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.     

Stability of ochratoxin A (OTA) during processing and decaffeination in commercial roasted coffee beans

The fate of ochratoxin A (OTA) during the processing of artificially contaminated green coffee beans, the effect of decaffeination on the production of OTA in green and roasted coffee beans, and the effect of caffeine on the growth and OTA production by Aspergillus ochraceus were studied. The data indicated that the roasting, milling and decoction (brewing and Turkish coffee making) processes caused different percentage reductions in OTA. Decaffeinated samples showed a significantly higher concentration of OTA production than the caffeinated ones. A significantly higher percentage of OTA was reduced when the decaffeination process was performed before roasting treatment. Caffeine at 1.0 and 2.0% concentrations completely prevented OTA production and completely inhibited A. ochraceus growth in YES medium after 3-21 days.     

Influence of roasting and different brewing processes on the ochratoxin A content in coffee determined by high-performance liquid chromatography-fluorescence detection (HPLC-FLD)

A rapid and reliable procedure has been developed for the determination of ochratoxin A (OTA) in green and roasted coffee. The method consists of extraction of the sample with methanol–5% aqueous sodium hydrogen carbonate/1% PEG8000 (20:80), followed by immunoaffinity column (IAC) clean-up and, finally, high-performance liquid chromatography (HPLC) determination with fluorimetric detection. Mean recoveries for green and roasted coffee spiked at different levels ranging from 94 and 105% were obtained. The limit of determination (S/N = 3) was 0.032 ng g^?1 and the precision (within-laboratory relative standard deviation) was 6%. The method described has been used to assess the influence of roasting and different brewing processes on OTA content in commercial lots of green and roasted coffee. The results provided evidence that roasting led to a significant drop on OTA levels (65–100%). Also, the way coffee is prepared affects the OTA content: brewing using a Moka Express (Italian coffee) led to a significant reduction of OTA concentration (50–75%) since hot water stays in contact with coffee for a short time. On the contrary, Turkish coffee-making (infusion for about 10 min) cause poor reduction in OTA.     

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.     

Post-harvest practices linked with ochratoxin A contamination of coffee in three provinces of Cordillera Administrative Region,Philippines

One of the emerging concerns in the Cordillera Administrative Region, Philippines is ochratoxin A (OTA) contamination in coffee. During 2015 to 2016, a total of 51 Arabica (Coffea arabica) coffee samples from Benguet province and 71 Robusta (Coffea canephora var. Robusta) coffee samples from the provinces of Ifugao and Kalinga were analysed for OTA contamination. The OTA-producing fungal contaminants during drying and storage of Arabica and Robusta coffee were Aspergillus niger and Aspergillus ochraceus. Ochratoxin A was more commonly detected in Robusta coffee (36.6%) than in Arabica coffee (21.6%). Among the contaminated samples, Robusta coffee cherries in the drying yard had the highest mean OTA level (120.2 μg kg^?1, n = 10) while roasted Robusta coffee beans had the lowest mean level (4.8 μg kg^?1, n = 9). The onset of contamination of Arabica coffee occurred during storage, with a mean OTA level of 46.7 μg kg^?1 (n = 9). Roasted coffee had lower OTA content although five samples had levels >5.0 μg kg^?1. Pearson Chi-square analysis (χ²) and Fisher’s exact test revealed that several post-harvest practices involving non-removal of the husk or hull and mixing of defective coffee were significantly associated with the occurrence of OTA during drying and storage (p < 0.05). No significant associations, however, were identified during roasting. This study suggests that the post-harvest practices in Cordillera Administrative Region should focus on the removal of defective coffee in all stages of post-harvest and rapid reduction of moisture content particularly during drying.     

Inactivation of A. ochraceus spores and detoxification of ochratoxin A in coffee beans by gamma irradiation

