American Journal of Food Technology

Year: 2011 | Volume: 6 | Issue: 8 | Page No.: 613-630
DOI: 10.3923/ajft.2011.613.630

Microbial Contamination and Mycotoxins from Nuts in Riyadh, Saudi Arabia

Suaad S. Alwakeel and Laila A. Nasser

Abstract: The occurrence of harmful aflatoxins from agricultural products varies with geographic location, farming practices and processing. To date, no data was reported from Saudi Arabia on mycotoxin content of nuts and edible seeds. Forty samples of edible nuts and dried seeds were randomly collected from different locations in Al-Riyadh, Saudi Arabia. Fungi were detected by seed-plate and dilutions plate method and were cultured on glucose-Czapek's agar, sucrose-Czapek's agar and starch yeast agar. Purified fungal isolates were identified morphologically. Mycotoxins were extractedusing chloroform and detected by thin layer chromatography. Bacterial analysis was done using total plate count method. There was a predominance of A. niger and A. flavus in all medium types. Aflatoxin B₁ (8.5 μg mL^-1) was detected in peanuts containing A. flavus. Aflatoxin B₁ (1.7 μg mL^-1) and B₂ (1.7 μg mL^-1) was detected in sunflower seeds containing A. terreus. T2 toxin (2.8 mg mL^-1) was detected in pumpkinseeds containing Stachybotrys chartarum and DAS (2.4 μg mL^-1) was detected in a salted peanut sample containing Trichthecium roseum. Four nut samples showed contamination with bacteria. Turkish pine seeds and American walnut had total plate counts of 12x10. Pakistani pine seeds and Iranian salted pistachio had TPC of 3x10. Listeria monocytogenes was isolated from American walnut samples. Government authorities for food safety consumption should continue to monitor and set appropriate guidelines and information initiatives for public knowledge on the safety of these agricultural products whole year round.

Keywords: Aflatoxin, mycotoxin, glucose-Czapek`s agar, Aspergillus species, osmotolerant fungi and osmophilic

INTRODUCTION

Mycotoxins are natural metabolism products of moulds which can have a toxic effect on humans and animals. They have most recently come to light over the toxic mold that has suddenly become an issue of the 21st century. Aflatoxins are the most toxic form of mycotoxins. Some types of food, such as dried fruit, spices and nuts, show an increased risk of aflatoxin release due to fungal infestation (Soubra et al., 2009; Wang and Liu, 2007). As mycotoxins are temperature-resistant they are usually not destroyed when the food is processed (Yazdanpanah et al., 2005).

Aflatoxins are detected occasionally in milk, cheese, corn, peanuts, cottonseed, nuts, almonds, figs, spices and a variety of other foods and feeds (Soubra et al., 2009; Wang and Liu, 2007; Pacheco and Scussel, 2007; Kenjo et al., 2007; Molyneux et al., 2007; Cheraghali et al., 2007; Abdulkadar et al., 2002). Milk, eggs and meat products are sometimes contaminated because of the animal consumption of aflatoxin-contaminated feeds. However, the commodities with the highest risk of aflatoxin contamination are corn, peanuts and cottonseed (Soubra et al., 2009; Wang and Liu, 2007; Pacheco and Scussel, 2007; Kenjo et al., 2007; Molyneux et al., 2007; Cheraghali et al., 2007; Abdulkadar et al., 2002; Mahmoud et al., 2001). Aflatoxins are detected in as much as 70% of corn products with more than 1000 μg kg^-1 level of aflatoxin (Wang and Liu, 2007). In peanuts, aflatoxin level is recorded to as much as 28.4 μg kg^-1 (Wang and Liu, 2007). Probably one of the worst mycotoxins (Aflatoxin) is the ones produced by at least three strains of Aspergillus found in nuts (Hedayati et al., 2007). Aflatoxins are toxic metabolites produced by certain fungi in/on foods and feeds (Ehrlich et al., 2007). They are probably the best known and most intensively researched mycotoxins in the world. Aflatoxin found in nuts is a carcinogenic toxin that has been linked to liver cancer in many countries (Wild and Gong, 2010; Caldas et al., 2002). Aflatoxin also causes other problems, for most people it is believed that the levels are low enough to not be harmful to an individual who occasionally has a few nuts (Alwakeel, 2009). Aflatoxins have been associated with various diseases, such as aflatoxicosis, in livestock, domestic animals and humans throughout the world (Williams et al., 2004). The occurrence of aflatoxins is influenced by certain environmental factors; hence, the extent of contamination will vary with geographic location (Mwanda et al., 2005) agricultural and agronomic practices and the susceptibility of commodities to fungal invasion during preharvest, storage and/or processing periods (Park, 2002). Processing per se reduces the amount of aflatoxins in foods by as much as 80% (Park, 2002). For these reasons, though most countries have adopted measures to control levels of mycotoxins specifically aflatoxins in agricultural products, environmental conditions affecting storage and consumption make it difficult or impossible to attain low concentrations of this aflatoxins (Dorner, 2008). Aflatoxins have received greater attention than any other mycotoxins because of their demonstrated potent carcinogenic effect in susceptible laboratory animals and their acute toxicological effects in humans. As it is realized that absolute safety is never achieved, many countries have attempted to limit exposure to aflatoxins by imposing regulatory limits on commodities intended for use as food and feed.

