Occurrence of patulin in various fruit juice marketed in Tunisia

Accepted Manuscript Occurrence of Patulin in various fruit Juice marketed in Tunisia Nidhal Zouaoui, Naira Sbaii, Hassen Bacha, Pr Salwa Abid-Essefi PII:

S0956-7135(14)00569-6

DOI:

10.1016/j.foodcont.2014.09.048

Reference:

JFCO 4097

To appear in:

Food Control

Received Date: 16 April 2014 Revised Date:

22 September 2014

Accepted Date: 30 September 2014

Please cite this article as: Zouaoui N., Sbaii N., Bacha H. & Abid-Essefi S., Occurrence of Patulin in various fruit Juice marketed in Tunisia, Food Control (2014), doi: 10.1016/j.foodcont.2014.09.048. 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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Occurrence of Patulin in various fruit Juice marketed in Tunisia Nidhal Zouaoui, Naira Sbaii, Hassen Bacha*, Salwa Abid-Essefi

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Laboratory for Research on Biologically Compatible Compounds (LRSBC), Faculty of Dentistry, Rue Avicenne, 5019

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Monastir, Tunisia

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ABSTRACT

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Patulin (PAT) is a secondary metabolite, which is mainly produced by certain species

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of Aspergillus and Penicillium fungi. The objective of this study is to investigate the presence

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of Patulin in various fruit products marketed and consumed in Tunisia. A total of 214 samples

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(30 concentrated juice samples, 42 apple juice samples, 42 pear juice samples, 34 mixed juice

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samples, 35 compote samples, 15 apple jams samples and 16 pear jam samples) were

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analyzed. The incidence of PAT contamination was 50% with a concentration ranging from 2

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to 889 µg/ l with an average of 89 µg/l and a median of 41 µg/l. Twenty two percent (22%)

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of total analyzed samples exceeded the limit recommended by the European Union. Our

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results showed that PAT seems to be a problem in fruit products marketed in Tunisia, which

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required and recommended a hard and fast surveillance of this toxin.

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Keywords: Patulin, fruit products, HPLC, Tunisia.

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* Corresponding author:

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Pr. Hassen BACHA. Head of the Laboratory for Research on Biologically Compatible

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Compounds (LRSBC), Faculty of Dentistry, Rue Avicenne, 5019 Monastir, Tunisia.

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Tel.: (+) 216 73 42 55 50

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Fax: (+) 216 73 42 55 50

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E-mail address: [email protected]

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1. Introduction Patulin (PAT) is a toxic secondary metabolite produced by a wide range of fungal species

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of the genera Penicillium, Aspergillius and Byssochlamys (Paster, Huppert, & Barkai-Golan,

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1995). Among the different genera, the most important PAT producer is Penicillium

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expansum (Moake, Padilla-Zakour, &Worobo, 2005). PAT was found as a contaminant in

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many mouldy fruits, vegetables, cereals and other foods. However, the major sources of

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contamination are apples and apple products, which are also the most important source of

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PAT in the human diet (Baert et al., 2007; Murillo-Arbizu, Amézqueta, González- Peñas, &

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de Cerain, 2009; Reddy et al., 2010).

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PAT is known to be mutagenic, immunotoxic, neurotoxic, and can cause adverse effects

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on the gastrointestinal tract; it also has adverse effects on the developing foetus (Wouters &

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Speijers, 1996; Shephard & Leggott, 2000). Due to its high toxicity, the European

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commission established the maximum recommended concentration of 50 µg/kg of PAT in

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apple fruit juice and apple juice, 50 µg/kg in spirit drinks, ciders and other fermented drinks,

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25 µg/kg for solid apple products such as apple puree and 10 µg/kg for solid apple products

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intended for infants and young children (Commission Regulation No 1881/2006, 2006). The

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International Agency for Research on cancer (IARC) classified PAT as group 3 or as “not

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carcinogenic to humans” (IARC, 1993). PAT maximum intake is esteemed to be 0.2 µg/kg

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bw/day for children and 0.4 µg/kg bw /day for adults, greatly below the tolerable intake

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established by the joint FAO/WHO expert committee on food Additives (JECFA) (World

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Health Organization, 1995).

