Natural occurrence of aflatoxins in dry fruits and edible nuts

Accepted Manuscript Natural occurrence of aflatoxins in dry fruits and edible nuts Muhammad Masood, Shahzad Zafar Iqbal, Muhammad Rafique Asi, Noeen Malik PII:

S0956-7135(15)00134-6

DOI:

10.1016/j.foodcont.2015.02.041

Reference:

JFCO 4332

To appear in:

Food Control

Received Date: 6 December 2014 Revised Date:

16 February 2015

Accepted Date: 24 February 2015

Please cite this article as: Masood M., Iqbal S.Z., Asi M.R. & Malik N., Natural occurrence of aflatoxins in dry fruits and edible nuts, Food Control (2015), doi: 10.1016/j.foodcont.2015.02.041. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Natural occurrence of aflatoxins in dry fruits and edible nuts

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Muhammad Masood a, Shahzad Zafar Iqbal b,c,†, Muhammad Rafique Asi d, Noeen Malik e

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Pakistan

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Faisalabad, 38000, Pakistan

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University of Putra Malaysia, 43400, Serdang, Selangor, Malaysia

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Department of Chemistry, Government Municipal Degree College Faisalabad, 38000,

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Food Toxicology Lab., Nuclear Institute for Agriculture and Biology (NIAB), P.O. Box

128, Faisalabad 38950, Pakistan

University Hospital, Ulm, Clinic for Nuclear Medicine, Ulm, Germany

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Food Safety Research Centre (FOSREC), Faculty of Food Science and Technology,

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Department of Applied Chemistry & Biochemistry, Government College University

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†Corresponding author: [email protected] (Iqbal, S.Z.)

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Cell: 0060-12-686-121

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Fax: 0060-3-8942 3552

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ACCEPTED MANUSCRIPT Abstract

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A total 307 samples of dry fruits and edible nuts from Northern areas and Khyber

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Pakhtunkhwa, Pakistan were evaluated for the presence of aflatoxins (AFs). The samples

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were analysed using a reversed phase HPLC, equipped with fluorescence detector. Results

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have revealed that 132 out of 307 samples of dry fruits and nuts were found positive with

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aflatoxin B1 (AFB1) and total AFs. The highest mean level of total AFs i.e. 7.89 ± 0.99 µg/kg

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was found in peanuts without shell and lowest mean level (2.45 ± 0.11µg/kg) was found in

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watermelon seeds without shell samples. Samples 75 (24%) were found contaminated with

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AFB1, ranged from 8-10µg/kg and 41 (13%) samples were found above the level of 10µg/kg

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for total AFs. The high occurrence of AFs may cause health hazards for consumers and limit

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exports.

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Keywords: aflatoxins; dry fruits; edible nuts; HPLC

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ACCEPTED MANUSCRIPT 1. Introduction

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Fruits are good sources of antioxidants, vitamins, minerals but low in fat contents. Pakistan

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has a rich heritage of fruit orchards, especially in the harvesting seasons, and dried fruits in

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the off-season. Due to their long shelf-life, dried fruits can provide a good alternate to fresh

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fruits; particularly in winter season (Waheed, & Siddique, 2009). Studies have shown that

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nuts are the most susceptible commodity for fungal attack and consequently, the production

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of AFs (Iqbal, Asi, Zuber, Akram, & Batool, 2013). Fungal contamination can attack in the

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field, during harvest, transport or storage (Kader & Hussein, 2009).

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Aflatoxins are a group of natural food toxins which are recognised as toxic, carcinogenic

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secondary metabolites mainly produced by certain strains of Aspergillus flavus, Aspergillus

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paraciticus and Aspergillus nomius (Iqbal, Mustafa, Asi, & Jinap, 2014). AFs are found as

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contaminants in various agricultural commodities including corn, peanut, cottonseed, Brazil

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nut, pistachio nut, fig, spices and copra (El-tawila, Neamatallah, & Serdar, 2013; Iqbal, Nisar,

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Asi, & Jinap, 2014a). The International Agency for Research on Cancer (IARC) has

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classified AFB1 as a group I carcinogen which primarily affects the liver (IARC, 2002; Iqbal,

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Asi, & Jinap, 2014b).

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In Pakistan the consumption of dry fruits and nuts is increased during winter season i.e.