Numerous reports from many countries on the occurrence of mycotoxins have been published, however, none has been reported from Saudi Arabia. Considering the fact that nuts consumption is very high in Saudi Arabia, we deemed it necessary to conduct this study to investigate mycotoxins and bacterial contamination in edible nuts in this region.

MATERIALS AND METHODS

Collection of samples: Forty samples of edible nuts and dried seeds were randomly collected from different locations in Al-Riyadh, Saudi Arabia between May 2008 and July 2010. English and Scientific names of nuts and dried seeds are enumerated in Table 1. Each sample was placed in a sterile polyethylene bag, sealed and double-sealed with another bag for storage.

Isolation and identification of fungi: Fungi were detected using two methods. The first is seed-plate method as described by Seo et al. (2008). Four seeds were placed on the surface of sterile media. Five plates were used for each sample and each medium; the plates were incubated for 5-7 days at 25°C. The second method is the dilution plate method as used by Kenjo et al. (2007). Five gram seeds of each sample were placed in a 500 mL sterilized distilled water in Erlenmeyer flask and shaken for 15 min. One mL of seed suspension was placed into each Petri dish, 12-15 mL of melted and cooled medium was poured. Five plates were used for each sample and for each medium. Glucophilic fungi were cultured on glucose-Czapek's agar medium in which glucose (10 g L^-1) replaced sucrose.

To determine cellulose decomposition by fungal species, glucose was replaced by powdered cellulose (20 g L^-1) in cellulose-Czapek's agar as medium. Osmophilic and osmotolerant fungi were allowed to grow on sucrose-Czapek's agar which contained 200 g L^-1 sucrose instead of glucose. Thermophilic and thermotolerant fungi were cultured on starch yeast agar (YpSS) which contained g L^-1: Soluble starch, 20; yeast extract, 4; KH₂PO₄, 1; Mgso₄.7H₂O, 0.5 and agar, 15 g. All types of media were supplemented with chloramphenicol (20 μg mL^-1) and Rose Bengal (30 ppm) as bacteriostatic agent. Pure cultures of fungi were kept in slant agar tubes which containing 0.5 g chloramphenicol.

Identification of fungal isolates: Purified fungal isolates were identified morphologically (based on macroscopic and microscopic characteristics) whenever possible, in the original Petri dishes culture (Kenjo et al., 2007; Seo et al., 2008).

Extraction of mycotoxins from samples: The samples were stored at 22°C for 1, 2, 3 and 4 months then extracted for the presence of aflatoxins B₁, B₂, T₂ Toxin and DAS. During these periods the rate of fungal growth was determined visually as described by Joosten et al. (2001). Twenty gram of each sample was defatted by extraction with cyclohexane for 10 h using a Soxhlet-type extractor. The defatted residue was extracted for another 10 h with chloroform. The chloroform extract was dried over anhydrous sodium sulphate, filtered and then evaporated under vacuum to near dryness. The residue was diluted with chloroform to 1 mL.

Detection and verification of mycotoxins: Caldas et al. (2002) did thin layer chromatographic technique of the clean extract on percolated silica gel plate type for the presence of mycotoxins according to standard procedures as used in the detection of mycotoxins in foods.

