Ochratoxin A Removal Research Articles

Occurrence of ochratoxin A in Sichuan bacon from different geographical regions and characterization and biocontrol of ochratoxigenic Aspergillus westerdijkiae strain 21G2-1A

Sichuan bacon represents the most prevalent dry-cured meat product across Southwest China, but it is vulnerable to fungal spoilage. In the present study, a total of 47 Sichuan bacons were obtained from different regions of the Sichuan Province and analyzed for the presence of ochratoxin A (OTA), yielding a positive rate of 23.4 % (11/47). All the observed OTA concentrations exceeded the maximum admissible dose in meat products (1 μg/kg) established by some EU countries, with the highest OTA concentration being 250.75 μg/kg, which raises a food safety concern and reveals the need for a standardized scientific processing protocol. Then, an OTA-producing fungus named 21G2-1A was isolated from positive samples and found to be Aspergillus westerdijkiae. Further characterization suggested a positive correlation between fungal growth and OTA production. The optimal temperature for the former was 25 °C, while it was 20 °C for the latter. Although the A. westerdijkiae strain 21G2-1A demonstrated greater mycelium growth in the presence of NaCl, OTA production was significantly dismissed when the salinity was greater than 5 %. Four lactic acid bacteria (LAB) were screened out as antagonists against the ochratoxigenic fungus. In vitro evaluation of the antagonists revealed that live cells inhibited fungal growth, and adsorption also contributed to OTA removal at different levels. This study sheds some light on OTA control in Sichuan bacon through a biological approach.

Practical Strategies to Reduce Ochratoxin A in Foods.

Ochratoxin A (OTA), a potent nephrotoxin, is one of the most deleterious mycotoxins, with its prevalence in agricultural crops and their processed foods around the world. OTA is a major concern to food safety, as OTA exposure through dietary intake may lead to a significant level of accumulation in the body as a result of its long half-life (about 35 days). Its potent renal toxicity and high risk of exposure as well as the difficulty in controlling environmental factors OTA production has prompted the need for timely information on practical strategies for the food industry to effectively manage OTA contamination during food processing. The effects of various food processes, including both nonthermal and thermal methods, on the reduction in OTA were summarized in this review, with emphasis on the toxicity of residual OTA as well as its known and unknown degradation products. Since complete removal of OTA from foodstuffs is not feasible, additional strategies that may facilitate the reduction in OTA in food, such as adding baking soda and sugars, was also discussed, so that the industry may understand and apply practical measures to ensure the safety of its products destined for human consumption.

Ochratoxin A: Overview of Prevention, Removal, and Detoxification Methods.

Ochratoxins are the secondary metabolites of Penicillium and Aspergillus, among which ochratoxin A (OTA) is the most toxic molecule. OTA is widely found in food and agricultural products. Due to its severe nephrotoxicity, immunotoxicity, neurotoxicity, and teratogenic mutagenesis, it is essential to develop effective, economical, and environmentally friendly methods for OTA decontamination and detoxification. This review mainly summarizes the application of technology in OTA prevention, removal, and detoxification from physical, chemical, and biological aspects, depending on the properties of OTA, and describes the advantages and disadvantages of each method from an objective perspective. Overall, biological methods have the greatest potential to degrade OTA. This review provides some ideas for searching for new strains and degrading enzymes.

Isolation and identification of a Cytobacillus oceanisediminis strain with ochratoxin A detoxification ability

Ochratoxin A (OTA) is a polyketide mycotoxin produced by several Aspergillus and Penicillium species that is of great concern as a contaminant in agricultural products. In this study, the bacterial strain CO29 with high OTA-detoxification ability was isolated from vineyards and was identified as Cytobacillus oceanisediminis based on morphological and physiological characteristics, combined with 16S rRNA gene sequencing and phylogenetic analysis. The removal of OTA by CO29 was partly attributed to physical absorption, but the main mechanism of OTA removal was degradation. The degradation activity was completely removed by boiling, and could be significantly reduced by proteinase K treatment, but was only partially inhibited by the metal chelator EDTA. Zn2+ significantly improved the degradation of OTA, while Ca2+ and Mg2+ significantly inhibited the degradation. In addition, organic solvents inhibited OTA degradation in a dose-dependent manner. Thus, the mechanism of degradation by CO29 cell lysates was deduced as being enzymatic. Two OTA degradation intermediates were observed, one of which was identified as OTα. The time-profiles of OTA degradation by cell lysates showed an increase of OTα corresponding to the decrease of OTA. To our best knowledge, this is the first report of a Cytobacillus oceanisediminis strain with the ability to detoxify OTA, with potential applications in the bioremediation of contaminated soils.

