Ochratoxin A in Brazilian roasted and instant coffees

Thirty-four samples of roast and ground coffee, 14 samples of instant coffee and two samples of decaffeinated instant coffee were collected in markets and supermarkets in the city of Campinas, Brazil, and analysed for ochratoxin A using immunoaffinity columns for clean-up and HPLC with fluorescence detection for quantification. The limit of detection was 0.2 ng/g ochratoxin A. Twenty-three samples of ground and roast coffee were found to be contaminated with the toxin at levels ranging between 0.3 and 6.5 ng/g. The average concentration in all 34 samples was 0.9 ng/g. All samples of instant coffee contained ochratoxin A at levels ranging from 0.5 to 5.1 ng/g, with an average figure of 2.2 ng/g. Roast and ground coffee is the type of coffee most used by Brazilians for the preparation of the beverage. Considering that an average Brazilian adult takes five cups of coffee per day, which corresponds to 30 g of roast and ground coffee, the probable daily intake of ochratoxin A by a 70 kg adult would be 0.4 ng/kg bw, which is far below the current Provisional Tolerable Daily Intake of 14 ng/kg bw for ochratoxin A as set by the Codex Alimentarius. To study the transfer of ochratoxin A into coffee brew, the beverage was prepared by two methods: (a) the drip method and (b) the Brazilian country style method. No significant difference was observed between the two methods in terms of extraction of the toxin using five contaminated samples containing between 0.8 and 6.5 ng/g ochratoxin A. The drip method extracted 86 +/- 15% and the Brazilian country style 74 +/- 20% of the ochratoxin A initially present in the roast and ground coffee.     

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.     

Survey for aflatoxins, ochratoxin A, zearalenone and fumonisins in maize imported into the United Kingdom

This survey examined 140 samples of raw maize as received at ports or at major maize mills in the UK and 12 after initial cleaning. Samples were examined for aflatoxins B₁, B₂, G₁ and G₂, ochratoxin A, zearalenone and fumonisins B₁, B₂ and B₃ using fully validated analytical HPL C methods with detection limits of 0.1 mu g/kg for each aflatoxin and ochratoxin A, 4 mu g/ kg for zearalenone and 10 mu g/kg for each fumonisin. 95.0% and 92.1% of samples met the new EC statutory maximum permissible level for total aflatoxins and aflatoxin B₁ respectively. The maximum concentration of ochratoxin A found was 1.5 mu g/kg. Zearalenone and fumonisins were detected in almost every sample with 41.7% of maize containing more than 100 mu g/kg of zearalenone and 48% of samples containing more than 1000 mu g/kg total fumonisins. Initial cleaning of raw maize reduced aflatoxin concentrations by about 40% and total fumonisins by 32%.     

Ochratoxin A contamination of cereal grain harvested in 2003 and 2004 years

The analysis of ochratoxin A--mycotoxin produced by widely distributed Aspergillus and Penicillium--of cereal grain harvested in 2003 - 2004 years were performed by immunoaffinity column clean-up and HPLC with fluorescence detection. This survey examined 282 samples of raw grain wheat, rye, barley and oat 13.8% of all samples contaminated by ochratoxin A in the range 0.2-33.3 mg/kg. Calculation made on the basis of the obtained means showed that the daily ochratoxin A intake of human from cereal grain were from 1.58 to 2.84 ng/kg b.w. Scientific Committee for Food of the European Commission suggested that it was prudent to reduce exposure to ochratoxin A as much as possible below 5 ng/kg bw/day. Codex Alimentarius and European Commission have established maximum permissible level of 5 mg/kg for ochratoxin A in raw cereal grains.     

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.     

Sample comminution for mycotoxin analysis: Dry milling or slurry mixing?

A comparison was made between dry milling and slurry mixing as a comminuting step preceding mycotoxin analysis. Sample schemes of up to 30 kg are mandated by European Commission legislation. Cocoa, green coffee, almonds and pistachio samples of 10 kg were milled by a Romer analytical sampling mill and all three subsamples were analysed for aflatoxin B₁ or ochratoxin A content. The homogenization process was evaluated in terms of the analytical results, coefficients of variation for different mills and particle size distributions. Coefficients of variation for the comminuting step were higher for dry milling than for slurry mixing. This difference was explained based on measured particle size distributions for both milling types. Measurements also showed slight differences in mycotoxin content of samples based on milling procedures. This might lead to lots being wrongly accepted or rejected based on an erroneous subsample result. It was concluded that sample comminution was best performed by slurry mixing, which produced smaller particles and, consequently, homogeneous samples with lowest coefficients of variation. Additional data are given on analytical results in 10-kg subsamples that originate from the aggregate 30-kg sample as described in Commission Directive 98/53/EC.     

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.     

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.     

