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Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Survey of the presence of patulin in fruit juices a
a
a
E. Bonerba , E. Ceci , R. Conte & G. Tantillo
a
a
Dipartimento di Sanità Pubblica e Zootecnia, Facoltà di Medicina Veterinaria , Università degli Studi di Bari , Valenzano, Strada Provinciale per Casamassima, km 3, I-70010 Bari (BA), Italy Published online: 09 Jun 2010.
To cite this article: E. Bonerba , E. Ceci , R. Conte & G. Tantillo (2010) Survey of the presence of patulin in fruit juices, Food Additives & Contaminants: Part B: Surveillance, 3:2, 114-119, DOI: 10.1080/19393210.2010.490882 To link to this article: http://dx.doi.org/10.1080/19393210.2010.490882
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives and Contaminants: Part B Vol. 3, No. 2, June 2010, 114–119
VIEW DATASET Survey of the presence of patulin in fruit juices E. Bonerba, E. Ceci*, R. Conte and G. Tantillo Dipartimento di Sanita` Pubblica e Zootecnia, Facolta` di Medicina Veterinaria, Universita` degli Studi di Bari, Valenzano, Strada Provinciale per Casamassima, km 3, I-70010 Bari (BA), Italy
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(Received 18 February 2010; final version received 30 April 2010) Patulin is a mycotoxin produced by fungi belonging to the Penicillium and Aspergillus genera. The occurrence of patulin in fruit juices marketed in Italy in 2008 and purchased from supermarkets and retail shops has been measured. The aim of this study was to assess the presence of patulin in order to evaluate the potential risk for the consumer and, at the end of this food chain, to determine the quality of the raw material used. One hundred and five fruit juices (35 apple juice, 35 mixed-taste juices, 35 pear juices), produced by various Italian and European companies, were analysed using a previously published method. The analytical investigation showed that apple juices had a concentration of patulin ranging between 6 and 30 mg l1, with a mean of 18 mg l1; mixed fruit juices had a concentration ranging between 1 and 45 mg l1, with a mean of 23 mg l1. Instead, pear juices had a concentration ranging between 5 and 92 mg l1, with a mean of 43 mg l1, and 14 samples of the 35 analysed juices showed a patulin level above the highest regulated limit of 50 mg l1, imposed by European Commission Regulation 1881/06. Keywords: fruit juices; mycotoxins; patulin
Introduction Patulin (PAT) is a mycotoxin produced by more than 60 species of fungi belonging to 30 genera (Davis and Diener 1978; Scott 1994), in particular Penicillium expansum (Firsvad and Thrane 1996), Penicillium griseofulvum, Aspergillus clavatus, and Byssochlamys nivea fungal species (Frank 1977; Sabater-Vilar et al. 2004), which are considered the most dangerous for human health. The products most affected are fruit pome and stone fruit (apples, pears, peaches, black currant, blackberries, etc.) (Scott et al. 1972, 1977; Buchanan et al. 1974; Frank 1977; Karabulut and Baykal 2002), particularly at post-harvest time, because contamination by PAT is often associated with the presence of green–blue rotting on fruit, as determined by development of P. expansum (Ueno 1987). PAT, discovered in 1942, is a substance that, depending on the dose ingested, can show multiple effects on human health. It induces in humans a shortterm toxicity, with particular symptoms (convulsions, dyspnoea, pulmonary oedema, lesions of the gastrointestinal tract, vomiting) (World Health Organization (WHO) 1996), but also a long-term toxicity, which involves the reduction of the immune system (Escoula et al. 1977; Escoula, Bourdiol et al. 1988; Escoula, Thomsen et al. 1988; Paucod et al. 1990), a slowdown
*Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2010 Taylor & Francis DOI: 10.1080/19393210.2010.490882 http://www.informaworld.com
in growth, and the development of fibrosarcomas (Dickens and Jones 1961). Moreover, PAT has neurotoxic effects (Devaraj et al. 1982), and its in vitro genotoxicity (Bu¨rger et al. 1988; Mori et al. 1994) and teratogenicity (Roll et al. 1990; Ciegler et al. 1976) have been demonstrated. The International Agency for Research on Cancer (IARC) entered PAT in Group 3, i.e. those substances ‘not classifiable as to their carcinogenicity to humans’ (IARC 1986). In 2006, European Commission Regulation 1881/06 imposed limits for the presence of PAT in food by establishing that ‘in fruit juices, concentrates fruit juice as reconstituted, fruit nectars, spirit drinks, cider and other fermented drinks derived from apples or containing apple juice the maximum level must be equal to 50.0 mg kg1’ for adults. The production of fruit juices is also regulated by stringent standards, which take into consideration the concentration of fruit and water, the ban on food dyes, the use of only permitted additives, and the addition of known quantities of sugar. These rules have the specific task of controlling commercial and healthcare frauds. PAT, which has the chemical structure of a lactone (4-hydroxy-4H-furo-[3,2c]pyran-2[6H]-one), is a thermally resistant molecule and, therefore, it shows a resistance to the processes needed for industrial
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Food Additives and Contaminants: Part B
Materials and methods Samples One hundred and five samples of fruit juices were purchased from various Italian retail outlets in 2008. The samples were split according to the following: . Matrix typology: 35 apple juices, 35 mixed fruit juices and 35 pear juices. . Source: 65 samples were produced by Italian companies (61.9%) and 40 samples by different European companies (38.1%: 15 apple juices, 15 pear juices and ten mixed fruit juices). . Percentage of fruit: less than or equal to 50% ¼ 54 samples (18 apple juices, 18 pear juices and 18 mixed fruit juices); and greater than or equal to 50% ¼ 51 samples (17 apple juices, 17 pear juices and 17 mixed fruit juices). Samples were stored below 4 C for less than 2 months until analysis.
