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.     

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.     

Assessment of dietary intake of patulin from baby foods

Patulin is a mycotoxin produced by microscopic fungi belonging to the Penicillium and Aspergillus genera, frequently detectable in moldy fruits and their derivatives fruit products. The EC Regulation 1881/06 has imposed the limit for the presence of patulin equal to 10 μg/kg or 10 μg/L in baby food on the basis of a PMTDI of 0.4 μg/kg bw set by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). A total of 120 homogenized baby foods were analyzed to evaluate the exposure of baby and children to patulin through the consumption of these products. None of examined samples has shown a toxin concentration above the limit imposed by the law, however a PAT concentration equal to 9 μg/kg was found in 22 samples, slightly below the fixed limit. The presence of patulin in marketed baby food can be regarded as a parameter indicative of the quality of raw materials used.     

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.     

Saccharomyces cerevisiae YE‐7 reduces the risk of apple blue mold disease by inhibiting the fungal incidence and patulin biosynthesis

The yeast strain, Saccharomyces cerevisiae YE‐7, was tested to control the apple blue mold disease caused by Penicillium expansum and the accumulation of patulin mycotoxin. Compared with control, pre‐treatment of pathogen‐infected apples with YE‐7 significantly reduced the incidence of blue mold diseases and patulin accumulation in rotten apple tissue by 48% and 42.6%, respectively. Interestingly, late treatment of pathogen‐infected apples with YE‐7 did not decrease patulin accumulation in the rotten tissue compared with pre‐treatment or simultaneous‐treatment. Independently, patulin amount and gene expression of isoepoxydon dehydrogenase were also measured. YE‐7 pre‐treated apples showed 75% and four times reduction in patulin accumulation and IDH gene expression, respectively. The results indicated that YE‐7 directly affected patulin biosynthesis and did not affect accumulated patulin. On the other hand, cold storage enhanced the effect of YE‐7 on apple blue mold disease incidence.

Practical applications

Postharvest blue mold decay caused by Penicillium expansum was important postharvest disease of apples. In addition, under favorable conditions, P. expansum can cause severe losses in apple fruits due to the production of patulin. In this study, a strain of S. cerevisiae (YE‐7) was assessed for its efficacy not only in controlling apple blue mold diseases, but also in reducing patulin accumulation in fruit tissue. The research will give a practical way to control the decay in a bio‐control way instead of the chemical.     

Reducing patulin contamination in apple juice by using inactive yeast

The mycotoxin, patulin (4-hydroxy-4H-furo[3,2c]pyran-2[6H]-one), is a secondary metabolite produced mainly in rotten parts of fruits and vegetables, most notably apples and apple products, by a wide range of fungal species in the genera Penicillium, Aspergillus, and Byssochlamys. Due to its mutagenic and teratogenic nature and possible health risks to consumers, many countries have regulations to reduce levels of patulin in apple products. In the present study, reduction of patulin contamination in apple juice by using 10 different inactivated yeast strains was assessed. Our results indicated that nearly twofold differences in biomass existed among the 10 yeast strains. Eight of the 10 inactivated yeast strains could provide >50% patulin reduction in apple juice within 24 h, with the highest reduction rate being >72%. Furthermore, juice quality parameters, i.e., degrees Brix, total sugar, titratable acidity, color value, and clarity, of the treated apple juice were very similar to those of the untreated patulin-free juice. Potential applications of using inactivated yeast strain for patulin control are also discussed.     

Some factors influencing patulin production by Penicillium expansum in pome fruits

BACKGROUND: The objective of our study was to examine the effects of Penicillium expansum on patulin production in relation to isolates, species and cultivar type, incidence and severity of decay. In addition, patulin production at different incubation times and its diffusion were also investigated. These factors were evaluated in pome fruits inoculated with P. expansum and kept at 20 °C for short periods of time. RESULTS: The ability of five P. expansum isolates to grow and produce patulin in inoculated Golden Delicious apples varied among the strains from below the limit of quantification to 662 µg kg^?1. Variety and species of pome fruits influenced patulin production. P. expansum isolate PE97.IT produced a higher patulin content in apples than in pears. The highest patulin production was 386 µg kg^?1 in Golden Delicious. No blue mould symptom appeared in pears inoculated with P. expansum and no patulin was detected after 3 days at 20 °C. However, patulin increased with incubation time after 6 and 8 days. No patulin was detected in healthy pear tissue but it was high in the decayed area. CONCLUSION: Since patulin production is associated primarily with infected rotten tissue, patulin control is possible by using healthy fruits, sorting damaged and rotten fruits before processing. Copyright © 2010 Society of Chemical Industry     

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.     