A promising innovative technique for mycotoxin detoxification from beverages using biofilms of lactic acid bacteria

This study assessed the removal of aflatoxin M1 (AFM1) and ochratoxin A (OTA) from artificially contaminated whole UHT milk and red grape juice, respectively, using biofilms from Lactobacillus rhamnosus GG. Using ELISA, the level of AFM1 and OTA removal from beverages was determined depending on various factors. Biofilms of various ages demonstrated varying degrees of AFM1 removal capacity from phosphate-buffered saline (PBS). Different levels of AFM1 contaminated whole UHT milk (0.1, 0.2, and 1 μg/L) and OTA contaminated red grape juice (2 and 4 μg/L) were tested in the detoxification process. The binding ability of mycotoxins was improved by increasing the biofilm surface area up to 70 cm2. L. rhamnosus GG biofilm was effective in removing mycotoxins within a short contact time ranging from 1 to 10 min. The proportion of bound AFM1 and OTA by L. rhamnosus GG biofilm was 64.6 and 98.3% respectively. A new machine has been proposed and used as a trial for detoxication purposes which would be a promising application in liquid food industries.

Adsorption removal of ochratoxin A from milk by Tibetan kefir grains and its mechanism

Tibetan kefir grains (TKG) are microbial symbiotic starters used in various fermented foods. Recent studies have focused on removing toxin contaminants from food using TKG. This work aims to investigate the ability of TKG to remove ochratoxin A (OTA) from milk, the adsorption mechanism of TKG, and the detoxification behavior after exposure to a simulated gastrointestinal tract. The results revealed that TKG and TK removed 90.7% and 92% of OTA, respectively, while the removal capacity of isolates was low and strain-dependent (3.7%–40.7%). The limited OTA removal of freeze-dried TKG (19.6%), TKG kefiran (3.1%), and TKG protein (9.3%) indicated that the matrix network structure was critical in TKG adsorption. The effects of pH, temperature, inoculation percentage, and initial concentration on OTA removal using TKG were investigated. OTA adsorption was consistent with the Freundlich model and the pseudo-second-order kinetic model. In simulated gastric and intestinal fluids, the removal of OTA by TKG reached a maximum at 4 h with 76.1% and 75.6%, respectively. Moreover, only 33.9% of OTA was released from the TKG-OTA complex after a series of washing treatments. These findings support TKG to effectively adsorb OTA contamination in milk while fermenting and can reduce gastrointestinal absorption.

Isolation and characterization of Bacillus amyloliquefaciens YL-1 with ochratoxin A degradation ability and biocontrol activity against Aspergillus westerdijkiae

Ochratoxin A (OTA), a toxic secondary metabolite mainly produced by Aspergillus and Penicillium species, is often found in food and agricultural products. Microbiological degradation appears to be a promising method for mycotoxins detoxification. In the present work, a bacterial strain YL-1 with OTA removal ability was isolated and identified to be Bacillus amyloliquefaciens based on 16S rRNA and gyrA gene sequences analyses. Up to 79.7% of OTA with an initial concentration of 2.5 μg/mL could be removed in liquid medium by this isolated bacterium in 48 h. Extracellular enzymatic degradation is suggested to be mainly responsible for the OTA removal ability of B. amyloliquefaciens YL-1, with lactone-opened ochratoxin α as the possible degradation product. Moreover, B. amyloliquefaciens YL-1 could inhibit the fungal growth of Aspergillus westerdijkiae both on agar plate and rice grains. These findings indicated that B. amyloliquefaciens YL-1 could be considered as a promising biological agent against OTA contamination and A. westerdijkiae growth in cereal grains.

