A ratiometric fluorescent biosensing platform based on CDs and AuNCs@CGO for patulin detection.

BACKGROUND: Patulin (PAT) is a mycotoxin, usually found in fruit and their products, that can potentially be harmful to human health. In order to achieve rapid detection of mycotoxins and ensure the safety of food. This study reported a novel ratiometric fluorescent aptasensor for PAT detection. In this study, we used aptamer as the recognition element, Hybrid double stranded modified with fluorescent substances as the fluorescent donor, and AuNCs@CGO as the fluorescent acceptor. After the addition of PAT, the ratiometric fluorescence "turn on" response was exhibited. RESULTS: The AuNCs@CGO are obtained by amide reaction between BSA-AuNCs and carboxylated graphene oxide (CGO). The prepared AuNCs@CGO can shorten the time of FRET effect and exhibit highly efficient quenching ability by adsorption effects (π-π stacking and electrostatic gravity) on the Aptamer-modified CDs. When the target PAT bound specifically to the CDs-apt, the fluorescence of the CDs-apt would recover, while the fluorescence of ROX modified cDNA remained unchanged. This ratiometric fluorescence response improved the accuracy of PAT detection. In addition, the proposed had good linearity for PAT in the range of 0.1-50 ng/mL with a limit of detection 0.16 ng/mL. The recovery of standard addition in grapes were 95.9%-105.4 %. SIGNIFICANCE: An effective fluorescent detection method for PAT was constructed based on aptamer and nanomaterials. This new fluorescent biosensor has the characteristics of simple synthesis, easy operation, high sensitivity, strong selectivity and a low LOD, which may be a promising idea and platform for the detection of food safety hazard factors.

A novel multimodal aptasensor for Patulin detection in fruit products based on high-performance RuMOF@hydrogel and versatile pericarp-derived carbonized polymer dots.

Patulin (PAT) is a widespread fruit toxin. Trace-level PAT exposure can cause serious harm to human health. Herein, a multimodal PAT aptasensor was designed based on Ru(bpy)32+-based metal organic framework composited hydrogel (RuMOF@hydrogel) and versatile banana peel-derived carbonized polymer dots (BPPDs). RuMOF@hydrogel modified magnetic-electrode exhibited excellent anodic and cathodic electrochemiluminescence (ECL) emission and stability. Meanwhile, the BPPDs could enhance anodic ECL of RuMOF@hydrogel, and also show excellent fluorescence (FL) and photothermal (PT) properties. With the aid of PAT-triggered hybridization chain reaction and magnetic separation, ECL, FL, and PT responses could be recorded concurrently. The detection limit can reach as low as 0.25 fg mL-1. The ratiometric ECL quantitation ensured the sensitivity and accuracy of this assay. And visual FL and portable PT modes contributed to the utility. Furthermore, this aptasensor demonstrated better performances than HPLC in fruit products and the protocol can be extended to determine various contaminants in foods.

An ultrasensitive dual-mode aptasensor for patulin based on the upconversion particles and G-Quadruplex-hemin DNAzyme.

Patulin (PAT) is a mycotoxin-produced secondary metabolite that can contaminate foods, causing toxic effects on animal and human health. Therefore, for the first time, we have constructed a "turn-on" dual-mode aptamer sensor for PAT using oleic acid-coated upconversion nanomaterials (OA-UCNPs) and G-Quadruplex-hemin DNAzyme (G4-DNAzyme) as fluorescent and colorimetry probes. The sensor employs aptamers binding to PAT as recognition elements for specific molecule detection. Mxene-Au can be used as a biological inducer to assist OA-UCNPs in controlling fluorescence intensity. In addition, colorimetric signal amplification was performed using the trivalent G4-DNAzyme to increase detection sensitivity and reduce false positives. Under optimal conditions, the dual-mode aptasensor has a detection limit of 5.3 pg mL-1 in fluorescence and 2.4 pg mL-1 in colorimetric methods, respectively, with the wider linear range and limit of detection (LOD) of the colorimetric assay. The combination aptasensor can detect PAT with high sensitivity and high specificity and has broad application prospects in the field of food safety detection.

A fluorescence aptamer sensor utilizing WS2 nanosheets for sensitive detection of patulin: enhanced specificity and wide applicability.