Isolation, identification and toxigenic potential of ochratoxin A-producing Aspergillus species from coffee beans grown in two regions of Thailand

In 2006 and 2007, 32 Thai dried coffee bean samples (Coffea arabica) from two growing sites of Chiang Mai Province, and 32 Thai dried coffee bean samples (Coffea canephora var. robusta) from two growing sites of Chumphon Province, Thailand, were collected and assessed for the distribution of fungi with the potential to produce ochratoxin A (OTA). The overall percentage of fungal contamination in coffee was 98% and reduced to 60% after surface disinfection. There were remarkable ecological differences in the composition of ochratoxigenic species present in these two regions. Arabica coffee bean samples from the North had an average of 78% incidence of colonization with Aspergillus of section Circumdati with Aspergillus westerdijkiae and A. melleus as the predominant species. Aspergillus spp. of section Nigri were found in 75% of the samples whereas A. ochraceus was not detected. Robusta coffee beans from the South were 98-100% contaminated with predominantly A. carbonarius and A. niger. A. westerdijkiae was only found in one sample. The diversity of the fungal population was probably correlated with the geographical origin of the coffee, coffee cultivar, and processing method. Representative isolates of section Circumdati (52) and Nigri (82) were examined for their OTA production using HPLC with fluorescence detection. Aspergillus westerdijkiae (42 isolates out of 42), A. steynii (13/13), and A. carbonarius (35/35) in general produced large amounts of OTA, while one isolate of A. sclerotiorum produced intermediate amounts of OTA. 13% of the A. niger isolates produced OTA in intermediate amounts. OTA levels in coffee bean samples were analyzed using the Ridascreen OTA ELISA kits. Of the 64 coffee bean samples analyzed, 98% were contaminated with OTA in levels of <0.6-5.5 microg/kg (Arabica) and 1-27 microg/kg (Robusta). Presence of OTA in representative coffee samples was also confirmed by LC-MS/MS after ion-exchange purification.     

Survey of Canadian retail coffees for ochratoxin A

One-hundred and one specimens of coffee were gathered from retail outlets across Canada and analysed for ochratoxin A. Seventy-one specimens were roasted beans or roasted ground coffee, and 30 were instant (or 'soluble') coffees. All samples were extracted with methanol-sodium bicarbonate. The extracts were cleaned up either by immunoaffinity column chromatography or by a combination of solid-phase extraction and immunoaffinity column chromatography. Ochratoxin A was quantified by liquid chromatography (LC) with fluorescence detection. The minimum quantifiable level was 0.1 ng g^-1. Ochratoxin A was present, above the minimum quantifiable level, in 42 (59%) of 71 beans and ground coffee and in 20 (67%) of 30 instant coffees. The mean ochratoxin A level in the positive samples of beans and ground coffee was 0.6 ng g^-1, and the mean level in the positive samples of instant coffee was 1.1 ng g^-1.     

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.     

Ochratoxin formation in Aspergillus ochraceus with particular reference to spoilage of coffee

Production of ochratoxin on media by eight isolates of Aspergillus ochraceus from coffee or its processing environment in India, Indonesia, Kenya, and Brazil, and seven Brazilian isolates from other commodities, has been compared with yields in shaken fermentation on shredded wheat and coffee (Coffea arabica). Shredded wheat most consistently allowed expression of biosynthesis of ochratoxins A and B in yields up to 3.5% of the dry product. Culture on artificial media was an unreliable predictor of ochratoxin yield on both shredded wheat and coffee. Coffee was a relatively poor substrate for ochratoxin production particularly when sterilised. Notably, two Asian coffee isolates produced 400 mg kg(-1) ochratoxin A on unsterilised ground green coffee, showing this to be a preferred substrate for further experimentation. The study focused on isolates of A. ochraceus, which from evidence of culture on media would not be expected to be suitable fungi for future studies to establish both the fact of spoilage of coffee by A. ochraceus and the dynamics of ochratoxin formation by isolates of this species.     

Plasma ochratoxin A levels in three Swedish populations surveyed using an ion-pair HPLC technique.

A new HPLC method for the analysis of ochratoxin A in plasma samples is described. The analysis is performed at an alkaline pH using an ion-pair technique, fluorescence detection at an excitation wavelength 380 nm, and an emission wavelength 420 nm. The detection and quantification limits are 0.02 ng and 0.05 ng ochratoxin A/ml plasma, respectively. The method was used to determine the ochratoxin A content of human plasma samples, collected in three districts of Sweden. The Visby district had a significantly higher proportion of ochratoxin A positive samples and higher levels than the other two districts--Uppsala and Ostersund. The calculated daily intake of ochratoxin A in the Visby district (0.35 ng/kg body weight), exceeds the lower tolerable daily intake (TDI) value suggested by Kuiper-Goodman and Scott (1989). The calculated daily intake by the population on the mainland of Sweden (0.04 ng/kg body weight) is below the proposed TDIs.     

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.     