Reduction of ochratoxin A from contaminated food by Lactobacillus rhamnosus Bm01

Ochratoxin A (OTA) is a secondary fungal metabolite produced by Aspergillus and Penicillium spp. OTA contamination of food and animal feed has been attracting increasing concern in view of its toxicity, such as nephrotoxic, teratogenic, embryotoxic, genotoxic, neurotoxic, carcinogenic, and immunosuppressive effects. The widespread contamination of OTA in food and multiple toxicity of OTA have promoted research on detoxifying OTA in food or feed. The use of lactic acid bacteria (LAB) to remove OTA in food is a highly innovative and promising method compared with traditional methods. In this research, we screened an LAB strain, namely, Lactobacillus rhamnosus Bm01, which can remove 83.58% of OTA (50 ng/mL) within 48 h. The physiological mechanism of OTA removal by L. rhamnosus Bm01 was investigated. L. rhamnosus Bm01 removed OTA mainly by absorption through the cell wall. Furthermore, we applied L. rhamnosus Bm01 to remove OTA in grape juice or ready-to-drink coffee. Approximately 83.87% of 50 ng/mL and 100% of 20 ng/mL OTA in grape juice was removed at 48 h after incubation when treated with L. rhamnosus Bm01, respectively. Results indicated the potential of L. rhamnosus Bm01 in the decontamination of OTA in food industries.

Removal of Ochratoxin A from Red Wine Using Alginate-PVA-L. plantarum (APLP) Complexes: A Preliminary Study.

The presence of ochratoxin A (OTA) in wines is a problem mainly due to the health damage it can cause to frequent drinkers. A method for removing these toxic substances from wine is the use of lactic acid bacteria with mycotoxin-adsorption capacities; however, their use is limited since a matrix in which they can be immobilized, to remove them after use, is needed. In this study, L. plantarum (LP) was encapsulated in a polymeric matrix composed of polyvinyl alcohol (PVA) and alginate, forming alginate–PVA–LP (APLP) complexes. Then, these complexes were characterized, and assays of OTA and phenol removal from wines were performed. As a result, it was observed that the APLP complexes at a concentration of 0.5 g mL−1 removed over 50% of the OTA without substantially affecting the concentration of total phenols. In addition, it was determined that the presence of L. plantarum directly affected the ability to adsorb OTA from wines and did not decrease the total phenols. In conclusion, an alginate–PVA matrix allows immobilizing LP, and the complexes formed are an alternative for removing ochratoxin from contaminated wines.

Assessment of traditional clarifiers on the adsorption of ochratoxin A in Cabernet Sauvignon red wine and their kinetics

The effect of clarification on the elimination of Ochratoxin A (OTA) in wine was studied and the adsorption behavior of different clarifiers was evaluated. The results showed that OTA in wine can be effectively eliminated by gelatin with an adsorption rate of 28.59%, followed by chitosan (24.7%), bentonite (22.5%) and polyvinylpyrrolidone (PVPP) (7.6%). The clarification process was significantly affected by the clarifiers and OTA concentration. In addition, the experimental kinetic data for OTA removal were also evaluated by different equations. It displayed that the adsorption of gelatin and PVPP can be simulated by Pseudo-first order equation and Elovich equation, while that of chitosan and bentonite followed Pseudo-second order equation. The adsorption behavior of gelatin, chitosan and bentonite can fit Freundlich equation, Temkin equation and Dubinin-Radushkevich, and that of PVPP can only fitted by Langmuir equation. The thermodynamic parameters further revealed that the adsorption of OTA in wine was non-spontaneous.

Electrostatic adsorption and removal mechanism of ochratoxin A in wine via a positively charged nano-MgO microporous ceramic membrane

Ochratoxin A (OTA) is a very important mycotoxin. However, there are few studies on the removal of OTA in wine because of the great influence on product quality and difficulty in practical application. A nano-MgO-modified diatomite ceramic membrane (MCM) with a high positive charge was prepared and applied to remove OTA in wine. The isotherm adsorption between the positively charged membrane and OTA was in accordance with the Langmuir model, with a maximum adsorption capacity of 806 ng/g at 25 °C. All of the changes in adsorption enthalpy (ΔH), adsorption free energy (ΔG) and adsorption entropy (ΔS) were negative, which indicated that the combination of nano-MgO MCM and OTA was a spontaneous exothermic and nonspecific physical adsorption process. The concentrations of OTA in adsorption-treated wines were lower than 2 μg/kg, and the removal rates exceeded 92%. After OTA removal, the composition of wines was preserved to some extent.

