Evaluation of Fungal Flora and Mycotoxin in Some Important Nut Products in Erbil Local Markets

In this study a wide range of moulds representing several genera and species was recorded. Ten samples of each pure and salted (almond, cashewnut, hazelnut, Peanut and pistachio) were collected from different markets (Shekhalla, Tayrawa and Qaysari) in Erbil city, Iraq. High infection of fungal genera was found in pure samples, while low infection was found in salted nut products samples, application of nuts with salts was found to increase the resistance of nuts to invasion and colonization by the fungi during storage. The total counts of fungi were 138×10 cfu/g. Samples by dilution method and represented twelve genera and twenty species of fungi by all method. Detection of Mycotoxin on Dichloran Rose Bengal Chloramphincol Agar (DRBC) for fungal isolation from nut samples showed, A. flavus, A. fumigatus, A. niger and A. ochraceous, positive results on the culture for mycotoxin production, change in colony diameter and color change in compare with control Czapek Dox Agar (CDA). Estimation of natural occurrence of Aflatoxin (AT) and Ochratoxin (OT) in nut product samples by using ELISA method, the results show low AF and OT content in most samples which is lower than the normal rang for human consumption. The high results of aflatoxin and ochratoxin show in peanut while the low results show in cashewnut.


INTRODUCTION
Various nuts are used as a raw material in many industries as well as for a direct consumption.They contain an important of protein and fat and their products have wide acceptances food throughout the world (Watt and Merrill, 1950).Due to the extremely high fat, protein and low water content of various nuts such as hazelnut, almonds, walnuts, pistachio and cashewnut, these products are quite refractory to spoilage by microorganisms.Mould can grow upon them if they are stored under conditions that permit sufficient moisture for their propagation (Phillips et al., 1979;Frank, 1981).Contamination of different kinds of nut by fungi may occur at three different stages.It may first occur prior to harvest while the nuts are on the tree.At this stage, particularly when the nuts have ripened and the hulls have opened, they are often attacked by airborne and insect-borne spores of fungal species.The second stage of contamination may occur after harvesting, when nuts are de-hulled, washed and sorted.The washing water can be a source of contamination and if the nuts are allowed to remain wet, they will be highly susceptible to mould growth and mycotoxin contamination.The third stage of contamination can be in storage, especially when nuts are stored under adverse conditions of temperature (Abdel-Hafez and Saber, 1993;Burdaspal et al., 1990).The economic loss resulting from fungal and mycotoxin contamination of nuts is difficult to estimate.However, judging from the widespread occurrence of fungal and mycotoxin contamination and the large number of nuts affected, one can assume that such losses must be large.These losses constitute direct nut losses, human illness and reduced productivity and livestock losses from deaths and lower growth rates (E1-Magraby and E1- Maraghy, 1988).Additional economic losses include indirect costs of various systems for control of mycoflora and mycotoxins in nuts reduced value of rejected nuts costs of detoxification to recover acceptable products and, occasionally, from loss of export markets.Although the natural contamination by mycoflora and mycotoxins of various kinds of nuts such as almond, brazil nut, cashew nut, coconut, hazelnut, peanut, pistachio nut and walnut and different nut products were investigated in many parts of the world (Jimenez et al., 1991).A major problem related to fungal attack in nuts is the production of toxic secondary metabolites (Scott, 1993).Fungi can grow on simple and complex food products and produce various metabolites.These microorganisms exist in the environment and distribute by wind, insects and raining (Brus et al., 2005).Up to now, more than 100000 fungal species are considered as natural contaminants of agricultural and food products (Kacaniova, 2003).There are more and more indications that primary liver carcinoma and other serious diseases may be induced by consuming food or using raw materials for food processing contaminated with fungi or mycotoxins.Aflatoxins, ochratoxin and sterigmatocystin proved resistant to heat and have an ability to accumulate in the organism.The majority of the toxic species belongs to the genera Aspergillus, Penicillium and Fusarium.Other species of molds are frequently isolated, including Scopulariopsis and Sporendonema (Galvano et al., 2005;Zinedine et al., 2006).
The aim of the present study was to determine mycoflora distribution of nuts such as pure and salted (almond, cashewnut, hazelnut, Peanut and pistachio) and to determine the level of aflatoxin and ochratoxin in nut products.

