Patulin Degradation Research Articles

Kinetics of patulin degradation in model solution, apple cider and apple juice by ultraviolet radiation

Patulin is a mycotoxin produced by a wide range of molds involved in fruit spoilage, most commonly by Penicillium expansum and is a health concern for both consumers and manufacturers. The current study evaluated feasibility of monochromatic ultraviolet (UV) radiation at 253.7 nm as a possible commercial application for the reduction of patulin in fresh apple cider and juice. The R-52G MINERALIGHT® UV bench top lamp was used for patulin destruction. It was shown that 56.5%, 87.5%, 94.8% and 98.6% reduction of patulin can be achieved, respectively, in the model solution, apple cider, apple juice without ascorbic acid addition and apple juice with ascorbic acid addition in 2-mm thickness sample initially spiked by 1 mg·L(-1) of patulin after UV exposure for 40 min at UV irradiance of 3.00 mW·cm(-2). A mathematic model to compare the degradation rate and effective UV dose was developed. The effective UV doses that were directly absorbed by patulin for photochemical reaction were 430, 674, 724 and 763 mJ·cm(-3), respectively. The fluence-based decimal reduction time was estimated to 309.3, 31.3, 28.9 and 5.1 mW·cm(-2)·min, respectively, in four media mentioned above. The degradation of patulin followed the first-order reaction model. The time-based and fluence-based reaction rate constants were determined to predict patulin degradation. The time-based reaction rate constant of samples treated in dynamic regime with constant stirring (model solution: 2.95E-4 s(-1), juice: 4.31E-4 s(-1)) were significantly higher than samples treated in static regime (model solution: 2.79E-4 s(-1), juice: 3.49E-4 s(-1), p < 0.05) when applied UV irradiance and sample thickness were consistent. The reaction rate constant of patulin degradation in apple juice was significantly higher than model solution (p < 0.05). Although further investigations are still needed, the results of this study demonstrated that UV radiation may be an effective method for treating patulin-containing apple cider and juice.

Searching for genes responsible for patulin degradation in a biocontrol yeast provides insight into the basis for resistance to this mycotoxin.

Patulin is a mycotoxin that contaminates pome fruits and derived products worldwide. Basidiomycete yeasts belonging to the subphylum Pucciniomycotina have been identified to have the ability to degrade this molecule efficiently and have been explored through different approaches to understand this degradation process. In this study, Sporobolomyces sp. strain IAM 13481 was found to be able to degrade patulin to form two different breakdown products, desoxypatulinic acid and (Z)-ascladiol. To gain insight into the genetic basis of tolerance and degradation of patulin, more than 3,000 transfer DNA (T-DNA) insertional mutants were generated in strain IAM 13481 and screened for the inability to degrade patulin using a bioassay based on the sensitivity of Escherichia coli to patulin. Thirteen mutants showing reduced growth in the presence of patulin were isolated and further characterized. Genes disrupted in patulin-sensitive mutants included homologs of Saccharomyces cerevisiae YCK2, PAC2, DAL5, and VPS8. The patulin-sensitive mutants also exhibited hypersensitivity to reactive oxygen species as well as genotoxic and cell wall-destabilizing agents, suggesting that the inactivated genes are essential for tolerating and overcoming the initial toxicity of patulin. These results support a model whereby patulin degradation occurs through a multistep process that includes an initial tolerance to patulin that utilizes processes common to other external stresses, followed by two separate pathways for degradation.

Efficacy of Pichia caribbica in controlling blue mold rot and patulin degradation in apples

The efficacy of Pichia caribbica in controlling postharvest blue mold and natural decay development of apples and degrading the patulin produced by Penicillium expansum was investigated. The decay incidence of the blue mold of apples treated by P. caribbica was significantly reduced compared with the control samples, and the higher the concentration of P. caribbica, the better the efficacy of the biocontrol. P. caribbica significantly controlled the natural decay development of apples following storage at 20°C for 35days or 4°C for 45days followed by 20°C for 15days. Germination of spores and growth of P. expansum were markedly inhibited by P. caribbica in in vitro testing. Rapid colonization of apple wounds by the yeast was observed in fruit stored at 20°C or 4°C. After incubation with P. caribbica at 20°C for 15days, patulin production by P. expansum in apples was significantly reduced compared with the control. In vitro testing indicated that P. caribbica can degrade patulin directly.