A tungsten disulfide (WS2) nanosheet-based aptamer sensor was developed to detect patulin (PAT). The 5'-end of the PAT aptamer was modified with a cyanine 3 (Cy3) fluorophore, which self-assembled on WS2 nanosheets. The interaction between the Cy3 fluorophore at the 5'-end of the PAT aptamer and the WS2 nanosheets resulted in reduced fluorescence (FL) intensity due to fluorescence resonance energy transfer (FRET). The introduction of PAT into this sensing system led to hybridization with the PAT aptamer, forming a G-quadruplex/PAT complex with low affinity for the WS2 nanosheet surface. This hybridization increased the distance between the Cy3 fluorophore and the WS2 nanosheets, inhibiting FRET and producing a strong FL signal. Under optimal experimental conditions, the FL intensity of the sensing system demonstrated an excellent linear correlation with PAT concentrations ranging from 0.5 to 40.0 ng mL-1, and it achieved a detection limit (S/N = 3) of 0.23 ng mL-1. This sensing system offers enhanced specificity for PAT detection and has the potential for broad application in detecting other toxins by substituting the sequence of the recognition aptamer.

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).

Molecular and chemical evaluation of patulin production of Aspergillus and Penicillium-like species isolated from Hungarian apples.

Mycotoxins are secondary fungal metabolites harmful to humans and animals. Patulin (PAT) is a toxin found in different food products but especially in apples and their derivative products. The most common fungi producers of this compound are Aspergillus clavatus and Penicillium expansum. The production of patulin, as other mycotoxins, can be impacted by diverse phenomena such as water and nutrient availability, UV exposure, and the presence of antagonistic organisms. Consequently, gaining a comprehensive understanding of climate and environmental conditions is a crucial step in combating patulin contamination. In this study, moulds were isolated from 40 apple samples collected from seven locations across Hungary: Csenger, Damak, Pallag, Lövopetri, Nagykálló, and Újfehértó. A total of 183 moulds were morphologically identified, with 67 isolates belonging to the Alternaria, 45 to the Aspergillus, and 13 to the Penicillium groups. The location possessed a higher influence than farming method on the distribution of mould genera. Despite the requirement of higher temperature, Aspergillus species dominated only for the region of Újfehértó with approximately 50% of the isolates belonging to the genus. Four of the seven locations assessed: Csenger, Debrecen-Pallag, Nyírtass and Nagykálló, were dominated by Alternaria species. All isolates belonging to the genera Aspergillus and Penicillium were tested for the presence of the isoepoxidone dehydrogenase (idh) gene, a key player in the patulin metabolic pathway. To guarantee patulin production, this ability was confirmed with TLC assays. The only Aspergillus strain that presented a positive result was the strain Aspergillus clavatus B9/6, originated from the apple cultivar Golden Reinders grown in Debrecen-Pallag by integrated farming. Of the Penicillium isolates only one strain, B10/6, presented a band of the right size (500-600 bp) for the idh gene. Further sequencing of the ITS gene showed that this strain should be classified as Talaromyces pinophilus. The TLC tests confirmed this microorganism as the only patulin producer under the studied conditions for its cluster.

Validation and screening of patulin in apple beverages marketed in Morocco.

Patulin is one of the mycotoxins frequently detected in apples and derivatives, representing a major food safety risk. This study aimed to validate a high-performance liquid chromatography (HPLC) method with an ultraviolet (UV) detector for patulin quantification and assess its occurrence in apple beverages marketed in Morocco. The validation parameters showed satisfactory results with adequate linearity (R > 0.997), a relative standard deviation below 2.5%, repeatability between 3.6 and 7.1%, reproducibility between 3.9 and 11.5%, a limit of quantification (LOQ) of 4 µg/L, and recoveries close to 100% for three levels. Analysis of 30 samples revealed patulin levels ranging from 0 to 16.36 µg/L, with 50% of samples showing negative levels. All positive results remained below the regulatory maximum limit of 50 µg/L. These findings affirm the efficacy of the HPLC proposed method in ensuring compliance with patulin regulations in apple beverages, underlining its importance in safeguarding food safety.

Integrated Photoelectrochemical-SERS Platform Based on Plasmonic Metal-Semiconductor Heterostructures for Multidimensional Charge Transfer Analysis and Enhanced Patulin Detection.

