Hygienic significance of patulin in food. 2. Occurrence of patulin in fruit and vegetables]

The analyses of more than 200 samples of various foods of plant origin showed that patulin was contained in 36% of the fresh and canned fruits infested with mould, and in 7% of the vegetables. Besides apples, pears, plums, peaches and tomatoes contained also patulin. In organoleptically impeccable fruit juices, the contamination rates were 40% (for apple juice) and 16% (for the other juices, such as sour cherry, currant, sea buckthorn juices). The patulin content varied from 20 to 200 microgram/l, the mean value being 80 microgram/l. It ranged from 0.1 to 5 microgram/g in apples and sterile apple preserves. The authors discuss the hygienic-toxicologic significance of these findings, and suggest to include patulin in the examination of foods for mycotoxins, stipulating a permissible value.     

Incidence of patulin in fruits and fruit juices marketed in Campinas, Brazil

One hundred and eleven samples of processed fruit juices (apple, grape, pineapple, papaya, guava, banana and mango) and 38 samples of sound fruits (apple, papaya, mango, pear and peach) produced and marketed in Brazil, were analysed for patulin by HPLC. Only one out of 30 samples of apple juice was found positive at 17 mug/l. Patulin was not detected in the other foodstuffs. It was found in 14 samples of spoiled fruit samples of apple (150-267 mug/kg), pear (134-245 mug/ kg) and peach (92-174 mug/kg). Confirmation of the identity of patulin was based on the UV spectrum obtained by the HPLC diode array detector, compared with thatof standard patulin, TLC developed by several solvent systems and sprayed with 3-methyl-2-benzothiazolinone hydrazone , and by acetylation with acetic anhydride.     

Incidence of patulin in apple juices marketed in Turkey.

The purpose of this study was to investigate the patulin contamination of apple juices consumed by the Turkish population. Patulin was detected using high-performance liquid chromatography (HPLC) with a UV detector at 280 nm, and the identification of patulin was further confirmed by thin-layer chromatography (TLC). Using HPLC, the recoveries were 79.9 +/- 6.7% and 83.7 +/- 4.6%, and the coefficients of variation were 8.4 and 5.5% for apple juices spiked with the known amounts of patulin (60 and 120 microg/liter. respectively). The minimum patulin level detected was 5 ng in a standard solution and 5 microg/liter in apple juices. The TLC method was used only to confirm patulin levels higher than 20 microg/liter (100 ng/spot) in apple juices. The total number of samples was 45. Patulin was present in detectable levels in 60% of apple juices at concentrations ranging from 19.1 to 732.8 microg/liter. Forty-four percent of the apple juice samples had patulin contamination levels higher than 50 microg/ liter, which is the allowable upper limit in Turkey.     

Occurrence of patulin in organic, conventional, and handcrafted apple juices marketed in Belgium

The aim of this research was to compare the occurrence of patulin in a large group of organic, conventional, and handcrafted apple juices marketed in Belgium. An analytical procedure based on high-performance liquid chromatography with UV detection was validated and used to analyze 177 apple juice samples: 65 organic, 90 conventional, and 22 handcrafted. Patulin was detected in 22 samples (12%), and quantification was possible in 10 (6%) of these samples. The patulin content was higher than the European legal limit of 50 microg/liter in two samples of organic apple juice. Although, the incidence of patulin in organic (12%), conventional (13%), and handcrafted (10%) apple juices was not significantly different (P = 0.863), the mean concentration of patulin in contaminated samples was significantly higher in organic (43.1 microg/liter) than in conventional (10.2 microg/liter) (P = 0.02) and handcrafted (10.5 microg/liter) (P = 0.037) apple juice. The highest patulin concentrations were found in the most expensive apple juices because of the higher price of organic apple juice. This relation was not observed when only conventional apple juices were analyzed.     

Long-term survey of patulin in apple juice concentrates produced in Turkey.

A liquid chromatographic method described by us elsewhere was evaluated for a long-term survey of patulin in apple juice concentrates. Patulin was separated on a reversed phase C18 LC column with water-acetonitrile (99:1) as the mobile phase and quantitated with a photodiode array (PDA) detector. Relatively low amounts of patulin (< 5 micrograms/l for single strength juice at 11.2 degrees Bx) were detected in apple juice concentrates and confirmed by PDA detector, comparing the corresponding UV spectra with that of patulin standard. Four hundred and eighty two apple juice concentrates produced through 1996-99 were analysed for their patulin contents. Year-to-year variations in patulin levels of apple juice concentrates were found out to be statistically significant. Patulin contamination levels of apple juice concentrates tended to decrease through the years and averaged 63, 43, 19 and 31 micrograms/l in 1996, 1997, 1998 and 1999, respectively. Percentages of concentrates exceeding the maximum permitted concentration of 50 micrograms/l were 52%, 34%, 8% and 8% for 1996, 1997, 1998 and 1999, respectively.     

