Survival of Penicillium expansum and Patulin Production on Stored Apples after Wash Treatments

ABSTRACT: Penicillium expansum is a widespread fungus found on apples that causes fruit decay and may lead to production of a toxic secondary metabolite, patulin. This study was undertaken to evaluate the effectiveness of several chemical sanitizers against P. expansum NRRL 2304 and to establish sanitizing wash treatments that would inhibit P. expansum growth and subsequent patulin production on Empire apples destined for cider. Wash treatments included 200 ppm NaOCl, 1% StorOx®, 0.5% potassium sorbate, 300 ppm SO₂, and 0% to 5% acetic acid. Spores of P. expansum or inoculated apple slices were dipped in sanitizing wash solution for 5 min, and mold growth and patulin production was monitored on subsequent storage. It was found that 0.5% potassium sorbate and 300 ppm SO₂ did not affect mold survival or patulin production; 1% StorOx® was effective against mold spores in solution (4 log Most Probable Number destruction of spores), but there was no significant reduction in spore count when the same solution was used to sanitize mold‐inoculated apple discs. Washing with 200 ppm NaOCl delayed growth of P. expansum on inoculated apple discs but failed to completely inhibit patulin production. Acetic acid solution (2% to 5%) was the most efficient chemical against P. expansum. A wash treatment with ≥2% acetic acid for more than 1 min is recommended to completely inhibit growth of P. expansum and subsequent patulin production on apples destined for cider.     

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.     

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     

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.     

Quantification of patulin in fruit leathers by ultra-high-performance liquid chromatography-photodiode array (UPLC-PDA)

Patulin is a mycotoxin commonly found in certain fruit and fruit products. For this reason many countries have established regulatory limits pertaining to, in particular, apple juice and apple products. Fruit leathers are produced by dehydrating fruit puree, leaving a sweet product that has a leathery texture. A recent report in the literature described the detection of patulin at substantial levels in fruit leathers. To investigate this further, an ultra-high-performance liquid chromatography-photodiode array (UPLC-PDA) method was developed for the sensitive detection of patulin in fruit leathers. Investigations were also made of the suitability of direct analysis in real time-mass spectrometry (DART-MS) for detection of patulin from the surface of fruit leathers. Results indicated DART-MS was insufficiently sensitive for quantification from the surface of home-style apple leathers, although patulin spiked onto the surface of leather or peel could be detected. The UPLC-PDA method was used to determine the fate of patulin during the preparation of home-made fruit leathers. Interestingly, when a home-style process was used, the patulin was not destroyed, but rather increased in concentration as the puree was dehydrated. The UPLC-PDA method was also used to screen for patulin in commercial fruit leathers. Of the 36 products tested, 14 were above the limit of detection (3.5 μg kg^–1) and nine were above the limit of quantification (12 μg kg^–1). Positive samples were confirmed by UPLC-MS/MS. Only one sample was found above the US regulatory limit for single-strength apple juice products (50 μg kg^–1). These results suggest patulin can be concentrated during preparation and can be found in fruit leathers. The limited survey suggests that patulin is fairly prevalent in such commercial products, but that the levels are usually low.     

Determination of patulin in apple and quince products by GC–MS using C5–7 patulin as internal standard

A reliable gas chromatographic mass spectrometric method has been validated for the determination of trace levels (<10 μgL⁻¹) of patulin in apple products and quince jam. The method was based on extraction of patulin with ethyl acetate-hexane, alkalinisation and silylation with N,O-bis-trimethylsilyltrifluoroacetamide with 1% of trimethylchloro-silane. The accurate determination of patulin was achieved by employing commercial ¹³C_5–7 patulin labelled as an internal standard, which allowed compensating target analyte losses and enhancement or suppression matrix effects. Limits of detection and quantification of method using real samples were 0.4 and 1.6 μgkg⁻¹, respectively. Recoveries of patulin from samples spiked at 8–50 μgkg⁻¹ levels ranged between 71% and 89%. The repeatability of measurements (expressed as relative standard deviation) was lower than 16%. The method was successfully applied to the determination of patulin in apple fruit and apple products including juice, cider and baby food, and also in quince fruit and quince jam. A new PCR system for the detection of Penicillium expansum in samples containing highly degraded DNA was developed which permitted the detection of the mould in 2/3 of the samples containing patulin, including juices and jams.     

Comprehensive Review of Patulin Control Methods in Foods

The mycotoxin, patulin (4‐hydroxy‐4H‐furo [3, 2c] pyran‐2[6H]‐one), is produced by a number of fungi common to fruit‐ and vegetable‐based products, most notably apples. Despite patulin's original discovery as an antibiotic, it has come under heavy scrutiny for its potential negative health effects. Studies investigating these health effects have proved inconclusive, but there is little doubt as to the potential danger inherent in the contamination of food products by patulin. The danger posed by patulin necessitates its control and removal from foods products, creating a demand for handling and processing techniques capable of doing so, preferably at low cost to industry. With this being the case, much research has been devoted to understanding the basic chemical and biological nature of patulin, as well as its interaction within foods and food production. While past resarch has elucidated a great deal, patulin contamination continues to be a challenge for athe food industry. Here, we review in depth the past research on patulin with an emphasis upon its influence within the food industry, including its regulation, health effects, biosynthesis, detection, quantification, distribution within foods, and control, during the various stages of apple juice production. Finally, key areas where future patulin research should focus to best control the patulin contamination problem within the food industry are addressed.     

