niger were known to frequently contaminate peanuts and were able to produce mycotoxins such as aflatoxins [51,52,53,54]. A. flavus contamination and aflatoxin production in sorghum is a serious problem in most of sorghum producing countries where the crop is grown under rain fed conditions [54]. Fungi isolated in this work were common to previous mycological works by some researchers [55,56,57] on sorghum grains.

Moisture Content (%) and pH
In the present study, moisture content ranged 18.21±0.78 -18.85±0.65% for sorghum grains irradiated at doses 10 kGy and 32 kGy and also for control, respectively, ( Table 2) which apparently supported growth of a wide range of fungal diversity as well as load of > 10 3 CFU/g. Higher moisture content makes a substrate favorable for fungal invasion [58]. This is in direct agreement with the findings of Quezada et al. [59] who reported a gradual increase in fungal load and diversity with an increase in moisture content of stored maize sample. Moisture content along with substrate type and nutrient availability and presence of secondary metabolites also affect the extent of fungal contamination [60,61]. Essentially, moisture content which is too high (> 65%) could cause oxygen depletion and losses of nutrients through leaching [44]. On the other hand, low moisture content below a critical level (< 30%), would decrease activities of microorganisms by restricting the motility and make them dormant [62]. Under drier conditions, the ammonium and ammonia present generate a higher vapor pressure; thus conditions are more favorable for nitrogen loss.
The hydrogen environment of fungi is difficult to study because fungi change the pH as they grow. Some species increase and others decrease pH of their medium. pH of the medium is important because it influences mineral availability, enzyme activity and membrane function. Generally speaking, fungi can tolerate a wide range of pH, though most media used to culture fungi are acidic. During present investigation, samples with a low pH range (5.61±0.05 -6.36±0.04) were found to harbor a good number of fungi. Reports of [12,44] indicate that low pH (acidic range pH 4-6) favors good fungal growth and recolonization of fungi [63]. Generally, there were significant differences (P<0.05) between the treatments. Aspergillus was recorded as the most dominant genus in samples of all pH ranges; this can be attributed to the ability of the Aspergilli to grow in a wide range of pH. Wheeler et al. [64] reported that Aspergillus species are more tolerant to alkaline pH, while Penicillium are more tolerant to acidic pH. This is in accordance with our findings where A. niger and Penicillium sp. were recorded as the most dominant fungal species in the pH ranges of 3.50 to 7.0. Some scientists [65,12] stated that optimum pH ranges are mainly related to different species, strains, enzymatic systems, important vitamin entry in the cell, mineral capture, and surface metabolic reactions. High pH tends to suppress the growth as well as antagonize certain fungi in compost thus reducing competition for the mushroom [66].

CONCLUSION
The use of gamma irradiation treatment is a vital tool for the control of fungal microorganisms in foods and seeds. These products are often consumed raw or in their natural state. Data obtained reveal the type of fungi and an estimate of microbial loads on the sorghum grains as well as the level of reduction obtained when pretreated with steam and gamma radiation. Gamma irradiation proved to be an effective method for the control of microbes and so could be used as an alternative method of sterilization for sorghum spawn preparation. Despite the existence of these sterilization technologies, it is necessary to have a monitoring Program of Good Manufacturing Produce (GMP) and Hygienic practices to avoid fungal contamination during manufacturing process, storage and exposure of products on the market.