Occurrence of fungal contamination in pistachio and peanut samples from retail shops in Sanandaj province , Iran

Aspergillus flavus produces secondary metabolites called aflatoxin that has powerful teratogenic, mutagenic and hepato-carcinogenic effects. The aim of this study was to evaluate the A. flavus of pistachio and peanuts from Sanandaj, Iran. Pistachio and peanut nuts samples were obtained from dried fruit retail shops of Sanandaj, 2011. One hundred grams from each sample were sterilized in a 0.4% sodium hypochlorite solution for 2 min. Subsequently, the sample was rinsed once in distilled water and allowed to dry. Each sample was grounded into powder by vortex and then 1 g was poured into 100 ml of sterile distilled water and stirred. Next, 1 ml of the supernatant was inoculated into Petri dishes containing Sabouraud dextrose agar then incubated at 25°C for five days. The grown fungi were identified by standard mycological techniques based macroscopic and microscopic morphology. Of the 132 samples (peanut: n = 81; pistachio: n = 51), fungi were detected in almost 72% of the samples. The A. flavus was the most predominant isolate from peanut (19%) and pistachio (22%) samples. There was a significant relationship between A. flavus contaminations in the peanuts and pistachio with high humidity. Therefore, due to the isolation of high percentage of A. flavus as the main aflatoxins producer in nature, we recommended the need for good storage practices in order to prevent the occurrence of aflatoxins in peanuts and pistachio.


INTRODUCTION
Pistachio and peanut kernels are usually contaminated by a range of fungi.Several fungi are capable of infecting growing nuts and causing damage to hulls and kernels (Denizel et al., 2006).Contamination of nut seeds by fungi occurs during growth, harvesting and storage, where climatic conditions and agricultural and storage practices especially when nuts are stored under adverse conditions of temperature and relative humidity are *Corresponding author.E-mail: e.ghahramani1@gmail.comTel: +98-918-3806512.
conducive to fungal growth and toxin production (Njobeh et al., 2009;Bhattacharya and Raha, 2002;Bankole et al., 2005;Abdel-Gawad and Zohri, 1993).The storage temperature, moisture content, presence of oxygen and gaseous composition are the most important factors influencing the development of fungi during storage in these nut seeds (Ayyasam and Baskaran, 2005).The fungi that produce mycotoxins are various species of Aspergillus, Penicillium, and Fusarium, but mycotoxin production is restricted to only a few species.
Aspergillus flavus are common contaminants of peanuts and pistachio nuts that is a serious problem as it has adverse effects on human health (Hedayati et al., 2007;Peraica and Domijan, 2001).Some 300 to 400 compounds are now recognized as mycotoxins, of which approximately a dozen groups regularly receive attention as threat to human and animal health.Species of the A. flavus produce highly carcinogenic aflatoxins that can cause hepatomas in human (Wogan, 1966).Studies showed that aflatoxins have been shown to increase the incidence of human liver cancer by acting synergistically with hepatitis (Mishra and Das, 2003).Hence, there is a great need for more extensive investigations, where nuts production and consumption are predominant.It has also been estimated that 25% of the world's crops are affected by mould or fungal growth (Bryden, 2007).The economic loss resulting from fungal contamination of nuts is difficult to estimate.Previous studies showed that 30.97 million tones of greasy seed products, mainly pistachio and peanut, of different Asian and African countries were contaminated by A. flavus and Aspergillus parasiticus (Dekoe et al., 2000;D'Mello and Macdonald, 1998).They had been infected during or after harvesting, storage and transition (Bruce et al., 2003).
Although the natural contaminants by mycoflora and mycotoxins of various kinds of nuts such as peanut and pistachio nut have been investigated in many parts of the Iran and world (Moghaddam et al., 2006;Kabirian et al., 2011;Hedayati et al., 2010;Shahidi, 1996;Khosravi et al., 2007), none of these studies reported on the contamination by fungi of nuts in Sanandaj.Therefore, this work was done to determine fungi, especially A. flavus of pistachio and peanuts, from retailers and dried fruit retail shops of Sanandaj province in Iran.

Collection of samples
Sampling was done randomly on some human food products such as pistachio and peanut.A total of 50 -100 g of each sample were taken in sterile pockets coincidentally among dried fruit retail shops from several geographic regions between December and April 2011 in Sanandaj, under standard conditions.Also the various factors, including relative humidity and temperature (TES, Taiwan) were measuring and reported.The date and place of the sampling were labeled on the pockets and immediately transferred to the laboratory.