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November till April. There are very limited reports for the presence of AFs contamination in

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dry fruits or edible nuts considering conducive environment for fungal proliferation.

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Therefore, the present study was designed to analyse the occurrence and levels of AFs

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contamination in these important commodities and disseminate the results to farmers, traders,

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local officers and law enforcement agencies.

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2. Materials and methods

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2.1 Sampling

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ACCEPTED MANUSCRIPT A total of 307 samples of dry fruits and edible nuts were collected from Northern areas and

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and Khyber Pakhtunkhwa province of Pakistan. The samples includes dried plums (21), dates

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(15), dried apricot (20), raisins (21), almonds (21), walnut with shell and without shell (20

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each), peanuts with shell and without shell (20 each), dried figs (22), watermelon seeds (15),

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melon seeds (13), pistachio with shell and without shell (20 each), pine nuts (22) and cashew

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nuts (18) were collected randomly from different retail markets and local shops, during

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January 2013 to March 2013. The sample size was not less than 1 kg for each sample. The

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samples were ground with a grinding mill (Retsch ZM 200, Germany) and stored in a plastic

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bag in a refrigerator at 4 ºC, until further analysis.

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2.2 Regents and chemicals

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The standards of AFs were purchased from Sigma Aldrich, Steinheim, Germany and the

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immunoaffinity columns (IAC) were obtained from VICAM, Watertown, MA, USA. The

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HPLC grade acetonitrile, methanol and analytical grade trifluoroacetic acid (TFA) were

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purchased from Merck, Darmstadt, Germany. De-ionized water (Millipore, Bedford, MA,

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USA) was used in the study and all other chemicals and reagents were at least of analytical

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grade.

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2.3. Extraction of aflatoxins

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The sample preparation and analysis was done according to our previously validated method

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(Iqbal, Asi, & Jinap, 2014c).

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2. 4. HPLC conditions

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The samples were analysed using HPLC (Shimadzu, Kyoto, Japan) in a reverse phase

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isocratic mode having Supelco C18 column (Discovery HS, Bellefonte, PA, USA) with a

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fluorescence detector (RF-530). The mobile phase (acetonitrile: methanol: water (20:20:60,

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v/v/v) was used at a flow rate of 1 ml/min. The temperature of column was maintained at

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40ºC. Furthermore, the excitation and emission wavelengths were set at 360 and 440 nm,

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ACCEPTED MANUSCRIPT respectively. The method has shown good resolution and separation of aflatoxin standards

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(Figure 1a) and natural occurrence of AFB1 in date (Fig. 1b), plum (Fig. 1c) and apricot (Fig.

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1d) samples.

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2. 5. Quality control parameters

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The method was validated by using seven point calibration curves of analytes, to assess the

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linearity in a range of 1-80 µg/ml for AFB1 and AFG1, 0.5-12 µg/ml for AFB2 and AFG2. The

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values of coefficient of determination (R2) for all analytes were found above 0.99. The

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sensitivity of the method was checked in terms of limit of detection (LOD) and limit of

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quantification (LOQ) i.e. signal-to noise (S/N) ratio of 3 and 10, respectively. The LOD of

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0.04 µg/kg and LOQ 0.12 µg/kg was found for AFB1 and AFG1, and 0.07 and 0.21 µg/kg for

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AFB2 and AFG2, respectively. The selectivity was determined by spiking samples at levels of

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1, 2, and 6 µg/kg for AFB1 and AFG1, 0.5, 1.5 and 3 µg/kg for AFB2 and AFG2. The

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recoveries of fortified samples in dry fruits and nuts were found in the range of 83 to 90%

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with RSD varies from 8 to 19%.

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2. 6. Statistical analysis

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The data of AFs contamination in dry fruits and edible nuts was statistically analysed and

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presented as mean ± standard deviation (S.D.) by using SPSS software (IBM, PASW

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Statistics 19, USA). Furthermore, the value of R2 was determined by using

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regression/correlation analysis.

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3.