Ochratoxin A (OTA) produced in food by Aspergillus ochraceus is known to cause adverse health effects. Among the plantation products, green coffee beans are prone to fungal attack and get contaminated with OTA frequently. A fungal strain isolated from green coffee beans was characterized by morphological analyses as well as internal transcribed spacer (ITS) and 5.8S rDNA sequencing, turned out to be A. ochraceus, however, nontoxigenic. Hence, additional strains of A. ochraceus were procured and characterized for toxin production. Presterilized green coffee beans were spiked with a toxigenic strain and treated with gamma radiation. Minimum inhibitory dose (MID) of gamma radiation for 10(4) and 10(8) spores of A. ochraceus strain per 10 g of green coffee beans was found to be approximately 1 and approximately 2.5 kGy, respectively. The radiation treatment (10 kGy) almost degraded the preformed or in vitro added OTA (50 ppb) in coffee beans. OTA degradation was found to be enhanced with increase in moisture content. Cytotoxicity in terms of cell viability was found to be reduced significantly for radiation treated OTA in MTT [3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide] assay as well as flow cytometric analysis when studied using human intestinal epithelial (Int-407) cells. Similar finding was also observed with E. coli MG1655 cells. Thus the inclusion of gamma radiation treatment in the postharvest processing chain of green coffee beans could help in eliminating toxigenic fungi as well as destroying preformed OTA without affecting the sensory attributes. PRACTICAL APPLICATION: In general, mycotoxins including ochratoxin A (OTA) are highly stable to detoxifying agents. Green coffee beans are prone to fungal attack and could get frequently contaminated with the OTA due to improper drying or rehydration during storage. Gamma radiation processing of green coffee beans was found to eliminate the A. ochraceus spores as well as inactivate OTA without affecting its sensory attributes. Thus inclusion of gamma radiation in the postharvest processing chain of green coffee beans would be very useful for consumer safety and coffee trade.     

Ochratoxin A (OTA) in coffee: nation-wide evaluation of data collected by German Food Control 1995-1999

The evaluation process involved data collected by Official Food Control Laboratories during the period 1995 until 1999. A total of 613 samples analysed for ochratoxin A and complying with a detection limit lower than 0.6 microg/kg were evaluated. With the assistance of statistical process analysis the median concentrations for green coffee (0.4 microg/kg), for roasted coffee (0.6 microg/kg), for decaffeinated roasted coffee along with low-acid decaffeinated roasted coffee (0.4 microg/kg) as well as for soluble coffee (0.7 microg/kg) were determined. The result is a mean daily total intake per consumer of 9 ng OTA.     

谷物类食品中赭曲霉毒素A分析方法的研究进展

赭曲霉毒素A是由真菌产生的次级代谢产物,由于其具有极强的肾脏毒性、致癌性、致畸性,且对食品污染的范围广泛,许多国家对其制定了严格的限量标准。目前对谷物中赭曲霉毒素A的检测方法主要有薄层色谱分析法、高效液相色谱法、酶联免疫吸附法、毛细管电泳法和胶体金试纸条法等。随着检测水平的提高,对食品中的赭曲霉毒素A限量标准的要求也在不断地提高。建立安全、准确、灵敏度高、重现性好的检测体系将成为今后研究的重点。     

Influence of roasting and different brewing processes on the ochratoxin A content in coffee determined by high-performance liquid chromatography-fluorescence detection (HPLC-FLD)

A rapid and reliable procedure has been developed for the determination of ochratoxin A (OTA) in green and roasted coffee. The method consists of extraction of the sample with methanol-5% aqueous sodium hydrogen carbonate/1% PEG8000 (20:80), followed by immunoaffinity column (IAC) clean-up and, finally, high-performance liquid chromatography (HPLC) determination with fluorimetric detection. Mean recoveries for green and roasted coffee spiked at different levels ranging from 94 and 105% were obtained. The limit of determination (S/N = 3) was 0.032 ng g(-1) and the precision (within-laboratory relative standard deviation) was 6%. The method described has been used to assess the influence of roasting and different brewing processes on OTA content in commercial lots of green and roasted coffee. The results provided evidence that roasting led to a significant drop on OTA levels (65-100%). Also, the way coffee is prepared affects the OTA content: brewing using a Moka Express (Italian coffee) led to a significant reduction of OTA concentration (50-75%) since hot water stays in contact with coffee for a short time. On the contrary, Turkish coffee-making (infusion for about 10 min) cause poor reduction in OTA.     