Comprehending the charge transfer mechanism at the semiconductor interfaces is crucial for enhancing the electronic and optical performance of sensing devices. Yet, relying solely on single signal acquisition methods at the interface hinders a comprehensive understanding of the charge transfer under optical excitation. Herein, we present an integrated photoelectrochemical surface-enhanced Raman spectroscopy (PEC-SERS) platform based on quantum dots/metal-organic framework (CdTe/Yb-TCPP) nanocomposites for investigating the charge transfer mechanism under photoexcitation in multiple dimensions. This integrated platform allows simultaneous PEC and SERS measurements with a 532 nm laser. The obtained photocurrent and Raman spectra of the CdTe/Yb-TCPP nanocomposites are simultaneously influenced by variable bias voltages, and the correlation between them enables us to predict the charge transfer pathway. Moreover, we integrate gold nanorods (Au NRs) into the PEC-SERS system by using magnetic separation and DNA biometrics to construct a biosensor for patulin detection. This biosensor demonstrates the voltage-driven ON/OFF switching of PEC and SERS signals, a phenomenon attributed to the plasmon resonance effect of Au NRs at different voltages, thereby influencing charge transfer. The detection of patulin in apples verified the applicability of the biosensor. The study offers an efficient approach to understanding semiconductor-metal interfaces and presents a new avenue for designing high-performance biosensors.

Portable self-powered electrochemical aptasensing platform for ratiometric detection of mycotoxins based on multichannel photofuel cell.

Self-powered electrochemical sensors based on photofuel cells have attracted considerable research interest because their unique advantage of not requiring an external electric source, but their application in portable and multiplexed targets assay is limited by the inherent mechanism. In this work, a portable self-powered sensor constructed with multichannel photofuel cells was developed for the ratiometric detection of mycotoxins, namely ochratoxin A (OTA) and patulin (PAT). The spatially resolved CdS/Bi2S3-modified photoanodes and a shared Prussian Blue cathode were integrated on an etched indium-tin oxide slide to fabricate the multichannel photofuel cell. The aptamers of OTA and PAT were covalently bonded to individual photoanode regions to build sensitive interfaces, and the specific recognition of analytes impaired the output performance of constructed PFC. Accordingly, ratiometric sensing of OTA and PAT was achieved by utilizing the output performance of a control PFC as a reference signal. This approach effectively eliminates the impact of light intensity on the accuracy of the detection. Under the optimal conditions, the proposed sensing chip exhibited linear ranges of 2.0-1000 nM and 5.0-500 nM for OTA and PAT, respectively. The detection limits (3 S/N) were determined to be 0.25 nM for OTA and 0.27 nM for PAT. The developed ratiometric sensing method demonstrated good selectivity and stability in the simultaneous detection of OTA and PAT. It was successfully utilized for the analysis of OTA and PAT real samples. This work provides a new perspective for construction of portable and ratiometric self-powered sensing platform.

Determination and analysis of patulin in apples, hawthorns, and their products by high-performance liquid chromatography.

This study aimed to establish a high-performance liquid chromatography (HPLC) method to investigate the residues of patulin in apples, hawthorns, and their products. A total of 400 samples were collected from online shopping plats and supermarkets in China, including apples (n = 50), hawthorns (n = 50), and their products (apple juice, apple puree, apple jam, hawthorn juice, hawthorn chips, and hawthorn rolls, n = 300). In this experiment, this method had good linearity and a recovery of 82.3-94.4% for patulin. The limit of detection (LOD) was 0.2 µg/kg for liquid samples, while it was 0.3 µg/kg for solid and semi-fluid samples. The frequencies of patulin were 79.8% in 400 samples, and the patulin concentration is from 0.6 to 126.0 µg/kg. Two samples (0.5%) for patulin exceeded the regulatory limit (50 µg/kg) in 400 samples. The frequencies of patulin in kinds of samples were 32.0-98.0% (p < 0.05), and the percentage of samples exceeding the limit was not more than 2.0%. The frequencies of patulin in domestic samples were 83.0%, while they were 57.7% in imported samples. Two domestic samples (0.6%) contained patulin above the regulatory limit, while none of the imported samples exceeded the limit. Among the online and offline samples, the frequencies of patulin were 76.4 and 82.1%. Two online samples (1.0%) for patulin exceeded the regulatory limit, whereas none of the offline samples exceeded the limit. These results showed it is important to monitor regularly the content of patulin in apples, hawthorns, and their products to ensure consumer food safety.

The Influence of Long-Term Storage on the Epiphytic Microbiome of Postharvest Apples and on Penicillium expansum Occurrence and Patulin Accumulation.