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Therefore, many methods were developed to analyse Patulin in apple juice such as thin

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layer chromatography (Harwig, Chen, Kennedy & Scott, 1973), mass spectrometry (Sheu &

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Shyu, 1999), colorimetry (Subramanian, 1982), gas chromatography/mass spectrometry

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(Rupp &Turnipseed, 2000). At the moment, the high performance liquid chromatography 2

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with ultra-violet light detection (HPLC-UV) is the most frequently used method (Brause,

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Trucksess, Frederick & Page, 1996; McDonald, Long & Gilbert, 2000; Baert et al., 2007). Tunisia is a North African country, with a humid climate that registers high temperature

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levels that seem to stimulate the toxigenic moulds growth and their mycotoxins production

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(Hadidane et al., 1985; Bacha, Hadidane, Regnault, Ellouz, &Dirheimer, 1986; Bacha et al.,

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1988; Zaied et al., 2009; 2010; Zaied et al., 2012a, 2012b; Oueslati, Romero-Gonzalez,

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Lasram, Frenich & Vidal, 2012; Zaied et al., 2013). Besides, the Tunisian diet is rich in fruit

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and fruit products. Indeed, many fruit products consumed in Tunisia are based on importation,

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and little is known about their eventual mycotoxin contamination. In addition to that, there are

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no applicable norms concerning these products contamination by PAT in the country.

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The objective of this study was to determine the prevalence of PAT in various fruit juice

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products marketed in Tunisia and to compare the levels of PAT contamination to the

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European norms. Furthermore, in this study, a validation of a method for a quantitative

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analysis of PAT is conducted by using High Performance Liquid Chromatography (HPLC)

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method coupled with a UV detection which focused mainly on extraction and clean up.

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2. Materials and Methods

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2.1. Sample Collection

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Juice concentrated samples were provided by a Tunisian fruit juice company in 2013.

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Correspond marketed brand, Compote and jams were bought at local supermarkets. Thirty

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(30) of these samples were concentrated juice samples, Forty two (42) apple juice samples,

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forty two (42) pear juice samples, thirty six (36) mixed juice samples, thirty five (35)

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compotes samples, fifteen (15) apple jam samples and sixteen (16) pear jam samples. The

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volume of the samples is between 130 ml and 1.0 L. Samples are stored in a refrigerator at

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4°C until analysis in their original packages. The samples are thoroughly homogenized and

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used at the same day of analysis.

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2.2. Reagents

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PAT standard was provided by Sigma Chemicals (St. Louis, MO, USA) and dissolved

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in methanol. All reagents (ethyl acetate, acetic acid and sodium carbonate) were obtained

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from Prolabo, Merck and Sigma-Aldrich (France). All solvents (acetonitrile, methanol and

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water) were purchased from Fisher Scientific (Fisher chemicals HPLC, France) with HPLC

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grade.

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2.3. Preparation of standard solutions

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The standard solutions of PAT were prepared by dissolving 5 mg of pure crystalline

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PAT in 1 ml of methanol. The concentration of the PAT stock solution was determined by

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measuring the UV absorbance at 276 nm and calculated by using the molar extinction

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coefficient ε of 14,600 L/mol/cm. The standard curve solutions were freshly prepared from

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appropriate dilutions of the stock solution with methanol (300, 200, 100, 50, 25 and 10

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µg/ml). The prepared solutions were stored at +4°C.

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2.4. Extraction and clean up step PAT analyses were performed according to the method described by Yuan, Zhuang, Zhang

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and Liu (2010) using HPLC with UV detector with some modifications. Ten milliliters of

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liquid sample or 5 grams of solid sample were added to 10 ml of water and extracted with 10

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ml of ethyl acetate by mixing vigorously for 10 min using a vortex mixer. The mixture was

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centrifuged at 4500 rpm at 25°C for 5 min. The organic upper layer was transferred to a

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centrifuge tube and the aqueous phase was twice re-extracted with 20 ml of ethyl acetate. The

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organic layers were combined and 2 ml of 1.5% sodium carbonate was added and the tube

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was mixed vigorously. Five milliliters of ethyl acetate were added to Na2CO3 solution and

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shacked vigorously for 3min. The pH value was adjusted to 4 by acetic acid glacial. The

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solution was evaporated to dryness at 60°C and 5ml of acetonitrile solution (5%) was

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dissolved to the residue. Then, the reconstituted extract was purified through a 0.22 µm

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syringe filter (Millipore). Finally, the purified extract was evaporated to dryness at 60°C for

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analysis in the HPLC system, 500 µl of methanol were added.

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2.5. HPLC quantification

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HPLC analyses of PAT were performed with an 1100 series HPLC system from

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Agilent Technologies equipped with a UV detector (λ=276 nm) controlled by the Chemstation

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3D software (Agilent Technologies Chemstation family software products) and equipped with

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an auto-injector (Injection Valve Assembly G1313A). The separation was carried out on a

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C18 reversed-phase (Spherisorb ODII, Leonberg) (250 × 4 mm, 6 µm). The system was run

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isocratically with a mobile phase consisting of a mixture of water: acetonitrile (90:10, v/v) at

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a flow rate of 1 ml/min. The injection volume of the standard and sample extract was 50 µl.

