Patulin In Food Research Articles

Cytotoxic effects induced by combined exposure to the mycotoxins sterigmatocystin, ochratoxin A and patulin on human tumour and healthy 3D spheroids

Humans are exposed to complex mixtures of mycotoxins through diet. Despite the serious threat they pose, mycotoxin risk assessment often overlooks co-exposure. With the aim of filling this gap, the present study investigates the combined cytotoxicity of sterigmatocystin (STE), ochratoxin A (OTA) and patulin (PAT) in human tumour Neuroblastoma and healthy Mesenchymal Stem Cells three-dimensional (3D) spheroids. The range of concentrations tested (1.56–50 μM for STE, 0.78–25 μM for OTA and 0.15–5 μM for PAT) was selected considering the IC50 values obtained in previous studies and the estimated dietary exposure of consumers. To ensure appropriate experimental conditions, assessments for single mycotoxins and their combinations were conducted simultaneously. The nature of the toxicological interactions among the mycotoxins was then defined using the isobologram analysis. Our results demonstrated increased cytotoxicity in mycotoxin mixtures compared to individual exposure, with abundance of synergistic interactions. These findings highlight that the co-occurrence of STE, OTA and PAT in food may increase their individual toxic effects and should not be underestimated. Moreover, the use of advanced culture models increased the reliability and physiological relevance of our results which can serve as a groundwork for formulating standardized regulatory approaches towards mycotoxin mixtures in food and feed.

Determination of Patulin in Apple Juice and Apple-Derived Products Using a Robotic Sample Preparation System and LC-APCI-MS/MS.

Patulin, a toxic mycotoxin, can contaminate apple-derived products. The FDA has established an action level of 50 ppb (ng/g) for patulin in apple juice and apple juice products. To effectively monitor this mycotoxin, there is a need for adequate analytical methods that can reliably and efficiently determine patulin levels. In this work, we developed an automated sample preparation workflow followed by liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS) detection to identify and quantify patulin in a single method, further expanding testing capabilities for monitoring patulin in foods compared to traditional optical methods. Using a robotic sample preparation system, apple juice, apple cider, apple puree, apple-based baby food, applesauce, fruit rolls, and fruit jam were fortified with 13C-patulin and extracted using dichloromethane (DCM) without human intervention, followed by an LC-APCI-MS/MS analysis in negative ionization mode. The method achieved a limit of quantification of 4.0 ng/g and linearity ranging from 2 to 1000 ng/mL (r2 > 0.99). Quantitation was performed with isotope dilution using 13C-patulin as an internal standard and solvent calibration standards. Average recoveries (relative standard deviations, RSD%) in seven spike matrices were 95% (9%) at 10 ng/g, 110% (5%) at 50 ng/g, 101% (7%) at 200 ng/g, and 104% (4%) at 1000 ng/g (n = 28). The ranges of within-matrix and between-matrix variability (RSD) were 3-8% and 4-9%, respectively. In incurred samples, the identity of patulin was further confirmed with a comparison of the information-dependent acquisition-enhanced product ion (IDA-EPI) MS/MS spectra to a reference standard. The metrological traceability of the patulin measurements in an incurred apple cider (21.1 ± 8.0 µg/g) and apple juice concentrate (56.6 ± 15.6 µg/g) was established using a certified reference material and calibration data to demonstrate data confidence intervals (k = 2, 95% confidence interval).

Patulin removal in fruit-based products using probiotics and potential probiotics

<p>Patulin (PAT) is a prevalent mycotoxin frequently found in fruit and its derivatives, such as apple, pear, and juices. Despite worldwide attempts to diminish the levels of PAT at every stage of the fruit production process, its contamination rate remains high. This mycotoxin is worrisome due to its potential adverse impacts on human health. Eating PAT-contaminated fruit can lead to acute and chronic health issues. It is established, by the Joint FAO/WHO, a maximum tolerable daily intake for PAT at 0.4 µg/kg/day. Therefore, monitoring for PAT contamination is essential for the safe consumption of fruits and fruit-related products such as juices, purees, ciders, jams, marmalades, vinegar, and dried fruits. PAT has physiochemical properties that enable its survival in cold, hypoxic, acidic, or high-temperature conditions. Ideally, detoxification procedures should aim to reduce the level of toxins to safe levels whilst preserving the nutritional and palatable values of the treated commodity. There are several physical, chemical, and biological techniques available for PAT detoxification. However, while physical and chemical methods can remove PAT, they may also lower the nutrient quality and organoleptic properties of the food. Biological detoxification is an effective, environmentally friendly, easy, and cost-effective method, as established by existing research. It has proven efficacy in food safety research and regulatory compliance programs. Probiotics have been studied for their potential to reduce PAT in foods via different mechanisms (such as adsorption, degradation, and transformation), as well as all their health-beneficial effects. In this review, the reduction of PAT in fruit-based products using probiotics or potential probiotics is widely discussed.<strong></strong></p>