Patulin is a secondary metabolite primarily synthesized by the fungus Penicillium expansum, which is responsible for blue mold disease on apples. The latter are highly susceptible to fungal infection in the postharvest stages. Apples destined to produce compotes are processed throughout the year, which implies that long periods of storage are required under controlled atmospheres. P. expansum is capable of infecting apples throughout the whole process, and patulin can be detected in the end-product. In the present study, 455 apples (organically and conventionally grown), destined to produce compotes, of the variety "Golden Delicious" were sampled at multiple postharvest steps. The apple samples were analyzed for their patulin content and P. expansum was quantified using real-time PCR. The patulin results showed no significant differences between the two cultivation techniques; however, two critical control points were identified: the long-term storage and the deck storage of apples at ambient temperature before transport. Additionally, alterations in the epiphytic microbiota of both fungi and bacteria throughout various steps were investigated through the application of a metabarcoding approach. The alpha and beta diversity analysis highlighted the effect of long-term storage, causing an increase in the bacterial and fungal diversity on apples, and showed significant differences in the microbial communities during the different postharvest steps. The different network analyses demonstrated intra-species relationships. Multiple pairs of fungal and bacterial competitive relationships were observed. Positive interactions were also observed between P. expansum and multiple fungal and bacterial species. These network analyses provide a basis for further fungal and bacterial interaction analyses for fruit disease biocontrol.

Optimization of Seleno-chitosan-phytic acid nanocomplex for efficient removal of patulin from apple juice.

A novel and effective adsorbent known as Seleno-chitosan-phytic acid nanocomplex (Se-CS-PA) has been developed specifically for efficiently removing patulin (PAT) from a simulated juice solution. The synthesis of Se-CS-PA nanocomplex was confirmed through Fourier transform infrared (FT-IR), scanning electron microscopy (SEM), and energy dispersive X-Ray (EDX) analyses. Response surface methodology (RSM) was employed using central composite design (CCD) to examine the impact of four independent variables (PA concentration, amount of nano-complex, duration of interaction between PAT and nano-complex, and initial concentration of PAT) on the removal of PAT. PA concentration of 0.1 % with 2.1 g Se-CS-PA nanocomplex according to RSM polynomial equation and apple juice with 25 µg.L-1 PAT yielded a remarkable adsorption rate of 94.23 % and 87.52 % respectively after 7 h. The process of PAT adsorption was explained using the pseudo-first-order model (R2 = 0.8858) for the kinetic model and the Freundlich isotherm (R2 = 0.9988) for the isotherm model.

Smartphone-Controlled Aptasensor for Voltammetric Detection of Patulin in Apple Juice.

Patulin (PAT) is a mycotoxin that adversely affects the health of humans and animals. PAT can be particularly found in products such as apples and apple juice and can cause many health problems if consumed. Therefore, accurate and sensitive determination of PAT is very important for food quality and human and animal health. A voltammetric aptasensor was introduced in this study for PAT determination while measuring the changes at redox probe signal. The limit of detection (LOD) was found to be 0.18 pg/mL in the range of 1-104 pg/mL of PAT in buffer medium under optimum experimental conditions. The selectivity of the PAT aptasensor against ochratoxin A, fumonisin B1 and deoxynivalenol mycotoxins was examined and it was found that the aptasensor was very selective to PAT. PAT determination was performed in an apple juice medium for the first time by using a smartphone-integrated portable device, and accordingly, an LOD of 0.47 pg/mL was achieved in diluted apple juice medium. A recovery range of 91.24-93.47% was obtained for PAT detection.

Patulin contamination of hard apple cider by Paecilomyces niveus and other postharvest apple pathogens: Assessing risk factors.

Hard apple cider is considered to be a low-risk product for food spoilage and mycotoxin contamination due to its alcoholic nature and associated food sanitation measures. However, the thermotolerant mycotoxin-producing fungus Paecilomyces niveus may pose a significant threat to hard cider producers. P. niveus is known to infect apples (Malus xdomestica), and previous research indicates that it can survive thermal processing and contaminate finished apple juice with the mycotoxin patulin. To determine if hard apple cider is susceptible to a similar spoilage phenomenon, cider apples were infected with P. niveus or one of three patulin-producing Penicillium species and the infected fruits underwent benchtop fermentation. Cider was made with lab inoculated Dabinett and Medaille d'Or apple cultivars, and patulin was quantified before and after fermentation. Results show that all four fungi can infect cider apples and produce patulin, some of which is lost during fermentation. Only P. niveus was able to actively grow throughout the fermentation process. To determine if apple cider can be treated to hinder P. niveus growth, selected industry-grade sanitation measures were tested, including chemical preservatives and pasteurization. High concentrations of preservatives inhibited P. niveus growth, but apple cider flash pasteurization was not found to significantly impact spore germination. This study confirms that hard apple cider is susceptible to fungal-mediated spoilage and patulin contamination. P. niveus is an important concern for hard apple cider producers due to its demonstrated thermotolerance, survival in fermentative environments, and resistance to sanitation measures.