Sampling:
Sampling was done randomly on some human food products such as nut products.A total of ten samples, representing various types of Nut Products salted (roasted and salted), unsalted (pure), shelled and unshelled were collected randomly from different markets (Shekhalla, Tayrawa and Qaysari) of different localities in the Erbil city during the months of November and December 2012.The nuts include Almonds (Prunus dulcis), Cashewnuts (Anacardium occidentale), Hazelnuts (Corylus colurna), Peanut (Arachis hypogaea) and Pistachio (Pistacia vera).A total of different markets were investigated and each sample was purchased from three different sellers.These products were chosen on the basis of their availability in the market and popularly of usage.Nut Products samples were usually found outside, kept in metal or plastic containers or wooden boxes.For each samples, 3 replicates were taken and mixed to prepare one composite sample.Each sample was sealed in a double sterile polyethylene bag and stored at 3-5°C until mycoflora determination and mycotoxin analysis were performed.In the laboratory, samples were individually finely ground in a common household blender.The blender's cup was rinsed in 85% alcohol between samples.The powder kept tightly packed in a new study bags and stored at 4°C for further analysis.The common names and scientific name of each sample are presented in Table 1.

Mycological analysis (isolation techniques): Isolation of fungi by direct plating method:
• Isolation of sample-borne mycoflora: The method used for isolation of fungi was previously described by Abdullah et al. (2002), 10 gm of each sample were surface sterilized by 6% sodium hypochlorite solution (NaOCl) in a sterile conical flask for 1-2 min and then washed by distill water 3 times for 1.5-2 min to removing the toxic activity of the chemical agent on the samples.The disinfected samples transferred with sterile forceps into Petri dish contain sterilized Czapek Dox Agar (CDA), at the rate of (5-10) pieces per plate, depending on the size of the particles, larger samples cut into small pieces.CDA supplemented with 0.5 mg chloramphenicol/mL to restrict bacterial growth.Three replicates were made and the plates were incubated at 25°C for 5-7 days.
Fungi colonies were identified according to morphological and microscopic characteristics.• Isolation of sample surface mycoflora: The samples transferred with sterile forceps into Petri dish contain sterilized CDA.Three replicates were made and the plates were incubated at 25°C for 5-7 days.Fungi colonies were identified according to morphological and microscopic characteristics (Pitt et al., 1992).

Standard dilution plate:
For fungal analysis, dilution method was used to determine total fungal counts in nut products samples.One grams of each composite sample (fine powder) were transferred into screw-capped medicinal bottle containing 9 mL of sterile distilled water and were mechanically homogenized at constant speed for 15 min.The sample-water suspension was allowed to stand for 10 min with intermittent shaking before being plated.Appropriate tenfold serial dilutions (1:10) were prepared and one mL portions of suitable dilutions of the resulting samples suspension (10 -3 ) were used to inoculate Petri dishes each containing 15 mL Potato Dextrose Agar (PDA).Plates were then incubated for 7 days at 28 o C. Three replicates plates per medium were used for each sample and the developing fungi were counted and identified according to several key processes.After incubation, the results were expressed in Colony-Forming Units (CFU) /g of samples; all plates were examined visually, directly and with a microscope (Aziz et al., 1998;Suleiman and Taiga, 2009).

Diagnosis: Identification of the fungal genera:
The fungal isolates were transferred to sterilized plates for purification and identification.The grown fungi were mounted on a slide, stained with lactophenol cotton blue to detect fungal structures (Basu, 1980), covered with a cover slip, examined under microscope and identified on the basis of their colony morphology and spore characteristics (Rajankar et al., 2007;Ronhede et al., 2005).

Dichloran rose bengal chloramphincol agar test:
DRBC is a selective medium that supports good growth of fungi.The substances in the DRBC are dichloran (is added to the medium to reduce colony diameters of spreading fungi), rose bengal (suppresses the growth of bacteria and restricts the size height of colonies of the more rapidly growing moulds) and chloramphenicol (is included in this medium to inhibit the growth of bacteria present in environmental and food samples).The reduced pH of the medium from (7.2 to 5.6) helps inhibition of the spreading fungi (Jarvis, 1973;King et al., 1979).The isolated fungi were inoculated in the solidified DRBC medium after incubation for 7 days at 25 o C search for pigmentation and color change observed due to toxigenic compound in compare to CDA medium control.DRBC containing compounds to inhibit or reduce spreading growth of moulds such as Mucor sp., Rhizopus sp., (Samson et al., 1992;Hocking et al., 2006).Dichloran and rose Bengal effectively slow down the growth of fast-growing fungi, thus readily allowing detection of other yeast and mold propagules, which have lower growth rates (Tournas et al., 2001).

