A superefficient ochratoxin A hydrolase with promising potential for industrial applications

As the most seriously controlled mycotoxin produced by Aspergillus spp. and Penicillium spp., ochratoxin A (OTA) results in various toxicological effects and widely contaminates agro-products. Biological detoxification of OTA is the most priority in food and feed industry, but currently available detoxification enzymes are relatively low effectiveness in time and cost. Here we show a superefficient enzyme ADH3 identified from Stenotrophomonas acidaminiphila with a strong ability to transform OTA into non-toxic ochratoxin-α by acting as an amidohydrolase. Recombinant ADH3 (1.2 μg/mL) completely degrades 50 μg/L OTA within 90 seconds, while the availably most efficient OTA hydrolases takes several hours. The kinetic constant showed that rADH3 ( Kcat/Km ) catalytic efficiency was 56.7-35000 times higher than those of previous hydrolases rAfOTase, rOTase and commercial carboxypeptidase A (CPA). Protein structure-based assay suggested that ADH3 has a preference for hydrophobic residues to form a larger hydrophobic area than other detoxifying enzymes at the cavity of the catalytic sites, and this structure makes the OTA easier to access to catalytic sites. In addition, ADH3 shows considerable temperature adaptability to exert hydrolytic function at the temperature down to 0°C or up to 70°C. Collectively, we report a superefficient OTA detoxifying enzyme with promising potential for industrial applications. IMPORTANCE Ochratoxin A (OTA) can result in various toxicological effects and widely contaminates agro-products and feedstuffs. OTA detoxifications by microbial strains and bio-enzymes are significant to food safety. Although previous studies showed OTA could be transformed through several pathways, the ochratoxin-α pathway is recognized as the most effective one. However, the most currently available enzymes are not efficient enough. Here, a superefficient hydrolase ADH3 which can completely transform 50 μg/L OTA into ochratoxin-α within 90 seconds was screened and characterized. The hydrolase ADH3 shows considerable temperature adaptability (0-70°C) to exert the hydrolytic function. Findings of this study supplied an efficient OTA detoxifying enzyme and predicted the superefficient degradation mechanism which lay a foundation for future industrial applications.

Ochratoxins in Wines: A Review of Their Occurrence in the Last Decade, Toxicity, and Exposure Risk in Humans

Ochratoxins (OTs) are mycotoxins frequently found in wines, and their contamination can occur during any stage of the winemaking process. Ochratoxin A (OTA) has been the most widely reported and the only one whose concentrations are legislated in this beverage. However, ochratoxin B, ochratoxin A methyl ester, ochratoxin B methyl ester, ochratoxin A ethyl ester, ochratoxin B ethyl ester, ochratoxin α, ochratoxin β, OTα methyl ester, OTA ethyl amide, and OTA glucose ester have also been reported in wines. Thus, detecting only OTA would lead to the underestimation of ochratoxin levels, which is a risk to human health. Considering the threat represented by the presence of ochratoxins in wines and the long-term health problems that they can cause in wine drinkers, this paper aims to review reports of the last 10 years regarding the presence of different ochratoxins in wines and how the winemaking process influences the degree of contamination, mainly by OTA. Additionally, toxicity from human exposure due to the consumption of contaminated wines is addressed.

Mycotoxins Exposure of French Grain Elevator Workers: Biomonitoring and Airborne Measurements

It is now recognized that additional exposure to mycotoxins may occur through inhalation of contaminated dust at a workplace. The aim of this study was to characterize the multi-mycotoxin exposure of French grain elevator workers using biomonitoring and airborne measurements. Eighteen workers participated in the study. Personal airborne dust samples were analyzed for their mycotoxin concentrations. Workers provided multiple urine samples including pre-shift, post-shift and first morning urine samples or 24 h urine samples. Mycotoxin urinary biomarkers (aflatoxin B1, aflatoxin M1, ochratoxin A, ochratoxin α, deoxynivalenol, zearalenone, α-zearalenol, β-zearalenol, fumonisin B1, HT-2 toxin and T-2 toxin) were measured using a liquid chromatography–high-resolution mass spectrometry method. Grain elevator workers were highly exposed to organic airborne dust (median 4.92 mg.m−3). DON, ZEN and FB1 were frequent contaminants in 54, 76 and 72% of air samples, respectively. The mycotoxin biomarkers quantified were DON (98%), ZEN (99%), α-ZEL (52%), β-ZEL (33%), OTA (76%), T-2 (4%) and HT-2 (4%). DON elimination profiles showed highest concentrations in samples collected after the end of the work shift and the urinary DON concentrations were significantly higher in post-shift than in pre-shift-samples (9.9 and 22.1 µg/L, respectively). ZEN and its metabolites concentrations did not vary according to the sampling time. However, the levels of α-/β-ZEL were consistent with an additional occupational exposure. These data provide valuable information on grain worker exposure to mycotoxins. They also highlight the usefulness of multi-mycotoxin methods in assessing external and internal exposures, which shed light on the extent and pathways of exposure occurring in occupational settings.

