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 is the highest priority regarding OTA in food and feed industry, but currently available detoxification enzymes have relatively low effectiveness in terms of time and cost. Here we show a superefficient enzyme, ADH3, identified from Stenotrophomonas acidaminiphila that has a strong ability to transform OTA into nontoxic ochratoxin-α by acting as an amidohydrolase. Recombinant ADH3 (1.2 µg/mL) completely degrades 50 µg/L OTA within 90 s, while the other most efficient OTA hydrolases available take several hours. The kinetic constant showed that rADH3 (Kcat/Km) catalytic efficiency was 56.7 to 35,000 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 allows OTA easier access to the 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 s was screened and characterized. The hydrolase ADH3 shows considerable temperature adaptability (0 to 70°C) to exert the hydrolytic function. Findings of this study supplied an efficient OTA detoxifying enzyme and predicted the superefficient degradation mechanism, laying a foundation for future industrial applications.

Determination of six groups of mycotoxins in Chinese dark tea and the associated risk assessment.

Chinese dark tea is widely enjoyed for its multiple health-promoting effects and pleasant taste. However, its production involves fermentation by microbiota in raw tea, some of which are filamentous fungi and thus potential mycotoxin producers. Accordingly, whether mycotoxins pose health risk on dark tea consumption has become a public concern. In this study, a cleaning method of multi-functional column (MFC) and immunoaffinity column (IAC) in tandem combined to HPLC detection was developed and validated for determining ten mycotoxins of six groups (i.e., aflatoxins of B1, B2, G1 and G2, ochratoxin A, zearalenone, deoxynivalenol, fumonisins of B1, B2, and T-2) in dark teas. The interferences from secondary metabolites were effectively reduced, and the sensitivities and recoveries of the method were qualified for tea matrices. Six groups mycotoxins were determined in 108 samples representing the major Chinese dark teas by using the new method. Subsequently, the dietary exposure and health risks were evaluated for different age and gender groups in Kunming and Pu'er in China and Ulan Bator in Mongolia. The occurrence of zearalenone was 4.63% and that of ochratoxin A was 1.85%, with the other four groups mycotoxins were below the limits of quantification. The hazard index values for the five groups' non-carcinogenic mycotoxins were far below 1.0. The deterministic risk assessment indicated no non-carcinogenic risks for dark tea consumption in the three areas. Probabilistic estimation showed that the maximum value of 95th percentile carcinogenic risk value for the aflatoxins was 2.12 × 10-8, which is far below the acceptable carcinogenic risk level (10-6). Hereby, six groups mycotoxins in Chinese dark tea showed no observed risk concern to consumers.