Phosphoribosyl Transferase (URA5) Enzyme-Mediated Detoxification of Patulin in Apple Juice and Its Effects on Nutritional Quality

The aim of this study was to investigate the patulin concentration in apple juice commercialized produced in Mashhad city of Iran after treatment with So 2 and heat. Apple Juice samples were analyzed for patulin using reversed-phase high performance liquidated chromatography. The results of this study showed that temperature (90, 107.5 and 125°C for 30&268 min) and SO 2 (0&350 ppm) have significant effect on reducing patulin amount. The most effective condition in decreasing patulin was observed at 125°C and 350 ppm which resulted in about 86% reduction of patulin. Results showed that heat and SO 2 are the most effortless methods that be recommended for fruit juice industries in preventing infection which has got a major role in the product of patulin in apple juice.

A simple and rapid binary solvents-based dispersive liquid–liquid microextraction (BS-DLLME) method has been developed for determination of patulin (PAT) in apple juice followed by high-performance liquid chromatography. This method involves the use of an appropriate mixture of miscible binary extraction solvents and disperser solvent to form fine droplets of extractant in a sample solution. Parameters affecting extraction efficiency such as the type and volume of high-density extraction solvent, the volume of ethyl acetate, the kind and volume of disperser solvent, and salt addition were investigated and optimized. The detection and quantification limits were 2.0 and 10.0 μg L−1, respectively. The relative standard deviation for five measurements of 25 μg L−1 of PAT was 3.8 %. The relative recoveries of PAT from apple juice samples at spiking levels of 25, 50, and 75 ng mL−1 were in the range of 91.3–95.2 %.

Patulin (PAT) is a mycotoxin produced in fruits, especially in apples, by diverse fungal species that can be transferred into industrial apple juice during processing. An accurate, effective, and selective method has been validated for the quantification of PAT in different commercial apple juices by combining a modified µ-QuEChERS procedure with high-pressure liquid chromatography (LC) equipped with a triple quadrupole mass spectrometer (QqQMS). This sample extraction procedure reduced interference from the sugar-rich matrix, and the separation was performed using the C18 Atlantis T3 column within 10 min. PAT was found by MS with electrospray negative ionization (ESI−) in the mode of multiple reaction monitoring (MRM). The correlation coefficient (R2 = 0.999) satisfied the prerequisite of linearity for PAT in the concentration range of 2–50 μg/kg. The limits of detection (LOD) and quantification (LOQ) of PAT were 0.32 and 1.15 μg/kg, respectively, which were compliant with the maximum levels settled in Commission Regulation (EC) No. 1881/2006. The recoveries were within the 92–103% range, at three fortified levels of 2, 20 and 50 μg/kg, with relative standard deviations lower than 7%. Based on analytical validation, it was confirmed that the µ-QuEChERS/HPLC-MS/MS method is an enhanced, reliable, and quick approach for determination of PAT in apple juice. The current approach proposes reduced sample preparation and analysis time. In addition, it is economical, environmentally friendly, and simpler to implement in comparison to traditional approaches.

Evaluation of dispersive liquid–liquid microextraction for the determination of patulin in apple juices using micellar electrokinetic capillary chromatography

Patulin (PAT), a mycotoxin produced primarily by Penicillium expansum, poses significant health risks and frequently contaminates apples and apple-derived products, often exceeding permissible safety limits. This study investigated the potential of orotate phosphoribosyl transferase (URA5) to degrade PAT in apple juice under controlled conditions. PAT degradation was assessed at initial concentrations of 100 µg/L and 250 µg/L, with enzymatic treatment using 0.2 mg/mL URA5. Samples were incubated for up to 24 h, and PAT degradation was monitored at time intervals of 3, 6, 9, 12, 18, and 24 h using liquid chromatography-mass spectrometry (LC-MS). The results demonstrated a time-dependent PAT degradation, with significant reductions observed as incubation time increased. After 6 h, PAT concentrations decreased to 57.30 µg/L and 112.69 µg/L for the 100 µg/L and 250 µg/L samples, respectively. At 12 h, PAT levels in the 100 µg/L sample fell just below the permissible limit (50 µg/kg), while substantial degradation was observed in the 250 µg/L sample. By 18 h, PAT concentrations dropped further to 47.22 µg/L and 40.10 µg/L, reaching safe consumption levels. After 24 h, degradation rates reached 96.36% and 98.25%, reducing PAT levels to 30.22 µg/L and 31.48 µg/L, confirming the efficacy of URA5 in detoxifying PAT-contaminated apple juice. The findings highlight the potential application of URA5 as a biocatalyst for PAT detoxification in the fruit juice industry. Compared to existing detoxification methods, enzyme-based degradation presents a promising, environmentally friendly, cost-effective, and non-toxic alternative. Further studies should explore its feasibility in large-scale processing and its interaction with other contaminants in commercial apple juice production.