Efflux at the Blood-Brain Barrier Reduces the Cerebral Exposure to Ochratoxin A, Ochratoxin α, Citrinin and Dihydrocitrinone

Recent studies have implied that environmental toxins, such as mycotoxins, are risk factors for neurodegenerative diseases. To act directly as neurotoxins, mycotoxins need to penetrate or affect the integrity of the blood-brain barrier, which protects the mammalian brain from potentially harmful substances. As common food and feed contaminants of fungal origin, the interest in the potential neurotoxicity of ochratoxin A, citrinin and their metabolites has recently increased. Primary porcine brain capillary endothelial cells were used to investigate cytotoxic or barrier-weakening effects of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone. The transfer and transport properties of the mycotoxins across the barrier formed by porcine brain capillary endothelial cell monolayers were analysed using HPLC-MS/MS. High levels of Ochratoxin A caused cytotoxic and barrier-weakening effects, whereas ochratoxin α, citrinin and dihydrocitrinone showed no adverse effects up to 10 µM. Likely due to efflux transporter proteins, the transfer to the brain compartment was much slower than expected from their high lipophilicity. Due to their slow transfer across the blood-brain barrier, cerebral exposure of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone is low and neurotoxicity is likely to play a subordinate role in their toxicity at common physiological concentrations.

Investigating Multi-Mycotoxin Exposure in Occupational Settings: A Biomonitoring and Airborne Measurement Approach

Investigating workplace exposure to mycotoxins is of the utmost importance in supporting the implementation of preventive measures for workers. The aim of this study was to provide tools for measuring mycotoxins in urine and airborne samples. A multi-class mycotoxin method was developed in urine for the determination of aflatoxin B1, aflatoxin M1, ochratoxin A, ochratoxin α, deoxynivalenol, zearalenone, α-zearalenol, β-zearalenol, fumonisin B1, HT2-toxin and T2-toxin. Analysis was based on liquid chromatography–high resolution mass spectrometry. Sample pre-treatments included enzymatic digestion and an online or offline sample clean-up step. The method was validated according to the European Medicines Agency guidance procedures. In order to estimate external exposure, air samples collected with a CIP 10 (Capteur Individuel de Particules 10) personal dust sampler were analyzed for the quantification of up to ten mycotoxins, including aflatoxins, ochratoxin A, deoxynivalenol, zearalenone, fumonisin B1 and HT-2 toxin and T-2 toxin. The method was validated according to standards for workplace exposure to chemical and biological agents EN 482. Both methods, biomonitoring and airborne mycotoxin measurement, showed good analytical performances. They were successfully applied in a small pilot study to assess mycotoxin contamination in workers during cleaning of a grain elevator. We demonstrated that this approach was suitable for investigating occupational exposure to mycotoxins.

Truncated Expression of a Carboxypeptidase A from Bovine Improves Its Enzymatic Properties and Detoxification Efficiency of Ochratoxin A

Ochratoxin A (OTA) is a toxic secondary metabolite produced mainly by Penicillium spp. and Aspergillus spp. and commonly found in foodstuffs and feedstuffs. Carboxypeptidase A (CPA) can hydrolyze OTA into the non-toxic product ochratoxin α, with great potential to realize industrialized production and detoxify OTA in contaminated foods and feeds. This study constructed a P. pastoris expression vector of mature CPA (M-CPA) without propeptide and signal peptide. The results showed that the degradation rate of OTA by M-CPA was up to 93.36%. Its optimum pH was 8, the optimum temperature was 40 °C, the value of Km was 0.126 mmol/L, and the maximum reaction rate was 0.0219 mol/min. Compared with commercial CPA (S-CPA), the recombinant M-CPA had an improve stability, for which its optimum temperature increased by 10 °C and stability at a wide range pH, especially at pH 3–4 and pH 11. M-CPA could effectively degrade OTA in red wine. M-CPA has the potential for industrial applications, such as can be used as a detoxification additive for foods and feeds.

