Mycoflora and Aflatoxin Content of Hazelnuts, Walnuts, Peanuts, Almonds and Roasted Chickpeas (LEBLEBI) Sold in Turkey

Abstract

In this study, 28 hazelnuts, 24 walnuts, 18 peanuts, 13 almonds, and 11 roasted chickpeas (leblebi) were analyzed for aflatoxin contamination using thin layer chromatography (TLC). Aflatoxin was found in 26 of 94 samples (27.66%) at concentrations ranging from 1 to 113 ppb. Detectable levels of aflatoxin were 33.4 ppb in hazelnuts, 22.1 ppb in walnuts, 43.0 ppb in peanuts, 7.4 ppb in almonds, and 1.7 ppb in roasted chickpeas. The highest level of aflatoxin was 113 ppb in a single hazelnut sample. Aspergillus and Penicillium species were frequently determined in all the samples.

Keywords:

• Aflatoxin
• Hazelnut
• Walnut
• Peanut
• Almond

INTRODUCTION

Aflatoxins, are carcinogenic secondary metabolites produced by a few fungi such as Aspergillus flavus (A. flavus) and A. parasiticus. Aflatoxins may occur in contaminated foods with these fungi. This situation causes both potential health hazards for humans and economic losses. Various factors operate interdependently to affect mould growth and aflatoxin production. These factors are generally physical, chemical, and biological. Physical factors include the environmental conditions conducive to fungal colonization and mycotoxin production such as temperature, relative humidity, and insect infestation. Chemical factors include the use of fungicides and/or fertilizers. Stresses such as drought, an increase in temperature, and an increase in relative humidity may selectively alter colonization and metabolism of mycotogenic fungi and thus alter mycotoxin production.[1,2,3] Aflatoxins have adverse effects on humans, animals, and crops that result in illnesses and economic losses. The largest risk of aflatoxins to humans is usually as the result of chronic dietary exposure. Such dietary aflatoxin exposures have been associated with human hepatocellular carcinomas.[4] The contamination with Aspergillus spp. of dried fruits such as peanut, hazelnut, walnut, and almond occurs generally during harvest, processing, and storage. Although these types of hard crust fruits are less susceptible to mould contamination, aflatoxin formation still may be seen. Therefore, it is important to prevent the pre-and post-harvest mould contamination and growth.[5] A range of legislative controls on mycotoxins in foods and feeds have been implemented worldwide. In January 1999, the European Union set the levels of maximum aflatoxins in agricultural commodities at 4 ppb with Aflatoxin B₁ at 2 ppb. In the US, the Federal Food Drug and Cosmetic Act Sec. 402 (a) has regulated aflatoxins as adulterants in foods and feeds. Action levels were set to 20 ppb for foods and feeds and 0.5 ppb in milk.[6] Many countries also determined the aflatoxin B₁ limits for some foods such as pistachio, peanut, walnut, and hazelnut. These limits are 0 ppb in The Netherlands, 1 ppb in Switzerland, 2 ppb in Germany, 2 ppb in Finland, and 20 ppb in The USA,[7] and 5 ppb in Turkey.[8] The European Union agreed on a figure of 2 ppb for groundnuts, nuts, dried fruit, and cereals with products intended for direct human consumption, or as an ingredient in foods.[9] The objective of this research was to determine the level of mould contamination and aflatoxin formation in hazelnuts, walnuts, peanuts, almonds, and roasted chickpeas (Leblebi) sold in the Erzurum province of Turkey.

MATERIAL AND METHODS

Sampling

All the samples were collected from retailers, markets, and dried fruit retail shops between March 2001 and March 2002 in Erzurum, Turkey and transferred to the laboratory in plastic bags of 2 kg. The samples were sold without packaging and kept in normal room conditions.

Identification of Mycoflora

For the identification of mycoflora, 10 g of each sample was mixed with 90 mL sterile distilled water in a Waring blender to a 10⁻¹ stock dilution, from which 10-fold serial dilutions—up to 10⁻⁶—were made. Duplicate 1 mL volumes of each dilution were added to Petri plates containing 10–15 mL of potato dextrose agar (Merck).[10] The plates were then incubated at 25°C for 5 days and observed daily. Representative mould colonies from the Petri plates were inoculated into Czapek agar (Merck) to confirm the identification of Penicillium and Aspergillus spp. Isolates were classified to the genus according to Raper and Fennell,[11] and Raper and Thom[12] with some modifications as described by Bullerman and Olivigni.[13]

Aflatoxin Analyses

Aflatoxin analyses were carried out as outlined by Majerus and Zakaria[14] for strong-shelled fruits, with a detection limit of 0.5 ppb for aflatoxin B₁. Aflatoxins were extracted in two portions with 25 mL dichloromethane and aqueous phase; they were then shaken for 1 minute. The dichloromethane phase resulting sample was filtered through Whatman No. 1 filter paper, on which 5 g of dehydrated sodium sulfate was placed 100mL, into a round-bottom flask. The filtrate was evaporated using a rotating evaporator (Heidolph-511, Germany). The extract was purified using a 65 × 10 mm glass column. The aflatoxins were eluted with chloroform/aceton (90/10). The eluates were concentrated on a steam bath and examined on thin-layer chromatographic plates (TLC) (Aldrich art. 805013) to detect the presence of aflatoxin B₁. The chromatograms were developed in the first direction with diethyl ether. The diethyl ether was evaporated, and the plates were examined under ultraviolet light at 365 nm wavelength (Camag, Switzerland). The top portion (about 55 mm) of the plates containing the interfering compounds was cut off. The plates were turned 180° and developed in chloroform/acetone (90/10). After the second development, 25% H₂SO₄ was sprayed on the plates and heated to 110°C for 10 minutes. Aflatoxins were determined visually by comparing the R_f values and color intensity with the aflatoxin B₁ standard under ultraviolet.