Ratiometric fluorescence aptasensor for the detection of patulin in apple juice based on the octahedral UiO-66-TCPP metal-organic framework and aptamer systems

Patulin (PAT) is a mycotoxin produced by Penicillium species, which often contaminates fruit and fruit-derived products, posing a threat to human health and food safety. This work aims to investigate the detoxification of PAT by Kluyveromyces marxianus YG-4 (K. marxianus YG-4) and its application in apple juice. The results revealed that the detoxification effect of K. marxianus YG-4 on PAT includes adsorption and degradation. The adsorption binding sites were polysaccharides, proteins, and some lipids on the cell wall of K. marxianus YG-4, and the adsorption groups were hydroxyl groups, amino acid side chains, carboxyl groups, and ester groups, which were combined through strong forces (ion interactions, electrostatic interactions, and hydrogen bonding) and not easily eluted. The degradation active substance was an intracellular enzyme, and the degradation product was desoxypatulinic acid (DPA) without cytotoxicity. K. marxianus YG-4 can also effectively adsorb and degrade PAT in apple juice. The contents of organic acids and polyphenols significantly increased after detoxification, significantly improving the quality of apple juice. The detoxification ability of K. marxianus YG-4 toward PAT would be a novel approach for the elimination of PAT contamination.

The purpose of this study was to evaluate the ability of Lactobacillus rhamnosus to bind patulin (PAT) in the buffer solution and apple juice. The binding of L. rhamnosus to PAT was reversible, which improved the stability of the bacterial complex. The ability to bind PAT can be enhanced with the inactivation of the strain by high temperature and acid treatment. Acid-treated bacteria had the highest PAT binding rate of 72.73±1.05%. The binding rates of acid and high temperature (121 °C) treatments were increased by 21.37% and 19.15%, respectively. L. rhamnosus showed the best detoxification ability to PAT at 37 °C, where the binding rate reached 50.9±1.03%. When the dose of inactivated bacteria powder was 0.02 g ml−1, the minimum concentration of PAT in apple juice was 0.37 µg ml−1. The addition of the L. rhamnosus inactivated powder did not affect the quality of the juice product and effectively bound the PAT in apple juice.

Fruit pigments are associated with the presence of antioxidants. This study determined the content of antioxidant components (total phenolic content (TPC), ascorbic acid, beta carotene and lycopene) and colour in selected local fruits (papaya, watermelon, dragon fruit, wax apple, and jackfruit). The TPC, ascorbic acid, beta carotene and lycopene were determined spectrophotometrically. Ascorbic acid content was determined using the 2,6-Dichlorophenolindophenol (DCPIP) titration method. Colour was assessed by tristimulus colorimetry based on the reflection spectrum. Total soluble solids (TSS) were also determined. Papaya has TPC (61.86 mg GAE/100 g), ascorbic acid (31.14 mg/100 mL), lycopene (4.82 mg/100 g), beta carotene (19.98 mg /100 g), TSS (10.17 °Brix) and colour profile L*(41.30), a*(24.07), b*(26.48), chroma value (35.79) and hue angle (47.70°). Wax apple has TPC (94.20 mg GAE/100 g), ascorbic acid (11.13 mg/100 mL), lycopene (1.62 mg/100 g), TSS (10.00 °Brix) and colour profile L*(63.42), a*(0.42), b*(7.64), chroma value (7.66) and hue angle (86.69°). Dragon fruit has TPC (179.985 mg GAE/100 g), ascorbic acid (5.94 mg/100 mL), beta carotene (3.98 mg/100 g), TSS (10.30 °Brix) and colour profile L*(21.45), b*(0.66), chroma value (6.86) and hue angle (5.52°). Jackfruit has TPC (75.16 mg GAE/100 g), lycopene (2.31 mg/100 g), beta carotene (9.25 mg/100 g), TSS (20.43 °Brix) and colour profile L*(71.74), a*(5.79) and hue angle (82.98°). Watermelon has ascorbic acid (19.28 mg/100 mL), lycopene (36.55 mg/100 g), beta carotene (14.37 mg/100 g) and colour profile b* (12.36), chroma value (26.98) and hue angle (27.22°). The a* value has a significant positive correlation (p<0.05) with the content of ascorbic acid, lycopene, and beta carotene while the chroma value shows a negative correlation with the total phenolic content (p<0.05). The b* value showing a negative correlation with the total phenolic content (p<0.05). Local fruits can be a good source of nutrient, phytochemicals, and antioxidants.