Elimination of ochratoxin A from white and red wines: Critical characteristics of activated carbons and impact on wine quality

Ochratoxin A (OTA) is a mycotoxin frequently found in wines, with an allowed maximum limit of 2.0 μg/L. To optimise OTA removal from white and red wines at high levels (10.0 μg/L) using activated carbons (ACs) different commercial deodorising ACs were tested. OTA elimination from white wines was less dependent on ACs characteristics, with 100% removal with all but one AC with the lowest total pore volume. For red wines, only one of the ACs with higher abundance of mesopores was successful in completely removing OTA. This decrease in performance was due to the competition of anthocyanins for ACs mesopores. ACs treatment impacted positively on white wine chromatic characteristics, decreasing the yellowness-brownish colour but for the red wine it was observed a higher but limited impact (maximum 24% decrease) on wine colour. ACs pore volume distribution, with pore sizes in the range 42.6–55.9 Å and 125.6–137.4 Å were important for efficient OTA removal. These results will allow the rational design of ACs with pore volume characteristics for OTA elimination with less impact on wine colour. Therefore, it was shown for the first time that AC with appropriate pore size distribution can remove completely OTA from white and also red wines.

Effects of pulsed ultrasound at 20 kHz on the sonochemical degradation of mycotoxins

Mycotoxins, such as aflatoxin B1 (AFB1), deoxynivalenol (DON), zearalenone (ZEA) and ochratoxin A (OTA) are toxic secondary compounds that can reduce the quality of many kinds of food and may lead to other ill effects, both in humans and animals. This study aimed to investigate the potential of the ultrasound (US) treatment in the removal of AFB1, DON, ZEA and OTA from aqueous solution and maize by examining degradation rates and influencing factors of ultrasonication, such as the initial concentrations, power intensity, sonication duration, and duty cycle. The results showed that US treatment could simultaneously reduce AFB1, DON, ZEA and OTA effectively in aqueous solution. The degradation of mycotoxins was significantly affected by the ultrasonic intensity (2.2-11 W/cm3) and sonication time range from 10 to 50 min. DON is more stable than AFB1, ZEA, and OTA in the US treatment. It was found that, for the first time to our knowledge, the highest degradation rates of AFB1, DON, ZEA and OTA were attained at a duty cycle of 25%, and they were 96.5, 60.8, 95.9 and 91.6%, respectively. US strategy can be considered as an effective treatment to degrade the mycotoxins in aqueous solutions and food matrix.

Isolation and identification of a Bacillus megaterium strain with ochratoxin A removal ability and antifungal activity

Ochratoxin A (OTA) is a type of mycotoxin mainly produced by fungus belonging to the genus Aspergillus and Penicillium and plays a critical role in food safety since it causes serious health problem. Biological control by using microorganisms is considered a promising approach for detoxification of this mycotoxin. In order to find bacteria with efficient OTA elimination capability, one bacteria strain JSW-B1 with OTA removal ability was isolated from soil samples and was identified as Bacillus megaterium based on morphological and 16S rRNA analysis. After incubation of isolate JSW-B1 in liquid culture medium containing 2.5 μg/mL OTA for 72 h, the OTA removal percentage was 80.3%. No degradation products of OTA could be detected by liquid chromatography. About 62.6% OTA could be removed by the viable cell fraction after incubation for 72 h compared to 13.7% by cell-free supernatant. Meanwhile, heat treatment significantly enhanced OTA removing capability of the cells, suggesting that the OTA removal activity of the isolate JSW-B1 was mainly attributed to the adsorption by bacterial cells. In addition, the antifungal experiments revealed that isolate JSW-B1 was able to suppress the mycelial growth of Aspergillus ochraceus on solid medium with an inhibition ratio of 41.9% and reduce the fungal incidence on maize corns with a reduction ratio of 15%. Taken together, our results indicated that B. megaterium JSW-B1 could be considered as potential biological agent to control toxigenic fungal growth and mycotoxin contamination in agricultural products.