Analytical reagents . PAT standard: (4-hydroxy-4H-furo-[3,2c] pyran-2[6H]-one) (Sigma-Aldrich).
Patulina
y = 0.0278x + 0.0157 R2 = 0.9975
1.5 Abs
production of fruit products, such as pasteurization (Harrison 1989). The presence of PAT in marketed fruit juices can be regarded as a parameter indicative of the quality of the raw material used (Burda 1992). Investigations into the incidence of PAT in apple juice have been published for countries such as South Africa (Leggott and Shephard 2001; Katerere et al. 2007), Brazil (Sylos and Rodriguez-Amaya 1999; Prado et al. 2000), Cuba (Fernandez Trevejo et al. 2001), Turkey (Gockmen and Acar 1998; Yurdun et al. 2001), the Basque Country (Spain) (Armentia et al. 2000), Sweden (Thuvander et al. 2001), Belgium (Tangni et al. 2003), Italy (Spadaro et al. 2008), and Iran (Cheraghali et al. 2003). Annually, the consumption of fruit juices increases, especially due to the placing on the market of a new generation of juices fortified with vitamins and fibres. The aim of this study was to evaluate the presence of PAT in fruit juices intended for adults, but which are also used by infants and adolescents, to assess the potential risk for all categories of consumers, and to determine, at the end of this food-chain, the quality of the raw material used. This parameter indicates if, in the line-production, the producers have adopted appropriate control measures and monitoring of both raw materials and suppliers, following the good manufacturing practice expected by the hazard analysis critical control point systems (HACCP) system.
115
1 0.5 0 0
10
20
30 40 Conc µg l–1
50
60
Figure 1. Calibration curve of PAT.
. Solvents: ethanol absolute, n-hexane, ethyl acetate, acetonitrile, water, high-performance liquid chromatography (HPLC) grade (SigmaAldrich, St. Louis, MO, USA). . Acetic acid (glacial) greater than or equal to 99% (Merck, Darmstadt, Germany). . Sodium bicarbonate (J.T. Baker, Phillipsburg, NJ, USA).
Apparatus and chromatographic conditions Liquid chromatography (LC) analysis comprised of an Agilent 1100 Series equipped with pumps, a Rheodyne Model 7125 injector (100 ml loop), and a diode array detection (DAD) detector. An LC column Restek C18 (5 mm) (250 4.6 mm internal diameter) was used with a mobile phase consisting of a mixture of water– acetonitrile (90:10, v/v), degassed at a flow rate of 0.9 ml min1. The eluate was monitored at 276 nm.
Calibration From the stock solution consisting of 5 mg of PAT dissolved in 25 ml of ethyl acetate, 1 ml was taken, transferred to a 10 ml flask, and the solvent evaporated to dryness under a nitrogen stream. Absolute ethanol was immediately added to dissolve the residue and to obtain a PAT work solution of 200 mg l1. The work solution was stored at 20 C and its absorbance was evaluated at 276 nm. The linearity study was carried out in the range 5–50 mg l1. Each solution was injected three times and the mean values were used to construct the calibration graph (Figure 1). To determine the recovery of the method applied, five juice samples were fortified with PAT at concentrations of 5, 10 and 25 mg l1 and were subjected to the extraction procedures. Figure 2 shows an HPLC chromatogram as an example of juice fortification.
Procedures The samples were analysed using the extractive method described by Iha and Sabino (2006). From each sample were collected and placed in centrifuge tubes of
E. Bonerba et al.
7.04
20.0
17.5
15.0
12.5
10.0
7.5
5.0
2.5
0.0
Time (min.)
10.55
8.13
5.60
4.14
2.42
2.87
0.075 0.000
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0.150
Abs 0.225
0.300
0.375
0.450
pat-15.09
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Figure 2. HPLC chromatogram of a PAT std of 25 mg l1 (std 3) added to a sample of fruit juice.
Nalgene 5 ml of product, to which were added 0.5 g of sodium bicarbonate to remove the aqueous component and the interference of other phenolic compounds most likely present in the fruit juice. The samples were extracted with 5 ml of a mixture of ethyl acetate– n-hexane (96:4), vigorously shaken on a vortex for 1 min and then placed in a centrifuge at 8000 rpm for 5 min. A total of 30 ml of glacial acetic acid were added to 3 ml of the recovered supernatant. The PAT extract was evaporated to dryness under a nitrogen stream; the dry residue was dissolved in 1 ml of acetic acid 0.1% and filtered on filters of cellulose (0.45 mm). Aliquots of 20 ml filtrate were then chromatographed.