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.     

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.     

Characterisation of genes encoding key enzymes involved in sugar metabolism of apple fruit in controlled atmosphere storage

Sugars are essential contributors to fruit flavour. Controlled atmosphere (CA) storage has been proved to be beneficial for maintaining harvested fruit quality. To explore regulatory mechanism of sugar metabolism in fruit stored in CA condition, we cloned several genes, encoding key enzymes, involved in sugar metabolism in apple fruit, and analyzed sugar contents, along with gene expression and enzyme activities in fruits stored in air and CA. The results indicated that CA could maintain higher contents of sugars, including sucrose, fructose and glucose. Expression levels of key genes, such as sucrose synthase (SS), sucrose phosphate synthase (SPS), fructokinase (FK) and hexokinase (HK), were shown to be correlated with the corresponding enzyme activities. We found that activities of neutral invertase (NI), vacuolar invertase (VI), FK and HK were inhibited, but SPS activity was promoted in apple fruit stored in CA, suggesting that CA storage could enhance sucrose synthesis and delay hydrolysis of sucrose and hexose. These findings provided molecular evidence to explain why higher sugar levels in harvested fruit are maintained under CA storage.     

Biomass and Patulin Production by Byssochlamys nivea in Apple Juice as Affected by Sorbate, Benzoate, SO2 and Temperature

The effects of potassium sorbate, sodium benzoate, SO₂ and incubation temperature on biomass and patulin production by Byssochlamys nivea in apple juice were determined. Growth at 21, 30 and 37°C over a 25-day incubation period was significantly retarded by 75 ppm SO₂, 150 ppm potassium sorbate and 500 ppm sodium benzoate. Biomass accumulated to approximately 500 mg/100 ml in control samples of apple juice. Patulin was produced in the highest concentrations at 21°C after 20 days incubation. After reaching a maximum concentration at 30 and 37°C, a rapid decline in patulin content was observed. Patulin production was also observed at 12°C. On the basis of concentration, SO₂ had the most significant effect on the rate of biomass and patulin production by B. nivea followed by potassium sorbate and sodium benzoate, respectively.     

Exposure to patulin from consumption of apple-based products.

Patulin is a mycotoxin produced by species of Penicillium, Aspergillus and Byssochylamys. Several Scientific Committees classify patulin as mutagenic, embryotoxic and immunotoxic. It has been found as a natural contaminant of processed apple products and its presence may be indicative of the quality of fruit used in production. In this work, a method for the analysis of patulin is described, based on a simple liquid-liquid extraction with acetonitrile; patulin is analyzed using liquid chromatography with UV detection. Patulin identity was confirmed by GC-MS after its reaction with N-methyl-N-(trimethylsilyl)trifluoroacetamide. Fifty-three apple-containing products were analyzed and patulin was detected in 14 samples in a range 1.5-50.9 microg l(-1); six of which were above the maximum permitted level of the European Union. Based on these results and juice consumption by the Spanish adult population, patulin estimated intake was 0.42 ng kg(-1) body weight per day.     

Combination of 1-methylcyclopropene treatment and controlled atmosphere storage retains overall fruit quality and bioactive compounds in mango

BACKGROUND: Postharvest application of fungicide prochloraz and hot‐water dip are commercially practiced to control postharvest diseases in mangoes. Owing to the increasing consumer demand for organically produced fruit, the search for natural environmentally friendly alternative products and processes has become important for the fruit industry. This study evaluated the combined effect of 1‐methylcyclopropene (1‐MCP) (500 nL L^?1) and controlled atmosphere storage conditions (CA‐1, 5% O₂ + 5% CO₂ or CA‐2, 3% O₂ + 8% CO₂) on the maintenance of fruit quality and bioactive compounds on hot‐water treated mangoes (cv. Kent) during postharvest storage. RESULTS: In comparison to the 1‐MCP + CA‐1 treatment, 1‐MCP + CA‐2 reduced the incidence of anthracnose, weight and firmness loss; delayed the skin and flesh colour development; prevented the increase of soluble solids concentration/titratable acidity ratio, ethanol and acetaldehyde content; and maintained the ascorbic acid, carotenoid, total phenolic and flavonoid contents, and antioxidant scavenging activity in hot‐water treated mangoes. The untrained panel preferred 1‐MCP + CA‐2 treated fruit to the fruit subjected to other postharvest treatments adopted in this investigation. CONCLUSION: Our investigation suggests that the combined effect of 1‐MCP and CA‐2 storage can be recommended as an alternative treatment to replace prochloraz application for hot‐water treated mangoes and can be adopted commercially for organic export markets. Copyright © 2011 Society of Chemical Industry     