Contamination of food products with patulin

Based on the method for patulin determination designed by the authors the rate and level of contamination of fruit, vegetables and foods manufactured from them (juices, purees, jams) with this mycotoxin were studied. Patulin was found in 8 out of 160 samples of fruit and vegetables, the highest concentration of patulin was identified in the berries of sea buckthorn (up to 54 000 micrograms/kg). Patulin was found in 28 out of 185 samples of foods manufactured from fruit. The concentration of patulin in juices did not exceed the allowable concentrations on the average. Patulin was largely identified in apple, sea buckthorn and plum juices.     

Occurrence of patulin in various fruit juices from South Korea: An exposure assessment

To determine patulin in various fruit juices, high performance liquid chromatography (HPLC) method was optimized and validated. For validation of HPLC method, a linearity, accuracy, precision, detection limit, and quantification limit were determined. Linearity (R² = 0.99995), accuracy (96.1–115.7%), precision (3.31–9.52), detection limit (6 ng/mL), and quantification limit (8 ng/mL) were in agreement with performance criteria for patulin as set by the European Commission hence proved that HPLC can be used to detect patulin in fruit juices. After validation, the method was applied to estimate the prevalence of patulin in fruit juices (apple, grape, and orange juices). Nine samples (12.5%, 3 apple, 2 orange, and 4 grape juices) of 72 samples were positive for patulin in the range 2.8 to 30.9 ng/mL. According to the monitoring results, daily intake was estimated to be 0.17 ng/kg BW/day which was lower than the provisional maximum tolerable daily intake (0.4 μg/kg) established by Joint Expert Committee on Food Additives. These results indicate that the detection method coincides with the performance criteria and is appropriate for analysis of patulin, and continuous monitoring of patulin in various fruit juices from Korea is necessary.     

Inter-laboratory study for validation of a Japanese official analytical method for determination of patulin in apple juice

To validate a modified version of AOAC official method of analysis 995.10 as an official standard in Japan for determination of patulin in apple juice, an inter-laboratory study was performed in 11 laboratories using a non-contaminated sample, 2 naturally contaminated samples and 2 spiked samples of apple juice. For naturally contaminated apple juices, the relative standard deviations for repeatability and reproducibility were 3.2, 7.1% and 10.0, 21.7%, respectively. HORRAT values were 0.4, 0.9. The average recovery of patulin from spiked sample was 83.7%. The limit of quantification was calculated as 10 microg/kg. From these results, the method was thought to be suitable as an official standard for determination of patulin in apple juice in Japan.     

CONTAMINATION OF PATULIN IN CLEAR APPLE JUICE IN MASHHAD, IRAN

Patulin is a frequent contaminant of moldy and rotten apples and apple products. The aim of this study was to evaluate patulin contamination in 58 apple juices collected from a retail market in Mashhad during winter and spring of 2006.
Samples were assayed for patulin by high-performance liquid chromatography. Fifty-four samples were positive for patulin at levels that ranged from 10.5 to 121.8 µg/L, and six samples had patulin levels higher than 50 µg/L. The overall mean of patulin concentration was 29.2  ±  19.5 µg/L. Forty-eight samples had patulin concentration between 5 and 50 µg/L. Although the mean concentration of patulin samples was lower than Iranian maximum tolerated level of 50 µg/L, contamination of 10% of the samples at levels higher than 50 µg/L indicated the need for improving production techniques by the industry.

PRACTICAL APPLICATIONS

Studies have shown that the concentration of patulin may exceed the determined limits in apple juice and in other fruit products. Its presence can be a potential threat to the health of consumers, particularly children. The results may help us in understanding what should be the level of patulin in apple juice. The awareness of the apple industry of patulin contamination in fruit and the implementation of improved techniques for the production of apple products with reduced patulin concentrations have contributed to the quality of apple juice that are available on the Iranian market.     