Simple configuration method of recorded mycotoxins on precoated silica gel plates was done. The TLC plates commonly used are normal phase silica gel plates. Some acidic metabolites like cyclopiazonic acid, citrinin and luteoskyrin can be useful to impregnate the plate with oxalic acid. This is simply done by dipping the plate in an 8% solution of oxalic acid in water or methanol followed by air-drying. After application the TLC-plate, a suitable TLC-procedure can be performed using the following solvents: TEF: Toluene/Ethyl Acetate/Formic acid (90%) 5:4:1, CAB: Chloroform/Acetone/Iso propanol 85:15:20 and CM: Chloroform/Methanol 97:3.

After elution and air drying in a dark fume hood, the TLC-plates are examined in visible light (VIS), long wave UV-light (UV-366) and short wave UV-light (UV-254) some metabolites are treated with 1/2 min in UV-254 followed by UV-366.

The following spray reagents are useful for visualizing and verification of secondary metabolites:

Extraction of mycotoxins from fungal isolates: Culture of selective 25 fungal isolates collected from the current study was examined. The tested samples were represented by 3 species of Aspergillus (A. flavus (2 isolates), A. tamarii (1 isolate) and A. terreus (1 isolate), 1 isolate of Acremonium strictum, one isolate each of Curvularia ovoidae and Paecilomyces variotii, 3 isolates of Penicillium (Penicillium chrysogenum (1 isolate) and Penicillium purpurogenum (2 isolates), two isolates of Stachybotrys chartarum and two species of Trichoderma (Trichoderma harizianum (1 isolate) and Trichthecium roseum (1 isolate).

Inocula were prepared from 7-days old culture of each isolate on PDA slope as spore suspensions in 0.2% aqueous tween 80 (v/v). Isolates were inoculated into 250 mL Erlenmeyer flasks each containing 50 mL Capek's liquid medium supplemented with 0.2% yeast extract and 1.0 peptone and incubated at 28°C for 10 days as static culture (PYCZ) (Youssef et al., 2008a).

After incubation, the control of each flask (medium+mycelium) was homogenized for 5 min in a high-speed blender with 100 mL chloroform. The extract procedure was repeated three times. The chloroform extracts were combined, washed, dried, filtered and concentrated to near dryness, cleaned and mycotoxins are detected as previously described by Nieminen et al. (2002).

Bacterial analysis: A total of 40 samples of nuts and dried seeds were analyzed for bacterial total plate count using a method employed by Freitas et al. (2009) and Hossain et al. (2004).

Data analysis: Data analysis was done using Total Count (TC of species/ 100 seeds), (TC%) Total Count of one fungi species/total count of all speciesx100, Incidence (I) is: (the occurrence of fungi in each number of a specimen of nuts) and Percentage Incidence (%I) was calculated by the fallowing equation: incidence (I)/ the number of nut specimensx100. This applies to all the tables (Youssef et al., 2008b).

RESULTS

In peanut samples, Aspergillus flavus was isolated in all 7 peanut samples with 34.3% total count and 184 counts/100 seeds. Aspergillus niger showed the highest total percentage count with 38.8% and 208 counts/100 seeds. A. niger was seen in 6 of 7 peanut samples (85.7%). Other fungal species isolated from peanut samples include; Aspergillus tamarii, Aspergillus terreus, Mucor hiemalis, Paecilomyces variotii. The percentage from the total count was (3%). The percentage from the total count of Aspergillus versicolor, Cladosporium sphaerospermum, Microascus cinereus, Neurospora crassa, Penicillium chrysogenum, Penicillium glabrum, Rhizopus, Syncephalastrum racemosum, Trichotecium roseum and unidentified yeasts was 1.5% (Table 2).

In 4 samples of chick peas, A. niger predominated with 45.7% total count followed by A. flavus (5.7%) and 2.9% total count each of Emericella acrestata, Emericella nidulans, Penicillium purpurogenum and R. oryzae. R. oryzae predominated with 42.1% total count in pine seeds samples together with 18.4% of A. niger and 13.2% of A. flavus. Similarly, A. niger and A. flavus and predominated in sunflower seed samples, hazelnut sample, walnut sample, karela seed samples, pumpkin seed samples, almond nut samples and pistachio samples. R. oryzae also was isolated in high total counts in walnut, cashew, kerala seeds, pumpkin, almond and pistachios. (Table 2).

