Occurrence of ochratoxin A-producing fungi in raw Brazilian coffee

Ochratoxin A (OA)-producing fungi were identified in coffee at different stages of maturation. The toxin was quantified in coffee during terrace drying and in coffee stored in barns. By direct plating, a high level of contamination (100%) was found in the coffee beans studied, with the genus Aspergillus representing 33.2%, of which Aspergillus ochraceus and Aspergillus niger represented 10.3 and 22.9%, respectively, of the strains isolated from the coffee beans. The capacity to produce ochratoxin was determined in 155 strains of A. ochraceus and A. niger using both the agar plug method and extraction with chloroform, giving positive results for 88.1% of the A. ochraceus strains and 11.5% of the A. niger strains. Analysis for OA in the terrace and barn coffee samples showed that, independent of cultivar, year harvested, or production region, all except one of the samples analyzed showed mycotoxin levels below the limit suggested by the European Common Market (8 microg/kg), thus indicating that the problem is restricted and due to severe faults in harvesting and storage practices.     

Immunoaffinity column clean-up and thin layer chromatography for determination of ochratoxin A in green coffee.

An immunoaffinity clean-up-based method for determining ochratoxin A (OTA) in green coffee aiming at one-dimensional thin layer chromatography (TLC) analysis was established. OTA was extracted with a mixture of methanol and aqueous sodium hydrogen carbonate solution, purified through an immunoaffinity column, separated on normal or reversed-phase (RP) TLC plates and detected and quantified by visual and densitometric analysis. The linear equation of the standard calibration curve by densitometric analysis gave R(2) > 0.999 (0.04-84 ng). The mean recovery (R) of OTA from spiked samples (1.8-109 microg kg(-1)) by densitometric and visual analyses were 98.4 and 103.8%, respectively. The relative standard deviations (RSD) for densitometric and visual analysis varied from 1.1 to 24.9% and from 0.0 to 18.8%, respectively. The RSD for naturally contaminated samples by densitometry (three levels of contamination, n = 3) varied from 11.1 to 18.1%. The correlation (R(2)) between high-performance liquid chromatography (HPLC) and densitometry, and between visual and densitometric analysis for spiked samples were > 0.99. The limit of detection (LOD) of the method was 0.5 microg kg(-1) for normal TLC. Toluene-ethyl acetate-88% formic acid (6:3:1 v/v/v) and acetonitrile-methanol-water-glacial acetic acid (35:35:29:10 v/v/v/v) were regarded as the suitable TLC solvents for eluting both standards and samples on normal and RP TLC plates, respectively. Toluene-acetic acid (99:1 v/v) was chosen as the spotting solvent among several others for giving the best sensitivity and resolution of OTA on TLC plates as well as the best recovery of OTA from standard and sample extract residues. Preliminary studies were carried out to investigate the reuse of the immunoaffinity column and the interference of caffeine in the OTA recovery.     

The impact of roasting on the ochratoxin A content of coffee

Roasting coffee led to a drop in the ochratoxin A (OTA) concentration, as measured by the reference method, especially for dark type roasts. The way the beverage was prepared also affected the OTA content, which could paradoxically be higher than that of the initial roasted coffee. Assays on the thermal stability of pure OTA showed that it ought to be found in larger quantities in roasted coffee. This suggested that OTA was masked by reactions with the substrate during roasting. The absence of OTA in green coffee is therefore the best guarantee of safety.     

Two-dimensional thin-layer chromatographic method for the analysis of ochratoxin A in green coffee

A low-cost thin-layer chromatographic method has been developed for the presumptive measurement of ochratoxin A (OTA) at 5 microg/kg in green coffee beans. The analytical method consisted of extracting OTA by shaking the beans with a mixture of methanol and aqueous sodium bicarbonate solution, which was then purified by liquid-liquid partition into toluene. OTA was separated by normal-phase two-dimensional thin-layer chromatography and detected by visual estimation of fluorescence intensity under a UV lamp at 365 nm. The chromatography solvents were toluene-methanol-formic acid (8:2:0.03) for the first development and petroleum ether-ethyl acetate-formic acid (8:10:1) for the second dimension development. This method was tested with uncontaminated green coffee bean samples spiked with an OTA standard at four different concentrations (5, 10, 20, and 30 microg/kg). The method is rapid, simple, and very easy to implement in coffee-producing countries. It is highly selective and does not involve the use of chlorinated solvents in the sample extraction step. This inexpensive method has been applied to different types of green coffee samples from various countries (Zimbabwe, Brazil, India, Uganda, Colombia, and Indonesia) and different manufacturers, and no OTA below the detection limit of 5 microg/kg was detected in any samples analyzed.     