Patulin Detoxification by Recombinant Manganese Peroxidase from Moniliophthora roreri Expressed by Pichia pastoris.

The fungal secondary metabolite patulin is a mycotoxin widespread in foods and beverages which poses a serious threat to human health. However, no enzyme was known to be able to degrade this mycotoxin. For the first time, we discovered that a manganese peroxidase (MrMnP) from Moniliophthora roreri can efficiently degrade patulin. The MrMnP gene was cloned into pPICZα(A) and then the recombinant plasmid was transformed into Pichia pastoris X-33. The recombinant strain produced extracellular manganese peroxidase with an activity of up to 3659.5 U/L. The manganese peroxidase MrMnP was able to rapidly degrade patulin, with hydroascladiol appearing as a main degradation product. Five mg/L of pure patulin were completely degraded within 5 h. Moreover, up to 95% of the toxin was eliminated in a simulated patulin-contaminated apple juice after 24 h. Using Escherichia coli as a model, it was demonstrated that the deconstruction of patulin led to detoxification. Collectively, these traits make MrMnP an intriguing candidate useful in enzymatic detoxification of patulin in foods and beverages.

Chromatographic Determination of the Mycotoxin Patulin in 219 Chinese Tea Samples and Implications for Human Health.

Patulin (PAT) is a mycotoxin, with several acute, chronic, and cellular level toxic effects, produced by various fungi. A limit for PAT in food of has been set by authorities to guarantee food safety. Research on PAT in tea has been very limited although tea is the second largest beverage in the world. In this paper, HPLC−DAD and GC−MS methods for analysis of PAT in different tea products, such as non-fermented (green tea), partially fermented (oolong tea, white tea, yellow tea), completely fermented (black tea), and post-fermented (dark tea and Pu-erh tea) teas were developed. The methods showed good selectivity with regard to tea pigments and 5-hydroxymethylfurfural (5-HMF) and a recovery of 90–102% for PAT at a 10–100 ppb spiking level. Limit of detection (LOD) and limit of quantification (LOQ) in tea were 1.5 ng/g and 5.0 ng/g for HPLC−UV, and 0.25 ng/g and 0.83 ng/g for GC−MS. HPLC was simpler and more robust, while GC−MS showed higher sensitivity and selectivity. GC−MS was used to validate the HPLC−UV method and prove its accuracy. The PAT content of 219 Chinese tea samples was investigated. Most tea samples contained less than 10 ng/g, ten more than 10 ng/g and two more than 50 ng/g. The results imply that tea products in China are safe with regard to their PAT content. Even an extreme daily consumption of 25 g of the tea with the highest PAT content (124 ng/g), translates to an intake of only 3 μg/person/day, which is still an order of magnitude below the maximum allowed daily intake of 30 µg for an adult.

Global trends for patulin adsorption: A review

Patulin is a toxic metabolite produced by several species of fungi. The species that are responsible for the production of patulin enter fruits through bruised and broken skin, causing contamination. Apple-derived products are considered to be by far the most significant dietary sources of patulin. According to the literature, three strategies have been used to break down or remove patulin in food, such as physical, chemical, and biological methods. Degradation of patulin by microorganisms or biodegradation enzymes is an efficient and promising method for the removal of patulin from food. The incidence of patulin contamination continues to be high, despite global efforts to reduce the levels of this mycotoxin at each stage of the fruit production process. Its transformation into other compounds has been reported. However, the toxicities of its byproducts as deoxypatulinic acid, ascladiol, and hydroascladiol should be subjected to an intensive study.