Co-occurrence of patulin and citrinin in Portuguese apples with rotten spots

Patulin and citrinin are mycotoxins produced by certain fungi mainly belonging to Penicillium and Aspergillus and may be detected in mouldy fruits and fruit products. The data presented here refer to the simultaneous occurrence of patulin and citrinin in 351 samples of seven different varieties of apples with small rotten areas (Casanova, Golden Delicious, Red Delicious, Reineta, Richared, Rome Beauty, Starking). A rapid multidetection thin layer chromatography (TLC) method was used. The minimum detectable concentrations of patulin and citrinin were 120-130 and 15-20 μg kg^-1 respectively. The percentage contamination with patulin only was higher (68.6%) than that with citrinin only (3.9%). Patulin and citrinin (19.6%) were also detected simultaneously. The highest mean patulin content was 80.50 mg kg^-1 for the Richared variety, but the mean level of citrinin was lower. The lowest mean contaminations of patulin were found in Rome Beauty, Red Delicious and Reineta, ranging from 3.06 to 5.37 mg kg^-1. All analysed apples varieties had low citrinin contamination, ranging from 0.32 to 0.92 mg kg^-1. These findings indicate that there may be a risk of human exposure to patulin through the consumption of juices and jams manufactured with apples with small rotten areas.     

Production of mycotoxins by Penicillium expansum inoculated into apples

We investigated the production of mycotoxins in apple fruits inoculated with spores of 40 strains of apple blue mold, Penicillium expansum. Patulin and citrinin contents in the extracts from apples stored at 25 degrees C for 12 days after inoculation were determined by high-performance liquid chromatography (HPLC) analysis with UV and fluorescence detection. Patulin and citrinin were produced by 90% (36) and 80% (32) of the 40 strains, indicating that P. expansum is a consistent producer of these mycotoxins. The patulin content in the extracts was substantially higher than the citrinin content. Other mycotoxins whose production in pure culture has been reported were simultaneously detected with high-resolution liquid chromatography-mass spectrometry (LC-MS) analysis with the positive ion mode of electrospray ionization. Along with patulin and citrinin, expansolides A and B were identified based on the HPLC and LC-MS spectral data and detected in 88% (35) of the extracts. The results indicate that P. expansum is a consistent producer of expansolides A and B in rotten areas of apple fruits. The findings raise the possibility that products from decayed apples might contain expansolides A and B in addition to patulin and citrinin.     

Patulin distribution in Fuji and Golden apples contaminated with Penicillium expansum

This work assesses the extent of patulin contamination in Penicillium expansum-infected apples stored at room temperature for short periods of time and its relationship with apple variety (Golden or Fuji), degree of ripeness and size of lesions. Inoculated apples were incubated at 20°C. Patulin was determined in both sound and decayed tissue from cylindrical samples taken around the lesions and cut into 0.5-cm thick sections. Higher accumulation of patulin occurred in Golden apples, with less ripened apples showing higher concentrations. Total accumulated patulin was similar or higher in 4-cm compared to 2-cm lesioned apples, although a decrease in patulin concentration was observed in older lesion sections. Patulin accumulation occurred over a short period of time at room temperature, thus the stand-by period before processing should be minimised. Of total patulin, 2-6% migrated to the surrounding sound tissue, thus trimming tissue around the rotten part may be a good preventive practice for apple derivative production.     