Analytical quality assurance Table 1 shows the results of the percentage recovery and analysis of variance on recovery percentages expressed as the average of results obtained by the fortification of the five samples for each concentration of std PAT added. The average percentage recovery obtained is 95%. Further confirmation of the accuracy of this method was the double analysis of each sample. The average of reproducibility relative standard deviation (RSD) for PAT was 2.87%. Precision was determined by analysing three fruit juices samples spiked at 5, 10 and 25 mg kg1. Within-day precision was determined by analysing six replicates of spiked
Table 1. Results for the recovery of the method applied for the determination of patulin in fruit juices. [PAT] (mg l1) 5 10 25
Recovery (%)
Coefficient of variation (%)
96.7 93.8 94.5
2.228 3.120 3.267
homogenized apple-based baby food; between-day precision was determined by analysing six replicates during 3 days. The precision of the method was calculated from the RSD (Table 2). The identity of PAT in contaminated samples was confirmed by comparing the ultraviolet (UV) light spectrum of the peak eluting at a retention time of PAT with that of authentic standard of PAT (Sigma-Aldrich). Additional confirmation was performed in selected positive samples that were reanalysed by HPLC after spiking the extracts with an equivalent amount of PAT by checking the symmetry of the peak eluting at a retention time of PAT. The limit of detection (LOD) was determined to be 0.3 mg kg1; the limit of quantification (LOQ) was 1.0 mg kg1; the repeatability of results for duplicate analysis ranged from 1% to 5%.
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Table 2. Precision of the method for the determination of patulin in fruit juices. Within-day Concentration [PAT] (mg kg1) 5 10 25
Between-day
(mg kg1)
n
RSD (%)
(mg kg1)
n
RSD (%)
4.83 9.38 23.62
6 6 6
2.5 2.8 2.9
4.75 9.25 23.12
3 3 3
2.9 2.8 2.6
Table 3. PAT concentrations in examined fruit juices. PAT concentration range (mg l1) Apple juices
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Less than or equal to 50% of fruit
Greater than or equal to 50% of fruit
Italian product, 11 samples 10–30 Italian product, 9 samples n.d.–28
European product, 7 samples 18–30 European product, 8 samples 10–26
Mixed fruit juices Italian product, 13 samples n.d.–45 Italian product, 12 samples n.d.–43
European product, 5 samples 28–45 European product, 5 samples 27–44
Pear juices Italian product, 11 samples 7–80 Italian product, 9 samples 5–65
European product, 7 samples 25–92 European product, 8 samples 10–90
Notes: Numbers shown in bold are out of law limit. n.d., Not determinable.
Results and discussion In the 35 apple juices analysed, the concentration of PAT found was less than the limit set by Regulation EC 1881/2006 of 50 mg l1 and is between 6 and 30 mg l1 (in four samples the PAT concentration was not determined (n.d.)), and the average was 18.06 mg l1 (Table 3). In 35 mixed fruit juices the range of PAT contamination was 1–45 mg l1 (in three samples the PAT concentration was n.d.), and the average was 22.97 mg l1, again less than the regulation limit (Table 2). Instead, in pear juices the range of PAT contamination was 5–92 mg l1, with an average of 42.62 mg l1, and 14 samples (40% of the 35 analysed) exceeded the limit of 50 mg l1 (Table 3). The results show that 79.33% of samples tested had PAT contamination, though below the limits imposed by the regulations. This widespread contamination by PAT is a parameter to judge the quality of the raw material used, the selection process of the fruit and the respect of the HACCP system. To ensure food quality and safety, it would be necessary to adopt an integrated approach to the production line which is able to control the hazards that arise from primary production to consumption. The hazards should be identified as critical control points (CCP) and controlled during the production steps (collection, storage, sorting and washing fruits) in which corrective actions are most effective, economical and feasible. The operators involved in primary production must follow the documented procedures to ensure the
selection of raw material. The European Union Recommendation of 11/08/2003 on ‘the prevention and reduction of contamination of patulin in apple juice and the ingredients of apple juice in other beverages’ requires that the apple processing industries should adopt good manufacturing practice. The presence of a PAT concentration higher than the legal limits (50 mg l1) in 14 pear juice samples (40% of the examined ones) emphasizes the need to extend these criteria of good manufacturing practice and to develop an HACCP system that recognizes more relevant additional CCP by taking into account the formation of PAT in the industries that produce juices other than apple juices. It was also found that the amount of contamination is related to the percentage of fruit in juice. The lower the amount of fruit used, the greater the possibility that there is contamination. Samples with a percentage of fruit lower than 50% showed an average concentration of PAT equal to 31.55 mg l1; those with more than 50% of fruit revealed an average concentration of PAT equal to 23.41 mg l1. This suggests that in fruit juices, such as those that have a lower market cost, the raw material used is not carefully selected, probably because producers trust that the ‘diluting effect’ will reduce the potential levels of contamination. However, in addition, it should be considered that the dilution of the juices analysed included the use of apple juice, which has a low cost on the market and is
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often used for its sweetening properties as a dilutent for juices and beverages. It becomes necessary, therefore, not only to assess the quality of the raw material, but also in equal measure to check the health standards of the dilutents. By splitting the samples according to their source of origin – Italian (61.9%) and European (38.1%) – it was estimated that the European samples have an average concentration of PAT equal to 39.92 mg l1, which is higher than the average concentration of 20.78 mg l1 detected in Italian samples. The data presented allow one to make an assessment on the potential health risks for consumers of all age groups. In fact, in 13.33% of 105 analysed samples, the concentration of PAT exceeding the limit fixed by European Regulation 1881/2006 demonstrates that the high production standards required in this food chain have not yet been reached by all producers. Even thought the regulation limit for the presence of PAT in fruit juices concerns only adults, this product is also highly represented in the diet of all groups of consumers, and there is certainly an increased ‘intake’ in the diet of children above 2 years of age. The current law is mostly focused on early childhood, that includes babies up to the first 24 months of age, infants and young children setting lower PAT limits only for apple juice and solid apple product, including apple compote and apple puree. For the other fruit juices the maximum limits set for contaminants in food are the same as for an adult of 70 kg of body weight. As regards the risk of accumulation of specific substances such as mycotoxins, it would be necessary for competent authorities to provide further differentiation in the law limits taking into account the fact that child consumers have not yet reached a body weight of 70 kg and have a metabolism that is not fully specialized to carry out appropriate detoxification. Moreover, it would be necessary to divide the population into different categories, as has already been done for other substances subject to accumulation, as established in the pharmaceutical regulation since the 1990s.