Production of ochratoxin A by Aspergillus carbonarius on coffee cherries

Robusta coffee cherries collected before and during sun drying from two coffee farms in Thailand were examined for moulds producing ochratoxin A (OA). Aspergillus ochraceus was only detected in one sample, whereas Aspergillus carbonarius was isolated from 7 out of 14 samples. On gamma-irradiated coffee cherries, each of the six tested A. carbonarius strains produced OA. More than 4800 microg kg(-1) of toxin were detected under optimal conditions (25 degrees C, a(w) 0.99). OA production was strongly reduced (230 microg kg(-1)) at an a(w) of 0.94.     

Effect of operating conditions on ochratoxin A extraction from roasted coffee

Operating conditions affect ochratoxin A (OTA) extraction from roasted coffee. The OTA content found in the beverage can thus be greater than that found in the roasted coffee used to prepare it. Three extraction parameters were studied for roasted coffee: type of extraction solvent (alkaline, neutral, acid), temperature (ambient temperature/23°C, 60°C and 85°C), and extraction time (5, 20, 30, 40, 50, 60 and 80 min). The alkaline solvent used in the method recommended by the European Union extracted OTA better, but a maximum content was obtained at 60°C after 50 min. At least a 100% improvement in extraction was obtained when compared with the European Union usual quantification method that is carried out at ambient temperature. It turned out that the OTA extraction parameters for roasted coffee, as defined by that method, were not optimum and needed to be modified. These results were verified in double-extraction experiments showing that OTA is not completely extracted by this method. Confirmation was obtained by comparison of extraction methods on several commercial samples of roasted coffee.     

Influence of roasting and brew preparation on the ochratoxin A content in coffee infusion

A study of the effect of coffee processing in the ochratoxin A (OTA) level has been carried out from the green beans to the drinking form. The analysis of OTA has been carried out by an in-house validated HPLC method with fluorescence detection. The limits of detection were 0.04 µg/kg for green and roasted coffee, and 0.01 µg/L for coffee brew. Thirty-six green coffee samples of different origin (Colombia, Costa Rica, Brazil, Vietnam, India and Uganda) were analysed. The highest concentrations of OTA were found in Vietnamese samples - Robusta species treated by dry processing - (range 0.64-8.05 µg/kg), that also showed the highest percentage of defective beans (7.6%). These contaminated samples were roasted in a process that controlled loss of weight and color, as in the industry. A mean reduction of 66.5% was obtained, but the reduction seems to be heterogeneous. Coffee brew was prepared by the three brewing processes more utilized in Europe: moka, auto-drip and espresso. A reduction of the OTA level has been attained, being greater when using a espresso coffee maker (49.8%) than when using auto-drip (14.5%) or moka brewing (32.1%). Therefore, the method of coffee brew preparation plays a key role in the final OTA human exposure.     

Effect of post-harvest treatments on the occurrence of ochratoxin A in raw cocoa beans

Cocoa beans are the principal raw material for chocolate manufacture. Moulds have an important place in the change in the quality of cocoa beans due to their role in the production of free fatty acids and mycotoxins, namely ochratoxin A (OTA). This study investigated the impact of the key post-harvest treatments, namely the fermentation and drying methods on OTA contamination of raw cocoa beans. Analytical methods for OTA detection were based on solid–liquid extraction, clean-up using an immunoaffinity column, and identification by reversed-phase HPLC with fluorescence detection. Of a total of 104 randomly selected cocoa samples analysed, 32% had OTA contents above 2 µg kg^–1. Cocoa sourced from pods in a bad state of health had a maximum OTA content of 39.2 µg kg^–1, while that obtained from healthy pods recorded 11.2 µg kg^–1. The production of OTA in cocoa beans increased according to the pod-opening delay and reached 39.2 µg kg^–1 after an opening delay of 7 days after harvest, while 6.1 and 11.2 µg kg^–1 were observed when pods were opened after 0 and 4 days. OTA production also seemed to depend considerably to the cocoa fermentation materials. When using plastic boxes for bean fermentation, the OTA production was enhanced and reached an average OTA content of about 4.9 µg kg^–1, while the raw cocoa treated in banana leaves and wooden boxes recorded 1.6 and 2.2 µg kg^–1 on average respectively. In parallel, the OTA production was not really influenced by either the mixing or the duration of the fermentation or the drying materials.     