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The quantification of PAT was performed by the measurement of the peak area at PAT

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retention time and the comparison with the relevant calibration curve (300, 200, 100, 50, 25

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and 10 µg/ml) (Fig.1).

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2.6. Statistical analysis The statistical analysis was performed using the SPSS software program (SPSS

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Institute, 2012, version 21.0) and the differences in PAT levels between groups were analysed

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by ANOVA test. p< 0.05 was considered to be statistically significant.

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3. Results and discussion

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3.1. Method of validation

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The validation of the method developed herein consists in determination of the

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recovery rate, linearity, reproducibility, repeatability of the method, and determination of the

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limits of detection and quantification of PAT in various fruit products consumed and

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marketed in Tunisia. To determine the recovery rate, blank samples were spiked with three

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different concentration of PAT (100, 150 and 300 µg/ml). Triplicate of each concentration

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were analysed and the mean recovery value was 86.46. The average relative standard

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deviation (RSD) of the whole method was 5%.

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Chromatograms obtained showed that PAT has a retention time (Rt) nearly 5.75 ± 0.5 (Fig. 2).

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Linearity was confirmed using calibration curve for each PAT concentration. It was

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linear in range of 10-300 µg/ml of PAT with a coefficient of correlation r=0.996 which

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showing a good calibration. The limit of detection (LOD) was defined as the smallest PAT

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amount detected with at least a 3:1 signal-to- noise ratio obtained with free sample, the LOD

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was 0.01 µg/ml. Concerning the limit of quantification (LOQ), it was defined as the smallest

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amount of PAT with at least a 10:1 signal-to-noise ratio for which the method was validated,

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the LOQ determined was 0.05 µg/ml.

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3.2. Occurrence of Patulin in fruit products

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Results of PAT occurrence in fruit products consumed in Tunisia are shown in Table

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1. In this study, a total of 214 samples analysed including apple juice, pear juice, mixed juice,

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concentrated juice, compote, apple jams and pear jams. The number of the sample, minimum,

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average, median, and maximum of values for Patulin levels in different apple products were

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also listed in Table1. Indeed, PAT was found in 107 of 214 analysed samples with an

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incidence of 50%. Percentage of positive samples were 64.28%, 47.61%, 50%, 80%, 20%,

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33.33%, 43.75% in apple juice, pear juice, mixed juice, concentrated juice, compote, apple

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jams and pear jams respectively. Levels of contamination in positive samples were important

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and ranged between 2 and 889µg/l with an averages of 45.71±6 µg/l; 62.5±12.35 µg/l;

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28.5±3.9 µg/l; 158.1±46.53 µg/l; 32.26±9.7 µg/l; 302±9.6 µg/l; 123.7±41 µg/l in apple juice,

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pear juice, mixed juice, concentrated juice, compote, apple jams and pear jams respectively.

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Our results showed that the highest level of contamination 889 µg/l was found in concentrated

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apple juice. It must be taken into account that fruit concentrates are usually reconstituted

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before direct consumption as fruit juices or soft drinks, or they are used for the production of

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fruit liqueurs, syrups and jellies, or as sweeteners in several products.

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The obtained levels exceed highly the recommended limits and they are similar to those found

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in India 845 µg/l (Saxena, Dwivedi, Ansari, & Das, 2008) and in Italy 1150 µg/l (Beretta,

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Gaiaschi, Galli, & Restani, 2000) but higher than those obtained in previous studies in Tunisia

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(Zaied, Abid, Hlel & Bacha, 2013) and than those in other countries such as in Malizia with

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33.7 µg/l (Lee et al., 2014), in Greek with 36.8 µg/l (Moukas et al., 2008), in Korea with 30.9

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µg/kg (Cho et al., 2010), in Turkey with 139.9 µg/l (Yurdun et al., 2001), in Spain with 41.3

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µg/l (Murillo, Gonzalez-Penas & Amézqueta,2008), in China with 28.6 µg/kg (Yuan et al.,

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2010) and in Saudi Arabia with 152.5 µg/kg (Al-Hazmi, 2010).

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In our study, PAT content in analysed samples exceeds the limit fixed by the European

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Commission Reg.1881/2006 which may cause serial health problems. The high level of PAT

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detected can be explained by the fact that Tunisian climate favors fungal growth and

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mycotoxin accumulation (Kammoun, Gargouri, Barreau, Richard-Forget, & Hajlaoui, 2010).

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Furthermore, the social and economic characteristics of Tunisia, the poor agricultural and

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manufacturing practices can increase the contamination by PAT and by other mycotoxins

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(Hadidane et al., 1985; Bacha, Hadidane, Regnault, Ellouz, & Dirheimer, 1986; Bacha et al.,

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1988; Zaied et al., 2009; 2010; Zaied et al., 2012a, 2012b, 2013).