In cellulose-Czapek agar, A. niger and A. flavus were the two predominant fungal specie which were isolated in peanuts, chick peas, pine seeds, hazelnuts, walnut, cashew, kerala seeds, pumpkin seeds, almond and pistachios. Pumpkin seeds contained the highest number of isolated fungal species (TC of 752 in 11 sample) followed by peanuts (TC of 384 in 7 sample), pistachios (TC of 368 in 5 sample) and chick peas (TC of 248 in 4 samples). The rest of the samples had total counts of less than 80 per sample (Table 3).

In 40% sucrose-Czapek agar, A. niger, A. flavus and Eurotium montevidensis were the three most predominant fungal specie which were isolated in peanuts, chick peas, pine seeds, hazelnuts, walnut, cashew, kerala seeds, pumpkin seeds, almond and pistachios (Table 4).

Among the thermophilic/thermotolerant fungi, A. fumigatus and A. niger predominated in almost all tested samples of peanuts, sunflower seeds and cashew nuts. E. nidulans was isolated from chickpeas, pine seeds and walnuts (Table 5).

Collectively comparing the total fungal counts isolated from 40 samples of nuts and dried seeds showed the predominance of A. niger (range: 36.9% TC on 40% sucrose-Czapek agar to as much as 59.8% TC on YPSS).

A. flavus was also isolated in all medium types (range: 4% TC on YPSS to as much as 33% on Cellulose-Cz agar). Six other species of Aspergillus were isolated from different medium types. A complete detailed fungal isolates collective total count is presented in Table 6.

Aflatoxin B₁ (8.5 μg mL^-1) was detected in a salted peanut sample containing A. flavus. Aflatoxin B₁ (1.7 μg mL^-1) and B₂ (1.7 μg mL^-1) were detected in sunflower seeds containing A. terreus. T2 toxin (2.8 mg mL^-1) was detected in pumpkin seeds containing Stachybotrys chartarum. DAS (2.4 μg mL^-1) was detected in a salted peanut sample containing Trichthecium roseum. No mycotoxins were detected in the chloroform extracts of the different samples analyzed (Table 7).

Four nut samples showed contamination with bacteria. Turkish pine seeds and American walnut had total plate counts of 12x10. Pakistani pine seeds and Iranian salted pistachio had TPC of 3x10. Listeria monocytogenes was isolated from American walnut samples (Table 8).

DISCUSSION

Present study confirmed the capability of Aspergillus species in producing mycotoxins which can be harmful for human consumption. This is in agreement with previous studies on this subject matter (Hedayati et al., 2007). In contrast to the findings made by Wang and Liu (2007), present results showed only 8.5 μg mL^-1 of mycotoxins from salted peanut contaminated with Aspergillus niger. Wang and Liu (2007) reported upto 28.4 μg kg^-1 of mycotoxins from peanuts. Furthermore, three strains of Aspergillus are identified from peanut samples, namely: A. flavus, A. niger and A. fumigatus. These have been mentioned by Hedayati et al. (2007).

In this study, we were able to demonstrate the diverse strains and species of fungi that can be isolated from nuts and edible seeds. Considering the fact that amongst these isolated fungi are strains or species that are capable of producing mycotoxins, to name a few, A. flavus, A. terreus and S. chartarum (Table 7). Unfortunately, these fungi are ubiquitous and widespread at all levels of the food chain. Their presence is considered unavoidable and it is not possible to predict or prevent entirely their occurrence during cultivation, harvest, storage and processing operations by current good agronomic and good manufacturing practices. As mentioned by Dorner (2008), measures to control levels of mycotoxins may not totally be a success story since a variety of environmental factors can affect storage and consumption thus making it very difficult to minimize mycotoxin porduction. Under favourable conditions of temperature and humidity, these fungi grow on certain foods especially on edible nuts and seeds resulting in the production of toxins. Much more, mycotoxins can also be metabolized by animals fed contaminated grains and pass into milk, eggs and other organs entering the food chain once again as previously reported by several researchers (Soubra et al., 2009; Wang and Liu, 2007; Pacheco and Scussel, 2007; Kenjo et al., 2007; Molyneux et al., 2007; Abdulkadar et al., 2002).