References Armentia A, Jalon M, Urieta I, Macho ML. 2000. Monitoring for presence of patulin in apple juices and ciders sold in the Basque Country. J Alimentaria. 310:65–70. Buchanan JR, Sommer NF, Fortlage RJ, Maxie EC, Mitchell FG, Hsieh DPH. 1974. Patulin from Penicillium expansum in stone fruits and pears. JASHS. 99:262–265. Burda K. 1992. Incidence of patulin in apple, pear, and mixed fruit products marketed in New South Wales. J Food Protect. 55:796–798. Bu¨rger MG, Brakhage AA, Creppy EE, Dirheimer G, Ro¨schenthaler RJ. 1988. Toxicity and mutagenicity of
patulin in different test systems. Arch Toxicol Suppl. 12:347–351. Cheraghali AM, Mohammadi HR, Amirahmadi M, Yazdanpanah H, Abouhossain G, Zamanian F, Khansari MG, Afshar M. 2005. Incidence of patulin contamination in apple juice produced in Iran. Food Control. 16:165–167. Ciegler A, Beckwith AC, Jackson LK. 1976. Teratogenicity of patulin and patulin adducts formed with cysteine. Appl Environ Microbiol. 5:664–667. Davis ND, Diener UL. 1978. Mycotoxins. In: Beuchat R, editor. Food and beverage mycology. Westport (CT): Avi. p. 397–444. Devaraj H, Radha-Shanmugasundaram K, Shanmugasun daram ER. 1982. Neurotoxic effect of patulin. IJEB. 20:230–231. Dickens F, Jones HEH. 1961. Carcinogenic activity of a series of reactive lactones and related substances. BJC. 15:85–100. Escoula L, Bourdiol D, Linas MD, Recco P, Seguela JP. 1988. Enhancing resistance and modulation of humoral immune response to experimental Candida albicans infection by patulin. Mycopathologia. 103:153–156. Escoula L, More J, Baradat C. 1977. The toxins of Byssochlamys nivea. Part I. Acute toxicity of patulin in adult rats and mice. Ann Rech Vet. 8:41–49. Escoula L, Thomsen M, Bourdiol M, Pipy B, Peuriere S, Roubinet S. 1988. Patulin immunotoxicology: effect on phagocyte activation and the cellular and humoral immune system of mice and rabbits. Int J Immunopharmacol. 10:983–989. European Commission. 2006. Commission regulation (EC) No. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Off J Eur Union. L364:5–24. Fernandez Trevejo EO, Aias Verdes JA, Sersa Espinosa R. 2001. Presence of patulin in fruit purees and juices. J Alimentaria. 321:133–135. Firsvad JC, Thrane U. 1996. Mycotoxin production by food-borne fungi. In: Samson RA, Hoekstra ES, Frisvad JC, Filtenborg O, editors. Introduction to foodborne fungi. 5th ed. The Netherlands: Centraalbureau voor Schimmelcultures Baarn. p. 251–260. Frank HK. 1977. Occurrence of patulin in fruit and vegetables. Annales Nutrition Alimentation. 31:459–465. Gockmen V, Acar J. 1998. Incidence of patulin in apple juice concentrates produced in Turkey. J Chromatogr A. 815:99–102. Harrison MA. 1989. Presence and stability of patulin in apple products: a review. J Food Safety. 9:147–153. International Agency for Research on Cancer (IARC). 1986. Patulin. Some naturally occurring toxins and synthetic food components. Monographs on the Evaluation of Carcinogenic risk to Humans. Lyon (France): IARC. 40:83–89. Iha MH, Sabino M. 2006. Determination of patulin in apple juice by liquid chromatography. J AOAC Int. 89:139–143. Karabulut OA, Baykal N. 2002. Evaluation of the use of microwave power for the control of postharvest diseases of peaches. Postharvest Biol Tec. 26:237–240.