Patulin Degradation in a Model Apple Juice System and in Apple Juice during Ultraviolet Processing

Factors affecting ultraviolet (UV) radiation-induced degradation of patulin in a malic acid model system, apple cider and apple juice were studied. First-order rate constants (cm 2 /J) in the model system were not significantly (P > 0.05) affected by pH (3.0‐3.6), initial patulin concentration (0‐1000 mg/L), glucose (0‐10%), sucrose (0‐10%) and ascorbic acid (0‐100 mg/L). Tannic acid (0‐1 g/L) reduced patulin degradation while fructose (0‐10%) accelerated it. Although suspended particles yielding nephelometric turbidity units values between 0 and 100 did not affect patulin degradation, higher turbidity levels in unfiltered apple cider reduced degradation rates. Even in clarified apple juice, the UV dose required for 50% patulin reduction in apple juice (4.62 J/cm 2 ) was higher than that required for a 5-log reduction of Escherichia coli O157:H7. Therefore, achieving efficient UV reduction of patulin in apple juice products, without quality losses, may require particulate filtration, phenolic adsorption treatments, and addition of compounds that accelerate patulin degradation. PRACTICAL APPLICATIONS This study demonstrates that apple juice constituents affect patulin degradation during ultraviolet (UV) processing. High turbidity levels in apple cider suggest that this technology may be impractical for this product. UV irradiation can be used to decrease patulin levels in clarified apple juice, although the required dose levels remain higher than that required for 5-log reduction of human pathogens. Filtration, adsorption treatments that lower levels of UV scattering and absorbing suspended particles and polyphenols can result in substantially greater patulin degradation during UV processing. Because fructose had an accelerating effect on patulin reduction in the model system studied, addition of high-fructose sweeteners to apple beverages prior to UV processing may result in further patulin reductions.

Application of pulsed light to patulin reduction in McIlvaine buffer and apple products

Numerous studies around the world have reported the occurrence of patulin in commercialized apple products. The persistence of this mycotoxin in apple products indicates that current methods used to reduce it during the manufacturing process are not entirely successful and reflects the need to evaluate new detoxification methods. The purpose of the present study was to investigate the effect of pulsed light (PL) dose on patulin degradation in McIlvaine buffer, apple juice and apple purée. The exposure of all samples to PL doses between 2.4 and 35.8 J/cm2 resulted in a significant decrease in patulin levels. Patulin reduction in McIlvaine buffer did not depend markedly on the initial concentration of the mycotoxin. At the maximum dose tested, the remaining average patulin level dissolved in McIlvaine buffer was approximately 5–15%, while in apple juice the values declined up to 22%. In apple purée naturally contaminated with 29 μg/kg of patulin, exposure to a PL dose of 12 J/cm2 provoked a 51% reduction in patulin concentration, while no residual contamination was detected for higher irradiation times.These results suggested that PL treatment would be a potential alternative method to reduce patulin contamination in apple products. However, further investigations need to be conducted to evaluate toxicological safety of patulin degradation product(s).

Comparative metabolomic analysis of Saccharomyces cerevisiae during the degradation of patulin using gas chromatography–mass spectrometry

A comparative metabolomic analysis was conducted on Saccharomyces cerevisiae cells with and without patulin treatment using gas chromatography-mass spectrometry-based approach. A total of 72 metabolites were detected and compared, including 16 amino acids, 29 organic acids and alcohols, 19 sugars and sugar alcohols, 2 nucleotides, and 6 miscellaneous compounds. Principle component analysis showed a clear separation of metabolome between the cells with and without patulin treatment, and most of the identified metabolites contributed to the separation. A close examination of the identified metabolites showed an increased level of most of the free amino acids, an increased level of the intermediates in the tricarboxylic acid cycle, a higher amount of glycerol, a changed fatty acid composition, and a decreased level of cysteine and glutathione in the cells with patulin treatment. This finding indicated a slower protein synthesis rate and induced oxidative stress in the cells with patulin treatment, and provided new insights into the effect of toxic chemicals on the metabolism of organisms.