Identification and pretreatment analysis of endogenous degradation products of patulin in zebrafish.

Identification and pretreatment analysis of endogenous metabolites of patulin (PAT) in zebrafish were successfully carried out using UHPLC-Q-Orbitrap-HRMS. Three major metabolites, namely hydroascladiol, E-ascladiol, and Z-ascladiol, were identified. They exhibited similar fragmentation pathways to PAT, with the structurally significant ions *b' and *c' generated through the cleavage of the side chains of *b and *c, respectively. These ions were crucial for confirming the modification site and have been confirmed as characteristic fragments for the identification of PAT metabolites. Furthermore, a pretreatment method for analyzing PAT and the three metabolites in zebrafish was proposed, using solid-phase-assisted liquid/liquid extraction (SLLE) and matrix solid-phase dispersion (MSPD) techniques. The initial purification process involved loading the aqueous phase onto a macroporous diatomaceous column, followed by elution with acetonitrile. Following this, neutral alumina powder was added to the organic phase, effectively eliminating interference from hydrophilic and lipid-soluble compounds through the optimization of this step. Due to their structural similarity, the three metabolites were semi-quantitatively analyzed using a PAT standard curve. The results demonstrated a good linear relationship in the concentration range of 0.001-0.02 µg/mL (r2 ≥ 0.999). The limit of detection for PAT and the three metabolites ranged from 0.01 to 0.03 mg/kg.

A sensitive monoclonal antibody-based ELISA integrated with immunoaffinity column extraction for the detection of zearalenone in food and feed samples.

Zearalenone (ZEN) is one of the most toxic mycotoxins widely found in agricultural products. In this study, a sensitive enzyme-linked immunosorbent assay (ELISA) integrated with immunoaffinity column extraction for the detection of ZEN in food and feed samples was developed. A ZEN derivative containing a carboxylic group was first synthesized and then linked to bovine serum albumin (BSA). The formed ZEN-BSA conjugate was used as the immunogen for the production of the monoclonal antibody (mAb) against ZEN. The hybridoma clones (1G5) capable of secreting antibodies against ZEN were successfully selected. Based on this mAb, the IC50 and LOD of the ELISA for ZEN were 0.37 ng mL-1 and 0.04 ng mL-1, respectively, which were 1.6-308.1 times lower than those in the published ELISAs, indicating the high sensitivity of our assay. There was no cross-reactivity of the mAb with other four mycotoxins (patulin, AFB1, DON, and OTA). Due to the high similarity in molecular structures among ZEN and its homologs (α-zearalanol, ß-zearalanol, zearalanone, α-zearalenol, ß-zearalenol), the CR values of the mAb with the homologs were within 3.59%-105.71%. Taking advantage of plenty of mAb, the immunoaffinity column was prepared by immobilizing the mAb on Sepharose-4B gel and filling it into an SPE column. ZEN spiked samples (corn, wheat, feed) were extracted using an immunoaffinity column and measured by ELISA and HPLC-FLD simultaneously. The recoveries of the ELISA for ZEN in the spiked samples were 92.46-105.48% with RSDs of 4.87-10.11%. A good correlation between ELISA (x) and HPLC-FLD (y) with the linear regression equation y = 1.0589x + 1.43815 (R2 = 0.998, n = 6) was obtained. To verify the applicability, the proposed ELISA was also applied to some real samples randomly collected from a local market. It was proven that the newly produced mAb-based ELISA was a feasible and sensitive method for the detection of ZEN in food and feed samples.

Dispersive solid phase extraction using a hydrophilic molecularly imprinted polymer for the selective extraction of patulin in apple juice samples.

A molecularly imprinted polymer with a specific selectivity for patulin was successfully synthesized. The molecularly imprinted material was prepared using the two functional monomers dopamine and melamine and formaldehyde as the cross-linker. The resulting material possessed a large number of hydrophilic groups, such as hydroxyls, imino groups, and ether linkages. For the first time, uric acid was used as a dummy template for its structural similarity to patulin. Comprehensive characterization and detailed studies of the adsorption process were carried out via adsorption isotherms, while the rate-limiting steps were investigated using adsorption kinetics. Separation, determination, and quantification of patulin were achieved by ultra-high performance liquid chromatography coupled with both photodiode array detection and tandem mass spectrometry. The latter was applied to patulin confirmation in the analysis of real samples. The methodology was validated in 20 apple juice samples. The results showed that the developed hydrophilic molecularly imprinted polymer had high selectivity and specific adsorption towards patulin, with mean recoveries ranging between 85 and 90% and a relative standard deviation lower than 15%. The developed molecularly imprinted polymer exhibited good linearity in the range 1-100 ng mL-1 with coefficient of determination (R2) > 0.99. The limit of detection was 0.5 ng mL-1, and the limit of quantification was 1 ng g-1. The developed method showed a good purification capacity for apple juices due to its hydrophilic nature and the polar interactions established with the target analyte.