Sample preparation for natural aflatoxin and ochratoxin determination by ELISA method:
Samples were homogenized and kept in glass bottle and stored at 2-8°C until further analysis.For the quantitative analysis of mycotoxins (aflatoxin and ochratoxin), Enzyme Linked Immounosorbent Assay technique (ELISA) was used.Samples (10 g) were taken in 50 mL of 70% methanol for aflatoxins and ochratoxin analysis.Sample powder was blended individually with high speed blender for three minutes.After blending the material was filtered with Whatman filter paper number 1, the filtrate was used for further analysis.Commercially available immunoassay kit Veratox for quantitative analysis of aflatoxin and ochratoxin test-NEOGEN Crop, Lansing, MI was used.The assay kit was based on Competitive Direct Enzyme Linked Immunosorbent Assay (CD-ELISA).The antibodies captured the analyte and conjugated to the enzyme (horse reddish peroxidase).Tetra methylbenzidine/hydrogen peroxide was used as a substrate for color development.Finally stopping solution was added to stop the reaction.The color intensity was inversely proportional to the mycotoxin concentration and measured with the ELISA reader.All necessary reagents were present in the kit.Concentration of mycotoxins was calculated by Log/logit Software Awareness Technology Inc. (Anonymous, 2000;Stoloff et al., 1991).