Removal of ochratoxin A by a carboxypeptidase and peptides present in liquid cultures ofBacillus subtilis CW14

Ochratoxin A (OTA) is an important mycotoxin that contaminates a variety of agricultural products. The cell-free supernatant ofBacillus subtilis CW14 liquid cultures were reported previously to be capable of removing OTA efficiently. In this work, we examined several substances that are probably involved in this removal of OTA usingin vitro experiments. The strain CW14 culture supernatant that was separated by ultrafiltration showed that the fractions collected at >10 kDa and <3 kDa had a significant ability to reduce OTA (84.9 and 74.8%, respectively) when incubated with 6 μg/ml OTA at 37 °C for 24 h. A putative metalloenzyme was responsible for the activity of the >10-kDa fraction, which was confirmed by the detrimental effects of heat treatments or addition of SDS, proteinase K, or EDTA. Subsequently, a carboxypeptidase (CP) gene that was likely related to the enzymatic conversion of OTA by the >10-kDa fraction was cloned from theB.subtilis CW14 genome, and over-expressed inEscherichia coli. The recombinant CP degraded 71.3% of OTA at 37 °C for 24 h, and ochratoxin α (OTα) was confirmed as a degradation product. From the <3-kDa fraction, some small peptides (1.7 kDa >Mw >0.7 kDa) were purified and decreased OTA by 45.0% under the same conditions, but no product was detected. These peptides were presumed to be capable of binding OTA due to their affinity with the OTA molecule, and the OTA-peptide complexes escaped from the extraction procedures for OTA quantification. These results indicated there was a probable synergistic effect that was involved in removal of OTA by the strain CW14 culture supernatant, which included enzymatic degradation by a CP and physical adsorption by some small peptides.

Anthocyanins enhance yeast’s adsorption of Ochratoxin A during the alcoholic fermentation

Ochratoxin A (OTA) is a mycotoxin that can be present in food products and beverages such as wine, in which the European Union has regulated its maximum concentration. Since yeasts can adsorb OTA during the alcoholic fermentation (AF), we have analyzed whether anthocyanins influence the reduction of OTA in wine during AF carried out by two Saccharomyces cerevisiae strains. Laboratory experiments were conducted with one white must and two red musts that were obtained by adding to the white must two different concentrations of anthocyanins extract. Musts were supplemented with OTA and tested at two pHs. A reduction of OTA was observed in all wines, being higher in red wines. These data point that a chemical bond between anthocyanins and OTA is probably responsible for enhanced adsorption of OTA by yeast. On the other hand, the removal of OTA between the two inoculated yeast strains was different. This result cannot be only attributed to differences in the yeasts’ cell wall composition, because differences were not detected in white wines.

Fermentation of table cream by Lactobacillus plantarum strains: effect on fungal growth, aflatoxin M1 and ochratoxin A

SummaryAflatoxin M1 (AFM1) and ochratoxin A (OTA) are two main mycotoxins in milk and dairy products. In the present work, the ability of four Lactobacillus strains (L. plantarum PTCC 1058, L. plantarum LP3, L. plantarum AF1 and L. plantarum LU5) to remove AFM1 and OTA in fermented cream was studied during 24 h fermentation. The antifungal activity of the mentioned lactobacilli against the defined fungi (Aspergillus flavus PTCC 5004, Aspergillus parasiticus PTCC 5018, Aspergillus nidulans PTCC 5014, Aspergillus ochraceus PTCC 5060) was also evaluated. The results showed that the cell counts of all strains were increased by 64–70% during fermentation. All Lactobacillus strains decreased the amount of AFM1 significantly (P ≤ 0.05) in the range of 26–52%, which the highest AFM1‐reducing effect was related to L. plantarum LU5 (from 0.5 to 0.24 μg kg−1). The mean OTA removal by Lactobacillus strains in fermented cream also ranged from 32 to 58%. Amongst Lactobacillus strains, the cell‐free culture supernatants of L. plantarum LU5 showed the highest (inhibition zone of 26.7 ± 1.2 mm) and L. plantarum LP3 and L. plantarum PTCC 1058 the lowest antifungal activities. The fermented creams contained Lactobacillus strains exhibited the highest and lowest antifungal activities against A. ochraceus and A. parasiticus, respectively. L. plantarum LU5, with the inhibition zone of 27.6 ± 0.9 mm, was the most effective fungal inhibitor, while L. plantarum PTCC 1058 had the lowest antifungal activity.