Visualization detection of mycotoxin patulin in fruit juices by a small-molecule fluorescent probe.

The mycotoxin patulin is a common contaminant in rotten fruits, posing severe food safety risks and threats to human health. Developing a convenient, sensitive and reliable method for patulin detection is of utmost importance but remains challenging. In this study, we have successfully designed and synthesized a small-molecule fluorescent probe, FITC-Lys, which demonstrates good sensitivity in detecting patulin. Upon contact with patulin, the terminal Lys group of the FITC-Lys probe reacts with patulin, resulting in the formation of the fluorescein dimer that subsequently quenches fluorescence. This variation of fluorescence enables the visualization and sensitive detection of patulin. The probe exhibits good sensitivity with a low LOD of 8 ng mL-1 for the fluorescence spectrum method and a LOD of 12 ng mL-1 for the fluorescence imaging method. Moreover, we have validated the probe's capability for patulin detection in apple and pear juices, achieving good recoveries ranging from 98.60% to 103.80%. Notably, the probe FITC-Lys is the first small-molecule fluorescent probe that has proven successful in visualizing patulin in juices derived from decayed apples and pears. Consequently, this probe holds great potential as a practical tool for monitoring patulin in foodstuffs, thereby contributing to enhanced food safety standards.

The histone demethylase KdmB is part of a trimeric protein complex and mediates virulence and mycotoxin production in Penicillium expansum.

Epigenetic modification of chromosome structure has increasingly been associated with alterations in secondary metabolism and sporulation defects in filamentous fungal pathogens. Recently, the epigenetic reader protein SntB was shown to govern virulence, spore production and mycotoxin synthesis in the fruit pathogen Penicillium expansum. Through immunoprecipitation-coupled mass spectrometry, we found that SntB is a member of a protein complex with KdmB, a histone demethylase and the essential protein RpdA, a histone deacetylase. Deletion of kdmB phenocopied some but not all characteristics of the ΔsntB mutant. KdmB deletion strains exhibited reduced lesion development on Golden Delicious apples and this was accompanied by decreased production of patulin and citrinin in host tissue. In addition, ΔkdmB mutants were sensitive to several cell wall stressors which possibly contributed to the decreased virulence observed on apples. Slight differences in spore production and germination rates of ΔkdmB mutants in vitro did not impact overall diameter growth in culture.

Diversity of Mycotoxins and Other Secondary Metabolites Recovered from Blood Oranges Infected by Colletotrichum, Alternaria, and Penicillium Species.

This study identified secondary metabolites produced by Alternaria alternata, Colletotrichum gloeosporioides, and Penicillium digitatum in fruits of two blood orange cultivars before harvest. Analysis was performed by UHPLC-Q-TOF-MS. Three types of fruits were selected, asymptomatic, symptomatic showing necrotic lesions caused by hail, and mummified. Extracts from peel and juice were analyzed separately. Penicillium digitatum was the prevalent species recovered from mummified and hail-injured fruits. Among 47 secondary metabolites identified, 16, 18, and 13 were of A. alternata, C. gloeosporioides, and P. digitatum, respectively. Consistently with isolations, indicating the presence of these fungi also in asymptomatic fruits, the metabolic profiles of the peel of hail-injured and asymptomatic fruits did not differ substantially. Major differences were found in the profiles of juice from hail-injured and mummified fruits, such as a significant higher presence of 5,4-dihydroxy-3,7,8-trimethoxy-6C-methylflavone and Atrovenetin, particularly in the juice of mummified fruits of the Tarocco Lempso cultivar. Moreover, the mycotoxins patulin and Rubratoxin B were detected exclusively in mummified fruits. Patulin was detected in both the juice and peel, with a higher relative abundance in the juice, while Rubratoxin B was detected only in the juice. These findings provide basic information for evaluating and preventing the risk of contamination by mycotoxins in the citrus fresh fruit supply chain and juice industry.