Development of an immuno-electrochemical glass carbon electrode sensor based on graphene oxide/gold nanocomposite and antibody for the detection of patulin

The presence of fungal-produced patulin in foods poses a high health risk to people because it can cause neurologic and gastrointestinal illnesses. A glass carbon electrode (GCE) sensor was developed for the rapid and sensitive detection of patulin. Anti-patulin-BSA IgG of a rabbit was produced and immobilised on a GCE coated with a graphene oxide/gold nanocomposite. The mycotoxin patulin in the samples could be captured by the anti-patulin-BSA IgG on the surface of the GCE sensor. The spatial hindrance effect of IgG on the GCE sensor was reduced by the reaction between IgG and patulin, resulting in a decrease in the electron transfer resistance. The current changes in the immobilised anti-patulin-BSA IgG GCE sensor exhibited a linear relationship with patulin concentration and facilitated the sensitive detection of patulin. This immuno-electrochemical GCE sensor could rapidly detect patulin in less than 1 min with a detection limit of 5 µg/L.

Recent trends in detecting, controlling, and detoxifying of patulin mycotoxin using biotechnology methods.

Patulin (PAT) is a mycotoxin that can contaminate many foods and especially fruits and fruit-based products. Therefore, accurate and effective testing is necessary to enable producers to comply with regulations and promote food safety. Traditional approaches involving the use of chemical compounds or physical treatments in food have provided practical methods that have been used to date. However, growing concerns about environmental and health problems associated with these approaches call for new alternatives. In contrast, recent advances in biotechnology have revolutionized the understanding of living organisms and brought more effective biological tools. This review, therefore, focuses on the study of biotechnology approaches for the detection, control, and mitigation of PAT in food. Future aspects of biotechnology development to overcome the food safety problem posed by PAT were also examined. We find that biotechnology advances offer novel, more effective, and environmental friendly approaches for the control and elimination of PAT in food compared to traditional methods. Biosensors represent the future of PAT detection and use biological tools such as aptamer, enzyme, and antibody. PAT prevention strategies include microbial biocontrol, the use of antifungal biomolecules, and the use of microorganisms in combination with antifungal molecules. PAT detoxification aims at the breakdown and removal of PAT in food by using enzymes, microorganisms, and various adsorbent biopolymers. Finally, biotechnology advances will be dependent on the understanding of fundamental biology of living organisms regarding PAT synthesis and resistance mechanisms.

The Possible Mechanisms Involved in Degradation of Patulin by Pichia caribbica.

In this work, we examined the mechanisms involved in the degradation of patulin by Pichia caribbica. Our results indicate that cell-free filtrate of P. caribbica reduced patutlin content. The heat-killed cells could not degrade patulin. However, the live cells significantly reduced the concentration of the patulin. In furtherance to this, it was observed that patulin was not detected in the broken yeast cells and cell wall. The addition of cycloheximide to the P. caribbica cells decreased the capacity of degradation of patulin. Proteomics analyses revealed that patulin treatment resulted in an upregulated protein which was involved in metabolism and stress response processes. Our results suggested that the mechanism of degradation of patulin by P. caribbica was not absorption; the presence of patulin can induce P. caribbica to produce associated intracellular and extracellular enzymes, both of which have the ability to degrade patulin. The result provides a new possible method that used the enzymes produced by yeast to detoxify patulin in food and feed.

Development and application of a quartz crystal microbalance sensor based on molecularly imprinted sol-gel polymer for rapid detection of patulin in foods

A simple quartz crystal microbalance (QCM) sensor based on a sol-gel molecularly imprinted polymer (MIP) film as the recognition element was developed for patulin (PAT) detection. The high adsorption capacity of the proposed MIP for PAT was demonstrated by Scatchard equation analysis. Through the evaluation, based on the QCM frequency change, the developed sensor exhibited a linear concentration range of PAT from 7.5×10−3μgmL−1 to 6×10−2μgmL−1 with a lower detection limit of 3.1×10−3μgmL−1 (S/N=3). The sensor showed good selective affinity for PAT (selectivity coefficient=3.82) as well as good reproducibility and long-term stability. The developed sensor was tested on its ability to detect PAT in real samples through a spiking and recovery study with the recoveries ranging from 76.9% to 91.3% (RSDs<8.4%). The good agreement results of the established MIP-QCM sensor and HPLC–MS method indicated that the developed method could accurately detect PAT in practical food samples.