Conversion of the Mycotoxin Patulin to the Less Toxic Desoxypatulinic Acid by the Biocontrol Yeast Rhodosporidium kratochvilovae Strain LS11

The infection of stored apples by the fungus Penicillium expansum causes the contamination of fruits and fruit-derived products with the mycotoxin patulin, which is a major issue in food safety. Fungal attack can be prevented by beneficial microorganisms, so-called biocontrol agents. Previous time-course thin layer chromatography analyses showed that the aerobic incubation of patulin with the biocontrol yeast Rhodosporidium kratochvilovae strain LS11 leads to the disappearance of the mycotoxin spot and the parallel emergence of two new spots, one of which disappears over time. In this work, we analyzed the biodegradation of patulin effected by LS11 through HPLC. The more stable of the two compounds was purified and characterized by nuclear magnetic resonance as desoxypatulinic acid, whose formation was also quantitated in patulin degradation experiments. After R. kratochvilovae LS11 had been incubated in the presence of (13)C-labeled patulin, label was traced to desoxypatulinic acid, thus proving that this compound derives from the metabolization of patulin by the yeast. Desoxypatulinic acid was much less toxic than patulin to human lymphocytes and, in contrast to patulin, did not react in vitro with the thiol-bearing tripeptide glutathione. The lower toxicity of desoxypatulinic acid is proposed to be a consequence of the hydrolysis of the lactone ring and the loss of functional groups that react with thiol groups. The formation of desoxypatulinic acid from patulin represents a novel biodegradation pathway that is also a detoxification process.

Low Occurrence of Patulin-Producing Strains of Penicillium in Grapes and Patulin Degradation during Winemaking in Chile

<i>Penicillium expansum</i> has emerged as the cause of storage decay of table grapes (<i>Vitis vinifera</i>) and has been frequently isolated from apparently healthy clusters of grapes in Chile. The objectives of this study were to identify patulin-producing strains of <i>Penicillium</i> associated with winegrapes and wineries in Chile and to determine the potential presence of patulin in wines made with grapes infected with <i>P. expansum</i>. In this study, <i>P. brevicompactum, P. expansum,</i> and <i>P. glabrum</i> were identified on apparently healthy grape clusters and in the air of vineyards and wineries. Of 132 <i>Penicillium</i> isolates, 4 <i>P. brevicompactum</i> and 11 <i>P. expansum</i> strains were patulin-producing, determined by HPLC-UV/DAD. Patulin was also detected in Cabernet Sauvignon musts produced with grapes contaminated with a patulin-producing strain of <i>P. expansum.</i> However, patulin concentrations decreased during fermentation by 67.3 to 83.3%. Overall, the frequency of <i>P. expansum</i> isolation from grapes was relatively low; thus, considering the rapid degradation of patulin produced during fermentation, the risk of patulin contamination of bottled wine appears to be low.

Effect of processing of apple puree on patulin content

Mycotoxin patulin represents potential health hazard especially in apple based baby food. There are already a lot of data to predict the fate of the toxin during the processing of fruits, but not enough for the apple puree and the existing data for apple puree are not usually consistent. The objective of the study was to evaluate the course of patulin degradation during the processing of apple puree under the conditions close to the real production process. The apple samples were spiked with patulin at four levels of concentration (539 μg/kg, 140 μg/kg, 23 μg/kg and <2 μg/kg). The patulin content changes during the processing were followed. The samples were taken after the homogenization, pulping, pasteurization and aseptic packaging. All operations of apple puree production contributed to patulin reduction. The principle operation was pulping, where the patulin levels were reduced from 29% to 80% of original content.