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Food Additives and Contaminants: Part B Katerere DR, Stockenstrom S, Thembo KM, Balducci G, Shepard GS. 2007. Investigation of patulin contamination in apple juice sold in retail outlets in Italy and South Africa. Food Addit Contam. 24(6):630–634. Leggott NL, Shephard GS. 2001. Patulin in South African commercial apple products. Food Control. 12:73–76. Mori H, Kawai K, Ohbayashi F, Kuniyasu T, Yamazaki M, Hamasaki T, Williams GM. 1994. Genotoxicity of a variety of mycotoxins in the hepatocyte primary, culture DNA repair test using rat and mouse hepatocytes. Cancer Res. 44:2918–2923. Paucod JC, Krivobok S, Vidal D. 1990. Immunotoxicity testing of mycotoxins T-2 and patulin on Balb/C mice. Acta Microbiol Hung. 37:331–339. Prado G, Oliveira MS, Cunha MR, Gomides MF, Abrantes FM, Santos LG, Veloso T, Barroso RES. 2000. Occurrence of patulin in apple juice [marketed in Brazil] by high-performance liquid chromatography. Revista Instituto Adolfo Lutz. 59:21–25. Roll R, Matthiaschk G, Korte A. 1990. Embryotoxicity and mutagenicity of mycotoxins. J Environ Path Tox Oncol. 10:1–7. Sabater-Vilar M, Maas RFM, De Bosschere H, Ducatelle R, Fink-Gremmels J. 2004. Patulin produced by an Aspergillus clavatus isolated from feed containing malting residues associated with a lethal neurotoxicosis in cattle. Mycopathologia. 158:419–426. Scott PM. 1994. Penicillium and Aspergillus toxins. In Miller JD, Trenholm HL, editors. Mycotoxins in grains: Compounds other than aflatoxin. St. Paul (MN): Eagan. p. 261–285.
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Scott PM, Fuleki T, Harwig J. 1977. Patulin content of juice and wine produced from moldy grapes. J Agr Food Chem. 25:434–437. Scott PM, Miles WF, Toft P, Dobe´ JG. 1972. Occurrence of patulin in apple juice. J Agr Food Chem. 20:450–451. Spadaro D, Garibaldi A, Gullino L. 2008. Occurrence of patulin and its dietary intake through pear, peach, and apricot juices in Italy. Food Addit Contam B. 1:134–139. Sylos CM, Rodriguez-Amaya DB. 1999. Incidence of patulin in fruits and fruit juices marketed in Campinas, Brazil. Food Addit Contam. 16:71–74. Tangni EK, Theys R, Mignolet E, Maudoux M, Michelet JY, Larondelle Y. 2003. Patulin in domestic and imported apple-based drinks in Belgium: occurrence and exposure assessment. Food Addit Contam. 83:482–489. Thuvander A, Moeller T, Enghardt Barbieri H, Jansson A, Salomonsson AC, Olsen M. 2001. Dietary intake of some important mycotoxins by the Swedish population. Food Addit Contam. 18:696–706. Ueno Y. 1987. Mycotoxins. In: Miller K, editor. Toxicological aspects of food. Amsterdam (the Netherlands): Elsevier. p. 139–204. World Health Organization (WHO). 1996. Patulin. Food Additives Series No. 35. Geneva (Switzerland): WHO; [cited 2007 Aug 7]. Available from: http://www.inchem. org/documents/jecfa/jecmono/v35je16.htm/ Yurdun T, Zehra Omurtag G, Ersov O. 2001. Incidence of patulin in apple juices markets in Turkey. J Food Protect. 64:1851–1853.
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Survey of the presence of patulin in fruit juices a
a
a
E. Bonerba , E. Ceci , R. Conte & G. Tantillo
a
a
Dipartimento di Sanità Pubblica e Zootecnia, Facoltà di Medicina Veterinaria , Università degli Studi di Bari , Valenzano, Strada Provinciale per Casamassima, km 3, I-70010 Bari (BA), Italy Published online: 09 Jun 2010.