Study of ochratoxin A-producing strains in coffee processing

Ochratoxin A (OTA) is the main mycotoxin that has been detected in coffee. The occurrence of OTA in coffee beans can be because of environmental conditions and/or processing conditions. Three coffee processes were evaluated (wet, mechanical and dry processes), at different stages from harvesting to storage, and fungi producing OTA were enumerated and identified. The frequency of potential OTA‐producing fungi and their ability to produce OTA was also studied. By direct plating, the levels of contamination found in the coffee processes were 80, 72 and 92%, respectively, for parchment and dry cherry coffee and 20, 34 and 15% for green coffee. Aspergillus ochraceus isolated from the three processes accounted for 6.6, 8.3 and 3.3%, and Aspergillus niger for 15, 13 and 25% of the strains isolated, respectively. The toxigenic potential of five A. ochraceus and two A. niger strains was tested in FDA medium and coffee medium using the HPLC technique. There was no difference between the processes studied in terms of isolation and occurrence of ochratoxigenic fungi.     

Design and implementation of an integrated management system for ochratoxin A in the coffee production chain

Coffee is an important export product of Ecuador. Producers are challenged by the implementation of regulatory limits for ochratoxin A. Ecuador has four coffee production areas and the potential for mycotoxin contamination varies due to different environmental conditions and cultural differences in harvesting, storage, processing and commercialization. The major contributors to contamination are the lack of selection during harvesting, delays in drying or rewetting, the lack of proper drying and storage conditions, the mixing of products with different levels of moisture, and the potential for cross-contamination. The long commercialization chain involves different intermediaries that use foreign materials to increase the weight of the product without consideration of quality. An integrated mycotoxin management system using the Hazard Analysis Critical Control Point Systems (HACCP) principles was developed to prevent mycotoxin contamination at each stage of production. Critical control points were developed based on the resources available at the different stages of the production chain. Training programmes helped increase awareness about the impact of contamination, but failed to transform knowledge into improved practices. Thus, different demonstrative models specific for each productive region at all production levels were developed to show the application of prevention mechanisms using limited resources and to demonstrate the increased commercial value of coffee produced using good practices throughout the chain so producers have a better disposition to adopt improved practices. Preliminary results show that coffee managed using the models had a better quality, a lower contamination, a higher yield and better commercial value. The use of local resources and low-cost technology was important in demonstrating the practical approach.     

A study of the contamination by ochratoxin A of green and roasted coffee beans

A study was performed to evaluate the contamination by ochratoxin A in coffee beans. Twenty-nine samples of green coffee were collected from large lots of material by representative sampling. The analyses of green coffee samples showed a significantly high contamination percentage (58%) ranging from 0.2 to 15 micrograms/kg. Naturally and artificially contaminated samples were roasted at different operation times (5-6 min) to verify the percentage of destruction of the mycotoxin. The percentage ranged from 48% to 87% and from 90% to 100% in artificially and naturally contaminated samples respectively. The beverages prepared from artificially contaminated coffee using the most common types of coffee makers showed no residues of ochratoxin A.     