食品中赭曲霉毒素A检测方法研究进展

赭曲霉毒素A(ochratoxin A, OTA)是曲霉属和青霉属等有毒真菌产生的一类次级代谢产物,是常见污染食品的五大真菌毒素之一,具有较强的肾毒性、肝毒性、神经毒性和免疫毒性,以及致畸、致癌和致突变作用。OTA广泛存在于各种谷物及其制品、葡萄与葡萄酒、咖啡等多种食品原料及其成品中,严重威胁人体健康,因此有必要建立快速、准确、灵敏的OTA检测方法。针对食品中OTA的检测,目前已经拥有许多方法,如薄层色谱法,高效液相色谱法,液相-质谱联用法以及酶联免疫吸附法等。本研究对赭曲霉毒素A不同检测方法的原理、优缺点等进行归纳总结,旨在为食品中OTA的检测提供支持。     

Ochratoxin A contamination of coffee batches from Kenya in relation to cultivation methods and post-harvest processing treatments

This study set out to assess the relative importance of sound and unsound beans in a batch of coffee with regard to ochratoxin A (OTA) contamination. Initially, unsound beans were found to account for 95% of contamination in a batch of coffee, whatever the methods used for post–harvest processing. It was also found that beans displaying traces of attacks by Colletotrichum kahawae were the greatest contributors to OTA contamination. In a second stage, the study compared the contamination of sound beans with that of beans attacked by Colletotrichum kahawae. On average, beans attacked by Colletotrichum kahawae had a statistically higher OTA content than sound beans (18.0 µg kg^?1 as opposed to 1.2 µg kg^?1). In addition, the average OTA content in unsound beans varied depending on growing conditions.     

Ecophysiology of ochratoxigenic Aspergillus ochraceus and Penicillium verrucosum isolates. Predictive models for fungal spoilage prevention - a review

Ochratoxin A (OTA) is a secondary metabolite produced by several species of Aspergillus and Penicillium; among them Aspergillus ochraceus and Penicillium verrucosum are two ochratoxigenic species capable of growing in different climates and thus contamination of food crops with OTA can occur worldwide. OTA can be found in a wide range of foods such as cereals, coffee, cocoa, spices, beer, wine, dried vine fruit, grapes and meat products. OTA is toxic to animals, it presents neurotoxic, immunotoxic and nephrotoxic effects. It has been implicated in a human kidney disorder known as Balkan Endemic Nephropathy. This review focuses on the ecophysiology of ochratoxin-producing Aspergillus ochraceus and Penicillium verrucosum, the effect of environmental factors on their germination, mycelial growth, and OTA production. Knowledge of environmental conditions required for sucessive stages of fungal development represent the first step towards preventing mycotoxin formation. Predictive models for different stages of fungal development are presented, which allow prediction of the time before spoilage as a function of the abiotic factors. Finally, the implications of these studies in management of barley, coffee and grapes are described. This can help to identify the critical control points in their production, storage and distribution processes.     

Performance of new clean-up column for the determination of ochratoxin A in cereals and foodstuffs by HPLC-FLD

The performance of the newly developed Mycosep® 229 Ochra and Multisep® 229 Ochra clean-up columns for ochratoxin A (OTA) determination was evaluated. OTA was subsequently analysed using RP-HPLC with fluorescence detection. Recoveries for frequently contaminated commodities, like cereals, red wine, raisins and green coffee, were estimated. The recoveries obtained for the Mycosep 229 Ochra column were in the range from 87.9 ± 12.5% (n = 6) for wheat to 99.4 ± 2.7% (n = 24) for raisins. For Multisep 229 Ochra, recoveries from 76.5 ± 8.0% (n = 6) for barley to 86.4 ± 1.4% (n = 24) for raisins were achieved. Limits of detection for all matrices investigated (maize, wheat, rice, barley, raisins, green coffee beans, red wine) were in the range 0.4-2.4 μg kg^-1. The trueness of the method was tested using a certified reference material.     

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.     

新疆葡萄酒中赭曲霉毒素A含量分析

赭曲霉毒素A（OTA）是葡萄及其深加工产品中主要的真菌毒素,同时也被国际癌症研究机构（IARC）定为2B类致癌物。 采用 酶联免疫法（ELISA）商业试剂盒对新疆四大产区葡萄酒中OTA含量进行调研分析,研究不同产区葡萄酒中OTA含量的差异;同时, 对不同葡萄品种酿造葡萄酒过程中发酵葡萄汁和葡萄酒的OTA含量进行测定,分析酿造过程中OTA的变化规律。 结果显示,34份葡 萄酒样品中OTA含量均未超过2 μg/L;其中,焉耆盆地和吐哈盆地葡萄酒中OTA含量较低,平均含量分别为0.19 μg/L、0.20 μg/L;在 白葡萄酒酿造过程中OTA含量呈显著的下降趋势,而红葡萄酒酿造中OTA含量呈先升高后降低的趋势。     