Optimization of clean-up procedure for patulin determination in apple juice and apple purees by liquid chromatography

Patulin (PAT) is a mycotoxin produced in fruits, mainly in apples, by several fungal species that can be carried into industrial apple juice by-products during factory processing. An analytical method for determination of PAT in apple juice and another one for determination of this compound in apple purees and apple compotes by liquid chromatography are proposed in the present paper. These methods have better precision and sensitivity than previously reported methods and focus mainly on extraction and clean-up. To accomplish analytical methods with higher accuracy, lower limits of detection and simpler procedures for application in quality control of the goods, different extraction and clean-up procedures for PAT were comparatively studied. PAT recoveries in apple juice spiked with 1.0 mg PAT/kg varied between 52.3% and 81.0%. The highest PAT recovery in apple puree spiked with 0.1 mg PAT/kg was 82.9%. Addition of NaH 2PO 4 during the extraction phase here reported for the first time has the advantage of keeping the pH slightly acidic, thus avoiding PAT degradation.

Effects of Some Metals and Chelating Agents on Patulin Degradation by Ozone

The efficiency of ozone treatment for degradation of the mycotoxin patulin in the presence of various metal ions was evaluated in model systems. An initial patulin concentration of 250 μg/L was ozonated and residual ozone concentration was about 0.17±0.01 mg/L at the end of the experiment. Patulin showed a weak resistance to ozone, because up to 98% of this toxin was oxidized in only one minute. Degradation rates in the presence of calcium, aluminum, copper and zinc were almost the same in the absence of these metals. However, degradation of patulin was reduced from 98 to 37% when the concentration of manganese increased from 0 to 3 mg/L. Patulin was almost completely degraded in the absence of iron, while the degradation was only 8.5% in the presence of 0.5 mg/L of iron. These results have revealed that manganese and iron significantly reduce the detoxification of patulin by ozone. Agents such as ethylenediaminetetraacetic acid and sodium polyphosphate effectively chelated iron and increased the degradation rate of patulin. None of the tested agents were able to chelate manganese and to enhance patulin degradation by ozone.

Penicillium expansum versus antagonist yeasts and patulin degradation in vitro

Taking into account the preliminary antagonistic/biodegradation property showed by Pichia membranifaciens and Sporobolomyces roseus, which decreased the initial patulin concentration of 588.4 to 290.0 µg/mL, ability of P. ohmeri 158 in biocontrol against Penicillium expansum and patulin decrease in vitro was performed. The culture supernatant of P. ohmeri 158 was effective against 66.17% micelial growth, indicating antibiosis related with the killer phenomenon. The initial patulin concentration of 223 µg in the presence of P. ohmeri 158 cells was decreased over 83% of the original concentration, when incubated at 25ºC/2 days and &gt; 99% after 5 days incubation time, with undetectable patulin level after 15 days. The initial pH 4.0 decreased to pH 3.3 along 15 days experiment, suggesting that patulin decrease was an active process and a consequence of yeast metabolism. The results suggested that P. ohmeri 158 could be a promising alternative for the inhibition of P. expansum growth and patulin degradation.

Stability of patulin in a juice-like aqueous model system in the presence of ascorbic acid

In the present study, the stability of patulin in an aqueous juice-like model system was investigated. At acidic pH, the presence of ascorbic acid reduced the stability of patulin. After 34 days, patulin was reduced to 30% of its initial concentration in the presence of ascorbic acid compared to 68–71% in samples without ascorbic acid. Conditions during storage (presence of light, oxygen and/or metal ions) influenced the stability of patulin. Furthermore, it was possible to induce degradation of patulin by either generating hydroxyl radicals or by adding the rather stable radical diphenyl-1-picrylhydrazyl (DPPH). Data from the present study indicate that patulin is decomposed by free radicals generated by oxidation of ascorbic acid to dehydroascorbic acid. Rapid oxidation of ascorbic acid in the presence of oxygen, catalysed by free metal ions, resulted in a decrease of patulin. After complete oxidation of ascorbic acid, no further patulin degradation was observed. In contrast, slow oxidation of ascorbic acid in the presence of metal-chelators induced a continuous, slow oxidation of patulin. Due to low oxygen content in the headspace of a food package, addition of ascorbic acid to products such as apple juice, prior to filling, cannot be considered as an effective decontamination strategy.