Factors affecting patulin production by Penicillium expansum

Patulin, a mycotoxin produced by Penicillium spp. during fruit spoilage, is a major concern with regard to human health because exposure can result in severe acute and chronic toxicity, including carcinogenic, mutagenic, and teratogenic effects. In this study, we investigated the effects of Penicillium expansum isolate, apple cultivar, storage temperature and time, and pH on the production of patulin. Patulin was analyzed by a previously developed micellar electrokinetic capillary electrophoresis method. P. expansum isolates originating from across Ontario produced widely differing levels of patulin, ranging from 0 to >6 mg/g by dry mycelial weight. The highest patulin levels were those for isolates displaying aggressive growth (characterized by rapidly increasing acidity) accompanied by profuse mycelial development. Distinct patterns in fungal growth rates and patulin production were evident among isolates grown in McIntosh, Empire, and Mutsu ciders. Extensive fungal growth and higher patulin levels (538 to 1,822 microg/ml on day 14) in apple ciders were associated with incubation at room temperature (25 degrees C), although potentially toxic patulin levels (75 to 396 microg/ml on day 24) were also found in refrigerated ciders (4 degrees C) inoculated with P. expansum.     

QUALITY OF ‘GALA’ APPLES AS INFLUENCED BY HARVEST MATURITY,STORAGE ATMOSPHERE AND CONCOMITANT STORAGE WITH ‘BARTLETT’ PEARS1

Controlled atmosphere (CA) storage for 45 or 90 days following harvest reduced quality losses for both ‘Gala’apples and ‘Bartlett’pears compared with fruit from regular atmosphere (RA) storage. Fruit stored in CA were firmer, had higher acidity and less color change than fruit from RA storage. Apples and pears stored together in CA maintained good quality and compared favorably with apples or pears stored separately. There was no difference in fruit quality between fruit stored at 1% O₂ and 1% CO₂ compared with fruit stored in 2% O₂ and 3% CO₂. Fruit harvested at a more advanced stage of maturity deteriorated more quickly in storage than earlier harvested fruit. A combination of 45 days in CA followed by 30 days RA resulted in apples that were superior in quality to apples stored for 75 days in RA alone.     

Delayed harvest and cold storage period influence ethylene production,fruit firmness and quality of ‘Cripps Pink’ apple

The effects of harvest date and cold storage period on ethylene production, fruit firmness and quality of ‘Cripps Pink’ apple were investigated. Fruit were harvested from Perth Hills and Donnybrook (Western Australia) at commercial maturity (CM), 2, 4 and 6 weeks after CM prior to 0, 45, 90 and 135 days cold storage in 2003 and 2004. Delayed harvest significantly increased ethylene production, CIE values a* and C* at both locations in 2 years. Delayed harvest and extended cold storage period increased the internal ethylene production and reduced fruit firmness, soluble solids concentration: titratable acidity (SSC:TA) ratio and reduced CIE values L*, b*, h° during both years and locations. Fruit harvested 2 weeks after CM can be cold‐stored for 90 days with acceptable fruit quality. If the fruit is intended to be sold fresh immediately in local markets, the harvest can be delayed up to 6 weeks after CM to harness substantially improved fruit colour with acceptable fruit quality.     

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.     

Analysis of patulin in apple products by liquid–liquid extraction,solid phase extraction and matrix solid-phase dispersion methods: a comparative study

Patulin is a marker of quality in the apple and apple juice industry and due to the potential risk for human health, reliable and potential methods for extracting patulin from a sample are therefore needed. In this study, the three methods with liquid–liquid extraction, matrix solid-phase dispersion (MSPD) and with solid-phase extraction (SPE) were studied for extracting patulin from different apple products. Result showed that for AJC and apple sample, MSPD method is most suitable for extracting patulin among the three methods. The recovery rates of AJC and apple sample were 80.35–114.46 and 79.68–94.32%, respectively, the coefficient variations were 3.18–4.90%; For dilute juice, SPE procedure is suitable for analysis of patulin and the recovery rates were and 85.35–90.14%.     

展青霉素检测方法的研究进展

展青霉素是由部分曲霉、青霉产生的有毒的代谢产物,主要污染水果及其制品。对目前常见的展青霉素的检测方法薄层色谱法、气-质相联机色谱法、液相色谱法、免疫学检测方法进行了综述,讨论了4种方法的优缺点,指出免疫检测方法是检测展青霉素的发展方向。     

REDUCTION OF PATULIN IN APPLE JUICE CONCENTRATES DURING STORAGE

In this study, the reduction of patulin content in apple juice concentrates during 6 months of storage at 22 and 30C was investigated. Results demonstrated that reduction in patulin content was dependent on the storage temperature and time. Patulin reductions after 1 month of storage at 22 and 30C were in the ranges of 45–64% and 66–86%, respectively. Levels of patulin were below detectable limits after 4 months of storage at 22 and 30C.