OTA Production and Its Detoxification

What Is OTA?

Ochratoxins (A, B, C) are secondary metabolites of Penicillium and Aspergillus, of which ochratoxin A (OTA) is the most common and prevalent toxin, posing a hazard to both humans and animals. The body absorbs OTA through the gastrointestinal tract, and OTA binds to high-affinity albumin, resulting in a long plasma half-life and difficulty in eliminating it cleanly. In healthy populations, plasma concentrations of OTA can range from a few hundred pmol/L to a few nmol/L. Under physiologic pH conditions, OTA manifests itself as monoanion (OTA^-) and dianion (OTA^2-). The structure of ochratoxins consists of a dihydro-isocoumarin portion linked to phenylalanine via an amide bond, and the OTA also contains a p-chlorophenol portion. OTA is a white, odorless, heat-stable crystal with poor water solubility.

Figure 1. Chemical structures of Ochratoxin A, B, and C
(Source: Kőszegi T, et al. 2016)

Detoxification of OTA

Physical methods

Physical methods can effectively remove OTA from contaminated food or feed, with low cost, simple operation, and environment-friendly. The commonly used physical methods are heating and extrusion, adsorption, ultraviolet radiation, gamma radiation, and so on, in which adsorption is considered to be an economical, environmentally friendly, and simple method. Both natural and synthetic adsorbents can remove OTA from contaminated food and agricultural products, such as activated carbon, chitosan, and β-cyclodextrin-polyurethane polymers. One study found that 1% activated carbon can absorb up to 87% of OTA (5 ng/mL) in white wine. Chitosan contains amino and carboxyl groups in its main chain, which makes it highly adsorbent to mycotoxins and environmental pollutants. Some researchers synthesized three different types of crosslinked chitosan based on natural chitosan and evaluated their adsorption capacity. 97% of OTA could be removed by crosslinked chitosan-glutaraldehyde complexes, and this adsorption behavior was affected by conditions such as pH, amount used, and reaction time. It is worth noting that non-specific adsorption may affect food texture or nutrients.

The traditional method to remove OTA is heat treatment. The melting point of OTA is 169℃, and the temperature above 180℃ will decrease the activity of OTA. Baking at 180°C for 60 minutes has been reported to reduce OTA in contaminated oats by 2%-18%. Some researchers have also found that baking and microwaving at 150°C reduces OTA in pistachios by more than 60%. However, in general, mycotoxins are generally thermally stable, and conservative food processing temperatures are generally between 80°C and 121°C, so treatment of contamination by temperature is not very effective. In addition, heat treatment consumes a lot of energy and causes other active ingredients to decompose, which can also destroy the nutrition of the food itself.

Radiation methods can safely and effectively reduce the level of OTA in food, including ultraviolet (UV), gamma radiation, and so on. Different wavelengths of ultraviolet rays significantly affect the production and degradation of OTA. It was found that the OTA standard could be degraded by up to 96.5% under UV-B radiation. The decrease in OTA yield may be related to the direct degradation of OTA and the inhibition of OTA biosynthesis by UV-B. The content of feed samples containing OTA could be reduced from 500 μg/kg to 100 μg/kg after 1 h of UV irradiation, and was completely removed after 8 h of irradiation.

Chemical methods

Chemical methods achieve detoxification by destroying toxic groups or changing the solubility of toxins. These methods generally hydrolyze the lactone ring or amide bond of OTA. Alkalis, oxidizing agents, and organic acids have been applied to detoxify OTA. In a strong alkaline environment, the amide bond in the OTA molecule is rapidly hydrolyzed to release non-toxic products, namely ochratoxin α (OTα) and phenylalanine. After treating artificially polluted black pepper with 2% sodium hydrosulphite for 15 s, the concentration of OTA decreased by 96%. In addition, OTA can be decomposed under acidic conditions. OTA in grape pomace can be resolved by acid treatment, and organic acids such as lactic acid and acetic acid degrade better than inorganic acids. The OTA reduction efficiency of the same acid varies with grape pomace varieties, and some researchers believe that the reason for this is related to the polyphenol content of grape pomace. However, because the chemical reagents themselves may be harmful to the human body, many countries prohibit the use of chemical methods in food processing, and strictly stipulate the allowable scope of application and safe dosage.

Biological methods

Biological methods remove OTAs by utilizing yeast fungi, non-toxic bacteria, or enzymes; these methods are low-cost, efficient, safe, have few side effects on nutrients, and are applicable to both liquid and solid foods. Biological methods inhibit the growth of toxin-producing fungi, bind the fungal toxins, and degrade the OTA to non-toxic products.

Lactic acid bacteria are widely used to remove mycotoxins from food. Some researchers investigated the OTA treatment capacity of three active or heat-inactivated lactic acid bacteria and found that the inactivated bacteria had a higher binding rate to OTA and considered that this was due to the adsorption of OTA on the surface of the cell wall through hydrophobic and electrostatic interactions. In addition, some studies have found that the degradation rate of OTA by active bacteria during the growth of liquid medium is between 8% and 28%, and no degradation products are observed, so it is proved that the removal of OTA is a phenomenon of cell binding. The surface characteristics of yeast cells treated by heat or acid were altered, including increased pore size, protein denaturation, and formation of Maillard reaction products, resulting in greater exposure of OTA-binding sites in dead cells, which increased the ability to remove OTA. The adsorption capacity of microorganisms to OTA is a polygenic genetic trait, and more effective OTA adsorption strains can be cultivated through breeding strategies.

The most common enzymes used for biodegradation of OTA are carboxypeptidase and amidase, which can degrade the OTA-amide bond or lactone ring into less toxic OTα or ring-opening OTA, thus reducing toxicity. There are also some enzymes or microorganisms that detoxify by degrading the amide bond in OTA to produce L-β-phenylalanine and non-toxic OTα, or by hydrolyzing the lactone ring. Brevibacterium strains were able to completely degrade OTA, with the degradation product being OTα. Similarly, Pediococcus parvulus was able to degrade approximately 90% of OTA within 19 hours, with the main degradation product being OTα. The removal efficiency of OTA by yeast was affected by culture conditions, including initial OTA, yeast concentration, pH, and temperature. After genome sequencing of Bacillus subtilis strain CW14, nine secreted enzymes related to OTA degradation were predicted, including four carboxypeptidases, two amidases, two hydrolases, and one lactamase. Among them, two carboxypeptidases contributed to OTA degradation, and the highest OTA degradation rate was 71.3% within 24 hours.

Figure 2. The biodegradation mechanism of OTA
(Source: Ding L, et al. 2023)

Some commercial enzymes are also used to detoxify OTA. Commercial peroxidase can simultaneously degrade OTA and zearalenone (ZEA) in beer samples within 6 hours, with degradation rates of 4.8% and 10.9%, respectively. In order to improve the biocatalytic activity and stability of the enzyme, recombinant DNA technology was adopted to solve the problems of instability, low activity, substrate, or product inhibition of the enzyme. For example, the natural carboxypeptidase A was modified by rounding off the propeptide and signal peptide, and it was found that the recombinant truncated enzyme had better acid-base resistance and thermal stability compared with the natural enzyme, and was able to efficiently degrade OTA in red wine. On the other hand, the zearalenone hydrolase and carboxypeptidase genes were combined, and the fusion enzyme expressed was able to degrade OTA and ZEA at the same time, and possessed bifunctional activity.

Table 2. OTA degradation by microorganisms and enzymes

(Source: Wang L, et al. 20222)

Ochratoxin ELISA Kits

Ochratoxin Antigens

Ochratoxin Antibodies