Determination and the hygienic-toxicologic significance of patulin in fruit and fruit products]

The combination of extracting, chromatographic and fluorescence densitometric steps permits to determine patulin in fruits and fruit products with great precision and sensitivity. The limit of detection is 10 microgram/kg; the recovery rates range from 86 to 92% with a variation coefficient lying between 5.6 and 13.6%. Interferences due to patulin-simulating substances are widely excluded. Patulin concentrations ranging from 0.3 to 42 mg/kg were found in the brown-rotten portions of apples. Juices domestically prepared from healthy fruits and fruits rid of rotten portions, respectively, contained no patulin. On the contrary, patulin concentrations ranging from 0.02 to 0.3 mg/l were observed in commercial apple juices. As to products with subsequent mould infection, patulin values up to 50 mg/l were found in apple juices, and up to 0.4 mg/kg in peach preserves. In a further 24 commercial fruit and vegetable juices, patulin was not detectable even not in cider, in cereals which had gone mouldy spontaneously, and in bread samples. The fruits and fruit products were analysed not only for patulin, but also for aflatoxin and ochratoxin; the latter two, however, were found in none of the samples examined. The possibilities of manufacturing patulin-free products are discussed.     

Patulin contamination in fruit derivatives,including baby food,from the Spanish market

The aim of this study was to provide a current overview on patulin (PAT) contamination of apple products in Spain. A second aim was to provide some data on the effect of processing stages of fruit juice concentrates on patulin levels. None of the analysed samples contained PAT concentrations exceeding the maximum levels fixed by the EU. Pear and apple concentrates showed the highest levels (up to 126 μg/kg). After milling, patulin levels did not exhibit important differences, except for at the concentration stage where a greater than fourfold mean increase was recorded. Moreover, a decrease was observed in those fruit juices that were decolourised after ultrafiltration. Thus, patulin presence in fruit products might not pose a health risk for the average consumer, although studies on patulin intake by populations consuming high amounts of fruit derivatives might be of importance.     

Effect of activated charcoal on patulin, fumaric acid and some other properties of apple juice

In this study, 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 g/l amounts of activated charcoal (AC) were added into apple juice with a patulin content of 62.3 ppb obtained from a well-established manufacturing company. Apple juice samples were then mixed for 0, 5, 10, 20, and 30 min, respectively. Considerable reduction in the patulin and HMF values was found while there is a dramatic improvement in the colour and clearness of apple juice. However, AC did not cause a significant decrease in the fumaric acid level of apple juice. The best result was obtained at 3.0 g/l AC mixed for 5 min. In addition, a negligible reduction in brix and pH values of samples was observed.     

Survey of South African fruit juices using a fast screening HILIC-MS method

Adulteration of fruit juices – by the addition of sugar or other less expensive fruit juices as well as preservatives, artificial sweeteners and colours – was tested for by using a developed screening method. The method employs hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) using electrospray ionisation in the negative mode and ultraviolet light detection. Different fruit juices can be differentiated by the content of marker compounds like sorbitol, certain phenolic molecules and their saccharide profile. This method was used to test 46 fruit juice samples from the retail market as well as 12 control samples. The study focused on the main types of fruit juices consumed on the South African market including apple, orange, grape and blends of these juices with other fruits like mango, pear and guava. Overall, the 46 samples tested mostly agreed with label claims. One grape juice sample was adulterated, probably with apple juice. Natamycin above the legal limits was found in two samples. In addition, two samples contained natamycin and one sample benzoate without it being indicated on the label. The method is well suited as a quick screening method for fruit juice adulteration and if used routinely would reduce fruit juice adulteration without the cost of the current array of tests needed for authenticity testing.     

Development and characterization of a carbon-based composite material for reducing patulin levels in apple juice

Patulin, a heterocyclic lactone produced by various species of Penicillium and Aspergillus fungi, is often detected in apple juices and ciders. Previous research has shown the effectiveness of granular activated carbon for reducing patulin levels in aqueous solutions, apple juices, and ciders. In this study, ultrafine activated carbon was bonded onto granular quartz to produce a composite carbon adsorbent (CCA) with a high carbonaceous surface area, good bed porosity, and increased bulk density. CCA in fixed-bed adsorption columns was evaluated for efficacy in reducing patulin levels from aqueous solutions and apple juice. Columns containing 1.0, 0.5, and 0.25 g of CCA were continuously loaded with a patulin solution (10 microg/ml) and eluted at a flow rate of 1 ml/min. Results indicated that 50% breakthrough capacities for patulin on 1.0-, 0.5-, and 0.25-g CCA columns were 137.5, 38.5, and 19.9 microg, respectively. The effectiveness of CCA to adsorb patulin and prevent toxic effects was confirmed in vitro using adult hydra in culture. Hydra were sensitive to the effects of patulin, with a minimal affective concentration equal to 0.7 microg/ml; CCA adsorption prevented patulin toxicity until 76% breakthrough capacity was achieved. Fixed-bed adsorption with 1.0 g of CCA was also effective in reducing patulin concentrations (20 microg/liter) in a naturally contaminated apple juice, and breakthrough capacities were shown to increase with temperature. Additionally, CCA offered a higher initial breakthrough capacity than pelleted activated carbon when compared in parallel experiments. This study suggests that CCA used in fixed-bed adsorption systems effectively reduced patulin levels in both aqueous solutions and naturally contaminated apple juice; however, the appearance and taste of apple juice may be affected by the treatment process.     