Apple quality,storage, and washing treatments affect patulin levels in apple cider

Patulin is a mycotoxin produced primarily by Penicillium expansum, a mold responsible for rot in apples and other fruits. The growth of this fungus and the production of patulin are common in fruit that has been damaged. However, patulin can be detected in visibly sound fruit. The purpose of this project was to determine how apple quality, storage, and washing treatments affect patulin levels in apple cider. Patulin was not detected in cider pressed from fresh tree-picked apples (seven cultivars) but was found at levels of 40.2 to 374 microg/liter in cider pressed from four cultivars of fresh ground-harvested (dropped) apples. Patulin was not detected in cider pressed from culled tree-picked apples stored for 4 to 6 weeks at 0 to 2 degrees C but was found at levels of 0.97 to 64.0 microg/liter in cider pressed from unculled fruit stored under the same conditions. Cider from controlled-atmosphere-stored apples that were culled before pressing contained 0 to 15.1 microg of patulin per liter, while cider made from unculled fruit contained 59.9 to 120.5 microg of patulin per liter. The washing of ground-harvested apples before pressing reduced patulin levels in cider by 10 to 100%, depending on the initial patulin levels and the type of wash solution used. These results indicate that patulin is a good indicator of the quality of the apples used to manufacture cider. The avoidance of ground-harvested apples and the careful culling of apples before pressing are good methods for reducing patulin levels in cider.     

Detection of patulin in apple juices marketed in the Tohoku district, Japan

Patulin is a mycotoxin mainly produced by Penicillium and Aspergillus. We investigated the incidence of patulin contamination in 179 samples of apple juice and 9 samples of mixed juice (containing apple juice concentrate as an ingredient) commercially available in the Tohoku district of Japan. Patulin was detected in 3 of 143 samples containing domestic fruits and in 6 of 45 samples containing imported products and products produced in Japan using imported apple juice concentrate. Patulin analyses were carried out using high-pressure liquid chromatography with a detection limit of 4 microg/liter. The patulin content of contaminated domestic samples (three samples with concentrations ranging from 6 to 10 microg/liter), imported samples (one sample with a concentration of 15 microg/liter), and domestic samples produced containing imported concentrate (five samples with concentrations ranging from 6 to 9 microg/liter) was lower than the maximum limit of 50 microg/liter currently adopted by many countries, including Japan.     

腐烂苹果中棒曲霉素的分布研究

为探讨腐烂苹果中棒曲霉素(patulin,Pat)的分布情况,用富士苹果作材料,高效液相色谱仪(HPLC)检测Pat 含量。结果表明：苹果腐烂程度越高,Pat 平均含量越大;腐烂部位Pat 含量最大,离腐烂部位越远,Pat含量随距离增加而降低;腐烂苹果中的Pat 大部分已经扩散至未腐烂部分。建议生产中将腐烂严重的苹果整个弃除。     

EFFECT OF APPLE DECAY PROPORTION ON THE PATULIN, FUMARIC ACID, HMF AND OTHER APPLE JUICE PROPERTIES

Starking apples, classified by the decay proportion on the fruit surface as sound, 30, 60 or 100% decayed were used in the production of apple juice. Then patulin, fumaric acid, HMF, pH, brix and Hunter L, a and b values of apple juice samples were determined. Patulin and fumaric acid analysis were done with HPLC. The patulin concentration in juice samples produced with Starking apples that are sound, 30, 60 and 100% decayed ranged between 0.0–15.9 μg/kg, 47.1–500.3 μg/kg, 156.4–2257.5 μg/kg, 54.9–2508.6 μg/kg, respectively. The fumaric acid concentration in the same juice samples ranged between 0.00–0.20 mg/kg, 0.23–0.69 mg/kg, 0.41–2.08 mg/kg and 1.40–4.14 mg/kg. In general, the color values (L, a, b) of the samples produced from 100% decayed apples measured by reflectance with a Hunter colorimeter were low when compared with the others.     

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.     