Determination and identification of fungi
Approximately 100 g from each sample were sterilized in a 0.4% sodium hypochlorite solution for 2 min.Subsequently, the sample was rinsed once in distilled water and allowed to dry.Each sample was ground into powder by vortex and then 1 g was poured into 100 ml of sterile distilled water and stirred.Next, 1 ml of the supernatant was inoculated into two Petri dishes containing Sabouraud dextrose agar (Scharlau, Spain) and then incubated at 25°C for five days.The grown fungi were identified by standard mycological techniques based macroscopic and microscopic morphology.The fungi that could not be identified by this manner were sub-cultured on potato dextrose agar (PDA, Merck, Germany), water agar and /or slide cultures for further study.Also, Aspergillus species were identified using Czapek-dox agar (CZA, Merck, Germany) media and according to the method of Raper and Fennell (Raper and Fennell, 1965).The yeast was identified with subculture onto corn meal agar and Tween 80 and CHROMagar Candida (Becton Dickinson, Spark, MD).

Statistical analysis
The mean percentage of recovery of each fungus was calculated as the ratio of the number of samples contaminated by each fungus over the total number of samples submitted to analysis.All data were analyzed using the software SPSS ver.16.Statements of significance were based on p<0.05.

RESULTS
A total number of 150 (peanut: n = 70; pistachio: n=80) nuts samples were obtained.In total, 1207 fungal colonies were isolated from the samples.The contamination mean value for positive samples was 1.25 × 10 3 CFU/g.The fungi were detected in almost 62% of the peanut and pistachio kernel samples.The pistachio (70%) was the most contaminated samples.The contamination mean values of peanut and pistachio were 0.93 and 1.6%, respectively.As shown in Tables 1 and 2, the genera isolated from different nuts included Aspergillus, Penicillium, Fusarium, and yeast.The most predominant isolated fungus was Aspergillus 77(70%), followed by Penicillium 11(10%), Fusarium 6(5%), and yeast 12(10%).The Aspergillus was the predominant genus in its count and frequency and was isolated from all samples of nuts tested; it comprised 69.2 and 71.1% of fungi in pistachio and peanut nut seeds, respectively.
A. flavus was the most predominant isolate from 56.2% pistachio and 44.3% peanut samples.This fungus had the highest contamination mean value in pistachio and peanut kernels were 0.33 and 0.29 ×10 3 CFU/g, respectively.The highest incidences of colonization by A. flavus generally occurred at seed relative humidity of 55% and temperatures of 35°C.There was a significant relationship between A. flavus contaminations in the peanuts and pistachio with high humidity (p<0.05).

DISCUSSION
Among the species of fungi, A. flavus was found as the commonly isolated species found in both consumption forms of peanut and pistachio kernel samples in our study.These findings are also similar to those of other studies (Abdel-Gawad and Zohri, 1993;Hedayati et al., 2007;Kabirian et al., 2011;Hedayati et al., 2010;Awuah and Kpod, 1996;Thomson and Mehdy, 1978).Research on Iranian pistachio in Kerman gardens showed that 25% of the pistachio products that have gapped or lost their shells during early period of growth and 12.5% of the immature splitted ones were contaminated by A. flavus and other Aspergillus species (Shahidi, 1996).In a study  by Hedayati et al. (2010), A. flavus also had the highest contamination mean value in dry roasted and raw form of peanut and pistachio kernels.The experimental results of Kabirian et al. (2011) showed that the rate of pistachio contamination to A. flavus in samples taken from Semnan Province is varied with the type of processing plants.
Presumably traditional processing plants and storing have more population density of A. flavus than semimechanized processing plants (Moradi and Javanshah, 2006).The results of our study showed significant relationship between A. flavus contaminations in the peanuts and pistachio with high humidity.Also previous studies have shown that relative humidity plays a vital role in the development and spread of fungal contaminations (Nawar, 2008).The report of different fungal contamination rate by different investigators may be a result of adverse pre-harvest conditions of temperature and humidity in the field and improper postharvest handling and storage (Nakai et al., 2008).The increase in the frequency of Aspergillus can also be explained by the fact that this genus is considered to be storage fungi, unlike Fusarium that exist in field.In addition, Aspergillus spp.grow well at lower moisture content in contrast to other fungi as they require very high moisture content in the substrate for growth and mycotoxin synthesis (Hedayati et al., 2010).This situation can cause the decay of stored products and potential health hazards for humans and economic losses.Nut growing areas have very similar meteorological conditions for A. flavus growth and aflatoxin production (Moghaddam et al., 2006).It is important to control A. flavus and its aflatoxin production to increase pistachio exportation in the country.It is also necessary to find various ways for control management of these fungi so as to control wilting and inhibit toxic production in pistachio.Soil solarization and biological control application are the best methods for controlling soil borne fungi and A. flavus, as currently used in many countries.

Conclusion
In summary, we recommend the storage of peanut and pistachio nuts in a controlled storage condition (e.g.storage in high air-permeable sacks, cool and dry condition, assisted drying) as soon as possible to control the infection caused by A. flavus.In addition, further research is needed to explore other means of preventing mould infection and aflatoxin production for each region of exporting countries.

Table 1 .
Frequency distribution (count, %) of fungi isolated from different nut products.
*Mean values for positive samples.

Table 2 .
Mean fungal contamination (viable count, × 10 3 CFU/g) and the frequency of fungi isolated from pistachio and peanut kernel samples.
*Mean values for positive samples.