Patulin in apple-based foods: occurrence and safety evaluation

Patulin is a mycotoxin produced by certain species of Penicillium and Aspergillus, often detectable in mouldy fruits and their derivatives. On the basis of a PMTDI of 0.4 mu g/kg bw, limit values of 50 mu g/kg or 50 mu g/l of patulin have been set in fruit derivatives. To estimate the quantity of patulin that can be taken in with the diet, we analysed by HPL C samples of apples and apple derivatives which are most likely to be contaminated with patulin. In apple juices and in homogenized babyfoods, the mycotoxin concentration was always below the established limits, while in some samples of juice with pulp the mycotoxin content exceeded the safe levels. In rotten apples, not only was the amount of patulin extraordinarily high in the rotten area, but the mycotoxin had also spread to the part unaffected by mould. The data presented in this study indicate that the intake of patulin with apple derivatives is usually below the tolerable level of 0.4 mu g/kg bw/day, but since the patulin content in apples can vary considerably, the quality of fruits used in the production of apple derivatives should be strictly controlled in order not to exceed the safe limits.     

Patulin in domestic and imported apple-based drinks in Belgium: occurrence and exposure assessment

Apple-based beverages are regularly consumed by adults and children in Belgium. They are locally produced or imported from other countries. The apples used as starting material for these productions are frequently contaminated by mycotoxin-producing moulds and damaged during transport and handling. The current study was undertaken to investigate whether patulin (PAT) is present in the industrial or handicraft-made apple juices and ciders consumed by the Belgian population and to assess the population's exposure to this mycotoxin through apple-based drinks. Belgian (n = 29) and imported (14) apple juices as well as ciders (7) were assayed for PAT by high-performance liquid chromatography with ultraviolet light detection. PAT was detected respectively in 79, 86 and 43% of these tested samples. However, no contaminated sample exceeded the safe level of 50 µg PAT l^-1. Levels of PAT contamination were comparable in Belgian and imported juice samples. The overall mean PAT concentrations were 9.0 and 3.4 µg l^-1 for contaminated apple juices and ciders, respectively. This study also indicates that there was no statistically significant difference in the mean PAT contamination between clear (7.8 µg l^-1) and cloudy (10.7 µg l^-1) apple juices, as well as between handicraft-made apple juices (14.6 µg l^-1) and industrial ones (7.0 µg l^-1). On the basis of the mean results, a consumer exposure assessment indicates that a daily intake of 0.2 litres apple juice contributes to 45% of the provisional maximum tolerable daily intake for a child of 10 kg body weight.     

Investigation of patulin contamination in apple juice sold in retail outlets in Italy and South Africa

A study of apple juice products sold in Italy and South Africa was initially carried out on 20 samples bought in Cesena, Italy, and Tygerberg in Cape Town, South Africa. The samples were bought at random and analysed for patulin contamination. All 12 of the Italian samples had no detectable levels of patulin, except one, which was just slightly above the lowest regulatory limit of 10 ng ml^-1. On the other hand, five of the eight South African samples were all contaminated with patulin levels above 10 ng ml^-1, with one showing a concentration of 75 ng ml^-1, well above the highest regulated limit of 50 ng ml^-1. This latter result led to a more targeted investigation with 14 samples being purchased in the low-income areas of Tygerberg where the initial samples were sourced. These samples confirmed that there might be a problem of mycotoxin contamination in apple juices products sold to low-income consumers because half of the samples showed patulin contamination of which four had levels well above the acceptable limits. This is the first study in South Africa to look at apple juice products in low-income areas and it points to a need to intervene and introduce quality systems in the supply chain of the manufacture and packaging of apple juice products by independent small business.     