Degradation of ochratoxin A by supernatant and ochratoxinase of Aspergillus niger W-35 isolated from cereals

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus spp. and Penicillium spp. and poses a threat to food safety. Biodegradation may be a promising strategy for reducing the OTA contamination in the future. In this study, Aspergillus niger strain W-35 was isolated from cereals and studied for its ability to degrade OTA. Results showed that the supernatant of W-35 could degrade OTA both in vitro and in commercial feeds after incubation at 37 °C for 12 h by 78.0 and 37.0%, respectively. Ochratoxin α (OTα) was assayed as a degradation product by HPLC-FLD. Furthermore, an enzyme specific for OTA degradation (ochratoxinase, OTase) obtained from W-35 was successfully expressed in Escherichia coli BL21, and degraded OTA at a rate of 85.1% for 12 h. These results indicated that this OTA degradation is enzymatic and that the responsible enzyme is extracellular OTase. Reliable degradation of OTA has the potential for wide-ranging applications in the food and feed industries.

Heterologous Expression and Characterization of A Novel Ochratoxin A Degrading Enzyme, N-acyl-L-amino Acid Amidohydrolase, from Alcaligenes faecalis

Ochratoxin A (OTA) is a well-known, natural contaminant in foods and feeds because of its toxic effects, such as nephrotoxicity in various animals. Recent studies have revealed that Alcaligenes faecalis could generate enzymes to efficiently degrade OTA to ochratoxin α (OTα) in vitro. In an effort to obtain the OTA degrading mechanism, we purified and identified a novel degrading enzyme, N-acyl-L-amino acid amidohydrolase (AfOTase), from A. faecalis DSM 16503 via mass spectrometry. The same gene of the enzyme was also encountered in other A. faecalis strains. AfOTase belongs to peptidase family M20 and contains metal ions at the active site. In this study, recombination AfOTase was expressed and characterized in Escherichia coli. The molecular mass of recombinant rAfOTase was approximately 47.0 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited a wide temperature range (30–70 °C) and pH adaptation (4.5–9.0) and the optimal temperature and pH were 50 °C and 6.5, respectively.

Analysis of the Carry-Over of Ochratoxin A from Feed to Milk, Blood, Urine, and Different Tissues of Dairy Cows Based on the Establishment of a Reliable LC-MS/MS Method

A rapid and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of ochratoxin A (OTA) and its metabolite ochratoxin α (OTα), for the first time, in dairy cow plasma, milk, urine, heart, liver, spleen, lung, and kidney. The established method was extensively validated by determining the linearity (R2 ≥ 0.990), sensitivity (lower limit of quantification, 0.1–0.2 ng mL−1), recovery (75.3–114.1%), precision (RSD ≤ 13.6%), and stability (≥83.0%). Based on the methodological advances, the carry-over of OTA was subsequently studied after oral administration of 30 μg/kg body weight OTA to dairy cows. As revealed, OTA and OTα were detected in urine, with maximal concentrations of 1.8 ng mL−1 and 324.6 ng mL−1, respectively, but not in milk, plasma, or different tissues, verifying the protection effects of rumen flora against OTA exposure for dairy cows. Moreover, 100 fresh milk samples randomly collected from different supermarkets in Shanghai were also analyzed, and no positive samples were found, further proving the correctness of the in vivo biotransformation results. Thus, from the currently available data, regarding OTA contamination issues on dairy cows, no significant health risks were related to OTA exposure due to the consumption of these products.

Simultaneous determination of mycotoxins in peruvian purple maize

ABSTRACT: Purple maize is an important foodstuff for the Peruvian people. Its unique nutritional and antioxidant characteristics makes it widely exported to other countries. However, when contaminated by fungi, it can trigger numerous health problems in the consumers. This study aimed to evaluate the presence of 27 mycotoxins in 63 samples of purple maize collected in Peru. Frequency of occurrence and mean concentration of the following mycotoxins were determined: alternariolmetileter (AME), alternariol (AOH), tentoxin, neosolaniol, nivalenol, wortmannin, deoxynivalenol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, zearalenone, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, fumonisin B1, fumonisin B2, fumonisin B3, ochratoxin A, ochratoxin α, T-2 toxin, HT-2 toxin, fusarenon x, cyclopiazonic acid, gliotoxin, agroclavin and citreoviridin. The main mycotoxins reported in purple maize were AME and AOH, with a frequency of occurrence of 14.3 and 7.9%, and mean concentration of 23.3% and 1.8%, respectively. AME and AOH do not have guidance levels in the Brazilian legislation. Contrastingly, levels of mycotoxins which are within the standards of the country’s regulations were below the limit of quantification. The present results suggested that purple maize is a raw material with a great potential for the production and industrialization of special products.