Surveillance of stored grain from the 1997 harvest in the United Kingdom for ochratoxin A

This survey examined 306 samples of farm-stored wheat, barley and oats as received at, or tested by, central grain depots in the UK. Samples were taken from lorries or from stored grain using the existing inhouse procedures used for quality checking and examined for ochratoxin A using a fully validated analytical HPLC method with a detection limit of 0.1 μg/kg. Ochratoxin A was detected in 21 % of the samples examined, with barley more frequently contaminated than wheat. Mean concentrations of ochratoxin A found for all samples were 0.69 μg/kg in barley, 0.29 μg/kg in wheat and 0.15 μg/kg in oats. The highest concentration found was 17.8 μg/kg in a barley feed although concentrations of 81 and 30 μg/kg were found in 'reject-grade' wheat samples whose results were excluded from the main survey. In summary, 2.7 and 0.3 % of samples exceeded concentrations of 5 and 10 μg/kg respectively. There appeared to be significant relationships between ochratoxin A concentrations and moisture content, storage time and geographical area. Although conditions at harvest in 1997 were quite variable countrywide and often wet, results were similar to those found in earlier surveys carried out in the UK.     

A survey of cereals, cereal products, feedstuffs and porcine kidneys for ochratoxin A by radioimmunoassay

A commercial kit for the radioimmunochemical determination of ochratoxin A has been validated for application to various foods and feedstuffs. Using this kit a survey was carried out for the occurrence of ochratoxin A in Czechoslovak cereals, cereal products, feedstuffs and pig kidneys. The results show that the most contaminated are feedstuffs and cereals, in 26.7% and 8.8% of the samples of which, respectively, the ochratoxin A level exceeded 20 micrograms/kg. None of the cereal products and 4.2% of the pig kidneys unfit for human consumption by visual examination possessed ochratoxin A concentration higher than 5 micrograms/kg. Of all the samples analysed, 57% contained no detectable toxin (less than 1 microgram/kg).     

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.     

Ochratoxin A in dried vine fruit: method development and survey

A method is described for the determination of concentrations of the mycotoxin ochratoxin A in dried vine fruits (currants, raisins and sultanas) using acidic methanolic extraction,immunoaffinity chromatography clean-up and HPLC determination. The limit of detection was estimated as 0.2mug/kg, and recoveries of 63-77% were achieved at 5mug/kg. HPLC-mass spectrometric confirmation of the identity of ochratoxin was obtained. Ochratoxin A and aflatoxins were determined in 60 samples of retail dried vine fruits purchased in the United Kingdom. Ochratoxin A was found in excess of 0.2mug/kg in 19 of 20 currant, 17 of 20 sultana and 17 of 20 raisin samples examined, an overall incidence of 88% . The maximum level found was 53.6mug/kg. No aflatoxin was found in any sample analysed, using a method with a detection limit of 0.2mug/kg for each of aflatoxin B1, B2, G1 and G2.     

Effect of the frequency of the mixing of coffee cherries put out for drying on the kinetics of drying and the relationship to ochratoxin A production

The effect of the frequency of the mixing of coffee cherries put out for sun drying on the kinetics of the drying, fungal growth and kinetics of ochratoxin A production was evaluated. The results showed that the more coffee cherries were mixed, the quicker they dried. This rapidity of drying led to a reduction of fungal development. Indeed, coffee cherries mixed eight and ten times a day, dried quickly and were free inside of fungi. However, infection by fungi gives little indication of ochratoxin A production in coffee cherries. Indeed, although coffee cherries mixed twice a day were more contaminated by fungi, the analysis of ochratoxin A content showed they were free of this mycotoxin. The coffee cherries that were more contaminated by ochratoxin A were those mixed four times a day (containing 0.35-5.46 µg kg^-1 ochratoxin A). Ochratoxin A contamination was essentially due to the presence of Aspergillus species capable of producing ochratoxin A inside the coffee cherries.     

Ochratoxin A in conventional and organic cereal derivatives: a survey of the Italian market, 2001-02.

Ochratoxin A is a mycotoxin produced mainly by Penicillium verrucosum and Aspergillus ochraceus. Although typically considered a cereal contaminant, it has also been detected in dried fruit, nuts, meat and derivatives. To estimate the quantity of ochratoxin A that might be ingested by Italian consumers from these foods, 211 cereal derivatives (flours and bakery products) were analysed by high-performance liquid chromatography. Products were from conventional and organic agriculture and from integrated pest management agriculture. All commercial flours and derivatives examined contained ochratoxin A at concentrations very much below the legal limit (3 microg kg(-1)): the highest value, 0.816 microg kg(-1), was detected in a sample of spelt whole flour from organic agriculture. In many samples, the ochratoxin content was below the limit of detection; only rarely did values exceed 0.5 microg kg(-1). In baby foods, four samples were above the particularly restrictive Italian legal limit of 0.5 microg kg(-1). Although some significant differences were found between samples from conventional and organic agriculture when some product categories were examined (namely, baby foods as semolina and rice creams), no important difference was found between the two types of agricultural practice when all types of cereal derivatives were considered together.