赭曲霉毒素A单克隆抗体免疫亲和柱的制备

制备了赭曲霉毒素A(OTA)单克隆抗体免疫亲和柱,并用间接竞争ELISA和HPLC法评价了免疫亲和柱的性能,其柱容量(结合OTA的能力)约为200ng,加标回收率为90.38%～100.1%,可反复使用3次。 建立了免疫亲和柱-HPLC联用分析谷物中OTA的方法,最低检出限为0.2μg/kg,线性范围为0.6～400μg/kg,OTA加标量为1～10μg/kg时谷物样品中的回收率为78.7%～87.1%,变异系数小于6.5%。用此法检测了大米、小麦、玉米和玉米饲料等15份市售样品,检出率为46.7%,其中OTA的最高含量为0.785μg/kg。     

Investigation of ochratoxin a, B and citrinin contamination in various commercial foods

Ochratoxin A (OTA), ochratoxin B (OTB) and citrinin (CIT) in commercial foods were simultaneously determined and confirmed with high-performance liquid chromatography (HPLC) and liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). The samples examined were made up of cereal, fruit, coffee, and cacao products. The limits of quantification (S/N> or =10) of OTA, OTB and CIT were 0.1 microg/kg or less. Aflatoxins (AF), deoxynivalenol (DON) and fumonisins were also surveyed. Of 157 samples examined, 44 were contaminated with OTA at levels of 0.11 to 4.0 microg/kg. At least 2 positive samples were labeled as domestics. In most positive samples, the OTA level was low, less than 1 microg/kg. The highest incidence of OTA was observed in cacao powder (10/12), followed by instant coffee (5/7), cocoa (5/8) and raisin (6/13). OTB was found in fruit and cacao products containing relatively high levels of OTA. Co-occurrence of OTA, CIT and DON was found in cereal products, and co-occurrence of OTA and AF was found in cacao products. Approximately 30% of naturally contaminated OTA in roasted coffee bean moved into the extract solution when brewed with paper filter.     

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).     

Ochratoxin A Removal by Yeasts after Exposure to Simulated Human Gastrointestinal Conditions

Yeasts can remove ochratoxin A (OTA) in foods; however, we do not know if they can experience this ability in the gut. Thus, the aim of this paper was to study OTA binding by 3 wine strains of Saccharomyces cerevisiae (W13, W28, and the commercial isolate BM45) and S. cerevisiae var. boulardii ATCC MYA‐796 (a probiotic yeast) after exposure to conditions simulating the passage through human gastrointestinal tract. Yeasts removed OTA by up to 44%, with a better ability found after gastrointestinal and sequential assays. However, the phenomenon was reversible, because ca. 18% to 28% of toxin was not stably bound. After the evaluation of the net amount of OTA bound by yeasts (that is, after toxin release), S. cerevisiae W13 and S. cerevisiae var. boulardii ATCC MYA‐796 were selected as the most promising strains for further studies (for example, clinical trials) due to their ability to remove 21% of OTA.     

植物乳杆菌QH06清除赭曲霉素A的作用及其初步应用

经过筛选、鉴定得到清除赭曲霉素A（Ochratoxin A,OTA）的植物乳杆菌QH06,并进一步研究其清除OTA的途径及其在食品中清除OTA的效果。通过研究植物乳杆菌QH06的活菌、死菌,胞内、胞外代谢物,细胞壁等对赭曲霉素A（OTA）的清除作用探究该乳酸菌清除OTA的途径,并通过分析QH06在葡萄汁中清除OTA的效果对其应用进行评价。结果表明,植物乳杆菌QH06活细胞和死细胞分别与质量浓度为100 ng/mL的OTA共培养48 h后,OTA的清除率分别为83.61%和83.51%;QH06胞内酶和胞外代谢物分别与质量浓度为100 ng/mL的OTA共培养72 h后,OTA质量浓度均在90 ng/mL之上;QH06细胞壁与质量浓度为100 ng/mL的OTA共孵育72 h后,OTA质量浓度由80.75 ng/mL（0 h）降低至13.81 ng/mL,清除率为82.90%。QH06与含质量浓度为100 ng/mL的OTA葡萄汁共培养72 h后,葡萄汁中OTA的质量浓度降低至15.23 ng/mL;在含质量浓度为20 ng/mL OTA的葡萄汁中,QH06对OTA清除率达到100%。以上结果表明植物乳杆菌QH06通过细胞壁吸附作用清除食品中OTA污染,为其在防治食品中OTA污染方面的应用提供了理论基础。