Design of a sampling plan to detect ochratoxin A in green coffee

The establishment of maximum limits for ochratoxin A (OTA) in coffee by importing countries requires that coffee-producing countries develop scientifically based sampling plans to assess OTA contents in lots of green coffee before coffee enters the market thus reducing consumer exposure to OTA, minimizing the number of lots rejected, and reducing financial loss for producing countries. A study was carried out to design an official sampling plan to determine OTA in green coffee produced in Brazil. Twenty-five lots of green coffee (type 7 - approximately 160 defects) were sampled according to an experimental protocol where 16 test samples were taken from each lot (total of 16 kg) resulting in a total of 800 OTA analyses. The total, sampling, sample preparation, and analytical variances were 10.75 (CV = 65.6%), 7.80 (CV = 55.8%), 2.84 (CV = 33.7%), and 0.11 (CV = 6.6%), respectively, assuming a regulatory limit of 5 µg kg^-1 OTA and using a 1 kg sample, Romer RAS mill, 25 g sub-samples, and high performance liquid chromatography. The observed OTA distribution among the 16 OTA sample results was compared to several theoretical distributions. The 2 parameter-log normal distribution was selected to model OTA test results for green coffee as it gave the best fit across all 25 lot distributions. Specific computer software was developed using the variance and distribution information to predict the probability of accepting or rejecting coffee lots at specific OTA concentrations. The acceptation probability was used to compute an operating characteristic (OC) curve specific to a sampling plan design. The OC curve was used to predict the rejection of good lots (sellers' or exporters' risk) and the acceptance of bad lots (buyers' or importers' risk).     

一种测定酿酒葡萄中赭曲霉毒素A的新方法

建立了一种新的测定酿酒葡萄中赭曲霉毒素A含量的方法——"葡萄皮-SPE-HPLC"法:将酿酒葡萄剥皮后用0.1 mol/L磷酸-二氯甲烷(1∶10)提取;取10 mL提取液进样固相萃取小柱,0.1 mol/L磷酸、双蒸水淋洗,乙酸乙酯洗脱后吹干,流动相溶解;高效液相色谱法测定条件为C18反相柱分离,乙腈-水-乙酸(体积比99∶99∶2)为流动相,荧光检测器(激发波长330 nm,发射波长460 nm)检测。该方法回收率为102%¹09%,变异系数为0.1%109%,变异系数为0.1%².0%;用此方法检测贺兰山东麓"玉泉营"和"万义山庄"两个产地酿酒葡萄中OTA含量,结果介于52.0%;用此方法检测贺兰山东麓"玉泉营"和"万义山庄"两个产地酿酒葡萄中OTA含量,结果介于5¹0μg/kg,均有一定程度赭曲霉毒素A污染。     

Enzyme-assisted extraction for the HPLC determination of ochratoxin A in pork and dry-cured ham

The extraction of ochratoxin A from meat products is generally carried out using chlorinated organic solvents, such as chloroform or methyl chloride, acidified with hydrochloric or o-phosphoric acid. In this study, an innovative method was developed to extract ochratoxin A from pork and dry-cured ham samples. The method was based on an enzyme-assisted extraction with pancreatin in phosphate buffer pH 7.5. Pancreatin hydrolyses the proteins, so that ochratoxin A, kept in the ionised form, is easily extracted by the aqueous solution. After purification through an immunoaffinity column, ochratoxin A is determined by HPLC with fluorescence detection. The average recovery values were higher than 90.0% and the relative standard deviations were below 5.5%. The limits of detection and of quantification were 0.06 and 0.12 μg kg(-1), respectively. A comparison between the new enzyme-assisted extraction and an established chloroform method was carried out on six naturally contaminated samples of pork and on 40 samples of dry-cured ham. Significantly higher (p<0.001) values of ochratoxin A were obtained on dry-cured ham samples by the enzyme-assisted method.     

Enzyme-assisted extraction for the HPLC determination of ochratoxin A in pork and dry-cured ham

The extraction of ochratoxin A from meat products is generally carried out using chlorinated organic solvents, such as chloroform or methyl chloride, acidified with hydrochloric or o-phosphoric acid. In this study, an innovative method was developed to extract ochratoxin A from pork and dry-cured ham samples. The method was based on an enzyme-assisted extraction with pancreatin in phosphate buffer pH 7.5. Pancreatin hydrolyses the proteins, so that ochratoxin A, kept in the ionised form, is easily extracted by the aqueous solution. After purification through an immunoaffinity column, ochratoxin A is determined by HPLC with fluorescence detection. The average recovery values were higher than 90.0% and the relative standard deviations were below 5.5%. The limits of detection and of quantification were 0.06 and 0.12?µg?kg^?1, respectively. A comparison between the new enzyme-assisted extraction and an established chloroform method was carried out on six naturally contaminated samples of pork and on 40 samples of dry-cured ham. Significantly higher (p?<?0.001) values of ochratoxin A were obtained on dry-cured ham samples by the enzyme-assisted method.     