Rapid degradation of the mycotoxin patulin in man quantified by stable isotope dilution assays

The absorption and degradation of the mycotoxin patulin in man was quantified by using a recently developed stable isotope dilution assay. Application of this currently most sensitive method revealed a patulin content less than 200 ng l−1 in the blood serum of five consumers of apple juice. Likewise, no patulin was found in the serum of a volunteer, whose blood was drawn shortly after consumption of a juice containing a maximum tolerable amount of patulin. In further in vitro experiments, the degradation of patulin by reacting it with whole blood was investigated. After addition of 100 µg patulin to 9 ml blood, only 6.1% of the mycotoxin was detected after 2 min. It was concluded, therefore, that even high naturally occurring concentrations of patulin in foods are quickly degraded before reaching other tissues than the gastrointestinal tract.

Fate of patulin in the presence of the yeast Saccharomyces cerevisiae

Patulin is known to become analytically non-detectable during the production of cider from contaminated apple juice. The fate of [14C]-labelled patulin during the alcoholic fermentation of apple juice was studied. Three commercial cider strains of Saccharomyces cerevisiae degraded patulin during active fermentative growth, but not when growing aerobically. The products of patulin degradation were more polar than patulin itself and remained in the clarified fermented cider. Patulin did not appear to bind to yeast cells or apple juice sediment in these model experiments. HPLC analysis of patulin-spiked fermentations showed the appearance of two major metabolites, one of which corresponded by both TLC and HPLC to E-ascladiol prepared by the chemical reduction of patulin using sodium borohydride. Using a diode array detector, both metabolites had a ⋋max = 271nm, identical to that of ascladiol. Thenmr spectrum of a crude preparation of these metabolites showed signals corresponding to those of the E-ascladiol prepared chemically and a weaker set of signals corresponding to those reported in the literature for Z-ascladiol.

Procedures for destruction of patulin in laboratory wastes.

Patulin is immunosuppressive and there is limited evidence of its carcinogenicity in experimental animals. The International Agency for Research on Cancer (IARC) initiated a programme for the development of degradation techniques for the commonly investigated mycotoxins. As a part of this programme, the following techniques were tested for the degradation of patulin: treatment with ammonia, treatment with ascorbic acid, and treatment with potassium permanganate in acidic or in alkaline conditions. Patulin analysis was performed by using HPLC with UV detection. Mutagenic activity of degradation residues was tested by in Salmonella typhimurium strains TA 97a, TA 98, TA 100, and TA 102. Complete disappearance of patulin was not achieved after 92 h of treatment with ascorbic acid. All the other methods tested led to complete removal of the molecule. However, the technique using potassium permanganate in acidic conditions produced residues which were mutagenic without activation to Salmonella typhimurium strains TA 100 and TA 102, which was attributed later to Mn2+. The two other techniques gave satisfactory results and were selected for further validation studies.

Effect of irradiation on the patulin content and chemical composition of apple juice concentrate.

The influence of ionizing radiation on the patulin content of apple juice concentrate was investigated. The results indicated that patulin, at an initial concentration of about 2 mg/kg, disappeared after irradiation of the concentrate with doses as low as 2.5 kGy. For lower doses, the extent of patulin degradation was proportional to the absorbed dose. Irradiation of the concentrate with doses sufficient for patulin disappearance did not change the titratable acidity, the content of reducing sugars and carbonyl compounds or the amino acid composition. The content of ascorbic acid slightly decreased and the colour of the concentrate brightened. The intensity of the patulin absorption spectra after irradiation of mycotoxin in aqueous solutions decreased.

Production and degradation of patulin by paecilomyces species, a common contaminant of silage

AbstractThe production and degradation of patulin by a common silage comtaminant, Paecilomyces sp., is described. Utilising protoplasts of the parent organism, 80% degradation of radiolabelled patulin occurs over the 3‐h period studied and it is proposed that this degradation effect may account for anomalies in the investigation of suspected mycotoxin poisoning.