Characterization and quantification of anthocyanins in grape juices obtained from the grapes cultivated in Korea by HPLC/DAD, HPLC/MS, and HPLC/MS/MS

The characterization and quantification of anthocyanins in grape cultivars of Oll-Meoru (Vitis coignetiaexVitis labrusca), Neut-Meoru (Vitis coignetiaexVitis labrusca), Muscal Bailey A. (Vitis labruscana), and Campbell Early (Vitis labruscaxV. vinifera) cultivated in Korea were carried out by partial purification through XAD-7 column chromatography followed by C-18 HPLC/diode array detector (DAD), HPLC/MS, and HPLC/MS/MS analyses. The column oven temperature during the reverse phase C-18 HPLC greatly affected the separation of individual anthocyanins. The result showed that the optimum column oven temperature was 35 degrees C. Sixteen different anthocyanins (11 nonacylated and 5 acylated anthocyanins) were identified in the grape juices. Oll-Meoru, Neut-Meoru, and Muscat Bailey A (MBA) grape juices contained only nonacylated anthocyanins. Oll-Meoru and Neut-Meoru grape juices had same anthocyanins, but their proportions were considerably different. Peonidin 3,5-diglucoside and malvidin 3,5-diglucoside were the major anthocyanins in Oll-Meoru grape juice. Delphinidin 3-glucoside was, however, the major anthocyanin in Neut-Meoru grape juice. Peonidin 3-glucoside and malvidin 3-glucoside were the most abundant anthocyanins in Muscal Bailey A grape juice. Campbell Early grape juice contained both nonacylated and acylated anthocyanins. Cyanidin 3-(p-coumaroyl)glucoside-5-glucoside and peonidin 3-(p-coumaroyl)glucoside-5-glucoside were the most abundant anthocyanins in Campbell Early grape juice. Total anthocyanin contents were greatly different in different grape jucies, with the highest in Neut-Meoru juice (1043.5 microg/mL), followed by Oll-Meoru (997.7 microg/mL), MBA (390.2 microg/mL), and Campbell Early (183.9 microg/mL) juices. The total anthocyanin content in Neut-Meoru grape juice was 5.67 times higher than that in Campbell Early grape juice. This represents the 1st report on the systematic characterization and quantification of anthocyanins in the juices of these grapes cultivated in Korea.     

Grape,apple and pineapple juice characterisation and detection of mixtures

Several multivariate statistical methods (principal component, cluster, stepwise discriminant and multiple linear regression analysis) were applied to analytical data obtained by conventional methods and to analytical data for free amino acids, in an attempt to distinguish between grape, apple and pineapple juice. Analysis for methionine, proline, asparagine, arginine and glutamic acid made it possible to differentiate among the 34 samples of grape, apple and pineapple juices studied. Multiple linear regression analysis of computer-simulated mixtures of grape and apple juices made it possible to detect the proportion of each fruit within the blend.     

Occurrence of patulin in conventional and organic fruit products in Italy and subsequent exposure assessment.

The occurrence of patulin was investigated in 100 conventional and 69 organic fruity foodstuffs samples commercially available in Italy by using an HPLC method with a limit of quantification of 0.5 microg kg(-1). Patulin was detected in 26 (26%) conventional and 31 (45%) organic products with a significantly higher (p<0.01) mean concentration in the organic products (4.78 vs. 1.15 microg kg(-1)). Mean patulin concentrations in conventional apple juices, pear juices, other juices and fruits purees were 3.14, 0.22, 0.19, 0.11 microg kg(-1), respectively, and 7.11, 11.46, 2.10, 0.18 microg kg(-1) in the relevant organic products. Four samples of juices (one conventional and two organic apple, and one organic pear) contained patulin at concentrations above the limit of 50 microg kg(-1), four at concentrations between 10 and 25 microg kg(-1), and the remaining ones below 10 microg kg(-1). Patulin was detected (<1 microg kg(-1)) in only three of the 23 fruity baby food samples tested (homogenized fruits, 11 conventional and 12 organic). Based on the available data on Italian intakes of fruit juices, the estimated daily intakes of patulin, were 0.38 and 1.57 ng kg(-1) body weight (bw) from conventional and organic products, respectively. Estimated daily intakes of patulin for children were higher, 3.41 ng kg(-1) bw from conventional and 14.17 ng kg(-1) bw from organic products, but largely below the provisional maximum tolerable daily intake (PMTDI) of 400 ng kg(-1) bw. Patulin was also found in two samples of organic apple vinegar (<5 microg kg(-1)) and in fresh apples with rotten spots (12 out of 24 samples) with maximum levels at 16,402 and 44,572 microg kg(-1) for conventional and organic apples, respectively.