Isolation of lactic acid bacteria from grape fruit: antifungal activities, probiotic properties, and in vitro detoxification of ochratoxin A

The aim of this study was to isolate lactic acid bacteria from two varieties of grape bunch cultivated in Tunisia (cardinal and red-globe) in order to assess their ability to inhibit the growth of their most widespread contaminant, Aspergillus niger aggregats and Aspergillus carbonarius. Antifungal activity of 18 isolates was investigated using overlay technique; selected isolates were than identified using 16s rDNA sequence analysis. Isolates with antifungal activities were screened out and studied for their probiotic properties using in vitro tests (tolerance to simulated gastric jus, bile salts, hydrophobicity properties).The most efficient strain was also investigated for its ability to reduce the concentration of ochratoxin A (OTA) on liquid medium. Determination of OTA content in media was released using HPLC analysis. Selected strains (RG7B) (C11C) and (RG8A) showing a good antifungal activities against Aspergillus niger aggregats and Aspergillus carbonarius were identified as Pediococcus pentosaceus and Lactobacillus plantarum, respectively. Pediococcus pentosaceus (RG7B) showed promising potential probiotic characteristics and had a high ability for OTA removal after 48 h of incubation in both MRS and PBS media. The OTA removal percentage was significantly higher in MRS than in PBS media (84 and 25%, respectively). This study provides evidence for the control of black Aspergillus growth in grape and OTA detoxifying by the use of autochthones LAB strains having antifungal effect and probiotic potential.

Polymeric substances for the removal of ochratoxin A from red wine followed by computational modeling of the complexes formed

Ochratoxin A (OTA) is a mycotoxin produced by filamentous-type fungi that contaminates a wide variety of foods and beverages such as wines. In these trials, we evaluated the capacity of the following polymers for the removal of OTA from acidic model solutions and red wine: polyvinylpolypyrrolidone (PVPP), resin of N-vinyl-2-pyrrolidinone with ethylene glycol dimethacrylate and triallyl isocyanurate (PVP-DEGMA-TAIC), and poly(acrylamide-co-ethylene glycol-dimethacrylate) (PA-EGDMA). In acidic model solution, PVP-DEGMA-TAIC and PA-EGDMA polymers removed up to 99.9% of OTA, but their trapping capacity was highly reduced by the presence of competing phenolic substances (i.e. gallic acid and 4-methylcathecol). In real red wine, PA-EGDMA polymer showed the most promising results, with more than 68.0% OTA removal and less than 14.0% reduction in total phenolic. Finally, computational chemistry analyses showed that the affinity between OTA and the polymers studied would be due to Van der Waals interactions.

Removal of Ochratoxin A in Saccharomyces cerevisiae Liquid Cultures

The capacity for removal of ochratoxin A (OTA) during alcoholic fermentation was evaluated in batch systems with one commercial strain and one wild strain of Saccharomyces cerevisiae. Batch alcoholic fermentations were carried out in yeast extract-malt extract broth (YM) medium, with 18.0% glucose and OTA added to final concentrations of 3.48 and 4.95 ng/mL respectively. The removal capacity of each yeast strain was examined after completion of fermentation in batch culture and after extended contact with yeast biomass. The removal capacity of the yeast strains was also examined in stationary phase cultures. Stationary phase yeasts were studied with biomass harvested from the stationary phase of anaerobic fermentation, by incubation in phosphate buffer, with the addition of 5.00 ng/ mL of OTA. Removal studies with stationary phase cells were performed with viable and non-viable cells inactivated with Na-azide. The study showed that in growing phase cultures, OTA removal was significant only after extended contact with yeast biomass; up to 29.7% and 25.4% for wild yeast ZIM 1927 and commercial yeast Lalvin EC-1118 respectively, but not during alcoholic fermentation. In stationary phase cultures, viable and non-viable cells were not significantly different in OTA removal from the medium. This demonstrated that OTA was not metabolised, but possibly adsorbed by the yeast cells. The presence of OTA in synthetic media influenced yeast metabolism, causing the production of higher volatile acidity by 0.08 and 0.13 g/L for Lalvin EC-1118 and ZIM 1927 respectively, and lower concentrations of reducing sugar, by 0.32 g/L, but only for ZIM 1927.