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Results of 307 dried fruits and nuts samples analysed for the presence of AFs contamination

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are presented in (Table 1). Total 16 out of 20 samples of peanut (without shell) have found

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positive with AFs contamination and shown the highest level of total AFs 7.89± 0.99µg/kg,

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ranging from LOD-21.34µg/kg. The lowest level (2.45 ± 0.11 µg/kg) of AFB1 was found in

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watermelon seeds without shell. It is evident from the results that watermelon seeds without

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Results and discussion

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ACCEPTED MANUSCRIPT shell, melon seeds without shell, pine nuts and cashew nuts have shown only the

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contamination of AFB1 while other AFs were not detected. The results have shown that 75

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(24%) samples of dry fruits and nuts were in the range of 8-10 µg/kg and 41 (13%) samples

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were found above the level of 10 µg/kg, (Table 2). Highest incidence level of AFs

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contamination was found in pistachios without shell samples where 55 % sample ranged in 8-

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10 µg/kg and 45% sample were found above the level of 10 µg/kg.

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The results of present study are comparatively high as compared to previous study from

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Pakistan by Luttfullah & Hussain (2011). They have found 2 samples of dates contaminated

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with AFs ranging from 2.1 to 3.2 µg/kg (compared to present finding range LOD to 18.79

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µg/kg) and in dried figs, 5 samples were contaminated with concentration ranging from 4.5 to

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12.5 µg/kg as compared to present results 7 samples of dried figs were contaminated with

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AFs ranging from LOD to 12.34 µg/kg. Furthermore, they have found 4 apricot kernels

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samples contaminated with AFs with 1 sample with concentration of 5.6 µg/kg, 3 samples of

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almonds without shell had concentration ranging from 1.2 to 3.4 µg/kg, 2 samples of walnut

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with shell had concentration ranging from 1.5 to 3.0 µg/kg and 2 samples had contamination

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levels ranging from 7.8 to 13.5 µg/kg. Similarly, Bankole, Ogunsanwo, Osho, & Adewuyi

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(2006) has found 27 out of 37 samples of melon seeds positive with AFB1 with mean level

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14.2µg/kg (ranged from 2.3 to 47.7µg/kg). In another study Milhome, Lima, Lima, Lima,

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Sousa, & Nascimento (2014) have analysed 70 samples of cashew nuts with 2 (2.8%)

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samples showed the levels above 4 µg/kg and 1 sample has total AFs above 20 µg/kg. The

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mean level of total AFs in dried dates was low as compared to our previous study (Iqbal et

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al., 2014c), which reported 38/96 samples positive with AFs with mean level of 4.11 ±

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0.80µg/kg ranged from LOD to 26.60µg/kg.

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However, studies with very high level of AFs contamination in dry fruits and nuts compared

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to present study were reported. Karaca & Nas (2006) from Turkey, have found AFs ranged

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ACCEPTED MANUSCRIPT from 117.9 to 471.9 µg/kg in dried figs. A very high incidence (78%) of AFs contamination

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in peanuts from Botswana was reported with concentration ranged from 12 to 329 µg/kg

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(Mphande, Siame, & Taylor, 2004). Wang & Liu (2006) from China have reported the

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average level 80.27 µg/kg in peanuts and the highest level was 437.09 µg/kg. In our previous

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study 13 out of 22 samples of peanut in shell and 16 out of 29 samples of peanut without

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shell were found contaminated with AFs, with mean levels of total AFs 6.4± 3.4, 9.6 ± 2.5

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µg/kg, ranged from LOD to 59.8 and LOD to 82.1µg/kg, respectively (Iqbal et al., 2013).

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In present study pistachio with shell and without shell were found highly contaminated after

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peanuts. Cheraghali et al. (2007) from Iran has documented very high levels of total AFs in

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pistachio. In another report published by Iranian Ministry of Health, 7926 pistachio samples

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were analysed during (2001-02), data indicated 761 samples contained AFB1 higher than 10

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µg/kg (FDCL, 2002). Abdulkadar, Al-Ali, & Al-Jedah, (2000) from Qatar, have reported that

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in 37% pistachio kernels an incidence of total AFs, which ranged from 0.53-289 µg/kg.

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In present study the levels of AFB1 and total AFs in dry fruits and edible nuts were found

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high and therefore urgent actions from law informant agencies are required. These dry fruits

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and nuts are extensively consumed in Pakistan during winter season and may pose a serious

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health hazards for consumers.

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4. Conclusions

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This study documented that 132 out of 307 analysed samples of dry fruits and nuts were

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found positive. The mean level of total AFs was ranged from LOD to 21.50µg/kg. The study

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urged the need of continuous monitoring and implementation of strict regulations in order to

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minimize or avoid fungal contamination in these commodities.