To cite this article: E. Bonerba , E. Ceci , R. Conte & G. Tantillo (2010) Survey of the presence of patulin in fruit juices, Food Additives & Contaminants: Part B: Surveillance, 3:2, 114-119, DOI: 10.1080/19393210.2010.490882 To link to this article: http://dx.doi.org/10.1080/19393210.2010.490882
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives and Contaminants: Part B Vol. 3, No. 2, June 2010, 114–119
VIEW DATASET Survey of the presence of patulin in fruit juices E. Bonerba, E. Ceci*, R. Conte and G. Tantillo Dipartimento di Sanita` Pubblica e Zootecnia, Facolta` di Medicina Veterinaria, Universita` degli Studi di Bari, Valenzano, Strada Provinciale per Casamassima, km 3, I-70010 Bari (BA), Italy
Downloaded by [New York University] at 06:48 17 June 2015
(Received 18 February 2010; final version received 30 April 2010) Patulin is a mycotoxin produced by fungi belonging to the Penicillium and Aspergillus genera. The occurrence of patulin in fruit juices marketed in Italy in 2008 and purchased from supermarkets and retail shops has been measured. The aim of this study was to assess the presence of patulin in order to evaluate the potential risk for the consumer and, at the end of this food chain, to determine the quality of the raw material used. One hundred and five fruit juices (35 apple juice, 35 mixed-taste juices, 35 pear juices), produced by various Italian and European companies, were analysed using a previously published method. The analytical investigation showed that apple juices had a concentration of patulin ranging between 6 and 30 mg l1, with a mean of 18 mg l1; mixed fruit juices had a concentration ranging between 1 and 45 mg l1, with a mean of 23 mg l1. Instead, pear juices had a concentration ranging between 5 and 92 mg l1, with a mean of 43 mg l1, and 14 samples of the 35 analysed juices showed a patulin level above the highest regulated limit of 50 mg l1, imposed by European Commission Regulation 1881/06. Keywords: fruit juices; mycotoxins; patulin
Introduction Patulin (PAT) is a mycotoxin produced by more than 60 species of fungi belonging to 30 genera (Davis and Diener 1978; Scott 1994), in particular Penicillium expansum (Firsvad and Thrane 1996), Penicillium griseofulvum, Aspergillus clavatus, and Byssochlamys nivea fungal species (Frank 1977; Sabater-Vilar et al. 2004), which are considered the most dangerous for human health. The products most affected are fruit pome and stone fruit (apples, pears, peaches, black currant, blackberries, etc.) (Scott et al. 1972, 1977; Buchanan et al. 1974; Frank 1977; Karabulut and Baykal 2002), particularly at post-harvest time, because contamination by PAT is often associated with the presence of green–blue rotting on fruit, as determined by development of P. expansum (Ueno 1987). PAT, discovered in 1942, is a substance that, depending on the dose ingested, can show multiple effects on human health. It induces in humans a shortterm toxicity, with particular symptoms (convulsions, dyspnoea, pulmonary oedema, lesions of the gastrointestinal tract, vomiting) (World Health Organization (WHO) 1996), but also a long-term toxicity, which involves the reduction of the immune system (Escoula et al. 1977; Escoula, Bourdiol et al. 1988; Escoula, Thomsen et al. 1988; Paucod et al. 1990), a slowdown
*Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2010 Taylor & Francis DOI: 10.1080/19393210.2010.490882 http://www.informaworld.com
in growth, and the development of fibrosarcomas (Dickens and Jones 1961). Moreover, PAT has neurotoxic effects (Devaraj et al. 1982), and its in vitro genotoxicity (Bu¨rger et al. 1988; Mori et al. 1994) and teratogenicity (Roll et al. 1990; Ciegler et al. 1976) have been demonstrated. The International Agency for Research on Cancer (IARC) entered PAT in Group 3, i.e. those substances ‘not classifiable as to their carcinogenicity to humans’ (IARC 1986). In 2006, European Commission Regulation 1881/06 imposed limits for the presence of PAT in food by establishing that ‘in fruit juices, concentrates fruit juice as reconstituted, fruit nectars, spirit drinks, cider and other fermented drinks derived from apples or containing apple juice the maximum level must be equal to 50.0 mg kg1’ for adults. The production of fruit juices is also regulated by stringent standards, which take into consideration the concentration of fruit and water, the ban on food dyes, the use of only permitted additives, and the addition of known quantities of sugar. These rules have the specific task of controlling commercial and healthcare frauds. PAT, which has the chemical structure of a lactone (4-hydroxy-4H-furo-[3,2c]pyran-2[6H]-one), is a thermally resistant molecule and, therefore, it shows a resistance to the processes needed for industrial
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Food Additives and Contaminants: Part B
Materials and methods Samples One hundred and five samples of fruit juices were purchased from various Italian retail outlets in 2008. The samples were split according to the following: . Matrix typology: 35 apple juices, 35 mixed fruit juices and 35 pear juices. . Source: 65 samples were produced by Italian companies (61.9%) and 40 samples by different European companies (38.1%: 15 apple juices, 15 pear juices and ten mixed fruit juices). . Percentage of fruit: less than or equal to 50% ¼ 54 samples (18 apple juices, 18 pear juices and 18 mixed fruit juices); and greater than or equal to 50% ¼ 51 samples (17 apple juices, 17 pear juices and 17 mixed fruit juices). Samples were stored below 4 C for less than 2 months until analysis.
Analytical reagents . PAT standard: (4-hydroxy-4H-furo-[3,2c] pyran-2[6H]-one) (Sigma-Aldrich).