Research on the origin, and on the impact of post-harvest handling and manufacturing on the presence of ochratoxin A in coffee

The major risk factors and processing steps that can lead to contamination of green coffee with ochratoxin A (OTA) have been identified. Surveys of the green coffee production chain indicate that Aspergillus ochraceus and A. carbonarius are the most potent OTA producers on coffee. Both have been successfully grown in vitro on green coffee and coffee cherries, respectively, producing high amounts of OTA (5-13 mg kg -1 ). The so-called dry processing of coffee, which is cherry drying, was identified as one of the steps during which OTA formation can take place, particularly under humid tropical conditions. Cherries contain sufficient amounts of water to support mould growth and OTA formation during the initial 3-5 days of drying on the outer part of the cherries. Not surprisingly, after dehulling, husks can be highly contaminated with OTA, as also indicated by its enhanced concentration in soluble coffees adulterated with husks and parchment. A minimum water activity of 0.80 (about 14% MC) is required for in vitro OTA production on green coffee, a fact that does not rule out the possibility of OTA contamination due to improper transportation and storage of green coffee. However, this appears not to be a major route for OTA contamination of coffee. OTA contamination can clearly be minimized by following good agricultural practice and a subsequent post-harvest handling consisting of appropriate techniques for drying, grading, transportation and storage of green coffee; these procedures are well established.     

Incidence of microflora and of ochratoxin A in green coffee beans (Coffea arabica).

Coffee is produced in tropical countries around the Equator where climatic conditions are favourable for fungal development and mycotoxin production; however, mycotoxins do not only occur in the tropical countries. The aim was to evaluate the mycoflora and possible incidence of ochratoxin A (OTA) in 60 samples of green coffee beans from Brazil. The mycological evaluation was carried out using a conventional method and the OTA was determined using sequential phenyl silane and immunoaffinity column cleanup followed by HPLC. The detection limit was 0.2 microg kg(-1). Practically all samples (91.7%) were contaminated with moulds. The dominant fungal genus was Aspergillus, including A. niger (83.3%), A. ochraceus (53.3%) and A. flavus (25.0%). The occurrence and the levels of the genus Cladosporium (16.6%) and Penicillium (10.0%) were substantially lower than Aspergilli. Twenty samples (33.3%) of 60 were contaminated with the toxin at levels ranging from 0.2 to 7.3microg kg(-1). The average concentration was 2.38 microg kg(-1). All positive samples showed OTA levels below the limit suggested by the European Union (8 microg kg(-1)).     

Metabolic fate of ochratoxin A as a coffee contaminant in a dynamic simulator of the human colon

Ochratoxin A (OTA) is a mycotoxin frequently encountered in coffee. The relevance of this contaminant in the colon upon digestion necessitates a study on its interaction with colon microbiota. Here, the fate of OTA during colon digestion was investigated using a dynamic simulator of the human gut. The influence of coffee as a food matrix was taken into account, as it may affect the colonic microbial ecosystem and, consequently, the fate of OTA. Biodegradation was followed by measuring OTA concentration over time, and by screening for several possible metabolites, using LC–ESI-MS and HRMS. The descending colon was found to be the main site of OTA biodegradation. Two metabolites, ochratoxin α and ochratoxin B, were identified, suggesting that biodegradation by gut microbiota is beneficial for the host, as they are considered less toxic than OTA. The extent of biodegradation was reduced in the presence of the coffee matrix, possibly due to competition for available carbon sources. Effects of OTA and the coffee matrix on the microbial ecosystem were contrasting. While OTA caused a specific, but lasting loss, of the beneficial species Lactobacillus reuteri, coffee temporarily altered the fermentation pattern towards lower ammonia and higher acetate and propionate production, likely due to its dietary fibre content.