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Acknowledgements

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The authors are thankful to Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad,

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Pakistan for providing the analytical facilities.

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ACCEPTED MANUSCRIPT Figure legends Figure 1: Chromatograms showing the retention times of individual retention times of

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aflatoxins standards (a), natural occurrence of AFB1 in date (b), in plum (c), and in apricot (d)

ACCEPTED MANUSCRIPT Table 1: Aflatoxin B1 (AFB1) and total aflatoxins (AFs) in dry fruits and edible nuts AFB1 (µg/kg) Positive (N)

Dried plums Dried dates Dried apricot Raisins Almonds Walnuts with shell Walnuts without shell Peanuts with shells Peanuts without shells Dried figs Watermelon seeds without shell Melon seeds without shell Pistachios with shell Pistachios without shell Pine nuts Cashew nuts Total

21 15 20 21 20 20 20 20 20 22 15 13 20 20 22 18 307

8 9 7 9 8 5 9 12 16 7 5 4 9 14 6 4 132

Mean ± S.D.

Limit

Mean ± S.D.

Limit

2.42 ± 0.23 4.50 ± 0.46 3.93 ± 0.58 3.89 ± 0.52 4.64 ± 0.98 3.22 ± 0.78 4.80 ± 0.93 5.87 ± 0.99 6.34 ± 1.02 2.76 ± 0.34 2.45 ± 0.11 2.91 ± 0.34 5.96 ± 0.89 6.47 ± 1.25 2.98 ± 0.34 3.11 ± 0.41

LOD – 7.45 LOD- 9.80 LOD- 7.15 LOD- 9.12 LOD- 8.70 LOD- 9.30 LOD- 11.50 LOD- 12.78 LOD- 14.50 LOD- 6.80 LOD- 8.90 LOD- 9.52 LOD- 10.56 LOD- 13.67 LOD- 6.88 LOD- 5.90 LOD- 14.50

3.72 ± 0.14 6.32 ± 0.78 4.75 ± 0.54 4.12± 0.43 4.97 ± 0.61 5.82 ± 0.88 5.43 ± 0.83 6.40 ± 0.97 7.89± 0.99 3.28 ± 0.45 2.45 ± 0.11 2.91 ± 0.34 6.80 ± 0.89 7.53 ± 1.12 2.98 ± 0.34 3.11 ± 0.41

LOD- 14.76 LOD- 18.79 LOD- 11.50 LOD- 13.45 LOD- 15.80 LOD- 16.89 LOD- 15.78 LOD- 17.80 LOD- 21.34 LOD- 12.34 LOD- 8.90 LOD- 9.52 LOD- 16.70 LOD- 21.50 LOD- 6.88 LOD- 5.90 LOD- 21.50

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Total AFs (µg/kg)

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Samples (N)

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ACCEPTED MANUSCRIPT Table 2: The incidence of AFB1 and total AFs in dry fruits and edible nuts

Samples

AFB1 (8-10 µg/kg)

AFs ˃ 10µg/kg

Dried plums Dried dates dried apricot Raisins Almonds Walnuts with shell Walnuts without shell Peanuts with shells Peanuts without shells Dried figs Watermelon seeds without shell Melon seeds without shell Pistachios with shell Pistachios without shell Pine nuts Cashew nuts Total

8/21 9/15 7/20 9/21 8/20 5/20 9/20 12/20 16/20 7/22 5/15 4/13 9/20 14/20 6/22 4/18 132/307

4 (19) 5 (33) 3 (15) 4 (19) 4 (20) 3 (15) 5 (25) 7 (35) 11 (55) 4 (18) 2 (13) 2 (15) 5 (25) 11 (55) 4 (18) 1 (6) 75 (24)

2 (10) 3 (20) 1 (5) -2 (10) 1 (5) 3 (15) 4 (20) 8 (40) 2 (9) --4 (20) 9 (45) 2 (9) -41 (13)

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The figure in parentheses represents the %age

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The EU permissible level for AFB1 and total AFs is 8 and 10µg/kg, respectively (for almonds, pistachios, and apricot kernels intended for direct human consumption)

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b AFB1 AFB1

AFG2

AFB2

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c AFB1

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AFG1

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AFB1