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In this study, results on PAT occurrence in fruit products consumed in Tunisia have shown a

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high contamination levels. PAT seems to be a problem in these products which recommend a

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surveillance of the occurrence of this toxin. Besides of the regulatory controls, many

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strategies have been adopted to decrease or even eliminate the presence of the mycotoxins in

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fruit products (De Souza Sant’Ana et al., 2008). These strategies aimed to inhibit mycotoxin

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formation in agricultural products avoiding then exposure of the consumer. Among them, a

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good farm management, methods of culture to improve plant vigour, use of insecticides,

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fungicides and biological control. Postharvest contamination can be avoided by controlling

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moisture, temperature and microbiological, insect and animal pests.

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Conclusion

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Mycotoxins and related pathologies are a worldwide preoccupation and they raise

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serious economic and sanitary problems (FAO, 1997). Such a situation is chiefly favored by

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the Mediterranean climate of the country, added to the social and economic conditions.

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Previous studies in Tunisia have shown that the alimentary chain was contaminated by

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several mycotoxins (Hadidane et al., 1985; Bacha et al., 1986; Bacha et al., 1988; Mhadhbi et

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al., 2007; Zaied et al., 2009; 2010; Zaied et al., 2012a, 2012b, Oueslati et al., 2012; Zaied et

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al., 2013).

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Our present study shows the contamination of fruit products marketed in Tunisia by

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PAT. The levels found in concentrated juice, apple juice, pear juice, mixed juice, compote,

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apple jam and pear jam exceed the PAT limit fixed by the European Commission Regulation.

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Improved monitoring programs should be encouraged by Tunisian authorities to set

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limit for mycotoxins, particularly for PAT in food.

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Acknowledgments

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This research is supported by the Tunisian Ministry of Higher Education within the

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Laboratory for Research on Biologically Compatible Compounds (LRSBC).

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Legend to figures:

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Figure 1: Calibration curve of patulin standard concentrations (300, 200 100, 50, 25 and 10 µg/ml).

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Figure 2: HPLC chromatograms: (A) patulin standard (150µg/ml); (B) naturally contaminated apple

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sample with patulin (231µg/kg); (C) free patulin sample.

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Table 1: Contamination levels of Patulin in various fruit products

Total

Contaminated

Incidence (%)

PAT content in Average positive samples contamination (µg/l) (µg/l)a

Median

Concentrated

12/30

24

80

4.5-889

158.1±46.53

66.75

Apple juice

12/42

27

64.28

4-122.36

45.71±6

39.9

Pear juice

9/42

20

47.61

5-231

62.5±12.35

47

Mixed juice

2/34

17

50

10-55.7

28.5±3.9

22.41

Compote

5/35

7

20

2-76.75

32.26±9.7

29

Apple jam

4/15

5

33.33

4.7-554

302±9.6

371

Pear jam

4/16

7

43.75

17-325

123.7±41

76.75

Total

48/214

107

50

2-889

89±13.6

41

M AN U

TE D

EP

Average contamination of positive samples

AC C

a

SC

juice

RI PT

Sample

ACCEPTED MANUSCRIPT

Figure 1 PAT, ADC1 A Area = 49.9941457*Amt +0 Area 16000

Rel. Res%(1): 108.970

RI PT

7

14000 12000 10000

6

8000

SC

6000 5 4000 1

4

0

M AN U

2000

3 2

Correlation: 0.99684

100

200

AC C

EP

TE D

0

Amount[µg/ml]

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Figure 2 (A) ADC1 A, ADC1 CHANNEL A (300PA.D) 5.756 - PAT

RI PT

mAU

400

300

SC

1.818

200

M AN U

100

0 2

4

(B) ADC1 A, ADC1 CHANNEL A (PAT15.D)

TE D

mAU

6

300

250

8

min

5.450 - PAT

EP

1.938 200

AC C

150

100

50

0 1

2

3

4

5

6

7

8

9

min

ACCEPTED MANUSCRIPT

(C) 2 .66 8

ADC1 A, ADC1 CHANNEL A (JN20.D) mAU

90

RI PT

80

70

60

50

SC

40

M AN U

20

0 .39 3

30

10

4

AC C

EP

TE D

2

6

8

min

ACCEPTED MANUSCRIPT

Highlights - We evaluate the PAT occurrence and levels many fruit products marketed in Tunisia. - Samples were analysed by HPLC-UV.

- The contamination average of the positive samples is 89µg/l.

RI PT

- 50% of the 214 fruit products samples contained PAT.

AC C

EP

TE D

M AN U

SC

- Exact situation of fruit products by PAT in Tunisia was provided.