RESULTS AND DISCUSSION

Aflatoxin B₁ was detected in 26 (27.66%) of the total 94 samples, in levels ranging between 1 and 113 ppb of all samples (Table 1). As seen in Table 1, 9 of 28 hazelnuts, 7 of 18 peanuts, 6 of 24 walnuts, 3 of 13 almonds, and 1 of 11 chickpeas were positive for aflatoxin B₁; its mean levels were 34.4 ppb, 21.1 ppb, 43.0 ppb, 7.4 ppb, and 1.7 ppb, respectively. Various investigators also reported the similar results. Pantovic and Adomovic[15] also investigated 666 samples of wheat, corn, bean, rice, coffee, almond, hazelnut, walnut, and peanut for aflatoxin B₁ and G₁; they found aflatoxin in 173 samples, of which less than 5 ppb in 22.9%, 5 to 10 ppb in 1.5%, and more than 10 ppb in 1.5%. Kershaw[16] found ≤ 50µg/kg aflatoxin B₁ in 74% of 188 dry crusted fruits imported to England. Abdulkadar et al.[17] examined 126 various food products collected from the markets of Qatar and analyzed for mycotoxins; they found aflatoxin in the range of 0.14–81.64 µg/kg in 28 of the samples. The highest frequency of occurrence for aflatoxin was in peanut butter, in the range of 0.32–13.26 µg/kg. They reported that the highest level of aflatoxin contamination (81.64 µg/kg) was detected in pistachio. Bankole et al.[18] also showed that the most of frequent fungi identified in the dried roasted groundnut samples were A. flavus (43.4%), and the total aflatoxin contamination ranged from 10 to 176 ppb in the samples and was above 20 ppb in 44% of total samples. In the current work, a total of 170 mould colonies were isolated from all the samples. Of the isolates, 49 (28.82%) belonged to the genus Aspergillus; 51 (30%) to the genus Penicillium; 53 (31.18) to the genus Mucor; and 2 (1.18%) were of the genus Fusarium. The remaining 15 isolates (8.82%) belonged to other genera. In the result of the morphological screening of Aspergillus isolates using the microscope, A. flavus is identified from all the samples containing aflatoxin. Sahin and Kalyoncuoglu[19] also reported that 45 of 58 fungal strains (78%) isolated from a total of 12 hazelnut, 4 walnut, 3 sunflower, and 2 chocolate samples were species of Aspergillus and Penicillium. In another study in Turkey, it was stated that 6 of 27 hazelnut samples were contaminated with A. flavus.[20]

Table 1 Presence of aflatoxin B₁ in hazelnuts, walnuts, peanuts, almonds and roasted chickpeas (Leblebi) sold in Turkey.

	Number of Samples			Contamination (ppb)
Types of dry fruits	Tested	Negative	Positive	Level ^a	Range
Hazelnut	28	19	9	34.4	1–113
Walnut	24	18	6	22.1	3–28
Peanut	18	11	7	43.0	8–94
Almond	13	10	3	7.4	1–13
Roasted Chickpea	11	10	1	1.7	1–2
Total	94	68	26
^aMean values for positive samples.

A significant result of current study presented that aflatoxin levels of 6 hazelnuts, 5 peanuts, and 3 walnuts samples were higher than the approved levels of European Union (2 ppb for groundnuts, nuts, and dried fruits) and FDA (20 ppb in foods and feeds) and not fit to human consumption. However, the aflatoxin content of roasted chickpea samples was in the allowable level.

CONCLUSION

The present study indicates that aflatoxin B₁ may be found in high levels in hazelnuts, walnuts, peanuts, and almonds—except roasted chickpeas (leblebi). This is a major public health concern and requires investigations into the reasons for these high levels and means of minimizing or eliminating them. On the other hand, this situation may be a result of adverse pre-harvest conditions of temperature and humidity in the field and improper post-harvest handling and storage. Therefore, the prevention of contamination with toxigenic fungi of foods during harvest, processing, and storage is the best way to control aflatoxin formation. In addition, the mold growth and toxin formation may be significantly limited by packaging, removal of the damaged and moldy fruits, and mechanical drying prior to storage.

Notes

8. Anonymous. Gida maddelerinde belirli bulaşanlarin maksimum seviyelerinin belirlenmesi hakkinda tebliğ. In (Tarim Orman ve Köyişleri ve Sağlik Bakanliği, Türk Gida Kodeksi, Tebliğ no: 2002/63); Resmi Gazete (23 Eylül 2002 ve 24885 sayili): Ankara, Turkey, 2002.