Mycotoxins may cause various adverse health effects from immediate toxic response and immune-suppression to the potential long-term carcinogenic effects. S. chartarum has been found to cause pulmonary diseases including pulmonary arterial hypertension (Shariat and Collard, 2007; Ochial et al., 2008; Al-Ahmad et al., 2010). The variety of symptoms also include dermatitis, recurring cold and flu-like symptoms, burning sore throat, headaches and excessive fatigue, diarrhea and impaired or altered immune function. Cladosporium and Aspergillus are commonly found fungi in ventilation systems and indoor environments making up to 75% of the particulates, also found in present study (Reboux et al., 2009; Hedayati et al., 2009; Bundy et al., 2009). These organisms can occur naturally in the exterior environment and enter as spores or active fungi attached to dust particles. These families of molds have been implicated in being causative agents in asthma, hypersensitivity pneumonitis and pulmonary mycosis, including toxic pneumonitis, tremors, chronic fatigue syndrome, kidney failure and cancer. Exposure to molds has become a significant health risk to an increasing number of workers in various occupations throughout the nations. Fungal antigens are able to cause occupational asthma, rhinoconjunctivitis, hypersensitivity pneumonitis and organic dust toxic syndrome.

Growth of commonly occurring filamentous fungi result in production of mycotoxins, the most dangerous aflatoxins, ochratoxin A, fumonisins, trichothecenes and zearalenone, a toxin known for causing infertility and endometriosis in animals (Meyer et al., 2000). Aflatoxins are potent carcinogens and in association with hepatitis B virus are responsible for many thousands of human deaths per annum, mostly in non-industrialized tropical countries. Ochratoxin A is a carcinogen and has caused urinary tract cancer and kidney damage in people from northern and eastern Europe. Fumonisins appear to be the cause of oesophageal cancer in southern Africa, parts of China and elsewhere. Trichothecenes are highly immunosuppressive and zearalenone causes oestrogenic effects in animals and man.

Surprisingly, only 4 samples of this study showed bacterial contamination with only 3 samples showing positivity for coliforms and 1 for listerial contamination. These levels of microbial contamination of food are influenced by harvesting/slaughtering technologies and by the processes applied during food manufacture. With current technologies it is impossible to guarantee the absence of pathogenic microorganisms on raw foods, both of plant and animal origin thus, increasing incidence of foodborne diseases and the resultant social and economic impact on the human population have brought food safety to the forefront of public health concerns. Many outbreaks are the consequence of a failed process, or inappropriate storage conditions (usually temperature abuse) during distribution, food service or by the consumer. Besides, mycotoxin-producing molds such as A. ochraceus and P. varidicatum can produce ochratoxin A 4-5 days after inoculation at 25°C to 46 μg g^-1 of any grain after 28 days.

Hope and Simon (2007) has reported the association between exposure to dampness and excess growth mold and the development of aeroirritant symptoms. Changes in temperature, relative humidity and moisture content of food products are important indicators of fungal mycotoxin production. For example, Ochratoxin A. production occur in products stored at the top of containers and in wet bags (Palacios-Cabrera et al., 2007). In present study, fungal growth occurred in all types of media, osmophiles with 2840 gross total count, followed by mesophiles (2,640 total count) and the thermopiles (1,392 total count) (Table 6). These findings state that fungi whether they are mycotoxin or non-mycotoxin producers grow in environmental conditions with adequate temperature, relative humidity and moisture.

CONCLUSION

The microbiological safety of food can never be achieved by end-product testing, which only detects that a failure has occurred and can only contribute indirectly to identification and control of the cause of the problem. Furthermore, the isolation of a bacterial pathogen from a food does not mean that the food necessarily is dangerous, e.g., the food may be cooked before consumption. Hence preventive approaches are required, often as simple as control of storage time and temperature. Nevertheless, microbiological testing, used appropriately, is one of the measures that can be used to achieve microbiological safety.

There is a continuous need to protect the health of humans and susceptible animals by limiting their exposure to mycotoxins because of their toxicological manifestations and agricultural products contaminated with harmful microorganisms. Long-term sequelae including the development of cancer and other fatal conditions should prompt health and safety authorities to regulate for or suggest permitted levels of mycotoxins in foods and feed because of the public health significance and commercial consequences. This should be carefully accounted for since monitoring such contamination and health hazard, this can have profound economic implications resulting in losses of foodstuff due to mycotoxins and bacterial contamination. In conclusion, government authorities for food safety consumption should continue to monitor and set appropriate guidelines and information initiatives for public knowledge on the safety of these agricultural products whole year round.

REFERENCES