Patulina
y = 0.0278x + 0.0157 R2 = 0.9975
1.5 Abs
production of fruit products, such as pasteurization (Harrison 1989). The presence of PAT in marketed fruit juices can be regarded as a parameter indicative of the quality of the raw material used (Burda 1992). Investigations into the incidence of PAT in apple juice have been published for countries such as South Africa (Leggott and Shephard 2001; Katerere et al. 2007), Brazil (Sylos and Rodriguez-Amaya 1999; Prado et al. 2000), Cuba (Fernandez Trevejo et al. 2001), Turkey (Gockmen and Acar 1998; Yurdun et al. 2001), the Basque Country (Spain) (Armentia et al. 2000), Sweden (Thuvander et al. 2001), Belgium (Tangni et al. 2003), Italy (Spadaro et al. 2008), and Iran (Cheraghali et al. 2003). Annually, the consumption of fruit juices increases, especially due to the placing on the market of a new generation of juices fortified with vitamins and fibres. The aim of this study was to evaluate the presence of PAT in fruit juices intended for adults, but which are also used by infants and adolescents, to assess the potential risk for all categories of consumers, and to determine, at the end of this food-chain, the quality of the raw material used. This parameter indicates if, in the line-production, the producers have adopted appropriate control measures and monitoring of both raw materials and suppliers, following the good manufacturing practice expected by the hazard analysis critical control point systems (HACCP) system.
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1 0.5 0 0
10
20
30 40 Conc µg l–1
50
60
Figure 1. Calibration curve of PAT.
. Solvents: ethanol absolute, n-hexane, ethyl acetate, acetonitrile, water, high-performance liquid chromatography (HPLC) grade (SigmaAldrich, St. Louis, MO, USA). . Acetic acid (glacial) greater than or equal to 99% (Merck, Darmstadt, Germany). . Sodium bicarbonate (J.T. Baker, Phillipsburg, NJ, USA).
Apparatus and chromatographic conditions Liquid chromatography (LC) analysis comprised of an Agilent 1100 Series equipped with pumps, a Rheodyne Model 7125 injector (100 ml loop), and a diode array detection (DAD) detector. An LC column Restek C18 (5 mm) (250 4.6 mm internal diameter) was used with a mobile phase consisting of a mixture of water– acetonitrile (90:10, v/v), degassed at a flow rate of 0.9 ml min1. The eluate was monitored at 276 nm.
Calibration From the stock solution consisting of 5 mg of PAT dissolved in 25 ml of ethyl acetate, 1 ml was taken, transferred to a 10 ml flask, and the solvent evaporated to dryness under a nitrogen stream. Absolute ethanol was immediately added to dissolve the residue and to obtain a PAT work solution of 200 mg l1. The work solution was stored at 20 C and its absorbance was evaluated at 276 nm. The linearity study was carried out in the range 5–50 mg l1. Each solution was injected three times and the mean values were used to construct the calibration graph (Figure 1). To determine the recovery of the method applied, five juice samples were fortified with PAT at concentrations of 5, 10 and 25 mg l1 and were subjected to the extraction procedures. Figure 2 shows an HPLC chromatogram as an example of juice fortification.
Procedures The samples were analysed using the extractive method described by Iha and Sabino (2006). From each sample were collected and placed in centrifuge tubes of
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7.04
20.0
17.5
15.0
12.5
10.0
7.5
5.0
2.5
0.0
Time (min.)
10.55
8.13
5.60
4.14
2.42
2.87
0.075 0.000
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0.150
Abs 0.225
0.300
0.375
0.450
pat-15.09
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Figure 2. HPLC chromatogram of a PAT std of 25 mg l1 (std 3) added to a sample of fruit juice.
Nalgene 5 ml of product, to which were added 0.5 g of sodium bicarbonate to remove the aqueous component and the interference of other phenolic compounds most likely present in the fruit juice. The samples were extracted with 5 ml of a mixture of ethyl acetate– n-hexane (96:4), vigorously shaken on a vortex for 1 min and then placed in a centrifuge at 8000 rpm for 5 min. A total of 30 ml of glacial acetic acid were added to 3 ml of the recovered supernatant. The PAT extract was evaporated to dryness under a nitrogen stream; the dry residue was dissolved in 1 ml of acetic acid 0.1% and filtered on filters of cellulose (0.45 mm). Aliquots of 20 ml filtrate were then chromatographed.
Analytical quality assurance Table 1 shows the results of the percentage recovery and analysis of variance on recovery percentages expressed as the average of results obtained by the fortification of the five samples for each concentration of std PAT added. The average percentage recovery obtained is 95%. Further confirmation of the accuracy of this method was the double analysis of each sample. The average of reproducibility relative standard deviation (RSD) for PAT was 2.87%. Precision was determined by analysing three fruit juices samples spiked at 5, 10 and 25 mg kg1. Within-day precision was determined by analysing six replicates of spiked
Table 1. Results for the recovery of the method applied for the determination of patulin in fruit juices. [PAT] (mg l1) 5 10 25
Recovery (%)
Coefficient of variation (%)
96.7 93.8 94.5
2.228 3.120 3.267
homogenized apple-based baby food; between-day precision was determined by analysing six replicates during 3 days. The precision of the method was calculated from the RSD (Table 2). The identity of PAT in contaminated samples was confirmed by comparing the ultraviolet (UV) light spectrum of the peak eluting at a retention time of PAT with that of authentic standard of PAT (Sigma-Aldrich). Additional confirmation was performed in selected positive samples that were reanalysed by HPLC after spiking the extracts with an equivalent amount of PAT by checking the symmetry of the peak eluting at a retention time of PAT. The limit of detection (LOD) was determined to be 0.3 mg kg1; the limit of quantification (LOQ) was 1.0 mg kg1; the repeatability of results for duplicate analysis ranged from 1% to 5%.