Evaluation of strategies for reducing patulin contamination of apple juice using a farm to fork risk assessment model

The numerous studies conducted so far on the issue of patulin contamination have focused mainly on aspects like growth of Penicillium expansum, patulin production under different conditions and the influence of processing on the patulin concentration in apple juice. The purpose of the present study was to collect the necessary information and to develop a quantitative risk assessment model (QRAM) in order to evaluate different strategies to reduce patulin contamination. For apple juice (AJ) production 3 types of apples are considered, namely fresh apples, apples stored under cold air (short term storage) and apples stored under controlled atmosphere (CA) (long term storage). The QRAM described the complete chain from the picking of apples until storage of produced AJ. In comparison to a traditional chemical analysis, the QRAM was found accurate in predicting the concentration of patulin in cloudy and clear AJs commercialised in Belgium. Simulation of the model demonstrated that the use of apples stored under CA contributes to a large extent to the patulin contamination of AJ. Since apples stored in CA are used from more or less January onwards, AJ with high patulin concentration can be produced from January onwards. It would be useful in this respect to take this into account when sampling plans are made by apple juice producers in the framework of their HACCP-system and by governments and control agencies when monitoring programmes are elaborated. The duration of deck storage between the delivery at the apple juice producer (AJP) and the processing of the apples had a large influence on the patulin concentration, and this effect was more pronounced for apples stored under controlled atmosphere compared to apples stored under cold air. The duration of the deck storage should therefore be considered as a Critical Control Point (CCP) within HACCP-systems. Also the application of a sorting step was evaluated to be efficient to reduce the high patulin concentration in AJ. Therefore, a combination of the 2 most effective measures (namely sorting out apples with an infection lesion larger than 10 cm(2) and a reduction of the volume of CA apples) was tested and resulted in a reduction to levels below 25 μg/kg in 99.7 to 99.9% of the clear and cloudy apple juices, respectively. It is therefore advisable to include a sorting step prior to processing, when apples stored in CA are used.     

Model studies on the diffusion behavior of the mycotoxin patulin in apples, tomatoes, and wheat bread

 Quantitative studies on the diffusion behavior of the mycotoxin patulin (Pat) performed by stable isotope dilution assays using a carbon-13-labeled analogue as the internal standard revealed that Pat did not diffuse into apples affected by the fungus Penicillium expansum. At a distance of more than 2 cm from the infected zone the mycotoxin was not detectable. However, in a similar experiment with tomatoes the mycotoxin was found to penetrate into the whole fruit. These different characteristics were related to the physical laws of diffusion and attributed to differences in the texture of the foods. Like in apple tissue, the patulin content in molded wheat bread crumb fell sharply at a longer distance from the fungal mycelium. A comparison with aflatoxins revealed that these mycotoxins show much faster diffusion into the crumb as compared to patulin. Received: 14 March / Revised version: 8 June 2000     

Effects of apple and pear varieties and pH on patulin accumulation by Penicillium expansum

BACKGROUND: Accumulation of patulin in apples and pears and subsequently in their by‐products is caused mostly by Penicillium expansum which causes blue mould. Fruit pH and other parameters are sometimes characteristic of a certain variety and thus the use of particular varieties might affect patulin content in the final products. The aim of this study was to evaluate the influence of fruit variety and pH on patulin accumulation. Patulin accumulation in both apple and pear juices at different pH and also in different apple varieties (Golden, Gala and Fuji) and pear varieties (Blanquilla and Conference) was assessed. RESULTS: The pH of juices significantly influenced patulin accumulation, especially in apple juice in which the highest amounts of patulin were detected at pH 3.5. In fruits, the pH values of the substrate were determinant only under cold storage. Thus, Golden apples, which presented a lower pH, accumulated more patulin at 1 °C. However, this trend was not observed at other temperatures in which varieties with higher amounts of organic acids (Golden and Fuji apples) accumulated most patulin. In the pear varieties, significant differences in pH did not lead to significant differences in patulin accumulation. CONCLUSION: Although pH influences patulin accumulation, other factors such as organic acid content may play an important role. Concerning the apple and pear varieties we studied, it seems that rather than variety, other parameters like pH or acidic content may be more important. These parameters vary significantly depending on the degree of ripeness of the fruit. Gala apple, which is used in Spanish juice production, was the only variety that accumulated dramatically higher amounts of patulin. Copyright © 2008 Society of Chemical Industry     

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.