Patulin (PAT), a toxic secondary metabolite produced mainly by Penicillium species that frequently contaminates fruit and fruit-derived products, poses serious health risks to humans and animals. In the present study, three short-chain dehydrogenases/reductases (SDRs) with PAT-degrading ability, designated BsSDR1, BsSDR2, and BsSDR3, were identified from the genome of Bacillus subtilis 168. BsSDR1 and BsSDR2 showed powerful PAT elimination abilities, which can completely convert PAT to nontoxic E-ascladiol. Moreover, BsSDR1, BsSDR2, and BsSDR3 shared the highest sequence identity of 36.03% with the reported PAT-degrading enzymes, indicating that they are novel PAT-degrading enzymes. BsSDR1, BsSDR2, and BsSDR3 exhibited the highest activity against PAT at 40, 40, and 35 °C, respectively. Additionally, BsSDR1, BsSDR2, and BsSDR3 displayed remarkable thermostability, retaining 32.50, 24.63, and 46.74% residual activity, respectively, after incubation at 50 °C for 1 h. Three-dimensional (3D) simulation and site-directed mutagenesis indicated that the catalytic triad formed by the residues (Ser, Tyr, and Lys) was the key for SDR activity, and this conserved catalytic mechanism was followed in the catalytic process of novel PAT-degrading enzymes BsSDR1, BsSDR2, and BsSDR3. More importantly, BsSDR1, BsSDR2, and BsSDR3 can degrade PAT in apple juice at rates of 86.90, 90.17, and 61.57%, respectively. The identification of BsSDR1, BsSDR2, and BsSDR3 enriched the PAT-degrading enzyme libraries, providing promising candidates for PAT decontamination in the food industry.

Magnetic solid phase extraction (MSPE) is a valuable tool for the extraction and preconcentration of analytes from environmental, food, and biological samples because of its convenience, promptness, and simplicity. In this study, a graphene oxide-based magnetic nanocomposite (MGO) was synthesized and characterized by various instrumental methods. An effective MSPE procedure coupled with high-performance liquid chromatography (MSPE-HPLC) was developed for the preconcentration and determination of the patulin (PAT) in apple juice samples. Critical experimental parameters of MSPE that could affect the extraction efficiencies, such as the amount of MGO, the pH, and ionic strength of the sample solution; the extraction time; and the desorption conditions, were investigated. After optimizing the conditions, the detection limit of this method provided was 2.3 μg/kg; the recoveries were in the range of 68.7–83.6 %, with relative standard deviations (RSDs) ranging between 1.8–8.7 %. It was confirmed that the graphene-based magnetic nanocomposite was a kind of effective MSPE material used for the PAT analyses in apple juice samples.

Surface molecularly imprinted polymer capped Mn-doped ZnS quantum dots as a phosphorescent nanosensor for detecting patulin in apple juice

In this study, ozone processing was used to degrade patulin in apple juice, and the ozonolysis efficiency of patulin and its effects on phenolic compounds and organic acids in apple juice were investigated. Ozone processing was performed using a self-developed ozonolysis reactor at the ozone concentration of 12mg/L and flow rate of 3L/min for increasing ozonation times ranged from 0 to 30min. Ozone processing significantly degraded patulin in apple juice, and decreased it from 201.06 to below 50μg/L within 15min, with a reduction of 75.36%. While major phenolic compounds and organic acids in apple juice were seriously destroyed by ozone processing compared with the control. Processors should consider the adverse effects of ozone processing on quality of apple juices and further studies are advised to optimize the ozone processing for remaining the phenols and organic acids in apple juices.

Thiol-functionalized inactivated yeast embedded in agar aerogel for highly efficient adsorption of patulin in apple juice

A method was developed and validated in-house for the determination of patulin (PAT), a toxic mold metabolite, in apple juice. The sample was extracted with ethyl acetate-hexane and analyzed by liquid chromatography equipped with a C18 column and diode array detector. The mobile phase used for the quantification was water-ethanol, at a flow rate of 0.5 mL/min. The method showed a mean recovery of 84.8%, the relative standard deviation obtained in the precision study was <7.7%, the quantification and detection limits were 7 and 3 microg/L, respectively, and the linear range for PAT in apple juice was 2.6-650 microg/L. The ruggedness was evaluated by an intralaboratory experiment, in which 5 factors were studied, and only one was found to influence the observed results. The developed method is fast, practical, and simple; the solvents (except hexane) and reagents used were nontoxic. The results of the validation confirmed the efficiency of the method, which is sensitive enough to be used in studies required to quantify PAT in apple juice.