RESULTS AND DISCUSSION
Totally 10 samples were analyzed for enumeration of fungal isolates.Fungal genera were isolated by different method on two types of media: Czapek Dox Agar (CDA) and Potato Dextrose Agar (PDA).Common and scientific names of the nut products were tabulated in Table 1.
The results presented in Table 2 show fungi were isolated by using Agar Plate Method (APM) plated on CDA medium.Agar plate methods was employed for this study and two sets of samples were analyzed i.e., unsterilized and surface sterilized nut samples.A total of seven different fungal genera and sixteen species were isolated.All fungi were identified on the basis of their cultural and morphological characteristics.These were identified as Aspergillus spp.Cladospoium sp., Mucor sp., Rhizopus stolonifer, Penicillium spp.sterile mycellium and yeasts.It was observed that treated samples (with sodium hypochlorite) yielded less population of samples-borne fungi than the untreated   The results agree with many of the investigators working on seed pathology.Khomeiri et al. (2008), it was shown that collected peanut samples from Golestan, Mazandaran and Gilan provinces of Iran were contaminated by Aspergillus flavus.Sejiny et al. (1989) isolated fungi such as: Aspergillus, Penicillium, Rhizopus, Mucor and Cladosporium.Doster and Michailides (1994) showed the predominance of Aspergillus spp.and Penicillium spp. in stored nuts.Denizel et al. (2006), shows that the predominantly encountered species from the infected nuts in Turkey were: Aspergillus niger, Cephalosporium sp. and Trichoderma viridae.These species are known to have strains that cause toxic metabolites (Cole and Cox, 1981;Suleiman, 2010).Khosravi et al. (2007), showed a total of 60 nuts samples were analyzed and mycological analyses revealed that the most frequent isolated fungi from different nuts were Aspergillus, Penicillium, Mucor, Fusarium, Paecilomyces and yeast.Jecfa (2000) and Weidonborner (2001) showed that Aspergillus, Penicillium and Fusarium were the most important toxigenic fungi that decay food products.In addition, fungal contaminations due to Aspergillus, Penicillium, Fusarium, Trichoderma and Cladosporium in nuts especially almond and pistachio and other greasy edible seeds were reported in America, Brazil and western African countries, most of American pistachio products were contaminated by Aspergillus (Schatzki, 1995;Doster et al., 2001).Research on Iranian pistachio in Kerman gardens showed that most of the pistachio products that have gapped or lost their shells during early period of growth and the immature splitted ones were contaminated by A. flavus and other Aspergillus sp., (Shahidi, 2004).
The results presented in Table 3 show the identity and the total Colony Forming Units (CFU) of fungi were found in all of the collected samples, they were serially diluted and plated on PDA medium.The total number of isolated fungi from the all 10 samples was (138×10 3 ) cfu/g.samples.A total of twelve different fungal genera and twenty species were isolated.This study showed that contamination by fungal genera in pure nut products samples were more than salted samples.The minimum number of fungi was detected in Cashews (Salted) (3×10 3 ) followed by Hazelnut (Salted) (6×10 3 ) and Pistachio (Salted) (8×10 3 ).In contrast, Peanut (pure) and Pistachio (pure) (24×10 3 ) for each one, represented the highest infections of fungi.Aspergillus, Rhizopus and Penicillium genera were more frequently detected than other genera of fungi.Aspergillus spp. was found in the most examined samples.Bispora antennata found only in Pistachio (Salted), Rhizoctonia sp., in Cashews (Salted) Gliocladium sp., in Cashews (pure), Trichodochium disseminatum in Hazelnut (pure) and Taeniolella exillis in Peanut (pure).
Our results were in well agreement with those found by Adebajo and Diyaolu (2003), who isolated fourteen fungal species, mostly toxigenic and belonging to 5 genera were Aspergillus, Penicillium, Rhizopus, Mucor and Syncephalastrum, the most predominant isolates were: A. niger, A. restrictus, A. flavus, A. fumigatus and Aspergillus sp., the mean and range of total Fungal Counts (CFU/g) in samples were: 3,368.Rostami et al. (2009) who isolated fungal genera from nut products, found that the most common fungi isolated were extracted from 20 salted and 16 pure peanut samples: Aspergillus flavus, Aspergillus niger, Penicillium, Rhizopus, Mucor, Alternaria and Nigrospora were the most dominant genera, contamination by Aspergillus flavus in pure peanut   Jimenez et al. (1991) found Rhizopus spp. as the predominant fungi from almond, peanut, hazelnut and pistachio seeds in Spain.Data represented in Table 4 show the result of mycotoxin detection on DRBC media for fungi isolated from nut samples, A. flavus, A. fumigatus, A. niger and A. ochraceous, show positive results on the culture for mycotoxin production and show change in colony diameter and color change in compare with control (CDA media), while Penicillium spp.show no any change in colony diameter and color.
Our results were in Contrary to this finding with Rajab (2011) show that the result of aflatoxin detection on DRBC media for fungi isolated from dried fruit samples, all the fungal culture were negative for aflatoxin production and show no any change in colony diameter or color change in compare with control (CDA media).
Table 5 shows results of Estimation of natural occurrence of Aflatoxin (AT) and Ochratoxin (OT) in pure nut sample products, by ELISA method, the concentrations of aflatoxin and ochratoxin were (1.7, 0.3, 5.3, 10.3 and 5.2), (6, 5.5, 1.5, 11.5 and 11.7) ppb.respectively.The results show low AF and OT content in most samples which is lower than the normal rang for mycotoxin (20 ppb) for human consumption, the high results of aflatoxin and ochratoxin show in peanut while the low results show in cashewnut.For the detection of mycotoxins, samples were analyzed quantitatively by CD ELISA technique (Competitive Direct Enzyme Linked Immunosorbent Assay).The quantity of mycotoxins in most of the samples was not detected within the detectable limits.However, in all samples, values of mycotoxins were below the permissible safe limits for human consumption and health.Mycotoxins can cause severe damage to liver, kidney and nervous system of man even in low dosages (Rodricks, 1976).Fusarium and Aspergillus species are common fungal contaminants of maize and also produce mycotoxins (Bakan et al., 2002;Verga and Teren, 2005).The presence of Aspergillus in stored nuts results in deterioration, discolouration and bad odour, couple with toxins, posses potential hazard to consumer's health; a confirmation of that aflatoxin produced by A. flavus is found in cashewnuts and is hazardous to human health (Abdel-Gwada and Zohri, 1993).

Table 1 :
English and scientific names of the tested nuts products

Table 2 :
Isolated fungi from nut products by using (APM) directly with and without sodium hypochlorite

Table 3 :
The isolated fungi from nut product samples by standard dilution plate method Sample Penicillium spp. was isolated from all samples and out of 2 samples, Aspergillus spp. was isolated from all samples.

Table 4 :
Determination of aflatoxin and ochratoxin content in fungal culture by DRBC agar media