A near-infrared fluorescence assay method to detect patulin in food

Patulin (PAT) is a toxic secondary metabolite (mycotoxin) of different fungal species belonging to the genera Penicillium, Aspergillus, and Byssochlamys. They can grow on a large variety of food, including fruits, grains, and cheese. The amount of PAT in apple derivative products is a crucial issue because it is the measure of the quality of both the used raw products and the performed production process. Actually, all current methodologies used for the quantification of PAT are time-consuming and require skilled personnel beyond the sample pretreatment methods (e.g., high-performance liquid chromatography, mass spectrometry, and electrophoresis techniques). In this work, we present a novel fluorescence polarization approach based on the use of emergent near-infrared (NIR) fluorescence probes. The use of these fluorophores coupled to anti-PAT antibodies makes possible the detection of PAT directly in apple juice without any sample pretreatment. This methodology is based on the increase of fluorescence polarization emission of a fluorescence-labeled PAT derivative on binding to specific antibodies. A competition between PAT and the fluorescence-labeled PAT derivative allowed detecting PAT. The limit of detection of the method is 0.06μg/L, a value that is lower than maximum residue limit of PAT fixed at 50μg/L from European Union regulation.

An optimized method for the accurate determination of patulin in apple products by isotope dilution-liquid chromatography/mass spectrometry.

Patulin, a mycotoxin produced by several molds in fruits, has been frequently detected in apple products. Therefore, regulatory bodies have established recommended maximum permitted patulin concentrations for each type of apple product. Although several analytical methods have been adopted to determine patulin in food, quality control of patulin analysis is not easy, as reliable certified reference materials (CRMs) are not available. In this study, as a part of a project for developing CRMs for patulin analysis, we developed isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC/MS/MS) as a higher-order reference method for the accurate value-assignment of CRMs. (13)C7-patulin was used as internal standard. Samples were extracted with ethyl acetate to improve recovery. For further sample cleanup with solid-phase extraction (SPE), the HLB SPE cartridge was chosen after comparing with several other types of SPE cartridges. High-performance liquid chromatography was performed on a multimode column for proper retention and separation of highly polar and water-soluble patulin from sample interferences. Sample extracts were analyzed by LC/MS/MS with electrospray ionization in negative ion mode with selected reaction monitoring of patulin and (13)C7-patulin at m/z 153→m/z 109 and m/z 160→m/z 115, respectively. The validity of the method was tested by measuring gravimetrically fortified samples of various apple products. In addition, the repeatability and the reproducibility of the method were tested to evaluate the performance of the method. The method was shown to provide accurate measurements in the 3-40 μg/kg range with a relative expanded uncertainty of around 1%.

Occurrence of Patulin its Dietary Intake through Consumption of Apple and Apple Products and Methods of its Removal

Patulin (4-hydroxy-4H-furo [3,2c] pyran-2[6H]-one), a mycotoxin, is a secondary metabolite produced by a number of fungi, primarily Penicillium and Aspergillus species common to fruit and vegetable based products. Most notably apple is of major concern from food safety considerations. Patulin 1st discovered as an antibiotic but later on scientific community realised its negative health effects. It is classified as a group-3 carcinogen as there is no evidence of its carcinogenicity in humans but based on a long-term investigation in rats, the World Health Organization (WHO) has set a tolerable weekly intake of 7 ppb (μg/Kg) body weight. The maximum limit of patulin in foods is restricted to 50 ppb in many countries of the world. Conventional analytical detection methods involve chromatographic analyses, such as HPLC, GC and more recently techniques such as LC/MS and GC/MS. The risk associated with the patulin necessitates its control and ultimately removal from the food products. It has been detected in several apple products viz., apple juice, apple puree, apple wine, apple cider and baby foods. The quantities range from 0.5 to 732.8 μg/L. Effort to understand the basic chemical and biological nature of patulin as well as interaction with the other food components are being made. It may occur in apples during harvest and postharvest stages. The principal risk arises when unfit/rotten fruit is used for the production of juices and other processed products. Process stages and conditions of process may effect concentration of patulin. Nature of processing such as fermentation, heat treatment and clarification applications or additional steps of production line such as the use of binding material can help in removing patulin from products or at least reduce its concentration employing low cost technology.