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Table 2. Precision of the method for the determination of patulin in fruit juices. Within-day Concentration [PAT] (mg kg1) 5 10 25
Between-day
(mg kg1)
n
RSD (%)
(mg kg1)
n
RSD (%)
4.83 9.38 23.62
6 6 6
2.5 2.8 2.9
4.75 9.25 23.12
3 3 3
2.9 2.8 2.6
Table 3. PAT concentrations in examined fruit juices. PAT concentration range (mg l1) Apple juices
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Less than or equal to 50% of fruit
Greater than or equal to 50% of fruit
Italian product, 11 samples 10–30 Italian product, 9 samples n.d.–28
European product, 7 samples 18–30 European product, 8 samples 10–26
Mixed fruit juices Italian product, 13 samples n.d.–45 Italian product, 12 samples n.d.–43
European product, 5 samples 28–45 European product, 5 samples 27–44
Pear juices Italian product, 11 samples 7–80 Italian product, 9 samples 5–65
European product, 7 samples 25–92 European product, 8 samples 10–90
Notes: Numbers shown in bold are out of law limit. n.d., Not determinable.
Results and discussion In the 35 apple juices analysed, the concentration of PAT found was less than the limit set by Regulation EC 1881/2006 of 50 mg l1 and is between 6 and 30 mg l1 (in four samples the PAT concentration was not determined (n.d.)), and the average was 18.06 mg l1 (Table 3). In 35 mixed fruit juices the range of PAT contamination was 1–45 mg l1 (in three samples the PAT concentration was n.d.), and the average was 22.97 mg l1, again less than the regulation limit (Table 2). Instead, in pear juices the range of PAT contamination was 5–92 mg l1, with an average of 42.62 mg l1, and 14 samples (40% of the 35 analysed) exceeded the limit of 50 mg l1 (Table 3). The results show that 79.33% of samples tested had PAT contamination, though below the limits imposed by the regulations. This widespread contamination by PAT is a parameter to judge the quality of the raw material used, the selection process of the fruit and the respect of the HACCP system. To ensure food quality and safety, it would be necessary to adopt an integrated approach to the production line which is able to control the hazards that arise from primary production to consumption. The hazards should be identified as critical control points (CCP) and controlled during the production steps (collection, storage, sorting and washing fruits) in which corrective actions are most effective, economical and feasible. The operators involved in primary production must follow the documented procedures to ensure the
selection of raw material. The European Union Recommendation of 11/08/2003 on ‘the prevention and reduction of contamination of patulin in apple juice and the ingredients of apple juice in other beverages’ requires that the apple processing industries should adopt good manufacturing practice. The presence of a PAT concentration higher than the legal limits (50 mg l1) in 14 pear juice samples (40% of the examined ones) emphasizes the need to extend these criteria of good manufacturing practice and to develop an HACCP system that recognizes more relevant additional CCP by taking into account the formation of PAT in the industries that produce juices other than apple juices. It was also found that the amount of contamination is related to the percentage of fruit in juice. The lower the amount of fruit used, the greater the possibility that there is contamination. Samples with a percentage of fruit lower than 50% showed an average concentration of PAT equal to 31.55 mg l1; those with more than 50% of fruit revealed an average concentration of PAT equal to 23.41 mg l1. This suggests that in fruit juices, such as those that have a lower market cost, the raw material used is not carefully selected, probably because producers trust that the ‘diluting effect’ will reduce the potential levels of contamination. However, in addition, it should be considered that the dilution of the juices analysed included the use of apple juice, which has a low cost on the market and is
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often used for its sweetening properties as a dilutent for juices and beverages. It becomes necessary, therefore, not only to assess the quality of the raw material, but also in equal measure to check the health standards of the dilutents. By splitting the samples according to their source of origin – Italian (61.9%) and European (38.1%) – it was estimated that the European samples have an average concentration of PAT equal to 39.92 mg l1, which is higher than the average concentration of 20.78 mg l1 detected in Italian samples. The data presented allow one to make an assessment on the potential health risks for consumers of all age groups. In fact, in 13.33% of 105 analysed samples, the concentration of PAT exceeding the limit fixed by European Regulation 1881/2006 demonstrates that the high production standards required in this food chain have not yet been reached by all producers. Even thought the regulation limit for the presence of PAT in fruit juices concerns only adults, this product is also highly represented in the diet of all groups of consumers, and there is certainly an increased ‘intake’ in the diet of children above 2 years of age. The current law is mostly focused on early childhood, that includes babies up to the first 24 months of age, infants and young children setting lower PAT limits only for apple juice and solid apple product, including apple compote and apple puree. For the other fruit juices the maximum limits set for contaminants in food are the same as for an adult of 70 kg of body weight. As regards the risk of accumulation of specific substances such as mycotoxins, it would be necessary for competent authorities to provide further differentiation in the law limits taking into account the fact that child consumers have not yet reached a body weight of 70 kg and have a metabolism that is not fully specialized to carry out appropriate detoxification. Moreover, it would be necessary to divide the population into different categories, as has already been done for other substances subject to accumulation, as established in the pharmaceutical regulation since the 1990s.
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