LC–MS/MS multi-method for mycotoxins after single extraction, with validation data for peanut, pistachio, wheat, maize, cornflakes, raisins and figs

Mycotoxin analysis is usually carried out by high performance liquid chromatography after immunoaffinity column cleanup or in enzyme-linked immunosorbent assay tests. These methods normally involve determination of single compounds only. EU legislation already exists for the aflatoxins, ochratoxin A and patulin in food, and legislation will come into force for deoxynivalenol, zearalenone and the fumonisins in 2007. To enforce the various legal limits, it would be preferable to determine all mycotoxins by routine analysis in different types of matrices in one single extract. This would also be advantageous for HACCP control purposes. For this reason, a multi-method was developed with which 33 mycotoxins in various products could be analysed simultaneously. The mycotoxins were extracted with an acetonitrile/water mixture, diluted with water and then directly injected into a LC–MS/MS system. The mycotoxins were separated by reversed-phase HPLC and detected using an electrospray ionisation interface (ESI) and tandem MS, using MRM in the positive ion mode, to increase specificity for quality control. The following mycotoxins could be analysed in a single 30-min run: Aflatoxins B1, B2, G1 and G2, ochratoxin A, deoxynivalenol, zearalenone, T-2 toxin, HT-2 toxin, α-zearalenol, α-zearalanol, β-zearalanol, sterigmatocystin, cyclopiazonic acid, penicillic acid, fumonisins B1, B2 and B3, diacetoxyscirpenol, 3- and 15-acetyl-deoxynivalenol, zearalanone, ergotamin, ergocornin, ergocristin, α-ergocryptin, citrinin, roquefortin C, fusarenone X, nivalenol, mycophenolic acid, alternariol and alternariol monomethyl ether. The limit of quantification for the aflatoxins and ochratoxin A was 1.0 µg kg−1 and for deoxynivalenol 50 µg kg−1. The quantification limits for the other mycotoxins were in the range 10–200 µg kg−1. The matrix effect and validation data are presented for between 13 and 24 mycotoxins in peanuts, pistachios, wheat, maize, cornflakes, raisins and figs. The method has been compared with the official EU method for the determination of aflatoxins in food and relevant FAPAS rounds. The multi-mycotoxin method has been proven by the detection of more than one mycotoxin in maize, buckwheat, figs and nuts. The LC–MS/MS technique has also been applied to baby food, which is subject to lower limits for aflatoxin B1 and ochratoxin A, ergot alkaloids in naturally contaminated rye and freeze-dried silage samples.

Patulin in food: state-of-the-art and analytical trends

Patulin is a mycotoxin produced by several fungal species of the genera Penicillium and Aspergillus, found on several fruit species and, remarkably, in apples and apple products. Patulin has a broad spectrum of toxicity, including carcinogenicity and teratogenicity in animals. Due to the stability of the molecule, considerable amounts of patulin still remain in apple products after processing. This paper reviews different analytical methods for patulin determination and methods to reduce levels of patulin in apple products as well.

A New Competitive Fluorescence Assay for the Detection of Patulin Toxin

Patulin is a toxic secondary metabolite of a number of fungal species belonging to the genera Penicillum and Aspergillus. It has been mainly isolated from apples and apple products contaminated with the common storage-rot fungus of apples, Penicillum expansum, but it has also been extracted from rotten fruits, moldy feeds, and stored cheese. Human exposure to patulin can lead to serious health problems, and according to a long-term investigation in rats, the World Health Organization has set a tolerable weekly intake of 7 ppb body weight. The content of patulin in foods has been restricted to 50 ppb in many countries. Conventional analytical detection methods involve chromatographic analyses, such as HPLC, GC, and, more recently, techniques such as LC/MS and GC/MS. However, extensive protocols of sample cleanup are required prior to the analysis, and to accomplish it, expensive analytical instrumentation is necessary. An immunochemical analytical method, based on highly specific antigen-antibody interactions, would be desirable, offering several advantages compared to conventional techniques, i.e., low cost per sample, high selectivity, high sensitivity, and high throughput. In this paper, the synthesis of two new derivatives of patulin is described, along with their conjugation to the bovine serum albumin for the production of polyclonal antibodies. Finally, a fluorescence competitive immunoassay was developed for the on-line detection of patulin.

Overview of natural occurrence of patulin in foods

Overview of natural occurrence of patulin in foods

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.